WO2019092027A1

WO2019092027A1 - Sapelovirus immunogenic compositions and uses thereof

Info

Publication number: WO2019092027A1
Application number: PCT/EP2018/080486
Authority: WO
Inventors: Marshall Allen HERRICK; Lea Ann Hobbs; Arun V. Iyer; Sean O'conner; Abby Rae Patterson; Joseph Gilbert Victoria
Original assignee: Boehringer Ingelheim Vetmedica Gmbh
Priority date: 2017-11-09
Filing date: 2018-11-07
Publication date: 2019-05-16

Abstract

The present invention relates to killed/inactivated and/or recombinant Sapelovirus immunogenic compositions and vaccines, and methods of preventing or treating animals in need of with such an immunogenic compositions and vaccines.

Description

SAPELOVIRUS IMMUNOGENIC COMPOSITIONS AND USES THEREOF

SEQUENCE LISTING

[0001] This application contains a sequence listing in accordance with 37 C.F.R. 1.821 - 1.825. The sequence listing accompanying this application is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

A. Field of the invention

[0002] The present invention relates to Sapelovirus and its use as an immunogenic composition or vaccine to treat animals affected by Sapelovirus.

B. Description of the related art

[0003] Sapeloviruses are a non-enveloped, positive-sense, single-stranded RNA virus within the family Picomaviridae. Viruses were previously identified as porcine enterovirus type 8, porcine enterovirus A and as a CPE type II porcine enterovirus, but have been since re-classified into a separate genus with three species (Avian Sapelovirus, Sapelovirus A and Sapelovirus B) [ictvonline.org/virustaxonomy.asp]. The species associated with disease in pigs has been named Sapelovirus A.

[0004] Clinical disease associated with enteroviruses was originally described in 1929 in Teschen, Czechoslovakia on small private farms which were using domestic refuse for pig feed; often without proper sterilization [Kouba, V., Teschen disease (Teschovirus encephalomyelitis) eradication in Czechoslovakia: a historical report. Veterinarini Medicina, 2009. 54(11): p. 550-560]. Throughout the 1940's and 1950's, epidemics of the disease occurred in most European countries [Teschovirus encephalomyelitis, in OIE Terrestrial Manual. 2008. p. 1146-1152]. Clinical signs in affected pigs included pyrexia, anorexia, uncoordinated movements, locomotive disorders, tremors, nystagmus, opisthotonos, convulsions, paralysis and death within 3-4 days [Lan, D., et al., Isolation and characterization of the first Chinese porcine Sapelovirus strain. Arch Virol, 2011. 156(9): p. 1567-74]. In the majority of cases, serotype PEV-1 resulted in severe clinical signs while the remaining serotypes led to milder disease [Donin, D.G., et al., First report of Porcine teschovirus (PTV), Porcine Sapelovirus (PSV) and Enterovirus G (EV-G) in pig herds of Brazil. Trop Anim Health Prod, 2014. 46(3): p. 523-8]. Clinical diseases associated specifically with Sapelovirus A have included reproductive failure, neurological disorders, pneumonia and diarrhea [Schock, A., et al., Investigation into an outbreak of encephalomyelitis caused by a neuroinvasive porcine Sapelovirus in the United Kingdom. Vet Microbiol, 2014. 172(3-4): p. 381-9; Kim, D.S., et al., Pathogenesis of Korean SapelovirusA in piglets and chicks. J Gen Virol, 2016. 97(10): p. 2566-2574; Huang, J., R.F. Gentry, and A. Zarkower, Experimental infection of pregnant sows with porcine enteroviruses. Am J Vet Res, 1980. 41 : p. 469; Arruda, P.H., et al., Detection of a novel Sapelovirus in central nervous tissue of pigs with polioencephalomyelitis in the USA. Transbound Emerg Dis, 2017; Dunne, H.W., et al., Porcine reproductive failure associated with a newly identified "SMEDI" group of picorna viruses. Am J Vet Res, 1965. 26(115): p. 1284-97; and Traub, E., Active immunization against Teschen disease using vaccines adsorbed on aluminium hydroxide. Arch. Tierheilkd, 1942. 77(52-66)].

[0005] While implicated in cases of reproductive failure as early as the 1960's [Mayr, A., the viruses of Teschen disease. Ann N Y Acad Sci, 1962. 101 : p423-7], outbreaks of polioencephalomyelitis in weaned pigs have been more frequently reported since 2011.

[0006] During periods of high clinical disease in Europe in the 1940's and 50' s, vaccine was commonly used [Traub, E., Active immunization against Teschen disease using vaccines adsorbed on aluminium hydroxide. Arch. Tierheilkd, 1942. 77(52-66)]; however, vaccination was discontinued when clinical disease became rare in the 1980's.

[0007] There are no vaccines currently available and due to the increase in reporting of

polioencephalomyelitis in weaned pigs, it is of interest to develop a vaccine to help control this virus.

SUMMARY OF THE INVENTION [0008] The present invention provides immunogenic compositions, vaccines, and related methods that overcome deficiencies in the art. The compositions and methods provide immunogenic compositions, which include an inactivated/killed of a non-enveloped (+) single-stranded RNA virus of Sapelovirus. In particular, the application provides a vaccine for generating an immune response in porcine for protection against diseases associated with Sapelovirus. The present Sapelovirus was isolated from the brain of a pig exhibiting neurological signs.

[0009] Immunogenic compositions and vaccines of the invention comprise SEQ ID NOs: 1 -12, and more preferably SEQ ID NOs: 9-12.

[0010] Exemplary compositions of the invention comprise the polypeptide sequences of SEQ ID NOs: 1 -12, or fragments thereof that are immunoreactive to Sapelovirus. [0011] In another aspect, the invention provides nucleic acid sequences that encode one or more polypeptides, antibody constructs, or antibody conjugates. The gene sequences coding for the polypeptides comprise a nucleic acid sequence that is at least 95%, 90%, 85%, or even 80% homologous to and/or identical with the sequence of SEQ ID NOs: 1-12, and more preferably, SEQ ID NOs: 9-12, or fragments thereof coding for a polypeptide that is immunoreactive to Sapelovirus. Exemplary nucleic acid sequences of the invention include any one of the sequences of SEQ ID NOs: 1-12, and fragments thereof that encode a polypeptide that is immunoreactive to Sapelovirus.

[0012] Moreover a polypeptide of the invention as used herein includes but is not limited to a polypeptide that comprises:

i) a polypeptide comprising the amino acid sequence of SEQ ID NOs: 4, 8, and 12; ii) a polypeptide that is at least 80% homologous to and/or identical with a polypeptide of i); iii) a fragment of the polypeptides of i) and/or ii);

iv) a polypeptide of i) or ii);

v) a fragment of iii) or iv) comprising at least 5, preferably 8, more preferably 10, even more preferably 15 contiguous amino acids included in the sequences of SEQ ID NOs: 4, 8, and 12;

vi) a polypeptide that is encoded by a polynucleotide comprising the sequence of SEQ ID NOs: 3, 7, and 11 ; vii) a polypeptide that is encoded by a polynucleotide that is at least 80% homologous to or identical with polynucleotides of vi);

viii) a protein fragment that is encoded by a polynucleotide that comprises at least 15, preferably 24, more preferably 30, even more preferably 45 contiguous nucleotides included in the sequences of SEQ ID

NOs: 1, 2, 5, 7, 9, and 11.

[0013] Immunogenic compositions of the invention, which comprise at least one or more Sapelovirus polypeptides as defined herein, may further comprise a physiologically-acceptable vehicle, such as a pharmaceutically or veterinarily acceptable carrier, adjuvant, or combination thereof. [0014] Any of the Sapelovirus polypeptides provided herewith or any immunogenic compositions comprising one or more of these Sapelovirus polypeptides provided herewith may be used as a medicament, preferably as a vaccine or immunogenic composition, most preferably for the prophylaxis or treatment of a subject against a Sapelovirus infection.

[0015] Those of skill in the art will understand that the compositions used herein may incorporate known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, e.g., saline or plasma protein solutions, are readily available. In addition, the immunogenic and vaccine compositions of the present invention can include veterinary-acceptable carriers, diluents, isotonic agents, stabilizers, or adjuvants.

[0016] Methods of the invention include, but are not limited to, a method of provoking an immune response against a Sapelovirus infection in a subject comprising the step of administering to the subject an immunogenic composition comprising one or more Sapelovirus polypeptides as defined herein. Compositions of the invention may be used to treat or alternatively to prevent a Sapelovirus infection. Preferably, such immune response reduces the incidence of or severity of one or more clinical signs associated with or caused by the infection with Sapelovirus serotypes. [0017] Herein, suitable subjects and subjects in need to which compositions of the invention may be administered include animals in need of either prophylactic or treatment for a viral associated infection, disease, or condition. Animals in which the immune response is stimulated by use of compositions or methods of the invention include livestock, such as swine, bovines, goats, and sheep. Preferred animals include porcines, murids, equids, lagomorphs, and bovids. Most preferably, an immune response is stimulated in swine. [0018] The invention also provides a method of reducing the incidence of or severity of one or more clinical signs associated with or caused by Sapelovirus infection, comprising the step of administering an immunogenic composition of the invention that comprises one or more Sapelovirus peptides as provided herewith and preferably a carrier molecule, such that the incidence of or the severity of a clinical sign of the Sapelovirus infection is reduced by at least 10%, preferably at least 20%, even more preferred at least 30%, even more preferred at least 50%, even more preferred at least 70%, most preferred at least 100% relative to a subject that has not received the immunogenic composition as provided herewith.

[0019] Such clinical signs include pyrexia, anorexia, ataxia, uncoordinated movements, locomotive disorders, tremors, nystagmus, opisthotonos (e.g., spasm of the muscles causing backward arching of the head, neck, and spine), convulsions, paralysis, paresis, mental dullness, and death within 3-4 days. Clinical diseases associated specifically with sapelovirus A have included reproductive failure, neurological disorders, pneumonia and diarrhea.

[0020] According to a further aspect, the present invention also relates to a method for the prophylaxis of a Sapelovirus infection, wherein said Sapelovirus infection may be caused by Sapelovirus, comprising the step of administering an immunogenic composition of the invention that comprises one or more Sapelovirus peptides as provided herewith. [0021] The invention also provides a method of preparing any of the immunogenic compositions provided herewith that method comprises mixing one or more Sapelovirus peptides as provided herewith with a carrier molecule, preferably such that the one or more Sapelovirus peptides and carrier molecule are covalently coupled or conjugated to one another. Such conjugates may be multivalent or univalent. Multivalent compositions or vaccines include an immuno-conjugation of multiple Sapelovirus peptides with a carrier molecule. In a further aspect, the invention provides a method of producing one or more Sapelovirus peptides that method comprises transforming a host cell, preferably a prokaryotic cell such as E. coli with a nucleic acid molecule that codes for any of the Sapelovirus peptides as provided herewith. Alternatively, the host cell may be a eukaryotic cell such as an animal cell, protist cell, plant cell, or fungal cell. Preferably the eukaryotic cell is a mammalian cell such as CHO, BHK or COS, or a fungal cell such as Saccharomyces cerevisiae, or an insect cell such as Sf . [0022] Another aspect of the invention provides a method of producing one or more Sapelovirus peptides that induce an immune response against Sapelovirus. This comprises culturing a transformed expression vector coding for and expressing one or more Sapelovirus peptides disclosed herein. The expressed proteins are either retained by the expression organism or secreted into the culture medium. Expression is conducted under conditions sufficient to produce a Sapelovirus peptide capable of inducing an immune response to Sapelovirus. [0023] Methods of making compositions of the invention may further comprise admixing the conjugate of one or more Sapelovirus peptides or inactivated whole-virus preparations and a carrier molecule with a physiologically-acceptable vehicle such as a pharmaceutically- or veterinary-acceptable carrier, adjuvant, or combination thereof. Those of skill in the art will recognize that the choice of vehicle, adjuvant, or combination will be determined by the delivery route, personal preference, and animal species among others. [0024] In another aspect, the invention provides a method of diagnosing a Sapelovirus infection in a subject. That method comprises providing one or more Sapelovirus peptides; contacting the one or more Sapelovirus peptides with a sample obtained from the subject; and identifying the subject as having a Sapelovirus infection if an antibody capable of binding the one or more Sapelovirus peptides is detected in the sample.

[0025] In another respect, the invention provides a method of ascertaining that a subject has been previously exposed to a Sapelovirus infection and is able to express an immune response to Sapelovirus. That method comprises providing one or more Sapelovirus peptides; contacting the one or more Sapelovirus peptides with a sample obtained from the subject; and identifying the subject as having a Sapelovirus infection if an antibody capable of binding the one or more Sapelovirus peptides is detected in the sample.

[0026] The invention also provides kits that comprise an immunogenic composition that comprises one or more Sapelovirus peptides, preferably together with a carrier molecule; a container for packaging the immunogenic composition; a set of printed instructions; and a dispenser capable of administering the immunogenic composition to an animal. Optionally, the one or more Sapelovirus peptides and the carrier molecule may be packaged as a conjugate or as separate compounds. When supplied separately, a means of conjugating the one or more Sapelovirus peptides and carrier molecule, as well as appropriate printed instructions, is also supplied. [0027] The invention also provides kits for vaccinating an animal comprising a set of printed instructions; a dispenser capable of administering the immunogenic composition provided herewith comprising one or more Sapelovirus peptides to an animal; and wherein at least one of Sapelovirus peptides effectively immunizes the animal against at least one disease associated with Sapelovirus infection. Preferably, the one or more

Sapelovirus peptides are selected from those provided herewith. Kits of the invention may further comprise a veterinary acceptable carrier, adjuvant, or combination thereof.

[0028] The dispenser in a kit of the invention is capable of dispensing its contents as droplets; and the immunogenic composition comprises the Sapelovirus peptides as provided herewith included in the kit is capable of reducing the severity of at least one clinical sign of a Sapelovirus infection when administered intranasally, orally, intradermally, or intramuscularly to an animal. Preferably, the severity of a clinical sign is reduced by at least 10% preferably by at least 20%, even more preferred by at least 30%, even more preferred by at least 50%, even more preferred by at least 70%, most preferred by at least 100% as compared to an untreated, infected animal.

[0029] The invention also provides methods for specific detection and quantification of viremia from infected animals. This method comprises specific polynucleotide sequences (SEQ ID NOs: 13-15) complementary to viral cDNA (SEQ ID NOS: 1, 5 and 9) and methodology to amplify and detect viral infection of the sample.

[0030] Methods for the treatment or prophylaxis of infections caused by Sapelovirus are also disclosed. The method comprises administering an effective amount of the immunogenic composition of the present invention to a subject, wherein said treatment or prophylaxis is selected from the group consisting of reducing signs of Sapelovirus infection, reducing the severity of or incidence of clinical signs of Sapelovirus infection, reducing the mortality of subjects from Sapelovirus infection, and combinations thereof. [0031] Compositions of the invention further comprise a veterinarily acceptable carrier, adjuvant, or combination thereof. Such compositions may be used as a vaccine and comprise an inactivated vaccine. Such vaccines elicit a protective immunological response against at least one disease associated with Sapelovirus.

[0032] Those of skill in the art will understand that the compositions used herein may incorporate known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, e.g., saline or plasma protein solutions, are readily available. In addition, the immunogenic and vaccine compositions of the present invention can include pharmaceutical- or veterinary-acceptable carriers, diluents, isotonic agents, stabilizers, or adjuvants.

[0033] Methods of the invention may also comprise admixing a composition of the invention with a veterinarily acceptable carrier, adjuvant, or combination thereof. Those of skill in the art will recognize that the choice of carrier, adjuvant, or combination will be determined by the delivery route, personal preference, and animal species among others.

[0034] Methods for the treatment or prophylaxis of infections caused by Sapelovirus are also disclosed. The method comprises administering an effective amount of the immunogenic composition of the present invention to an animal, wherein said treatment or prophylaxis is selected from the group consisting of reducing signs of Sapelovirus infection, reducing the severity of or incidence of clinical signs of Sapelovirus infection, reducing the mortality of animals from Sapelovirus infection, and combinations thereof.

[0035] Preferred routes of administration include intranasal, oral, intradermal, and intramuscular.

Administration via the intramuscular route, most preferably in a single dose, is preferred. The skilled artisan will recognize that compositions of the invention may also be administered in two or more doses, as well as, by other routes of administration. For example, such other routes include subcutaneously, intracutaneously, intravenously, intravascularly, intraarterially, intraperitnoeally, intrathecally, intratracheally, intracutaneously, intracardially, intralobally, intramedullarly, intrapulmonarily, or intravaginally. Depending on the desired duration and effectiveness of the treatment, the compositions according to the invention may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months and in different dosages.

[0036] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 shows the isolates of the present invention that were sequenced and an approximate -9% nucleotide difference (whole genome comparison) was identified between the two isolates.

[0038] FIG. 2 is a graph showing total and neurological mortality percentage by wean group. [0039] FIG. 3 illustrates the total neurologic mortality and percent of neurologic mortality by days post placement.

[0040] FIG. 4 shows Virus Neutralizing (VN) percent positives data in weaned pigs and late finishing pigs.

DETAILED DESCRIPTION [0041] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology, protein chemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Vols. I, II and III, Second Edition (1989); DNA Cloning, Vols. I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. K. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL press, 1986); Perbal, B., A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Protein purification methods - a practical approach (E.L.V. Harris and S. Angal, eds., IRL Press at Oxford University Press); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific Publications).

[0042] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular DNA, polypeptide sequences or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an antigen" includes a mixture of two or more antigens; reference to "an excipient" includes mixtures of two or more excipients, and the like.

A. Definitions [0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs at the time of filing. The meaning and scope of terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Herein, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms such as "includes" and "included" is not limiting. All patents and publications referred to herein are incorporated by reference herein.

[0044] "Protection against disease", "protective immunity", "functional immunity" and similar phrases, means a response against a disease or condition generated by administration of one or more therapeutic compositions of the invention, or a combination thereof, that results in fewer deleterious effects than would be expected in a non-immunized subject that has been exposed to disease or infection. That is, the severity of the deleterious effects of the infection is lessened in a vaccinated subject. Infection may be reduced, slowed, or possibly fully prevented, in a vaccinated subject. Herein, where complete prevention of infection is meant, it is specifically stated. If complete prevention is not stated then the term includes partial prevention. [0045] Herein, "reduction of the incidence and/or severity of clinical signs" or "reduction of clinical symptoms" means, but is not limited to, reducing the number of infected subjects in a group, reducing or eliminating the number of subjects exhibiting clinical signs of infection, or reducing the severity of any clinical signs that are present in one or more subjects, in comparison to wild-type infection. For example, it should refer to any reduction of pathogen load, pathogen shedding, reduction in pathogen transmission, or reduction of any clinical sign symptomatic of Sapelovirus infection. Preferably, these clinical signs are reduced in one or more subjects receiving the therapeutic composition of the present invention by at least 10% in comparison to subjects not receiving the composition and that become infected. More preferably, clinical signs are reduced in subjects receiving a composition of the present invention by at least 20%, preferably by at least 30%, more preferably by at least 40%, and even more preferably by at least 50%. [0046] The term "increased protection" herein means, but is not limited to, a statistically significant reduction of one or more clinical symptoms which are associated with infection by an infectious agent, preferably Sapelovirus, respectively, in a vaccinated group of subjects vs. a non-vaccinated control group of subjects. The term "statistically significant reduction of clinical symptoms" means, but is not limited to, the frequency in the incidence of at least one clinical symptom in the vaccinated group of subjects is at least 10%, preferably 20%, more preferably 30%, even more preferably 50%, and even more preferably 70% lower than in the non-vaccinated control group after the challenge the infectious agent.

[0047] "Long-lasting protection" shall refer to "improved efficacy" that persists for at least 3 weeks, but more preferably at least 3 months, still more preferably at least 6 months. In the case of livestock, it is most preferred that the long lasting protection shall persist until the average age at which animals are marketed for meat.

[0048] An "immunogenic or immunological composition" refers to a composition of matter that comprises at least one Sapelovirus immunogenic composition, or immunogenic portion thereof, that elicits an immunological response in the host of a cellular or antibody-mediated immune response to the composition. In a preferred embodiment of the present invention, an immunogenic composition induces an immune response and, more preferably, confers protective immunity against one or more of the clinical signs of a Sapelovirus infection. [0049] An "immunogenic" or "antigen" as used herein refer to a polypeptide or protein that elicits an immunological response as described herein. An "immunogenic" Sapelovirus protein or polypeptide includes the full-length sequence of any of the Sapelovirus identified herein or analogs or immunogenic fragments thereof. The term "immunogenic fragment" or "immunogenic portion" refers to a fragment or truncated and/or substituted form of a Sapelovirus that includes one or more epitopes and thus elicits the immunological response described herein. In general, such truncated and/or substituted forms, or fragments will comprise at least six contiguous amino acids from the full-length Sapelovirus protein. More preferably, the truncated or substituted forms, or fragments will have at least 10, more preferably at least 15, and still more preferably at least 19 contiguous amino acids from the full-length Sapelovirus protein. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known and described in the art, see e.g., U.S. Patent No. 4,708,871 ; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81 :3998-4002; and Geysen et al. (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and two-dimensional nuclear magnetic resonance. See Epitope Mapping Protocols, supra. Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al. (1993) Eur. J. Immunol. 23:2777-2781 ; Bergmann et al. (1996), J. Immunol. 157:3242-3249; Suhrbier, A. (1997), Immunol, and Cell Biol. 75:402-408; and Gardner et al., (1998) 12th World AIDS Conference, Geneva, Switzerland, June 28-July 3, 1998. (The teachings and content of which are all incorporated by reference herein.)

[0050] An "immune response" or "immunological response" means, but is not limited to, the development of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, an immune or immunological response includes, 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, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or a protective immunological (memory) response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction in number of symptoms, severity of symptoms, or the lack of one or more of the symptoms associated with the infection of the pathogen, a delay in the of onset of viremia, reduced viral persistence, a reduction in the overall viral load and/or a reduction of viral excretion.

[0051] Herein, "specifically immunoreactive" refers to an immunoreactive protein or polypeptide that recognizes an antigen characteristic of Sapelovirus infection but does not react with an antigen characteristic of a strict challenge control. [0052] As used herein, "a pharmaceutical- or veterinary-acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. In some preferred embodiments, and especially those that include lyophilized immunogenic compositions, stabilizing agents for use in the present invention include stabilizers for lyophilization or freeze-drying.

[0053] In some embodiments, the immunogenic composition of the present invention contains an adjuvant. "Adjuvants" as used herein, can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di- (caprylate/caprate), glyceryl 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 blocks, in particular the Pluronic products, especially L121 (commercially available from BASF Corporation). See Hunter et al., The Theory and Practical Application of Adjuvants (Ed.Stewart-Tull, D. E. S.), John Wiley and Sons, NY, pp51-94 (1995) and Todd et al, Vaccine 15:564-570 (1997). Exemplary adjuvants are the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach" edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of this same book.

[0054] A further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative. Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid, which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Patent No. 2,909,462, which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name CARBOPOL® (Lubrizol)) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned Carbopol 974P, 934P and 97 IP. Most preferred is the use of Carbopol 97 IP. Among the copolymers of maleic anhydride and alkenyl derivative, are the copolymers EMA (Monsanto), which are copolymers of maleic anhydride and ethylene. The dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated. [0055] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid- amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide, or naturally occurring or recombinant cytokines or analogs thereof or stimulants of endogenous cytokine release, among many others.

[0056] It is expected that an adjuvant can be added in an amount of about 100 μg to about 10 mg per dose, preferably in an amount of about 100 μg to about 10 mg per dose, more preferably in an amount of about 500 μg to about 5 mg per dose, even more preferably in an amount of about 750 μg to about 2.5 mg per dose, and most preferably in an amount of about 1 mg per dose. Alternatively, the adjuvant may be at a concentration of about 0.01 to 50%, preferably at a concentration of about 2% to 30%, more preferably at a concentration of about 5% to 25%, still more preferably at a concentration of about 7% to 22%, and most preferably at a concentration of 10% to 20% by volume of the final product.

[0057] "Diluents" can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid (EDTA), among others.

[0058] "Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. [0059] "Safety" refers to the absence of adverse consequences in a vaccinated animal following vaccination, including but not limited to: potential reversion of an attenuated virus vaccine to virulence and resistance to viral inactivation, clinically significant side effects such as persistent, systemic illness or unacceptable inflammation at the site of vaccine administration.

[0060] The terms "vaccination" or "vaccinating" or variants thereof, as used herein means, but is not limited to, a process which includes the administration of an immunogenic composition of the invention that, when administered to an animal, elicits, or is able to elicit - directly or indirectly - an immune response in the animal against Sapelovirus.

[0061] "Mortality", in the context of the present invention, refers to death caused by Sapelovirus infection, and includes the situation where the infection is so severe that an animal is euthanized to prevent suffering and provide a humane ending to its life.

[0062] "Killed" or "inactivated" means treated with a physical or chemical agent which renders the Sapelovirus dead and/or otherwise incapable of reproduction. Sapelovirus may be killed by conventional means, such as, for example, heat, radiation or psoralen in the presence of ultraviolet light. Sapelovirus can be inactivated by conventional means such as, for example, through chemical inactivation using one or more chemical inactivating agents including, but not limited to, one or more of binary ethyleneimine (BEI), beta- propiolactone, formalin, gluteraldehyde, and/or sodium dodecyl sulfate. Methods of attenuating virulent strains of these viruses and methods of making an inactivated viral preparation are known in the art and are described in, e.g., U.S. 4,567,042 and 4,567,043. Antigens from Sapelovirus for use in the vaccine compositions of the present invention may thus be in the form of a whole virus which is a modified and/or attenuated live viral preparation or a killed or inactivated viral preparation, inter alia.

[0063] "Antibodies" as used herein includes anti-Sapelovirus antibodies, e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, humanized, human, porcine, and CDR -grafted antibodies, including compounds which include CDR sequences which specifically recognize a Sapelovirus polypeptide of the invention. The term "specific for" indicates that the variable regions of the antibodies of the invention recognize and bind a Sapelovirus polypeptide exclusively (i.e., are able to distinguish a single

Sapelovirus polypeptide from related polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), and which are permitted (optionally) to interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the antibody molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual: Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6. Antibodies that recognize and bind fragments of the Sapelovirus polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, a Sapelovirus polypeptide of the invention from which the fragment was derived. For the purposes of clarity, "antibody" refers to an immunoglobulin molecule that can bind to a specific antigen as the result of an immune response to that antigen. Immunoglobulins are serum proteins composed of "light" and "heavy" polypeptide chains having "constant" and "variable" regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions. Antibodies can exist in a variety of forms including, for example, as, Fv, Fab', F(ab')2, as well as in single chains, and include synthetic polypeptides that contain all or part of one or more antibody single chain polypeptide sequences.

[0064] Herein, "effective dose" means, but is not limited to, an amount of antigen that elicits, or is able to elicit, an immune response that yields a reduction of clinical symptoms in an animal to which the antigen is administered. [0065] As used herein, the term "effective amount" means, in the context of a composition, an amount of an immunogenic composition capable of inducing an immune response that reduces the incidence of or lessens the severity of infection or incident of disease in an animal. Particularly, an effective amount refers to measured inclusion by TCID₅₀ per dose or by qPCR based determination of viral load per dose. Alternatively, in the context of a therapy, the term "effective amount" refers to the amount of a therapy which is sufficient to reduce or ameliorate the severity or duration of a disease or disorder, or one or more symptoms thereof, prevent the advancement of a disease or disorder, cause the regression of a disease or disorder, prevent the recurrence, development, onset, or progression of one or more symptoms associated with a disease or disorder, or enhance or improve the prophylaxis or treatment of another therapy or therapeutic agent.

[0066] The term "fragment" refers to a fragment or truncated and/or substituted form of a Sapelovirus peptide or a gene coding for such Sapelovirus peptide that includes one or more epitopes and thus elicits the immunological response against Sapelovirus. Preferably, such fragment is a fragment or truncated and/or substituted form of any of the Sapelovirus peptides or any of the Sapelovirus genes provided herewith. In general, such truncated and/or substituted forms, or fragments will comprise at least six contiguous amino acids from the full-length Sapelovirus sequence. More preferably, the truncated or substituted forms, or fragments will have at least 10, more preferably at least 15, and still more preferably at least 19 contiguous amino acids from the full-length Sapelovirus sequence. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by concurrently synthesizing large numbers of peptides on solid supports, the peptides

corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known and described in the art, see e.g., U.S. Patent No. 4,708,871 ; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81 :3998-4002; and Geysen et al. (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and two-dimensional nuclear magnetic resonance. See Epitope Mapping Protocols, supra. Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al. (1993) Eur. J. Immunol. 23:2777-2781 ; Bergmann et al. (1996), J. Immunol. 157:3242-3249; Suhrbier, A. (1997), Immunol, and Cell Biol. 75:402-408; and Gardner et al., (1998) 12th World AIDS Conference, Geneva, Switzerland, June 28-July 3, 1998. (The teachings and content of which are all incorporated by reference herein.) [0067] The term "variant" with respect to sequences (e.g., a polypeptide or nucleic acid sequence) is intended to mean substantially similar sequences. For nucleotide sequences comprising an open reading frame, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein for the purposes of codon optimization. Generally, nucleotide sequence variants of the invention will have at least at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91 %, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 96.1 %, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1 %, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1 %, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1 % , 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%. 99.9% sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters. [0068] The term "immunoreactive to Sapelovirus" as used herein means that the peptide or fragment elicits the immunological response against Sapelovirus.

[0069] "Sequence homology", as used herein, refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned, and gaps are introduced if necessary. However, in contrast to "sequence identity", conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 85%, preferably 90%, even more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 15%, preferably up to 10%, even more preferably up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence. Preferably the homo log sequence comprises at least a stretch of 50, even more preferred of 100, even more preferred of 250, even more preferred of 500 nucleotides.

[0070] A "conservative substitution" refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, hydrophobicity, etc., such that the overall functionality does not change significantly.

[0071] "Sequence Identity" as it is known in the art refers to a relationship 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 determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are "identical" at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular 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); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence 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 package (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, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings 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% "sequence 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 mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 85%, preferably 90%, even more preferably 95% identity relative to the reference nucleotide sequence, up to 15%, preferably 10%, even more preferably 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides 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 nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. 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 reference 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%, preferably 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%, preferably up to 10%, even more preferably 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 terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence 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.

[0072] The terms "sequence identity" or "percent identity" are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid for optimal alignment with a second amino or nucleic acid sequence). The amino acid or nucleotide residues at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical positions/total number of positions (i.e.

overlapping positions) x 100). Preferably, the two sequences are the same length.

[0073] A sequence comparison may be carried out over the entire lengths of the two sequences being compared or over fragment of the two sequences. Typically and preferred in the scope of the present invention, the comparison will be carried out over the full length of the two sequences being compared. However, sequence identity may be carried out over a region of, for example, twenty, fifty, one hundred or more contiguous amino acid residues.

[0074] As used herein, it is in particular understood that the term "having at least X% sequence identity with the nucleic acid/amino acid sequence according to SEQ ID NO:Y" (or, alternatively, the term "having at least X% sequence identity with the nucleic acid/amino acid sequence of/set forth in SEQ ID NO:Y") is equivalent to the term "having at least X% sequence identity with the nucleic acid/amino acid sequence according to SEQ ID NO:Y over the length of SEQ ID NO:Y" or to the term "having at least X% sequence identity with the nucleic acid/amino acid sequence according to SEQ ID NO:Y over the whole length of SEQ ID NO:Y", respectively.

[0075] Vectors and methods for making and/or using vectors (or recombinants) for expression can be by or analogous to the methods disclosed in: U.S. Pat. Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807, 4,722,848, 5,942,235, 5,364,773, 5,762,938, 5,770,212, 5,942,235, 382,425, PCT publications WO 94/16716, WO 96/39491, WO 95/30018; Paoletti, "Applications of pox virus vectors to vaccination: An update, "PNAS USA 93: 11349-11353, October 1996; Moss, "Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety," PNAS USA 93: 11341-11348, October 1996; Smith et al., U.S. Pat. No. 4,745,051 (recombinant baculovirus); Richardson, C. D. (Editor), Methods in Molecular Biology 39,

"Baculovirus Expression Protocols" (1995 Humana Press Inc.); Smith et al., "Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector", Molecular and Cellular Biology, December, 1983, Vol. 3, No. 12, p. 2156-2165; Pennock et al., "Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus vector, "Molecular and Cellular Biology March 1984, Vol. 4, No. 3, p. 406; EPA0 370 573; U.S. application No. 920,197, filed Oct. 16, 1986; EP Patent publication No. 265785; U.S. Pat. No. 4,769,331 (recombinant herpesvirus); Roizman, "The function of herpes simplex virus genes: A primer for genetic engineering of novel vectors," PNAS USA 93: 11307-11312, October 1996; Andreansky et al., "The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors," PNAS USA 93: 11313-11318, October 1996; Robertson et al., "Epstein-Barr virus vectors for gene delivery to B lymphocytes", PNAS USA 93: 11334-11340, October 1996; Frolov et al.,

"Alphavirus-based expression vectors: Strategies and applications," PNAS USA 93: 11371-11377, October 1996; Kitson et al., J. Virol. 65, 3068-3075, 1991 ; U.S. Pat. Nos. 5,591,439, 5,552,143; WO 98/00166; allowed U.S. application Ser. Nos. 08/675,556, and 08/675,566 both filed Jul. 3, 1996 (recombinant adenovirus);

Grunhaus et al., 1992, "Adenovirus as cloning vectors," Seminars in Virology (Vol. 3) p. 237-52, 1993; Ballay et al. EMBO Journal, vol. 4, p. 3861-65, Graham, Tibtech 8, 85-87, April, 1990; Prevec et al., J. Gen Virol. 70, 42434; PCT WO 91/11525; Feigner et al. (1994), J. Biol. Chem. 269, 2550-2561, Science, 259: 1745-49, 1993; and McClements et al., "Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease", PNAS USA 93: 11414-11420, October 1996; and U.S. Pat. Nos. 5,591,639, 5,589,466, and 5,580,859, as well as WO 90/11092, W093/19183, W094/21797, WO95/11307, WO95/20660; Tang et al., Nature, and Furth et al., Analytical Biochemistry, relating to DNA expression vectors, inter alia. See also WO 98/33510; Ju et al., Diabetologia, 41 : 736-739, 1998 (lentiviral expression system); Sanford et al., U.S. Pat. No. 4,945,050;

Fischbachet al. (Intracel); WO 90/01543; Robinson et al., Seminars in Immunology vol. 9, pp. 271-283 (1997), (DNA vector systems); Szoka et al., U.S. patent No. 4,394,448 (method of inserting DNA into living cells); McCormick et al., U.S. Pat. No. 5,677,178 (use of cytopathic viruses); and U.S. Pat. No. 5,928,913 (vectors for gene delivery); as well as other documents cited herein. [0076] Preferred viral vectors include baculovirus such as BaculoGold (BD Biosciences Pharmingen, San Diego, Calif.), in particular provided that the production cells are insect cells. Although the baculovirus expression system is preferred, it is understood by those of skill in the art that other expression systems will work for purposes of the present invention.

B. Carriers Molecules [0077] The carrier molecules to which the Sapelovirus peptides of the invention can be conjugated or covalently linked are preferably those described above. Preferred carriers for animal use are bovine serum albumin (BS A) and Keyhole Limpet Hemocyanin (KLH). Protein carriers suitable for human use include tetanus toxoid, diphtheria toxoid, acellular pertussis vaccine (LPF toxoid), cross-reacting materials (CRM's) which are antigenically similar to bacterial toxins but are non-toxic by means of mutation. For example, CRM 197 obtained according to Pappenheimer, et al, Immunochemistry, 9, 891-906 (1972), and other bacterial protein carriers, for example meningococcal outer membrane protein may be used. Preferably, the carrier protein itself is an immunogen.

[0078] The Sapelovirus peptides of the invention may be covalently coupled to the carrier by any convenient method known to the art. While use of a symmetric linker such as adipic acid dihydrazide, as described by Schneerson et al, J. Experimental Medicine, 152, 361 -376 (1980), or a heterobifunctional linker such as N- succinimidyl 3-(2-pyridyldithio) propionate as described by Fattom et al, Infection and Immunity, 56, 2292- 2298 (1988) are within the scope of the invention, it is preferred to avoid the use of any linker but instead couple a Sapelovirus peptide of the invention directly to the carrier molecule. Such coupling may be achieved by means of reductive amination as described by Landi et al J. Immunology, 127, 1011-1019 (1981). [0079] The size of the immunogenic composition, as defined by average molecular weight, is variable and dependent upon the chosen Sapelovirus peptide(s) and the method of coupling of the Sapelovirus peptide(s) to the carrier. Therefore, it can be as small as 1,000 daltons (10³) or greater than 10⁶ daltons. With the reductive amination coupling method, the molecular weight of the Sapelovirus peptide(s) is usually within the range of 5,000 to 500,000, for example 300,000 to 500,000, or for example 5,000 to 50,000 daltons. [0080] Carrier molecules, i.e. peptides, derivatives and analogs thereof, and peptide mimetics that specifically bind a Sapelovirus peptide of the invention can be produced by various methods known in the art, including, but not limited to solid-phase synthesis or by solution (Nakanishi et al., 1993, Gene 137:51-56; Merrifield, 1963, J. Am. Chem. Soc. 15:2149-2154; Neurath, H. et al., Eds., The Proteins, Vol II, 3d Ed., p. 105- 237, Academic Press, New York, N.Y. (1976), incorporated herein in their entirety by reference).

[0081] The Sapelovirus peptides of the invention or the antibodies or binding portions thereof of the present invention may be administered in injectable dosages by solution or suspension of in a diluent with a pharmaceutical or veterinary carrier.

[0082] Toxicity and therapeutic efficacy of such molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population). [0083] The vaccines of the invention may be multivalent or monovalent. Multivalent vaccines are made from immuno-conjugation of multiple Sapelovirus peptides with a carrier molecule.

[0084] In one aspect, the Sapelovirus peptide compositions comprise an effective immunizing amount of the immunogenic conjugate, preferably in combination with an immunostimulant; and a physiologically acceptable vehicle. As used in the present context, "immunostimulant" is intended to encompass any compound or composition, which has the ability to enhance the activity of the immune system, whether it is a specific potentiating effect in combination with a specific antigen, or simply an independent effect upon the activity of one or more elements of the immune response. Immunostimulant compounds include but are not limited to mineral gels, e.g., aluminum hydroxide; surface active substances such as lysolecithin, PLURONIC® polyols; polyanions; peptides; oil emulsions; alum, and MDP. Methods of utilizing these materials are known in the art, and it is well within the ability of the skilled artisan to determine an optimum amount of stimulant for a given vaccine. More than one immunostimulant may be used in a given formulation. The immunogen may also be incorporated into liposomes, or conjugated to polysaccharides and/or other polymers for use in a vaccine formulation.

[0085] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration preferably for administration to a mammal, especially a pig. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

C. Adjuvants

[0086] In order to further increase the immunogenicity of the immunogenic compositions provided herewith, and which contain one or more Sapelovirus peptides may also comprise one or more adjuvants.

[0087] The adjuvant may be purified by any of the techniques described previously or known in the art. The preferred purification technique is silica gel chromatography, in particular the "flash" (rapid) chromatographic technique, as described by W. Clark Still et al, J. Organic Chemistry, 43, 2923-2925 (1978). However, other chromatographic methods, including HPLC, may be used for purification of the adjuvant. Crystallization may also be used to purify the adjuvant. In some cases, no purification is required as a product of analytical purity is obtained directly from the synthesis. [0088] The vaccine compositions of the invention are prepared by physically mixing the adjuvant with the Sapelovirus peptide(s) under appropriate sterile conditions in accordance with known techniques to produce the adjuvanted composition. Complexation of the Sapelovirus peptide(s) and the adjuvant is facilitated by the existence of a net negative charge on the conjugate which is electrostatically attracted to the positive charge present on the long chain alkyl compound adjuvant. [0089] It is expected that an adjuvant can be added in an amount of about 100 μg to about 10 mg per dose, preferably in an amount of about 100 μg to about 10 mg per dose, more preferably in an amount of about 500 μg to about 5 mg per dose, even more preferably in an amount of about 750 μg to about 2.5 mg per dose, and most preferably in an amount of about 1 mg per dose. Alternatively, the adjuvant may be at a concentration of about 0.01 % to 75%, preferably at a concentration of about 2% to 30%, more preferably at a concentration of about 5% to 25%, still more preferably at a concentration of about 7% to 22%, and most preferably at a concentration of 10% to 20% by volume of the final product.

D. Physiologically- Acceptable Vehicles

[0090] The vaccine compositions of this invention may be formulated using techniques similar to those used for other pharmaceutical polypeptide compositions. Thus, the adjuvant and Sapelovirus peptide(s), preferably conjugated to carrier molecule and/or admixed with an adjuvant may be stored in lyophilized form and reconstituted in a physiologically acceptable vehicle to form a suspension prior to administration. Alternatively, the adjuvant and conjugate may be stored in the vehicle. Preferred vehicles are sterile solutions, in particular, sterile buffer solutions, such as phosphate buffered saline. Any method of combining the adjuvant and the conjugate in the vehicle such that improved immunological effectiveness of the immunogenic composition is appropriate.

[0091] The volume of a single dose of the vaccine of this invention may vary but will be generally within the ranges commonly employed in conventional vaccines. The volume of a single dose is preferably between about 0.1 ml and about 3 ml, preferably between about 0.2 ml and about 1.5 ml, more preferably between about 0.2 ml and about 0.5 ml at the concentrations of conjugate and adjuvant noted above. [0092] The vaccine compositions of the invention may be administered by any convenient means.

E. Formulations

[0093] Immunogenic conjugates comprising a Sapelovirus peptide coupled to a carrier molecule can be used as vaccines for immunization against Sapelovirus. The vaccines, comprising the immunogenic conjugate in a physiologically acceptable vehicle, are useful in a method of immunizing animals, preferably swine, for treatment or prevention of infections by Sapelovirus. [0094] Antibodies generated against immunogenic conjugates of the present invention by immunization with an immunogenic conjugate can be used in passive immunotherapy and generation of anti-idiotypic antibodies for treating or preventing infections of Sapelovirus.

[0095] The subject to which the composition is administered is preferably an animal, including but not limited to cows, horses, sheep, pigs, poultry (e.g. chickens), goats, cats, dogs, hamsters, mice and rats, most preferably the mammal is swine.

[0096] The formulations of the invention comprise an effective immunizing amount of one or more immunogenic compositions or antibodies thereto and a physiologically acceptable vehicle. Vaccines comprise an effective immunizing amount of one or more immunogenic compositions and a physiologically acceptable vehicle. The formulation should suit the mode of administration.

[0097] The immunogenic composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The immunogenic composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

F. Effective Dose

[0098] The compounds described herein can be administered to a subject at therapeutically effective doses to treat Sapelovirus-associated diseases. The dosage will depend upon the host receiving the vaccine as well as factors such as the size, weight, and age of the host. [0099] The precise amount of immunogenic conjugate or antibody of the invention to be employed in a formulation will depend on the route of administration and the nature of the subject (e.g., species, age, size, stage/level of disease), and should be decided according to the judgment of the practitioner and each subject's circumstances according to standard clinical techniques. An effective immunizing amount is that amount sufficient to treat or prevent a Sapelovirus infectious disease in a subject. An example of an appropriate dose is about 5 to 9 log TCID50/mL. Alternatively, effective doses may also be extrapolated from dose-response curves derived from animal model test systems and can vary from 0.001 mg/kg to 100 mg/kg.

[00100] Toxicity and therapeutic efficacy of compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀ /ED₅₀.

Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[00101] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in animals, especially swine. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in subjects. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[00102] Immunogenicity of a composition can be determined by monitoring the immune response of test subjects following immunization with the composition by use of any immunoassay known in the art. Generation of a humoral (antibody) response and/or cell-mediated immunity, may be taken as an indication of an immune response. Test subjects may include animals such as pigs, mice, hamsters, dogs, cats, rabbits, cows, horses, sheep, poultry (e.g. chickens, ducks, geese, and turkeys).

[00103] The immune response of the test subjects can be analyzed by various approaches such as: the reactivity of the resultant immune serum to the immunogenic conjugate, as assayed by known techniques, e.g., enzyme linked immunosorbent assay (ELISA), immunoblots, immunoprecipitations, etc.; or, by protection of immunized hosts from infection by the pathogen and/or attenuation of symptoms due to infection by the pathogen in immunized hosts as determined by any method known in the art, for assaying the levels of an infectious disease agent, e.g., the levels of viremia (for example, by isolation or qPCR), or other technique known in the art. The levels of the infectious disease agent may also be determined by measuring the levels of the antigen against which the immunoglobulin was directed. A decrease in the levels of the infectious disease agent or an amelioration of the symptoms of the infectious disease indicates that the composition is effective.

[00104] The therapeutics of the invention can be tested in vitro for the desired therapeutic or prophylactic activity, prior to in vivo use in animals or humans. For example, in vitro assays that can be used to determine whether administration of a specific therapeutic is indicated include in vitro cell culture assays in which appropriate cells from a cell line or cells cultured from a subject having a particular disease or disorder are exposed to or otherwise administered a therapeutic, and the effect of the therapeutic on the cells is observed.

[00105] Alternatively, the therapeutic may be assayed by contacting the therapeutic to cells (either cultured from a subject or from a cultured cell line) that are susceptible to infection by the infectious disease agent but that are not infected with the infectious disease agent, exposing the cells to the infectious disease agent, and then determining whether the infection rate of cells contacted with the therapeutic was lower than the infection rate of cells not contacted with the therapeutic. Infection of cells with an infectious disease agent may be assayed by any method known in the art.

[00106] In addition, the therapeutic can be assessed by measuring the level of the molecule against which the antibody is directed in the animal model or human subject at suitable time intervals before, during, or after therapy. Any change or absence of change in the amount of the molecule can be identified and correlated with the effect of the treatment on the subject. The level of the molecule can be determined by any method known in the art.

[00107] After vaccination of an animal to a Sapelovirus infection using the methods and compositions of the present invention, any binding assay known in the art can be used to assess the binding between the resulting antibody and the particular molecule. These assays may also be performed to select antibodies that exhibit a higher affinity or specificity for the particular antigen.

G. Detection and Diagnostic Methods

[00108] Antibodies, or binding portions thereof, resulting from the use of Sapelovirus peptides of the present invention are useful for detecting in a sample the presence of Sapelovirus virus. This detection method comprises the steps of providing an isolated antibody or binding portion thereof raised against an Sapelovirus peptide of the invention, adding to the isolated antibody or binding portion thereof a sample suspected of containing a quantity of Sapelovirus, and detecting the presence of a complex comprising the isolated antibody or binding portion thereof bound to Sapelovirus.

[00109] The antibodies or binding portions thereof of the present invention are also useful for detecting in a sample the presence of a Sapelovirus peptide. This detection method comprises the steps of providing an isolated antibody or binding portion thereof raised against a Sapelovirus peptide, adding to the isolated antibody or binding portion thereof a sample suspected of containing a quantity of the Sapelovirus peptide, and detecting the presence of a complex comprising the isolated antibody or binding portion thereof bound to the Sapelovirus peptide. [00110] Immunoglobulins, particularly antibodies, (and functionally active fragments thereof) that bind a specific molecule that is a member of a binding pair may be used as diagnostics and prognostics, as described herein. In various embodiments, the present invention provides the measurement of a member of the binding pair, and the uses of such measurements in clinical applications. The immunoglobulins in the present invention may be used, for example, in the detection of an antigen in a biological sample whereby subjects may be tested for aberrant levels of the molecule to which the immunoglobulin binds, and/or for the presence of abnormal forms of such molecules. By "aberrant levels" is meant increased or decreased relative to that present, or a standard level representing that present, in an analogous sample from a portion of the body or from a subject not having the disease. The antibodies of this invention may also be included as a reagent in a kit for use in a diagnostic or prognostic technique. [00111] In one aspect, an antibody of the invention that immunospecifically binds to a Sapelovirus peptide may be used to diagnose, prognosis or screen for a Sapelovirus infection.

[00112] In another aspect, the invention provides a method of diagnosing or screening for the presence of a Sapelovirus infection or immunity thereto, comprising measuring in a subject the level of immunospecific binding of an antibody to a sample derived from the subject, in which the antibody immunospecifically binds a Sapelovirus peptide in which an increase in the level of said immunospecific binding, relative to the level of said immunospecific binding in an analogous sample from a subject not having the infectious disease agent, indicates the presence of Sapelovirus.

[00113] Examples of suitable assays to detect the presence of Sapelovirus peptides or antagonists thereof include but are not limited to ELISA, radioimmunoassay, gel-diffusion precipitation reaction assay, immunodiffusion assay, agglutination assay, fluorescent immunoassay, protein A immunoassay, or

Immunoelectrophoresis assay.

[00114] Immunoassays for the particular molecule will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cultured cells, in the presence of a detectably labeled antibody and detecting the bound antibody by any of a number of techniques well-known in the art.

[00115] The binding activity of a given antibody may be determined according to well-known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

[00116] An additional aspect of the present invention relates to diagnostic kits for the detection or measurement of Sapelovirus. Kits for diagnostic use are provided, that comprise in one or more containers an anti-Sapelovirus peptide antibody, and, optionally, a labeled binding partner to the antibody. Alternatively, the anti-Sapelovirus peptide antibody can be labeled (with a detectable marker, e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive moiety). Accordingly, the present invention provides a diagnostic kit comprising, an anti-Sapelovirus peptide antibody and a control immunoglobulin. In a specific embodiment, one of the foregoing compounds of the container can be detectably labeled. A kit can optionally further comprise, in a container, a predetermined amount of a Sapelovirus peptide recognized by the antibody of the kit, for use as a standard or control.

[00117] Preferred routes of administration include but are not limited to intranasal, oral, intradermal, and intramuscular. Administration via the intramuscular route, most preferably in a single dose, is desirable. The skilled artisan will recognize that compositions of the invention may also be administered in one, two or more doses, as well as, by other routes of administration. For example, such other routes include subcutaneously, intracutaneously, intravenously, intravascularly, intraarterially, intraperitoneally, intrathecally, intratracheally, intracutaneously, intracardially, intralobally, intramedullary, intrapulmonarily, and intravaginally. Depending on the desired duration and effectiveness of the treatment, the compositions according to the invention may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months and in different dosages.

[00118] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

SEQUENCE LISTING

[00119] This application contains a sequence listing. The sequence listing comprises the following sequences:

[00120] SEQ ID NO: 1 denotes the cDNA (complementary DNA copy of viral RNA) sequence of the low passage isolate from clinical sample (fecal) of the present invention.

[00121] SEQ ID NO:2 denotes the RNA sequence of the low passage isolate from clinical sample (fecal) of the present invention.

[00122] SEQ ID NO:3 denotes the DNA sequence encoding the polyprotein for the low passage isolate from clinical sample (fecal) of the present invention.

[00123] SEQ ID NO:4 denotes the amino acid sequence of the polyprotein for the low passage isolate from clinical sample (fecal) of the present invention.

[00124] SEQ ID NO:5 denotes the cDNA (complementary DNA copy of viral RNA) sequence of the mid- passage isolate from clinical sample (fecal) of the present invention.

[00125] SEQ ID NO:6 denotes the RNA sequence of the mid-passage isolate from clinical sample (fecal) of the present invention.

[00126] SEQ ID NO:7 denotes the DNA sequence encoding the polyprotein for the mid-passage isolate from clinical sample (fecal) of the present invention.

[00127] SEQ ID NO:8 denotes the amino acid sequence of the polyprotein for the mid-passage isolate from clinical sample (fecal) of the present invention.

[00128] SEQ ID NO:9 denotes the cDNA (complementary DNA copy of viral RNA) sequence of the mid- passage isolate from clinical sample (brain) of the present invention.

[00129] SEQ ID NO: 10 denotes the RNA sequence of the mid-passage isolate from clinical sample (brain) of the present invention.

[00130] SEQ ID NO: 11 denotes the DNA sequence encoding the polyprotein for the mid-passage isolate from clinical sample (brain) of the present invention.

[00131] SEQ ID NO: 12 denotes the amino acid sequence of the polyprotein for the mid-passage isolate from clinical sample (brain) of the present invention.

[00132] SEQ ID NO: 13 denotes specific polynucleotide sequence primer complementary to viral cDNA SEQ ID NO 1, 5, and 9. [00133] SEQ ID NO: 14 denotes specific polynucleotide sequence primer complementary to viral cDNA SEQ ID NO 1, 5, and 9.

[00134] SEQ ID NO: 15 denotes specific polynucleotide sequence probe complementary to viral cDNA SEQ ID NO 1, 5, and 9.

[00135] The invention further includes the following clauses:

[00136] 1. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of a killed Sapelovirus comprising:

(a) a nucleic acid sequence of SEQ ID NO: 1, 2, 5, 6, 9, or 10, or

(b) a nucleic acid sequence 97% identical to SEQ ID NO:3, 7, or 11, which encodes a polypeptide having immunologically-effective activity of a polypeptide of SEQ ID NO:4, 9, or 12.

[00137] 2. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of a killed Sapelovirus comprising:

(a) having the amino acid sequence of SEQ ID NO:4, 8, or 12;

(b) having an amino acid sequence 80% identical to SEQ ID NO:4, 8, or 12 and having a biological or immunologically-effective activity of a polypeptide encoded by SEQ ID NO:4, 8, or 12; or

(c) that is a fragment of the amino acid sequence of SEQ ID NO:4, 8, or 12 comprising at least 15 contiguous amino acids of SEQ ID NO:4, 8, or 12 and having an immunologically-effective activity.

[00138] 3. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of a killed Sapelovirus comprising:

(a) a nucleic acid sequence of SEQ ID NO: 1, 2, 5, 6, 9, or 10, or

(b) a nucleic acid sequence 97% identical to SEQ ID NO:3, 7, or 11, which encodes a polypeptide having immunologically-effective activity of a polypeptide of SEQ ID NO:4, 8, or 12.

[00139] 4. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of an immunogenic compositions according to clause 1.

[00140] 5. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of an immunogenic compositions according to clause 2.

[00141] 6. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of an immunogenic composition according to clause 3. [00142] 7. A method according to clause 1, wherein the mammal is a swine, and the immune response provides protective immunity to disease caused by Sapelovirus infection. [00143] 8. A method according to clause 2, wherein the mammal is a swine, and the immune response provides protective immunity to disease caused by Sapelovirus infection.

[00144] 9. A method according to clause 3, wherein the mammal is a swine, and the immune response provides protective immunity to disease caused by Sapelovirus infection. [00145] The invention further includes the following additional clauses:

[00146] 10. A nucleic acid comprising a polynucleotide having a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1, 2, 3, 5, 6, 7, 9, 10, 11 ;

(b) a nucleotide sequence being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identical to any of SEQ ID NO: 1, 2, 3, 5, 6, 7, 9, 10, 11 ;

(c) a nucleotide sequence encoding a polypeptide comprising, preferably having, an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, 8 and 12;

(d) a nucleotide sequence encoding a polypeptide comprising, preferably having, an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identical to any of SEQ ID NO: 4, 8, 12; (e) the complementary strand of any of the nucleotide sequences of (a) to (d);

(f) a DNA equivalent of any of the nucleotide sequences of SEQ ID NO: 2, 6 and 10.

[00147] 11. The nucleic acid according to clause 10, wherein the nucleic acid encodes a Sapelovirus.

[00148] 12. The nucleic acid according to clause 10 or clause 1 1, wherein said Sapelovirus is able to induce an infection in a subject, particularly an infection in a subject with clinical signs associated with or caused by such infection, wherein the clinical signs associated with or caused by such infection are selected from the group consisting of: pyrexia, anorexia, ataxia, uncoordinated movements, locomotive disorders, tremors, nystagmus, opisthotonos (e.g., spasm of the muscles causing backward arching of the head, neck, and spine), convulsions, paralysis, paresis, mental dullness, death within 3-4 days; or where such infection is a clinical disease associated specifically with Sapelovirus A selected from the group consisting of: reproductive failure, neurological disorders, pneumonia, diarrhea; preferably the subject being a swine. [00149] 13. A Sapelovirus the genome of which comprises a ribonucleic acid selected from the group consisting of: SEQ ID NO: 2, 6 and 10.

[00150] 14. The Sapelovirus according to clause 13, wherein the Sapelovirus is attenuated or

killed/inactivated.

[00151] 15. A vector comprising the nucleic acid according to any one of clauses 10 to 12. [00152] 16. A host cell comprising the vector according to clause 15.

[00153] 17. A polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) a polypeptide comprising, preferably having, an amino acid sequence that is encoded by a nucleotide selected from the group consisting of: SEQ ID NO: 3, 7 and 11 ;

(b) a polypeptide comprising, preferably having, an amino acid sequence that is encoded by a nucleotide which is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identical to any of SEQ ID NO: 3, 7, 11 ;

(c) a polypeptide comprising, preferably having, an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, 8 and 12;

(d) a polypeptide comprising, preferably having, an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identical to SEQ ID NO: 4, 8 and 12;

(e) a polypeptide fragment of the polypeptides according to (a), (b), (c) and/or (d);

(f) a polypeptide fragment comprising at least 5, preferably 8, more preferably 10, even more preferably 15 contiguous amino acid residues included in the sequences of SEQ ID NO: 4, 8 and 12;

(g) a polypeptide fragment that is encoded by a polynucleotide that comprises at least 15, preferably 24, more preferably 30, even more preferably 45 contiguous nucleotides included in the sequences of any of SEQ ID NO: 1, 2, 3, 5, 6, 7, 9, 10, 11 ; preferably included in the sequences of SEQ ID NOS: 1, 2,

5, 7, 9, and 11.

[00154] 18. An antibody that specifically binds to a polypeptide according to clause 17.

[00155] 19. An immunogenic composition comprising:

(a) the Sapelovirus according to any one of clauses 13 to 14; and/or

(b) the nucleic acid according to any one of clauses 10 to 12; and/or

(c) a polypeptide according to clause 17;

and optionally at least one pharmaceutically acceptable carrier and/or excipient.

[00156] 20. A vaccine, preferably a recombinant vaccine or a killed vaccine, comprising:

(a) the Sapelovirus according to any one of clauses 13 to 14; and/or

(b) the nucleic acid according to any one of clauses 10 to 12; and/or

(c) a polypeptide according to clause 17;

and at least one pharmaceutically acceptable carrier and/or excipient; optionally said vaccine further comprising at least one adjuvant.

[00157] 21. The immunogenic composition according to clause 19 or the vaccine according to clause 20 for use as a medicament. [00158] 22. Use of the immunogenic composition according to clause 19 or the vaccine according to clause 20 for the preparation of a medicament.

[00159] 23. The immunogenic composition according to clause 19 or the vaccine according to clause 20 for use in a method of prophylaxis and/or treatment of a subject against a Sapelovirus infection. [00160] 24. The immunogenic composition according to clause 19 or the vaccine according to clause 20 for use according to clause 23, wherein the subject is a swine.

[00161] 25. Use of the immunogenic composition according to clause 19 or the vaccine according to clause 20 for the preparation of a medicament for prophylaxis and/or treatment of a subject against a Sapelovirus infection. [00162] 26. The use according to clause 25, wherein the subject is a swine.

[00163] 27. Method of prophylactic ally treating and/or treating a subject against a Sapelovirus infection, said method comprising administering to such subject in need thereof the immunogenic composition according to clause 19 or the vaccine according to clause 20.

[00164] 28. The method according to clause 27, wherein the subject is a swine. [00165] 29. The immunogenic composition according to clause 19 or the vaccine according to clause 20 for use in a method of provoking an immune response against a Sapelovirus infection in a subject comprising the step of administering to the subject the immunogenic composition according to clause 19 or the vaccine according to clause 20.

[00166] 30. The immunogenic composition according to clause 19 or the vaccine according to clause 20 for use according to clause 29, wherein the subject is a swine.

[00167] 31. Use of the immunogenic composition according to clause 19 or the vaccine according to clause 20 for the preparation of a medicament for provoking an immune response against a Sapelovirus infection in a subject.

[00168] 32. The use according to clause 31, wherein the subject is a swine. [00169] 33. Method of provoking an immune response against a Sapelovirus infection in a subject, said method comprising the step of administering to a subject in need thereof the immunogenic composition according to clause 19 or the vaccine according to clause 20.

[00170] 34. The method according to clause 33, wherein the subject is a swine.

[00171] 35. The immunogenic composition according to clause 19 or the vaccine according to clause 20 for use in a method of reducing the incidence of or severity of one or more clinical signs associated with or caused by a Sapelovirus infection in a subject. [00172] 36. The immunogenic composition according to clause 19 or the vaccine according to clause 20 for use according to clause 35, wherein the clinical signs are selected from the group consisting of: pyrexia, anorexia, ataxia, uncoordinated movements, locomotive disorders, tremors, nystagmus, opisthotonos (e.g., spasm of the muscles causing backward arching of the head, neck, and spine), convulsions, paralysis, paresis, mental dullness, death within 3-4 days.

[00173] 37. The immunogenic composition according to clause 19 or the vaccine according to clause 20 for use according to clause 35 or clause 36, wherein the subject is a swine.

[00174] 38. Use of the immunogenic composition according to clause 19 or the vaccine according to clause 20 for the preparation of a medicament for reducing the incidence of or severity of one or more clinical signs associated with or caused by a Sapelovirus infection in a subject.

[00175] 39. The use according to clause 38, wherein the clinical signs are selected from the group consisting of: pyrexia, anorexia, ataxia, uncoordinated movements, locomotive disorders, tremors, nystagmus, opisthotonos (e.g., spasm of the muscles causing backward arching of the head, neck, and spine), convulsions, paralysis, paresis, mental dullness, death within 3-4 days. [00176] 40. The use according to clause 38 or clause 39, wherein the subject is a swine.

[00177] 41. Method of reducing the incidence of or severity of one or more clinical signs associated with or caused by a Sapelovirus infection in a subject, said method comprising the step of administering to a subject in need thereof the immunogenic composition according to clause 19 or the vaccine according to clause 20.

[00178] 42. The method according to clause 41, wherein the clinical signs are selected from the group consisting of: pyrexia, anorexia, ataxia, uncoordinated movements, locomotive disorders, tremors, nystagmus, opisthotonos (e.g., spasm of the muscles causing backward arching of the head, neck, and spine), convulsions, paralysis, paresis, mental dullness, death within 3-4 days.

[00179] 43. The method according to clause 41 or clause 42, wherein the subject is a swine.

[00180] 44. A kit for vaccinating a subject against a disease associated with a Sapelovirus infection in a subject comprising:

(a) a dispenser capable of administering a vaccine to the subject; and

(b) the immunogenic composition or the vaccine according to clause 19 or clause 20, and

(c) optionally an instruction leaflet.

[00181] 45. The kit according to clause 44, wherein the subject is a swine. [00182] 46. A kit for reducing the incidence or the severity of one or more clinical signs associated with or caused by a Sapelovirus infection in a subject comprising:

(a) a dispenser capable of administering a vaccine to the subject; and

(b) the immunogenic composition or the vaccine according to clause 19 or clause 20, and (c) optionally an instruction leaflet.

[00183] 47. The kit according to clause 46, wherein the subject is a swine.

[00184] 48. A method for detecting the Sapelovirus according to clause 13 or clause 14 in a sample, comprising the steps of:

(i) contacting the sample with one or more oligonucleotide primers and/or probes which are specific for a nucleic acid according to any one of clauses 10 to 12, and

(ii) detecting binding between said Sapelovirus and said one or more oligonucleotide primers and/or probes.

[00185] 49. A method for detecting the Sapelovirus according to clause 13 or clause 14 in a sample, comprising the steps of:

(iii) contacting the sample with an antibody according to clause 17, and

(iv) detecting binding between said Sapelovirus and said antibody; or

(v) detecting the presence or absence of an antibody against said Sapelovirus in said sample.

[00186] 50. A method for diagnosing an infection with the Sapelovirus according to clause 13 or clause 14 in a sample from a subject, comprising a method for detecting the Sapelovirus according to clause 48, wherein the presence of said Sapelovirus is indicative for an infection.

[00187] 51. The method according to clause 50, wherein the subject is a swine.

[00188] 52. A method for diagnosing an infection with the Sapelovirus according to clause 13 or clause 14 in a sample from a subject, comprising a method for detecting an antibody against said Sapelovirus according to clause 49, wherein the presence of said antibody is indicative for an infection with said Sapelovirus.

[00189] 53. The method according to clause 52, wherein the subject is a swine.

EXAMPLES

[00190] Example 1 [00191] The Sapelovirus of the present invention was isolated from brain and fecal samples. Brain tissues were frozen and ground using sterile mortar and pestle. Fecal samples were thoroughly mixed, diluted using serum free media (DMEM) (1 :5-1 :20). Sample suspensions spun down at 1000-5000 rpm for 10 mins at 4 °C. Supernatants were filtered through 0.2μιη filter and used as inoculum for virus isolation.

[00192] About 60-80% monolayers of BHK-21 cells (baby hamster kidney) and EU-Vero cells (African green monkey kidney) were used for initial virus isolation. Briefly, growth media from cell monolayers in 6-well plates were aspirated and cells washed 1-2 times with serum free media (DMEM). Cells were inoculated with 0.1-0.5 mL inoculum and samples were adsorbed for 1-2 hours at 37 °C+1 °C on a rocker. Post adsorption, 2.0 to 2.5 mL serum free media (DMEM) was added to each well and cells incubated at 37 °C+1 °C in a 5% CO₂ atmosphere. Plates were observed daily for cytopathic effects (CPE) and plates were frozen at <-70 °C. Three blind passages were done and virus CPE observed within 24-48 hours post infection was confirmed by real time qPCR. Sapelovirus was isolated from brain tissue on BHK21 and then adapted to EU-Vero cells. Sapelovirus from fecal samples were isolated on EU-VERO cells. Pass 6 brain derived Sapelovirus (SEQ ID NO: 9) and Pass 5 fecal derived Sapelovirus (SEQ ID NO: 6) were subjected to three rounds of limiting dilutions. These viruses also adapted to grow in AI-ST clone Al cells (Swine testes cells). Virus strains were scaled up in 850cm² roller bottles and pre -master seed virus was generated in EU-VERO cells.

[00193] Example 2

[00194] This study utilized conventional animals to determine the preliminary feasibility of induction of a serological response following vaccine administration. The primary purpose of this study was to evaluate if inactivated whole-virus preparations using Sapelovirus would result in seroconversion to Sapelovirus in conventional pigs.

[00195] Tissues from conventional 9-12 week old pigs with a history of neurologic signs and lameness were received (NAC#20160923). Sapelovirus was isolated from both the brain and a fecal sample from the same animal. Both isolates were sequenced and an approximate -9% nucleotide difference (whole genome comparison) was identified between the two isolates; see FIG. 1. Additional preliminary virus neutralization work indicated a lack of serological cross-protection between the two isolates.

[00196] A total of 30 pigs will be used for the study. Pigs will be randomized into five treatment groups (n=6/group) and co-mingled in one room throughout the study. On DO, pigs will be administered a 2mL intramuscular (IM) dose of the appropriate vaccine or placebo; see Table 1 for details. On D21, all animals will receive a booster administration of the appropriate vaccine. For a minimum of three days following each vaccination, injection areas will be monitored. Rectal temperatures will also be collected on selected days as a measure of vaccine safety. Blood will be collected weekly and tested for evidence of seroconversion to Sapelovirus. On D35, all animals will be humanely euthanized and terminal blood will be collected from selected animals based on the serological response. If vaccine site reactions are observed grossly at the time of necropsy, sections will be collected for microscopic examination. The study design is described in Table 1.

[00197] Table 1. Study design

[00198] Table 2. Schedule of Events Study Day Study Event

TBD Collection of pre-purchase serum from pigs

Pigs transferred to Sioux Center

TBD

General Health Observations through DO

D-l, 0, 0+4hrs, 1, 2,

Rectal temperatures

D20, 21, 21+4hrs, 22, 23

DO, D21 Vaccination

D0-D35,

D0+4hrs, General Health Observations

D21+4hrs

Injection site observations

DO-3; D21-23 Any visible lesions should be followed

through resolution

DO, 7, 14, 21, 28, and 35 Collection of blood

Necropsy

Terminal serum (two 250mL bottles/animal*)

D35

Collection of fixed tissue if gross lesions are

observed at injection site

* Animals were selected by study monitor prior to necropsy based on the serological response

[00199] The treatments consist of Sapelovirus vaccines generated at two different concentrations (lx and 5x) from two different isolates (brain and fecal). A placebo will also be included. The treatments are outlined in Table 3.

[00200] Table 3. Treatments

[00201] The vaccines will be administered on DO intramuscularly into the right side of the neck, midway between the base of the ear and point of the shoulder, using appropriately-sized sterile needles and syringes. The administration of the treatments will be documented on a Product Dosing Record. The second dose will be administered on D21 intramuscularly into the left side of the neck as described above. [00202] The pig is the experimental unit. The randomization of pigs to pen and treatment will be conducted by a Statistician. Prior to the start of the study, the available pigs, litter Treatments will be randomly assigned within litter, with 5-6 pigs/litter from each of 6 litters expected for randomization. Animals will be housed by litter with 2-3 litters per pen. Personnel involved with collecting data or performing laboratory assays will be blinded to the allocation of pigs to groups throughout the study. Treatments will be administered by an individual not involved with data collection.

[00203] Pigs will be observed daily to ensure access to an adequate supply of non-medicated feed and water and to determine the animals' general health. Veterinary care will be provided as needed. Adequate floor and feeder space will be provided in accordance with acceptable animal husbandry practices. [00204] Each animal will receive a dose of EXCEDE® per the manufacturer's recommendations prior to shipment and at the time of arrival. Vaccination prior to arrival for PCV2 and administration of vitamin E/selenium is acceptable if deemed necessary to prevent disease based on prior herd history. No other biological or pharmaceutical products will be administered to the test animals without prior approval by the study monitor. The animals will be under veterinary supervision upon arrival at the facility until the end of the study. Any animals exhibiting injury or illness unrelated to challenge administration will be given appropriate veterinary care. Documentation provided by the Investigator will include a description of the observed clinical signs, the outcome of any diagnostic examination, and the outcome of administered treatments.

[00205] The rectal temperature of each pig will be taken on the days specified in the schedule of events. On blood collection dates, eight to 15 mL of venous whole blood will be collected by the Investigator via the anterior vena cava from each pig using a sterile 18-20g x 1 inch (2.54 cm) to 1.5 inch (3.81 cm)

VACCUTAINER® needle, a VACCUTAINER® needle holder and 9 or 13 mL serum separator tubes (SST). The blood will be centrifuged. Serum will be decanted into two screw-cap cryogenic vials labeled with at least study number, day of study, and animal ID. Serum samples will be stored at -70+10°C. Serum will be tested by virus neutralization. [00206] Following euthanasia, ~500mL of blood will be collected from a minimum of 5 animals (one per group). The animals will be selected by the study monitor prior to necropsy. The preferred storage container is a 250mL centrifuge bottle (Fisher cat#05-538-53; Corning cat#430776).

[00207] If animals have evidence of clinical disease, a necropsy should be performed. Appropriate tissues should be collected to determine the cause of clinical disease. [00208] At the time of off -test, musculature on the right and left side of the neck should be palpated. If an injection site reaction is felt, it should be excised. The material should be placed into an appropriate amount of 10% buffered formalin and labeled with a minimum of animal id, study number and date.

[00209] The study will be considered valid if animals in T05 remain seronegative throughout the study and there are no confounding disease processes diagnosed during the course of the study that would interfere with assessment of the study objective. [00210] Evidence of seroconversion as determined by virus neutralization assay by 35 days post vaccination will be considered the primary outcome parameter. General Health Observations, rectal temperature and injection site reactions will be considered as supportive data (safety) for the vaccines. VN responses on day 35 will be evaluated to determine the GMT and proportion of animals exhibiting a 4-fold increase for each group. [00211] Statistical analysis of data will be conducted by a statistician. All data will be imported into SAS version 9.4 or later (SAS, Cary, USA/North Carolina, SAS Institute Inc.) for management and analyses. Data listings and summary statistics by treatment group including minimum, 25% percentile, median,75% percentile, and maximum (5 number summary) will be generated for serology and rectal temperatures. If appropriate (based on serologic response to vaccination), serology data will be analyzed using a linear mixed model with group as a fixed effect and litter as a random effect. If serologic responses are abundant prior to day 35, the model will be adjusted to incorporate the repeated measurements reflective of data collected over multiple days. Least-square means, standard errors and contrasts of interest with corresponding 95% confidence intervals will be estimated as appropriate. Frequency distributions of titers by group will be reported. A detailed description of the study analysis, including any changes from the current plan, will be provided in the final study report. [00212] In regards to the Sapelovirus vaccine, pigs vaccinated with the inactivated Sapelovirus vaccine are able to generate a neutralizing antibody response. In conclusion, this study was able to demonstrate reasonable expectation of efficacy for the inactivated Sapelovirus vaccine.

[00213] Example 3

[00214] The described case occurred on a 1, 100-head farrow-to-fmish farm with two sites 3.5 miles apart (Site 1 : Sows and nursery; Site 2: Wean-finish, finisher and gilt development). The farm is positive for field infection PRRS, Mycoplasma hyopneumoniae and influenza-A. In addition, the farm experienced pestivirus- associated congenital tremors in 2012.

[00215] The farm experienced neurological cases in growing pigs since at least 2008. In 2016, the incidence of cases began being more closely monitored and it was observed that nearly 100% of all weekly 500 head groups had individual neurologic cases in pigs between nine to twelve weeks of age. A typical case description included: Day 1 - Pig is observed in the pen as swaying, staggering, leaning on the gate or wall while walking. The pig is pulled into the alley for treatment. Day 2 - Neurologic signs progress, pig starts to "bunny-hop" when encouraged to rise and walk (front legs are curled underneath and the back legs extend simultaneously pushing the pig forward). The pig stops and lies down when not prompted to move. Day 3 - The pig becomes recumbent, paddling of the legs is not often noticeable, nystagmus may or may not be present. Pig succumbs this day or on day 4 regardless of antibiotic or palliative care; all morbid pigs (100%) die. Neurologic tissues from mortalities were submitted to multiple laboratories. Histopathology results were suggestive of a viral infection due to the severe, non-suppurative, encephalomyelitis with perivascular cuffing observed in multiple sections of spinal cord and brain stem. Further testing by PCR confirmed the presence of porcine Sapelovirus (PSV); other viral differentials (porcine enterovirus and porcine teschovirus) were not detected. Since 2014, the average total mortality in all groups was 5.3%. Within total mortalities, those animals which showed clinical neurological signs prior to death were considered neurologic mortalities; mortality associated with these cases was 0.8% (FIG. 2). On the farm, 95% of neurologic mortalities occurred in the first 90 days post- weaning (FIG. 3). To determine the prevalence of PSV on the farm, a multi-age fecal PSV PCR survey was conducted. Results indicated that PSV was ubiquitous in weaned to market age pigs, but less prevalent in gestating sows (33%), farrowing sows (0%) and suckling pigs (16%) (Table 4). In addition, 15 serum samples from two age groups were collected for serological evaluation. Virus neutralizing (VN) antibodies were detected in 20% and 100% of weaned pigs and late finishing pigs, respectively. The average VN titers were approximately 20x higher in finisher pigs (FIG. 4).

[00216] Table 4: Sapelovirus PCR results of fecal swab pools, by age group and location

Designation % detected*

1 4 vvks 6/6 (100%)

2 7 vvks 9/9 (100%)

3 11 vvks 9/9 (100%)

4 15 vvks 9/9 (100%)

£

5 19 vv ks 9/9 (100%)

6 23 vv ks 9/9 (100%)

7 Gilts (finisher stage) 8/9 (89 %)

8 Gestating sows 3/9 (33%)

9 Farrowing sows 0/9 (0%)

10 Suckling piglets 1/6 (17%)

11 Nursery (4-6 weeks of age) 9/9 (100%)

*Number of positive fecal swab pools / total number of fecal swab pools tested; ~5 swabs/pool were tested

[00217] To further investigate the role of Sapelovirus on the farm, an autogenous vaccine program was implemented. The protocol included two, 2mL doses of vaccine (Newport Labs, Worthington, MN) administered at three and six weeks of age. Vaccinates (-83%) and non-vaccinates (-17%) were commingled. Clinical signs and mortality were monitored for thirteen groups (~500pigs/group). Brain and fibrin swabs were collected as supportive data. Sapelovirus is highly suspected to be the cause of the majority of the neurologic diseases in this farm. However, the wide distribution of PSV found on the farm is inconsistent with the low number of clinical cases. Possible reasons include: 1) PSV is present but not the primary etiologic agent, 2) lack of uniform herd immunity leading to endemic circulation of the virus with clinical signs being observed in susceptible animals, or 3) the impact of PSV on pigs ranges from clinical to subclinical presentations.

[00218] Example 4

[00219] This study utilizes conventional animals to determine the preliminary feasibility of induction of a serological response following vaccine administration. The primary purpose of this study is to evaluate if inactivated whole-virus preparations using Sapelovirus would result in seroconversion to Sapelovirus in conventional pigs.

[00220] Tissues from conventional 9-12 week old pigs with a history of neurologic signs and lameness are received (NAC#20160923). Sapelovirus is isolated from both the brain and a fecal sample from the same animal. Both isolates are sequenced and an approximate -9% nucleotide difference (whole genome comparison) is identified between the two isolates; see FIG. 1. Additional preliminary virus neutralization work indicates a lack of serological cross-protection between the two isolates.

[00221] A total of 34 pigs are used for the study. Pigs are randomized into five treatment groups (n=6- 7/group) and co-mingled in one room throughout the study. On DO, pigs are administered a 2mL intramuscular (IM) dose of the appropriate vaccine or placebo; see Table 5 for details. On D21, all animals receive a booster administration of the appropriate vaccine. For a minimum of three days following each vaccination, injection areas are monitored. Rectal temperatures are collected on selected days as a measure of vaccine safety. Blood is collected weekly and tested for evidence of seroconversion to Sapelovirus A. On D35, all animals are humanely euthanized and terminal blood is collected from two randomly selected animals per group. [00222] Table 5. Study design

[00223] Table 6. Schedule of Events

Study Day Study Event

D-15 Collection of pre -purchase serum from pigs

Pigs transferred to Sioux Center

D-5

General Health Observations through DO

D-l, 0, 0+4hrs, 1, 2,

Rectal temperatures

D20, 21, 21+4hrs, 22, 23

DO, D21 Vaccination

D0-D35, General Health Observations

Injection site observations

D0-3; D21-23 Any visible lesions were followed through until

resolution DO, 7, 14, 21, 28, and 35 Collection of blood

Necropsy

D35

Terminal serum (two 250mL bottles/animal*)

*Two animals per group are randomly selected by study monitor prior to necropsy

[00224] The treatments consist of Sapelovirus vaccines generated at two different concentrations (lx and 5x) from two different isolates (brain and fecal). A placebo is also included. The treatments are outlined in Table 7.

[00225] Table 7. Treatments

[00226] The vaccines are administered on DO intramuscularly into the right side of the neck, midway between the base of the ear and point of the shoulder, using appropriately-sized sterile needles and syringes. The administration of the treatments is documented on a Product Dosing Record. The second dose is administered on D21 intramuscularly into the left side of the neck as described above. [00227] The pig is the experimental unit. The randomization of pigs to pen and treatment is conducted by a BIAH Statistician or designee. Prior to the start of the study, the available pigs, litter information, and housing facility set-up are provided to the study monitor and/or BIAH Statistician or designee. Treatments are randomly assigned within litter. A total of four litters of six pigs and two litters of five pigs are used for the study. Animals are housed by litter with 2 litters per pen. Personnel involved with collecting data or performing laboratory assays are blinded to the allocation of pigs to groups throughout the study. Treatments are administered by an individual not involved with data collection.

[00228] Pigs are observed daily to ensure access to an adequate supply of non-medicated feed and water and to determine the animals' general health. Veterinary care is provided as needed. Adequate floor and feeder space is provided in accordance with acceptable animal husbandry practices. [00229] Each animal receives a dose of Excede® (Lot #182978, exp 9/2018) and Circoflex® (Lot

#3091088A, exp 27Sepl8) per the manufacturer's recommendations prior to shipment at Wilson's Prairie View Farm, Inc. Upon arrival at Sioux Center, animals are again treated with Excede® (Lot #182978, exp 9/2018). No other biological or pharmaceutical products are administered to the test animals throughout the study. [00230] The rectal temperature of each pig is taken on the days specified in the schedule of events. On blood collection dates, eight to 15 mL of venous whole blood is collected by the Investigator via the anterior vena cava from each pig using a sterile 18-20g x 1 inch (2.54 cm) to 1.5 inch (3.81 cm) VACCUTAINER® needle, a VACCUTAINER® needle holder and 9 or 13 mL serum separator tubes (SST). The blood is centrifuged. Serum is decanted into two screw-cap cryogenic vials labeled with at least study number, day of study, and animal ID. Serum samples are stored at -70+10°C. Serum is tested by virus neutralization.

[00231] Following euthanasia, approximately 500mL of blood is collected from two randomly selected animals per group No evidence of clinical disease is apparent throughout the study; therefore, no necropsies are done and no tissues are collected.

[00232] At the time of off-test, musculature on the right and left side of the neck is palpated. If an injection site reaction is felt, it iss excised and placed into an appropriate amount of 10% buffered formalin. If an injection cannot be palpated, each side of the neck is incised several times at one inch increments and the deeper tissues assessed for presence of an injection site lesion. If abnormal tissue is identified, it is resected and placed into an appropriate amount of 10% buffered formalin. Tissues are collected from 18 animals.

[00233] Evidence of seroconversion as determined by virus neutralization assay by 35 days post vaccination is considered the primary outcome parameter. General Health Observations, rectal temperature and injection site reactions are considered as supportive data (safety) for the vaccines. VN responses on day 35 was evaluated to determine the GMT.

[00234] Serum samples are tested by virus neutralization assay against both the brain and fecal isolate. The frequency of detection by group, assay, and study day is presented in Table 8. [00235] Table 8. Frequency of anti-Sapelovirus A neutralizing antibody detection by group, assay, and study day.

Assay Group DO D7 D14 D21 D28 D35

Fecal -lx 2/7 2/7 2/7 0/7 3/7 0/7

Fecal -5x 1/7 1/7 0/7 0/7 2/7 1/7

Fecal Brain -lx 1/7 0/7 0/7 0/7 0/7 1/7

Brain -5x 1/7 1/7 1/7 0/7 0/7 0/7

Placebo 2/6 0/6 0/6 0/6 0/6 0/6

Fecal -lx 0/7 0/7 0/7 0/7 1/7 0/7

Fecal -5x 0/7 0/7 0/7 0/7 0/7 0/7

Brain Brain -lx 0/7 0/7 0/7 0/7 4/7 2/7

Brain -5x 0/7 0/7 0/7 0/7 7/7 3/7

Placebo 0/6 0/6 0/6 0/6 1/7 0/7 [00236] The primary objective of this study is to evaluate the serological response in three week old conventional pigs following two doses of inactivated sapelovirus prototype vaccines. Of the vaccine prototypes that are evaluated, the whole-virus preparation based on the brain isolate (5x concentration) appears the most promising as 7/7 animals have a detectable virus neutralization titer at D28. In conclusion, this study is able to demonstrate reasonable expectation of efficacy for the inactivated Sapelovirus vaccine.

[00237] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the following claims.

Claims

A nucleic acid comprising a polynucleotide having a nucleotide sequence selected from the group consisting of:

(d) a nucleotide sequence encoding a polypeptide comprising, preferably having, an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.

2%, at least 99.

3%, at least 99.

4%, at least 99.

5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identical to any of SEQ ID NO: 4, 8, 12;

(e) the complementary strand of any of the nucleotide sequences of (a) to (d);

The nucleic acid according to claim 1, wherein the nucleic acid encodes a Sapelovirus.

The nucleic acid according to claim 1 or claim 2, wherein said Sapelovirus is able to induce an infection in a subject, particularly an infection in a subject with clinical signs associated with or caused by such infection, wherein the clinical signs associated with or caused by such infection are selected from the group consisting of: pyrexia, anorexia, ataxia, uncoordinated movements, locomotive disorders, tremors, nystagmus, opisthotonos (e.g., spasm of the muscles causing backward arching of the head, neck, and spine), convulsions, paralysis, paresis, mental dullness, death within 3-4 days; or wherein such infection is a clinical disease associated specifically with Sapelovirus A selected from the group consisting of: reproductive failure, neurological disorders, pneumonia, diarrhea; preferably the subject being a swine.

A Sapelovirus the genome of which comprises a ribonucleic acid selected from the group consisting of: SEQ ID NO: 2,

6 and 10.

The Sapelovirus according to claim 4, wherein the Sapelovirus is attenuated or killed/inactivated. A vector comprising the nucleic acid according to any one of claims 1 to 3.

7. A host cell comprising the vector according to claim 6.

8. A polypeptide comprising an amino acid sequence selected from the group consisting of: (a) a polypeptide comprising, preferably having, an amino acid sequence that is encoded by a nucleotide selected from the group consisting of: SEQ ID NO: 3, 7 and 11 ;

(g) a polypeptide fragment that is encoded by a polynucleotide that comprises at least 15, preferably 24, more preferably 30, even more preferably 45 contiguous nucleotides included in the sequences of any of SEQ ID NO: 1, 2, 3, 5, 6, 7, 9, 10, 11 ; preferably included in the sequences of SEQ ID NOS: 1, 2, 5, 7, 9, and 11.

9. An antibody that specifically binds to a polypeptide according to claim 8.

10. An immunogenic composition comprising:

(a) the Sapelovirus according to any one of claims 4 to 5; and/or

(b) the nucleic acid according to any one of claims 1 to 3; and/or

(c) a polypeptide according to claim 8;

11. A vaccine, preferably a recombinant vaccine or a killed vaccine, comprising:

(a) the Sapelovirus according to any one of claims 4 to 5; and/or

(b) the nucleic acid according to any one of claims 1 to 3; and/or

(c) a polypeptide according to claim 8;

12. The immunogenic composition according to claim 10 or the vaccine according to claim 11 for use as a medicament, preferably for use in a method of prophylaxis and/or treatment of a subject against a Sapelovirus infection or for use in a method of provoking an immune response against a Sapelovirus infection in a subject comprising the step of administering to the subject the immunogenic composition according to claim 10 or the vaccine according to claim 11, more preferably the subject being a swine. The immunogenic composition according to claim 10 or the vaccine according to claim 11 for use in a method of reducing the incidence of or severity of one or more clinical signs associated with or caused by a Sapelovirus infection in a subject, the clinical signs preferably being selected from the group consisting of: pyrexia, anorexia, ataxia, uncoordinated movements, locomotive disorders, tremors, nystagmus, opisthotonos (e.g., spasm of the muscles causing backward arching of the head, neck, and spine), convulsions, paralysis, paresis, mental dullness, death within 3-4 days; more preferably the subject being a swine.

A kit for vaccinating a subject against a disease associated with and/or reducing the incidence or the severity of one or more clinical signs associated with or caused by a Sapelovirus infection in a subject comprising:

(a) a dispenser capable of administering a vaccine to the subject; and

(b) the immunogenic composition or the vaccine according to claim 10 or 11, and

(c) optionally an instruction leaflet;

preferably the subject being a swine.

A method for detecting the Sapelovirus according to claims 4 or 5 in a sample, comprising the steps of:

(i) contacting the sample with one or more oligonucleotide primers and/or probes which are specific for a nucleic acid according to any one of claims 1 to 3, and

(ii) detecting binding between said Sapelovirus and said one or more oligonucleotide primers and/or probes; or

(iii) contacting the sample with an antibody according to claim 9, and

(iv) detecting binding between said Sapelovirus and said antibody; or

A method for diagnosing an infection with the Sapelovirus according to claims 4 or 5 in a sample from a subject, preferably a swine, comprising a method for detecting the Sapelovirus according to claim 15, wherein the presence of said Sapelovirus is indicative for an infection, or comprising a method for detecting an antibody against said Sapelovirus according to claim 15, wherein the presence of said antibody is indicative for an infection with said Sapelovirus.