WO2019068905A2

WO2019068905A2 - Feline vaccines conferring early protection

Info

Publication number: WO2019068905A2
Application number: PCT/EP2018/077215
Authority: WO
Inventors: Cynthia LESBROS; Jules Minke
Original assignee: Virbac
Priority date: 2017-10-06
Filing date: 2018-10-05
Publication date: 2019-04-11
Also published as: EP3691679A2; WO2019068905A3

Abstract

The present invention relates to the use of a vaccine composition comprising at least one feline calicivirus antigen and/or at least one feline leukemia virus antigen and/or at least one feline herpesvirus-1 antigen, for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus and/or a feline leukemia virus and/or a feline herpesvirus-1 respectively, in a feline, wherein said vaccine composition is administered in one single administration to the feline.

Description

Feline vaccines conferring early protection

The present invention relates to the use of a vaccine composition comprising at least one feline calicivirus antigen and/or at least one feline leukemia virus antigen and/or at least one feline herpesvirus-1 (FHV-1 ) antigen, in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus and/or a feline leukemia virus and/or a FHV-1 respectively, in a feline. Said vaccine composition provides a protection of said feline after the first administration, and said vaccine can be administered in one single administration to the feline.

Feline calicivirus (FCV) is a RNA virus which can cause various symptoms that affect mainly the oral cavity and, occasionally, the upper respiratory tract. It is one of the main pathogens responsible for coryza (cat flu), a very common disease in cats. Symptoms possibly induced by FCV include oral ulcerations, salivation, gingivitis, stomatitis, conjunctivitis, sneezing and nasal discharge. Some atypical FCV can cause lameness or skin ulcerations while some hypervirulent strains can lead to systemic diseases (Radford AD, Coyne KP, Dawson S, Porter CJ, Gaskell RM. Feline calicivirus. Vet Res. 2007;38(2):319-35; Radford AD, Addie D, Belak S, Boucraut-Baralon C, Egberink H, Frymus T, Gruffydd-Jones T, Hartmann K, Hosie MJ, Lloret A, Lutz H, Marsilio F, Pennisi MG, Thiry E, Truyen U, Horzinek MC. Feline calicivirus infection. ABCD guidelines on prevention and management. J Feline Med Surg. 2009;1 1 (7):556-64).

Vaccines against FCV are available and widely used. They generally contain one or two FCV strains that are either live attenuated or inactivated. Although regulatory agency approved vaccines should trigger protective immunity, their efficacy can differ according to the type of immunity stimulated (Saalmuller A. New understanding of immunological mechanisms. Vet Microbiol. 2006;1 17(1 ):32-8; Greene CE, Schultz RD. Immunoprophylaxis. In: Greene CE (Ed). Infectious Diseases of the Dog and Cat. 3^rd ed. Saunders Elsevier, St Louis; 2006. p.1069-1 1 19). Inactivated vaccines are known to elicit the production of neutralising antibodies (NAb) directed against specific extracellular antigens (humoral immunity). Live vaccines, however, mimic the natural infection and stimulate the cellular component of the immune system (cell-mediated immunity) in addition to the humoral response. Since live attenuated viruses can replicate, live vaccines generally induce a quicker, longer lasting and more efficient immune response than inactivated vaccines; nevertheless, in the case of FCV and FHV-1 , protection would be maximized using live attenuated viruses by administering two doses (Greene CE, Schultz RD. Immunoprophylaxis. In: Greene CE (Ed). Infectious Diseases of the Dog and Cat. 3^rd ed. Saunders Elsevier, St Louis; 2006. p.1069-1 1 19; Day MJ, Horzinek MC, Schultz RD, Squires RA; Vaccination Guidelines Group (VGG) of the World Small Animal Veterinary Association (WSAVA). WSAVA Guidelines for the vaccination of dogs and cats. J Small Anim Pract. 2016;57(1 ):E1 -E45).

Among other common feline viruses, feline leukaemia virus (FeLV) is an enveloped retrovirus belonging to the oncovirus family. Said FeLV can infect domestic cats of any age throughout the world, especially kittens and juvenile cats. The virus is transmitted in the saliva or nasal secretions resulting from prolonged intimate contacts (e.g., mutual grooming), biting or sharing of food and water utensils. The virus may also be transmitted by transfusion of blood from an infected cat, in utero or through milk. Clinical signs of FeLV infection are numerous and non-specific. They include pyrexia, failure to thrive, chronic or recurrent respiratory tract and gastrointestinal disease, neoplasia, anaemia, and concurrent diseases resulting from immunosuppression. Efficacious FeLV vaccines are of great importance as they protect cats from persistent viraemia and thus from FeLV- associated fatal disease. Nevertheless, they do not prevent minimal viral replication and proviral integration. Feline herpesvirus-1 (FHV-1 ), also called feline viral rhinotracheitis virus (FVR), is a feline DNA herpesvirus of serotype 1 from the family of Herpesviridae causing upper respiratory infections in cats, especially the feline viral rhinotracheitis which infects nose and/or throat in cats. Cats get infected by contact with virus particles either by direct contact with infected nasal, oral and/or conjunctival secretions from a cat, or indirect contact through the environment of an infected cat. Said infection occurs in cats of all age but kittens are particularly susceptible to contract such infection. It is therefore of particular importance to vaccinate young cats at least to reduce clinical signs and viral excretion of the virus.

As explained above, the guidelines of the World Small Animal Veterinary Association (WSAVA) on feline vaccines recommend the administration of two doses of vaccine 2-4 weeks apart for vaccines against feline calicivirosis and feline viral rhinotracheitis, and 3-4 weeks apart for vaccines against feline leukemia, to ensure optimal protection as a primary/initial vaccination (Vaccination Guidelines Group (VGG) of the World Small Animal Veterinary Association (WSAVA). WSAVA Guidelines for the vaccination of dogs and cats. J Small Anim Pract. 2016;57(1 ):E1 -E45). Typically a first administration of primary vaccination is performed at day 0, and then a second administration is performed at day 21 (i.e. 3 weeks later).

Onset of immunity is commonly admitted to be obtained for each of said vaccines after the second administration of the primary vaccination.

A vaccine enabling a protection from the first administration would enable to provide an early protection to vaccinated felines. Decreasing the frequency of administration would also be of most interest. Indeed, the compliance would be increased as the number of visits to the veterinarian would be alleged, and it would be more convenient for the animal.

Consequently, there is still a need for feline vaccines, especially a feline calicivirosis vaccine and/or a feline leukemia vaccine and/or feline rhinotracheitis vaccine, which would be early efficient and/or possibly administered at a minimal frequency.

There is a need for an efficient feline calicivirosis vaccine and/or a feline leukemia vaccine and/or feline rhinotracheitis vaccine from its first administered dose. Said vaccine would have to be protective for the feline against the targeted virus from its first administered dose. The inventors have now discovered that the response of the immune system to a FCV vaccine is directly initiated with the administration of the first dose (i.e. the first dose of the primary or initial vaccination. The terms "primary" and "initial" may be used interchangeably).

By "primary vaccination", it is meant the very first administration scheme of a given vaccine composition. As mentioned above, for example, the primary vaccination against FCV typically comprises an administration scheme of two doses, 2-4 weeks apart.

Indeed, as shown in the examples, as soon as the administration of the first dose of FCV vaccine is performed, the antibodies which are produced reach a maximal amount which is not increased further to the second administration. Thus, the clinical protection against FCV is maximal after the first dose of primary vaccination; a single dose of FCV vaccine is enough to be immunoprotective. Also, in the case of a primary vaccination administration scheme in two doses, the first dose of the vaccine enables the immunoprotection of the feline animal. Said surprising efficacy after the first dose of primary vaccination has also been demonstrated by the inventors with a feline leukemia virus (FeLV) vaccine as well as with a feline herpesvirus-1 (FHV-1 ) vaccine. According to a first embodiment, the present invention thus relates to the use of a vaccine composition comprising at least one feline calicivirus (FCV) antigen and/or at least one feline leukemia virus (FeLV) antigen and/or at least one feline herpesvirus-1 (FHV-1 ) antigen, in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus and/or a feline leukemia virus and/or a feline herpesvirus-1 respectively, in an animal, wherein said vaccine composition is administered in one single administration to the animal. Of course, as the vaccine composition comprises at least one FCV antigen and/or at least one FeLV antigen and/or at least one FHV-1 antigen, the animal is a feline. By "one single administration" or "single administration" or "one administration" or "one single dose" or "one dose" of the vaccine composition, it is understood a unique administration, or a unique dose, of said vaccine composition for the primary vaccination. Such a vaccine composition is administered in one shot. Thus, it explicitly excludes any second and/or further administration of said vaccine composition.

According to a second embodiment, the invention also relates to the use of a vaccine composition comprising at least one feline calicivirus antigen and/or at least one feline leukemia virus antigen and/or at least one feline herpesvirus-1 antigen, in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus and/or a feline leukemia virus and/or a feline herpesvirus-1 respectively, in a feline, wherein an initial administration of said vaccine composition provides an onset of immunity in the feline.

According to the second embodiment, an "initial administration" or a "first administration" of the vaccine composition is to be understood as the first administration of the primary vaccination. Said administration is sufficient for an onset of immunity against feline calicivirosis and/or feline leukaemia and/or feline viral rhinotracheitis respectively, in the feline. Said initial administration may be followed by a second and/or further administration of said vaccine composition; however, said second and/or further administration will have few or no effect on the onset of immunity in the feline. By "onset of immunity" against a virus, it is meant the time period after which the feline is protected against said virus following the administration of the vaccine. Said onset of immunity may correspond to the maximal amount of produced anti-virus antibodies after the initial administration. Typically this amount is not significantly increased after a second and/or further administration of a vaccine according to the invention.

Preferably, the onset of immunity arises from 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 days after the initial administration of said vaccine composition. More preferably, the onset of immunity arises from 16, 17, 18, 19, 20, 21 or 22 days after the initial administration of said vaccine composition.

Preferably according to the second embodiment, the initial administration of the vaccine composition consists of a single administration (as in the first embodiment above), and the second embodiment excludes any second and/or further administration of said vaccine composition.

The following definitions are applicable to the present invention.

"Antibody" refers to an immunoglobulin molecule that can bind to a specific antigen as the result of an immune response to that antigen. Immunoglobulins are serum proteins composed of light and heavy polypeptide chains having constant and variable regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions. An antibody that is specific for a given antigen indicates that the variable regions of the antibody recognize and bind a specific antigen exclusively - e.g., the antibody is able to distinguish a particular capsid protein from other known proteins by virtue of measurable differences in binding affinity, despite the existence of localized sequence identity, homology, or similarity between capsid proteins and other polypeptides. Specific antibodies may also interact with other proteins through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant regions of the molecule. Screening assays to determine binding specificity of an antibody are well known in the art. Antibodies may also recognize and bind fragments of FCV capsid proteins, provided that the antibodies are specific for FCV capsid proteins. Antibodies can be produced using methods known in the art.

"Antigen" or "immunogen" refers to a molecule that contains one or more epitopes (linear, conformational or both) that upon exposure to a subject will induce an immune response that is specific for that antigen. An epitope is the specific site of the antigen which binds to a T-cell receptor or specific antibody, and typically comprises about 3 amino acid residues to about 20 amino acid residues. The antigen refers to subunit antigens (antigens separate and discrete from a whole organism with which the antigen is associated in nature) as well as killed, attenuated or inactivated bacteria, viruses, fungi, parasites or other microbes. The antigen also refers to an oligonucleotide or polynucleotide that expresses an antigen or antigenic determinant in vivo, such as in DNA immunization applications.

"Immune response" in a subject refers to the development of a humoral immune response, a cellular immune response, or a humoral and a cellular immune response to an antigen. A "humoral immune response" refers to one that is mediated by antibodies. A "cellular immune response" is one mediated by T-lymphocytes or other white blood cells or both, and includes the production of cytokines, chemokines and similar molecules produced by activated T-cells, white blood cells, or both. Immune responses can be determined using standard immunoassays and neutralization assays, which are known in the art.

"Immunologically protective amount" or "effective amount" of an antigen is an amount effective to induce an immunogenic response in the animal (feline) that is adequate to prevent or ameliorate (i.e. significantly reduce) signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with the calicivirus antigen and in particular with FCV. Either humoral immunity or cell-mediated immunity or both may be induced. The immunogenic response of an animal to a vaccine composition may be evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain. The protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, temperature, overall physical condition and overall health of the subject. The amount of a vaccine that is therapeutically effective may vary depending on the particular FCV used, or the condition of the cat, and can be determined by a veterinary physician.

"Feline" designates an animal of the Felidae family. This includes cats and wild species such as tigers, cougars, wildcats or ocelots. Preferably, the feline is a cat. By "controlling" a disease or disorder, it is meant monitoring the occurrence and/or evolution of said disease or disorder, in order to avoid its progression. By "treating" a disease or disorder in an animal, it is meant reversing, alleviating or inhibiting the progress of said disease or disorder in said animal. By "preventing" a disease or disorder in an animal, it is meant keeping from occurring, or hindering, defending from, or protecting from the occurrence of said disease or disorder, including a symptom, in said animal.

Preferably, the vaccine composition of the invention comprising at least one feline calicivirus antigen and/or at least one feline leukemia virus antigen and/or at least one feline herpesvirus-1 antigen is used to protect a feline or to provide a protective immunity to a feline against a disease or disorder caused by an infection with a feline calicivirus and/or a feline leukemia virus and/or a feline herpesvirus-1 respectively. Preferably, the vaccine composition of the invention comprises an effective amount of at least one FCV antigen and/or an effective amount of at least one FeLV antigen and/or an effective amount of at least one FHV-1 antigen. Thus, preferably, the invention relates to the use of a vaccine composition comprising an effective amount of at least one feline calicivirus (FCV) antigen and/or an effective amount of at least one FeLV antigen and/or an effective amount of at least one FHV-1 antigen, in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus and/or a feline leukemia virus and/or a feline viral rhinotracheitis virus respectively, in a feline, wherein said vaccine composition is administered in one administration (i.e. one dose) to the feline.

FCV antigen

In a first preferred embodiment, the vaccine composition of the invention comprises at least one FCV antigen. The FCV antigen which may be present in the vaccine composition of the invention may be a FCV strain, a protein of the FCV, for example a capsid protein derived from a FCV strain, or an immunogenic fragment thereof, a virus-like particle, a recombinant virus vector encoding a protein of the FCV such as the FCV capsid protein or a specific immunogenic fragment thereof, or a DNA or RNA molecule encoding a protein of the FCV, such as the FCV derived capsid protein or a specific immunogenic fragment thereof. In a preferred embodiment, the feline calicivirus antigen present in the vaccine composition of the invention is a feline calicivirus strain (FCV strain). The FCV strain may be live attenuated or inactivated. Preferably, the feline calicivirus antigen present in the vaccine composition of the invention is a feline calicivirus strain which is live attenuated.

Preferably, the FCV strain is chosen from FCV strains F9, 431 , G1 , 255, LLK, M8 and 2280. These FCV strains are notably disclosed in "the calicivirus pages" available online from The Pirbright Institute, UK, at the address:

http://www.caliciviridae.com/vesivirus/fcv/fcv seqs.htm.

A preferred FCV strain is the FCV F9 strain available under the accession number ATCC VR-782. The genome of said F9 strain may be found in Genbank under accession number M86379. Said strain is quoted in US3937812. The FCV strain F9 is a live attenuated strain which is present in several commercial vaccines against feline calicivirosis. Said commercial vaccines include for example the vaccine composition Leucofeligen™ FeLV/RCP of Virbac.

Other preferred strains of FCV include the FCV strains 431 and G1 , which may be found at the CNCM (Collection Nationale de Cultures de Microorganismes of the Institut Pasteur, 28, rue du Dr Roux, 75724 Paris Cedex 15, France) respectively under accession numbers 1-2166 and 1-2167, and both deposited on March 12, 1999 under the terms of the Budapest Treaty, as disclosed in US6534066. The genomes of FCV strains 431 and G1 are described in FR2796397. Live attenuated vaccines

The vaccine composition may comprise a live attenuated feline calicivirus strain.

Particularly, the live attenuated FCV F9 strain is preferred for the preparation of a live vaccine which stimulates the feline's immune system without causing disease.

Methods for attenuating the viruses further are well known in the art and include methods such as serial passage in cell culture on a suitable cell line, or ultraviolet or chemical mutagenesis.

Inactivated vaccines

The vaccine composition may comprise an inactivated or killed FCV strain. The inactivated vaccine is made by methods well known in the art. For example, once the virus is propagated to high titers, it would be readily apparent by those skilled in the art that the virus antigenic mass could be obtained by methods well known in the art. For example, the virus antigenic mass may be obtained by dilution, concentration, or extraction. All of these methods have been employed to obtain appropriate viral antigenic mass to produce vaccines. The FCV is inactivated by treatment with formalin, betapropriolactone, or with binary ethyleneimine (BEI), or other methods known to those skilled in the art.

Inactivation by formalin may be performed by mixing the calicivirus suspension with 37% formaldehyde to a final formaldehyde concentration of 0.05%. The calicivirus- formaldehyde mixture is mixed by constant stirring for approximately 24 hours at room temperature. The inactivated calicivirus mixture is then tested for residual live virus by assaying for growth on a suitable feline cell line such as CRFK cells.

Inactivation by BEI is performed by mixing the calicivirus suspension of the present invention with 0.1 M bromoethylamine hydrobromide (i.e. 2-bromo-ethylamine in 0.175 N NaOH) to a final BEI concentration of 1 mM. The calicivirus-BEI mixture is mixed by constant stirring for approximately 48 hours at room temperature, followed by the addition of 1 .0 M sodium thiosulfate to a final concentration of 0.1 mM. Mixing is continued for an additional two hours. The inactivated calicivirus mixture is tested for residual live calicivirus by assaying for growth on a suitable feline cell line such as NLFK cells.

Preferred inactivated strains include the inactivated strains FCV 431 and G1 as described here above, which are present in commercial vaccine compositions such as in Purevax™ RCP vaccine of Merial.

Recombinant vaccines

The vaccine composition may comprise an FCV antigen in the form of a protein of the FCV, such as the capsid protein derived from a FCV strain or an immunogenic fragment thereof.

Preferably, said vaccine comprises a capsid protein of a feline calicivirus.

Examples of capsid proteins include the capsid protein gene of the FCV 255 strain, which may be found in Genbank under accession number U06646.

Other examples of capsid protein genes, such as the ones of the FCV strains LLK and 2280, may respectively be found in Genbank under accession numbers U06649 and X99445. The FCV-2280 strain has been deposited at the A.T.C.C. on Dec. 9, 1982, and given accession Number VR 2057, as disclosed in US4522810.

The capsid protein may also come from strain FCV-M8; said strain and its use as a vaccine are described by Davis and Beckenhauer, VM/SAC (1976) 71 :1405-1410. More preferably, the feline calicivirus antigen present in the vaccine composition of the invention is the live attenuated F9 strain.

As effective amount of at least one FCV antigen, one can quote a dose of from 10³ to 10⁸ cell culture infecting dose, 50% endpoint (CCID₅₀) per ml, particularly when the FCV antigen is a FCV strain.

Preferably, the feline calicivirus antigen is present in the vaccine composition of the invention in an amount of from 10⁴ to 10⁷ cell culture infecting dose, 50% endpoint (CCID₅₀) per ml, even more preferably in an amount of from 10^4,5 to 10^6,5 cell culture infecting dose, 50% endpoint (CCID₅₀) per ml.

Preferably, a vaccine composition according to the invention comprising at least one feline calicivirus antigen is used for active immunisation of the animal against feline calicivirosis to reduce the clinical signs of the feline calicivirus infection. Said vaccine composition comprising at least one feline calicivirus antigen may also be used for active immunisation of the animal against feline calicivirosis to reduce the excretion of the feline calicivirus.

FeLV antigen

In another preferred embodiment, the vaccine composition of the invention comprises at least one FeLV antigen.

The FeLV antigen which may be present in the vaccine composition of the invention may be a FeLV strain, a protein from a FeLV strain (such as an envelope protein derived from a FeLV strain or an immunogenic fragment thereof, a capsid protein derived from a FeLV strain or an immunogenic fragment thereof), a recombinant virus vector encoding a protein from a FeLV strain such as the envelope protein of a FeLV strain the FeLV capsid protein or a specific immunogenic fragment thereof, a virus-like particle, a DNA or RNA molecule encoding a protein from a FeLV strain such as the FeLV derived envelope protein or a specific immunogenic fragment thereof or the FeLV derived capsid protein or a specific immunogenic fragment thereof.

Preferably, the FeLV antigen is a protein, preferably a recombinant protein, of said FeLV. The FeLV genome comprises amongst the gag and po/ genes, the env gene that encodes the envelope protein composed of a surface glycoprotein of 70,000 dalton (gp 70) and a 15,000 dalton protein 1/21 (p15E). gp 70 is essential for binding of the virus to the cellular receptors for FeLV. Preferably, the FeLV antigen is a recombinant antigen derived from the entire gp 70 protein. Preferably, the FeLV antigen is a non-glycosylated version of gp 70 from FeLV subgroup A. Preferably the FeLV antigen is a fusion protein between the entire exterior envelope protein gp 70 and the first 34 amino acids of p15E; such a FeLV antigen is called p45. A preferred p45 protein according to the invention is encoded at least by the nucleic acid sequence of SEQ ID NO:1 . The corresponding amino acid sequence is SEQ

ID NO:2. Thus, a preferred p45 protein according to the invention comprises at least the amino acid sequence of SEQ ID NO:2. Said amino acid sequence may comprise, in N- terminal and/or C-terminal, any additional sequence useful for cloning and/or expression of p45. Notably said additional sequence(s) may be a linker.

SEQ ID NO:1 and 2 are summarized below:

SEQ ID NO:1 :

gccaatcctagtccacaccaaa

tatataatgtaacttgggtaataaccaatgtacaaactaacacccaagctaatgccacctctatgttagg

aaccttaaccgatgcctaccctaccctacatgttgacctatgtgacctagtgggaaacacctgggaacct

atagtcctagatccaaccaatgtaaaacacggggcacgttactcctcctcaaagtatggatgtaaaacta

cagatagaaaaaaacagcaacaaacataccccttttacgtctgccccggacatgccccctcgctggggcc

aaagggaacacactgtggaggggcacaagatgggttttgtgccgcatggggatgtgagaccaccggagaa gcttggtggaagccctcctcctcatgggactatatcacagtaaaaagagggagtagtcaggacaatagct

gtgagggaaaatgcaaccccctgattttgcagttcacccagaagggaagacaagcctcttgggacggacc

taagatgtggggattgcgactataccgtacaggatatgaccctatcgccttattcacggtgtcccggcag

gtgtcaaccattacgccgcctcaggcaatgggacccaacctagtcttacctgatcaaaaacccccatccc

gacaatcccaaacagggtccaaagtggcgacccagaggctccaaacgaatgaaagcgcctcaaggtctgt tgcccccaccaccgtggttcccaaacggattgggaccggagataggttaataaatttagtacaagggaca

tacctagccttaaatgccaccgaccccaacaaaactaaagactgttggctctgcctggtttctcgaccac

cctattacgaagggattgcaatcttaggtaactacagcaaccaaacaaacccccccccatcctgcctatc

tactccgcaacacaaactgaccatatctgaagtatcagggcaaggactgtgcatagggactgttcctaag

acccaccaggctttgtgcaatgagacacaacagggacatacaggggcgcactatctagccgcccccaatg

gcacctattgggcctgtaacactggactcaccccatgcatttccatggcggtgctcaattggacctctga

tttttgtgtcttaatcgaattatggcccagagtgacttaccatcaacccgaatatgtgtacacacatttt

gccaaagctgtcaggttccgaagagaaccaatatcactaactgttgccctcatgttgggaggactcactg

tagggggcatagccgcgggggtcggaacagggactaaagccctccttgaaacagcc SEQ ID NO:2:

ANPSPHQIYNVTWVITNVQTNTQANATSMLGTLTDAYPTLHVDLCDLVGNTWEPIVLDPT NVKHGARYSSSKYGCKTTDRKKQQQTYPFYVCPGHAPSLGPKGTHCGGAQDGFCAAW GCETTGEAWWKPSSSWDYITVKRGSSQDNSCEGKCNPLILQFTQKGRQASWDGPKM WGLRLYRTGYDPIALFTVSRQVSTITPPQAMGPNLVLPDQKPPSRQSQTGSKVATQRLQ TNESASRSVAPTTVVPKRIGTGDRLINLVQGTYLALNATDPNKTKDCWLCLVSRPPYYEG IAILGNYSNQTNPPPSCLSTPQHKLTISEVSGQGLCIGTVPKTHQALCNETQQGHTGAHY LAAPNGTYWACNTGLTPCISMAVLNWTSDFCVLIELWPRVTYHQPEYVYTHFAKAVRFR REPISLTVALMLGGLTVGGIAAGVGTGTKALLETA Preferably the FeLV antigen is expressed in bacteria, preferably Escherichia coli. Said expression may be performed in inclusion bodies before extraction and purification. Preferably the antigen is adsorbed on an adjuvant (see below), more preferably chosen from aluminium hydroxide, extracts of Quillaja saponaria and mixtures thereof.

As effective amount of at least one FeLV recombinant antigen, one can quote a dose of at least 10 μg per ml, preferably at least 30 μg per ml and even more preferably at least 80μg per ml. An even most preferred dose for a FeLV recombinant antigen is comprised between 80 to 120 μg, for example 102 μg per ml.

In another preferred embodiment, said FeLV antigen is a FeLV strain, which may be killed, live attenuated or inactivated.

Another preferred FeLV antigen is a FeLV recombinant canarypox virus.

As effective amount of at least one FeLV antigen in the form of a strain or recombinant canarypox virus, one can quote a dose of from 10³ to 10^s cell culture infecting dose, 50% endpoint (CCID₅₀) per ml, preferably a dose from 10⁴ to 10^7,5 cell culture infecting dose, 50% endpoint (CCID₅₀) per ml.

Preferably, a vaccine composition according to the invention comprising at least one feline leukemia virus antigen is used for active immunisation of the animal against feline leukemia to prevent persistent viraemia. Said vaccine composition comprising at least one feline leukemia virus antigen may also be used for active immunisation of the animal against feline leukemia to reduce the clinical signs of the related disease.

FHV-1 (or FVR) antigen

Preferably, the vaccine composition of the invention comprises at least one FVR antigen. Feline rhinotracheitis virus (FVR) and feline herpesvirus-1 (FHV-1 ) may be used interchangeably.

The FVR antigen which may be present in the vaccine composition of the invention may be a FVR strain, a protein of the FVR such as a capsid protein derived from a FVR strain or an immunogenic fragment thereof, a recombinant virus vector encoding a protein of the FVR such as the FVR capsid protein or a specific immunogenic fragment thereof, a viruslike particle, or a DNA or RNA molecule encoding a protein of the FVR such as the FVR derived capsid protein or a specific immunogenic fragment thereof. Preferably, the FVR antigen is a FVR strain. The FVR strain may be live attenuated or inactivated. Inactivated strains may be prepared as described above for the FCV strains. Preferably, the FVR antigen present in the vaccine composition of the invention is a FVR strain which is live attenuated.

Preferably the FVR strain is the F2 strain, preferably live attenuated. Preferably, the live attenuated FVR strain F2 is the F2 feline herpesvirus-1 strain which was isolated in 1958 from ocular secretions of a cat with clinical disease by BITLE J.L. and al., Feline Practice, (1975), 5, 13-15. 89 passages were carried out on primary feline kidney cells, with an additional passage for the master strain establishment.

As effective amount of at least one FVR antigen, one can quote a dose of from 10³ to 10⁸ cell culture infecting dose, 50% endpoint (CCID₅₀) per ml, particularly when the FVR antigen is a FVR strain.

Preferably, the FVR antigen is present in the vaccine composition of the invention in an amount of from 10⁴ to 10⁷ cell culture infecting dose, 50% endpoint (CCID₅₀) per ml, even more preferably in an amount of from 10⁵ to 10⁷ cell culture infecting dose, 50% endpoint (CCIDso) per ml.

Preferably, a vaccine composition according to the invention comprising at least one feline herpesvirus-1 antigen is used for active immunisation of the animal against feline rhinotracheitis to reduce the clinical signs of the related disease. Said vaccine composition comprising at least one feline herpesvirus-1 antigen may also be used for active immunisation of the animal against feline rhinotracheitis to reduce the viral excretion. In a preferred embodiment, the vaccine composition according to the invention comprises at least one feline calicivirus antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus in a feline, wherein said vaccine composition is administered in one single administration to the feline.

In another preferred embodiment, the vaccine composition according to the invention comprises at least one feline leukemia virus antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline leukemia virus in a feline, wherein said vaccine composition is administered in one single administration to the feline.

In another preferred embodiment, the vaccine composition according to the invention comprises at least one feline herpesvirus-1 antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline herpesvirus-1 in a feline, wherein said vaccine composition is administered in one single administration to the feline. In another preferred embodiment, the vaccine composition according to the invention comprises at least one feline calicivirus antigen and at least one feline leukemia virus antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus and a feline leukemia virus respectively, in a feline, wherein said vaccine composition is administered in one single administration to the feline.

In another preferred embodiment, the vaccine composition according to the invention comprises at least one feline calicivirus antigen and at least one feline herpesvirus-1 antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus and a feline herpesvirus-1 respectively, in a feline, wherein said vaccine composition is administered in one single administration to the feline.

In another preferred embodiment, the vaccine composition according to the invention comprises at least one feline leukemia virus antigen and at least one feline herpesvirus-1 antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline leukemia virus and a feline herpesvirus-1 respectively, in a feline, wherein said vaccine composition is administered in one single administration to the feline.

In another preferred embodiment, the vaccine composition according to the invention comprises at least one feline calicivirus antigen, at least one feline leukemia virus antigen and at least one feline herpesvirus-1 antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus, a feline leukemia virus and a feline herpesvirus-1 respectively, in a feline, wherein said vaccine composition is administered in one single administration to the feline. In another preferred embodiment, the vaccine composition according to the invention comprises at least one feline calicivirus antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus in a feline, wherein an initial administration of said vaccine composition provides an onset of immunity in the feline.

In another preferred embodiment, the vaccine composition according to the invention comprises at least one feline leukemia virus antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline leukemia virus in a feline, wherein an initial administration of said vaccine composition provides an onset of immunity in the feline.

In another preferred embodiment, the vaccine composition according to the invention comprises at least one feline herpesvirus-1 antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline herpesvirus-1 , in a feline, wherein an initial administration of said vaccine composition provides an onset of immunity in the feline.

In another preferred embodiment, the vaccine composition according to the invention comprises at least one feline calicivirus antigen and at least one feline leukemia virus antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus and a feline leukemia virus respectively, in a feline, wherein an initial administration of said vaccine composition provides an onset of immunity in the feline.

In another preferred embodiment, the vaccine composition according to the invention comprises at least one feline calicivirus antigen and at least one feline herpesvirus-1 antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus and a feline herpesvirus-1 respectively, in a feline, wherein an initial administration of said vaccine composition provides an onset of immunity in the feline.

In another preferred embodiment, the vaccine composition according to the invention comprises at least one feline leukemia virus antigen and at least one feline herpesvirus-1 antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline leukemia virus and a feline herpesvirus-1 respectively, in a feline, wherein an initial administration of said vaccine composition provides an onset of immunity in the feline. In another preferred embodiment, the vaccine composition according to the invention comprises at least one feline calicivirus antigen, at least one feline leukemia virus antigen and at least one feline herpesvirus-1 antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus, a feline leukemia virus and a feline herpesvirus-1 respectively, in a feline, wherein an initial administration of said vaccine composition provides an onset of immunity in the feline.

Preferably, the vaccine composition of the invention comprises:

- the live attenuated FCV strain F9, especially in an amount of 10^{4 6}-10^{6 1} CCID50^* per 1 ml;

- the live attenuated feline herpesvirus-1 strain F2, especially in an amount of 10^{5 0}- 10^{6 6} CCID50^* per l ml; and

- the p45 FeLV-envelope antigen, preferably encoded by SEQ ID NO:2, preferably obtained in recombinant form (such as produced in E.coli), especially in an amount of at least 102 μg/m\.

In an embodiment, the present invention provides a vaccine composition further including a vaccine for immunizing a feline (particularly a cat) against one or more other feline pathogens.

Preferably, said other feline pathogen is selected from feline influenza virus, rabies virus, feline immunodeficiency virus, Chlamydia, Bordetella, feline panleukopenia virus.

More preferably, the vaccine composition of the invention further comprises at least one additional antigen selected from a feline panleukopenia virus antigen, a feline influenza virus antigen, a rabies virus antigen, a Chlamydia antigen such as Chlamydia pssittaci, a Bordetella antigen such as Bordetella bronchiseptica, a feline immunodeficiency virus antigen and their mixtures.

More preferably, the vaccine composition of the invention further comprises the additional antigen which is a feline panleukopenia virus antigen. The feline panleukopenia virus antigen may be strain(s). Preferably the feline panleukopenia virus antigen is the live attenuated LR 72 strain.

More preferably, the vaccine composition of the invention comprises:

- the live attenuated FCV strain F9, especially in an amount of 10⁴

per 1 ml; - the live attenuated feline herpesvirus-1 strain F2, especially in an amount of 10^{5 0}- 10^{6 6} CCID50^* per 1 ml;

- the p45 FeLV-envelope antigen, preferably encoded by SEQ ID NO:2, preferably obtained in recombinant form (such as produced in E.coli), especially in an amount of at least 102 μg/ml; and

- the live attenuated feline panleukopenia virus strain LR 72, especially in an amount of 10^{3 7}-10⁴⁵ CCID50^* per 1 ml/.

The vaccine composition of the invention may additionally comprise or be administered with at least one adjuvant and/or at least one excipient.

The vaccine compositions may include vaccine-compatible pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles or excipients. "Pharmaceutically acceptable" refers to substances, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio, and effective for their intended use. "Excipient" refers to any component of a vaccine that is not an antigen and that is not an adjuvant. Typically, the excipients will be sterile and pyrogen-free. The excipient may be a diluent and/or a stabilizer.

Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, potassium hydroxide, glutamic acid, potassium dihydrogen phosphate, dipotassium phosphate, disodium phosphate, gelatin, dextrose, mannitol, sorbitol, and lactose. Stabilizers include albumin, among others.

The vaccine can further be mixed with an adjuvant that is pharmaceutically acceptable. Any adjuvant known in the art may be used alone or in combination in the vaccine composition, including oil-based adjuvants such as Freund's Complete Adjuvant and Freund's Incomplete Adjuvant, carbomer-based adjuvants, mycolate-based adjuvants (such as trehalose dimycolate), bacterial lipopolysaccharide, peptidoglycans (i.e. mureins, mucopeptides, or glycoproteins such as N-Opaca, muramyl dipeptide or its analogs), proteoglycans (e.g. extracted from Klebsiella pneumoniae), streptococcal preparations (e.g. OK432), alum, aluminium hydroxide, saponin, DEAE-dextran, neutral oils (such as Miglyol), vegetable oils (such as arachid oil), liposomes, Pluronic® polyols, the RIBI adjuvant system (Ribi Inc.), cholesterol, Quillaja saponaria extracts such as QS-21 (Cambridge Biotech Inc., Cambridge MA), other saponins and fractions thereof such as GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), monophosphoryl lipid A or avridine lipid-amine adjuvant.

Preferably the adjuvant is selected in the group consisting of a Quillaja saponaria extract, aluminium hydroxide and their mixtures. Even most preferably, the adjuvant according to the invention comprises aluminium hydroxide gel and a Quillaja saponaria extract.

The amounts and concentrations of adjuvants and excipients useful in the context of the present invention can readily be determined by the skilled artisan. Preferably, when the adjuvant comprises a Quillaja saponaria extract, said extract may be present in an amount of at least 1 μg, preferably from 1 to 100 μg, preferably from 5 to 50 μg. Preferably, when the adjuvant comprises aluminium hydroxide, it may be present in the form of a gel comprising around 3% of aluminium by weight of the total weight of said gel.

The vaccine compositions of the present invention can be made in various forms depending upon the route of administration. For example, the vaccine compositions can be made in the form of sterile aqueous solutions or dispersions suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized vaccine compositions are typically maintained at about 4°C, and can be reconstituted in a stabilizing solution, with or without adjuvant.

The vaccine composition according to the present invention may be administered subcutaneously, intramuscularly, intradermal^, transdermal^, ocularly, mucosally, or orally. Preferably it is administered subcutaneously. The antigens according to the invention may be administered in various formulations. Examples of various formulations include virus-like particles, microspheres and nanospheres.

The present invention also relates to products containing:

a) at least one vaccine composition according to the invention, called vaccine composition A, and

b) at least one vaccine composition B selected from a feline calicivirus vaccine composition, an herpesvirus (for example a rhinotracheitis virus) vaccine composition, a panleukopenia vaccine composition, a feline leukemia vaccine composition, a feline influenza virus composition, a rabies virus vaccine composition, a Chlamydia vaccine composition, a Bordetella vaccine composition and a feline immunodeficiency virus vaccine composition, or combinations thereof,

as a combination preparation for simultaneous, separate or sequential use for controlling and/or preventing and/or treating viral and/or bacterial infections in a feline,

provided that vaccine composition A is different from vaccine composition B,

or provided that vaccine composition A targets a viral infection different from the one of vaccine composition B.

The vaccine composition according to the invention (composition A) comprises at least one FCV antigen and/or at least one FeLV antigen and/or at least one FHV-1 antigen, as described above.

Such products correspond to combinations of the vaccine of the invention with other feline vaccines (i.e. composition B), to produce a polyvalent vaccine product that can protect the feline against a wide variety of diseases caused by other feline pathogens.

Thus, the present invention also relates to products containing:

as a combination preparation for simultaneous, separate or sequential use as polyvalent vaccines in a feline,

provided that vaccine composition A is different from vaccine composition B,

Preferably, the vaccine composition B of the products of the invention comprises at least one live attenuated strain, at least one inactivated strain and/or at least one recombinant vaccine. In a preferred embodiment, the present invention provides a polyvalent vaccine which immunizes a feline against feline calicivirus and at least one other feline pathogen, preferably selected from feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia and feline panleukopenia virus.

The invention will be better understood on reading the following illustrative and non- limiting examples.

In the following examples, reference is made to the following figures:

Figure 1 : Unrooted tree of the partial capsid sequences of 4 feline calicivirus strains (FCV), the FR4 01 challenge strain and the 3 vaccine strains FCV F9 (GenBank accession No. M86379), FCV 431 (disclosed in FR2796397), FCV G1 (disclosed in FR2796397)

This phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program (v3.1/3.0 aLRT). The percentage of replicate trees in which the associated taxa clustered together in bootstrap tests (1000 replicates) is shown newt to the branches. Only bootstrap values >95 are shown. Distances are drawn to scale and relate to the distance bar.

Figure 2: Titration of total IgG and neutralising antibodies directed against the FCV- FR4 01 strain, during the course of the study after two vaccinations at 3 weeks of intervals

Titration of total IgG was assessed by immunofluorescence (IF; A) while neutralising antibodies were assessed by a serum neutralisation (SN) assay (B). Titrations were performed from samples collected on days 0, 21 , 42, 49 (DPC 0) and 63 (DPC 14) in group 1 (Leucofeligen™ FeLV/RCP, green, n = 10), group 2 (Purevax™ RCP FeLV, green, n = 10) and group 3 (control, black, n = 9). Mean values (and SD as error bars) are reported. (A) A value of 0 was attributed to titration < 2.1 log 10 (positive threshold) and 3.9 log 10 was the maximum value which could be measured. In the vaccinated groups, all values from day 21 were significantly different from day 0 and from values in control group (p < 0.05, Tukey adjusted multiple comparisons), a, b and c: statistically significant difference between group 1 (Leucofeligen™ FeLV/RCP) and control (a); between group 2 (Purevax™ RCP FeLV) and control (b); and between group 1 (Leucofeligen™ FeLV/RCP) and group 2 (Purevax™ RCP FeLV) (c). ^* p < 0.05; ^** p < 0.01 ; ^*** p < 0.001 . (B) A value of 0 was attributed to titration < 0.9 log 10 (positive threshold) and 2.56 log 10 was the maximum value. No difference between groups was observed.

Example 1 : Comparative efficacy of the Leucofeliqen™ FeLV/RCP and Purevax™ RCP FeLV vaccines against infection with circulating Feline Calicivirus

Background

Feline calicivirus (FCV) is a common virus, found worldwide, mainly responsible for chronic ulceroproliferative faucitis and periodontitis. This virus has a high mutation rate, leading to the presence of numerous FCV strains in the field. The objectives of this study was to evaluate and compare the efficacy of two vaccines (Leucofeligen™ FeLV/RCP and Purevax™ RCP FeLV), which differ by their nature (live vs. inactivated) and the vaccinal strains, against circulating FCV strains. Thirty 9-week-old Specific Pathogen Free (SPF) kittens were thus randomised into 3 groups and were either not vaccinated (control) or vaccinated (2 injections, 3 weeks apart) with one of the vaccines. Four weeks after the second injection of primary vaccination, the cats were inoculated with a pathogenic strain representative of the ones circulating in Europe (FCV-FR4 01 ) and followed for 2 weeks. Results

After challenge, significant differences (p < 0.05) between control cats and cats vaccinated with Leucofeligen™ FeLV/RCP or Purevax™ RCP FeLV were observed for body weight variation; rectal temperature rise and maximum clinical scores, reflecting the intensity of the signs (83% and 67% lower in the respective vaccinated groups than in the control group). Significant differences were observed between the vaccinated groups, as cats vaccinated with Leucofeligen™ FeLV/RCP had a lower temperature rise (p < 0.05 at DPC 3 to 5) and lower virus shedding titres (p < 0.05 at DPC 8, 9 and 1 1 ) than cats vaccinated with Purevax™ RCP FeLV. Finally, only cats vaccinated with Leucofeligen™ FeLV/RCP had a significantly lower cumulative score, reflecting the intensity and duration of calicivirosis clinical signs, than the control cats (77% lower vs. 62% lower for cats vaccinated with Purevax™ RCP FeLV).

Conclusions

Both vaccines, Leucofeligen™ FeLV/RCP and Purevax™ RCP FeLV, were found to be efficacious in reducing clinical signs induced by FCV-FR4 01 , a FCV strain representative of the circulating ones. However, cats vaccinated with Leucofeligen™ FeLV/RCP were able to control the infection more efficiently than those vaccinated with Purevax™ RCP FeLV, as evidenced by the shorter duration of clinical signs and lower viral titre in excretions. Methods

This controlled, randomised, not blinded, experimental study was carried out under the supervision of the Ethical Committee of Virbac, in accordance with the recommendations issued in the European Pharmacopoeia ("Feline calicivirosis vaccine (live)" European Pharmacopoeia Monograph 04/2013:1 102, p 5350-5351 ).

Animals and Study Protocols. Thirty specific-pathogen free European kittens (9 weeks old; average ± SD body weight of 2 ± 0.25 kg, ranging from 0.87 to 1 .34 kg) obtained from a commercial supplier (Liberty Research, USA), were randomly assigned to 3 groups: group 1 (vaccinated with Leucofeligen™ FeLV/RCP), group 2 (vaccinated with Purevax™ RCP FeLV) and group 3 (not vaccinated). Cats were acclimatised for 7 days to the animal housing conditions (12 h light/dark cycle, 18 ± 3°C, 55 ± 10% humidity with free access to water and commercial dried food daily feeding). During the acclimation and post-challenge phases, a commercial humid food was proposed in addition to ease the acclimation and limit the effect of oral-ulcer-related anorexia.

Cats included in groups 1 and 2 were vaccinated twice, 3 weeks apart (day 0 and day 21 ), by subcutaneous injection according to the recommendations of the manufacturers. Cats in group 3 did not receive any vaccine.

Four weeks after the second injection of primary vaccination, on day 49, equivalent to day post-challenge (DPC) 0, all cats were inoculated with a virulent heterologous strain of calicivirus (FCV-FR4 01 ) and observed for 14 days. All cats of each group were housed together but the groups were separated to avoid contaminations. Vaccines. Leucofeligen™ FeLV/RCP was granted a pan-European marketing authorisation (centralised procedure) in 2009. It is packaged as a freeze-dried fraction containing the live attenuated viruses, that is, FCV (F9 strain), Feline Herpes Virus-1 (FHV-1 ; F2 strain), and Feline Panleukopenia Virus (FPV; LR72 strain), and a liquid fraction containing the recombinant FeLV-envelope antigen p45 (derived from FeLV gp70) with aluminium hydroxide and QA-21 (from Quillaja saponaria) adjuvants. Purevax™ RCP FeLV is presented as a freeze-dried fraction containing inactivated strains of FCV (431 and G1 ) and attenuated strains of FHV (F2) and FPV (PLI IV), and a liquid fraction containing a recombinant virus canarypox FeLV (vCP97).

The vaccine vials were stored between +2°C and +8°C and were reconstituted immediately prior to use by rehydrating the freeze-dried fraction with the liquid fraction. Challenge. The FCV-FR4 01 strain (Genbank accession number MF882991 .1 ) was isolated during the field epidemiology study performed by Hou et al. This strain was chosen for the challenge since it was identified as a single isolate (unique sequence) with a high titration from a single cat presenting various clinical signs typical of a calicivirus infection. The strain was amplified through two passages on CRFK cells and controlled in order to ensure the absence of any interfering pathogens. Regulatory compliant reproducible experimental infections were developed (internal data). The same clinical signs, identical to the ones observed in the original cat, were constantly reproduced: a marked weight loss in the first week, hyperthermia, a depression of the general status and apparition of oral and nasal ulcers, typical of FCV infections. Minor nasal and ocular discharge could also be observed. The FCV-FR4 01 challenge strain was therefore considered as representative of the initial FCV-FR4 01 isolate and thus representative also of the FCV circulating strains.

Further analyses of the sequence identities, performed on partial capsid sequences, showed that the FCV-FR4_01 strain had 73% homology with FCV-F9, 75% with FCV-G1 and 76% with FCV-431 while the three vaccination strains shared between 70% and 74% identities. These levels of homology were in accordance with the homology found between circulating strains (Hou et al). None of the vaccine strains clustered with the FCV-FR4 01 challenge strain (pairwise genetic distance > 20%, Figure 1 ) (Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M, Claverie JM, Gascuel O. Phylogeny.fr: robust phylogenetic analysis for the non- specialist. Nucleic Acids Res. 2008; doi: 10.1093/nar/gkn180; Dereeper A, Audic S, Claverie JM, Blanc G. BLAST-EXPLORER helps you building datasets for phylogenetic analysis. BMC Evol Biol. 2010; doi: 10.1 186/1471 -2148-10-8).

The challenge was performed on anesthetised cats inoculated intranasally with 10^7,5 cell culture infecting dose, 50% endpoint (CCID₅₀) per cat of FCV-FR4 01 suspension using a volume of 0.25 mL/nostril.

Monitoring. During the vaccination phase, cats were checked daily for appearance of any clinical signs. The animals were examined and weighed weekly. In the post-challenge phase, clinical examinations were performed daily, starting with Group 2 then 1 and ending with Group 3 to avoid contamination between groups, and included rectal temperature measurement and observation of clinical signs (general status, nasal and ocular discharge, ulcers) until DPC 14. Weighing was performed twice a week. Scoring. The clinical status was evaluated according to a scoring system based on that specified in the pharmacopoeia monograph ("Feline calicivirosis vaccine (live)" European Pharmacopoeia Monograph 04/2013:1 102, p 5350-5351 ) (see

Table 1 ). A score was given for each parameter, each cat, on every assessment day. For comparison of scores between groups, two methods were used. The first method, the cumulative score, consisted of adding up all daily scores recorded for each cat and for each parameter (cumulative score per cat for parameter x = sum of scores recorded from DPC 0 to DPC 14). The median of the cumulative scores per parameter was then calculated in each group as well as the median of the total cumulative scores recorded per cat, taking into account all parameters.

For the second method, the maximum score recorded per cat and per parameter during the period was taken as a reference to calculate the median maximum scores for each parameter and the total of maximum scores recorded for each cat. The median of the total of maximum scores was used to assess differences in the severity of the clinical picture while the median of the total cumulative scores was used to evaluate the severity and duration of each sign. For both methods, scores for general status, nasal discharge, ocular discharge, ulcers and rectal temperature were included. The score for weight loss (scored once on DPC 7 to evaluate the loss over the first week) was also taken into account for the calculation of the maximum scores.

Table 1 : Scoring system for the different signs observed, based on the European Pharmacopoeia monograph 1 102

Observed signs Description Score

Death 10

Depressed state 2

≥ 39.5°C 1

Temperature

< 37°C 2

Small and few in number 1

Ulcer (nasal or oral)

Large and numerous 3

slight 1

Nasal discharge

copious 2

Ocular discharge 1

Weight loss≥ 3% 2 Efficacy assessment. A vaccine was evaluated according to the European monography; it was judged to be efficacious if the median of the total maximum scores was significantly lower in the vaccinated group than in the control group. Viral shedding. Viral shedding was assessed from nasal washings performed daily from DPC 2. Successive dilutions (10-fold steps) of samples were mixed with plated CRFK cells and incubated for one hour at 37°C before adding more medium. They were then further incubated for 6 days and the cytopathogenic effect of FCV viruses was assessed microscopically. Cats were considered negative when the titre was < 10° ³ DICC₅₀/mL, which was the detection threshold.

Serological Assessments. The serological assessments included evaluation of the titres of total immunoglobulin G (IgG) and of neutralising antibodies (NAb) directed against the FCV-FR4_01 strain. Blood samples were collected on days 0, 21 , 35, 49 (DPC 0), 56 (DPC 7) and 63 (DPC 14).

Total IgG. Titres of IgG reactive to FCV antigens were assessed using an immunofluorescent antibody assay. Briefly, two-fold dilutions of each serum (from 1/8 to 1/8192) were added to a 96-well plate containing acetone-fixed CRFK cells infected with FCV-FR4 01 . Positive and negative sera were diluted the same way and used as controls. They were incubated for 1 hour at 37°C and revealed with a fluorescein- conjugated anti-feline IgG antibody and a solution of Evans Blue. The positivity threshold was 1 /128 (equivalent to a dilution of 10^{2 1}). Neutralising Ab. Titres of NAb were determined by serum neutralisation (SN) of the FCV- FR4 01 strain. Briefly, each serum was diluted to provide six 2-fold dilution steps (1/8 to 1/256). Diluted sera were incubated for 1 hour with FCV-FR4_01 suspension (100 - 502 DICC₅₀/test) to allow viral neutralisation. Each mixture was then added to six 96-well plates containing 70% confluent CRFK cells. After 6 days of incubation, the characteristic cytopathic effect was assessed. The titre was determined by the Spearman and Karber method (Lorenz RJ, Bogel K. Laboratory techniques in rabies: methods of calculation. Monogr Ser World Health Organ. 1973;23:321 -35) and considered negative when inferior to 10° ⁹ which was the detection threshold. Statistical Tests. All statistical analyses were performed using SAS 9.3 software. For the comparison of parameters with repeated measures (antibody titration, rectal temperature, body weight, and viral shedding), a mixed model analysis of variance was used to compare the 3 groups over time. For weight and temperature, the baseline values were used as covariates. In case of significant interaction, adjusted group comparisons (Tukey- Kramer) at each significant time point were performed. Assumptions of normality and homogeneity of variances of residuals were checked. If the Levene's test of homogeneity of variances was significant, an ANOVA model allowing for different variances in each group using Kenward & Roger method was used to compute the denominator degree of freedom for the tests involving fixed effects.

For other parameters (scores, days of hyperthermia, area under the hyperthermia curve and weight change between DPC 0 and DPC 7), a Kruskal-Wallis test was performed to compare groups, followed in case of a significant p-value by a Dunn's test for multiple comparisons. The area under the hyperthermia (≥ 39.5°C) curve was calculated using the trapezoidal rule (using NCSS 2004 software).

A p value < 0.05 was considered significant.

Results

Vaccination phase (day 0 - day 49)

Clinical monitoring and safety

During the vaccination phase, one control cat was found dead while all other cats remained healthy. Weight gain was similar between groups and rectal temperature remained stable in all groups (mean temperatures between 38.5°C and 39°C in all groups). No abnormal general or local reaction was observed with any vaccine. Immune response

The presence of FCV antibodies was assessed using IF or SN against the FCV-FR4 01 challenge strain. Titration of total FCV antibodies (IgGs) assessed by IF showed a detectable seroconversion from day 21 in 10/10 (100%) cats in group 1 (Leucofeligen™ FeLV/RCP) but only in 5/10 (50%) cats in group 2 (Purevax™ RCP FeLV). Mean FCV- FR4 01 IgGs titre was significantly higher in group 1 (Leucofeligen™ FeLV/RCP) than in group 2 (Purevax™ RCP FeLV) three weeks after the first injection of primary vaccination: mean Iog10 (± SD) of 2.9 ± 0.2 vs. 1 .1 ± 1 .1 (p < 0.001 , n = 10 in each group, Figure 2A). Mean titration was similar in both vaccinated groups 3 weeks after the second injection of primary vaccination. All vaccinated cats (20/20) had seroconverted at that time. No antibodies were found in the serum of cats in the control group before inoculating cats with the FCV-FR4 01 strain (n = 10). During the vaccination phase, neutralising antibodies were not present in any group or at a very low level on days 21 and 42 (Figure 2B).

Post-challenge phase (day 49 - day 63 or DPC 0 - DPC 14)

Clinical monitoring

Weight

Before challenge on DPC 0, mean (± SD) body weights were not significantly different between groups: 2.1 1 ± 0.20 kg in the control group; 1 .98 ± 0.25 kg in cats vaccinated with Purevax™ RCP FeLV and 1 .92 ± 0.29 kg in cats vaccinated with Leucofeligen™ FeLV/RC. A decrease in mean body weight was observed during the first week of the challenge phase in the control group but not in the vaccinated groups (data not shown). Indeed, all kittens (9/9) in the control group lost more than 3% of their body weight (a loss considered significant at this age and taken as a reference for severity (Lesbros C, Martin V, Najbar W, Sanquer A, McGahie D, Eun HM, Gueguen S. Protective Efficacy of the Calicivirus Valency of the Leucofeligen™ FeLV/RCP Vaccine against a Virulent Heterologous Challenge in Kittens. Vet Med Int. 2013;2013:232397)) between DPC 0 and DPC 7 compared to only 2 cats in each vaccinated group. Mean weight (± SD) in the control group went from 2.1 1 ± 0.20 kg to 1 .96 ± 0.19 kg and 1 .86 ± 0.21 kg from DPC 0 to DPC 4 (p < 0.001 ) and DPC 7 (p < 0.001 ), respectively, and was of 2.09 ± 0.24 kg at DPC 14 (NS).

Temperature

An increase in rectal temperature was observed in all groups with a peak of hyperthermia (≥ 39.5 °C) on days 3 to 5 after challenge (data not shown). However, compared to DPC 0, cats in the control group had, on average, a higher increase in temperature (p < 0.05) than cats in the vaccinated group 1 (Leucofeligen™ FeLV/RCP) on DPC 2 to 6, and cats in the vaccinated group 2 (Purevax™ RCP FeLV) on DPC 2, 3 and 6 (data not shown). A greater mean increase in rectal temperature was also observed in cats vaccinated with Purevax™ RCP FeLV than in cats vaccinated with Leucofeligen™ FeLV/RCP, on DPC 3, 4 and 5 (p < 0.05, data not shown). The number (%) of cats with severe hyperthermia (> 40 °C) was of 5/10 (50%), 8/10 (80%) and 9/9 (100%) in group 1 , 2 and 3 (control), respectively. Furthermore, cats in the control group remained, on average ± SD, longer in hyperthermia than those in vaccinated groups 1 and 2 (5.2 ± 1 .6 vs. 2.1 ± 1 .3 and 3 ± 1 .4 days, respectively, p < 0.05). Cats vaccinated with Purevax™ RCP FeLV remained longer, on average ± SD, in severe hyperthermia than cats vaccinated with Leucofeligen™ FeLV/RCP (3.2 ± 1 .2 vs. 0.8 ± 1 .0 days, p < 0.05). Overall, by comparing the areas under the hyperthermia curves (> 39.5 °C), the cats in group 1 (Leucofeligen™ FeLV/RCP), unlike those in group 2 (Purevax™ RCP FeLV), responded differently than the cats in the control group (1 .10 ± 1 .01 ; 2.26 ± 1 .37 and 3.77 ± 1 .29 °C.day, mean ± SD respectively, p < 0.05). These data suggest that control cats had a more severe and longer lasting hyperthermia than cats vaccinated with Leucofeligen™ FeLV/RCP.

Clinical signs

Concerning general status, 7/9 (78%) cats in the control group showed apathy or depression while the cats in the vaccinated groups 1 and 2 showed no general signs. Rare, minor and transient ocular and nasal discharges were observed in very few cats, in group 1 (Leucofeligen™ FeLV/RCP, 1 cat with ocular discharge for 1 day) and group 3 (control, 1 cat with ocular discharge and 1 with nasal discharge for 1 day each). Fewer cats vaccinated with Leucofeligen™ FeLV/RCP had buccal and nasal ulcers (3/10: 30%) than cats in the other groups (7/10: 70% of cats vaccinated with Purevax™ RCP FeLV and 7/9: 78% of control cats). Large ulcers (≥ 5 mm) were found in 0/10 (0%), 1/10 (10%) and 2/9 (22%) cats in groups 1 , 2 and 3, respectively. Ulcers lasted for up to 3 days in group 1 but up to 10 days in groups 2 and 3.

Scores

Cumulative clinical scores take into account the scores recorded daily for each parameter (general status, nasal discharge, ocular discharge, ulcers and rectal temperature). The median (inter-quartile range or IQR) of the total cumulative clinical scores, indicative of the clinical severity and duration of signs, was significantly higher in the control group [13.0 (1 1 .0 - 14.0); n = 9] than in the group vaccinated with Leucofeligen™ FeLV/RCP [3.0 (1 .0 - 5.0); n = 10; p < 0.05] but was not significantly different than in the group vaccinated with Purevax™ RCP FeLV [5.0 (5.0 - 8.8); n = 10, data not shown]. Compared to unvaccinated cats, the median of the total cumulative clinical scores was 77% lower in cats vaccinated with Leucofeligen™ FeLV/RCP and was 62% lower in those vaccinated with Purevax™ RCP FeLV.

The median (IQR) of the total maximum scores (all parameters/cat: general status, nasal discharge, ocular discharge, ulcers, rectal temperature and weight) was significantly higher (p < 0.05) in the control group [6.0 (6.0 - 7.0); n = 9] than in the group vaccinated with Leucofeligen™ FeLV/RCP [1 .0 (1 .0 - 1 .8); 83% lower; n = 10] and the group vaccinated with Purevax™ RCP FeLV [2.0 (1 .0 - 3.5); 67% lower; n = 10; data not shown). Viral shedding

A peak of viral shedding was observed in all groups between DPC 2 and DPC 5 (data not shown). However, viral elimination seemed to occur earlier and faster in some animals vaccinated with Leucofeligen™ FeLV/RCP than in the control group or the group of cats vaccinated with Purevax™ RCP FeLV. Indeed, viral titres were significantly lower in cats vaccinated with Leucofeligen™ FeLV/RCP than in the control group on DPC 7 to 1 1 (p < 0.05, n = 10 and 9, respectively, data not shown) and significantly lower than in group 2 on DPC 8, 9 and 1 1 (p < 0.05, n = 10 in each group, data not shown No statistical differences were observed between groups 2 (Purevax™ RCP FeLV) and 3 (control).

Immune response

As shown in Figure 2, all cats had seroconverted at the end of the study. The titre of total IgG was, however, significantly lower in the control group than in both vaccinated groups (p < 0.01 , Figure 2A).

Both vaccines were deemed efficacious. However, some differences were observed between the cats vaccinated with Purevax™ RCP FeLV and those vaccinated with Leucofeligen™ FeLV/RCP. First, the cats vaccinated with Leucofeligen™ FeLV/RCP seroconverted faster (within the 3 weeks after the first primary injection) than some cats vaccinated with Purevax™ RCP FeLV (two injections of primary vaccination required for seroconversion of all cats) (Figure 2A). The difference in the types of vaccine used, live versus killed, may provide a partial explanation for this result (Saalmuller A. New understanding of immunological mechanisms. Vet Microbiol. 2006;1 17(1 ):32-8; Greene CE, Schultz RD. Immunoprophylaxis. In: Greene CE (Ed). Infectious Diseases of the Dog and Cat. 3^rd ed. Saunders Elsevier, St Louis; 2006. p.1069-1 1 19). For example, live organisms induce a greater up-regulation of activation molecules and granulocyte- macrophage colony-stimulating factors than do killed organisms (Tizard IR. Veterinary Immunolgy: An Introduction. 8th edition. W.B. Saunders Company; 2008 ; 258-259). This difference may explain both the delay in producing IgGs and the requirement for two injections of primary vaccination for the cats vaccinated with Purevax™ RCP FeLV (Saalmuller A. New understanding of immunological mechanisms. Vet Microbiol. 2006;1 17(1 ):32-8; Greene CE, Schultz RD. Immunoprophylaxis. In: Greene CE (Ed). Infectious Diseases of the Dog and Cat. 3^rd ed. Saunders Elsevier, St Louis; 2006. p.1069-1 1 19; Day MJ, Horzinek MC, Schultz RD, Squires RA; Vaccination Guidelines Group (VGG) of the World Small Animal Veterinary Association (WSAVA). WSAVA Guidelines for the vaccination of dogs and cats. J Small Anim Pract. 2016;57(1 ):E1 -E45). On the contrary, the Leucofeligen™ FeLV/RCP vaccine may require only one injection as a primary vaccination since IgGs were detected after the first injection.

This study therefore showed that both vaccines have a good efficacy against circulating strains but that Leucofeligen™ FeLV/RCP may bring a better protection than Purevax™ RCP FeLV against a heterologous strain.

Example 2 : Comparative efficacy of the FCV valence of the Leucofeligen™ FeLV/RCP and Purevax™ RCP FeLV vaccines after one single administration

This trial was performed to compare the efficacy of the calicivirus valence from the Leucofeligen™ FeLV/RCP and Purevax™ RCP FeLV vaccines 3 weeks after one administration in primary vaccination. Methods

Vaccines. As explained in example 1 , Leucofeligen™ FeLV/RCP is packaged as a freeze- dried fraction containing the live attenuated viruses, that is, FCV (F9 strain), Feline Herpes Virus-1 (FHV-1 ; F2 strain), and Feline Panleukopenia Virus (FPV; LR72 strain), and a liquid fraction containing the recombinant FeLV-envelope antigen p45 (derived from FeLV gp70) with aluminium hydroxide and QA-21 (from Quillaja saponaria) adjuvants. Purevax™ RCP FeLV is presented as a freeze-dried fraction containing inactivated strains of FCV (431 and G1 ) and attenuated strains of FHV (F2) and FPV (PLI IV), and a liquid fraction containing a recombinant virus canarypox FeLV (vCP97).

The vaccine vials were stored between +2°C and +8°C and were reconstituted immediately prior to use by rehydrating the freeze-dried fraction with the liquid fraction.

Animals and Study Protocols. Thirty specific-pathogen free European kittens of 9 to 1 1 weeks old were randomly assigned to 3 groups of 10 animals: one control group (Group 3), one vaccinated group with Leucofeligen™ FeLV/RCP (Group 1 ) and one vaccinated group with Purevax™ RCP FeLV (Group 2).

The animals were vaccinated with one subcutaneous injection on day (D) 0, as follows: Group 1 : 10 cats received on DO one dose of Leucofeligen™ FeLV/RCP vaccine.

Group 2: 10 cats received on DO one dose of Purevax™ RCP FeLV vaccine.

Group 3: 10 cats were kept unvaccinated and constituted the control group. A challenge was performed 3 weeks after the vaccination (i.e. on day 21 , equivalent to day post challenge (DPC) 0) with a virulent heterologous FCV strain, and then monitored for 14 days (i.e. DPCO to DPC14). Challenge. The virulent FCV strain was the same as in example 1 , in the "Challenge" paragraph (i.e. FCV-FR4 01 strain of Genbank accession number MF882991 .1 ). More specifically, on DPCO, the challenge was performed on anesthetised cats inoculated intranasally with 10^7,5 cell culture infecting dose, 50% endpoint (CCID₅₀) per cat of FCV- FR4 01 suspension using a volume of 0.25 mL/nostril.

Monitoring. Clinical signs and viral excretion were followed during 14 days post-challenge (i.e. DPCO - DPC14). A weight loss≥ 3% per week is considered as relevant. Normal rectal temperature for the cat ranges from 37.5°C to 39.5°C. Mild hyperthermia is considered for temperature between 39.6°C and 40.0°C and severe beyond 40.0°C.

Scoring. The clinical status used was the same as in example 1 .

Viral shedding. Viral shedding was assessed on all cats of the three groups. Nasal washings were carried out in both nostrils of sedated (DPC3 to DPC13 except on week- ends) or anaesthetised (on DPCO and DPC14) animals for FCV titration. FCV titration was made on CRFK cells. Successive dilutions of sample (10 fold serial dilutions) are inoculated on CRFK cells. After one hour at 35 - 37°C, 5% C02, medium is added and plates are incubated during 6 days at 35 - 37°C, 5% C02. Then, direct reading of cytopathogenic effect (CPE) due to FCV is done on inversed microscopy. The titre is calculated by Spearman and Karber method. Cats were considered as negative when the titre was inferior to 10° ³ CCID₅₀/ml (positivity threshold).

Detection of FCV virus by PCR. One control animal presented abnormal FCV serological results in regard to the SPF status of the cats during the vaccinal phase. FCV was therefore sought by PCR in all nasal washings collected on D21 in the control group.

Serological Assessments. Blood samples were taken on DO, D6, D14, D21 (=DPC0) and DPC14. The serological assessment included evaluation of the titres of total immunoglobulin G (IgG) and of neutralizing antibodies (Nab) directed against the two following FCV strains: • the FCV-F9 strain (strain contained in the Leucofeligen™ FeLV/RCP vaccine) in the samples collected from DO to D21 in the three groups to perform homologous serological assessments;

• the FCV-FR4 strain, different from the strains contained in vaccines and homologous to the challenge strain, on the samples collected from DO to D21 and on DPC14 in the three groups to measure the corresponding anti-FCV antibody response.

Neutralizing Ab. A determined quantity of FCV virus (100 to 502 CCID50/test) is added to successive dilutions of serum to be tested (two fold serial dilutions: 1/8th to 1 /256th). After one step of neutralisation, consisting in incubating the mixture virus-serum for 1 hour at 35-37°C, the mixture is then inoculated on CRFK cells to titrate the residual virus. After a 6-day incubation, the cytopathogenic effect (CPE) induced by residual infectious virus particles (not neutralised by the antibodies) is observed by direct reading of the plates. The titre is calculated according to the Spearman and Karber method to determine the endpoint dilution, at which cytopathogenic effect induced by the virus is reduced by half. Cats were considered as negative when the titre was inferior to 10° ⁹ (positivity threshold). For calculation purposes, titres were transformed in Log10 units. Means and standard deviations were calculated on transformed values. Values below the positive threshold were considered as nil.

Total IgG. Titration of antibodies against FCV by immunofluorescence test (IFA test) A suspension of CRFK cells (1 x106 cells/ml) is mixed with a FCV suspension (5x10³ CCID50/ml) V/V, and added in each well of a 96-well microtitration plate. After 16/18-hour incubation at 35-37°C, non-specific sites are blocked with PBS-milk 5% for 1 hour at 35- 37°C. Then, successive dilutions of serum to be tested (two fold serial dilutions in PBS milk 5%: 1 /8th to 1 /8192th) are added to the infected cells. After one-hour incubation at 35-37°C, the antibodies specifically bound are revealed by immunofluorescence using a specific antibody. The titre is equal to the last dilution showing fluorescence. Cats were considered as negative when the titre was inferior to 1 /128 (positivity threshold). For calculation purposes, titres were transformed in Log10 units. Means and standard deviations were calculated on transformed values. Values below the positive threshold were considered as nil. Statistical tests. For the comparison of repeated measures parameters (rectal temperature, body weight, and viral excretion), a mixed model analysis of variance was used to compare the 3 groups over time. For weight and temperature, the baseline values were used as covariates. In case of significant interaction, adjusted group's comparisons (Tu key- Kramer) at each significant time point were performed. Assumptions of normality and homogeneity of variances of residuals were checked. If the Levene's test of homogeneity of variances was significant, an ANOVA model allowing for different variances in each group using Kenward & Roger method was used. For the other parameters (maximal scores and area under the viral excretion curve), a Kruskal-Wallis test followed in case of significant p-value by Dunn's test for pairwise comparisons was performed to compare groups. Area under the viral excretion curve was calculated using the trapezoidal rule (using NCSS 2004 software). A p value < 0.05 was considered as significant.

Results Vaccination phase (day 0 - day 21)

Clinical monitoring

The growth was normal, there was no abnormal clinical sign. Immune response

All animals were free from FCV antibodies before vaccination (on DO). All control cats (group 3) remained seronegative against FCV all along the vaccination phase.

Antibodies against FCV-F9 strain (strain used in Leucofeligen™ FeL V/RCP vaccine): On DO, before vaccination, all cats were free from antibodies against FCV-F9 except for one control animal, tested positive on DO, D6 and D14. Considering the negative results for PCR detection of FCV performed on D21 on the control cats, this data was considered as an aberrant result. All other control cats remained negative during the vaccination phase. Total IgG directed against FCV-F9:

In group 1 (Leucofeligen™ FeLV/RCP), all animals seroconverted to FCV-F9 after the single injection on DO - 5/10 cats from D14 and 100% of the cats on D21 . No anti-FCV-F9 response was observed in group 2.

FCV-F9 neutralising antibodies As observed for total antibodies measured by immunofluorescence, all cats of group 1 (Leucofeligen™ FeLV/RCP) developed an FCV-F9 seroneutralising response after the injection: 4/10 from D6, and 10/10 on D14 with high titres maintained until D21 . In group 2, a weak and transitory seroneutralising anti-FCV-F9 response was detected in two cases, with a titer equal to positive threshold on D6 for one cat and on D14 for another cat.

Antibodies against FCV-FR4 strain (challenge strain, heterologous to the FCV strains of the two tested vaccines):

On DO, all cats were free from antibodies against FCV-FR4 and all control cats remained negative during the vaccination phase.

Total IgG directed against FCV-FR4:

In group 1 (Leucofeligen™ FeLV/RCP), all animals except two seroconverted to FCV-FR4 after the vaccine injection - 4/8 cats from D14 and the 4 other responding cats on D21 . No anti-FCV-FR4 response was observed in group 2.

FCV-FR4 neutralising antibodies:

A moderate seroconversion to FCV-FR4 was observed in four cats from group 1 (Leucofeligen™ FeLV/RCP), including one cat with a transitory positive titre on D14 only. The three other cats were tested positive on D14 and D21 . No neutralising response to FCV-FR4 was observed in group 2.

Post-challenge phase (DPC0 - DPC14)

Clinical monitoring

Weight

An important weight loss was observed over the first week post inoculation in 10/10 cats of the control group and 5/10 cats of group 2. The range of individual body weight variation was -3.2% to -12.6% and -3.5% to -17.7% respectively. For both groups, mean bodyweight increased form DPC 5 to DPC 14.

In contrast, no meaningful individual weight loss was observed in the group of cats vaccinated with Leucofeligen FeLV/RCP. In this group, all cats (10/10) showed a normal growth during the post-challenge period.

The statistical analysis revealed that the body weight of the cats vaccinated with Leucofeligen™ FeLV/RCP was significantly higher than the bodyweight of the cats of group 2 and of the control group at each time point (DPC5, DPC7 and DPC14). Temperature

After challenge, mild and severe hyperthermia were observed in 10/10 cats of the control group from DPC2 to DPC7. A severe hyperthermia lasting from 2 to 4 days (consecutive or not) was reported for 10/10 non-vaccinated cats. After DPC7, the temperature values were within the normal range for 9/10 cats, but just above the upper threshold for the remaining 1/10 cat. In this cat, the rectal temperature was 39.6°C on DPC9.

Mild to severe hyperthermia was also recorded from DPC2 to DPC5 in 10/10 cats of group 2. Severe hyperthermia lasting from 1 to 2 days (consecutive or not) was recorded in 8/10 animals. Hyperthermia was mild in the 2 remaining cats. After DPC5, rectal temperatures were within the normal range for 9/10 cats. A mild hyperthermia of 39.7°C was recorded for the remaining cat on DPC9.

In group 1 , individual mild to severe hyperthermia was observed from DPC3 to DPC5 in five cats out of 10. Three of them presented a severe hyperthermia during 24h. After DPC5, the individual rectal temperature recorded were within in the normal range for 10/10 cats.

The rectal temperatures were significantly higher in control group than in vaccinated groups:

- from DPC3 to DPC6 for group 1 (group of cats vaccinated with Leucofeligen™ FeLV/RCP) and

- on DPC3, DPC5 and DPC6 for group 2.

Rectal temperatures were also significantly higher in group 2 than in group 1 (Leucofeligen FeLV/RCP) on DPC3 and DPC4.

Clinical signs

A mild to moderate alteration of general health was observed in all (10/10) control cats between DPC 3 and DPC9. Buccal ulceration(s), typical of feline calicivirus infection, were reported in 6/10 cats (Table 2). The ulcers were associated with a lack of vitality developing into apathy during 2 to 3 days from DPC3 to DPC5, and in 4/6 cases with slight to established dehydration during 2 to 4 days from DPC3 to DPC9. Ptyalism was also observed once on DPC5 in 1 /6 cat. One cat showed transitory slight and mucous nasal discharge during 24h, on DPC3. The 4/10 other cats only showed lack of vitality and apathy (or depression in one case) during 2 to 3 days, associated in 2/4 cases with slight to established dehydration during 2 to 6 days. In group 2, a mild to moderate deterioration of the general condition was observed in 10/10 cats between DPC3 and DPC9, associated in 5/10 cases with ulceration(s), oral and/or nasal, related to calicivirus infection (Table 2). In the 5 cats with ulcers, a slight to established dehydration during 1 to 3 days from DPC3 to DPC9, and lack of vitality developing into apathy during 2 to 3 days from DPC3 to DPC5 were also reported.

The five other cats without ulcers showed a lack of vitality and apathy during 2 to 4 days, associated in three cases with slight to established dehydration during 1 to 5 days.

In group 1 (cats vaccinated with Leucofeligen™ FeLV/RCP), Oral or nasal ulcers were recorded in 5/10 cats (Table 2). These ulcers were associated with ptyalism on DPC5 and DPC6 for one cat, apathy on DPC5 for another cat and glossitis on DPC8 for a last cat. A lack of vitality on DPC3 was reported for the only 1 /5 cat presenting a nasal ulcer.

Table 2 : Ulcers in Groups 1 to 3

Small ulcers are < 5 mm. Scores

The maximal scores of group 1 (Leucofeligen™ FeLV/RCP) were significantly lower than those of the group 3 (control). The statistical analysis did not demonstrate any significant difference however between group 2 (Purevax™ RCP FeLV) and group 3 (control) (figure not shown). Viral shedding

All cats were negative before FCV inoculation.

All 30/30 cats excreted the virus from DPC3 and the peak of viral excretion was detected between DPC3 and DPC7. Titres of excreted FCV then decreased until nil values for most of the cats, particularly in the vaccinated groups.

However, the mean titre for viral excretion were significantly lower in group 1 , ie for the cats vaccinated with Leucofeligen FeLV/RCP than in control group on DPC10, DPC1 1 , DPC13 and DPC14. Immune response

The FCV-FR4 experimental infection induced a seroconversion against FCV-FR4 (total and seroneutralising) in all animals negative on DPC0: control cats, vaccinated cats of group 2, and respectively two and seven cats from group 1 (Leucofeligen FeLV/RCP) for total and neutralising antibodies. Vaccinated animals responded with higher titres than control animals.

A strong boost of total and neutralising antibody titres against FCV-FR4 was observed in the animals of that had serologically responded to vaccination with Leucofeligen FeLV/RCP (group 1 ), i.e. 8/10 cats for total antibodies and 3/10 cats for neutralising antibodies.

On DPC14, maximum titres for total antibodies against FCV-FR4 were obtained for respectively nine cats out of 10 in group 1 and 5/10 cats in group 2.

Conclusion

Leucofeligen™ FeLV/RCP vaccine administered as a single dose in 9/1 1 week-old cat significantly reduced both clinical signs relative to calicivirosis and viral excretion compared with the control group. Moreover, the maximal scores for the cats vaccinated with Leucofeligen™ FeLV/RCP were significantly lower than those of the non-vaccinated cats.

Similar results were not obtained for group 2.

These results enabled to conclude to the efficacy of protection of the Leucofeligen™ FeLV/RCP vaccine against the calicivirus valence 3 weeks after only one injection in primary vaccination, whereas the Purevax™ RCP FeLV vaccine did not provide such a protection in the same conditions. Example 3 - Efficacy of a vaccine against feline leukaemia (FeLV) after one single administration

The aim of this study was to assess the efficacy of a Leucogen vaccine 3 weeks after one injection of vaccine on kittens, through a virulent challenge.

Leucogen vaccine is a vaccine against FeLV, and comprises a liquid fraction containing the recombinant FeLV-envelope antigen p45 (derived from FeLV gp70) with aluminium hydroxide and a purified Quillaja saponaria extract adjuvants.

The vaccine vials were stored between +2°C and +8°C prior to use.

Animals and study protocol. 25 Serum Pathogenic Free (SPF) kittens of an age comprised between 9 and 10 weeks (males and females) were divided in 2 groups:

Group 1 (Leucogen): 15 cats at 9/10 weeks of age the day of vaccination (DO) received, by subcutaneous route, one dose of Leucogen vaccine.

Group 2 (control): 10 cats at 9/10 weeks of age did not receive any vaccine and were kept as controls.

Challenge phase. Three weeks after the vaccine injection (D21 =W0), cats were challenged via oronasal route with a virulent FeLV challenge strain FeLVA.13.04.

The origin of FeLVA.13.04 strain comes from Fea cells infected with FeLV-A, provided by Cambridge Biosciences Corporation and received in 1988. They were passaged ten times in 2013. The cell culture supernatant from the last passage was harvested and aliquoted constituting FeLVA.13.04. They are stored in 10 % FCSi - DMEM medium.

During the challenge phase, after clinical examination, weighing and blood sampling, the cats were anesthetised. Before each inoculation, the suspension was mixed. 0.25 ml of the suspension was injected slowly in each nostril and 0.5 ml of the suspension was administered at the back of the throat or at the base of the tongue.

In total, each cat will receive 1 ml of suspension at 1 .10⁶ PFU/ml. All the cats were then followed during 15 weeks (W15) after challenge strain inoculation.

The vaccine efficacy was assessed through the antigenaemia follow-up during the challenge phase (on W0 and weekly from W3 to W15). Said follow-up included clinical examination of the animals, clinical observation of the animals (behavior), and weighing of the animals. Said follow-up further included a weekly blood testing to determine the antigenaemia. Results

The number of animals which where persistently infected by the virus was calculated according to the result of the antigenaemia test P27. A cat is considered persistently infected if it shows positive viraemia or antigenaemia for 3 consecutive weeks or on 5 occasions, consecutively or not, between the 3rd and the 15th week.

Among Group 1 (vaccinated animals), only 4 out of 15 animals were identified as persistently infected by the FeLV (27%). 9 animals showed no persistent infection (73%). Among Group 2 (control animals), 9 out of 10 animals were persistently infected with the virus (90%).

Conclusions

3 weeks after the vaccination with a unique administration, the Leucogen vaccine enabled to protect 73% of the animals after an experimental infection without requiring a second injection of the vaccine.

These results enabled to conclude to the efficacy of protection of the Leucogen vaccine against the FeLV valence 3 weeks after only one injection in primary vaccination.

Example 4 - Efficacy of a vaccine against feline rhinotracheitis after one single administration

As for example 3, the aim of this study is to assess the efficacy of a vaccine against FVR 3 weeks after one injection of vaccine on kittens, through a virulent challenge. Animals and study protocol. Cats between approximately between 9 and 10 weeks old kittens (males and females) will be divided in 2 groups:

Group 1 : cats at approximately 9/10 weeks of age the day of vaccination (DO) will receive, by subcutaneous route, one dose of FVR vaccine. Said vaccine comprises the live attenuated F2 strain.

Group 2 (control): cats at approximately 9/10 weeks of age will not receive any vaccine and be kept as controls.

Challenge phase. Three weeks after the vaccine injection (D21 =W0), cats will be challenged via oronasal route or intranasal route with a virulent FVR challenge strain. During the challenge phase, after clinical examination, weighing and blood sampling, the cats will be anesthetised. Before each inoculation, the suspension will be mixed and administered to the animals of Group 1 .

All the cats will then be followed up during few weeks after challenge strain inoculation. The vaccine efficacy will be assessed through the antigenaemia follow-up during the challenge phase.

Claims

1 . A vaccine composition comprising at least one feline calicivirus antigen and/or at least one feline leukemia virus antigen and/or at least one feline herpesvirus-1 antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus and/or a feline leukemia virus and/or a feline herpesvirus-1 respectively, in a feline, wherein said vaccine composition is administered in one single administration to the feline.

2. A vaccine composition comprising at least one feline calicivirus antigen and/or at least one feline leukemia virus antigen and/or at least one feline herpesvirus-1 antigen for use in the control and/or the treatment and/or the prevention of a disease or disorder caused by an infection with a feline calicivirus and/or a feline leukemia virus and/or a feline herpesvirus-1 respectively, in a feline, wherein an initial administration of said vaccine composition provides an onset of immunity in the feline.

3. The vaccine composition for use according to claim 2, wherein the onset of immunity arises from 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 days after the initial administration of said vaccine composition.

4. The vaccine composition for use according to any one of claims 2 to 3, wherein the initial administration consists of a single administration, and excludes any second and/or further administration.

5. The vaccine composition for use according any one of the preceding claims, wherein said feline calicivirus antigen is a feline calicivirus strain.

6. The vaccine composition for use according to claim 5, wherein said feline calicivirus strain is a live attenuated strain or an inactivated strain, preferably a live attenuated feline calicivirus strain, more preferably the live attenuated F9 strain.

7. The vaccine composition for use according to any one of claims 1 to 6, wherein said feline leukemia virus (FeLV) antigen is the gp70 of the FeLV-envelope protein, preferably protein p45.

8. The vaccine composition for use according to any one of claims 1 to 7, wherein said feline herpesvirus-1 antigen is a feline herpesvirus-1 strain, preferably a live attenuated strain or an inactivated strain, preferably a live attenuated feline herpesvirus-1 strain, more preferably the live attenuated F2 strain.

9. The vaccine composition for use according to any one of the preceding claims, wherein each of said feline calicivirus antigen and/or said feline herpesvirus-1 antigen is present in an amount of from 10³ to 10⁸ cell culture infecting dose, 50% endpoint (CCID₅₀) per ml, and/or said feline leukemia virus antigen is present in an amount of at least 10 μg/ml.

10. The vaccine composition for use according to any one of the preceding claims, wherein it is administered subcutaneously, intramuscularly, intradermal^, transdermal^, ocularly, mucosally or orally.

1 1 . The vaccine composition for use according to any one of the preceding claims, wherein it is administered subcutaneously.

12. The vaccine composition for use according to any one of the preceding claims, wherein it further comprises at least one adjuvant and/or at least one excipient, preferably wherein said adjuvant is chosen from a Quillaja saponaria extract, aluminium hydroxide and their mixtures.

13. The vaccine composition for use according to any one of the preceding claims, wherein it further comprises at least one additional antigen selected from a feline panleukopenia virus antigen, a feline influenza virus antigen, a rabies virus antigen, a Chlamydia antigen, a Bordetella antigen, a feline immunodeficiency virus antigen and their mixtures.

14. Products containing:

a) at least one vaccine composition A according to any one of the preceding claims, and b) at least one vaccine composition B selected from a feline calicivirus vaccine composition, an herpesvirus (for example a rhinotracheitis virus) vaccine composition, a feline leukemia vaccine composition, a panleukopenia vaccine composition, a feline influenza virus composition, a rabies virus vaccine composition, a Chlamydia vaccine composition, a Bordetella vaccine composition and a feline immunodeficiency virus vaccine composition, or combinations thereof, as a combination preparation for simultaneous, separate or sequential use for controlling and/or preventing and/or treating viral and/or bacterial infections in a feline,

provided that vaccine composition A is different from vaccine composition B,

15. The products according to claim 14, wherein the vaccine composition B comprises at least one live attenuated strain, at least one inactivated strain and/or at least one recombinant vaccine.