NZ708320B2

NZ708320B2 - Equine Rhinitis vaccine

Info

Publication number: NZ708320B2
Application number: NZ708320A
Authority: NZ
Inventors: Andres Diazmendez; Phillip Wayne Hayes; Kristina J Hennessy; Laurent Viel
Original assignee: Boehringer Ingelheim Animal Health Usa Inc
Priority date: 2011-03-14
Filing date: 2012-03-12
Publication date: 2016-11-29

Abstract

immunogenic composition comprising one or more strains of inactivated Equine Rhinitis B Virus (ERBV) and a method for producing said immunogenic composition.

Description

Eguine Rhinitis Vaccine Seguence Listing The instant application contains a Sequence g which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 26, 2012, is named 10PCT-SEQ.txt and is 33,877 bytes in size. ound of the Invention Equine viral respiratory infections are commonly associated with movement of horses and ly respiratory outbreaks are reported throughout the world. Equine Inﬂuenza 2 (AE2—H3N8), Equine Herpesvirus 1 and 4 (EHV1/4), and Equine is A and B Viruses (ERAV and ERBV, tively) are among the most ant viruses isolated from the upper respiratory tract during respiratory outbreaks.

In particular, ERAV has been reported in acute febrile respiratory disease in horses (Li et al., J. Clin. Microbiol. 35:937—943; 1997, incorporated by reference). Similarly, a recent study in Ontario found ERBV and ERAV to be highly prevalent in the horse population (Diaz— Mendez et al., The an Journal of Veterinary Research 74:271—278; 2010, incorporated by reference). Clinical signs of ERAV infection are non-specific and difficult to differentiate from other atory viral ions, ing equine inﬂuenza and herpes virus infections. Non— cytopathic strains of this virus have been identified in equine respiratory outbreaks (Li et al., J.

Clin. Microbiol. 35:937—943; 1997) making its diagnosis challenging. er, ERAV and ERBV, being single stranded RNA viruses, have the potential for mutation, rendering the ability of the immune system to protect an animal against disease caused by a given ERAV/ERBV strain unclear.

A need exists for methods and medicaments for preventing respiratory diseases or for reducing the incidence or lessening the severity of clinical symptoms associated with such diseases, including those associated with Equine Rhinitis A and B Viruses.

Brief Summary of the Invention The ors have determined that immunogenic compositions comprising one or more strains of inactivated or live, attenuated ERAV, which when live and active and unattenuated are virulent (i.e., at least 50%, 60%, 70%, 80%, 90% or even 100% of seronegative horses, when purposely exposed to the virus, present with observable respiratory disease, particularly nasal and/or ocular discharge), can be grown to high titers in culture to yield a vaccine that is able to induce high titers of serum dies against ERAV when administered, for example, to an equine, for example resulting in a serum titer of at least 1:112, and preferably, at least 1:200, 1:500, 1:750 or 1:1000. In addition, the strains grow well in culture and are highly efficient to produce, for example, to a titer of at least 106 TCID50/mL, more ably at least 107 TCID50/mL, 108 TCID50/mL, or even 109 TCID50/mL. Similarly, compositions comprising one or more strains of inactivated ERBV, which when live and active and not attenuated are also virulent (as defined above for ERAV), were also found to grow well in culture, to a titer of at least 106 TCID50/mL, more preferably 107 TCID50/mL, 108 TCID50/mL or even 109 TCID50/mL, to be highly efficient to produce, and to induce high titers of serum antibodies in animals following immunization, for e, resulting in a serum titer of at least 1:120 and preferably at least 1:200, 1:500, 1:750 or 1:1000. Both the ERAV and ERBV compositions, and combinations thereof, are e of reducing the duration, severity, and incidence of disease in an animal such as a horse that has been immunized with the compositions and subsequently challenged.

Accordingly, the present invention provides an immunogenic ition comprising one or more strains of inactivated or live, attenuated ERAV or ERBV, wherein the ERAV strain or the ERBV strain, prior to inactivation or attenuation, causes able respiratory disease in at least 50% of gative horses exposed to the strain, or grows in cell culture to 106 TCID50/mL or higher, or, when used as a vaccine in equines at a dose of 106 TCID50 or higher results in a serum titer of at least 1:112. [0005a] According to a first aspect of the present invention there is provided an immunogenic ition comprising one or more s of vated Equine Rhinitis B Virus (ERBV). [0005b] According to a second aspect of the present invention there is provided use of an genic composition according to any one of claims 1 to 9 for the manufacture of a (11178654_1):JJP medicament for reducing the incidence or lessening the severity of clinical symptoms associated with or caused by ERBV in an animal or a herd of s. [0005c] According to a third aspect of the t invention there is provided a method for reducing the incidence or lessening the severity of clinical symptoms associated with or caused by ERBV in an animal or a herd of animals, said method comprising the step of administering an immunogenic composition according to the first aspect to an animal in need thereof, wherein the animal is a non-human animal. [0005d] According to a fourth aspect of the present invention there is provided a method for producing an immunogenic composition comprising one or more strains of inactivated ERBV, the method comprising: a) infecting a susceptible cell line with ERBV; b) growing the infected cell line in growth media until a cytopathic effect (CPE) is attained; c) harvesting the media; d) filtering the media to yield a filtered media; and e) contacting the filtered media with an inactivating agent to obtain the inactivated ERBV.

In n embodiments, the , when alive and nuated, causes detectable respiratory disease (e.g., detectable ocular and/or nasal discharge) in at least 50%, 60%, 70%, 80%, 90% or even 100% of seronegative horses upon exposure to the strain. (11178654_1):JJP The strain can be grown in cell culture to a titer of at least 106 TCleo/mL, more preferably 107 TCIDso/mL, 108 TCIDso/mL, or even 109 TCID50/mL. Administration of an genic composition containing the strain results in a serum titer of at least 1:120 and preferably at least 1:200, 1:500, 1:750 or 1:1000.

In the immunogenic compositions of the invention, the one or more strains of ERAV or ERBV preferably include ERAV/ON/OS (ATCC Accession No. PTA-11828) and/or ERBV strain 07-103042 (ATCC ion PTA-11829). In addition, the immunogenic compositions of the invention contain at least an ERAV strain which comprises a genomic sequence whose reverse transcript has greater than 95% identity to SEQ ID NO: 2 or encodes a polyprotein with an amino acid sequence with greater than 95% identity to SEQ ID NO: 3, wherein said ERAV strain, when not inactivated, is active to infect and ate in host cells.

The immunogenic composition of the ion may also include an ERAV strain which comprises a genomic sequence whose reverse transcript has a nucleotide sequence comprising SEQ ID NO: 2 or encodes a polyprotein with an amino acid sequence of SEQ ID NO: 3.

The immunogenic compositions of the invention may also e an ERBV strain which comprises a genomic sequence whose reverse transcript has greater than 95% identity to the reverse transcript of the genomic sequence of the ERBV strain having ATCC Accession no.

PTA—11829 or encodes a polyprotein with an amino acid ce with greater than 95% ty to the polyprotein encoded by the genome of the ERBV strain having ATCC Accession no. 829, wherein said ERBV , when not inactivated, is active to infect and replicate in host cells. The ERBV strain may also comprise a genomic sequence that is the genomic sequence of the ERBV strain having ATCC Accession no. PTA—11829 or encodes a polyprotein that has the amino acid sequence of the polyprotein encoded by the genome of the ERBV strain having ATCC Accession no. PTA-11829.

In a specific embodiment, the immunogenic composition of the invention comprises Equine Rhinitis A Virus (ERAV) and Equine Rhinitis B Virus (ERBV), wherein the ERAV strain is ERAV/ON/05 and the ERBV strain has ATCC Accession No. PTA—11829.

In addition, the invention further es multivalent immunogenic itions sing inactivated or live attenuated viruses or antigens from viruses other than ERAV or ERBV that cause disease in Equidae. In particular, the invention provides immunogenic itions comprising, in addition to inactivated or live, attenuated ERAV and/or ERBV, at least one antigen or one inactivated or live, attenuated strain of Equine Herpes Virus (EHV), and, in particular ments, the EHV is ed from the group consisting of EHV—1 and EHV—4, and a combination thereof, more specifically, the Equine Herpes Virus is selected from the group consisting of EHV-1, EHV-4, strains deposited with the ATCC under accession Nos. 25 and PTA-9526, and a combination thereof.

The invention r provides immunogenic composition, which, in addition to the inactivate or live, attenuated strain of ERAV and/or ERBV, at least one inactivated or live, attenuated strain of or at least one antigen of Equine Inﬂuenza Virus. In specific embodiments, the Equine Inﬂuenza Virus is selected from the group consisting of Clade l viruses, Clade 2 viruses, Inﬂuenza A/South Africa/2003, Inﬂuenza A/equine—Z/Ohio/03, Inﬂuenza A/equine— 2/New Market/2/93 Inﬂuenza Inﬂuenza , A/equine—Z/Kentucky/95 , A/equine— 2/Richmond/1/2007, strains deposited with the ATCC under accession Nos. PTA—9522, PTA— 9523, and PTA—9524, and combinations f. The immunogenic compositions may include, in addition to inactivated or live, ated ERAV and/or ERBV, at least one antigen or one inactivated or live, attenuated strain of Equine Herpes Virus and at least one antigen or one inactivated or live, attenuated strain of Equine Inﬂuenza Virus.

The immunogenic compositions of the invention may include, in addition to inactivated or live, attenuated ERAV and/or ERBV, at least one inactivated or live, attenuated Virus or at least one antigen of one or more strains selected from the group consisting of West Nile Virus, Eastern Equine Encephalomyelitis Virus, n Equine Encephalomyelitis Virus, Venezuelan Equine Encephalomyelitis Virus, and s Toxoid, and combinations thereof.

Alternatively, the immunogenic composition, in addition to vated or live, attenuated ERAV and/or ERBV, comprises one or more vated or live, ated strains of or antigens of strains of Eastern Equine Encephalomyelitis, Western Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis Virus, and Tetanus Toxoid. In specific embodiments, the West Nile Virus is one of the strains selected from the group consisting of Horse Origin 2005, deposited with the ATCC under accession number PTA—9409; NAEE159, deposited at the United States ment of Agriculture Isolate under accession number 405330; NY2002Nassau; NY2002Clinton; NY2002Queens; GA20021; GA20022; TX20021; TX20022; IN2002; NY2003Albany; NY2003Suffolk; NY2003Chatauqua; C020031; C020032; TX2003; TX2003Harris4; TX2003Harris6; TX2003Harris7; Harrile; A22004; and TX2004Harris4; and ation thereof. In immunogenic compositions comprising Western Equine Encephalomyelitis Virus, the strain may be the strain deposited with the ATCC under accession number PTA-9410. In compositions sing Venezuelan Equine Encephalomyelitis Virus, the strain may be the strain deposited with the ATCC under accession number PTA—9411. In immunogenic compositions comprising Eastern Equine Encephalomyelitis Virus, the strain may be the strain deposited with the ATCC under accession number 12. And, in immunogenic compositions comprising Equine Herpes Virus, the strain may be selected from the group consisting of the strains ted with the ATCC under accession Nos. PTA—9525 or PTA—9526, and combinations thereof.

In specific embodiments, one or more of the strains in the immunogenic composition are t in an amount from about 102'0TCID50/mL to about 1010‘0TCID50/mL per dose. The composition may further include a suitable pharmaceutical carrier, such as a diluent, adjuvant, antimicrobial agent, preservative, inactivating agent, or combination thereof. In particular embodiments, the immunogenic composition comprises an adjuvant, specifically, HRA-S.

The invention further provides methods for reducing the incidence or lessening the ty of al symptoms associated with or caused by Equine is A Virus or Equine Rhinitis B Virus in an animal or a herd of animals sing the step of administering an immunogenic composition that comprises one or more strains of inactivated or live, attenuated ERAV or ERBV, wherein the ERAV strain or the ERBV strain, prior to inactivation or attenuation, causes detectable atory disease in at least 50% of seronegative horses exposed to the strain, or grows in cell culture to 106 TCIDsO/mL or higher, or, when used as a vaccine in equines at a dose of 106 TCID50 or higher results in a serum titer of at least 1:112. In particular, the one or more strains of ERAV or ERBV preferably include ERAV/ON/OS (ATCC Accession No. PTA—11828) and/or ERBV strain 071103042 (ATCC Accession PTA—11829). In addition, the ERAV strain may comprise a genomic sequence whose reverse transcript has greater than 95% identity to SEQ ID NO: 2 or encodes a polyprotein with an amino acid sequence with greater than 95% identity to SEQ ID NO: 3, n said ERAV strain, when not inactivated or attenuated, is active to infect and replicate in host cells, or the ERAV strain comprises a genomic sequence whose reverse ript has a tide sequence comprising SEQ ID NO: 2 or encodes a polyprotein with an amino acid sequence of SEQ ID NO: 3. In addition, or alternatively, the ERBV strain may comprise a genomic sequence whose e transcript has greater than 95% identity to the reverse transcript of the genomic ce of the ERBV strain having ATCC Accession no. PTA—11829 or encodes a polyprotein with an amino acid sequence with greater than 95% identity to the polyprotein encoded by the genome of the ERBV strain having ATCC Accession no. PTA—11829, wherein said ERBV strain, when not inactivated or attenuated, is active to infect and replicate in host cells. The ERBV strain may also comprise a genomic sequence that is the genomic sequence of the ERBV strain having ATCC Accession no.

PTA—11829 or encodes a polyprotein that has the amino acid sequence of the otein encoded by the genome of the ERBV strain having ATCC Accession no. PTA—11829.

In on to providing methods for reducing the incidence or lessening the severity of clinical symptoms associated with or caused by ERAV or ERBV in an animal or a herd of animals, the methods of the invention may further reduce the nce or lessening the severity of clinical ms associated with or caused by one or more of the pathogens ed from the group consisting of West Nile Virus, n Equine Encephalomyelitis Virus, Western Equine Encephalomyelitis Virus, Venezuelan Equine Encephalomyelitis Virus, and Clostridium tetani in an animal or a herd of animals by administering an immunogenic composition of the invention. The methods of the invention also include methods of reducing the incidence or lessening the severity of clinical symptoms associated with or caused by ERAV or ERBV in an animal or a herd of animals along with reducing the incidence or lessening the severity of al symptoms associated with or caused by one or more of the pathogens selected from the group consisting of: Eastern Equine Encephalomyelitis Virus, Western Equine Encephalomyelitis Virus, Venezuelan Equine Encephalomyelitis Virus, Equine Herpes Virus, and Clostridium tetani in an animal or a herd of animals by administering an immunogenic composition of the invention.

The invention also provides s for reducing the nce or lessening the severity of al symptoms associated with or caused by ERAV and/or ERBV as well as one or more of the pathogens selected from the group consisting of: West Nile Virus, Eastern Equine Encephalomyelitis Virus, Western Equine Encephalomyelitis Virus, Venezuelan Equine Encephalomyelitis Virus, Equine Herpes Virus, Equine Inﬂuenza Virus, and Clostridium tetani in an animal or a herd of animals, comprising the step of administering an immunogenic composition of the invention.

In connection with the methods of the invention, the incidence of clinical symptoms caused by one or more of said pathogens in a herd of animals is reduced from about 10% — 50% as compared to a herd not receiving the immunogenic composition. The s of the invention, in particular embodiments, e a duration of immunity of at least 12 months against one or more of the pathogens present in the genic composition. In the s of the ion, the immunogenic composition is administered to an Equidae, preferably a horse.

The dosing scheme may include administration of the immunogenic composition in one or more doses. The doses for the methods of the invention may be formulated in 0.5 mL to 2.5 mL dosage forms. Preferably, the methods of the invention administer immunogenic compositions which are safe for use in foals or horses 4 months of age or older.

The invention also es methods for producing an immunogenic composition comprising one or more strains of inactivated Equine Rhinitis A Virus (ERAV) or Equine Rhinitis B Virus (ERBV) as follows: a) infecting a susceptible cell line with ERAV or ERBV; b) growing the infected cell line in growth media until a cytopathic effect (CPE) is attained; c) harvesting the media; d) filtering the media to yield a ed media; and e) contacting the filtered media with an inactivating agent to obtain the inactivated ERAV or ERBV.

Brief Description of the Drawings Fig. 1 is a graphical representation of the proportion of virus positive across time.

Fig. 2 is a graphical representation of the proportion of buffy coat positive across time.

Fig. 3 is a graphical entation of the serum neutralization titers across time.

Fig. 4 is a graphical representation of the mean nasal scores across time.

Fig. 5 is a graphical representation of the mean ocular scores across time.

Fig. 6 is a ClustalW alignment of a portion of equine rhinitis A virus N/05 (5’ UTR portion, SEQ ID NO: 1) with ERAV/PERV—l sion number: DQ272578 (SEQ ID NO: 14)). ERAV/ON/05 nucleotide insertions shown in bold and deletions in g.

Fig. 7 is a graph of total clinical score means from control, infected, and re—infected groups from Example 4.

Fig. 8 is a graph of body temperature means from control, infected, and re—infected groups from Example 4.

Fig. 9 is a Table showing titers to equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV) in control, infected, and re—infected groups of Example 4. The virus neutralization test (VN) was used to measure antibody titers on serum samples. ed Description of the Invention The inventors have determined that immunogenic itions comprising one or more strains of inactivated ERAV, which when live and active are virulent (i.e., at least 50%, 60%, 70%, 80%, 90% or even 100% of seronegative , when ely exposed to the virus, present with observable respiratory disease, particularly nasal and/or ocular discharge), can be grown to high titers in culture to yield a e that is able to induce high titers of serum antibodies against ERAV when administered, for example, to an equine, for example resulting in a serum titer of at least 1:112, and preferably, at least 1:200, 1:500, 1:750 or 1:1000. In addition, the strains grow well in e and are highly efficient to produce, for example, to a titer of at least 106 TCID50/mL, more preferably 107 TCID50/mL, 108 TCID50/mL, or even 109 TCID50/mL.

Similarly, compositions comprising one or more strains of inactivated ERBV, which when live and active and not attenuated are also virulent (as defined above for ERAV), were also found to grow well in culture, to a titer of at least 106 TCID50/mL, more preferably 107 TCleo/mL, 108 TCID50/mL or even 109 TCID50/mL, to be highly efficient to produce, and to induce high titers of serum antibodies in animals following immunization, for example, resulting in a serum titer of at least 1:120 and ably at least 1:200, 1:500, 1:750 or . Both the ERAV and ERBV itions, and combinations f, are capable of reducing the duration, severity, and incidence of disease in an animal such as a horse that has been immunized with the compositions and subsequently nged.

In one embodiment, provided is an genic composition comprising one or more strains of vated ERAV and/or ERBV. Alternatively, the s may be attenuated by routine means and the live, attenuated virus used in the vaccine composition. In some embodiments, the ERAV strain comprises a genomic sequence whose e transcript has a ’UTR comprising the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of SEQ ID NO: 2 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or encodes functional ERAV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of SEQ ID NO: 3, which polyprotein contains functional ERAV proteins (i.e., active in viral infection and replication). In some embodiments, the ERAV strain comprises a genomic sequence which, when reverse transcribed, has a nucleotide sequence of SEQ ID NO: 2 or which encodes a polyprotein with an amino acid sequence of SEQ ID NO: 3. In certain ments the ERAV strain is ERAV/ON/05 having ATCC Accession No. PTA—11828, deposited with the American Type Culture Collection on April 14, 2011, which is hereby orated by reference.

In some embodiments, the ERBV is strain 042, having ATCC Accession No: PTA-11829, deposited with the American Type Culture Collection on April 14, 2011, which is hereby incorporated by reference. In some embodiments, the ERBV strain comprises a genomic sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, r than 98% or greater than 99% identity to the nucleotide sequence of the reverse transcript of the genome of the ERBV strain having ATCC ion No. PTA— ll829 and, when not inactivated or attenuated, is active to infect and ate in host cells and/or encodes functional ERBV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of the polyprotein of the strain with ATCC Accession No. PTA-11829, which polyprotein contains functional ERBV proteins (i.e., active in viral infection and replication).

In one embodiment provided is an immunogenic composition comprising inactivated (or, alternatively, live, attenuated) ERAV and ERBV. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse transcript has a 5’UTR comprising the nucleotide ce of SEQ ID NO: 1. In some embodiments, the ERAV strain comprises a genomic sequence whose e transcript has a tide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of SEQ ID NO: 2 and, when not inactivated, is active to infect and replicate in host cells and/or encodes onal ERAV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of SEQ ID NO: 3, which polyprotein contains functional ERAV proteins (i.e., active in viral ion and replication). In some embodiments, the ERAV strain comprises a genomic sequence which, when reverse transcribed, has a nucleotide sequence of SEQ ID NO: 2 or which encodes a polyprotein with an amino acid sequence of SEQ ID NO: 3. In some embodiments, the ERAV strain is ERAV/ON/05 (ATCC Accession No. PTA—11828). In some embodiments, the ERBV is a strain having ATCC Accession No: PTA-11829. In some ments, the ERBV strain comprises a genomic ce whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, r than 98% or greater than 99% identity to the nucleotide sequence of the reverse ript of the genome of the ERBV strain having ATCC ion No. PTA—11829 and, when not inactivated, is active to infect and replicate in host cells and/or encodes functional ERBV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of the polyprotein of the strain with ATCC Accession No.PTA—l 1829, which polyprotein contains functional ERBV proteins (i.e., active in viral infection and replication).

In one embodiment, along with the inactivated or live, attenuated one or more strains of ERAV and/or ERBV, the immunogenic compositions provided herein further comprise at least one n or one additional inactivated or live, attenuated strain of Equine Herpes Virus (EHV). In some embodiments the itions comprise at least one antigen of EHV. In some embodiments the EHV is selected from the group consisting of EHV-l, EHV—4, strains deposited with the ATCC under accession Nos. PTA—9525 and PTA—9526, and combinations thereof. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse ript has a 5’UTR comprising the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the tide sequence of SEQ ID NO: 2 and, when not inactivated, is active to infect and replicate in host cells and/or encodes functional ERAV ns, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of SEQ ID NO: 3, which polyprotein contains functional ERAV proteins (i.e., active in viral infection and replication). In some embodiments, the ERAV strain comprises a genomic sequence which, when reverse transcribed, has a nucleotide sequence of SEQ ID NO: 2 or which encodes a polyprotein with an amino acid ce of SEQ ID NO: 3. In some embodiments, the ERAV strain is ERAV/ON/05 (ATCC Accession No. PTA—11828). In some ments, the ERBV is a strain having ATCC Accession No: PTA—11829. In some embodiments, the ERBV strain ses a genomic sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, r than 97%, greater than 98% or r than 99% identity to the nucleotide sequence of the reverse transcript of the genome of the ERBV strain having ATCC Accession No. PTA—11829 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or encodes functional ERBV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, r than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of the polyprotein of the strain with ATCC Accession No. PTA—11829, which polyprotein contains functional ERBV ns (i.e., active in viral infection and replication).

In one ment, along with the inactivated (or attenuated) one or more strains of ERAV and/or ERBV, the immunogenic compositions provided herein further comprise at least one antigen or one additional vated or attenuated strain of Equine za Virus (EIV). In some embodiments the compositions comprise at least one antigen of EIV. In some embodiments the EIV is selected from the group consisting of Clade 1 Viruses, Clade 2 Viruses, Inﬂuenza A/South Africa/2003, Inﬂuenza A/equine—2/Ohio/03, Inﬂuenza A/equine—2/New Market/2/93, Inﬂuenza A/equine—2/Kentucky/95, Inﬂuenza A/equine—2/Richmond/l/2007 and combinations thereof. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse transcript has a 5’UTR comprising the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse transcript has a nucleotide sequence with r than 95%, greater than 96%, greater than 97%, greater than 98% or r than 99% identity to the nucleotide sequence of SEQ ID NO: 2 and, when not inactivated or ated, is active to infect and ate in host cells and/or encodes functional ERAV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, r than 98% or greater than 99% identity to the amino acid sequence of SEQ ID NO: 3, which polyprotein contains functional ERAV proteins (i.e., active in viral infection and replication). In some embodiments, the ERAV strain comprises a genomic sequence which, when reverse transcribed, has a nucleotide sequence of SEQ ID NO: 2 or which encodes a polyprotein with an amino acid sequence of SEQ ID NO: 3. In some embodiments, the ERAV strain is ERAV/ON/05 (ATCC Accession No.PTA—11828).

In some embodiments, the ERBV is a strain having ATCC Accession No: PTA-11829. In some ments, the ERBV strain ses a genomic sequence whose e transcript has a nucleotide sequence with r than 95%, greater than 96%, r than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of the reverse transcript of the genome of the ERBV strain having ATCC Accession No. PTA—11829 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or encodes functional ERBV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, r than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid ce of the polyprotein of the strain with ATCC Accession No. PTA—11829, which polyprotein contains functional ERBV proteins (i.e., active in viral infection and replication In one embodiment, along with the inactivated (or live, attenuated) one or more strains of ERAV and/or ERBV, the immunogenic compositions provided herein r comprise at least one antigen or one additional inactivated or live, attenuated strain of Equine Inﬂuenza Virus and at least one n or one additional inactivated or live, attenuated strain of Equine Herpes Virus. In some embodiments the compositions comprise at least one antigen of EHV and at least one antigen of EIV. In some embodiments the EHV is EHV—1 or EHV—4 or a combination thereof and the EIV is selected from the group consisting of Clade 1 Viruses, Clade 2 Viruses, Inﬂuenza A/South Africa/2003, za A/equine—2/Ohio/03, Inﬂuenza A/equine— 2/New Market/2/93, Inﬂuenza ne—2/Kentucky/95, Inﬂuenza A/equine—2/Richmond/1/2007 and combinations thereof. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse ript has a 5’UTR comprising the nucleotide sequence of SEQ ID NO: 1. In some ments, the ERAV strain comprises a genomic sequence whose reverse ript has a nucleotide ce with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of SEQ ID NO: 2 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or s functional ERAV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of SEQ ID NO: 3, which polyprotein contains functional ERAV proteins (i.e., active in viral infection and replication). In some embodiments, the ERAV strain comprises a genomic sequence which, when reverse transcribed, has a tide sequence of SEQ ID NO: 2 or which encodes a polyprotein with an amino acid sequence of SEQ ID NO: 3. In some embodiments, the ERAV strain is ERAV/ON/OS (ATCC Accession No. PTA- 11828). In some embodiments, the ERBV is a strain having ATCC Accession No: PTA—11829.

In some embodiments, the ERBV strain comprises a genomic sequence whose reverse transcript has a tide sequence with greater than 95%, greater than 96%, r than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of the reverse transcript of the genome of the ERBV strain having ATCC Accession No. PTA—11829 and, when not inactivated, is active to infect and replicate in host cells and/or encodes functional ERBV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, r than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of the polyprotein of the strain with ATCC Accession No. PTA-11829, which polyprotein contains functional ERBV proteins (i.e., active in viral infection and replication).

In one embodiment, along with the vated (or live, ated) one or more strains of ERAV and/or ERBV, the immunogenic compositions provided herein further se at least one antigen or vated virus of one or more additional strains selected from the group ting of Equine Inﬂuenza Virus, Equine Herpes Virus, West Nile Virus, Eastern Equine Encephalomyelitis Virus, Western Equine Encephalomyelitis Virus, and Venezuelan Equine Encephalomyelitis Virus, and/or Tetanus Toxoid, and combinations f. In some embodiments, the ERAV strain comprises a genomic ce whose reverse transcript has a ’UTR comprising the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse ript has a nucleotide ce with greater than 95%, greater than 96%, greater than 97%, r than 98% or greater than 99% identity to the nucleotide sequence of SEQ ID NO: 2 and, when not inactivated is attenuated, is active to infect and replicate in host cells and/or encodes functional ERAV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of SEQ ID NO: 3, which polyprotein contains functional ERAV proteins (i.e., active in viral infection and replication). In some embodiments, the ERAV strain comprises a c sequence which, when reverse transcribed, has a nucleotide sequence of SEQ ID NO: 2 or which encodes a polyprotein with an amino acid sequence of SEQ ID NO: 3. In some embodiments, the ERAV strain is N/OS (ATCC Accession No. PTA-11828). In some embodiments, the ERBV is a strain having ATCC Accession No: PTA—11829. In some embodiments, the ERBV strain comprises a genomic sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of the e transcript of the genome of the ERBV strain having ATCC Accession No. PTA—11829 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or encodes functional ERBV ns, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, r than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of the polyprotein of the strain with ATCC Accession No. PTA-11829, which polyprotein contains functional ERBV proteins (i.e., active in viral infection and replication).

In one embodiment, along with the inactivated (or live, attenuated) one or more strains of ERAV and/or ERBV, the immunogenic compositions provided herein further comprise at least one additional inactivated or live, attenuated virus of a strain selected from the group consisting of West Nile Virus, Eastern Equine Encephalomyelitis Virus, Western Equine Encephalomyelitis Virus, and Venezuelan Equine Encephalomyelitis Virus, and/or Tetanus Toxoid, and combinations thereof. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse transcript has a 5’UTR comprising the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse ript has a nucleotide sequence with r than 95%, greater than 96%, r than 97%, greater than 98% or greater than 99% identity to the tide sequence of SEQ ID NO: 2 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or encodes functional ERAV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of SEQ ID NO: 3, which otein ns functional ERAV proteins (i.e., active in viral infection and replication). In some ments, the ERAV strain comprises a genomic sequence which, when reverse transcribed, has a nucleotide sequence of SEQ ID NO: 2 or which s a polyprotein with an amino acid sequence of SEQ ID NO: 3. In some embodiments, the ERAV strain is ERAV/ON/OS (ATCC ion No. PTA- 11828). In some embodiments, the ERBV is a strain having ATCC Accession No: 829.

In some embodiments, the ERBV strain comprises a genomic sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of the reverse transcript of the genome of the ERBV strain having ATCC Accession No. PTA—11829 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or encodes functional ERBV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of the polyprotein of the strain with ATCC Accession No. PTA—11829, which polyprotein contains functional ERBV ns (i.e., active in viral infection and replication).

In one embodiment, provided is a method of making the immunogenic composition of the present invention. The method generally comprises the steps of combining an vated or live, attenuated ERAV and/or ERBV and a pharmaceutically acceptable carrier. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse transcript has a ’UTR comprising the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, r than 98% or greater than 99% identity to the nucleotide sequence of SEQ ID NO: 2 and, when not inactivated or ated, is active to infect and replicate in host cells and/or encodes functional ERAV ns, or which encodes a otein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of SEQ ID NO: 3, which polyprotein ns functional ERAV proteins (i.e., active in viral infection and replication). In some embodiments, the ERAV strain comprises a genomic sequence which, when reverse transcribed, has a nucleotide sequence of SEQ ID NO: 2 or which s a polyprotein with an amino acid sequence of SEQ ID NO: 3. In some embodiments, the ERAV strain is ERAV/ON/OS (ATCC Accession No. PTA—11828). In some embodiments, the ERBV is a strain having ATCC ion No: PTA—11829. In some embodiments, the ERBV strain comprises a genomic sequence whose reverse transcript has a tide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or r than 99% identity to the nucleotide sequence of the reverse transcript of the genome of the ERBV strain having ATCC Accession No. PTA—11829 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or encodes onal ERBV proteins, or which s a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of the polyprotein of the strain with ATCC Accession No. PTA—11829, which polyprotein contains functional ERBV proteins (i.e., active in viral infection and replication). In some embodiments the method further comprises the step of adding one or more additional equine virus antigens or inactivated or live, attenuated equine viruses. In another embodiment, the method further comprises the step of adding a suitable adjuvant to the composition.

In one embodiment, provided is a method for reducing the incidence of or lessening the severity of clinical symptoms associated with or caused by ERAV or ERBV in an animal or a herd of animals sing administering an immunogenic composition as disclosed herein to an animal in need thereof. In some embodiments, the animal is a horse.

The entioned embodiments may further n one or more of the ing features described below.

In one embodiment, provided is a composition comprising at least one strain of inactivated (or, alternatively, live, attenuated) ERAV and/or ERBV and further containing many or all relevant antigenic components and proteins of pathogenic West Nile Virus (WNV) or an inactivated or live, attenuated strain of WNV.

In one embodiment, provided is a vaccine composition comprising one or more strains of vated (or live, attenuated) ERAV and/or ERBV in combination with one or more immunologically ive amounts of antigenic ents or one or more inactivated or live, attenuated strains selected from the group consisting of West Nile Virus (WNV), Venezuelan Equine Encephalomyelitis (VEE), Eastern Equine Encephalomyelitis (EEE), Western Equine Encephalomyelitis (WEE), s toxoid (T), Equine herpes viruses (EHV) including types 1 and 4, Equine inﬂuenza viruses (EIV), and ations thereof, along with a pharmaceutically able carrier. Preferably such embodiments will include an adjuvant, such as a carbomer, and a pharmaceutically acceptable carrier. In other embodiments the adjuvant is HRA-S, a carbomer, or mineral oil.

In some embodiments, the compositions also e inactivated or live, attenuated ERAV and/or ERBV in combination with the following inactivated or live, attenuated viral strains or ns and combinations of strains and ns: West Nile Virus; Eastern Equine Encephalomyelitis; Western Equine Encephalomyelitis; Venezuelan Equine Encephalomyelitis; Tetanus Toxoid; Eastern Equine Encephalomyelitis and Western Equine Encephalomyelitis; Eastern Equine Encephalomyelitis and elan Equine Encephalomyelitis; Eastern Equine Encephalomyelitis and Tetanus Toxoid; Eastern Equine alomyelitis, Western Equine alomyelitis, and elan Equine Encephalomyelitis; Eastern Equine Encephalomyelitis, Western Equine Encephalomyelitis, and Tetanus Toxoid; Eastern Equine Encephalomyelitis, Western Equine Encephalomyelitis, elan Equine Encephalomyelitis and Tetanus Toxoid; Western Equine Encephalomyelitis and elan Equine Encephalomyelitis; Western Equine Encephalomyelitis and Tetanus Toxoid; Western Equine Encephalomyelitis, Venezuelan Equine alomyelitis, and Tetanus Toxoid; Venezuelan Equine Encephalomyelitis and Tetanus Toxoid; and Eastern Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis and Tetanus Toxoid, or antigens or antigenic components thereof. A red combination of these specified combinations es ERAV and/or ERBV in combination with antigens or nic components of inactivated viruses of West Nile Virus, Eastern Equine Encephalomyelitis, Western Equine Encephalomyelitis, Venezuelan Equine alomyelitis, and Tetanus . Another preferred combination includes ERAV and/or ERBV in combination with antigens or antigenic components of Eastern Equine Encephalomyelitis, Western Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis, and Tetanus Toxoid. Preferred ERAV include ERAV/ON/05(ATCC Accession No. PTA— 11828), ERAV strain which comprises a genomic sequence whose reverse transcript has a S’UTR comprising the nucleotide sequence of SEQ ID NO: 1, which comprises a genomic sequence whose reverse transcript has a nucleotide sequence with r than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the tide sequence of SEQ ID NO: 2 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or s functional ERAV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of SEQ ID NO: 3, which polyprotein contains functional ERAV proteins (i.e., active in viral infection and replication). The ERAV strain may also comprise a genomic sequence which, when reverse transcribed, has a nucleotide sequence of SEQ ID NO: 2 or which encodes a otein with an amino acid sequence of SEQ ID NO: 3. Preferred ERBV is a strain having ATCC ion No: PTA—11829, or which comprises a genomic sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of the reverse transcript of the genome of the ERBV strain having ATCC Accession No. PTA-11829 and, when not vated, is active to infect and replicate in host cells and/or encodes functional ERBV ns, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of the polyprotein of the strain with ATCC Accession No. PTA—11829, which otein contains functional ERBV proteins (i.e., active in Viral infection and replication). In each such specified combination, an adjuvant or combination of adjuvants can be used such as, HRA—S, carbomer or with carbopol. The NJO strain of Eastern Equine Encephalomyelitis, the Fleming strain of Western Equine Encephalomyelitis strain, and the TC—83 strain of Venezuelan Equine Encephalomyelitis strain are all representative strains of these e components.

Further preferred embodiments of the present invention include immunogenic compositions made using each of the specified combination vaccines listed above and adding ns or inactivated or attenuated viruses from Equine Herpesvirus, preferably type 1, type 4, (EHVl and/or EHV4) or combinations thereof.

Still further variations of each of the specified combination vaccines or immunogenic itions listed above, including those that include EHVl and/or EHV4 can be made by adding in antigens or inactivated or attenuated viruses from Equine inﬂuenza Virus (EIV). Preferred ments incorporating Equine inﬂuenza virus include inactivated or live, attenuated: ERAV and/or ERBV and at least one vated or live, attenuated strain of each of WNV, Equine za Virus, and Tetanus Toxoid; ERAV and/or ERBV and at least one inactivated or live, attenuated strain of each of WNV, Equine Inﬂuenza Virus, Tetanus Toxoid, and Eastern Equine Encephalomyelitis; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Tetanus Toxoid, Eastern Equine Encephalomyelitis, and Western Equine Encephalomyelitis; ERAV and/or ERBV and at least one strain of each of WNV, Equine za Virus, Tetanus Toxoid, Eastern Equine Encephalomyelitis, Western Equine Encephalomyelitis; and Venezuelan Equine Encephalomyelitis; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, and Eastern Equine Encephalomyelitis; ERAV and/or ERBV and, at least one strain of each of WNV, Equine Inﬂuenza Virus, and Western Equine Encephalomyelitis; ERAV and/or ERBV and at least one strain of each of WNV, Equine za Virus, and Venezuelan Equine Encephalomyelitis; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, n Equine Encephalomyelitis, and Western Equine Encephalomyelitis; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Eastern Equine Encephalomyelitis, and Venezuelan Equine Encephalomyelitis; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Western Equine Encephalomyelitis, and Venezuelan Equine Encephalomyelitis; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Western Equine Encephalomyelitis, and tetanus toxoid; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Venezuelan Equine Encephalomyelitis, and tetanus toxoid; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Venezuelan Equine Encephalomyelitis, Western Equine alomyelitis, and s toxoid; and ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Venezuelan Equine Encephalomyelitis, Eastern Equine Encephalomyelitis, and tetanus toxoid, wherein the aforementioned strains are inactivated or live, attenuated. In each specified embodiment one or more inactivated or live, ated strains of Equine Inﬂuenza Virus may be present. Preferred strains of Equine Inﬂuenza virus include Inﬂuenza A/equine—2/Ohio/03, Inﬂuenza A/equine—2/New Market/2/93, Inﬂuenza A/equine— 2/Kentucky/95, and combinations thereof. In all of the combinations listed above, it is preferred to use at least two inactivated or live, attenuated s of Equine Inﬂuenza Virus and still more preferred to use at least 3 strains of Equine za Virus. red embodiments incorporating Equine Herpes Virus include: ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Tetanus Toxoid, and Equine Herpes Virus; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Tetanus Toxoid, n Equine Encephalomyelitis, and Equine Herpes Virus; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, s , Eastern Equine Encephalomyelitis, Western Equine Encephalomyelitis, and Equine Herpes Virus; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Tetanus Toxoid, Eastern Equine Encephalomyelitis, Western Equine Encephalomyelitis; Venezuelan Equine Encephalomyelitis, and Equine Herpes Virus; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, and Eastern Equine Encephalomyelitis; ERAV and/or ERBV and at least one strain of each of WNV, Equine za Virus, Western Equine Encephalomyelitis and Equine Herpes Virus; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Venezuelan Equine alomyelitis, and Equine Herpes Virus; ERAV and/or ERBV and at least one strain of each of WNV, Equine za Virus, Eastern Equine Encephalomyelitis, Western Equine Encephalomyelitis, and ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Eastern Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis, and Equine Herpes Virus; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Western Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis, and Equine Herpes Virus; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Western Equine Encephalomyelitis, Tetanus Toxoid, and Equine Herpes Virus; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Venezuelan Equine Encephalomyelitis, tetanus toxoid, and Equine Herpes Virus; ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Venezuelan Equine Encephalomyelitis, Western Equine Encephalomyelitis, Tetanus Toxoid, and Equine Herpes Virus; and ERAV and/or ERBV and at least one strain of each of WNV, Equine Inﬂuenza Virus, Venezuelan Equine Encephalomyelitis, Eastern Equine Encephalomyelitis, Tetanus Toxoid, and Equine Herpes Virus, wherein the aforementioned strains are vated or live, ated. In all of the combinations listed above, it is preferred to use at least two strains of vated or live, attenuated Equine Inﬂuenza Virus and still more preferred to use at least 3 strains of inactivated or live, attenuated Equine Inﬂuenza Virus. onally, in all combinations above, the “at least one” strain of Equine Herpes Virus is preferred to be selected from the group consisting of inactivated EHV—l and EHV—4. In some preferred forms, both inactivated or live, attenuated strains, EHV—l and EHV—4, will be included in the immunogenic composition. In other preferred forms, just EHV—l will be included. In a preferred combination, the inactivated or live, attenuated ERAV strain is ERAV/ON/05, an ERAV strain which comprises a genomic sequence whose reverse transcript has a 5’UTR comprising the nucleotide sequence of SEQ ID NO: 1, which comprises a genomic sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or r than 99% identity to the nucleotide sequence of SEQ ID NO: 2 and, when not inactivated, is active to infect and replicate in host cells and/or encodes functional ERAV ns, or which encodes a otein having an amino acid sequence with greater than 95%, r than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of SEQ ID NO: 3, which otein contains functional ERAV proteins (i.e., active in viral infection and replication). The ERAV strain may also comprise a genomic sequence which, when reverse transcribed, has a nucleotide ce of SEQ ID NO: 2 or which encodes a polyprotein with an amino acid sequence of SEQ ID NO: 3.

In other preferred ations, the ERBV is a strain having ATCC Accession No: PTA—11828, or which comprises a genomic sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, r than 97%, greater than 98% or greater than 99% identity to the tide sequence of the reverse transcript of the genome of the ERBV strain having ATCC Accession No. PTA—11828 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or encodes functional ERBV ns, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of the polyprotein of the strain with ATCC Accession No. PTA—11828, which otein contains functional ERBV proteins (i.e., active in viral infection and replication).

The immunogenic compositions as disclosed herein can be administered in any genically effective dose. In a preferred embodiment, the immunogenic composition is administered as a single dose. Preferably, the dose has a total volume between about 0.5 mL and about 2.5 mL, more ably between about 0.6 mL and about 2.0 mL, even more preferably between about 0.7 mL and about 1.75 mL, still more preferably between about 0.8 mL and about 1.5 mL, even more preferably between about 0.9 mL and about 1.25 mL, with a single dose about 1.0 mL being the most preferred.

In r embodiment, the immunogenic composition is stered with a first dose being administered prior to the administration of a second (booster) dose. Preferably, the second dose is administered at least about 15 days after the first close. More preferably, the second dose is administered between about 15 and about 28 days after the first dose. Even more preferably, the second dose is administered at least about 17 days after the first dose. Still more preferably, the second dose is administered n about 17 and about 25 days after the first dose. Even more ably, the second dose is administered at least about 19 days after the first dose. Still more preferably, the second dose is administered between about 19 and about 23 days after the first dose. Most preferably the second dose is administered at least about 21 days after the first dose. In a preferred embodiment, both the first and second doses of the immunogenic composition are in the same . Preferably, each dose is in the preferred amounts specified above, with a dose of about 1 mL for the first and second dose being most preferred. In addition to the first and second dose n, an alternate embodiment comprises further subsequent doses. For example, a third, fourth, or fifth dose could be administered in these embodiments.

Preferably, subsequent third, fourth, and fifth dose regimens are administered in the same amount as the first dose, with the time frame between the doses being consistent with the timing between the first and second doses mentioned above, although the timing may also vary.

In one embodiment the immunogenic composition is administered in three doses.

In some embodiments the three doses are administered at three week intervals.

In an embodiment that comprises ERAV, preferably ERAV/ON/OS, the amount of ERAV in the immunogenic composition is at least about 102‘0TCID50/dose. More preferably, the amount of ERAV is between about 102‘0TC1D50/dose to about 1010‘0TCID50/dose. Still more preferably, the amount of ERAV is at least about 102'5TCID50/dose. Even more preferably, the amount of ERAV is between about 102‘5TCleo/dose to about 109'5TCID50/dose. Still more preferably, the amount of ERAV is at least about CID50/dose. Even more preferably, the amount of ERAV is between about 103'0TClD5o/dose to about 109'0TCID50/dose. Still more ably, the amount of ERAV is at least about ClD5o/dose. Even more preferably, the amount of ERAV is between about CID50/dose to about 109'0TCID50/dose. Sill more preferably, the amount of ERAV is between about 106‘5TCID50/dose and about CID50/dose.

More preferably, the amount of ERAV is between about 107‘0TCID50/dose and about 109‘0TCID50/dose. The TCID50 values of an vated or attenuated ERAV or any other inactivated or attenuated vaccine refer in general to the viral content in the final vaccine that however is equivalent to the viral content calculated for the vaccine composition prior to the inactivation of its virus. Preferably, the immunogenic composition of the present invention stimulates serum neutralizing antibodies to ERAV at a titer of at least 1:112, 1:300, 1:500, 1:700, 1:900, 1:1000, or 1:1500 In an embodiment that comprises ERBV, ably a strain having ATCC Accession NO: PTA—11829, the amount of ERBV is at least about 102'0TCID50/dose. More preferably, the amount of ERBV is between about 102'0TCIDso/dose to about lOlO'OTCID50/dose.

Still more preferably, the amount of ERBV is at least about 102'5TCID50/dose. Even more preferably, the amount of ERBV is between about 102'5TCIDso/dose to about 109‘5TCID50/dose.

Still more ably, the amount of ERBV is at least about 103‘0TCID50/dose. Even more ably, the amount of ERBV is between about 103'0TCID50/dose to about 109‘0TCID50/dose.

Still more preferably, the amount of ERBV is at least about 103‘5TCID50/dose. Even more preferably, the amount of ERBV is between about 103‘5TCIDso/dose to about 109‘0TCID50/dose.

Sill more preferably, the amount of ERBV is between about 106‘5TCID50/dose and about 108‘5TCID50/dose. More preferably, the amount of ERBV is between about 107'0TCID50/dose and about 109‘0TCID50/dose. The TCIDsO values of an vated ERBV or any other inactivated vaccine refer in general to the viral t in the final vaccine that however is equivalent to the viral content calculated for the vaccine composition prior to the inactivation of its virus. ably, the immunogenic composition of the present invention stimulates serum neutralizing antibodies to ERBV at a titer of at least 1:4 or higher. In some embodiments, the titer is at least 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11; 1:12, 1:13, 1:14, or 1:15 or higher. In some embodiments, the titer is at least 1:64, 1:256, or 1:512 or higher. In some embodiments, the titer is at least 1:1024 or higher. In some embodiments, the titer is at least 1:2048, 1:1536, 1:3072, 1:4096, 1:6144, 1:8192, 1:12288, or 9 or higher. In some embodiments, the titer is no more than 1:300, 1:1050, 1:32000, 1:70000, or 1:140000.

In one embodiment, in each dose of an embodiment of the t invention that comprises one or more additional equine antigens, the amount of Eastern Equine Encephalomyelitis or Venezuelan Equine alomyelitis in any dose is ably at least about CID50/dose. Even more ably, the dose is between about 105'5TCID50/dose and about 109‘5TCID50/dose. Still more preferably, the dose is at least about 106'0TCID50/dose. Still more ably, the dose is between about 106'0TCID50/dose and about 109'0TCID50/dose. Even more preferably, the dose is at least about 106'5TCID50/dose. Still more preferably, the dose is between about 106‘5TCID50/dose and about 109'5TC1D50/dose. Even more preferably, the dose is at least about 107‘0TCID50/dose. Most preferably, the dose is between about 106'7TCID50 and about 109‘2TCID50/dose.

In an embodiment that comprises inactivated or killed WNV or antigen, the amount of WNV or antigen is at least about 102'0TCID50/dose. More preferably, the WNV or antigen is between about 102‘0TCID50/dose to about 1010'0TCID50/dose. Still more preferably, the WNV or antigen is at least about 102‘5TCID50/dose. Even more preferably, the WNV or antigen is between about CID50/dose to about 109‘5TCIDso/dose. Still more preferably, the WNV or antigen is at least about 103‘0TCID50/dose. Even more preferably, the WNV or antigen is between about CID50/dose to about 109‘0TCIDso/dose. Still more preferably, the WNV or antigen is at least about 103‘5TCID50/dose. Even more preferably, the WNV or antigen is between about 103‘5TCID50/dose to about 109‘0TCIDsO/dose. Most preferably, the WNV or antigen is between about 107‘0TCID50/dose and about 109‘0TCID50/dose. The TCID50 values of an inactivated WNV vaccine or any other vated vaccine refer in general to the antigen content in the final vaccine that however is equivalent to the antigen content calculated for the e composition prior to the inactivation of its antigen. Preferably, the immunogenic composition of the t invention stimulates serum neutralizing antibodies to WNV at a titer of at least 1:4 or higher. In some embodiments the titer is at least 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11; 1:12, 1:13, 1:14, or 1:15 or higher. In some embodiments, the titer is no more than 1:300, 1:1050, 1:32000, 1:70000, or 1:140000. In a preferred embodiment, in each dose of an embodiment of the present invention that comprises additional equine n, the amount of Eastern Equine alomyelitis or elan Equine alomyelitis in any dose is preferably at least about lOS‘STCID50/dose. Even more preferably, the dose is between about 105‘5TCID50/dose and about 109‘5TC1D50/dose. Still more preferably, the dose is at least about 106‘0TCID50/dose. Still more preferably, the dose is between about 106‘0TCIDso/dose and about 109‘0TCID50/dose. Even more ably, the dose is at least about 106‘5TCID50/dose. Still more preferably, the dose is between about 106'5TCID50/dose and about 109‘5TCID50/dose. Even more preferably, the dose is at least about 107'0TCID50/dose. Most preferably, the dose is between about 106-7TC1D50 and about C1D50/dose.

Preferably, the Western Equine Encephalomyelitis antigen, when present in the composition of the present invention, is in an amount of at least about 106'2PFU/mL. Even more preferably, the amount is between about 106'2PFU/mL and about lOlO'ZPFU/mL. Still more preferably, the amount is at least about 106'7PFU/mL. Even more preferably, the amount is between about 106‘5PFU/mL and about 109'7PFU/mL. Still more preferably, the amount is at least about 107‘2PFU/mL. Even more preferably, the amount is between about 107‘2PFU/mL and about FU/mL. Still more preferably, the amount is at least about 107‘7PFU/mL with between about 106‘5 PFU/dose and about 109‘OPFU/mL being the most preferred.

In another preferred embodiment, the amount of tetanus toxoid, if present in the composition of the present ion, is in an amount of at least about 3 CPU, more preferably, between about 3 CPU and about 20 CPU, still more preferably, at least about 4 CPU, and most preferably, at least about 5 CPU but not more than about 20 CPU.

In an alternate embodiment, where one or more strains of Equine Inﬂuenza Virus is present, the amount of Equine Inﬂuenza present in the composition is in an amount of at least about 105‘0 TCIDso/mL. More ably, the Equine Inﬂuenza is in an amount of between about 105‘0 TCIDso/mL to about 109'0 TCIDso/mL, and, more preferably, at least about 106‘0 TCID50/mL.

Still more preferably, the amount is between about 106‘0 TCIDso/mL to about 108‘0 TCIDso/mL and, more ably, the amount is at least about 106'5 TCID50/mL. Still more preferably, the amount is between about 106‘5 TCID50/mL to about 107'5 TClD50/mL, with the most preferred amount being between about 106‘7 TCID50/mL to about 107'3 TCID50/mL.

In an embodiment that comprises Equine Herpes Virus, the amount of Equine Herpes Virus in each dose is at least about 106‘0 /mL. More preferably, Equine Herpes Virus is present in the composition in an amount of between about 106‘0 TCID50/mL to about 109‘5 TCIDso/mL and, more preferably, in an amount of about 107‘0 TCleo/mL. Still more preferably, Equine Herpes Virus is present in an amount between about 107‘5 TCIDso/mL to about 109‘0 TCIDso/mL and, more preferably, in an amount of about 108‘0 TCID50/mL. Still more preferably, Equine Herpes Virus is present in an amount of between about 108‘0 TCIDsO/mL to about 109‘0 TCID50/mL and, most preferably, in an amount of about 108'50 TCID50/mL.

In yet another preferred embodiment, a vaccine composition comprising the chronologically contemporary and epidemiologically prevalent s of ERAV and/or ERBV is provided. Preferably the composition comprises ERAV/ON/05 and/or a ERBV strain having ATCC Accession NO: PTA—11829. In specific embodiments, the ERAV strain comprises a genomic sequence whose e transcript has a 5’UTR comprising the nucleotide ce of SEQ ID NO: 1. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse transcript has a nucleotide ce with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of SEQ ID NO: 2 and, when not inactivated, is active to infect and ate in host cells and/or encodes functional ERAV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, r than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid ce of SEQ ID NO: 3, which polyprotein contains onal ERAV proteins (i.e., active in viral infection and replication). In some embodiments, the ERAV strain comprises a genomic sequence which, when reverse transcribed, has a nucleotide sequence of SEQ ID NO: 2 or which encodes a polyprotein with an amino acid ce of SEQ ID NO: 3. In some embodiments, the ERBV is a strain having ATCC Accession No: PTA-11829. In some embodiments, the ERBV strain comprises a genomic sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of the reverse ript of the genome of the ERBV strain having ATCC Accession No. PTA—11829 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or encodes functional ERBV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, r than 96%, greater than 97%, greater than 98% or r than 99% identity to the amino acid sequence of the otein of the strain with ATCC Accession No. PTA—11829, which polyprotein contains functional ERBV proteins (i.e., active in viral infection and replication).

Such a composition will generally improve the cy of the composition.

The present invention additionally provides for a method of reduction of the incidence of and/or severity of clinical signs associated with, ERAV and/or ERBV infection in an animal, preferably a horse. Such methods generally se the step of administering a vaccine composition comprising an inactivated or live, ated strain of an ERAV and/or ERBV and a pharrnaceutically acceptable r. In particular embodiments, the ERAV is ERAV/ON/05, or the ERAV strain comprises a genomic sequence whose reverse transcript has a ’UTR comprising SEQ ID NO: I. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of SEQ ID NO: 2 and, when not inactivated, is active to infect and replicate in host cells and/or encodes functional ERAV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or r than 99% identity to the amino acid ce of SEQ ID NO: 3, which polyprotein ns functional ERAV proteins (i.e., active in viral infection and replication). In some embodiments, the ERAV strain ses a c sequence which, when reverse transcribed, has a nucleotide sequence of SEQ ID NO: 2 or which encodes a polyprotein with an amino acid sequence of SEQ ID NO: 3. In some embodiments, the ERBV is a strain having ATCC Accession No: PTA-11829. In some embodiments, the ERBV strain comprises a genomic sequence whose e transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of the reverse transcript of the genome of the ERBV strain having ATCC Accession No. PTA- 11829 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or encodes onal ERBV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of the polyprotein of the strain with ATCC Accession No. PTA—11829, which polyprotein contains functional ERBV proteins (i.e., active in Viral infection and replication). In some preferred embodiments, an adjuvant, particularly HRA— , is added to the composition, and in other preferred forms, no adjuvant is provided.

In an alternate preferred embodiment, the method ses administering a vaccine composition comprising one or more inactivated or live, attenuated strains of ERAV and/or ERBV in combination with immunologically effective amounts of antigenic components or vated strains from other equine pathogens. Preferably, the ERAV strain is ERAV/ON/05 (ATCC Accession No. PTA—11828) and the ERBV strain has ATCC ion NO: PTA—11829.

In certain such embodiments, the ERBV strain comprises a genomic sequence whose e ript has a 5’UTR comprising SEQ ID NO: 1. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse transcript has a tide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of SEQ ID NO: 2 and, when not inactivated or live, attenuated, is active to infect and replicate in host cells and/or encodes functional ERAV proteins, or which encodes a polyprotein haVing an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of SEQ ID NO: 3, which polyprotein contains functional ERAV ns (i.e., active in Viral ion and replication). In some embodiments, the ERAV strain ses a genomic ce which, when reverse ribed, has a nucleotide sequence of SEQ ID NO: 2 or which encodes a otein with an amino acid sequence of SEQ ID NO: 3. In some embodiments, the ERBV is a strain having ATCC Accession No: PTA—11829. In some embodiments, the ERBV strain comprises a genomic sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of the reverse transcript of the genome of the ERBV strain having ATCC Accession No. PTA-11829 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or s functional ERBV proteins, or which encodes a otein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid sequence of the polyprotein of the strain with ATCC ion No. PTA—11829, which polyprotein contains functional ERBV proteins (i.e., active in viral ion and replication).

In some embodiments of the method, the pathogens in combination with the ERAV and/or ERBV strains, are selected from the group consisting of antigens or inactivated or attenuated strains of EHV and EIV and combinations thereof. In some embodiments the pathogens are antigens. In some embodiments the EHV is EHV-1 or EHV-4 or a combination thereof. In other embodiments the EIV is selected from the group consisting of Clade 1 viruses, Clade 2 s, za A/South Africa/2003, Inﬂuenza A/equine—2/Ohio/03, Inﬂuenza A/equine—2/New Market/2/93, Inﬂuenza A/equine—2/Kentucky/95, Inﬂuenza A/equine— 2/Richmond/l/2007 and combinations thereof.

In still other embodiments of the method, the pathogens in combination with the ERAV and/or ERBV strains, are selected from the group consisting of antigens or inactivated or attenuated strains of WNV, n Equine Encephalomyelitis, Western Equine Encephalomyelitis, and Venezuelan Equine Encephalomyelitis, and tetanus toxoid, and combinations thereof, and more preferably being those combinations described above. In another preferred embodiment, the vaccine of the present invention is combined with a suitable nt and/or pharmaceutically able carrier.

The present invention provides for ion of the incidence of and/or severity of clinical symptoms ated with, ERAV and/or ERBV infection in a herd. Preferably, the ty and/or incidence of clinical symptoms in animals receiving the immunogenic composition of the present invention are reduced at least 10% in comparison to animals not receiving such an administration when both groups (animals receiving and animals not receiving the composition) are challenged with or exposed to infection by ERAV and/or ERBV. More preferably, the incidence or severity is reduced at least 20%, even more preferably, at least 30%, still more ably, at least 40%, even more ably, at least 50%, still more preferably, at least 60%, even more preferably, at least 70%, still more preferably, at least 80%, even more preferably, at least 90%, still more preferably, at least 95%, and most preferably, at least 100%, wherein the animals receiving the composition of the present invention exhibit no clinical symptoms, or alternatively exhibit clinical ms of reduced severity. Advantageously, the present invention also provides protection from heterologous strains ive to the strain used in the composition) of pathogens.

The present invention further provides a method of stimulating serum neutralizing or serum hemagglutination antibodies to a pathogen selected from the group consisting of ERAV, ERBV, WNV, WEE, VEE, EEE, EHV, EIV, and combinations thereof by administering a composition in accordance with the present invention bed herein. In particular embodiments, the ERAV is ERAV/ON/OS, or the ERAV strain comprises a genomic sequence whose reverse transcript has a 5’UTR comprising SEQ ID NO: 1. In some embodiments, the ERAV strain comprises a genomic sequence whose reverse transcript has a nucleotide ce with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide ce of SEQ ID NO: 2 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or encodes functional ERAV proteins, or which encodes a polyprotein having an amino acid ce with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% ty to the amino acid sequence of SEQ ID NO: 3, which polyprotein contains functional ERAV proteins (i.e., active in viral infection and replication). In some embodiments, the ERAV strain comprises a genomic sequence which, when reverse transcribed, has a nucleotide sequence of SEQ ID NO: 2 or which encodes a polyprotein with an amino acid sequence of SEQ ID NO: 3. In some embodiments, the ERBV is a strain having ATCC Accession No: PTA—11829. In some embodiments, the ERBV strain ses a c sequence whose reverse transcript has a nucleotide sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the nucleotide sequence of the reverse transcript of the genome of the ERBV strain having ATCC Accession No. PTA—11829 and, when not inactivated or attenuated, is active to infect and replicate in host cells and/or encodes onal ERBV proteins, or which encodes a polyprotein having an amino acid sequence with greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the amino acid ce of the otein of the strain with ATCC Accession No. PTA—11829, which polyprotein ns functional ERBV proteins (i.e., active in viral infection and replication).

Preferably the compositions of the present invention stimulate serum neutralizing dies to ERAV and/or ERBV at a titer of at least 1:112, 1:120, 1:300, 1:500, 1:1000, or 1:1024, or higher.

The immunogenic ition of the present ion provides an extended duration of immunity against all strains t in the vaccine. Preferably, the duration of ty against ERAV and/or ERBV is at least 1 month, more preferably, the duration of immunity is at least 2 months, still more ably, the duration of immunity is at least 3 months, even more ably, the duration of immunity is at least 4-24 months, still more preferably, the duration of immunity is at least 6—24 months, even more preferably, the duration of immunity is at least 7—24 months, still more ably, the duration of ty is at least 8— 24 months, even more preferably, the duration of immunity is at least 9—24 months, still more preferably, the duration of immunity is at least 10—24 months, and most preferably, the duration of immunity is at least 12—24 months.

The immunogenic composition of the t invention also provides an extended duration of immunity against all antigens present in the vaccine. Preferably, the duration of ty against West Nile is at least 1 month, more preferably, the duration of immunity is at least 2 months, still more preferably, the duration of immunity is at least 3 months, even more preferably, the duration of immunity is at least 4—24 months, still more preferably, the on of immunity is at least 6—24 months, even more preferably, the duration of immunity is at least 7—24 months, still more preferably, the duration of immunity is at least 8—24 months, even more preferably, the on of immunity is at least 9—24 months, still more preferably, the duration of immunity is at least 10—24 months, and most preferably, the duration of immunity is at least 12— 24 months.

Preferably, the duration of immunity against EIV is at least 1 month, more preferably, the duration of immunity is at least 2 months, still more preferably, the duration of immunity is at least 3 , even more preferably, the duration of immunity is at least 4- 24 months, still more preferably, the duration of immunity is at least 6 -24 months, even more preferably, the duration of immunity is at least 7—24 months, still more preferably, the duration of immunity is at least 8—24 months, even more preferably, the duration of immunity is at least 9—24 months, still more ably, the duration of immunity is at least 10—24 months, and most preferably, the duration of immunity is at least 12—24 months. ably, the duration of immunity against EHV is at least 1 month, more ably, the duration of immunity is at least 2 months, still more ably, the duration of immunity is at least 3 months, even more preferably, the duration of immunity is at least 4— 24 months, still more preferably, the duration of immunity is at least 6 -24 months, even more preferably, the duration of immunity is at least 7-24 months, still more preferably, the duration of immunity is at least 8—24 months, even more preferably, the duration of immunity is at least 9—24 months, still more preferably, the duration of immunity is at least 10—24 months, and most preferably, the duration of immunity is at least 12—24 months.

Preferably, the duration of ty t Western Equine Encephalomyelitis is at least 1 month, more preferably, the duration of immunity is at least 2 months, still more preferably, the duration of immunity is at least 3 months, even more preferably, the duration of immunity is at least 4— 24 months, still more preferably, the duration of immunity is at least 6 —24 months, even more preferably, the on of immunity is at least 7—24 , still more preferably, the duration of immunity is at least 8—24 months, even more preferably, the duration of immunity is at least 9—24 months, still more preferably, the duration of immunity is at least 10— 24 months, and most preferably, the duration of immunity is at least 12—24 months. ably, the duration of immunity against Eastern Equine Encephalomyelitis is at least 1 month, more preferably, the duration of immunity is at least 2 months, still more preferably, the duration of immunity is at least 3 months, even more preferably, the duration of immunity is at least 4— 24 months, still more preferably, the duration of immunity is at least 6 —24 months, even more preferably, the duration of ty is at least 7—24 months, still more preferably, the duration of immunity is at least 8-24 months, even more preferably, the duration of ty is at least 9-24 months, still more preferably, the duration of ty is at least 10- 24 months, and most preferably, the duration of immunity is at least 12-24 .

Preferably, the duration of immunity against Venezuelan Equine Encephalomyelitis is at least 1 month, more ably, the duration of immunity is at least 2 months, still more preferably, the duration of immunity is at least 3 months, even more preferably, the duration of immunity is at least 4— 24 months, still more preferably, the duration of immunity is at least 6 —24 , even more preferably, the duration of immunity is at least 7—24 months, still more preferably, the duration of immunity is at least 8—24 months, even more preferably, the duration of immunity is at least 9-24 months, still more preferably, the duration of immunity is at least 10— 24 months, and most preferably, the duration of immunity is at least 12—24 months.

Preferably, the duration of immunity against Tetanus Toxoid is at least 1 month, more preferably, the duration of ty is at least 2 months, still more preferably, the duration of immunity is at least 3 months, even more preferably, the duration of immunity is at least 4— 24 months, still more preferably, the on of ty is at least 6 —24 months, even more preferably, the duration of immunity is at least 7—24 months, still more preferably, the duration of ty is at least 8—24 months, even more preferably, the duration of immunity is at least 9—24 months, still more preferably, the duration of immunity is at least 10—24 , and most preferably, the on of immunity is at least 12—24 months.

Preferably, the duration of immunity of at least 12 months further relates to any combination of antigens forming the immunogenic composition of the t invention.

In one embodiment comprising an inactivated (or, alternatively live attenuated) ERAV and/or ERBV as disclosed herein, the immunogenic composition ameliorates shedding of infectious ERAV and/or ERBV to prevent spread of the virus to other susceptible animals. In some embodiments the compositions prevent shedding of the virus.

In one embodiment comprising EIV and/or EHV antigen, as described above, the immunogenic composition ameliorates shedding of infectious EIV or EHV to prevent spread of the virus to other susceptible s.

In one embodiment, compositions in accordance with the present invention described herein me interference from passively acquired maternal immunity and stimulate active immunity and a reduction in the incidence of or severity of clinical signs of EIV infection in vaccinated animals against EIV.

In one embodiment of the present invention, an immunogenic composition comprising ERAV and/or ERBV, VEE, WEE, EEE, tetanus, WNV, equine rhinopneumonitis and equine inﬂuenza, all as bed herein, demonstrates cy against ERAV, ERBV, VEE, WEE, EEE, tetanus, WNV, equine rhinopneumonitis and equine za after administration in accordance with the present invention. Preferably, such a composition will further include an adjuvant, preferably HRA—S, mineral oil and/or a carbomer, and a pharmaceutically acceptable carrier. In preferred forms, the composition will be stered in a single, 1 mL dose. In some embodiments composition is administered in two doses or preferably three doses, with each dose separated by 1, 2, 3, and 4 weeks.

Each of the immunogenic compositions described herein that include ERAV, particularly ERAV/ON/OS (ATCC Accessions NO: PTA—11828) and other strains as bed supra, and/or ERBV, particularly a ERBV strain having ATCC Accession NO: PTA—11829, or others as also described supra, can be administered as described such that they reduce the incidence of or lessen the severity of clinical symptoms associated with ERAV and/or ERBV, such as pyrexia, elevations in temperature, increased lung sounds, lymphadenopathy, nasal discharge, ocular discharge, pharyngitis, edema of legs, cough, and in the case of ERAV, increased incidence of abortion in nt mares. In some aspects, the compositions lessen the amount or length of nasal or ocular discharge or the length of time that such ms are presented. In some aspects, animals inoculated with the compositions show no clinical symptoms of ERAV and/or ERBV infection one week or longer after exposure to ERAV and/or ERBV. In other aspects, animals inoculated with the compositions show no clinical symptoms of ERAV and/or ERBV infection when d to ERAV and/or ERBV. al symptoms of ERAV and ERBV may be scored such as according to Table 3 in e 2, Table 14 in Example 4, or Table 17 in Example 5.

Each of the immunogenic compositions described herein that include EIV n or inactivated EIV and can be administered as described such that they reduce the incidence of or lessen the severity of clinical symptoms associated with Equine Inﬂuenza Virus.

The t invention also provides a method for reducing the incidence of or lessening the severity of clinical symptoms associated with Equine Herpes Virus comprising the step of administering any one of the immunogenic compositions described above containing EHV antigen or inactivated or attenuated EHV to an animal.

The present invention also es a method for reducing the incidence of clinical symptoms associated with West Nile Virus comprising the step of administering any one of the immunogenic compositions that includes WNV antigen or inactivated or attenuated WNV, as described herein, to an animal.

The present invention also provides a method for ng the incidence of clinical symptoms associated with Equine Inﬂuenza Virus comprising the step of administering any one of the immunogenic itions described above, that includes an EIV antigen or inactivated or attenuated EIV, to an animal.

The present invention further provides a method for reducing the incidence of clinical symptoms associated with Equine Herpes Virus comprising the step of administering any one of the immunogenic compositions described above that es an EHV antigen or inactivated or attenuated EHV, to an animal.

The present invention provides a method of reducing the incidence of viral infection in a herd comprising the step of administering any one of the genic compositions described above to an animal, wherein the reduction of nce of infection, ed to herds not receiving the immunogenic composition, is from about 10% to about 50% reduction. In one embodiment the compositions provided herein reduce ERAV infection by 10% to 50%. In other embodiments the compositions provided herein reduce ERBV ion by 10% to 50%.

The present invention also provides a method of reducing the incidence of clinical symptoms ated with Equine Inﬂuenza Virus comprising the step of administering any one of the genic compositions described above to an animal, wherein the reduction in clinical signs, compared to animals not receiving the immunogenic ition, is at least a 10% reduction in clinical signs.

The t invention es a method of reducing the incidence and severity of clinical symptoms of ERAV or ERBV in a herd, wherein the clinical symptoms are selected from the group consisting of pyrexia, elevations in temperature, increased lung sounds, lymphadenopathy, nasal discharge, ocular discharge, pharyngitis, cough, edema of legs, and in the case of ERAV, increased incidence of abortion in pregnant mares.

The present invention es a method of reducing the incidence and severity of al symptoms of EHV in a herd, wherein the al symptoms are ed from the group consisting of respiratory disease, abortion, reproductive complications, ogical disease, central nervous system e, and combinations thereof.

The present invention es a method for reducing the incidence of or lessening the severity of clinical symptoms associated with Equine Herpes Virus comprising the step of stering any one of the immunogenic compositions disclosed herein, that includes an EHV antigen or inactivated or ated EHV, to an animal.

The present invention provides a method for reducing the incidence of or lessening the severity of clinical ms associated with Equine Inﬂuenza Virus in a herd, comprising the step of administering any one of the immunogenic compositions disclosed herein, that includes an EIV antigen or inactivated or attenuated EIV, to an animal.

The present invention provides a method for reducing the incidence of or lessening the severity of clinical symptoms associated with West Nile Virus in a herd, comprising the step of administering any one of the immunogenic compositions sed herein, that includes a WNV antigen or inactivated or attenuated WNV, to an animal.

The present invention provides a method for reducing the incidence of or lessening the severity of clinical symptoms associated with Eastern Equine Encephalomyelitis in a herd, comprising the step of administering any one of the immunogenic compositions disclosed herein that includes an EEE virus antigen or an inactivated or attenuated EEE Virus, to an animal.

The present invention further provides a method for reducing the incidence of or lessening the severity of clinical symptoms associated with Western Equine Encephalomyelitis in a herd, sing the step of administering any one of the immunogenic itions disclosed , that includes a WEE virus antigen or an inactivated or attenuated WEE virus, to an animal.

The present invention further es a method for reducing the incidence of or lessening the severity of clinical symptoms associated with Venezuelan Equine Encephalomyelitis in a herd, comprising the step of administering any one of the immunogenic compositions disclosed herein, that includes a VEE virus antigen or an inactivated attenuated VEE virus, to an animal.

The aforementioned embodiments may be used in a combination therapy or as part of a immunization schedule in combination with other genic agents and vaccines. In one embodiment, the compositions provided herein are used in combination with the immunogenic agents and vaccines bed in , which is incorporated herein by nce in its entirety.

The present invention also provides a method of making any one of the immunogenic composition as described above and herein, comprising the steps of combining an inactivated or live, attenuated ERAV or ERBV with a suitable pharmaceutical carrier. In preferred forms, this method further comprises the step of adding one or more equine antigens or inactivated or attenuated s. A red group of equine antigens and viruses are ed from the group consisting of West Nile Virus, Western Equine alomyelitis, Eastern Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis, EHV, and EIV, and tetanus toxoid, and combinations thereof. In some preferred forms, the methods described herein can further comprise a filtration step, wherein the final product is in a more pure form.

“About” refers to i10% of the specified quantity.

“Animals” as used herein includes domesticated animals including dogs and hooved animals including equidae, and specifically, horses. In some embodiments, the term also refers to a human.

“Adjuvants” as used , can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-Zl (Cambridge Biotech Inc., dge MA), GPI—OlOO (Galenica ceuticals, Inc., Birmingham, AL), tabolizable oil, mineral and/or plant/vegetable and/or animal oils, polymers, carbomers, tants, natural organic compounds, plant extracts, carbohydrates, cholesterol, lipids, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, HRA-3 (acrylic acid saccharide cross-linked polymer), HRA-3 with cottonseed oil (CSO), or preferably HRA—S (acrylic acid polyol linked polymer). The emulsion can be based in particular on light liquid paraffin oil (European copeia type); isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in ular of isobutene or decene; esters of acids or of alcohols ning a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di—(caprylate/caprate), yl tri— (caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene—polyoxyethylene copolymer , in particular the Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed.Stewart—Tull, D. E. S.) John Wiley and Sons, NY, 4 (1995) and Todd et al., Vaccine :564—570 (1997). In a preferred ment the adjuvant is at a concentration of about 0.01 to about 50%, preferably at a concentration of about 2% to 30%, more preferably at a concentration of about 5% to about 25%, still more ably at a concentration of about 7% to about 22%, and most preferably at a concentration of about 10% to about 20% by volume of the final product.

As used herein, “a pharmaceutically acceptable carrier” or “pharmaceutical carrier” includes any and all excipients, solvents, growth media, dispersion media, coatings, adjuvants, izing , diluents, preservatives, inactivating agents, antimicrobial, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Such ients include those that are safe and appropriate for use in veterinary applications. In some preferred embodiments, and especially those that include lyophilized immunogenic compositions, stabilizing agents for use in the present invention e stabilizers for lization or freeze— drying.

“Diluents” can include water, saline, dextrose, ethanol, glycerol, and the like.

Isotonic agents can e sodium de, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid, among others.

In a preferred embodiment, the immunogenic composition of the present invention is prepared comprising a preservative and a stabilizer; and, more preferably, the immunogenic ition of the present invention is prepared comprising Amphotericin, formaldehyde, gentamycin, EDTA, glycerol, and combinations thereof.

An “immunogenic or immunological composition” refers to a composition of matter that comprises at least one antigen, which elicits an immunological response in the host of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest.

Usually, an “immunological se” es but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or gamma—delta T cells, directed specifically to an n or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that ance to new infection will be enhanced and/or the clinical severity of the disease d. Such protection will be demonstrated by either a reduction or lack of clinical signs normally displayed by an infected host, a quicker recovery time and/or a lowered duration or bacterial titer in the tissues or body ﬂuids or ions of the infected host.

The term “in need of such administration” or “in need of such administration treatment,” as used herein means that the administration/treatment is associated with the boosting or improvement in health or any other positive medicinal effect on health of the animals which receive the genic composition in accordance with the present invention, such as reducing the incidence or ty of a viral infection or disease.

“Equine rhinitis A virus (ERAV)” refers to an Aphthovims in the family aviridae, and was previously known as Equine rhinovirus l. “ERAV” as used herein includes inactivated forms. In one embodiment, the ERAV is strain ERAV/ON/OS having ion number PTA-11829 deposited on April 14, 2011 with the ATCC (American Type e Collection, PO. Box 1549 Manassas, VA 20108 USA) in accordance with the Budapest Treaty, and that was recovered from Rabbit-kidney-13 (RK-13) cell culture from a nasal swab from a horse in Ontario Canada in 2005. The ERAV/ON/OS when reverse transcribed and sequenced has SEQ ID NO: 1 in its 5’ UTR region.

The reverse ribed genomic sequence of ERAV/ON/OS is SEQ ID NO:2.

The polyprotein encoded by the genomic sequence of ERAV/ON/05 is SEQ ID NO:3.

The ERAV strains useful in the immunogenic compositions of the invention have reverse transcribed 5’UTR nucleotide sequences that have greater than 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:1. In some embodiments, the ERAV strains have genomic ces that when reverse transcribed into DNA are greater than 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:2 or have polyprotein coding sequences that are more than 97%, 98%, 99%, or 100% identical to the polyprotein coding sequence in SEQ ID NO:2. In some embodiments, the ERAV have polyproteins with an amino acid sequence greater than 95%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:3 or to the VP1 ce within SEQ ID NO:3. In yet other embodiments, the ERAV has an L protein, VP2, VP3, VP4, VPO, 2A, 2B, 2C, 3A, 3B, 3C or 3D, that have an amino acid sequence with r than 80%, greater than 90%, greater than 99%, or 100% identity to the same protein found in SEQ ID NO:3. All of the ERAV strains are, when not inactivated or attenuated, infective and able to ate in host cells. e rhinitis B virus (ERBV)” refers to an Erbovirus in the family Picornaviridae, and was previously known as Equine rhinovirus 2. “ERBV” as used herein es inactivated forms. In one embodiment, the ERBV is a strain deposited with the ATCC that was recovered from Rabbit—kidney—l3 (RK—l3) cell culture from a nasal swab from a horse in Ontario Canada (ATCC Accession NO: PTA—11828) that was deposited with the ATCC (American Type Culture Collection, PO. Box 1549 Manassas, VA 20108 USA) on April 14, 2011 under the Budapest Treaty. The ERBV strains useful in the immunogenic compositions of the invention have c sequences that when e transcribed into DNA are greater than 85%, 90%, 95%, 98%, 99% or 100% identical to the reverse transcript of the genomic sequence of the ERBV strain having ATCC Accession No. PTA-11829, or have polyprotein coding sequences that are more than 97%, 98%, 99%, or 100% identical to the polyprotein coding sequence of the ERBV strain having ATCC Accession No. PTA—11829. In some embodiments, the ERBV strains useful in the invention have polyproteins with an amino acid sequence greater than 95%, 95%, 97%, 98%, 99%, or 100% cal to the polyprotein sequence of the ERBV strain having ATCC Accession No. PTA—11829. In yet other embodiments, the ERBV has an L n, VP4. VPZ. V1333, Vl’l. 2A, 2.8, ’2 T, 3A (Vpg). 38, 3Cpro, 3?le that have an amino acid sequence with greater than 80%, greater than 90%, greater than 99%, or 100% identity to the same protein found in the ERBV strain having ATCC Accession No. PTA—11829. All of the ERBV strains are, when not inactivated or attenuated, infective and able to replicate in host cells.

The term “West Nile Virus” antigen means, but is not limited to the components of the WNV virion that are genic when present in an animal, and most particularly protein components, such as envelope and ructural proteins, of the WNV that e humoral or cellular immune responses When present in an animal. Such antigens can include DNA, protein subunits, modified live virus, and inactivated Virus. In preferred forms of the ion, the WNV antigen or antigens comprise inactivated or killed, and even more preferably, North American dominant, WNV strains.

The term “North American West Nile Virus ns)” refers to, but is not limited to any West Nile Virus strain that has ever been discovered on the North American continent.

Preferably, a North American West Nile Virus strain has a sequence identity to the NY99 strain (GenBank accession no. AF196835 or NCBI reference sequence NC_00942.1 of at least 97%, even more preferably, at least 98%, still more preferably, at least 98.5%, more preferably, at least 99%, even more preferably, at least 99.2%, and, most preferably of at least 99.4%. WN02 is a representative example of a WNV strain that can be referred to as a North American Dominant West Nile Virus strain. Specifically, North American nt strains are those having at least 1 nucleotide change resulting in an amino acid change from the WN99 isolates. Strain NY99 (GenBank accession no. AF196835) serves as a reference strain for ining if a strain is North American Dominant. In addition, these strains may have one or more silent amino acid changes. In some embodiments, the nucleotide change results in an amino acid change in an envelope protein of the strain and, more ably, the tide change results in an amino acid change from valine to alanine. ably, this amino acid change is associated with a greater ability to replicate in the intermediate host, , the mosquito. More preferably, North American Dominant strains include either (and preferably both) a U to C mutation and a C to U mutation at positions 1442 and 2466 (in comparison to a North American strain, e. g., NY 99), respectively. Still more preferably, North American Dominant strains further include a mutation in the nucleotide sequence encoding the E n and the C to U mutation at position 9352 in the sequence encoding the NSS protein (again in comparison to a North American , e.g., NY 99). These preferred mutations are shown in Phylogenetic is of North American West Nile Virus Isolates, 2001-2004: Evidence For the Emergence of a Dominant Genotype, C.

Todd Davis, et. al, Virology 342, p. 252-265 (2005), the teaching and content of which is hereby incorporated by reference. West Nile Virus strains, for purposes of the present invention, are not limited to horse and equine West Nile Virus strains but encompass, while not being limited to, those West Nile Virus strains of bird origin, donkey origin, pig origin, human origin, mammal origin, and equine origin.

“Sequence Identity” as it is known in the art refers to a onship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as ined by the match between strings of such sequences. Upon such ent, sequence identity is ascertained on a position—by—position basis, e.g., the sequences are “identical” at a ular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position ties is then divided by the total number of nucleotides or residues in the reference sequence to give % ce identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational lar Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); er Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence is in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and ux, J ., eds., M. Stockton Press, New York (1991); and o, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to ine the ce identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program e (Devereux, J ., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J.

Molec. Biol, 215 :403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, ul, S. F. et al., J. Molec. Biol, 215:403-410 (1990), the ngs of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 85%, preferably 90%, even more preferably 95% nce identity” to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 15, preferably up to 10, even more preferably up to 5 point ons per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide ce having at least 85%, preferably 90%, even more preferably 95% identity relative to the reference nucleotide sequence, up to 15%, preferably %, even more preferably 5% of the tides in the reference sequence may be deleted or tuted with r nucleotide, or a number of tides up to 15%, preferably 10%, even more preferably 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference tide sequence or anywhere n those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference ce. Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the nce sequence except that the given polypeptide sequence may include up to 15, preferably up to 10, even more preferably up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, even more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, ably up to 10%, even more preferably up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 15%, ably up to 10%, even more ably up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxy al positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among es in the reference ce or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity. In the present disclosure, it is understood that SEQ ID NO:1 (5’ UTR) and SEQ ID NO:2 are the DNA sequences that result from reverse transcription of the 5’ UTR and the entire genome of ERAV/ON/05, respectively. Likewise, SEQ ID NO: 3 refers to the amino acid sequence corresponding to the polyprotein encoded by the genomic sequence of ERAV/ON/05. Percent identity of a given ERAV strain in comparison to SEQ ID NO:1 or SEQ ID NO:2 is thus meant to refer to the corresponding DNA ce ing from reverse transcription and sequencing.

“TCID50” refers to tissue culture infective dose ing 50% of cells in a culture inoculated with the virus.

For purposes of the present invention the terms “strain” and “isolate” have the same g and are used interchangeably.

“Clinical signs” or “clinical symptoms” for ERAV and ERBV e but are not limited to pyrexia, elevation in temperature, increased lung sounds, lymphadenopathy, nasal rge, ocular rge, and cough, and pharyngitis. Still other signs or symptoms include anemia, anorexia, lymphadenitis of the head and neck, edema of the legs, lethargy, and pain.

Additionally, clinical signs of ERAV and/or ERBV infections may include those associated with Equine Herpes virus and Equine Inﬂuenza virus. In one embodiment the clinical signs for ERAV include cough, pharyngitis, pyrexia, elevations in temperature, increased submandibular lymph node size, nasal discharge, and ocular discharge. In n embodiments, the clinical signs of ERAV include an increased incidence of abortion in pregnant mares. In another ment the clinical signs to be addressed by an immunological composition disclosed herein are those of respiratory ions, such as those cause by one or more of ERAV, ERBV, EIV, EHV-l, and EHV-4.

“Clinical signs” or “clinical symptoms” of West Nile Virus, for purposes of this invention, include, but are not limited to, symptoms or lesions associated with encephalitis, Viremia, anorexia, depression, fever, weakness, abnormal gait, sis of hind limbs, impaired vision, ataxia, aimless wandering, convulsions, inability to swallow, coma, posterior weakness, paralysis, poor coordination, depression and related behavior, tremors, convulsions, paddling of the limbs, neurological problems, swelling of the central nervous system, death, and combinations f. The al signs exhibited by an infected animal vary depending on the ty of infection.

“Clinical Signs” or “clinical symptoms” of Equine Herpes virus, for purposes of this invention e, but are not limited to, abortion, neurological encies, respiratory disease, reproductive system encies and failure, and symptoms relating to the central nervous system. Additionally, clinical symptoms of EHVl include, but are not limited to, the enon of foals infected with EHVl, exhibiting atory complications, passing the virus to the older members of the herd who then exhibit uctive deficiencies, including abortion, and neurological deficiencies, normally exhibited in the central nervous system.

“Clinical Signs” or “clinical symptoms” of Eastern Equine Encephalomyelitis, Western Equine Encephalomyelitis, and Venezuelan Equine alomyelitis, for purposes of the present invention are those symptoms normally known to be associated with encephalomyelitis, including, but are not limited to fever, nervous signs such as sensitivity to sound, periods of excitement, and restlessness, brain lesions, drowsiness, drooping ears, circling, abnormal gait, paralysis, loss of appetite, depression, head pressing, lack of coordination, long- term disability, brain damage, death, and combinations thereof. “Safety” as used herein, refers to the absence of adverse consequences in the vaccinated animal following vaccination, including but not limited to, potential reversion of e virus to virulence and clinically significant side effects, such as persistent systemic illness or unacceptable inﬂammation at the site of vaccine administration.

“Reduction of the incidence and/or severity of clinical signs” or “reduction in the incidence and/or severity of clinical symptoms,” as referred to herein, means reducing the number of infected animals in a group, reducing or eliminating the number of animals exhibiting al signs of infection, or reducing the severity of any al signs that are present in the animals, in comparison to ype infection. For example, such clinical signs included Viremia, fever, dy response, ocular discharge, nasal discharge, and histopathology.

Preferably, these are reduced in animals receiving the composition of the present invention by at least 10% in comparison to animals not receiving the vaccination which may become infected.

More preferably, clinical signs are reduced in animals receiving the composition of the present invention by at least 20%, more ably by at least 30%, even more preferably by at least 40%, and even more preferably by at least 50%.

“Duration of Immunity,” as used herein, refers to the m number of days during which an animal produces an immunogenic response such that the animal will be vely immune from contracting a Virus and/or benefit from reduction of incidence and/or severity of al signs, as described herein.

The term “inactivated” and “inactivated virus” refers to a previously virulent Virus that has been heated or chemically treated to inactivate, kill, or otherwise modify the Virus to substantially eliminate its virulent properties while retaining its immunogenicity. In a preferred embodiment, the inactivated Viruses disclosed herein are inactivated by treatment with an inactivating agent. Suitable inactivating agents include beta—propiolactone, binary ethyleneimine, aldenyde, and formaldehyde. In some ments, the inactivating agent is formaldehyde.

The terms ne” and ogenic composition”, when used herein, are meant to be used interchangeably.

Any West Nile Virus strain(s) or isolate(s) can be used in accordance with the present invention. In a preferred embodiment, the isolate is selected from one or more of the following: New York (Northeastern North American) Isolate (WN—NY 99), Horse Origin, 1999, New York (Northeastern North American) Isolate (WN—NY 99), Crow Origin, 1999, United States Department of Agricultures Isolate 292206 (USDA 2004), Donkey Origin, United States Department of Agriculture Isolate 405330 (USDA 2005), Horse Origin, North American e (WN—Texas—2002/2003), Southeast Texas Coastal Isolate 2002, Mexico (Tabasco) Isolate 2003, and combinations thereof, and in a more preferred embodiment the isolate is ed from one or more of the following: United States Department of Agricultures Isolate 292206 (USDA 2004), Donkey Origin, United States Department of Agriculture Isolate 405330 (USDA 2005), Horse Origin, North American Isolate (WN—Texas—2002/2003), Southeast Texas Coastal Isolate 2002, Mexico (Tabasco) Isolate 2003, and combinations thereof. In a most preferred embodiment, the isolate is United States Department of Agriculture Isolate 405330 (USDA 2005), Horse Origin singularly or in ation with one or more isolates as listed above. In an additionally preferred embodiment, those isolates which are part of the North an West Nile Virus isolates are included. In yet another preferred embodiment North American Dominant West Nile Virus isolates are included. In on to those listed above, specific isolates include, but are not limited to, WN02 and isolates which have at least 1, preferably at least 2, and even more preferably at least 3 nucleotide s resulting in at least one amino acid change from the WN NY99 isolates, and most red are s with the amino acid change from valine to alanine at position 159 of the envelope protein. Most preferred North American Dominant strains include, but are not limited to: NY2002Nassau, NY2002Clinton, NY2002Queens, GA20021, GA20022, TX20021, TX20022, IN2002, NY2003Albany, NY2003Suffolk, NY2003Chatauqua, CO20031, CO20032, , TX2003Harris4, TX2003Harris6, TX2003Harris7, TX2003Harris10, AZ2004, and TX2004Harris4, and combinations thereof. The strains of West Nile Virus useful in the vaccine or immunogenic composition of the present invention can be any strain or isolate. In a red embodiment, the North American Dominant West Nile Virus strain used is either E-159 (Horse Origin) or E-159 (Donkey Origin). A representative strain of such a North American Dominant WNV strain includes the Horse Origin 2005 strain ted with the ATCC (ATCC Accession NO: PTA—9409), located at 10801 University Boulevard, Manassas, VA, 20110—2209, on August 14, 2008, under the provisions of the Budapest Treaty.

Equine Inﬂuenza strains useful in the e or immunogenic ition of the present invention can be any strain or isolate. Representative strains e Equi—2/Ohio/03, deposited as ATCC Accession NO: PTA—9522, Equi—2/Kentucky/95, deposited as ATCC Accession NO: PTA—9523, and Equi—2/New Market/2/93, deposited as ATCC Accession NO: PTA—9524.

Representative strains ATCC Accession Nos. PTA—9522, PTA—9523, and PTA—9524 were each deposited with the ATCC at 10801 University ard, Manassas, VA, 20110—2209 on September 23, 2008, under the provisions of the Budapest Treaty.

Equine Herpes Virus (“EHV”) strains useful in the vaccine or immunogenic composition of the present invention can be any strain or isolate. Representative strains e EHV Subtype l, deposited as ATCC Accession NO: PTA—9525, and EHV Subtype 4, deposited as ATCC Accession NO: PTA—9526. Representative strains ATCC Accession Nos. PTA—9525 and PTA—9526 were each deposited with the ATCC at 10801 University Boulevard, Manassas, VA, 20110—2209 on ber 23, 2008, under the provisions of the Budapest Treaty. n Equine Encephalomyelitis strains useful in the vaccine or immunogenic composition of the present invention can be any strain or isolate. A entative strain includes the Fleming Strain, deposited with the ATCC (ATCC Accession NO: PTA—9410), d at 10801 University Boulevard, Manassas, VA, 20110—2209, on August 14, 2008, under the provisions of the Budapest Treaty.

Venezuelan Equine Encephalomyelitis strains useful in the e or immunogenic composition of the present invention can be any strain or isolate. A entative strain includes the TC—83 , deposited with the ATCC (ATCC Accession NO: 11), located at 10801 University Boulevard, Manassas, VA, 20110—2209, on August 14, 2008, under the provisions of the Budapest Treaty.

Eastern Equine Encephalomyelitis strains useful in the vaccine or immunogenic composition of the present invention can be any strain or isolate. A representative strain includes the NJO strain, deposited with the ATCC (ATCC Accession NO: PTA-9412), located at 10801 University Boulevard, Manassas, VA, 20110-2209, on August 14, 2008, under the provisions of the Budapest Treaty.

Tetanus toxoid strains useful in the vaccine or immunogenic composition of the present ion can be any strain or isolate. A representative strain is that taken from a master seed of Clostridium tetani from The Massachusetts ment of Public Health Institute of Laboratories in Boston, Massachusetts.

The vaccine or immunogenic composition as disclosed herein is safe for administration in ERAV or ERBV susceptible species, particularly equidae, at any age. In a preferred embodiment, the present invention is safe for administration to foals 12 months of age or older, more preferably, it is safe for administration to foals 10 months of age or older, more ably, it is safe for administration to foals 8 months or older, more preferably, it is safe for administration to foals 6 months of age or older, more preferably, is safe for administration to foals 4 months of age or older, more preferably, it is safe for administration to foals 2 months of age or older, more preferably, it is safe for administration to foals 1 month of age or older, even more preferably, it is safe for administration to foals between 1 day and 1 month of age, and, most ably, it is safe for administration to foals 1 day of age or older.

The compositions as disclosed herein can be administered in any conventional manner. Examples of administration methods include any that afford access by cells of the immune system to the immunogenic ition including oral, transdermal/intradermal, intravenous, subcutaneous, intramuscular, intraocular, intraperitoneal, ectal, intravaginal, intranasal, astrical, racheal, ulmonarial, or any combination thereof. In a preferred embodiment, the vaccine is administered parenterally, preferably intranasally, subcutaneously, or intramuscularly, and in the most preferred embodiment the vaccine is administered intramuscularly.

In one embodiment, provided is a method for preparing and immunogenic composition comprising an ERAV and/or ERAB as disclosed herein. In one embodiment, the method comprises: a) infecting a susceptible cell line with ERAV or ERBV; b) g the infected cell line in growth media until a cytopathic effect (CPE) is attained; c) harvesting the media; d) filtering the media to yield a filtered media; and e) contacting the filtered media with an inactivating agent to obtain the inactivated ERAV or ERBV.

In one embodiment, the method comprises providing strain ERAV/ON/OS or a ERBV strain having ATCC Accession NO: PTA—11829. These strains are used to infect a susceptible cell line having advantageous growth and secretion properties useful for vaccine preparation. A preferred susceptible cell line is a Vero cell line such as a Vero76 or an E-Vero cell line.

Suitable growth media e E199. This media may be supplemented with L— glutamine solution and an antibiotic such as gentamicin sulfate solution. In some embodiments the growth media is E199 supplemented with L—glutamine solution (up to 2 mM), and gentamicin sulfate solution (up to 30 ug/mL). In some ments, viral ﬂuids are harvested when the thic effect CPE reaches 75% or r. The harvested media are pooled and filtered such as through 5.0 micron pore—size rated opylene filter cartridges. The strains are inactivated chemically such as by treatment with 01—02% formaldehyde solution over a suitable period of time, for example 48 hours to 72 hours and the resulting ﬂuids are concentrated. In some embodiments, preservatives and adjuvants are next added. Suitable preservatives include one or more of ericin B, gentamicin sulfate, and formaldehyde. In other embodiments the adjuvant is HRA—S. In still other embodiments the adjuvant is HRA—3 with cottonseed oil (C80).

In one embodiment and in accordance with the methods disclosed herein, provided is an immunogenic composition comprising ERAV/ON/05 and/or a ERBV strain having ATCC Accession NO: 829 and Amphotericin B, gentamicin e, formaldehyde, and HRA—S prepared according to the methods disclosed herein.

Examples The following examples are set forth below to illustrate specific embodiments of the present ion. These examples are merely illustrative and are understood not to limit the scope or the ying principles of the present invention.

Example 1 This example illustrates one embodiment of a Equine Rhinitis A Virus composition in accordance with the present invention.

Materials and Methods Equine Rhinitis A Virus strain ERAV/ON/05 (ATCC Accession No. PTA-11828) was recovered from Rabbit-kidney-l3 ) cell e from a nasal swab from a horse in Ontario Canada. The virus was passaged once on E-Vero cells to produce a high pre-titered master stock, and was then diluted with cell culture media to produce the Master Seed Virus.

Master Cell Stock is an E—Vero cell line grown and maintained using E199 mented with L— Glutamine solution (up to 2 mM) and Gentamicin sulfate solution (up to 30 ug/mL). Inactivated ERAV/ON/05 was produced according to the following l procedure.

Frozen Master Cell Stock is thawed at room temperature °C) and used to inoculate a range of pre—sterilized T—25 cm2 up to T—150 cm2 ﬂasks or pre—sterilized 850 cm2 or 1050 cm2 PETG roller bottles. Thawed cells are suspended in growth medium at the rate of 0.15 to 0.40 mL per cmz. Cells are incubated at 36—38°C for up to seven days. Cultures planted from frozen stock may be re—fed with medium to remove residual Dimethyl Sulfoxide (DMSO), to remove excessive debris, to stimulate the growth of cultures which have not reached conﬂuency, or to maintain viability of nt cultures. Cells are passaged by decanting the spent medium and adding 1—10 mL (depending on the size of the vessel), of 25% trypsin—EDTA solution to each vessel. The vessels are agitated gently until the cells slough from the surface. The cells are removed from the vessels by rinsing with growth medium and pooled er. The range of cell culture passages is one to .

Prior to inoculation, cell growth medium is decanted from cells that are at least 90% conﬂuent. The cell sheet is rinsed with 20-50 mL of virus infection media (E199 supplemented with L-Glutamine solution (up to 2 mM) and Gentamicin sulfate solution (up to 30 , then re—fed with virus infection media at the rate of 0.15 to 0.40 mL/cmz. Vessels are then ated at a Multiplicity of Infection (MOI) of 0.0005 to 0.005. Roller bottle cultures are incubated at 36—38°C for two to five days at 0.2—0.4 rpm.

During the growth period, cultures are checked for CPE microscopically and for gross contamination macroscopically. Unsuitable cultures are discarded after sterilization.

Virus ﬂuids are harvested when CPE reaches 75% or greater. Roller s are swirled to remove loose cells, and ﬂuids are pooled into sterile 2—20 L glass, plastic, or PETG bottles, 20 L sterile polypropylene containers or 2—200 L sterile stainless steel tanks appropriate for clarification. Pooled ﬂuids may be held for up to seven days at 2-80C prior to clarification.

Only ﬂuids from monolayer es showing evidence of viral ion are harvested. Bottles ting contamination are discarded. A sample of pooled, clarified ﬂuids is collected before inactivation for titration by TCID50. Fluids with a titer of 106'2 TCIDso/mL or greater may be used in the preparation of final product. Multiple lots can be blended to achieve the minimum titers.

Harvested lots are ied by filtration using 5.0 micron pore—size rated polypropylene filter cartridges. Post—clarified harvest lots may be stored for up to seven days at 2—8°C. Clarified ﬂuids are then inactivated with formaldehyde solution, USP, 01—02% by volume, transferred to a secondary ner, and held at room temperature (18—26°C) with agitation for a minimum of 48 to 72 hours. A sample of inactivated ﬂuids is taken for inactivation nce testing prior to concentration. Inactivated lot material is held at 2—8°C prior to concentration. Clarified, inactivated virus ﬂuids are concentrated by a factor of 5X to 50X using tangential ﬂow ultra—filtration membrane cartridges with molecular weight cut—off ratings of not more than 10,000 Dalton MW.

A number of suitable adjuvants may be added to the vaccine formulation, most preferably HRA—5. Other adjuvants include HRA—3 with cottonseed oil (CSO). Typical processing steps may be employed such as mixing, blending, microﬂuidization, and emulsification, of the adjuvant and/or the harvested virus antigens with other ingredients.

The product is then assembled to ﬁnal formulation. In a batching method in one exemplary ment, the required amounts of adjuvant and MEME Diluent are combined in a sterile container and the pH is adjusted to approximately 7 with Sodium Hydroxide.

ERAV, gentamicin sulfate, formaldehyde, and Amphotericin B are added one at a time during constant mixing. The pH is ed to 6.8—7.0 with sodium hydroxide or hydrochloric acid and the serial is mixed for a minimum of 18 hours at 2—8°C. The completed bulk serial is then transferred to a suitable storage container and stored at 2—8°C. Amphotericin B may be added at a concentration of up to 2.5 ug/mL of the diluent volume as a preservative. Gentamicin sulfate is added at a concentration of up to 30 ug/mL of the diluent volume as a preservative dehyde is added at a concentration of 0.1% of the diluent volume as a preservative. An adjuvant such as HRA-S is added at a concentration of 10% v/v of the final serial volume.

The vaccine is given by typical hypodermic injection, with booster vaccinations if desired.

This investigation was carried out to obtain an cy evaluation of a Equine Rhinitis A Virus vaccine to protect horses from challenge With Equine Rhinitis A Virus.

Materials and Methods A high dose A—9 (107-5 TCID50/mL) and low dose A—10 (107-0 TCID50/mL) ERAV vaccine were prepared according to Example 1.

Table l: A—9 e formulation (1 mL): N/OS 107- TCIDso/mL HRA-S 100 uL t, MEM-E+ containing Gentamicin, q.s. ug/mL of diluent volume and Formaldehyde, 0.1% of diluent volume Table 2: A—10 1 vaccine formulation (1 mL): ERAV/ON/OS 107“O TCIDSO/mL HRA-5 100 uL Diluent, MEM-E+ containing Gentamicin q.. ug/mL of diluent volume and Formaldehyde, 0.1% of diluent volume A V—05 placebo 1 mL formulation was prepared as in the A—9 and A—lO vaccines but without the ERAV/ON/05 antigen.

A total of 44 horses were randomly divided into one of three treatment groups ting of a 15 horse V-05 placebo control group, a 14 horse A—10 se group, and a 15 horse A—9 high-dose group. Horses were vaccinated with a 1 mL dose product adjuvanted with HRA-5 for three total doses, with a 21 day interval in between doses. Horses were subsequently challenged 21 days after the third vaccination by intranasal aerosolization with a 107‘0 TCID50/mL nebulized Rhinitis A dose over a four minute period. Horses were evaluated for clinical signs of signs of ature, nasal exudate, ocular discharge daily. Blood for serum neutralization (SN) was taken weekly.

Challenge Virus The challenge virus was produced in tissue culture on E—Vero cells. The titer of the challenge virus was determined to be 1 x 106'9 TCIDso/mL on the day of challenge. Challenge virus was diluted on the morning of challenge 1: 10 with tissue culture media to effect a titer of l x 105-9 TCID50/mL.

Intranasal Challenge Method Sedivet® (romifidine hydrochloride), a sedative and analgesic, was administered intravenously to each horse prior to challenge at a dosage of 50 [Lg/kg of body . Each horse was then challenged with imately 106‘2 TCID50 of equine is A virus. The challenge virus was administered intranasally as an aerosol produced by a nebulizer into an Equine AeroMask (Trudell Medical ational, Ontario, Canada) by the following method.

Four milliliters of 105‘9 /mL challenge virus were placed into the nebulizer cup in the AeroMask . A pressure hose was fitted from an air compressor to the inlet port of the nebulizer. The outlet tube was then inserted into the Aero Mask attached to the head of the horse being challenged and approximately 10 psi of air re was applied to the inlet port for three minutes. During this time approximately two milliliters of challenge Virus ﬂuid was aerosolized directly into the nostrils of the horse being challenged.

Serum Neutralization A standard microtiter serum neutralization test was employed in this study. A standard microtiter serum neutralization test was employed in this study. All sera were tested in sterile ﬂat bottom microtiter plates using five wells per dilution and an 8 well dilution series for each of the 5 test wells. Each of the 5 test wells contained 25 ul of serum dilution mixed with 25 pl of the indicator virus and 150 pl of a freshly planted eVero cell suspension containing approximately 5 X 104 cells. The test indicator virus used was either Equine Rhinitis Virus Type I. Serum neutralizing antibody titers are expressed as Reed—Muench ID50 titers.

For performance of the test, two—fold dilutions of each test serum was made in a sterile ﬂat bottom microtiter plate using five replicate wells per test serum and an 8 well dilution series. Dilutions were made with an able volume single or multi—channel pipeting instrument using e microtiter tips. The volume of serum added each of 5 wells of the first row was 50 Ml. All other wells contained 25 ul of DMEM (no FBS). ing serial dilution down the plate, 25 ml was ded from the last row. 25 pl of a pre—determined dilution of the indicator Virus was added to each test well. Plates were then mixed and incubated for one hour at 37°C in 5% C02. On conclusion of the incubation period, 150 ul of a suspension containing 4 x 106 /mL eVero cells was added to each test and cell control well. The plates were incubated at 37° C in a C02 tor for 5 days, at which time plates were microscopically examined for CPE typical of equine rhinitis virus.

Nasal Exudate Evaluation All nasal e observations were made prior to collection of nasopharyngeal swabs. On the Day of Challenge and for 10 days hallenge, the nasal passages and muzzle of each of the 44 vaccinated and control horses were ed and graded using the grading and scoring description listed below.

The scoring grades of 0 through 6 were assigned on the basis of the severity of the disease indicated by each of the following classiﬁcation: Table 3: Scoring Grades Score Descri tion of s mtoms Essentially normal indicates the horse was clean and ially free of nasal exudate Slight clear serous discharge that may be frequently observed in both diseased and normal horses Very slight mucopurulent discharge indicates that mucus was definitely present in small s in either one or both nostrils Moderate clear serous discharge is indicative of a definite increase in volume over that normally observed Slightly mucopurulent is a discharge easily observed in one or both nostrils Copious clear serous discharge that is generally observed only in diseased horses Moderately mucopurulent indicates that mucoid discharges were present in large quantities in both nostrils Heavy mucopurulent indicates that copious amounts of a mucoid rge filled both nostrils Ocular tion Ocular discharge was evaluated daily at the time of nasal exudate tion.

Ocular discharge scores were recorded as 0=normal; l=mild to moderate ocular discharge, and 2=severe ocular discharge.

Nasopharyngeal Viral Isolation On each observation test day each nasal passage of each vaccinated and control horse was swabbed deeply by means of a sterile WECK-CELTM surgical spear (Edward Weck and Company, Inc., Research Triangle Park, NC. 27709) attached to an ll—inch long e c pipette. On collection, each of two surgical spears was immediately placed in a single tube containing 4 mL of chilled transport medium (E—l99 supplemented with gentamicin, L— glutamine, 2X Pen/Strep, 2X Amphotericin B).

For isolation of virus, the tubes were mixed, the swabs aseptically removed, and the medium centrifuged at 1500 rpm for 10 to 15 minutes to remove particulates. Medium was ed through a 0.2 ML syringe filter prior to inoculation on tissue culture cells. After filtration, 4—6% of e 85% sucrose solution was added to each sample for freezing at —80°C in order for all samples to be tested concurrently.

For ion of virus, the tubes were mixed, the swabs aseptically removed, and the medium centrifuged at 1500 rpm for 10 minutes to remove particulates. Medium was filtered through a 0.2 ML syringe filter prior to inoculation on tissue culture cells. One mL of the clarified transport medium was used to inoculate a 2 cm2 two day old monolayer of E—Vero cells grown in a 24 well tissue culture plate from which the growth medium had been aseptically removed.

Following ation, the inoculum was allowed to adsorb on the cell monolayer for one hour at 37°C in a humidified tor containing a 5% C02 atmosphere. After the adsorption period, an additional 1 mL of re—feed medium (E—199 containing 7% fetal bovine serum (FBS), 2mM L— glutamine, Gentamicin 2X Pen—Strep and 2X Amphotericin B) was added to each well.

Following addition of re—feed media the plates were then incubated at 37°C in a C02 incubator.

Each test and l tissue culture well was examined microscopically for 7 days for signs of cytopathic effect (CPE) typical of the ERAV challenge virus. Wells that were negative at the end of the 7 day observation period were subcultured onto fresh cells and observed for an additional 7 days.

STATISTICAL EVALUATION METHODS Data from all horses vaccinated with either A—9 or A—10 were combined for tical evaluation. The inﬂuence of vaccination on the duration of disease (number of days with nasal scores> 0) was evaluated using the Kruskal-Wallis and Hodges—Lehmann test (the NPARlWAY procedure in SAS, SAS Institute, Cary NC). ty of disease was ted by comparing the maximum disease status between the vaccinated horses and the control horses.

Nasal scores were dichotomized to g 1.5 and> 1.5 based on the distribution of outcomes. The prevented fraction (PF) and 95% confidence als (CI) were estimated. Ocular scores were evaluated as present or absent and the prevented fraction and 95% confidence intervals were estimated (the FREQ procedure in SAS). Repeated es analysis appropriate for continuous data was used to assess the effect of vaccination on body temperature and serum neutralization titers (the MIXED procedure in SAS). The proportion of horses which were virus positive over time and buffy coat positive over time was evaluated using Fisher's exact test at each time point (the Freq procedure). Nasal and ocular scores were ed by Wilcoxon's rank sum test at each time point (the NPARlWAY procedure).

Results and Conclusions VIRUS ISOLATION FROM NASAL SWABS (Virus Shedding) and BUFFY COATS (Viremia) The proportion of animals which were virus positive was significantly lower in the vaccinated groups on days 2 through 7 (Table 4 below and Figure 1) as compared to control group (P<0.05).

Table 4. Proportion of nasal swab virus positive over time (* P<0.05 versus control, Fisher’s Exact test on each day) Day Control A—9 A—10 A-9 + A-lO 0 0 0 0 0 1 25 7.1 7.7 7.4 2 100 20* 231* 21.4* 3 100 467* 57.1* 51.7* 4 100 60* 57.1* 58.6* 93.3 53.3* 35.7* 448* 6 60 40 0* 20.7* 7 100 46.7* 78.6 62.1* 8 26.7 13.3 0 13.8 9 0 6.7 0 3.5 0 6.7 0 3.5 11 0 0 0 0 12 0 0 0 0 13 0 0 0 0 14 0 0 0 0 Buffy coat positive animals were less frequent in the ated group on days 4—7 as compared to the l group (Table 5 below and Figure 2).

Table 5: Proportion of Buffy Coat positive over time (* P<0.05 versus control, Fisher’s Exact test on each day) 4 73.3 35* 80 39* 6 8O 35* 7 33.3 0* 8 o 3.5 9 o 0 1o 0 0 11 o o 12 o o 13 o o 14 o 3-5 Serum Neutralization Table 6: Summary of body temperature and serum neutralization P-Values ——-E-Vaccine * Da Temoerature 0.9269 < 0.0001 0.0255 SN titer In transformed < 0.0001 < 00001 < 0.0001 Mean body temperature values (rectal temperature measured with ated GSA Electronics meter probe) on each challenge day were always within normal body temperature parameters. Large increases in SN titers resulting from vaccination were found (see also Fig. 3) following challenge.

Nasal And Ocular Evaluation Mean ranks for nasal scores were lower on days 4 though 10 and days 12—13 in the vaccinated group compared to the l group (Table 7 below and Figure 4). The mean ranks for ocular scores were lower on day 7 in the vaccinated group as compared to the control group (see also Figure 5).

Table 7: Mean rank for nasal and ocular score across time (*P<0.05 versus control; Wilcoxon’s rank sum test on each day, A-9 and A-lO combined). __A-9+A-10 A-9+A-10 Disease Duration: Nasal Exudate Evaluation Table 8: Summary of the effect of vaccination on the duration of disease (Number of days with nasal score > 0) Group Minimum 25th quantile 50Lh quantile 75th quantile Maximum Control 8 9 10 l l l 3 Vaccinated 0 1 3 9 l 3 Table 9: Effect of ation on the duration of disease (nasal scores; >“significantly lower than control group by l—Wallis test P<0.05) Control Vaccinate Shift in days 95% confidence interval Duration 10 3 7* 3, 8 days Long duration of nasal rge in controls was found following challenge (Table 8, see also Figure 4). Vaccinated group showed a significant reduction in duration of nasal discharge. Controls experienced a minimum of 8 days of nasal discharge vs 11 vaccinates (38%) with 1 day or less. Two thirds of vaccinates had r duration of nasal discharge than the minimum of 8 days in the control group. Duration was from first to last abnormal observation, even if horse had normal days in between. The number of days animals were sick with al signs of atory disease (nasal score > 0) was significantly shorter (shift of 7 days) in the vaccinated group ed to the control group.

Table 10: Maximum Nasal Scores —Signif1cant difference between the two distributions of scores (Kruskal—Wallis test, P = 0.0157).

Grouo Maximum Nasal Score _I-I-Imn 13 10 Vaccinated 4 14% 2 7% 45% 34% Table 11: Nasal scores (3 1.5 and > 1.5) Group % with score P-value Prevented Fraction 95% confidence interval Control 66.7% < 0.0588 0.48 0.042 Vaccinate 34.5% 0.72 Vaccination also reduced severity of nasal discharge (maximum score on any day post—challenge). Maximum nasal scores were compared n groups (Table 10). The m nasal score for horses in the control group was 1.5. Results were thus dichotomized to scores 3 1.5 and > 1.5 for the evaluation of disease severity (Table 11). The prevented on was 48% with a lower confidence limit greater than 0. The overall bution of maximum nasal scores was significantly reduced by vaccination al—Wallis test, P=0.0157).

Ocular Evaluation Table 12: Ocular scores (present or absent) Group % with score P—value Prevented Fraction 95% confidence interval Control 66.7% < 0.0001 0.897 0.587 Vaccinate 6.9% 0.9741 Since only two values were reported for ocular scores (0 or 2), results were dichotomized to present or absent within an individual. The prevented fraction was then used to evaluate the effect of vaccination on the presence of ocular signs. The vaccine significantly d the severity of ocular discharge resulting from infection with ERAV. Mean ocular scores over time are also shown in Figure 5.

Data from this study demonstrate that administration of intramuscular doses of the inactivated Virus is capable of immunizing an animal to high levels of antibody detection which prevent ocular disease and reduce severity and duration of nasal discharge. Vaccine was highly effective in preventing any ocular discharge following ERAV challenge. Nasal discharge persisted for a relatively long period hallenge, while ocular rge peaked and abated quickly. Hence the vaccine showed a significant improvement in nasal discharge for a prolonged period (9 of 10 consecutive days), whereas vaccine significantly reduced ocular discharge for 1 day during which ocular signs were most severe. In addition, vaccination significantly reduced nasal virus shedding throughout the successive six days of peak viral shedding, and viremia also was significantly reduced during the four day of peak viremia within the challenge period.

No unacceptable adverse reactions either at the injection sites or by manifestation of signs of ic illness were observed. The vaccine is safe and well tolerated for stration in Equine rhinitis A susceptible species, particularly equidae. This study thus demonstrates that 3 x 1 mL intramuscular ions of the ERAV compositions of the example significantly reduced severity and duration of atory disease caused by ERAV virulent challenge.

Example 3 This example illustrates sequencing and analytical studies carried out on an Equine is A Virus strain as disclosed herein.

Cells And Virus Rabbit—kidney—l3 (RK—l3) cells (passages 100—160) were grown in Dulbbeco’s modified eagles medium nutrient mixture F12 HAM (DMEM F12) (Sigma—Aldrich Canada Ltd.

Oakville, Ontario) with 2—5% fetal bovine serum (FBS) (Sigma). Cell growth and viral propagation were performed in a C02 (5%) incubator at 37 0C. The ERAV isolate N/OS was propagated in RK—l3 cells and aliquots were stored at —70 0C for later work. RK—l3 yers were inoculated at 90% conﬂuence and RNA was ted before cytopathic effects (CPE) was observed.

Virus Titration And Plague Purification RK—l3 cells were grown on 3 cm round dishes, using DMEM F12 with 2% fetal calf serum. Cells were infected at 90% conﬂuence and plates were ted at 37 0C for 72 hours. After 24 hours, the medium was replaced and a 0.7 % agarose layer was added. Plaques were counted and recorded every 24 hours. At 72 hours the agarose layer was removed and plaques were d with crystal violet and counted.

For plaque purification RK—l3 cells were infected with ERAV/ON/OS, adsorption was allowed for 45 minutes and the inoculum was removed and replaced with a 0.7 % agarose layer. Plates were checked every 12 hours and plaques were classified as small, medium and large. Five s of each size were selected, picked into 300 uL of DMEM F12, and frozen at -70 OC. Viruses from each plaque size were ated in RK-13 cells and RNA was extracted from the small and large plaques for genome sequencing of the 5’ UTR.

Viral Growth Kinetics To study the growth characteristics of this strain, RK—l3 cells were infected with ERAV/ON/OS and supernatant samples were collected at s times for titration. RK—l3 cells were grown on 3 cm individual round plates and infected at 90% conﬂuence. Plates were incubated at 37 OC and atant samples were taken every 4 hours starting at 0 hours for a period of 28 hours. All samples were titrated using the plaque forming unit (PFU) que as previously described here.

Immunofluorescence RK—l3 cells were grown on glass tissue culture chamber/slides (Miles Scientific, Inc., Naperville, Illinois), and inoculated with ERAV/ON/05. Twenty eight hours post—infection, the cell culture medium was removed and cells were fixed in acetone. The slides were kept at 4 0C until processing. Sera from experimentally infected horses were used as a source of ERAV antibodies.

Rna Extraction And Sequencing RNA was extracted from infected cell yers. Cells were treated with 1 mL of TRIzol (Invitrogen) 18-20 hours post-infection and extraction was performed according to manufacture’s endations. RNA pellets were eluted in 30 uL of RNAse free water and kept at —70 0C for later use. First strand cDNA was synthesized using superscript lI (Invitrogen) following manufacturer’s recommendations. A SOuL PCR reaction was carried out using a set of sense and antisense primers. For genome sequencing, the primer walking approach was used, and primers were designed based on eight ERAV sequences available on GenBank. PCR conditions were: 4 minutes at 94 OC, followed by 30 cycles of 30 s at 94 0C, 30 seconds at 55 OC and 30 seconds at 72 0C with a final ion at 72 0C for l0 s.

Sequencing of the 5’ and 3’ ends were ted with the 5’ RACE and 3’ RACE kits (Invitrogen) as recommended by the manufacturer. Several nested PCRs were required to amplify the 5’ UTR end.

Seguence Analysis The preliminary identification of the virus was completed by partial sequencing of the structural protein VPl using primers designed based on other sequences available on GenBank.

Table 13: Primers used to amplify some of the ERAV s.

NAME PRIMER SEQUENCE SEQ ID NO: GENOMIC SITE foerP1 last 5’ to aataoocaa o onccttt 3’ 3087 revVP1 last 5’ acctttaaaa act ocaca 3’ 3671 foerC112 5’ gtcagtaaaacgcaacaaccat 3’ 1 12 foerC805 5’ tgtgaagaatgtcctgaaggca 3’ 805 749 5’ accatccacctaaaccao am a 3’ 1749 foerC5217 5’ atto ooocttttca ooottt aa 3’ revPC5952 5’ gtttctaactttgggacccgaa 3’ 10 5952 foerC6915 5’ tggatttgagattggttctgca 3’ 1 1 6915 revPC7511 5’ gcgaacgaaactgaggattg 3’ 12 7511 All primers were designed on Gene Runner version 3.05 (Hastings Software Inc.).

Sequencing reactions were set and run by the Laboratory Services Division at the sity of Guelph.

All sequences were assembled and edited with EditSeq and SeqMan DNASTAR Lasergene 8 (DNASTAR Inc., Madison, WI, USA). Sequencing results were entered into the BLAST software [National Center for Biotechnology Information, Bethesda, MD (NCBI)] and compared to similar entries on GenBank. ClustalW2 [European Bioinformatics ute, Dublin, Ireland (EBI)] was used for le sequence alignment and preliminary construction of the phylogenetic tree. The final phylogenetic tree was d on MEGA 4.0 by using the Maximum Composite Likelihood method and the reliability was evaluated by bootstrapping with 1000 replications. Analysis of the nucleotide sequences were d on t n 3.5.1 (Baltimore, MD, USA). In order to investigate the possibility of viral recombination between ERAV isolates, we completed a Bootscan analysis on SimPlot (Version 3.5.1) comparing the genomic sequences of all reported ERAV available on GenBank. Polyprotein cleavage sites were predicted based on sequences reported on GenBank with accession numbers: DQ272578 and NC003982.

Results Initial Characterization The viral protein (VPl) of the virus was amplified, partially sequenced and compared to sequences available on GenBank sion numbers: NC003982, DQ272577, DQ272128, DQ272127, DQ268580, DQ272578, ). Results from this initial comparison demonstrated a maximum identity of 95% to Equine rhinitis A Virus VPl.

Virus Titration The ERAV isolate was initially propagated in cell e and ed by the plaque g unit method. The stock virus titre obtained was 5X107 PFU on the initial titration. uent experiments showed no dramatic s in viral titre after 3 years of initial freezing. For animal experiments the stock virus with the initial titre was employed.

GROWTH KINETICS All supernatant samples were titrated using the PFU technique and results were entered on a spread sheet to construct a growth curve. As seen in other picornaviruses, the Ontario isolate showed an increased titre at 4 hours post—infection, reaching a plateau by 12 hours post—infection.

Immunofluorescence Infected RK-13 cells were visualised under the immunoﬂuorescence microscope.

Bright green ﬂuorescence signals were detected in the cytoplasm of ERAV infected cells compared to negative signals on mock infected cells.

Plague Purification Sequencing of a 426 tide fragment in the 5’ UTR on the small and large plaques showed no difference n plaque sizes at the nucleotide level. However, morphological characteristics were evidently different on RK—13 cell cultures.

Full Genome Seguencing Genome sequencing of the ERAV isolate resulted in 7839 nucleotides in length with a GC content of 47% including the poly (A) tail. Four cal repeats (CTGTAGCGTCAGTAAAACGC SEQ ID NO: 13) separated by 18, 21 and 18 nucleotides were identified on the 5’ UTR. The ERAV 5’ UTR was composed of 940 nucleotide with a 54% GC content. A single polyprotein with 6747 nucleotides (2248 amino acids) composes the viral genome with a 46.8% GC content. The protein starting sis was detected on nucleotide 940 with the AUG start codon and the ending on nucleotide 7686 with the UAA stop codon. Of great interest, a second AUG sequence was identified following the initiation codon.

A subsequent AUGAUG sequence was also identified 58—nucleotides downstream from the polyprotein start codon. Analysis with Blastx (NCBI) of the polyprotein on this isolate showed a 96% nucleotide identity with respect to other ERAV reported, however when the full—length genome sequence of the Ontario isolate was aligned and compared to others using ClustalW2 (EBI) and BlastX (NCBI), only a maximum identity of 80 % was observed. The amino acid composition showed an identical protein (structural and non structural) organization and length along the entire genome. These comparisons were made with PERV—l and PERV reported genome sequences (accession s DQ272578, and NC003982).

The 3’ UTR was composed of 110—nucleotides with a 24.3 % GC content, and a poly—A tail. Alignments of the 5’ UTR of all ERAV available demonstrated a lower identity percentage, g from 73% to 81%. Similarly, the 3’ UTR was analysed by the same method and the ty percentage ranged from 75% to 81%. Interestingly, various ions (1- tide, 2—nucleotides, and l3—nucleotides) and two small deletions (2—nucleotides, and 3— nucleotides) in the 5’ UTR were identified (Fig. 4). No other major s were observed throughout the entire genome.

Simplot Genome Analysis t analysis showed a comparable similarity (percentage) among all ERAV isolates when ed to ERAV/ON/OS. However, the mayor disparities were fied at nucleotide 1500 (59% similarity) and nucleotide 4700 (65% similarity). Nevertheless, this analysis showed that the similarity along the genome was between 70% and 82%. To further investigate the major divergence in the genomes, a scan (Bootscan) to identify possible ination sites was performed. These analyses demonstrated that the Ontario isolate has not had predicted recombination sites with other ERAV isolates. heless, when the Ontario isolate was removed from the analysis, possible recombination between other ERAV isolates may have happened in the pass.

Discussion ERAV is not routinely sought and recovered from clinical cases during equine respiratory outbreaks. Therefore ERAV isolation from clinical cases has been incidental and in most cases a challenge. For these reasons the recovery and sequencing rate of these viruses may be small.

During previous years, equine rhinitis viruses were classified within the Picornaviridae family, but were not clearly assigned to a ic genus. In 1996 Li and coworkers (Li F, Browning GF, Studdert MJ, and Crabb BS. Equine irus l is more closely related to foot—and—mouth disease virus than to other picornaviruses. Proc Natl Acad Sci U S A. 1996 6; 93:990—995) demonstrated that ERAV was closely related to foot and mouth disease virus (FMDV) based on the phylogenetic characteristics and nucleotide sequencing. In agreement with their findings and others (Wutz G, Auer H, Nowotny N, Grosse B, Skern T and er E. Equine rhinovirus serotypes 1 and 2: relationship to each other and to aphthoviruses and cardioviruses. J Gen Virol 1996, 77:1719-1730) the ERAV e has been found to be closely related to es L43052, DQ272578, and NC003982.

The ERAV/ON/05 isolate resulted in 7839 nucleotides including the 5’ UTR, the polyprotein, 3’ UTR and the poly—A tail. In comparison to previously reported ERAV isolates, the ERAV/ON/05 is one of the most complete ERAV sequences ed to date. Most of the other ces lack some information either from the 5’ UTR or are partial sequences of individual viral proteins. Data from the 5’ UTR revealed the presence of 3 repeats that have been previously reported in other ERAV and commonly found on the FMDV. It has been suggested that these repeats may be required in the formation of the ary structures found in the 5’ UTR, the internal ribosome entry site (IRES). The ty is showed that the ERAV/ON/05 isolate’s polyprotein bears a highly conserved nucleotide sequence. As an RNA virus, the ERAV is prone to constant mutations due to the lack of proofreading by the polymerase. This may indicate, that even though this RNA virus has been under natural evolution and constant replication no significant genomic changes have been introduced since its first genomic sequence was reported. It is evident that the structural and non structural ns, which are encoded within the polyprotein, represent the most conserved regions in the genome.

Interestingly, the viral ation rate in cell culture was rapid and efficient. A complete viral replication cycle was detected in less than 4 hours, reaching a plateau in 12 hours.

These characteristics are typical of picornaviruses and may reﬂect the viral activity in viva. Such features might explain the severity and speed of clinical signs in some equine respiratory viral infections. As observed in other viruses, such as irus and FMDV, the presence of small deletions and/or insertions in the 5’UTR have been associated with differences in plaque size in cell culture, and excel virulence in vivo. We found that the ERAV/ON/05 isolate generates different plaque sizes when infecting RK—l3 cells. To investigate and correlate the presence of these insertions and deletions in the 5’ UTR in the ERAV/ON/05 e, we sequenced this region from plaque purified viruses from different plaque sizes, and found no discrepancy at the nucleotide level. This finding may indicate that the plaque size difference may be due to a growth and replication characteristic of the RK—l3 cells and perhaps not associated with the ce or absence of the ces we found in this region.

Example 4 This e shows results of a study designed to develop a reliable ERAV infection model for investigating the clinical characteristics of N/OS.

Materials and Methods On the first phase a pony foal was infected with ERAV/ON/05 to adjust the infectious dose and sampling collection techniques. A second pilot study was designed to compare infected and control animals, and to master sampling techniques. The main infection study was conducted in two phases due to animal ng and time sampling constraints. One year after the first infection, four previously infected ponies were selected for a re—infection study based on their titre to ERAV. Animals with an intermediate and a high titre to ERAV were selected to be re-exposed to the Ontario isolate.

Experimental Animals A total of 12 pregnant pony mares were selected and their foals were kept for the experimental ERAV infection. All mares and foals were kept in a separate group away from the main barn and biosecurity measurements were put into place to prevent Viral respiratory infection. Blood samples from all foals were taken regularly and titers to ERAV were checked ically. Foals were kept until they reached 12 months of age. These animals were not vaccinated against any respiratory s during this period and general practices were ined to ensure healthy living conditions. All animals were de—wormed according to the herd management protocol. Animal ization and handling was performed regularly. Ponies were trained for pulmonary function test (PFT). Once the animals were conditioned for the experiments, they were transported in groups to the isolation unit and acclimatized for at least one week prior to the Viral infection.

Isolation unit All ion trials were conducted at the animal isolation unit in the Department of Pathobiology, at the University of Guelph. This isolation unit contains dual stalls that are equipped with controlled ature, ty, airﬂow, and lighting. Access to the stalls was restricted to the researchers and care personnel.

Selection criteria All foals were handled frequently, and health status was checked periodically.

Based on health status and serological parameters, animals to be included in these experiments were chosen. At that time, all ponies remained seronegative to equine rhinitis A Virus , equine rhinitis B virus (ERBV), equine virus 1 and 4 (EHVl/4), and equine inﬂuenza 2 Virus (AE2). Even thought AE2 antibody titers were detected at birth, a steady decreased was observed during the first 5 months and were not able by the single radial haemolysis test (SRH) at six months of age. For the ERAV experimental infection trial, one pony was selected to be used. Subsequently, two other ponies were selected for a second pilot study, and a group of 8 ponies (four infected and four controls) were selected for the main infection study. After a year from the first infection trial, 4 ponies with intermediate and high titers to ERAV were chosen for a re-infection trial.

ERAV isolate (ERAV/ON/05) recovered from a horse during an equine viral respiratory outbreak in o 2005 was propagated in rabbit kidney—l3 cells (RK—l3) and aliquots were stored at —70 0C for viral characterization and animal viral infection experiments. , for inoculum preparation, the isolate was propagated in RK—l3 cells. 30 mL round dishes containing 90% conﬂuent yers were infected with 500 uL of ERAV/ON/05 and incubated in the presence of C02 (5%) at 37 0C for 24—36 hours. All dishes were removed from the incubator and freeze/thawed four times to provoke cell rupture and viral release from intact cells. Supematants from all dishes were pooled into one ﬂask and centrifuged at 6000 RPMS for 15 minutes to clarify and discard cell debris. The clarified supernatant was tted in 10 mL vials to be used as an inoculum in the animal viral infection experiments.

Additionally, small 1 mL—aliquots were separated and kept for viral titration. The virus was ed using the plaque forming unit method (PFU).

Animal model Ponies were chosen as an animal model due to the nature of the experimental agent (ERAV), animal size, availability and handling.

Infection protocol Ponies between 8 and 12 months of age were selected, trained and used in the infection experiments. Due to the large number of samples to be taken, these experiments were divided in five sections (two pilot studies, two main infection experiments, and one re—infection experiment). During the pilot studies, equipment, animal handling, inoculum dose, route of stration, and sample collection were optimized. The re-infection study was conducted one year after the initial infection trial. Ponies in the re—infected group were exposed to the same viral strain at the same dose used during the ﬁrst infection. Only clinical examination, blood sampling for titers assessment and nasopharyngeal swabs for Virus isolation were collected from this group.

Face mask and nebulization For viral infection, a small size Equine AeroMaskTM was fitted with a rubber seal on the nostril ending. Size was adjusted depending on the pony’s head and the breathing windows were not modified. The mask was fitted with an inhaler connector and a one way “T” valve for virus nebulization. Conventional 6 mL nebulizer cups were ed to deliver the inoculum.

Nebulization was performed using a PM14 compressor (Precision Medical Inc. Northampton, PA) with a gas ﬂow of 9 LPM (liters per minute). This ﬂow rate and delivery cups allow for consistent delivery of breathable particles of more or less 5 microns. Each pony was nebulized for 45 min (taking a 5 minute break every 15 minutes) with 15 mL (total volume) of either inoculum or o. A nasopharyngeal swab per pony was collected nfection to ensure viability of the inoculum when delivered. Ponies that were re—infected a year latter were exposed to the virus by the same protocol. al examination All ponies were clinically evaluated on a regular basis from birth. Prior to the pilot and infection studies, all ponies were evaluated daily and during trial ments twice daily for the first 10 days and once daily from day 11 up to day 21 nfection. Clinical examination included: Body temperature (temp) (Celsius degrees), heart rate (hr), respiratory rate (rr), capillary refill (ref), gastrointestinal ty (gi), lung sounds (ls), nasal discharge (nd), ocular rge (od) [presence or absence and characteristics], lymph nodes (In) [size and teristics], tion, and general physical condition. Physical examination was performed at around the same times on each pony every day, commencing with the control animals and moving onto the infected group. Approximately 10 minutes were spent on each animal examination every time and all individual data were recorded on a daily check up form.

Pulmonary function test (PFT) The PFT was carried out as usly described by Hare and coworkers (Hare JE, Viel L. Pulmonary eosinophilia associated with sed airway responsiveness in young racing horses. J Vet Intern Med 1998;12(3):163—?0). The test was performed on all control and infected ponies prior to infection (day 0) and on days 1, 7, 14, and 21 post-infection. , on testing day, ponies were off feed in the morning and were mildly sedated as previously described here for endoscopy procedures. A rubber face mask was fashioned to snugly fit the ponies’ .

A pneumotachograp # 4 (Gould Electronics, Biltholven, The Netherlands) was attached to the face mask, and connected to a set of transducers that t the ﬂow and pressure signals into breath loops that are recorded on a computer (Pulmonary Mechanics Analyzer, Buxco onics Inc, Sharon, CT, USA). Flow was measure at the pneumotachograp level and the pleural pressure was assessed by an esophageal balloon (10cm long) that was placed in the mid thorax Via esophageal tubing. A total volume of 3ml of air were introduced into the balloon.

The pressure difference between the pleural pressure and the atmospheric pressure (measured at the nostril level) was considered as the transpulmonary pressure (APpl).

Bronchoprovocation g Bronchoprovocation challenge was carried out as part of the PFT testing. To determine the hyperresponsiveness of the airways post ERAV/ON/OS infection, control and infected animals were exposed to increasing histamine doses (doubling dose) by nebulization with a PMl4 compressor with a gas ﬂow of 9 LPM (Precision Medical Inc. Northampton, PA).

Pulmonary physiological parameters were assessed initially by administering 0.9% physiological saline solution (base line), followed by increased histamine doses. After each administration (2 minutes) data was recorded for 3 s and respiratory physiology was later analyzed. ine doses were started at 0.5 mg/ml and were increased gradually to a maximum of 32 mg/ml. Dynamic compliance (Cdyn) and APpl were used as parameters to discontinue ine nebulization. When Cdy11 was decreased by two thirds or APpl was d during histamine administration, nebulization was suspended. Histamine triggering dose was later plotted and calculated. All ponies were mildly sedated and physically restrained during these procedures.

Sampling technigues Blood collection Blood samples were collected from either the right or left jugular vein.

Approximately 10 ml of blood were collected on a red top tube (serum) from each pony according to the sample collection schedule. onally, 3 to 5 ml of blood were also collected for CBC (complete blood count) and proﬁle. All samples were collected in the morning hours and processed within the same day.

Blood samples for serum separation were kept at room temperature for at least 30 minutes and then centrifuged at 3000 rpm on a table centrifuge. Serum was separated within 6 hours from the tion time and aliquots were labelled and frozen at —700 C for later analysis (antibodies to ERAV, ERBV, AE2, and EHVl/4). Blood samples for CBC and profile were sed within the same day.

Nasopharyngeal swabbing Nasopharyngeal swabs were collected for virus ion on days previous to the infection trial and on days 0, l, 3, 5, 7, 10, 12, l4, l7 and 21. Each pony was ined with a muzzle twitch and a 30 cm long swab (Kalayjian Industries, Inc. Signal Hill, California) was passed into either the right or left nostril until it reached the pharynx. Swabbing was performed by ng the swab for about 5 to 10 seconds. The swab was removed carefully, and the tips were cut off into a vial containing 3ml of virus transport medium (VTM). The vials containing the swabs were shaken and kept on ice until processing. To release the viral les and cells attached to the swabs, vials were vortexed for about 20 seconds and the medium was transfer to a 1.5 mL Eppendorf tube and frozen at -?0 0C for later analysis.

Urine and fecal sampling Virus isolation was attempted on urine and fecal samples pre and post-infection with ERAV/ON/OS. Urine was collected in the morning after al examination and/or stall cleaning by holding a collection cup while the animals were urinating. When the sample could not be collected by hand, the animal was fitted with a plastic collection bag around the genitals.

This bag was removed after the animal urinated and a 10 mL aliquot was saved for virus isolation. Fecal samples were hand collected from fresh manure on the stall’s ﬂoor. About 5 mL of manure were collected into a collection cup and 10 mL of e saline were added to ve the sample. Urine and fecal samples were kept on ice until sed.

Upper and lower endoscopy Bronchoscopy and BAL were performed as previously described (Hare et al., 1998). A sterile ﬂexible fiberoptic endoscope, 140 cm length with a 0.8 mm OD (Olympus, Corp., Tokyo, Japan) was advance through the right or left nostril into the nasal cavity to the larynx level. At this point the upper s conformation was evaluated. Followed, the bronchoscope was advance into the trachea and the presence or absence of inﬂammation and/or mucus and its teristics were recorded. Data were recorded on the daily evaluation sheet and all the endoscopies were video recorded for later analysis.

Pharyngeal and al brush biopsies In order to assess viral replication in the upper and lower airways, a brush biopsy was taken from the pharynx, mid trachea, and the carina of infected and control animals on days 0, l, 3, 5, 7, 10, 14, and 21. During endoscopy examination, a 200 cm guarded (protective sleeve) cytology brush (Hobbs l Inc. Connecticut) was e through the biopsy channel at the predetermined sample location and a sample was collected. Brushes were retracted into the protective sleeve and removed from the bronchoscope. To remove the sampled tissue from the brush, this was put into 600 uL of VTM and vortexed for l0 to 20 seconds. All samples were kept on ice and transported to the laboratory for further analysis (virus isolation).

Bronchoalveolar lavage [BAL] Brieﬂy, all ponies were mildly d with Romifidine (0.04 mg/kg, IV) and a sterile 0.8 mm OD bronchoscope us, Corp., Tokyo, Japan) was advance through the right or left nostril into the trachea to the carina level. As the bronchoscope was advanced, a 0.2% warmed lidocaine solution was administered to reduce cough and distress. Once the cough reﬂex was reduced the bronchoscope was advanced and wedged into a proximal terminal bronchus. A total of 250 mL of warmed sterile saline solution was administered h the biopsy channel divided in two aliquots. BAL ﬂuid was retrieved by manual suction with a 60 cc syringe through the biopsy channel and placed on ice. The ﬂuid was filtered and aliquots for virus isolation, cell count, and cytospin slides were made.

Clinical samples culturing Collected samples were transported on ice and frozen at —70 0C for later inoculation on cell cultures.

RK-13 cells (passages 100-160) were grown in Dulbbeco’s modified eagles medium nutrient mixture F12 HAM (DMEM F12) (Sigma—Aldrich Canada Ltd. le, Ontario) with 2—5% fetal calf serum (Sigma). Cell growth and isolation were med in a C02 (5%) incubator at 37 OC. RK—l3 cells monolayers were grown on 6 well plates to a 90 % conﬂuence and infected with the clinical samples ted from the infection trial. In brief, 90% of the growing medium was removed from each of the wells and 200 uL of the specimen to be tested were added to the yer. Plates were put on the rocker platform for one hour and 3 mL of medium were added after the time elapsed. Plates were incubated and checked every 24 hours for cytopathogenic effects (CPE). If CPE was detected, the supernatant was removed from the well and frozen for later analysis. Results from the virus isolation test were recorded on a spread sheet. Plates were checked for up to 7 days and if CPE was not developed, a second e was attempted using 200 uL of supernatant from the first passage. After a second passage if CPE was not detected the sample was classified as negative. atant from positive and negative samples was saved to be confirmed by reverse transcriptase polymerase chain on RT—PCR). tical analysis Results from virus isolation and all clinical scores (Table 7) were entered on a spread sheet.

Table 14. Scoring system for experimental infection with ERAV/ONOS.

Clinical sign Degree Score Cough None 0 Intermittent l Frequent 2 Mucus membranes Pink 0 Pale 1 Gastro intestinal Normal 0 auscultation Abnormal 1 Feces/Urine Normal 0 Abnormal 1 Lung Sounds Normal 0 Slightly Increased 1 Marked Increased throughout 2 chest Crackles and wheezes 3 Nasal Discharge None 0 Moderate/severe serous 1 Mucopurulent 2 Ocular Discharge None 0 Serous 1 purulent 2 Adenitis not palpable 0 le (<lcm) 1 Enlarged (>1 cm) 2 Anorexia None 0 Mild to te 1 Severe 2 Temperament Bright, alert and responsive 0 Dull (head down, 1 disinterested) Perfusion Time Normal (2s) 0 3-4s 1 5s 2 Virus isolation results were categorized as positive or negative and results were compared between groups. To determine if there was a statistical difference between locations on virus isolation (in the infected group) an exact conditional logistic regression was used at each day and site. The same test was used to determine if there was a statistical difference between groups depending on the ion day. Clinical scores were summarized and the totals were analysed and compared between .

A generalized linear mixed—model was employed to analyze all clinical parameters.

Factors included in the model were: pony, ent, and time as well as their interactions. Since animals were measured over time, the AKAIKE information criterion (AIC) was used to determine an error structure for the auto-regression. The assumptions of the ANOVA were assessed by comprehensive residual analyses. A Shapro—Wilk test, a orov—Smirnov test, a Cramer—von Mises test, and an Anderson—Darling test were ted to assess overall normality. Residuals were plotted against predicted values and explanatory les (pony, ent and time) to look for patterns that suggested outliers, l variance or other problems. If residual analyses suggested a need for data transformation or data was presented as a percent, analyses were done on a logit or log scale. If the overall f test was significant, a Dunnetts test going back to baseline within a ent or a tukey test between treatments and sites at each time was applied. gical response was defined as a four fold increase in antibody levels from baseline (day 0) to any of the time points in sampling collection (days 7, 14, or 21).

Statistical analysis was carried out on SAS 9.1.3 (SAS institute Inc., 2004, Cary, NC). Statistical significance was set at P < 0.05.

Results This study was designed to consistently reproduce ERAV clinical disease in ponies and to study its in viva characteristics. Pilot studies demonstrated that nebulized ERAV/ON/OS was able to cause clinical respiratory disease in healthy ponies (age 10—12 months). Mild immunosuppression was induced in all ponies except in the ected animals to mimic natural conditions under stressful events (e.g. movement for sales, field relocation, vaccination programs, etc.). Results from the main infection study confirmed the results observed during the pilot studies. Ponies in the ed group (n=4) developed respiratory clinical disease that consisted with increased body temperature, lymphadenopathy, increased lung sounds, increased tracheal mucus, and increased nasal discharge (Fig. 7—8). None of these clinical signs were significantly extreme to require additional animal care or supportive treatment. Neither respiratory rate, nor heart rate was significantly different between groups. None of the al signs developed by the infected group were observed on the control s (n24), which remained healthy throughout the extent of these trials. The etiological agent (ERAV) was only recovered from ponies in the infected group for up to seven days (Table 8). Ponies in the re— infected group (n=4) did not developed clinical respiratory signs and remained healthy throughout the experiments. The statistical analysis of the data collected during these trials demonstrated a significant difference between infected and control animals.

Table 15. Equine rhinitis A Virus (ERAV) shedding summary. Results of Virus isolation from samples ted during the ERAV/ON/OS experimental infection.

Sample collection day Sample location x brush biopsy Mid trachea brush biopsy Carina brush biopsy Plasma Urine + Virus was red from clinical sample on cell culture (RK—l3 cells) — Virus was not recovered from al sample BAL Bronchoalveolar lavage Neither depression, nor appetite lost was recorded in either group. As well hydration, urination, defecation, and gastrointestinal movements remained unchanged on all groups during the infection trials. There was no difference between re-infected and control animals on the clinical scores and Virus isolation test, since no al disease was observed on the re—infected animals and the Virus could not be recovered from this group.

Clinical findings All ponies were considered to be y prior to these experiments. ERAV/ON/OS exposure induced clinical respiratory e in infected animals compared to controls and re— infected animals. The main clinical signs able by physical examination were pyrexia, nasal discharge, and lymphadenopathy. opic examination also revealed the presence of large volumes of mucus and emia in the mid a and lower airways of the infected animals.

Rectal temperature No significant differences in daily rectal ature were found between groups prior to Viral or placebo exposure. A significant treatment effect was identified when infected animals were compared to controls and re—infected (p = 0.002). When the body temperature from the three groups was compared at different times, the infected group was significantly different compared to controls and re—infected animals ([9 = 0.028). Increased body temperature was detected 24 hours post—infection only in the infected animals and was significantly different from day 2.5 to day 6 when compared to control animals at the same time points ([9 2 <0.005).

The body temperature increase picked on day 4 with a mean body temperature of 38.450C ) (p = 0.001) and persisted for two more consecutive days (Fig. 8). No statistical difference was found when body temperatures from control and re—infected groups were compared at different times. Animals in the l and re—infected groups did not have a significant change in their body’s temperature when comparing baseline to individual points in time within the groups (days 1, 7, l4 and 21).

Lymph nodes Submandibular and retropharyngeal lymph nodes were examined daily and classified as non palpable, palpable and enlarge. Palpability or enlargement of the lymph nodes was only recorded on the infected and re-infected animals. In all infected , the submandibular area became sensitive to palpation on day two and in most cases persisted for up to two weeks. The submandibular lymph nodes size in the infected animals varied from 3 to 5 cm in length by 2 to 3 cm in ess, and the analysis of the total scores demonstrated a statistical ence between groups (p = <.0001) (Fig.7). Interestingly, the retropharyngeal lymph nodes were not consistently palpable in all infected animals, but a significant change in size was recorded in one pony. This lymphadenopathy did not appear to interfere with food or water consumption and the sensitivity to palpation became less pronounce as the days progressed. Submandibular lymph nodes were palpable in three animals from the re—infected animals with an average size of less than one cm in length and 0.5 cm in thickness approximately. Control s had no detectable lymph nodes changes at palpation throughout the ion experiments.

Heart and atory rate Respiratory rate (RR) and heart rate (HR) means were not significantly different between treatment groups (P = 0.1) at any time points. In general RR and HR were within the normal physiological parameters and small changes were only associated with handling and sample collection. The highest RR mean among all groups was fied on day 0 and the lowest mean from all the groups was recorded on day 21.

Endoscopic examination On the endoscopic examination, infected animals had an increase amount of tracheal mucus detectable on day one that persisted up to day 21. Neither the control nor the re— infected animals had mucus secretions detectable at endoscopic examination throughout these experiments.

Characteristics of the mucus varied from clear and serous on day one to mucoid on days 7—21. Mucus patches were consistently distributed from the upper trachea to the bifurcation of the carina. Localized tracheal hyperaemia was observed in all ed and in some control animals throughout these experiments. The carina on all infected animals was blunted and in some cases emic starting on day three. Sensitivity to endoscopic ation was noticeably increased by day seven on infected animals and bronchoconstriction was observed during BAL. Nasal discharge varied from mild to moderate in all infected ponies and was not present in the control and re—infected . Serous nasal discharge was ed during clinical examination for about 8 days on the ed animals starting n 36 and 48 hours post—infection. However this discharge was not a reﬂection of the mucus (characteristics and volume) observed during endoscopic ation. Mild ocular rge was observed inconsistently in the infected animals.

Serology A total of 12 ponies (age 10 to 12 months) were infected with ERAV/ON/OS or placebo by nebulization. All ponies were serologically negative to ERAV, ERBV, AE2, and EHV1/4 and in a healthy condition prior to the infection experiments. Following exposure to ERAV/ON/OS all infected animals (100%) seroconverted (four fold increase) to ERAV determined by the virus neutralization test (VN) (Fig. 9). A significant treatment by day interaction was observed in infected animals (P = <0.0001). A statistical difference between infected and re—infected animals was found on baseline and day 7 (P 2 <0.001). In the re— infected group no statistical differences were found when titers to ERAV on days 7, 14, and 21 were compared back to baseline.

Antibody titers against ERAV were significantly elevated in infected animals from day 7 and in most cases peaked by day 14 and maintained to day 21 (P 2 <0.001). In st, all control animals remained seronegative to ERAV and no detectable changes were identified by the VN test. s in all groups did not show an se in antibody levels or serological conversion to any other atory viruses (ERBV, AE2, and EHV1/4) during these ments (Fig. 9). Animals in the re—infected group (n=4) did not have a significant difference in antibody titers to ERAV between baseline and day 21 post—infection. However, a small change in antibody levels to ERAV was detected in 3 ponies and a four fold increase in one pony from the same group (Fig. 9).

Virus Isolation aryngeal swabbing, laryngeal brushing, tracheal brushing, BAL, fecal and urine samples were negative for virus isolation (equine respiratory viruses) on all ponies prior to infection (Table 8). Swabs obtained from the nasopharynx from infected and l animals after completing nebulization were cultured on RK—13 cells and ERAV was recovered on a first passage from all infected animals only. RT—PCR using primers that targeted the VP1 gene confirmed the positive and negative diagnoses from both groups.

Virus isolation results from the infection and re—infection trials are summarized on Table 8. A significant difference between infected, l and re—infected animals was fied when comparing virus isolation between groups (P = < 0.05). ERAV was only recovered from animals in the infected group on specific days and specific areas of the respiratory tract (Table 8). No other viruses were recovered from samples collected during theses experiments. All ponies in the control group were negative in the virus ion tests throughout the study. A treatment by day interaction on infected animals was first detected on day 7 and persisted on days 14 and 21 (P = < 0.05). on for virus recovery varied from the lower airways on days 1, 3, and 5 to mid and upper airways on days 1, 3, 5, and 7. On day one ERAV was isolated from the pharynx and carina from all the ponies in the infected group and the mid trachea and BAL from 3 ponies from the same group. All attempts for virus recovery from feces in all groups were unsuccessful.

Virus isolation from urine and plasma was achieved only in rare occasions (Table 8). Virus ry was gradually decreased from day 1 up to day 7 when ERAV was consistently red from al samples. This last viral recovery was associated with an increase in antibody titre to ERAV and a decrease in the clinical signs scoring (Figs. 7 and 9).

Pulmonary function testing [PFT] Hyperactivity of the airways was assessed based on the physiological and clinical response to histamine provocation. Data from infected and control s were plotted and triggering histamine doses were calculated. Interestingly, ponies from both groups (infected and control) responded on day 0 to a low dose of histamine (<6 mg of histamine). Overall, the triggering histamine doses did not go beyond 13 mg. The clinical histamine reaction (dose— dependant) was ed as hyperventilation associated with abdominal lift and breathing difficulty. The physiological reaction was detected in the PFT by a 35% drop in lung c ance (Cdyn) or a doubling in the transpulmonary pressure (APpl) when comparing saline and histamine administration. Ponies in the infected group showed a small reduction to the histamine triggering dose from day 0 to day 1. However, this was not significantly different n groups. A significant difference between infected and controls was detected on day 21 (P = 0.02).

BAL ﬂuid differential cell counts Differential cell counts were carried out on the cytospin slides prepared from BAL ﬂuid aliquots. No significant differences in the cell counts were found among horses in the different treatment groups prior to the infection trial. No treatment by day effect was detected on the macrophage and epithelial cells percentages throughout the experiments. A significant increase in the number of neutrophils was observed on day 7 post-infection in the infected group (P = <0.05). These numbers were not significantly different on the control or ected animals when comparing base line to days 7‘, 14, and 21. The mean tages of lymphocytes, eosinophils and mast cells were proportionally decreased on day 7 nfection in the infected animals and a statistical difference was detected (P 2 <0.05). Ciliated epithelial cells were ly observed on the slides from infected and control animals, however, no significant differences were detected, except on one of the infected s that had a high count on day 7.

In general, a non—septic suppurative inﬂammation with the presence of epithelial cells and ic giant cells was detected in the infected ponies. Interestingly, no major s in the cytological examination were observed on the samples from the re—infected animals.

This study demonstrates that N/05 d clinical respiratory disease in infected ponies. Serology demonstrated that no other respiratory viruses were present during these trials. The disease is characterized by pyrexia, nasal discharge, increased lung sounds, and increased submandibular lymph nodes size. Additionally, large volumes of mucus were opically detected in the lower airways that persisted up to day 21. The virus was isolated from the lower and upper airways up to day 7 corresponding with the ance of detectable equine rhinitis A virus (ERAV) antibodies. None of the re—infected animals developed clinical disease and only one pony from this group had a four fold increase in the antibody titers to ERAV. Ponies with pre-existing ERAV antibodies did not develop clinical disease when exposed to the virus.

Example 5 This example rates one embodiment of a Equine is B Virus composition in accordance with the present invention.

Materials and Methods Equine Rhinitis B Virus strain 07-10342 (ATCC Accession NoPTA—11829) was red from Rabbit—kidney-l3 ) cell culture from a nasal swab from a horse in Ontario Canada. Inactivated 07-10342 was produced following the general procedure described in Example 1 for ERAV/ON/OS.

The following compositions containing ERBV alone or in combination with ERAV were prepared.

Table 16. Vaccine Groups Vaccine Group Vaccine Titers A fB Adjuvant Volume 1 (N=8) Monovalent B - High 7.5 HRA-S 200 mL 2 (N=8) lent B - High 7.5 HRA-3 with CS0 100 mL 3 (N=8) Monovalent B - Low 7.0 HRA-S 200 mL 4 Bivalent A and B - High 8.0 i 7.5 HRA-S 200 mL Bivalent A and B — High 8.0 / 7.5 HRA-3 with C50 100 mL Eight horses from each of vaccine groups 1, 2, and 3, were challenged along with eight l horses with a 106‘6 TCID50 challenge dose. Disease status based on nasal discharge and conjunctivitis scores as indicated in Table 10. Effect of vaccination on duration, severity, and incidence of disease is shown in Tables 17—22.

Table 17. Disease status scoring ‘ Disease Status ‘ Nasal Score Conjunctivitis Score ‘ Normal (0) O or 1 0 or 1 Vaccinated group showed a icant reduction (Table 18) in duration of nasal discharge including in some cases no signs of mild respiratory disease.

Table 18. Number of days with mild, moderate, or severe respiratory disease Vaccine Group Minimum 25th quantile 50th quantile 75th quantile Control (N:8) 1 ‘9Maximum The number of days animals were sick with clinical signs of respiratory disease (nasal score > 0) was significantly r in the ated group ed to the control group.

Table 19. Effect of vaccination on duration of disease Control Vaccinate Shift in days 95% confidence interval Duration vs Group 1 ‘ 8 ‘ 1* ‘ 1, 8 days Duration vs Group 2 ‘ 8 ‘ 1* ‘ 0, 8 days on vs Group 3 1, 7 days *Significantly lower than the control group by Kruskal—Wallis test (P<0.05) Immunization with ERBV also lessened the severity of the disease with a lower percentage of vaccinates than controls demonstrating mild and moderate clinical signs of respiratory disease throughout the study (Table 20).

Table 20. Summary of the severity of disease based on the m respiratory score Vaccine Group ‘ Normal Mild ‘ Moderate Control (N=8) ‘ 0% (0/8) 87.5% (7/8) t 12.5% (1/8) 1 (N=8) 37.5% (318) 50.0% (48) 12.5% (1/8) 2 (N=8) 25.0% (2I8) 75.0% (6.8) 0.0% (0/8) 3 (N=8) 12.5% (1i8) 87.5% (778) 0.0 % (0/8) Immunization with ERBV was found to icantly reduce the incidence of disease, by 37.5%, 25%, and 12.5% in vaccinated groups 1, 2, and 3, respectively (Tables 21 and 22).

Table 21. Effect of vaccination on incidence of disease (disease statuses of mild and moderate were pooled for the purposes of this evaluation) Control Group Prevented 95% confidence interval (n=8) (n=8) fraction Control vs. Group 1 1 62.5% 0.3?5 —0.069, 0.6346 Control vs. Group 2 —0.119, 0.4973 Control vs. Group 3 0.125 —0.137, 0.3266 Vaccination also reduced the shedding of virus from the nares indicating lesser clinical disease in the vaccinated horses as well as demonstrating the effectiveness of the vaccine in preventing spread of the infectious disease to other ially susceptible horses (Table 22).

Table 22. Incidence of Virus positive over time Day Control | Group 1 Group 2 Group 3 0 0.250 0.25 0.000 0 7 0.125 0.00 0.125 0 Results and Discussion Inactivated ERBV vaccines were found to be capable of immunizing an animal to high levels of antibody detection. The vaccines reduced the duration, severity, and incidence of disease in immunized animals challenged with ERBV. Vaccination also d the shedding of infectious virus from ill . No unacceptable adverse ons either at the injection sites or by manifestation of signs of systemic illness were observed. The vaccines are safe and well tolerated for administration in Equine rhinitis B tible species, particularly equidae.

Example 6 This example illustrates a guinea pig model for use in a release y assay. e 5 (A/B—l) and Vaccine 6 (A/B—2) were each administered to guinea pigs (five guinea pigs per vaccine, 0.5 mL per intramuscular vaccination). A booster vaccination shot was administered three weeks later. After 19 days, the pigs were bled and blood was analyzed using serum neutralization tests for ERAV and ERBV.

Table 23. Vaccine 5 (A/B-l) d with 108-0 A/107-5 B, with HRA—S adjuvant Guinea Pig ERVA Titer ERVB Titer >1414 2 1300 180 3 448 | 120 4 1123 360 1120 Table 24. Vaccine 6 (A/B-2) Batched with 103~0 14/107“5 B, with HRA-3 + cottonseed oil adjuvant ERVA Titer ERVB Titer 1 194 220 2 326 | 280 194 560 No Sample No Sample Back titer 191 | 260 Example 7 This example illustrates a challenge evaluation of Rhinitis Virus A after vaccineation with a 2 dose equine Rhinitis A/Rhinopneumonitis/Inﬂuenza Killed Virus e and protention against Equine Rhinitis A respiratory infection after vaccination with the Rhinitis A/Rhinopneumonitis/Inﬂuenza Killed Virus e.

Objective The objective of this vaccination—challenge study was to demonstrate efficacy of Equine Rhinitis A (ATCC Accession No. PTA—11828) for Equine Rhinitis A/Rhinopneumonitis/Inﬂuenza Killed Virus Vaccine. The primary outcome variable used to evaluate the efficacy of vaccination was reduction of respiratory disease caused by Equine Rhinitis A Virus.

Material and Methods The e used in this study is an Equine Rhinitis A/Rhinopneumonitis/ za e of the present invention, Killed Virus vaccine.

A. EQUINE RHINITIS A VIRUS The original Equine Rhinitis Virus A Strain (ERhA V) was obtained from University of Guelph, Animal Health Laboratory, as Isolate number 04—54188, and was received on September 9, 2008 under Import Permit No. 106930. The virus was passed once on E—Vero cells to produce a high titered pre-master stock and was then diluted with cell culture media to produce the Master Seed Virus (MSV). The MSV is designated as ERhA V (04-54188), MSV, Lot 091508A-diluted, 24Sept08, and was approved for use for Establishment 597 by USDA on June 18, 2010.

The Equine Rhinitis A Viral antigen used in vaccines evaluated in this study was a MSV+5 virus produced on the twentieth passage of APHIS ed E—Vero cells. Following , viral ﬂuids were filtered, in inactivated, and concentrated in accordance with the Outline of Production for Product Code A522.20. The inactivated viral ﬂuids were tested for residual live virus after inactivation. The s were satisfactory. Inactivated viral ﬂuids were used to formulate e at an antigen inclusion level of 107'5 TCIDso/mL. 1. Experimental Vaccine Rhinitis Combo Lot 122110 Experimental vaccine Rhinitis Combo Lot 122110 was formulated based on pre— inactivation titers.

The final formulated vaccine ned the following ingredients per 1 mL dose: Equine Rhinitis A 107-5 TCID50/mL EHV—l 107-0 TCID50/mL EHV-4 106-5 TCIDso/mL Inﬂuenza A2/Ohio/03 107-0 TCIDso/mL Inﬂuenza 95 107-0 /mL Inﬂuenza A2/NewMarket/2/93 107-O TCIDso/mL Adjuvant (MVP Laboratories, SO. #25) 100 uL Diluent, MEM-E containing q.s.

Gentamicin, 30 ug/mL of diluent volume Formaldehyde, 0.1% of t volume Amphotericin B, 2.5 ug/mL B. mental Horses 1. Description of Experimental Horses Forty (40), six to eight—month old, draft—cross horses purchased from Steve Waagen, Bottineau, North Dakota were microchipped upon arrival at Equine Resources, LLC, Butler, Mo. and were assigned to either IVP (Investigational Veterinary Product) or Control Product (CP) based on random number generator after being pre-screened for rhinitis A titers of 1:4.

During the entire study, horses were quartered in a single large paddock with common feed bunks, waterers and hay racks. Upon challenge, each animal was assigned a 2 digit “barn code” by laboratory nel that was attached to a halter worn throughout the post— nge time period and used to identify horses when clinical signs and samples were taken each day. During each observation day, horses were corralled into holding pens and worked randomly h individual restraining .

Table 25 summarizes the study design: Table 25: Study Design 1 ‘ 20 ‘IVP 2x1mL IM D46 2 ‘20 ‘CP 2x1mL IM D46 2. Vaccination /Challenge and Sampling Schedule On January 28, 2011 and February 18, 2011, experimental vaccine Rhinitis Combo Lot 122110 was administered intramuscularly in a 1 mL dose volume to each of 20 horses (vaccinate group, IVP). Twenty horses (control group, CP) received a 1 mL dose of adjuvanted MEM-E (Experimental product 005) containing the excipients used in the Lot 122110 vaccine (adjuvant, gentamicin, amphotericin B, and formaldehyde) but no antigens. All horses were challenged by intranasal aerosolization of nt Equine Rhinitis A virus at Study Day 46 (25 days post—booster vaccination) on March 15, 2011. Table 26 shows the schedule of events.

Table 26: Schedule of Events Jan 28,2011 Randomize horses to groups Collected serum samples all horses IVP administered to Group 1 CP administered to Group 2 February 18, 2011 ted serum samples all horses IVP administered to Group 1 CP administered to Group 2 March 15, 2011 Challenge Groups 1 and 2 Body Temperatures Whole blood samples (virus isolation) Nasal Swabs (virus ng) Clinical observations March 16-21, Body Temperatures 2011 Whole blood samples (virus isolation) Nasal Swabs (virus shedding) Clinical observations March 22, 2011 Serum samples Body Temperatures Whole blood samples (virus isolation) Nasal Swabs (virus shedding) Clinical observations March 23-28, Body atures 201 1 Whole blood samples (virus isolation) Nasal Swabs (virus ng) Clinical observations March 29, 2011 Serum samples Body Temperatures Whole blood samples (Virus isolation) Nasal Swabs (virus shedding) Clinical observations March 30-April 4, 61-66 Body Temperatures 2011 Whole blood samples (virus isolation) Nasal Swabs (virus shedding) Clinical observations April 5 , 201 1 67 Serum s Body atures Whole blood samples (virus isolation) Nasal Swabs (virus shedding) Clinical observations End of Study 3. Intranasal Challenge Inoculation of Horses a. Challenge Virus The challenge virus Equine Rhinitis A lot 112108A was produced in tissue culture on E—Vero cells. The titer of the challenge virus was determined to be 1 X 107‘3 TCID50/mL on the day of challenge. b. Intranasal Challenge Method Sedivet® (romifidine hydrochloride), a sedative and analgesic, was administered intravenously to each horse prior to nge at a dosage of 50 ug/kg of body weight. The challenge virus was stered asally as an aerosol produced by a nebulizer into an Equine AeroMask (Trudell Medical International, Ontario, Canada) by the ing method: Four milliliters of 107‘3 TCID50/mL challenge virus were placed into the nebulizer cup in the AeroMask device. A pressure hose was fitted from an air compressor to the inlet port of the nebulizer. The outlet tube was then ed into the AeroMask attached to the head of the horse being challenged and approximately 10 psi of air pressure was applied to the inlet port for three minutes. During this time approximately two milliliters of challenge Virus ﬂuid was lized directly into the nostrils of the horse being challenged. Challenge Virus was administered to horses undiluted, effecting a Challenge amount of 1 x 107'6 TCID50 in a 2 mL dose.

C. PRE AND POST CHALLENGE EVALUATION PARAMETERS l. Nasal Exudate Evaluation All nasal exudate observations were made prior to collection of nasopharyngeal swabs. On the Day of Challenge (D46) and for 21 days post challenge, the nasal passages and muzzle of each of the 40 vaccinate and control horses were examined and graded using the grading and scoring ption listed below.

The scoring grades of 0 through 6 were assigned on the basis of the severity of the disease indicated by each of the following classification: Essentlally normal 1nd1cates the horse was clean and essentially free of nasal exudate Slight clear serous rge that may be frequently observed in both ed and normal horses Very slight mucopurulent discharge indicates that mucus was definitely present in small amounts in either one or both nostrils Moderate clear serous discharge is tive of a definite increase in volume over that ly observed/slightly mucopurulent is a discharge easily observed in one or both nostrils Copious clear serous discharge that is generally ed only in diseased horses Moderately mucopurulent indicates that mucoid discharges were present in large quantities in both nostrils Heavy mucopurulent indicates that copious amounts of a HM 6 mucoid discharge filled both nostrils 2. Ocular Discharge Ocular rge was evaluated daily at the time of nasal exudate tion.

Ocular discharge scores were recorded as 0 = normal; 1: mild to moderate ocular discharge and 2 2 severe ocular discharge. 3. Temperature Daily rectal temperatures were recorded for each of the 40 vaccinate and l horses on Day of Challenge and for 21 days post challenge by means of a calibrated, electronic thermometer (GSA onics) probe. The daily rectal atures were recorded in degrees Fahrenheit (OF). 4. Nasopharyngeal Viral Isolation On each observation test day each nasal passage of each vaccinated and control horse was swabbed deeply by means of a e WECK—CEL® surgical spear (Edward Weck and Company, Inc., Research Triangle Park, NC. 27709) attached to an ll—inch long e plastic pipette. On collection, each of two surgical spears was immediately placed in a single tube containing 4 mL of chilled transport medium (E—l99 supplemented with gentamicin, L— glutamine, 2X Pen/Strep, 2X Amphotericin B).

For isolation of virus, the tubes were mixed, the swabs aseptically removed, and the medium centrifuged at 1500 rpm for 10 to 15 minutes to remove particulates. Medium was ed through a 0.2 u syringe filter prior to inoculation on tissue culture cells. After filtration, 4—6% of sterile 85% sucrose solution was added to each sample for freezing at —80°C in order for all samples to be tested rently.

On the day of testing, one mL of the thawed, clarified transport medium was used to inoculate a 2 cm2 two day old monolayer of E—Vero cells grown in a 24 well tissue culture plate from which the growth medium had been aseptically removed. Following inoculation, the inoculum was allowed to adsorb on the cell yer for at least one hour at 37°C in a humidified incubator containing a 5% C02 atmosphere. After the adsorption period, an additional 1 mL of re-feed medium (E-199 containing 2 mM L-glutamine, Gentamicin 2X Pen- Strep and 2X Amphotericin B) was added to each well. Following addition of re—feed media the plates were then incubated at 37°C in a C02 incubator. Each test and control tissue culture well was examined microscopically for 7 days for signs of cytopathic effect (CPE) typical of the Equine Rhinitis A nge virus. Wells that were negative at the end of the 7 day observation period were subcultured onto fresh cells and observed for an additional 7 days.

Statistical tion Methods Horses were classified to a range of respiratory disease status, on a daily basis. The fication to disease status included combining the nasal and ocular tions. The algorithm used is detailed below: 33211 Score Normal (0) Mild (1) ‘ 0 or 1 Mild (1) ‘ 1.5 Moderate (2) ‘ 2 or 3 Severe (3) The inﬂuence of vaccination on the duration of disease (number of days with at least moderate disease) was evaluated using the Hodges—Lehman (exact) estimate (the NPARlWAY procedure in SAS, SAS Institute, Cary NC).

Severity of e was evaluated by comparing the maximum disease status between the vaccinated horses and the placebo horses. The mitigated fraction and 95% confidence intervals (CI, asymmetric standard error) were calculated (the FREQ procedure in SAS).

Rectal temperature and serum neutralization titers were analyzed by repeated measures analysis appropriate for continuous data (temperature and log ormed SN titers; ANOVA). SN titers were assumed to be 0 if the titer was reported as < 4 and 709 if the titer was reported as > 709. SN titers were log transformed prior to the analysis. Buffy coat and nasal swab results on each day were ed using Fisher’s exact test.

Results One horse, #39 (IVP group) died on Day 62 of the study. Upon necropsy at University of Missouri College of nary Medicine, the diagnosis was made of chronic— active, diffuse, severe izing and emphysematous submucosal esophagitis, gastritis, and pharyngitis with intralesional coccobacilli bacterial colonies and plant material, as well as a diffuse severe fibrinosuppurative pleuropneumonia of the lung. The most likely explanation of this death suggests a previous mucosal breach, such as a mucosal ulcer in the cardia of the stomach which allowed seeding of plant material in the associated connective s (pathology report attached).

A. DURATION The bution of the duration of disease (number of days with moderate or severe respiratory disease) in days is ized in Table 27. The median number of days animals in the placebo group were observed with disease was 14. In the vaccinated group, the median number of days with disease was 1. The duration of disease was significantly lower in the vaccinated animals as compared to the ls (Table 28; P < 0.0001).

Table 27: Summary of the effect of ation on the duration of disease (Number of days with moderate or severe respiratory disease) Control (N = 20) Vaccinated (N = 20) Table 28: Effect of ation on the duration of disease (Number of days with moderate or severe respiratory disease) Duratlon 14 1 1 1 (median days) Severity The distribution of the severity of disease is summarized in Table 29. The severity of disease was significantly lower in the vaccinated horses as compared to the placebo horses (Table 30; mitigated on 2 0.7550, 95% CI = 0.5518, 0.9582). Individual outcomes are provided in Table 31.

In Table 31 the values for horse #39 which died on Day 62 of the study are reported as 0’s from Day 62 through 67, but in the analysis only values > 1 are considered, therefore not affecting the outcome. Duration is 1 day for this horse and the max score is 3.

Table 29: Summary of the severity of disease based on the maximum nasal rge score Control 0% 5% 20% 75% (N = 20) (0/20) ( 1/20) (4/20) ) Vaccinated 5% 35% 55% 5% (N = 20) (1/20) (7/20) (11/20) (1/20) Table 30: Effect of ation on the severity of disease Severity 28.05 1295* 0.7550 0.5518, 0.9582 (mean rank) *Significantly lower than the placebo group by Wilcoxon’s rank sum test (P < 0.05). o o o o o o o o o H H o o o o o o o o o H o o o o o o o o o o o o o o o o o o o o OH o o o o. o o o o o o o o o o o o .88ch H o o o o H o 82,8 o o H o 830on o H o o Ho o o 8333 o o H o :06 o o H o o o o o o o H o o H o o o o o H o o o o o :2on o o H o o o o o o o o o o o o o H5923 o H o o H o H o H o o H H o o o o o o o o o o o o o o o o o o o o o o H o o o o me8qu o o o o o o o o H o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 1.5339: o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 89¢ 06—800 06—800 0:809 U 09:00 0:800 09:50 onEoU onEoU onEoU onEoU onEoU 09:50 09:50 03:50 09:50 09:50 09:50 09:50 09:50 £30m ”Hm mwmovmwmv Nwmomwmv ﬁnchmwmv Nwmoowmv HmoHowNw Nooobwmw omowgmv Emowwmv mmoowwmv omwoowmv meHmmv ammommon mmOnmmon wwNHomon Nmmmomon mwmoomon on thNHmon mBOmHmon ooommmon .88me 89,8 BSovoS 832% :06 32on 8923 22:5“.

E5339: £53m OOOO BREE 88E 80an Boos—m 882m 30an 8035 Boos—m 383m O noon—n— 882m 30an 30an 0982; 23an 80an 80an 80an 882; 882m “@025qu ﬁ owomoowﬁ Mama? macaw? 2 2 Gwmv mewmowmw @853? mw $33? 8ng cowvbwmv 38%? 309me mmmmmwmv wwmmwwmv 550%? 32%? 008%? :3?on 303on £23on B. RECTAL TEMPERATURE The results of the statistical analysis are summarized in Table 32. The vaccine by day interaction was statistically significant. Within day analyses are provided in Table 33 (appendix). On Days 3, 4, and 5, horses in the vaccinated group had statistically significantly lower temperatures than those in the placebo group. On Day 20, horses in the vaccinated group had significantly higher temperatures than those in the placebo group. However, on all days any differences between treatment groups were not clinically meaningful and only one horse (#39) had a rectal temperature greater than 102°F (39°C) on any challenge study day.

Table 32: y of the effect of ation on rectal ature and serum neutralization titers 0.5754 < 0.0001 0.0006 < 0.0001 < 0.0001 < 0.0001 1. Serum Neutralization Titers The results of the statistical analysis are summarized in Table 32. On Days 46 and 53, horses in the vaccinated group had significantly higher titers than those in the placebo group.

On Day 67, horses in the vaccinated group had significantly lower than those in the o group.

Discussion This study was conducted to demonstrate efficacy of the Equine Rhinitis A virus component of an inactivated Equine Rhinitis A virus vaccine in ation with inactivated Equine Herpesvirus types 1 and 4 and Equine Inﬂuenza Viruses. Vaccination was given in a 2— dose format, and challenge of virulent Equine Rhinitis A virus was performed 25 days post- booster vaccination. Vaccinated and placebo treated control horses were evaluated to assess effect of vaccination on reduction of severity and duration of clinical respiratory disease. al signs, nasal swabs and buffy coats were evaluated daily for evidence of Equine Rhinitis A e and presence of the virus in the challenged horses.

Results from this challenge study show a statistically significant and clinically important reduction of both the ty and of the duration of respiratory disease in vaccinates following challenge. As med by mitigated fraction analysis of respiratory disease scores in horses vaccinated with the IVP as compared to horses vaccinated with the control product, moderate/severe signs of respiratory disease were reduced 75.5% (95% ence interval 96% to 55%). Duration of moderate/severe disease was also significantly reduced by 11 days in vaccinated horses as compared to l horses (95% confidence interval 14 days to 8 days shorter disease duration). In addition, vaccination significantly reduced nasal virus shedding throughout the successive 5 days of peak viral shedding, and viremia also was significantly reduced during the 4 days of peak viremia within the challenge period. Importantly, virus was recovered from buffy coat samples of l horses a total of 54 times over 5 study days in ison to only 8 total days of viremia over 4 study days in vaccinated horses.

Conclusions Data from this study clearly demonstrate that 2 X 1 mL uscular doses of Equine Rhinitis A/Rhinopneumonitis/Inﬂuenza Killed Virus Vaccine ct Code TBD, unlicensed), administered at 21 day intervals between doses to , significantly reduced ty and duration of respiratory disease caused by Equine Rhinitis A virulent challenge.

The study results illustrate the use of this vaccine for the immunization of horses six months of age or older against respiratory disease caused by Equine Rhinitis A virus. These data also establish Vaccine Lot 122110 as an acceptable nce vaccine for potency testing of subsequent serials of Equine Rhinitis A/Rhinopneumonitis/ Inﬂuenza Vaccine, Killed Virus and of Equine Rhinitis A Vaccine, Killed Virus (Product Code 1522.21, unlicensed).

Claims

WE CLAIM:

1. An immunogenic composition comprising one or more strains of inactivated Equine is B Virus (ERBV).

2. The immunogenic composition according to claim 1, wherein the ERBV strain grows in cell culture to 106 /mL or higher, or, when used as a vaccine in s at a dose of 106 TCID50 or higher results in a serum titer of 1:112 to 1:1000.

3. The genic composition according to claim 1 or claim 2, wherein the immunogenic composition further comprises one or more strains of inactivated Equine Rhinitis A Virus (ERAV).

4. The immunogenic composition according to claim 3, wherein the ERAV strain comprises a genomic ce whose reverse transcript has greater than 95% identity to SEQ ID NO: 2 or encodes a polyprotein with an amino acid sequence with greater than 95% identity to SEQ ID NO: 3, wherein said ERAV strain, prior to inactivation or attenuation, is active to infect and replicate in host cells.

5. The immunogenic composition according to any one of claims 1 to 4, wherein the ERBV strain comprises a genomic sequence whose reverse transcript has r than 95% identity to the reverse transcript of the genomic sequence of the ERBV strain having ATCC Accession no. PTA-11829 or encodes a otein with an amino acid sequence with greater than 95% identity to the polyprotein encoded by the genome of the ERBV strain having ATCC ion no. PTA-11829, wherein said ERBV strain, prior to inactivation or attenuation, is active to infect and replicate in host cells.

6. The immunogenic composition according to any one of claims 1 to 5 in which the strain of ERBV is strain 07-103042 (ATCC Accession PTA-11829).

7. The immunogenic composition according to any one of claims 1 to 6, wherein said genic composition comprises antigens or an inactivated or live, attenuated strain of Equine Herpes Virus. 11178739_1:JJP

8. The immunogenic composition according to any one of claims 1 to 7, wherein said immunogenic composition comprises antigens or an vated or live, attenuated strain of Equine Influenza Virus.

9. The immunogenic composition according to any one of claims 1 to 8, wherein said immunogenic ition further comprises antigens of one or more strains selected from the group ting of West Nile Virus, Eastern Equine Encephalomyelitis Virus, Western Equine Encephalomyelitis Virus, and Venezuelan Equine Encephalomyelitis Virus, or antigens of Tetanus Toxoid, or combinations thereof.

10. Use of an immunogenic composition according to any one of claims 1 to 9 for the manufacture of a medicament for reducing the incidence or lessening the severity of clinical symptoms associated with or caused by ERBV in an animal or a herd of animals.

11. Use of an immunogenic composition according to any one of claims 3 to 9 for the manufacture of a medicament for reducing the incidence or lessening the severity of clinical symptoms associated with or caused by ERAV in an animal or a herd of animals.

12. Use of an immunogenic composition according to any one of claims 7 to 9 for the manufacture of a medicament for ng the incidence or lessening the severity of clinical ms associated with or caused by ERBV and one or more of the pathogens selected from the group ting of: West Nile Virus, Eastern Equine Encephalomyelitis Virus, Western Equine Encephalomyelitis Virus, Venezuelan Equine Encephalomyelitis Virus, Equine Herpes Virus, Equine Influenza Virus, and Clostridium tetani in an animal or a herd of animals.

13. The use ing to any one of claims 10 to 12, wherein the incidence of clinical symptoms caused by one or more of said ens in a herd of animals is reduced as compared to a herd not receiving said genic composition.

14. The use according to any one of claims 10 to 13, wherein the administration of one dose or two doses of said immunogenic composition provides a on of immunity of at least 12 months against one or more of said pathogens.

15. The use according to any one of claims 10 to 14, wherein said immunogenic composition is safe for use in foals or horses 4 months of age or older. 11178739_1:JJP

16. The immunogenic ition according to any one of claims 1 to 9 for use as a ment.

17. A method for reducing the incidence or lessening the ty of clinical symptoms associated with or caused by ERBV in an animal or a herd of animals, said method comprising the step of administering an immunogenic composition according to any one of claims 1 to 9 to an animal in need thereof, wherein the animal is a non-human animal.

18. A method for reducing the incidence or ing the ty of clinical symptoms associated with or caused by ERAV in an animal or a herd of animals, said method comprising the step of administering an immunogenic composition according to any one of claims 3 to 9 to an animal in need thereof, wherein the animal is a non-human animal.

19. A method for ng the incidence or lessening the severity of clinical symptoms associated with or caused by ERBV and one or more of the pathogens selected from the group consisting of: West Nile Virus, n Equine Encephalomyelitis Virus, Western Equine Encephalomyelitis Virus, Venezuelan Equine alomyelitis Virus, Equine Herpes Virus, Equine Influenza Virus, and Clostridium tetani in an animal or a herd of animals, said method comprising the step of administering an immunogenic composition according to any one of claims 7 to 9 to an animal in need thereof, wherein the animal is a non-human animal.

20. The method according to any one of claims 17 to 19, wherein the incidence of clinical symptoms caused by one or more of said pathogens in a herd of animals is reduced as compared to a herd not receiving said immunogenic composition.

21. The method according to any one of claims 17 to 20, wherein the administration of one dose or two doses of said immunogenic composition provides a duration of immunity of at least 12 months against one or more of said pathogens.

22. The method of any one of claims 17 to 21, wherein said immunogenic composition is safe for use in foals or horses 4 months of age or older.

23. A method for producing an immunogenic ition comprising one or more s of inactivated ERBV, the method comprising: a) infecting a susceptible cell line with ERBV; 11178739_1:JJP b) growing the infected cell line in growth media until a cytopathic effect (CPE) is attained; c) harvesting the media; d) filtering the media to yield a filtered media; and e) contacting the ed media with an inactivating agent to obtain the inactivated ERBV.

24. The method of claim 23 wherein the ERBV is strain 07-103042C ATCC Accession NO: PTA-11829. Boehringer Ingelheim Vetmedica, Inc. By the Attorneys for the Applicant