NZ741950B2 - Vaccines against hepatitis b virus - Google Patents

Abstract

The present application provides immunotherapies for Hepatitis B virus infections. Provided herein are genetically modified arenaviral vectors suitable as vaccines for prevention and treatment of Hepatitis B virus infections. Also provided herein are pharmaceutical compositions and methods for the treatment of Hepatitis B virus infections. Specifically, provided herein are pharmaceutical compositions, vaccines, and methods of treating Hepatitis B virus infection.

Description

VACCINES AGAINST HEPATITIS B VIRUS This application claims benefit of U.S. Provisional Patent Application No. 62/250,639, filed November 4, 2015, the disclosure of which is incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE G SUBMITTED ELECTRONICALLY This application orates by reference a Sequence Listing submitted with this application as text file entitled "Sequence_Listing_13194228.TXT" created on November 2, 2016 and having a size of 128,899 bytes. A copy of this sequence listing is also appended to this ication. 1. INTRODUCTION Provided herein are genetically ed arenaviruses suitable as vaccines for prevention and treatment of Hepatitis B virus infections. Also ed herein are pharmaceutical itions and methods for the treatment of tis B virus infections.

Specifically, provided herein are pharmaceutical compositions, es, and methods of treating Hepatitis B virus infections. As such, the present application provides immunotherapies for Hepatitis B virus infections. [0003a] In particular the present invention provides: 1 An infectious arenavirus viral vector, wherein an arenavirus open reading frame is d and replaced by a nucleotide sequence selected from the group consisting a. a nucleotide sequence encoding an HBV pre-S2/S protein or an antigenic fragment thereof; b. a tide sequence encoding an HBV HBc protein or an antigenic fragment thereof; and c. a nucleotide sequence encoding a fusion of HBV HBc and HBs proteins or antigenic fragments thereof. (followed by 1A) 2. The viral vector of claim 1 wherein the pre-S2/S protein or the antigenic nt thereof comprises an amino acid ce that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1. 3. The viral vector of 1 wherein the HBc protein or the nic fragment thereof comprises an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence d by the nucleotide sequence of SEQ ID NO: 2. 4. The viral vector of 1 wherein fusion of HBV HBc and HBs proteins or antigenic fragments thereof comprises an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid ce encoded by the nucleotide sequence of SEQ ID NO: 3.

. The viral vector of 1 comprising at least two of: a. a nucleotide sequence encoding an HBV pre-S2/S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding an HBV HBc protein or an antigenic fragment thereof; and c. a nucleotide sequence encoding a fusion of HBV HBc and HBs proteins or antigenic fragments thereof. 6. The viral vector of 5 wherein expression of the nucleotide sequences es an antigenic protein complex that s higher titers of neutralizing antibodies than expression of the protein x components individually. 7. The viral vector of any one of claims 1 to 6 wherein the arenavirus is lymphocytic choriomeningitis virus, Junin virus, or Pichinde virus. 8. The viral vector as of any one of 1 to 7 wherein the open reading frame that s the glycoprotein of the arenavirus is deleted or functionally inactivated. (followed by 1B) 9. The viral vector of any one of 1 to 9 wherein the genomic information encoding the infectious arenavirus viral vector is derived from the lymphocytic choriomeningitis virus Clone 13 strain.

. The viral vector of any one of claims 1 to 9 wherein the viral vector comprises a genomic segment, wherein the genomic segment comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% cal to the sequence of nucleotide 1639 to 3315 of SEQ ID NO: 11 or 1640 to 3316 of SEQ ID NO: 11. The viral vector of any one of 1 to 10 wherein the viral vector ses a genomic segment comprising a nucleotide sequence encoding an expression product whose amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence d by nucleotides1639 to 3315 of SEQ ID NO: 11 or 1640 to 3316 of SEQ ID NO: 12. 12. The viral vector of any one of 1 to 11 wherein the arenavirus is bi-segmented and replication-deficient. 13. The viral vector of any one of 1 to 7 and 9 to 11, n the arenavirus is trisegmented and replication-competent. 14. The viral vector of any one of 1 to 13, wherein the growth or infectivity of the arenavirus is not affected by the nucleotide sequence.

. A pharmaceutical ition, immunogenic composition, or e, comprising a viral vector as described above and a pharmaceutically acceptable carrier. 16. Use of the viral vector of any one of 1 to 14, the pharmaceutical composition, the immunogenic composition, or the vaccine of 15 in the manufacture of a ment for treating or preventing a Hepatitis B virus infection in a patient by administration of the viral vector of any one of 1 to 14, the pharmaceutical composition, the immunogenic ition, or the vaccine of 15 to the patient. 17. The use of 16, wherein the administration of the viral , pharmaceutical composition, immunogenic composition, or vaccine is by intramuscular injection or intravenous injection. (followed by 1C) 18. An isolated nucleic acid, wherein the nucleic acid comprises an arenavirus genomic segment n one open reading frame of the genomic segment is d or functionally inactivated and wherein the genomic segment comprises one or more of: a. a nucleotide sequence encoding an HBV pre-S2/S protein or an antigenic fragment thereof; b. a nucleotide sequence ng an HBV HBc protein or an antigenic fragment thereof; and c. a nucleotide sequence encoding a fusion of HBV HBc and HBs proteins or antigenic nts thereof. 19. The isolated nucleic acid of 18, wherein the arenavirus genomic segment is the short segment, wherein the open reading frame ng the glycoprotein is deleted.

. A method for generating an infectious, replication-deficient arenavirus viral vector comprising: a. transfecting into a host cell, provided said host cell is not within a human, the nucleic acid of 18 or 19; b. maintaining the host cell under conditions suitable for virus formation; and c. harvesting the infectious, replication-deficient arenavirus viral vector; wherein the host cell expresses the open reading frame that is deleted or functionally inactivated of the genomic t. 21. The arenavirus viral vector of 1, wherein the arenavirus open reading frame is the glycoprotein open reading frame. 22. An infectious, replication-deficient arenavirus viral vector ered to contain a genome with the ability to y and s its genetic information in infected cells but unable to produce further infectious y particles in normal, not genetically (followed by 1D) engineered cells, wherein one arenavirus open reading frame is removed and replaced by a tide sequence encoding an HBV antigen or an antigenic fragment thereof, wherein the HBV antigen or antigenic nt f is selected from the group ting of: i. an HBV /S protein or an antigenic fragment f; ii. an HBV HBc protein or an antigenic fragment thereof; and iii. a fusion of HBV HBc and HBs proteins or antigenic fragments thereof; and a. wherein administration of the irus viral vector to a subject induces a longlasting immune response against the HBV antigen or an antigenic fragment thereof, wherein (i) the long-lasting immune response induces a detectable antibody titer against the HBV antigen or the antigenic fragment thereof; or (ii) the long-lasting immune response s a detectable antibody titer against the HBV antigen or the nic fragment f for at least a minimum of 4 weeks; or b. wherein administration of the arenavirus viral vector to a subject infected with an HBV infection increases the antibody titer against the HBV antigen or the antigenic fragment thereof by at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%. 23. A pharmaceutical composition comprising a first infectious, replicationdeficient arenavirus viral vector engineered to contain a genome with the ability to amplify and express its genetic information in ed cells but unable to produce further infectious progeny particles in normal, not genetically engineered cells, wherein one arenavirus open reading frame is removed and replaced by a first nucleotide sequence selected from the group consisting of: a. a tide sequence ng an HBV pre-S2/S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding an HBV HBc protein or an antigenic fragment thereof; and c. a nucleotide sequence encoding a fusion of HBV HBc and HBs proteins or antigenic fragments thereof; and a second infectious, ation-deficient arenavirus viral vector engineered to contain a genome with the y to amplify and express its genetic information in infected cells but unable to produce further infectious progeny particles in normal, not genetically (followed by 1E) engineered cells, wherein one irus open reading frame is removed and replaced by a second nucleotide sequence selected from the group consisting of: a. a nucleotide sequence encoding an HBV pre-S2/S protein or an antigenic fragment thereof; b. a tide sequence encoding an HBV HBc protein or an antigenic fragment thereof; and c. a nucleotide sequence encoding a fusion of HBV HBc and HBs proteins or antigenic fragments thereof; wherein the first and second nucleotide sequences are ent. 24. The pharmaceutical composition of 23, wherein: a. the first nucleotide sequence encodes the HBV pre-S2/S protein or the antigenic fragment thereof, and wherein the second nucleotide sequence s the HBV HBc protein or a fragment thereof; or b. the first tide sequence encodes the HBV pre-S2/S protein or the antigenic fragment thereof, and wherein the second nucleotide sequence s the fusion of the HBV HBc and HBs proteins or antigenic fragments thereof; or c. the first nucleotide sequence encodes the HBV HBc protein or the nic fragment thereof, and wherein the second nucleotide sequence encodes the fusion of the HBV HBs and HBc proteins or antigenic fragments thereof.

. The pharmaceutical composition of 23 or 24, wherein the composition is suitable for intramuscular administration or intravenous administration. 26. The method of 20 wherein the method r comprises in step a. transfecting into the host cell: a cDNA of the second arenavirus genomic segment, a nucleic acid comprising the L protein ORF, and/or a nucleic acid sing the NP n ORF. (followed by 1F) 27. The use of 16, wherein: a. said administration s in a ion in liver damage in the patient; b. said administration results in a reduction in one or more of HBsAg, HBeAg, and HBcAg levels in the blood of the patient; or c. said administration results in a reduction in the level of an antibody against an HBV antigen in the blood of the patient. 28. The viral vector of any one of 1 to 12 or 14, wherein the arenavirus is replication-deficient and is engineered to contain a genome with the ability to amplify and express its c information in infected cells but unable to produce further infectious progeny particles in normal, not genetically engineered cells. 29. The viral vector of any one of 1 to 7, wherein the viral vector is replicationcompetent.

. An infectious arenavirus viral vector according to 1, wherein an arenavirus open reading frame is removed and replaced by a nucleotide sequence encoding a fusion of HBV HBc and HBs proteins or antigenic fragments thereof. 31. The viral vector of 30, wherein the arenavirus is cytic choriomeningitis virus or Pichinde virus. 32. The viral vector of 30 or 31, n the open g frame that encodes the glycoprotein of the arenavirus is deleted or functionally inactivated. 33. The viral vector of any one of 30 to 31, wherein the viral vector is replicationdeficient. 34. The viral vector of 30 or 31, wherein the viral vector is replication-competent. (followed by 1G) . Use of the viral vector of any one of 30 to 34 in the cture of a medicament for treating or preventing a tis B virus infection or cancer in a t.

] These and other embodiments are described herein in more detail below.

Certain aspects and embodiments are not the subject of the present application, although the disclosure is retained herein for completeness. 2. BACKGROUND 2.1 The pathogen and the disease Hepatitis B virus (HBV) is a double-stranded enveloped virus of the Hepadnaviridae family. The virus particle consists of an outer lipid envelope and an icosahedral nucleocapsid core composed of protein. The nucleocapsid encloses the viral DNA and a DNA polymerase that has reverse transcriptase activity. The outer envelope contains embedded proteins which are involved in viral binding of, and entry into, susceptible cells. HBV ates in the hepatocytes of humans and other higher primates, but does not grow in artificial cell es.

The outcomes of HBV infection are age-dependent and e asymptomatic infection, acute hepatitis B, chronic HBV infection, cirrhosis and hepatocellular carcinoma (HCC). Acute hepatitis B occurs in approximately 1% of perinatal infections, 10% of early (followed by 2) childhood infections (children aged 1—5 years) and 30% of late infections (people aged >5 years). Fulminant hepatitis develops in 01—06% of acute hepatitis cases; mortality from fulminant hepatitis B is approximately 70%. The development of chronic HBV infection is inversely related to the age of acquisition, occurring in approximately 80—90% ofpeople infected perinatally, about 30% of children infected before the age of 6 years, and in <5% of infections occurring in otherwise healthy adults (Hyams et al., 1995, Clinical Infections Diseases - 1000). Comorbidities, including concurrent HIV infection and ingestion of alcohol or a?otoxins, or both, may have an important role in the development of morbidity d to hepatitis B. It is estimated that 10% of the 40 million people infected with HIV worldwide are cted with People with chronic HBV infection have a 15—25% risk of dying prematurely from HBV-related cirrhosis and HCC (Beasley and Hwang, 1991, Proceedings of the 1990 International Symposium on Viral Hepatitis and Liver Disease: Contemporary Issues and Future Prospects 532-535). Acute HBV infection is characterized by the presence of HBsAg, the surface n of HBV, and immunoglobulin M (IgM) antibody to the core antigen, HBcAg.

During the l, highly replicative phase of infection, patients are also seropositive for HBeAg, the extracellular and secreted form of HBcAg which can be found in the serum of ts where it serves as a marker of active replication in chronic hepatitis. Antibody to HBsAg (anti-HBs) is discernible after a few weeks and is followed by clearance of the HBsAg. Chronic infection is terized by the persistence (>6 months) of HBsAg (with or without concurrent HBeAg).

Persistence of HBsAg is the principal marker of risk for developing chronic liver disease and HCC later in life. The presence of HBeAg indicates that the blood and body ?uids of the infected dual are highly contagious. 2.2 Epidemiology and public health Diseases caused by the hepatitis B virus have a worldwide distribution. It is estimated that two billion people have at some time been infected with HBV. Of these, approximately 360 million individuals are chronically infected and at risk of serious s and death, mainly from liver cirrhosis and hepatocellular carcinoma (HCC). Mathematical modeling for the year 2000 estimated the number of deaths from lated diseases at about 600 000 each year worldwide (Goldstein et al., 2005, ational J. iology 34: 1329-1339).

Humans are the only reservoir of HBV. The virus is transmitted by percutaneous and permucosal exposure to infected blood and other body ?uids, mainly semen and l ?uid. The incubation period is 75 days on average, but may vary from about 30 days to 180 days. The surface antigen ofHBV (HBsAg) may be detected in serum 30—60 days following infection and may persist for widely variable s of time. The endemicity of hepatitis B is described by the prevalence of HBsAg in the general population of a de?ned geographical area, and it varies considerably globally: HBsAg prevalences of >8% are typical of highly endemic areas, prevalences of 2—7% are found in areas of intermediate endemicity, whereas in areas with low endemicity <2% ofthe population is HBsAg-positive.

In highly endemic areas, HBV is most commonly spread from mother to child at birth, or from person to person in early childhood (Goldstein et al., 2005, International J.

Epidemiology 34:1329-1339; Wong et al., 1984, Lancet 12921-926; de la Hoz et al., 2008 International J. Infectious Diseases 12:183-189). Perinatal or early childhood transmission may also account for more than one third of chronic infections in areas of low endemicity (Margolis et al., 1995, JAMA 274: 1201-1208) although in those settings, sexual transmission and the use of contaminated needles, especially among injecting drug users, are the major routes of infection (Goldstein et al., 2002, J. Infectious Diseases 185:713-719). 2.3 t treatment Universal hepatitis B vaccination has been shown to reduce the rates of HBV infection and HCC significantly. However, once chronic HBV infection is established, treatment still poses a major nge as traditional therapies usually fail to provide sustained control of viral replication and liver damage in most patients.

Currently approved antiviral treatments for chronic hepatitis B include pegylated (PEG) recombinant interferon-0t and viral DNA polymerase inhibitors. These agents decrease viral ation and have been shown to delay progression of cirrhosis, reduce the incidence of HCC and improve long-term survival. However, treatment is complicated by the ty of the agents and it can only cure a small subset of cally infected duals. Although viral levels in the blood plummet to almost ctable levels in individuals receiving rd therapies, reductions of intrahepatic viral DNA are only modest. As a uence, rebound of viraemia frequently occurs after tinuation of treatment and people with chronic HBV infections must stay on lifelong ent. However, even after ten years on antiviral therapy, drugs reduce liver e by only 40—70%, and ity from cirrhosis and liver cancer remains high. 2.4 Hepatitis B and the immune system Chronic hepatitis B infection is characterized by ctional innate and adaptive antiviral immunity (Bertoletti & Ferrari, 2012, Gut 61 :1754-1764). In st, HBV- specific immunity in patients with resolved HBV infection is robust and multi?inctional. Several mechanisms might contribute to the dysfunction of HBV-speci?c T-cell immunity in chronic tis B patients, including high levels of viral antigenaemia, and the tolerizing microenvironment of the liver (Jenne & Kubes, 2013, Nat. Immunol. 14:996-1006). Previous studies have demonstrated that suppression of viral replication can transiently and partially restore antiviral T-cell immunity, which supports the hypothesis that long-term exposure to high levels of antigenaemia might cause dysfunction of antiviral T cells (Boni et al., 2003, J. Hepatol. 39:595—605).

Therapeutic vaccines that could reverse the dysfunctional immune state of chronic hepatitis B and restore ral ty, would theoretically have the potential to eliminate viremia and reduce intrahepatic levels ofHBV DNA to zero, thus holding great promise for HBV cure.

Recently, HBV vaccines have been identi?ed as a promising therapeutic strategy for treatment and l of HBV ion in HBV carriers and persistently infected patients (Michel & Tiollais, 2010, Pathol. Biol. (Paris) 582288-295; Liu et al., 2014, Virol. Sin. 29:10- 16). In about 50% of chronic active HBV patients speci?c therapy by conventional anti-HBV vaccination effectively d the replication of HBV and inhibited the immune tolerance to HBsAg protein (Couillin et al., 1999, J. Infect. Dis. 180:15-26). However, so far monotherapy with HBsAg based vaccines did not lead to sustained control ofHBV replication and/or liver damage (Akbar et al., 2013, Hepatobiliary Pancreat. Dis. Int. -369) and new therapy strategies are needed to provide potent and durable antiviral immune responses and long—term control ofHBV replication.

The failure of previous therapeutic vaccine approaches ghts the challenges and limitations of current knowledge regarding immune responses in chronic HBV infection (Michel et al., 2011, J. Hepatol. 54:1286-1296). The combination of a high viral load condition such as c hepatitis B with the tolerizing liver microenvironment might make it dif?cult to achieve ?lll ry of antiviral T-cell immunity.

Intensive ch is currently trated on a better understanding of immune responses in hepatocytes, on mechanisms by which HBV evades innate immunity and on proper selection of patients tible to bene?t from immune therapy, which could increase the ef?cacy oftherapeutic vaccination (Michel et al., 2015, Med. Microbiol. Immunol. 204:121- 129). 3. SUMMARY OF THE INVENTION The present application es immunotherapies for Hepatitis B virus infections. Provided herein is an infectious arenavirus viral vector comprising a nucleotide sequence selected from the group ting of: a. a nucleotide sequence encoding an HBV /S protein or an antigenic fragment thereof; b. a nucleotide ce encoding an HBV HBc n or an antigenic fragment thereof; 0. a tide sequence encoding an HBV HBs protein or an antigenic fragment thereof; d. a nucleotide sequence encoding a fusion ofHBV HBs and HBc proteins or antigenic fragments thereof; and e. a nucleotide sequence encoding an HBV HBe protein or an antigenic fragment thereof.

In certain embodiments, the infectious arenavirus viral vector is replication-de?cient (See Section 6. 1(a)). In certain embodiments, the infectious irus viral vector is replication- competent (See Section 6.1(b)). In certain embodiments, the infectious, replication-de?cient arenavirus viral vector is bisegmented. In certain embodiments, the infectious, replication- de?cient arenavirus viral vector is trisegmented. In certain embodiments, the infectious, replication-competent arenavirus viral vector is trisegmented.

In certain embodiments, provided herein is an arenavirus viral vector comprising a nucleotide sequence selected from the group consisting of: a. a nucleotide sequence encoding an HBV /S protein or an antigenic fragment f; b. a nucleotide sequence encoding an HBV HBc protein or an antigenic fragment thereof; 0. a nucleotide sequence encoding an HBV HBs protein or an antigenic fragment thereof; d. a nucleotide sequence ng a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and e. a nucleotide sequence encoding an HBV HBe protein or an antigenic nt thereof.

In certain embodiments, the arenavirus viral vector is replication-deficient. In certain embodiments, the arenavirus viral vector is replication-competent.

In n embodiments, a viral vector as provided herein is infectious, i.e., is capable of entering into or injecting its genetic material into a host cell. In certain more speci?c embodiments, a viral vector as provided herein is infectious, i.e., is capable of entering into or injecting its genetic al into a host cell followed by amplification and expression of its genetic information inside the host cell. In certain ments, the viral vector is an infectious, replication-de?cient irus Viral vector ered to contain a genome with the ability to amplify and express its genetic information in infected cells but unable to produce further infectious progeny particles in normal, not genetically engineered cells. In certain embodiments, provided herein is a cell line that supports viral growth of a wild type virus but does not express the complementing viral n, thus is unable to produce further infectious viral progeny particles. In certain embodiments, the infectious arenavirus viral vector is replication-competent and able to produce further infectious progeny particles in normal, not genetically engineered cells.

In certain embodiments, the /S protein or the antigenic fragment thereof comprises an amino acid ce that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the nucleotide ce of SEQ ID NO: 1. In certain embodiments, the fragment is antigenic when it is capable of (i) eliciting an antibody immune se in a host (e.g., mouse, rabbit, goat, or donkey) wherein the resulting antibodies bind speci?cally to human HBV pre-S2/S protein; and/or (ii) eliciting a speci?c T cell immune response.

In certain embodiments, the HBc protein or the antigenic fragment thereof comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence d by the nucleotide sequence of SEQ ID NO: 2. In certain embodiments, the fragment is antigenic when it is capable of (i) eliciting an antibody immune response in a host (e.g., mouse, rabbit, goat, or donkey) wherein the resulting antibodies bind speci?cally to human HBV HBc protein; and/or (ii) eliciting a speci?c T cell immune se.

In certain embodiments, the ?ision ofHBV HBs and HBc proteins or antigenic fragments thereof comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% cal to an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3. In certain embodiments, the fragment is nic when it is capable of (i) eliciting an antibody immune response in a host (e.g., mouse, rabbit, goat, or donkey) wherein the resulting antibodies bind cally to human HBV HBs, HBc or both HBs and HBc; and/or (ii) eliciting a speci?c T cell immune response.

In certain embodiments, the HBe protein or the nic fragment thereof comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the nucleotide ce of SEQ ID NO: 26. In certain embodiments, the fragment is antigenic when it is capable of (i) ing an antibody immune response in a host (e.g., mouse, rabbit, goat, or donkey) wherein the ing antibodies bind speci?cally to human HBV HBe protein; and/or (ii) eliciting a speci?c T cell immune response.

In certain embodiments, the Viral vector comprises at least two of: a. a nucleotide sequence encoding an HBV pre—S2/S protein or an antigenic fragment thereof; b. a tide sequence encoding an HBV HBc protein or an antigenic nt thereof; c. a nucleotide sequence encoding an HBV HBs protein or an antigenic fragment thereof; d. a nucleotide sequence ng a fusion ofHBV HBs and HBc proteins or antigenic fragments thereof; and e. a nucleotide sequence encoding an HBV HBe protein or an antigenic fragment thereof.

In certain embodiments, the viral vector comprises at least three of: a. a nucleotide sequence encoding an HBV /S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding an HBV HBc protein or an antigenic fragment thereof; 0. a nucleotide sequence encoding an HBV HBs protein or an antigenic nt thereof; (1. a nucleotide sequence ng a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and e. a nucleotide sequence encoding an HBV HBe protein or an antigenic fragment thereof.

In certain embodiments, an open reading frame (ORF) of the irus is deleted or ?anctionally inactivated and replaced with a nucleic acid encoding an HBV antigen as described herein. In a speci?c embodiment, the ORF that encodes the glycoprotein GP ofthe irus is deleted or functionally inactivated. In certain embodiments, functional inactivation of a gene eliminates any ation product. In certain embodiments, functional inactivation refers to a genetic tion that allows some ation, the translation product, r, is not longer ?inctional and cannot replace the wild type protein.

In certain embodiments, the viral vector can amplify and express its genetic information in a cell that has been infected by the viral vector but the viral vector is unable to produce further infectious progeny particles in a non-complementing cell. In certain ments, a viral vector as provided herein is infectious, i.e., is capable of entering into or injecting its genetic material into a host cell. In certain more speci?c embodiments, a viral vector as provided herein is ious, i.e., is capable of entering into or injecting its genetic al into a host cell followed by ampli?cation and expression of its genetic information inside the host cell.

In certain embodiments, the genomic information encoding the ious arenavirus particle is derived from the lymphocytic choriomeningitis virus (LCMV) Clone 13 strain or the LCMV MP strain. The nucleotide sequence of the S segment and of the L segment of Clone 13 are set forth in SEQ ID NOs: 12 and 7, respectively.

In certain embodiments, provided herein is a viral vector whose genome is or has been d from the genome of Clone 13 (SEQ ID NOs: 12 and 7) by deleting an ORF of the Clone 13 genome (e.g., the ORF of the GP protein) and replacing it with a heterologous ORF that encodes an antigen (e.g., an HBV antigen) such that the remaining LCMV genome is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the nucleotide sequence of Clone 13 (SEQ ID N03: 12 and 7).

In certain embodiments, provided herein is a viral vector whose genome has been derived from the genome of the LCMV strain MP (SEQ ID NOs: 13 and 14) by deleting an ORF ofthe LCMV strain MP genome (e.g., the ORF of the GP n) and replacing it with a heterologous ORF that encodes an antigen (e.g., an HBV antigen) such that the remaining LCMV genome is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, at least 99.9% or 100% identical to the nucleotide sequence ofLCMV strain MP (SEQ ID NOS: 13 and 14).

In a more speci?c embodiment, the Viral vector ses a genomic segment, wherein the genomic segment comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the sequence of nucleotide 1639 to 3315 of SEQ ID NO: 11 or 1640 to 3316 of SEQ ID NO: 12. In certain ments, the viral vector comprises a genomic segment sing a nucleotide sequence encoding an expression product whose amino acid sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence encoded by 1639 to 3315 of SEQ ID NO: 11 or 1640 to 3316 ofSEQ ID NO: 12.

Also provided herein are isolated nucleic acids, wherein the nucleic acid is a cDNA of an arenavirus c segment wherein one ORF of the genomic segment is deleted or functionally inactivated and wherein the genomic segment comprises one or any combination of: a. a nucleotide sequence ng an HBV pre-SZ/S protein or an antigenic fragment f; b. a nucleotide sequence encoding an HBV HBc protein or an nic fragment thereof; c. a tide sequence encoding an HBV HBs protein or an antigenic fragment thereof; (1. a nucleotide ce ng a fusion ofHBV HBs and HBc proteins or antigenic fragments thereof; and e. a nucleotide ce ng an HBV HBe protein or an antigenic fragment thereof.

In certain embodiments, the genomic segment is the short segment, wherein the ORF encoding the GP is deleted.

In one aspect, provided herein are methods for generating an infectious, replication-de?cient arenavirus particle comprising: a. transfecting into a host cell a nucleic acid bed herein; b. maintaining the host cell under conditions suitable for virus formation; and c. harvesting the infectious, replication-de?cient arenavirus le; wherein the host cell expresses the ORF that is deleted or ?mctionally inactivated on the genomic segment. In certain embodiments, any additional nucleic acids required for the rescue of a viral particle are also transfected into the host cell in step a. Such additional nucleic acids can be: the cDNA of the second arenavirus genomic segment, a nucleic acid comprising the L ORF, and/or a nucleic acid comprising the NP ORF.

In another aspect, provided herein are compositions, e.g., pharmaceutical, immunogenic or e compositions, comprising a viral vector described herein and a pharmaceutically acceptable r. Also provided herein are compositions (e. g., vaccine compositions) that comprise two or more different viral vectors described herein (i.e., wherein the viral vectors encode ent HBV antigens). In certain embodiments, the pharmaceutical composition comprises a c acid or fusion protein described herein.

In a ?lrther aspect, provided herein are methods of treating or preventing HBV infection in a patient, comprising administering to the patient a viral vector, a pharmaceutical composition, an genic composition, or a vaccine described herein. In yet another aspect, ed herein is use of a viral vector, a pharmaceutical composition, an immunogenic composition, or a vaccine bed herein for the treatment or prevention of HBV. In certain embodiments, an infectious arenavirus expressing an HBV antigen or a fragment thereof is capable of preventing transmission and/or infection of HBV from a mother to an unborn child.

In certain embodiments, one or more infectious arenaviruses expressing an HBV antigen or a fragment thereof are capable of preventing transmission and/or infection of HBV from a mother to an unborn child. In certain embodiments, the infectious arenavirus viral vector is replication- de?cient (See Section 6.1(a)). In certain embodiments, the infectious irus viral vector is replication-competent (See Section 6.1(b)).

In certain embodiments, administering to a t an infectious arenavirus expressing an HBV antigen or a fragment thereof induces a long-lasting immune se. In certain embodiments, the infectious arenavirus viral vector is replication-de?cient (See Section 6.1(a)). In certain embodiments, the infectious arenavirus Viral vector is replication—competent (See Section 6.1(b)).

In n embodiments, provided herein are methods of treating and or preventing HBV ion in a patient, comprising stering to the patient two or more arenaviruses expressing an HBV antigen or fragment thereof. In a more speci?c embodiment, each arenavirus expresses a ent HBV antigen or fragment thereof In other embodiments, each irus expresses an HBV antigen or a derivative thereof. In some embodiments the derivative thereof is an HBV antigen fragment. In yet another embodiment ed herein are itions that comprise two or more arenaviruses each expressing a different HBV antigen or fragment thereof. In certain embodiments, the infectious arenavirus viral vector is replication- deficient (See Section 6. 1(a)). In certain embodiments, the infectious arenavirus viral vector is replication-competent (See Section 6.1(b)).

In certain embodiments, the irus is lymphocytic choriomeningitis virus (LCMV) or Junin virus (JUNV).

In certain embodiments, provided herein is an infectious arenavirus Viral vector, wherein an arenavirus open g frame is removed and replaced by a nucleotide sequence encoding a fusion ofHBV HBs and HBc proteins or antigenic fragments thereof In speci?c embodiments, the arenavirus is cytic choriomeningitis virus. In speci?c embodiments, the open reading frame that encodes the rotein of the arenavirus is d or functionally inactivated. In speci?c embodiments, the viral vector is replication-de?cient. In speci?c embodiments, the viral vector is replication-competent. In speci?c embodiments, the viral vector is trisegmented. In certain embodiments, provided herein is a method of treating or preventing a Hepatitis B virus infection in a patient, n said method ses administering to the patient the viral vector from which an arenavirus open reading frame is removed and replaced by a nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or nic fragments thereof. 3.1 Conventions and Abbreviations AFP Alpha-fetoprotein ALT Alanine aminotransferase APC Antigen presenting cells AST Aspartate aminotransferase C-cell Complementing cell line CD4 Cluster of Differentiation 4 CD8 Cluster of Differentiation 8 CMI Cell-mediated immunity GS-plasmid Plasmid expressing genome segments HBc or HBcAg HBV core antigen HBe or HBeAg Extracellular HBV core n HBs or HBsAg HBV (large) surface antigen HBV Hepatitis B virus HCC Hepatocellular carcinoma HRP Horse radish peroxidase IFN-y Interferon-y IGR Intergenic region JUNV Junin virus LCMV Lymphocytic choriomeningitis virus LDH Lactate dehydrogenase MHC Major Histocompatibility Complex NP protein ORF Open reading frame Pre-SZ/S HBV middle surface antigen TF-plasmid Plasmid expressing transacting factors TNF-or Tumor necrosis factor-a UTR Untranslated region Z Matrix Protein from LCMV 4. DESCRIPTION OF THE SEQUENCE LISTING The following sequences are illustrative amino acid sequences and nucleotide sequences that can be used with the methods and compositions described herein. In some ces a DNA sequence is used to describe the RNA sequence of a viral genomic segment.

The RNA sequence can be readily deduced from the DNA sequence. The sequences themselves may also be found in Table 3 of Section 6.10.

SEQ ID NO: 1 is the tide sequence of the HBV /S ORF.

SEQ ID NO: 2 is the nucleotide sequence of the HBV HBc ORF.

SEQ ID NO: 3 is the nucleotide sequence of the HBV HBs—HBc fusion protein SEQ ID NO: 4 is the nucleotide ce of the LCMV S t expressing HBV HBs-HBc fusion protein in cDNA form. The genomic segment is RNA, the sequence in SEQ ID NO:4 is shown for DNA; however, exchanging all thymidines ("T") in SEQ ID NO:4 for uridines ("U") provides the RNA sequence.

SEQ ID NO: 5 is the nucleotide sequence of the LCMV S segment expressing the HBc ORF, in cDNA form. The genomic segment is RNA, the sequence in SEQ ID NO:5 is shown for DNA; however, exchanging all thymidines ("T") in SEQ ID N025 for uridines ("U") provides the RNA sequence.

SEQ ID NO: 6 is the nucleotide sequence of the LCMV S segment expressing the /S ORF, in cDNA form. The genomic segment is RNA, the sequence in SEQ ID NO:6 is shown for DNA; however, exchanging all ines ("T") in SEQ ID NO:6 for uridines ("U") provides the RNA sequence.

SEQ ID NO: 7 is the lymphocytic choriomeningitis virus clone 13 segment L, complete sequence (GenBank: DQ361066.l). The genomic segment is RNA, the sequence in SEQ ID NO: 7 is shown for DNA; however, exchanging all ines ("T") in SEQ ID NO: 7 for es ("U") es the RNA sequence.

SEQ ID NO: 8 is the amino acid sequence of an HBV HBs protein-derived epitope.

SEQ ID NO: 9 is the amino acid sequence of an HBV HBs protein-derived epitope.

SEQ ID NO: 10 is the amino acid sequence of an HBV HBc protein-derived epitope.

SEQ ID NO: 11 is the lymphocytic choriomeningitis virus segment S, complete ce. The genomic segment is RNA, the sequence in SEQ ID NO: 11 is shown for DNA; however, exchanging all thymidines ("T") in SEQ ID NO: 11 for uridines ("U") provides the RNA sequence.

SEQ ID NO: 12 is the lymphocytic choriomeningitis virus clone 13 segment S, complete sequence nk: DQ361065.2). The genomic segment is RNA, the sequence in SEQ ID NO: 12 is shown for DNA; however, exchanging all thymidines ("T") in SEQ ID NO: 12 for uridines ("U") provides the RNA sequence.

SEQ ID NO: 13 is the lymphocytic choriomeningitis strain MP segment L, complete sequence. The genomic t is RNA, the sequence in SEQ ID NO:13 is shown for DNA; however, exchanging all thymidines ("T") in SEQ ID NO:l3 for es ("U") provides the RNA sequence.

SEQ ID NO: 14 is the cytic choriomeningitis strain MP segment S, te sequence. The genomic segment is RNA, the sequence in SEQ ID NO:14 is shown for DNA; however, exchanging all thymidines ("T") in SEQ ID NO:14 for uridines ("U") provides the RNA sequence.

SEQ ID NO: 15 is the amino acid sequence of the NP protein of the MP strain of LCMV.

SEQ ID NO: 16 is the amino acid sequence of the GP protein of the MP strain of LCMV.

SEQ ID NO: 17 is the amino acid sequence of the L protein of the MP strain of LCMV.

SEQ ID NO: 18 is the amino acid sequence of the Z protein of the MP strain of LCMV.

SEQ ID NO: 19 is Junin Virus Candid #1 strain segment L, complete sequence.

SEQ ID NO: 20 is Junin Virus Candid #1 strain segment S, complete ce.

SEQ ID NO: 21 is the amino acid sequence of the NP protein of the Clone 13 strain of LCMV.

SEQ ID NO: 22 is the amino acid sequence of the GP protein of the Clone 13 strain of LCMV.

SEQ ID NO: 23 is the amino acid sequence of the L protein of the Clone 13 strain ofLCMV.

SEQ ID NO: 24 is the amino acid sequence ofthe Z protein of the Clone 13 strain ofLCMV SEQ ID NO: 25 is the amino acid sequence of the GP protein of the WE strain of LCMV.

SEQ ID NO: 26 is the nucleotide sequence of the HBV HBe antigen.

. BRIEF DESCRIPTION OF THE FIGURES Fig. 1: The genome ofwild type arenaviruses consists of a short (1; ~3.4 kb) and a large (2; ~72 kb) RNA t. The short segment s ORFs encoding the nucleoprotein (3) and rotein (4). The large segment encodes the RNA-dependent RNA polymerase L (5) and the matrix protein Z (6). Wild type arenaviruses can be rendered replication-de?cient vaccine vectors by deleting the glycoprotein gene and inserting, instead of the glycoprotein gene, antigens of choice (7) against which immune responses are to be induced.

Figs. 2A-C: Schematic representation of the genomic organization of bi- and tri- segmented LCMV. The bi—segmented genome of ype LCMV consists of one S segment encoding the GP and NP and one L segment encoding the Z protein and the L protein (A). Both segments are ?anked by the respective 5’ and 3’ UTRs. The genome of recombinant tri- segmented LCMVs (r3LCMV) consists of one L and two S segments with one position where to insert a gene of interest (here GFP) into each one of the S segments. -GFPnatural (nat) has all viral genes in their natural position (B), whereas the GP ORF in r3LCMV—GFPa?ifiCial (art) is arti?cially juxtaposed to and expressed under control of the 3’ UTR (C).

Fig. 3: Hepatitis B virus-specific CD8+ T cells, expressed as a percentage of the total CD8+B220— T cell pool in peripheral blood of C57BL/6 mice (5 mice per group) ten days after intravenous immunization with 105 FFU V/HBs-HBc (group 1), rLCMV/HBc (group 3), rLCMV/Pre-SZ (group 4), or with 104 FFU V/HBs-HBc (group 2). Control mice were left untreated.

Fig. 4A-B: Hepatitis B virus-speci?c CD8+ T cells, expressed as (A) a tage of the total CD8+B220— T cell pool in peripheral blood or, (B) as a percentage of the circulating lymphocytes in the blood, of C57BL/6 mice (5 mice per group) eight days after intravenous zation with 105 FFU MV/HBs-HBc (group 1), r3LCMV/HBc (group 2), r3LCMV/Pre—S2 (group 3), or with 105 FFU ofrLCMV/HBs-HBc (group 4). Control mice were left untreated. 6. DETAILED DESCRIPTION OF THE INVENTION The present application provides immunotherapies for Hepatitis B virus infections. Provided herein are methods and compositions for the treatment or prevention of infection of a subject with HBV. More specifically, provided herein are infectious arenaviruses that comprise a nucleotide ce encoding an HBV antigen. In certain ments, the infectious arenavirus is replication-deficient. In certain embodiments, the infectious arenavirus is replication-competent. These viruses can be administered to a subject for the treatment or prevention ofHBV ion. The generation of infectious arenavirus s for use with the present invention is bed in more detail in Section 6.3.

Provided herein is a genetically modified arenavirus, where the arenavirus: is infectious; cannot form infectious progeny virus in a non-complementary cell (i.e., a cell that does not express the ?inctionality that is missing from the replication-de?cient arenavirus and causes it to be replication-de?cient); is capable of replicating its genome and expressing its genetic information; and encodes an HBV antigen or a fragment thereof A genetically d arenavirus described herein is infectious, i.e., it can attach to a host cell and release its genetic material into the host cell. A genetically modi?ed arenavirus described herein may be replication-de?cient, i.e., the arenavirus is unable to e ?arther infectious progeny particles in a non-complementing cell. In particular, to create a ation- de?cient arenavirus, the genome ofthe arenavirus is modi?ed (e.g., by deletion or functional inactivation of an ORF) such that a virus carrying the modi?ed genome can no longer produce infectious progeny viruses. A non-complementing cell is a cell that does not provide the functionality that has been ated from the replication—de?cient arenavirus by modi?cation ofthe virus genome (e.g., if the ORF encoding the GP protein is deleted or onally inactivated, a non-complementing cell does not provide the GP protein). However, a genetically modi?ed replication-de?cient arenavirus provided herein is capable of producing infectious progeny Viruses in complementing cells. Complementing cells are cells that provide (in trans) the onality that has been eliminated from the replication-de?cient arenavirus by modi?cation ofthe virus genome (e.g., if the ORF encoding the GP protein is deleted or onally inactivated, a complementing cell does e the GP protein). Expression of the complementing functionality (e.g., the GP protein) can be accomplished by any method known to the skilled artisan (e.g., transient or stable expression). A genetically modi?ed irus bed herein can amplify and express its genetic information in a cell that has been infected by the Virus. A genetically modi?ed arenavirus provided herein comprises a nucleotide sequence that encodes an HBV antigen such as but not limited to the HBV antigens described in Section In certain embodiments, provided herein is a genetically modi?ed arenavirus in which an ORF of the arenavirus genome is d or ?anctionally inactivated such that the resulting virus cannot produce ?irther infectious progeny virus particles in non-complementing cells. An irus le comprising a cally modi?ed genome in which an ORF is deleted or functionally inactivated can be produced in complementing cells (i.e., in cells that express the arenaviral ORF that has been deleted or ?anctionally inactivated) (see Section 6.3).

The c material of the resulting irus particles can be transferred upon infection of a host cell into the host cell, wherein the genetic material can be sed and ampli?ed. In addition, the genome of the genetically modified irus particles provided herein encodes an HBV antigen that can be expressed in the host cell.

In certain embodiments, the ORF that encodes the glycoprotein (GP) of the arenavirus is deleted to generate a ation-de?cient arenavirus for use With the t ion. In a speci?c embodiment, the replication-de?cient arenavirus comprises a genomic segment comprising a nucleotide sequence ng an HBV antigen. Thus, in certain embodiments, a genetically modified arenavirus particle provided herein ses a genomic segment that a) has a deletion or functional inactivation of an ORF that is present in the Wild type form ofthe genomic segment; and b) encodes (either in sense or antisense) an HBV antigen (see Section 6.3).

In certain ments, the antigen encoded by the nucleic acid that is inserted into the genome of the irus can encode, for example, an HBV antigen or combinations of HBV antigens including, but not limited to: a. a nucleotide sequence encoding an HBV pre-S2/S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding an HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding an HBV HBs protein or an antigenic fragment thereof; d. a nucleotide sequence encoding a fusion ofHBV HBs and HBc proteins or antigenic fragments f; e. a nucleotide sequence encoding an HBV HBe protein or an antigenic fragment thereof.

In certain embodiments, the infectious arenavirus Viral vector is replication- nt (See Section 6.1(a)). In certain embodiments, the infectious arenavirus viral vector is replication-competent (See Section 6.1(b)) A detailed description of the antigens bed herein is provided in Section 6.2.

In certain embodiments, the arenaviruses used according to the invention described herein can be Old World s, for example, cytic choriomeningitis virus . More detailed description of the arenaviruses described herein is provided in Section 6. 1. In certain embodiments, the iruses used ing to the invention bed herein can be New World viruses.

Provided herein are c acids comprising the genome of such replication- de?cient arenaviruses. In certain aspects, an infectious, replication-de?cient arenavirus particle comprises a genomic segment comprising a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Provided herein is an expression plasmid that s one or more components required for the generation of a viral vector described herein. Speci?cally, provided herein is an expression vector that encodes an LCMV S segment wherein the ORF for the GP protein has been deleted from the S segment and has been replaced with the ORF of human HBV pre-S2/S protein (e.g., having an amino acid sequence encoded by the nucleotide ce of SEQ ID NO: 1 or an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1).

Provided herein is an expression d that encodes one or more ents required for the generation of a viral vector described herein. Speci?cally, provided herein is an expression vector that encodes an LCMV S segment wherein the ORF for the GP n has been deleted from the S segment and has been replaced with the ORF of human HBV HBc protein (e.g., having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2 or an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2).

Provided herein is an expression plasmid that encodes one or more components required for the generation of a viral vector described herein. Speci?cally, provided herein is an expression vector that encodes an LCMV S segment wherein the ORF for the GP protein has been deleted from the S segment and has been replaced with the ORF of human HBV HBs and the ORF of human HBV HBc (e.g., having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3 or an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% cal to an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3).

Provided herein are kits comprising one or two of the vector plasmids described herein. In certain embodiments, provided herein is a kit that comprises a) an expression plasmid that comprises the nucleotide sequence of the S segment of an LCMV vector; b) an expression plasmid that ses the nucleotide sequence of the L segment of an LCMV vector; and c) an sion plasmid that encodes the complementing functionality. In a speci?c embodiment, provided herein is a kit comprising a) an expression vector that comprises the nucleotide sequence of an LCMV S segment n the ORF for the GP protein has been deleted from the S segment and has been replaced with the ORF of human HBV pre-S2/S protein (e.g., having an amino acid sequence encoded by the tide sequence of SEQ ID NO: 1 or an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the tide sequence of SEQ ID NO: 1); b) an expression plasmid that comprises the nucleotide sequence of the L segment of an LCMV ; and c) an expression plasmid that s the LCMV GP protein (or a cell line that expresses LCMV GP protein).

Provided herein are kits comprising one or two of the vector plasmids described herein. In certain embodiments, provided herein is a kit that comprises a) an expression d that ses the nucleotide sequence of the S segment of an LCMV vector; b) an expression plasmid that comprises the nucleotide sequence of the L segment of an LCMV vector; and c) an expression plasmid that encodes the complementing functionality. In a speci?c embodiment, provided herein is a kit comprising a) an expression vector that comprises the tide sequence of an LCMV S segment wherein the ORF for the GP protein has been deleted from the S segment and has been replaced with the ORF of human HBV HBc protein (e.g., having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2 or an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid ce encoded by the nucleotide sequence of SEQ ID NO: 2); b) an expression plasmid that comprises the nucleotide sequence of the L segment of an LCMV ; and c) an expression plasmid that encodes the LCMV GP protein (or a cell line that expresses LCMV GP protein).

Provided herein are kits comprising one or two of the vector plasmids described herein. In certain embodiments, ed herein is a kit that comprises a) an expression plasmid that comprises the nucleotide sequence of the S segment of an LCMV vector; b) an expression plasmid that comprises the nucleotide sequence of the L segment of an LCMV vector; and c) an expression plasmid that encodes the complementing functionality. In a speci?c embodiment, provided herein is a kit comprising a) an expression vector that comprises the nucleotide sequence of an LCMV S t wherein the ORF for the GP protein has been deleted from the S segment and has been replaced with the ORF of human HBV HBs and the ORF of human HBV HBc (e.g., having an amino acid sequence encoded by the tide sequence of SEQ ID NO: 3 or an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3); b) an expression plasmid that comprises the nucleotide sequence of the L segment of an LCMV vector; and c) an expression plasmid that encodes the LCMV GP protein (or a cell line that expresses LCMV GP protein).

Also provided herein are cell lines, cultures and methods of culturing cells infected with nucleic acids, vectors, and compositions provided . More detailed ption of the nucleic acids, vector s and cell lines described herein is provided in Section 6.4.

In one aspect, provided herein are such genetically modi?ed replication-de?cient arenaviruses suitable as vaccines and methods ofusing such arenaviruses in vaccination and treatment or prevention of infections by HBV. More detailed description of methods of using such arenaviruses described herein is provided in Section 6.5.

In certain embodiments, immunization with an infectious arenavirus that expresses an HBV antigen or a fragment thereof, as described herein es a long-lasting immune response. In n embodiments, maximal antibody levels can be achieved after two immunizations. In another ment, a third immunization can be administered for a boosting effect. In more speci?c embodiments, provided herein are administration schedules using the infectious arenavirus in a vaccination for the treatment and/or tion of infections by HBV.

A more detailed description of administration les using an infectious arenavirus as bed herein is provided in Section 6.6. In certain embodiments, the infectious arenavirus viral vector is replication-de?cient (See Section 6.1(a)). In n embodiments, the infectious arenavirus viral vector is replication-competent (See Section 6.1(b)).

In certain embodiments, administering to a seronegative subject an infectious irus sing an HBV antigen or a nt thereof, as described herein s a detectable antibody titer for a minimum of at least 4 weeks. In another embodiment, stering to a subject infected with an HBV infection an infectious arenavirus expressing an HBV antigen or a fragment f, as bed herein increases the antibody titer by at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%. In certain embodiments, primary antigen exposure, by ?rst zation with an infectious arenavirus expressing an HBV antigen, elicits a functional, (neutralizing) and minimum dy titer of at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% ofmean control sera from infection-immune human subjects. In more speci?c embodiments, the primary neutralizing geometric mean antibody titer increases up to a peak value of at least 1:50, at least 1:100, at least 1:200, or at least 1:1000 within at least 4 weeks post-immunization. In another embodiment, immunization with an infectious arenavirus expressing an HBV antigen or a fragment thereof, as described herein produces high titers of antibodies that last for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years post- immunization following a single administration of the vaccine. In certain embodiments, the infectious arenavirus Viral vector is replication-de?cient (See Section 6.1(a)). In certain embodiments, the infectious irus viral vector is replication-competent (See Section 6. 1 (b)).

In yet another embodiment, secondary antigen exposure by second immunization with an infectious arenavirus expressing an HBV antigen or a fragment thereof increases the antibody titer by at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%. In another embodiment, ary antigen exposure elicits a functional, (neutralizing) and minimum antibody titer of at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% of mean l sera from infection- immune human subjects. In more speci?c embodiments, the secondary neutralizing geometric mean antibody titer increases up to a peak value of at least 1:50, at least 1:100, at least 1:200, or at least 1:1000 within at least 4 weeks post-immunization. In another embodiment, a second immunization with an infectious arenavirus expressing an HBV antigen or a fragment thereof, as described herein produces high titers of antibodies that last for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years post-immunization. In certain embodiments, the infectious arenavirus viral vector is replication-deficient (See n 6.1(a)). In certain embodiments, the infectious arenavirus viral vector is replication-competent (See Section 6.1(b)).

In yet another embodiment, a third boosting immunization increases the antibody titer by at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%. In another embodiment, the boosting immunization elicits a onal, alizing) and minimum antibody titer of at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% ofmean l sera from infection-immune human subjects. In more speci?c embodiments, the neutralizing geometric mean antibody titer after the third boosting immunization increases up to a peak value of at least 1:50, at least 1:100, at least 1:200, or at least 1:1000 within at least 4 weeks post-immunization. In r embodiment, a third boosting immunization prolongs the antibody titer by at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years post-immunization.

In n embodiments, the infectious arenavirus expressing an HBV antigen or fragment f, elicits a T cell independent or T cell dependent response. In other embodiments, the infectious arenavirus expressing an HBV antigen or a fragment thereof, elicits a T cell se. In other embodiments, the infectious irus expressing an HBV antigen or a fragment thereof, as bed herein elicits a T helper response. In another embodiment, the infectious arenavirus expressing an HBV antigen or a fragment thereof, as described herein elicits a Thl-orientated response or a ientated response. In certain embodiments, the infectious arenavirus viral vector is replication-deficient (See Section 6.1(a)). In n embodiments, the infectious arenavirus viral vector is replication-competent (See n 6. 1 (b)).

In more speci?c embodiments, the Thl-orientated response is indicated by a predominance of IgG1 antibodies versus IgG2. In other embodiments the ratio of IgG1 :IgG2 is greater than 1:1, greater than 2: 1, greater than 3:1, or greater than 4: 1. In another embodiment the ious arenavirus expressing an HBV antigen or a fragment thereof, as described herein is indicated by a predominance of IgG3 antibodies. In certain embodiments, the infectious arenavirus Viral vector is replication-de?cient (See Section 6. 1(a)). In n embodiments, the infectious arenavirus Viral vector is replication-competent (See Section 6.1(b)).

In some embodiments, the infectious arenavirus expressing an HBV antigen or a fragment thereof elicits a CD8+ T cell response. In other embodiments, the infectious arenavirus sing an HBV antigen or a fragment thereof elicits a regulatory T cell response.

In more speci?c embodiments, the regulatory T cell response maintains immune tolerance. In another embodiment, the infectious arenavirus expressing an HBV antigen or a nt thereof elicits both CD4+ and CD8+ T cell responses. In certain embodiments, the infectious arenavirus Viral vector is replication-de?cient (See Section 6.1(a)). In n embodiments, the infectious arenavirus Viral vector is replication-competent (See Section 6.1(b)).

In certain embodiments, the infectious arenavirus sing one or more HBV antigens or fragment thereof, as described herein, elicits high titers of lizing antibodies. In another embodiment, the infectious arenavirus sing two or more HBV antigens or nts f, as described herein, elicits higher titers of neutralizing antibodies than expression of the protein complex components individually. In certain embodiments, the infectious arenavirus Viral vector is replication-de?cient (See n 6.1(a)). In n embodiments, the infectious irus Viral vector is replication—competent (See Section 6. l (b)).

In other embodiments, two or more infectious iruses expressing an HBV antigen elicit high titers of lizing antibodies. In a more c embodiment, two or more infectious arenaviruses expressing an HBV antigen elicit higher titers of neutralizing antibodies than an infectious arenavirus expressing one HBV antigen or fragment thereof. In certain embodiments, the infectious arenavirus Viral vector is replication-de?cient (See Section 6.1(a)).

In certain embodiments, the infectious arenavirus Viral vector is ation-competent (See Section 6.1(b)).

In another embodiment, the infectious arenavirus expressing two, three, four, ?ve, or more HBV antigens elicits higher titers of neutralizing antibodies than an infectious arenavirus expressing one HBV antigen or fragment thereof. In certain embodiments, the infectious arenavirus Viral vector is replication—de?cient (See Section 6. 1(a)). In certain embodiments, the infectious irus viral vector is ation-competent (See Section 6.1(b)). 6.1 Arenavirus Vectors Expressing an HBV Antigen Arenaviruses for use with the methods and compositions provided herein can be Old World viruses, for example Lassa virus, Lymphocytic choriomeningitis virus (LCMV), Mobala Virus, Mopeia Virus, or Ippy Virus, or New World Viruses, for example Amapari virus, Flexal virus, Guanarito virus, Junin virus, Latino virus, o virus, Oliveros virus, Parana virus, Pichinde virus, l virus, Sabia virus, Tacaribe virus, Tamiami virus, Bear Canyon virus, or Whitewater Arroyo virus. The genetically modi?ed arenavirus can be generated as described in Section 6.3.

The wild type arenavirus genome consists of a short (~3.4 kb) and a large (~72 kb) RNA segment. The short segment carries the ORFs encoding the protein NP and glycoprotein GP genes. The large segment comprises the RNA-dependent RNA polymerase L and the matrix protein Z genes. (3) Replication-de?cient arenavirus vectors In n embodiments, the arenavirus vector is a replication-de?cient, ented arenavirus vector. In certain embodiments, the arenavirus vector is a replication- de?cient, trisegmented arenavirus vector. Wild type arenaviruses can be rendered replication- de?cient to generate vaccine vectors by substituting the glycoprotein gene for one or more HBV antigens, against which immune responses are to be induced.

Infectious arenavirus vectors expressing an HBV antigen, or a combination of HBV antigens as described herein, can be used to immunize (in a preventive manner) or treat (in an immunotherapeutic manner) subjects against HBV infection. In a speci?c embodiment, a combination of HBs and HBc is used.

Arenavirus disease and immunosuppression in wild type arenavirus infection are known to result from unchecked viral replication. By abolishing replication, i.e., the ability to produce infectious progeny virus particles, of irus vectors by ng from their genome, e.g., the Z gene which is ed for particle release, or the GP gene which is required for infection of target cells, the total number of infected cells can be limited by the inoculum stered, e.g., to a vaccine recipient, or ntally transmitted to personnel involved in medical or biotechnological applications, or to animals. Therefore, abolishing replication of arenavirus vectors prevents pathogenesis as a result of intentional or accidental transmission of vector particles. Provided herein, one important aspect consists in exploiting the above necessity of abolishment of replication in a ial way for the purpose of expressing an HBV antigen.

In certain embodiments, an arenavirus le is rendered replication de?cient by genetic modi?cation of its genome. Such modi?cations to the genome can include: deletion of an ORF (e.g., the ORF encoding the GP, NP, L, or Z protein); onal inactivation of an ORF (e.g., the ORF encoding the GP, NP, L, or Z protein).

For example, this can be ed by introducing a missense or a nonsense mutation; change of the sequence of the ORF (e. g., the exchange of an SlP cleavage site with the cleavage site of r se); mutagenesis of one of the 5 ’ or 3’ termini of one of the genomic segments; mutagenesis of an intergenic region (i.e., of the L or the S genomic segment).

] In certain embodiments, an infectious arenavirus expressing an HBV antigen described herein is a Lymphocytic choriomeningitis virus (LCMV) wherein the S segment of the Virus is modi?ed by substituting the ORF encoding the GP protein with an ORF encoding an HBV antigen.

In certain embodiments, a wild type arenavirus vector genome ( can be designed to retain at least the essential regulatory elements on the 5 ’ and 3’ untranslated regions (UTRs) of both segments, and/or also the intergenic regions (IGRs). Without being bound by theory, the minimal transacting factors for gene expression in infected cells remain in the vector genome as ORFs that can be sed, yet they can be placed differently in the genome and can be placed under control of a ent er than naturally, or can be expressed from internal ribosome entry sites. In certain embodiments, the nucleic acid ng an HBV antigen is transcribed from one of the endogenous arenavirus promoters (i.e., 5’ UTR, 3’ UTR of the S segment, 5 ’ UTR, 3 ’ UTR of the L segment). In other embodiments, the nucleic acid encoding an HBV antigen is sed from a heterologous introduced promoter ces that can be read by the viral pendent RNA polymerase, by cellular RNA polymerase I, RNA polymerase II or RNA polymerase 111, such as duplications of viral promoter sequences that are naturally found in the viral UTRs, the 28S ribosomal RNA promoter, the beta-actin promoter or the 5S ribosomal RNA promoter, respectively. In certain ments ribonucleic acids coding for HBV antigens are transcribed and translated either by themselves or as read-through by fusion to arenavirus n ORFs, and expression of proteins in the host cell may be enhanced by ucing in the viral transcript sequence at the appropriate s) one or more, e.g., two, three or four, al ribosome entry sites. [001 11] In certain embodiments, for use with the compositions and methods provided herein is a tri-segmented arenavirus particle comprising one L segment and two S ts in which (i) an ORF is in a position other than the wild-type position of the ORF; and (ii) an ORF encoding GP or NP has been removed or functionally inactivated, such that the resulting virus cannot produce further infectious progeny virus particles. In a speci?c embodiment, one ORF is removed and replaced with a heterologous ORF (e.g, encoding an HBV antigen) from an organism other than an irus. In another speci?c embodiment, two ORFs are removed and replaced with a heterologous ORF from an organism other than an arenavirus. In other speci?c embodiments, three ORFs are removed and replaced with a heterologous ORF (e.g., encoding an HBV antigen) from an organism other than an arenavirus. In speci?c embodiments, the ORF encoding GP is removed and replaced with a heterologous ORF (e.g., encoding an HBV antigen) from an organism other than an irus. In other speci?c embodiments, the ORF encoding NP is removed and replaced with a heterologous ORF (e.g, encoding an HBV antigen) from an organism other than an arenavirus. In yet more speci?c embodiments, the ORF encoding NP and the ORF encoding GP are removed and replaced with one or two heterologous ORFs (e.g., encoding one or two HBV ns) from an organism other than an arenavirus particle. Thus, in certain ments the tri-segmented arenavirus particle comprises (i) one L segment and two S segments; (ii) an ORF in a position other than the ype position of the ORF; (iii) one or more heterologous ORFs (e.g., encoding one or more HBV antigens) from an organism other than an arenavirus.

In certain ments, for use with the compositions and methods provided herein is a gmented arenavirus particle comprising two L segments and one S segment in which (i) an ORF is in a position other than the wild-type position of the ORF; and (ii) an ORF encoding the Z protein, and/or the L n has been removed or functionally inactivated, such that the resulting virus cannot produce further infectious progeny virus le. In a c embodiment, one ORF is removed and replaced with a heterologous ORF (e.g, encoding an HBV antigen) from an organism other than an arenavirus. In another speci?c embodiment, two ORFs are removed and replaced with a heterologous ORF (e.g, encoding an HBV antigen) ?om an organism other than an arenavirus. In speci?c embodiments, the ORF encoding the Z protein is d and replaced with a logous ORF (e.g., encoding an HBV antigen) from an organism other than an arenavirus. In other speci?c embodiments, the ORF encoding the L n is removed and replaced with a heterologous ORF (e.g. , encoding an HBV antigen) from an organism other than an arenavirus. In yet more speci?c embodiments, the ORF encoding the Z protein and the ORF encoding the L protein is removed and replaced with a heterologous ORF (e.g., encoding an HBV antigen) from an organism other than an arenavirus particle. Thus, in certain embodiments the tri-segmented arenavirus particle comprises (i) two L segments and one S segment; (ii) an ORF in a position other than the ype position of the ORF; (iii) a heterologous ORF (e.g., encoding an HBV antigen) from an organism other than an arenavirus.

Thus, in n embodiments, the tri-segmented arenavirus particle for use with the compositions and methods provided herein ses a tri-segmented arenavirus particle (z'.e., one L segment and two S ts or two L segments and one S segment) that i) is engineered to carry an ORF in a non-natural position; ii) an ORF encoding GP, NP, Z protein, or L protein is removed; iii) the ORF that is removed is replaced with one or more heterologous ORFS (e.g. one or more HBV antigens) from an organism other than an arenavirus. , encoding In certain embodiments, the vector generated to encode one or more HBV antigens may be based on a c strain of LCMV. Strains ofLCMV include Clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009, 200501927, 810362, 811316, 810316, 810366, 20112714, Douglas, GROl, SNOS, CABN and their derivatives. In certain embodiments, the vector generated to encode one or more HBV antigens may be based on LCMV Clone 13. In other embodiments, the vector generated to encode one or more HBV antigens may be based on LCMV MP strain. The sequence of the S segment ofLCMV Clone 13 is listed as SEQ ID NO: 12. In certain embodiments, the sequence of the S t ofLCMV Clone 13 is the sequence set forth in SEQ ID NO: 11. The ce of the L segment of LCMV Clone 13 is listed as SEQ ID NO: 7.

The ce ofthe S segment ofLCMV strain MP is listed as SEQ ID NO: 14. The sequence ofthe L segment ofLCMV strain MP is listed as SEQ ID NO: 13.

In certain embodiments, the vector generated to encode one or more HBV antigens may be based on a speci?c strain of Junin Virus. Strains of Junin virus include e strains XJ13, XJ#44, and, Candid#l as well as IV4454, a human isolate. In certain embodiments, the vector generated to encode one or more HBV antigens is based on Junin virus Candid #1 strain.

In certain embodiments, described herein is an ious, replication-de?cient arenavirus particle comprising a nucleotide sequence or fragment thereof ed from SEQ ID NO: 13, SEQ ID NO: 14, or a combination thereof.

In certain embodiments, bed herein is an infectious, replication-de?cient arenavirus particle comprising a nucleotide sequence, or a combination of nucleotide sequences, ed from the group ting of : o a nucleotide sequence encoding a Hepatitis B virus pre-S2/S protein or an antigenic fragment f; 0 a nucleotide sequence encoding a Hepatitis B virus HBc protein or an antigenic fragment thereof; 0 a nucleotide sequence encoding a Hepatitis B virus HBs protein or an nic fragment thereof; 0 a nucleotide sequence encoding a fusion of Hepatitis B virus HBs and HBc proteins or antigenic fragments thereof; 0 a nucleotide sequence encoding a Hepatitis B Virus HBe n or an antigenic fragment thereof.

In certain ments, the infectious, replication-de?cient arenavirus vector is trisegmented. (b) Replication-competent mented arenavirus vectors In certain embodiments, for use with the compositions and methods provided herein is a replication-competent, trisegmented arenavirus vector. In certain embodiments, the arenavirus vector is a tri-segmented arenavirus particle comprising one L segment and two S segments or two L segments and one S segment that do not ine into a replication- competent bi—segmented arenavirus particle.

In certain embodiments, an infectious arenavirus expressing an HBV n for use with the compositions and methods described herein is engineered to carry a viral ORF in a position other than the wild-type position ofthe ORF. In some embodiments, the arenavirus genomic segment is selected from the group ting of: (i) an S segment, wherein the ORF encoding the NP is under control of an arenavirus 5’ UTR; (ii) an S segment, wherein the ORF encoding the Z protein is under control of an arenavirus 5 ’ UTR; (iii) an S segment, wherein the ORF encoding the L protein is under control of an arenavirus 5’ UTR; (iv) an S segment, wherein the ORF encoding the GP is under control of an arenavirus 3’ UTR; (V) an S segment, wherein the ORF encoding the L protein is under control of an arenavirus 3 ’ UTR; (vi) an S segment, wherein the ORF encoding the Z protein is under control of an arenavirus 3 ’ UTR; (vii) an L segment, n the ORF ng the GP is under control of an arenavirus 5’ UTR; (viii) an L t, wherein the ORF encoding the NP is under control of an arenavirus 5’ UTR; (ix) an L segment, n the ORF encoding the L protein is under control of an arenavirus 5’ UTR; (x) an L t, wherein the ORF encoding the GP is under control of an arenavirus 3’ UTR; (xi) an L segment, wherein the ORF encoding the NP is under control of an arenavirus 3’ UTR; and (xii) an L segment, wherein the ORF encoding the Z n is under control of an arenavirus 3 ’ UTR.

In some embodiments, the arenavirus 3’ UTR is the 3’ UTR of the arenavirus S segment or the irus L segment. In certain embodiments, the arenavirus 5 ’ UTR is the 5 ’ UTR of the arenavirus S segment or the irus L segment.

For use with the compositions and methods ed herein are tri-segmented arenavirus particles with rearrangements of their ORFs. In one aspect, for use with the compositions and methods provided herein is a tri-segmented arenavirus particle comprising one L segment and two S segments or two L segments and one S segment. In certain embodiments, the tri-segmented arenavirus particle does not recombine into a replication competent bi- segmented arenavirus particle. In speci?c embodiments, the tri-segmented arenavirus particle comprises an ORF in a position other than the wild-type position of the ORF. In yet another speci?c embodiment, the tri-segmented arenavirus le comprises all four arenavirus ORFs.

Thus, in certain embodiments, the gmented irus particle is replication competent and infectious. Figure 2 shows exemplary schematic representations of the genomic organization of a replication-competent mented LCMV vector (Figs. 2B-C). Figure 2C shows an exemplary schematic representation of the genomic organization of replication-competent trisegmented LCMV vector which cannot recombine into a replication-competent bisegmented arenavirus particle. In ison, Figure 2A shows the wildtype bisegmented LCMV vector.

In n embodiments, the ORF encoding GP, NP, Z protein, or the L protein of the tri-segmented arenavirus particle described herein can be under the control of an arenavirus 3’ UTR or an arenavirus 5’ UTR. In more speci?c embodiments, the tri—segmented arenavirus 3’ UTR is the 3’ UTR of an irus S segment(s). In another speci?c embodiment, the tri— segmented arenavirus 3’ UTR is the 3’ UTR of an arenavirus L segment(s). In more speci?c embodiments, the gmented arenavirus 5 ’ UTR is the 5 ’ UTR of an arenavirus S segment(s).

In other speci?c embodiments, the 5 ’ UTR is the 5 ’ UTR of an arenavirus L segment(s).

] In other embodiments, the ORF encoding GP, NP, Z protein, or the L n of an tri-segmented arenavirus particle described herein can be under the control of the irus conserved terminal sequence element (the 5’- and 3'-terminal l9nt regions) (see e.g., Perez & de la Torre, 2003, J Virol. 77(2): 1184—1194).

In certain embodiments, the ORF encoding GP, NP, Z protein or the L protein of the tri-segmented arenavirus particle can be under the control of the promoter element of the 5 ’ UTR (see e.g., no et al., 2011, J Virol., 85(8):4020-4). In another embodiment, the ORF encoding GP, NP Z protein, L protein ofthe gmented arenavirus particle can be under the control of the promoter element of the 3’ UTR (see e.g., no et al., 2011, J Virol., 85(8):4020-4). In more speci?c ments, the promoter element of the 5’ UTR is the 5’ UTR promoter element of the S segment(s) or the L segment(s). In another speci?c embodiment, the promoter element of the 3 ’ UTR is the 3’ UTR the promoter element of the S segment(s) or the L segment(s).

In n embodiments, the ORF that encoding GP, NP, Z protein or the L protein of the tri-segmented arenavirus particle can be under the control of a truncated arenavirus 3’ UTR or a truncated arenavirus 5’ UTR (see e.g., Perez & de la Torre, 2003, J Virol. 77(2): 1184—1194; Albarino et al., 2011, J Virol, 85(8):4020-4). In more speci?c embodiments, the truncated 3 ’ UTR is the 3 ’ UTR of the arenavirus S t or L segment. In more speci?c embodiments, the truncated 5 ’ UTR is the 5 ’ UTR of the arenavirus S segment(s) or L segment(s).

In one aspect, for use with the compositions and methods provided herein is a tri- segmented arenavirus particle comprising one L segment and two S segments. In certain embodiments, propagation of the tri-segmented arenavirus particle sing one L segment and two S segments does not result in a replication-competent bi-segmented Viral particle. In c embodiments, propagation of the tri-segmented irus particle comprising one L segment and two S segments does not result in a replication-competent bi—segmented Viral particle after at least 10 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, or at least 100 days of persistent infection in mice lacking type I interferon receptor, type II interferon receptor and recombination activating gene (RAGl), and having been ed with 104 PFU of the tri- segmented arenavirus particle. In other embodiments, propagation of the tri-segmented irus particle comprising one L segment and two S segments does not result in a replication-competent bi—segmented Viral particle after at least 10 passages, at least 20 passages, at least 30 passages, at least 40 passages, or at least 50 passages.

] The tri-segmented arenavirus particle with all Viral genes in their respective wild- type position is known in the art (e.g., Emonet et al., 2011 J. Virol, 85(4):1473; Popkin et al., 2011, J. Virol, :7928). In particular, the tri-segmented arenavirus genome consists of one L segment and two S segments, in which a heterologous ORF (eg, a GFP) is inserted into one position on each S segment. More speci?cally, one S segment encodes GP and GFP, respectively. The other S segment encodes GFP and NP, respectively. The L segment encodes the L protein and Z protein. All segments are ?anked by the respective 5’ and 3’ UTRs.

In n embodiments, inter-segmental recombination of the two S segments of the tri-segmented arenavirus particle for use with the compositions and methods ed , that unities the two arenaviral ORFs on one instead of two separate segments results in a non functional promoter (i.e., a genomic segment of the structure: 5’ UTR-----------5’ UTR or a 3’ UTR------------3’ UTR), wherein each UTR forming one end of the genome is an inverted repeat ce of the other end of the same genome.

In certain embodiments, the tri-segmented arenavirus particle comprising one L segment and two S segments has been engineered to carry an arenavirus ORF in a position other than the wild—type position of the ORF. In other embodiments, the tri-segmented arenavirus particle sing one L segment and two S segments has been engineered to carry two arenavirus ORFs, or three arenavirus ORFs, or four arenavirus ORFs, or ?ve arenavirus ORFs, or six arenavirus ORFs in a position other than the ype position. In speci?c embodiments, the tri-segmented arenavirus particle comprising one L segment and two S ts comprises a ?ll complement of all four arenavirus ORFs. Thus, in some embodiments, the tri—segmented arenavirus particle is an infectious and replication ent tri—segmented arenavirus particle.

In speci?c embodiments, the two S segments of the gmented arenavirus le have been engineered to carry one of their ORFs in a position other than the wild-type position. In more c embodiments, the two S segments comprise a full ment of the S segment ORF’s.

In certain speci?c embodiments, the L segment has been engineered to carry an ORF in a position other than the wild-type position or the L segment can be the wild-type genomic segment.

In certain embodiments, one of the two S segments can be: (i) an arenavirus S segment, wherein the ORF encoding the Z protein is under control of an arenavirus 5’ UTR; (ii) an arenavirus S segment, wherein the ORF encoding the L protein is under control of an arenavirus 5’ UTR; (iii) an arenavirus S segment, n the ORF encoding the NP is under control of an arenavirus 5’ UTR; (iV) an arenavirus S segment, wherein the ORF encoding the GP is under control of an arenavirus 3’ UTR; (V) an arenavirus S segment, wherein the ORF encoding the L is under control of an arenavirus 3’ UTR; and (Vi) an arenavirus S segment, wherein the ORF encoding the Z protein is under control of an arenavirus 3’ UTR.

] In certain embodiments, the tri-segmented arenavirus particle comprising one L segment and two S segments can comprise a duplicate ORF (z'.e., two wild-type S segment ORFs e.g, GP or NP). In speci?c embodiments, the tri-segmented arenavirus particle sing one L segment and two S segments can comprise one ate ORF (e.g., (GP, GP)) or two duplicate ORFs (e.g, (GP, GP) and (NP, NP)).

Table 1A, below, is an exemplary illustration of the genome organization of a tri— segmented irus particle comprising one L segment and two S segments, wherein intersegmental recombination of the two S segments in the tri-segmented arenavirus genome does not result in a replication-competent bi-segmented Viral particle and abrogates arenaviral promoter ty (z'.e., the resulting recombined S segment is made up of two 3 ’UTRs instead of a 3’ UTR and a 5’ UTR).

Table 1A Tri—segmented arenavirus particle sing one L segment and two S segments on 1 is under the control of an arenavirus S segment 5’ UTR; Position 2 is under the control of an arenavirus S segment 3’ UTR; Position 3 is under the control of an arenavirus S segment 5’ UTR; Position 4 under the control of an arenavirus S t 3’ UTR; Position 5 is under the control of an arenavirus L segment 5’ UTR; Position 6 is under the control of an arenavirus L segment 3’ UTR.

*ORF indicates that a logous ORF, for example, a heterologous ORF encoding an HBV antigen, has been inserted.

Position 1 Position 2 Position 3 Position 4 Position 5 Position 6 *ORF GP *ORF NP Z L *ORF NP *ORF GP Z L *ORF NP *ORF GP L Z *ORF NP *ORF Z L GP *ORF NP Z GP *ORF Z *ORF NP Z GP Z *ORF *ORF NP *ORF L Z GP *ORF L *ORF NP GP *ORF L Z NP *ORF GP *ORF *ORF GP Z l\P *ORF L Z GP *ORF NP *ORF Z *ORF GP L NP L GP *ORF NP *ORF Z L GP *ORF *ORF Z l\P L GP *ORF Z *ORF NP L *ORF Z GP *ORF l\P L GP *ORF NP *ORF Z L GP *ORF Z *ORF l\P L GP Z NP *ORF *ORF L GP Z NP *ORF *ORF Position 1 Position 2 Position 3 Position 4 Position 5 Position 6 L *ORF Z NP *ORF GP L NP *ORF Z *ORF GP L NP Z *ORF GP *ORF L *ORF Z *ORF GP NP L NP Z GP *ORF *ORF L NP *ORF Z *ORF GP L *ORF Z NP *ORF GP L Z *ORF GP *ORF NP L Z *ORF NP *ORF GP Z GP *ORF NP *ORF L Z GP *ORF *ORF L NP Z GP *ORF L *ORF NP Z *ORF L GP *ORF NP Z GP *ORF NP *ORF L Z GP *ORF L *ORF NP Z GP L NP *ORF *ORF Z GP NP *ORF *ORF Z *ORF NP *ORF GP Z \IP *ORF *ORF L GP Z NP *ORF GP *ORF L Z \IP *ORF *ORF L GP Z NP *ORF L *ORF GP Z \IP L GP *ORF *ORF Z *ORF L GP *ORF NP Z \IP *ORF GP *ORF L Z NP *ORF L *ORF GP Z *ORF L NP *ORF GP Z L *ORF GP *ORF NP In n embodiments, the IGR between position one and position two can be an arenavirus S segment or L segment IGR; the IGR between on two and three can be an arenavirus S segment or L segment IGR; and the IGR between the position ?ve and six can be an arenavirus L t IGR. In a speci?c embodiment, the IGR between on one and position two can be an arenavirus S segment IGR; the IGR between on two and three can be an arenavirus S segment IGR; and the IGR n the on ?ve and six can be an arenavirus L segment IGR. In certain embodiments, other combinations are also possible. For example, a tri—segmented arenavirus particle sing one L segment and two S segments, wherein intersegmental ination of the two S segments in the tri-segmented arenavirus genome does not result in a replication-competent bi-segmented viral particle and abrogates arenaviral promoter activity (i.e., the resulting recombined S segment is made up of two 5’UTRs instead of a 3’ UTR and a 5’ UTR).

In certain embodiments, intersegmental recombination of an S segment and an L segment in the tri-segmented arenavirus particle comprising one L segment and two S segments, restores a ?anctional segment with two viral genes on only one segment instead of two te segments. In other embodiments, intersegmental recombination of an S segment and an L segment in the tri-segmented arenavirus particle comprising one L segment and two S segments does not result in a replication-competent bi—segmeneted Viral le.

Table 1B, below, is an exemplary ration ofthe genome organization of a tri- segmented arenavirus particle comprising one L segment and two S segments, wherein intersegmental recombination of an S segment and an L segment in the tri—segmented arenavirus genome does not result in a replication-competent bi-segmented Viral particle and abrogates arenaviral promoter ty (i.e., the resulting recombined S segment is made up of two 3’UTRs instead of a 3’ UTR and a 5’ UTR).

Table 1B Tri—segmented arenavirus particle comprising one L segment and two S segments Position 1 is under the control of an arenavirus S segment 5’ UTR; Position 2 is under the control of an arenavirus S segment 3’ UTR; Position 3 is under the control of an arenavirus S segment 5’ UTR; Position 4 under the control of an arenavirus S segment 3’ UTR; Position 5 is under the control of an arenavirus L segment 5’ UTR; Position 6 is under the l of an arenavirus L t 3’ UTR.

*ORF indicates that a heterologous ORF, for example, a heterologous ORF encoding an HBV antigen, has been inserted.

Position 1 Position 2 Position 3 on 4 Position 5 Position 6 L GP *ORF NP Z *ORF L GP Z *ORF *ORF NP L GP *ORF NP Z *ORF on 1 Position 2 Position 3 Position 4 on 5 Position 6 L GP Z *ORF *ORF NP L NP *ORF GP Z *ORF L NP Z *ORF *ORF GP L NP *ORF GP Z *ORF L NP Z *ORF *ORF GP Z GP *ORF NP L *ORF Z GP L *ORF *ORF NP Z GP *ORF NP L *ORF Z NP L *ORF *ORF GP Z NP *ORF GP L *ORF Z NP L *ORF *ORF GP In certain embodiments, the IGR between position one and position two can be an arenavirus S segment or L segment IGR; the IGR between position two and three can be an irus S segment or L segment IGR; and the IGR n the position ?ve and six can be an arenavirus L segment IGR. In a speci?c embodiment, the IGR n position one and position two can be an arenavirus S segment IGR; the IGR between position two and three can be an arenavirus S segment IGR; and the IGR between the position ?ve and six can be an arenavirus L segment IGR. In certain embodiments, other combinations are also possible. For example, a tri-segmented arenavirus particle comprising one L segment and two S segments, wherein intersegmental recombination of the two S segments in the tri-segmented arenavirus genome does not result in a replication-competent bi-segmented viral particle and abrogates arenaviral promoter activity (i.e., the resulting recombined S segment is made up of two 5’UTRs d of a 3’ UTR and a 5’ UTR).

In one aspect, for use with the compositions and methods provided herein is a tri- segmented arenavirus particle comprising two L segments and one S segment. In certain embodiments, propagation of the tri-segmented arenavirus particle comprising two L segments and one S segment does not result in a replication-competent bi-segmented viral particle. In speci?c ments, propagation of the gmented arenavirus particle comprising two L segments and one S segment does not result in a replication-competent bi-segmented viral particle after at least 10 days, at least 20 days, at least 30 days, at least 40 days, or at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days of persistent in mice lacking type I interferon receptor, type II interferon receptor and recombination activating gene (RAGl), and having been infected with 104 PFU of the tri- segmented arenavirus particle. In other embodiments, propagation of the tri-segmented arenavirus particle comprising two L segments and one S segment does not result in a replication-competent bi—segmented viral particle after at least 10 passages, 20 passages, 30 es, 40 passages, or 50 es.

In certain embodiments, inter-segmental recombination of the two L segments of the tri—segmented arenavirus particle for use with the compositions and methods ed herein, that unities the two arenaviral ORFs on one instead of two separate segments s in a non onal promoter (z'.e., a genomic segment of the structure: 5’ UTR-----------5’ UTR or a 3’ ---------3’ UTR), wherein each UTR forming one end ofthe genome is an inverted repeat sequence of the other end of the same genome.

In certain embodiments, the tri-segmented arenavirus le comprising two L segments and one S segment has been engineered to carry an arenavirus ORF in a position other than the wild-type position of the ORF. In other embodiments, the tri-segmented arenavirus le comprising two L segments and one S segment has been engineered to carry two arenavirus ORFS, or three arenavirus ORFs, or four arenavirus ORFs, or ?ve arenavirus ORFs, or six irus ORFs in a position other than the wild-type on. In c embodiments, the tri-segmented arenavirus particle comprising two L segments and one S segment comprises a full complement of all four arenavirus ORFs. Thus, in some embodiments, the tri-segmented irus particle is an infectious and replication ent tri-segmented arenavirus particle.

In speci?c embodiments, the two L segments of the tri-segmented arenavirus particle have been engineered to carry one of their ORFs in a position other than the ype position. In more speci?c embodiments, the two L segments comprise a full complement of the L segment ORF’s.

In certain speci?c embodiments, the S segment has been engineered to carry one of their ORFs in a position other than the wild-type position or the S segment can be the wild-type genomic segment.

In certain embodiments, one of the two L segments can be: (i) an L segment, wherein the ORF ng the GP is under control of an arenavirus 5’ UTR; (ii) an L segment, wherein the ORF encoding NP is under control of an arenaVirus 5 ’ UTR; (iii) an L segment, wherein the ORF encoding the L protein is under control of an arenaVirus 5’ UTR; (iV) an L segment, wherein the ORF encoding the GP is under control of an arenaVirus 3 ’ UTR; (V) an L segment, wherein the ORF encoding the NP is under control of an irus 3’ UTR; and (Vi) an L segment, wherein the ORF encoding the Z protein is under control of an arenaVirus 3’ UTR.

] In certain embodiments, the tri-segmented arenaVirus particle comprising one L segment and two S ts can comprise a duplicate ORF (i.e., two wild—type L segment ORFs e.g, Z protein or L protein). In c embodiments, the tri-segmented arenaVirus particle comprising two L segments and one S segment can se one ate ORF (e.g., (Z protein, Z protein)) or two duplicate ORFs (e.g., (Z protein, Z protein) and (L protein, L protein)).

Table 2A, below, is an exemplary illustration of the genome organization of a tri- segmented arenavirus particle comprising two L segments and one S segment, wherein intersegmental recombination ofthe two L segments in the tri-segmented arenaVirus genome does not result in a replication-competent bi—segmented Viral particle and abrogates arenaviral promoter activity (i.e., the the S segment is made up of two 3’UTRs d of a 3’ UTR and a 5’ UTR). Based on Table 3 simiar combinations could be predicted for generating an arenavirus particle made up of two 5’ UTRs instead of a 3’ UTR and a 5’ UTR..

Table 2A Tri—segmented arenavirus particle comprising two L segments and one S segment *Position 1 is under the control of an irus L segment 5’ UTR; on 2 is under the control of an arenavirus L segment 3’ UTR; position 3 is under the control of an arenaVirus L t 5’ UTR; position 4 is under the control of an irus L segment 3’ UTR; position 5 is under the control of an arenavirus S segment 5’ UTR; position 6 is under the control of an irus S segment 3’ UTR.

* ORF indicates that a heterologous ORF, for example, a heterologous ORF encoding an HBV antigen, has been ed.

Position 1 Position 2 Position 3 Position 4 Position 5 Position 6 *ORF Z *ORF L NP GP Position 1 Position 2 Position 3 Position 4 Position 5 Position 6 *ORF Z *ORF L GP NP *ORF Z GP L *ORF NP *ORF Z *ORF GP NP L *ORF Z GP *ORF NP L *ORF Z NP *ORF GP L *ORF *ORF NP Z GP L *ORF Z GP NP *ORF L *ORF Z NP GP *ORF L *ORF L *ORF Z NP GP *ORF L *ORF Z GP NP *ORF L *ORF GP NP Z *ORF L GP Z *ORF NP *ORF L *ORF GP NP Z *ORF L NP Z *ORF GP *ORF L GP NP *ORF Z *ORF L NP GP *ORF Z *ORF GP *ORF L NP Z *ORF GP NP L *ORF Z *ORF GP *ORF Z NP L *ORF GP NP Z *ORF L *ORF NP *ORF L GP Z *ORF NP GP L *ORF Z *ORF NP GP Z *ORF L *ORF NP *ORF Z GP L *ORF L *ORF Z NP GP *ORF L *ORF Z GP NP *ORF L *ORF NP GP Z *ORF L *ORF GP NP Z *ORF L NP Z *ORF GP *ORF Z *ORF GP NP L *ORF Z GP L *ORF NP *ORF Z NP GP *ORF L Position 1 Position 2 Position 3 Position 4 Position 5 Position 6 *ORF Z GP NP *ORF L *ORF GP *ORF L NP Z *ORF GP *ORF L Z NP *ORF GP *ORF Z GP L *ORF GP NP L *ORF Z GP L *ORF Z *ORF NP GP L *ORF NP *ORF Z GP Z *ORF L *ORF NP GP Z *ORF L *ORF NP GP Z *ORF NP *ORF L GP NP *ORF Z *ORF L NP *ORF Z *ORF GP NP *ORF GP *ORF Z NP *ORF Z *ORF GP In certain ments, the IGR between position one and position two cab be an arenavirus S segment or L segment IGR; the IGR between position two and three can be an arenavirus S t or L segment IGR; and the IGR between the position ?ve and six can be an arenavirus L segment IGR. In a speci?c embodiment, the IGR between position one and position two can be an irus L t IGR; the IGR between position two and three can be an arenavirus L segment IGR; and the IGR between the on ?ve and six can be an arenavirus S segment IGR. In certain embodiments, other combinations are also le.

In certain embodiments intersegmental recombination of an L segment and an S segment from the tri-segmented arenavirus le comprising two L segments and one S segment restores a ?mctional segment with two Viral genes on only one segment instead of two separate segments. In other embodiments, intersegmental recombination of an L segment and an S segment in the the tri-segmented arenavirus particle comprising two L ts and one S segment does not result in a replication-competent bi-segmeneted Viral particle.

Table 2B, below, is an exemplary illustration ofthe genome organization of a tri- segmented arenavirus particle comprising two L segments and one S segment, wherein intersegmental recombination of an L segment and an S segment in the tri—segmented arenavirus genome does not result in a replication-competent bi-segmented viral particle and tes arenaviral promoter activity (i.e., the resulting recombined S segment is made up of two 3’UTRs instead of a 3’ UTR and a 5’ UTR).

Table 2B Tri—segmented arenavirus particle comprising two L segments and one S segment *Position 1 is under the control of an arenavirus L segment 5’ UTR; position 2 is under the control of an arenavirus L segment 3’ UTR; position 3 is under the control of an arenavirus L segment 5’ UTR; position 4 is under the control of an arenavirus L t 3’ UTR; position 5 is under the control of an irus S segment 5’ UTR; on 6 is under the control of an arenavirus S segment 3’ UTR.

*ORF indicates that a heterologous ORF, for example, a heterologous ORF ng an HBV antigen, has been inserted. on 1 Position 2 Position 3 Position 4 Position 5 Position 6 \P Z *ORF GP L *ORF RP Z GP *ORF *ORF L \P Z *ORF GP L *ORF RP Z GP *ORF *ORF L \‘P L *ORF GP Z *ORF RP L GP *ORF *ORF Z \‘P L *ORF GP Z *ORF NP L GP *ORF *ORF Z GP Z *ORF NP L *ORF GP Z NP *ORF *ORF L GP Z *ORF NP L *ORF GP L NP *ORF *ORF Z GP L *ORF NP Z *ORF GP L NP *ORF *ORF Z In certain embodiments, the IGR between position one and position two cab be an arenavirus S segment or L segment IGR; the IGR between position two and three can be an arenavirus S segment or L segment IGR; and the IGR between the on ?ve and six can be an arenavirus L segment IGR. In a speci?c embodiment, the IGR between position one and position two can be an arenavirus L segment IGR; the IGR between position two and three can be an arenavirus L segment IGR; and the IGR n the position ?ve and six can be an arenavirus S segment IGR. In certain embodiments, other combinations are also possible.

In certain embodiments, the tri-segmented arenavirus particle as described herein results in a infectious and replication competent arenavirus particle. In speci?c embodiments, the arenavirus particle described herein is attenuated. In a particular embodiment, the tri- segmented arenavirus particle is attenuated such that the virus remains, at least partially, replication-competent and can replicate in viva, but can only te low Viral loads resulting in subclinical levels of infection that are non-pathogenic. Such attenuated s can be used as an genic composition. In other embodiments, the arenavirus particle is infectious but unable to produce ?arther infectious progeny in non-complementing cells.

In certain embodiments, the arenavirus c segment, and the respective arenavirus le or tri—segmented arenavirus le can comprise a heterologous ORF. In other embodiments, the arenavirus genomic segment and the tive arenavirus particle or tri- segmented arenavirus particle can comprise a gene of interest. In more c embodiments, the heterologous ORF or the gene of interest encodes an antigen. In more speci?c embodiments, the heterologous ORF or the gene or st encodes an HBV antigen or an antigenic nt f (see Section 6.2).

In certain embodiments, the arenavirus genomic segment, the arenavirus particle or the tri-segmented arenavirus particle can comprise one or more heterologous ORFs or one or more genes of interest. In other embodiments, the arenavirus c segment, the arenavirus particle or the tri-segmented arenavirus particle can comprise at least one heterologous ORF, at least two heterologous ORFs, at least three heterologous ORFs, or more logous ORFs. In other embodiments, the arenavirus particle or the tri-segmented arenavirus particle comprises at least one gene of interest, at least two genes of interest, at least three genes of interest, or more genes of interest. In more speci?c embodiments, the one or more heterologous ORFs or the genes of interest encode one or more HBV antigens or antigenic fragments thereof (see Section 6.2).

] In certain embodiments, an infectious arenavirus expressing an HBV antigen described herein is a tri-segmented irus particle comprising one L segment and two S segments. In certain embodiments, an infectious arenavirus expressing an HBV antigen described herein is a tri-segmented arenavirus particle comprising two L segments and one S segment. 6.2 HBV Antigens In certain embodiments, antigens for use with the methods and compositions bed herein are HBV antigens.

In certain embodiments, the ORFs of two or more HBV antigens described are transcribed as a single transcript.

] In certain ments, any genotype or subgenotype of human HBV or any clinical isolate ofhuman HBV can be used with the present invention to obtain the antigens for generation of the arenaViral s described herein. Such HBV genotypes and subgenotypes include genotypes A-J, and subgenotypes A1-A6, B1-B4, C1-C6, D1-D7, and F1-F4.

In certain embodiments, the HBV antigen can be an HBV antigen ortholog, e.g., a mammalian (i.e., non-human primate, pig, dog, cat, or horse) HBV antigen. (3) pre—S2/S protein antigens In certain embodiments, the antigen is the HBV pre-S2/S n or a nt thereof. In certain embodiments, the antigen is a nt of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or more amino acids of HBV pre-S2/S protein. In certain embodiments, the antigen is an antigenic fragment of HBV pre-S2/S protein. In certain embodiments, the antigen is encoded by a nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In certain embodiments, the antigen comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1. (b) HBc protein antigens In n embodiments, the antigen is the HBV HBc protein or a fragment f. In certain embodiments, the antigen is a fragment of at least 10, 15, 20, 25, 50, 75, 100, 125, 150 or more amino acids of the HBV HBc n. In certain embodiments, the antigen is an antigenic fragment of HBc. In certain embodiments, the antigen is encoded by a nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In n embodiments, the antigen comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2. (c) HBs protein antigens In certain embodiments, the antigen is the HBV HBs protein or a fragment thereof. In certain embodiments, the antigen is a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the HBV HBs protein. In n embodiments, the antigen is an antigenic fragment of HBs.

In certain embodiments, the antigen is the HBV HBs small polypeptide (e.g. "S") or a fragment thereof. In certain embodiments, the antigen is the HBV HBs medium polypeptide (e. g., "pre-S2/S") or a fragment thereof. In certain embodiments, the antigen is the HBV HBs large polypeptide (e.g., "pre-S 1/pre-S2/S") or a nt thereof. In certain embodiments, the antigen is a fragment ofat least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or more amino acids ofthe HBV HBs small polypeptide. In n embodiments, the antigen is a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or more amino acids ofthe HBV HBs medium polypeptide. In certain embodiments, the antigen is a fragment ofat least 10, 15, 20, 25, 30,35, 40,45, 50, 100, 150, 200, 250, 300, 350 or more amino acids of the HBV HBs large ptide. (d) HBs and HBc fusion proteins In certain embodiments, the antigen is a fusion protein of the HBV HBs and HBc proteins or nic fragments thereof. In certain ments, the antigen is a fragment of at least 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225 or more amino acids ofa fusion protein ofHBs and HBc. In certain embodiments, the antigen is d by a nucleic acid ce that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3. In certain embodiments, the antigen comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3. (e) HBe protein antigens In certain embodiments, the antigen is the HBV HBe protein or a fragment thereof. In certain embodiments, the antigen is a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or more amino acids of the HBV HBe protein. In certain embodiments, the antigen is an antigenic fragment of HBe. In certain embodiments, the antigen is encoded by a nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26. In n embodiments, the antigen comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 26. (t) Polymerase n antigens ] In certain embodiments, the antigen is an HBV rase protein or antigenic fragment thereof. In certain embodiments, the antigen is a fragment of at least 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, 500, 600, 700 or more amino acids of an HBV polymerase protein.

Nucleic acid sequences encoding an HBV antigen can be introduced in the genome of an ious arenavirus by tution ofthe nucleic acid ce of the ORF of rotein GP, the matrix protein Z, the nucleoprotein NP, or the polymerase protein L. In other embodiments, the nucleic acid sequence encoding the HBV antigen is fused to the ORF of glycoprotein GP, the matrix protein Z, the nucleoprotein NP, or the polymerase protein L. The tide sequence encoding the HBV antigen, once inserted into the genome of an infectious arenavirus, can be transcribed and/or expressed under control ofone of the four arenavirus promoters (5’ UTR and 3’ UTR of the S t, and 5’ UTR and 3’ UTR of the L segment), as well as ribonucleic acids that can be inserted with regulatory elements that can be read by the viral RNA-dependent RNA polymerase, cellular RNA polymerase I, RNA polymerase II or RNA polymerase 111, such as duplications of viral promoter sequences that are naturally found in the viral UTRs, the 28S ribosomal RNA promoter, the beta-actin promoter or the 5S ribosomal RNA promoter, tively. The nucleic acids encoding the HBV antigen can be ribed and/or expressed either by themselves or as read-through by ?lSiOIl to arenavirus ORFs and genes, respectively, and/or in combination with one or more, e.g., two, three or four, internal ribosome entry sites.

In one embodiment, the antigen is one that is useful for the prevention and/or treatment of infectious disease. In a speci?c embodiment, the antigen is derived from HBV. In certain embodiments, the ORF that encodes the rotein of the arenavirus is substituted by a nucleic acid ce encoding HBV pre-SZ/S n. In n embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by a c acid sequence ng HBV HBc protein. In n embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by a nucleic acid sequence encoding HBV HBs protein. In certain embodiments, the ORF that s the glycoprotein of the arenavirus is substituted by a nucleic acid sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof. (g) Substitution of the ORF ng the glycoprotein of the arenavirus In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by a nucleic acid sequence encoding one, two, or more HBV antigens described herein.

In one embodiment, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an HBV antigen. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding antigen that is a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids of a gene product of a gene of the pre-SZ/S protein of HBV or a fragment thereof. In n embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigenic nt of /S. In n embodiments, the ORF that encodes the rotein of the arenavirus is substituted by nucleic acid sequences encoding antigens including, but not limited to /S or a fragment ofpre-SZ/S.

In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences ng antigen that is a fragment of at least 10, 15, 20, , 50, 75, 100, 125, 150 or more amino acids of a gene product of a gene of the HBc protein of HBV or a fragment thereof In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigenic fragment of HBc.

In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding antigens including, but not limited to HBc or a fragment of In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is tuted by nucleic acid sequences encoding antigen that is a fragment of at least 10, 15, 20, , 30, 35, 40, 45, 50 or more amino acids of a gene product of a gene of the HBs protein of HBV or a fragment thereof In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigenic fragment of HBs.

In n embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding antigens including, but not limited to HBs or a fragment of In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by a nucleic acid sequence encoding two or more HBV proteins or fragments of at least 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225 or more amino acids thereof. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by a c acid sequence encoding HBs and HBc.

In certain embodiments, the ORF that s the glycoprotein of the arenavirus is substituted by a nucleic acid sequence encoding one or more ofpre-S2/S protein or an antigenic fragment thereof, HBc protein or an antigenic fragment thereof, HBs protein or an antigenic nt thereof, and Hbe protein or an antigenic fragment thereof. 6.3 Generation of Infectious Arenavirus sing an HBV Antigen ] Generally, arenavirus particles can be recombinantly produced by standard reverse genetic ques as described for LCMV (L. Flatz, A. aler, J. C. de la Torre, and D. D. Pinschewer, Proc Natl Acad Sci USA 103:4663-4668, 2006; A. B. Sanchez and J. C. de la Torre, Virology 3502370, 2006; E. Ortiz-Riano, B.Y. Cheng, J. C. de la Torre, L. Martinez- Sobrido. J Gen Virol. 94: 1 175-88, 2013). (a) Replication-de?cient arenaviruses To generate infectious, replication-de?cient arenaviruses for use with the present invention these techniques can be used, however, the genome of the rescued Virus is modi?ed as described in Section 6.1. These modi?cations can be: i) one or more, e.g., two, three or four, of the four arenavirus ORFS (glycoprotein (GP); nucleoprotein (NP); the matrix n Z; the RNA-dependent RNA polymerase L) are removed or functionally inactivated to prevent formation of infectious particles in normal cells albeit still allowing gene expression in arenavirus -infected host cells; and ii) nucleic acids coding for HBV antigens can be introduced. Infectious, replication-de?cient s as described herein can be produced as described in International Patent Application ation No. number ) and International Patent Application Publication No. W0 2014/140301 (application number ), each of which is incorporated by reference herein in its entirety.

Once generated from cDNA, the infectious, replication-de?cient arenaviruses provided herein can be ated in complementing cells. Complementing cells are cells that provide the ?anctionality that has been eliminated from the replication-de?cient arenavirus by modi?cation of its genome (e. g., if the ORF encoding the GP protein is deleted or functionally vated, a menting cell does provide the GP protein).

Owing to the removal or functional inactivation of one or more of the viral genes in irus vectors (here deletion of the glycoprotein, GP, will be taken as an example), irus vectors can be generated and expanded in cells providing in trans the deleted viral gene(s), e.g., the GP in the present e. Such a menting cell line, henceforth referred to as C—cells, is ted by transfecting a mammalian cell line such as BHK—Zl, HEK 293, VERO or other (here BHK—2l will be taken as an example) with one or more plasmid(s) for expression of the viral gene(s) of interest (complementation plasmid, referred to as C-plasmid).

The C-plasmid(s) express the viral gene(s) deleted in the irus vector to be generated under control of one or more expression cassettes suitable for expression in mammalian cells, e.g., a ian polymerase 11 promoter such as the CMV or EFlalpha promoter with a polyadenylation signal. In addition, the complementation plasmid features a mammalian selection marker, e.g., puromycin ance, under control of an expression cassette suitable for gene expression in mammalian cells, e.g., polymerase II expression cassette as above, or the viral gene transcript(s) are followed by an internal ribosome entry site, such as the one of encephalomyocarditis Virus, ed by the mammalian resistance marker. For production in E. coli, the plasmid additionally features a bacterial ion marker, such as an ampicillin resistance cassette.

Cells that can be used, e.g., BHK-Zl, HEK 293, MC57G or other, are kept in culture and are transfected with the complementation plasmid(s) using any of the commonly used gies such as calcium-phosphate, liposome-based protocols or electroporation. A few days later the suitable selection agent, e.g., puromycin, is added in titrated concentrations.

Surviving clones are isolated and subcloned following standard procedures, and high-expressing C-cell clones are identi?ed using Western blot or ?ow cytometry procedures with antibodies directed against the viral protein(s) of interest. As an alternative to the use of stably transfected C-cells transient transfection of normal cells can complement the missing viral ) in each of the steps where C-cells will be used below. In addition, a helper virus can be used to provide the missing ?anctionality in trans.

] Plasmids that can be used can be of two types: i) Two plasmids, referred to as TF- plasmids for expressing intracellularly in C-cells the minimal transacting factors of the arenavirus, is derived from e.g., NP and L proteins ofLCMV in the present example; and ii) Plasmids, referred to as GS-plasmids, for expressing intracellularly in C—cells the irus vector genome segments, e. g., the segments with designed modi?cations. TF-plasmids express the NP and L proteins of the respective arenavirus vector under control of an sion cassette suitable for protein sion in mammalian cells, typically e.g., a mammalian polymerase II promoter such as the CMV or EFlalpha promoter, either one of them preferentially in combination with a polyadenylation signal. GS-plasmids express the small (S) and the large (L) genome segments of the vector. Typically, polymerase en expression cassettes or T7 bacteriophage RNA rase (T7-) driven expression cassettes can be used, the latter entially with a 3 ’-terminal me for processing of the primary transcript to yield the correct end. In the case of using a T7-based system, expression of T7 in C—cells must be provided by either including in the recovery process an additional expression plasmid, constructed analogously to TF-plasmids, providing T7, or C-cells are constructed to additionally express T7 in a stable manner. In certain embodiments, TF and GS plasmids can be the same, i.e. the genome sequence and transacting factors can be ribed by T7, poll and polII promoters from one plasmid.

For recovering of the arenavirus vector, the following procedures can be used.

First day: C-cells, typically 80% con?uent in l plates, are transfected with a e of the two TF-plasmids plus the two GS-plasmids. In certain embodiments, the TF and GS plasmids can be the same, i.e. the genome sequence and transacting factors can be transcribed by T7, p011 and polII promoters from one plasmid. For this one can exploit any of the ly used strategies such as calcium-phosphate, liposome-based protocols or electroporation. 3-5 days later: The culture supernatant (arenavirus vector ation) is harvested, aliquoted and stored at 4°C, -20°C or —80°C depending on how long the arenavirus vector should be stored prior to use. Then the arenavirus vector preparation’s infectious titer is assessed by an immunofocus assay on C—cells.

The invention furthermore relates to expression of an HBV antigen in a cell culture wherein the cell culture is infected with an infectious arenavirus expressing an HBV antigen. When used for expression of an HBV antigen in cultured cells, the following two procedures can be used: i) The cell type of interest is infected with the arenavirus vector preparation described herein at a multiplicity of infection (MOI) of one or more, e.g., two, three or four, resulting in production ofthe HBV antigen in all cells already shortly after infection. ii) atively, a lower MOI can be used and individual cell clones can be selected for their level of virally driven HBV antigen expression. Subsequently individual clones can be expanded ely owing to the non-cytolytic nature of arenavirus vectors. Irrespective of the approach, the HBV antigen can subsequently be collected (and puri?ed) either from the e supernatant or from the cells lves, depending on the properties of the HBV antigen produced. However, the invention is not limited to these two strategies, and other ways of driving expression ofHBV n using infectious, replication-de?cient iruses as vectors may be considered.

Alternatively, a rescue system consisting of three plasmids can be used: (1) the ?rst plasmid expresses the protein NP by transcription via Polymerase II and subsequent translation in transfected cells; (2) the second plasmid gives rise to the (negative-stranded) L- Segment of the LCMV genome by transcription via Polymerase I as well as the L protein by transcription via Polymerase II from the same template in the opposite direction of the Polymerase I er; (3) the third plasmid gives rise to the S-segment of the LCMV genome (encoding the antigen coding sequence instead of the LCMV glycoprotein) via ription by Polymerase I. 3ug of each plasmid is used for electroporation of C-cells, followed by seeding of cells in 6-we11 plates and incubation at 37°C. After incubation, cells and supernatant from transfections are combined with freshly seeded C-cells, and vectors are harvested and d from cells & debris at a de?ned timepoint post infection. Once the vector has been generated, a nucleic acid encoding an antigen of an nic virus and/or an immunomodulatory peptide, polypeptide, or protein (see n 6.2) can be inserted into a d from which a genomic segment of an infectious replication-de?cient vector is ribed by any que known to the skilled artisan.

Owing to the removal or functional inactivation of one or more of the viral genes in arenavirus vectors (here on of the glycoprotein, GP, will be taken as an example) arenavirus vectors can be generated and expanded in cells that provide the deleted or functionally inactivated viral gene(s) (e.g., the GP) in trans. The resulting virus itself is infectious but is unable to produce further infectious progeny particles in non-complementing cells due to the lack of the deleted or ?anctionally vated viral gene(s) (e.g., the GP). The complementing cell can provide the missing functionality either by stable transfection, transient transfection, or by infection with a helper virus that expresses the g functionality.

In certain embodiments, the complementing cell provides the viral gene that has been deleted or functionally inactivated from the arenavirus vector genome. In a speci?c embodiment, the complementing cell provides the viral gene from a viral strain that is the same as the viral strain that was used to generate the genome of the arenavirus vector. In another embodiment, the complementing cell provides the viral gene from a viral strain that is different from the viral strain that was used to generate the genome of the arenavirus vector. For example, the viral gene provided in the complementing cell is obtained from the MP strain ofLCMV and encodes a n having the amino acid sequence of SEQ ID NO: 15, l6, 17, or 18. In another example, the viral gene ed in the menting cell is ed from the Clone 13 strain ofLCMV and encodes a protein having the amino acid sequence of SEQ ID NO: 21, 22, 23, or 24. In another example, the viral gene provided in the complementing cell is obtained from the WE strain ofLCMV and encodes a protein having the amino acid sequence of SEQ ID NO: 25.

In a speci?c embodiment, the complementing cell provides the GP of the MP strain of LCMV and the arenavirus vector comprises an ORF of a human HBV n as described herein in place of the ORF encoding the GP protein. In an even more speci?c embodiment, the complementing cell provides the GP of the MP strain ofLCMV and the arenavirus vector is obtained from LCMV Clone l3 and comprises an ORF of a human HBV antigen as described herein in place of the ORF encoding the GP n. In an even more speci?c embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16.

In a speci?c embodiment, the complementing cell provides the GP of the Clone 13 strain of LCMV and the irus vector comprises an ORF of a human HBV antigen as described herein in place of the ORF encoding the GP protein. In an even more speci?c embodiment, the complementing cell provides the GP of the Clone 13 strain ofLCMV and the irus vector is obtained from LCMV MP strain and comprises an ORF of a human HBV antigen as described herein in place of the ORF encoding the GP n. In an even more speci?c embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

In a speci?c embodiment, the complementing cell provides the GP of the WE strain of LCMV and the arenavirus vector comprises an ORF of a human HBV antigen as described herein in place of the ORF ng the GP protein. In an even more speci?c embodiment, the complementing cell provides the GP of the WE strain ofLCMV and the arenavirus vector is obtained from LCMV Clone 13 and comprises an ORF of a human HBV n as described herein in place of the ORF encoding the GP protein. In an even more speci?c embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% cal to the amino acid sequence of SEQ ID NO: 25.

In a speci?c embodiment, the complementing cell es the GP of the WE strain of LCMV and the arenavirus vector comprises an ORF of a human HBV antigen as described herein in place of the ORF encoding the GP protein. In an even more speci?c embodiment, the complementing cell provides the GP of the WE strain ofLCMV and the arenavirus vector is obtained from LCMV MP strain and comprises an ORF of a human HBV antigen as described herein in place of the ORF encoding the GP protein. In an even more speci?c embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25.

In certain embodiments, the infectious, replication-de?cient irus is trisegmented. (b) Replication-competent, trisegmented iruses For use with the methods and compositions provided herein are methods of generation of replication-competent arenavirus vectors. Infectious, replication-competent trisegmented viruses as described herein can be produced as described in United States Provisional Patent ation No. 62/079,493, which is incorporated by reference herein in its entirety.

] In certain ments, the method of generating a tri-segmented arenavirus particle comprises (i) transfecting into a host cell the cDNAs of the one L segment and two S segments or two L segments and one S segment; (ii) transfecting into a host cell plasmids sing the arenavirus’ minimal trans—acting factors NP and L; (iii) maintaining the host cell under conditions suitable for virus formation; and (iv) harvesting the arenavirus particle.

Once generated from cDNA, the tri-segmented arenavirus particle (i.e., infectious and replication competent) can be ated. In certain embodiments tri-segmented arenavirus les can be propagated in any host cell that allows the virus to grow to titers that permit the uses of the virus as described herein. In one embodiment, the host cell allows the tri-segmented arenavirus particle to grow to titers able to those determined for the corresponding wild- type.

In certain embodiments, the tri-segmented arenavirus particle may be propagated in host cells. Speci?c examples of host cells that can be used include BHK-Zl, HEK 293, VERO or other. In a speci?c embodiment, the tri-segmented arenavirus particle may be ated in a cell line.

In n embodiments, the host cells are kept in culture and are transfected with one or more plasmid(s). The plasmid(s) express the irus c segment(s) to be generated under control of one or more sion cassettes suitable for expression in mammalian cells, e.g., consisting of a polymerase I promoter and terminator.

In speci?c ments, the host cells are kept in culture and are transfected with one or more plasmid(s). The plasmid(s) express the viral gene(s) to be generated under control of one or more expression cassettes suitable for expression in mammalian cells, e.g., consisting of a polymerase I promoter and terminator.

Plasmids that can be used for generating a tri-segmented arenavirus comprising one L segment and two S segments can include: i) two ds each encoding the S genome segment e.g., pol-I driven S segment expression plasmids, ii) a plasmid encoding the L genome segment e.g., a pol-I driven L segment expression plasmid. Plasmids needed for the tri- segmented arenavirus sing two L segments and one S ts are: i) two plasmids each encoding the L genome segment e.g., pol-L, ii) a plasmid encoding the S genome segment e. g., pol-I S.

In certain embodiments, plasmids encoding an arenavirus polymerase that direct intracellular synthesis of the Viral L and S segments can be incorporated into the transfection mixture. For example, a plasmid encoding the L protein and a plasmid encoding NP (pC-L and pC-NP, respectively). The L protein and NP are the minimal trans-acting factors necessary for viral RNA ription and replication. Alternatively, intracellular synthesis of viral L and S segments, together with NP and L n can be performed using an expression cassette with pol-I and pol-II promoters reading from opposite sides into the L and S segment cDNAs of two te plasmids, respectively.

In on, the plasmid(s) features a mammalian selection marker, e.g., puromycin resistance, under control of an expression cassette suitable for gene expression in mammalian cells, e.g., rase II sion cassette as above, or the viral gene transcript(s) are followed by an internal me entry site, such as the one of encephalomyocarditis virus, followed by the mammalian resistance marker. For production in E.coli, the plasmid onally features a bacterial selection , such as an ampicillin resistance cassette.

Transfection of BHK—21 cells with a plasmid(s) can be performed using any of the commonly used strategies such as m-phosphate, liposome-based protocols or electroporation. A few days later the suitable selection agent, e. g., puromycin, is added in titrated concentrations. Surviving clones are isolated and ned following standard procedures, and high-expressing clones are identi?ed using Western blot or ?ow cytometry procedures with antibodies directed against the viral protein(s) of interest.

Typically, RNA polymerase I-driven expression cassettes, RNA polymerase II- driven cassettes or T7 bacteriophage RNA rase driven cassettes can be used, the latter preferentially with a 3 ’-terminal ribozyme for processing of the primary transcript to yield the correct end. In certain embodiments, the plasmids encoding the arenavirus genomic segments can be the same, i.e., the genome sequence and transacting factors can be transcribed by T7, poll and po III promoters from one plasmid.

For recovering the tri-segmented arenavirus vector, the following procedures are envisaged. First day: cells, typically 80% con?uent in l plates, are transfected with a mixture of the plasmids, as described above. For this one can exploit any commonly used strategies such as calcium-phosphate, liposome-based protocols or electroporation. ] 3-5 days later: The cultured supernatant (arenavirus vector preparation) is harvested, aliquoted and stored at 4°C, -20°C, or -80°C, depending on how long the arenavirus vector should be stored prior use. The arenavirus vector preparation’s infectious titer is assessed by an focus assay. Alternatively, the transfected cells and supernatant may be passaged to a larger vessel (e.g., a T75 tissue culture ?ask) on day 3-5 after ection, and culture supernatant is harvested up to ?ve days after passage.

The present application furthermore relates to expression of a heterologous ORF (e.g., an HBV antigen), wherein a plasmid encoding the c segment is modi?ed to orate a heterologous ORF. The logous ORF can be incorporated into the plasmid using restriction enzymes. In certain embodiments, the heterologous ORF encodes an HBV antigen. In certain embodiments, the plasmid encoding the genomic segement is d to incorporate one or more heterologous ORFs. In certain embodiments, the heterologous ORFs encode one or more HBV antigens. 6.4 Nucleic Acids, Vector Systems and Cell Lines In one ment, described herein is a nucleic acid sequence which is the cDNA ofthe large genomic segment (L t) of an infectious arenavirus described herein, in which one ORF of the genomic segment is deleted or ?inctionally inactivated, and the genomic segment comprises a nucleotide sequence ng an HBV antigen. In certain embodiments, the infectious arenavirus viral vector is replication-de?cient (See Section 6.1(a)). In certain embodiments, the infectious arenavirus viral vector is replication—competent (See Section 6. 1 (b)).

In one embodiment, described herein is a nucleic acid ce that s the short genomic segment (S segment) of an infectious irus described herein, in which one ORF of the genomic t is deleted or functionally inactivated and wherein the short genomic segment ses a nucleotide sequence encoding an HBV antigen. In another embodiment, described herein is a nucleic acid sequence that encodes the short genomic segment (S segment) of an ious arenavirus described herein, in which the ORF of the glycoprotein gene is d or functionally inactivated and wherein the short genomic segment comprises a nucleotide sequence encoding an HBV antigen. In certain, more c embodiments, the HBV antigen is an antigen described in Section 6.2.

In certain embodiments, the nucleic acid sequences provided herein can be derived from a particular strain of LCMV. Strains ofLCMV include Clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009, 200501927, 810362, 811316, 810316, 810366, 20112714, Douglas, GROl, SN05, CABN and their derivatives. In speci?c embodiments, the nucleic acid is derived from LCMV Clone 13. In other speci?c embodiments, the nucleic acid is derived from LCMV MP strain.

In a more speci?c embodiment, provided herein is a nucleic acid comprising an arenavirus genomic segment comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the ce of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In r embodiment, ed herein is a nucleic acid that comprises an arenavirus genomic t comprising (i) a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the sequence of nucleotide 1639 to 3315 of SEQ ID NO: 11; and (ii) a nucleotide sequence encoding an HBV n.

In another embodiment, provided herein is a nucleic acid that comprises an arenavirus genomic segment comprising (i) a nucleotide sequence encoding an expression product Whose amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid ce encoded by 1639 to 3315 of SEQ ID NO: 11; and (ii) a nucleotide sequence encoding an HBV antigen.

In r embodiment, provided herein is a nucleic acid that comprises an arenavirus c segment comprising (i) a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the sequence of nucleotide 1640 to 3316 of SEQ ID NO: 12; and (ii) a nucleotide sequence encoding an HBV antigen.

] In another embodiment, ed herein is a nucleic acid that comprises an arenavirus genomic segment sing (i) a nucleotide ce encoding an expression product Whose amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% cal to the amino acid sequence encoded by 1640 to 3316 of SEQ ID NO: 12; and (ii) a nucleotide sequence encoding an HBV antigen In one embodiment, described herein is a vector system comprising one or more vectors that together comprise the genome of an infectious arenavirus particle described herein.

Speci?cally, provided herein is a vector system wherein the one or more vectors comprise two arenavirus genomic segments, namely an L segment and an S segment, of an infectious arenavirus described herein. Such a vector system can comprise (on one or more separate DNA molecules): An arenavirus S genomic segment that is modi?ed such that an arenavirus particle carrying this modi?ed S genomic segment cannot produce infectious progeny Virus particles and an arenavirus L genomic segment that comprises a nucleotide sequence encoding (in sense or antisense) an HBV antigen; An arenavirus L genomic segment that is modi?ed such that an irus particle carrying this modi?ed L genomic segment cannot produce infectious progeny Virus particles and an arenavirus S genomic segment that comprises a nucleotide sequence encoding (in sense or antisense) an HBV antigen; An arenavirus S c segment that is modi?ed such that an arenavirus particle carrying this modi?ed S genomic segment cannot produce infectious progeny Virus particles and wherein the arenavirus S genomic segment comprises a nucleotide sequence encoding (in sense or antisense) an HBV n and comprising a wild type arenavirus L c segment; or ] An arenavirus L genomic segment that is modi?ed such that an arenavirus particle ng this modi?ed L genomic segment cannot produce infectious progeny Virus particles and wherein the arenavirus L genomic segment comprises a nucleotide sequence encoding (in sense or antisense) an HBV antigen and comprising a wild type arenavirus S genomic segment.

In certain ments, described herein is a nucleic acid sequence comprising an arenavirus (e.g., LCMV) genomic segment in which the ORF encoding the GP of the S genomic segment is substituted with a nucleotide sequence comprising: a tide sequence encoding a tis B pre-SZ/S protein or an antigenic fragment thereof; a nucleotide sequence encoding a tis B Virus HBc protein or an antigenic fragment thereof; a tide sequence encoding a Hepatitis B Virus HBs protein or an antigenic fragment a nucleotide sequence encoding a fusion of Hepatitis B Virus HBs and HBc ns or antigenic fragments thereof; a tide sequence encoding a Hepatitis B Virus HBe n or an antigenic fragment thereof.

In certain embodiments, described herein is a nucleic acid sequence comprising an arenavirus (e.g., LCMV) genomic segment in which the ORF encoding the GP of the S genomic segment is substituted with a nucleotide sequence encoding one or more HBV antigens (e.g., one or more of those listed in the above paragraph).

In another embodiment, provided herein is a cell wherein the cell comprises a nucleic acid or a vector system bed above in this section. Cell lines derived from such cells, cultures comprising such cells, and methods of culturing such cells infected with c acids or vector systems are also provided herein. In certain embodiments, provided herein is a cell wherein the cell comprises a nucleic acid comprising the large genomic segment (L segment) of an ious arenavirus described herein, in which one ORF of the genomic segment is d or functionally inactivated, and the genomic segment comprises a nucleotide ce encoding an HBV antigen.

In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that comprises the short genomic t (S t) of an infectious arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a nucleotide sequence encoding HBV pre-SZ/S protein or an antigenic fragment thereof.

In other ments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that comprises the short genomic segment (S segment) of an infectious arenavirus described herein, in which one ORF of the genomic segment is deleted or onally inactivated and wherein the short genomic segment ses a nucleotide sequence encoding HBV HBc protein or an nic fragment thereof.

In other embodiments, provided herein is a cell n the cell comprises a nucleic acid sequence that comprises the short genomic segment (S t) of an infectious irus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic t ses a nucleotide sequence encoding HBV HBs protein or an nic fragment thereof.

In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that comprises the short genomic segment (S segment) of an infectious arenavirus described herein, in which one ORF of the genomic segment is deleted or onally inactivated and wherein the short genomic segment comprises a nucleotide sequence encoding a fusion protein comprising at least one domain from HBV HBs protein and HBV HBc protein.

In other embodiments, provided herein is a cell n the cell comprises a nucleic acid sequence that comprises the short genomic segment (S segment) of an infectious arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a tide sequence encoding one or more ofHBV antigens.

In another embodiment, ed herein is a cell wherein the cell comprises two c acids or vector systems bed herein. Cell lines derived from such cells, cultures comprising such cells, and methods of culturing such cells infected with nucleic acids or vector systems are also provided herein.

In certain embodiments, provided herein is a nucleic acid sing a tide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13 or SEQ ID NO: 14. In certain embodiments, provided herein is an expression vector comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% cal to SEQ ID NO: 13 or SEQ ID NO: 14. In certain embodiments, provided herein is a host cell comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13 or SEQ ID NO: 14.

In certain embodiments, provided herein is a c acid comprising a nucleotide sequence encoding an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15, 16, 17, or 18. In certain embodiments, provided herein is an expression vector comprising a nucleotide sequence encoding an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15, 16, 17, or 18. In certain embodiments, provided herein is a host cell comprising a nucleotide sequence that encodes an amino acid ce that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15, 16, 17, or In certain embodiments, provided herein is an isolated protein sing an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15, 16, 17, or 18. In certain embodiments, provided herein is a host cell that expresses a protein comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15, 16, 17, or 18. In certain embodiments, the host cell is cultured in cell culture In certain embodiments, provided herein is a nucleic acid comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12 or SEQ ID NO: 7. In certain embodiments, provided herein is an expression vector comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12 or SEQ ID NO: 7. In certain embodiments, provided herein is a host cell sing a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12 or SEQ ID NO: 7.

] In certain ments, provided herein is a c acid comprising a nucleotide sequence encoding an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21, 22, 23, or 24. In certain embodiments, provided herein is an expression vector comprising a nucleotide sequence encoding an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21, 22, 23, or 24. In certain embodiments, provided herein is a host cell comprising a nucleotide sequence that s an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% cal to SEQ ID NO: 21, 22, 23, or In certain embodiments, provided herein is an isolated protein comprising an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21, 22, 23, or 24. In certain embodiments, provided herein is a host cell that expresses a protein comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21, 22, 23, or 24. In certain embodiments, the host cell is cultured in cell culture medium. 6.5 Methods of Use Provided herein are immunotherapies for Hepatitis B virus infections. In one embodiment, provided herein are s of ng an infection in a subject comprising administering to the subject one or more infectious arenaviruses expressing an HBV antigen as described herein or a composition thereof. In certain embodiments, the infectious arenaviruses are replication—de?cient. In certain embodiments, the infectious arenaviruses are replication- competent. In a specific embodiment, a method for treating an infection described herein comprises administering to a subject in need thereof an effective amount of one or more infectious arenaviruses expressing an HBV antigen described herein or a composition thereof.

The subject can be a mammal, such as but not limited to a human being, a mouse, a rat, a guinea pig, a icated animal, such as, but not limited to, a cow, a horse, a sheep, a pig, a goat, a cat, a dog, a hamster, a donkey. In a speci?c embodiment, the subject is a human.

In another embodiment, provided herein are methods for inducing an immune response against HBV in a subject comprising administering to the subject an infectious arenavirus expressing an HBV antigen or a ition thereof.

In r ment, the subjects to whom an ious arenavirus expressing an HBV antigen bed herein or a composition thereof is administered have, are susceptible to, or are at risk for an HBV ion. In r speci?c embodiment, the subjects to whom an infectious arenavirus expressing an HBV antigen described herein or a ition f is administered are infected with, are susceptible to, or are at risk for, an infection with HBV.

In another ment, the subjects to whom an infectious arenavirus expressing an HBV antigen described herein or a composition thereof is administered are ing from, are susceptible to, or are at risk for, an infection with HBV, e.g., in the liver. In a speci?c embodiment, the subjects to whom an infectious arenavirus expressing an HBV antigen described herein or a composition thereof is administered are ing from, are susceptible to, or are at risk for, an infection with HBV in one or more organs of the body, e.g., the liver.

In another embodiment, the subjects to whom an ious arenavirus expressing an HBV antigen described herein or a composition f is administered have test results (e.g., blood test s) indicating liver . In certain embodiments, the subjects have alanine aminotransferase (ALT) levels in the blood indicating liver damage. In certain embodiment, the subjects have aspartate aminotransferase (AST) levels in the blood indicating liver . In certain embodiments, the subjects have alkaline phosphatase levels in the blood indicating liver damage. In certain embodiments, the subjects have lactate dehydrogenase (LDH) levels in the blood indicating liver damage. In certain embodiments, the subjects have one or more of ALT, AST, alkaline phosphatase, and LDH levels in the blood indicating liver damage.

] In certain embodiments, the subjects have alpha-fetoprotein (AFP) levels in the blood indicating liver cancer or susceptibility thereto. In certain embodiments, the subjects have bilirubin (e.g. levels in the blood indicating liver . In certain , ated bilirubin) embodiments, the ts have albumin levels in the blood indicating liver damage.

In certain embodiments, the subjects have abdominal ultrasound results indicating liver damage. In certain embodiments, the subjects have CAT scan results ting liver damage. In certain embodiments, the subjects have MRI results indicating liver damage.

In another embodiment, the subjects to Whom an infectious arenavirus sing an HBV antigen described herein or a composition thereof is administered have detectable levels ofHBs antigen ) in the blood. In certain embodiments, the subjects have detectable levels of IgM antibody against HBc antigen (HBcAg) in the blood. In certain embodiments, the subjects have detectable levels of HBe antigen (HBeAg, the extracellular/secreted version of the HBc protein) in the blood. In certain ments, the subjects have detectable levels of antibody to HBsAg in the blood.

In another ment, the subjects to whom an infectious arenavirus expressing an HBV antigen described herein or a composition f is administered have persistent levels ofHBsAg, indicative of chronic hepatitis. In certain embodiments, the subjects have persistent levels of HBeAg, indicative of chronic hepatitis. In certain embodiments, the subjects have persistent levels ofHBsAg and HBeAg, indicative of chronic hepatitis.

In another embodiment, the ts to Whom an infectious arenavirus expressing an HBV antigen described herein or a composition thereof is administered are suffering from symptoms ofHBV infection, including but not limited to loss of appetite, fatigue, nausea, vomiting, itchiness, abdominal pain, abdominal swelling, or jaundice.

] In another embodiment, the subjects to whom an infectious arenavirus expressing an HBV antigen described herein or a composition thereof is stered are ing from stations of HBV, including but not limited to acute tis B, chronic HBV infection, cirrhosis, and cellular carcinoma (HCC). In another embodiment, the infectious arenavirus expressing an HBV n described herein or a composition thereof is administered to a subject with asymptomatic HBV.

In another embodiment, an infectious arenavirus expressing an HBV antigen as described herein or a composition thereof is administered to a subject of any age group suffering from, tible to, or are at risk for, an infection with HBV. In a speci?c embodiment, an infectious arenavirus expressing an HBV antigen as described herein or a composition thereof is administered to a subject with a mised immune system, a pregnant subject, a subject undergoing an organ or bone marrow transplant, a subject taking immunosuppressive drugs, a t undergoing hemodialysis, a subject who has cancer, or a subject who is suffering from, susceptible to, or at risk for, an infection with HBV. In a more speci?c embodiment, an infectious arenavirus sing an HBV antigen as described herein or a composition thereof is administered to a t with a compromised immune system due to HIV infection, who is suffering from, is susceptible to, or is at risk for, an infection with HBV. In yet another specific embodiment, an ious arenavirus expressing an HBV antigen as described herein or a composition thereof is administered to a subject who is a child of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years of age suffering from, susceptible to, or at risk for, an infection with HBV. In yet another speci?c embodiment, an infectious arenavirus expressing an HBV antigen described herein or a composition thereof is administered to a subject who is an infant suffering from, susceptible to, or at risk for, an infection with HBV. In yet another specific embodiment, an infectious arenavirus expressing an HBV antigen described herein or a ition f is administered to a subject who is an infant of 0, l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months of age suffering from, susceptible to, or at risk for, an infection with HBV. In yet another specific embodiment, an infectious arenavirus expressing an HBV antigen described herein or a composition f is administered to an elderly subject who is suffering from, is susceptible to, or is at risk for, an infection with HBV.

In another embodiment, an infectious arenavirus expressing an HBV antigen described herein or a ition thereof is administered to subjects with a heightened risk of disseminated HBV infection. In a speci?c embodiment, an infectious arenaVirus expressing an HBV antigen described herein or a composition thereof is administered to ts in neonatal period with immature neonatal immune system. In another ment, an infectious arenaVirus expressing an HBV antigen described herein or a composition thereof is administered to a subject who uses intravenous drugs with a heightened risk ofHBV infection.

In another embodiment, an infectious arenavirus expressing an HBV antigen described herein or a composition f is administered to subjects infected with one or more genotypes or subgenotypes ofHBV. In certain embodiments, the genotype is one or more of genotypes A-J, or another genotype. In certain embodiments, the subgenotype is one or more otypes Al-A6, Bl-B4, C1—C6, Dl—D7, F1—F4, or another otype.

In another embodiment, administering an infectious arenavirus expressing an HBV antigen as bed herein or a ition f to subjects confer cell-mediated ty (CMI) against an infection with HBV. Without being bound by theory, in another embodiment, an infectious arenavirus expressing an HBV antigen as described herein or a composition thereof infects and ses antigens of interest in antigen presenting cells (APC) ofthe host (e.g., macrophages) for direct presentation of antigens on Major Histocompatibility Complex (MHC) class I and II. In another embodiment, administering an infectious arenaVirus expressing an HBV antigen as described herein or a composition thereof to subjects induces plurifunctional IFN—y and TNF-(x co-producing HBV-speci?c CD4+ and CD8+ T cell responses (IFN—y is produced by CD4+ and CD8+ T cells and TNF-OL is produced by CD4+ T cells) of high magnitude to treat or t an infection with HBV.

In another ment, administering an infectious arenavirus expressing an HBV antigen or a composition thereof s the risk that an dual will develop an infection with HBV by at least about 10%, at least about 20%, at least about 25%, at least about %, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the risk of developing an infection with HBV in the absence of such treatment.

In another embodiment, administering an infectious arenaVirus expressing an HBV antigen or a composition thereof reduces the symptoms of an infection with HBV by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the manifestation of the symptoms of an infection HBV in the absence of such treatment.

In r embodiment, administering an infectious arenavirus expressing an HBV antigen or a composition thereof in subjects with immature neonatal immune system induces cell-mediated immunity (CMI) se against an infection with HBV by at least about %, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to cell-mediated immunity (CMI) response against an infection with HBV in the absence of such a treatment.

In certain embodiments, administering an infectious arenavirus expressing an HBV n or a composition thereofreduces ALT levels in the blood. In certain ments, administering an ious arenavirus expressing an HBV antigen or a composition thereof s AST levels in the blood. In certain embodiments, administering an infectious arenavirus expressing an HBV antigen or a composition thereof reduces alkaline phosphatase levels in the blood. In certain embodiments, stering an infectious arenavirus expressing an HBV antigen or a composition thereof reduces LDH levels in the blood. In certain embodiments, administering an ious arenavirus expressing an HBV antigen or a composition thereof reduces one or more ofALT, AST, alkaline atase, and LDH levels in the blood.

In certain embodiments, administering an infectious arenavirus expressing an HBV antigen or a composition thereof reduces AFP levels in the blood. In n embodiments, administering an infectious arenavirus expressing an HBV antigen or a composition thereof reduces bilirubin (e.g. , conjugated bilirubin) levels in the blood. In certain embodiments, administering an infectious irus expressing an HBV antigen or a composition thereof increases albumin levels in the blood.

In certain embodiments, administering an ious arenavirus expressing an HBV n or a composition thereof reduces levels of HBsAg in the blood. In certain embodiments, administering an infectious arenavirus expressing an HBV antigen or a composition thereof reduces levels of IgM antibody against HBcAg in the blood. In certain embodiments, stering an infectious arenavirus expressing an HBV antigen or a composition thereof reduces levels of HBeAg in the blood. In certain embodiments, administering an infectious arenavirus expressing an HBV antigen or a composition thereof reduces levels of antibody to HBsAg in the blood.

In certain embodiments, administering an infectious arenavirus expressing an HBV antigen or a composition f reduces the number of inclusion bodies detected in salivary glands or another histological sample. In n embodiments, administering an infectious arenavirus expressing an HBV antigen or a composition thereofreduces the number of anti-HBV antibodies detected in a patient blood sample. In certain embodiments, administering an infectious arenavirus expressing an HBV antigen or a composition thereof reduces the amount ofHBV detected in urine, saliva, blood, tears, semen, or breast milk. In certain ments, administering an infectious irus expressing an HBV antigen or a composition thereof reduces the level of virus cultured from a urine, throat swab, bronchial lavage, or tissue .

In certain ments, administering an infectious arenavirus sing an HBV antigen or a ition thereof reduces the level of virus detected through quantitative or qualitative PCR tests. s in cell-mediated immunity (CMI) response function against an ion with HBV induced by administering an infectious irus expressing an HBV antigen or a composition thereof in subjects can be measured by any assay known to the skilled artisan including, but not limited to ?ow cytometry (see, e.g., Perfetto S.P. et al., Nat Rev Immun. 2004; 4(8):648—55), lymphocyte proliferation assays (see, e.g., Bonilla EA. et al., Ann Allergy Asthma Immunol. 2008; 101 :101-4; and Hicks M.J. et al., Am J Clin Pathol. 1983; 80: ), assays to e lymphocyte activation including determining changes in e marker expression following activation of measurement of cytokines ofT lymphocytes (see, e.g., Caruso A. et al., Cytometry. 1997;27:71-6), ELISPOT assays (see, e.g., Czerkinsky C.C. et al., J l Methods. 1983; 65:109-121; and Hutchings P.R. Et al., J Immunol Methods. 1989; 120: 1-8), or Natural killer cell cytotoxicity assays (see, e.g., Bonilla EA. et al., Ann Allergy Asthma Immunol. 2005 May; 94(5 Suppl 1):S1-63).

In another embodiment, described herein is a method of use with an infectious arenavirus (e. g., LCMV) expressing an HBV antigen as described herein in which the ORF ng the GP ofthe S genomic segment is substituted with a nucleotide sequence comprising: a. a nucleotide sequence encoding an HBV pre-SZ/S protein or an antigenic fragment thereof; b. a tide sequence encoding an HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding an HBV HBs protein or an antigenic fragment thereof; d. a nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; e. a nucleotide sequence encoding an HBV HBe protein or an antigenic fragment thereof In r embodiment, provided herein are methods of preventing transmission and/or ion of HBV from a mother to an unborn child comprising administering to a subject of child-bearing age an infectious arenavirus expressing an HBV antigen as described herein.

See n 6.2. In speci?c ments, provided herein are methods of ting transmission and/or infection ofHBV from a mother to an unborn child comprising stering to a seronegative subject of bearing age an infectious arenavirus expressing an HBV antigen as described herein. In yet another embodiment provided herein are s of preventing transmission and/or infection ofHBV from a mother to an unborn child comprising administering to a subject of child-bearing age with the intention to procreate an infectious arenavirus expressing an HBV antigen as described herein.

In another embodiment, provided herein are methods of preventing transmission and/or infection of HBV from a mother to an unborn child comprising administering to a subject of child-bearing age one or more ious arenaviruses expressing an HBV antigen as described herein. See Section 6.2. In speci?c embodiments, provided herein are methods of preventing ission and/or infection ofHBV from a mother to an unborn child comprising administering to a gative subject of child-bearing age one or more infectious arenaviruses expressing an HBV antigen as described herein. In yet another embodiment, ed herein are methods of preventing transmission and/or infection ofHBV from a mother to an unborn child comprising administering to a subject of child-bearing age with the intention to procreate one or more ious arenaviruses expressing an HBV n as described herein.

In another embodiment, provided herein are methods of preventing transmission and/or infection of HBV from a mother to an unborn child comprising administering to a pregnant t an infectious arenavirus expressing an HBV antigen as described . In speci?c embodiments, provided herein are methods of preventing transmission and/or infection ofHBV from a mother to an unborn child comprising administering to a pregnant t an effective amount of an infectious arenavirus expressing an HBV antigen described herein.

In another embodiment, provided herein are methods of preventing ission and/or infection of HBV from a mother to an unborn child comprising administering to a pregnant subject one or more infectious iruses expressing an HBV antigen as described herein. In speci?c embodiments, provided herein are s of preventing transmission and/or ion of HBV from a mother to an unborn child comprising administering to a pregnant subject an effective amount of one or more infectious iruses expressing an HBV antigen described herein.

In another embodiment, administering an infectious irus expressing an HBV antigen reduces congenital HBV infection. In another embodiment, stering one or more infectious arenaviruses expressing an HBV antigen reduces congenital HBV infection.

In another ment, administering an infectious arenavirus expressing an HBV antigen reduces manifestations of ital HBV infection by at least about 10%, at least about 20%, at least 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least 80%, at least 90%, or more. In another speci?c embodiment, administering an infectious arenavirus expressing an HBV antigen reduces mortality ofnewborn infants with congenital HBV infection.

In another embodiment, administering one or more infectious arenaviruses expressing an HBV antigen reduces manifestations of congenital HBV infection by at least about %, at least about 20%, at least 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least 80%, at least 90%, or more.

In another speci?c ment, administering one or more infectious arenaviruses expressing an HBV antigen reduces mortality of n infants with congenital HBV infection.

Such manifestations of congenital HBV include but are not limited to acute hepatitis B, chronic HBV infection, cirrhosis, and hepatocellular carcinoma (HCC). 6.6 Compositions, Administration and Dosage The invention ?thhermore relates to vaccines, immunogenic itions, and pharmaceutical compositions comprising a genetically engineered arenavirus as described herein.

Such vaccines and pharmaceutical compositions can be formulated according to standard procedures in the art.

In another embodiment, provided herein are compositions comprising an infectious irus described herein. Such compositions can be used in methods of treatment and tion of disease. In a speci?c embodiment, the compositions described herein are used in the ent of subjects infected with, or susceptible to, an infection with HBV. In another specific embodiment, the immunogenic compositions provided herein can be used to induce an immune response in a host to whom the composition is administered. The immunogenic compositions described herein can be used as vaccines and can accordingly be formulated as pharmaceutical compositions. In a speci?c embodiment, the immunogenic compositions described herein are used in the prevention of infection of subjects (e.g., human subjects) by HBV. In certain embodiments, the infectious arenavirus viral vector is replication—de?cient (See Section 6. 1(a)). In certain embodiments, the infectious arenavirus viral vector is replication- competent (See Section 6.1(b)).

In certain embodiments, provided herein are immunogenic compositions comprising an irus vector (or a combination of different arenavirus vectors) as described herein. In certain ments, such an immunogenic composition further comprises a pharmaceutically acceptable excipient. In certain embodiments, such an genic composition ?thher comprises an adjuvant. The adjuvant for administration in combination with a composition described herein may be administered before, concomitantly with, or after stration of said composition. In some embodiments, the term "adjuvant" refers to a nd that when administered in ction with or as part of a composition described herein augments, enhances and/or boosts the immune response to an infectious arenavirus particle, but when the compound is administered alone does not generate an immune response to the infectious arenavirus le. In some embodiments, the adjuvant generates an immune response to the infectious irus particle and does not produce an allergy or other adverse reaction. Adjuvants can enhance an immune se by several isms including, e.g., lymphocyte recruitment, ation of B and/or T cells, and stimulation of macrophages. When a e or immunogenic composition of the invention comprises adjuvants or is administered together with one or more adjuvants, the adjuvants that can be used include, but are not limited to, mineral salt nts or mineral salt gel adjuvants, particulate adjuvants, microparticulate nts, mucosal adjuvants, and immunostimulatory adjuvants. Examples of adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), A803 SmithKline), ASO4 (GlaxoSmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT/USZOO7/064857, hed as International ation No. W02007/109812), imidazoquinoxaline compounds (see International Application No. PCT/U$2007/06485 8, published as International Publication No. WO2007/109813) and saponins, such as QSZl (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); US. Pat. No. 5,057,540). In some embodiments, the adjuvant is Freund’s nt (complete or incomplete). Other adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)).

The compositions comprise the infectious arenaviruses described herein alone or together with a pharmaceutically acceptable carrier. Suspensions or dispersions of genetically engineered arenaviruses, especially isotonic aqueous suspensions or dispersions, can be used.

The pharmaceutical compositions may be sterilized and/or may comprise excipients, e.g., preservatives, izers, g agents and/or emulsi?ers, solubilizers, salts for regulating osmotic pressure and/or buffers and are ed in a manner known per se, for e by means of conventional dispersing and suspending processes. In certain embodiments, such sions or sions may comprise viscosity-regulating . The suspensions or dispersions are kept at temperatures around 2-8°C, or preferentially for longer storage may be frozen and then thawed shortly before use. For injection, the vaccine or immunogenic preparations may be formulated in aqueous solutions, preferably in physiologically ible buffers such as Hanks’s solution, Ringer’s on, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

In certain embodiments, the compositions described herein additionally se a preservative, e.g., the mercury derivative thimerosal. In a speci?c embodiment, the pharmaceutical compositions described herein comprise 0.001% to 0.01% thimerosal. In other embodiments, the pharmaceutical compositions described herein do not comprise a preservative.

] The pharmaceutical compositions se from about 103 to about 1011 focus g units of the genetically engineered arenaViruses. Unit dose forms for parenteral administration are, for example, ampoules or Vials, e.g., Vials containing from about 103 to 1010 focus forming units or 105 to 1015 physical particles of genetically engineered arenaviruses.

In another embodiment, a vaccine or immunogenic composition ed herein is administered to a subject by, including but not d to, oral, intradermal, intramuscular, intraperitoneal, enous, topical, aneous, percutaneous, intranasal and inhalation routes, and Via scarification (scratching through the top layers of skin, e.g., using a bifurcated needle). cally, subcutaneous, intramuscular or intravenous routes can be used.

For administration intranasally or by inhalation, the preparation for use according to the present invention can be conveniently red in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodi?uoromethane, trichloro?uoromethane, dichlorotetra?uoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e. g., gelatin for use in an inhaler or ators may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The dosage of the active ingredient depends upon the type of vaccination and upon the subject, and their age, weight, individual condition, the individual pharmacokinetic data, and the mode of administration.

Also provided herein are processes and uses of genetically engineered arenaviruses for the manufacture of vaccines in the form of pharmaceutical preparations, which comprise genetically engineered arenaviruses as active ingredient. The pharmaceutical itions ofthe present invention are ed in a manner known per se, for example by means of conventional mixing and/or dispersing processes. 6.7 Optimized tion of LCMV s Owing to the removal or functional inactivation of one or more of the Viral genes in arenaVirus vectors (here deletion of the glycoprotein, GP, will be taken as an example) arenavirus vectors can be generated and expanded in cells that provide the deleted or ?Jnctionally inactivated viral gene(s) (e.g., the GP) "in trans." The resulting virus itself is infectious but is unable to produce further infectious progeny particles in non-complementing cells due to the lack of the deleted or ?Jnctionally vated viral gene(s) (e.g., the GP). The complementing cell can provide the missing ?lnctionality either by stable transfection, transient transfection, or by infection with a helper virus that expresses the g onality.

] In certain embodiments, the complementing cell provides the viral gene that has been deleted or functionally inactivated from the arenavirus vector . In a speci?c embodiment, the complementing cell es the viral gene from a viral strain that is the same as the Viral strain that was used to te the genome of the arenavirus vector. In another embodiment, the complementing cell provides the viral gene from a viral strain that is ent from the viral strain that was used to generate the genome of the irus vector. For example, the viral gene ed in the complementing cell is obtained from the MP strain ofLCMV and s a protein having the amino acid sequence of SEQ ID NO: 15, 16, 17, or 18. In another example, the viral gene provided in the complementing cell is obtained from the Clone 13 strain ofLCMV and encodes a protein having the amino acid sequence of SEQ ID NO: 21, 22, 23, or 24. In another example, the viral gene provided in the complementing cell is obtained from the WE strain ofLCMV and encodes a protein having the amino acid sequence of SEQ ID NO: 25.

In a speci?c embodiment, the complementing cell provides the GP of the MP strain of LCMV and the arenavirus vector comprises an ORF of a human HBV antigen as described herein in place of the ORF encoding the GP protein. In an even more specific embodiment, the complementing cell provides the GP of the MP strain of LCMV and the arenavirus vector is obtained from LCMV Clone 13 and comprises an ORF of a human HBV antigen as described herein in place of the ORF encoding the GP protein. In an even more speci?c embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16.

In a speci?c embodiment, the complementing cell provides the GP of the Clone 13 strain of LCMV and the arenavirus vector comprises an ORF of a human HBV antigen as described herein in place of the ORF encoding the GP protein. In an even more specific embodiment, the complementing cell provides the GP of the Clone 13 strain of LCMV and the irus vector is obtained from LCMV MP strain and comprises an ORF of a human HBV antigen as described herein in place of the ORF encoding the GP protein. In an even more speci?c embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

In a speci?c embodiment, the complementing cell provides the GP of the WE strain of LCMV and the arenavirus vector comprises an ORF of a human HBV antigen as described herein in place of the ORF encoding the GP protein. In an even more speci?c embodiment, the complementing cell provides the GP of the WE strain ofLCMV and the arenavirus vector is obtained from LCMV Clone 13 and comprises an ORF of a human HBV antigen as described herein in place of the ORF encoding the GP protein. In an even more c embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25.

In a speci?c embodiment, the complementing cell provides the GP of the WE strain of LCMV and the arenavirus vector comprises an ORF of a human HBV antigen as described herein in place of the ORF encoding the GP protein. In an even more speci?c embodiment, the complementing cell provides the GP of the WE strain ofLCMV and the arenavirus vector is obtained from LCMV MP strain and ses an ORF of a human HBV antigen as described herein in place of the ORF ng the GP n. In an even more speci?c embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25. 6.8 Combination therapy 6.8 (a) Methods In one embodiment, provided herein are s of treating and/or preventing an HBV infection in a subject comprising administering to the subject two or more infectious arenaviruses expressing an HBV antigen as bed . See, e.g., Section 6.2. In speci?c embodiments, a method for treating and/or ting an HBV infection comprises administering a ?rst ious irus sing an HBV antigen as described herein, e.g., in which the ORF encoding the GP of the S genomic segment is substituted with a nucleotide sequence encoding the HBV antigen, wherein the HBV antigen can be but is not limited to: a) a nucleotide sequence encoding an HBV pre-S2/S protein or an antigenic fragment thereof; b) a nucleotide sequence encoding an HBV HBc protein or an antigenic fragment thereof; 0) a nucleotide sequence encoding an HBV HBs protein or an antigenic nt thereof; d) a tide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; e) a tide sequence encoding an HBV HBe protein or an antigenic fragment thereof; and a second infectious arenavirus expressing an HBV antigen as described herein, e.g., in which the ORF encoding the GP of the S genomic segment is tuted with a nucleotide sequence encoding the HBV antigen, wherein the HBV n can be but is not limited to: a) a tide sequence encoding an HBV pre-S2/S protein or an antigenic fragment thereof; b) a nucleotide sequence encoding an HBV HBc n or an antigenic fragment thereof; 0) a nucleotide sequence encoding an HBV HBs protein or an antigenic fragment thereof; d) a nucleotide sequence encoding a fusion ofHBV HBs and HBc proteins or nic fragments thereof; e) a nucleotide sequence ng an HBV HBe protein or an antigenic fragment thereof.

In certain embodiments, the ?rst and second ious arenaviruses are replication-de?cient. In certain embodiments, the ?rst and second infectious arenaviruses are replication-competent. In certain embodiments, either the ?rst or second infectious arenavirus is replication-de?cient. In certain embodiments, the ?rst and second infectious iruses are bisegmented. In certain embodiments, the ?rst and second infectious arenaviruses are trisegmented. In certain embodiments, either the ?rst or second infectious arenavirus is bisegmented, and the other is trisegmented.

In speci?c embodiments, provided herein are methods for treating and/or preventing an HBV ion comprising administering a ?rst infectious irus expressing a ?rst HBV antigen, selected from: an HBV /S protein or an antigenic fragment thereof; an HBV HBc protein or an antigenic fragment thereof, an HBV HBs protein or an antigenic fragment f, or an HBV HBe protein or an antigenic fragment thereof as described herein and a second infectious arenavirus expressing a second HBV antigen, selected from: a nucleotide sequence encoding an HBV /S protein or an antigenic fragment thereof; an HBV HBc protein or an antigenic fragment thereof, an HBV HBs protein or an antigenic fragment thereof, or an HBV HBe protein or an antigenic fragment thereof.

In certain embodiments, provided herein are methods for treating and/or preventing an infection comprising stering two arenavirus vector constructs expressing an HBV antigen as described herein. In a c embodiment, the two arenavirus vector constructs express a different HBV antigen.

] In certain embodiments, provided herein are methods for treating and/or preventing an infection comprising administering two or more arenavirus vector constructs expressing an HBV antigen as described herein. In a c embodiment, provided herein are methods for treating and/or preventing an infection sing administering three or more arenavirus vector constructs expressing an HBV antigen as described herein. In certain embodiments, the arenavirus vector construct can be based on LCMV.

In certain embodiments, provided herein are methods for treating and/or preventing an infection comprising stering two or more arenavirus vector constructs each expressing a different HBV n as described herein. In a speci?c embodiment, provided herein are methods for treating and/or preventing an infection comprising administering three or more arenavirus vector constructs, each expressing a different HBV antigen as described herein.

In certain embodiments, the arenavirus vector construct can be based on LCMV.

In speci?c embodiments, the antigen is the HBV /S protein or a fragment f. (See, e.g., n 6.2(a)).

In certain embodiments, the antigen is the HBV HBc protein or a fragment thereof. (See, e.g., Section 6.2(b)).

In certain embodiments, the antigen is the HBV HBs protein or a fragment thereof. (See, e.g., Section 62(0)).

] In certain embodiments, the antigen is a fusion of HBV HBs and HBc proteins or antigenic fragments thereof. (See, e.g., Section 6.2(d)).

In certain embodiments, the antigen is the HBV HBe protein or a fragment thereof. (See, e.g., n 6.2(e)).

] In certain ments, the vector generated to encode one or more HBV antigens as described herein comprises one or more nucleic acids encoding an HBV antigen and combinations thereof as described. In c embodiments the HBV antigens as bed herein are separated by various linkers, spacers, and cleavage sites as described herein.

] In another embodiment, the vector generated to encode one or more HBV antigens as described herein of the ?rst ious arenavirus may be based on LCMV Clone 13 or LCMV MP strain. (See, e.g., Section 7.1).

In another ment, the vector generated to encode one or more HBV ns as described herein of the second infectious arenavirus may be based on LCMV Clone 13 or LCMV MP strain. (See, e.g., Section 7.1).In another embodiment, the vector ted to encode one or more HBV antigens as described herein of the ?rst infectious arenavirus may be based on Junin virus.

In another embodiment, the vector generated to encode one or more HBV ns as described herein of the second infectious arenavirus may be based on Junin virus.

] In a speci?c embodiment, provided herein are methods of treating and/or ting an infection in a subject comprising administering to the subject a ?rst infectious arenavirus expressing an HBV pre-SZ/S protein or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV HBc protein or an antigenic fragment thereof In a speci?c embodiment, provided herein are methods of ng and/or preventing an infection in a subject sing administering sequentially to the subject a ?rst infectious arenavirus sing an HBV pre-SZ/S protein or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV HBs protein or an antigenic fragment thereof.

In a speci?c embodiment, provided herein are methods of treating and/or preventing an infection in a subject comprising stering simultaneously to the subject a ?rst infectious arenavirus expressing an HBV HBc protein or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV HBs protein or an antigenic fragment thereof.

In a speci?c embodiment, provided herein are methods of treating and/or preventing an infection in a subject comprising administering sequentially to the subject a ?rst infectious arenavirus expressing an HBV pre-SZ/S protein or an antigenic fragment thereof and a second infectious irus expressing a fusion ofHBV HBs and HBc ns or antigenic fragments thereof In a speci?c embodiment, provided herein are methods of treating and/or ting an ion in a subject comprising administering sequentially to the subject a ?rst infectious arenavirus expressing an HBV HBc protein or an antigenic fragment thereof and a second infectious arenavirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof.

In a speci?c embodiment, provided herein are s of treating and/or preventing an infection in a subject comprising administering sequentially to the subject a ?rst infectious irus expressing an HBV HBc protein or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV /S protein or an antigenic nt ] In a speci?c embodiment, provided herein are methods of treating and/or preventing an infection in a subject comprising administering sequentially to the subject a ?rst infectious arenavirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof and a second infectious arenavirus sing an HBV pre-SZ/S protein or an antigenic fragment thereof.

In a speci?c embodiment, provided herein are methods of treating and/or preventing an infection in a subject comprising administering tially to the subject a ?rst infectious arenavirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof and a second infectious arenavirus expressing an HBV HBe protein or an antigenic fragment thereof.

In a speci?c embodiment, provided herein are methods of treating and/or preventing an infection in a subject comprising administering sequentially to the subject a ?rst infectious arenavirus sing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof and a second ious arenavirus expressing an HBV HBc protein or an antigenic fragment thereof.

In a speci?c embodiment, provided herein are methods of treating and/or preventing an infection in a subject comprising administering to the subject a ?rst infectious arenavirus expressing an HBV pre-SZ/S protein or an antigenic fragment f and a second infectious arenavirus expressing an HBV HBe protein or an antigenic fragment thereof.

] In a c embodiment, provided herein are methods of treating and/or preventing an infection in a subject comprising administering aneously to the subject a ?rst infectious arenavirus expressing an HBV HBe protein or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV HBs n or an antigenic fragment thereof.

In a speci?c embodiment, provided herein are methods of treating and/or preventing an infection in a subject sing administering sequentially to the subject a ?rst ious arenavirus sing an HBV HBe protein or an antigenic fragment thereof and a second infectious arenavirus expressing a fusion of HBV HBs and HBc proteins or nic fragments thereof.

In a speci?c embodiment, provided herein are methods of treating and/or preventing an infection in a subject comprising administering sequentially to the subject a ?rst infectious arenavirus expressing an HBV HBs protein or an antigenic fragment thereof and a second infectious arenavirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof.

In a speci?c embodiment, provided herein are methods of treating and/or ting an ion in a subject comprising administering sequentially to the subject a ?rst infectious arenavirus expressing an HBV HBe n or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV pre-SZ/S protein or an antigenic fragment thereof.

In a speci?c embodiment, provided herein are methods of treating and/or preventing an infection in a subject comprising administering sequentially to the subject a ?rst infectious arenavirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof and a second infectious arenavirus expressing an HBV HBs n or an antigenic fragment thereof.

In a speci?c embodiment, provided herein are methods of treating and/or preventing an infection in a subject comprising stering sequentially to the subject a ?rst infectious arenavirus expressing an HBV HBs protein or an antigenic fragment thereof and a second ious irus expressing an HBV pre-SZ/S protein or an antigenic fragment thereof.

In a speci?c ment, provided herein are methods of treating and/or preventing an infection in a subject comprising stering simultaneously to the subject a ?rst infectious arenavirus expressing an HBV HBc protein or an antigenic fragment thereof and a second infectious arenavirus sing an HBV HBe protein or an antigenic fragment thereof In a speci?c embodiment, provided herein are methods of ng and/or preventing an infection in a subject comprising administering simultaneously to the subject a ?rst infectious arenavirus expressing an HBV HBs protein or an nic fragment thereof and a second infectious arenavirus expressing an HBV HBe n or an antigenic fragment thereof.

In a speci?c embodiment, provided herein are methods of treating and/or preventing an infection in a subject comprising administering simultaneously to the subject a ?rst infectious arenavirus expressing an HBV HBs protein or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV HBc protein or an nic fragment thereof.

] In a speci?c embodiment, provided herein are methods of treating and/or preventing an infection in a subject comprising administering simultaneously to the subject a ?rst infectious irus sing an HBV HBe n or an antigenic fragment thereof and a second infectious irus expressing an HBV HBc protein or an antigenic fragment f.

In r embodiment, the ?rst infectious arenavirus expressing an HBV antigen is a primary vaccine antigen and the second infectious arenavirus expressing another HBV antigen is a secondary vaccine antigen.

In certain embodiments, administering a ?rst infectious arenavirus expressing an HBV pre-SZ/S protein or a fragment thereof or an HBV HBc protein and a second infectious arenavirus expressing an HBV pre-SZ/S protein or an HBV HBc protein provides a better protective effect to HBV after vaccination than administering a single infectious irus expressing an HBV antigen, e.g., expressing only the pre—SZ/S protein (or a fragment thereof) or only the HBc protein. In other embodiments, administering a ?rst infectious arenavirus sing an HBV pre—SZ/S protein or a fragment thereof or an HBV HBc protein and a second infectious arenavirus sing an HBV pre-SZ/S protein or a fragment thereof or an HBV HBc protein elicits a greater immune response than administering a single infectious irus expressing an HBV antigen e.g., expressing only the pre-SZ/S protein (or a nt thereof) or only the HBc protein. In another embodiment, administering a ?rst infectious irus expressing an HBV pre-SZ/S protein or a fragment thereof or an HBV HBc protein and a second infectious arenavirus expressing an HBV pre-SZ/S protein or a nt thereof, or an HBV HBc protein elicits a larger CD8+ T cell response than administering a single infectious arenavirus expressing an HBV n e.g., expressing only the pre-SZ/S protein (or a fragment thereof) or only the HBc protein. In other ments, administering a ?rst infectious arenavirus expressing an HBV pre—SZ/S protein or a fragment thereof or an HBV HBc protein and a second infectious arenavirus expressing an HBV pre-SZ/S protein or a fragment thereof or an HBV HBc protein elicits higher titers of neutralizing antibodies than administering a single infectious arenavirus expressing an HBV antigen e.g., expressing only the pre-SZ/S protein (or a fragment thereof) or only the HBc protein.

In certain ments, administering a ?rst infectious arenavirus expressing an HBV /S protein or a fragment thereof or an HBV HBs protein and a second infectious arenavirus expressing an HBV pre-SZ/S protein or an HBV HBs protein provides a better protective effect to HBV after vaccination than administering a single infectious arenavirus sing an HBV antigen, e.g., expressing only the pre—SZ/S protein (or a fragment thereof) or only the HBs protein. In other embodiments, stering a ?rst infectious arenavirus expressing an HBV pre—SZ/S protein or a fragment thereof or an HBV HBs protein and a second infectious arenavirus expressing an HBV pre-SZ/S protein or a fragment thereof or an HBV HBs protein s a greater immune response than administering a single infectious arenavirus expressing an HBV n e.g., expressing only the pre—SZ/S protein (or a fragment thereof) or only the HBs n. In another embodiment, stering a ?rst infectious arenavirus expressing an HBV /S protein or a fragment thereof or an HBV HBs n and a second infectious arenavirus sing an HBV pre-SZ/S protein or a fragment thereof, or an HBV HBs protein elicits a larger CD8+ T cell response than administering a single infectious arenavirus expressing an HBV antigen e.g., expressing only the pre—SZ/S protein (or a fragment thereof) or only the HBs protein. In other embodiments, administering a ?rst infectious arenavirus expressing an HBV pre—SZ/S protein or a nt thereof or an HBV HBs protein and a second infectious irus expressing an HBV pre-SZ/S protein or a fragment thereof or an HBV HBs protein elicits higher titers of neutralizing antibodies than administering a single infectious arenavirus sing an HBV antigen e.g., expressing only the pre-SZ/S protein (or a fragment thereof) or only the HBs n.

In certain embodiments, administering a ?rst infectious arenavirus expressing an HBV pre-SZ/S protein or a fragment thereof or a fusion ofHBV HBs and HBc proteins and a second infectious arenavirus expressing an HBV pre-SZ/S protein or a fusion ofHBV HBs and HBc ns provides a better protective effect to HBV after vaccination than administering a single infectious arenavirus expressing an HBV n, e. g., expressing only the pre-SZ/S protein (or a fragment thereof) or only the fusion of HBV HBs and HBc proteins. In other embodiments, administering a ?rst infectious arenavirus expressing an HBV pre-SZ/S protein or a fragment thereof or a fusion ofHBV HBs and HBc proteins and a second ious arenavirus expressing an HBV pre—SZ/S protein or a fragment thereof or a fusion ofHBV HBs and HBc proteins elicits a greater immune response than administering a single infectious arenavirus expressing an HBV antigen e.g., expressing only the pre-SZ/S n (or a nt thereof) or only the fusion ofHBV HBs and HBc proteins. In another embodiment, administering a first infectious irus sing an HBV pre-SZ/S protein or a fragment thereof or a fusion of HBV HBs and HBc proteins and a second infectious arenavirus expressing an HBV pre-S2/S protein or a fragment thereof, or a fusion of HBV HBs and HBc proteins elicits a larger CD8+ T cell response than administering a single infectious arenavirus expressing an HBV antigen e.g., expressing only the pre—SZ/S n (or a fragment thereof) or only the fusion of HBV HBs and HBc ns. In other embodiments, administering a ?rst ious arenavirus expressing an HBV pre-SZ/S protein or a fragment thereof or a fusion ofHBV HBs and HBc proteins and a second infectious arenavirus expressing an HBV pre-SZ/S protein or a fragment thereof or a fusion ofHBV HBs and HBc proteins elicits higher titers of neutralizing antibodies than administering a single infectious arenavirus expressing an HBV antigen e.g., sing only the pre-SZ/S protein (or a nt thereof) or only the fusion of HBV HBs and HBc proteins.

In certain embodiments, administering a ?rst infectious arenavirus expressing an HBV pre—SZ/S protein or a fragment thereof or an HBV HBe protein and a second infectious arenavirus expressing an HBV pre-SZ/S protein or an HBV HBe protein provides a better tive effect to HBV after vaccination than administering a single infectious arenavirus expressing an HBV antigen, e.g., expressing only the pre—SZ/S protein (or a fragment thereof) or only the HBe n. In other embodiments, administering a ?rst infectious arenavirus expressing an HBV pre-SZ/S protein or a fragment thereof or an HBV HBe protein and a second infectious arenavirus sing an HBV pre-SZ/S protein or a fragment thereof or an HBV HBe protein elicits a r immune response than administering a single infectious arenavirus expressing an HBV n e.g., expressing only the pre-S2/S protein (or a fragment thereof) or only the HBe protein. In another embodiment, administering a first infectious arenavirus expressing an HBV pre—SZ/S protein or a fragment thereof or an HBV HBe n and a second infectious arenavirus expressing an HBV pre-SZ/S protein or a fragment thereof, or an HBV HBe protein elicits a larger CD8+ T cell response than administering a single infectious arenavirus expressing an HBV antigen e.g., expressing only the pre-SZ/S protein (or a fragment thereof) or only the HBe protein. In other ments, administering a ?rst infectious irus expressing an HBV pre—SZ/S protein or a fragment thereof or an HBV HBe protein and a second infectious arenavirus expressing an HBV pre-SZ/S protein or a fragment thereof or an HBV HBe protein elicits higher titers of neutralizing antibodies than administering a single infectious arenavirus expressing an HBV antigen e.g., expressing only the /S protein (or a fragment thereof) or only the HBe protein.

In yet another embodiment, provided herein is the combined use of the replication-de?cient arenavirus expressing an HBV antigen described herein and one or more replication-defective Virus vectors. In a more speci?c embodiment the replication-defective Virus vector is ed from the group comprising of poxviruses, adenoviruses, alphaviruses, herpes simplex Viruses, paramyxoviruses, rhabdoviruses, poliovirus, associated Virus, and sendai Virus, and mixtures thereof. In a speci?c embodiment, the poxvirus is a modi?ed e Ankara.

In yet another embodiment, provided herein is the combined use of the replication-de?cient arenavirus expressing an HBV antigen described herein and one or more replication-defective Virus vectors expressing an HBV antigen. In a more speci?c embodiment the replication-defective Virus vector is selected from the group comprising of poxviruses, adenoviruses, alphaviruses, herpes simplex Viruses, paramyxoviruses, rhabdoviruses, poliovirus, adeno-associated Virus, and sendai Virus, and es thereof. In a speci?c embodiment, the poxvirus is a modi?ed vaccine Ankara.

In another embodiment, the ?rst infectious arenavirus sing an HBV antigen as described herein is administered before or after the second infectious arenavirus expressing an HBV n as described herein. For example the ?rst infectious arenavirus expressing an HBV antigen is administered around 30-60 minutes before or after the ?rst administration of the second infectious arenavirus.

In another embodiment, the ?rst infectious irus expressing a vaccine antigen is administered before the second infectious arenavirus expressing a e antigen. In certain embodiments there is a period of about 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 1 month, 2 , 3 months, 4 months, 5 months, 6 months, 7 , 8 months, 9 months, 10 months, 11 , 1 year between the administration ofthe ?rst infectious irus and the second infectious arenavirus.

In r embodiment, two infectious arenaviruses are administered in a treatment regime at molar ratios ranging from about 1:1 to 1:1000, in particular including: 1:1 ratio, 1:2 ratio, 1:5 ratio, 1:10 ratio, 1:20 ratio, 1:50 ratio, 1:100 ratio, 1:200 ratio, 1:300 ratio, 1:400 ratio, 1:500 ratio, 1:600 ratio, 1:700 ratio, 1:800 ratio, 1:900 ratio, 1:1000 ratio.

In r embodiment, the subjects whom two or more infectious arenaviruses expressing an HBV antigen bed herein are stered have, are susceptible to, or are at risk for an HBV infection. In another embodiment, the subjects whom two or more infectious arenaviruses sing an HBV antigen described herein are administered are infected with, are tible to, or are at risk for, an infection with HBV.

In another embodiment, the subjects whom two or more infectious arenaviruses expressing an HBV antigen bed herein, are administered simultaneously have, are susceptible to, or are at risk for an HBV infection. In another embodiment, the ts whom two or more infectious arenaviruses expressing an HBV n described herein are stered simultaneously are infected with, are tible to, or are at risk for, an infection with HBV.

In another embodiment, the subjects whom two or more infectious arenaviruses expressing an HBV antigen described herein, are administered sequentially have, are susceptible to, or are at risk for an HBV infection. In another embodiment, the ts whom two or more infectious arenaviruses expressing an HBV antigen described herein are administered tially are infected with, are susceptible to, or are at risk for, an infection with HBV.

In another embodiment, said two or more infectious arenaviruses expressing an HBV antigen as described herein are further administered in combination with at least one other medicament for treating and/or preventing HBV. Therapeutic medicaments for treating and/or preventing HBV include, but are not limited to entecaVir (BARACLUDE®; Bristol-Myers Squibb), lamivudine (EPIVIR HBV®; GlaxoSmithKline), adefovir dipivoxil (HEPSERA®; Gilead Sciences), interferon alpha 2b N A®; Schering), pegylated interferon (PEGASYS®; Roche), telbivudine (TYZEKA®, Novartis), and tenofovir (VIREAD®; Gilead Sciences).

In another embodiment, said two or more infectious arenaviruses expressing an HBV antigen as described herein are further administered in a combination with at least one other modulator. In a more specific embodiment, said two or more infectious arenaviruses expressing an HBV antigen as described herein are further stered in a combination with at least one Thl-speci?c adjuvant. In a more speci?c embodiment the Th—l speci?c adjuvant is Bacillus Calmette-Guerin (BCG).

In another embodiment, the administration regime can involve administering to a symptomatic subject a second infectious arenavirus expressing an HBV antigen as described herein. In yet r embodiment, the administration regime can involve administering to an subject with a compromised immune system, especially transplant recipients, fected persons, a pregnant subject, a subject who has cancer, a second infectious arenavirus expressing an HBV antigen as described . In another embodiment, two or more infectious arenaviruses expressing an HBV antigen as described herein are administered to a subject who is a child ofO, 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16, or 17 years ofage suffering from or susceptible to, or at risk for, an ion with HBV.

In another ment, the administration regime can e administering to a subject who is a child, a ?rst arenavirus expressing an HBV antigen, and administering to the same subject who is an adolescent a second arenavirus expressing an HBV antigen. In a speci?c ment, the stration regime can involve administering to a subject who is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years of age a ?rst arenavirus expressing an HBV antigen as described herein, and to the same subject who is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 years of age a second infectious arenavirus sing an HBV antigen.

In another embodiment, the administration regime can involve administering to a prepubescent subject a second infectious arenavirus expressing an HBV antigen. In another embodiment, the administration regime can involve administering to an adolescent male, aged 12 to 18 years a second infectious irus expressing an HBV antigen as bed herein. In another ment, the administration regime can involve administering to a female, aged 12 to 18 years a second infectious irus expressing an HBV antigen.

In another embodiment, administering two or more infectious arenaviruses expressing an HBV antigen reduces the risk that an individual will develop an ion with HBV by at least 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the risk of developing an infection with HBV in the absence of such treatment.

In another embodiment, stering two or more infectious arenaviruses sing an HBV antigen, administered separately, reduces the risk that an individual will develop an infection with HBV by at least 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the risk of developing an infection with HBV in the absence of such treatment.

In another ment, administering two or more infectious iruses expressing an HBV antigen, administered sequentially, reduces the risk that an individual will develop an infection with HBV by at least 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the risk of developing an infection with HBV in the absence of such treatment. ] t being limited by theory, stration of a ?rst ious arenavirus and uently of a second infectious arenavirus vector results in a prime-boost effect.

In certain embodiments, provided herein are methods for treating and/or preventing an HBV infection comprising administering two or more arenavirus vector constructs each expressing the same or a different HBV antigen sequentially. The time interval between each administration can be about 1 week, about 2 weeks, about 3 week, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, or about 24 months.

In certain embodiments, the ?rst infectious arenavirus and the second infectious arenavirus are homologous. In certain embodiments, the ?rst infectious arenavirus and the second infectious arenavirus are heterologous.

In certain speci?c embodiments, the ?rst infectious arenavirus is an Old World arenavirus, and the second infectious arenavirus is an Old World arenavirus. In certain speci?c embodiments, the ?rst infectious arenavirus is an Old World arenavirus, and the second infectious arenavirus is a New World irus. In certain c embodiments, the ?rst infectious arenavirus is a New World irus, and the second infectious arenavirus is a New World arenavirus. In certain speci?c embodiments, the ?rst infectious arenavirus is a New World arenavirus, and the second infectious arenavirus is an Old World arenavirus.

In certain speci?c embodiments, the ?rst infectious arenavirus is derived from LCMV, and the second infectious arenavirus is derived from LCMV. In certain speci?c embodiments, the ?rst infectious arenavirus is derived from LCMV, and the second infectious arenavirus is derived from Junin virus. In certain c embodiments, the ?rst infectious arenavirus is derived from Junin virus, and the second infectious arenavirus is derived from Junin virus. In certain speci?c embodiments, the ?rst infectious arenavirus is derived from Junin virus, and the second infectious irus is derived from LCMV.

] In certain ments, provided herein is a method of treating and/or preventing an HBV infection wherein a ?rst infectious arenavirus is administered ?rst as a ," and a second infectious arenavirus is administered as a "boost." The ?rst and the second infectious arenavirus vectors can express the same or different HBV antigens. In n speci?c embodiments, the "prime" administration is performed with an infectious arenavirus derived from LCMV, and the "boost" is performed with an infectious arenavirus derived from Junin Virus. In certain speci?c embodiments, the "prime" administration is performed with an infectious arenavirus derived from Junin virus, and the "boost" is performed with an ious arenavirus derived from LCMV.

In certain embodiments, administering a ?rst infectious arenavirus expressing an HBV antigen or a fragment thereof, followed by administering a second infectious arenavirus expressing an HBV antigen or a fragment thereof results in a greater n speci?c CD8+ T cell response than administering a single infectious arenavirus expressing an HBV antigen or a fragment f. In certain embodiments, the antigen speci?c CD8+ T cell count increases by 50%, 100%, 150% or 200% after the second administration compared to the ?rst administration.

In certain ments, administering a third infectious arenavirus expressing an HBV n results in a r antigen speci?c CD8+ T cell response than administering two consecutive infectious arenaviruses expressing an HBV antigen. In n embodiments, the antigen speci?c CD8+ T cell count increases by about 50%, about 100%, about 150%, about 200% or about 250% after the third administration compared to the ?rst administration.

In certain ments, provided herein are methods for treating and/or preventing an infection comprising administering two or more irus vector constructs, wherein the two or more arenavirus vector constructs are homologous, and wherein the time interval n each administration is about 1 week, about 2 weeks, about 3 week, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 , about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about months, about 11 months, about 12 months, about 18 months, or about 24 months.

In certain embodiments, administering a ?rst infectious arenavirus expressing an HBV antigen or a fragment thereof and a second, heterologous, infectious arenavirus sing an HBV antigen or a fragment thereof elicits a greater CD8+ T cell response than stering a ?rst infectious irus expressing an HBV antigen or a fragment thereof and a second, homologous, infectious arenavirus expressing an HBV antigen or a fragment thereof.

In certain speci?c embodiments, the ?rst infectious arenavirus expressing an HBV pre-S2/S protein is LCMV, and the second, heterologous, infectious arenavirus expressing an HBV /S protein is Junin Virus. In certain speci?c embodiments, the ?rst infectious arenavirus sing an HBV pre-S2/S protein is Junin Virus, and the second, heterologous, infectious arenavirus expressing an HBV pre-S2/S protein is LCMV.

In certain speci?c embodiments, the ?rst infectious arenavirus expressing an HBV HBc protein is LCMV, and the second, logous, infectious arenavirus expressing an HBV HBc protein is Junin Virus. In certain speci?c embodiments, the ?rst infectious arenavirus expressing an HBV HBc protein is Junin Virus, and the second, heterologous, infectious arenavirus expressing an HBV HBc protein is LCMV.

In certain c embodiments, the ?rst infectious arenavirus expressing an HBV HBs and HBc fusion protein is LCMV, and the second, heterologous, infectious arenavirus expressing an HBV HBs and HBc fusion n is Junin Virus. In certain speci?c embodiments, the ?rst ious arenavirus expressing an HBV HBs and HBc fusion protein is Junin Virus, and the second, heterologous, infectious arenavirus expressing an HBV HBs and HBc fusion n is LCMV.

In certain speci?c embodiments, the ?rst infectious arenavirus expressing an HBV HBe protein is LCMV, and the second, heterologous, infectious arenavirus expressing an HBV HBe protein is Junin Virus. In certain speci?c embodiments, the ?rst infectious arenavirus sing an HBV HBe protein is Junin Virus, and the , heterologous, infectious arenavirus expressing an HBV HBe protein is LCMV.

In certain specific ments, administering a ?rst infectious arenavirus expressing an HBV /S protein and a second, heterologous, infectious arenavirus expressing an HBV pre—S2/S protein elicits a greater CD8+ T cell response than stering a ?rst ious irus expressing an HBV pre-S2/S protein and a second, gous, ious arenavirus expressing HBV pre-S2/S protein. In certain speci?c embodiments, administering a ?rst infectious arenavirus expressing an HBV /S protein and a second, heterologous, infectious arenavirus expressing an HBV pre-S2/S protein elicits a CD8+ T cell se that is about 20%, about 40%, about 60%, about 80%, about 100%, about 120%, about 140%, about 160%, about 180%, or about 200% greater than administering a ?rst infectious arenavirus expressing an HBV pre-S2/S protein and a second, homologous, infectious arenavirus expressing an HBV pre—S2/S protein.

In certain speci?c embodiments, administering a ?rst infectious arenavirus expressing an HBV HBc protein and a second, heterologous, infectious arenavirus expressing an HBV HBc protein elicits a greater CD8+ T cell response than administering a ?rst infectious arenavirus expressing an HBV HBc protein and a second, homologous, infectious arenavirus expressing HBV HBc protein. In certain speci?c embodiments, stering a ?rst infectious arenavirus expressing an HBV HBc protein and a second, heterologous, infectious arenavirus expressing an HBV HBc protein s a CD8+ T cell response that is about 20%, about 40%, about 60%, about 80%, about 100%, about 120%, about 140%, about 160%, about 180%, or about 200% greater than administering a ?rst infectious arenavirus expressing an HBV HBc protein and a second, gous, infectious irus expressing an HBV HBc protein.

In certain speci?c embodiments, administering a ?rst infectious arenavirus sing an HBV HBs and HBc fusion protein and a second, heterologous, infectious irus expressing an HBV HBs and HBc fusion protein elicits a greater CD8+ T cell response than administering a ?rst infectious arenavirus expressing an HBV HBs and HBc fusion protein and a second, homologous, ious arenavirus expressing HBV HBs and HBc fusion protein. In certain speci?c embodiments, administering a ?rst infectious arenavirus expressing an HBV HBs and HBc fusion protein and a second, heterologous, infectious arenavirus expressing an HBV HBs and HBc fusion protein elicits a CD8+ T cell response that is about 20%, about 40%, about 60%, about 80%, about 100%, about 120%, about 140%, about 160%, about 180%, or about 200% greater than administering a ?rst infectious irus expressing an HBV HBs and HBc fusion protein and a second, homologous, infectious arenavirus expressing an HBV HBs and HBc fusion protein.

In certain speci?c embodiments, administering a ?rst infectious arenavirus expressing an HBV HBe protein and a second, heterologous, infectious arenavirus expressing an HBV HBe protein elicits a greater CD8+ T cell response than administering a ?rst infectious arenavirus expressing an HBV HBe protein and a second, homologous, ious arenavirus sing HBV HBe protein. In certain speci?c embodiments, administering a ?rst infectious arenavirus expressing an HBV HBe protein and a second, heterologous, infectious arenavirus expressing an HBV HBe protein elicits a CD8+ T cell response that is about 20%, about 40%, about 60%, about 80%, about 100%, about 120%, about 140%, about 160%, about 180%, or about 200% greater than administering a ?rst infectious arenavirus expressing an HBV HBe protein and a second, gous, infectious arenavirus expressing an HBV HBe protein.

] In certain embodiments, provided herein are methods for treating and/or preventing an infection comprising administering two or more irus vector constructs, wherein the two or more arenavirus vector ucts are heterologous, and wherein the time interval n each administration is about 1 week, about 2 weeks, about 3 week, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about months, about 11 months, about 12 months, about 18 months, or about 24 months.

In yet another embodiment, provided herein is the combined use of the replication-de?cient arenavirus expressing an HBV antigen bed herein and one or more replication-defective Virus vectors. In a more speci?c embodiment the replication—defective virus vector is selected from the group comprising of poxviruses, adenoviruses, alphaviruses, herpes simplex viruses, paramyxoviruses, rhabdoviruses, poliovirus, associated Virus, and sendai virus, and es f. In a speci?c embodiment, the poxvirus is a modi?ed vaccine Ankara.

In yet another embodiment, provided herein is the combined use of the replication-de?cient arenavirus expressing an HBV antigen described herein and one or more ation-defective virus vectors expressing an HBV antigen. In a more speci?c embodiment the replication-defective virus vector is selected from the group comprising of poxviruses, adenoviruses, iruses, herpes simplex viruses, xoviruses, rhabdoviruses, poliovirus, adeno—associated virus, and sendai virus, and mixtures thereof. In a speci?c embodiment, the us is a modi?ed vaccine Ankara.

In another embodiment, the ?rst ious arenavirus expressing an HBV antigen as described herein is administered before or after the second infectious arenavirus sing an HBV antigen as bed herein. For example the ?rst infectious arenavirus expressing an HBV antigen is administered around 30-60 minutes before or after the ?rst administration of the second infectious arenavirus.

In r embodiment, the ?rst infectious arenavirus expressing a vaccine antigen is administered before the second infectious arenavirus expressing a vaccine antigen. In certain embodiments there is a period of about 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 , 9 months, 10 months, 11 months, 1 year between the administration ofthe ?rst infectious arenavirus and the second infectious arenavirus.

In another ment, two infectious arenaviruses are stered in a treatment regime at molar ratios ranging from about 1:1 to 1:1000, in particular including: 1:1 ratio, 1:2 ratio, 1:5 ratio, 1:10 ratio, 1:20 ratio, 1:50 ratio, 1:100 ratio, 1:200 ratio, 1:300 ratio, 1:400 ratio, 1:500 ratio, 1:600 ratio, 1:700 ratio, 1:800 ratio, 1:900 ratio, 1:1000 ratio.

In another embodiment, the subjects to whom two or more infectious arenaviruses expressing an HBV n described herein are administered have, are susceptible to, or are at risk for an HBV infection. In another embodiment, the subjects to whom two or more infectious arenaviruses expressing an HBV antigen described herein are administered are infected with, are susceptible to, or are at risk for, an infection with HBV.

The subjects who can be treated with the methods provided herein are susceptible to, or are at risk for an HBV infection.

In another embodiment, said two or more infectious arenaviruses expressing an HBV antigen as described herein further express at least r stimulatory peptide, polypeptide or protein. In certain embodiments, the immunostimulatory peptide, polypeptide or protein is Calreticulin (CRT), or a fragment thereof; Ubiquitin or a fragment thereof; Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), or a fragment thereof; Invariant chain (CD74) or an nic fragment thereof; Mycobacterium tuberculosis Heat shock protein 70 or an antigenic fragment f; Herpes simplex virus 1 protein VP22 or an antigenic fragment thereof; CD40 ligand or an antigenic fragment thereof; or Fms-related tyrosine kinase 3 (Flt3) ligand or an antigenic fragment thereof.

Heterologous prime-boost methods with infectious replication-defective arenavirus vectors n the two infectious replication—defective arenavirus vectors are derived from different arenaviruses (e.g, LCMV and Junin virus) are also provided. These ious replication-defective arenavirus vectors can express an antigen, such as an antigen of HBV.

Heterologous prime-boost methods with infectious replication-competent arenavirus vectors wherein the two infectious ation—competent arenavirus vectors are derived from ent arenaviruses (e.g., LCMV and Junin virus) are also provided. These infectious replication-competent arenavirus vectors can express an antigen, such as an antigen of 6.8 (b) Compositions ] The invention furthermore relates to vaccines, immunogenic itions, and pharmaceutical itions comprising a genetically engineered arenavirus as described herein.

Such vaccines and pharmaceutical compositions can be formulated according to standard procedures in the art.

In one embodiment, provided herein are compositions comprising two or more infectious arenaviruses expressing an HBV antigen as described . See, e. g., Section 6.2.

In a specific embodiments, the compositions described herein comprises administering to a subject a ?rst ious arenavirus expressing an HBV antigen as described herein, e.g., in which the ORF encoding the GP of the S genomic segment is tuted with a nucleotide sequence encoding the HBV antigen. The HBV n can be but is not limited to: a) a nucleotide sequence ng an HBV pre-S2/S protein or an antigenic fragment thereof; b) a nucleotide sequence encoding an HBV HBc protein or an antigenic fragment thereof; 0) a nucleotide sequence encoding an HBV HBs protein or an antigenic fragment thereof; d) a nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic nts thereof; e) a nucleotide sequence encoding an HBV HBe protein or an antigenic fragment thereof; and a second infectious arenavirus composition expressing an HBV antigen as bed herein, e.g., in which the ORF encoding the GP of the S genomic segment is tuted with a nucleotide sequence encoding the HBV n. The HBV antigen can be but is not limited to: a) a nucleotide ce encoding an HBV pre-S2/S protein or an antigenic fragment thereof; b) a nucleotide sequence encoding an HBV HBc protein or an antigenic fragment f; 0) a tide sequence encoding an HBV HBs protein or an antigenic nt f; d) a nucleotide sequence encoding a fusion ofHBV HBs and HBc proteins or antigenic fragments thereof; e) a nucleotide sequence encoding an HBV HBe protein or an nic fragment thereof.

In certain embodiments, the ?rst and second infectious iruses are replication-de?cient. In certain embodiments, the ?rst and second infectious arenaviruses are replication—competent. In certain embodiments, either the ?rst or second infectious arenavirus is replication—de?cient.

In speci?c embodiments, provided herein are methods for treating and/or preventing an HBV infection sing administering a ?rst infectious arenavirus expressing a ?rst HBV antigen, selected from: an HBV pre-SZ/S protein or an antigenic fragment thereof; an HBV HBc protein or an antigenic fragment thereof; an HBV HBs protein or an antigenic fragment thereof, a fusion of HBV HBs and HBc proteins or antigenic fragments thereof, or an HBV HBe protein or an antigenic fragment thereof, as described herein and a second infectious arenavirus expressing a second HBV antigen, selected from: an HBV pre—SZ/S protein or an antigenic fragment thereof; an HBV HBc protein or an antigenic nt thereof; or an HBV HBs protein or an antigenic fragment thereof, a fusion ofHBV HBs and HBc proteins or antigenic fragments thereof, or an HBV HBe protein or an antigenic fragment thereof.

] In certain embodiments, provided herein are compositions suitable for a method oftreating and/or preventing an HBV infection comprising administering two arenavirus constructs expressing an HBV n as described herein. In a speci?c embodiment, the two arenavirus vector ucts s an HBV antigen.

In certain embodiments, provided herein are compositions comprising two or more arenavirus vector constructs expressing an HBV antigen as described herein. In speci?c embodiments, ed herein are compositions comprising three or more arenavirus vector constructs expressing an HBV antigen as described herein. In certain embodiments, the arenavirus can be LCMV.

] In speci?c embodiments, the antigen is the HBV pre-SZ/S n or a fragment thereof. (See, e.g., Section 6.2(a)).

In certain embodiments, the antigen is the HBV HBc protein or a fragment thereof. (See, e.g., Section 6.2(b)).

In certain embodiments, the antigen is the HBV HBs protein or a fragment thereof. (See, e.g., Section 6.2(e)).

In certain embodiments, the n is a fusion of the HBV HBs and HBc proteins or antigenic fragments thereof (See, e. g., Section 6.2(d)).

In certain embodiments, the antigen is the HBV HBe protein or a fragment f. (See, e.g., Section 6.2(e)).

In n embodiments, the vector generated to encode one or more HBV antigens as described herein comprises one or more c acids encoding an HBV antigen and combinations thereof as described. In speci?c embodiments the HBV antigens as described herein are separated by various linkers, spacers, and cleavage sites as described herein.

] In another embodiment, the vector generated to encode one or more HBV antigens as described herein of the ?rst infectious arenavirus may be based on LCMV Clone 13 or LCMV MP strain. (See, e.g., Section 7.1).

In another embodiment, the vector generated to encode one or more HBV ns as described herein of the second infectious arenavirus may be based on LCMV Clone 13 or LCMV MP strain. (See, e.g., Section 7.1).

] In a c embodiment, provided herein are itions suitable for a method oftreating and/or preventing an HBV infection in a subject comprising administering to the subject a ?rst infectious arenavirus composition expressing an HBV pre-SZ/S protein or an antigenic fragment thereof and a second infectious irus composition expressing an HBV HBc protein or an antigenic fragment thereof.

In a c embodiment, provided herein are itions suitable for a method ting and/or preventing an infection in a subject comprising administering sequentially to the t a ?rst infectious arenavirus sing an HBV pre-S2/S protein or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV HBs protein or an antigenic fragment thereof.

In a speci?c embodiment, provided herein are compositions suitable for a an infection in a subject comprising administering simultaneously to the subject a ?rst infectious arenavirus expressing an HBV HBc protein or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV HBs n or an antigenic fragment thereof In a speci?c embodiment, provided herein are compositions suitable for a method ting and/or preventing an ion in a subject comprising administering sequentially to the subject a ?rst infectious arenavirus expressing an HBV pre-S2/S protein or an antigenic fragment thereof and a second infectious arenavirus expressing a fusion ofHBV HBs and HBc proteins or antigenic fragments thereof.

In a speci?c embodiment, provided herein are compositions suitable for a method oftreating and/or preventing an infection in a t comprising administering simultaneously to the subject a ?rst infectious arenavirus expressing an HBV HBc protein or an antigenic fragment thereof and a second infectious arenavirus expressing a ?rsion ofHBV HBs and HBc proteins or antigenic fragments thereof.

In a speci?c embodiment, provided herein are compositions suitable for a method oftreating and/or preventing an infection in a subject sing administering sequentially to the subject a ?rst infectious arenavirus expressing an HBV HBc protein or an nic fragment thereof and a second infectious arenavirus expressing an HBV pre—S2/S protein or an antigenic fragment thereof.

In a speci?c embodiment, ed herein are itions suitable for a method oftreating and/or preventing an infection in a subject comprising administering sequentially to the t a ?rst infectious arenavirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof and a second infectious arenavirus expressing an HBV pre-S2/S protein or an nic fragment thereof.

] In a speci?c ment, provided herein are itions suitable for a method oftreating and/or preventing an infection in a subject comprising administering sequentially to the subject a ?rst infectious irus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof and a second infectious arenavirus expressing an HBV HBe protein or an antigenic fragment thereof.

In a speci?c embodiment, ed herein are compositions suitable for a method oftreating and/or preventing an infection in a subject comprising stering sequentially to the subject a ?rst infectious arenavirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof and a second infectious arenavirus expressing an HBV HBc protein or an antigenic fragment thereof.

In a speci?c embodiment, provided herein are compositions suitable for a method ting and/or ting an infection in a subject sing stering to the subject a ?rst infectious arenavirus expressing an HBV pre-S2/S protein or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV HBe protein or an antigenic fragment thereof.

In a c embodiment, provided herein are compositions suitable for a method oftreating and/or preventing an infection in a subject comprising administering simultaneously to the subject a ?rst infectious arenavirus expressing an HBV HBe protein or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV HBs protein or an antigenic fragment thereof.

In a speci?c embodiment, provided herein are compositions suitable for a method oftreating and/or preventing an infection in a subject comprising administering sequentially to the t a ?rst infectious arenavirus expressing an HBV HBe protein or an antigenic fragment thereof and a second infectious arenavirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof In a c embodiment, provided herein are compositions suitable for a method oftreating and/or preventing an infection in a t comprising administering sequentially to the subject a ?rst infectious arenavirus expressing an HBV HBs protein or an nic fragment thereof and a second infectious arenavirus expressing a fusion ofHBV HBs and HBc proteins or antigenic fragments thereof.

In a speci?c embodiment, ed herein are compositions suitable for a method oftreating and/or preventing an infection in a subject comprising administering sequentially to the subject a ?rst ious arenavirus expressing an HBV HBe protein or an antigenic fragment f and a second infectious arenavirus expressing an HBV pre-SZ/S protein or an antigenic nt thereof.

In a speci?c ment, provided herein are compositions suitable for a method oftreating and/or preventing an infection in a subject sing administering sequentially to the subject a ?rst infectious arenavirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof and a second infectious arenavirus sing an HBV HBs protein or an antigenic fragment thereof.

In a speci?c embodiment, provided herein are compositions le for a method oftreating and/or preventing an infection in a subject comprising administering sequentially to the subject a ?rst infectious arenavirus expressing an HBV HBs protein or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV pre-SZ/S protein or an antigenic fragment thereof.

In a c embodiment, provided herein are itions suitable for a method oftreating and/or preventing an infection in a subject comprising administering aneously to the subject a ?rst infectious irus expressing an HBV HBc protein or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV HBe protein or an antigenic fragment thereof ] In a speci?c embodiment, provided herein are compositions suitable for a method oftreating and/or preventing an infection in a subject comprising administering simultaneously to the subject a ?rst infectious arenavirus sing an HBV HBs protein or an antigenic fragment f and a second ious arenavirus expressing an HBV HBe protein or an antigenic nt thereof.

In a speci?c embodiment, provided herein are compositions suitable for a method oftreating and/or preventing an infection in a subject comprising administering simultaneously to the subject a ?rst infectious arenavirus expressing an HBV HBs protein or an antigenic fragment thereof and a second infectious arenavirus expressing an HBV HBc protein or an antigenic fragment thereof.

In a c embodiment, provided herein are compositions suitable for a method oftreating and/or preventing an infection in a subject comprising administering simultaneously to the subject a ?rst infectious arenavirus expressing an HBV HBe protein or an antigenic nt f and a second infectious arenavirus expressing an HBV HBc protein or an antigenic fragment thereof.

In another embodiment, the ?rst infectious arenavirus composition expressing an HBV antigen is a primary vaccine antigen and the second infectious arenavirus expressing another HBV antigen is a secondary vaccine antigen.

In yet another ment, provided herein is the combined use of the replication-de?cient arenaviruses compositions expressing an HBV antigen as described herein and one or more replication-defective virus vector compositions. In a more speci?c embodiment the replication-defective virus vector composition can be but is not limited to: uses, adenoviruses, alphaviruses, herpes simplex viruses, paramyxoviruses, rhabdoviruses, poliovirus, associated virus, and Sendai virus, and es thereof. In a c embodiment, the poxvirus is a modi?ed vaccine Ankara.

In another embodiment, two infectious arenaviruses compositions have molar ratios ranging from about 1:1 to 1:1000, in particular including: 1:1 ratio, 1:2 ratio, 1:5 ratio, 1:10 ratio, 1:20 ratio, 1:50 ratio, 1:100 ratio, 1:200 ratio, 1:300 ratio, 1:400 ratio, 1:500 ratio, 1:600 ratio, 1:700 ratio, 1:800 ratio, 1:900 ratio, 1:1000 ratio.

In another embodiment, two or more infectious arenavirus compositions expressing an HBV antigen described herein are suitable for administration to subjects who have, are susceptible to, or are at risk for an HBV ion. In r embodiment, the subjects, to whom two or more infectious arenaviruses compositions expressing an HBV antigen described herein or a composition thereof is administered, are infected with, are susceptible to, or are at risk for, an infection with HBV.

In another embodiment, said two or more infectious arenavirus compositions r comprise at least one other medicament for treating and/or preventing HBV ion.

Therapeutic medicaments include, but are not limited to, entecavir (BARACLUDE®; Bristol- Myers Squibb), dine (EPIVIR HBV®; GlaxoSmithKline), adefovir dipivoxil (HEPSERA®; Gilead Sciences), interferon alpha 2b (INTRON A®; Schering), pegylated interferon (PEGASYS®; Roche), telbivudine (TYZEKA®, Novartis), and tenofovir (VIREAD®; Gilead es).

In another ment, compositions are suitable for administrating to a symptomatic subject a second infectious arenavirus composition expressing an HBV antigen or a fragment thereof as described herein. In yet another embodiment, the compositions are suitable for administration to a subject with a compromised immune system, especially transplant recipients, HIV—infected persons, a pregnant t, or a subject who has , a second infectious irus composition expressing an HBV antigen described herein or a fragment thereof. In another embodiment, two or more infectious arenavirus compositions expressing an HBV antigen as described herein or a nt thereof are suitable for strating to a subject who is a child of0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years ofage suffering from or susceptible to, or are at risk for, an infection with HBV.

In another embodiment, compositions are suitable for administrating to a subject who is a child, a ?rst arenavirus sing an HBV antigen, and administering to the same subject who is an adolescent a second arenavirus expressing an HBV antigen. In a speci?c embodiment, the administration regime can involve administering to a subject who is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years of age a ?rst arenavirus expressing an HBV antigen as described herein, and to the same subject who is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 years of age a second ious arenavirus expressing an HBV antigen.

In r embodiment, compositions are suitable for administering to a prepubescent subject a second infectious arenavirus sing an HBV n. In another embodiment, the administration regime can involve administering to an adolescent male, aged 12 to 18 years a second infectious arenavirus expressing an HBV antigen as described herein. In another embodiment, the administration regime can involve administering to a , aged 12 to 18 years a second infectious arenavirus expressing an HBV antigen.

In another embodiment, two or more infectious arenavirus compositions expressing an HBV antigen or a fragment thereof, as described herein reduce the risk that an individual will develop an infection with HBV by at least 10%, at least about 20%, at least about %, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the risk of developing an infection with HBV in the absence of such treatment.

In another ment, two or more infectious arenavirus compositions expressing an HBV antigen or a fragment thereof, as described herein, administered separately, reduce the risk that an individual will develop an infection with HBV by at least 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the risk of developing an infection with HBV in the absence of such treatment.

In r embodiment, two or more infectious arenavirus itions expressing an HBV antigen or a fragment f, as described , administered sequentially, reduce the risk that an individual will develop an infection with HBV by at least 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the risk of developing an infection with HBV in the absence of such treatment.

In another embodiment, provided herein the invention provides a e composition comprising a synergistic combination of two or more infectious replication-de?cient arenaviruses expressing an HBV antigen.

In another ment, ed herein the invention provides a vaccine composition comprising a synergistic ation of two or more infectious replication- competent arenaviruses sing an HBV antigen. 6.9 Assays Assay for Measuring irus Vector Infectivity Any assay known to the skilled artisan can be used for measuring the infectivity of an irus vector preparation. For example, determination of the virus/vector titer can be done by a "focus forming unit assay" (FFU assay). In brief, complementing cells, e.g. HEK 293 cells expressing LCMV GP protein, are plated and inoculated with different dilutions of a virus/vector sample. After an incubation period, to allow cells to form a mono layer and virus to attach to cells, the mono layer is covered with Methylcellulose. When the plates are further incubated, the original infected cells e viral progeny. Due to the Methylcellulose overlay the spread of the new viruses is restricted to oring cells. uently, each infectious particle produces a circular zone of infected cells called a Focus. Such Foci can be made visible and by that countable using antibodies t LCMV- NP and a HRP-based color reaction. The titer of a Virus / vector can be calculated in focus-forming units per milliliter (FFU/mL).

To determine the ious titer (FFU/mL) of transgene-carrying vectors this assay is modi?ed by the use of the respective transgene-speci?c antibody instead of anti-LCMV- NP antibody.

Serum ELISA ination of the humoral immune response upon vaccination of animals (e.g. mice, guinea pigs) can be done by antigen-speci?c serum ELISAs (enzyme- linked immunosorbent assays). In brief, plates are coated with antigen (e.g. recombinant protein), blocked to avoid unspeci?c binding of antibodies and incubated with serial dilutions of sera. After incubation, bound serum-antibodies can be detected, e.g., using an enzyme-coupled anti-species (e.g. mouse, guinea pig)—specif1c antibody (detecting total IgG or IgG subclasses) and subsequent color reaction. Antibody titers can be determined as, e.g., endpoint geometric mean titer.

Imrnunocapture ELISA (IC-ELISA) may also be performed (see Shanmugham et al., 2010, Clin. Vaccine Immunol. 17(8):1252-l260), wherein the capture agents are cross-linked to beads.

Neutralizing Assay in ARPE-l9 cells Determination of the neutralizing ty of induced antibodies in sera is performed with the following cell assay using ARPE-l9 cells from ATCC and a GFP-tagged virus. In addition supplemental serum as a source of exogenous complement is used. The assay is started with seeding of 6.5x103 cells/well (SOul/well) in a 384 well plate one or two days before using for neutralization. The neutralization is done in l e tissue culture plates t cells for lh at 37°C. After the neutralization incubation step the mixture is added to the cells and incubated for additional 4 days for GFP-detection with a plate reader. A positive neutralizing human sera is used as assay positive control on each plate to check the reliability of all results. Titers (ECSO) are determined using a 4 parameter logistic curve ?tting. As additional testing the wells are d with a cence microscope.

Plaque Reduction Assay In brief, plaque reduction (neutralization) assays for Hepatitis B virus are performed by use of an isolate ofHBV tagged with green ?uorescent protein, 5% rabbit serum was used as a source of exogenous ment, and plaques are enumerated by cence microscopy. Neutralization titers are de?ned as the highest dilution of serum that s in a 50% reduction in plaques, ed with that in control (pre-immune) serum samples.

] Neutralization Assay in guinea pig lung ?broblast (GPL) cells In brief, serial dilutions of test and control (pre-vaccination) sera were prepared in GPL complete media with supplemental rabbit serum (1%) as a source of exogenous complement. The dilution series spanned 1:40 through 1:5120. Serum dilutions were incubated with eGFP tagged virus (100-200 p?a per well) for 30 min at 37°C, and then transferred to 12-well plates containing con?uent GPL cells. Samples were processed in triplicate. After 2 hours incubation at 37°C the cells were washed with PBS, re-fed with GPL complete media and incubated at 37°C / 5% C02 for 5 days.

Plaques were visualized by ?uorescence microscopy, counted, and compared to control wells.

That serum dilution resulting in a 50% reduction in plaque number compared to controls was ated as the neutralizing titer. qPCR LCMV RNA genomes are isolated using QIAamp Viral RNA mini Kit (QIAGEN), ing to the protocol provided by the manufacturer. LCMV RNA genome equivalents are detected by quantitative PCR carried out on an StepOnePlus Real Time PCR System (Applied Biosystems) with SuperScript® III Platinum® One-Step qRT-PCR Kit rogen) and primers and probes (FAM reporter and NFQ-MGB Quencher) speci?c for part ofthe LCMV NP coding region. The temperature pro?le of the reaction is : 30 min at 60°C, 2 min at 95°C, ed by 45 cycles of 15 s at 95°C, 30 s at 56°C. RNA is quanti?ed by comparison of the sample results to a standard curve prepared from a loglO dilution series of a spectrophotometrically ?ed, in vitro-transcribed RNA fragment, corresponding to a fragment of the LCMV NP coding sequence containing the primer and probe binding sites.

Western Blotting Infected cells grown in tissue culture ?asks or in suspension are lysed at indicated timepoints post infection using RIPA buffer o Scienti?c) or used directly without cell-lysis. Samples are heated to 99°C for 10 minutes with reducing agent and NuPage LDS Sample buffer (NOVEX) and chilled to room temperature before loading on 4- 12% SDS-gels for electrophoresis. Proteins are d onto membranes using Invitrogens iBlot Gel transfer Device and visualized by Ponceau staining. Finally, the preparations are probed with an primary antibodies directed against proteins of interest and alkaline phosphatase conjugated secondary antibodies followed by staining with l-Step NBT/BCIP solution (INVITROGEN).

MHC-Peptide Multimer Staining Assay for Detection of Antigen-Speci?c CD8+ T-cell proliferation Any assay known to the skilled artisan can be used to test antigen- c CD8+ T-cell responses. For example, the MHC-peptide er staining assay can be used (see, e.g., Altman J.D. et al., Science. 1996; 274:94-96; and Murali-Krishna K. et al., Immunity. 1998 ; 8: 177-187). Brie?y, the assay comprises the following steps, a er assay is used to detect the presence of antigen speci?c T-cells. In order for a T-cell to detect the e to which it is speci?c, it must both recognize the peptide and the tetramer ofMHC molecules custom made for an antigen speci?c T-cell (typically ?uorescently labeled). The tetramer is then ed by ?ow cytometry via the ?uorescent label.

ELISPOT Assay for Detection of n-Speci?c CD4+ T-cell Proliferation Any assay known to the skilled artisan can be used to test antigen—speci?c CD4+ T-cell responses. For example, the ELISPOT assay can be used (see, e.g., Czerkinsky C.C. et al., J Immunol Methods. 1983; 65:109-121; and Hutchings P.R. Et al., J Immunol Methods. 1989; 120: 1—8). Brie?y, the assay comprises the following steps: An immunospot plate is coated with an ytokine antibody. Cells are incubated in the spot plate. Cells e cytokines and are then washed off. Plates are then coated with a second biotyinlated-anticytokine antibody and visualized with an avidin-HRP system.

Intracellular Cytokine Assay for Detection of Functionality of CD8+ and CD4+ T-cell Responses Any assay known to the skilled artisan can be used to test the nality ofCD8+ and CD4+ T cell responses. For example, the intracellular cytokine assay combined with ?ow cytometry can be used (see, e.g., Suni MA. et al., J Immunol Methods. 1998; 212:89-98; Nomura L.E. et a1., Cytometry. 2000; 40:60-68; and Ghanekar S.A. et al., Clinical and Diagnostic Laboratory Immunology. 2001; 63). Brie?y, the assay comprises the following steps: activation of cells via speci?c peptides or protein, an tion of protein transport (e.g., brefeldin A) is added to retain the cytokines within the cell. After washing, antibodies to other cellular markers can be added to the cells. Cells are then ?xed and permeabilized. The anti—cytokine antibody is added and the cells can be analyzed by ?ow cytometry.

Assay for Con?rming ation-De?ciency of Viral Vectors Any assay known to the skilled artisan that determines concentration of infectious and replication- competent virus particles can also be used as a to measure replication-de?cient Viral particles in a sample. For example, FFU assays (as described in [00408]) with non-complementing cells can be used for this purpose.

Furthermore, -based assays are the rd method used to ine virus concentration in terms ofplaque forming units (PFU) in a virus sample. Speci?cally, a con?uent monolayer ofnon-complementing host cells is infected with the virus at varying dilutions and covered with a semi-so lid medium, such as agar to t the virus infection from spreading indiscriminately. A viral plaque is formed when a virus successfully infects and replicates itself in a cell within the ?xed cell monolayer (see, e. g., Kaufmann, S.H.; Kabelitz, D. (2002). Methods in Microbiology Vol.32:lmmunology ofInfection. Academic Press. ISBN 0- 120). Plaque formation can take 3 — 14 days, depending on the virus being analyzed.

Plaques are generally counted manually and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per sample unit volume (PFU/mL). The PFU/mL result represents the number of infective replication— competent particles within the sample.

Measuring Viral Load in the Blood or Liver Any assay known to the skilled n that determines the viral load may be used to detect the number ofHBV particles per volume in the blood or liver (see, e. g., Mendy et al., 2010, J. Viral Hepat. 17(2): 2). Non- limiting examples of such assays include c acid-based tests such as PCR, as well as non- nucleic ased tests.

Liver Biopsy Any ure known to the skilled artisan that performs a liver biopsy may be used to determine the degree of liver damage, for example, to test a patient for chronic HBV infection or liver cancer. Non-limiting es oftypes of liver es include percutaneous needle biopsies, laparoscopic biopsies, and enous biopsies. In certain embodiments, a liver biopsy is used to determine the presence of ground glass hepatocytes when the cells are examined under a light microscope. The ance of ground glass hepatocytes is tive of the presence of HBsAg in the liver cells.

Assay for Expression of Viral Antigen Any assay known to the skilled artisan can be used for measuring expression of Viral antigens. For example, FFU assays (as described in [00408]) can be performed. For detection, mono- or polyclonal antibody preparation(s) against respective Viral antigens are used (transgene-speci?c FFU).

Furthermore, Western Blotting (as described in [00415]) can be performed.

Microparticle Enzyme Immunoassay The AXSYM® HbsAg (Abbott) is a microparticle enzyme immunoassay (MEIA) to detect HBsAg in adult, pediatric, and neonatal serum or plasma, including in pregnant women. This assay can be used as an aid in the sis of acute or chronic HBV. This assay may also be used to con?rm the presence ofHBV infection.

] To perform the assay, a sample of the t’s blood is placed into reaction wells containing detector antibodies and microparticles coated with antibodies to HBV (e.g. , to HBV antigens). If the blood sample contains HBV proteins (e.g. will bind to the , HBsAg), they microparticles in the reaction wells. This reaction is detected by another substance that produces light, which is then measured to determine the presence ofHBV (e.g. HBV antigens) in the blood. If the ?rst test is positive, the patient’s blood is re—tested to con?rm the ce ofHBV (e.g., HBV antigens). Any microparticle enzyme immunoassay known to the skilled artisan may be used to measure the presence of HBsAg or other HBV antigens.

Other HBV Assays A sample of the patient’s blood is placed in contact with either HBV dies or HBV antigens. The antibodies and/or ns include HBsAg, antibodies to HBeAg, antibodies to HBsAg, HBeAg, IgM antibodies to HBcAg, and antibodies to HBcAg. If the t is infected with HBV, antigens and/or dies present in the blood will cause a chemical reaction to occur when the test is run. This assay allows for the detection ofthe stage of HBV, according to what HBV antigens and/or antibodies are present in the patient’s blood.

Any assay known to one of skill in the art may be used to evaluate levels of HBV, HBV antigens, or HBV antibodies. For non-limiting examples of such assays, see, e. g., Mayer et al., 2012, BMC Clin. Pathol. 12:8, Van Helden et al., 2004, Clin. Lab. 50(1-2):63—73, and Villar et al., 2011, J. Med. Virol. 1522-1529., Animal Models The safety, nce and immunogenic effectiveness of vaccines comprising of an infectious irus expressing an HBV antigen described herein or a composition thereof can be tested in animals models. In certain embodiments, the animal models that can be used to test the safety, nce and genic effectiveness of the es and compositions thereof used herein include mouse, guinea pig, rat, , and chimpanzee. In a preferred embodiment, the animal models that can be used to test the safety, tolerance and immunogenic effectiveness of the vaccines and compositions thereofused herein include mouse.

In a speci?c example, a transgenic mouse model may be used to assess the antiviral potential of pharmacological agents, such as immunotherapies or vaccines, and to assess physiological processes, including the immune response (see, e. g., Guidotti et al., 1995, J. Virol. 69(10):6158-69). Such transgenic mouse models may express human les, such as human class I and II HLA molecules, and/or the hepatitis B surface antigen (HBsAg) (see, e.g., Bourgine et al., 2012, Virology 430(1): 10-9).

In another speci?c example, the woodchuck (Marmota monax) can be used as an animal model for developing and testing treatment and prevention approaches to chronic hepadnaviral infections, such as chronic hepatitis B (see, e.g., Kosinska et al., Hepat. Res. Treat. 2010:8175 80). The uck model is applicable for evaluation of the immunogenicity and other immune responses of potential immunotherapies such as vaccines (see, e.g, Vaccine 27(25-26):3271-3275). 6.10 Sequences The ces in Table 3 are illustrative amino acid sequences and nucleotide sequences that can be used with the methods and compositions described herein. In some ces a DNA sequence is used to describe the RNA sequence of a viral c segment.

The RNA sequence can be readily deduced from the DNA sequence.

Table 3. Illustrative amino acid sequences.

ID Description ce 1 nucleotide sequence of ATGCAGTGGAATTCCACAACCTTCCACCAAACTCT the HBV pre-SZ/S ORF GCAAGATCCCAGAGTGAGAGGCCTGTATTTCCCT GCTGGTGGCTCCAGTTCAGGAACAGTCAACCCTG TTCTGACCACTGCCTCTCCCTTGTCATCAATCTTCT CCAGGATTGGGGACCCTGCTCTGAACATGGAGAA CATCACATCAGGATTCCTGGGACCCCTTCTTGTGT TGCAGGCAGGGTTTTTCTTGTTGACAAGAATCCTC ACAATCCCTCAGAGTCTGGACTCTTGGTGGACTTC TCTCAATTTTCTGGGGGGAACCACAGTGTGTCTTG GCCAAAATTCTCAGTCCCCAACCTCCAATCACTCA CCAACCTCTTGTCCTCCAACTTGTCCTGGTTACAG ATGGATGTGTCTGAGGAGATTCATCATCTTCCTCT TCATCCTGCTGCTGTGCCTCATCTTCTTGTTGGTTC TTCTGGACTATCAAGGAATGTTGCCAGTTTGTCCT CTGATTCCAGGATCCTCAACAACCAGCACTGGAC CATGCAGGACCTGCATGACCACTGCTCAAGGAAC CTCAATGTATCCCTCCTGTTGCTGCACCAAACCTT CAGATGGAAATTGCACCTGCATTCCCATCCCATCA TCCTGGGCTTTTGGAAAATTCCTTTGGGAGTGGGC CTCAGCCAGATTCTCCTGGCTCAGTTTGCTGGTGC CATTTGTTCAGTGGTTTGTTGGGCTTTCCCCCACT CTTTCAGTGATTTGGATGATGTGGTATTG GGGGCCAAGTCTGTACAGCATCTTGAGTCCCTTTT TGCCTCTGTTGCCAATTTTCTTTTGTCTTTGGGTCT ACATTTAA 2 nucleotide sequence of ATGGACATTGACCCTTACAAAGAATTTGGAGCAA the HBV HBC ORF CTGTGGAGTTGCTCTCCTTTTTGCCTTCTGACTTCT TTCCTTCAGTGAGAGATCTTCTTGACACTGCCTCA GCTCTGTACAGGGAAGCCTTGGAGTCTCCTGAGC ATTGTTCACCTCACCACACTGCACTCAGGCAAGC TTGCTGGGGGGAACTCATGACTCTGGCA ACCTGGGTGGGTGTCAATTTGGAAGATCCAGCCT CAAGAGACCTTGTGGTCAGTTATGTCAACACAAA CATGGGCCTGAAGTTCAGGCAACTCTTGTGGTTTC ACATTTCTTGTCTCACTTTTGGAAGAGAAACAGTC ATTGAGTATTTGGTGTCTTTTGGAGTGTGGATCAG GACTCCTCCAGCTTACAGACCACCAAATGCCCCA ATCCTGTCAACACTTCCAGAGACCACTGTTGTCAG AGGCAGGTCCCCCAGAAGAAGAACTCC CTCACCAAGAAGAAGAAGGTCTCAATCTCCCAGA AGGAGAAGATCTCAATCAAGGGAATCTCAATGTT 3 nucleotide sequence of ATGGGGCAGAATCTTTCCACCAGCAATCCTCTGGGATTCTT the HBV HBS-HBC TCCAGACCACCAGTTGGATCCAGCCTTCAGAGCAAACACTG ?lsion protein ORF CAAATCCAGATTGGGACTTCAATCCCAACAAGGACACCTGG CCAGATGCCAACAAGGTGGGAGCTGGAGCATTTGGGCTGGG TTTCACCCCACCCCATGGAGGCCTTTTGGGGTGGAGCCCTC AGGCTCAGGGCATTCTGCAAACTTTGCCAGCAAATCCACCT CCTGCCTCCACCAACAGGCAGTCAGGAAGGCAGCCCACCCC TCTGTCTCCACCTTTGAGAAACACTCATCCTCAGGCCATGC AGTGGAATTCCACAACCTTCCACCAAACTCTGCAAGATCCC AGAGTGAGAGGCCTGTATTTCCCTGCTGGTGGCTCCAGTTC AGGAACAGTCAACCCTGTTCTGACCACTGCCTCTCCCTTGT CATCAATCTTCTCCAGGATTGGGGACCCTGCTCTGAACATG GAGAACATCACATCAGGATTCCTGGGACCCCTTCTTGTGTT GCAGGCAGGGTTTTTCTTGTTGACAAGAATCCTCACAATCC CTCAGAGTCTGGACTCTTGGTGGACTTCTCTCAATTTTCTG GGGGGAACCACAGTGTGTCTTGGCCAAAATTCTCAGTCCCC CAATCACTCACCAACCTCTTGTCCTCCAACTTGTC CTGGTTACAGATGGATGTGTCTGAGGAGATTCATCATCTTC ATCCTGCTGCTGTGCCTCATCTTCTTGTTGGTTCT TCTGGACTATCAAGGAATGTTGCCAGTTTGTCCTCTGATTC CAGGATCCTCAACAACCAGCACTGGACCATGCAGGACCTGC ATGACCACTGCTCAAGGAACCTCAATGTATCCCTCCTGTTG CTGCACCAAACCTTCAGATGGAAATTGCACCTGCATTCCCA TCCCATCATCCTGGGCTTTTGGAAAATTCCTTTGGGAGTGG GCCTCAGCCAGATTCTCCTGGCTCAGTTTGCTGGTGCCATT TGTTCAGTGGTTTGTTGGGCTTTCCCCCACTGTTTGGCTTT CAGTGATTTGGATGATGTGGTATTGGGGGCCAAGTCTGTAC AGCATCTTGAGTCCCTTTTTGCCTCTGTTGCCAATTTTCTT TTGTCTTTGGGTCTACATTATGGACATTGACCCTTACAAAG AATTTGGAGCAACTGTGGAGTTGCTCTCCTTTTTGCCTTCT GACTTCTTTCCTTCAGTGAGAGATCTTCTTGACACTGCCTC AGCTCTGTACAGGGAAGCCTTGGAGTCTCCTGAGCATTGTT CACCTCACCACACTGCACTCAGGCAAGCAATTCTTTGCTGG GGGGAACTCATGACTCTGGCAACCTGGGTGGGTGTCAATTT GGAAGATCCAGCCTCAAGAGACCTTGTGGTCAGTTATGTCA ACACAAACATGGGCCTGAAGTTCAGGCAACTCTTGTGGTTT CACATTTCTTGTCTCACTTTTGGAAGAGAAACAGTCATTGA GTATTTGGTGTCTTTTGGAGTGTGGATCAGGACTCCTCCAG CTTACAGACCACCAAATGCCCCAATCCTGTCAACACTTCCA GAGACCACTGTTGTCAGAAGAAGAGGCAGGTCCCCCAGAAG AAGAACTCCCTCACCAAGAAGAAGAAGGTCTCAATCTCCCA GAAGATCTCAATCAAGGGAATCTCAATGTTAG nucleotide sequence of GCGCACCGGGGATCCTAGGCTTTTTGGATTGCGCT the LCMV S segment TTCCTCTAGATCAACTGGGTGTCAGGCCCTATCCT expressing HBV HBs- ACAGAAGGATGGGGCAGAATCTTTCCACCAGCAA HBC fusion protein in TCCTCTGGGATTCTTTCCAGACCACCAGTTGGATC cDNA form (The CAGCCTTCAGAGCAAACACTGCAAATCCAGATTG genomic segment is GGACTTCAATCCCAACAAGGACACCTGGCCAGAT RNA, the sequence in GCCAACAAGGTGGGAGCTGGAGCATTTGGGCTGG SEQ ID N024 is shown GTTTCACCCCACCCCATGGAGGCCTTTTGGGGTGG for DNA; r, AGCCCTCAGGCTCAGGGCATTCTGCAAACTTTGCC exchanging all AGCAAATCCACCTCCTGCCTCCACCAACAGGCAG thymidines ("T") in TCAGGAAGGCAGCCCACCCCTCTGTCTCCACCTTT SEQ ID N024 for GAGAAACACTCATCCTCAGGCCATGCAGTGGAAT uridines ("U") provides TCCACAACCTTCCACCAAACTCTGCAAGATCCCA the RNA sequence.) GAGTGAGAGGCCTGTATTTCCCTGCTGGTGGCTCC AGTTCAGGAACAGTCAACCCTGTTCTGACCACTG CCTCTCCCTTGTCATCAATCTTCTCCAGGATTGGG GACCCTGCTCTGAACATGGAGAACATCACATCAG TGGGACCCCTTCTTGTGTTGCAGGCAGGG TTTTTCTTGTTGACAAGAATCCTCACAATCCCTCA GAGTCTGGACTCTTGGTGGACTTCTCTCAATTTTC TGGGGGGAACCACAGTGTGTCTTGGCCAAAATTC TCAGTCCCCAACCTCCAATCACTCACCAACCTCTT GTCCTCCAACTTGTCCTGGTTACAGATGGATGTGT CTGAGGAGATTCATCATCTTCCTCTTCATCCTGCT GCTGTGCCTCATCTTCTTGTTGGTTCTTCTGGACTA TCAAGGAATGTTGCCAGTTTGTCCTCTGATTCCAG GATCCTCAACAACCAGCACTGGACCATGCAGGAC CTGCATGACCACTGCTCAAGGAACCTCAATGTAT CCCTCCTGTTGCTGCACCAAACCTTCAGATGGAAA TTGCACCTGCATTCCCATCCCATCATCCTGGGCTT TTGGAAAATTCCTTTGGGAGTGGGCCTCAGCCAG ATTCTCCTGGCTCAGTTTGCTGGTGCCATTTGTTC AGTGGTTTGTTGGGCTTTCCCCCACTGTTTGGCTT TCAGTGATTTGGATGATGTGGTATTGGGGGCCAA GTCTGTACAGCATCTTGAGTCCCTTTTTGCCTCTG TTGCCAATTTTCTTTTGTCTTTGGGTCTACATTATG GACATTGACCCTTACAAAGAATTTGGAGCAACTG TGGAGTTGCTCTCCTTTTTGCCTTCTGACTTCTTTC CTTCAGTGAGAGATCTTCTTGACACTGCCTCAGCT CTGTACAGGGAAGCCTTGGAGTCTCCTGAGCATT GTTCACCTCACCACACTGCACTCAGGCAAGCAAT TCTTTGCTGGGGGGAACTCATGACTCTGGCAACCT GGGTGGGTGTCAATTTGGAAGATCCAGCCTCAAG TGTGGTCAGTTATGTCAACACAAACATG GGCCTGAAGTTCAGGCAACTCTTGTGGTTTCACAT TTCTTGTCTCACTTTTGGAAGAGAAACAGTCATTG AGTATTTGGTGTCTTTTGGAGTGTGGATCAGGACT CCTCCAGCTTACAGACCACCAAATGCCCCAATCCT GTCAACACTTCCAGAGACCACTGTTGTCAGAAGA AGAGGCAGGTCCCCCAGAAGAAGAACTCCCTCAC CAAGAAGAAGAAGGTCTCAATCTCCCAGAAGGAG AAGATCTCAATCAAGGGAATCTCAATGTTAGAGA CCTCCCTGACTCTCCACCTCGAAAGAGG TGGAGAGTCAGGGAGGCCCAGAGGGTCTTAGAGT GTCACAACATTTGGGCCTCTAAAAATTAGGTCAT GTGGCAGAATGTTGTGAACAGTTTTCAGATCTGG GAGCCTTGCTTTGGAGGCGCTTTCAAAAATGATG ATGAGTGCACAGTGCGGGGTGATCTCTTT CTTCTTTTTGTCCCTTACTATTCCAGTATGCATCTT ACACAACCAGCCATATTTGTCCCACACTTTATCTT CATACTCCCTCGAAGCTTCCCTGGTCATTTCAACA TCGATAAGCTTAATGTCCTTCCTATTTTGTGAGTC CAGAAGCTTTCTGATGTCATCGGAGCCTTGACAG CTTAGAACCATCCCCTGCGGAAGAGCACCTATAA CTGACGAGGTCAACCCGGGTTGCGCATTGAAGAG GTCGGCAAGATCCATGCCGTGTGAGTACTTGGAA TTGAATTGTTTTTGATCAACGGGTTCCCT GTAAAAGTGTATGAACTGCCCGTTCTGTGGTTGG AAAATTGCTATTTCCACTGGATCATTAAATCTACC CTCAATGTCAATCCATGTAGGAGCGTTGGGGTCA ATTCCTCCCATGAGGTCTTTTAAAAGCATTGTCTG GCTGTAGCTTAAGCCCACCTGAGGTGGACCTGCT GCTCCAGGCGCTGGCCTGGGTGAGTTGACTGCAG GTTTCTCGCTTGTGAGATCAATTGTTGTGTTTTCCC ATGCTCTCCCCACAATCGATGTTCTACAAGCTATG TATGGCCATCCTTCACCTGAAAGGCAAACTTTATA GAGGATGTTTTCATAAGGGTTCCTGTCCCCAACTT GGTCTGAAACAAACATGTTGAGTTTTCTCTTGGCC CCGAGAACTGCCTTCAAGAGATCCTCGCTGTTGCT TGGCTTGATCAAAATTGACTCTAACATGTTACCCC CATCCAACAGGGCTGCCCCTGCCTTCACGGCAGC ACCAAGACTAAAGTTATAGCCAGAAATGTTGATG CTGGACTGCTGTTCAGTGATGACCCCCAGAACTG GGTGCTTGTCTTTCAGCCTTTCAAGATCATTAAGA TTTGGATACTTGACTGTGTAAAGCAAGCCAAGGT CTGTGAGCGCTTGTACAACGTCATTGAGCGGAGT CTGTGACTGTTTGGCCATACAAGCCATAGTTAGAC TTGTGCCAAATTGATTGTTCAAAAGTGAT GAGTCTTTCACATCCCAAACTCTTACCACACCACT TGCACCCTGCTGAGGCTTTCTCATCCCAACTATCT GTAGGATCTGAGATCTTTGGTCTAGTTGCTGTGTT GTTAAGTTCCCCATATATACCCCTGAAGCCTGGGG AGACCTCATGATCTTGGCCTTCAGCTTCT CAAGGTCAGCCGCAAGAGACATCAGTTCTTCTGC ACTGAGCCTCCCCACTTTCAAAACATTCTTCTTTG ATGTTGACTTTAAATCCACAAGAGAATGTACAGT CTGGTTGAGACTTCTGAGTCTCTGTAGGTCTTTGT CATCTCTCTTTTCCTTCCTCATGATCCTCTGAACAT TGCTGACCTCAGAGAAGTCCAACCCATTCAGAAG GTTGGTTGCATCCTTAATGACAGCAGCCTTCACAT CTGATGTGAAGCTCTGCAATTCTCTTCTCAATGCT TGCGTCCATTGGAAGCTCTTAACTTCCTTAGACAA GGACATCTTGTTGCTCAATGGTTTCTCAAGACAAA ATCAAATGCCTAGGATCCACTGTGCG nucleotide sequence of GCGCACCGGGGATCCTAGGCTTTTTGGATTGCGCTTTCCTC the LCMV S segment TAGATCAACTGGGTGTCAGGCCCTATCCTACAGAAGGATGG expressing the HBc ACATTGACCCTTACAAAGAAT TTGGAGCAACTGTGGAGTTG ORF, in cDNA form CTCTCCTTTTTGCCTTCTGACTTCTTTCCTTCAGTGAGAGA (The c segment TCTTCTTGACACTGCCTCAGCTCTGTACAGGGAAGCCTTGG is RNA, the sequence in AGTCTCCTGAGCATTGTTCACCTCACCACACTGCACTCAGG SEQ ID N015 is shown CAAGCAATTCTTTGCTGGGGGGAACTCATGACTCTGGCAAC for DNA; however, CTGGGTGGGTGTCAATTTGGAAGATCCAGCCTCAAGAGACC exchanging all TTGTGGTCAGTTATGTCAACACAAACATGGGCCTGAAGTTC thymidines ("T") in AGGCAACTCTTGTGGTTTCACATTTCTTGTCTCACTTTTGG SEQ ID N015 for AAGAGAAACAGTCATTGAGTATTTGGTGTCTTTTGGAGTGT uridines ("U") provides GGATCAGGACTCCTCCAGCTTACAGACCACCAAATGCCCCA ATCCTGTCAACACT TCCAGAGACCACTGT TGTCAGAAGAAG the RNA sequence.) AGGCAGGTCCCCCAGAAGAAGAACTCCCTCACCAAGAAGAA GAAGGTCTCAATCTCCCAGAAGGAGAAGATCTCAATCAAGG GAATCTCAATGTTAGAGAACAGCGCCTCCCTGACTCTCCAC CTCGAAAGAGGTGGAGAGTCAGGGAGGCCCAGAGGGTCTTA GAGTGTCACAACATTTGGGCCTCTAAAAATTAGGTCATGTG GCAGAATGTTGTGAACAGTTTTCAGATCTGGGAGCCTTGCT TTGGAGGCGCTTTCAAAAATGATGCAGTCCATGAGTGCACA GTGCGGGGTGATCTCTTTCTTCTTTTTGTCCCTTACTATTC CAGTATGCATCTTACACAACCAGCCATATTTGTCCCACACT TTATCTTCATACTCCCTCGAAGCTTCCCTGGTCATTTCAAC ATCGATAAGCTTAATGTCCTTCCTATTTTGTGAGTCCAGAA TGATGTCATCGGAGCCTTGACAGCTTAGAACCATC CCCTGCGGAAGAGCACCTATAACTGACGAGGTCAACCCGGG TTGCGCATTGAAGAGGTCGGCAAGATCCATGCCGTGTGAGT ACTTGGAATCTTGCTTGAATTGTTTTTGATCAACGGGTTCC CTGTAAAAGTGTATGAACTGCCCGTTCTGTGGTTGGAAAAT TTCCACTGGATCATTAAATCTACCCTCAATGTCAA TCCATGTAGGAGCGTTGGGGTCAATTCCTCCCATGAGGTCT TTTAAAAGCATTGTCTGGCTGTAGCTTAAGCCCACCTGAGG TGCTGCTCCAGGCGCTGGCCTGGGTGAGTTGACTG CAGGTTTCTCGCTTGTGAGATCAATTGTTGTGTTTTCCCAT GCTCTCCCCACAATCGATGTTCTACAAGCTATGTATGGCCA TCCTTCACCTGAAAGGCAAACTTTATAGAGGATGTTTTCAT AAGGGTTCCTGTCCCCAACTTGGTCTGAAACAAACATGTTG AGTTTTCTCTTGGCCCCGAGAACTGCCTTCAAGAGATCCTC GCTGTTGCTTGGCTTGATCAAAATTGACTCTAACATGTTAC CCCCATCCAACAGGGCTGCCCCTGCCTTCACGGCAGCACCA AGACTAAAGTTATAGCCAGAAATGTTGATGCTGGACTGCTG TTCAGTGATGACCCCCAGAACTGGGTGCTTGTCTTTCAGCC TTTCAAGATCATTAAGATTTGGATACTTGACTGTGTAAAGC AAGCCAAGGTCTGTGAGCGCTTGTACAACGTCATTGAGCGG AGTCTGTGACTGTTTGGCCATACAAGCCATAGTTAGACTTG GCATTGTGCCAAATTGATTGTTCAAAAGTGATGAGTCTTTC ACATCCCAAACTCTTACCACACCACTTGCACCCTGCTGAGG CTTTCTCATCCCAACTATCTGTAGGATCTGAGATCTTTGGT CTAGTTGCTGTGTTGTTAAGTTCCCCATATATACCCCTGAA GCCTGGGGCCTTTCAGACCTCATGATCDUGGCCTTCAGCTT GTCAGCCGCAAGAGACATCAGTTCTTCTGCACTGA GCCTCCCCACTTTCAAAACATTCTTCTEEGATGTTGACTTT AAATCCACAAGAGAATGTACAGTCTGGTTGAGACTTCTGAG TCTCTGTAGGTCTTTGTCATCTCTCTTTTCCTTCCTCATGA TCCTCTGAACATTGCTGACCTCAGAGAAGTCCAACCCATTC AGAAGGTTGGTTGCATCCTTAATGACAGCAGCCTTCACATC TGATGTGAAGCTCTGCAATTCTCTTCTCAATGCTTGCGTCC ATTGGAAGCTCTTAACTTCCTTAGACAAGGACATCTTGTTG CTCAATGGTTTCTCAAGACAAATGCGCAATCAAATGCCTAG GATCCACTGTGCG nucleotide sequence of GCGCACCGGGGATCCTAGGCTTTTTGGATTGCGCT the LCMV S segment TTCCTCTAGATCAACTGGGTGTCAGGCCCTATCCT expressing the pre-S2/S ACAGAAGGATGCAGTGGAATTCCACAACCTTCCA ORF, in cDNA form CCAAACTCTGCAAGATCCCAGAGTGAGAGGCCTG (The genomic segment TATTTCCCTGCTGGTGGCTCCAGTTCAGGAACAGT is RNA, the sequence in CAACCCTGTTCTGACCACTGCCTCTCCCTTGTCAT SEQ ID NO:6 is shown CAATCTTCTCCAGGATTGGGGACCCTGCTCTGAAC for DNA; however, ATGGAGAACATCACATCAGGATTCCTGGGACCCC exchanging all TTCTTGTGTTGCAGGCAGGGTTTTTCTTGTTGACA thymidines ("T") in CTCACAATCCCTCAGAGTCTGGACTCTTG SEQ ID NO:6 for TTCTCTCAATTTTCTGGGGGGAACCACAG uridines ("U") provides TGTGTCTTGGCCAAAATTCTCAGTCCCCAACCTCC the RNA sequence.) TCACCAACCTCTTGTCCTCCAACTTGTCC TGGTTACAGATGGATGTGTCTGAGGAGATTCATC ATCTTCCTCTTCATCCTGCTGCTGTGCCTCATCTTC GTTCTTCTGGACTATCAAGGAATGTTGCC AGTTTGTCCTCTGATTCCAGGATCCTCAACAACCA GCACTGGACCATGCAGGACCTGCATGACCACTGC TCAAGGAACCTCAATGTATCCCTCCTGTTGCTGCA CCAAACCTTCAGATGGAAATTGCACCTGCATTCCC ATCCCATCATCCTGGGCTTTTGGAAAATTCCTTTG GGAGTGGGCCTCAGCCAGATTCTCCTGGCTCAGTT TGCTGGTGCCATTTGTTCAGTGGTTTGTTGGGCTT TCCCCCACTGTTTGGCTTTCAGTGATTTGGATGAT GTGGTATTGGGGGCCAAGTCTGTACAGCATCTTG AGTCCCTTTTTGCCTCTGTTGCCAATTTTCTTTTGT CTTTGGGTCTACATTTAAAGAACAGCGCCTCCCTG ACTCTCCACCTCGAAAGAGGTGGAGAGTCAGGGA GGCCCAGAGGGTCTTAGAGTGTCACAACATTTGG GCCTCTAAAAATTAGGTCATGTGGCAGAATGTTG GTTTTCAGATCTGGGAGCCTTGCTTTGGA GGCGCTTTCAAAAATGATGCAGTCCATGAGTGCA CAGTGCGGGGTGATCTCTTTCTTCTTTTTGTCCCTT ACTATTCCAGTATGCATCTTACACAACCAGCCATA TTTGTCCCACACTTTATCTTCATACTCCCTCGAAG CTTCCCTGGTCATTTCAACATCGATAAGCTTAATG TCCTTCCTATTTTGTGAGTCCAGAAGCTTTCTGAT GTCATCGGAGCCTTGACAGCTTAGAACCATCCCCT GCGGAAGAGCACCTATAACTGACGAGGTCAACCC CGCATTGAAGAGGTCGGCAAGATCCATG CCGTGTGAGTACTTGGAATCTTGCTTGAATTGTTT TTGATCAACGGGTTCCCTGTAAAAGTGTATGAACT GCCCGTTCTGTGGTTGGAAAATTGCTATTTCCACT GGATCATTAAATCTACCCTCAATGTCAATCCATGT AGGAGCGTTGGGGTCAATTCCTCCCATGAGGTCTT TTAAAAGCATTGTCTGGCTGTAGCTTAAGCCCACC TGAGGTGGACCTGCTGCTCCAGGCGCTGGCCTGG GTGAGTTGACTGCAGGTTTCTCGCTTGTGAGATCA ATTGTTGTGTTTTCCCATGCTCTCCCCACAATCGA TGTTCTACAAGCTATGTATGGCCATCCTTCACCTG GTTCCTGTCCCCAACTTGGTCTGAAACAAACATGT TGAGTTTTCTCTTGGCCCCGAGAACTGCCTTCAAG AGATCCTCGCTGTTGCTTGGCTTGATCAAAATTGA CTCTAACATGTTACCCCCATCCAACAGGGCTGCCC CTGCCTTCACGGCAGCACCAAGACTAAAGTTATA GCCAGAAATGTTGATGCTGGACTGCTGTTCAGTG ATGACCCCCAGAACTGGGTGCTTGTCTTTCAGCCT TTCAAGATCATTAAGATTTGGATACTTGACTGTGT AGCCAAGGTCTGTGAGCGCTTGTACAAC GTCATTGAGCGGAGTCTGTGACTGTTTGGCCATAC TAGTTAGACTTGGCATTGTGCCAAATTG ATTGTTCAAAAGTGATGAGTCTTTCACATCCCAAA CCACACCACTTGCACCCTGCTGAGGCTTT CTCATCCCAACTATCTGTAGGATCTGAGATCTTTG GTCTAGTTGCTGTGTTGTTAAGTTCCCCATATATA CCCCTGAAGCCTGGGGCCTTTCAGACCTCATGATC TTGGCCTTCAGCTTCTCAAGGTCAGCCGCAAGAG ACATCAGTTCTTCTGCACTGAGCCTCCCCACTTTC AAGAGAATGTACAGTCTGGTTGAGACTTCTGAGT CTCTGTAGGTCTTTGTCATCTCTCTTTTCCTTCCTC ATGATCCTCTGAACATTGCTGACCTCAGAGAAGT CCAACCCATTCAGAAGGTTGGTTGCATCCTTAATG ACAGCAGCCTTCACATCTGATGTGAAGCTCTGCA ATTCTCTTCTCAATGCTTGCGTCCATTGGAAGCTC TTAACTTCCTTAGACAAGGACATCTTGTTGCTCAA CTCAAGACAAATGCGCAATCAAATGCCT AGGATCCACTGTGCG lymphocytic GCGCACCGGGGATCCTAGGCGTTTAGTTGCGCTG choriomeningitis Virus TTTGGTTGCACAACTTTCTTCGTGAGGCTGTCAGA clone 13 segment L, AGTGGACCTGGCTGATAGCGATGGGTCAAGGCAA complete sequence GTCCAGAGAGGAGAAAGGCACCAATAGTACAAA (GenBank: CAGGGCCGAAATCCTACCAGATACCACCTATCTT DQ36 l 066. l) GGCCCTTTAAGCTGCAAATCTTGCTGGCAGAAATT (The genomic segment TGACAGCTTGGTAAGATGCCATGACCACTACCTTT is RNA, the sequence in GCAGGCACTGTTTAAACCTTCTGCTGTCAGTATCC SEQ ID NO: 7 is shown TGTCCTCTTTGTAAATATCCATTACCAAC for DNA; however, CAGATTGAAGATATCAACAGCCCCAAGCTCTCCA exchanging all CCTCCCTACGAAGAGTAACACCGTCCGGCCCCGG thymidines ("T") in CCCCGACAAACAGCCCAGCACAAGGGAACCGCAC SEQ ID NO: 7 for GTCaCCCAACGCACACAGACACAGCACCCAACAC uridines ("U") provides AGAACACGCACACACACACACACACACACCCACA the RNA sequence.) CGCACGCGCCCCCACCACCGGGGGGCGCCCCCCC CCGGGGGGCGGCCCCCCGGGAGCCCGGGCGGAG CCCCACGGAGATGCCCATCAGTCGATGTCCTCGG CCACCGACCCGCCcAGCCAATCGTCGCAGGACCTC CCCTTGAGTCTAAACCTGCCCCCCACTgTTTCATA CATCAAAGTGCTCCTAGATTTGCTAAAACAAAGT TCCTTAAAGGCGAACCAGTCTGGCAAAA GCGACAGTGGAATCAGCAGAATAGATCTGTCTAT ACATAGTTCCTGGAGGATTACACTTATCTCTGAAC CCAACAAATGTTCACCAGTTCTGAATCGATGCAG GAAGAGGTTCCCAAGGACATCACTAATCTTTTCAT AGCCCTCAAGTCCTGCTAGAAAGACTTTCATGTCC TTGGTCTCCAGCTTCACAATGATATTTTGGACAAG GTTTCTTCCTTCAAAAAGGGCACCCATCTTTACAG TCAGTGGCACAGGCTCCCACTCAGGTCCAACTCTC TCAAAGTCAATAGATCTAATCCCATCCAGTATTCT TTTGGAGCCCAACAACTCAAGCTCAAGAGAATCA CCAAGTATCAAGGGATCTTCCATGTAATCCTCAA ACTCTTCAGATCTGATATCAAAGACACCATCGTTC AAGACAGAGTCTGTCCTCAGTAAGTGGA GGCATTCATCCAACATTCTTCTATCTATCTCACCC TTAAAGAGGTGAGAGCATGATAAAAGTTCAGCCA CACCTGGATTCTGTAATTGGCACCTAACCAAGAA TATCAATGAAAATTTCCTTAAACAGTCAGTATTAT TCTGATTGTGCGTAAAGTCCACTGAAATTGAAAA TACCCCTTTTGTGTAGTTGAGCATGTAGT CCCACAGATCCTTTAAGGATTTAAATGCCTTTGGG TTTGTCAGGCCCTGCCTAATCAACATGGCAGCATT ACACACAACATCTCCCATTCGGTAAGAGAACCAC CCAAAACCAAACTGCAAATCATTCCTAAACATAG GCCTCTCCACATTTTTGTTCACCACCTTTGAGACA AATGATTGAAAGGGGCCCAGTGCCTCAGCACCAT CTTCAGATGGCATCATYKHWTATGAGGGAACCAT GAAAAATTGCCTAATGTCCTGGTTGTTGCAACAA GAACAAATGATTCAAAATACACCTGTTTT AAGAAGTTCTTGCAGACATCCCTCGTGCTAACAA CAAATTCATCAACCAGACTGGAGTCAGATCGCTG ATGAGAATTGGCAAGGTCAGAAAACAGAACAGT GTAATGTTCATCCCTTTTCCACTTAACAACATGAG AAATGAGTGACAAGGATTCTGAGTTAATATCAAT TAAAACACAGAGGTCAAGGAATTTAATTCTGGGA CTCCACCTCATGTTTTTTGAGCTCATGTCAGACAT AAATGGAAGAAGCTGATCCTCAAAGATCTTGGGA TATAGCCGCCTCACAGATTGAATCACTTGGTTCAA ATTCACTTTGTCCTCCAGTAGCCTTGAGCTCTCAG GCTTTCTTGCTACATAATCACATGGGTTTAAGTGC TTAAGAGTTAGGTTCTCACTGTTATTCTTCCCTTTG TCTGCTAGGACCCAAACACCCAACTCAA AAGAGTTGCTCAATGAAATACAAATGTAGTCCCA AAGAAGAGGCCTTAAAAGGCATATATGATCACGG TGGGCTTCTGGATGAGACTGTTTGTCACAAATGTA CAGCGTTATACCATCCCGATTGCAAACTCTTGTCA CATGATCATCTGTGGTTAGATCCTCAAGCAGCTTT TTGATATACAGATTTTCCCTATTTTTGTTTCTCACA CACCTGCTTCCTAGAGTTTTGCAAAGGCCTATAAA GCCAGATGAGATACAACTCTGGAAAGCTGACTTG TTGATTGCTTCTGACAGCAGCTTCTGTGCACCCCT TGTGAATTTACTACAAAGTTTGTTCTGGAGTGTCT TGATCAATGATGGGATTCTTTCCTCTTGGAAAGTC ATCACTGATGGATAAACCACCTTTTGTCTTAAAAC CATCCTTAATGGGAACATTTCATTCAAATTCAACC AGTTAACATCTGCTAACTGATTCAGATCTTCTTCA AGGAGGTCTCCCAATTGAAGAATGGCCT CCtTTTTATCTCTGTTAAATAGGTCTAAGAAAAATT CTTCATTAAATTCACCATTTTTGAGCTTATGATGC AGTTTCCTTACAAGCTTTCTTACAACCTTTGTTTCA TTAGGACACAGTTCCTCAATGAGTCTTTGTATTCT GTAACCTCTAGAACCATCCAGCCAATCTTTCACAT CAGTGTTGGTATTCAGTAGAAATGGATCCAAAGG GAAATTGGCATACTTTAGGAGGTCCAGTGTTCTCC TTTGGATACTATTAACTAGGGAGACTGGGACGCC ATTTGCGATGGCTTGATCTGCAATTGTATCTATTG AAAGTTGATGTGGCTCTTTACACTTGACA TTGTGTAGCGCTGCAGATACAAACTTTGTGAGAA GAGGGACTTCCTCCCCCCATACATAGAATCTAGA TTTAAATTCTGCAGCGAACCTCCCAGCCACACTTT TTGGGCTGATAAATTTGTTTAACAAGCCGCTCAGA TGAGATTGGAATTCCAACAGGACAAGGACTTCCT CACTTACAACCAGGTCACTCAGCCTCCTA TCAAATAAAGTGATCTGATCATCACTTGATGTGTA AGCCTCTGGTCTTTCGCCAAAGATAACACCAATG TTGATGAACCTCTCGCTAAGCAAACCAT AGAAGTCAGAAGCATTATGCAAGATTCCCTGCCC CATATCAATAAGGCTGGATATATGGGATGGCACT ATCCCCATTTCAAAATATTGTCTGAAAATTCTCTC AGTAACAGTTGTTTCTGAACCCCTGAGAAGTTTTA GCTTCGACTTGACATATGATTTCATCATTGCATTC ACAACAGGAAAGGGGACCTCGACAAGCTTATGCA AAGTTAACAAAGTGCTAACATGATCTTTC CCGGAACGCACATACTGGTCATCACCTAGTTTGA GATTTTGTAGAAACATTAAGAACAAAAATGGGCA CATCATTGGTCCCCATTTGCTGTGATCCATACTAT AGTTTAAGAACCCTTCCCGCACATTGATAGTCATT GACAAGATTGCATTTTCAAATTCCTTATCATTGTT TAAACAGGAGCCTGAAAAGAAACTTGAAAAAGA CTCAAAATAATCTTCTATTAACCTTGTGAACATTT TTGTCCTCAAATCTCCAATATAGAGTTCTCTATTT CCCCCAACCTGCTCTTTATAAGATAGTGCAAATTT CAGCCTTCCAGAGTCAGGACCTACTGAGGTGTAT GATGTTGGTGATTCTTCTGAGTAGAAGCACAGATT TTTCAAAGCAGCACTCATACAITgTGTCAACGACA GAGCTTTACTAAGGGACTCAGAATTACTTTCCCTC TCACTGATTCTCACGTCTTCTTCCAGTTTGTCCCA GTCAAATTTGAAATTCAAGCCTTGCCTTTGCATAT GCCTGTATTTCCCTGAGTACGCATTTGCATTCATT TGCAACAGAATCATCTTCATGCAAGAAAACCAAT CATTCTCAGAAAAGAACTTTCTACAAAGGTTTTTT TCATCGAGGCCACACTGATCTTTAATGAC TGAGGTGAAATACAAAGGTGACAGCTCTGTGGAA CCCTCAACAGCCTCACAGATAAATTTCATGTCATC ATTGGTTAGACATGATGGGTCAAAGTCTTCTACTA AATGGAAAGATATTTCTGACAAGATAACTTTTCTT AAGTGAGCCATCTTCCCTGTTAGAATAAGCTGTA AATGATGTAGTCCTTTTGTATTTGTAAGTTTTTCTC CTTTGTCATTGGCCCTCCTACCTCTTCTGT ACCGTGCTATTGTGGTGTTGACCTTTTCTTCGAGA CTTTTGAAGAAGCTTGTCTCTTCTTCTCCATCAAA ACATATTTCTGCCAGGTTGTCTTCCGATCTCCCTG TCTCTTCTCCCTTGGAACCGATGACCAATCTAGAG ACTAACTTGGAAACTTTATATTCATAGTCTGAGTG GCTCAACTTATACTTTTGTTTTCTTACGAAACTCTC CGTAATTTGACTCACAGCACTAACAAGCAATTTGT TAAAGTCATATTCCAGAAGTCGTTCTCCATTTAGA TGCTTATTAACCACCACACTTTTGTTACTAGCAAG ATCTAATGCTGTCGCACATCCAGAGTTAGTCATGG GATCTAGGCTGTTTAGCTTCTTCTCTCCTTTGAAA ATTAAAGTGCCGTTGTTAAATGAAGACACCATTA GGCTAAAGGCTTCCAGATTAACACCTGGAGTTGT ATGCTGACAGTCAATTTCTTTACTAGTGAATCTCT TCATTTGCTCATAGAACACACATTCTTCCTCAGGA GTGATTGCTTCCTTGGGGTTGACAAAAAAACCAA ATTGACTTTTGGGCTCAAAGAACTTTTCAAAACAT TTTATCTGATCTGTTAGCCTGTCAGGGGTCTCCTT TGTGATCAAATGACACAGGTATGACACATTCAAC ATAAATTTAAATTTTGCACTCAACAACACCTTCTC ACCAGTACCAAAAATAGTTTTTATTAGGAATCTA AGCAGCTTATACACCACCTTCTCAGCAGGTGTGAT CAGATCCTCCCTCAACTTATCCATTAATGATGTAG ATGAAAAATCTGACACTATTGCCATCACCAAATA TCTGACACTCTGTACCTGCTTTTGATTTCTCTTTGT TGGGTTGGTGAGCATTAGCAACAATAGGGTCCTC AGTGCAACCTCAATGTCGGTGAGACAGTCTTTCA GACATGATCTAATCCATGAAATCATGAT GTCTATCATATTGTATAAGACCTCATCTGAAAAAA TTGGTAAAAAGAACCTTTTAGGATCTGCATAGAA GGAAATTAAATGACCATCCGGGCCTTGTATGGAG TAGCACCTTGAAGATTCTCCAGTCTTCTGGTATAA GTATTCTTCAGAGTCCAGTTTTATTACTT GGCAAAACACTTCTTTGCATTCTACCACTTGATAT CTCACAGACCCTATTTGATTTTGCCTTAGTCTAGC AACTGAGCTAGTTTTCATACTGTTTGTTAAGGCCA GACAAACAGATGATAATCTTCTCAGGCTCTGTAT GTTCTTCAGCTGCTCTGTGCTGGGTTGGAAATTGT AATCTTCAAACTTCGTATAATACATTATCGGGTGA GCTCCAATTTTCATAAAGTTCTCAAATTCAGTGAA TGGTATGTGGCATTCTTGCTCAAGGTGTTCAGACA GTCCGTAATGCTCGAAACTCAGTCCCACCACTAA CAGGCATTTTTGAATTTTTGCAATGAACTCACTAA TAGAtGCCCTAAACAATTCCTCAAAAGACACCTTT CTAAACACCTTTGACTTTTTTCTATTCCTCAAAAG TCTAATGAACTCCTCTTTAGTGCTGTGAAAGCTTA CCAGCCTATCATTCACACTACTATAGCAACAACCC ACCCAGTGTTTATCATTTTTTAACCCTTTGAATTTC GACTGTTTTATCAATGAGGAAAGACACAAAACAT CCAGATTTAACAACTGTCTCCTTCTAGTATTCAAC AGTTTCAAACTCTTGACTTTGTTTAACATAGAGAG GAGCCTCTCATATTCAGTGCTAGTCTCACTTCCCC GCCCATGGGTCTCTGCAGTTATGAATCTC ATCAAAGGACAGGATTCGACTGCCTCCCTGCTTA ATGTTAAGATATCATCACTATCAGCAAGGTTTTCA TAGAGCTCAGAGAATTCCTTGATCAAGCCTTCAG GGTTTACTTTCTGAAAGTTTCTCTTTAATTTCCCAC TTTCTAAATCTCTTCTAAACCTGCTGAAAAGAGAG TTTATTCCAAAAACCACATCATCACAGCTCATGTT GGGGTTGATGCCTTCGTGGCACATCCTCATAATTT CATCATTGTGAGTTGACCTCGCATCTTTCAGAATT TTCATAGAGTCCATACCGGAGCGCTTGTCGATAGT AGTCTTCAGGGACTCACAGAGTCTAAAATATTCA GACTCTTCAAAGACTTTCTCATTTTGGTTAGAATA CTCCAAAAGTTTGAATAAAAGGTCTCTAAATTTG GCCCACTCTGGCATAAAACTATTATCATA ATCACAACGACCATCTACTATTGGAACTAATGTG ACACCCGCAACAGCAAGGTCTTCCCTGATGCATG CCAATTTGTTAGTGTCCTCTATAAATTTCTTCTCA AAACTGGCTGGaGtGCTCCTAACAAAACACTCAAG AAGAATGAGAGAATTGTCTATCAGCTTGTAACCA TCAGGAATGATAAGTGGTAGTCCTGGGCATACAA TTCCAGACTCCACCAAAATTGTTTCCACAGACTTA TGGTTGTGTGTGCAGCCACTCTTGTCTGC ACTGTCTATTTCAATGCAGCGTGACAGCAACTTGA GTCCCTCAATCAGAACCATTCTGGGTTCCCTTTGT CCCAGAAAGTTGAGTTTCTGCCTTGACAACCTCTC ATCCTGTTCTATATAGTTTAAACATAACTCTCTCA ATTCTGAGATGATTTCATCCATTGCGCATCAAAAA GCCTAGGATCCTCGGTGCG amino acid sequence of VWLSVIWM an HBV HBs protein- derived epitope 9 amino acid ce of IPQSLDSWWTSL an HBV HBs protein- derived epitope amino acid sequence of MGLKFRQL an HBV HBc protein— derived epitope ll lymphocytic CGCACCGGGGATCCTAGGCTTTTTGGATTGCGCTTTCCTC choriomeningitis Virus TAGATCAACTGGGTGTCAGGCCCTATCCTACAGAAGGATG segment S, complete GGTCAGATTGTGACAATGTTTGAGGCTCTGCCTCACATCA sequence TCGATGAGGTGATCAACATTGTCATTATTGTGCTTATCGT (The genomic segment GATCACGGGTATCAAGGCTGTCTACAATTTTGCCACCTGT is RNA, the sequence in GGGATATTCGCATTGATCAGTTTCCTACTTCTGGCTGGCA SEQ ID NO: ll is GGTCCTGTGGCATGTACGGTCTTAAGGGACCCGACATTTA shown for DNA; AGTTTACCAATTTAAGTCAGTGGAGTTTGATATG however, ging all TCACATCTGAACCTGACCATGCCCAACGCATGTTCAGCCA thymidines ("T") in ACAACTCCCACCATTACATCAGTATGGGGACTTCTGGACT SEQ ID NOzll for AGAATTGACCTTCACCAATGATTCCATCATCAGTCACAAC uridines ("U") provides TTTTGCAATCTGACCTCTGCCTTCAACAAAAAGACCTTTG the RNA sequence.) ACCACACACTCATGAGTATAGTTTCGAGCCTACACCTCAG TATCAGAGGGAACTCCAACTATAAGGCAGTATCCTGCGAC TTCAACAATGGCATAACCATCCAATACAACTTGACATTCT CAGATCGACAAAGTGCTCAGAGCCAGTGTAGAACCTTCAG AGGTAGAGTCCTAGATATGTTTAGAACTGCCTTCGGGGGG AAATACATGAGGAGTGGCTGGGGCTGGACAGGCTCAGATG GCAAGACCACCTGGTGTAGCCAGACGAGTTACCAATACCT GATTATACAAAATAGAACCTGGGAAAACCACTGCACATAT GCAGGTCCTTTTGGGATGTCCAGGATTCTCCTTTCCCAAG AGAAGACTAAGTTCTTCACTAGGAGACTAGCGGGCACATT CACCTGGACTTTGTCAGACTCTTCAGGGGTGGAGAATCCA GGTGGTTATTGCCTGACCAAATGGATGATTCTTGCTGCAG AGCTTAAGTGTTTCGGGAACACAGCAGTTGCGAAATGCAA TCATGATGCCGAATTCTGTGACATGCTGCGACTA ATTGACTACAACAAGGCTGCTTTGAGTAAGTTCAAAGAGG ACGTAGAATCTGCCTTGCACTTATTCAAAACAACAGTGAA TTCTTTGATTTCAGATCAACTACTGATGAGGAACCACTTG AGAGATCTGATGGGGGTGCCATATTGCAATTACTCAAAGT TTTGGTACCTAGAACATGCAAAGACCGGCGAAACTAGTGT CCCCAAGTGCTGGCTTGTCACCAATGGTTCTTACTTAAAT GAGACCCACTTCAGTGATCAAATCGAACAGGAAGCCGATA ACATGATTACAGAGATGTTGAGGAAGGATTACATAAAGAG GAGTACCCCCCTAGCATTGATGGACCTTCTGATG TTTTCCACATCTGCATATCTAGTCAGCATCTTCCTGCACC TTGTCAAAATACCAACACACAGGCACATAAAAGGTGGCTC ATGTCCAAAGCCACACCGATTAACCAACAAAGGAATTTGT AGTTGTGGTGCATTTAAGGTGCCTGGTGTAAAAACCGTCT GGAAAAGACGCTGAAGAACAGCGCCTCCCTGACTCTCCAC CTCGAAAGAGGTGGAGAGTCAGGGAGGCCCAGAGGGTCTT AGAGTGTCACAACATTTGGGCCTCTAAAAATTAGGTCATG TGGCAGAATGTTGTGAACAGTTTTCAGATCTGGGAGCCTT GCTTTGGAGGCGCTTTCAAAAATGATGCAGTCCATGAGTG CACAGTGCGGGGTGATCTCTTTCTTCTTTTTGTCCCTTAC TATTCCAGTATGCATCTTACACAACCAGCCATATTTGTCC CACACTTTGTCTTCATACTCCCTCGAAGCTTCCCTGGTCA TTTCAACATCGATAAGCTTAATGTCCTTCCTATTCTGTGA GTCCAGAAGCTTTCTGATGTCATCGGAGCCTTGACAGCTT AGAACCATCCCCTGCGGAAGAGCACCTATAACTGACGAGG TCAACCCGGGTTGCGCATTGAAGAGGTCGGCAAGATCCAT GCCGTGTGAGTACTTGGAATCTTGCTTGAATTGTTTTTGA TCAACGGGTTCCCTGTAAAAGTGTATGAACTGCCCGTTCT GTGGTTGGAAAATTGCTATTTCCACTGGATCATTAAATCT ACCCTCAATGTCAATCCATGTAGGAGCGTTGGGGTCAATT CCTCCCATGAGGTCTTTTAAAAGCATTGTCTGGCTGTAGC TTAAGCCCACCTGAGGTGGACCTGCTGCTCCAGGCGCTGG CCTGGGTGAATTGACTGCAGGTTTCTCGCTTGTGAGATCA ATTGTTGTGTTTTCCCATGCTCTCCCCACAATCGATGTTC TACAAGCTATGTATGGCCATCCTTCACCTGAAAGGCAAAC TTTATAGAGGATGTTTTCATAAGGGTTCCTGTCCCCAACT TGGTCTGAAACAAACATGTTGAGTTTTCTCTTGGCCCCGA GAACTGCCTTCAAGAGGTCCTCGCTGTTGCTTGGCTTGAT CAAAATTGACTCTAACATGTTACCCCCATCCAACAGGGCT GCCCCTGCCTTCACGGCAGCACCAAGACTAAAGTTATAGC CAGAAATGTTGATGCTGGACTGCTGTTCAGTGATGACCCC CAGAACTGGGTGCTTGTCTTTCAGCCTTTCAAGATCATTA AGATTTGGATACTTGACTGTGTAAAGCAAGCCAAGGTCTG TGAGCGCTTGTACAACGTCATTGAGCGGAGTCTGTGACTG TTTGGCCATACAAGCCATAGTTAGACTTGGCATTGTGCCA AATTGATTGTTCAAAAGTGATGAGTCTTTCACATCCCAAA CTCTTACCACACCACTTGCACCCTGCTGAGGCTTTCTCAT TATCTGTAGGATCTGAGATCTTTGGTCTAGTTGC GTTAAGTTCCCCATATATACCCCTGAAGCCTGGG GCCTTTCAGACCTCATGATCTTGGCCTTCAGCTTCTCAAG GTCAGCCGCAAGAGACATCAGTTCTTCTGCACTGAGCCTC CCCACTTTCAAAACATTCTTCTTTGATGTTGACTTTAAAT CCACAAGAGAATGTACAGTCTGGTTGAGACTTCTGAGTCT CTGTAGGTCTTTGTCATCTCTCTTTTCCTTCCTCATGATC CTCTGAACATTGCTGACCTCAGAGAAGTCCAACCCATTCA GAAGGTTGGTTGCATCCTTAATGACAGCAGCCTTCACATC TGATGTGAAGCTCTGCAATTCTCTTCTCAATGCTTGCGTC CATTGGAAGCTCTTAACTTCCTTAGACAAGGACATCTTGT TGCTCAATGGTTTCTCAAGACAAATGCGCAATCAAATGCC TAGGATCCACTGTGCG 12 lymphocytic GCGCACCGGGGATCCTAGGCTTTTTGGATTGCGCTTTCCT choriomeningitis Virus CTAGATCAACTGGGTGTCAGGCCCTATCCTACAGAAGGAT clone 13 segment S, GATTGTGACAATGTTTGAGGCTCTGCCTCACATC complete sequence ATCGATGAGGTGATCAACATTGTCATTATTGTGCTTATCG (GenBank: TGATCACGGGTATCAAGGCTGTCTACAATTTTGCCACCTG DQ36 l 065 .2) TGGGATATTCGCATTGATCAGTTTCCTACTTCTGGCTGGC (The genomic segment AGGTCCTGTGGCATGTACGGTCTTAAGGGACCCGACATTT is RNA, the sequence in ACAAAGGAGTTTACCAATTTAAGTCAGTGGAGTTTGATAT SEQ ID NO: 12 is GTCACATCTGAACCTGACCATGCCCAACGCATGTTCAGCC shown for DNA; AACAACTCCCACCATTACATCAGTATGGGGACTTCTGGAC however, exchanging all TAGAATTGACCTTCACCAATGATTCCATCATCAGTCACAA thymidines ("T") in CTTTTGCAATCTGACCTCTGCCTTCAACAAAAAGACCTTT SEQ ID NO: 12 for ACACTCATGAGTATAGTTTCGAGCCTACACCTCA uridines ("U") es GTATCAGAGGGAACTCCAACTATAAGGCAGTATCCTGCGA the RNA ce.) CTTCAACAATGGCATAACCATCCAATACAACTTGACATTC TCAGATGCACAAAGTGCTCAGAGCCAGTGTAGAACCTTCA GAGGTAGAGTCCTAGATATGTTTAGAACTGCCTTCGGGGG GAAATACATGAGGAGTGGCTGGGGCTGGACAGGCTCAGAT GGCAAGACCACCTGGTGTAGCCAGACGAGTTACCAATACC TGATTATACAAAATAGAACCTGGGAAAACCACTGCACATA TGCAGGTCCTTTTGGGATGTCCAGGATTCTCCTTTCCCAA GAGAAGACTAAGTTCCTCACTAGGAGACTAGCGGGCACAT TCACCTGGACTTTGTCAGACTCTTCAGGGGTGGAGAATCC AGGTGGTTATTGCCTGACCAAATGGATGATTCTTGCTGCA GAGCTTAAGTGTTTCGGGAACACAGCAGTTGCGAAATGCA ATGTAAATCATGATGAAGAATTCTGTGACATGCTGCGACT AATTGACTACAACAAGGCTGCTTTGAGTAAGTTCAAAGAG GACGTAGAATCTGCCTTGCACTTATTCAAAACAACAGTGA ATTCTTTGATTTCAGATCAACTACTGATGAGGAACCACTT GAGAGATCTGATGGGGGTGCCATATTGCAATTACTCAAAG TTTTGGTACCTAGAACATGCAAAGACCGGCGAAACTAGTG TCCCCAAGTGCTGGCTTGTCACCAATGGTTCTTACTTAAA TGAGACCCACTTCAGTGACCAAATCGAACAGGAAGCCGAT AACATGATTACAGAGATGTTGAGGAAGGATTACATAAAGA GGCAGGGGAGTACCCCCCTAGCATTGATGGACCTTCTGAT CACATCTGCATATCTAGTCAGCATCTTCCTGCAC CTTGTCAAAATACCAACACACAGGCACATAAAAGGTGGCT CATGTCCAAAGCCACACCGATTAACCAACAAAGGAATTTG TAGTTGTGGTGCATTTAAGGTGCCTGGTGTAAAAACCGTC TGGAAAAGACGCTGAAGAACAGCGCCTCCCTGACTCTCCA CCTCGAAAGAGGTGGAGAGTCAGGGAGGCCCAGAGGGTCT TAGAGTGTCACAACATTTGGGCCTCTAAAAATTAGGTCAT GTGGCAGAATGTTGTGAACAGTTTTCAGATCTGGGAGCCT TGCTTTGGAGGCGCTTTCAAAAATGATGCAGTCCATGAGT GCACAGTGCGGGGTGATCTCTTTCTTCTTTTTGTCCCTTA CTATTCCAGTATGCATCTTACACAACCAGCCATATTTGTC CCACACTTTGTCTTCATACTCCCTCGAAGCTTCCCTGGTC ATTTCAACATCGATAAGCTTAATGTCCTTCCTATTCTGTG AGTCCAGAAGCTTTCTGATGTCATCGGAGCCTTGACAGCT TAGAACCATCCCCTGCGGAAGAGCACCTATAACTGACGAG GTCAACCCGGGTTGCGCATTGAAGAGGTCGGCAAGATCCA TGCCGTGTGAGTACTTGGAATCTTGCTTGAATTGTTTTTG ATCAACGGGTTCCCTGTAAAAGTGTATGAACTGCCCGTTC TGTGGTTGGAAAATTGCTATTTCCACTGGATCATTAAATC TACCCTCAATGTCAATCCATGTAGGAGCGTTGGGGTCAAT TCCTCCCATGAGGTCTTTTAAAAGCATTGTCTGGCTGTAG CCCACCTGAGGTGGACCTGCTGCTCCAGGCGCTG GCCTGGGTGAATTGACTGCAGGTTTCTCGCTTGTGAGATC TGTGTTTTCCCATGCTCTCCCCACAATCGATGTT CTACAAGCTATGTATGGCCATCCTTCACCTGAAAGGCAAA CTTTATAGAGGATGTTTTCATAAGGGTTCCTGTCCCCAAC TTGGTCTGAAACAAACATGTTGAGTTTTCTCTTGGCCCCG AGAACTGCCTTCAAGAGGTCCTCGCTGTTGCTTGGCTTGA TCAAAATTGACTCTAACATGTTACCCCCATCCAACAGGGC TGCCCCTGCCTTCACGGCAGCACCAAGACTAAAGTTATAG CCAGAAATGTTGATGCTGGACTGCTGTTCAGTGATGACCC CCAGAACTGGGTGCTTGTCTTTCAGCCTTTCAAGATCATT AAGATTTGGATACTTGACTGTGTAAAGCAAGCCAAGGTCT GTGAGCGCTTGTACAACGTCATTGAGCGGAGTCTGTGACT GTTTGGCCATACAAGCCATAGTTAGACTTGGCATTGTGCC AAATTGATTGTTCAAAAGTGATGAGTCTTTCACATCCCAA ACTCTTACCACACCACTTGCACCCTGCTGAGGCTTTCTCA CTATCTGTAGGATCTGAGATCTTTGGTCTAGTTG CTGTGTTGTTAAGTTCCCCATATATACCCCTGAAGCCTGG GGCCTTTCAGACCTCATGATCTTGGCCTTCAGCTTCTCAA GGTCAGCCGCAAGAGACATCAGTTCTTCTGCACTGAGCCT CCCCACTTTCAAAACATTCTTCTTTGATGTTGACTTTAAA TCCACAAGAGAATGTACAGTCTGGTTGAGACTTCTGAGTC TCTGTAGGTCTTTGTCATCTCTCTTTTCCTTCCTCATGAT CCTCTGAACATTGCTGACCTCAGAGAAGTCCAACCCATTC TTGGTTGCATCCTTAATGACAGCAGCCTTCACAT CTGATGTGAAGCTCTGCAATTCTCTTCTCAATGCTTGCGT CCATTGGAAGCTCTTAACTTCCTTAGACAAGGACATCTTG TTGCTCAATGGTTTCTCAAGACAAATGCGCAATCAAATGC CTAGGATCCACTGTGCG 13 lymphocytic GCGCACCGGGGATCCTAGGCATTTTTGTTGCGCATTTTGT choriomeningitis strain ATTTGTTGCACAGCCCTTCATCGTGGGACCTTCA MP segment L, CAAACAAACCAAACCACCAGCCATGGGCCAAGGCAAGTCC complete sequence AAAGAGGGAAGGGATGCCAGCAATACGAGCAGAGCTGAAA (The c segment TTCTGCCAGACACCACCTATCTCGGACCTCTGAACTGCAA is RNA, the sequence in GTCATGCTGGCAGAGATTTGACAGTTTAGTCAGATGCCAT SEQ ID NO:13 is GACCACTATCTCTGCAGACACTGCCTGAACCTCCTGCTGT shown for DNA; CAGTCTCCGACAGGTGCCCTCTCTGCAAACATCCATTGCC r, exchanging all AACCAAACTGAAAATATCCACGGCCCCAAGCTCTCCACCC thymidines ("T") in CCTTACGAGGAGTGACGCCCCGAGCCCCAACACCGACACA SEQ ID NO:13 for AGGAGGCCACCAACACAACGCCCAACACGGAACACACACA uridines ("U") provides CACACACCCACACACACATCCACACACACGCGCCCCCACA the RNA ce.) ACGGGGGCGCCCCCCCGGGGGTGGCCCCCCGGGTGCTCGG GCGGAGCCCCACGGAGAGGCCAATTAGTCGATCTCCTCGA CCACCGACTTGGTCAGCCAGTCATCACAGGACTTGCCCTT AAGTCTGTACTTGCCCACAACTGTTTCATACATCACCGTG TTCTTTGACTTACTGAAACATAGCCTACAGTCTTTGAAAG TGAACCAGTCAGGCACAAGTGACAGCGGTACCAGTAGAAT GGATCTATCTATACACAACTCTTGGAGAATTGTGCTAATT TCCGACCCCTGTAGATGCTCACCAGTTCTGAATCGATGTA GAAGAAGGCTCCCAAGGACGTCATCAAAATTTCCATAACC CTCGAGCTCTGCCAAGAAAACTCTCATATCCTTGGTCTCC AGTTTCACAACGATGTTCTGAACAAGGCTTCTTCCCTCAA AAAGAGCACCCATTCTCACAGTCAAGGGCACAGGCTCCCA TTCAGGCCCAATCCTCTCAAAATCAAGGGATCTGATCCCG TCCAGTATTTTCCTTGAGCCTATCAGCTCAAGCTCAAGAG AGTCACCGAGTATCAGGGGGTCCTCCATATAGTCCTCAAA CTCTTCAGACCTAATGTCAAAAACACCATCGTTCACCTTG AAGATAGAGTCTGATCTCAACAGGTGGAGGCATTCGTCCA AGAACCTTCTGTCCACCTCACCTTTAAAGAGGTGAGAGCA TGATAGGAACTCAGCTACACCTGGACCTTGTAACTGGCAC AAAAAGATCAATGAAAACTTCCTCAAACAATCAG TGTTATTCTGGTTGTGAGTGAAATCTACTGTAATTGAGAA CTCTAGCACTCCCTCTGTATTATTTATCATGTAATCCCAC AAGTTTCTCAAAGACTTGAATGCCTTTGGATTTGTCAAGC CTTGTTTGATTAGCATGGCAGCATTGCACACAATATCTCC CAATCGGTAAGAGAACCATCCAAATCCAAATTGCAAGTCA TTCCTAAACATGGGCCTCTCCATATTTTTGTTCACTACTT TTAAGATGAATGATTGGAAAGGCCCCAATGCTTCAGCGCC ATCTTCAGATGGCATCATGTCTTTATGAGGGAACCATGAA AAACTTCCTAGAGTTCTGCTTGTTGCTACAAATTCTCGTA CAAATGACTCAAAATACACTTGTTTTAAAAAGTTTTTGCA CCTTGTACTAACGACAAATTCATCAACAAGGCTT GAGTCAGAGCGCTGATGGGAATTTACAAGATCAGAAAATA GAACAGTGTAGTGTTCGTCCCTCTTCCACTTAACTACATG AGAAATGAGCGATAAAGATTCTGAATTGATATCGATCAAT ACGCAAAGGTCAAGGAATTTGATTCTGGGACTCCATCTCA TGTTTTTTGAGCTCATATCAGACATGAAGGGAAGCAGCTG ATCTTCATAGATTTTAGGGTACAATCGCCTCACAGATTGG ATTACATGGTTTAAACTTATCTTGTCCTCCAGTAGCCTTG AACTCTCAGGCTTCCTTGCTACATAATCACATGGGTTCAA GTGCTTGAGGCTTGAGCTTCCCTCATTCTTCCCTTTCACA GGTTCAGCTAAGACCCAAACACCCAACTCAAAGGAATTAC TCAGTGAGATGCAAATATAGTCCCAAAGGAGGGGCCTCAA GAGACTGATGTGGTCGCAGTGAGCTTCTGGATGACTTTGC CTGTCACAAATGTACAACATTATGCCATCATGTCTGTGGA TTGCTGTCACATGCGCATCCATAGCTAGATCCTCAAGCAC TTTTCTAATGTATAGATTGTCCCTATTTTTATTTCTCACA CTTCCCAAAGTTTTGCAAAGACCTATAAAGCCTG ATGAGATGCAACTTTGAAAGGCTGACTTATTGATTGCTTC TGACAGCAACTTCTGTGCACCTCTTGTGAACTTACTGCAG AGCTTGTTCTGGAGTGTCTTGATTAATGATGGGATTCTTT CCTCTTGGAAAGTCATTACTGATGGATAAACCACTTTCTG CCTCAAGACCATTCTTAATGGGAACAACTCATTCAAATTC AGCCAATTTATGTTTGCCAATTGACTTAGATCCTCTTCGA GGCCAAGGATGTTTCCCAACTGAAGAATGGCTTCCTTTTT ATCCCTATTGAAGAGGTCTAAGAAGAATTCTTCATTGAAC TTCTTGAGCTTATGATGTAGTCTCCTTACAAGCC TTCTCATGACCTTCGTTTCACTAGGACACAATTCTTCAAT AAGCCTTTGGATTCTGTAACCTCTAGAGCCATCCAACCAA TCCTTGACATCAGTATTAGTGTTAAGCAAAAATGGGTCCA AGGGAAAGTTGGCATATTTTAAGAGGTCTAATGTTCTCTT CTGGATGCAGTTTACCAATGAAACTGGAACACCATTTGCA ACAGCTTGATCGGCAATTGTATCTATTGTTTCACAGAGTT GCTCTTTACACTTAACGTTGTGTAATGCTGCTGA CACAAATTTTGTTAAAAGTGGGACCTCTTCCCCCCACACA TAAAATCTGGATTTAAATTCTGCAGCAAATCGCCCCACCA CACTTTTCGGACTGATGAACTTGTTAAGCAAGCCACTCAA ATGAGAATGAAATTCCAGCAATACAAGGACTTCCTCAGGG TCAACCAGTTCACTCAATCTCCTATCAAATAAGG TGATCTGATCATCACTTGATGTGTAAGATTCTGGTCTCTC ACCAAAAATGACACCGATACAATAATTAATGAATCTCTCA CTGATTAAGCCGTAAAAGTCAGAGGCATTATGTAAGATTC CCTGTCCCATGTCAATGAGACTGCTTATATGGGAAGGCAC TATTCCTAATTCAAAATATTCTCGAAAGATTCTTTCAGTC ACAGTTGTCTCTGAACCCCTAAGAAGTTTCAGCTTTGATT TGATATATGATTTCATCATTGCATTCACAACAGGAAAAGG GACCTCAACAAGTTTGTGCATGTGCCAAGTTAATAAGGTG CTGATATGATCCTTTCCGGAACGCACATACTGGTCATCAC CCAGTTTGAGATTTTGAAGGAGCATTAAAAACAAAAATGG GCACATCATTGGCCCCCATTTGCTATGATCCATACTGTAG TTCAACAACCCCTCTCGCACATTGATGGTCATTGATAGAA TTGCATTTTCAAATTCTTTGTCATTGTTTAAGCATGAACC TGAGAAGAAGCTAGAAAAAGACTCAAAATAATCCTCTATC AATCTTGTAAACATTTTTGTTCTCAAATCCCCAATATAAA TGTTTCCTCCAACCTGCTCTTTGTATGATAACGC AAACTTCAACCTTCCGGAATCAGGACCAACTGAAGTGTAT GACGTTGGTGACTCCTCTGAGTAAAAACATAAATTCTTTA AAGCAGCACTCATGCATTTTGTCAATGATAGAGCCTTACT TAGAGACTCAGAATTACTTTCCCTTTCACTAATTCTAACA TCTTCTTCTAGTTTGTCCCAGTCAAACTTGAAATTCAGAC CTTGTCTTTGCATGTGCCTGTATTTCCCTGAGTATGCATT TGCATTCATTTGCAGTAGAATCATTTTCATACACGAAAAC CAATCACCCTCTGAAAAAAACTTCCTGCAGAGGTTTTTTG CATCCAGACCACATTGTTCTTTGACAGCTGAAGT GAAATACAATGGTGACAGTTCTGTAGAAGTTTCAATAGCC TCACAGATAAATTTCATGTCATCATTGGTGAGACAAGATG GGTCAAAATCTTCCACAAGATGAAAAGAAATTTCTGATAA GATGACCTTCCTTAAATATGCCATTTTACCTGACAATATA GTCTGAAGGTGATGCAATCCTTTTGTATTTTCAAACCCCA CCTCATTTTCCCCTTCATTGGTCTTCTTGCTTCTTTCATA TATTGTGGAGTTGACCTTATCTTCTAAATTCTTG AAGAAACTTGTCTCTTCTTCCCCATCAAAGCATATGTCTG CTGAGTCACCTTCTAGTTTCCCAGCTTCTGTTTCTTTAGA GCCGATAACCAATCTAGAGACCAACTTTGAAACCTTGTAC TCGTAATCTGAGTGGTTCAATTTGTACTTCTGCTTTCTCA TGAAGCTCTCTGTGATCTGACTCACAGCACTAACAAGCAA TTTGTTAAAATCATACTCTAGGAGCCGTTCCCCATTTAAA TGTTTGTTAACAACCACACTTTTGTTGCTGGCAAGGTCTA ATGCTGTTGCACACCCAGAGTTAGTCATGGGATCCAAGCT ATTGAGCCTCTTCTCCCCTTTGAAAATCAAAGTGCCATTG TTGAATGAGGACACCATCATGCTAAAGGCCTCCAGATTGA CACCTGGGGTTGTGCGCTGACAGTCAACTTCTTTCCCAGT GAACTTCTTCATTTGGTCATAAAAAACACACTCTTCCTCA GGGGTGATTGACTCTTTAGGGTTAACAAAGAAGCCAAACT CACTTTTAGGCTCAAAGAATTTCTCAAAGCATTTAATTTG ATCTGTCAGCCTATCAGGGGTTTCCTTTGTGATTAAATGA CACAGGTATGACACATTCAACATGAACTTGAACTTTGCGC TCAACAGTACCTTTTCACCAGTCCCAAAAACAGTTTTGAT CAAAAATCTGAGCAATTTGTACACTACTTTCTCAGCAGGT GTGATCAAATCCTCCTTCAACTTGTCCATCAATGATGTGG ATGAGAAGTCTGAGACAATGGCCATCACTAAATACCTAAT GTTTTGAACCTGTTTTTGATTCCTCTTTGTTGGGTTGGTG AGCATGAGTAATAATAGGGTTCTCAATGCAATCTCAACAT CATCAATGCTGTCCTTCAAGTCAGGACATGATCTGATCCA TGAGATCATGGTGTCAATCATGTTGTGCAACACTTCATCT ATTGGTAAAAAGAACCTTTTTGGGTCTGCATAAA AAGAGATTAGATGGCCATTGGGACCTTGTATAGAATAACA CCTTGAGGATTCTCCAGTCTTTTGATACAGCAGGTGATAT TCCTCAGAGTCCAATTTTATCACTTGGCAAAATACCTCTT TACATTCCACCACTTGATACCTTACAGAGCCCAATTGGTT TTGTCTTAATCTAGCAACTGAACTTGTTTTCATACTGTTT GTCAAAGCTAGACAGACAGATGACAATCTTTTCAAACTAT GCATGTTCCTTAATTGTTCCGTATTAGGCTGGAAATCATA ATCTTCAAACTTTGTATAATACATTATAGGATGAGTTCCG GACCTCATGAAATTCTCAAACTCAATAAATGGTATGTGGC ACTCATGCTCAAGATGTTCAGACAGACCATAGTGCCCAAA ACTAAGTCCCACCACTGACAAGCACCTTTGAACTTTTAAA ATGAACTCATTTATGGATGTTCTAAACAAATCCTCAAGAG ATACCTTTCTATACGCCTTTGACTTTCTCCTGTTCCTTAG GATGAACTCTTCCTTGGTGCTATGAAAGCTCACC AACCTATCATTCACACTCCCATAGCAACAACCAACCCAGT GCTTATCATTTTTTGACCCTTTGAGTTTAGACTGTTTGAT CAACGAAGAGAGACACAAGACATCCAAATTCAGTAACTGT CTCCTTCTGGTGTTCAATAATTTTAAACTTTTAACTTTGT TCAACATAGAGAGGAGCCTCTCATACTCAGTGCTAGTCTC ACTTCCTCTCTCATAACCATGGGTATCTGCTGTGATAAAT CTCATCAAAGGACAGGATTCAACTGCCTCCTTGCTTAGTG CTGAAATGTCATCACTGTCAGCAAGAGTCTCATAAAGCTC AGAGAATTCCTTAATTAAATTTCCGGGGTTGATTTTCTGA CTCTTGAGCTTCCCAGTTTCCAAGTCTCTTCTAA ACCTGCTGTAAAGGGAGTTTATGCCAAGAACCACATCATC GCAGTTCATGTTTGGGTTGACACCATCATGGCACATTTTC WO 76988 2016/076591 ATAATTTCATCATTGTGAAATGATCTTGCATCTTTCAAGA TTTTCATAGAGTCTATACCGGAACGCTTATCAACAGTGGT CTTGAGAGATTCGCAAAGTCTGAAGTACTCAGATTCCTCA AAGACTTTCTCATCTTGGCTAGAATACTCTAAAAGTTTAA ACAGAAGGTCTCTGAACTTGAAATTCACCCACTCTGGCAT AAAGCTGTTATCATAATCACACCGACCATCCACTATTGGG ACCAATGTGATACCCGCAATGGCAAGGTCTTCTTTGATAC AGGCTAGTTTATTGGTGTCCTCTATAAATTTCTTCTCAAA ACTAGCTGGTGTGCTTCTAACGAAGCACTCAAGAAGAATG AGGGAATTGTCAATCAGTTTATAACCATCAGGAATGATCA AAGGCAGTCCCGGGCACACAATCCCAGACTCTATTAGAAT TGCCTCAACAGATTTATCATCATGGTTGTGTATGCAGCCG CTCTTGTCAGCACTGTCTATCTCTATACAACGCGACAAAA GTTTGAGTCCCTCTATCAATACCATTCTGGGTTCTCTTTG CCCTAAAAAGTTGAGCTTCTGCCTTGACAACCTCTCATCT TGTTCTATGTGGTTTAAGCACAACTCTCTCAACTCCGAAA TAGCCTCATCCATTGCGCATCAAAAAGCCTAGGATCCTCG GTGCG 14 lymphocytic CGCACCGGGGATCCTAGGCTTTTTGGATTGCGCTTTCCTC choriomeningitis strain AGCTCCGTCTTGTGGGAGAATGGGTCAAATTGTGACGATG MP segment S, TTTGAGGCTCTGCCTCACATCATTGATGAGGTCATTAACA complete sequence TTGTCATTATCGTGCTTATTATCATCACGAGCATCAAAGC (The genomic segment TGTGTACAATTTCGCCACCTGCGGGATACTTGCATTGATC is RNA, the sequence in AGCTTTCTTTTTCTGGCTGGCAGGTCCTGTGGAATGTATG SEQ ID NO:l4 is GTCTTGATGGGCCTGACATTTACAAAGGGGTTTACCGATT shown for DNA; CAAGTCAGTGGAGTTTGACATGTCTTACCTTAACCTGACG however, ging all ATGCCCAATGCATGTTCGGCAAACAACTCCCATCATTATA thymidines ("T") in TAAGTATGGGGACTTCTGGATTGGAGTTAACCTTCACAAA SEQ ID NOzl4 for TGACTCCATCATCACCCACAACTTTTGTAATCTGACTTCC uridines ("U") provides GCCCTCAACAAGAGGACTTTTGACCACACACTTATGAGTA the RNA ce.) TAGTCTCAAGTCTGCACCTCAGCATTAGAGGGGTCCCCAG CTACAAAGCAGTGTCCTGTGATTTTAACAATGGCATCACT TACAACCTGTCATTTTCTAATGCACAGAGCGCTC AATGTAAGACCTTCAGGGGGAGAGTCCTGGATAT AACTGCTTTTGGAGGAAAGTACATGAGGAGTGGC TGGGGCTGGACAGGTTCAGATGGCAAGACTACTTGGTGCA GCCAGACAAACTACCAATATCTGATTATACAAAACAGGAC TTGGGAAAACCACTGCAGGTACGCAGGCCCTTTCGGAATG TCTAGAATTCTCTTCGCTCAAGAAAAGACAAGGTTTCTAA CTAGAAGGCTTGCAGGCACATTCACTTGGACTTTATCAGA CTCATCAGGAGTGGAGAATCCAGGTGGTTACTGCTTGACC AAGTGGATGATCCTCGCTGCAGAGCTCAAGTGTTTTGGGA ACACAGCTGTTGCAAAGTGCAATGTAAATCATGATGAAGA GTTCTGTGATATGCTACGACTGATTGATTACAACAAGGCT GCTTTGAGTAAATTCAAAGAAGATGTAGAATCCGCTCTAC ATCTGTTCAAGACAACAGTGAATTCTTTGATTTCTGATCA GCTTTTGATGAGAAATCACCTAAGAGACTTGATGGGAGTG CCATACTGCAATTACTCGAAATTCTGGTATCTAGAGCATG CAAAGACTGGTGAGACTAGTGTCCCCAAGTGCTGGCTTGT CAGCAATGGTTCTTATTTGAATGAAACCCATTTCAGCGAC CAAATTGAGCAGGAAGCAGATAATATGATCACAGAAATGC TGAGAAAGGACTACATAAAAAGGCAAGGGAGTACCCCTCT AGCCTTGATGGATCTATTGATGTTTTCTACATCAGCATAT TTGATCAGCATCTTTCTGCATCTTGTGAGGATACCAACAC ACAGACACATAAAGGGCGGCTCATGCCCAAAACCACATCG GTTAACCAGCAAGGGAATCTGTAGTTGTGGTGCATTTAAA GTACCAGGTGTGGAAACCACCTGGAAAAGACGCTGAACAG CAGCGCCTCCCTGACTCACCACCTCGAAAGAGGTGGTGAG TCAGGGAGGCCCAGAGGGTCTTAGAGTGTTACGACATTTG GACCTCTGAAGATTAGGTCATGTGGTAGGATATTGTGGAC AGTTTTCAGGTCGGGGAGCCTTGCCTTGGAGGCGCTTTCA AAGATGATACAGTCCATGAGTGCACAGTGTGGGGTGACCT CTTTCTTTTTCTTGTCCCTCACTATTCCAGTGTGCATCTT GCATAGCCAGCCATATTTGTCCCAGACTTTGTCCTCATAT TCTCTTGAAGCTTCTTTAGTCATCTCAACATCGATGAGCT TAATGTCTCTTCTGTTTTGTGAATCTAGGAGTTTCCTGAT GTCATCAGATCCCTGACAACTTAGGACCATTCCCTGTGGA AGAGCACCTATTACTGAAGATGTCAGCCCAGGTTGTGCAT TGAAGAGGTCAGCAAGGTCCATGCCATGTGAGTATTTGGA GTCCTGCTTGAATTGTTTTTGATCAGTGGGTTCTCTATAG AAATGTATGTACTGCCCATTCTGTGGCTGAAATATTGCTA TTTCTACCGGGTCATTAAATCTGCCCTCAATGTCAATCCA AGCGTTAGGGTCAATACCTCCCATGAGGTCCTTC AGCAACATTGTTTGGCTGTAGCTTAAGCCCACCTGAGGTG GGCCCGCTGCCCCAGGCGCTGGTTTGGGTGAGTTGGCCAT AGGCCTCTCATTTGTCAGATCAATTGTTGTGTTCTCCCAT GCTCTCCCTACAACTGATGTTCTACAAGCTATGTATGGCC ACCCCTCCCCTGAAAGACAGACTTTGTAGAGGATGTTCTC GTAAGGATTCCTGTCTCCAACCTGATCAGAAACAAACATG TTGAGTTTCTTCTTGGCCCCAAGAACTGCTTTCAGGAGAT CCTCACTGTTGCTTGGCTTAATTAAGATGGATTCCAACAT GTTACCCCCATCTAACAAGGCTGCCCCTGCTTTCACAGCA GCACCGAGACTGAAATTGTAGCCAGATATGTTGATGCTAG ACTGCTGCTCAGTGATGACTCCCAAGACTGGGTGCTTGTC CCTTTCAAGGTCACTTAGGTTCGGGTACTTGACT GTGTAAAGCAGCCCAAGGTCTGTGAGTGCTTGCACAACGT CATTGAGTGAGGTTTGTGATTGTTTGGCCATACAAGCCAT TGTTAAGCTTGGCATTGTGCCGAATTGATTGTTCAGAAGT GATGAGTCCTTCACATCCCAGACCCTCACCACACCATTTG CACTCTGCTGAGGTCTCCTCATTCCAACCATTTGCAGAAT CTGAGATCTTTGGTCAAGCTGTTGTGCTGTTAAGTTCCCC ATGTAGACTCCAGAAGTTAGAGGCCTTTCAGACCTCATGA TTTTAGCCTTCAGTTTTTCAAGGTCAGCTGCAAGGGACAT CAGTTCTTCTGCACTAAGCCTCCCTACTTTTAGAACATTC TTTTTTGATGTTGACTTTAGGTCCACAAGGGAATACACAG TTTGGTTGAGGCTTCTGAGTCTCTGTAAATCTTTGTCATC CCTCTTCTCTTTCCTCATGATCCTCTGAACATTGCTCACC TCAGAGAAGTCTAATCCATTCAGAAGGCTGGTGGCATCCT TGATCACAGCAGCTTTCACATCTGATGTGAAGCCTTGAAG CTCTCTCCTCAATGCCTGGGTCCATTGAAAGCTTTTAACT TCTTTGGACAGAGACATTTTGTCACTCAGTGGATTTCCAA GTCAAATGCGCAATCAAAATGCCTAGGATCCACTGTGCG mnmoaddsemumceof MSLSKEVKSFQWTQALRRELQGFTSDVKAAVIKDATSLLN theNPpn?dnof?w GLDFSEVSNVQRIMRKEKRDDKDLQ'LRSLNQTVYSLVDL N?’?n?nofLChJV NVLKVGRLSAEELUSLAADLEKLKAKIMRSERPL TSGVYMGNLTAQQLDQRSQ:LQMVGMQRPQQSANGVVRVW DVKDSSLLNNQFGT"?SLT"ACMAKQSQTSLNDVVQALTD LGLLYTVKYPNLSDLERLKJKHPVPGVIT?QQSS N SGY NFSLGAAVKAGAALLDGGNULESILIKPSNSEDLLKAVLG AKKKLNMFVSDQVGDQNPYENILYKVCLSG?GWPY ACRT SVVGRAWENTTiDLTNERBUANSBKPABGAAGPPQVGLSY SQTMLLKJLMGG:DPVA?TWIDIEGRFN3?V£IA EQPQN GQYiHEYREPTJQKQEKQJSKYSHGMDLADLFNAQPGLTS SVIGALPQGMVLSCQGSDD"{KLLDSQNR{D KP DVRMT KEASREYEDKVWDKYGWLCKUHTGIVRDKKKKEVTPHCAL MDC IF?SASKAQLPDL{TV{N"PP{DL"FRGPNVVTL 16 amino acid sequence of MGQ_VTV1FRALPHTTDFVTNTV'TV'.TT'TSTKAVYNFAT the GP protein ofthe CG T.AL Sh'LFLAG'RSCGMYGLDGPDIYKGVYRFKSVEFD MP strain ofLCMV MSYLNLTMPNACSAWSHHYI SMGTSGLELTFTNDS I ITH SALNKRTFDHTLMSIVSSLHLSn DFNNGITIQYNLSTSNAQSALSQCKTFRGRVLDMFQTAFG GKYMRSGWGWTGSDGKTTWCSQTNYQYL NHCK YAGPFGMSRILFAQEKTRFLTRRLAGTFTWTLSDSSGVEW PGGYCLTKWMILAAELKCFGNTAVAKCNVNHDEEFCDMLR LIDYNKAAPSKFK?DVFSAWHLFKTTVNSLISDQLLMRNH LRDLMGVPYCNYS{EWYLj{AKTGETSVPKCWLVSNGSYL DQ'RQFADWMTT?WRRKDY'KRQGSTPLALMDLL MFSTSAYLISIFL{LVRI?THRH:KGGSCPKPHRLTSKGI CSCGAFKVPGVETTWKRR 17 amino acid sequence of MDEAISELRELCLNHLEQDERLSRQKLNFLGQREPKMVL- the L protein of the MP EGLKLLSRCIEIDSADKSGCIHNHDDKSV?ATLT?SGTVC strain of LCMV PGLPLIIPDGYKLLDNSLiLLECEVKSTBASEEKKiiEDT NKPACIKWDLATAGJ. TLVP VDGRCDYDNSFMPEWVNFKF RDLLFKLLEYSSQDEKVFEESEYFRLCESLKTTVDKRSGI DSMKILKDARSFHNDEIMKMCHDGVNPNMNCDDVVLGINS LYSRFRRDLETGKLKKSEQKiNBGNLiKEESjLYETLADS DDISALS LSHLNKVKSLKLLNTRRRQLLNLDVLCLSSLIKQSKLKGS KNDKHWVGCCYGSVNDRLVSEHSTK?RF {PLRNRRKSKA EDLERTSiNEEiLKVQRCLSVVGLSFGHYGLSEH L?€?CH ?ETEF?NEMRSGTHP:MYYTKFEDYDFQPNTEQJ.

LRNUHSLKRLSSVCLALTNSMKTSSVARLRQNQLGSVRYQ VVECKEVFCQVIKLDSEEYHLLYQKTGESSRCYS:QGPNG HL_SEYADPKREFLPiESDEVLHNMIDTMISWIRSCPDLK DS DDV? ALRTPLPLMPTNPTKRNQKQVQN:RYLVMAIV SDFSSTSLMDKLKEDLITPAEKVVYKLLRFLIKTVFGTGE KFKFMLNVSYLCHL TK?T?DRPTDQIKCFEKFF EPKSEEGEFVNBKESiTBEEECVFYJQMKKETGKEVJCQR TTPGVNLEAFSMMVSSFWNGTLIFKGEKRLNSLDP TNSG CATALDLASNKSVVVNKHLNGERLLEYDFNKLLVSAVSQI TESFMRKQKYKLNHSDYEYKVSKLVSRLV"GSKFT?AGKL EGDSADICFDGEEETSFFKNLEDKVNSTIKRYERSKKTNE G?N?VGE?NTKGPHHLQTILSGKMAYLRKV ESE SEHLV EDFDPSCTTNDUMKFTCFATRTSTFLSPPYFTSAVKEQCG LIL-lJ AKNLCRKEESjGDWESCMKM:LLQMNANAYSGKYRH MQRQGLNFKTDWDKP?FDVRTSFR?SNSFSLSKAPSWTKC MSAALKNPCEYS??SPTSYTSVGPDSGRLKFALSYKEQVG GWRELYIGDLRTKWFTRLIEDYFESFSSFFSGSCLNWDKE EjNAiLSMTiNVRjGLLNYSMDHS{WGPMMCPFLFLHLLQ WLKLGDDQYVRSG KSYIKSKLKLLRGSiTUVT?XIER?YEFLG VPS? SSLi DWGQGILiNASDFYGT’S?<5INYCTGVTFG?RP?SYTSS DDQITLFDRRLSELVDSD?ijLVLLEFHSHLSGLLNKFI G?FAAEFKSQFYVWGEEVPLLTKFVSAAL{NVKC KEPHQLCETIDTIADQAVANGVPVSLVNC:QKRTLDLLKY AWFPLDPFLLNTNTDVKDWLDGSRGYRIQQLIEELCPSET KVMRxLVKRLHHKLKNGEENjEEELDLFNKDKKEAiLQLG N"PGTFFDPSQPANTNWVNPWEPFPVRMVPQQKVVYPSVM IPSLIKTLQNKLCSKFTRGAQKLLSEAINKSAFQ SC SSGE GPCKTPGSRCVRNKNRDNWY—R HVTAIHRHDGIMLYICDRQSHPEAACDH:SLLRPLLWDYI C"SPSNSF VGVWVPAFPVKGKNEGSSSLK€LNPCDYVAR:‘J LLZDKISLNHVIQSVRRLYPKIYEDQLLPFMSDM.L SSKNMRWS??IKFPDLCVP DINS?SLSP SHVVKWKRDE HYTVLFSDLVNSHQRSDSSLVDEFVVSTRDVCKNFLKQVY F1SEVREEVATSRTLGSESWFPHKJMMPSEDGAEALGPFQ SFILKVVNKNMERPMFRNDLQFGEGWESYRLGD_VCNAAM LIKQGLTN?KAFKSPRNPWDYMTNNT?GVP?FS’TVDFTH NQNNTDCLKKFSLLELVKCQLQGPGVAEFLSCSHLFKGEV DRRFLDECLHLLRSDSIFKVNDGVEDIRS??EEDYMjDPL ILGDSLELELIGSRKiLDGiRSLDEiKiGPjWEBVPLTVR MGAPFFGRSPVQN VVKL?TKDM&VFWA?L?GYGNEJDVL GSLLLHRFRTGEHLQGSEISTILQELCIDRSILLVPLSLV PDWFTFKJCRLCFSKSKNTVMYETVVGKYRLKGKSCDDWL TKSVVEEID 18 amino acid sequence of MGQGKSK1G{DASNTSRA? PPDTTYLGPLNCKSCWQRFD the Z protein of the MP SLVRCHDHYLCRHCLNLLLSVSDRCPLCKHPLPTKLKIST strain of LCMV APSSPPPY?? 19 Junin Virus Candid#1 L GCGCACCGGGGATCCTAGGCGTAACTTCATCATTAAAATCT segment CAGATTCTGCTCTGAGTGTGACTTACTGCGAAGAGGCAGAC AAATGGGCAACTGCAACGGGGCATCCAAGTCTAACCAGCCA GACTCCTCAAGAGCCACACAGCCAGCCGCAGAATTTAGGAG GGTAGCTCACAGCAGTCTATATGGTAGATATAACTGTAAGT GCTGCTGGTTTGCTGATACCAATTTGATAACCTGTAATGAT CACTACCTTTGTTTAAGGTGCCATCAGGGTATGTTAAGGAA TCTCTGCAATATCTGCTGGAAGCCCCT GCCCACCACAATCACAGTACCGGTGGAGCCAACAGCACCAC CACCATAGGCAGACTGCACAGGGTCAGACCCGACCCCCCGG GGGGCCCCCATGGGGACCCCCCGTGGGGGAACCCCGGGGGT GATGCGCCATTAGTCAATGTCTTTGATCTCGACTTTGTGCT TCAGTGGCCTGCATGTCACCCCTTTCAATCTGAACTGCCCT TGGGGATCTGATATCAGCAGGTCATTTAAAGATCT GCTGAATGCCACCTTGAAATTTGAGAATTCCAACCAGTCAC CAAATTTATCAAGTGAACGGATCAACTGCTCTTTGTGTA AAACGAGGACAAAGTCCTCTTGCTGAAATAATATT GTTTGTGATGTTGTTTTTAGATAAGGCCATAGTTGGCTT AATAAGGTTTCCACACTATCAATGTCCTCTAGTGCTCCAAT TGCCTTGACTATGACATCCCCAGACAACTCAACTCTATA TGTTGACAACCTTTCATTACCTCTGTAAAAGATACCCTCTT TCAAGACAAGAGGTTCTCCTGGGTTATCTGGCCCAATGA GGTCATATGCATACTTGTTACTTAGTTCAGAATAAAAGTCA CCAAAGTTGAACTTAACATGGCTCAGAATATTGTCATCA TTTGTCGCAGCGTAGCCTGCATCAATAAACAAGCCAGCTAG GTCAAAGCTCTCATGGCCTGTGAACAATGGTAGGCTAGC GATAACCAGTGCACCATCCAACAATGAGTGGCTTCCCTCAG ACCCAGAAACACATTGACTCATTGCATCCACATTCAGCT CTAATTCAGGGGTACCGACATCATCCACTCCTAGTGAACTG ACAATGGTGTAACTGTACACCATCTTTCTTCTAAGTTTA AATTTTGTCGAAACTCGTGTGTGTTCTACTTGAATGATCAA TTTCACAGCTTCTTGGCAAGCAACATTGCGCAA CACAGTGTGCAGGTCCATCATGTCTTCCTGAGGCAACAAGG AGATGTTGTCAACAGAGACACCCTCAAGGAAAACCTTGA TATTATCAAAGCTAGAAACTACATAACCCATTGCAATGTCT TCAACAAACATTGCTCTTGATACTTTATTATTCCTAACT GTAAAATCTGTGAGTTCAGCTAGATCTACTTGACT GTCATCTTCTAGATCTAGAACTTCATTGAACCAAAAGAA GGATTTGAGACACGATGTTGACATGACTAGTGGGTTTATCA TCGAAGATAAGACAACTTGCACCATGAAGTTCCTGCAAA CTTGCTGTGGGCTGATGCCAACTTCCCAATTTGTATACTCT GACTGTCTAACATGGGCTGAAGCGCAATCACTCTGTTTC ACAATATAAACATTATTATCTCTTACTTTCAATAAGTGACT TATAATCCCTAAGTTTTCATTCATCATGTCTAGAGCCAC ATCTAGAAACTTGAGTCTTCCACTATCCAAAGATC TGTTCACTTGAAGATCATTCATAAAGGGTGCCAAATGTT CTTCAAATAGTTTGGGGTAATTTCTTCGTATAGAATGCAAT ACATGGTTCATGCCTAATTGGTCTTCTATCTGTCGTACT GCTTTGGGTTTAACAGCCCAGAAGAAATTCTTATTACATAA GACCAGAGGGGCCTGTGGACTCTTAATAGCAGAAAACAC CCACTCCCCTAACTCACAGGCATTTGTCAGCACCAAAGAGA AGTAATCCCACAAAATTGGTTTAGAAAATTGGTTAACTT CTTTAAGTGATTTTTGACAGTAAATAACTTTAGGCTTTCTC TCACAAATTCCACAAAGACATGGCATTATTCGAGTAAAT ATGTCCTTTATATACAGAAATCCGCCTTTACCATCCCTAAC ACACTTACTCCCCATACTCTTACAAAACCCAATGAAGCC TGAGGCAACAGAAGACTGAAATGCAGATTTGTTGATTGACT CTGCCAAGATCTTCTTCACGCCTTTTGTGAAATTTCTTG ACAGCCTGGACTGTATTGTCCTTATCAATGTTGGCATCTCT TCTTTCTCTAACACTCTTCGACTTGTCATGAGTTTGGTC CTCAAGACCAACCTCAAGTCCCCAAAGCTCGCTAAATTGAC CCATCTGTAGTCTAGAGTTTGTCTGATTTCATCTTCACT ACACCCGGCATATTGCAGGAATCCGGATAAAGCCTCATCCC CTCCCCTGCTTATCAAGTTGATAAGGTTTTCCTCAAAGA TTTTGCCTCTCTTAATGTCATTGAACACTTTCCTCGCGCAG TTCCTTATAAACATTGTCTCCTTATCATCAGAAAAAATA GCTTCAATTTTCCTCTGTAGACGGTACCCTCTAGACCCATC AACCCAGTCTTTGACATCTTGTTCTTCAATAGCTCCAAA CGGAGTCTCTCTGTATCCAGAGTATCTAATCAATTGGTTGA CTCTAATGGAAATCTTTGACACTATATGAGTGCTAACCC CATTAGCAATACATTGATCACAAATTGTGTCTATGGTCTCT GACAGTTGTGTTGGAGTTTTACACTTAACGTTGTGTAGA GCAGCAGACACAAACTTGGTGAGTAAAGGAGTCTCTTCACC CATGACAAAAAATCTTGACTTAAACTCAGCAACAAAAGTTC CTATCACACTCTTTGGGCTGATAAACTTGTTTAATTTAGAA AATTCATGGAAGCACACCATTTCCAGCAGTT CTGTCCTGTCTTGAAACTTTTCATCACTAAGGCAAGGAATT TTTATAAGGCTAACCTGGTCATCGCTGGAGGTATAAGTG ACAGGTATCACATCATACAATAAGTCAAGTGCATAACACAG AAATTGTTCAGTAATTAGCCCATATAAATCTGATGTGTT GATTCCCTGGCCCATGTCCAAGACAGACATTATAT GGCTGGGGACCTGGTCCCTTGACTGCAGATACTGGTGAA AAAACTCTTCACCAACACTAGTACAGTCACAACCCATTAAA CCTAAAGATCTCTTCAATTTCCCTACACAGTAGGCTTCT GCAACATTAATTGGAACTTCAACGACCTTATGAAGATGCCA AATGTTCATTACTGGTTCAAGATTCACCTTTGT TCTATCTCTGGGATTCTTCAATTCTAATGTGTACAAAAAAG AAAGGAAAAGTGCTGGGCTCATAGTTGGTCCCCATTTGG AGTGGTCATATGAACAGGACAAGTCACCATTGTTAACAGCC ATTTTCATATCACAGATTGCACGTTCGAATTCCTTTTCT GAATTCAAGCATGTGTATTTCATTGAACTACCCACAGCTTC TGAGAAGTCTTCAACTAACCTGGTCATCAGCTTAGTGTT GAGGTCTCCCACATACAGTTCTCTATTTGAGCCAACCTGCT CCTTATAACTTAGTCCAAATTTCAAGTTCCCTGTATTTG AGCTGATGCTTGTGAACTCTGTAGGAGAGTCGTCTGAATAG AAACATAAATTCCGTAGGGCTGCATTTGTAAAATAACTT TTGTCTAGCTTATCAGCAATGGCTTCAGAATTGCTTTCCCT GGTACTAAGCCGAACCTCATCCTTTAGTCTCAGAACTTC ACTGGAAAAGCCCAATCTAGATCTACTTCTATGCTCATAAC TACCCAATTTCTGATCATAATGTCCTTGAATTAAAAGAT AGCATTCAAAGAATTCATCTTCTTGGTAGGCTATT GTTGTCAAATTTTTTAATAACAAACCCAAAGGGCAGATG TCCTGCGGTGCTTCAAGAAAATAAGTCAATTTAAATGGAGA TAGATAAACAGCATCACATAACTCTTTATACACATCAGA CCTGAGCACATCTGGATCAAAATCCTTCACCTCATGCATTG ACACCTCTGCTTTAATCTCTCTCAACACTCCAAAAGGGG CCCACAATGACTCAAGAGACTCTCGCTCATCAACAGATGGA TTTTTTGATTTCAACTTGGTGATCTCAACTTTTGTCCCC TCACTATTAGCCATCTTGGCTAGTGTCATTTGTACGTCATT TCTAATACCCTCAAAGGCCCTTACTTGATCCTCTGTTAA ACTCTCATACATCACTGATAATTCTTCTTGATTGGTTCTGG TTCTTGAACCGGTGCTCACAAGACCTGTTAGATTTTTTA AGTAGTCCATGGAATCAGGATCAAGATTATACCTG GTTTTAAACCTCTCAGCCATAGTAGAAACGCAT GTTGAAACAAGTTTCTCCTTATCATAAACAGAAAGAATATT TCCAAGTTCGTCGAGCTTGGGGATTACCACACTTTTATT GCTTGACAGATCCAGAGCTGTGCTAGTGATGTTAGGCCTGT AGGGATTGCTTTTCAGTTCACCTGTAACTTTAAGTCTTC CTCTATTGAAGAGAGAAATGCAGAAGGACAAAATCTCTTTA CACACTCCTGGAATTTGAGTATCTGAGGAAGTCTTAGCC TCTTTGGAAAAGAATCTGTCCAATCCTCTTATCATGGTGTC CTCTTGTTCCAGTGTTAGACTCCCACTTAGAGGGGGGTT TACAACAACACAATCAAACTTGACTTTGGGCTCAATAAACT TCTCAAAACACTTTATTTGATCTGTCAGGCGATCAGGTG TCTCTTTGGTTACCAAGTGACACAGATAACTAACATTTAAT AGATATTTAAACCTTCUDGCAAAGTAAAGATCTGCATCT TCCCCTTCACCCAAAATTGTCTGGAAAAGTTCCACAGCCAT CCTCTGAATCAGCACCTCTGATCCAGACATGCAGTCGAC CTTTGACATCAAATCCACATGATGGATTTGATTTG CATATGCCATCAAGAAATATCTTAGACCTTGTAAAAATG TCCTTTTGGAAGGGGAACAGAGTACAGCTAACACT AACAATCTTAATATTGGCCTTGTCATTGTCATGAGTTCG TGGCTAAAATCCAACCAGCTGGTCATTTCCTCACACATTTC AATTAACACATCCTCCGAAAATATAGGCAGGAAAAATCT CTTTGGATCACAGTAAAAAGAGCCTTGTTCTTCCAATACCC CATTGATGGATAGATAGATAGAATAGCACCTTGACTTCT CACCTGTTTTTTGGTAAAACAAGAGACCAAATGTATTCTTT GTCAGATGAAATCTTTGTACATAACACTCTCTTAGTCTA ACATTCCCAAAATATCTAGAATACTCTCTTTCATTGATTAA CAATCGGGAGGAAAATGATGTCTTCATCGAGTTGACCAA TGCAAGGGAAATGGAGGACAAAATCCTAAATAATTTCTTCT GCTCACCTTCCACTAAGCTGCTGAATGGCTGATGTCTAC AGATTTTCTCAAATTCCTTGTTAATAGTATATCTCATCACT GGTCTGTCAGAAACAAGTGCCTGAGCTAAAATCATCAAG ATATCAGGGTGTTTTATTAGTTTTTCCAGCTGTGA CCAGAGATCTTGATGAGAGTTCTTCAATGTTCTGGAACA CGCTTGAACCCACTTGGGGCTGGTCATCAATTTCTTCCTTA TTAGTTTAATCGCCTCCAGAATATCTAGAAGTCTGTCAT TGACTAACATTAACATTTGTCCAACAACTATTCCCGCATTT CTTAACCTTACAATTGCATCATCATGCGTTTTGAAAAGA TCACAAAGTAAATTGAGTAAAACTAAGTCCAGAAACAGTAA AGTGTTTCTCCTGGTGTTGAAAACTTTTAGACCTTTCAC TTTGTTACACACGGAAAGGGCTTGAAGATAACACCTCTCTA CAGCATCAATAGATATAGAATTCTCATCTGACTGGCTTT CCATGTTGACTTCATCTATTGGATGCAATGCGATAGAGTAG ACTACATCCATCAACTTGTTTGCACAAAAAGGGCAGCTG GGCACATCACTGTCTTTGTGGCTTCCTAATAAGATCAAGTC ATTTATAAGCTTAGACTTTTGTGAAAATTTGAATTTCCC CAACTGCTTGTCAAAAATCTCCTTCTTAAACCAAAACCTTA ACTTTATGAGTTCTTCTCTTATGACAGATTCTCTAATGT CTCCTCTAACCCCAACAAAGAGGGATTCATTTAACCTCTCA TCATAACCCAAAGAATTCTTTTTCAAGCATTCGATGTTT TCTAATCCCAAGCTCTGGTTTTTTGTGTTGGACAAACTATG TCGCTGGTATTCTTGTTCTTCAATATTAATCTC TTGCATAAATTTTGATTTCTTTAGGATGTCGATCAGCAACC ACCGAACTCTTTCAACAACCCAATCAGCAAGGAATCTAT TGCTGTAGCTAGATCTGCCATCAACCACAGGAACCAACGTA ATCCCTGCCCTTAGTAGGTCGGACTTTAGGTTTAAGAGC TTTGACATGTCACTCTTCCATTTTCTCTCAAACTCATCAGG ATTGACCCTAACAAAGGTTTCCAATAGGATGAGTGTTTT CCCTGTGAGTTTGAAGCCATCCGGAATGACTTTTGGAAGGG TGGGACATAGTATGCCATAGTCAGACAGGATCACATCAA CAAACTTCTGATCTGAATTGATCTGACAGGCGTGTGCCTCA CAGGACTCAAGCTCTACTAAACTTGACAGAAGTTTGAAC AACAACAGAGAGCTGGGGTGATGTTGAGATAAAAA GATGTCCCTTTGGTATGCTAGCTCCTGTCTTTCTGGAAA ATGCTTTCTAATAAGGCTTTTTATTTCATTTACTGATTCCT CCATGCTCAAGTGCCGCCTAGGATCCTCGGTGCG Junin Virus Candid#1 GCGCACCGGGGATCCTAGGCGATTTTGGTTACGCTATAATT S segment GTAACTGTTTTCTGTTTGGACAACATCAAAAACATCCATTG CACAATGGGGCAGTTCATTAGCTTCATGCAAGAAATACCAA CCTTTTTGCAGGAGGCTCTGAACATTGCTCTTGTTGC AGTCAGTCTCATTGCCATCATTAAGGGTATAGTGAACTTGT ACAAAAGTGGTTTATTCCAATTCTTTGTATTCCTAGCGC TTGCAGGAAGATCCTGCACAGAAGAAGCTTTCAAAATCGGA CTGCACACTGAGTTCCAGACTGTGTCCTTCTCAATGGTG GGTCTCTTTTCCAACAATCCACATGACCTACCTTTGTTGTG TACCTTAAACAAGAGCCATCTTTACATTAAGGGGGGCAA TGCTTCATTTCAGATCAGCTTTGATGATATTGCAGTATTGT TGCCACAGTATGATGTTATAATACAACATCCAGCAGATA TGAGCTGGTGTTCCAAAAGTGATGATCAAATTTGGTTGTCT CAGTGGTTCATGAATGCTGTGGGACATGATTGGCATCTA GACCCACCATTTCTGTGTAGGAACCGTGCAAAGACAGAAGG CTTCATCTTTCAAGTCAACACCTCCAAGACTGGTGTCAA TGGAAATTATGCTAAGAAGTTTAAGACTGGCATGCATCATT TATATAGAGAATATCCTGACCCTTGCTTGAATGGCAAAC TGTGCTTAATGAAGGCACAACCTACCAGTTGGCCTCTCCAA CTCGACCACGTTAACACATTACACTTCCTTACA AGAGGTAAAAACATTCAACTTCCAAGGAGGTCCTTGAAAGC ATTCTTCTCCTGGTCTTTGACAGACTCATCCGGCAAGGA TACCCCTGGAGGCTATTGTCTAGAAGAGTGGATGCTCGTAG CAGCCAAAATGAAGTGTTTTGGCAATACTGCTGTAGCAA AATGCAATTTGAATCATGACTCTGAATTCTGTGACATGTTG AGGCTCTTTGATTACAACAAAAATGCTATCAAAACCCTA AATGATGAAACTAAGAAACAAGTAAATCTGATGGGGCAGAC AATCAATGCCCTGATATCTGACAATTTATTGATGAAAAA CAAAATTAGGGAACTGATGAGTGTCCCTTACTGCAATTACA CAAAATTTTGGTATGTCAACCACACACTTTCAGGACAAC ACTCATTACCAAGGTGCTGGTTAATAAAAAACAACAGCTAT TTGAACATCTCTGACTTCCGTAATGACTGGATATTAGAA AGTGACTTCTTAATTTCTGAAATGCTAAGCAAAGAGTATTC GGACAGGCAGGGTAAAACTCCTTTGACTTTAGTTGACAT CTGTATTTGGAGCACAGTATTCTTCACAGCGTCACTCTTCC TTCACTTGGTGGGTATACCCTCCCACAGACACATCAGGG GCGAAGCATGCCCTTTGCCACACAGGTTGAACAGCTTGGGT GGTTGCAGATGTGGTAAGTACCCCAATCTAAAGAAACCA ACAGTTTGGCGTAGAGGACACTAAGACCTCCTGAGGGTCCC CACCAGCCCGGGCACTGCCCGGGCTGGTGTGGCCCCCCAGT CCGCGGCCTGGCCGCGGACTGGGGAGGCACTGCTTACAGTG CATAGGCTGCCTTCGGGAGGAACAGCAAGCTCGGTGGTAAT AGAGGTGTAGGTTCCTCCTCATAGAGCTTCCCATCTAGCAC TGACTGAAACATTATGCAGTCTAGCAGAGCACAGTGTGGTT CACTGGAGGCCAACTTGAAGGGAGTATCCTTTTCCCTCTTT TTCTTATTGACAACCACTCCATTGTGATATTTG CATAAGTGACCATATTTCTCCCAGACCTGTTGATCAAACTG CCTGGCTTGTTCAGATGTGAGCTTAACATCAACCAGTTT TCTTCTTCCATGGAGGTCAAACAACTTCCTGATGT CATCGGATCCTTGAGTAGTCACAACCATGTCTGGAGGCA GCAAGCCGATCACGTAACTAAGAACTCCTGGCATTGCATCT TCTATGTCCTTCATTAAGATGCCGTGAGAGTGTCTGCTA CCATTTTTAAACCCTTTCTCATCATGTGGTTTTCTGAAGCA GTGAATGTACTGCTTACCTGCAGGTTGGAATAATGCCAT CTCAACAGGGTCAGTGGCTGGTCCTTCAATGTCGAGCCAAA GGGTGTTGGTGGGGTCGAGTTTCCCCACTGCCTCTCTGA TGACAGCTTCTTGTATCTCTGTCAAGTTAGCCAATCTCAAA TTCTGACCGTTTTTTTCCGGCTGTCTAGGACCAGCAACT GGTTTCCTTGTCAGATCAATACTTGTGTTGTCCCATGACCT GCCTGTGATTTGTGATCTAGAACCAATATAAGGCCAACC ATCGCCAGAAAGACAAAGTTTGTACAAAAGGTTTTCATAAG GATTTCTATTGCCTGGTTTCTCATCAATAAACATGCCTT CTCTTCGTTTAACCTGAATGGTTGATTTTATGAGGGAAGAG TCTGGGGTGACTCTGATTGTTTCCAACATGTTT CCACCATCAAGAATAGATGCTCCAGCCTTTACTGCAGCTGA AAGACTGAAGTTGTAACCAGAAATATTGATGGAGCTTTC AGTCACAATCTGAAGGCAGTCATGTTCCTGAGTCA GTCTGTCAAGGTCACTTAAGTTTGGATACTTCACAGTGT ATAGAAGCCCAAGTGAGGTTAAAGCTTGTATGACACTGTTC ATTGTCTCACCTCCTTGAACAGTCATGCATGCAATTGTC AATGCAGGAACAGAGCCAAACTGATTGTTTAGCTTTGAAGG GTCTTTAACATCCCATATCCTCACCACACCATTTCCCCC AGTCCCTTGCTGTTGAAATCCCAGTGTTCTCAATATCTCTG ATCTTTTAGCAAGTTGTGACTGGGACAAGTTACCCATGT AAACCCCCTGAGAGCCTGTCTCTGCTCTTCTTATCTTGTTT TTTAATTTCTCAAGGTCAGACGCCAACTCCATCAGTTCA TCCCTCCCCAGATCTCCCACCTTGAAAACTGTGTTTCGTTG AACACTCCTCATGGACATGAGTCTGTCAACCTCTTTATT CAGGTCCCTCAACTTGTTGAGGTCTTCTTCCCCCTTTTTAG TCTTTCTGAGTGCCCGCTGCACCTGTGCCACTTGGTTGA AGTCGATGCTGTCAGCAATTAGCTTGGCGTCCTTCAAAACA TTGACAGTCTGAGTGAATTGGCTCAAACCTCTC CTTAAGGACTGAGTCCATCTAAAGCTTGGAACCTCCTTGGA GTGTGCCATGCCAGAAGTTCTGGTGATTTTGATCTAGAA TAGAGTTGCTCAGTGAAAGTGTTAGACACTATGCCTAGGAT CCACTGTGCG 21 amino acid sequence of MSLSKEVKSFQWTQALRRELQSFTSDVKAAVIKDATNLLNG the NP n of the LDFSEVSNVQRIMRKEKRDDKDLQRLRSLNQTVHSLVDLKS Clone 13 strain of TSKKNVLKVGRLSA??LMSPAADL?{PKAK"MRSFRPQASG LCMV VYMGNLTTQQLDQRSQiLQLVGMRKPQQGASGVVRVWDVKD (GenBank Accession SSLLNNQFGTMPSLTMACMAKQSQTPLNDVVQALTDLGLLY No. ABC96002.1; TVKYPNLNDPFRPKWKHPVPGVITHQQSS N SGYWESLGA GI:86440166) AVKAGAALLDGGNML?SIP KPSNSEDLLKAVLGA GDRVPYFNTTYKVCLSGFGWPYTACRTS:VGRAWE NTT PAVNS?RPA?GAAGP?QVGLSYSQT LLKDL MGG:DPNAPTWIDIEGRFNDPVEIAIFQPQNGQFIHFYREP VDQKQFKQDSKYS{GMDLADLFNAQPGLTSSVIGALPQGMV LSCQGSDDIRKLLDSQNRKUTKLTDVFMTRFASRFYRDKVW DKYGWLCKMHTGIVRDKKK LPDLKTVHNILPHDLIFRGPNVVTL 22 amino acid sequence of MGQLV'JMb'JL'ALPHL i DEVLNLVI LVLLVLTGLKAVYNFATC theGPpnndnof?m GTFAP'SFPPVAGRSCGMYGLKGPD:YKGVYQFKSVEFDMS Cbne13s?mnof HLNLTMPNACSANNSHHYISMGTSGLELTFTNDSIIS?NFC LONE] NLTSAFNKKTFDHTLMSIVSSLHLSn{GNSWYKAVSCDFNN (GenBank Accession GI T I QYNLT FS DAQSAQS QCRTb'RGXVLDMiRTAb'GGKYMR No. ABC96001.2; SGWGWTGSDGKTTWCSQTSYQYLI :QNRTWENHCTYAGPFG G121 16563462) MSR-LLSQEKTKh'LTRRLAGTb'TWTLSDSSGVENPGGYCLT AELKCFGNTAVAKCNVNHD??FCDMPQLTDYNKAA LSKFKEDVESALHLFKTTVNSLISDQLLMRNHLRDLMGVPY CNYSKFWYLEHAKTGETSVPKCWLVTNGSYLNETHFSDQIE ITEULRKDYIKRQGSTPLALMDLLMFSTSAYLVSI FLHLVKIPT{RHIKGGSCPKPHRLTNKGICSCGAFKV?GVK TVWKRR 23 amino acid sequence Of MD *1 SFT.R*1T.CLNY EQD RQKT.NFT.GQREPRMVLIE the L protein of the GLKLLSRCIEIDSADKSGCTHNHDDKSVETILVESGIVCPG Clone 13 strain of LP? PDGYKLIDNSLILLECFVRSTPASE?KKFi?DTNKL LCMV ACIREDLAVAGVTLVPIVDGRCDYDNSFMPEWANFKFRDLL (GenBank Accession FKLLEYSNQW?KVF??SFYERLCFSPKTT DK No. ABC96004.1; LKDARSTHWDEIMRWCHEGINPNMSCDDVVFGINSLFSRFR G1286440169) RDLESGKL RFAVFSCPVWRFTTA?THGHRRGSFTSTFYFRTLSWTNKVK SLKLLNTR< DRLVSFHSTKEEFIRLLRNRKKSKVTQKVSFEELF RAS SEE AKLQKCLLVVGLSFEHYGLSFAP?QECi PETH FENFWKIGAHPIMYYTKFEDYNFQPSTRQTKVTQSVRRTSS VCLALTNSMKTSSVARLRQNQ-GSV{YQVVjCKEVECQVI? LDSEEYHLLYQKTGESSRCYS:QGPDGHLISTYADPKRFFL PIFSDEVLYNMID M SW {SCPDL LMLTWPTKRNQKQVQSVRYLVMAIVSDFSSTSTMDKTR?DT IT?AEKVVYKLLRFLIKTIFGTGEKVLLSAKFKFMLNVSYL CHLITKETPDRLTDQIKCFEKFFEPKSQFGFFVNPKEAITP YEQMKRETSKEiDCQHTTPGVNLEAFSLMVSSFNN GTLIFKGEKKLNSL DPMTNSGCATALDLASWKSVVVNKHLN GEKLLjYDENKLLVSAVSQIITESFVRKQKY SKLVSQLV"GSKGF?TGRSF3NLAF"C?DG??RTSFFKSLE EKVNTTLARYXRGR {ANDKGDGEKLTNTKGL{HLQLILTGK MAHPRKV—PS?ISF {EVEDFDPSCLTNDDMKFICEAVEGST ELSPLYFTSVIKDQCGLDEMAKNLC {KEFSjWDWESCHKMI LLQMNANAYSGKYRHMQRQGLNFKFDWDKP?{DVRTS?RFS NSESLSKALSLTQCMSAALKNLCEYSijSPTSYTSVGPDSG RLKFALSYKEQVGGNRQLY"GDLRT FFSGSCLNNDKEEENALLSMTiNVRiG3LNYSMDHS MC?FLFLMFLQNLKLGDDQYVQSGK3 {VSTLLTWHM{KLVE VPFPVVNAMHKSYVKSKLKLLXGSETTVTERLERQYEEMGL VPS{ISSL:DMGQG:LHNASDTYGPPS?RF"NYCTGVTFGF RPEAYTSSDDQITLFDRRLSDLVVSDPEEVLVLLEFQSHLS GLLNKFIS? LHNVKCKEP?QLCETIDTIADQAIANGVPVSLVNSiQXXTL DLLKYANF?LDPFLLNTNTDVKDWLDGSRGY PNETKVVRKLVRKLHHKLKNGEFNE:EELDLENRDKKLALL QLGDPWGP??DLNQPADVNWLNLNIL-lJ FPLRMVLRQKVVYPS ER-PSLiKTLQNKLCSKFTRGAQKLLSEAINKSAF QSC SSGE GPCKTPGSRCVRNKNR *iN—X KKVLFJPTTDD NRDGITLYICDKQSHPEAHRD?ICLLRPLLWDYIC ISLSNSFELGVWVLAEPTKGKNNSENLTLKHLWPCDYVARK PESSRLLEDKVNLNQVIQSVRRLYPKIFEDQLLPFUSDMSS KNMRWSPR"KFLDPCVEID"NSESPSPIS {VV4WK{31HYT VLFSDLANSHQRSDSSLVDEFVVSTRDVCKNFLKQVYFESF VREFVATTRTLGNFSWFP{K?MMPS?DGA?APGPFQSEVSK VVNKNVERBMERNDLQEGEGWESYRUGDVVCNAAMLIRQGL TNPKAFKSLK DLWDYMLNYTKGVLEFSISVJFTHNQNNTDC LRKFSLI FLVRCQLQN?GVAELLSCSHLFKGE I DRRMLDEC LHLLRTDSVFKVNDGVSDiRSiEb'jDYMEDPL:LGDSLELE LT.GSKRT TDGTRS T DFTRVGEWWP. ?VP'.TVKMGALFEGQNL VQN iVKL *iTKDMKVh'LAGT. *IGYHK S DVLGNLFLH {h'x'i'G EHT.T.GSF. T SVTLQFITCTDRS T'.LT ?T.S'.LPDWFAFK3CQLC FSKSXSTL YJL'TVGGRE'RLKGXSCDDWLGGSVAEDI D 24 an?noaddseqw?we REEKGTNSTNRAEIL?DTTYLGPLSCKSCWQKFDS o?hean?dnofme LVRC?DHYLCRHCLNLLLSVSDRCPLCKYPLPTRLKISTAP Clone 13 strain of SSPP'9YEE (GenBank Accession No. ABC96003.1; GIS6440168) mnmoaddsemkmceof MFRALPHTTDRV'NTVITVLTTTTSTKAVYWFATC n?dnof?w SFLFLAGRSCGMYGLNGPDIYKGVYQFKSVEFDMS VVEsHMnofLChJV HLNLTMPNACSANNSHHY:SMGSSGLELTFTNDSILwaWFC NLTSAFNKKTFDHTLMSIVSSLHLSn{GNSNHKAVSCDFNN GITIQYNLSFSDPQSAISQCRTFRGRVLDMFRTAFGGKYMR SGWGWAGSDGKTTWCSQTSYQYLL"QNXTW1N{CRYAGPFG MSR:LFAQEKTKFLTQRLAGTFTWTLSUsSGVENPGGYCLT AELKCFGNTAVAKCNVNHD??fCDMP LSKFKQDVESALHVFKTTVNSLIsooiiMRNHZRoiMGVPY CNYSKFWYLEAAKTGETSVPKCWLVTNGSYLNETHFSDQIE QRADNM'TFWTRKDYTKRQGSTPLALMDLLMFSTSAYLISI FLHLVK-PT{xHiKGGSCPKPHRLTNKGICSCGAFKV?GVK TIWKRR 26 nucbo?desapwnceof ATGGACATTGACACGTATAAAGAATTTGGAGCTACTGTGGA the HBV HBe antigen GTTACTCTCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCA (GenBank Accession GAGATCTCCTAGACACCGCCTCAGCTCTGTATCGAGAAGCC No. E15688.1; GI: TTAGAGTCTCCTGAGCATTGCTCACCTCACCATACTGCACT 5710371) CAGGCAAGCCATTCTCTGCTGGGGGGAATTGATGACTCTAG CTACCTGGGTGGGTAATAATTTGGAAGATCCAGCATCCAGG GATCTAGTAGTCAATTATGTTAATACTAACATGGGTTTAAA GATCAGGCAACTATTGTGGTTTCATATATCTTGCCTTACTT TTGGAAGAGAGACTGTACTTGAATATTTGGTCTCTTTCGGA GTGTGGATTCGCACTCCTCCAGCCTATAGACCACCAAATGC CTTATCAACACTTCCGGAAACTACTGTTGTTTAA 7. EXAMPLES 7.1 Design of Arenavirus Vector Genome / Vector Construction Based on established approaches (US. Patent Application Publication No. US 2010/0297172 A1; and Flatz L. et al., Nat Med. 2010 March; 16(3): 339—345), LCMV- and Junin Virus -based vaccine s expressing the respective HBV antigens or certain domains thereof are designed (. 7.2 Vaccines Against Hepatitis B Virus Candidate vaccines against hepatitis B virus (HBV) comprise rLCMV-based and rJUNV (Junin vaccine strain Candid#l) vectors expressing pre-S2/S (rLCMV/pre—SZ/S, Pre-SZ/S), HBC (rLCMV/HBc, rJUNV/HBe), a fusion protein consisting ofthe full length HBs and HBc ORFs (rLCMV/HBsHBc), and HBe /HBe, rJUNV/HBe). Vectors will be replication-de?cient (r2LCMV, also referred to as rLCMV, r2JUNV, also referred to as rJUNV) and replication-competent trisegmented constructs (r3LCMV, r3JUNV; see, e.g, Emonet et al., 2009, PNAS, 106(9):3473—3478), wherein the transgenes are arranged in a so- called "arti?cial" way (r3LCMVm, r3JUNVa"). Mice (e.g., C57BL/6 mice) are immunized with one of these ucts, or With combinations thereof in a homologous or heterologous prime- boost vaccination. Administration is performed via the intraperitoneal, uscular, or intravenous route. The dose will be in the range of 104 to 107 focus forming units (FFU). At time points ranging from 7 to 100 days after immunization, HBV-speci?c CD8+ T cells are measured in the blood and/or spleen. T cells may be measured, for example, by using MHC class I tetramers in combination with D8 antibodies in order to identify the magnitude of the CD8+ T cell response to rived epitopes.

In a complementary approach, synthetic peptides are used to selectively stimulate directly ex vivo blood and/or spleen-derived CD8+ T cells by means of intracellular cytokine assays. The intracellular cytokine assays measure the frequency of interferon (IFN)-y, tumor necrosis factor (TNF)-0L, and/or interleukin (IL)producing CD8+ T cells. Surface expression of CD107a serves as a marker of cytolytic degranulation in ?ow cytometry (FACS). Peptide speci?cities are ed, including: HBs-derived epitope VWLSVIWM (SEQ ID NO: 8), HBs- derived epitope IPQSLDSWWTSL (SEQ ID NO: 9), and rived epitope QL (SEQ ID NO: 10). 7.3 Immunogenicity of Replication-De?cient irus—Based Vectors Expressing HBV Antigens C57BL/6 mice (5 mice per group) were immunized once with 105 FFU of rLCMV/HBs-HBc (group 1), rLCMV/HBc (group 3), rLCMV/Pre—S2 (group 4), or with 104 FFU ofrLCMV/HBs-HBc (group 2), via the intravenous route. Control mice were left untreated. days after immunization CD8+ T cells were measured in the blood by using MHC class I multimers. H-2Kb dextramers complexed with the HBs-derived e VWLSVIWM and H- 2Kb dextramers complexed with the HBc—derived epitope MGLKFRQL were used in combination with anti-CD80t antibody to identify hepatitis B virus-speci?c CD8+ T cells. The enumerated cells were expressed as a percentage of the total CD8+B220’ T cell pool in peripheral blood.

The results, as shown in Figure 3, te that vaccination with rLCMV/HBS- HBc, rLCMV/HBc and rLCMV/Pre-S2 induces substantial antigen-speci?c CD8+ T cell responses t the antigens expressed by the respective s. The anti-HBs and anti-HBc CD8+ T cell responses induced by vaccination with rLCMV/HBs—HBC showed a clear dose dependency. Higher frequencies of anti-HBc CD8+ T cells upon HBs-HBc immunization as compared to rLCMV/HBc zation indicate that fusion to HBs results in augmented immunogenicity ofHBc.

Anti-HBs CD8+ T cell frequencies were somewhat higher after immunization with rLCMV/Pre-S2 than after immunization with rLCMV/HBs-HBc, raising the ility that anti-HBc CD8+ T cell responses competed with Bs responses for antigen availability. 7.4 Immunogenicity of Attenuated Replication-Competent Arenavirus—Based Vectors sing HBV Antigens C57BL/6 mice (5 mice per group) were immunized once with 105 FFU of r3LCMV/HBs—HBc (group 1), r3LCMV/HBc (group 2), r3LCMV/Pre-S2 (group 3), or with 105 FFU ofrLCMV/HBs-HBc (group 4), via the intravenous route. Control mice were left t ation. 8 days after immunization HBs- and HBc-epitope-specific CD8+ T cells were measured in the blood by using MHC class I multimers. H-2Kb dextramers complexed with the HBs-derived epitope VWLSVIWM and H-2Kb dextramers complexed with the HBc-derived epitope QL were used in combination with anti—CD8d antibody to identify hepatitis B virus-speci?c CD8+ T cells. The enumerated cells were expressed in two different ways, either as a percentage of the total CD8+B220’ T cell pool in peripheral blood () or as a percentage of circulating lymphocytes in blood ().

The results, as shown in Figure 4, indicate that all r3LCMV-based constructs as well as the replication-de?cient rLCMV/HBs-HBc reference vector were immunogenic, eliciting epitope—speci?c CD8+ T cells against their vectorized antigens, respectively. Moreover, when enumerating epitope-speci?c CD8+ T cells as a percentage of circulating lymphocytes, the replicating r3LCMV/HBs—HBC is shown to be more genic than its replication-de?cient rpart rLCMV/HBs-HBc.

Equivalents and Incorporation by Reference: The ments described herein are ed to be merely exemplary, and those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the speci?c ures described herein. All such equivalents are considered to be within the scope of the present invention and are covered by the ing embodiments. All references (including patent applications, patents, and publications) cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was speci?cally and dually indicated to be incorporated by reference in its entirety for all purposes.

Claims (44)

WHAT WE CLAIM IS:

1. An infectious arenavirus viral vector, wherein an arenavirus open reading frame is removed and ed by a nucleotide sequence selected from the group consisting a. a nucleotide sequence encoding an HBV /S n or an antigenic fragment thereof; b. a nucleotide ce encoding an HBV HBc protein or an antigenic fragment thereof; and c. a tide sequence encoding a fusion of HBV HBc and HBs proteins or antigenic fragments thereof.

2. The viral vector of claim 1 wherein the pre-S2/S protein or the antigenic fragment thereof comprises an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the tide sequence of SEQ ID NO: 1.

3. The viral vector of claim 1 n the HBc protein or the antigenic fragment thereof comprises an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2.

4. The viral vector of claim 1 wherein fusion of HBV HBc and HBs proteins or antigenic nts thereof comprises an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3.

5. The viral vector of claim 1 comprising at least two of: a. a nucleotide sequence encoding an HBV pre-S2/S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding an HBV HBc protein or an antigenic fragment thereof; and c. a nucleotide sequence encoding a fusion of HBV HBc and HBs proteins or antigenic fragments thereof.

6. The viral vector of claim 5, n expression of the nucleotide sequences produces an antigenic protein complex that elicits higher titers of neutralizing antibodies than sion of the protein complex components individually.

7. The viral vector of any one of claims 1 to 6 wherein the arenavirus is lymphocytic choriomeningitis virus, Junin virus, or Pichinde virus.

8. The viral vector of any one of claims 1 to 7 wherein the open reading frame that s the glycoprotein of the arenavirus is deleted or functionally inactivated.

9. The viral vector of any one of claims 1 to 8 wherein the genomic ation encoding the infectious arenavirus viral vector is derived from the lymphocytic choriomeningitis virus Clone 13 strain.

10. The viral vector of any one of claims 1 to 9 wherein the viral vector comprises a genomic t, wherein the genomic segment comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the sequence of tide 1639 to 3315 of SEQ ID NO: 11 or 1640 to 3316 of SEQ ID NO:

11. The viral vector of any one of claims 1 to 10, wherein the viral vector comprises a genomic segment comprising a nucleotide sequence encoding an expression product whose amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid ce encoded by nucleotides 1639 to 3315 of SEQ ID NO: 11 or 1640 to 3316 of SEQ ID NO: 12.

12. The viral vector of any one of claims 1 to 11, wherein the arenavirus is bisegmented and replication-deficient.

13. The viral vector of any one of claims 1 to 7 and 9 to 11, n the arenavirus is tri-segmented and replication-competent.

14. The viral vector of any one of claims 1 to 13, wherein the growth or infectivity of the arenavirus is not affected by the nucleotide sequence.

15. A pharmaceutical composition, genic composition, or vaccine, comprising a viral vector of any one of the preceding claims and a pharmaceutically acceptable carrier.

16. Use of the viral vector of any one of claims 1 to 14, the pharmaceutical composition, the immunogenic ition, or the e of claim 15 in the manufacture of a medicament for treating or preventing a Hepatitis B virus infection in a patient by administration of the viral vector of any one of claims 1 to 14, the pharmaceutical composition, the immunogenic composition, or the vaccine of claim 15 to the patient.

17. The use of claim 16, wherein the administration of the viral vector, pharmaceutical ition, immunogenic composition, or vaccine is by intramuscular injection or intravenous injection.

18. An isolated nucleic acid, wherein the nucleic acid comprises an arenavirus genomic segment wherein one open reading frame of the genomic segment is deleted or functionally inactivated and wherein the genomic t comprises one or more of: a. a nucleotide sequence encoding an HBV pre-S2/S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding an HBV HBc protein or an antigenic fragment thereof; and c. a tide sequence encoding a fusion of HBV HBc and HBs ns or antigenic fragments thereof.

19. The ed c acid of claim 18, wherein the arenavirus c segment is the short t, wherein the open reading frame encoding the glycoprotein is deleted.

20. A method for generating an infectious, replication-deficient arenavirus viral vector comprising: a. transfecting into a host cell, provided said host cell is not within a human, the nucleic acid of claim 18 or 19; b. maintaining the host cell under conditions suitable for virus formation; and c. harvesting the infectious, replication-deficient arenavirus viral vector; wherein the host cell ses the open reading frame that is deleted or functionally inactivated of the c segment.

21. The arenavirus viral vector of claim 1, wherein the arenavirus open reading frame is the glycoprotein open reading frame.

22. An ious, replication-deficient arenavirus viral vector engineered to contain a genome with the y to amplify and express its genetic information in infected cells but unable to produce further infectious progeny particles in normal, not genetically engineered cells, wherein one arenavirus open reading frame is removed and replaced by a nucleotide sequence encoding an HBV antigen or an antigenic nt thereof, wherein the HBV antigen or antigenic fragment thereof is selected from the group consisting of: i. an HBV pre-S2/S protein or an antigenic fragment thereof; ii. an HBV HBc protein or an antigenic nt f; and iii. a fusion of HBV HBc and HBs proteins or antigenic fragments thereof; and a. wherein administration of the arenavirus viral vector to a subject induces a longlasting immune response against the HBV antigen or the nic fragment thereof, wherein (i) the long-lasting immune response induces a detectable antibody titer against the HBV antigen or the antigenic fragment thereof; or (ii) the long-lasting immune response s a detectable antibody titer against the HBV n or the antigenic nt thereof for at least a minimum of 4 weeks; or b. wherein administration of the arenavirus viral vector to a subject infected with an HBV infection increases the antibody titer against the HBV antigen or the antigenic fragment thereof by at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%.

23. A pharmaceutical composition sing a first infectious, replicationdeficient arenavirus viral vector engineered to contain a genome with the ability to amplify and express its genetic information in infected cells but unable to e further infectious progeny particles in normal, not genetically engineered cells, wherein one arenavirus open reading frame is removed and replaced by a first nucleotide sequence ed from the group consisting of: a. a tide sequence encoding an HBV pre-S2/S protein or an antigenic fragment f; b. a nucleotide sequence encoding an HBV HBc protein or an antigenic fragment thereof; and c. a nucleotide ce encoding a fusion of HBV HBc and HBs proteins or antigenic fragments thereof; and a second infectious, replication-deficient arenavirus viral vector engineered to n a genome with the ability to amplify and express its genetic information in infected cells but unable to produce further infectious y particles in normal, not genetically engineered cells, wherein one arenavirus open g frame is removed and ed by a second nucleotide sequence ed from the group consisting of: a. a nucleotide sequence encoding an HBV pre-S2/S protein or an nic fragment thereof; b. a nucleotide sequence encoding an HBV HBc protein or an antigenic fragment thereof; and c. a nucleotide ce encoding a fusion of HBV HBc and HBs proteins or antigenic fragments thereof; wherein the first and second nucleotide sequences are different.

24. The pharmaceutical composition of claim 23, wherein: a. the first tide sequence encodes the HBV pre-S2/S protein or the antigenic fragment thereof, and wherein the second nucleotide sequence encodes the HBV HBc protein or the antigenic fragment thereof; or b. the first nucleotide sequence encodes the HBV pre-S2/S protein or the antigenic fragment f, and wherein the second nucleotide sequence encodes the fusion of the HBV HBc and HBs proteins or antigenic fragments thereof; or c. the first nucleotide sequence encodes the HBV HBc protein or the antigenic fragment thereof, and wherein the second nucleotide sequence encodes the fusion of the HBV HBs and HBc proteins or antigenic fragments thereof.

25. The pharmaceutical composition of claim 23 or 24, wherein the composition is suitable for intramuscular administration or intravenous administration.

26. The method of claim 20, wherein the method further comprises in step a. transfecting into the host cell: a cDNA of the second arenavirus genomic segment, a nucleic acid comprising the L protein ORF, and/or a nucleic acid comprising the NP protein ORF.

27. The use of claim 16, wherein: a. said administration results in a reduction in liver damage in the t; b. said administration results in a reduction in one or more of HBsAg, HBeAg, and HBcAg levels in the blood of the patient; or c. said administration results in a reduction in the level of an antibody against an HBV antigen in the blood of the patient.

28. The viral vector of any one of claims 1 to 12 or 14, wherein the arenavirus is replication-deficient and is engineered to contain a genome with the ability to amplify and s its genetic ation in infected cells but unable to produce further infectious progeny particles in , not genetically engineered cells.

29. The viral vector of any one of claims 1 to 7, n the viral vector is replication-competent.

30. An infectious arenavirus viral vector according to claim 1, n an arenavirus open reading frame is removed and replaced by a nucleotide sequence encoding a fusion of HBV HBc and HBs proteins or antigenic fragments thereof.

31. The viral vector of claim 30, wherein the arenavirus is lymphocytic choriomeningitis virus or de virus.

32. The viral vector of claim 30 or 31, wherein the open reading frame that encodes the glycoprotein of the arenavirus is deleted or functionally vated.

33. The viral vector of any one of claims 30 to 32, wherein the viral vector is replication-deficient.

34. The viral vector of claim 30 or 31, n the viral vector is replicationcompetent and tri-segmented.

35. Use of the viral vector of any one of claims 30 to 34 in the cture of a medicament for treating or preventing a Hepatitis B virus infection or cancer in a t.

36. A viral vector according to claim 1 substantially as herein described or exemplified.

37. A pharmaceutical composition according to claim 15 substantially as herein described or exemplified.

38. An immunogenic composition ing to claim 15 substantially as herein described or exemplified.

39. A vaccine according to claim 15 substantially as herein described or exemplified.

40. A use according to claim 16 substantially as herein described or exemplified.

41. An isolated nucleic acid ing to claim 18 substantially as herein described or exemplified.

42. A method according to claim 20 substantially as herein described or exemplified.

43. A pharmaceutical composition according to claim 23 substantially as herein described or exemplified.

44. A use according to claim 35 substantially as herein described or exemplified > wt LCMV B r3LCMV—GFPnat C —GFP3” -,iC:.133 I n I _SUTR