AU2022209359B2 - Multivalent feline vaccine - Google Patents

Abstract

MULTIVALENT FELINE VACCINE The present invention provides new multivalent vaccines for felines. The present invention also provides methods of making and using the multivalent vaccines alone or in combinations with other protective agents. 2022 MULTIVALENT FELINE VACCINE

Description

MULTIVALENTFELINE FELINEVACCINE VACCINE 2022209359 30 Jul 2022

MULTIVALENT

CROSS-REFERENCETOTORELATED CROSS-REFERENCE RELATEDAPPLICATIONS APPLICATIONS This applicationisisa adivisional This application divisionalof of Australian Australian Patent Patent Application Application No. 2018383915 No. 2018383915 (the (the national phase national application of phase application of PCT/EP2018/080106) PCT/EP2018/080106) and and claims claims priority priority of of provisional provisional

application application U.S. U.S. Serial Serial No. No. 62/599,401 filed 15 62/599,401 filed 15 December 2017, December 2017, U.S.Serial U.S. SerialNo. No.62/596,508 62/596,508 filed 88December filed 2017,U.S. December 2017, U.S.Serial SerialNo. No.62/582,050 62/582,050 filed6 6November filed November 2017, 2017, and and U.S. U.S. Serial Serial

No. 62/581,955 No. 62/581,955filed filed 66 November November 2017, 2017, thethe contents contents of of each each of of which which areare hereby hereby

incorporatedby by incorporated reference reference in their in their entireties. entireties.

FIELD OF FIELD OF THE INVENTION THE INVENTION The present The presentinvention invention relates relates to to new multivalent vaccines new multivalent vaccinesfor for felines. felines. Methods of Methods of

makingand making andusing usingthe themultivalent multivalentvaccines vaccinesalone aloneororinin combination combinationwith withother otherprotective protective agents are also agents are also provided. provided.

BACKGROUND BACKGROUND Feline respiratory Feline respiratorydisease disease includes includes those those illnesses illnesses typified typified by rhinosinusitis, by rhinosinusitis,

conjunctivitis, conjunctivitis,lacrimation, salivation, lacrimation, andand salivation, oraloral ulcerations. The The ulcerations. two two most common most common pathogens pathogens

associated with associated with upper upper respiratory respiratory disease disease in catsinare cats are calicivirus feline feline calicivirus (FCV) (FCV) and felineand feline viral viral

rhinotracheitisvirus rhinotracheitis virus(FVR), (FVR), which which is also is also knownknown as feline as feline herpesvirus herpesvirus type(FHV-1). type 1 virus 1 virus (FHV-1). Thesetwo These twofeline feline viruses viruses are are considered consideredresponsible responsiblefor forapproximately approximately80% 80% of of allallfeline feline respiratory disease, respiratory disease, worldwide. bacterium, Chlamydophila A bacterium, worldwide. A Chlamydophila felis,is felis, is aa third third pathogen that pathogen that

also canplay also can playa role a role in in felinerespiratory feline respiratory disease. disease. Accordingly, Accordingly, vaccines vaccines against against these these three three

pathogensare pathogens arenow now commercially commercially available. available.

FVRisis an FVR an alphaherpesvirus alphaherpesvirusthat thatisis related related to to canine herpesvirus-1 and canine herpesvirus-1 andisis perhaps perhapsthe the most important most importantof of respiratory respiratory pathogens. FVRisisa alarge pathogens. FVR largeenveloped envelopedDNADNA virus virus that that is is extremely contagiousand extremely contagious andcan can lead lead totosevere severe disease disease in in kittens,asaswell kittens, well as as cats. cats. Accordingly, most Accordingly, mostcats cats are are exposed exposedtotoFVR FVR during during theirlifetime. their lifetime. The Thecommercially commercially available available

Nobivac© Nobivac® Feline-1 Feline-1 vaccine vaccine contains contains a modified a modified liveviral live feline felinerhinotrachesitis viral rhinotrachesitis virus. virus.

1

2

Themost The most common common characteristic characteristic and clinical and clinical signs signs of FCV of FCV infection infection are the are the developmentofofvesicles development vesicles (ulcers) (ulcers) on on the the tongue tongue and and oral oral mucosa, which begin mucosa, which begin as as small, individual, small, individual, ulcers ulcers but but can canspread spreadandand affect affect a large a large partpart of the of the tongue. tongue. Fever Fever often also often also is is observed observed in ininfected infected cats. cats. Certain Certain strains strains of FCV of FCV also cause also cause a disease a disease

55 in cats in knownasaslimping cats known limping syndrome, syndrome, which which is characterized is characterized by fever, fever,and byjoint joint and musclesoreness muscle soreness (limping), (limping), and and occasional occasional lingual/oral lingual/oral ulceration. ulceration. In addition, In addition, some some strains of strains of FCV FCVhave have been been associated associated with chronic with chronic stomatitis stomatitis in infected in infected cats. cats. Cats Cats infected with infected with FCV maybecome FCV may become persistentlyinfected, persistently infected, and and may mayshed shedinfectious infectiousvirus virus for long for periodsofoftime. long periods time. 00 FCVisolates FCV isolatesareare antigenically antigenically highly highly variable, variable, andand antibodies antibodies from from cats cats vaccinatedwith vaccinated witholder oldervaccine vaccine strains strains of FCV, of FCV, such such as FCVas FCV F9, F9, efficiently do not do not efficiently neutralize all neutralize all current field isolates. current field Moreover,newnew isolates. Moreover, FCV FCV strains strains associated associated with with systemicdisease systemic diseaseandand highhigh mortality mortality have have been identified been identified [seeU.S.

[see e.g., e.g.,7,449,323 U.S. 7,449,323 55 B2]. These B2]. These "virulent "virulent systemic" systemic" (VS-FCV) (VS-FCV) isolates isolates are responsible are responsible for localized for localized

outbreaks,and outbreaks, and current current vaccines vaccines also also doappear do not not appear to protect to protect catsdisease cats from from disease caused caused byby these these strains. strains.

FCVcomprises FCV comprisesa asingle-stranded, single-stranded,positive-sense positive-senseRNA RNA genome genome consisting consisting of of 10 :0 three open three reading frames open reading (ORFs).The frames (ORFs). The genome genome is polyadenylated is polyadenylated at the at the 3'end 3' end

and bound and bound by by a virally-encoded a virally-encoded protein protein at 5'-end. at the the 5'-end. The open The first first reading open reading frame frame encodes viral protease encodes a aviral protease and an RNA-dependent and an RNA-dependent RNARNA polymerase, polymerase, whichwhich are are

expressedon on expressed a single a single polypeptide. polypeptide. This This polypeptide polypeptide then isthen is post-translationally post-translationally

cleaved by the cleaved by the viral viralprotease. protease. The The second openreading second open readingframe frameencodes encodesthethe major major

25 25 capsid protein(i.e., capsid protein (i.e., the FCVcapsid the FCV capsid protein), protein), which which has has six regions six regions denoted denoted as A-F as A-F

[Scott et

[Scott etal., al.,60 60 Am.Am.J. J. Vet.Vet. Res.:652-658 (1999)].Region :652-658 (1999)]. Region A is is cleaved A cleaved to to produce produce

the mature the capsid protein. mature capsid protein. Whereas regionsB,B,D,D,and Whereas regions andF FofofORF2 ORF2areare relatively relatively

conserved betweenFCVFCV conserved between regionsC Cand isolates,regions isolates, and E are E are variable, with region variable,with region EE of of ORF2 the the containing ORF2 containing major major B-cell B-cell epitopes epitopes [see, [see, Radford Radford et al., et al., Vet 38(2) 38(2) Vet Res. Res.:319 :319-

30 30 335 (2007)]. ORF 335 (2007)]. encodes ORF3 3encodes a minor a minor structuralprotein structural [Sosnovtsevand protein [Sosnovtsev Green, andGreen, 277 277

Virology: 193-203 Virology: 193-203 (2000)]. (2000)].

3

Chlamydophilafelis Chlamydophila felis is is aabacterium bacterium that thatisis endemicamong endemic domestic among domestic

cats worldwide. cats worldwide.C.C.felis feliscan canlead leadto toinflammation inflammation of conjunctiva, of conjunctiva, rhinitis, rhinitis, and and

respiratory problems respiratory problems in in the the infected infected catcat C. C. felis felis hashas a relatively a relatively small small genome genome that that encodesonly encodes onlyabout aboutaathousand thousandproteins. proteins. This Thisbacterium bacteriumalso alsocontains containsa aplasmid plasmid 55 comprising 75,000 comprising 75,000base basepairs. pairs. The Thecommercially commerciallyavailable availableNobivac® Nobivac* Feline-1 Feline-1

vaccinecontains vaccine contains a modified a modified livelive Chlamydophila Chlamydophila felis. felis.

In addition In to the addition to three pathogens the three pathogens related related to feline to feline respiratory respiratory disease disease

discussedabove, discussed above, cats cats often often alsoalso are are vaccinated vaccinated against against threeviruses: three other other viruses: feline feline 00 leukemiavirus leukemia virus(FeLV), (FeLV), feline feline panleukopenia panleukopenia (FPV (FPV or or and FPLV), FPLV), andvirus. rabies rabies virus. Feline leukemia Feline leukemiavirus virusis isa aretrovirus retrovirusthat thatinfects infectsdomestic domestic cats, cats, resulting resulting in significant in significant

morbidity and morbidity mortality worldwide. and mortality worldwide. Though predominantlytransmitted Though predominantly transmittedthrough through saliva, FeLV saliva, has FeLV has been been reported reported also also to betospread be spread throughthrough contact contact with bodywith body fluids fluids

[Pacitti et

[Pacitti etal., al.,Vet VetRec Rec 118:381-384 (1986) 118:381-384 (1986) doi:10.1136/vr.118.14.381; doi:10.1136/vr.118.14.381; Levy et Levy al., Jet al., J 55 Feline Med Surg 10:300-316 (2008) doi:10.1016/j.jfms.2008.03.002]. TheTheclinical FelineMedSurg10:300-316(2008)doi:10.1016/j.jfms.2008.03.002]. clinical

signs in signs in cats cats observed observed during during FeLVFeLV infections infections include: include: cytoproliferative cytoproliferative disorders disorders

(lymphoidorormyeloid (lymphoid myeloid tumors), tumors), cytosuppressive cytosuppressive disorders disorders (infectious (infectious diseasesdiseases

associated with associated with immunosuppression, anemia, immunosuppression, anemia, myelosuppression), myelosuppression), inflammatory inflammatory

disorders,neurological disorders, neurologicaldisorders, disorders, abortions, abortions, and and enteritis enteritis [Hoover

[Hoover et al., et al., Am J Am JVet Vet 10 :0 MedAssoc Med Assoc199:1287-1297 199:1287-1297 (1991); (1991); Levy Levy and Crawford, and Crawford, Textbook Textbook of Veterinary of Veterinary

Internal Medicine, Internal Medicine, 6th 6thed ed(Ettinger (EttingerSJ, SJ,Feldman Feldman EC., EC.,eds.) eds.)WB WB Saunders, Saunders,

Philadelphia, PA. Philadelphia, PA. (2005)]. (2005)]. The The prevalence of antigenemia prevalence of mayvary antigenemia may varyfrom from1-5% 1-5%in in healthy cats healthy catstoto 15-30% 15-30% in afflicted in afflicted cats cats [Hosie

[Hosie et al. et al. Veterinary Veterinary Records, Records, 128: 128: 293- 293 297(1989); 297 (1989);Braley, Braley,Feline Feline Practice Practice 22: 22: 25-29 25-29 (1994); (1994); Malik Malik et Australian et al., al., Australian 25 25 VeterinaryJournal Veterinary Journal75:323-327 (1997); 75:323-327 (1997); Arjona Arjona et Journal et al., al., Journal of Clinical of Clinical Microbiology Microbiology

38:3448-3449 38:3448-3449 (2000)]. (2000)]. FeLV FeLV frequently frequently establishes establishes lasting infection a infection a lasting with a with a concomitant concomitant persistent persistent viremia, viremia, often often leading leading to the to the death death of theofhost the cat. host cat.

The single The single stranded stranded RNA RNAgenome genome of FeLV of FeLV encodes encodes only only threethree genes: genes: (i) (i) an an 30 30 gene,which ENVgene, ENV whichencodes encodes thethe envelope envelope glycoprotein, glycoprotein, (ii) aa GAG (ii) GAGgene, gene, which which

encodes encodes thethe major major structural structural components components of the of the virus, virus, anda (iii) and (iii) POLwhich POL agene, gene, which encodesthe encodes theRNA RNA polymerase polymerase [Thomsen

[Thomsen et al., et al., Journal Journal of of General General Virology Virology

73:1819-1824(1992)]. 73:1819-1824 (1992)]. The TheFeLV FeLV envelope envelope (ENV) (ENV) gene gene encodes encodes gp85 precursor a gp85a precursor

4

protein which protein whichisisproteolytically proteolytically processed processedby by one one or more or more cellular cellular enzymes enzymes to yieldto yield the major the major envelope glycoprotein gp70 envelope glycoprotein gp70and andthe theassociated associatedtransmembrane transmembrane protein protein

p15E [DeNoronha, p15E [DeNoronha,et et al., Virology al., Virology 85:617-621 (1978); Nunberg 85:617-621 (1978); Nunbergetetal., al., PNAS PNAS

81:3675-3679(1983)]. 81:3675-3679 (1983)]. The Thetransmembrane transmembrane protein protein p15Ep15E contains contains a sequence a sequence

55 conservedamong conserved among gammaretroviruses gammaretroviruses withwith immunosuppressive immunosuppressive properties properties [Mathes

[Mathes

et al., et al., Nature Nature274:687-689 (1978)]. Recently, 274:687-689 (1978)]. Recently, The TheEuropean EuropeanMedicines Medicines Agency's Agency's

Committeefor Committee for Medicinal Medicinal Products Productsfor for Veterinary Veterinary Use (CVMP)hashas Use (CVMP) adopted adopted a a positive opinion positive opinionfor vaccine fora a vaccinecomprising comprisinga arecombinant recombinant p45 p45 FeLV-envelope antigen FeLV-envelope antigen

derivedfrom derived fromthe thegp70 gp70 surface surface glycoprotein glycoprotein of theofFeLV the subgroup FeLV subgroup A that isA that is 00 expressedinin Escherichia expressed Escherichia coli colias as active activesubstance. substance. The FeLVenvelope The FeLV envelopeglycoprotein glycoprotein is the is the target target of of FeLV-specific cytotoxicT T FeLV-specific cytotoxic cellresponses, cell responses, as well as well as neutralizing as neutralizing

antibodiesand antibodies andaccordingly, accordingly, one one of the of the major major immunogens immunogens of FeLV of FeLV [Flynn [Flynn et al., J. et al., J. Virol. 76(5): Virol. 76(5): 2306-2315 (2002)]. 2306-2315 (2002)].

55 Feline panleukopenia Feline panleukopenia (FPV(FPV or FPLV) or FPLV) is a highly is a highly contagious contagious viral disease viral disease of of cats that cats that is is often often fatal. fatal.The namepanleukopenia The name panleukopenia derives derives from from the lowthe lowblood white white blood cell count cell (leucocytes)exhibited count (leucocytes) exhibitedby by affected affected animals. animals. FPLV infects FPLV infects and destroys and destroys

actively dividing actively cells in dividing cells in lymphoid tissues,bone lymphoid tissues, bone marrow, marrow, intestinal intestinal epithelium, epithelium, and and in in very young very youngkittens, kittens,the theretina retinaandand cerebellum. cerebellum. The virus The virus also also may may inspread spread in D 0 pregnantcats pregnant catstransplacentally transplacentally to to cause cause embryonic embryonic resorption, resorption, fetal mummification, fetal mummification,

stillbirth, oror stillbirth, abortion. abortion. Infected Infected cats cats shed thevirus shed the virusinin their their urine, urine, stool, stool, and nasal and nasal

secretionsresulting secretions resultinginin infection infectioninin susceptible susceptiblecats cats when when theythey come come in contact in contact with with thesesecretions these secretionsororfleas fleasfrom from infected infected cats. cats. The The clinical clinical signs signs due due to an to an FPLV FPLV infection have infection havealso alsobeen been labeled labeled as feline as feline distemper distemper or feline or feline parvo. parvo.

25 25

Feline panleukopenia Feline panleukopenia virus virus is aismember a member of theofgenus the genus Parvovirus, Parvovirus, in the in the family Parvoviridae. family Parvoviridae.Accordingly, Accordingly, FPLVFPLV is closely is closely related related to enteritis to mink mink enteritis virus virus and and canine Type canine Type22 parvovirus parvovirus (CPV-2). (CPV-2). FPLV FPLV hashas a single a single stranded stranded DNADNA genome genome that that has been has been sequenced sequenced [see,Liu

[see, Liuetetal., al., Genome Announc. Genome Announc. Mar-Apr; Mar-Apr; 3(2) 3(2) (2015): (2015):

30 30 Thecommercially e01556-14]. The e01556-14]. commercially available Nobivac*Feline-i availableNobivac® vaccine Feline-1 vaccine contains contains a a modifiedlive modified live feline feline panleukopenia panleukopenia virus. virus.

5

Rabiesisisaapreventable Rabies preventable zoonotic zoonotic disease disease that leads that leads to inflammation to inflammation of the of the brain brain in inhumans and other humans and other mammals. mammals. Clinicalrabies Clinical rabiesisisan an acute, acute, progressive progressive encephalitisthat encephalitis thatisis typically typically classified classified as either furious as either furious or or paralytic paralytic rabies. rabies. Furious Furious rabies is characterized rabies is characterized byby restlessness, restlessness, irritabilityand irritability andaggression. aggression. Paralytic Paralytic rabies rabies

55 is characterized is characterized bybyexcessive excessive salivation, salivation, deep, deep, labored labored breathing, breathing, paralysis, paralysis, and and eventuallycoma. eventually coma.The The causative causative agent agent of rabies of rabies is the is the rabies rabies virus, virus, which which is is capable capable

of infecting of infecting most mostmammals, mammals, including including felines, felines, and maintains and maintains a reservoir a reservoir of disease of disease in in wild and wild and susceptible susceptible domestic animals. domestic animals.

00 Therabies The virusisisananenveloped, rabiesvirus enveloped,RNA RNA virus virus that encodes that encodes five structural five structural

proteins: aa nucleoprotein proteins: nucleoprotein(N), (N),a aphosphoprotein phosphoprotein (P), (P), a a matrix matrix protein protein (M), a(M), a glycoprotein (G), glycoprotein (G), and and an an RNA-dependent RNA-dependent RNARNA polymerase polymerase [Dietzschold

[Dietzschold et al., et al., CritCrit RevImmunol Rev Immunol10:427-439 10:427-439 (1991)].TheThe (1991)]. glycoprotein glycoprotein (G)(G) is is considered considered theprotective the protective antigen whichinduces antigen which induces virus virus neutralizing neutralizing antibodies antibodies [Cox [Cox et Infect et al., al., Infect immun Immun 16:754-16:754

55 759 (1977)]. 759 (1977)]. Several types of Several types rabies vaccines of rabies vaccines have been produced have been combat producedtotocombat this this

disease.Inactivated disease. Inactivated cellculture cell culture derived derived whole-virus whole-virus killed killed rabies rabies virusvirus vaccines vaccines are are the most the commonlyused most commonly used vaccines vaccines in in theUnited the UnitedStates. States.These These whole-virus whole-virus killed killed

rabies virus rabies virus vaccines vaccinesrequire require high high levels levels of of antigen antigen and and therefore, therefore, require require an an adjuvant. Unfortunately, adjuvant. Unfortunately, this this useuse of adjuvant of an an adjuvant is associated is associated with injection with injection site site 10 :0 reactivity, hypersensitivity, reactivity, hypersensitivity, and evenwith and even perceived withthetheperceived riskrisk of injection of injection site site

sarcomasinin cats. sarcomas cats. Recently, Recently, aa modified modified live live vaccine vaccine has has been used successfully been used successfully with oral with oral vaccine vaccinebaits baitsfor forthe theimmunization immunization of wild of wild animals animals [Mahl[Mahl et Vet et al., Vet al., Res Res 45(1):77(2014)]. 45(1):77 (2014)].In Inaddition, recombinant addition,a arecombinant vaccine vaccine expressing expressing the glycoprotein the glycoprotein

(G) is currently (G) is marketed being marketed currently being in the in the United United States States for in for use usecats. in cats. acid acid Nucleic Nucleic

25 25 vaccinesalso vaccines alsohave have used used been been in laboratory in laboratory studies, studies, thoughthough none arenone are currently currently

licensedinin the licensed the United States. UnitedStates.

numberofofvector A number A vector strategies strategies have beenemployed have been employedthrough through thethe years years for for

vaccinesininananeffort vaccines efforttoto protect protectagainst againstcertain certainpathogens. pathogens. Onevector One such such strategy vector strategy 30 30 includesthe includes theuse alphavirus-derived useofofalphavirus-derived replicon replicon RNA particles RNA particles (RP) [Vander (RP) [Vander Veen, et Veen, et al. al.Anim Anim Health Health Res 13(1):1-9. (2012) Rev. 13(1):1-9. Res Rev. (2012) doi: doi: 10.1017/S1466252312000011; 10.1017/S1466252312000011;,

Kamrudetetal., Kamrud al., JJ Gen Virol. 91(Pt Gen Virol. 91(Pt7):1723-1727 (2010)] which 7):1723-1727 (2010)] which have have been developed been developed from several different alphaviruses, including Venezuelan equine encephalitis virus 17 Sep 2025

(VEE) [Pushko et al., Virology 239:389-401 (1997)], Sindbis (SIN) [Bredenbeek et al., Journal of Virology 67:6439-6446 (1993)], and Semliki Forest virus (SFV)

[Liljestrom and Garoff, Biotechnology (NY) 9:1356- 1361 (1991)]. RP vaccines deliver propagation-defective alphavirus RNA replicons into host cells and result in the expression of the desired antigenic transgene(s) in vivo [Pushko et al., Virology 239(2):389-401 (1997)]. RPs have an attractive safety and efficacy profile when 2022209359

compared to some traditional vaccine formulations [Vander Veen, et al. Anim Health Res Rev. 13(1):1-9. (2012) ]. The RP platform has been used to encode pathogenic antigens and is the basis for several USDA-licensed vaccines for swine and poultry.

Notably, alphavirus RNA replicon particles, and in particular Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particles, have been reported to catalyze a systemic antiviral state and protect against lethal virus challenge

[Konopka et al., J. Virol., 83 (29):12432-12442 (2009)] and more particularly, induce rapid protection against foot-and-mouth disease virus [Segundo et al., J. Virol., 87 (10):5447-5460 (2013)]. Accordingly, because alphavirus RNA replicon particles enhance the innate immune response against live virus it would appear that they should not be combined with a modified live virus in a vaccine because it would be expected to be detrimental to the immune response.

The citation of any reference herein should not be construed as an admission that such reference is available as "prior art" to the instant application.

SUMMARY OF THE INVENTION In a first aspect, the present invention provides a vaccine to aid in the prevention of disease due to feline leukemia virus (FeLV) comprising an alphavirus RNA replicon particle that encodes one or more FeLV glycoproteins and one or more modified live feline pathogens selected from the group consisting of a modified live feline calicivirus (FCV), a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), a modified live Chlamydophila felis, and any combination thereof, and a pharmaceutically acceptable carrier.

6a

In a second aspect, the present invention provides a vaccine to aid in the 17 Sep 2025

prevention of disease due to feline leukemia virus (FeLV) comprising an alphavirus RNA replicon particle that encodes one or more FeLV glycoproteins and modified live feline pathogens comprising a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), and a modified live Chlamydophila felis, and a pharmaceutically acceptable carrier. 2022209359

In a third aspect, the present invention provides a vaccine to aid in the prevention of disease due to feline leukemia virus (FeLV) comprising an alphavirus RNA replicon particle that encodes one or more FeLV glycoproteins and modified live feline pathogens comprising a modified live feline calicivirus (FCV), a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), and a modified live Chlamydophila felis, and a pharmaceutically acceptable carrier.

In a fourth aspect, the present invention provides a vaccine to aid in the prevention of disease due to feline leukemia virus (FeLV) comprising a modified live feline calicivirus (FCV), a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), a modified live Chlamydophila felis, and a Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle encoding an FeLV glycoprotein gp85 that comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 6.

In a fifth aspect, the present invention provides a method of immunizing a feline against a pathogenic FeLV comprising administering to the feline an immunologically effective amount of the vaccine of the first, second, third or fourth aspect.

In a sixth aspect, the present invention provides a method of immunizing a feline against a pathogenic FeLV comprising administering to the feline an immunologically effective amount of the vaccine of the first aspect.

6b

In a seventh aspect, the present invention provides a method of immunizing a 17 Sep 2025

feline against a pathogenic FeLV comprising administering to the feline an immunologically effective amount of the vaccine of the second aspect.

In an eighth aspect, the present invention provides a method of immunizing a feline against a pathogenic FeLV comprising administering to the feline an immunologically effective amount of the vaccine of the third aspect. 2022209359

In a ninth aspect, the present invention provides a method of immunizing a feline against a pathogenic FeLV comprising administering to the feline an immunologically effective amount of the vaccine of the fourth aspect. Accordingly, the present invention includes immunogenic compositions that comprise alphavirus RNA replicon particles that encode one or more antigens from one or more feline pathogens along with one or more modified live feline pathogens. All of the immunogenic compositions of the present invention also may be used in multivalent vaccines. In a particular embodiment of this type, the vaccinated subject is a feline. In a more particular embodiment, the vaccinated subject is a domestic cat. Methods of making and using the immunogenic compositions and vaccines of the present invention are also provided.

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In particular In particularembodiments, embodiments, the the immunogenic compositions immunogenic compositions comprise comprise

alphavirus RNA alphavirus RNA replicon replicon particles particles thatthat encode encode one orone moreorfeline more calicivirus feline calicivirus (FCV) (FCV)

antigens and aa modified antigens and modified live live feline felinepathogen. pathogen. InInother otherembodiments, embodiments, the the

55 immunogeniccompositions immunogenic compositions comprise comprise alphavirus alphavirus RNARNA replicon replicon particles particles thatencode that encode oneorormore one more felineleukemia feline leukemia virus virus (FeLV) (FeLV) antigens antigens and a modified and a modified live live feline feline pathogen. InIn yet pathogen. yet other other embodiments, theimmunogenic embodiments, the immunogenic compositions compositions comprise comprise

alphavirus RNA alphavirus RNA replicon replicon particles particles thatthat encode encode one orone moreorrabies more virus rabiesantigens virus antigens and and modifiedlive a modified live feline feline pathogen. pathogen.In certain In certain embodiments, embodiments, the modified the modified live feline live feline

00 pathogenis isa afeline pathogen felineviral viral rhinotracheitis rhinotracheitisvirus virus(FVR). (FVR).In In other other embodiments, embodiments, the the modifiedlive modified live feline feline pathogen pathogen is is a a felinepanleukopenia feline panleukopenia virusvirus (FPLV). (FPLV). In yet In yet other other embodiments, embodiments, the the modified modified live live feline feline pathogen pathogen is a modified is a modified live Ch/amydophila live Chlamydophila

felis. In felis. In still stillother embodiments, other themodified embodiments, the modified live live felinepathogen feline pathogen is a ismodified a modified live live F9-like feline F9-like feline calicivirus calicivirus (FCV F9-Like).In Inyetyetother (FCV F9-Like). other embodiments, embodiments, the modified the modified live live 55 feline pathogen feline pathogen isisa amodified modified live live Bordetella Bordetella bronchiseptica. bronchiseptica. The present The present invention invention

further provides further provides immunogenic compositionsthat immunogenic compositions thatcomprise compriseany anycombination combination of of these these

alphavirus RNA alphavirus RNA replicon replicon particles particles and and modified modified live feline live feline pathogens. pathogens. In specific In specific

embodimentsofofthis embodiments this type, type, the the immunogenic compositioncomprises immunogenic composition comprises alphavirus alphavirus RNARNA

replicon particles replicon particles encoding encodingan an FCVFCV antigen, antigen, alphavirus alphavirus RNA replicon RNA replicon particlesparticles

.0 0 encoding an FeLV encoding an FeLVantigen, antigen,a amodified modifiedlive live FVR, FVR, aa modified modified live live FPLV, and aa FPLV, and

modifiedlive modified live Chlamydophila Ch/amydophila felis. felis.

In certain In certainembodiments of this embodiments of this type, type,the theimmunogenic compositionscomprise immunogenic compositions comprise alphavirus alphavirus RNA replicon particles RNA replicon particles that thatencode encode an an FCV capsidprotein. FCV capsid protein. In In more more 25 25 particular embodiments, particular the FCV embodiments, the FCVcapsid capsidprotein protein is is an FCVF9-Like an FCV F9-Likecapsid capsidprotein. protein. In other In otherembodiments, the alphavirus embodiments, the alphavirus RNA RNAreplicon replicon particle particle encodes an antigenic encodes an antigenic fragment of an fragment of FCVF9-Like an FCV F9-Likecapsid capsidprotein. protein. InIn yet yet other other embodiments, theFCV embodiments, the FCV capsid proteinisisaa virulent capsid protein virulent systemic systemicFCVFCV (VS-FCV) (VS-FCV) capsid capsid protein.protein. In still In still other other

embodiments,the embodiments, thealphavirus alphavirusRNA RNA repliconparticle replicon particle encodes encodesananantigenic antigenicfragment fragment 30 30 of aa VS-FCV of capsidprotein. VS-FCV capsid protein. InIn yet yet other other embodiments, thealphavirus embodiments, the alphavirus RNA RNA replicon replicon

particle encodes particle both encodes both an an FCV FCV F9-Like F9-Like capsidcapsid proteinprotein or antigenic or antigenic fragmentfragment thereof, thereof, and an VS-FCV and an VS-FCVcapsid capsid proteinororantigenic protein antigenicfragment fragmentthereof. thereof.

In other In otherembodiments, the immunogenic embodiments, the immunogenic compositions compositions comprise comprise alphavirus alphavirus

RNAreplicon RNA replicon particles particles that thatencode encode aa VS-FCV capsidprotein VS-FCV capsid proteinoror antigenic antigenic fragment fragment

thereof ininwhich thereof which the theVS-FCV capsidprotein VS-FCV capsid protein comprises comprisesananamino aminoacid acidsequence sequence comprising 95% comprising 95%identity identity or or more with the more with the amino acid sequence amino acid sequenceofofSEQ SEQID ID NO: NO: 2. 2. In In 55 more specific more specific embodiments, theVS-FCV embodiments, the VS-FCV capsid capsid protein protein comprises comprises thethe amino amino acidacid

sequenceofofSEQ sequence SEQID ID NO: NO: 2. 2. In In even even more more specific specific embodiments, embodiments, the the VS-FCV VS-FCV

capsid protein capsid protein isisencoded encoded by by the the nucleotide nucleotide sequence of SEQ sequence of SEQIDIDNO: NO:1 1ororSEQ SEQID ID NO: 12. NO: 12. InIn related related embodiments, embodiments, ananalphavirus alphavirusRNA RNA repliconparticle replicon particleof of the the present present

invention encodes invention FCVF9-Like encodes aa FCV F9-Likecapsid capsidprotein proteinoror antigenic antigenic fragment fragment thereof. thereof. In In 00 specific embodiments specific ofthis embodiments of this type, type, the theFCV F9-Like capsid FCV F9-Like protein comprises capsid protein an comprises an

amino acid amino acid sequence sequencecomprising comprising95%95% identityorormore identity more withthe with theamino amino acid acid

sequenceofofSEQ sequence SEQID ID NO: NO: 4. 4. In In more more specific specific embodiments, embodiments, the the FCV FCV F9-Like F9-Like capsid capsid

protein comprises protein the amino comprises the acid sequence amino acid sequenceofofSEQ SEQID ID NO:NO: 4. 4. In even In even more more specific specific

embodimentsof ofthis embodiments this type, type, the the FCV F9-Like capsid FCV F9-Like capsidprotein protein is is encoded by the encoded by the 55 nucleotide sequence nucleotide ofSEQ sequence of SEQIDIDNO: NO: 3 orSEQ 3 or SEQ ID NO: ID NO: 13. 13.

In related In relatedembodiments, the immunogenic embodiments, the immunogeniccompositions compositions comprise comprise alphavirus alphavirus

RNAreplicon RNA replicon particles particles that that encode encode onemore one or or more feline feline leukemia leukemia virus virus (FeLV) (FeLV) antigens. InIncertain antigens. certainembodiments, embodiments, the antigen the FeLV FeLV antigen is glycoprotein is a FeLV a FeLV glycoprotein (e.g., (e.g., 10 :0 gp85). In gp85). In other other embodiments, thealphavirus embodiments, the alphavirusRNA RNA repliconparticle replicon particle encodes encodesanan antigenic fragment antigenic fragment of of the the FeLV gp85. InIn more FeLV gp85. morespecific specific embodiments embodiments ofof this type, this type, the antigenic the antigenicfragment fragmentof of thethe FeLV FeLV glycoprotein glycoprotein is FeLV is FeLV gp70. gp70. In In a a related related embodiment,the embodiment, theantigenic antigenicfragment fragmentofofthe the FeLV FeLVglycoprotein glycoproteinisis FeLV FeLVgp45. gp45.InIn more specific more specific embodiments embodiments ofofthis this type, type, the the FeLV gp85comprises FeLV gp85 comprisesananamino amino acid acid

25 25 sequencecomprising sequence comprising95% 95% identityorormore identity morewith withthe theamino aminoacid acidsequence sequenceof of SEQSEQ ID ID NO: 6. NO: 6. In In more more specific specific embodiments embodiments ofofthis this type, type, the the FeLV gp85comprises FeLV gp85 comprisesthe the amino acid amino acid sequence sequenceofofSEQ SEQID ID NO:NO: 6. 6. In even In even more more specific specific embodiments embodiments of this of this

type, the type, the FeLV gp85is FeLV gp85 is encoded encodedbybythe thenucleotide nucleotide sequence sequenceofofSEQ SEQID ID NO:NO: 5 5 or or SEQIDIDNO: SEQ NO:14.14.In Inrelated relatedembodiments, embodiments,thethe FeLV FeLV gp70gp70 comprises comprises an amino an amino acid acid 30 30 sequencecomprising sequence comprising95% 95% identityorormore identity morewith withthe theamino aminoacid acidsequence sequenceof of SEQSEQ ID ID NO: 8. NO: 8. In In more more specific specific embodiments embodiments ofofthis this type, type, the the FeLV gp70comprises FeLV gp70 comprisesthe the amino acid amino acid sequence sequenceofofSEQ SEQID ID NO:NO: 8. 8. In even In even more more specific specific embodiments embodiments of this of this

9

type, the type, the FeLV gp70 is FeLV gp70 is encoded encodedbybythe thenucleotide nucleotide sequence sequenceofofSEQ SEQID ID NO:NO: 7 7 or or SEQID SEQ ID NO: NO: 15. 15.

In yet In yetother otherembodiments the immunogenic embodiments the immunogenic compositions compositions comprise comprise

55 alphavirusRNA alphavirus RNA replicon replicon particles particles thatthat encode encode a rabies a rabies virus glycoprotein virus glycoprotein (G). In (G). stillIn still other embodiments, other thealphavirus embodiments, the alphavirusRNA RNA repliconparticles replicon particles encode encodeananantigenic antigenic fragmentofofthe fragment therabies rabies virusG. G. virus In more In more specific specific embodiments embodiments of thisthe of this type, type, the rabies virus rabies virus GG comprises an amino comprises an aminoacid acid sequence sequencecomprising comprising 95%95% identity identity orormore more with the with the amino acid sequence amino acid ofSEQ sequence of SEQID IDNO: NO: 10.10. In In still more still specific embodiments more specific embodiments

00 of this of thistype, thethe type, rabies virus rabies G comprises virus thethe G comprises amino aminoacid acidsequence sequence of ofSEQ ID SEQ ID

NO:10. NO: 10.In Ineven even more more specific specific embodiments embodiments of this of thisthe type, type, the virus rabies rabies virus G is G is encodedbybythe encoded thenucleotide nucleotidesequence sequenceofofSEQ SEQID ID NO:NO: 9 or 9 or SEQSEQ ID NO: ID NO: 16. 16.

In particular In particular embodiments, embodiments, the the alphavirus alphavirus RNA replicon RNA replicon particle particle is a is a 55 VenezuelanEquine Venezuelan EquineEncephalitis Encephalitis(VEE) (VEE) alphavirus alphavirus RNA RNA replicon replicon particle.InInmore particle. more specific embodiments specific embodiments of this of this type, type, the the VEE VEE alphavirus alphavirus RNA replicon RNA replicon particle particle is a TC- is a TC 83 VEE 83 alphavirusRNA VEE alphavirus RNA particle. In repliconparticle. replicon In other other embodiments, thealphavirus embodiments, the alphavirus RNAreplicon RNA replicon particleis isa aSindbis particle Sindbis (SIN) (SIN) alphavirus alphavirus RNA replicon RNA replicon particle. particle. In In still still other embodiments, other embodiments,the the alphavirus alphavirus RNA replicon RNA replicon particleparticle is a Semliki is a Semliki Forest Forest virus virus 10 :0 (SFV) alphavirus (SFV) RNAreplicon alphavirus RNA repliconparticle. particle. In In an alternativeembodiment, an alternative embodiment, aa naked naked

DNAvector DNA vectorencodes encodes one one or or more more antigens antigens that that originatefrom originate oneorormore fromone morefeline feline pathogens. InIn particular pathogens. particular embodiments embodiments ofofthis this type, type, the the naked naked DNA vectorsencode DNA vectors encode an FCV an FCVcapsid capsidprotein protein or or antigenic antigenic fragment thereof. In fragment thereof. In specific specificembodiments of embodiments of

this type, this type,the thenaked naked DNA vectors encode DNA vectors encodeananVS-FCV VS-FCV capsid capsid protein protein or or antigenic antigenic

25 25 fragment thereof. In fragment thereof. In other other embodiments embodiments ofofthis this type, type, the the naked naked DNA vectors DNA vectors

encodeananFeLV encode FeLV gp85, gp85, or or antigenicfragment antigenic fragmentthereof. thereof.

In certain In certainembodiments, the immunogenic embodiments, the immunogeniccompositions compositions cancan include include at at least least

onemodified one modifiedlive livefeline felinepathogen pathogenand and alphavirus alphavirus RNA replicon RNA replicon particles particles that that encode encode 30 30 one or one or more more FCV FCVcapsid capsidproteins proteinsororantigenic antigenic fragments fragmentsthereof, thereof, one one or or more more FeLV FeLV glycoproteinsororantigenic glycoproteins antigenic fragments fragments thereof, thereof, and/or and/or one one or orrabies more more virus rabiesG virus G proteins or proteins or antigenic antigenicfragments fragments thereof. thereof. In particular In particular embodiments embodiments of this of thisthe type, type, the alphavirus RNA alphavirus replicon particles RNA replicon particles encode both aa VS-FCV encode both VS-FCVcapsid capsidprotein proteinororanan

10

fragment antigenicfragment antigenic andand thereof, thereof, an FCV an FCV F9-Like capsid capsid F9-Like protein protein or an antigenic or an antigenic

fragment thereof. fragment thereof. In In related relatedembodiments, the alphavirus embodiments, the RNAreplicon alphavirus RNA repliconparticles particles encodeboth encode bothaaVS-FCV VS-FCV capsid capsid proteinororananantigenic protein antigenicfragment fragmentthereof, thereof,and andanan FeLVgp85 FeLV gp85ororananantigenic antigenicfragment fragmentthereof. thereof. InIn other other embodiments, embodiments,the thealphavirus alphavirus 55 RNAreplicon RNA replicon particles particles encode both aa FCV encode both FCVF9-Like F9-Likecapsid capsidprotein proteinor or an an antigenic antigenic fragmentthereof, fragment thereof,andand an an FeLVFeLV gp85 gp85 or an antigenic or an antigenic fragmentfragment thereof. thereof. In alternative In alternative

embodiments,the embodiments, thealphavirus alphavirusRNA RNA repliconparticles replicon particlesencode encodeboth botha aVS-FCV VS-FCV capsid capsid

protein or protein or an an antigenic antigenicfragment fragment thereof, thereof, and and a rabies a rabies virusvirus G protein G protein or an or an antigenic antigenic

fragmentthereof. fragment thereof.In In yetyet other other embodiments, embodiments, the alphavirus the alphavirus RNA particles RNA replicon replicon particles 00 encodeboth encode bothaaFCV FCVF9-Like F9-Likecapsid capsid proteinororan protein anantigenic antigenic fragment fragmentthereof, thereof, and and aa rabies virus rabies virus GGprotein proteinororananantigenic antigenic fragment fragment thereof. thereof. In still In still otherother embodiments, embodiments,

the alphavirus the alphavirus RNA replicon particles RNA replicon particles encode a VS-FCV encode a capsidprotein VS-FCV capsid proteinororan an antigenicfragment antigenic fragment thereof, thereof, an an FeLVFeLV gp85 gp85 or an or an antigenic antigenic fragment fragment thereof, thereof, and a and a rabies virus rabies virus GGprotein proteinororananantigenic antigenic fragment fragment thereof. thereof. In alternative In alternative embodiments, embodiments,

55 the alphavirus the alphavirus RNA particles encode replicon particles RNA replicon a VS-FCV encode a VS-FCV capsid proteinororan capsidprotein an antigenicfragment antigenic fragment thereof, thereof, a FCV a FCV F9-Like F9-Like capsidcapsid proteinprotein or an antigenic or an antigenic fragmentfragment

thereof, and thereof, andananFeLV FeLV gp85gp85 or anorantigenic an antigenic fragment fragment thereof.thereof. In yet In yet other other embodiments,the embodiments, thealphavirus alphavirusRNA RNA repliconparticles replicon particlesencode encodea aVS-FCV VS-FCV capsid capsid

protein or protein or an an antigenic antigenicfragment fragment thereof, thereof, an FeLV an FeLV gp85 gp85 or or an antigenic an antigenic fragmentfragment

0 0 thereof, aa rabies thereof, rabiesvirus virusGGprotein proteinororanan antigenic antigenic fragment fragment thereof, thereof, and and an FCVan FCV F9-Likecapsid F9-Like capsidprotein proteinor or an an antigenic antigenic fragment fragment thereof. thereof. In still In still other other embodiments, embodiments,

the alphavirus the alphavirus RNA replicon particles RNA replicon particles encode an FeLV encode an FeLVgp85 gp85ororananantigenic antigenic fragmentthereof, fragment thereof,andand a rabies a rabies virus virus G protein G protein or anorantigenic an antigenic fragment fragment thereof. thereof. In In yet other yet other embodiments, the alphavirus embodiments, the alphavirus RNA RNAreplicon repliconparticles particles encode anFeLV encode an FeLVgp85 gp85 25 25 or an or antigenicfragment an antigenic fragment thereof, thereof, an an FCV FCV F9-Like F9-Like capsid capsid protein protein or an antigenic or an antigenic

fragmentthereof, fragment thereof,andand a rabies a rabies virus virus G protein G protein or anorantigenic an antigenic fragment fragment thereof. thereof.

Accordingly, the Accordingly, the present present invention invention provides provides immunogenic compositionsthat immunogenic compositions that include at include at least least one onemodified modified livefeline live felinepathogen pathogen and and anyorone any one orofmore more the of the 30 30 alphavirusRNA alphavirus RNA replicon replicon particles particles of the of the present present invention invention that encode that encode multiple multiple feline feline pathogenantigens, pathogen antigens, and/or and/or any any one oneoror more morealphavirus alphavirusRNA RNA repliconparticles replicon particles of of the the presentinvention present inventionthat thatencode encode a single a single feline feline pathogen pathogen antigen. antigen. In particular In particular

embodiments, embodiments, the the modified modified live live feline feline pathogen pathogen is a modified is a modified liveInFVR. live FVR. other In other

11

embodiments, embodiments, the the modified modified live live feline feline pathogen pathogen is a modified is a modified live In live FPLV. FPLV. In yet other yet other

embodiments, embodiments, the the modified modified live live feline feline pathogen pathogen is a modified is a modified live Chlamydophila live Chlamydophila

felis. In felis. In still stillother embodiments, other themodified embodiments, the modified live live felinepathogen feline pathogen is a ismodified a modified live live FCVF9-Like. FCV F9-Like. In yet In yet other other embodiments, embodiments, the modified the modified live pathogen live feline feline pathogen is a is a 55 modifiedlive modified live Bordetella Bordetellabronchiseptica. bronchiseptica. In particular In particular embodiments, embodiments, two or two more or more modified live modified live feline felinepathogens pathogensare areincluded includedinin thethe immunogenic immunogenic composition. In composition. In

specific embodiments specific ofthis embodiments of this type, type,the theimmunogenic compositioncomprises immunogenic composition comprisesa a modified live modified live FVR and aa modified FVR and modified live live Chlamydophila. In other Chlamydophila. In other embodiments, embodiments,three three or more or modified live more modified live feline felinepathogens pathogens are are included included ininthe immunogenic the immunogenic

00 composition. In specific composition. In specific embodiments ofthis embodiments of this type, type, the the immunogenic composition immunogenic composition

comprises a modified comprises a modified livelive FVR, FVR, a modified a modified live Chlamydophila, live Chlamydophila, and a modified and a modified live live feline pathogen feline pathogen isisanan FPLV. FPLV. In still In still other other embodiments, embodiments, four four or ormodified more more modified live live feline pathogens feline are included pathogens are included in in the theimmunogenic composition. InInspecific immunogenic composition. specific embodiments embodiments ofofthis this type, type, the the immunogenic compositioncomprises immunogenic composition comprises a modified a modified live live

55 FVR,a amodified FVR, modified liveChlamydophila, live Chlamydophila, a modified a modified live FPLV, live FPLV, and a modified and a modified live live F9-LikeFCV. F9-Like FCV.In particular In particular embodiments, embodiments, all of all theofalphavirus the alphavirus RNA replicon RNA replicon particlesparticles

in the in theimmunogenic compositionare immunogenic composition areVenezuelan Venezuelan Equine Equine Encephalitis Encephalitis (VEE) (VEE)

alphavirusRNA alphavirus RNA replicon replicon particles. particles. In yet In yet moremore specific specific embodiments, embodiments, all all of VEE of VEE alphavirus alphavirus RNA replicon particles RNA replicon particles ininthe theimmunogenic compositionsare immunogenic compositions are TC-83 TC-83VEE VEE .0 :0 alphavirus RNA alphavirus RNA replicon replicon particles. particles.

In additional In additionalembodiments, alphavirus RNA embodiments, alphavirus RNA replicon particles can replicon particles can encode encode

protein antigens protein antigens(or (orantigenic antigenicfragments fragments thereof) thereof) that that originate originate from from otherother felinefeline

pathogens.In In pathogens. particular particular embodiments embodiments of thisoftype, this type, the protein the protein antigenantigen originates originates

25 25 from aa feline from feline pneumovirus pneumovirus (FPN). (FPN). In still In still otherother embodiments, embodiments, the protein the protein antigen antigen originates from originates fromfeline felineparvovirus parvovirus(FPV). (FPV). In yet In yet other other embodiments, embodiments, the the protein protein antigen originatesfrom antigen originates fromfeline felineinfectious infectiousperitonitis peritonitisvirus virus(FIPV). (FIPV).In yet In yet other other

embodiments, embodiments, the the protein protein antigen antigen originates originates from feline from feline immunodeficiency immunodeficiency virus. In virus. In

still other still otherembodiments, the embodiments, the protein protein antigen antigen originates originates fromfrom bornaborna disease disease virus virus 30 30 (BDV). In Inyet (BDV). yetother otherembodiments, embodiments, the protein the protein antigen antigen originates originates from from feline feline influenza virus. influenza virus. InIn still still other other embodiments, embodiments, thethe protein protein antigen antigen originates originates from from felinefeline

coronavirus (FCoV). coronavirus (FCoV).

12

The present invention The present invention further provides immunogenic further provides immunogenic compositions and/or compositionsand/or vaccines(multivalent vaccines (multivalentvaccines) vaccines) thatthat include include the the alphavirus alphavirus RNA replicon RNA replicon particles particles of of the presentinvention the present inventiontogether together with with oneone or more or more modified modified live (e.g., live (e.g., attenuated) attenuated)

feline pathogens feline pathogens of of thepresent the present invention, invention, together together with with a killed a killed feline feline pathogen. pathogen. In In 55 particular embodiments, particular the immunogenic embodiments, the immunogeniccompositions compositions cancan furthercomprise further comprise a a killed Chlamydophila killed felis,and/or Chlamydophila felis, and/orkilled killedFVR, FVR, and/or and/or killed killed F9-like F9-like FCV,FCV, and/or and/or killedkilled

VS-FCV, and/orkilled VS-FCV, and/or killed FeLV, FeLV, and/or and/or killed killed FPLV. In certain FPLV. In certain embodiments, vaccines embodiments, vaccines

comprisean comprise an immunologically immunologicallyeffective effective amount amountofofone oneorormore moreofofthese theseimmunogenic immunogenic compositions. compositions.

0 0 The present The invention further present invention further comprises comprises vaccines and multivalent vaccines and multivalent vaccines vaccines

that comprise that comprisethetheimmunogenic immunogenic compositions compositions of the present of the present invention. invention. In particular In particular

embodiments,the embodiments, themultivalent multivalent vaccines vaccinesare are nonadjuvanted nonadjuvanted vaccines.In Incertain vaccines. certain embodiments,the embodiments, thevaccine vaccineaids aidsinin the the prevention prevention of of disease disease due to FCV, due to and/or FCV, and/or

FeLV, and/or FeLV, and/or FVR, FVR,and/or and/orFPLV, FPLV,and/or and/orrabies rabiesvirus, virus, and/or and/or Chlamydophila Chlamydophilafelis. felis. In In 55 related embodiments, related embodiments, antibodies antibodies are induced are induced in a feline in a feline subject subject when when the theisfeline feline is immunized immunized with with thethe vaccine. vaccine. The present The present invention invention further further includesincludes all all of the of the alphavirus RNA alphavirus RNAreplicon replicon particles particles and and the the naked DNAvectors naked DNA vectorsofofthe the present present invention. invention.

.0 0 Thepresent The presentinvention inventionalso also provides provides methods methods of immunizing of immunizing a feline a feline against against a a feline pathogen, feline e.g., FCV, pathogen, e.g., FCV,comprising comprising administering administering to the to the feline feline an an immunologically immunologically

effective effective amount amount ofofaavaccine vaccineorormultivalent multivalentvaccine vaccine of of thethe present present invention. invention. In In

particular embodiments particular embodiments thethe vaccine vaccine is administered is administered via intramuscular via intramuscular injection. injection. In In alternative embodiments alternative embodiments thethe vaccine vaccine is administered is administered via subcutaneous via subcutaneous injection. injection. In In 25 25 other embodiments other embodiments the the vaccine vaccine is administered is administered via intravenous via intravenous injection. injection. In still In still other other embodiments embodiments the the vaccine vaccine is administered is administered via intradermal via intradermal injection. injection. In yetIn other yet other embodiments embodiments the the vaccine vaccine is administered is administered via transdermal via transdermal injection. injection. In still In still other other embodiments embodiments the the vaccine vaccine is administered is administered via oral via oral administration. administration. In yetInother yet other embodiments embodiments the the vaccine vaccine is administered is administered via intranasal via intranasal administration. administration. In specific In specific

30 30 embodiments, embodiments, thethe feline feline is is domestic a adomestic cat. cat.

Thevaccines The vaccines and and multivalent multivalent vaccines vaccines of the of the present present invention invention can can be be administeredasasa aprimer administered primer vaccine vaccine and/or and/or as aas a booster booster vaccine. vaccine. Inspecific In specific

13

embodiments, a vaccine embodiments, a vaccine of the of the present present invention invention is administered is administered as ashot as a one onevaccine shot vaccine (one dose), (one dose),without withoutrequiring requiringsubsequent subsequent administrations. administrations. In certain In certain embodiments, embodiments, in in the case the caseofofthe the administration administrationofofboth botha aprimer primervaccine vaccine andand a booster a booster vaccine, vaccine, the the primer vaccine primer vaccineand andthethebooster booster vaccine vaccine can can be administered be administered by the by the identical identical route.route. In In 55 certain embodiments certain of this embodiments of this type, type, theprimer the primer vaccine vaccine and and the booster the booster vaccine vaccine are are both both administeredbybysubcutaneous administered subcutaneous injection. injection. In alternative In alternative embodiments, in the in embodiments, theofcase case the of the administration of both administration of both aa primer primervaccine vaccineandand a booster a booster vaccine, vaccine, the the administration administration of the of the

primer vaccine primer vaccinecan canbebe performed performed by one by one routeroute andbooster and the the booster vaccinevaccine by by another another route. InIn certain route. certain embodiments of this embodiments of this type, type, thethe primer primer vaccine vaccine can can be administered be administered by by 00 subcutaneous subcutaneous injection injection andand thethe booster booster vaccine vaccine canadministered can be be administered orally.orally.

Theinvention The inventionfurther furtherprovides provides forfor a method a method of immunizing of immunizing a feline a feline againstagainst

FCVcomprising FCV comprising injecting injecting the the feline feline with with an immunologically an immunologically effective effective amount amount of the of the above describedinventive above described inventive vaccines. vaccines. InIn particular particular embodiments, the vaccines embodiments, the vaccines can can 55 include from about include from about 11 Xx 104 10 to about 1 to about 1 xX 101 10¹ RPs RPsororhigher, higher, for example. In for example. In more more particular embodiments, particular the vaccines embodiments, the can include vaccines can include from from about about 11 XX 10 to about 105to about 11 Xx 109 RPs. 10° In even RPs. In even more moreparticular particular embodiments embodimentsthethevaccines vaccines can can includefrom include fromabout about 1 1 x X 106 10 to toabout about1 X1 x10 108 RPs.RPs.In particular In particular embodiments, the feline embodiments, theisfeline a domestic a is domestic cat. cat.

00 In particular In particular embodiments embodiments the the vaccines vaccines of present of the the present invention invention are are administered in 0.05 administered in 0.05 mL to 33 mL mL to doses. InIn more mL doses. moreparticular particular embodiments embodiments thedose the dose administeredis is0.1 administered 0.1mLmL to to 2 mLs. 2 mLs. In still In still moremore particular particular embodiments embodiments the dosethe dose administered is administered is 0.2 0.2 mL mL to to 1.5 1.5 mLs. In even mLs. In more particular even more particular embodiments thedose embodiments the dose 25 25 administeredis is0.3 administered 0.3toto1.0 1.0mLs. mLs. In stillmore In still more particular particular embodiments embodiments the the dose dose administeredis is0.4 administered 0.4mLmL to to 0.80.8 mLs. mLs.

Theseand These and other other aspects aspects of the of the present present invention invention willbetter will be be better appreciated appreciated

by reference by referencetotothe thefollowing followingDetailed Detailed Description. Description.

30 30

DETAILED DESCRIPTION DETAILED DESCRIPTION OF OF THE INVENTION THE INVENTION Thepresent The presentinvention invention provides provides safesafe and efficacious and efficacious multivalent multivalent vaccines. vaccines. In In particular embodiments particular embodiments the the vaccine vaccine is nonadjuvanted. is nonadjuvanted. In this of In this aspect aspect the of the

14

invention, the invention, the vaccines vaccinesdodo notnot induce induce feline feline injection-site injection-site sarcomas, sarcomas, yet still yet still aid aid in in the protection the protectionofofthe thevaccinates vaccinates from from disease disease caused caused by infections by infections by by feline feline calicivirus calicivirus (FCV) and/orfeline (FCV) and/or felineleukemia leukemia virus virus (FeLV), (FeLV), and infections and infections by feline by feline viral viral

rhinotracheitis virus rhinotracheitis virus (FVR), (FVR),and/or and/or felinepanleukopenia feline panleukopenia virusvirus (FPLV), (FPLV), and/or and/or live live 55 Chlamydophila Chlamydophila felis. felis.

Despite the Despite the known knownenhancement enhancementof of thethe innateimmune innate immune system system by alphavirus by alphavirus

RNAreplicon RNA replicon particles, particles, thethe multivalent multivalent vaccines vaccines of present of the the present invention, invention, which which compriseboth comprise both alphavirus alphavirus RNA RNA replicon replicon particles particles and modified and modified live viruses, live viruses, are are 00 unexpectedly unexpectedly safe safe andand efficacious, efficacious, without without the alphavirus the alphavirus RNA replicon RNA replicon particlesparticles

significantly interfering significantly interfering with with the immunological the immunological effect effect of of thethe modified modified livelive viruses. viruses.

Indeed,alphavirus Indeed, alphavirus RNA RNA replicon replicon particles, particles, andparticular and in in particular Venezuelan Venezuelan Equine Equine Encephalitis (VEE) Encephalitis alphavirus RNA (VEE) alphavirus replicon particles, RNA replicon particles, have have previously previously been been shown shown

to catalyze to systemic catalyze aa systemic antiviralstate antiviral stateand and protect protect against against lethal lethal virus virus challenge challenge

55 [Konopka

[Konopka et et al.,J.J. Virol., al., Virol., 83 (29):12432-12442 83 (29):12432-12442 (2009)]. (2009)]. Moreover, Moreover, VEE alphavirus VEE alphavirus

RNAreplicon RNA replicon particles particles have have beenbeen reported reported to induce to induce rapid protection rapid protection against against foot- foot and-mouth disease and-mouth disease virus virus [Segundo

[Segundo et al.,etJ.al., J. Virol., Virol., 87 (10):5447-5460 87 (10):5447-5460 (2013)].(2013)].

Therefore,itit would Therefore, wouldhave have been been anticipated anticipated that that vaccines vaccines comprising comprising both alphavirus both alphavirus

RNAreplicon RNA replicon particles particles andand modified modified live live viruses viruses wouldwould lead lead to to a substantial a substantial

D 0 inhibition of inhibition of the the immunological effectofofthe immunological effect themodified modified livelive viruses. viruses. However, However, the the enhancement enhancement of of theinnate the innateimmune immune response response due due to the to the presence presence of the of the alphavirus alphavirus

RNAreplicon RNA replicon particles particles surprisingly surprisingly proved proved notbetodetrimental not to be detrimental to thetoimmune the immune responseinduced response inducedbybythe theaccompanying accompanying modified modified liveviruses. live viruses.

25 25 Accordingly,inina aparticular Accordingly, particularaspect, aspect,thethepresent present invention invention provides provides vaccines vaccines

that comprise that alphavirus RNA comprise alphavirus replicon particles RNA replicon particles that thatencode encode an an FCV capsidprotein FCV capsid protein or antigenic or fragment antigenic fragment thereof thereof and/or and/or an FeLV an FeLV gp85 gp85 or or antigentic antigentic fragmentfragment thereof, thereof, along withaamodified along with modifiedlive livefeline felineFVR, FVR, and/or and/or a modified a modified live live FPLV,FPLV, and/orand/or a modified a modified

live Chlamydophila live felis.In Inyetyetanother Chlamydophila felis. another aspect aspect of the of the present present invention, invention, the the 30 30 vaccines comprise vaccines comprisenaked nakedDNA DNA vectors vectors that that encode encode an an FCV FCV capsid capsid protein protein or or antigenic fragment antigenic fragment thereof thereof and/or and/or an FeLV an FeLV gp85 gp85 or or antigentic antigentic fragmentfragment thereof, thereof,

along witha amodified along with modifiedlive livefeline felineFVR, FVR, and/or and/or a modified a modified live live FPLV,FPLV, and/orand/or a modified a modified

live Chlamydophila live felis. Chlamydophila felis.

15

Thevaccines The vaccinesof of thethe present present invention invention canadministered can be be administered to a in to a feline feline the in the absence absence of of anan adjuvant adjuvant and and stillstill effectively effectively aidaid in in the the protection protection of the of the vaccinated vaccinated

feline against feline FCV. against FCV.

55 In order In to more order to morefully fully appreciate appreciatethetheinvention, invention, thethe following following definitions definitions areare

provided. provided.

Theuse The useofofsingular singularterms terms forfor convenience convenience in description in description is in is no in no way way 00 limiting.Thus, intendedtotobebesosolimiting. intended Thus, forfor example, example, reference reference to a composition to a composition

comprising comprising "a"apolypeptide" polypeptide" includes includes reference reference to onetoor one orofmore more such of such polypeptides. polypeptides.

In addition, In addition, reference toanan"alphavirus reference to "alphavirusRNARNA replicon replicon particle" particle" includes includes reference reference to to plurality of aa plurality of such alphavirusRNA such alphavirus RNA replicon replicon particles, particles, unless unless otherwise otherwise indicated. indicated.

55 As used As usedherein herein thethe term term "approximately" "approximately" is interchangeably is used used interchangeably with the with the term "about" term "about"and and signifies signifies thata value that a value is is within within fiftypercent fifty percentofofthetheindicated indicated value value

i.e., i.e., a acomposition composition containing containing"approximately" 1 X 10 "approximately" 1 xalphavirus RNA replicon 108 alphavirus RNA replicon particles per particles per milliliter milliliter contains contains from from 0.5 108 0.5 Xx10 to to 1.5X 10 1.5 x 108 alphavirus alphavirus RNA replicon RNA replicon

particles per milliliter. particles per milliliter.

10 0 As used As usedherein, herein,thethe term term "feline" "feline" refers refers to to anyany member member of theof the Felidae Felidae family.family.

Domestic cats, Domestic cats, pure-bred pure-bred and/or and/or mongrel mongrel companion companion cats, and cats, andferal wild or wildcats or feral are cats are

all all felines. felines.

25 25 As used As usedherein, herein,thethe term term "replicon" "replicon" refers refers tomodified to a a modified RNA genome RNA viral viral genome that lacks that lacks one oneorormore more elements elements (e.g., (e.g., coding coding sequences sequences for structural for structural proteins) proteins) that that if they if they were present,would were present, would enable enable the the successful successful propagation propagation of the parental of the parental virus virus in cell in cellcultures cultures or or animal hosts.InInsuitable animal hosts. suitablecellular cellularcontexts, contexts,thethe replicon replicon willamplify will amplify itself and itself andmay may produce produce one or more one or sub-genomicRNARNA more sub-genomic species. species.

30 30

As used As usedherein, herein, thethe term term "alphavirus "alphavirus RNA replicon RNA replicon particle", particle", abbreviated abbreviated

"RP", is "RP", is an an alphavirus-derived alphavirus-derivedRNARNA replicon replicon packaged packaged in structural in structural proteins, proteins, e.g., e.g., the capsid the capsidand andglycoproteins, glycoproteins, which which also also are derived are derived from from an an alphavirus, alphavirus, e.g., ase.g., as

16

described by described by Pushko Pushkoetetal., al., [Virology

[Virology239(2):389-401 239(2):389-401 (1997)]. (1997)]. An An RP cannot RP cannot

propagatein incell propagate cellcultures culturesororanimal animal hosts hosts (without (without a helper a helper plasmid plasmid or analogous or analogous

component),because component), because thereplicon the replicondoes doesnot notencode encodethethe alphavirusstructural alphavirus structural components(e.g., components (e.g., capsid capsid and andglycoproteins). glycoproteins). 55 The terms The terms "FCV "FCVF9-Like" and"F9-Like F9-Like"and "F9-LikeFCV" FCV" are are used used interchangeably interchangeably with with

each other each other and and with with the the term term "classical "classicalFCV" FCV" and and as as used herein is used herein is an an FCV that can FCV that can

be characterizedas as be characterized an an older older and and formerly, formerly, universal universal vaccine vaccine strain strain of FCV,offor FCV, for whichthe which theFCV FCV F9 strain F9 strain is considered is considered a typical a typical representative. representative. In direct In direct contrast, contrast,

00 the FCV the termedvirulent FCV termed systemic "VS-FCV" virulent systemic "VS-FCV"ororasasused usedherein interchangeably"(VS) hereininterchangeably "(VS) FCV",isis aa newer FCV", newer class class of of FCV, FCV, which which is unusually is unusually virulent, virulent, and cannot and cannot be be neutralized bybyantibodies neutralized antibodies from from thethe FCV FCV F9-Like F9-Like strains strains [see,7,449,323;

[see, U.S. U.S. 7,449,323; Radfordetetal., Radford al., 38(2) Vetres. 38(2)Vet res.319-335 319-335 (2007)]. (2007)].

5 5 Theterms The terms"originate "originatefrom", from", "originates "originates from" from" and and "originating "originating from" from" are are used used interchangeably interchangeably with with respect respect to atogiven a given protein protein antigen antigen andpathogen and the the pathogen or strainorofstrain of that pathogen that pathogenthat thatnaturally naturally encodes encodes it, and it, and as used as used hereinherein signify signify that that the the unmodifiedand/or unmodified and/or truncated truncated amino amino acid sequence acid sequence of thatprotein of that given given antigen proteinisantigen is encodedbybythat encoded that pathogen pathogenororstrain strain of of that thatpathogen. The coding pathogen. The coding sequence, sequence,within within aa D 0 nucleic acid nucleic acid construct constructofofthe thepresent present invention invention for for a protein a protein antigen antigen originating originating from from

pathogen,maymay a pathogen, a havehave been been genetically genetically manipulated manipulated so as to so as toin result result in a modification a modification

and/ortruncation and/or truncationofofthe theamino amino acid acid sequence sequence of theof the expressed expressed protein protein antigen antigen relative to relative to the the corresponding sequence corresponding sequence of that of that protein protein antigen antigen in theinpathogen the pathogen or or strain of strain of pathogen (includingnaturally pathogen (including naturally attenuated attenuated strains) strains) it originates it originates from. from.

25 25

As used As usedherein, herein, the the terms terms "protecting", "protecting", or "providing or "providing protection protection to", to", or or "eliciting protective "eliciting protective immunity to", "aids immunity to", "aids inin prevention preventionofofdisease", disease",andand "aids "aids in the in the

protection" do protection" donot notrequire requirecomplete complete protection protection from from any indication any indication of infection. of infection. For For example,"aids example, "aidsin inthe theprotection" protection" cancan meanmean thatprotection that the the protection is sufficient is sufficient such such that, that, 30 30 after challenge, after symptoms challenge, symptoms of the of the underlying underlying infection infection are atare at least least reduced, reduced, and/or and/or that one that ormore one or moreofofthethe underlying underlying cellular, cellular, physiological, physiological, or biochemical or biochemical causescauses or or mechanisms mechanisms causing causing thethe symptoms symptoms are are reduced reduced and/or and/or eliminated. eliminated. It isIt understood is understood that "reduced," that "reduced,"asasused used in in thiscontext, this context, means means relative relative to state to the the state of infection, of the the infection,

17

including the including the molecular molecular state state of of thethe infection,notnot infection, justthe just thephysiological physiological state state of the of the

infection. infection.

As used As usedherein, herein, a "vaccine" a "vaccine" is ais composition a composition that that is suitable is suitable for application for application to to 55 an animal, an animal,e.g., e.g.,feline feline (including, (including, in in certain certain embodiments, embodiments, humans, humans, while while in in other other embodimentsbeing embodiments being specifically not specifically not for for humans) comprising one humans) comprising oneorormore moreantigens antigens typically combined typically with combined with a pharmaceutically a pharmaceutically acceptable acceptable carriercarrier such assuch as a a liquid liquid containing water, containing water, which which upon administration to upon administration to the theanimal animal induces induces an an immune immune

responsestrong response strong enough enough to minimally to minimally aid inaid theinprotection the protection from afrom a disease disease arising arising 0 0 from an from aninfection infectionwith wild-type witha awild-type micro-organism, micro-organism, i.e.,i.e., strong strong enough enough for aiding for aiding in in the prevention the preventionofofthe thedisease, disease, and/or and/or preventing, preventing, ameliorating ameliorating or curing or curing the disease. the disease.

As used As usedherein, herein, a multivalent a multivalent vaccine vaccine is a isvaccine a vaccine that that comprises comprises two or two or moredifferent more differentantigens. antigens.In In a particular a particular embodiment embodiment oftype, of this this type, the multivalent the multivalent

55 vaccinestimulates vaccine stimulatesthethe immune immune of the of system system the recipient recipient againstagainst two or more or more different two different pathogens. pathogens.

The terms The terms "adjuvant" "adjuvant" and and "immune "immunestimulant" stimulant"are areused usedinterchangeably interchangeably herein, andare herein, and aredefined definedas as oneone or more or more substances substances thatstimulation that cause cause stimulation of the of the 0 0 immunesystem. immune system.In Inthis thiscontext, context, an an adjuvant adjuvant is is used to enhance used to animmune enhance an immune responsetoto one response one or or more morevaccine vaccineantigens/isolates. antigens/isolates. Accordingly, Accordingly, "adjuvants" "adjuvants" are are

agentsthat agents thatnonspecifically nonspecifically increase increase an immune an immune response response to a particular to a particular antigen, antigen,

thus reducing thus reducingthe thequantity quantity of of antigen antigen necessary necessary in anyingiven any given vaccine, vaccine, and/or and/or the the frequency of frequency of injection injection necessary necessary in inorder ordertoto generate generateananadequate adequate immune immune

25 25 responseto tothetheantigen response antigen of of interest. interest. In this In this context, context, an an adjuvant adjuvant is used is used to enhance to enhance

an immune an immuneresponse response to to one one or or more more vaccine vaccine antigens/isolates.TheThe antigens/isolates. American American

AssociationofofFeline Association FelinePractitioners Practitioners Feline Feline Vaccination Vaccination Guidelines, Guidelines, for example, for example,

suggest the suggest the use use of of nonadjuvanted FeLVvaccines nonadjuvanted FeLV vaccines [AAFP

[AAFP Feline Feline Advisory Advisory Panel, Panel, 15: 15:

785-808 (2013)]. 785-808 (2013)].

30 30

As used As usedherein, herein, a "nonadjuvanted a "nonadjuvanted vaccine" vaccine" is a vaccine is a vaccine or a multivalent or a multivalent

vaccinethat vaccine thatdoes doesnotnot contain contain an adjuvant. an adjuvant.

18

As used As usedherein, herein, thethe term term "pharmaceutically "pharmaceutically acceptable" acceptable" is used is used adjectivally adjectivally to to mean thatthe mean that themodified modified nounnoun is appropriate is appropriate forinuse for use in a pharmaceutical a pharmaceutical product. product.

Whenit itisisused, When used,for forexample, example,to to describe describe an excipient an excipient in a pharmaceutical in a pharmaceutical vaccine,vaccine,

it characterizes it the excipient characterizes the excipientasasbeing being compatible compatible with with the other the other ingredients ingredients of theof the 55 compositionandand composition notnot disadvantageously disadvantageously deleterious deleterious to the intended to the intended recipientrecipient animal, animal, e.g., feline. e.g., feline.

Parenteral administration" Parenteral administration"includes includessubcutaneous injections, submucosal subcutaneous injections, submucosal

injections, intravenous injections, injections,intramuscular intravenous injections, intramuscular injections, injections, intradermal intradermal injections, injections,

00 and infusion. and infusion.

As used As usedherein herein thethe term term "antigenic "antigenic fragment" fragment" in regard in regard to a particular to a particular protein protein

(e.g., aa protein (e.g., protein antigen) is aa fragment antigen) is fragment ofofthat thatprotein proteinthat thatisisantigenic, antigenic,i.e., i.e., capable capableofof specifically interacting specifically with an interacting with an antigen antigenrecognition recognition molecule molecule of immune of the the immune system,system,

55 such as such as an an immunoglobulin (antibody)oror TT cell immunoglobulin(antibody) cell antigen antigen receptor. receptor. For For example, an example, an

antigenic fragment antigenic fragmentof of an an FCVFCV capsid capsid protein protein is a fragment is a fragment of the of the capsid capsid protein protein that that is antigenic. is Preferably,ananantigenic antigenic. Preferably, antigenic fragment fragment of the of the present present invention invention is is immune-dominant immune-dominant for antibody for antibody and/orand/or cell receptor T cell Treceptor recognition. recognition. In particular In particular

embodiments, embodiments, an antigenic an antigenic fragment fragment with respect with respect to aprotein to a given given protein antigen antigen is a is a 10 :0 fragmentofofthat fragment thatprotein proteinthat thatretains retainsatatleast least25% 25% of the of the antigenicity antigenicity of the of the fullfull length length

protein. InIn preferred protein. preferredembodiments, embodiments, an antigenic an antigenic fragment fragment retains retains at leastat50% least of 50% of the antigenicity the antigenicity of of the the full full length length protein. In more protein. In morepreferred preferred embodiments, embodiments, an an antigenicfragment antigenic fragmentretains retains at at least least 75%75% of the of the antigenicity antigenicity of the of the full full length length protein. protein.

Antigenic fragments Antigenic can be fragments can beas as small small as as 20 20 amino aminoacids acidsoror at at the the other other extreme, extreme, be be

25 25 large fragments large fragmentsthat thatareare missing missing as littleasas as little a single a single amino amino acid acid from from the full-length the full-length

protein. In protein. In particular particularembodiments, embodiments, the the antigenic antigenicfragment fragment comprises comprises 25 to 150 25 to 150

amino acid amino acid residues. residues. In In other other embodiments, theantigenic embodiments, the antigenicfragment fragmentcomprises comprises5050toto 250 amino 250 aminoacid acidresidues. residues. For Forexample, example,for forFeLV, FeLV,the theFeLV FeLV gp45 gp45 glycoprotein glycoprotein andand

the FeLV the gp70glycoprotein FeLV gp70 glycoproteinare areantigenic antigenic fragments fragments of of the the FeLV FeLVgp85 gp85glycoprotein, glycoprotein, 30 30 whereas, for whereas, for FCV, FCV, one oneantigenic antigenic fragment fragmentofof an an FCV FCVcapsid capsidprotein proteincomprises comprises region EE of region of the the ORF2. ORF2.

19

As used As used herein, herein, one one amino aminoacid acidsequence sequenceis is100% 100% "identical"oror has "identical" has100% 100% "identity" to to "identity" a second a secondamino aminoacid acidsequence sequence when the amino when the aminoacid acidresidues residuesofof both both sequencesare sequences areidentical. identical. Accordingly, Accordingly, an an amino aminoacid acid sequence sequenceisis50% 50% "identical" to "identical" to aa second aminoacid second amino acidsequence sequence when when 50% 50% of the of the amino amino acid acid residues residues of the of the twotwo

55 amino acid sequences amino acid sequencesare areidentical. identical. The Thesequence sequence comparison comparison is performed is performed overover a a

contiguousblock contiguous block of of amino amino acidacid residues residues comprised comprised by aprotein, by a given given protein, e.g., a e.g., a protein, protein, or portion of a portion or a of the the polypeptide polypeptidebeing being compared. compared. In a particular In a particular embodiment, embodiment,

selecteddeletions selected deletionsororinsertions insertionsthat thatcould could otherwise otherwise alteralter the the correspondence correspondence

betweenthe between thetwo two amino aminoacid acidsequences sequencesareare taken taken intoaccount. into account. 00 As used As used herein, herein, nucleotide nucleotide and aminoacid and amino acid sequence sequence percent percent identity can identity can be be determined using determined usingC, C, MacVector MacVector(MacVector, (MacVector, Inc.Cary, Inc. Cary,NCNC 27519), 27519), Vector Vector NTINTI

(Informax, Inc. (Informax, MD), Oxford Inc.MD), Oxford Molecular Molecular Group PLC(1996) Group PLC (1996)and andthe theClustal ClustalWW algorithm withthe algorithm with thealignment alignment default default parameters parameters and default and default parameters parameters for identity. for identity.

55 Thesecommercially These commerciallyavailable availableprograms can programscan alsobebeused also used to to determine determine sequence sequence

similarity using similarity the same using the sameor or analogous analogous default default parameters. parameters. Alternatively, Alternatively, an an Advanced Advanced Blast Blast search search underunder the default the default filterfilter conditions conditions can becan be e.g., used, used,using e.g., using the GCG the (GeneticsComputer GCG (Genetics Computer Group, Group, Program Program Manual Manual forGCG for the the Package, GCG Package, Version 7, Version 7, Madison, Wisconsin)pileup Madison, Wisconsin) pileup program programusing usingthe thedefault default parameters. parameters. 10 0 As used As used herein, herein, the the term term "inactivated" "inactivated"microorganism is used microorganism is used interchangeably interchangeably

with the with the term term"killed" "killed" microorganism. microorganism. For For the purposes the purposes of thisofinvention, this invention, an an "inactivated" microorganism "inactivated" microorganism is organism is an an organism which which is capable is capable of eliciting of eliciting an an immune immune responsein inanan response animal, animal, but but is not is not capable capable of infecting of infecting the animal. the animal. An antigen An antigen of the of the 25 25 present invention(e.g., present invention (e.g.,ananinactivated inactivated felinecalicivirus) feline calicivirus)may maybe be inactivated inactivated by an by an

agent selectedfrom agent selected from thethe group group consisting consisting of binary of binary ethyleneimine, ethyleneimine, formalin, formalin, beta- beta

propiolactone,thimerosal, propiolactone, thimerosal,or or heat. heat. In aInparticular a particular embodiment, embodiment, inactivated inactivated feline feline calicivirus isolates calicivirus isolates combined with combined with an an RPthe RP of of the present present invention invention are inactivated are inactivated by by binary ethyleneimine. binary ethyleneimine. 30 30

Thealphavirus The alphavirusRNARNA replicon replicon particles particles of present of the the present invention invention may be may be lyophilized and lyophilized andrehydrated rehydrated with with a sterile a sterile water water diluent. diluent. On other On the the other hand, hand, when when the the alphavirus RNA alphavirus RNA replicon replicon particles particles are are stored stored separately, separately, but intended but intended to be mixed to be mixed

20

with other with other vaccine vaccinecomponents components prior prior to administration, to administration, the alphavirus the alphavirus RNA RNA replicon replicon particles can particles bestored can be storedininthe thestabilizing stabilizingsolution solutionofofthose those components, components, e.g., e.g., a a high high sucrosesolution. sucrose solution.

55 A vaccine A vaccineofofthe thepresent present invention invention can can be readily be readily administered administered by any by any standardroute standard routeincluding including intravenous, intravenous, intramuscular, intramuscular, subcutaneous, subcutaneous, oral, intranasal, oral, intranasal,

intradermal, and/or intradermal, and/orintraperitoneal intraperitonealvaccination. vaccination. The The skilled skilled artisan artisan will will appreciate appreciate

that the that vaccinecomposition the vaccine composition is preferably is preferably formulated formulated appropriately appropriately fortype for each eachoftype of recipient animal recipient animaland androute route of of administration. administration.

00 Thus,the Thus, thepresent present invention invention also also provides provides methods methods of immunizing of immunizing a feline a feline against feline against felinepathogens. Onesuch pathogens. One suchmethod method comprises comprises injectinga afeline injecting feline with with an an immunologically immunologically effective effective amount amount of a of a vaccine vaccine of theofpresent the present invention, invention, so thatso thethat the feline produces feline appropriate produces appropriate anti-pathogen anti-pathogen antibodies. antibodies.

5 5

MULTIVALENT VACCINES MULTIVALENT VACCINES Accordingly,the Accordingly, thepresent present invention invention provides provides multivalent multivalent vaccines vaccines comprising comprising at at least one least one modified modified live livefeline pathogen feline pathogenand andone one or ormore more alphavirus alphavirus RNA replicon RNA replicon

particles. For particles. For example, example,thethe coding coding sequence sequence of a protein of a protein antigenantigen or antigenic or antigenic

00 fragment thereof, fragment thereof, or or combination combination of of such such coding coding sequences sequences ofofprotein protein antigens antigens useful in useful in aa feline feline vaccine canbebeadded vaccine can added to alphavirus to an an alphavirus RNA replicon RNA replicon particleparticle (RP) (RP) or combined or in the combined in the same RPasasone same RP one thatencodes that encodes e.g.,ananFCV e.g., FCV capsid capsid protein protein

and/orthe and/or theFeLV FeLV glycoprotein glycoprotein (gp85) (gp85) in multivalent in the the multivalent vaccine. vaccine.

25 25 In specific In specific embodiments, vaccines embodiments, vaccines comprise comprise at one at least least one modified modified live live feline feline pathogen and pathogen andananalphavirus alphavirusRNA RNA repliconparticle replicon particle that that encodes encodesananFCV FCV F9-Like F9-Like

capsidprotein capsid proteinororananantigenic antigenic fragment fragment thereof, thereof, and/or and/or a VS-FCV a VS-FCV capsidorprotein capsid protein or an antigenicfragment an antigenic fragment thereof, thereof, with with another another alphavirus alphavirus RNA replicon RNA replicon particleparticle that that encodes anFeLV encodes an FeLV gp85 gp85 or or an an antigenic antigenic fragment fragment thereof.InInother thereof. other embodiments, embodiments, 30 30 vaccinescomprise vaccines comprise at least at least one one modified modified live feline live feline pathogen pathogen and oneand one alphavirus alphavirus

RNAreplicon RNA replicon particle particle that thatencodes encodes aa VS-FCV capsidprotein VS-FCV capsid proteinoror an an antigenic antigenic fragment thereof, fragment thereof, another another alphavirus alphavirus RNA replicon particle RNA replicon particle that thatencodes encodes an an FeLV FeLV

gp85ororananantigenic gp85 antigenic fragment fragment thereof, thereof, and still and still a third a third alphavirus alphavirus RNA replicon RNA replicon

21

particle that particle that encodes encodes anan FCVFCV F9-Like F9-Like capsid capsid protein protein or an antigenic or an antigenic fragmentfragment

thereof. InIn yet thereof. yetother otherembodiments, embodiments, vaccines vaccines comprise comprise at leastatone least one modified modified live live feline pathogen feline pathogen and an alphavirus and an alphavirus RNA RNAreplicon repliconparticle particle that thatencodes encodes an FCV an FCV

F9-Likecapsid F9-Like capsidprotein proteinor or an an antigenic antigenic fragment fragment thereof, thereof, a VS-FCV a VS-FCV capsidorprotein capsid protein or 55 an antigenic fragment an antigenic thereof, and fragment thereof, and an an FeLV gp85ororan FeLV gp85 an antigenic antigenic fragment fragment thereof. thereof.

Examplesofofpathogens Examples pathogensthat thatone oneorormore moreofofsuch suchprotein proteinantigens antigenscan can originate from originate frominclude includefeline felineviral viral rhinotracheitis rhinotracheitisVirus Virus(FVR), (FVR), felineleukemia feline leukemia virus virus

(FeLV), feline (FeLV), felinepanleukopenia Virus (FPL) panleukopenia Virus feline herpesvirus (FPL) feline herpesvirus (FHV), (FHV), other other FCV FCV

00 strains, feline strains, feline parvovirus (FPV),feline parvovirus (FPV), felineinfectious infectiousperitonitis peritonitisvirus virus(FIPV), (FIPV),feline feline immunodeficiency immunodeficiency virus, virus, borna borna disease disease virus virus (BDV),(BDV), rabies feline rabies virus, virus, feline influenza influenza

virus, canine virus, influenzavirus, canine influenza virus,avian avianinfluenza, influenza, canine canine pneumovirus, pneumovirus, felinefeline

pneumovirus,Chlamydophila pneumovirus, Chlamydophila felis(FKA felis (FKAChlamydophila Chlamydophila psittaci),Bordetella psittaci), Bordetella bronchiseptica, and bronchiseptica, and Bartonella Bartonella spp.spp. (e.g., (e.g., B. henselae). B. henselae). In particular In particular embodiments, embodiments,

55 aa coding sequencefor coding sequence for aa capsid capsid protein protein or or analogous protein from analogous protein from one or more one or of more of

these feline these feline ororcanine caninepathogens pathogens can be inserted can be inserted into intothe thesame same RP as the RP as the FCV FCV antigen. Alternatively,ororinin combination antigen. Alternatively, combination therewith, therewith, a coding a coding sequence sequence for a capsid for a capsid

protein or protein or analogous analogous protein protein from from one one or more or more of these of these feline feline or canine or canine pathogens pathogens

can be inserted can be inserted into intoone one or ormore more other other RPs, RPs, which can be which can be combined combinedininaavaccine vaccine .0 0 with an with an RP that encodes RP that the FCV encodes the FCVF9-Like F9-Likecapsid capsidprotein proteinororan anantigenic antigenic fragment fragment thereof and/or thereof and/orthe theVS-FCV VS-FCV capsid capsid protein protein or an or an antigenic antigenic fragment fragment thereof. thereof.

Accordingly, the Accordingly, the present present invention inventionprovides provides vaccines vaccines comprising comprising one or more one or more

alphavirus RNA alphavirus RNA replicon replicon particles particles (RP)(RP) of present of the the present invention invention [e.g., [e.g., a VS-FCV a VS-FCV

25 25 capsid proteinororananantigenic capsid protein antigenic fragment fragment thereof] thereof] along along with with one orone ormodified more more modified live live (attenuated)virus (attenuated) virusisolates, isolates,e.g., e.g., aa live live attenuated attenuatedolder oldervaccine vaccine strain strain of of FCV, FCV, such such

as live attenuated as aa live FCV attenuated FCV F9, F9, and/or and/or a live a live attenuated attenuated feline feline herpesvirus herpesvirus and/or and/or a live a live

attenuated felineparvovirus attenuated feline parvovirus and/or and/or a live, a live, attenuated attenuated feline feline leukemia leukemia virus,virus, and/or and/or a a live, attenuated live, feline infectious attenuated feline infectious peritonitis peritonitis virus and/ora alive, virus and/or live,attenuated attenuatedfeline feline 30 30 immunodeficiency immunodeficiency virus virus and/or and/or a live, a live, attenuated attenuated borna borna diseasedisease virus aand/or virus and/or live, a live, attenuated rabiesvirus, attenuated rabies virus,and/or and/or a live,attenuated a live, attenuated feline feline influenza influenza virus virus and/or and/or a live, a live,

attenuatedcanine attenuated canine influenza influenza virus, virus, and/or and/or a live, a live, attenuated attenuated avianavian influenza, influenza, and/orand/or a a live, attenuated live, caninepneumovirus, attenuated canine pneumovirus, and/or and/or a live, a live, attenuated attenuated felinefeline pneumovirus. pneumovirus.

22

In addition, In addition, a live, attenuated a live, Chlamydophila attenuated Chlamydophila felis, felis, and/or and/or a live, a live, attenuated attenuated

Bordetellabronchiseptica Bordetella bronchiseptica and/or and/or a live, a live, attenuated attenuated Bartonella Bartonella spp. (e.g., spp. (e.g., B. B. henselae)also henselae) alsocancan be be included included in such in such multivalent multivalent vaccines. vaccines.

55 Furthermore, the Furthermore, the vaccines of the vaccines of the present present invention comprising one invention comprising one or or more more

alphavirusRNA alphavirus RNA replicon replicon particles particles of the of the present present invention invention [e.g.,[e.g., encoding encoding a VS-FCV a VS-FCV

capsidprotein capsid proteinororananantigenic antigenic fragment fragment thereof] thereof] with with one one or or modified more more modified live, live, virus virus isolates, further isolates, further can comprise can comprise oneone or more or more killed killed virus virus isolates isolates such such as a killed as a killed FCV FCV strain, and/or strain, killed feline and/or aa killed feline herpesvirus and/ora akilled herpesvirus and/or killedfeline felineparvovirus parvovirus and/or and/or a a 00 killed feline killed feline leukemia virus, and/or leukemia virus, and/ora akilled killed feline feline infectious infectious peritonitis peritonitis virus virus and/or and/oraa killed feline killed feline immunodeficiency virus immunodeficiency virus and/or and/or a killed a killed borna borna disease disease virus virus and/orand/or a a killed rabies killed rabies virus, virus, and/or killed feline and/or aa killed feline influenza virus and/or influenza virus and/ora akilled killed canine canine influenza virus, influenza virus, and/or and/ora akilled killed avian avianinfluenza influenzavirus, virus,and/or and/or a killedcanine a killed canine pneumovirus, pneumovirus, and/or and/or a killed a killed feline feline pneumovirus. pneumovirus. In addition, In addition, bacterins bacterins of of 55 Chlamydophila Chlamydophila felis,and/or felis, and/or Bordetella Bordetella bronchiseptica bronchiseptica and/orand/or Bartonella Bartonella spp. spp. (e.g., (e.g., B. henselae) B. henselae)cancan also also be included be included in such in such multivalent multivalent vaccines. vaccines.

It isisalso It alsototobe be understood thatthis understood that this invention inventionisisnot notlimited limitedtotothe theparticular particular configurations,process configurations, process steps, steps, andand materials materials disclosed disclosed hereinherein as suchas such 00 configurations,process configurations, process steps, steps, andand materials materials maysomewhat. may vary vary somewhat. It is alsoItto is be also to be understood that understood that the the terminology terminology employed hereinisis used employed herein used for for the the purpose of purpose of

describingparticular describing particularembodiments embodimentsonly only and isand notisintended not intended to be limiting, to be limiting, since since the the scopeofofthe scope thepresent present invention invention willbe be will limited limited only only by the by the appended appended claims claims and and equivalentsthereof. equivalents thereof. 25

23

TABLE SEQUENCE TABLE SEQUENCE

SEQ NO: ID NO: Description Description Type Type SEQ ID

1 1 Feline Calicivirus Feline Calicivirus(VS-FCV) (VS-FCV) nucleic acid nucleic acid DNA DNA 22 Feline Calicivirus Feline Calicivirus(VS-FCV) (VS-FCV) amino acid amino acid

33 Calicivirus(F9-like) Feline Calicivirus Feline (F9-like) nucleic acid nucleic acid DNA DNA 4 4 Feline Calicivirus Feline Calicivirus (F9-like) (F9-like) amino acid amino acid

5 5 FeLVviral FeLV viralglycoprotein glycoprotein (gp85) (gp85) nucleic acid nucleic acid DNA DNA 66 FeLVviral FeLV viralglycoprotein glycoprotein (gp85) (gp85) amino acid amino acid 7 7 FeLVviral FeLV viralglycoprotein glycoprotein (gp70) (gp70) nucleic acid nucleic acid DNA DNA 88 FeLVviral FeLV viralglycoprotein glycoprotein (gp70) (gp70) amino acid amino acid

9 9 Rabies virus Rabies virus Glycoprotein Glycoprotein nucleic acid nucleic acid DNA DNA 10 10 Rabies virus Glycoprotein Rabies virus Glycoprotein amino acid aminoacid 11 11 GGCGCGCCGCACC nucleicacid nucleic acid GGCGCGCCGCACC 12 12 Feline Calicivirus Feline Calicivirus (VS-FCV) (VS-FCV) nucleic acid nucleic acid RNA RNA 13 13 Feline Calicivirus Feline Calicivirus(F9-like) (F9-like) nucleic acid nucleic acid RNA RNA 14 14 FeLVviral FeLV viralglycoprotein glycoprotein (gp85) (gp85) nucleic acid nucleic acid RNA RNA 15 15 FeLVviral FeLV viralglycoprotein glycoprotein(gp70) (gp70) nucleic acid nucleic acid RNA RNA 16 16 Rabies virus Rabies virus Glycoprotein Glycoprotein nucleic acid nucleic acid RNA RNA TTAATTAA TTAATTAA nucleic acid nucleicacid

SEQUENCES SEQUENCES 55 Feline Calicivirus Feline Caliciviruscapsid capsid(VS-FCV) (VS-FCV) SEQ ID NO: SEQ ID NO:1 1 atggctgacgacggatctgtgaccaccccagaacaaggaacaatggtcggaggagtgatt atggctgacgacggatctgtgaccaccccagaacaaggaacaatggtoggaggagtgatt gccgaacccagcgctcagatgtcaactgcggcggacatggcctccggaaagtcggtggac gccgaacccagcgctcagatgtcaactgcggcggacatggcctccggaaagtcggtggad tccgagtgggaagccttcttctcgttccacacgtccgtgaactggagcacctccgaaacc ccgagtgggaagccttcttctcgttccacacgtccgtgaactggagcacctccgaaacc caaggaaagatcctcttcaagcagtccctgggtcccctgctgaacccgtacctggagcac caaggaaagatcctcttcaagcagtccctgggtcccctgctgaacccgtacctggagcac 10 atcagcaagctgtacgtcgcttggagcgggtcgatcgaagtgcgattttccatctcggga atcagcaagctgtacgtcgcttggagcgggtcgatcgaagtgcgattttccatctoggga agcggcgtgttcggtggtaaactggccgccatcgtcgtgccgcctggtgtcgaccctgtc agcggcgtgttcggtggtaaactggccgccatcgtcgtgccgcctggtgtcgaccctgtc cagtcaacctccatgctgcagtacccgcacgtcctgttcgacgcaagacaagtggagcca cagtcaacctccatgctgcagtacccgcacgtcctgttcgacgcaagacaagtggagcca gtgatcttctccatcccggacctccgcaacagcctgtatcacttgatgtccgataccgat gtgatcttctccatcccggacctccgcaacagcctgtatcacttgatgtccgataccgat accacttccctcgtgatcatggtgtacaacgatctgatcaacccgtacgccaatgactcc accacttccctcgtgatcatggtgtacaacgatctgatcaacccgtacgccaatgactcc 15 15 aacagctcgggttgcatcgtgaccgtcgaaacgaagcctggcatcgatttcaagtttcat aacagctcgggttgcatcgtgaccgtcgaaacgaagcctggcatcgatttcaagtttcat ctgctgaaaccgcccggatccatgcttactcacgggtccatcccttccgatctgatcccc ctgctgaaaccgcccggatccatgcttactcacgggtccatcccttccgatctgatcccc aagagctcctccctgtggattgggaaccgccactggaccgatattaccgatttcgtgatt aagagctcctccctgtggattgggaaccgccactggaccgatattaccgatttcgtgatt

24

cggcctttcgtgttccaagccaaccggcacttcgacttcaaccaggagactgccggctgg 66106600610e6e66e00ee0110e60110e36600ee006ee0011616311100660 tcaactccacggttccgcccattggccgtgactgtgtcgcagtcaaagggagccaagctc gggaacggcatcgccaccgactacattgtgcctggaatccccgacggatggcctgatact accatccccaccaagctgacccctaccggagattacgccatcacctcctccgacggcaat 5 S gatattgaaaccaagctggaatacgagaacgcggacgtgattaagaacaacaccaacttc 0170ee00e0ee0ee6ee11e660e66060ee6e60eqee66106eec0pee611e1eb cgctccatgtatatctgcggaagcctccagagggcttggggcgacaagaagatcagcaac 0ee06e07e6ee6ee0e606666110666e6e001006ee6606101e1e161e001060 accgggttcatcactaccggagtgatttctgacaactccatcagcccttcgaacacaatt 2022209359 gaccagtccaagatcgtggtgtaccaggacaaccatgtcaattcggaggtccagactagc gacatcactcttgccatcctgggctacaccggaattggagaagaggccataggcgccaac 0 0 cgggactccgtcgtgagaatttccgtgcttccggaaatggagaaggggcggaaatcac 0e01eee6656666ee56e6610eee660011061630111ee6e6160160010e6660 cccatcttctacaaaaattccatgaagctgggctacgtgatctcctccattgacgtgttc aactcccaaatcctccacacctcgcgccagctgtcactgaacaactacttgttgccccct gactccttcgcggtgtaccggattattgacagcaacggatcatggttcgacattgggatt 11e66611e0e6011661e0qe665ee06e0e611e11e6600e16166060110010e6 gacagcgatgggttttcattcgtgggcgtgtcgtcatttccaaagctggagtttccgctg 6106001116e66106eee00111e01601616066616011e011116661e606e3e6 5 S tccgcctcatacatgggcatccagctcgcaaagatccggctggcgtccaacatccggtca e0t66001e0ee00160661066001e6eee060106e001e06661e0e1e0q006001 tccatgactaagctgtga tccatgactaagctgtga

Feline Calicivirus Feline Caliciviruscapsid capsid(VS-FCV) (VS-FCV) SEQ SEQ IDIDNO: NO:2 2 MADDGSVTTPEQGTMVGGVIAEPSAQMSTAADMASGKSVDSEWEAFFSFHTSVNWSTSET .0 0: QGKILFKQSLGPLLNPYLEHISKLYVAWSGSIEVRFSISGSGVFGGKLAAIVTJPPGVDPV QSTSMLQYPHVLFDARQVEPVIFSIPDLRNSLYHLMSDTDTTSLVIMVYNDLINPYANDS NSSGCIVTVETKPGIDFKFHLLKPPGSMLTHGSIPSDLIPKSSSLWIGNRHWTDITDFVI RPFVFQANRHFDFNQETAGWSTPRFRPLAVTVSQSKGAKLGNGIATDYIVPGIPDGWPDT TIPTKLTPTGDYAITSSDGNDIETKLEYENADVIKNNTNFRSMYICGSLQRAWGDKKISN Si .5 TGFITTGVISDNSISPSNTIDQSKIVTJYQDNHVNSEVQTSDITLAILGYTGIGEEAIGAN RDSVTJRISVLPETGARGGNHPIFYKNSMKLGYVISSIDVFNSQILHTSRQLSLNNYLLPP DSFAVYRIIDSNGSWFDIGIDSDGFSFVGVSSFPKLEFPLSASYMGIQLAKIRLASNIRS SMTKL

.0 0' Feline Calicivirus Feline Calicivirus (VS-FCV) capsid (SEQ (VS-FCV) capsid (SEQIDIDNO: NO:12)12) auggcugacgacggaucugugaccaccccagaacaaggaacaauggucggaggagugauu nne6n6e66e66on66neeoee66eeoee6e3033eooe6n6none66oe6oe6n3f6ne gccgaacccagcgcucagaugucaacugcggcggacauggccuccggaaagucgguggac uccgagugggaagccuucuucucguuccacacguccgugaacuggagcaccuccgaaacc boeee60onooeo6e66noee6n6oon6oeoeoonn6ononnonnoobee666n6e630n caaggaaagauccucuucaagcagucccuggguccccugcugaacccguaccuggagcac 15 S' aucagcaagcuguacgucgcuuggagcgggucgaucgaagugcgauuuuccaucucggga e666ononeoonnnne6o6n6ee6one6on666o6e66nno6on6oen6nobeeobeone agcggcguguucggugguaaacuggccgccaucgucgugccgccuggugucgacccuguc on6noooe6on6n66noo6oo6n6on6oneoo6o366noeeenb6n66onn6n636606e cagucaaccuccaugcugcaguacccgcacguccuguucgacgcaagacaaguggagcca cagucaaccuccaugcugcaguacccgcacguccuguucgacgcaagacaaguggagcca gugaucuucuccaucccggaccuccgcaacagccuguaucacuugauguccgauaccgau ne6ooene6oon6ne6nnoeonen6noo6eoeeofoono3ef6o3oneoononnone6n6 accacuucccucgugaucaugguguacaacgaucugaucaacccguacgccaaugacucc oonoe6neeoo6oen6oooeeone6none6oeeoen6nf6neonefnfonooonnoeooe 40 40 aacagcucggguugcaucgugaccgucgaaacgaagccuggcaucgauuucaaguuucau neonnn6eeonnne6oneo66noobee6oeee6onbooebn6oneobnn6663nobeoee cugcugaaaccgcccggauccaugcuuacucacggguccaucccuuccgaucugaucccc aagagcuccucccuguggauugggaaccgccacuggaccgauauuaccgauuucgugauu nne6n6onnne6ooennene6ooe66noe336ooee66bnnef6n6no3onoonobebee cggccuuucguguuccaagccaaccggcacuucgacuucaaccaggagacugccggcugg 66no66oo6noe6e66eooeeonnoebonnoeo663oeeoofeeoonnfn6onnnoo660 ucaacuccacgguuccgcccauuggccgugacugugucgcagucaaagggagccaagcuc 45 45 gggaacggcaucgccaccgacuacauugugccuggaauccccgacggauggccugauacu accauccccaccaagcugaccccuaccggagauuacgccaucaccuccuccgacggcaau gauauugaaaccaagcuggaauacgagaacgcggacgugauuaagaacaacaccaacuuc onnoeeooeoeeoee6eenne6n6oe6636bee6e6oenee66nobeeoopeefnnene6 cgcuccauguauaucugcggaagccuccagagggcuuggggcgacaagaagaucagcaac cgcuccauguauaucugcggaagccuccagagggcuuggggcgacaagaagaucagcaac accggguucaucacuaccggagugauuucugacaacuccaucagcccuucgaacacaauu nneeoeoee6onn0oo6eoneson3eeoebnonnne6nbe6653enoeoneonn66600e 50 0S gaccaguccaagaucgugguguaccaggacaaccaugucaauucggagguccagacuagc gacaucacucuugccauccugggcuacaccggaauuggagaagaggccauaggcgccaac cgggacuccgucgugagaauuuccgugcuuccggaaacuggagcaaggggcggaaaucac cgggacuccgucgugagaauuuccgugcuuccggaaacuggagcaaggggcggaaaucac cccaucuucuacaaaaauuccaugaagcugggcuacgugaucuccuccauugacguguuc onn6n6oe6nneoonoonone6n6oeno666nobeebneoonnpppeeopnonnoneooo aacucccaaauccuccacaccucgcgccagcugucacugaacaacuacuuguugcccccu 55 SS gacuccuucgcgguguaccggauuauugacagcaacggaucaugguucgacauugggauu nne666nneoe6onn66neone66oeeobeoefnnenne6booenfn66ofonnoanoe6 gacagcgauggguuuucauucgugggcgugucgucauuuccaaagcuggaguuuccgcug 6no6oonnn6e66no6eeeoonnneon6onfn6o666nbonneonnnnf66ne63be0e6 uccgccucauacaugggcauccagcucgcaaagauccggcuggcguccaacauccgguca uccaugacuaagcuguga e6n6nobeenoebneoon

25

Feline Calicivirus Feline Calicivirus (F9-like) (F9-like) capsid capsid(SEQ (SEQ ID NO: ID NO: 3) 3) atgactgccccggaacaaggaacgatggtcggaggagtgattgcagaaccgtcagcacag atgactgccccggaacaaggaacgatggtcggaggagtgattgcagaaccgtcagcacag atgtccaccgctgccgacatggccactggaaagagcgtggactccgaatgggaagccttc atgtccaccgctgccgacatggccactggaaagagcgtggactccgaatgggaagcctto ttctccttccacacttcggtcaactggtcgactagcgaaacccaggggaagattttgttc ttctccttccacacttcggtcaactggtcgactagogaaacccaggggaagattttgttc 5 5 aagcaatccctcggccctctgctgaacccctacctggagcatctggccaagctgtacgtg agcaatccctcggccctctgctgaacccctacctggagcatctggccaagctgtacgto gcatggtcgggcagcatcgaagtgcgctttagcatttccggctccggagtgttcggggga gcatggtcgggcagcatcgaagtgcgctttagcatttccggctccggagtgttcggggga aagcttgctgccattgtcgtgccgccaggagtggacccggtgcagtccacttctatgctc aagcttgctgccattgtcgtgccgccaggagtggacccggtgcagtccacttctatgctc 2022209359 caatacccgcatgtcctgttcgacgccagacaggtggagcctgtgatcttttgcctgccg caatacccgcatgtcctgttcgacgccagacaggtggagcctgtgatcttttgcctgccg gatctcaggtccaccctgtatcacctcatgtccgacaccgacaccacctcgctcgtgatc gatctcaggtccaccctgtatcacctcatgtccgacaccgacaccacctcgctcgtgatc 0 0 atggtgtacaacgacctgatcaacccctacgctaacgacgccaacagctcaggttgcatt atggtgtacaacgacctgatcaacccctacgctaacgacgccaacagctcaggttgcatt gtgactgtcgaaaccaagccaggccctgacttcaagtttcatttgctgaagccgcccggt gtgactgtcgaaaccaagccaggccctgacttcaagtttcatttgctgaagcogcccggt tccatgctgacccacggctcgatcccatccgacctgatccccaagacgagctccctgtgg tccatgctgacccacggctcgatcccatccgacctgatccccaagacgagctccctgtgg atcggaaaccgctactggtccgatattaccgacttcgtgatcagaccattcgtgttccaa atcggaaaccgctactggtccgatattaccgacttcgtgatcagaccattcgtgttccaa gccaaccgccatttcgacttcaaccaggaaaccgcaggatggtcgacccctcgattccgc gccaaccgccatttcgacttcaaccaggaaaccgcaggatggtcgacccctcgattccgo 55 ccgatttcagtgaccatcaccgaacagaacggcgcgaagctgggaattggcgtggcgacc ccgatttcagtgaccatcaccgaacagaacggcgcgaagctgggaattggcgtggcgaco gactacatcgtgccgggaatcccggatggatggcctgatacgaccattcccggggagctg gactacatcgtgccgggaatcccggatggatggcctgatacgaccattcccggggagctc atccctgccggggactacgccatcaccaacggtactggaaacgacatcaccactgccacc atccctgccggggactacgccatcaccaacggtactggaaacgacatcaccactgccaco ggttacgacaccgccgacatcataaagaacaacaccaacttcagaggaatgtacatttgc ggttacgacaccgccgacatcataaagaacaacaccaacttcagaggaatgtacatttgo ggctccctgcaacgcgcttggggtgacaaaaagatctcgaacactgccttcatcacaaca ggctccctgcaacgcgcttggggtgacaaaaagatctogaacactgccttcatcacaaca 10 0 gcgactctggacggcgataacaacaacaagatcaatccttgtaataccatcgaccagtcc gcgactctggacggcgataacaacaacaagatcaatccttgtaataccatcgaccagtcc aaaatcgtggtgttccaggataaccacgtgggaaagaaggcgcagacctccgacgacact aaaatcgtggtgttccaggataaccacgtgggaaagaaggcgcagacctccgacgacact ctggcgctgcttggctacaccgggatcggcgagcaggccattggaagcgatcgggatcgg ctggcgctgcttggctacaccgggatcggcgagcaggccattggaagcgatcgggatcgg gtcgtgcggatctccaccctccccgagactggagcaaggggaggcaaccaccccatcttt gtcgtgcggatctccaccctccccgagactggagcaaggggaggcaaccaccccatcttt tacaaaaacagcattaagctcggatacgtcatccgctccatcgatgtgttcaactctcaa tacaaaaacagcattaagctcggatacgtcatccgctccatcgatgtgttcaactotcaa '5 :5 atcctgcacacttcgcggcagctgtccctgaaccactacctcttgccgcccgactccttc atcctgcacacttcgcggcagctgtccctgaaccactacctottgccgcccgactccttc gccgtctaccggatcattgattcgaacgggagctggttcgacatcggcattgatagcgat gccgtctaccggatcattgattcgaacgggagctggttcgacatcggcattgatagcgat ggcttctcgtttgtgggcgtgtcgggcttcgggaagctggagttcccactgagcgcctca ggcttctcgtttgtgggcgtgtcgggcttcgggaagctggagttcccactgagcgcctca tacatgggtatccagctggccaagatcaggctggcctccaacatccgctcacctatgact tacatgggtatccagctggccaagatcaggctggcctccaacatccgctcacctatgact aagctgtga aagctgtga .0 0 Feline Calicivirus Feline Calicivirus (F9-like) (F9-like) capsid capsid(SEQ (SEQ ID NO: ID NO: 4) 4) MTAPEQGTMVGGVIAEPSAQMSTAADMATGKSVDSEWEAFFSFHTSVNWSTSETQGKILF MTAPEQGTMVGGVIAEPSAQMSTAADMATGKSVDSEWEAFFSFHTSVNWSTSETQGKILF KQSLGPLLNPYLEHLAKLYVAWSGSIEVRFSISGSGVFGGKLAAIVVPPGVDPVQSTSML KQSLGPLLNPYLEHLAKLYVAWSGSIEVRFSISGSGVFGGKLAAIVVPPGVDPVQSTSML QYPHVLFDARQVEPVIFCLPDLRSTLYHLMSDTDTTSLVIMVYNDLINPYANDANSSGCI QYPHVLFDARQVEPVIFCLPDLRSTLYHLMSDTDTTSLVIMVYNDLINPYANDANSSGC1 15 5 VTVETKPGPDFKFHLLKPPGSMLTHGSIPSDLIPKTSSLWIGNRYWSDITDFVIRPFVFQ VTVETKPGPDFKFHLLKPPGSMLTHGSIPSDLIPKTSSLWIGNRYWSDITDFVIRPFVFQ ANRHFDFNQETAGWSTPRFRPISVTITEQNGAKLGIGVATDYIVPGIPDGWPDTTIPGEL ANRHFDFNQETAGWSTPRFRPISVTITEQNGAKLGIGVATDYIVPGIPDGWPDTTIPGEI IPAGDYAITNGTGNDITTATGYDTADIIKNNTNFRGMYICGSLQRAWGDKKISNTAFITT ATLDGDNNNKINPCNTIDQSKIVVFQDNHVGKKAQTSDDTLALLGYTGIGEQAIGSDRDR ATLDGDNNNKINPCNTIDQSKIVVFQDNHVGKKAQTSDDTLALLGYTGIGEQAIGSDRDR VVRISTLPETGARGGNHPIFYKNSIKLGYVIRSIDVFNSQILHTSRQLSLNHYLLPPDSF VVRISTLPETGARGGNHPIFYKNSIKLGYVIRSIDVFNSQILHTSRQLSLNHYLLPPDSI 40 40 AVYRIIDSNGSWFDIGIDSDGFSFVGVSGFGKLEFPLSASYMGIQLAKIRLASNIRSPMT AVYRIIDSNGSWFDIGIDSDGFSFVGVSGFGKLEFPLSASYMGIQLAKIRLASNIRSPM KL KL

Feline Calicivirus Feline Calicivirus (F9-like) (F9-like) capsid capsid(SEQ (SEQ ID NO: ID NO: 13) 13) augacugccccggaacaaggaacgauggucggaggagugauugcagaaccgucagcacag augacugccccggaacaaggaacgauggucggaggagugauugcagaaccgucagcacac 45 45 auguccaccgcugccgacauggccacuggaaagagcguggacuccgaaugggaagccuuc auguccaccgcugccgacauggccacuggaaagagcguggacuccgaaugggaagccuuc uucuccuuccacacuucggucaacuggucgacuagcgaaacccaggggaagauuuuguuc uucuccuuccacacuucggucaacuggucgacuagcgaaacccaggggaagauuuuguu aagcaaucccucggcccucugcugaaccccuaccuggagcaucuggccaagcuguacgug aagcaaucccucggcccucugcugaaccccuaccuggagcaucuggccaagcuguacgug gcauggucgggcagcaucgaagugcgcuuuagcauuuccggcuccggaguguucggggga gcauggucgggcagcaucgaagugcgcuuuagcauuuccggcuccggaguguucggggga aagcuugcugccauugucgugccgccaggaguggacccggugcaguccacuucuaugcuc aagcuugcugccauugucgugccgccaggaguggacccggugcaguccacuucuaugcuc 50 50 caauacccgcauguccuguucgacgccagacagguggagccugugaucuuuugccugccg caauacccgcauguccuguucgacgccagacagguggagccugugaucuuuugccugcct gaucucagguccacccuguaucaccucauguccgacaccgacaccaccucgcucgugauc gaucucagguccacccuguaucaccucauguccgacaccgacaccaccucgcucgugauc augguguacaacgaccugaucaaccccuacgcuaacgacgccaacagcucagguugcauu augguguacaacgaccugaucaaccccuacgcuaacgacgccaacagcucagguugcauu gugacugucgaaaccaagccaggcccugacuucaaguuucauuugcugaagccgcccggu gugacugucgaaaccaagccaggcccugacuucaaguuucauuugcugaagccgcccggu uccaugcugacccacggcucgaucccauccgaccugauccccaagacgagcucccugugg uccaugcugacccacggcucgaucccauccgaccugauccccaagacgagcucccugugg 55 55 aucggaaaccgcuacugguccgauauuaccgacuucgugaucagaccauucguguuccaa aucggaaaccgcuacugguccgauauuaccgacuucgugaucagaccauucguguuccaa gccaaccgccauuucgacuucaaccaggaaaccgcaggauggucgaccccucgauuccgc gccaaccgccauuucgacuucaaccaggaaaccgcaggauggucgaccccucgauuccgo ccgauuucagugaccaucaccgaacagaacggcgcgaagcugggaauuggcguggcgacc ccgauuucagugaccaucaccgaacagaacggcgcgaagcugggaauuggcguggogacc gacuacaucgugccgggaaucccggauggauggccugauacgaccauucccggggagcug gacuacaucgugccgggaaucccggauggauggccugauacgaccauucccggggagcug aucccugccggggacuacgccaucaccaacgguacuggaaacgacaucaccacugccacc aucccugccggggacuacgccaucaccaacgguacuggaaacgacaucaccacugccaco

26

gguuacgacaccgccgacaucauaaagaacaacaccaacuucagaggaauguacauuugc gguuacgacaccgccgacaucauaaagaacaacaccaacuucagaggaauguacauuugc ggcucccugcaacgcgcuuggggugacaaaaagaucucgaacacugccuucaucacaaca ggcucccugcaacgcgcuuggggugacaaaaagaucucgaacacugccuucaucacaaca gcgacucuggacggcgauaacaacaacaagaucaauccuuguaauaccaucgaccagucc gcgacucuggacggcgauaacaacaacaagaucaauccuuguaauaccaucgaccagucc aaaaucgugguguuccaggauaaccacgugggaaagaaggcgcagaccuccgacgacacu aaaaucgugguguuccaggauaaccacgugggaaagaaggcgcagaccuccgacgacacu 5 5 cuggcgcugcuuggcuacaccgggaucggcgagcaggccauuggaagcgaucgggaucgg cuggcgcugcuuggcuacaccgggaucggcgagcaggccauuggaagcgaucgggaucgg gucgugcggaucuccacccuccccgagacuggagcaaggggaggcaaccaccccaucuuu gucgugcggaucuccacccuccccgagacuggagcaaggggaggcaaccaccccaucuuu uacaaaaacagcauuaagcucggauacgucauccgcuccaucgauguguucaacucucaa uacaaaaacagcauuaagcucggauacgucauccgcuccaucgauguguucaacucucaa 2022209359 auccugcacacuucgcggcagcugucccugaaccacuaccucuugccgcccgacuccuuc uccugcacacuucgcggcagcugucccugaaccacuaccucuugccgcccgacuccuuc gccgucuaccggaucauugauucgaacgggagcugguucgacaucggcauugauagcgau gccgucuaccggaucauugauucgaacgggagcugguucgacaucggcauugauagcgau 0 0 ggcuucuguuugugggeguguagggcuuegggaageuggaguucccacugagegccuca ggcuucucguuugugggcgugucgggcuucgggaagcuggaguucccacugagcgccuca uacauggguauccagcuggccaagaucaggcuggccuccaacauccgcucaccuaugacu uacauggguauccagcuggccaagaucaggcuggccuccaacauccgcucaccuaugacu aagcuguga aagcuguga

Feline Leukemia Feline Virus envelope Leukemia Virus envelopeglycoprotein glycoprotein(gp85) (gp85)SEQ SEQID ID NO:NO: 5 5 5 5 atggagtcaccaacacaccctaaaccttctaaagacaaaaccctctcgtggaatctcgccttccttgt atggagtcaccaacacaccctaaaccttctaaagacaaaaccctctcgtggaatctcgccttccttgt gggcatcctgttcacaatcgacatcggcatggccaacccttcgccgcatcagatctacaatgtgacat gggcatcctgttcacaatcgacatcggcatggccaacccttcgccgcatcagatctacaatgtgaca gggtcattactaatgtgcagacaaacacccaggcaaatgctacttctatgcttggtactctgactgat gggtcattactaatgtgcagacaaacacccaggcaaatgctacttctatgcttggtactctgactgat gcttatccaaccctgcacgtcgacctttgcgatctcgtcggtgacacatgggagcccatcgtgctgaa gcttatccaaccctgcacgtcgacctttgcgatctogtcggtgacacatgggagcccatcgtgctgaa tccaactaatgtcaaacatggtgccaggtattcttctagcaaatacgggtgtaagaccactgatcgga tccaactaatgtcaaacatggtgccaggtattcttctagcaaatacgggtgtaagaccactgatogga .0 0 agaaacagcaacaaacctacccattctacgtgtgcccgggtcacgcaccgtccctgggtccgaaggga agaaacagcaacaaacctacccattctacgtgtgcccgggtcacgcaccgtccctgggtccgaaggga acacattgtgggggagcccaagacggtttttgcgctgcttggggttgtgaaacaaccggagaagcctg acacattgtgggggagcccaagacggtttttgcgctgcttggggttgtgaaacaaccggagaagcctg gtggaagcctacctcatcttgggactacattactgtgaaaagaggctctagccaggataacagctgcg gtggaagcctacctcatcttgggactacattactgtgaaaagaggctctagccaggataacagctgcg aaggaaagtgtaatcccctggtgcttcaattcacccagaaaggccggcaggcatcatgggatggaccg aaggaaagtgtaatcccctggtgcttcaattcacccagaaaggccggcaggcatcatgggatggaccg aaaatgtggggacttagactctatcgcaccggatacgaccccatcgctctgtttactgtgtcacgcca aaaatgtggggacttagactctatcgcaccggatacgaccccatcgctctgtttactgtgtcacgcca .5 :5 agtctccaccattactccgccacaggccatggggccgaatctggtcctccccgatcagaagccaccct agtctccaccattactccgccacaggccatggggccgaatctggtcctccccgatcagaagccaccct cacggcaaagtcaaaccggctcaaaagtggccacccaacggccccagacaaatgagtccgcacctagg cacggcaaagtcaaaccggctcaaaagtggccacccaacggccccagacaaatgagtccgcacctagg tcagtggcacctacaacaatgggtccaaagcggatcggaaccggagacaggctcattaacctcgtgca tcagtggcacctacaacaatgggtccaaagcggatcggaaccggagacaggctcattaacctcgtgca agggacttatctggcccttaacgctactgaccccaacaagaccaaggattgctggctctgccttgtga agggacttatctggcccttaacgctactgaccccaacaagaccaaggattgctggctctgccttgtga gcagacctccttactatgaggggatcgccattctcggaaactactcaaatcagaccaacccccctccg gcagacctccttactatgaggggatcgccattctcggaaactactcaaatcagaccaacccccctccg 0 0 tcgtgtctgagcaccccccagcacaagcttactatttcagaagtcagtggacagggaatgtgcatcgg tcgtgtctgagcaccccccagcacaagcttactatttcagaagtcagtggacagggaatgtgcatogg aaccgtgccaaagactcatcaagccctttgcaacaaaactcaacaagggcacactggagctcattatc aaccgtgccaaagactcatcaagccctttgcaacaaaactcaacaagggcacactggagctcattatc tcgccgcacctaacgggacctactgggcttgcaatactggattgaccccgtgtatctctatggccgtg tcgccgcacctaacgggacctactgggcttgcaatactggattgaccccgtgtatctctatggccgtg ctgaattggacttccgacttctgcgtgcttattgagctttggcctagagtgacataccatcagcctga ctgaattggacttccgacttctgcgtgcttattgagctttggcctagagtgacataccatcagcctga gtacgtctatacccatttcgccaaggcagtcagattccggcgggagcctatctccctgactgtggcct gtacgtctatacccatttcgccaaggcagtcagattccggcgggagcctatctccctgactgtggcct 5 5 tgatgctcggtggactgacagtgggaggaattgcagctggagtcggaactggaaccaaggccctgctc tgatgctcggtggactgacagtgggaggaattgcagctggagtcggaactggaaccaaggccctgctc gaaactgctcagttccggcagctgcagatggccatgcacactgacatccaggctctggaggaatcaat gaaactgctcagttccggcagctgcagatggccatgcacactgacatccaggctctggaggaatcaat ttcagcccttgagaaaagcttgacctcgctgtctgaagtggtcctccaaaacaggcgcggtttggaca ttcagcccttgagaaaagcttgacctogctgtctgaagtggtcctccaaaacaggcgcggtttggaca tcctgttccttcaagagggtggtctgtgcgccgctctcaaggaggaatgctgtttctacgctgaccat tcctgttccttcaagagggtggtctgtgcgccgctctcaaggaggaatgctgtttctacgctgaccat accgggctggtgcgcgataacatggcaaagctgcgggaacgcttgaaacagaggcagcaactgttcga accgggctggtgcgcgataacatggcaaagctgcgggaacgcttgaaacagaggcagcaactgttcga 40 40 ctctcagcagggatggttcgagggctggtttaacaagagcccatggtttaccactctgatctcttcaa ctctcagcagggatggttcgagggctggtttaacaagagcccatggtttaccactctgatctcttcaa tcatgggtccactgctcatcctgcttctgattcttctcttcggaccgtgtattctcaacaggctggtg tcatgggtccactgctcatcctgcttctgattcttctcttcggaccgtgtattctcaacaggctggtg cagtttgtcaaggacagaatctcggtggtccaggccctgattcttactcagcagtatcagcagattaa cagtttgtcaaggacagaatctcggtggtccaggccctgattcttactcagcagtatcagcagattaa gcagtacgaccccgatcggccttga gcagtacgaccccgatcggccttga

45 45 Feline Leukemia Feline Virus envelope Leukemia Virus envelopeglycoprotein glycoprotein(gp85) (gp85)SEQ SEQID ID NO: NO: 6 6 MESPTHPKPSKDKTLSWNLAFLVGILFTIDIGMANPSPHQIYNVTWVITNVQTNTQANAT MESPTHPKPSKDKTLSWNLAFLVGILFTIDIGMANPSPHOIYNVTWVITNVOTNTOANA7 SMLGTLTDAYPTLHVDLCDLVGDTWEPIVLNPTNVKHGARYSSSKYGCKTTDRKKQQQTY SMLGTLTDAYPTLHVDLCDLVGDTWEPIVLNPTNVKHGARYSSSKYGCKTTDRKKQQOT PFYVCPGHAPSLGPKGTHCGGAQDGFCAAWGCETTGEAWWKPTSSWDYITVKRGSSQDNS PFYVCPGHAPSLGPKGTHCGGAQDGFCAAWGCETTGEAWWKPTSSWDYITVKRGSSODNS CEGKCNPLVLQFTQKGRQASWDGPKMWGLRLYRTGYDPIALFTVSRQVSTITPPQAMGPN CEGKCNPLVLQFTQKGRQASWDGPKMWGLRLYRTGYDPIALFTVSRQVSTITPPQAMGPN 50 50 LVLPDQKPPSRQSQTGSKVATQRPQTNESAPRSVAPTTMGPKRIGTGDRLINLVQGTYLA LNATDPNKTKDCWLCLVSRPPYYEGIAILGNYSNQTNPPPSCLSTPQHKLTISEVSGQGM LNATDPNKTKDCWLCLVSRPPYYEGIAILGNYSNQTNPPPSCLSTPQHKLTISEVSGQGM CIGTVPKTHQALCNKTQQGHTGAHYLAAPNGTYWACNTGLTPCISMAVLNWTSDFCVLIE CIGTVPKTHQALCNKTQQGHTGAHYLAAPNGTYWACNTGLTPCISMAVLNWTSDFCVLIE LWPRVTYHQPEYVYTHFAKAVRFRREPISLTVALMLGGLTVGGIAAGVGTGTKALLETAQ WPRVTYHQPEYVYTHFAKAVRFRREPISLTVALMLGGLTVGGIAAGVGTGTKALLETAO FRQLQMAMHTDIQALEESISALEKSLTSLSEVIVLQNRRGLDILFLQEGGLCAALKEECCF FRQLQMAMHTDIQALEESISALEKSLTSLSEVVLONRRGLDILFLQEGGLCAALKEECCF 55 55 YADHTGLVRDNMAKLRERLKQRQQLFDSQQGWFEGWFNKSPWFTTLISSIMGPLLILLLI YADHTGLVRDNMAKLRERLKQRQQLFDSQQGWFEGWFNKSPWFTTLISSIMGPLLILLLI LLFGPCILNRLVQFVKDRISIVQALILTQQYQQIKQYDPDRP* LLFGPCILNRLVQFVKDRISVVQALILTQQYQQIKQYDPDRP

Feline Leukemia Feline Virus envelope Leukemia Virus envelopeglycoprotein glycoprotein(gp85) (gp85)SEQ SEQID ID NO:NO: 14 14

27

auggagucaccaacacacccuaaaccuucuaaagacaaaacccucucguggaaucucgccuuccuugu auggagucaccaacacacccuaaaccuucuaaagacaaaacccucucguggaaucucgccuuccuugu gggcauccuguucacaaucgacaucggcauggccaacccuucgccgcaucagaucuacaaugugacau gggucauuacuaaugugcagacaaacacccaggcaaaugcuacuucuaugcuugguacucugacugau gggucauuacuaaugugcagacaaacacccaggcaaaugcuacuucuaugcuugguacucugacugau gcuuauccaacccugcacgucgaccuuugcgaucucgucggugacacaugggagcccaucgugcugaa 5 5 uccaacuaaugucaaacauggugccagguauucuucuagcaaauacggguguaagaccacugaucgga e66one6noeooe6eenfn666oeneeeobenonnonnen6beoo6n6bneoppeonbnpenoepoof agaaacagcaacaaaccuacccauucuacgugugcccgggucacgcaccgucccuggguccgaaggga acacauugugggggagcccaagacgguuuuugcgcugcuugggguugugaaacaaccggagaagccug 6noo6ee6e66ooeeoeee6n6nn6666nno6no636nnnnn66oe6eeooo6e66666nbnne0e0e 2022209359 guggaagccuaccucaucuugggacuacauuacugugaaaagaggcucuagccaggauaacagcugcg 6o6no6eoeene66eo56enono66e6peee6n6noenneoenoe666nnoneonooenoo6ee66n6 aaggaaaguguaauccccuggugcuucaauucacccagaaaggccggcaggcaucaugggauggaccg 0 0 aaaauguggggacuuagacucuauogeaceggauacgaccccauegcucuguuuacugugucacgeca agucuccaccauuacuccgccacaggccauggggccgaaucugguccuccccgaucagaagccacccu cacggcaaagucaaaccggcucaaaaguggccacccaacggccccagacaaaugaguccgcaccuagg ucaguggcaccuacaacaauggguccaaagcggaucggaaccggagacaggcucauuaaccucgugca agggacuuaucuggcccuuaacgcuacugaccccaacaagaccaaggauugcuggcucugccuuguga 5 S gcagaccuccuuacuaugaggggaucgccauucucggaaacuacucaaaucagaccaaccccccuccg ucgugucugagcaccccccagcacaagcuuacuauuucagaagucaguggacagggaaugugcaucgg ucgugucugagcaccccccagcacaagcuuacuaiuucagaagucaguggacagggaaugugcaucgg aaccgugccaaagacucaucaagcccuuugcaacaaaacucaacaagggcacacuggagcucauuauc onenneono6e66noeoeo666eeoeeonoepeeoeeobnnnooofeeoneonopbeeeoobn6oope ucgccgcaccuaacgggaccuacugggcuugcaauacuggauugaccccguguaucucuauggccgug 6n6oo66nenononen6n6ooooe6nne66noeneeo6nno666n3enooe6660eenooeo6o06o1 cugaauuggacuuccgacuucugcgugcuuauugagcuuuggccuagagugacauaccaucagccuga e6noobeoneooeneoe6n6e6enoo66nnno6ebnnenno6n6obnannoe6oonnoe6bnnee6na 0 guacgucuauacccauuucgccaaggcagucagauuccggcgggagccuaucucccugacuguggccu noo66n6noe6nooononenoo6e666o66oonne6eonbeo6beeo3fonnneoooenenonboen6 ugaugcucgguggacugacagugggaggaauugcagcuggagucggaacuggaaccaaggcccugcuc ugaugcucgguggacugacagugggaggaauugcagcuggagucggaacuggaaccaaggcccugcuc gaaacugcucaguuccggcagcugcagauggccaugcacacugacauccaggcucuggaggaaucaau uucagcccuugagaaaagcuugaccucgcugucugaagugguccuccaaaacaggcgcgguuuggaca eoe66nnn6606o66eoeeeeoonoon66n6ee6non6no6onooebnnobeeee6ebnnooobeann uccuguuccuucaagaggguggucugugcgccgcucucaaggaggaaugcuguuucuacgcugaccau 55 accgggcuggugcgcgauaacauggcaaagcugcgggaacgcuugaaacagaggcagcaacuguucga cucucagcagggaugguucgagggcugguuuaacaagagcccaugguuuaccacucugaucucuucaa eeonnonone6nonoeooennn66neooo6ebeeoeennn66no666e6onn66ne666eobeanono ucauggguccacugcucauccugcuucugauucuucucuucggaccguguauucucaacaggcuggug 5n66no66eoeeononnen6n6ooe66onnonannonne6nonnobnooneonobnoeoonb66ne0n caguuugucaaggacagaaucucggugguccaggcccugauucuuacucagcaguaucagcagauuaa eenne6eo6eonen6eo6eonoennonne6noo3f6eoon66nb6ononeebeoe6beeonfnnnbe0 gcaguacgaccccgaucggccuuga 00 Feline Leukemia Feline Virus envelope Leukemia Virus envelopeglycoprotein glycoprotein(gp70) (gp70)SEQ SEQID ID NO:NO: 7 7 aatcctagtccacaccaaatatataatgtaacttgggtaataaccaatgtacaaactaacacc aatcctagtccacaccaaatatataatgtaacttgggtaataaccaatgtacaaactaacacc caagctaacgccacctctatgttaggaaccttaaccgatgcctaccctaccctacatgttgac ttatgtgacctagtgggagacacctgggaacctatagtcctaaacccaaccaatgtaaaacac 0e0eeee161ee00ee000eee10016e1e100ee666100e0ebe666q6e100e6161e11 5 S' ggggcacgttactcctcctcaaaatatggatgtaaaactacagatagaaaaaaacagcaacag ggggcacgttactcctcctcaaaatatggatgtaaaactacagatagaaaaaaacagcaacag acataccccttttacgtctgccccggacatgccccctcgttggggccaaagggaacacattgt 7611e0e0ee666eee006666116010000061e0e660000610160e11110000e1e3e ggaggggcacaagatgggttttgtgccgcatggggatgtgagaccaccggagaagcttggtgg 661661106ee6e6600e00e6e6161e66661eo6c061611116661e6eeoeo6666e66 aagcccacctcctcatgggactatatcacagtaaaaagagggagtagtcaggacaatagctgt 76706e1ee0e66e016e16e666e6eeeee16e3e01e1e10e666qe0100103e0006ee gagggaaaatgcaaccccctggttttgcagttcacccagaagggaagacaagcctcttgggac 0e666110006ee0e6ee666ee6e000e0116e06111166100300ee061eeee666e6 40 40 ggacctaagatgtggggattgcgactataccgtacaggatatgaccctatcgctttattcacg 60e011e1110601e1000e61e1e66e0e1600e1e10e60611e6666161e6ee100e66 gtgtcccggcaggtatcaaccattacgccgcctcaggcaatgggaccaaacctagtcttacct gatcaaaaacccccatcccgacaatctcaaacagggtccaaagtggcgacccagaggccccaa acgaatgaaagcgccccaaggtctgttgcccccaccaccatgggtcccaaacggattgggacc ggagataggttaataaatttagtacaagggacatacctagccttaaatgccaccgaccccaac ggagataggttaataaatttagtacaagggacatacctagccttaaatgccaccgaccccaac 45 45 aaaactaaagactgttggctctgcctggtttctcgaccaccctattacgaagggattgcaatc ttaggtaactacagcaaccaaacaaacccccccccatcctgcctatctactccgcaacacaaa ctaactatatctgaagtatcagggcaaggaatgtgcatagggactgttcctaaaacccaccag 6e00e000eeeet0011610e666e1e36161ee66ee0666e01e16ee6101e1e10ee10 gctttgtgcaataagacacaacagggacatacaggggcgcactatctagccgcccccaacggc 0660ee000006006e10e10e0606666e0e1e0e666e0ee0e0ebee1pe061611106 acctattgggcctgtaacactggactcaccccatgcatttccatggcggtgctcaattggacc 50 os tctgatttttgtgtcttaatcgaattatggcccagagtgacttaccatcaacccgaatatgtg 6167e1ee6000ee01e00e110e616e6e000661e11ee601ee110161611171e6101 tacacacattttgccaaagctgtcaggttccgaaga Feline Leukemia Feline Virus envelope Leukemia Virus envelopeglycoprotein glycoprotein(gp70) (gp70)SEQ SEQID ID NO:NO: 8 8 NPSPHQIYNVTWVITNVQTNTQANATSMLGTLTDAYPTLHVDLCDLVGDTWEPIVLNPTNVKHGARYSSS KYGCKTTDRKKQQQTYPFYVCPGHAPSLGPKGTHCGGAQDGFCAAWGCETTGEAWWKPTSSWDYITVKRG 55 SS SSQDNSCEGKCNPLVLQFTQKGRQASWDGPKMWGLRLYRTGYDPIALFTVSRQVSTITPPQAMGPNLVLP DQKPPSRQSQTGSKVATQRPQTNESAPRSVAPTTMGPKRIGTGDRLINLVQGTYLALNATDPNKTKDCWL CLVSRPPYYEGIAILGNYSNQTNPPPSCLSTPQHKLTISEVSGQGMCIGTVPKTHQALCNKTQQGHTGAH YLAAPNGTYWACNTGLTPCISMAVLNWTSDFCVLIELWPRVTYHQPEYVYTHFAKAVRFRR

Jul 2022 28 28

Feline Leukemia Feline Virus envelope Leukemia Virus envelopeglycoprotein glycoprotein(gp70) (gp70)SEQ SEQID ID NO: NO: 15 15 aauccuaguccacaccaaauauauaauguaacuuggguaauaaccaauguacaaacuaacacc aauccuaguccacaccaaauauauaauguaacuuggguaauaaccaauguacaaacuaacaco caagcuaacgccaccucuauguuaggaaccuuaaccgaugccuacccuacccuacauguugac caagcuaacgccaccucuauguuaggaaccuuaaccgaugccuacccuacccuacauguugad 2022209359 30 uuaugugaccuagugggagacaccugggaaccuauaguccuaaacccaaccaauguaaaacac uuaugugaccuagugggagacaccugggaaccuauaguccuaaacccaaccaauguaaaacac 5 5 ggggcacguuacuccuccucaaaauauggauguaaaacuacagauagaaaaaaacagcaacag ggggcacguuacuccuccucaaaauauggauguaaaacuacagauagaaaaaaacagcaacag acauaccccuuuuacgucugccccggacaugcccccucguuggggccaaagggaacacauugu acauaccccuuuuacgucugccccggacaugcccccucguuggggccaaagggaacacauugu ggaggggcacaagauggguuuugugccgcauggggaugugagaccaccggagaagcuuggugg ggaggggcacaagauggguuuugugccgcauggggaugugagaccaccggagaagcuuggugg aagcccaccuccucaugggacuauaucacaguaaaaagagggaguagucaggacaauagcugu aagcccaccuccucaugggacuauaucacaguaaaaagagggaguagucaggacaauagcugu gagggaaaaugcaacccccugguuuugcaguucacccagaagggaagacaagccucuugggac gagggaaaaugcaacccccugguuuugcaguucacccagaagggaagacaagccucuugggac 0 0 ggaccuaagauguggggauugogacuauaccguacaggauaugaccouaucgcuuuauucacg ggaccuaagauguggggauugcgacuauaccguacaggauaugacccuaucgcuuuauucacg gugucccggcagguaucaaccauuacgccgccucaggcaaugggaccaaaccuagucuuaccu gugucccggcagguaucaaccauuacgccgccucaggcaaugggaccaaaccuagucuuacct gaucaaaaacccccaucccgacaaucucaaacaggguccaaaguggcgacccagaggccccaa gaucaaaaacccccaucccgacaaucucaaacaggguccaaaguggcgacccagaggccccaa acgaaugaaagcgccccaaggucuguugcccccaccaccaugggucccaaacggauugggacc acgaaugaaagcgccccaaggucuguugcccccaccaccaugggucccaaacggauugggacc ggagauagguuaauaaauuuaguacaagggacauaccuagccuuaaaugccaccgaccccaac ggagauagguuaauaaauuuaguacaagggacauaccuagccuuaaaugccaccgaccccaac 55 aaaacuaaagacuguuggcucugccugguuucucgaccacccuauuacgaagggauugcaauc aaaacuaaagacuguuggcucugccugguuucucgaccacccuauuacgaagggauugcaauc uuagguaacuacagcaaccaaacaaacccccccccauccugccuaucuacuccgcaacacaaa uuagguaacuacagcaaccaaacaaacccccccccauccugccuaucuacuccgcaacacaaa cuaacuauaucugaaguaucagggcaaggaaugugcauagggacuguuccuaaaacccaccag cuaacuauaucugaaguaucagggcaaggaaugugcauagggacuguuccuaaaacccaccag gcuuugugcaauaagacacaacagggacauacaggggcgcacuaucuagccgcccccaacggc gcuuugugcaauaagacacaacagggacauacaggggcgcacuaucuagccgcccccaacggc accuauugggccuguaacacuggacucaccccaugcauuuccauggcggugcucaauuggacc accuauugggccuguaacacuggacucaccccaugcauuuccauggcggugcucaauuggacc .0 0 ucugauuuuugugucuuaaucgaauuauggcccagagugacuuaccaucaacccgaauaugug ucugauuuuugugucuuaaucgaauuauggcccagagugacuuaccaucaacccgaauaugu uacacacauuuugccaaagcugucagguuccgaaga ebee6oonn66eon6no6eepoo6nnnneoeoeoen

RABIESVIRUS RABIES VIRUSG G(SEQ (SEQ ID IDNO: NO:9)9) atggtgccgcaggctctcctgtttgtcccccttctggtctttccattgtgttttgggaaattccctatctacacaattc atggtgccgcaggctctcctgtttgtcccccttctggtctttccattgtgttttgggaaattccctatctacacaatto .5 :5 cggacaagttgggaccctggagcccaattgacattcatcatctcagctgcccgaacaatttggtcgtggaggacgaagg cggacaagttgggaccctggagcccaattgacattcatcatctcagctgcccgaacaatttggtcgtggaggacgaagg atgcaccaacctgtcggggttctcctacatggaattgaaagtcggatacatcagtgccattaagatgaacgggttcact atgcaccaacctgtcggggttctcctacatggaattgaaagtcggatacatcagtgccattaagatgaacgggttcac tgcacaggcgtcgtgactgaagctgagacatacactaacttcgtgggatatgtcactaccactttcaaaagaaagcatt tgcacaggcgtcgtgactgaagctgagacatacactaacttcgtgggatatgtcactaccactttcaaaagaaagcatt tccgccctactcctgatgcttgtagggccgcatacaactggaagatggccggtgaccccagatatgaggaatcacttca ccgccctactcctgatgcttgtagggccgcatacaactggaagatggccggtgaccccagatatgaggaatcacttca caatccgtaccctgactaccactggcttcggactgtcaaaaccaccaaggagtcactcgtgatcattagtccaagtgtg caatccgtaccctgactaccactggcttcggactgtcaaaaccaccaaggagtcactogtgatcattagtccaagtgtQ .0 0 gctgatcttgacccatacgaccggtcacttcactcacgggtgttcccgggggggaattgctctggtgtcgcagtgtcgt gctgatcttgacccatacgaccggtcacttcactcacgggtgttcccgggggggaattgctctggtgtcgcagtgtcgt caacctactgctccacaaaccacgattacaccatttggatgccagaaaatcctcggcttggtatgtcatgtgacatttt caacctactgctccacaaaccacgattacaccatttggatgccagaaaatcctcggcttggtatgtcatgtgacatttt caccaattctcgggggaagagggcttccaaagggtctgaaacttgcggctttgtcgatgagcggggcttgtataagtca caccaattctcgggggaagagggcttccaaagggtctgaaacttgcggctttgtogatgagcggggcttgtataagtca cttaaaggtgcttgcaaactcaagctttgtggtgtcttgggattgagattgatggatggaacttgggtcgcaatgcaga cttaaaggtgcttgcaaactcaagctttgtggtgtcttgggattgagattgatggatggaacttgggtcgcaatgcaga cttctaacgaaaccaaatggtgccctcccggacagcttgtgaatttgcatgactttcgctctgacgaaattgagcatct cttctaacgaaaccaaatggtgccctcccggacagcttgtgaatttgcatgactttcgctctgacgaaattgagcatc 15 5 tgtcgtcgaggagttggtcaagaagcgggaagagtgtctggatgctttggaatcaatcatgaccaccaagtcagtgtct cgtcgtcgaggagttggtcaagaagcgggaagagtgtctggatgctttggaatcaatcatgaccaccaagtcagtgtot ttcagacggctctcacatcttaggaaattggtgccaggttttggaaaagcatataccattttcaacaagacccttatgg ttcagacggctctcacatcttaggaaattggtgccaggttttggaaaagcatataccattttcaacaagacccttatgg aagccgatgctcactacaagtctgtcaggacttggaatgagatcatcccgtctaaagggtgtcttagggtcggagggag aagccgatgctcactacaagtctgtcaggacttggaatgagatcatcccgtctaaagggtgtcttagggtcggagggag atgtcatcctcatgtcaacggagtctttttcaatggtatcattcttggacctgacggaaatgtccttatccctgagatg atgtcatcctcatgtcaacggagtctttttcaatggtatcattcttggacctgacggaaatgtccttatccctgagatg caatcttccctcctccagcaacacatggaacttcttgtctcatcggtcatcccccttatgcaccccctggctgacccat caatcttccctcctccagcaacacatggaacttcttgtctcatcggtcatcccccttatgcaccccctggctgacccat 40 40 caaccgtgttcaagaacggtgacgaggcagaggattttgtcgaggtccaccttcccgatgtgcatgaacggatctctgg caaccgtgttcaagaacggtgacgaggcagaggattttgtcgaggtccaccttcccgatgtgcatgaacggatctctgg tgtcgaccttggactccctaactggggaaagtatgtccttctgtcggcaggagccctgactgccttgatgttgattatc tgtcgaccttggactccctaactggggaaagtatgtccttctgtcggcaggagccctgactgccttgatgttgattata ttcctgatgacttgttggaggagagtcaatcggtcggagccaacacaacataatctcagaggaacaggaagggaggtgt ttcctgatgacttgttggaggagagtcaatcggtcggagccaacacaacataatctcagaggaacaggaagggaggtgt cagtcacaccccaaagcgggaagatcatttcgtcttgggagtcatacaagagcggaggtgaaaccggactgtga cagtcacaccccaaagcgggaagatcatttcgtcttgggagtcatacaagagoggaggtgaaaccggactgtga

45

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RABIESVIRUS RABIES VIRUSG G(SEQ (SEQ ID IDNO: NO: 10) 10) MVPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVJEDEGCTNLSGF MVPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGE SYMELKVGYISAIKMNGFTCTGVTJTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWK SYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWK MAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPGGNCSG MAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPGGNCSG 55 VAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKG VAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKG ACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDFRSDEIEHLVJEELVKKRE ACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDFRSDEIEHLVVEELVKKRE ECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPS 2022209359 KGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLMHPLAD KGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLMHPLAD PSTVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNWGKYVLLSAGALTALMLIIFLMTCW PSTVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNWGKYVLLSAGALTALMLIIFLMTCA 0 RRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESYKSGGETGL* RRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESYKSGGETGL*

RABIESVIRUS RABIES VIRUSG G(SEQ (SEQ ID IDNO: NO: 16) 16) auggugccgcaggcucuccuguuugucccccuucuggucuuuccauuguguuuugggaaauucccuaucuacacaauuc a cggacaaguugggacccuggagcccaauugacauucaucaucucagcugcccgaacaauuuggucguggaggacgaagg ggacaaguugggacccuggagcccaauugacauucaucaucucagcugcccgaacaauuuggucguggaggacgaagg 55 augcaccaaccugucgggguucuccuacauggaauugaaagucggauacaucagugccauuaagaugaacggguucacu augcaccaaccugucgggguucuccuacauggaauugaaagucggauacaucagugccauuaagaugaacggguucacy ugcacaggcgucgugacugaagcugagacauacacuaacuucgugggauaugucacuaccacuuucaaaagaaagcauu uccgcccuacuccugaugcuuguagggccgcauacaacuggaagauggccggugaccccagauaugaggaaucacuuca uccgcccuacuccugaugcuuguagggccgcauacaacuggaagauggccggugaccccagauaugaggaaucacuuca caauccguacccugacuaccacuggcuucggacugucaaaaccaccaaggagucacucgugaucauuaguccaagugug caauccguacccugacuaccacuggcuucggacugucaaaaccaccaaggagucacucgugaucauuaguccaagugug gcugaucuugacccauacgaccggucacuucacucacggguguucccgggggggaauugcucuggugucgcagugucgu jcugaucuugacccauacgaccggucacuucacucacggguguucccgggggggaauugcucuggugucgcagugycg1 '0 0 caaccuacugcuccacaaaccacgauuacaccauuuggaugccagaaaauccucggcuugguaugucaugugacauuuu aaccuacugcuccacaaaccacgauuacaccauuuggaugccagaaaauccucggcuugguaugucaugugacauuus caccaauucucgggggaagagggcuuccaaagggucugaaacuugcggcuuugucgaugagcggggcuuguauaaguca cuuaaaggugcuugcaaacucaagcuuuguggugucuugggauugagauugauggauggaacuugggucgcaaugcaga cuuaaaggugcuugcaaacucaagcuuuguggugucuugggauugagauugauggauggaacuugggucgcaaugcaga cuucuaacgaaaccaaauggugcccucccggacagcuugugaauuugcaugacuuucgcucugacgaaauugagcaucu cuucuaacgaaaccaaauggugcccucccggacagcuugugaauuugcaugacuuucgcucugacgaaauugagcauct ugucgucgaggaguuggucaagaagcgggaagagugucuggaugcuuuggaaucaaucaugaccaccaagucagugucu gucgucgaggaguuggucaagaagcgggaagagugucuggaugcuuuggaaucaaucaugaccaccaagucagugucu ,5 :5 uucagacggcucucacaucuuaggaaauuggugccagguuuuggaaaagcauauaccauuuucaacaagacccuuaugg iucagacggcucucacaucuuaggaaauuggugccagguuuuggaaaagcauauaccauuuucaacaagacccuuaugg aagccgaugcucacuacaagucugucaggacuuggaaugagaucaucccgucuaaagggugucuuagggucggagggag aagccgaugcucacuacaagucugucaggacuuggaaugagaucaucccgucuaaagggugucuuagggucggagggag augucauccucaugucaacggagucuuuuucaaugguaucauucuuggaccugacggaaauguccuuaucccugagaug augucauccucaugucaacggagucuuuuucaaugguaucauucuuggaccugacggaaauguccuuaucccugagaug caaucuucccuccuccagcaacacauggaacuucuugucucaucggucaucccccuuaugcacccccuggcugacccau aaucuucccuccuccagcaacacauggaacuucuugucucaucggucaucccccuuaugcacccccuggcugacccau caaccguguucaagaacggugacgaggcagaggauuuugucgagguccaccuucccgaugugcaugaacggaucucugg 66nonone66oee6neo6n6ne6ooonnooeoon66e6on6nnnne66e6e066e6oe6n66oee6eeonn0n6ooee 10 ugucgaccuuggacucccuaacuggggaaaguauguccuucugucggcaggagcccugacugccuugauguugauuauc 0 igucgaccuuggacucccuaacuggggaaaguauguccuucugucggcaggagcccugacugccuugauguugauuaud uuccugaugacuuguuggaggagagucaaucggucggagccaacacaacauaaucucagaggaacaggaagggaggugu uuccugaugacuuguuggaggagaqucaaucggucggagccaacacaacauaaucucagaggaacaggaagggaggugu cagucacaccccaaagcgggaagaucauuucgucuugggagucauacaagagcggaggugaaaccggacuguga cagucacaccccaaagcgggaagaucauuucgucuugggagucauacaagagcggaggugaaaccggacuquga

Thefollowing The followingexamples examples serve serve to provide to provide further further appreciation appreciation of the of the invention invention

15 5 but are but are not not meant meantin inanyany wayway to restrict to restrict thethe effective effective scope scope of invention. of the the invention.

EXAMPLES EXAMPLES

EXAMPLE 11 EXAMPLE 40 40

INCORPORATION OF INCORPORATION OF THE THE CODING CODING SEQUENCES SEQUENCESFORFOR FCV CAPSID PROTEINS INTO THE ALPHAVIRUS RNA REPLICON PARTICLES FCV CAPSID PROTEINS INTO THE ALPHAVIRUS RNA REPLICON PARTICLES

INTRODUCTION INTRODUCTION 45 45 RNAviruses RNA viruseshave havebeen beenused usedas as vector-vehicles vector-vehicles forintroducing for introducing vaccine vaccine antigens, which antigens, have been which have been genetically genetically engineered engineered into into their their genomes. However, genomes. However,

their use their to date use to date has hasbeen been limited limited primarily primarily to to incorporating incorporating viral viral antigens antigens into into the the RNAvirus RNA virusandand then then introducing introducing the virus the virus into into a recipient a recipient host.host. The result The result is the is the inductionof induction of protective protectiveantibodies antibodiesagainst against thethe incorporated incorporated viralviral antigens. antigens. Alphavirus Alphavirus

30

RNAreplicon RNA particles have replicon particles been used have been usedtoto encode pathogenicantigens. encodepathogenic antigens.Such Such alphavirus repliconplatforms alphavirus replicon platforms have have beenbeen developed developed from several from several differentdifferent

alphaviruses, includingVenezuelan alphaviruses, including Venezuelan equine equine encephalitis encephalitis virus[Pushko virus (VEE) (VEE) et

[Pushko al., et al., Virology239:389-401 Virology 239:389-401 (1997)], (1997)], Sindbis Sindbis (SIN)(SIN) [Bredenbeek

[Bredenbeek et al., Journal et al., Journal of Virology of Virology

55 67:6439-6446 67:6439-6446 (1993) (1993) the contents the contents of which of which are hereby are hereby incorporated incorporated herein in herein their in their entireties], and entireties], SemlikiForest and Semliki Forestvirus virus(SFV) (SFV) [Liljestrom

[Liljestrom andand Garoff, Garoff, Biotechnology Biotechnology

(NY) 9:1356- (NY) 9:1356-1361 1361 (1991), (1991), the the contents contents of which of which are hereby are hereby incorporated incorporated herein in herein in their entireties]. their entireties].Moreover, alphavirusRNARNA Moreover, alphavirus replicon replicon particles particles are basis are the the basis for for several USDA-licensed several vaccinesfor USDA-licensed vaccines forswine swineand andpoultry. poultry. These These include:Porcine include: Porcine 00 Epidemic Diarrhea Epidemic Diarrhea Vaccine, Vaccine,RNA RNA Particle(Product Particle (ProductCode Code 19U5.P1), 19U5.P1), Swine Swine Influenza Influenza

Vaccine, RNA Vaccine, RNA(Product (ProductCode Code 19A5.DO), 19A5.D0), Avian Avian Influenza Influenza Vaccine, Vaccine, RNA RNA (Product (Product

Code19O5.D0), Code 1905.DO),and and PrescriptionProduct, Prescription Product,RNA RNA Particle(Product Particle (ProductCode Code 9PPO.00). 9PP0.00).

ALPHAVIRUS ALPHAVIRUS RNA RNA REPLICON REPLICON PARTICLE PARTICLE CONSTRUCTION CONSTRUCTION 55 RP-FCV: RP-FCV: Aminoacid Amino acid sequences sequences forFCV for FCV capsid capsid proteinswere proteins were used used to to generate generate

codon-optimized(feline codon-optimized (feline codon usage) nucleotide codon usage) nucleotide sequences sequencesininsilico. silico. Optimized Optimized sequenceswere sequences wereprepared prepared as as syntheticDNADNA synthetic bycommercial by a a commercial vendor vendor (ATUM, (ATUM,

Newark, CA). Newark, CA). Accordingly, Accordingly,synthetic synthetic genes geneswere weredesigned designed based based on on thethe amino amino acidacid

10 :0 sequencesofofaa VS-FCV sequences VS-FCV capsid capsid protein protein and and an an FCVFCV F9-like F9-like capsid capsid protein, protein,

respectively. The respectively. constructs encoded The constructs aminoacid encoded amino acidsequence sequence [SEQ

[SEQ ID NO: ID NO: 2] for 2] for thethe

VS-FCVcapsid VS-FCV capsid protein,oror [SEQ protein, [SEQIDIDNO: NO:4]4]for for the the FCV FCVF9-like F9-like capsid capsid protein, protein, were were

codon-optimized codon-optimized forfor feline,with feline, withflanking flanking sequence sequence appropriate appropriate for cloning for cloning into the into the

alphavirus repliconplasmid. alphavirus replicon plasmid. 25 25

The VEE The VEEreplicon repliconvectors vectorsdesigned designedtotoexpress expressFCV FCV capsid capsid proteins proteins were were

constructedasaspreviously constructed previously described described [see,[see, U.S. U.S. 9,441,247 9,441,247 B2; theB2; the contents contents of which of which are herebyincorporated are hereby incorporated herein herein by reference], by reference], with with the following the following modifications. modifications. The The TC-83-derived replicon TC-83-derived replicon vector vector "pVEK" "pVEK"[disclosed

[disclosed and and described described inin 30 30 U.S. 9,441,247 U.S. B2] was 9,441,247 B2] wasdigested with restriction digestedwith restriction enzymes enzymes Ascl and Pacl. Asc/ and Pacl. AADNA DNA plasmid containing plasmid containing the the codon-optimized openreading codon-optimized open readingframe framenucleotide nucleotidesequence sequenceof of the FCV the capsid genes, FCV capsid genes,with with5'flanking sequence 5' flanking (5'-GGCGCGCCGCACC-3') sequence (5'-GGCGCGCCGCACC-3') [SEQ

[SEQ ID NO: ID 11] and NO: 11] and 3' 3'flanking flankingsequence (5'-TTAATTAA-3'),were sequence (5'-TTAATTAA-3'), weresimilarly similarly digested digested with with

31

restriction enzymes restriction enzymes Asc/ Ascl and Thesynthetic Pac. The and Pacl. synthetic gene gene cassette wasthen cassette was thenligated ligated into the into the digested pVEK digested pVEK vector. vector.

Production of Production of TC-83 RNAreplicon TC-83 RNA repliconparticles particles (RP) (RP) was wasconducted conductedaccording according toto

55 methodspreviously methods previouslydescribed described[U.S.

[U.S. 9,441,247 9,441,247B2B2and andU.S. U.S.8,460,913 8,460,913 B2; B2; thethe

contentsofofwhich contents whichareare hereby hereby incorporated incorporated hereinherein by reference]. by reference]. Briefly,Briefly, pVHV pVHV replicon vector replicon vectorDNA DNAandand helper helper DNA plasmids DNA plasmids were linearized were linearized with Notl with Not/ restriction restriction

enzyme prior to enzyme prior to in in vitro vitrotranscription using transcription MegaScript using T7T7 MegaScript RNA RNA polymerase and cap polymerase and cap analog (Promega,Madison, analog (Promega, Madison,WI). WI).Importantly, Importantly,the thehelper helperRNAs RNAs used used in in thethe 00 production lack production lack the the VEE subgenomic VEE subgenomic promoter promoter sequence, sequence, as previously as previously described described

[Kamrudetet al.,

[Kamrud al., JJGen Gen Virol. Virol.91(Pt 7):1723-1727 7):1723-1727 (2010)]. (2010)]. Purified Purified RNAthe RNA for for the replicon and replicon and helper helper components werecombined components were combined andand mixed mixed withwith a suspension a suspension of of Verocells, Vero cells, electroporated electroporatedin in4 4mmmm cuvettes, cuvettes, and returned and returned to OptiPro* to OptiPro® SFM cellSFM cell culture culture media (ThermoFisher, media (Thermo Fisher, Waltham WalthamMA). MA).Following Following overnight overnight incubation, incubation,

55 alphavirus RNA alphavirus RNA replicon replicon particles particles werewere purified purified from from the cells the cells and by and media media by passing passing

the suspension the through aa ZetaPlus suspension through ZetaPlusBioCap BioCap depth depth filter (3M, filter (3M, Maplewood, MN), Maplewood,MN), washingwith washing withphosphate phosphate buffered buffered salinesaline containing containing 5% sucrose 5% sucrose (w/v), and(w/v), and finally finally eluting eluting the theretained retainedRP RP with with400 400 mM NaCIbuffer. mM NaCI buffer. Eluted RPwere Eluted RP wereformulated formulatedtotoa a final 5% final 5% sucrose (w/v), passed sucrose (w/v), passed through a 0.22 through a 0.22 micron membrane micron membrane filter, and filter, and

0D dispensedinto dispensed intoaliquots aliquots forstorage. for storage. Titer Titer of of functional functional RP determined RP was was determined by by immunofluorescenceassay immunofluorescence assay on on infectedVero infected Vero cellmonolayers. cell monolayers. RP-FeLV: RP-FeLV: An amino An aminoacid acid sequence sequence forFeLV for FeLV gp85 gp85 were were usedused to generate to generate codon codon-

optimized (feline optimized (feline codon codon usage) usage) nucleotide nucleotide sequences sequences inin silico. silico. Optimized Optimized

25 25 sequenceswere sequences wereprepared prepared as as syntheticDNADNA synthetic bycommercial by a a commercial vendor vendor (ATUM, (ATUM,

Newark, CA). Newark, CA). Accordingly, Accordingly,a asynthetic synthetic gene genewas wasdesigned designed based based on on thethe amino amino acidacid

sequenceofofgp85. sequence gp85.The The construct(gp85 construct (gp85 wt)wt) waswas wild-type wild-type amino amino acid acid sequence sequence

[SEQIDIDNO:NO:

[SEQ 2],2], codon-optimized codon-optimized for feline, for feline, with with flanking flanking sequence sequence appropriate appropriate for for cloning into the cloning into the alphavirus alphavirusreplicon repliconplasmid. plasmid. 30 30

The VEE The VEEreplicon repliconvectors vectorsdesigned designedtotoexpress expressFeLV FeLV gp85 gp85 were were constructed constructed

as previouslydescribed as previously described [see,

[see, U.S. U.S. 9,441,247 9,441,247 B2;contents B2; the the contents ofare of which which are hereby hereby

incorporatedherein incorporated herein by by reference reference in their in their entireties], entireties], with with thethe following following modifications. modifications.

32

The TC-83-derived The TC-83-derivedreplicon repliconvector vector "pVEK" "pVEK"[disclosed

[disclosed and anddescribed describedinin U.S. 9,441,247 U.S. B2] was 9,441,247 B2] wasdigested digestedwith with restriction restriction enzymes Asc/ and enzymes Ascl and Pacl. Pac/. AADNA DNA plasmid containing plasmid containing the the codon-optimized openreading codon-optimized open readingframe framenucleotide nucleotidesequence sequenceof of the FeLV the FeLV gp85 gp85 genes, genes, with with5'flanking sequence 5' flanking (5'-GGCGCGCCGCACC-3') sequence (5'-GGCGCGCCGCACC-3') [SEQ

[SEQ 55 ID NO: ID 11] and NO: 11] and 3' 3'flanking flankingsequence (5'-TTAATTAA-3'),was sequence (5'-TTAATTAA-3'), wassimilarly similarly digested digested with with restriction enzymes restriction enzymes Asc/ Ascl and Pac/. The and Pacl. Thesynthetic synthetic gene gene cassette cassettewas wasthen thenligated ligated into the into thedigested digestedpVEK vector, and pVEK vector, and the the resulting resultingclone clonewas was re-named "pVHV-FeLV re-named "pVHV- FeLV gp85". The gp85". The"pVHV" "pVHV" vectornomenclature vector nomenclature waswas chosen chosen to refer to refer to to pVEK-derived pVEK-derived

replicon vectors replicon vectorscontaining containing transgene transgene cassettes cassettes clonedcloned via thevia theand Ascl Asc/ Pacland Pac/ sites sites 00 in the in the multiple cloning site multiple cloning site of of pVEK. pVEK.

Production of Production of TC-83 RNAreplicon TC-83 RNA repliconparticles particles (RP) (RP) was wasconducted conductedaccording accordingtoto methodspreviously methods previouslydescribed described[U.S.

[U.S. 9,441,247 9,441,247B2B2and andU.S. U.S.8,460,913 8,460,913 B2; B2; thethe

contentsofofwhich contents whichareare hereby hereby incorporated incorporated hereinherein by reference by reference in theirinentireties]. their entireties]. 55 pVHV replicon Briefly, pVHV Briefly, replicon vector vectorDNA and helper DNA and helper DNA plasmids DNAplasmids were were linearizedwith linearized with restriction enzyme Notl restriction Notl prior enzyme prior to to ininvitro vitrotranscription transcriptionusing usingMegaScript MegaScript T7 T7 RNA RNA polymeraseand polymerase andcap capanalog analog (Promega, (Promega, Madison, Madison, WI).WI). Importantly, Importantly, the the helper helper RNAs RNAs

used in used in the the production production lack lack the theVEE VEE subgenomic promoter subgenomic promoter sequence, sequence, as as previously previously

described[Kamrud described [Kamrud et al., et al., J Gen J Gen Virol. Virol. 91(Pt 91 (Pt 7):1723-1727 7):1723-1727 (2010)]. (2010)]. Purified Purified RNA for RNA for

00 the replicon the replicon and and helper helper components werecombined components were combined andand mixed mixed withwith a suspension a suspension of of Verocells, Vero cells, electroporated electroporatedin in4 4mmmm cuvettes, cuvettes, and returned and returned to OptiPro* to OptiPro® SFM cellSFM cell culture media culture (ThermoFisher, media (Thermo Fisher, Waltham WalthamMA). MA).Following Following overnight overnight incubation, incubation,

alphavirusRNA alphavirus RNA replicon replicon particles particles werewere purified purified from from the cells the cells and by and media media by passing passing the suspension the through aa ZetaPlus suspension through ZetaPlusBioCap BioCap depth depth filter (3M, filter (3M, Maplewood, MN), Maplewood,MN), 25 25 washingwith washing withphosphate phosphate buffered buffered salinesaline containing containing 5% (w/v), 5% sucrose sucrose and(w/v), and finally finally eluting eluting the theretained retainedRP RP with with400 400 mM NaCIbuffer. mM NaCI buffer. Eluted RPwere Eluted RP wereformulated formulatedtotoa a final 5% final 5% sucrose (w/v), passed sucrose (w/v), passed through a 0.22 through a 0.22 micron membrane micron membrane filter, and filter, and

dispensedinto dispensed intoaliquots aliquots forstorage. for storage. Titer Titer of of functional functional RP determined RP was was determined by by immunofluorescence immunofluorescence assay assay on on infectedVero infected Vero cellmonolayers. cell monolayers. 30 30

RP-RV RP-RV vaccine was A vaccine A wasprepared preparedcomprising comprisingananalphavirus alphavirusRNARNA replicon replicon particle particle

encoding therabies encoding the rabies virus virus glycoprotein glycoprotein (G) (G) fromfrom rabies rabies virus virus (RV) packaged (RV) packaged with the with the

33

capsid proteinand capsid protein andglycoproteins glycoproteins of the of the avirulent avirulent TC-83 TC-83 strainstrain of Venezuelan of Venezuelan Equine Equine

EncephalitisVirus. Encephalitis Virus.TheThe nucleotide nucleotide sequence sequence for thefor the rabies rabies virus Gvirus G protein protein was was codon-optimized for humans. codon-optimized for humans.TheThe resultingsequence resulting sequence hashas only only -85% ~85% nucleotide nucleotide

identity to identity to aa live live rabies rabies virus virus glycoprotein (G) sequence, glycoprotein (G) sequence, despite despite having having 100% 100% amino amino 55 acid identity. The acid identity. Thevaccine vaccinecancan be used be used as a as a single single dose administered dose administered to a to a subject,e.g., mammaliansubject, mammalian e.g., subcutaneously subcutaneouslytotocats anddogs cats and dogsaged aged1212 weeks weeks or or older older

or alternatively, or alternatively, in in aa multiple multiple dose comprising dose comprising a primary a primary administration administration followed followed by by one or one or more more booster boosteradministrations. administrations.

0 0 An amino An aminoacid acidsequence sequence forRabies for Rabiesglycoprotein glycoprotein(G) (G)was was used used to to generate generate

codon-optimized(human codon-optimized (human codon codon usage) usage) nucleotide nucleotide sequences sequences in siico.Optimized in silico. Optimized sequenceswere sequences wereprepared prepared as as syntheticDNA synthetic DNA bycommercial by a a commercial vendor vendor (ATUM, (ATUM,

Newark,CA). Newark, CA). Accordingly, Accordingly,a asynthetic synthetic gene gene[SEQ

[SEQID IDNO:NO: 9] 9] was was designed designed based based

on the on the amino acid sequence amino acid sequenceofofRabies RabiesGlycoprotein. The Glycoprotein.The construct construct (RABV-G) (RABV-G) was was

55 a wild-type a wild-type amino amino acid acid sequence [SEQIDIDNO: sequence [SEQ NO: 10],codon-optimized 10], codon-optimized forfor humans, humans,

with flanking with flanking sequence sequence appropriate appropriate for cloning for cloning into into the alphavirus the alphavirus replicon replicon plasmid. plasmid.

The VEE The VEEreplicon repliconvectors vectorsthat that were were designed designedtoto express expressRabies RabiesG G were were

constructedasas constructed previously previously described described [see,[see, U.S. U.S. 9,441,247 9,441,247 B2; theB2; the contents contents of which of which 00O are hereby are herebyincorporated incorporated herein herein by reference], by reference], with with the following the following modifications. modifications. The The TC-83-derivedreplicon TC-83-derived replicon vector vector "pVEK" "pVEK"[disclosed

[disclosed and and described describedinin U.S. 9,441,247 U.S. B2] was 9,441,247 B2] wasdigested digestedwith with restriction restriction enzymes Asc/ and enzymes Ascl and Pacl. Pac/. AADNA DNA plasmid containing plasmid containing the the codon-optimized openreading codon-optimized open readingframe nucleotidesequence framenucleotide sequenceof of the Rabies the Rabies GG gene, gene,with with5'flanking sequence 5' flanking (5'-GGCGCGCCGCACC-3') sequence [SEQID (5'-GGCGCGCCGCACC-3) [SEQ ID 25 25 NO: 11] NO: 11] and and 3' 3'flanking flanking sequence (5'-TTAATTAA-3')was sequence (5'-TTAATTAA-3') was similarlydigested similarly digestedwith with restriction enzymes restriction enzymes Asc/ Ascl and Pac/. The and Pacl. Thesynthetic gene cassette synthetic gene cassettewas wasthen thenligated ligated into the into thedigested digestedpVEK vector, and pVEK vector, and the the resulting resultingclone clonewas was re-named "pVHV-RABV- re-named "pVHV- RABV G". The G". The "pVHV" "pVHV" vectornomenclature vector nomenclaturewaswas chosen chosen to refer to refer to to pVEK-derived pVEK-derived replicon replicon

vectorscontaining vectors containingtransgene transgene cassettes cassettes cloned cloned via thevia theand Ascl Asc/ and Pacl Pac/ sites in sites the in the 30 30 multiple cloning multiple site of cloningsite of pVEK. pVEK.

Production of TC-83 Production of RNAreplicon TC-83 RNA repliconparticles particles (RP) (RP) was wasconducted conductedaccording according to to

methodspreviously methods previouslydescribed described[U.S.

[U.S. 9,441,247 9,441,247B2B2and andU.S. U.S.8,460,913 8,460,913 B2; B2; thethe

34

contents contents ofofwhich whichareare hereby hereby hereinherein incorporated incorporated by reference by reference in theirinentireties]. their entireties]. Briefly, pVHV Briefly, pVHV replicon replicon vector vectorDNA and helper DNA and helper DNA DNAplasmids plasmids were were linearizedwith linearized with Notl restriction Notl restriction enzyme priortotoininvitro enzyme prior vitrotranscription transcriptionusing usingMegaScript MegaScript T7 T7 RNA RNA polymeraseand polymerase andcap capanalog analog (Promega, (Promega, Madison, Madison, WI).WI). Importantly, Importantly, the the helper helper RNAs RNAs

55 used in used in the the production production lack lackthe theVEE VEE subgenomic promotersequence, subgenomic promoter sequence, as as previously previously

described [Kamrud described [Kamrudetetal., al., JJ Gen Virol. 91(Pt Gen Virol. 7):1723-1727(2010)]. (Pt 7):1723-1727 (2010)]. Purified Purified RNA RNAfor for the replicon the replicon and and helper helper components werecombined components were combined andand mixed mixed withwith a suspension a suspension of of Vero cells, Vero cells, electroporated electroporatedin in4 4mmmm cuvettes, cuvettes, and returned and returned to OptiPro* to OptiPro® SFM cellSFM cell culture culture media (ThermoFisher, media (Thermo Fisher, Waltham, Waltham,MA). MA).Following Following overnight overnight incubation, incubation,

00 alphavirus RNA alphavirus RNA replicon replicon particles particles werewere purified purified from from the cells the cells and by and media media by passing passing

the suspension the through aa ZetaPlus suspension through ZetaPlusBioCap® BioCap* depth depth filter (3M, filter (3M, Maplewood, MN), Maplewood,MN), washingwith washing withphosphate phosphate buffered buffered salinesaline containing containing 5% (w/v), 5% sucrose sucrose and(w/v), and finally finally eluting eluting the theretained retainedRP RP with with400 400 mM NaCIbuffer. mM NaCI buffer. Eluted Eluted RP RPwere wereformulated formulatedtotoa a final 5% final 5% sucrose (w/v), passed sucrose (w/v), passed through a 0.22 through a 0.22 micron membrane micron membrane filter, and filter, and

55 dispensedinto dispensed intoaliquots aliquots forstorage. for storage. Titer Titer of of functional functional RP determined RP was was determined by by immunofluorescenceassay immunofluorescence assay on on infectedVero infected Vero cellmonolayers. cell monolayers.

EXAMPLE 22 EXAMPLE EVALUATION OF EVALUATION OF THE THE SAFETY SAFETY OF OF AA COMBINATION COMBINATION VACCINE VACCINE CONTAINING CONTAINING 0D TWO RP CONSTRUCTS AND THREE MODIFIED LIVE FRACTIONS IN TWO RP CONSTRUCTS AND THREE MODIFIED LIVE FRACTIONS IN CATS CATS

Variousformulations Various formulationsandand reconstitution reconstitution methods methods of a lyophilized of a lyophilized pentavalent pentavalent

feline combination feline vaccine combination vaccine were were evaluated evaluated in regard in regard to safety to their their safety in cats. in cats. The The desiredpresentation desired presentationof of thethe pentavalent pentavalent vaccine vaccine is a mL is a 0.5 0.5dose, mL the dose, the optimal optimal

25 25 formulationand formulation andfill fill method methodis isa a0.50.5mLmL fill. The fill. The stabilizer stabilizer contained contained 1.1%1.1% NZ-amine NZ-amine

(a casein (a enzymatic casein enzymatic hydrolysate), hydrolysate), 1.1% 1.1% gelatin, gelatin, and sucrose, and 7.5% 7.5% sucrose, with the with the percentagesprovided percentages providedrepresenting representingthe thefinal final concentrations. concentrations. Should the volume Should the and volume and

final potency final constraintsfor potency constraints forthe theaddition additionofofthe thefive fivefractions fractionsnecessitate necessitate a volume a volume

greater than greater than0.5 0.5mL, mL, the the product product can can be formulated be formulated and to and filled filled 1.0to mL1.0 andmL and 30 30 reconstitutedwith reconstituted with0.5 of of 0.5mLmL diluent.ThisThis diluent. doubles doubles the concentration the concentration of stabilizer of stabilizer

componentsininthe components theadministered administereddose. dose.TheThe safety safety ofofsuch sucha aconcentrated concentrated dose dose of of

stabilizer with stabilizer with these antigenshad these antigens had notnot previously previously beenbeen tested tested in cats. in cats.

35

third option A third A option of of aa 1.0 1.0 mL mLfill fill volume rehydrated volume rehydrated withwith 1.0 1.0 mLdiluent mL of of diluent also also wastested was testedininthe theevent event that that thethe 1.01.0 mL mL 0.5mLmL fill/0.5 fill/ rehydration rehydration format format wassafe was not not safe in cats. in cats. The The vaccines vaccines were blendedand were blended andthen thenlyophilized. Thevaccines lyophilized. The vaccinescontained contained an alphavirusRNA an alphavirus RNA replicon replicon particle particle thatthat encodes encodes a feline a feline leukemia leukemia virus virus

55 glycoprotein (RP-FeLV) glycoprotein andananalphavirus (RP-FeLV) and alphavirusRNA RNA repliconparticle replicon that encodes particle that encodesaa feline calicivirus feline calicivirus capsid protein (RP-FCV), capsid protein (RP-FCV), together together withwith the the components components of a of a commercially commercially available available vaccine, vaccine, Feline-1, i.e., ai.e., Nobivac*Feline-1, Nobivac® a modified modified live (MLV) live (MLV) feline feline panleukopenia panleukopenia virus virus (FPL), (FPL), a modified a modified live live feline feline viral viral rhinotracheitis rhinotracheitis virus virus (FVR) (FVR) and and modifiedlive a modified live Chlamydophila Chlamydophila felis felis in ain stabilizer a stabilizer containing containing 1.1%1.1% gelatin, gelatin, 1.1% 1.1% NZ- NZ 00 amine and7.5% amine and 7.5%sucrose. sucrose.TheThe various various formulations formulations of of thepentavalent the pentavalentfeline feline combination vaccine combination vaccinewere wereprepared preparedand and reconstitutedasasdescribed reconstituted describedininthe theTable Table1 1 below: below:

TABLE TABLE 11 55 FORMULATIONS OFTHE FORMULATIONS OF THE PENTAVALENT PENTAVALENTCOMBINATION VACCINE COMBINATIONVACCINE Freeze-Dried Freeze-Dried Diluent Diluent Treatment Treatment No. of No. of Test Product Test Product Cake (Fill) Cake (Fill) (Rehydration) (Rehydration) Group Group Cats Cats Antigens Antigens Vlm Volume Volume Volume Volume 1 1 6 6 1.0 mL 1.0 mL 1.0 mL 1.0 mL - ~RP-FCV, RP-FeLV, RP-FCV, RP-FeLV, 2 2 6 6 1.0 mL 1.0 mL 0.5 mL 0.5 mL FPL, FVR FPL, FVR& & C.C.felis felis 33 66 0.5 mL 0.5 mL 0.5 mL 0.5 mL 4 4 2 2 Diluent only Diluent only NA NA 1.0 mL 1.0 mL

The vaccines The vaccineswere wereformulated formulatedtotocontain contain the the same samedose doseofofeach eachantigen antigen(the (the 0.5 mL 0.5 mLcake cake vaccine vaccine was was formulated formulated with the with twice twice the concentration concentration of each of each antigen), antigen), the stabilizer the stabilizer was usedat ata aconstant was used constant concentration concentration in formulations in all all formulations (themL1.0 (the 1.0 mL 20 20 cakevaccine cake vaccine rehydrated rehydrated withwith 0.5 0.5 mL diluent mL diluent contained contained twice twice the the concentration concentration of of stabilizer upon stabilizer rehydration). upon rehydration).

The experimental The experimental cat cat subjects subjects were were vaccinated vaccinatedwith with the the indicated indicated volume of volume of

the respective the respectivetest testvaccine vaccineat at 7-8 7-8 weeks weeks of age of age (typically (typically having having a minimum a minimum age of age of 25 25 vaccinationfor vaccination forthe thefeline felinecore corevaccine) vaccine)andand again again 21 days 21 days later.later. Thewere The cats cats were observedforfora aperiod observed period of of 15 15 minutes minutes immediately immediately aftervaccination after each each vaccination for reactions for reactions

36

to the to the test test vaccine, whichmight vaccine, which might indicate indicate pain pain or discomfort, or discomfort, such such as vocalization, as vocalization,

stinging, itching, biting, stinging, itching, biting,sudden movement sudden movement upon upon the injection the injection of theof the vaccine, vaccine, or any or any

unusual reaction unusual reaction (see (see Tables and 33 below). Tables 22 and below). AAclinical clinical assessment wasperformed assessment was performed by aa veterinarian by veterinarian4 4toto66hours hoursafter afterthe thevaccination vaccination and and eacheach daythree day for for three days days 55 post-vaccination. post-vaccination.

Theclinical The clinical assessment assessment included included palpation palpation ofinjection of the the injection site site and and observationsforforany observations any localreactions local reactions including including painpain to touch, to touch, swelling, swelling, redness redness or or abscess. Catswere abscess. Cats werealso alsoobserved observed forany for anysystemic systemicreactions reactionssuch suchasasdepression, depression, 00 lethargy, lameness, lethargy, lameness, vomiting, vomiting, tremors, tremors, agitation, agitation, and and diarrhea. diarrhea. Body temperatures Body temperatures

were also were also measured measuredand and recorded recorded at at 4 4toto6 6hours hourspost-vaccination andeach post-vaccination and eachday day for for

two days two daysfollowing followingeach each vaccination. vaccination. Palpation Palpation of injection of injection sites sites for local for local reactions reactions

wasadditionally was additionally performed three times performed three times each weekbetween each week between 7 days 7 days andand 21 21 days days

after after each vaccination. each vaccination.

55 All vaccine All formulationsandand vaccine formulations rehydration rehydration protocols protocols were were found found to to beNosafe. be safe. No local or local or systemic reactionswere systemic reactions were observed observed in anyin of any theofcats. the cats. Body temperatures Body temperatures at at eachmeasurement each measurementperiodperiod forcats for all all cats were were normal, normal, with with the the exception exception of in of one cat one cat in Treatment Group Treatment Group3 3(0.5 (0.5mLmLcake/ cake!0.5 0.5mLmLdiluent) diluent)which whichpresented presentedwith witha abody body

00 temperatureof of temperature 103.6°C 103.6°C fivefive hours hours post-second post-second vaccination. vaccination. Therefore, Therefore, all three all three preparationsofofthe preparations thepentavalent pentavalent vaccine vaccine were were found found to be acceptable. to be acceptable.

2022209359 2022 37 37

TABLE 22 TABLE 30 Jul

IMMEDIATE IMMEDIATE REACTIONS REACTIONS TO TO VACCINATION VACCINATION WITH WITH PENTAVALENT COMBINATION VACCINE PENTAVALENT COMBINATION VACCINE Sudden Sudden Scratching Scratching Treatment Treatment Vaccine ID Vaccine ID Vocalization Vocalization Bite (during Bite movement movement at injection at injection

Group Group injection) injection) away from away from site (post site (post-

injection injection injection) injection) Ist 1st 2" 2nd 1st 14 S 2" 2nd 14 IS2" 14 IS2"'~ 1st 2nd 1st 2nd

Vac. Vac. Vac. Vac. Vac. Vac. Vac. Vac. Vac. Vac. Vac. Vac. Vac. Vac. Vac. Vac.

1 1 Pentavalent Feline Pentavalent Feline 0/6 0/6 1/6 1/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 Vaccine Vaccine 1.0 1.0 mL cake/ mL cake/ 1.0 mL 1.0 diluent mL diluent 2 2 Pentavalent Feline Pentavalent Feline 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 Vaccine Vaccine 1.0 1.0 mL cake/ mL cake/ 0.5 mL 0.5 diluent mL diluent 3 3 Pentavalent Feline Pentavalent Feline 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 Vaccine Vaccine 0.5 mL 0.5 cake/ mL cake/ 0.5 mL 0.5 diluent mL diluent 4 4 Diluent only Diluent only 1/2 1/2 1/2 1/2 0/2 0/2 0/2 0/2 1/2 1/2 1/2 1/2 0/2 0/2 0/2 1.0 1.0mLmL

38

TABLE3 TABLE 3 LOCAL/SYSTEMIC REACTIONS LOCAL/SYSTEMIC REACTIONS TO TO VACCINATION VACCINATION WITH PENTAVALENT WITH COMBINATION VACCINE PENTAVALENT COMBINATION VACCINE

Treatment Treatment Vaccine Vaccine ID ID Local Reactions Local Reactions Systemic Reactions Systemic Reactions Body Temperature Body Temperature Group Group (Redness, pain (Redness, pain to to (vomiting, (vomiting, 103.5°C Post- >103.5°C Post touch, swelling or touch, swelling or lameness, lameness, Vaccination Vaccination abscess) abscess) depression, depression, lethargy, tremors, lethargy, tremors,

agitation or agitation or

diarrhea) diarrhea) Vac. 1I" Vac. 1st 2"d Vac. 2 Vac. 1st Vac. 1st Vac. 2"dVac. 2 Vac. 1stVac. 1I" Vac. 2"dVac. 2 Vac. 1 1 Pentavalent Pentavalent 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 Feline Feline Vaccine Vaccine 1.0 1.0 mL cake/ mL cake/ 1.0 1.0 mLmL diluent diluent

2 2 Pentavalent Pentavalent 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 Feline Feline Vaccine Vaccine 1.0 1.0 mL cake/ mL cake/ 0.5 mL 0.5 mL diluent diluent

3 3 Pentavalent Pentavalent 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 1/6 1/6

Feline Feline Vaccine Vaccine 0.5 mL 0.5 cake/ mL cake/ 0.5 mL 0.5 mL diluent diluent

4 4 Diluent only Diluent only 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 1.0 1.0 mL mL

39

The present Thepresent invention invention is not is not be be to to limited in in limited scope scope by the by the specific specific

embodiments described embodiments described herein. herein. Indeed,Indeed, various various modifications modifications of the invention of the invention in in addition to those addition to thosedescribed described herein herein will will become become apparent apparent to skilled to those those skilled in the in artthe art

from the from theforegoing foregoingdescription. description. SuchSuch modifications modifications are intended are intended to fall to fall within within the the 55 scope of the scope of the appended claims. appended claims.

It isisfurther It furthertotobebeunderstood thatall understood that all base basesizes sizesororamino amino acid acid sizes, sizes, and and all all molecularweight molecular weightor or molecular molecular massmass values, values, given given for for nucleic nucleic acids acids or or polypeptides polypeptides

are approximate, are approximate, andand are are provided provided for description. for description.

40

STATEMENTS OF STATEMENTS OFTHE THE INVENTION INVENTION

1. 1. An immunogenic An immunogenic composition composition comprising comprising an alphavirus an alphavirus RNA RNA replicon replicon particle particle

that encodes that encodesa feline a felinecalicivirus calicivirus(FCV) (FCV) antigen, antigen, and and a modified a modified live feline live feline pathogen pathogen

55 selectedfrom selected fromthe thegroup group consisting consisting of aof a modified modified live live feline feline viral viral rhinotracheitis rhinotracheitis virus virus

(FVR), modifiedlive (FVR), a amodified livefeline felinepanleukopenia panleukopeniavirusvirus (FPLV), (FPLV), a modified a modified live live Chlamydophila Chlamydophila felis,andand felis, anyany combination combination thereof. thereof.

2. 2. The immunogenic The immunogenic composition composition of of statement statement 1, 1, wherein wherein thethe FCVFCV antigen antigen is ais a 00 capsid proteinororananantigenic capsid protein antigenic fragment fragment thereof. thereof.

3. 3. The immunogenic The immunogenic composition composition of of statement statement 2, 2, wherein wherein thethe capsid capsid proteinisis protein

selectedfrom selected fromthe thegroup group consisting consisting of aof a FCV FCV F9-Like F9-Like capsid capsid protein,protein, an antigenic an antigenic

fragment of fragment of the the FCV F9-Likecapsid FCV F9-Like capsidprotein, protein, aa virulent virulentsystemic systemicFCV FCV (VS-FCV) (VS-FCV)

55 capsid protein,and capsid protein, andanan antigenic antigenic fragment fragment of VS-FCV of the the VS-FCV capsid protein. capsid protein.

4. 4. The immunogenic The immunogenic composition composition of of statement statement 3, 3, wherein wherein thethe capsid capsid proteinisisaa protein

VS-FCVcapsid VS-FCV capsid proteinororananantigenic protein antigenic fragment fragment thereof. thereof.

00 5. 5. The immunogenic The immunogenic composition composition of of statement statement 4, 4, wherein wherein thethe alphavirus alphavirus RNA RNA

replicon particle replicon particle also also encodes encodes an an antigen antigen selected selected from from the group the group consisting consisting of an of an FCVF9-Like FCV F9-Likecapsid capsidprotein, protein, an an antigenic antigenic fragment of the fragment of the FCV F9-Like capsid FCV F9-Like capsid protein, aa feline protein, feline leukemia virus(FeLV) leukemia virus (FeLV) glycoprotein glycoprotein (gp85), (gp85), an antigenic an antigenic fragment fragment of of the gp85, the gp85,a arabies rabiesvirus virusG G protein, protein, an an antigenic antigenic fragment fragment ofrabies of the the rabies virus virus G G 25 25 protein, and protein, andany anycombination combination thereof. thereof.

6. 6. The immunogenic The immunogenic composition composition of of statement statement 3, 3, wherein wherein thethe capsid capsid proteinisisaa protein

FCVF9-Like FCV F9-Like capsid capsid protein protein orantigenic or an an antigenic fragment fragment thereof. thereof.

30 30 7. 7. Theimmunogenic The immunogenic composition composition of statement of statement 1, 2, 3, 1, 5,3,or4,6,5,that 4,2, or 6,comprises that comprises oneorormore one moreadditional additional alphavirus alphavirus RNA RNA replicon replicon particles particles that encode that encode an an antigen antigen selectedfrom selected fromthe thegroup group consisting consisting of FeLV of an an FeLV glycoprotein glycoprotein (gp85), (gp85), an antigenic an antigenic

41

fragmentofofthe fragment thegp85, gp85, a rabies a rabies virus virus G protein, G protein, an antigenic an antigenic fragment fragment of the of the rabies rabies virus GG protein, virus protein, and andanyany combination combination thereof. thereof.

8. 8. The immunogenic The immunogenic composition composition of of statement 4, 4, statement thatfurther that further comprises comprisesanan 55 additional alphavirus additional alphavirus RNA particlethat replicon particle RNA replicon encodes that encodesan an FCV F9-Like capsid FCV F9-Like capsid protein or protein or an an antigenic antigenicfragment fragment thereof. thereof.

9. 9. The immunogenic The immunogenic composition composition of statement of statement 3, 4, 3, 4, 5, 5, 7, 7, or or whereinthetheVS- 8,8,wherein VS FCVcapsid FCV capsidprotein proteincomprises comprises an amino an amino acid acid sequence sequence comprising comprising at 95% at least least 95% 00 identity with identity withthe amino the aminoacid acidsequence sequence of ofSEQ ID NO: SEQ ID NO: 2. 2.

10. TheThe 10. immunogenic immunogenic composition composition of statement of statement 3, 5, 3, 6,5,7,6,8, 7, owBerein 8, owerein the the FCVF9-Like FCV F9-Likecapsid capsidprotein protein comprises comprisesananamino aminoacid acidsequence sequence comprising comprising at least at least

95%identity 95% identity with with the theamino amino acid acid sequence of SEQ sequence of SEQ IDIDNO: NO:4.4. 55 11. TheThe 11. immunogenic immunogenic composition composition of statement of statement 5, 7, 5, 9,7,or9,10, or 10, wherein wherein the the FeLVFeLV

glycoprotein (gp85) glycoprotein (gp85) comprises an amino comprises an aminoacid acidsequence sequence comprising comprising at at least95% least 95% identity with identity withthe amino the aminoacid acidsequence sequence of ofSEQ ID NO: SEQ ID NO: 6. 6.

0O 0 12. TheThe 12. immunogenic immunogenic composition composition of statement of statement 5, 10, 5, 7, 9, 7, 9, or 10, 11, or 11, wherein wherein the the rabies virus rabies virus G protein comprises G protein an amino comprises an aminoacid acidsequence sequence comprising comprising at least at least 95%95%

identity with identity withthethe amino aminoacid acidsequence sequence of ofSEQ ID NO: SEQ ID 10. NO: 10.

13. The The 13. immunogenic immunogenic composition composition of statement of statement 1, 2, 3, 4, 1, 5, 2, 6,3, 7,4,8,5,9,6,10, 7, 8, 11,9,or10, 11, or 25 25 12, 12, wherein wherein the alphavirus RNA the alphavirus replicon particle RNA replicon a Venezuelan Equine particle isisa Venezuelan Equine

Encephalitis(VEE) Encephalitis (VEE)alphavirus alphavirus RNA RNA replicon replicon particle. particle.

14. TheThe 14. immunogenic immunogenic composition composition of statement of statement 7, 8, 7, 9,8,10, 9, 10, 11, 11, 12, 12, or or 13,13, wherein wherein

the additional the additional alphavirus alphavirusRNARNA replicon replicon particle particle is aisVenezuelan a Venezuelan Equine Equine Encephalitis Encephalitis

30 30 (VEE)alphavirus (VEE) alphavirus RNARNA replicon replicon particle. particle.

42

15. 15. A vaccine A vaccine to to aidaid ininthe theprevention prevention of of disease disease due duetoto FCV FCVcomprising comprisingthe the immunogenic immunogenic composition composition of statement of statement 1, 4, 1, 2, 3, 2, 5, 3, 6, 4, 7, 5, 6, 8, 7, 9, 8, 10,9,11, 10,12, 11,13, 12,or13, 14,or 14, and pharmaceutically and aa pharmaceutically acceptable acceptable carrier. carrier.

55 16. A vaccine 16. A vaccine to to aidaid ininthe theprevention preventionof of disease disease due duetoto FCV FCVcomprising comprisinganan alphavirus RNA alphavirus RNA replicon replicon particle particle thatthat encodes encodes a virulent a virulent systemic systemic feline feline calicivirus calicivirus

(VS-FCV) capsid (VS-FCV) capsid protein protein or antigenic or an an antigenic fragment fragment thereof, thereof, a modified a modified live viral live feline feline viral rhinotracheitis virus rhinotracheitis virus (FVR), (FVR),a amodified modified livefeline live felinepanleukopenia panleukopenia virusvirus (FPLV), (FPLV), and a and a modifiedlive modified live Chlamydophila Chlamydophila felis; felis; wherein wherein the alphavirus the alphavirus RNA replicon RNA replicon particleparticle is a is a 00 VenezuelanEquine Venezuelan Equine Encephalitis(VEE) Encephalitis (VEE)alphavirus alphavirusRNARNA replicon replicon particle particle

17. 17. TheThe vaccine vaccine of statement of statement 16, 16, furthercomprising further comprising an an alphavirus alphavirus RNA RNA replicon replicon

particle that particle that encodes encodes a a felinecalicivirus feline calicivirusF9-Like F9-Likecapsid capsid protein protein or antigenic or an an antigenic fragment thereof. fragment thereof.

55 18. 18. TheThe vaccine vaccine of statement of statement 16,16, wherein wherein the the alphavirus alphavirus RNARNA replicon replicon particle particle

further further encodes a felinecalicivirus encodes a feline calicivirusF9-Like F9-Like capsid capsid protein protein orantigenic or an an antigenic fragment fragment

thereof. thereof.

.0 0 19. 19. The The vaccine vaccine of statement of statement 16, further 16, further comprising comprising a killed a killed or or modified modified live F9- live F9 Like feline calicivirus. Like feline calicivirus.

20. TheThe 20. vaccine vaccine of statement of statement 16, 16, 17, 17, 18, 18, or 19, or 19, further further comprising comprising an an alphavirus alphavirus

RNA replicon RNA replicon particle particle that that encodes encodes feline feline leukemia leukemia virus (FeLV) virus (FeLV) glycoprotein glycoprotein (gp85) (gp85) 25 25 or an or antigenicfragment an antigenic fragment thereof. thereof.

21. TheThe 21. vaccine vaccine of statement of statement 16, 16, 17,17, 18,18, or or 19,wherein 19, whereinthethealphavirus alphavirusRNA RNA replicon particle replicon particle further further encodes encodesa feline a felineleukemia leukemia virus virus (FeLV) (FeLV) glycoprotein glycoprotein (gp85) (gp85)

or an or antigenicfragment an antigenic fragment thereof. thereof.

30 30

22. The The 22. vaccine vaccine composition composition of statement of statement 15, 16, 15, 17, 16, 17, 20, 18, 19, 18, or 19,21, 20,that or 21, is that a is a nonadjuvanted vaccine. nonadjuvanted vaccine.

43

23. A method 23. A method of immunizing of immunizing a feline a feline againsta apathogenic against pathogenicFCV FCV comprising comprising

administering to the administering to the feline feline an an immunologically immunologicallyeffective effectiveamount amount of the of the vaccine vaccine of of

statement15, statement 15,16, 16,17, 17,18, 18,19, 19,20, 20,21, 21,oror22. 22.

Claims (18)

CLAIMS: 17 Sep 2025

1. A vaccine to aid in the prevention of disease due to feline leukemia virus (FeLV) comprising an alphavirus RNA replicon particle that encodes one or more FeLV glycoproteins and one or more modified live feline pathogens selected from the group consisting of a modified live feline calicivirus (FCV), a modified live feline viral rhinotracheitis virus (FVR), a modified live feline 2022209359

panleukopenia virus (FPLV), a modified live Chlamydophila felis, and any combination thereof, and a pharmaceutically acceptable carrier.

2. The vaccine of claim 1, wherein the vaccine comprises at least two modified live pathogens selected from the group consisting of the modified live FCV, the modified live FVR, the modified live FPLV, and the modified live Chlamydophila felis.

3. The vaccine of claim 1, wherein the vaccine comprises at least three modified live pathogens selected from the group consisting of the modified live FCV, the modified live FVR, the modified live FPLV, and the modified live Chlamydophila felis.

4. A vaccine to aid in the prevention of disease due to feline leukemia virus (FeLV) comprising an alphavirus RNA replicon particle that encodes one or more FeLV glycoproteins and modified live feline pathogens comprising a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), and a modified live Chlamydophila felis, and a pharmaceutically acceptable carrier.

5. A vaccine to aid in the prevention of disease due to feline leukemia virus (FeLV) comprising an alphavirus RNA replicon particle that encodes one or more FeLV glycoproteins and modified live feline pathogens comprising a modified live feline calicivirus (FCV), a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), and a modified live Chlamydophila felis, and a pharmaceutically acceptable carrier.

6. The vaccine of anyone of Claims 1 to 5, wherein the one or 17 Sep 2025

more FeLV glycoprotein is selected from the group consisting of an FeLV glycoprotein gp85, an FeLV glycoprotein gp70, an FeLV glycoprotein gp45, and any combination thereof.

7. The vaccine of any one of Claims 1 to 6, wherein the alphavirus RNA replicon particle is a Venezuelan Equine Encephalitis (VEE) alphavirus RNA 2022209359

replicon particle.

8. The vaccine of Claim 7, wherein the FeLV glycoprotein is selected from the group consisting of an FeLV glycoprotein gp85 that comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 6, an FeLV glycoprotein gp70 that comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 8, and a combination thereof.

9. A vaccine to aid in the prevention of disease due to feline leukemia virus (FeLV) comprising a modified live feline calicivirus (FCV), a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), a modified live Chlamydophila felis, and a Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle encoding an FeLV glycoprotein gp85 that comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 6.

10. The vaccine of any one of Claims 7 to 9, wherein the VEE alphavirus RNA replicon particle further encodes one or more non-FeLV antigen selected from the group consisting of an FCV F9-Like capsid protein, a virulent systemic FCV (VS-FCV) capsid protein, a rabies virus G protein, and any combination thereof.

11. The vaccine of any one of Claims 7 to 9, that further comprises an additional VEE alphavirus RNA replicon particle that encodes one or more non- FeLV antigen selected from the group consisting of an FCV F9-Like capsid protein, a VS-FCV capsid protein, a rabies virus G protein, and any combination thereof.

12. The vaccine of Claim 10 or 11, wherein the one or more non- FeLV antigen is selected from the group consisting of an FCV F9 Like capsid protein that comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 4, a VS FCV capsid protein that comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID 2022209359

NO: 2, a rabies virus G protein that comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 10, and any combination thereof.

13. The vaccine of any one of Claims 7 to 10, further comprising an additional VEE alphavirus RNA replicon particle that encodes an FeLV glycoprotein gp70 that comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 8.

14. A method of immunizing a feline against a pathogenic FeLV comprising administering to the feline an immunologically effective amount of the vaccine of any one of Claims 1 to 13.

15. A method of immunizing a feline against a pathogenic FeLV comprising administering to the feline an immunologically effective amount of the vaccine of Claim 1.

16. A method of immunizing a feline against a pathogenic FeLV comprising administering to the feline an immunologically effective amount of the vaccine of Claim 4.

17. A method of immunizing a feline against a pathogenic FeLV comprising administering to the feline an immunologically effective amount of the vaccine of Claim 5.

18. A method of immunizing a feline against a pathogenic FeLV 17 Sep 2025

comprising administering to the feline an immunologically effective amount of the vaccine of Claim 9. Intervet International B.V.

Patent Attorneys for the Applicant/Nominated Person

SPRUSON & FERGUSON

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GSIPSDLIPKSSSLWIGNRHWTDITDFVIRPFVFQANRHFDFNQETAGWSTPRFRPLAVTVS 13 Sep 2022

QSKGAKLGNGIATDYIVPGIPDGWPDTTIPTKLTPTGDYAITSSDGNDIETKLEYENADVIKNN TNFRSMYICGSLQRAWGDKKISNTGFITTGVISDNSISPSNTIDQSKIVVYQDNHVNSEVQTS DITLAILGYTGIGEEAIGANRDSVVRISVLPETGARGGNHPIFYKNSMKLGYVISSIDVFNSQIL HTSRQLSLNNYLLPPDSFAVYRIIDSNGSWFDIGIDSDGFSFVGVSSFPKLEFPLSASYMGIQ LAKIRLASNIRSSMTKL</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData xmlns:xml='http://www.w3.org/XML/1998/namespace' sequenceIDNumber='3'> <INSDSeq xmlns:xml='http://www.w3.org/XML/1998/namespace'> 2022209359

<INSDSeq_length xmlns:xml='http://www.w3.org/XML/1998/namespace'>1629</INSDSeq_length> <INSDSeq_moltype xmlns:xml='http://www.w3.org/XML/1998/namespace'>DNA</INSDSeq_moltype> <INSDSeq_division xmlns:xml='http://www.w3.org/XML/1998/namespace'>PAT</INSDSeq_division> <INSDSeq_feature-table xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>misc_feature</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1629</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q4'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>note</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>codon optimized for feline</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>source</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1629</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>other DNA</INSDQualifier_value>

</INSDQualifier> 13 Sep 2022

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<INSDFeature_location 13 Sep 2022

xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..542</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>protein</INSDQualifier_value> </INSDQualifier> 2022209359

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<INSDFeature_location 13 Sep 2022

xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1929</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q7'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>note</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>codon optimized for feline</INSDQualifier_value> 2022209359

</INSDQualifier> </INSDFeature_quals> </INSDFeature> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>source</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1929</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q8'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>organism</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>synthetic construct</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence xmlns:xml='http://www.w3.org/XML/1998/namespace'>atggagtcaccaacacaccctaaaccttctaaag acaaaaccctctcgtggaatctcgccttccttgtgggcatcctgttcacaatcgacatcggcatggccaacccttcgccgcatcaga tctacaatgtgacatgggtcattactaatgtgcagacaaacacccaggcaaatgctacttctatgcttggtactctgactgatgcttat ccaaccctgcacgtcgacctttgcgatctcgtcggtgacacatgggagcccatcgtgctgaatccaactaatgtcaaacatggtgc caggtattcttctagcaaatacgggtgtaagaccactgatcggaagaaacagcaacaaacctacccattctacgtgtgcccgggt cacgcaccgtccctgggtccgaagggaacacattgtgggggagcccaagacggtttttgcgctgcttggggttgtgaaacaaccg gagaagcctggtggaagcctacctcatcttgggactacattactgtgaaaagaggctctagccaggataacagctgcgaaggaa agtgtaatcccctggtgcttcaattcacccagaaaggccggcaggcatcatgggatggaccgaaaatgtggggacttagactcta tcgcaccggatacgaccccatcgctctgtttactgtgtcacgccaagtctccaccattactccgccacaggccatggggccgaatct ggtcctccccgatcagaagccaccctcacggcaaagtcaaaccggctcaaaagtggccacccaacggccccagacaaatga 13 Sep 2022 gtccgcacctaggtcagtggcacctacaacaatgggtccaaagcggatcggaaccggagacaggctcattaacctcgtgcaag ggacttatctggcccttaacgctactgaccccaacaagaccaaggattgctggctctgccttgtgagcagacctccttactatgagg ggatcgccattctcggaaactactcaaatcagaccaacccccctccgtcgtgtctgagcaccccccagcacaagcttactatttca gaagtcagtggacagggaatgtgcatcggaaccgtgccaaagactcatcaagccctttgcaacaaaactcaacaagggcaca ctggagctcattatctcgccgcacctaacgggacctactgggcttgcaatactggattgaccccgtgtatctctatggccgtgctgaa ttggacttccgacttctgcgtgcttattgagctttggcctagagtgacataccatcagcctgagtacgtctatacccatttcgccaaggc agtcagattccggcgggagcctatctccctgactgtggccttgatgctcggtggactgacagtgggaggaattgcagctggagtcg gaactggaaccaaggccctgctcgaaactgctcagttccggcagctgcagatggccatgcacactgacatccaggctctggag gaatcaatttcagcccttgagaaaagcttgacctcgctgtctgaagtggtcctccaaaacaggcgcggtttggacatcctgttccttc aagagggtggtctgtgcgccgctctcaaggaggaatgctgtttctacgctgaccataccgggctggtgcgcgataacatggcaaa 2022209359 gctgcgggaacgcttgaaacagaggcagcaactgttcgactctcagcagggatggttcgagggctggtttaacaagagcccatg gtttaccactctgatctcttcaatcatgggtccactgctcatcctgcttctgattcttctcttcggaccgtgtattctcaacaggctggtgca gtttgtcaaggacagaatctcggtggtccaggccctgattcttactcagcagtatcagcagattaagcagtacgaccccgatcggc cttga</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData xmlns:xml='http://www.w3.org/XML/1998/namespace' sequenceIDNumber='6'> <INSDSeq xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDSeq_length xmlns:xml='http://www.w3.org/XML/1998/namespace'>642</INSDSeq_length> <INSDSeq_moltype xmlns:xml='http://www.w3.org/XML/1998/namespace'>AA</INSDSeq_moltype> <INSDSeq_division xmlns:xml='http://www.w3.org/XML/1998/namespace'>PAT</INSDSeq_division> <INSDSeq_feature-table xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>source</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..642</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>protein</INSDQualifier_value> </INSDQualifier> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q9'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>organism</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>Feline leukemia virus</INSDQualifier_value>

</INSDQualifier> 13 Sep 2022

</INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence xmlns:xml='http://www.w3.org/XML/1998/namespace'>MESPTHPKPSKDKTLSWNLAFLVGIL FTIDIGMANPSPHQIYNVTWVITNVQTNTQANATSMLGTLTDAYPTLHVDLCDLVGDTWEPIV LNPTNVKHGARYSSSKYGCKTTDRKKQQQTYPFYVCPGHAPSLGPKGTHCGGAQDGFCA AWGCETTGEAWWKPTSSWDYITVKRGSSQDNSCEGKCNPLVLQFTQKGRQASWDGPKM WGLRLYRTGYDPIALFTVSRQVSTITPPQAMGPNLVLPDQKPPSRQSQTGSKVATQRPQTN ESAPRSVAPTTMGPKRIGTGDRLINLVQGTYLALNATDPNKTKDCWLCLVSRPPYYEGIAILG 2022209359

NYSNQTNPPPSCLSTPQHKLTISEVSGQGMCIGTVPKTHQALCNKTQQGHTGAHYLAAPNG TYWACNTGLTPCISMAVLNWTSDFCVLIELWPRVTYHQPEYVYTHFAKAVRFRREPISLTVA LMLGGLTVGGIAAGVGTGTKALLETAQFRQLQMAMHTDIQALEESISALEKSLTSLSEVVLQN RRGLDILFLQEGGLCAALKEECCFYADHTGLVRDNMAKLRERLKQRQQLFDSQQGWFEGW FNKSPWFTTLISSIMGPLLILLLILLFGPCILNRLVQFVKDRISVVQALILTQQYQQIKQYDPDRP </INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData xmlns:xml='http://www.w3.org/XML/1998/namespace' sequenceIDNumber='7'> <INSDSeq xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDSeq_length xmlns:xml='http://www.w3.org/XML/1998/namespace'>1233</INSDSeq_length> <INSDSeq_moltype xmlns:xml='http://www.w3.org/XML/1998/namespace'>DNA</INSDSeq_moltype> <INSDSeq_division xmlns:xml='http://www.w3.org/XML/1998/namespace'>PAT</INSDSeq_division> <INSDSeq_feature-table xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>misc_feature</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1233</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q10'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>note</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>codon optimized for feline</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>source</INSDFeature_key> 13 Sep 2022

<INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1233</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>other DNA</INSDQualifier_value> 2022209359

</INSDQualifier> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q11'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>organism</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>synthetic construct</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence xmlns:xml='http://www.w3.org/XML/1998/namespace'>aatcctagtccacaccaaatatataatgtaacttgg gtaataaccaatgtacaaactaacacccaagctaacgccacctctatgttaggaaccttaaccgatgcctaccctaccctacatgtt gacttatgtgacctagtgggagacacctgggaacctatagtcctaaacccaaccaatgtaaaacacggggcacgttactcctcct caaaatatggatgtaaaactacagatagaaaaaaacagcaacagacataccccttttacgtctgccccggacatgccccctcgtt ggggccaaagggaacacattgtggaggggcacaagatgggttttgtgccgcatggggatgtgagaccaccggagaagcttggt ggaagcccacctcctcatgggactatatcacagtaaaaagagggagtagtcaggacaatagctgtgagggaaaatgcaaccc cctggttttgcagttcacccagaagggaagacaagcctcttgggacggacctaagatgtggggattgcgactataccgtacagga tatgaccctatcgctttattcacggtgtcccggcaggtatcaaccattacgccgcctcaggcaatgggaccaaacctagtcttacctg atcaaaaacccccatcccgacaatctcaaacagggtccaaagtggcgacccagaggccccaaacgaatgaaagcgcccca aggtctgttgcccccaccaccatgggtcccaaacggattgggaccggagataggttaataaatttagtacaagggacatacctag ccttaaatgccaccgaccccaacaaaactaaagactgttggctctgcctggtttctcgaccaccctattacgaagggattgcaatctt aggtaactacagcaaccaaacaaacccccccccatcctgcctatctactccgcaacacaaactaactatatctgaagtatcagg gcaaggaatgtgcatagggactgttcctaaaacccaccaggctttgtgcaataagacacaacagggacatacaggggcgcact atctagccgcccccaacggcacctattgggcctgtaacactggactcaccccatgcatttccatggcggtgctcaattggacctctg atttttgtgtcttaatcgaattatggcccagagtgacttaccatcaacccgaatatgtgtacacacattttgccaaagctgtcaggttcc gaaga</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData xmlns:xml='http://www.w3.org/XML/1998/namespace' sequenceIDNumber='8'> <INSDSeq xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDSeq_length xmlns:xml='http://www.w3.org/XML/1998/namespace'>411</INSDSeq_length> <INSDSeq_moltype xmlns:xml='http://www.w3.org/XML/1998/namespace'>AA</INSDSeq_moltype>

<INSDSeq_division 13 Sep 2022

xmlns:xml='http://www.w3.org/XML/1998/namespace'>PAT</INSDSeq_division> <INSDSeq_feature-table xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>source</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..411</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> 2022209359

<INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>protein</INSDQualifier_value> </INSDQualifier> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q12'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>organism</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>Feline leukemia virus</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence xmlns:xml='http://www.w3.org/XML/1998/namespace'>NPSPHQIYNVTWVITNVQTNTQANA TSMLGTLTDAYPTLHVDLCDLVGDTWEPIVLNPTNVKHGARYSSSKYGCKTTDRKKQQQTY PFYVCPGHAPSLGPKGTHCGGAQDGFCAAWGCETTGEAWWKPTSSWDYITVKRGSSQD NSCEGKCNPLVLQFTQKGRQASWDGPKMWGLRLYRTGYDPIALFTVSRQVSTITPPQAMG PNLVLPDQKPPSRQSQTGSKVATQRPQTNESAPRSVAPTTMGPKRIGTGDRLINLVQGTYL ALNATDPNKTKDCWLCLVSRPPYYEGIAILGNYSNQTNPPPSCLSTPQHKLTISEVSGQGMC IGTVPKTHQALCNKTQQGHTGAHYLAAPNGTYWACNTGLTPCISMAVLNWTSDFCVLIELW PRVTYHQPEYVYTHFAKAVRFRR</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData xmlns:xml='http://www.w3.org/XML/1998/namespace' sequenceIDNumber='9'> <INSDSeq xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDSeq_length xmlns:xml='http://www.w3.org/XML/1998/namespace'>1575</INSDSeq_length> <INSDSeq_moltype xmlns:xml='http://www.w3.org/XML/1998/namespace'>DNA</INSDSeq_moltype> <INSDSeq_division xmlns:xml='http://www.w3.org/XML/1998/namespace'>PAT</INSDSeq_division>

<INSDSeq_feature-table 13 Sep 2022

xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>misc_feature</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1575</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q13'> 2022209359

<INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>note</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>codon optimized for human</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>source</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1575</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q14'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>organism</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>synthetic construct</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence xmlns:xml='http://www.w3.org/XML/1998/namespace'>atggtgccgcaggctctcctgtttgtcccccttctggt ctttccattgtgttttgggaaattccctatctacacaattccggacaagttgggaccctggagcccaattgacattcatcatctcagctg cccgaacaatttggtcgtggaggacgaaggatgcaccaacctgtcggggttctcctacatggaattgaaagtcggatacatcagt gccattaagatgaacgggttcacttgcacaggcgtcgtgactgaagctgagacatacactaacttcgtgggatatgtcactaccac tttcaaaagaaagcatttccgccctactcctgatgcttgtagggccgcatacaactggaagatggccggtgaccccagatatgagg 13 Sep 2022 aatcacttcacaatccgtaccctgactaccactggcttcggactgtcaaaaccaccaaggagtcactcgtgatcattagtccaagtg tggctgatcttgacccatacgaccggtcacttcactcacgggtgttcccgggggggaattgctctggtgtcgcagtgtcgtcaaccta ctgctccacaaaccacgattacaccatttggatgccagaaaatcctcggcttggtatgtcatgtgacattttcaccaattctcggggg aagagggcttccaaagggtctgaaacttgcggctttgtcgatgagcggggcttgtataagtcacttaaaggtgcttgcaaactcaa gctttgtggtgtcttgggattgagattgatggatggaacttgggtcgcaatgcagacttctaacgaaaccaaatggtgccctcccgga cagcttgtgaatttgcatgactttcgctctgacgaaattgagcatcttgtcgtcgaggagttggtcaagaagcgggaagagtgtctgg atgctttggaatcaatcatgaccaccaagtcagtgtctttcagacggctctcacatcttaggaaattggtgccaggttttggaaaagc atataccattttcaacaagacccttatggaagccgatgctcactacaagtctgtcaggacttggaatgagatcatcccgtctaaagg gtgtcttagggtcggagggagatgtcatcctcatgtcaacggagtctttttcaatggtatcattcttggacctgacggaaatgtccttatc cctgagatgcaatcttccctcctccagcaacacatggaacttcttgtctcatcggtcatcccccttatgcaccccctggctgacccatc 2022209359 aaccgtgttcaagaacggtgacgaggcagaggattttgtcgaggtccaccttcccgatgtgcatgaacggatctctggtgtcgacc ttggactccctaactggggaaagtatgtccttctgtcggcaggagccctgactgccttgatgttgattatcttcctgatgacttgttggag gagagtcaatcggtcggagccaacacaacataatctcagaggaacaggaagggaggtgtcagtcacaccccaaagcggga agatcatttcgtcttgggagtcatacaagagcggaggtgaaaccggactgtga</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData xmlns:xml='http://www.w3.org/XML/1998/namespace' sequenceIDNumber='10'> <INSDSeq xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDSeq_length xmlns:xml='http://www.w3.org/XML/1998/namespace'>524</INSDSeq_length> <INSDSeq_moltype xmlns:xml='http://www.w3.org/XML/1998/namespace'>AA</INSDSeq_moltype> <INSDSeq_division xmlns:xml='http://www.w3.org/XML/1998/namespace'>PAT</INSDSeq_division> <INSDSeq_feature-table xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>source</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..524</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>protein</INSDQualifier_value> </INSDQualifier> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q15'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>organism</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>Rabies virus</INSDQualifier_value> </INSDQualifier>

</INSDFeature_quals> 13 Sep 2022

</INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence xmlns:xml='http://www.w3.org/XML/1998/namespace'>MVPQALLFVPLLVFPLCFGKFPIYTIP DKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKMNGFTCTGVVTEAET YTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKE SLVIISPSVADLDPYDRSLHSRVFPGGNCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIF TNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKW CPPGQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGK AYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEM 2022209359

QSSLLQQHMELLVSSVIPLMHPLADPSTVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNW GKYVLLSAGALTALMLIIFLMTCWRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESYKS GGETGL</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData xmlns:xml='http://www.w3.org/XML/1998/namespace' sequenceIDNumber='11'> <INSDSeq xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDSeq_length xmlns:xml='http://www.w3.org/XML/1998/namespace'>13</INSDSeq_length> <INSDSeq_moltype xmlns:xml='http://www.w3.org/XML/1998/namespace'>DNA</INSDSeq_moltype> <INSDSeq_division xmlns:xml='http://www.w3.org/XML/1998/namespace'>PAT</INSDSeq_division> <INSDSeq_feature-table xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>misc_feature</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..13</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q16'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>note</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>5 'flanking sequence</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>source</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..13</INSDFeature_location>

<INSDFeature_quals 13 Sep 2022

xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q17'> 2022209359

<INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>organism</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>synthetic construct</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence xmlns:xml='http://www.w3.org/XML/1998/namespace'>ggcgcgccgcacc</INSDSeq_sequence > </INSDSeq> </SequenceData> <SequenceData xmlns:xml='http://www.w3.org/XML/1998/namespace' sequenceIDNumber='12'> <INSDSeq xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDSeq_length xmlns:xml='http://www.w3.org/XML/1998/namespace'>1638</INSDSeq_length> <INSDSeq_moltype xmlns:xml='http://www.w3.org/XML/1998/namespace'>RNA</INSDSeq_moltype> <INSDSeq_division xmlns:xml='http://www.w3.org/XML/1998/namespace'>PAT</INSDSeq_division> <INSDSeq_feature-table xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>misc_feature</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1638</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q18'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>note</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>codon optimized for feline</INSDQualifier_value> 13 Sep 2022

</INSDQualifier> </INSDFeature_quals> </INSDFeature> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>source</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1638</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> 2022209359

<INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>other RNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q19'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>organism</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>synthetic construct</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence xmlns:xml='http://www.w3.org/XML/1998/namespace'>atggctgacgacggatctgtgaccaccccagaac aaggaacaatggtcggaggagtgattgccgaacccagcgctcagatgtcaactgcggcggacatggcctccggaaagtcggt ggactccgagtgggaagccttcttctcgttccacacgtccgtgaactggagcacctccgaaacccaaggaaagatcctcttcaag cagtccctgggtcccctgctgaacccgtacctggagcacatcagcaagctgtacgtcgcttggagcgggtcgatcgaagtgcgat tttccatctcgggaagcggcgtgttcggtggtaaactggccgccatcgtcgtgccgcctggtgtcgaccctgtccagtcaacctccat gctgcagtacccgcacgtcctgttcgacgcaagacaagtggagccagtgatcttctccatcccggacctccgcaacagcctgtat cacttgatgtccgataccgataccacttccctcgtgatcatggtgtacaacgatctgatcaacccgtacgccaatgactccaacagc tcgggttgcatcgtgaccgtcgaaacgaagcctggcatcgatttcaagtttcatctgctgaaaccgcccggatccatgcttactcac gggtccatcccttccgatctgatccccaagagctcctccctgtggattgggaaccgccactggaccgatattaccgatttcgtgattc ggcctttcgtgttccaagccaaccggcacttcgacttcaaccaggagactgccggctggtcaactccacggttccgcccattggcc gtgactgtgtcgcagtcaaagggagccaagctcgggaacggcatcgccaccgactacattgtgcctggaatccccgacggatg gcctgatactaccatccccaccaagctgacccctaccggagattacgccatcacctcctccgacggcaatgatattgaaaccaag ctggaatacgagaacgcggacgtgattaagaacaacaccaacttccgctccatgtatatctgcggaagcctccagagggcttgg ggcgacaagaagatcagcaacaccgggttcatcactaccggagtgatttctgacaactccatcagcccttcgaacacaattgac cagtccaagatcgtggtgtaccaggacaaccatgtcaattcggaggtccagactagcgacatcactcttgccatcctgggctaca ccggaattggagaagaggccataggcgccaaccgggactccgtcgtgagaatttccgtgcttccggaaactggagcaagggg cggaaatcaccccatcttctacaaaaattccatgaagctgggctacgtgatctcctccattgacgtgttcaactcccaaatcctccac acctcgcgccagctgtcactgaacaactacttgttgccccctgactccttcgcggtgtaccggattattgacagcaacggatcatggt tcgacattgggattgacagcgatgggttttcattcgtgggcgtgtcgtcatttccaaagctggagtttccgctgtccgcctcatacatgg gcatccagctcgcaaagatccggctggcgtccaacatccggtcatccatgactaagctgtga</INSDSeq_sequence> 13 Sep 2022

</INSDSeq> </SequenceData> <SequenceData xmlns:xml='http://www.w3.org/XML/1998/namespace' sequenceIDNumber='13'> <INSDSeq xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDSeq_length xmlns:xml='http://www.w3.org/XML/1998/namespace'>1629</INSDSeq_length> <INSDSeq_moltype xmlns:xml='http://www.w3.org/XML/1998/namespace'>RNA</INSDSeq_moltype> <INSDSeq_division 2022209359

xmlns:xml='http://www.w3.org/XML/1998/namespace'>PAT</INSDSeq_division> <INSDSeq_feature-table xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>misc_feature</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1629</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q20'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>note</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>codon optimized for feline</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>source</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1629</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>other RNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q21'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>organism</INSDQualifier_name>

<INSDQualifier_value 13 Sep 2022

xmlns:xml='http://www.w3.org/XML/1998/namespace'>synthetic construct</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence xmlns:xml='http://www.w3.org/XML/1998/namespace'>atgactgccccggaacaaggaacgatggtcgga ggagtgattgcagaaccgtcagcacagatgtccaccgctgccgacatggccactggaaagagcgtggactccgaatgggaag ccttcttctccttccacacttcggtcaactggtcgactagcgaaacccaggggaagattttgttcaagcaatccctcggccctctgctg 2022209359

aacccctacctggagcatctggccaagctgtacgtggcatggtcgggcagcatcgaagtgcgctttagcatttccggctccggagt gttcgggggaaagcttgctgccattgtcgtgccgccaggagtggacccggtgcagtccacttctatgctccaatacccgcatgtcct gttcgacgccagacaggtggagcctgtgatcttttgcctgccggatctcaggtccaccctgtatcacctcatgtccgacaccgacac cacctcgctcgtgatcatggtgtacaacgacctgatcaacccctacgctaacgacgccaacagctcaggttgcattgtgactgtcg aaaccaagccaggccctgacttcaagtttcatttgctgaagccgcccggttccatgctgacccacggctcgatcccatccgacctg atccccaagacgagctccctgtggatcggaaaccgctactggtccgatattaccgacttcgtgatcagaccattcgtgttccaagcc aaccgccatttcgacttcaaccaggaaaccgcaggatggtcgacccctcgattccgcccgatttcagtgaccatcaccgaacag aacggcgcgaagctgggaattggcgtggcgaccgactacatcgtgccgggaatcccggatggatggcctgatacgaccattcc cggggagctgatccctgccggggactacgccatcaccaacggtactggaaacgacatcaccactgccaccggttacgacacc gccgacatcataaagaacaacaccaacttcagaggaatgtacatttgcggctccctgcaacgcgcttggggtgacaaaaagatc tcgaacactgccttcatcacaacagcgactctggacggcgataacaacaacaagatcaatccttgtaataccatcgaccagtcca aaatcgtggtgttccaggataaccacgtgggaaagaaggcgcagacctccgacgacactctggcgctgcttggctacaccggg atcggcgagcaggccattggaagcgatcgggatcgggtcgtgcggatctccaccctccccgagactggagcaaggggaggca accaccccatcttttacaaaaacagcattaagctcggatacgtcatccgctccatcgatgtgttcaactctcaaatcctgcacacttc gcggcagctgtccctgaaccactacctcttgccgcccgactccttcgccgtctaccggatcattgattcgaacgggagctggttcga catcggcattgatagcgatggcttctcgtttgtgggcgtgtcgggcttcgggaagctggagttcccactgagcgcctcatacatgggt atccagctggccaagatcaggctggcctccaacatccgctcacctatgactaagctgtga</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData xmlns:xml='http://www.w3.org/XML/1998/namespace' sequenceIDNumber='14'> <INSDSeq xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDSeq_length xmlns:xml='http://www.w3.org/XML/1998/namespace'>1929</INSDSeq_length> <INSDSeq_moltype xmlns:xml='http://www.w3.org/XML/1998/namespace'>RNA</INSDSeq_moltype> <INSDSeq_division xmlns:xml='http://www.w3.org/XML/1998/namespace'>PAT</INSDSeq_division> <INSDSeq_feature-table xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>misc_feature</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1929</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q22'> 13 Sep 2022

<INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>note</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>codon optimized for feline</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key 2022209359

xmlns:xml='http://www.w3.org/XML/1998/namespace'>source</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1929</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>other RNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q23'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>organism</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>synthetic construct</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence xmlns:xml='http://www.w3.org/XML/1998/namespace'>atggagtcaccaacacaccctaaaccttctaaag acaaaaccctctcgtggaatctcgccttccttgtgggcatcctgttcacaatcgacatcggcatggccaacccttcgccgcatcaga tctacaatgtgacatgggtcattactaatgtgcagacaaacacccaggcaaatgctacttctatgcttggtactctgactgatgcttat ccaaccctgcacgtcgacctttgcgatctcgtcggtgacacatgggagcccatcgtgctgaatccaactaatgtcaaacatggtgc caggtattcttctagcaaatacgggtgtaagaccactgatcggaagaaacagcaacaaacctacccattctacgtgtgcccgggt cacgcaccgtccctgggtccgaagggaacacattgtgggggagcccaagacggtttttgcgctgcttggggttgtgaaacaaccg gagaagcctggtggaagcctacctcatcttgggactacattactgtgaaaagaggctctagccaggataacagctgcgaaggaa agtgtaatcccctggtgcttcaattcacccagaaaggccggcaggcatcatgggatggaccgaaaatgtggggacttagactcta tcgcaccggatacgaccccatcgctctgtttactgtgtcacgccaagtctccaccattactccgccacaggccatggggccgaatct ggtcctccccgatcagaagccaccctcacggcaaagtcaaaccggctcaaaagtggccacccaacggccccagacaaatga gtccgcacctaggtcagtggcacctacaacaatgggtccaaagcggatcggaaccggagacaggctcattaacctcgtgcaag ggacttatctggcccttaacgctactgaccccaacaagaccaaggattgctggctctgccttgtgagcagacctccttactatgagg ggatcgccattctcggaaactactcaaatcagaccaacccccctccgtcgtgtctgagcaccccccagcacaagcttactatttca gaagtcagtggacagggaatgtgcatcggaaccgtgccaaagactcatcaagccctttgcaacaaaactcaacaagggcaca ctggagctcattatctcgccgcacctaacgggacctactgggcttgcaatactggattgaccccgtgtatctctatggccgtgctgaa 13 Sep 2022 ttggacttccgacttctgcgtgcttattgagctttggcctagagtgacataccatcagcctgagtacgtctatacccatttcgccaaggc agtcagattccggcgggagcctatctccctgactgtggccttgatgctcggtggactgacagtgggaggaattgcagctggagtcg gaactggaaccaaggccctgctcgaaactgctcagttccggcagctgcagatggccatgcacactgacatccaggctctggag gaatcaatttcagcccttgagaaaagcttgacctcgctgtctgaagtggtcctccaaaacaggcgcggtttggacatcctgttccttc aagagggtggtctgtgcgccgctctcaaggaggaatgctgtttctacgctgaccataccgggctggtgcgcgataacatggcaaa gctgcgggaacgcttgaaacagaggcagcaactgttcgactctcagcagggatggttcgagggctggtttaacaagagcccatg gtttaccactctgatctcttcaatcatgggtccactgctcatcctgcttctgattcttctcttcggaccgtgtattctcaacaggctggtgca gtttgtcaaggacagaatctcggtggtccaggccctgattcttactcagcagtatcagcagattaagcagtacgaccccgatcggc cttga</INSDSeq_sequence> </INSDSeq> 2022209359

</SequenceData> <SequenceData xmlns:xml='http://www.w3.org/XML/1998/namespace' sequenceIDNumber='15'> <INSDSeq xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDSeq_length xmlns:xml='http://www.w3.org/XML/1998/namespace'>1233</INSDSeq_length> <INSDSeq_moltype xmlns:xml='http://www.w3.org/XML/1998/namespace'>RNA</INSDSeq_moltype> <INSDSeq_division xmlns:xml='http://www.w3.org/XML/1998/namespace'>PAT</INSDSeq_division> <INSDSeq_feature-table xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>misc_feature</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1233</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q24'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>note</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>codon optimized for feline</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>source</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1233</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'>

<INSDQualifier_name 13 Sep 2022

xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>other RNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q25'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>organism</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>synthetic 2022209359

construct</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence xmlns:xml='http://www.w3.org/XML/1998/namespace'>aatcctagtccacaccaaatatataatgtaacttgg gtaataaccaatgtacaaactaacacccaagctaacgccacctctatgttaggaaccttaaccgatgcctaccctaccctacatgtt gacttatgtgacctagtgggagacacctgggaacctatagtcctaaacccaaccaatgtaaaacacggggcacgttactcctcct caaaatatggatgtaaaactacagatagaaaaaaacagcaacagacataccccttttacgtctgccccggacatgccccctcgtt ggggccaaagggaacacattgtggaggggcacaagatgggttttgtgccgcatggggatgtgagaccaccggagaagcttggt ggaagcccacctcctcatgggactatatcacagtaaaaagagggagtagtcaggacaatagctgtgagggaaaatgcaaccc cctggttttgcagttcacccagaagggaagacaagcctcttgggacggacctaagatgtggggattgcgactataccgtacagga tatgaccctatcgctttattcacggtgtcccggcaggtatcaaccattacgccgcctcaggcaatgggaccaaacctagtcttacctg atcaaaaacccccatcccgacaatctcaaacagggtccaaagtggcgacccagaggccccaaacgaatgaaagcgcccca aggtctgttgcccccaccaccatgggtcccaaacggattgggaccggagataggttaataaatttagtacaagggacatacctag ccttaaatgccaccgaccccaacaaaactaaagactgttggctctgcctggtttctcgaccaccctattacgaagggattgcaatctt aggtaactacagcaaccaaacaaacccccccccatcctgcctatctactccgcaacacaaactaactatatctgaagtatcagg gcaaggaatgtgcatagggactgttcctaaaacccaccaggctttgtgcaataagacacaacagggacatacaggggcgcact atctagccgcccccaacggcacctattgggcctgtaacactggactcaccccatgcatttccatggcggtgctcaattggacctctg atttttgtgtcttaatcgaattatggcccagagtgacttaccatcaacccgaatatgtgtacacacattttgccaaagctgtcaggttcc gaaga</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData xmlns:xml='http://www.w3.org/XML/1998/namespace' sequenceIDNumber='16'> <INSDSeq xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDSeq_length xmlns:xml='http://www.w3.org/XML/1998/namespace'>1575</INSDSeq_length> <INSDSeq_moltype xmlns:xml='http://www.w3.org/XML/1998/namespace'>RNA</INSDSeq_moltype> <INSDSeq_division xmlns:xml='http://www.w3.org/XML/1998/namespace'>PAT</INSDSeq_division> <INSDSeq_feature-table xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>misc_feature</INSDFeature_key>

<INSDFeature_location 13 Sep 2022

xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1575</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q26'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>note</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>codon optimized for human</INSDQualifier_value> 2022209359

</INSDQualifier> </INSDFeature_quals> </INSDFeature> <INSDFeature xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDFeature_key xmlns:xml='http://www.w3.org/XML/1998/namespace'>source</INSDFeature_key> <INSDFeature_location xmlns:xml='http://www.w3.org/XML/1998/namespace'>1..1575</INSDFeature_location> <INSDFeature_quals xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>mol_type</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>other RNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier xmlns:xml='http://www.w3.org/XML/1998/namespace' id='q27'> <INSDQualifier_name xmlns:xml='http://www.w3.org/XML/1998/namespace'>organism</INSDQualifier_name> <INSDQualifier_value xmlns:xml='http://www.w3.org/XML/1998/namespace'>synthetic construct</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence xmlns:xml='http://www.w3.org/XML/1998/namespace'>atggtgccgcaggctctcctgtttgtcccccttctggt ctttccattgtgttttgggaaattccctatctacacaattccggacaagttgggaccctggagcccaattgacattcatcatctcagctg cccgaacaatttggtcgtggaggacgaaggatgcaccaacctgtcggggttctcctacatggaattgaaagtcggatacatcagt gccattaagatgaacgggttcacttgcacaggcgtcgtgactgaagctgagacatacactaacttcgtgggatatgtcactaccac tttcaaaagaaagcatttccgccctactcctgatgcttgtagggccgcatacaactggaagatggccggtgaccccagatatgagg aatcacttcacaatccgtaccctgactaccactggcttcggactgtcaaaaccaccaaggagtcactcgtgatcattagtccaagtg tggctgatcttgacccatacgaccggtcacttcactcacgggtgttcccgggggggaattgctctggtgtcgcagtgtcgtcaaccta ctgctccacaaaccacgattacaccatttggatgccagaaaatcctcggcttggtatgtcatgtgacattttcaccaattctcggggg aagagggcttccaaagggtctgaaacttgcggctttgtcgatgagcggggcttgtataagtcacttaaaggtgcttgcaaactcaa gctttgtggtgtcttgggattgagattgatggatggaacttgggtcgcaatgcagacttctaacgaaaccaaatggtgccctcccgga 13 Sep 2022 cagcttgtgaatttgcatgactttcgctctgacgaaattgagcatcttgtcgtcgaggagttggtcaagaagcgggaagagtgtctgg atgctttggaatcaatcatgaccaccaagtcagtgtctttcagacggctctcacatcttaggaaattggtgccaggttttggaaaagc atataccattttcaacaagacccttatggaagccgatgctcactacaagtctgtcaggacttggaatgagatcatcccgtctaaagg gtgtcttagggtcggagggagatgtcatcctcatgtcaacggagtctttttcaatggtatcattcttggacctgacggaaatgtccttatc cctgagatgcaatcttccctcctccagcaacacatggaacttcttgtctcatcggtcatcccccttatgcaccccctggctgacccatc aaccgtgttcaagaacggtgacgaggcagaggattttgtcgaggtccaccttcccgatgtgcatgaacggatctctggtgtcgacc ttggactccctaactggggaaagtatgtccttctgtcggcaggagccctgactgccttgatgttgattatcttcctgatgacttgttggag gagagtcaatcggtcggagccaacacaacataatctcagaggaacaggaagggaggtgtcagtcacaccccaaagcggga agatcatttcgtcttgggagtcatacaagagcggaggtgaaaccggactgtga</INSDSeq_sequence> </INSDSeq> 2022209359

</SequenceData> </ST26SequenceListing>