NZ616283B2 - Pharmaceutical compositions for preventing and/or treating an hiv disease in humans - Google Patents

Abstract

Disclosed is a pharmaceutical composition comprising a mixture of a particulate antigen having one or more epitopes from HIV Gag and/or Pol proteins and a non-pathogenic bacterium. Also disclosed is its use in treating HIV.

Description

PHARMACEUTICAL COMPOSITIONS FOR PREVENTING AND/OR TREATING AN HIV DISEASE IN HUMANS The present invention relates to pharmaceutical itions comprising a mixture of a specific HIV antigen and a non-pathogenic bacterium. Said ic HIV antigen comprises one or more epitopes from Gag and/or Pol ns and is ably under a particulate form. Said bacterium is preferably Lactobacillus plantarum. These compositions are useful for preventing and/or ng an HIV disease in humans.

Background to the invention More than twenty five years after the ery of human immunodeficiency virus (HIV), recent projections from the World Health Organization and the Joint United Nations Program on HIV/AIDS indicate that if the pandemic progresses at its current rate, there will be more than 30 million ions by 201 1.

However, despite considerable research efforts for finding effective treatments for preventing HIV infections. the two recently tested preventive vaccines either have failed (Mc Elrath et al., 2008) or produced modest results (Rerks—Ngarm et al., 2009).

Jae-Sung Yu et al. (Clinical and Vaccine Immunology, Nov. 2006, vol 13, No. 11, 1204-121 1) described recombinant Mycobacterium smegmatis vectors constructed to express the HIV-1 group M consensus env gene CON6 either as a surface, intracellular, or secreted protein. The authors could demonstrate that, in mice, recombinant M. smegmatis was immunogenic for the induction of HIV-1 T—cell responses at l es.

Ke-Qin Xin et al. (Blood, 1 July 2003, vol 102, No. 1, 8) described a recombinant Lactococcus lactis vector expressing the V2-V4 loop of HIV-1 Env on its cell surface. Oral immunization of mice with this vector induced: - both mucosal and humoral immune responseS'as shown by detecting high levels of HIV-specific serum lgG and fecal lgA antibodies; and - a cellular immune response as shown by an increased number of ecific lFN-gamma-secreting cells.

To be properly expressed on-the L. lactis cell surface, gene segments of 1 kb or less could be used.

WO 37071 PCT/[320121000857 Most scientists involved in HIV pathogenesis and prevention feel that before testing .HlV preventive vaccines or other biological compositions for preventing or treating HIV ion in human beings, it would be more constructive to test their counterparts in non human primates (Morgan C, et al., 2008). The non human primate of choice is the e rhesus and among macaques, it has now been conclusively shown that macaques of Chinese origin infected by the Simian Immunodeficiency Virus (SIV) 239 are the best model mimicking most of the clinical, virologic and immunologic aspects of the evolution of HIV infection in humans (Marcondes MC, et al. 2006; Stahl- Hennig C, et al. 2007; Chen S, et al. 2008).

Finally, the scientific community now agrees that, once an effective preventive biological composition or vaccine against SIV 239, is discovered in the macaque, it should in all probability be successfully adaptable to humans to protect them from AIDS.

Despite constant research efforts of the scientific community, tive and therapeutic efficient strategies remain d to combat the worldwide AIDS pandemic.

Various bacteria have been described to have interesting adjuvanticity and immunomodulating properties upon administration to subjects. In particular, lactic acid bacteria have been reported to promote a tolerance effect on the immune system.

For example, published on 23 November 2006 in the name of Stallergenes S.A., bes the use of a ium selected from Bifidobacteria and lactic acid bacteria as an adjuvant in an immunogenic composition e of inducing antigen-specific tolerance upon sublingual, perlingual or oral administration to a t. The immunogenic composition is proposed to be used for treating allergies, auto-immune diseases or for ting graft rejections.

Yet for example, published on 30 July 2009 in the name of Stichting Top ute Food and Nutrition, describes a tolerogenic composition containing a substantial amount of lactic acid bacteria in the mid-log phase. This l composition induces a non antigen-specific immune tolerance when administered to a subject. The composition is proposed to be used for preventing, delaying and/or treating conditions or diseases ated with inflammatory responses that can lead to tissue damage such as ies, mune diseases, and inflammatory diseases of the intestine.

Summam of the invention The Inventors were able to show that, singly, original pharmaceutical compositions as described in the Examples below induced an efficient antigen-specific immune protection against SIV in macaques. Moreover,.when said SlV—specific immune protection was induced, the Inventors showed that it prevented SIV replication/dissemination and the subsequent establishment of the infection in vivo.

Indeed, the Inventors have surprisingly shown that upon administering a pharmaceutical composition as disclosed here either mucosally or by the intradermal or intraepithelial route, virus replication was significantly inhibited, or even abrogated or prevented.

Actually, the Inventors could observe forthe first time that a non-cytotoxic CD8+T cell se suppressed the early activation of SN antigen-presenting CD4+T cells in macaques. Thus, without wishing to be bound by theory, the pharmaceutical compositions according to the present ion induce an unexpected new type of virus-specific immunotolerance upon mucosa] or intradermal or intraepitheliai administration to subjects.

This immunotolerance appears to be 3 HIV Gag and/or Pol n-specific suppressive CD8+T cell-induced immunotolerance (also named herein "Ts" immunotolerance for “I suppressive” immunotolerance), which is MHC (for "Major Histocompatibility Complex")—|b/E- cted and non-cytotoxic.

In the light of the results reported herein, the t invention provides a novel pharmaceutical composition capable of achieving a "Ts" immunotolerance as d above for preventing and/or treating an HIV disease in humans.

In one aspect the present invention provides a pharmaceutical composition comprising a mixture of an antigen and a non—pathogenic Iiving bacterium, n, ably, said antigen is particulate and/or it has one or more epitopes from HIV Gag and/or Pol proteins, and wherein said bacterium is preferably Lactobacillus plantarum.

In a particular aspect, the invention provides a pharmaceutical ition comprising a mixture of a particulate antigen having one or more epitopes from HIV Gag and/or Pol proteins and a non-pathogenic ium.

In another aspect the t invention provides a pharmaceutical ition as bed herein, for use as a vaccine.

In another aspect the present invention provides a method for preventing and/or treating an HIV disease in a human in need thereof, comprising at least the step of mucosally (preferably orally) or intradermally or intraepithelially administering an effective amount of a pharmaceutical composition as mentioned above to said human.

In yet another aspect the present invention provides a method for protecting a human against HIV, comprising at least the step of mucosally (preferably orally) or intradermal or intraepithelially administering an effective amount of a pharmaceutical composition as mentioned above to said human.

In yet another aspect the present invention provides a method for protecting a human from HIV seroconversion, comprising at least the step of mucosally rably orally) or intradermally or intraepithelially administering an effective amount of a pharmaceutical composition as mentioned above to said human.

In a particular aspect, the ion provides use of a particulate antigen having one or more epitopes from HIV Gag and/or Pol proteins and a non-pathogenic bacterium in the manufacture of a medicament for preventing and/or treating an HIV disease in a human.

In yet another aspect the present invention provides a ceutical kit for preventing and/or treating an HIV disease in a human in need thereof, comprising: - in a first ner. an antigen; and - in a second container, a non-pathogenic bacterium, wherein said antigen and said bacterium are in pharmaceutically acceptable carriers for mucosal or ermal or intraepithelial administration, wherein preferably said antigen is particulate and/or it has one or more epitopes from HIV Gag and/or Pol proteins, and wherein said bacterium is preferably Lactobacillus plantarum.

In one aspect, the invention provides a pharmaceutical kit for preventing and/or treating an HIV disease in a human in need thereof, sing: - in a first container, a particulate antigen having one or more epitopes from HIV Gag and/or Pol proteins; and - in a second container, a non—pathogenic living bacterium, said antigen and said ium being in ceutically able carriers for l or intradermal or intraepithelial administration. msnvovcmNRPonhRDCCNFMTififiééz73_l idoc» ”09/20 [4 Brief description of the drawings The present invention is illustrated by the following figures to which reference is made in the non-limiting examples below.

Figure 1 : Intravenous (i.v.) SleacZ39 challenge of rhesus macaques pretreated with an intravaginal iSlVlBCG.

Figure 2: Intrarectal (i.r.) Sleac239 challenge of rhesus es pretreated with an intravaginal iSlV/BCG.

Figure 3: Repeated 39 challenges (3 times by i.v. and 2 times by i.r. ) of rhesus macaques pretreated with an aginal iSlV/BCG.

Figure 4: Intravenous Sleac239 challenge of rhesus macaques pretreated with an intravaginal iSlV/BCG plus an intradermal booster.

Figure 5: Intrarectal Sleac239 challenge of rhesus macaques pretreated with an intravaginal iSlV/BCG plus an intradermal booster.

PCT/[82012/000857 Figure 6: ectal SleacZ39 challenge of rhesus macaques pretreated with an oral iSlV/BOG.

Figure 7: In vitro ral activity of 008+ T cells obtained from rhesus macaques pretreated with an intravaginal iSlV/BOG.

Figure 8: In vitro antiviral activity of CD8+ T cells obtained from the 4 rhesus macaques pretreated with an oral iSlV/BOG.

Figure 9: SlV—specific suppression of CD4+ T—cell activation by autologous CD8+ T cells obtained from the 4 rhesus macaques pretreated with an oral iSlV/BOG.

Figure 10a: Anti-SIV lgG antibody titers in plasma samples taken from the rhesus macaques pretreated with iSlV/LP, iSlV or LP.

Figure 10b: SlV—specific T-cell proliferation in PBMC samples taken from the rhesus macaques pretreated with iSlV/LP, iSlV or LP.

Figure 10c: SlV—specific lFN-gamma-secreting T cells upon in vitro stimulation in the presence or the absence of CD8 or CD25 T cells. [5 Figure 10d: SlV—specific suppression of CD4+ T-cell activation by autologous CD8+ T cells obtained from the 8 rhesus macaques pretreated with an oral iSlV/LP as compared to animals pretreated with an oral LP (n = 4) or iSlV (n = 3).

Figure 10e: SlV-specific CD8+ T cells after 60 days following intragastric administration of an iSlV/LP preparation: cytotoxicity of AT-2 SlV—pulsed CD4+ T cells in the presence of 008+ T cells or of K562 in the presence of human nature killer cells (hNK) (controls) with or without SEB and anti-CD3/CD28 ation.

Figure 11a: In vitro antiviral ty (in CD4 cells) of autologous CD8+ T cells obtained from the 8 rhesus macaques pretreated with an oral iSlV/LP as compared to animals pretreated with an oral LP (n = 4) or iSlV (n = 3).

Figure 11b: In vitro antiviral activity (in CD4 cells) of heterologous or allogenic CD8+ T ! cells obtained from 4 out of the 8 rhesus es 80 days after the treatment of an oral P.

Figure 11c-g: IV ty of 008+ T cells after 60 days following oral : 30 immunization in a delayed (0), insert (cf), allogenic (a) culture , in the presence W0 2012/137071 PCT/[BZOIZ/000857 of anti-MHC-la/ABC or anti-MHC-lb/E antibodies (0, and in the CD8+ T cells depleted of TC R75+orvj38 +subset (9).

Figure 12a: Plasma viral load levels (SIV RNA copies per ml of plasma) following ectal and intravenous Slea0239 challenges in the rhesus macaques pretreated with an oral iSlV/LP as compared to animals pretreated with an oral LP or iSIV.

Figure 12b:'Ce|lu|ar viral load levels (3 IV DNA copies per million PBMCs) following ectal and intravenous Sleac239 challenges in the rhesus macaques pretreated with an oral iSlV/LP as compared to animals pretreated with an oral LP or iSlV.

Figure 13: Depletion of eral blood and lymph node CD8+T cells of the 8 iSlV/LP- treated es by infusion of the anti-CD8 antibody cMT807. a, Peripheral blood CD8+ T-cell counts before and after receiving three injections of cMT807; b, % of lymph node CD8+ T cells before and after ing three injections of cMT807; c, Plasma viral load before and after receiving three injections of cMT807; d, PBMC DNA SIV load before after receiving three injections of cMT807; e, Lymph node SIV DNA load before and after receiving three injections of cMT807.

Figure 14: Plasma (a) and PBMC (b) viral loads following athird intrarectal challenge performed intrarectally with SIVBBYO in 8 rhesus macaques immunized with an oral preparation made of iSIV and LP and 2 onal naive monkeys.

Figure 15: In vitro and in vivo CD8+ T cell-mediated antiviral activity following intragastric immunization with iSlV and LP (iSlV/LP immunization No. 2). a, IV ty (fold of viral suppression) of CD8+ T cells during 60-420 days post- immunization in 8 rhesus macaques that will be challenged intrarectally; b and 0, Plasma and cellular viral loads ing intrarectal Sleac239 challenge of those 8 rhesus macaques immunized with an oral iSlV/LP and of 8 control monkeys treated with LP alone (n = 4) or iSlV (n = 4) alone.

Figure 16: SIV DNA and RNA loads in rectal mucosa intraepithelial cytes (IPLs) (a-b), lamina propria cells (LPC) (c-d), and in pelvic lymph nodes (PLN) (e) post intrarectal challenge of SleacZ39 in 8 macaques (iSlV/LP immunization No. 2).

Detailed description of the invention The present invention is directed to a pharmaceutical composition comprising a e of an antigen and a non-pathogenic living bacterium. 2012/000857 The antigen Due to the great variability in the HIV genome, which‘results from on, recombination. insertion and/or deletion, HIV has been fied in groups, subgroups, types, subtypes and genotypes. There are two major HIV groups (HIV-1 and HIV-2) and many subgroups e the HIV genome mutates constantly. The major difference between the groups and subgroups is associated with the viral envelope.

HIV~1 is classified into a main subgroup (M), said subgroup M being divided into nine es (clades or subtypes) ed gh J (Hu et al., JAMA 275:210-216, 1996 ;Korber et al., Science 280:1868-1871 and a 10th outlier subgroup , 1998), (O).

Many other subgroups resulting from in vivo recombinations of the previous ones also exist (Papathanasopoulos MA, et al. Virus Genes 2003, 26:151-163). Preferably, the HIV virus is HIV-1 or HIV-2, including all known and so far n clades thereof. Yet preferably, it is HIV-1 .

In the context of the present invention, an "antigen" is from HIV origin, which means that it is related to a specific HIV group, subgroup, type, e orto a combination of several subtypes. Preferably, said HIV antigen is a HIV-1 or HIV-2 antigen.

Said antigen is non-infectious.

It was ted for a long time by the scientific community that the activation of CD4+ T cells, the principal target of both HIV-1 and SIV, contributed directly to viral replication (Andrieu and Lu, 1995; Korin and Zack, 1999). However, it was only recently that the interplay between CD4+T cell activation and the successive steps of the SN or HIV infectious process was clarified. In quiescent CD4+ T cells, virus penetration was followed within 2 hours post entry by the presentation at the plasma membrane of Gag and Pol n-derived epitopes of incoming virions while Env and Nef proteins needed de novo synthesis (Sacha et al., 2007). However, the subsequent phases of the infectious process, i.e. reverse , transcription followed by virus integration, developed very inefficiently in quiescent cells (Vatakis et al., 2009a and 2009b). In contrast, when CD4+T cells were activated before or within the 48 hours following the presentation of Gag and Pol epitopes at the plasma membrane, HIV/SIV reverse transcription and DNA integration were extremely active which allowed very efficient virus replication and release (Vatakis et al., 2009a and 2009b).

W0 2012/137071 PCT/1132012/000857 Hence, the Inventors postulated that specifically blocking in vivo the early development of HlV/SlV Gag or Pol-specific CD4+ T-cell activation after HIV/SIV exposure will result in the prevention of active viral replication.

Bearing this in mind, in order to induce the suppression of the activation of HIV Gag and/or Pol antigen-presenting CD4+ T cells, and in turn to prevent in vivo HIV replication and dissemination in exposed humans, the pharmaceutical composition of the t invention comprises an HIV antigen that preferably has one or more es from HIV Gag and/or Pol proteins. Such an antigen ageously either contains or is d from HIV Gag and/or Pol.

The terms "an antigen ning, or derived from, Gag and/or Pol of a HIV virus" thus mean an HIV antigen: - that comprises at least Gag and/or Pol (as an en containing Gag and/or Pol"); or - that ses one or more proteins encoded by GAG such as the capsid protein (p24) and the matrix protein (p17), and/or one or more proteins encoded by POL such as the integrase, the reverse transcriptase and the protease (as an "antigen derived from Gag and/or Pol"); or - that comprises one or more epitopes from those proteins (also as an "antigen derived from Gag and/or Pol").

In particular, any other viral proteins or epitopes thereof selected in the group consisting of ENV, VIF, VPR, VPU for HIV-1 ,VPX for HIV-2, REV, NEF, TAT, and the like, are not essential components of the antigen comprised in the pharmaceutical composition disclosed here. Anyone of these proteins, if t, is only an optional component of the antigen to be used in the pharmaceutical composition disclosed herein.

The antigen is preferabty a particulate antigen. This means that it is preferably selected from virus particles, recombinant virus particles, virus-like particles, Gag and/or Pol-expressing recombinant bacteria or fungi, polymeric microparticles presenting on their surface one or more viral proteins or peptides or epitopes (containing or derived from HIV Gag and/or Pol). Preferably, one or more epitopes from Gag and/or Pol are produced by or expressed by or contained in said antigen. When inant virus particles or virus les or Gag and/or Pol-expressing recombinant bacteria orfungi are used, these are preferably vated microorganisms.

PCT/132012/000857 The n may be a virus particle, a recombinant virus particle, a virus-like particle or a Gag and/or Pol-expressing recombinant bacterium orfungus. It also may be one or more viral proteins or peptides (containing or d from HIV Gag and/or Pol), recombinant or not, either in the form of conjugates or of concatemers. The antigen is then viral nucleic acid independent, that is to say it is non viral DNA- or non viral RNA-dependent.

The antigen may result from the expression of a viral nucleic acid sequence advantageously contained into an appropriate recombinant microorganism.

If the antigen contained into the ceutical composition of the present invention is a Gag and/or Pol-expressing recombinant bacterium, then said inant bacterium is preferably different from the non-pathogenic living bacterium that is also sed in the composition.

When the antigen inthe pharmaceutical composition according to the present invention is one or more viral proteins or peptides (containing or derived from HIV Gag and/or Pot), it is preferably under a particulate form. In practice, riate particulate antigens may be produced by living microorganisms such as yeasts, in the same manner as for recombinant DNA tis B vaccines wherein the expressed HBsAg polypeptide self-assembles into immunogenic spherical particles closely resembling the natural 22-nm les found in the serum of patients with chronic HBV infection (Plotkin et al., 2008).

Alternatively, when the antigen in the pharmaceutical composition according to the present invention is one or more viral proteins or peptides (containing or d from HIV Gag and/or Pol), it is in the form of conjugates. In such an embodiment, as it is well known in the art. proteins or es of interest are convalently conjugated to an appropriate carrier. Conventional carriers that are commercially ble are inter alia proteins such as the KLH (Keyhole Limpet Hemocyanin) protein, the BSA (Bovine serum Albumin) protein, the OVA (ovalbumin) protein, and the like (which can ably be safely administrable orally to humans). Methods for producing appropriate conjugates are familiar to a person skilled in the art.

Yet alternatively, when the antigen in the pharmaceutical ition according tothe present invention is one or more viral proteins or peptides (containing or derived from HIV Gag and/or Pol), it is in the form of concatemers. As it is well known in the art, concatemers are made of multiple copies of proteins or peptides of st that are physically linked together in one macromolecule. In concatemers, a copy of the protein PCT/[82012/000857 or peptide of st can be linked to another either directly or they can be separated by a synthetic arm. A emer thus comprises at least two copies, preferably up to copies or more, of the protein or peptide of interest. Methods for producing appropriate concatemers belong to the general knowledge of a person skilled in the art.

As used herein, a "virus-like particle" (VLP) means a particle that closely resemble mature virions, but that does not contain viral c material of said virus.

More precisely, VLPs, which are also called pseudo-virions, represent subunit structures composed of le copies of a viralcapsid and/or other viral proteins.

These viral proteins are capable to ssemble into VLPs of defined spherical symmetry in vivo. These VLPs do not comprise any nucleic acid molecules coding for virus proteins, and more precisely do not contain any c acid molecules.

Therefore, VLPs are non-replicative and non-infectious in nature, which make them safe for administration in the form of a pharmaceutical composition. s for producing VLPs are well known from one of skill in the art (see, e.g., Liew et al., 2010; Plummer and Manchester, 2010). Non-limiting examples of appropriate methods for producing VLPs are described in US 458, EP 386882, WO 91/07425, US ,861,282 and WO 91/05864 disclosing HIV VLPs (pseudovirions) which do not se HIV genome nor any nucleic acid molecule.

As used herein, "a recombinant virus particle" means a virus particle which contains, or which exposed at its e, proteins from different viruses. Besides. a recombinant virus particle can also mean a bacterium or another host cell which contains, which produces or which exposed at its surface, one or more viral proteins or peptides or es containing or derived from HIV Gag and/or Pol.

Actually, most of the recombinant virus particles are virus particles in which part of original structural proteins (i.e., mainly envelope ns and core proteins) is ed by counterpart proteins from another virus. As an example, the envelope proteins can be exchanged. In such a case, recombinant virus particles n a "chimeric" genome consisting in genome ofa virus having the sequence encoding envelope proteins exchanged with sequence coding for envelope proteins from another virus. Most of the recombinant virus particles are replicative and infectious.

As used herein, a inant virus comprising proteins from another virus means that the recombinant virus particle contains one or more viral proteins or peptides orepitopes containing or derived from HIV Gag and/or Pol, either internally or W0 2012/137071 PCT/132012/000857 present at its surface. Non-limiting examples of methods for ing recombinant virus particles are described for: *Alphavirus: in WC 02/053757 disclosing a recombinant alphavirus expressing HIV (ENV protein) S * Retrovirus :in EP 6 disclosi ng iral vectors expressing chimeric glycoproteins.

* Adenovirus (such as type 5, 7, or 35): in US 2007/077257, US 2007/054395, JP 2007037402, 20034, US 2004/2532 10, US 2004/1 70647, US 2005/07001 7.

US 2003/2 28329, US 2004/ 101957, US 2003/219458, US 2004/0 09936, US 2004/028652 ,WO 03/050238, W0 03/038057 ,WO 03/020893 ,WO 02/31168, WO 80 ,WO 01/02607 , and U86716823 which se recombinant adenovirus expressing HIV proteins.

* Pox virus (can arypox, vaccinia ,vacci nia Ankara, and fowlpox virus): in US 598, EP 6, US 2007/04886 1, US 2006/188961, US 2006/1 34133, EP 1789438, W0 2005/01 7208, , US 2004/146528, JP 2003321391 , EP 1378516,WO 95l07099, JP 7170982 EP 0449116, JP 1148183, JP , DE4141741, 1085072, EP 6, EP 9, US 2005/2 87162, JP 2004105187, JP 200408 9185, WC 03/09 5656, EP 0592546, WO 96/408 80, US 6,136,318, US ,670, 367 which disclose recombinant pox virus expressing viral proteins including HIV proteins.

* Bacteria which contain, which produce or which expose at their surface, at least one protein from a virus: in US 7,189,402 and W0 96/1 1708 which disclose Salmonella or E. coli expressing HIV glycoproteins (i.e., envelope proteins).

Preferably, a recombinant virus particle corresponds to a poxvirus, which pox virus is preferably selected in the group comprising canarypox (e.g.,ALVAC viral s such as the one disclosed in patent US 5,766,598 and EP 0592546), vaccinia (e.g. the vaccinia , virus disclosed in International patent application WO 99), vacci nia Ankara (e.g., NWAC viral vectors such as the one disclosed in pate nt application EP 1789438), and fowlpox virus (e.g., TROVAC viral vectors such as the one disclosed in International patent application W0 03/095656).

More prefe rably, said poxvirus is a canarypoxvirus. As an example of recom bin ant virus particle corresponding to pox virus and expressi ng HIV PCT/[32012/000857 e/protein, one can cite the ALVAC viral vectors disclosed in patent US 5,766,598, (incorporated herein by reference from column 6, line 18to column 82, line 36), which ALVAC vectors express as an exam ple HIV-1 gp120, HIV-1 gp160, non ble secreted form of HIV-1 env, HIV-1 gp120 anchored with membrane ce, HIV-1 l , HlV-1 gag/pol and env (gp120), HIV-1 gag/pol and env (gp 160), and HIV-1 l and env (gp1 20 with transmembrane anchor). Preferably, said ALVAC vector express HIV-1 gag/pol and env (gp120), and most preferably said ALVAC vector is ALVAC VCP‘I 521 .

A "virus particle" is ably an SIV or a HIV particle such as an SIV or a HIV virus particle that may contain a mutated viral genome (e.g., by nucleic acid mutation, substitution or insertion) ing in the production of non-infectious virus particles.

Virus particles containing a mutated viral genome are disclosed in US 7,229,625, US 6,121,021, US 6,923,970, US 6,544,527, US 6,451 .322, and US 6,080,408.

Advantageously, and to have virus les or recombinant virus particles safe for stration toa human said virus particles or recombinant , virus particles are inactivated befo re being administered. Such inactivati on may be necessa ry for recombinant virus particles, even for non-replicative ones.

As used herein "an inactivated virus particle", said virus particle being recombinant or not, means a viral particle, which is no longer infectious and, preferably, no longer replicative.

Methods for inactivation of viral particles or recombinant virus particles are well known from one of skill in the art. Non-limiting examples of viral inactivation include chemical inactivation such as formalin, taurine chloramine, formaldehyde, paraformaldehyde, lactene, beta-propiolactone (REMU NE) or aldrithiol-2 (AT-2, see US 6,001 ,155) treatment, thermal inactivation, physical inactivation such as U.V or gamma irradiation or microwave exposure, and combinations thereof. For a reference for HIV inactivation , see RAVIV er al. (J. Virol., vo|.79(1 9), p: 12394-1 2400, 2005).

According to an embodiment, said inactivation is a chemical inactivation selected in the group comprising formalin, taurine mine, formaldehyde, paraformaidehyde, propiolactene, beta-propiolactone (REMUNE) or aldrithiol-2 inactivation.

Alternatively or additi onal ly, said inactivation is a thermal inactivation . Such inactivation is well known from the skilled person and, as an example of such method, PCT/1132012/000857 one can cite the one disclosed in the examples. indeed, the Inventors have surprisingly ished'in macaques that chemically (i.e., AT-2) and/or thermally inactivated virus induces a protective immunotoierance when associated to a thogenic living bacterium.

Advantageously, for the purposes of administration to humans, virus particles are at least inactivated twice, typically using at least two methods of inactivation mentioned above. ably, as yet mentioned above, the virus particles (recombinant or not, VLPs or not) that are used as antigens in the pharmaceutical compositions of the present invention, are not nucleic acid (i.e., DNA or RNA) dependent, which means that the virus particles do not contain any viral DNA or RNA, or if they n DNA or RNA, it has no role in the immunogenicity.

Alternatively, polymeric micropaiticles (under the form of microcapsules, microspheres, and the like) of various structures and presenting on their surface one or more viral proteins or peptides or epitopes containing or derived from HIV Gag and/or Pol, may be used as antigens in the pharmaceutical compositions according tothe present invention. Such microparticles may be made of riate biological or chemical polymers, such as methacrylated dextran, methacrylated poly(ethyieneglycol) and/or gelatin, onto which the HIV virus or viral ns or peptides or epitopes containing or derived from HIV Gag and/or Pol can adhere. Examples of polymeric microparticles can be found in the literature (for example, in Wei Li Lee et al. (2010), Sandri et al. (2007), Goldberg et al. (2003), Delia F. (1998), Ponchel et al. , Mathiowitz et al. (1997), Fasano et al. (1997), Chickering et al. (1997)).

In a preferred embodiment, the antigen in an HIV-1 pharmaceutical composition according to the present invention is one or more viral particles capable of expressing one or more viral proteins or peptides or epitopes containing or derived from HIV-1 Gag and/or Pol. Alternatively, the n in an HIV-1 pharmaceutical composition according to the present invention is one or more polymeric microparticies presenting on their surface one or more viral ns or peptides or epitopes containing or derived from HIV-1 Gag and/or Pol. ably, the antigen to be used in the ceutical ition according to the present invention is at least about 110 kDa in size. It is preferably at least about 120, 130, 140, 150, 160, 170, 180, 190, 200 kDa or even more, in size.

W0 2012/137071 PCT/[32012/000857 An effective amount of the viral antigen to be used in the context of the invention can easily be ined by the skilled person, using the common general knowledge and in the light of the Examples disclosed hereafter, in connection with SIV or HIV virus.

As an example, when said antigen is a particulate antigen and is more specifically a virus le, the amount of virus particles is from about 10° to about 1012 per ml of said mixture.

The non-pathogenic bacterium As shown by the Inventors with SIV in macaques, when administered by the mucosal orthe intradermal orthe intraepithelial route together with an riate antigen as defined above, the non-pathogenic living bacterium comprised in the pharmaceutical composition is capable of inducing and preferably maintaining a state of immunotoierance to the above-mentioned antigen. In humans, this makes it possible to t and/or treat an HIV disease.

Said bacterium can thus be regarded as a particular adjuvant which can herein be designated as a "tolerogenic adjuvant" or a "tolerogenic carrier" or a "tolerogenic vehicle" or a "carrier of tolerance" or a "carrier of tolerization" or a "vehicle for nce", these terms being synonymous. ably, all these equivalent terms refer to a thogenic living bacterium that is used in combination with an HIV n as defined above in order to achieve a specific immune protection (preferably, immunotoierance) to the antigen, thereby preventing and/or treating an HIV disease in .

More ably, a "tolerogenic vehicle" is a non-pathogenic living bacterium that is administered in admixture with an HIV antigen as defined above, in order to achieve one or more, preferably 2 or more, yet preferably 3 or more, of the following immunoprotecting effects: 1) A "tolerogenic vehicle" does not induce significant production of systemic HIV antigen-specific antibodies: In particular, no significant production of systemic anti-HIV IgM and/or IgG antibodies is observed. For e, there is no significant systemic humoral response that is to say either no specific detectable systemic dy response can be ed by classical clinical laboratory methods such as ELISA, or if systemic antibodies are detected, they are not protective against HIV virus infection. 2012/000857 2) A "tolerogenic vehicle" does not induce significant HIV antigen-specific proliferation of CD4+T cells: In particular, no significant proliferation of HIV antigen-specific CD4 cells is observed upon in vitro HIV antigen stimulation as measured by standard assays such as that described in the accompanying examples. 3) A "tolerogenic vehicle" does not induce significant production of gammainterferon by CD8+ T cells upon in vitro HIV antigen ation: In particular, the level of gamma interferon ion by CD8+T cells which is observed upon in vitro HIV antigen stimulation is below the threshold level for an ELlspot assay. 4)A "tolerogenic Vehicle" induces a significant CD8+ T cell response suppressing the activation of HIV antigen-presenting CD4+T cells: In ular, this se can be determined by an in vitro test measuring the level of inhibition of viral ation by CD8+T cells (indicating a "significant" CD8+ T cell response) as shown in the accompanying examples. These CD8+ T cells are also called CD8+ "regulatory" T-cells. Yet in particular, this response is totoxic given that, e.g., it does not induce significant production of gamma-interferon. Yet in particular, this response is MHC-lb/E-restricted. Yet in particular, TCRap appear to be involved in the CD8+T cell response suppressing viral replication. Yet in particular, this response suppresses the activation of HIV antigen-presenting CD4+T cells compared to the same cell population depleted of CD8+T cells. Preferably, said response sses the early activation of HIV antigen-presenting CD4+T cells, wherein said "early" activation is measured by the Ki67+ marker (Scholzen and Gerdes. J. Cell Physiol. 182,31 1-322 (March .

By the terms "does not induce" as used above in 1), 2) and 3), it is meant a result below the threshold level for an appropriate quantitative detecting assay, wherein said "threshold level" is a value determined in the assay on the basis of the negative control(s): under this value, the result is a ve result. This value may vary from an assay to another and from a method of detection to another.

Advantageously, the tolerogenic vehicle is selected from living: - non-pathogenic bacteria, especially probiotics and commensal bacteria; - attenuated pathogenic bacteria; and - inactivated (optionally, also previously attenuated) pathogenic bacteria.

The tolerogenic e may be recombinant or not; "Non—pathogenic ia" to be used as tolerogenic vehicles in the context of the present invention do not generally induce any pathology in humans. This is the W0 2012/137071 PCT/[82012/000857 l6 ' reason why they are Generally Recognized As Safe (GRAS). Of course, such ia have to be administrable to humans.

Preferred non-pathogenic bacteria to be used as tolerogenic vehicles are commensal bacteria. Such bacteria are well-known to the skilled artisan. Non-limiting examples include Bacillus sp. (e.g., B. coagulans), Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium is, Bifidobacterium longum, Bifidobacten‘um bifidum, bacterium Iactis, Escherichia coli, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus sei, Lactobacillus johnSOnii, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus brevis, Lactobacillus gasseri, Lactobacillus salivarius, Lactococcus lactis, Streptococcus thermophilus, and the like.

A "commensal bacterium" for use as a tolerogenic vehicle in the context of the present invention is advantageously a lactic acid bacterium or a bifidobacterium which is more particularly selected in the list above, including also combinations thereof. A red commensal bacterium is Lactobacillus sp., and more preferably Lactobacillus rum. The Examples reported below show for the first time that Lactobacillus plantarum is a tolerogenic vehicle, g to viral immunotolerance when administered er with an antigen as defined above.

Advantageously, a combination of non-pathogenic bacteria, such as two or more commensal bacteria, may be used as the toierogenic vehicle.

As used , the terms "pathogenic bacteria" refer to bacteria inducing J pathologies in humans. Such bacteria are well known from the skilled person and include inter alia Listeria species (e.g., ia monocytogenes), Corynebacterium species, Mycobacterium species, Rhococcus species, Eubacteria species, Borradella species and Nocardia species. ably, a pathogenic bacterium is selected among Mycobacterium species, and is more preferably Mycobacterium bovis.

As used herein, "attenuated enic bacteria" are pathogenic bacteria which are less virulent compared to their ype counterpart because of one or several ons or of one or more ation treatments (e.g., chemical ent and/or successive es on specific media). Such attenuated pathogenic bacteria are well known from the one of skill in the art. Non-limiting examples of attenuated pathogenic bacteria include attenuated Salmonella typhimurium and Mycobacteria with a preference for attenuated Mycobacteria. As an e of attenuated Mycobacteria, one can cite the "Bacille de Calmette Guerin", also known as "BCG", and, more PCT/1132012/000857 especially, among others, the six widely used BCG strains - the evolutionarily early strain BCG Japanese, the two evolutionarily late s in DU2 Group III (BCG Danish and Glaxo), and the three evolutionarily late strains in DU2 Group IV (BCG Connaught, Pasteur, and Tice). As another example of attenuated Mycobacteria, one can also cite recombinant BCG such as the strain rBCG30 disclosed in HOFT et al. (2008), the recombinant BCG disclosed in WANG ef al (2008), and also the inant BCG disclosed in International patent applications W0 2005/1 11205 and W0 02/102409, and disclosed in patents US 195 and US 6,261 ,568.

Advantageously, instead of or additionally to being attenuated, enic bacteria may be inactivated to be used as tolerogenic es in the context of the present invention, but attenuated pathogenic bacteria may also be used after having been vated. "lnactivated pathogenic bacteria" are well known from the one of skill in the art.

Methods of preparation of such inactivated enic bacteria form part of the common general knowledge in the art. As an example of such methods, one can cite phage mediated lysis, chemical inactivation such as formalin treatment (see US 7,393,541), thermal inactivation, physical inactivation such as lyophilisation (e.g., Extended Freeze Drying) or U.V or gamma ation (see 28065) or microwave exposure, and combinations thereof. ably, said tolerogenic vehicle is an attenuated derivative of pathogenic bacteria like BCG. The Examples reported below show for the first time that BCG is a genic vehicle, leading to viral immunotolerance when administered together with an antigen as defined above.

When recombinant, the tolerogenic e according to the t invention does not express any HIV protein or peptide or epitope.

Preferably, at least a significant amount of the living bacteria used as a tolerogenic vehicle is in the mid—log phase. More preferably, at least about 50%, 55%. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even about 100% of the total number of bacterial cells is in the mid-log phase.

An effective amount for a tolerogenic vehicle can be easily determined by the skilled person and examples of such an effective amount are disclosed hereinafter.

PCT/[32012/000857 As an example, the amount of said bacterium in the pharmaceutical composition of the present ion is from about 104 to about 1014 CFU per ml of said mixture.

The pharmaceutical composition Said composition is also referred to herein as a "tolerogenic composition" or a "tolero—immunogenic composition“, these terms being equivalent.

The tolerogenic vehicle and the antigen containing, or derived from, Gag and/or Pol of 3 HIV virus are two separate and distinct components that are contained as a mixture into the pharmaceutical composition of the present invention. This means that said tolerogenic vehicle and said antigen are present as distinct ents in said ition.

Advantageously, the pharmaceutical composition of the invention does not comprise any oligonucleotide (e.g., CpG or dsRNA) as nt.

Since the tolerogenic vehicle is a ium, a bacterium of the same genus and/or species may be separately used under a recombinant form as a source of antigen. For instance, the recombinant bacterium will contain a nucleic acid ng the antigen placed under the control of appropriate regulatory sequences (including promoters -inducible or constitutive-), either on a nucleic acid vector contained into the cell or as an ated nucleic acid sequence into the ial chromosome. Thereby, the inant bacterium will be able to express or produce said antigen. Thus, according to a particular embodiment, the pharmaceutical composition of the present invention incorporates a tolerogenic vehicle which is a bacterium and an antigen which is one or more viral proteins or es or epitopes ning or derived from HIV Gag and/or Pol, and which has been separately produced by a recombinant bacterium belonging to the same genus and/or species as the tolerogenic vehicle.

In a pharmaceutical composition according to the present invention, when said antigen is a particulate antigen and more specifically a virus particle, the ratio in said mixture of said virus particle (expressed in particles per ml of said mixture) to said bacterium (expressed in CFU per ml of said mixture) is from about 1:10to about 1:1000, preferably from about 1:25 to about 1:750, yet preferably from about 1:50 to about 1:500, even yet preferably from about 1:75 to about 1:250, and yet further preferably about 1:100.

Administering the pharmaceutical composition of the invention W0 2012/137071 PCT/[32012/000857 It may be possible to administer the tolerogenic vehicle and the antigen either simultaneously, or separately, or sequentially.

It is thus an object of the present invention to provide a pharmaceutical kit for preventing and/or treating an HIV disease in a human in need thereof, comprising: - in a first ner, an antigen as defined above; and - in a second container, a non-pathogenic living bacterium as defined above, n said antigen and said bacterium are in pharmaceutically acceptable carriers for mucosal or intradermal or intraepithelial administration.

It is also an object of the present invention to provide products containing: - a non-pathogenic living bacterium as a genic vehicle as defined above; and - a particulate antigen or an antigen having one or more epitopes from HIV Gag and/or Pol proteins as defined above, as a ed pharmaceutical ition for simultaneous, te or sequential use in ting and/or treating an HIV disease in a human in need thereof. Said prevention and/or treatment is(are) achieved via mucosally or intradermally or pithelially administering said combined pharmaceutical composition to said human. To do so, it may be possible to administer the tolerogenic vehicle and the antigen either simultaneously, or separately, or sequentially.

As an example, the non-pathogenic living bacterium may be administered orally (e.g., as an oral drug or a food supplement), s the n is administered mucosally, or intradermally or intraepithelially.

Of course, appropriate pharmaceutical vehicles may be used in order to ensure a le delivery of each to the expected site (e.g., a mucosal surface). The time and dose for administering each of the tolerogenic vehicle and the antigen will be easily adapted by the skilled n.

Preferably, the pharmaceutical ition according to the present invention is a mucosal or intradermal or intraepithelial pharmaceutical composition. Yet preferably, it is an oral pharmaceutical composition.

As used herein ,a "mucosa! or intradermal or intraepithelial pharmaceutical composition" is a pharmaceutical composition for mucosal or intradermal or intraepithelial administration, which means that it is formulated for such an administration.

W0 2012/137071 PCT/132012/000857 In particular, the pharmaceutical composition may further comprise one or more appropriate ceutical vehicles (or ts) for l or intradermal or intraepithelial delivery of said antigen and of said bacterium.

Preferably, a "mucosal delivery" is herein selected from nasal, oral, sub-lingual, tracheal, pharyngeal, ial, esophageal, gastric, duodenal, intestinal, rectal, ial and vaginal deliveries. A "mucosal delivery" is a delivery to a mucosal surface, such as nasal, oral, sub-lingual, al, bronchial, pharyngeal, esophageal, gastric, and mucosae of the duodenum, small and large intestines, including the rectum, as well as ial and vaginal mucosae. In the present context, the l surface also includes the external surface of the eye, i.e., the mucosa of and that surrounding the eye. Yet preferably, the mucosal surface refers to vaginal and digestive mucosa, and more preferably to digestive . Yet preferably, the mucosal delivery is an oral delivery.

Thus, the pharmaceutical composition may also comprise one or more pharmaceutical vehicles depending on the route of administration. Those of ordinary skill in the pharmaceutical art are ar with, or can readily ascertain, vehicles for drug delivery to a mucosal surface orfor an intradermal or intraepithelial delivery.

Useful references in this regard are Chien (Novel Drug delivery system, Chapters 3 through 6 and 9, Marcel Dekker, 1992), Ullmann's Encyclopedia of Industrial Chemistry, 6‘" Ed. us editors, 1989-1998, Marcel Dekker); and Pharmaceutical Dosage Forms and Drug Delivery Systems (ANSEL et a/., 1994, WILLlAMS & WILKINS).

Exemplary methods and routes for drug delivery useful in the invention are briefly described below.

Administration to the bronchial, bronchiolar, tracheal, nasal, oral, preputial or pharyngeal mucosa can be obtained by formulating the pharmaceutical composition as inhalable, spray and the like (e.g., nasal spray, aerosol spray or pump spray and the like), solution, gel, etc. Nebulizer devices suitable for delivery of pharmaceutical compositions to the nasal mucosa, trachea and bronchioli are nown in the art and will therefore not be described in detail here. The pharmaceutical composition may then comprise a vehicle selected in the group comprising solutions, emulsions, mulsions, oil-in-water emulsions, anhydrous lipids and -water ons, other types of emulsions.

Administration to the l mucosa can be obtained by formulating the pharmaceutical composition as solution, enema, foam, suppository, vaginal tablet or W0 2012/137071 PCT/lBZOlZ/000857 topical gel. Preferred vehicles for vaginal delivery include hydrophilic and hydrophobic vehicles such as those commonly used in formulating emulsion or gel preparations (e.g., ter emulsion gel).

Administration to the digestive tract mucosa can be obtained by formulating the pharmaceutical ition as e, microcapsule. Preferred vehicles for digestive delivery pond to capsules and microcapsules (e.g., capsules and microcapsules of pectin and/or alginate) generally given per as such as those commonly used in formulating ations for digestive ry (e.g., the microcapsules disclosed in International patent application ). Alternatively, digestive delivery may be obtained by consuming or administering appropriate liquids and/or foodstuffs, such as beverages, yoghourts, and the like.

Intradermal or intraepithelial administration is well-known to the skilled artisan.

Intradermal administration (e.g., injection) can for ce be done with needle- devices such as those disclosed in patent US 6,933,31 9 and in international patent application W0 2004/1 01025, or with appropriate -free devices.

The pharmaceutical composition may r comprise at least one absorption agent. "Absorption agents" are well known from the one of skill in the art. As examples, one can cite surfactants such as polyoxyethylene derivatives of fatty acid partial esters of sorbitol anhydrides (e.g., Tween® 80, Polyoxyl 40 Stearate, Polyoxyethylene 50 Stearate, polyoxyethylenelauryl ether and Octoxynol), bile salts such as sodium glycocholate. mixed micelles, enamines, nitric oxide donors (eg., S—nitroso-N-acetyl- DL-penicillamine, NOR1,NOR4-which are preferably inistered with an NO scavenger such as carboxy-PITO or doclofenac sodium), sodium salicylate, glycerol este rs of aceto acet ic acid (eg., glyeerym,3-diacetoacetate or 1,2- isopropylideneglycerineacetoacetate), extrin or beta-cyclodextrin derivatives (e g . , 2-hydroxypropyi-beta-cyclodextrin and heptakis(2,6-dimethyl-beta— cyclodextrin», medium-chain fatty acid such as mono- and diglycerides (eg., sodium caprate—extracts of coconut oil, Capmul), or triglycerides (eg., amylodextrin, Estaram 299, Migiyol 810), rs such as ymethylcellulose, carbopol, polycarbophil, tragacanth and sodium alginate, and other absorption agents adapted for mucosal or intradermal or intraepithelial delivery. For a reference concerning general principles regarding absorption agents, which have been used with success in mucosal or intradermal or intraepithelial delivery of drugs, see Chien, Novel Drug Delivery Systems, Ch. 4 l Dekker, 1992).

PCT/132012/000857 The pharmaceutical composition may further comprise one or more additives (e.g., diluents, excipients, stabilizers, preservatives, and the like). See, generally, Ullmann's Encyclopedia of Industrial Chemistry, 6‘“ Ed. (various editors, 1989-1998, Marcel Dekker); and Pharmaceutical Dosage Forms and Drug Delivery Systems (ANSEL et al., 1994, WILLIAMS NS).

As disclosed below, appropriate dosages of the pharmaceutical composition according to the present invention to be administered to a human subject may be determined depending on one or more characteristics of said subject such as sex, age, weight, health, etc.

As an example, when the antigen is particulate and, more specifically, when it is a virus particle, a dose of about 108 to about 1014 virus particles per day may be administered to said human. As another example, a dose of thogenic living bacterium of about 106 to about 1016 CFU per day may be administered to said human.

Applications of the pharmaceutical composition of the invention It is an object of the present ion to e a ceutical composition as described above, for use as a medicament, preferably as a vaccine.

The present invention also relates to a method for preventing and/or treating an HIV disease in a human in need f, comprising at least the step of mucosally or intradermal" or intraepithelially administering an effective amount of a pharmaceutical composition as defined above'to said human.

According to the present invention, for tive purposes, a"human in need thereof" can be any human, preferably having at least about 2 years old. For therapeutic purposes, a "human in need thereof is a human to be treated because he/she is suffering from an HIV disease.

An "HIV e" refers to any HIV-related immune er, including AIDS as well as earlier stages of e progression, including seroconversion (establishment of chronic infection).

The present invention r relates to a method for ting a human against HIV, comprising at least the step of mucosally or intradermal" or intraepithelially administering an effective amount of a pharmaceutical composition as defined above to said human.

In particular, such a method enables to protect a human from an HIV infection if lly exposed to HIV and/or from HIV replication if intravenously exposed to HIV.

PCT/132012/000857 The present invention yet further relates to a method for ting a human from HIV seroconversion, comprising at least the step of mucosally or intradermally or pithelially administering an effective amount of a pharmaceutical ition as defined above to said human. y, said human will not become seropositive and will not exhibit a significant level of HIV antibodies.

The term "vaccination" refers to the action(s) ially, administering the pharmaceutical composition of the present invention) that is(are) taken for preventing and/or treating an HIV disease in a human. Preferably, the pharmaceutical composition of the invention is useful for ng and, ably, maintaining immunotolerance to an antigen containing, or derived from, Gag and/or Pol of a HIV virus in a human that is to say, in other words, for vaccinating (or "tolerizing") said human. Thus, vaccinating a human using the pharmaceutical of the present invention is regarded as a "tolerogenic vaccination" (or a ization" or a "tolerisation").

If. after the mucosal or the intradermal orthe pithelial administration of the pharmaceutical composition of the invention (i.e., after tolerogenic vaccination), immunotolerance has been successfully induced in a human, said human is considered as being "vaccinated" (or "tolerized" or ant"). The response, i.e., the viral replication as evaluated by the plasma viral RNA load of a "vaccinated" human to an in vivo viral infectious challenge is reduced by at least about 50%, more preferably by at least about 70%, yet more preferably by at least about 75% or 80% or 85% or 90% or 95% or 98% or 99%, or even more (99.5%, 99.8%, 99.9%, 100%), relative to the plasma viral RNA load of a control human to which either the antigen alone orthe antigen associated with a standard adjuvant (as defined above) or no pharmaceutical composition ora placebo, was administered.

According to the present invention, atolerogenic vaccination may comprise one or several consecutive administrations of the pharmaceutical composition. Preferably, the tolerogenic vaccination may comprise at least two or more consecutive administrations (i.e., vaccinations), and more preferably more than two consecutive administrations of said composition.

Advantageously, the interval between consecutive tolerogenic vaccinations is comprised between 1 minute and 3 months, preferably between 15 minutes and 2 months.

W0 2012/137071 Yet advantageously, the tolerogenic vaccinations of the invention may also include recall tolerogenic vaccinations one or several years after the first l or intradermal or pithelial tolerogenic vaccination (e.g., Ho 10 years).

The new tolerogenic vaccinations following the first mucosal or intradermal or pithelial tolerogenic vaccination may be selected from mucosal, intradermal and intraepithelial tolerogenic vaccinations. Noticeably, if the new tolerogenic vaccinations are intraepithelial or intradermal injections, then a specific systemic humoral and/or a cytotoxic (gamma interferon producing) se may be detectable but having no role on the prevention ortreatment of the disease.

According to the present ion, an effective amount of the pharmaceutical composition is administered to a human in need thereof. The terms "effective amount" mean a sufficient amount to achieve the desired biological effect, which is here a ve or protective effect (in other words, an immunoprotecting effect) through induction of an immunotolerance, preferably a "Ts" immunotolerance. It is tood that the effective dosage will be dependent upon the age, sex, health, and weight of the subject to be treated, the kind of concurrent treatment, if any, the frequency of treatment, and the nature of the expected effect. The ranges of ive doses provided below are not intended to limit the invention and represent preferred dose ranges. However, the preferred dosage can be adapted to the t, as it is understood and determinable by the one of skill inthe art, t undue experimentation. See, e.g., Ebadi, cology, Little, Brown and 00., Boston, Mass. (1985).

For ce, with respect to HIV, a typical dosage for a human adult will be from about 106 — 1012 HIV virus particles (i.e., VLP, recombinant or non-recombinant virus particles) per dose, with 108- 1010 preferred. Of course, whatever dosage is used, it should be a safe and effective amount as determined by known methods, as also described herein.

Moreover, the one of skill in the art can also determine in the light of his/her general knowledge the effective amount of tolerogenic vehicle to be administered to a human in order to achieve the desired biological effect.

As an example, said effective amount for attenuated derivative of pathogenic bacteria (e.g., BCG) is comprised in the range of 104 to 1012, preferably 105 to 101° CFU (colony forming unit), and more preferably 106 to 108 CFU per dose. As another example, said ive amount for attenuated derivative of pathogenic bacteria or PCTflB2012/000857 inactivated pathogenic bacteria (e.g., BCG) is comprised in the range of 0.001 mg to 1 9, preferably 0.01 to 100 mg, and more preferably 0.1 to 10 mg per dose.

As another e, said effective amount for non-pathogenic bacteria (e.g., Lactobacillus sp.) is comprised in the range of about 105-1014 CFU, and more preferably about 1010-1012 CFU per dose.

As described above, the pharmaceutical compositions of the present invention are suitable for preventing a future HIV disease in a human, orfor treating a human yet suffering from an HIV e.

For therapeutic purposes, the "antigen containing, or derived from, Gag and/or Pol of a HIV virus" as defined above may be autologous, that is to say it may be derived from the HIV virus infecting the human to be treated. In such a case, for example, the HIV virus may be isolated from the human, then it may be cultured and inactivated (preferabiy at least inactivated twice), to be finally associated with a genic vehicle so as to obtain a pharmaceutical composition as described above.

Yet for example, the pharmaceutical composition comprising an autologous or non-autologous antigen containing or derived from HIV Gag and/or Pol may be administered to the human during a conventional antiviral treatment which would have first led to an ctable viral load. The conventional antiviral treatment may then be stopped after one or more tolerogenic vaccinations using the pharmaceutical ition, ed appropriate ex vivo viral replication suppression of non- gous acutely infected CD4 cells is achieved by autologous virus—specific CD8 cells, or provided appropriate suppression of CD4 T cell tion induced by CD8 T cells is achieved.

In particular, for therapeutic purposes, the pharmaceutical ition may be administered once only during the life of the human to be treated. Alternatively, it may be administered twice or more times during the life of the human to be treated, on the same day or on different days separated by a period g for example from about 1 day to about 1 year, or more. More particularly, it may be administered every day or periodically, for periods ranging for example from about 1 day to about 1 year, or more.

If necessary, the pharmaceutical composition may be administered all along the life of the human to be treated.

The present invention further es an in vitro method for determining whether a human is protected against a HIV virus, comprising: 201211100857 a) isolating peripheral blood CD8 T cells from a blood sample of said vaccinated human; b) cultivating under appropriate conditions: (i) said isolated CD8 T cells with allogenic or autologous CD4+T cells which were in vitro acutely infected by a viral strain equivalent to said HIV virus; and (ii) said in vitro acutely infected allogenic or autologous CD4+T cells; 0) recovering the culture supernatants; d) measuring the viral load in said supernatants; and e) determining whether said human is protected against said HIV virus, or not.

By "a viral strain equivalent to a HIV virus to be tested", it is meant that said viral strain originates from a wild virus and has essential characteristics similar to those of the HIV virus to be tested (for e, one can cite the viral strain HTLVIIIB originating from an individual HIV-1: HTLVIIIB can be considered as "a viral strain equivalent to" . Preferably, said viral strain will originate from awild virus which is the HIV virus to be tested. Said viral strain thus represents an riate model for s involving a HIV virus, especially a wild HiV virus. The viral strain is of course dapted for such studies, especially in terms of safety.

All the steps above can be performed using standard techniques that are well- known from the person skilled in the art. In particular, the appropriate culture conditions for step b) are part of the general knowledge in the field of the invention (such as the conventional methods described in the Examples below).

The viral load can be measured in step d) by conventional methods such as those described in the Examples below.

The viral load in the supernatant recovered from the culture of said in vitro acutely infected allogenic or autologous CD4+T cells according to sub-step b)(ii) will be used as a nce for the determination in step 9). One will advantageously calculate the "percent suppression (%)" or "suppressive ratio" or iral effect" by, e.g., W0 2012/137071 PCT/[32012/000857 comparing the geometric mean of viral concentration in the supernatants from duplicate (or triplicate or quadruplicate or more) wells containing only cells from the in vitro acutely infected allogenic or autologous CD4+T cells with the geometric mean of viral concentration in the supernatants from duplicate (or triplicate or quadruplicate or more) wells containing CD8 T cells, and cells from the in vitro acutely infected allogenic or gous CD4+T cells.

Then, said determination in step e) is preferably performed as s: - If the suppressive ratio is higher than about 100, one can conclude that said human is protected. Typically, this will be the case if a HIV-non infected human has been administered an efficient preventive ral ent or ifa HIV- ed human has been stered an efficient therapeutic treatment, said efficient preventive or therapeutic treatment comprising preferably a pharmaceutical composition according to the present invention, and it will thus not be necessary to further administer any preventive ortherapeutic treatment to the human, as long as it remains protected.

- If the suppressive ratio is lower than about 100, one can conclude that said human is not protected against said virus. Then, the human, either a HIV-non infected human ora HIV-infected human, will advantageously be stered a preventive ortherapeutic treatment comprising a pharmaceutical composition according to the present ion, respectively, and the in vitro method above will be performed once or more at appropriate time intervals to make sure the human become protected.

Also, the present invention provides a kit for in vitro determining whether a human is protected against a HIV virus, comprising allogenic or autologous CD4+T cells that can be infected by a viral , said viral strain being, as defined above, equivalent to said HIV virus to be tested. The kit may also e an adequate viral strain in appropriate concentration to infect the above-mentioned nic or autologous CD4+T cells and/or appropriate l reagents and/or controls and/or media (such as media for cell suspension, cell culture, cell storage, etc.). The kit of the present invention may i 30 be specific to a particular type of HIV virus, or it may be adapted to various types of virus, said types of virus being close (in particular, phylogenetically close). i The present invention can easily be d in order to be used for preventing 1 and/or ng any chronic infectious diseases. Non-limiting examples of such W0 2012/137071 PCT/IBZ012/000857 diseases are: hepatitis B and C, human papilloma virus (HPV), EBV and other herpes viruses, tuberculosis, leprosis, leishmaniosis, etc.

Globally, each time where one or several pathogenic antigens associated with the above-mentioned infections or diseases are involved in the specific activation of CD4+T cells which present epitopes d from above-mentioned pathogenic proteins or es, the specific suppression/prevention of activation of CD4+T cell can be raised by non-cytotoxic CD8+T cells generated by mucosal or intraepithelial or intradermal pharmaceutical compositions associating the above-mentioned antigen(s) and atolerogenic vehicle as described herein. it is herein shown that viral infections and associated diseases can be prevented and/or treated in mammals/humans using pharmaceutical compositions of the present invention. Based on this ng, it is possible to provide other pharmaceutical compositions sing (i) tolerogenic vehicles as disclosed herein; and (ii) any antigens from viral, bacterial, fungal. protozoal or parasitic origin. Such pharmaceutical compositions are formulated for riate delivery (preferably, mucosal or intradermal orintraepithelial) of said tolerogenic vehicles and of said ns. They are useful for preventing and/or treating chronic infections in mammals caused by the virus, bacteria, fungi, protozoa or tes which the ns are derived from. An example is a bacterial pharmaceutical ition comprising (i) a tolerogenic e '20 as described ; and (ii) an antigen derived from cterium tuberculosis. This bacterial pharmaceutical composition is formulated for appropriate ry (preferably, mucosal or intradermal or intraepithelial) of said antigen and of said tolerogenic vehicle.

Of particular interest for preventing and/or treating tuberculosis in humans is such a bacterial pharmaceutical composition wherein the mycobacterial antigen is derived from the Koch's bacillus.

The immune protection achieved by the pharmaceutical composition of the invention Tolerance is the physiological capacity of the immune system to recognize antigens taken in through the l system and to develop anergy generally associated with other immunological modifications to a subsequent encounter with the same antigens. Tolerance had been frequently shown to elicit mucosal slgA permitting antibody containment of l antigens without stimulating the ic immune compartment. TGF-beta, a regulatory cytokine had also been sometime invoived in the development of nce. The active suppression by CD25+ regulatory T cells had W0 2012/137071 ‘ ZOI2/000857 also been ntly suggested as a potential mechanism of mucosa! nce (Faria and Weiner, 2005; Mestecky etal., 2007). However, none of these immunological modifi catio ns was obse rve d in the pharmaceutical composition-induced immunotolerance described in the present invention, which is principally characterized by the activity of CD8+T cells which suppress the activation of virus-epitope antigen - presenting CD4+T cells, atype of immune reaction so far unrecognized and, more specifically, a completely new type of immune tolerance.

The expressions "tolerance", "immunological tolerance", "immunotolerance". “immunotolerance to a virus", "new type of virus-specific tolerance", "immunotolerance to viral antigens", "immunotolerance to viral immunogens", and "Ts" tolerance" are synonymous. This has been shown by the Inventors to correspond in macaques to an actively-induced strong non-cytotoxic, /E-restricted CD8+T cell response suppressing the early activation of HIV Gag and/or Pol antigen-presenting CD4+T cells associated with an e of proliferation of CD4+T cells, together with a lack of gamma interferon secretion by CD8+T cells upon inactivated SIV antigen stimulation and a lack of production of systemic anti-SIV IgM and IgG antibodies. Also, the ors could show that TCRya and V38 were not involved in CD8+T ceil suppression of viral replication, suggesting that TCRoiB should play a central role in the recognition of MHC-Ib/E-peptide presentation on infected CD4+T cells.

A "usual immune response to an antigen derived from a virus" can be observed inter alia upon vaccination with a conventional preventive vaccine ition comprising an antigen containing, or derived from, Gag and/or Pol of a HIV virus and a standard or conventional adjuvant (i.e., any form of physical, chemical or biological i nt aimed at stimulating and/or facilitating and/or increasing the immune response associated with the antigen, such as those described in the Chapter entitled "Adjuvants" in "Vaccines" by S. Piotkin et at). Such a "usual immune response to an antigen d from a virus" es humoral, cellular, or both humoral and cellular immune responses, and is conventionally characterized by: (i) the proliferation of virus-specific CD4 cells upon specific in vitro stimulation; and/or (ii) the induction of a specific systemic humoral response via the production of systemic antibodies against viral antigenic proteins and/or peptides; and/or PCT/m2012/000857 (iii) the induction of a c ar response associated with the production of gamma interferon by CD8 T cells, and/or (iv) the absence of non-cytotoxic, CD8+T cell response suppressing the activation of HIV Gag and/or Pol antigen-presenting CD4+T cells.

By st, a pharmaceutical composition comprising an n containing, or derived from, Gag and/or Pol of a HlV virus and a tolerogenic vehicle, as provided by the present invention, generates an "immunotolerance to a virus" and, more particularly, a ""Ts" immunotolerance" which is characterized in the Examples below with respect to SIV in macaques, by: (i) the absence of proliferation of virus-specific CD4 cells upon specific in vitro stimulation; and (ii) the absence of any significant systemic humoral response, that is to say either no specific able systemic dy response can be detected by classical clinical laboratory methods such as ELISA, orif systemic dies are detected, they are not protective against SIV virus infection; and (iii) the absence of any cytotoxic 008+ T cell response associated with the production of gamma interferon (e.g., detectable by t) upon adequate in vitro stimulation, or if a cytotoxic CD8 T cell response is detected, it is not protective against SIV virus infection; and (iv) as an essential feature, the actively-induced strong non-cytotoxic, non CD25 MHC-Ib/E-restricted CD8+T cell response suppressing the early activation of SN antigen-presenting CD4+T cells.

As mentioned above, the pharmaceutical composition of the present invention comprises a tolerogenic e and an antigen containing, or derived from, Gag and/or Pol of a HIV virus. Actually, a tolerogenic vehicle ed to the viral antigen induces a state of virus-specific immunotolerance in a human, instead of eliciting a usual immune response as defined above. Thus, the antigen administered by the mucosal or the intradermal orthe intraepithelial route, alone or ated with a standard adjuvant is lly capable of eliciting a usual immune response, excepting when it is ated with a tolerogenic vehicle as in the pharmaceutical composition of the present invention. Under these specific circumstances, the association tolerogenic PCTfl32012/000857 vehicle I antigen in the ceutical composition of the present invention induces an immunotoierance as defined above. This means that tolerance can only be achieved upon administering (by the mucosa! orthe intradermai or the intraepithelial route) an appropriate mixture of a tolerogenic vehicle and an antigen containing, or derived from, Gag and/or Pol of 3 HIV virus. If one is administered to a human in the absence of the other, the human will not be "vaccinated" or "tolerized".

As shown inthe es below (using SIV in the macaque model), the immunotolerance (also called "Ts" immunotolerance) induced or achieved upon administering the pharmaceutical composition ing to the present invention is characterized by a CD8+T cell response (in particular, non-cytotoxic and/or MHC-lb/E- restricted) that suppresses the activation (especially the early activation) of SIV Gag and/or Pol antigen-presenting CD4+T cells; and advantageously by one or more of: 1) an absence of proliferation of CD4+T cells; 2) a lack of significant gamma interferon secretion by CD8+T cells upon SIV antigen stimulation; and 3) a lack of significant production of systemic anti-SIV lgM and lgG antibodies.

By the terms "lack of significant gamma interferon ion by CD8+T cells upon HIV or SIV antigen stimulation", it is meant herein that the level of gamma interferon secretion by CD8+T cells which is observed upon HIV or SIV antigen stimulation is zero or weak. A "weak" gamma interferon secretion by CD8+T cells upon HIV or SIV antigen stimulation is typically less than about 80 SFCs per 2X1 05 PBMCs.

By the terms "lack of significant production of systemic anti-HIV or anti-SIV lgM I and lgG antibodies", it is meant herein that the level of production of systemic IV or IV lgM and lgG antibodies which is observed is zero or weak. A "weak" production of ic anti-HIV or IV antibodies is typically a titer of IV or anti-SIV of about 300 or less.

Thus, the pharmaceutical composition according to the t invention induces and preferably maintains an antigen-specific immune protection against HIV in a human, wherein said immune protection is preferably characterized by: - the reduction and preferably the suppression of an HIV viral load in said human compared to appropriate experimental controls; and/or - CD8+T cells that suppress the activation of HIV Gag and/or Pol antigen- presenting CD4+T cells in said human compared to appropriate experimental controls.

Advantageously, said immune protection is determined in said human by in vitro detecting the presence orthe activity of CD8+ tory s from said human.

Such detection may be med by standard in vitro techniques, such as those described in the Examples below.

PCT/1132012/000857 All patents, patent applications and publications referred to above are hereby orated by reference.

EXAMPLES PART A - GENERAL MATERIALS 8: METHODS A-l Animals. Colony-bred Chinese rhesus es a mulatta) were housed in accordance with the regulations of the National utes of Health 'Guide for the Care and Use of tory Animals'. All animals were in goodhealth, 2-4 years old._ weighed 4-6 kg and were seronegative for SIV, SRV, simian T cells lymphotropic virus 1, hepatitis B virus, and Bvirus. X ray and skin test (PPD) were performed at entry for all animals to exclude ial carriers of tuberculosis.

A-ll MHC class Ityping. Rhesus macaque classical MHC class Ialleles were genotyped in peripheral blood mononuclear cells (PBMC) samples using sequence— specific primers (SSP) PCR assays for representative MamU-A and Mamu-B sequences as previously described (Muhl etal., 2002; Loffredo etal., 2007).

A-lll Antigenic viral preparations.

Ill-1.The SIV production was performed on CEM174 cells inoculated with Slea0239 (gift of PA. Marx). The e supernatants were collected at pick viral production.

Ill-2. nactivated Sleac239: SIVmac239 was inactivated by 250 uM aldrithiol (AT—) (Sigma) for 2 hours and was washed three times by ultracentrifugation. The AT2- inactivated virus was used in a final dose of 109 viral les for each administration (i.e., vaccination).

III-3. Heat-inactivated Sleac239: Sleac239 was inactivated at 56°C for 30 s. The heat-inactivated virus was used in a final dose of 109 viral particles for each administration.

III-4. ATHeat-inactivated SleacZ39: SIVmacZSQ was inactivated by 250 uM aldrithioi (AT-2) (Sigma) for 2 hours and was washed three times by ultracentrifugation.

Then, the virus was subjected to a temperature of 56°C for 30 minutes. The inactivated virus is used in afinal dose of 109 viral particles for each administration.

III-5. The inactivated virus preparations were inoculated to CEM174 cells to verify the 100% inhibition ofviral infectivity.

W0 2012/137071 PCT/132012/000857 A-IV Assay for antibody responses to SIV. Anti-SIV IgG, 19M, and IgA antibodies in plasma were titrated by an immunofluorescence antibody (IFA) assay (Mederle et al., 2003). Briefly, serial twofold dilutions of test plasma were incubated with SlV-infected CEM174 cell-attached slides at 37°C for 30 minutes. After washing with Hanks, FITC- conjugated goal anti—macaque IgG (Sigma), lgM (ADI, San Antonio, , or lgA (ADI) were added for additional 30 minutes (at 37° C). Antibody titers were determined as reciprocal of the highest on to reach a positive immunofluorescence staining.

The sensitivity of IFA assay was a titer of 20 for lgG and a titer of 5 for IgM and lgA.

When a plasma sample was negative for the IFA (below the assay sensitivity), a value of 1 was ed for facilitating data analysis.

Mucosal secretions were collected by washing of the rectum with PBS using a catheter for gastric lation as described previously (Tsai et al., 1993). , trypsin tor (10 ug/ml) and EDTA (5 x 10-4 M) (Sigma) were added to the samples which were then centrifuged for 10 minutes at 10000 x g at 4°C. Supernatants were collected and supplemented with phenylmethylsulfonyl e (10-3 M) and sodium azide (0.01%) (Sigma). Samples were stored at -80°C until use. Anti-SIV IgA titers in rectum were detected by the above IFA assay.

A-V Flow cytometry. ytometry analysis was carried out with FACScalibur (BD Biosciences, San Jose, California) using fluorescence-labeled monoclonal antibodies against the following: CD3-PE-Cy7 (clone SP34-2), CD4—PE (clone MT477), CD8- PerCP (clone RPA—T8), and secondary rabbit anti-mouse-APC (BD Biosciences). The Ki-67—PE (BD Biosciences) and FITC-conjugated anti-P27 onal antibody (Fitzgerald, Concord, MA) or biotin-conjugated anti-P27 onal antibody (Fitzgerald) coupled with APC-SAV (BD Biosciences) were used for ellular staining after permeabilization.

PE—conjugated monoclonal antibodies against TCRyS (clone B1),\/138, and CD antigens (CD7, CD16, CD28, CD62L, CD95, CD122, CD137, CD150, CD183, CD184.

CD195, CD196, CD197, CD226, CD272, and CD305) were purchased from BD Biosciences; PE—conjugated monoclonal antibodies against CD antigens (CD11a, CD25, CD27, CD39, CD101,CD129, CD215, 00277, and CD357) were purchased from BioLegend (San Diego, CA, USA); and jugated monoclonal antibodies against CD antigens (CD127, CD247, and CD279) were purchased from eBioscience (San Diego, CA, USA).

A-Vl Cell proliferation. PBMCs were obtained as described previously (Lu et al. 2003). The proliferation of ecific CD4+ or CD8+ T cells was evaluated by carboxy-fluorescein diacetate, imidyl ester (CFSE) labeling assay (Molecular PCTfl32012/000857 Probes, Eugene, Oregon) according the manufacturer's instruction. PBMC were stained with 3 uM CFSE for 15 minutes at 37°C. After washing, the CFSE-labeled cells were stimulated for 5 days with 10 ug/ml recombinant SlV core protein P27 oDiagnostics, Wobun, MA), 2 pg/ml SIV gag 15-mer peptides (GLS, Shanghai, China). 109/ml ATinactivated SIV or medium alone. After ng with anti-CD3 and anti-CD4 or anti-CDB antibodies, PBMC were fixed in 1% paraformaldehyde for flow try.

A-Vll Cell activation. Fresh PBMCs, depleted (or not) with CBS or CD25 by magnetic beads were single-round infected with reated SleacZ39 for 2 hours at a viral concentration of l. Infected cells were stimulated overnight with staphylococcal enterotoxin B (2.5 pg/ml) and anti-CD3 (2.5 Mg/ml)/anti-CD28 (2.5 Mg/ml) antibodies. intracellular ng of SIV P27 and Ki-67 was performed 48 hours after ation in order to determine the percentage of activation (Ki-67+) within ed (P27+) CD4+cells.

A-VIII ELISPOT assay. The rhesus e lFN-y and lL-10 ELISPOT assays were carried out in uncultured PBMC in the presence orthe e of P27 or AT inactivated SIV using a commercial kit (Cell Sciences, , MA). A TGF-b1 ELISPOT kit was purchased from R&D Systems (Minneapolis, MN). The data were read with an automated ELISPOT reader (AID, GmbH, Stra3berg, Germany). The number of SIV-specific spot forming cells (SFCs) was calculated by subtracting the nonspecific SPCs in the presence of medium alone.

A-lX Antiviral assay. Autologous CD4+ T cells from each animal purified by magnetic ve-labeling (MicroBeads. Miltenyi Biotec) were acutely infected with SleacZ39 (10'3 MOl) in the presence orthe absence of magnetically purified CD8+ T cells at a CD4/CD8 ratio of 1:2 and then stimulated with SEB (Sigma) for 16 hours. After washing, the cells were cultured in quadruplicates in a final volume of 200 pl per well of RPMI 1640 medium (lnvitrogen, Shanghai, China) containing 100 IU of human rlL2 in 96—well plates for 5 days at 37°C in the presence of 5% CO The cell cultures were . replaced once with half of fresh medium at day 3. The culture supernatants collected at day 5 were used for the measurement of viral load by a real-time RT-PCR (see below).

; Percent suppression (%) was calculated by comparing the geometric mean of viral concentration in the culture supernatants from duplicate wells containing only CD4+ infected cells with the ric mean of viral concentration in the supernatants from quadruplicate wells containing the mixed CD8+ and CD4+ cells. CD4+ T cells were ! 35 also co-cultured with allogenic CD8+ T cells in order to determine the correlation between viral suppression and HLA restriction.

PCT/132012/000857 A-X Viral load measures. SIV RAN in plasma or ssociated SIV DNA was fied by a real-time RT-PCR or PCR using primers (sense, SEQ ID No. 1: 5'- GAGGAAAAGAAATTTGGAGCAGAA-3'; antisense, SEQ ID No. 2 : 5'- GCTTGATGGTCTCCCACACAA—S') and probe (SEQ ID No. 3: 5’- FAM- AAAGTTGCACCCCCTATGACATTAATCAGATGTTA—TAMRA-3') cally optimized for Slea0239 and for Slea0251 .

A-XI SIV-specific suppressive T—cell assay. Fresh PBMCs, depleted (or not) with either CD8 or CD25 by magnetic bead-conjugated anti-CD8 or D25 antibodies according to the protocol provided by the manufacturer (Miltenyi ) were infected with SIVmac239 for 2 hours at 0.5 multiplicity of infection (MOI). Infected cells were dovernight with staphylococcal enterotoxin B (SEB) (2.5 pg/ml) (Sigma) and anti- CD3 (2.5 pg/ml)/anti~CD28 (2.5 pg/ml) antibodies (BD Biosciences). Simultaneous intracellular staining of SIV P27 and Ki-67 were performed 48 hours after in vitro stimulation in order to ine the percentage (%) of T—cell activation (Ki-67+) within infected (P27+) cell tions.

A-Xll Viral challenges.

XII-1. The SIV production was performed on macaques PBMC inoculated with Slea0239 (gift of PA. Marx). The culture atants were collected at pick viral production. .20 XII-2.!ntrarectal challenge (lRC): Following ation, the animals were inoculated (repeatedly) intrarectally with 5000 MID 100 Le. 5 x 105 TCID50 of pathogenic SIVmac239. This infectious dose generally results in a systemic infection of 100% Chinese rhesus macaques with a peak plasma viral load (105-107 copies/ml) between day 10 and day 14. All SlV~challenged animals were evaluated clinically and biologically every 2-week for 1 month and every 1-month thereafter.

XII-3. Intravenous challenge (IVC): Following vaccination, the animals were inoculated (repeatedly) intravenously with 5 MID 100 is. 500 TCID50 (titrated in CEM174 cell line) of pathogenic Sleac239 (gift of Dr. P.A. Marx from Aaron Diamond AIDS Research Center, New York, USA). This infectious dose generally results in a systemic infection of 100% Chinese rhesus macaques with a peak plasma viral load (105-107 copies/ml) between day 10 and day 14. All SIV-challenged animals were evaluated clinically and biologically every 2-week for 1 month and every 1-month thereafter.

A-XIII Statistical analysis. ed data n different groups of animals or paired data before and after immunization were compared by the hitney orthe Wilcoxon test, respectively.

W0 2012/137071 PCT/[BZOIZ/000857 PART B - SPECIFIC MATERIALS & METHODS B-I- USE OF BCG AS ATOLEROGENIC VEHICLE B-l-l ation of BCG 1-1 . Live BCG: Live BCG prepared in Copenhagen at the Statens Serum Institut n SSI 1331 )was purchased from Laboratories Sanofi-Pasteur Merck, Sharp and Dome (SPMSD) and was used at a final tration of 5x106 cfu for intestinal or intravaginal administration or at a final concentration of 5x1 05 cfu for each intradermal boost administration. 1-2. Extended freeze drying (EFD) inactivated BCG: The live SSI 133 BCG strain was killed by 5 days extended freeze-drying (EFD) under a vacuum of less than um Hg and is used at a final dose ponding to 5x106 cfu for each intestinal or intravaginal administration or 5x1050fu for each intradermal administration. l-3. Heat inactivated BCG: The live SSI 133 BCG strain was autoclaved for 15 minutes at 115°C in borate buffer and is used at afinal dose corresponding to 5x10‘5 cfu for each intestinal or intravaginal administration or 5x1 05 for each intradermal administration.

B-l- il Pharmaceutical compositions The composition was prepared freshly with the use of RPMI 1640 (lnvitrogen, Shanghai. China) containing one of the SN antigens and the tolerogenic vehicle.

B-l- Ill Animal immunization At the time of zation, animals were anesthetized with mine hydrochloride and zolazepan (0.7 mg/kg) injected intramuscularly. 111-1 . lntravaginal immunization : Female s were immunized under anesthesia by intravaginal injection for 4 hours of one milliliter of pharmaceutical E composition or of one tolerogenic vehicle disclosed previously as a control. A booster f immunization with the pharmaceuticai composition or with the tolerogenic vehicle was W0 2012/137071 PCT/[BZ012/000857 given with the same dose at the same site at 8weeks. All animals were evaluated clinically and biologically every two weeks after the first immunization. 111-2. Oral (intra-gastric) zation (lGl): Male orfemale animals under esia were administered intragastrally with 15 mlof 0.1 M sodium bicarbonate 15- minutes before ingestion of pharmaceutical composition or of one tolerogenic vehicle disclosed above as a control. Additional 15 ml of the sodium bicarbonate solution was given immediately after administration. The same tolerogenic vaccination than the initial one was repeated two times at 1-month interval to each animal. All animals were ted clinically and biologically every two weeks after the first immunization. 1143.lntradermal boost immunization (IDI): Female intravaginally immunized animals (see above |V| n) were given at 90 days after the first immunization under anesthesia an intradermal booster with 0.1 mlof ceutical composition containing 109 copies of nactivated SN and 5x105 cfu of live BCG. All animals were evaluated clinically and biologically every two Weeks after the first zation.

B-I-IV Antiviral assay. The threshold corresponding to sterile immunity after intrarectal challenge is at least 20.

B-II- USE OF Lactobacillus plantarum AS A TOLEROGENIC VEHICLE B-ll-l Bacterial preparation (tolerogenic vehicle preparation). Lactobacillus plantarum (LP) (ATCC8014) was cultured at 37°C in MRS medium with a rotation rate of 200 rpm. To obtain LP at the logarithmic (midlog) phase of bacterial culture, bacteria were cultured until reaching an optical density of 1.0 at 600 nm with afinal LP concentration of around 1010 cfu/ml (obtained in about 3.5 hours).

B-II-Il Animal immunization by oral (intra-gastric) delivery. Animals were fasted overnight (without ast). At the time of oral stration, animals were anesthetized with tiletamine hydrochloride and zolazepan (0.7 mg/kg) injected intramuscularly.

Immunisation No.1: Eight animals were administered intragastrically 30 ml of a made ofa viral-bacterial preparation containing 4 x 107 copies/ml of Dl-SIV and 3 x 109 cfu/ml of living LP in extrin (20%) on. After this first immunization, monkeys were ing intragastrically 25 ml of the same viral-bacterial preparation (i.e., pharmaceutical ition) each 30 minutes for 3 hours. This oral delivery protocol W0 2012/137071 PCT/[32012/000857 was performed 5 times over 5 consecutive working days. As controls 4 animals were stered living LP alone and other 3 received only twice inactivated SIV in parallel. lmmu nisation No. 2: Twelve animals (iSlV/LP#9-20) were intragastrically administered 30 mi of a preparation of 4 x 107 copies/ml of iSlV (AT—2/heat-inactivated Slea0239) and 3 x 109 cfu/ml of living LP in maltodextrin (20%) solution. Then, animals were receiving 25 ml of the same preparation every 30 minutes for 3 hours (6 times) on 5 consecutive days. Six animals (LP#5-10) were intragastrically stered ml of 3 x 109 cfu/ml of living LP in extrin (20%) solution. Then, animals were receiving 25 ml of the same ation every 30 minutes for 3 hours (6 times) on 5 consecutive days. Finally, another 6 animals (iSlV#5-10) were intragastrically administered 30 ml of a preparation of 4 x 107 copies/ml of iSlV alone. Then, animals were receiving 25 ml of the same preparation every 30 minutes for 3 hours (6 times) on consecutive days.

B-ll-ll! Depletion of 608* T cells in vivo. Macaques were first anesthetized and then given an intravenous injection of a chimeric anti-CD8 monoclonal antibody (cMT-807, or Research & Development, Inc., n, lvania, USA) at 5 mg/kg on days 0,4, and 7) as described earlier (Schmitz et al., 1999). Peripheral blood samples (5 ml) were taken from each animal at day 0 and at various time points after antibody injection.

B-ll-IV Antiviral assay. The old corresponding to sterile immunityafter intrarectal challenge is at least 100.

B-ll-V CD8+ T cell SIV suppression assay. Autologous CD4+ T cells from each animal purified by magnetic positive-labeling (MicroBeads, Miltenyi ) were acutely infected with Sleac239 (10'3 multiplicity of infection) in the presence orthe absence of magnetically purified CD8+ T cells at a CD4/CD8 ratio of 1:3 and then stimulated with SEB and anti—CD3/anti-CD28 antibodies for 16 hours. After washing, the cells were cultured in quadruplicates in 96-well plates. Cultures were maintained in a final volume of 200 p1 per well of RPMI 1640 medium ning 100 IU of human r|L2 (Roche Diagnostics GmbH, Mannheim, Germany) for 5 days. Culture supernatants collected at day'5were used for the measurement of viral load by a real-time RT-PCR (see below). Fold suppression was ated as follows: the geometric means of viral concentration in the culture supernatants from the infected CD4+ target cells only/ the geometric means of viral concentration in the supernatants from the mixed CD8+ and CD4+ T cells).

PCT/[32012/000857 In some experiments, CD8+ and CD4+ T cells were cultured without o-cell contact by using a Multiwell insert System (BD Biosciences) (CD8 in the insert well and CD4 in the bottom well); CD4+ T cells were cocultured with allogenic CD8+ T cells in order to determine the correlation between viral suppression and MHC restriction; and 008+ and CD4+ T cells were also co—cultured in the presence of anti-MHC—ABC (BioLegend) or anti-MHC-E (Cell Science) antibodies to define the modes of MHC ction. To define the subsets of CDB+T cells associated with antiviral activity, CD8+T cells were purified from PBMCs immediately after their depletion with PE-conjugate anti-TCRy5. anti-v85 ,or other anti-CD antigen antibodies using anti-PE eads through a LD column nyi Biotec).

I SIV-specific 608+ T cell's cytotoxicity assay. Both purified CD8+ T cells (effector cells) and purified CD4+ T cells pulsed with 101° reated SIVma0239 (target cells) were labeled with 40 nM 3,3'dihexyloxacarbocyanine (DiOCs) (Marchetti et al., 1996) (Molecular Probes) for 10 min at 37°C. Target cells were labeled with PerCP-Cy5-conjugated anti-CD4 (BD Bioscience) for 20 min on ice. After washing 3 times, effector cells were mixed with target cells in a U-bottomed 96-well plate at ent E/T ratios (3:1 , 1:1, 0.3:1) in triplicate. K562 cells (target) with APO-conjugated anti-CD32 (BD Bioscience) and purified CD56+ (NK) cells (effector) from 4 healthy donors were included as an assay control. After 4 hrs incubation at 37°C in the presence of SEB and Dslanti-CD28, cells were harvested and analyzed by flow cytometry. Percent cytotoxicity was calculated as follows: 100 x (% of total apoptotic target cells - % of spontaneous apoptotic target cells) / (100 - % of spontaneous apoptotic target .

B-II-Vll Viral nges.

First study: Four months after the oral administration of the vaccine or the controls in the first batch of experiment (immunization No. 1), the 8 immunized animals and their 7 controls were inoculated intra-rectally with 2500 MIDm (100.000 TCIDso)of pathogenic 239. Two months later, 4 vaccinated and already protected monkeys were rechallenged by ectal route (100.000 TC|D50)while the 4 other protected monkeys were intravenously rechallenged with 5 MID100 (200 TCIDSD) of SIVmac239. As controls, 2 monkeys received an intrarectal challenge and other 2 an intravenous challenge. These infectious doses generally result in a systemic infection of 100% Chinese rhesus macaques with a peak plasma viral load (107- 109vp/ml) between day and day 14.

PCTfl32012/000857 Second study (immunization No. 2): On day 420 mmunization in the second set of study, 16 animals (8 s immunized with iSlV and LP) and 8 controls (4 iSlV and 4 LP) were intrarectal" challenged with 100,000 TClD5[J of Sleac239.

PART C - RESULTS 6-! 306 IS A TOLEROGENIC VEHICLE C-l-l Protection against the enous Slea0239 challenge following intravaginal administration: Six animals (Compositions_1 ,2, 3, 4, 5, and 6) were administered intravaginally one milliliter of a tolerogenic composition comprising AT2-inactivated virus as an antigen and live BCG. A booster stration was given with the same tolerogenic composition at the same site at 8 weeks.

Simultaneously, 5 other animals (controls_1 ,2, 3, 4, and 5) were given intravaginally one milliliter of a composition comprising only iive BCG. A booster administration was also given with the same composition at the same site at 8 weeks.

Four months after the l administration, all 11 animals (Compositions_1-6 and controls_1-5) were challenged by an enous viral inoculation.

The viral loads were determined regularly in the plasma of the treated animals.

Figure 1 shows the virus loads (plasma SIV RNA copies/ml) as a function of time (days) in animals which have received the composition (Compositions_1-6) and in control animals (controls_1-5) following a single intravenous viral challenge.

The results show that, after intravenous viral challenge, the 5 control animals ols_1-5) showed a typical primary infection with a peak plasma viral load (106- 107 copies/ml) between days 10-14 post-challenge as expected. The plasma viral load of this group of control animals ed still high (>1 05 vp/ml) over the 60 days post viral-challenge and thereafter.

In contrast, 4/6 animals which had received intravaginally the tolerogenic composition made of an nactivated Sleac239 plus BCG showed a very low plasma viral load peak (<1000 vp/ml; between days 10-14), which became PCT/[32012/000857 undetectable (<10 vp/ml) rapidly (one month after viral challenge). The 2 animals with a high plasma viral load peak (> 106 copies/ml) had a lower set-point viral load level (<1000 /ml) than the control group (> 105 copies/ml) at day 60.

C-l-ll— Protection against the intrarectal SleacZ39 challenge ing intravaginal administration of the composition: Seven s (Compositionsj, 8, 9, 10, 11, 12, and 13) were administered intravaginally one milliliter ofatolerogenic composition comprising AT2-inactivated virus as an antigen and live 806 as a tolerogenic vehicle. A booster administration was given with the same composition at the same site at 8 weeks.

Simultaneously, five other animals (controls_6, 7, 8, 9, and 10) were given intravaginally one milliliter of a composition comprising only live BCG. A booster administration was also given with the same composition at the same site at 8 weeks.

Four months after the initial administration, all 12 animals (Compositionsj-13 and controls_6-1 0) were challenged with 39 through an ectal viral inoculation.

The viral loads were determined regularly in the plasma of the treated and l animals.

Figure 2 shows the virus loads (plasma SlV RNA copies/ml) as a function of time (days) in animals which received the ition (Compositions_7—1 3) and in control animals ols_6-10) following intrarectal viral challenges.

The results show that, ing intrarectal viral challenges, the 5 animals which received the intravaginal administration of live BCG alone (Controls_6-1 0) showed a typical primary infection with a peak plasma viral load (106-107vp/ml) between days 10- 14 post-challenge as ed. The plasma set-point viral load of this group of control s remained still high (>1 05 copies/ml) over the 60 days post challenge.

In contrast, 4/7 animals which received intravaginally ATinactivated SIVmacZSQ plus live BCG showed surprisingly undetectable viral load level (<10 copies/ml) over a period of 60 days post-challenge. The 3 other animals showed a typical primary infection with a peak plasma viral load between days 10-14 post- challenge. However, their set-point viral load (103-105 copies/ml) was significantly lower than the control animals' level (>105).

W0 2012/137071 PCT/lBZOlZ/000857 C-l-III- Protection against repeatedly intravenous or intrarectal Sleac239 challenges following aginal administration of the pharmaceutical composition: Two and eight months later, the 3 animals with an undetectable viral load following intravenous challenge (Compositions__1 ,_2, and _3) were subjected to a second and a third intravenous challenge with the same dose of viral a.

After the second and third intravenous viral challenges of this group of monkeys, a similar low peak plasma viral load was observed at day 10. However, by 30 days after viral nge, viral loads became again undetectable (Figure 3).

Sixteen and twenty three months after the initial administration of the composition, the 3 animals which already had atotal of 3 intravenous nges (Compositions_1 ,_2, and _3) were further challenged by intrarectal inoculation.

As expected, these 3 animals (which initially received intravaginally AT inactivated Sleac239 plus BCG) showed again no able (<10 copies/ml) plasma viral load peak after 2 successive intrarectal viral challenges (Figure 3).

These results have established that efficiency on inhibiting viral replication is stable since this inhibition is still observed more than 20 months after the initial administration of the ition C-l-IV- Protection against the intravenous or ectal SIVma0239 challenge following intravaginal administration of the pharmaceutical composition plus an intradermal booster: As expected, after following intravenous (controls 17 and 18,figure 4) or intrarectal (controls 19 and 20, Figure 5) viral challenges, the 4 animals which had received intravaginal administration of live BCG alone showed al primary infection with a peak plasma viral load 07 vp/ml) between days 10-14 post- nge as expected. The plasma set-point viral load of this group of control animals remained still high (>105 copies/ml) by 60 days post viral challenge.

In st, the 3/4 (75%) animals (Compositions 14, 15, and 17) which received intravaginally the composition made of AT—2-inactivated Slea0239 and live BCG plus an intradermal booster with the same composition showed undetectable plasma viral load (<10 copies/ml) over a period of 60 days post-intravenous challenge (see Figure 4). The remaining one animal (composition 16) showed a primary infection with a peak W0 2012/137071 PCT/[32012/000857 plasma viral load (>105 copies/ml) between days 10-14 post-challenge (see Figure 4).

However, its set-point viral load reached relatively low level (104 copies/ml) at day 60.

Moreover, the 4/4 (100%) animals (compositions 18-21 )which received intravaginally the composition made of ATinactivated Sleac239 plus live BCG plus an intradermal booster of the same composition showed undetectable plasma viral load (<10 copies/ml) over a period of 60 days post-intrarectal challenge (see Figure 5).

C—l—V- Protection against the ectal Slea0239 challenge following oral administration of the pharmaceutical composition: Four animals (Compositions_22, 23, _24, and _25) were administered intragastrically one milliliter of a ition comprising AT2-inactivated virus and live BCG.

Simultaneously, four other animals (controls 21-24) were intragastrically given one milliliter of live BCG alone.

The same stration given initially to each animal was repeated three times at day 15, 30 and 60 following the first administration step.

The results show that after intrarectal viral nge (performed at day 90), the 4 animals which received live BCG alone (controls 21-24) showed al primary infection with a peak plasma viral load (106-107 copies/ml) between days 10-14 post- nge whereas the 4 animals (Compositions_22-25) which received the AT inactivated SIV plus live BCG composition showed surprisingly an undetectable plasma viral load (<10 copies/ml; between days 10-14) (Figure 6).

C-l-Vl- Immune correlates and protection against Slea0239 nge following the administration of the composition made of AT2—inactivated virus plus live BCG: No systemic antibody directed against SIV was detected in the blood of the treated animals. However, some specific ic humoral se has been detected when intradermal boost composition administration has been used.

Consequently, the observed protection against SIV infection for the treated s does not result from a systemic humoral response.

Moreover, no conventional SIV—specific gamma eron-producing cytotoxic T lymphocytes were detectable by ELlspot (data not shown). For the purpose of wo 2012/137071 ZOIZ/000857 evaluating whether a SIV-specific non-conventional cellular response existed, blood samples were taken for each treated or l , and CD4+ and CD8+ cells were purified from each sample according to tional methods. The previously obtained CD4+ cells were cultured and then infected with SIV ma0239 according to conventional methods. The SIV-infected CD4+ cells were then cultured in the ce or in the absence of the previously obtained autologous CD8+ cells for 5 days. The supernatant SIV concentration was assayed by a quantitative real-PCR.

Figure 7 shows the fold of suppression of viral replication in SIV-infected CD4+ obtained in the presence or in the e of autologous 008+ cells obtained in the course of the experiments presented in Figure 2. The tested CD8 were obtained from animals which received, by the intravaginal route, the composition of AT2-inactivated virus plus BCG (Compositions_7-13) or from control animals (controls 6-10).

The results show that the CD8 T cells from animals ted against virus ion (composition_7, composition _8, composition _10, and composition _11) provide a level of viral suppression in SIV-infected CD4 cells greater than 20 fold, whereas the CD8 T cells from animals non-protected against virus infection (composition_9, composition_1 2, and composition_1 3) e a level of viral suppression inferior or equal to 10 fold (Figure 7). Moreover, a more than 20 fold viral suppression has been also observed in the 4 animals protected against intravenous viral nges presented in Figure 1 (data not shown).

Figure 8 shows the levels of viral suppression in SIV-infected CD4+ obtained in the presence or in the absence of autologous CD8+ cells ed in the course of the experiments presented in Figure 6. The tested CD8 were obtained from the 4 animals which received the composition sitions_22—25) by oral administration of AT2- inactivated virus plus ECG or from the 4 control animals (controls 21-24).

Figure 9 shows the levels of T-cell activation (KT-67+) in SIV (P27+)—infected CD4 cell population obtained in the presence or in the absence of autologous CD8+ cells obtained in the course of the experiments presented in Figure 6. The tested CD8 were obtained from the 4 s which received the composition (compositions_22-25) by oral administration of AT2-inactivated virus plus BCG orfrom the 4 control animals (controls 21-24). A SIV-specific suppression of CD4+ T-cell activation by autologous CD8+ T cells was observed in the 4 animals which received the composition.

The results confirm that the prevention of systemic or mucosal SIV ion obtained by intravaginal or oral administration of AT2-inactivated plus BCG induced a W0 2012/137071 ' PCT/[32012/000857 state of immunotolerance characterized by a non—cytotoxic CD8+ T-cell response associated with an fected CD4 cell anergy. In view of these results, BCG can thus be identified as a tolerogenic adjuvant.

Taken together, these findings have demonstrated that a steady state of immunotolerance to SIV antigens is for the first time achieved by aginal or oral (or intragastric) administration of a composition made of inactivated SIV virus plus live BCG. At the same time, it was shown for the first time that an intravaginal or oral administration of a pharmaceuticalcomposition comprising activated SIV virus plus live BCG according to the invention is effective (>50%) to prevent chronic viral infection following intrarectal or intravenous challenge.

CJI- USE OF Lactobacillus QIantarum AS A GENIC VEHICLE C-ll-i- induction of SIV-specific immunotolerance by oral co-administration of double inactivated SIV and Lactobacillus plantarum (iSlV/LP) On the one hand, SIV-specific antibodies (IgG, lgM, and lgA) were not detected in animals d with oral iSlV/LP (Fig. 10a). 0n the other hand, no significant SlV P27- specific peripheral blood CD4+ T cell proliferation was observed in iSlV/LP-treated animals while iSIV-treated animals did show significant P27-specific peripheral blood CD4+ T cell proliferation.

C-II-II- Anti-activation and antiviral activities of non-c otoxic CD8+cells SIV P27-specific peripheral blood CD8+ T cell proliferation was observed both in iSlV/LP—treated and iSlV-treated animals (Fig. 10b). However, no interferon-gamma secreting T cells (upon to In vitro ation) were detected in iSlV/LP-treated animals and the depletion of either CD8+ or 0025+ cells did not alter the unresponsiveness of ecific or T cells (Fig. 100). Moreover, a strong suppression of tion (Ki-67+) of infected (P27+) CD4+ T cells by non-cytotoxic CD8+ T cells was also ed in acutely in vitro infected PBMCs taken from iSlV/LP-treated animals and the depletion of CD25+ cells did not alter the potent suppression operated by CD25- CD8+T cells on the activation of infected CD4+ T cells (Fig. 10d).

Of note the fact that no cell lysis was detected by a high-sensitive cytotoxicity assay (Marchetti et al., 1996) after co—incubating 008+ T cells and CD4+ T cells pulsed with WO 2012113707] PCT/1320121000857 non-replicative Sleac239 in the presence or the absence of SEB and anti-CDS/anti- CD28 antibodies (Fig. 109).

Finally, the peripheral blood 008+ T cells taken from animals d since 2 2 months by iSlV/LP showed a strong inhibiting activity against viral replication in acutely in vitro infected autologous CD4+ T cells (Fig. 11a). Furthermore ,such a strong ral activity of CD8+ T cells was also observed equally in acutel y in vitro infected heterologous CD4+ T cells (Fig. 11b), suggesting that a non classical HLA1 cted mechanism is involved in the suppressive/inhibiting activity of 008+ T cells.

Purified peripheral blood CD8+ T cells taken from macaques immunized with LP/iSlV 2 2 months earlier had a strong antiviral activity on autologous y SleacZ39- infected CD4+ T cells stimulated overnight with SEB and anti-CD3Ianti-CD28 antibodies and then co-cultured for 5 days. Once SlV-specific CD4+ T cells activation is established (48 hours post~stimulation), adding CD8+T cells can no longer inhibit viral ation (Fig. 11c). This observation argues against the potential lysis of target (productively infected) CD4+ T cells by 008+ T cells in prolon ged culture, as suggested by a previous study in human autoimmune type 1 diabetes (Jiang et al.. 201 0). This 008+ T cell-mediated antiviral activity needed cell-to-cell contact (Fig. 11d) and also was classical MHC1a-unrestricted as shown by the strong inhibition of viral replication 'operated by 008+ T cells on acutely infected CD4+ T cells from other immunized animals or from l animals (Fig. 11a). y, the CBS-mediated antiviral activity was blocked by an anti-MHC-lb/E antibody but not by the anti-MHC- la/ABC antibody, indicating a non-classical MHC-lb/E-restricted 008+ T cell activity (Fig 11f). it is established that a CD8+ T cell TCR expression is necessary to recognize MHC- Ib/E-peptide complexes carried by target CD4+ T cells (Sarantopoulos et al. Van , 2004; Kaer, 2010). Using an in vitro depletion by antibody-conjugated magnetic microbeads, TCR‘yS and Vp8 were shown not to be involved in 008+ T cell ssion of viral 1 replication (Fig. 119). TCRaB thus appears to play a central role in the recognition of MHC-lb/E-peptide presentation on ed CD4+ T cells. Moreover, by depleting CD8+ i 30 T cells with available uman dies cross-reacting with membrane CD (for "Cluster Differentiation") antigens of non-human primates (CD7, CD11a, C016, CD25 A), 0027, 0028, C039, CD62L, 0095, CD101, CD122 (lL-2RB), CD127 (iL-7R), CD129 (IL-9R), CD137, CD150, CD183 (CXC R3), 00184 ), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), C0215 (IL-1 5Ra), 00218 (lL-18Ra), 00223 (LAG3), i 35 00226, C0247, CD272, 00277, 00279 (PD-1 ), CD305 (LAIR1), and 00357), no CD antigen associated with MHC—lb/E-restricted 008* T cells activity could be identified 2012/000857 (Table 1). Table 1 below shows the antiviral activity (fold suppression, geometric mean 1 SE) of CD8+ T cells taken from 8 iSlV/LP-im munized animals before and after depletions of CD antigen-defined subsets" in the first immunisation study (immunisation No. 1).

Table 1 CD antigens eted CD8+ T cells Depleted CD8+ T cells P value CD7 1387 4; 301 964 i- 326 0.313 CD1 1a 941 :t 377 0.568 CD1 6 529 .1: 152 533 i 99 0.772 CD25 691 i 258 0.490 CD27 704 :t 242 761 :l: 122 0.867 CD28 1021 i 177 0.407 CD39 970 i 361 1256 i 354 0.71 0 CD62L 1013 i 302 0.832 CD95 813 i 238 775 t 239 0.954 CD101 980 :l: 197 0.613 CD1 22 997 -|_- 411 784 t 265 0.41 2 CD127 715 i 339 0.545 CD1 29 872 i 325 855 i 252 0.81 3 CD137 868 i 306 0.852 CD1 50 889 i 223 924 i 231 0.959 CD1 83 633 i 198 0.354 CD1 84 1452 i 253 1265 t 447 0.841 CD1 95 1083 t 295 0.374 CD 196 789 i 245 652 t 280 0.882 CD1 97 878 t 247 0.789 CD215 1221 i213 1214 i445 0.621 CD218 739 t 371 0.477 CD223 623 t 293 1208 :1: 248 0.197 CD226 1234 i 192 0.237 CD247 914 i 288 940 i 279 0.991 CD272 1056 :I: 231 0.846 CD277 1247 i 216 957 i 282 0.523 CD279 1197 1 151 0.616 CD305 798 i 245 1157 i 241 0.233 CD357 820 i 127 0.807 *In each batch of experiment, antiviral activity (fold suppression, geometric mean t SE) 40 of CD8+ T cells taken from 8 iSlV/LP-immunized animals depleted (or not) with 2 anti- CD antigen antibodies was performed.

C-II-l ll Protection of animals from intra-rectal challenges by oral immunotolerance Three months after the administration of iSlV/LP or l preparations, the 8 iSlV/LP- 45 immunized and the 7 control animals were intrarectally challenged with a single high PCT/1132012/000857 dose (100,000 TC|D50) of SleacZ39. Eight out of 8 iSlV/LP-treated animals were protected from infra-rectal challenge of pathogenic Sleac239 while the 4 iSlV—treated and the 4 LP-treated animals were infected by the same rectal viral challenge ] (Fig. 12a and b, left part of the Figures).

C-ll-lV Protection of s from intravenous challenge by oral immunotolerance E Two months after this first challenge, 4 out of the 8 monkeys received a second challenge via the intravenous route (200 TCIDSO). All of them showed a slight peak of replication (5 200 SIV DNA /million PBMC and 200 SIV RNA /ml of plasma) at day 10 post—challenge; however by day 30, PBMC SIV DNA had decreased to s 10 copies/million cells and plasma SIV RNA was ctable (s 10 copies/ml), indicatingthe lack of in vivo active replication of the virus (Fig. 12a and b, right part of the Figures). In contrast, 2 naive animals which ed the same intravenous Slea0239 challenge (200 TCID50) were successfully infected. The 4 remaining monkeys were intrarectally re-challenged (100.000 TCIDSO) and all of them ed fully protected (Fig. 12a and b, right part of the Figures).

C-Il-V Confirmation in vivo of the role of CD8+ T cells Five months afterthis second challenge, in order to confirm in vivo the role of CD8+ T cells, 3 intravenous injections of a mouse-human chimeric onal anti-CD8 antibody (cMT-807, Centocor) were given over a period of one week (days 300, 304 and 307 post-immunization) to the 8 already—challenged monkeys to temporarily deplete their 0138+ T cells from peripheral blood and lymphoid organs (Fig. 13a & b).

No viral RNA or DNA emergence was detected in the 4 macaques llenged by intrarectal route, demonstrating again their full sterile protection; in contrast, a strong viral replication accompanied the depletion of CD8+ T cells from lymphoid organs of the 4 intravenously challenged animals as shown by their plasma viral loads that peaked at 106 RNA copies/ml and their PBMC and lymph node proviral loads that reached 104 DNA copies/106 cells by day 15 (the nadir of CD8+ T cells depletion); by . 30 days 60-90, when the 4 monkeys had recovered baseline CD8+ T cells concentrations, plasma SIV RNA and PBMC and lymph nodes SIV DNA red also baseline levels (Fig. 13 c, d & e ). This confirmed the unique role of iSlV/LP-induced CD8+ T cells in the control of in vivo viral replication in intravenously SIV-challenged animals in which replication-competent virus remained latent in presumably in quiescent memory CD4+ T cells.

PCT/[32012/000857 Eight months after the second challenge, the 4 intrarectally rechallenged monkeys as well asthe 4 intravenously rechallenged ones received a third nge, this time via the intrarectal route with SIVBBYO (100,000 TC|D50), a distinct infectious SIV strain.

The 8 animals remained fully protected over the next 12 months as shown by their undetectable 0 DNA and RNA levels whereas 2 naive animals were successfully infected by the same SIVBS70 challenge, demonstrating that LP/iSlea0239-generated MHC-lb/E—restricted CD8+ T cells were protective through preventing the activation of CD4+ T cells infected by other SIV s es 14a & b ).

To determine the duration of efficacy for preventing SIV es in the P-treated animals, a second immunization with iSlV/LP was conducted in 8 new es of Chinese origin and the in vitro antivirai activity of their CD8+ T cells was d overtime without SIV challenge. Such an in vitro antiviral ty was detected as from 60 days post-immunization as compared to the control animals either treated with LP (n = 4) or iSlV (n = 4) alone.

Ex-Vivo anti-SIV activity levels were maintained until day 420 in 7 out of 8 s while the antiviral activity of one monkey progressively decreased from day 360 to reach baseline levels of control monkeys by day 420 (Fig. 15a). On day 420 post- immunization. the 16 animals were intrarectally challenged with 100,000 TCIDs0 of SIVmac239. Seven out of the 8 iSlV/LP-immunized animals acquired a sterile immunity without any SIV RNA and DNA emergence in plasma and PBMC (Fig. 15b & c), as well as in rectal mucosa lymphocytes (where they were measured from day 1 post challenge) and pelvic lymph nodes (Fig. 16a to 16a) while one immunized monkey was fully infected. lmportantly, the evolution of the ex-vivo antiviral activity of the 8 vaccinated monkeys allowed to t from day 360 post immunization (i.e., 60 days before their challenge) the 7 protected monkeys and the unprotected one (Fig. 15a to C-II-Vl Conclusions it is disclosed herein, in the macaque model, that the administration of inactivated SIVmac239 (iSIV) and commensal Lactobacillus plantarum (LP) (referred to as a tolerogenic adjuvant) generates MHC-lbIE-restricted CD8+ T cells that induced the suppression of activation of SIV antigen-presenting CD4+ T cells and thereby the suppression of SIV replication and the protection of es from SIV challenges. 2012/000857 A mixture made of inactivated iSIV and LP was administered intragastrically to a total of 16 animals and 15 controls. Four to 14 months later, all animals were challenged intrarectally with pathogenic SleacZ39.

Full protection t SIV infection was observed in 15 out of 16 iSlV/LP-administered animals; in contrast, infection was established in all control animals and one vaccinated monkey. The unprotected monkey can be predicted by an ex vivo antiviral assay 60 days before the intrarectal challenge. Eight protected animals remained protected afler a second Sleac239 nge given intravenously in 4 monkeys and intrarectally in the other4.

The 8 iSlV/LP-delivered animals had complete lack of SIV-specific peripheral blood CD4+ T cell proliferation and did not raise any systemic SIV-specific dies (lgG, lgM, or lgA).

Moreover, their SIV-specific peripheral blood CD8+ T cell had several particularities: 1) they proliferated well but t interferon-y secretion upon to in vitro stimulation; 2) they strongly suppressed the activation of acutely infected autologous CD4+T cell; 3) both functions remained unchanged after depletion of CD25+ cells; 4) they ted also SIV replication in acutely infected allogenic CD4+ T cells; and ) their suppressive/inhibiting action was MHC—lb/E-restricted.

These results show that intra-gastric co-administration of iSlV and LP altows macaques to p virus-specific totoxic /E-restricted CD8+ tory T cells which generate an SIV-specific tolerance and that very surprisingly such a virus-specific immunotolerance is associated with vaccine protection of animals t the establishment of SIV infection.

It is shown hereinabove that the ceutical composition according to the present invention prevents HIV and SIV infections in humans/mammals. This preventive action is obtained in macaques by inducing a "Ts'I immunotolerance in the tolerogenically- vaccinated subjects (i.e., the mammals having been administered the pharmaceutical com position) . Said "Ts" immunotolerance is herein demonstrated to involve virus- specific non-cytotoxic MHC-lb/E-restricted suppressive CD8 regulatory T cells,the presence and the activity of which being shown to: - inhibit SIV replication in acutely-infected CD4+T cells of es having been administered the pharmaceutical composition of the present invention (in vitro); and/or - preve nt SIV replication in toleroge nically-va cci nated macaques that are challenged with infectious SIV (in vivo).

The reference in this specification to any prior publication (or ation d from it), orto any matterwhich is known, is not, and should not be taken as an acknowledgment or admission or any form of tion thatthat prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Throughout this specification and the claims which follow, unless the context requires othen~ise1 the word “comprise”, and variations such as “comprises" and “comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

, PCT/[32012I000857 NCES Morgan 0, et al. Plos Medicine 2008, 5:1200—1204 des MC, et al. Viral lmmunol 2006,19:679-689 Stahl-Hennig C, et al. J Med Primatol 2007,36z195-205 Chen S, et al. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2008,30:156—160 Hu et al., JAMA 0—216, 1996 Korber et al., Science 280:1868-1871, 1998 Papathanasopoulos MA, et al. Virus Genes 2003,262151-163 RAVIV etal. J. Virol., vol.79(19), p: 12394-12400, 2005 Chien, Novel Drug Delivery Systems, Ch. 4 (Marcel Dekker, 1992) Ullmann's Encyclopedia of Industrial Chemistry, 6’" Ed. (various editors, 1989-1998, Marcel Dekker) Pharmaceutical Dosage Forms and Drug Delivery Systems (ANSEL et al., 1994, WILLIAMS &W|LK|NS) Ebadi, Pharmacology, Little, Brown and 00., Boston, Mass. (1985) WONG at al. Proc. Natl. Acad. Sci. USA, 4(8), p:2591-2595, 2007 ; Fuller. J.Appl. Bacterial. 66:365-378 (1989) I. Mederle, R. Le Grand, B. Vaslin et al., e 21 (27-30), 4153 (2003) W. Lu, X. Wu, Y. Lu et al., Nature medicine 9 (1), 27 (2003) A. S. Fauci, Nature 384 (6609), 529 (1996) Faria and Weiner, lmmunoi Rev 206, 232 (2005) Mestecky et al., J lmmunol 179 (9), 5633 (2007) HOFT etal. (J infect Dis, vol. 198(10), -1 501 .2008) WANG et al (Med Microbiol Immunol. , 2008 May 20) Rerks—Ngarm et al. N. Engl. J. Med. Vaccination with ALVAC and AiDSVAX to prevent HIV-1 infection in Thailand, 2009 Oct. 20 (epub) McEIrath et al. The Lancet, Volume 372, Issue 9653, Pages 1894 - 1905, 29 November 2008 Tsai et al., tory animal science 43 (5), 411 (1993) Wei Li Lee et al. Small, (p 1003—1011) Published Online: Mar 31 2010 12:16PM DOI: 10.1 OO2/smll.200901 985 PCT/[32012/000857 Delie F. Adv Drug Deliv Rev. 1998;34:221-233 Mathiowitz et al. Nature. 86:410-414 Goldberg et al. Nat Rev Drug Discov. 2003;2z289-295 Fasano et al. J Clin Invest. 1997;99:1 158-1 164 Sandri et al. EurJ Pharm Biopharm. 2007;65:68-77 Chickering et al. J Control Release. 1997;48:35"*6 Ponchel et al. Adv Drug Deliv Rev. 1998;34:191-219 Plotkin et al. Vaccines, Saunders Elsevier Fifth n, 2008, pages 215 & 216 Korin and Zack, Journal of virology 73 (8), 6526 (1999) Vatakis et al., Journal of virology 83 (12), 6222 (2009a) Vatakis et al., Journal of virology 83 (7), 3374 (2009b) Andrieu and Lu, l Today 16 (1), 5 (1995) i Sacha et al., J lmmunoi 178 (5), 2746 (2007) Schmitz et al., The Americanjournal ofpathology 154 (6), 1923 (1999) Liew et al., 2010. Journal of Biotechnology 150:224-231 Plummer and Manchester, iral nanoparticles and virus-like particles: platforms for porary vaccine design. John Wiley & Sons, Inc. WIREs Nanomedicine and Nanobiotechnology. DOI: 2/wnan.119 Ke-Qin Xin et al. Blood 102 (1), 223-228 (1 July 2003) Jae-Sung Yu et al. Clinical and Vaccine Immunology 13(11), 1204-121 1 (Nov. 2006) Scholzen and Gerdes. J. Cell Physiol. 182,31 1-322 (March 2000) Muhl, et al. MHC class lalleles influence set-point viral load and survival time in simian immunodeficiency virus—infected rhesus monkeys. J Immunol 169, 3438-3446 (2002) Loffredo etal.

; Mamu-B*08-positive es control simian immunodeficiency virus .! 25 replication. Journal of virology 81, 832 (2007) Sarantopoulos et al. Qa-1 ction of CD8+ ssor T cells. The Journal of clinical investigation 114, 1218-1221 (2004) Jiang ei al. HLA-E-restricted regulatory CD8(+) T cells are involved in development and control of human autoimmune type 1 diabetes. The Journal of clinical investigation 120, 3641-3650 (2010) WO 37071 PCT/[32012/000857 Van Kaer, L. Comeback kids: CDB(+) suppressor T cells are back in the game. The Journal of clinical investigation 120, 3432-3434 (2010) Marchetti, P., eta]. Mitochondrial permeability transition is a central coordinating event of apoptosis. The Journal of experimental medicine 184, 1155-1 160 (1996)

Claims (11)

1. CLAIMS 1.
2. A pharmaceutical ition comprising a mixture of a ulate antigen having one or more epitopes from HIV Gag and/or Pol proteins and a non-pathogenic bacterium.
3. The pharmaceutical composition according to claim 1, wherein said particulate antigen is selected from virus particles, virus-like les, recombinant virus particles, conjugate viral proteins and concatemer viral ns.
4. The pharmaceutical composition according to claim 2, wherein said recombinant virus particles or said virus particles are inactivated.
5. The pharmaceutical composition according to any one of claims 1 to 3, wherein said 15 ium is selected from attenuated pathogenic bacteria, inactivated pathogenic bacteria, and non-pathogenic lactic acid bacteria.
6. The pharmaceutical composition according to claim 4, wherein said bacterium is a Lactobacillus bacterium.
7. The pharmaceutical composition according to claim 4, wherein said bacterium is BCG.
8. The ceutical composition according to any one of claims 1 to 6, wherein the 25 composition is a mucosal or intradermal or intraepithelial ceutical composition.
9. The pharmaceutical composition according to claim 7, wherein the composition is an oral pharmaceutical composition. 30 The pharmaceutical composition according to any one of claims 1 to 8, wherein the composition induces an antigen-specific immune protection against HIV in a human.
10. 10. The pharmaceutical composition ing to claim 9, wherein the composition maintains said antigen-specific immune protection against HIV in a human.
11. 11. The pharmaceutical composition according to claim 9 or 10, wherein said immune tion ses the reduction of an HIV viral load in said human.