Porcine reproductive and respiratory syndrome virus, compositions, vaccine and methods of use

Abstract

The present invention relates to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) mutant strains having increased stability; compositions containing the strains or antigens derived therefrom; vaccines containing the strains, including killed, attenuated or subunit vaccines; nucleic acids encoding PRRSV polypeptides, polypeptides encoded by the nucleic acids, including antigenic fragments thereof; antibodies which specifically bind to said polypeptides, and methods making and using thereof.

Claims

1. An immunogenic composition of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), comprising at least one of a) a composition comprising SEQ ID NO:2 (attenuated Wetzel p41); or b) a polypeptide selected from the group consisting of i) SEQ ID NO:9 (M protein of Wetzel p3 and of Wetzel p41), ii) SEQ ID NO:10 (N protein of Wetzel p3and of Wetzel p41), iii) SEQ ID NO:11 (ORF1a protein of Wetzel p41), iv) SEQ ID NO:12 ORF1b protein of Wetzel p41), v) SEQ ID NO:13 (GP2 protein of Wetzel p41), vi) SEQ ID NO:14 (GP3 protein of Wetzel p41), and vii) SEQ ID NO:15 (GP5 protein of Wetzel p41).

2. An isolated Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), comprising a) SEQ ID NO:2, or b) a nucleic acid sequence 97% identical to SEQ ID NO:2, which encodes a polypeptide selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.

3. The PRRSV of claim 2, comprising an attenuated, non-virulent form of a PRRSV.

4. A vaccine for generating an immune response by a virulent PRRSV, comprising a killed or attenuated form of a PRRSV of claim 2.

5. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of a PRRSV of claim 2.

6. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of a vaccine of claim 4.

7. The method of claim 6, wherein the mammal is a swine.

8. The method of claim 6, wherein the vaccine provides protective immunity to a disease caused by PRRSV infection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

(2) FIG. 1 is a chart of the Lung Pathology from the respiratory trial of Example 1. Pig safety study average lung scores as collected on day 14. The letter a indicates the average lung score was higher than the negative control group while the letter b indicates the scores were lower than the Isolate X wild type group.

(3) FIG. 2 is a graph of the IDEXX ELISA from the respiratory trial of Example 1. Samples are considered positive for PRRSV specific antibodies if (S/P) ratio was at least 0.4.

(4) FIG. 3 is a graph of the Gilt Viremia of the reproductive trial as determined by RT-PCR via the AgPath-ID NA and EU PRRSV Multiplex kit with relatively short period of viremia detected of Example 1.

(5) FIG. 4 is a graph of the piglet viremia as determined by RT-PCR via the AgPath-ID NA and EU PRRSV Multiplex kit. Variably levels of piglet viremia at birth followed by horizontal transmission within litters from the reproductive trial of Example 1.

(6) FIG. 5 is a comparison of the nucleotide sequences (DNA equivalent of the RNA viral sequences) of SEQ ID NO: 1 (pre-attenuated Wetzel p3) and SEQ ID NO: 2 (attenuated Wetzel p41).

(7) FIG. 6 is a graph of the average PRRSV ELISA S/P from Example 3.

(8) FIG. 7 is a graph showing percent positive by PCR for PPRSV from Example 3.

(9) FIG. 8 shows graphs of respiratory scores, behavior scores and cough scores by percentage affected from Example 3.

(10) FIG. 9 shows average rectal temperatures from Example 3.

(11) FIG. 10 shows average weight gain (in pounds) from Example 3.

DETAILED DESCRIPTION

(12) The invention provides attenuated PRRSV strains having enhanced stability. Specifically polypeptides of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, and fragments thereof (including but not limited at least 5, preferably 8, more preferably 10, even more preferably 15 contiguous amino acids included in the above described sequences) are disclosed. In addition, polypeptides which are at least 95% homologous to and/or identical with the above-described polypeptides. Further, associated polynucleotides of the above described polypeptides are also disclosed.

(13) 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 methodsa 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).

(14) 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.

(15) A. Definitions

(16) 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.

(17) 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.

(18) 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 malaria. 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%.

(19) 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 a PRRSV, 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 at least 20%, more preferably at least 30%, even more preferably at least 50%, and even more preferably at least 70% lower than in the non-vaccinated control group after the challenge the infectious agent.

(20) 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.

(21) An immunogenic or immunological composition or vaccine refers to a composition of matter that comprises at least one PRRSV, 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 PRRSV infection. The term thus encompasses both subunit immunogenic compositions, as described below, as well as compositions containing whole killed, or attenuated, and/or inactivated PRRSV.

(22) The term subunit immunogenic composition or subunit vaccine as used herein refers to a composition containing at least one immunogenic polypeptide or antigen, but not all antigens, derived from or homologous to an antigen from PRRSV. Such a composition is substantially free of intact PRRSV. Thus, a subunit immunogenic composition or subunit vaccine is prepared from at least partially purified or fractionated (preferably substantially purified) immunogenic polypeptides from PRRSV, or recombinant analogs thereof. A subunit immunogenic composition can comprise the subunit antigen or antigens of interest substantially free of other antigens or polypeptides from PRRSV, or in fractionated form. A preferred immunogenic subunit composition comprises PRRSV proteins or fragments thereof, e.g., those which are incorporated into the envelope of the virus, e.g., gp2, gp4, gp5 and/or matrix (M) proteins. Also included are subunit immunogenic compositions which are modified analogs of the PRRSV virus, wherein the amino acid sequences of the antigen have been modified to enhance stability or immunogenicity.

(23) An immunogenic PRRSV polypeptide, or antigen, as used herein refer to a polypeptide or protein that elicits an immunological response as described herein. An immunogenic PRRSV protein or polypeptide includes the full-length sequence of any of the PPRSV 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 PRRSV 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 PRRSV 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 or more contiguous amino acids from the full-length PRRSV protein. Analogs include modifications to the antigen which confer enhanced properties to the molecule such as stability or immunogenicity.

(24) The term epitope means a segment or fragment of a composition of matter, e.g., a protein or polypeptide, which is recognized by the immune system, specifically by antibodies, B cells, or T cells. In the present invention, the epitope is generally a fragment or fragments of a polypeptide sequence of a viral protein.

(25) 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, N.J. 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. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. U.S Pat No. 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-Jul. 3, 1998. (The teachings and content of which are all incorporated by reference herein.)

(26) 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.

(27) Herein, specifically immunoreactive refers to an immunoreactive protein or polypeptide that recognizes an antigen characteristic of PRRSV infection but does not react with an antigen characteristic of a strict challenge control. To determine the specificity of a potential PRRSV immunoreactive protein or other polypeptide, various immunoassays (ELISA, IFA, WesternBlot) would be used to test the protein against animal sera containing genetically similar viruses. The protein would also be tested in various immunoassays against material containing proteins related to the expression method (Baculovirus, Sf9 cells, etc.).

(28) As used herein, a pharmaceutically- 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.

(29) 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 Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), 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. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed.Stewart-Tull, D. E. S.), John Wiley and Sons, NY, pp 51-94 (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.

(30) 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 (Pharmeuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Pat. 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; (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with allyl sucrose or with allyl pentaerythritol. Among them, there may be mentioned CARBOPOL 974P, 934P and 971P. Most preferred is the use of CARBOPOL 971P. 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.

(31) 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 Calif.), 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.

(32) It is expected that an adjuvant can be added in an amount of about 100 g to about 10 mg per dose, preferably in an amount of about 100 g to about 10 mg per dose, more preferably in an amount of about 500 g to about 5 mg per dose, even more preferably in an amount of about 750 g 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.

(33) 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, among others.

(34) 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.

(35) Safety refers to the absence of adverse consequences in a vaccinated animal following vaccination, including but not limited to: potential reversion of a bacterium-based vaccine to virulence, clinically significant side effects such as persistent, systemic illness or unacceptable inflammation at the site of vaccine administration.

(36) 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 elicitdirectly or indirectlyan immune response in the animal against PRRSV.

(37) Mortality, in the context of the present invention, refers to death caused by PRRSV 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.

(38) Attenuation means reducing the virulence of a pathogen. In the present invention attenuation is synonymous with avirulent. In the present invention, an attenuated virus is one in which the virulence has been reduced so that it does not cause clinical signs of a PRRSV infection but is capable of inducing an immune response in the target mammal, but may also mean that the clinical signs are reduced in incidence or severity in animals infected with the attenuated PRRSV in comparison with a control group of animals infected with non-attenuated PRRSV and not receiving the attenuated bacterium. In this context, the term reduce/reduced means a reduction of at least 10%, preferably 25%, even more preferably 50%, still more preferably 60%, even more preferably 70%, still more preferably 80%, even more preferably 90% and most preferably of 100% as compared to the control group as defined above. Thus, an attenuated, avirulent PRRSV strain is one that suitable for incorporation into an immunogenic composition comprising a modified live PRRSV virus.

(39) Killed or inactivated means treated with a physical or chemical agent which renders the PRRSV virus dead and/or otherwise incapable of reproduction. PRRSV may be killed by conventional means, such as, for example, heat, radiation or psoralen in the presence of ultraviolet light. PRRSV 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. Pat. Nos. 4,567,042 and 4,567,043. Antigens from PRRSV for use in the vaccine compositions of the present invention can 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.

(40) Antibodies as used herein includes anti-PRRSV 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 PRRSV polypeptide of the invention. The term specific for indicates that the variable regions of the antibodies of the invention recognize and bind a PRRSV polypeptide exclusively (i.e., are able to distinguish a single PRRSV 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, N.Y. (1988), Chapter 6. Antibodies that recognize and bind fragments of the PRRSV polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, a PPRSV 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 functionally active fragments such 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. Genetic constructs and methods for making antibodies of the invention, including cloning vectors, host cells transformed with cloning vectors and hybridomas, are well-known in the art.

(41) 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.

(42) 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 colony forming units (CFU) 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.

(43) The term immunoreactive to PRRSV as used herein means that the peptide or fragment elicits the immunological response against PRRSV.

(44) As used herein, complementary means a nucleic acid molecule has a sequence which complements a reference template sequence, whereby the two sequences can specifically hybridize. Preferably, the term refers to exact complementarity, e.g., as is found between the two strands of a nucleotide sequence in a naturally-occurring DNA-encoded gene.

(45) 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, with gaps introduced if necessary. 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 BLASTX (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, at least 85%, preferably at least 90%, even more preferably at least 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 at least 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.

(46) 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 also counted as a match when determining sequence homology. In other words, to obtain a polypeptide having 95% sequence homology with a reference sequence, 85%, preferably 90%, even more preferably 95% of the amino acid residues in the reference sequence must match or comprise a conservative substitution 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 amino acid residues, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence. Preferably the homologous sequence comprises at least a stretch of 50, even more preferred of 100, even more preferred of 250, even more preferred of 500 nucleotides encoding homologous amino acids.

(47) A conservative substitution refers to the substitution of an amino acid residue with another amino acid residue having similar characteristics or properties including size, hydrophobicity, etc., such that the overall functionality does not change significantly. It can also mean a nucleotide substitution which results in a conservative amino acid substitution.

(48) B. Carriers Molecules

(49) The carrier molecules to which the PRRSV peptides of the invention can be conjugated or covalently linked are preferably those described above. Preferred carriers for animal use are bovine serum albumin and Keyhole Limpet Hemocyanin. Protein carriers suitable for human use but which may also be used in animals 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.

(50) The PRRSV 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), all incorporated by reference, are within the scope of the invention, it is preferred to avoid the use of any linker but instead couple a S. suis 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), expressly incorporated by reference herein.

(51) The size of the immunogenic composition, as defined by average molecular weight, is variable and dependent upon the chosen PRRSV peptide(s) and the method of coupling of the PRRSV peptide(s) to the carrier. Therefore, it can be as small as 1,000 daltons (10.sup.3) or greater than 10.sup.6 daltons. With the reductive amination coupling method, the molecular weight of the PRRSV 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.

(52) Carrier molecules, i.e. peptides, derivatives and analogs thereof, and peptide mimetics that specifically bind a PRRSV 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), all incorporated herein by reference in their entirety).

(53) The PRRSV 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.

(54) Safety and efficacy of such molecules are determined by standard procedures in cell cultures or experimental animals as described and regulated by the Center for Veterinary Biologics (CVB). 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 LD.sub.50 (the dose lethal to 50% of the population).

(55) The vaccines of the invention may be multivalent or univalent. Multivalent vaccines are made from immuno-conjugation of multiple PRRSV peptides with a carrier molecule.

(56) In one aspect, the PRRSV 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.

(57) 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 administration.

(58) C. Adjuvants

(59) In order to further increase the immunogenicity of the immunogenic compositions provided herewith, and which contain one or more PRRSV peptides may also comprise one or more adjuvants.

(60) 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.

(61) The vaccine compositions of the invention are prepared by physically mixing the adjuvant with the PRRSV peptide(s) under appropriate sterile conditions in accordance with known techniques to produce the adjuvanted composition. Complexation of the PRSV 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.

(62) It is expected that an adjuvant can be added in an amount of about 100 g to about 10 mg per dose, preferably in an amount of about 100 g to about 10 mg per dose, more preferably in an amount of about 500 g to about 5 mg per dose, even more preferably in an amount of about 750 g 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.

(63) D. Physiologically-Acceptable Vehicles

(64) The vaccine compositions of this invention may be formulated using techniques similar to those used for other pharmaceutical polypeptide compositions. Thus, the adjuvant and PRRSV 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.

(65) 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.

(66) The vaccine compositions of the invention may be administered by any convenient means.

(67) E. Formulation

(68) Immunogenic conjugates comprising a PRRSV peptide(s) coupled to a carrier molecule can be used as vaccines for immunization against one or more serotypes of PRRSV. 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 PRRSV.

(69) Antibodies generated against immunogenic conjugates of the present invention by immunization with an immunogenic conjugate can be used in passive immunotherapy and generation of antiidiotypic antibodies for treating or preventing infections of PRRSV.

(70) 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 a swine.

(71) 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.

(72) 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.

(73) F. Effective Dose

(74) The compounds described herein can be administered to a subject at therapeutically effective doses to treat PRRSV-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.

(75) 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 PRRSV infectious disease in a subject. 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.

(76) Toxicity and therapeutic efficacy of compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD.sub.50 (the dose lethal to 50% of the population) and the ED.sub.50 (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 LD.sub.50/ED.sub.50. 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.

(77) 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 ED.sub.50 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 IC.sub.50 (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.

(78) 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, and poultry (e.g. chickens, ducks, geese and turkeys).

(79) 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 bacterial levels (for example, by culturing of a sample from the subject), 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.

(80) The therapeutics of the invention can be tested in vitro for the desired therapeutic or prophylactic activity, prior to in vitro use in animals. 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.

(81) 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.

(82) In addition, the therapeutic can be assessed by measuring the level of the molecule against which the antibody is directed in the animal model 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.

(83) After vaccination of an animal to a PRRSV 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.

(84) G. Detection And Diagnostic Methods

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

(86) The antibodies or binding portions thereof of the present invention are also useful for detecting in a sample the presence of a PRRSV peptide. This detection method comprises the steps of providing an isolated antibody or binding portion thereof raised against a PRRSV peptide, adding to the isolated antibody or binding portion thereof a sample suspected of containing a quantity of the PRRSV peptide, and detecting the presence of a complex comprising the isolated antibody or binding portion thereof bound to the PRRSV peptide.

(87) 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.

(88) In one aspect, an antibody of the invention that immunospecifically binds to a PRRSV peptide may be used to diagnose, prognose or screen for a PRRSV infection.

(89) In another aspect, the invention provides a method of diagnosing or screening for the presence of a PRRSV 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 PRRSV 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 PRRSV.

(90) Examples of suitable assays to detect the presence of PRRSV 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.

(91) 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.

(92) 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.

(93) An additional aspect of the present invention relates to diagnostic kits for the detection or measurement of PRRSV. Kits for diagnostic use are provided, that comprise in one or more containers an anti-PRRSV peptide antibody, and, optionally, a labeled binding partner to the antibody. Alternatively, the anti-PRRSV 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-PRRSV 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 PRRSV peptide recognized by the antibody of the kit, for use as a standard or control.

(94) H. Administration To A Subject

(95) Preferred routes of administration include but are not limited to intranasal, oral, intradermal, and intramuscular. Administration in drinking water, 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, intraperitnoeally, intrathecally, intratracheally, intracutaneously, intracardially, intralobally, intramedullarly, 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.

(96) 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.

(97) The entire disclosures of all applications, patents and publications, cited herein are expressly incorporated by reference herein.

EXAMPLES

(98) The strains of the invention used in the examples are SEQ ID NO: 1 (Wetzel p3 (pre-attenuated)) and SEQ ID NO: 2 (Wetzel p41 (attenuated)).

Example 1

Respiratory and Reproductive Safety of Cell Passage-Attenuated PRRS Virus

(99) The following disclosure relates to experiments performed on different PRRSV isolates; however, similar methods were used to prepare the Wetzel isolates of the present invention, and can be used by one of skill in the art to prepare these and similar attenuated isolates.

(100) Cell Passage

(101) With the goal of creating isolates with naturally occurring deletions, a unique passage process was used. A 96 well cell culture plates were planted with MA-104 cells (ATCC CRL2621) and incubated with minimum essential medium (EMEM, SAFC Biosciences M56416) with 10% fetal bovine serum (Invitrogen) and 5% CO.sub.2 at 37 C. Once cells had incubated for 3 days, 100 L of parent virus, either MN184 (MN/01/A2) or Isolate X, were added to each well of an entire row on the culture plate. Each well was then used to make four 1/10 dilutions down each column. After dilution, 100 L of fresh media was added over the top of each well. Plates were incubated for 3-8 days prior to being checked for cytopathic effect. After reading the plates, the highest positive dilution of each column was passed to another plate with 3 day old MA-104 cells. On this new plate four 1/10 dilutions were performed, media was overlayed, and plates were again incubated. This process was performed continuously through in vitro cell passage 51. Occasionally, if a column would display no growth the lowest dilution was passed in an attempt to harbor enough virus to maintain the passing infection. Subsequently, if there were still no signs of CPE on the next plate another column was used to plant the next plate.

(102) Isolate X is an isolated virulent field virus from a 2007 PRRS outbreak that exhibits an ORFS 1-7-4 RFLP pattern and has a JA-142 like nsp2 region. MN184 is a very pathogenic isolate that appeared in North America in 2002 causing high mortality and severe reproductive disorders. This isolate has a 131 amino acid deletion in the nsp2 region as compared to VR-2332, the type 2 prototype strain. Furthermore, MN184 has been found to have the shortest PRRS genome found to date at 15,019 kb [12] [16]. Deletion mutants created through the passage process were referred to as 96 well deletion mutants while Isolate X-9, related to Isolate X, contained no significant deletions in the nsp2 region but was selected for comparison purposes.

(103) Nucleotide Sequence Analysis

(104) Nucleotide sequence analysis was performed from both in vitro cell cultures and from study animal serum samples. For the in vitro samples, QIAamp Viral RNA Mini kit (Qiagen Inc, Valencia, Calif.) was used for the ribonucleic acid (RNA) extractions while the Total Nucleic Acid MagNA Pure kit (Roche Applied Science) was used to extract from the collected swine serum. SUPERSCRIPT III First-Strand Synthesis (Invitrogen Corp, Carlsbad, Calif.) was used to create complementary deoxyribonucleic acid (cDNA) per the manufacturer's recommendations. The cDNA product then underwent polymerase chain reaction (PCR) in a GENEAMP PCR System 9700 (Applied Biosystems) 96 well machine with the following configuration: an initializing step for 5 minutes (min.) at 95 C., 35 cycles of a denaturation step at 95 C. for 30 seconds (sec.) an annealing step at 53 C. for 30 sec. followed by an extension step at 72 C. for 150 sec., and finally a single elongation step at 72 C. for 5 min. Samples were held at 4 C. until removal from the thermocycler. Each PCR well was set up accordingly: 25 L, of 2AmpliTaq Gold Mastermix (Applied Biosystems), 21 L, DEPC Water (EMD Chemicals, Gibbstown, N.J.), 2 L, of cDNA template, and 1 L, of each respective primer (see Table 1).

(105) TABLE-US-00001 TABLE1 PrimersusedinpreparationofPRRSVisolates Primer Target Sequence SEQIDNO: PCRAmplificationofcDNA NamNsp2U Nsp2 CTGCGGCCTTRGACAGGAACGG 16 NamNsp2L Nsp2 TGTCHACCCKATCCCACATGCG 17 PRRS-Out1 ORF5 GTACGGCGATAGGGACACC 18 PRRS-Out2 ORF5 CCAGAATGTACTTGCGGCC 19 Letter Key: A - Adenosine; T - Thymidine; K - G or T; G - Guanosine; R - A or G; Y - C or T; C - Cytidine; H - A, C or T

(106) The PCR products were then purified with the WIZARD DNA Clean-Up System (Promega, Madison Wis.) per the manufacturer's recommendations. Samples were then placed into a Savant Speedvac DNA 110 (Global Medical Instrumentation, Ramsey, Minn.) at medium speed for 10 min. to remove any residual isopropanol. Samples were then mixed with 10 L of loading dye and loaded onto a 1.5% agarose gel. Electrophoresis was performed by applying 120 volts for 45 min. Bands of the appropriate size were visualized and underwent purification with the WIZARD SV Gel and PCR Clean-Up System (Promega).

(107) Samples were then sent to Iowa State University (ISU) DNA Facility for sequence analysis where the sequencing reactions were set up with the appropriate primers. At ISU an Applied Biosystems 37301 DNA Analyzer was used. Sequences were analyzed with GeneTool Version 2.0 (BioTools Inc.). The ORFS sequences were classified according to their restriction fragment length polymorphism cut pattern as outlined by the University of Minnesota Veterinary Diagnostic Laboratory.

(108) Respiratory Safety Trial

(109) For this study, twenty-five 3 week old commercial crossbred pigs nave to PRRSV were randomly split by weight into groups of 5 for a 14 day respiratory safety trial. All animals were housed at a research facility and blinded to all individuals working with the animals throughout the study. Each animal within a study group, with the exception of the negative controls, received a 2 mL intramuscular (IM) injection in the neck that contained either an attenuated clone of MN184 (Isolate Y) (4.86 logs delivered), Isolate X-2 (another attenuated isolate of Isolate X) (6.13 logs delivered), Isolate X-9 (6.31 logs delivered), or wild type Isolate X (3.59 logs delivered). Viral titers were determined by TCID.sub.50/ml (Johnson W, Roof M, Vaughn E, Christopher-Hennings J, Johnson C R, Murtaugh M P. Pathogenic and humoral immune responses to porcine reproductive and respiratory syndrome virus (PRRSV) are related to viral load in acute infection. Vet Immunol Immunopathol 2004 December. 8; 102(3):233-47 [17] and Reed L J, Muench H. A simple method of estimating fifty percent end-points. Amer J Hyg 1938; 27:493-7 [18], both hereby incorporated by reference).

(110) The study concluded at day 14 upon which all animals underwent humane euthanasia and necropsy by a licensed veterinarian. Blood samples were collected prior to treatment administration on study day 0 as well as on day 7, and 14.

(111) Reproductive Safety Trial

(112) The reproductive safety trial included 16 PRRS ELISA negative commercial crossbred gilts at 904 days of gestation housed in a research facility and blinded to all animal caretakers and investigators. Animals were randomly divided into four treatment groups consisting of the same three cell passaged test articles from the respiratory trial and one negative control group. The study officially began at day 0 when the females were inoculated intranasally with 2.0 mL per nostril with one of the following: Isolate Y, Isolate X-2, Isolate X-9, or phosphate buffered saline (PBS). All viruses were diluted with minimum essential medium (MEM; Sigma, St. Louis, Mo.) containing 2% fetal bovine serum (FBS) (Sigma, St. Louis, Mo.) in order to deliver a target of 5.00 logs of virus in 4 ml to each animal. Viral titers were determined by TCID.sub.50/dose. The study ended for each gilt and her piglets 21 days after their respective day of farrowing (DOF). All gilts had blood drawn at day 0, 7, 14, 21, day of farrowing (DOF), 7 days after farrowing (DOF+7), 14 days after farrowing (DOF+14), and 21 days after farrowing (DOF+21). In addition, live piglets had blood collected on DOF, DOF+7, DOF+14, and DOF+21 while dead piglets had blood or thoracic fluid collected immediately upon examination regardless of study day.

(113) Respiratory Safety Trial Clinical Lung Evaluation

(114) The lungs of all animals in the pig safety study were evaluated at necropsy for percent consolidation consistent with PRRSV infection. Lungs were scored for each individual lobe and total gross lung pathology using a standardized scoring system. See, Halbur P G, Miller L D, Paul P S, Meng X J, Huffman E L, Andrews J J. Immunohistochemical identification of porcine reproductive and respiratory syndrome virus (PRRSV) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs. Vet Pathol 1995 March; 32(2):200-4 [19]. The final observation score equaled the weighted sum of all the individual lobe scores.

(115) Reproductive Safety Trial Reproductive Performance

(116) Abortions and farrowing data were recorded for each female. The day of farrowing was defined as the day the first live born pig of the litter is delivered. Gilts were observed periodically from at least 6:00 AM to 10:00 PM daily. Litter observations included the number of abortions, stillbirths, mummies, live piglets, and weak born live piglets for each female. Piglets found dead due to being crushed by the dam were recorded and confirmed with a necropsy.

(117) Serology

(118) Whole blood (5-10 mL) was collected from each animal via venipuncture. Samples were returned to the laboratory for separation from the clot by centrifugation. Serum was then decanted into screw-cap cryogenic vials and stored for a maximum of 24 hours at 4 C. or frozen at 70 C. until testing could be performed. Serum samples were analyzed for PRRSV specific antibody using the IDEXX HERDCHEK PRRS ELISA 2XR (IDEXX Laboratories Inc, Westbrook, Me.). All tests were performed as described by the manufacturer's instructions. Samples were considered positive for PRRSV antibodies if the sample-to-positive (S/P) ratio was at least 0.4.

(119) Average Daily Weight Gain

(120) As another parameter to gauge the health status of study animals, average daily weight gain (ADWG) was recorded. Animal weights were recorded on a scale that was calibrated prior to each use. For the respiratory safety study, animals were weighed 3 days prior to vaccination (Day 3), and at necropsy (Day 14) for average daily weight gain analysis. For the reproductive safety study, all piglets delivered were weighed within 8 hours of farrowing and all remaining live piglets were again weighed 21 days later for analysis.

(121) Reproductive Safety Trial Viremia

(122) Both gilt and piglet serum was tested via reverse transcriptase polymerase chain reaction (RT-PCR) for the presence of PRRSV nucleic acid. Serum was extracted by the Roche MagNA pure extraction robot with the Total Nucleic Acid MagNA Pure kit (Roche Applied Science). The RT-PCR was performed with the AgPath-ID NA and EU PRRSV Multiplex (Applied Biosystems) per the manufacturer's instructions. The reactions were run on a Roche Lightcycler 480 96 well machine with the following configuration: an annealing step at 45 C. for 10 min., denaturation at 95 C. for 10 min., and 40 cycles of denaturation at 97 C. for 2 sec. followed by an annealing step at 60 C. for 40 sec., and finally a cooling step at 40 C. for 40 seconds.

(123) Reproductive Safety Study Animal Clinical Observations

(124) All study animals were observed daily for overall health and clinical signs associated with PRRS. Each animal was evaluated for respiration, behavior, and cough with scores assigned for each category. Possible scores ranged from 0 (normal) to 1 (abnormal). Abnormal behaviors were defined as abnormal respiration, abnormal behavior such as lethargy, and the presence of a cough. A total daily score was recorded based on the sum of the 3 individual scores. Any dead piglets were assigned a score of 3 and then were weighed and subsequently had a necropsy performed to determine the likely cause of death. In addition to the daily observations, gilt rectal temperatures were taken periodically beginning with the day prior to treatment (day 1) through study termination (DOF+21).

(125) Statistics/Biometrics

(126) All data for the respiratory safety and reproductive safety studies were imported into SAS version 9.1 for management and analysis. Summary statistics by treatment group including mean, median, standard deviation, standard error, minimum, maximum, coefficient of variation, and frequency distributions were generated for all variables, where appropriate. All parameters were compared between groups (pairwise) 1-4 vs. 5 for both the pig and reproductive studies and groups 1-3 vs. 4 for the respiratory study. In compliance with the methods recommended by APHIS, only two-sided results were reported and all comparisons were at =0.05. The specific methods used to analyze data are shown in Table 2.

(127) TABLE-US-00002 TABLE 2 Statistical methods of analysis for each parameter evaluated Specific Statistical Evaluation To Method of Safety Study Parameter Be Conducted Analysis Respiratory Mean Lung Mean lung scores ANOVA Scores for each group Respiratory Serology Number of animals Fisher's and in each group Exact Test Reproductive serologically positive/ total number of animals in each group Respiratory Weights and Mean Data ANOVA and ADWG ANCOVA Reproductive Reproductive Gilt 1. Total Piglets 1-6. Kurskal- Reproductive 2. Stillborn Wallis/Wilcoxon Performance 3. Healthy Live Piglets Two Sample Test 4. Weak Live Piglets 5. Mummies 6. Live Pigs at DOF 21 Reproductive Gilt and Pig- Gilts/Piglets Fisher's let Clinical with score >0 for Exact Test Observations at least one day Reproductive Gilt Rectal Mean Data 1. ANOVA Temperatures Pyrexia Fisher's Exact Test Reproductive Gilt/Piglet Animals Fisher's Viremia positive Exact Test
Results:
Sequence Analysis

(128) The viruses undergoing continuous cell passage had genomic sequence analysis performed at approximately every 10 passages on the nsp2 region and on the ORFS region at passage 50. As expected, several isolates had natural deletions occur throughout the process. At cell passage 50 Isolate Y had a continuous 6 nucleotide deletion in the nsp2 region, expected to result in the loss of 2 amino acids. Isolate X-2 at cell passage 50 exhibited a continuous 63 nucleotide deletion in the nsp2 region which results in the loss of a continuous 21 amino acid sequence. Isolate X-9 remained similar to JA-142 and had no large deletions in the nsp2 region. Results of sequencing of the ORF5 region displayed that the MN184 isolates still had a 1-8-4 RFLP cut pattern while the Isolate X isolates maintained a 1-7-4 RFLP cut pattern.

(129) Samples from the reproductive safety trial also underwent sequence analysis to determine how stable the viruses were in vitro. All PCR positive piglet blood samples, from the final day of the study (DOF+21), underwent attempted sequence analysis and at least one sequence was successfully attained from each vaccinate group. All viruses remained relatively stable after in vitro replication. The nsp2 region sequencing from each individual animal revealed only a few substitutions for each virus. Substitutions resulting in amino acid changes are summarized in Table 3 and are described in comparison to MN184A and JA142 AY424271. The ORF5 regions were also sequenced and all isolates maintained their original cut pattern.

(130) TABLE-US-00003 TABLE 3 Nsp2 mutations which resulted in corresponding amino acid changes from positive piglets at three weeks after farrowing. Isolate Nucleotide Change MN184A Position* Isolate Y A A .fwdarw. G Isolate Y B A .fwdarw. G Isolate Y C T .fwdarw. C Isolate Y D A .fwdarw. G JA142 AY424271 Position* Isolate X-2 Position A .fwdarw. G Isolate X-2 F G .fwdarw. A Isolate X-9 G G .fwdarw. A Isolate X-9 H C .fwdarw. T Isolate X-9 I T .fwdarw. G Isolate X-9 J G .fwdarw. A
Respiratory Safety Trial Lung Pathology

(131) Upon completion of the study on day 14, all animals were necropsied and assessed for lung pathology (FIG. 1). Using ANOVA to compare group mean lung scores, only the wild type Isolate X group had a statistically significant increase in clinical lung scores as compared to the negative control group. The Isolate Y group and Isolate X-9 group had statistically significant lower lung scores than the Isolate X wild type group. The lung scores indicate that the cell passage process has indeed significantly reduced the virulence of the Isolate Y and Isolate X-9 isolates while Isolate X-2 may still be capable of causing some degree of respiratory disease.

(132) Average Daily Weight Gain

(133) For the respiratory safety study, average daily weight gain analysis via ANOVA revealed that there were no statistically significant differences for either group mean initial body weight or average daily gain for the duration of the study amongst all treatment groups. The wild type Isolate X treatment had a slightly higher mean initial body weight than the other groups which may account for the lack of statistical differences in ADWG as compared to the other groups.

(134) For the reproductive safety study, ANOVA analysis revealed piglets in the Isolate X-2 group had a statistically significant lower mean body weight at farrowing than the negative control group. Interestingly, piglets in all treatment groups had a statistically significant lower ADWG as compared to the negative control group. However, with the use of another statistical test, ANCOVA, it was determined that initial piglet mean body weight and the initial piglet mean body weighttreatment group interaction were significant contributors to piglet ADWG while the treatment group each piglet belonged to was not a significant contributor to ADWG.

(135) Herdchek Elisa

(136) For the respiratory safety study, on day 0 and 7 all animals were negative via the IDEXX HERDCHEK PRRS ELISA 2XR. However, on day 14 all animals in the Isolate Y group and most animals in group 4 (wild type Isolate X) had seroconverted while the treatment groups administered Isolates X-2 and X-9, after about 50 passages, displayed poor seroconversion (FIG. 2). Fisher's Exact Test was used to compare the number of animals that had seroconverted in each group. The results indicated that only the Isolate Y group and the wild type Isolate X group had significantly higher numbers of animals positive by ELISA as compared to the negative control group. The wild type Isolate X treatment group was statistically different from animals in the Isolate X-2 group, but not the Isolate X-9 group.

(137) For the reproductive safety study, all animals were again negative on days 0 and 7. However, by day 21 nearly all gilts that received virus as a treatment were positive. Gilts in the MN184 treatment group all remained positive by ELISA for the duration of the study. All treatment groups had a statistically significant increase in positive animals as compared to the negative control group at day 21, DOF 0, and DOF+14.

(138) The piglets from the reproductive safety study were found to have a considerable amount of PRRSV specific antibody. The Isolate X-9 and Isolate Y groups displayed statistically significant increases in positive ELISA tests as compared to the piglets belonging to the negative control group for all sample collection dates. The Isolate X-2 treatment group piglets were found to only be statistically distinct from the negative control group at DOF+7.

(139) ELISA findings for both studies echoed the same results. While all isolates proved to be capable of inducing a PRRSV specific antibody response as detected by the HERDCHEK PRRS ELISA 2XR, the onset and duration of antibody detected in the MN184 treatment group seemed to be earlier and longer lasting than the response of the Isolate X treatment groups. Within the Isolate X isolates, the Isolate X-9 groups consistently had more animals test positive than the groups that received Isolate X-2.

(140) Gilt Reproductive Performance

(141) All females included in the study farrowed relatively normal and healthy litters as outlined in Table 4.

(142) TABLE-US-00004 TABLE 4 Mean gilt reproductive performance per litter Total Piglets Healthy Live Weak Live Live Pigs at Farrowed Stillborn Piglets Piglets Mummies DOF 21 Isolate X-9 11.25 0.25 9.5 1 0.5 9.75 Isolate Y 9 1.75 6.25 0.25 0.5 6.5 Isolate X-2 8.25 2.5 5.75 0 0 5.25 Negative 8.25 1.25 6.5 0 0.25 6.5

(143) Due to facility and labor limitations, only 4 gilts were used for each isolate. There were no dramatic signs of virulence in any of the tested isolates. For statistical analysis, a Wilcoxon two sample test was used to make comparisons between each treatment group and the negative control group. The number of total piglets born, stillborns, healthy live piglets, weak live piglets, mummies, and pigs still alive 21 days after farrowing were compared to the negative control group. No statistical differences were found for any of the reproductive performance variables. These findings suggest that the described attenuation process has been successful in reducing the amount of reproductive failure caused by each isolate.

(144) Mortality of Piglets, Pigs, and Gilts

(145) For the respiratory safety study no animals were lost during the study. However, for the reproductive safety study 6 piglets were lost from the DOF 0 to DOF+21. These losses still fall within the expected range of piglet mortality prior to weaning. One gilt in the Isolate X-2 treatment group was euthanized due to illness resulting from a retained placenta. This gilt delivered just 3 stillborn piglets so no piglet performance data was compromised.

(146) Viremia

(147) Gilt viremia as detected by RT-PCR was relatively short for most groups (FIG. 3). Each treatment group was compared to the negative control group using Fischer's exact test for statistical analysis. The Isolate X-9 group, Isolate Y group, and Isolate X-2 group had a statistically significant increase in animals positive for PRRS RNA at study day 14 while the Isolate Y group was also statistically distinct at day 7. The negative control group had 1 animal test positive at DOF+7. This was likely the result of sample contamination sometime along processing as this was the only positive test out of this group.

(148) An important safety indicator for PRRS is transplacental transfer from dam to piglet. For the Isolate X treatment groups, the majority of the piglets were negative on the day of farrowing. However, by DOF+21 most piglets belonging to the Isolate X-9 and Isolate Y treatment groups were positive, presumably due to horizontal transmission of virus. Fisher's exact test was used for statistical analysis. All treatment groups exhibited a statistically significant increase in the number of piglets testing positive for PRRS RNA, with the exception of Isolate X-2 at DOF+7 which lost significance due to 4 out of the 26 piglets in the negative control group testing positive on that day. The negative control positive samples were likely the results of contamination somewhere along the line of sample collection, handling, and processing as no other negative control group samples were PCR positive prior to or after DOF+7 (FIG. 4).

(149) Reproductive Safety Study Clinical Observations

(150) Clinical observations were recorded for the reproductive safety study as supporting data for remaining isolate virulence. With the exception of the euthanized gilt, only a few isolated events of abnormal scores of 1 were documented. No groups were statistically different than the negative control group when using Fisher's exact test to compare the number of animals per group with a clinically abnormal score for at least one day. Also, mean gilt group rectal temperatures were collected and compared to the negative control group using ANOVA. Statistically significant higher mean rectal temperatures were noted at day 7 (Isolate X-9 and Isolate X-2) and day 14 (Isolate Y). Body temperatures for gilts in all treatment groups remained normal for the duration of the study.

(151) For the piglet clinical observation scores there were statistically significant increases in piglet clinical abnormalities in the Isolate X-9 and Isolate X-2 treatment groups as compared to the negative control group. Piglets in the Isolate Y treatment group had a P value of 0.0547 which is approaching statistical significance but is above the 0.05 cutoff used for all statistical analysis for this study. As previously mentioned 8 piglets (4 from the Isolate X-9 group, 1 from each MN184 group, and 2 from the Isolate X-2 group) were found dead throughout the study. No piglets in the negative control group were found dead during the study. All other clinically abnormal animals were not severally affected and no abnormalities compromised the outcome of the study.

(152) In this study, a passage process is described in which single isolates were passed multiple times by limiting dilution. Frequent sequence analysis was used as a selection tool to test individual clones in vitro for vaccine potential. Genomic deletions within the nsp2 gene were targeted, as these isolates are believed to be more genomically stable in the animal host while also capable of providing sufficient immunological protection against wild type virus. The selected isolates were screened for genomic stability and safety through the use of both a respiratory and reproductive animal model.

(153) The results of this trial serve as evidence that all isolates are attenuated to some degree. When compared to the negative control group, all cell passage isolates had no statistical differences for lung pathology or average daily weight gain. Furthermore, the wild type Isolate X group had statistically significant higher scores for lung pathology than the Isolate Y and the Isolate X-9 group. A wild type MN184 was not used in this study, but has been tested previously [17]. Johnson et al. inoculated ten 2-3 week old pigs with MN184 at cell culture passage 1 and documented the death of 2 pigs in that group by the first 14 days of the study. Lung pathology was not collected for the MN184 group, but the group mortality rate speaks for the general virulence of the parent wild type isolate. Here, in the pig respiratory trial, no animals were lost for any treatment group over the course of the study. Clearly, the ability to cause respiratory disease has been decreased with all isolates, but perhaps less so with Isolate X-2 group. This group's lung scores were not statistically different when compared to the wild type Isolate X treatment group.

(154) Results for the reproductive safety trial mimicked those from the respiratory trial. When compared to the negative control group, all reproductive performance parameters and gilt clinical observations had no statistical differences for any of the tested isolates. ELISA results for both trials indicated all isolates are capable of inducing the production of PRRSV specific antibodies by day 21 in the gilts, although seroconversion in the MN184 group seems to be more consistent and longer lasting than that in the Isolate X groups. Viremia detected by RT-PCR showed that the virus attained detectable levels within the blood for only short periods of time in the gilts while there was transplacental transmission of virus with subsequent horizontal spread to littermates in all groups. While congenital transmission of virus with the transmission to littermates did occur, it didn't negatively affect the performance of all groups. The Isolate Y group had no statistical differences in piglet clinical observations when compared to the negative control group which is a phenomenon that has been previously described with currently available attenuated vaccines See, Mengeling W L, Lager K M, Vorwald A C. Clinical effects of porcine reproductive and respiratory syndrome virus on pigs during the early postnatal interval. Am J Vet Res 1998 January; 59(1):52-5 [20], hereby incorporated by reference. With the rising concern of modified live vaccines possibly reverting back to virulent forms, increased and proven stability of vaccine isolates will become increasingly important. Our genomic stability data indicates these isolates appear to be sufficiently stable with relatively few changes in both the nsp2 and ORF 5 regions which are to be expected and especially so with PRRS. See, Wesley R D, Mengeling W L, Lager K M, Vorwald A C, Roof M B. Evidence for divergence of restriction fragment length polymorphism patterns following in vitro replication of porcine reproductive and respiratory syndrome virus. Am J Vet Res 1999 April; 60(4):463-7 [21], hereby incorporated by reference.

(155) The given results indicate the isolates are capable of inducing seroconversion in vitro and are much less virulent than the parent viruses, yet may still be capable of causing some disease or losses in production. Therefore, these isolates can be further passed in cell culture in an attempt to further attenuate and stabilize the genome. The 96 well deletion passage process is an effective and efficient method to develop novel modified live vaccines with increased efficacy and genomic stability.

Example 2

Safety of Cell Passage-Attenuated PRRS Virus in 5 Week Old Piglets

(156) In this study, three PRRSV clones were evaluated for safety: Isolate X-6, Wetzel Clone 3 p51, and Wetzel Clone 9 p48. A 2 mL dose of each clone was administered intramuscularly (IM) to one of three groups of 6 cesarean delivered-colostrum deprived (CD-CD) pigs at 5 weeks of age. A fourth group of 5 pigs received a placebo and served as controls. The safety of the clones was evaluated based on PRRSV seroconversion, viremia and PRRSV-associated lung lesions.

(157) On Day 0 (D0) of the study, twenty-three (23) healthy 5-week old CD-CD piglets, susceptible to PRRSV (serologically negative; <0.4, for PRRSV antibodies by ELISA), were inoculated with PRRSV Isolate X-6 (Group T1), Wetzel Clone 3 p51 (Group T2), Wetzel Clone 9 p48 (Group T3), or a placebo (Group C).

(158) Serum samples were collected on D0, 7 and 14 from all pigs. On D14, all pigs were euthanized at which time gross pathological examinations and lung lesion scoring was performed.

(159) Clinical observations were made daily from D-1 to D14 for overall health and signs associated with PRRS disease, specifically, respiration, behavior, and cough. No clinical signs were observed in any pig throughout the entire study. Additionally, no lung lesions (score=0) were observed in any pig at necropsy.

(160) Polymerase Chain Reaction (PCR) testing for the presence of PRRSV viremia was completed on all serum samples. PCR results for pigs in all groups were negative on D0 indicating a lack of exposure to PRRSV at the time of inoculation. Results on the remaining time points for the control Group C were also negative indicating a lack of environmental exposure to PRRSV during the study. On D7, all pigs in Group T1 (6/6, 100%) and T2 (6/6, 100%) were PCR positive while all pigs in Group T3 (0/6, 0.0%) were negative. On D14, Group T1 (6/6, 100%) and T2 (6/6, 100%) remained at the same levels, while Group T3 (2/6, 33.3%) showed two pigs positive for PRRS viremia. To verify that PRRSV in these last two pigs was the clone they received at inoculation, ORF 5 sequence analysis was performed with results matching Wetzel Clone 9 p48 indicating no cross contamination and/or environmental infection.

(161) Antibody titers for PRRSV were determined by IDEXX ELISA. All pigs were sero-negative (<0.4) on D0. This indicates a lack of maternal antibodies at the time of inoculation. All pigs remained negative on D7. On D14, Group T1, T2, and T3 had 4/6 (66.7%), 5/6 (83.3%), and 1/6 (16.7%) positive pigs, respectively. Controls remained negative at D14 showing a lack of exposure to PRRSV.

(162) Despite the fact that piglets developed viremia by Day 14, the data indicates that the PRRSV clones used in this study caused no lung lesions and/or clinical signs of PRRSV infection. These clones are deemed to be safe in 5-week old piglets.

(163) In this study, three clones were evaluated for safety: Isolate X-6, Wetzel Clone 3 p51, and Wetzel Clone 9 p48. A 2 mL dose of each clone was administered intramuscularly (IM) to one of three groups of 6 cesarean delivered-colostrum deprived (CDCD) pigs at 5 weeks of age. A fourth group of 5 pigs received a placebo and served as controls. The safety of the clones was evaluated based on PRRSV seroconversion, viremia and PRRSV-associated lung lesions to help determine the feasibility of their use as a MLV candidate.

(164) The objective of this study was to evaluate the safety of the PRRSV clones Isolate X-6, Wetzel Clone 3 p51, and Wetzel Clone 9 p48 in 5-week old piglets. Clinical evaluation of all piglets was conducted daily from Day-1 to 14. Evaluations included incidence of mortality, clinical scores (respiratory distress, lethargy, behavior, and cough), viremia, lung lesions, sero-status, and virus isolation (VI). A schedule of events is shown in Table 5.

(165) TABLE-US-00005 TABLE 5 Schedule of events Day Event Samples Testing 0 Pigs to be 5 weeks Serum PCR, ELISA, old +/ 5 d VI Clinical Evaluations, Serum collection, Inoculate with IVP's 1-14 Clinical evaluation None None 7 Serum Collection Serum PCR, ELISA, VI 14 Necropsy, serum collection, Serum, Lung PCR, ELISA, lavage collection Scores, Lavages VI
Study Design:

(166) Twenty-three (23) PRRS-susceptible (ELISA <0.4), CD-CD study animals were purchased, transported, and then housed together 1 week prior to vaccination at Veterinary Resources, Inc.'s (VR1) Cambridge, Iowa facility. The test animals were individually identified and examined by the study investigator for overall health.

(167) Piglets were randomly divided into four groups as shown in Table 6.

(168) TABLE-US-00006 TABLE 6 Treatment Groups Number of Group Animals Treatment Dose/Route T1 6 PRRSV Isolate X-6 2 mL/IM T2 6 PRRSV Wetzel Clone 3 p51 2 mL/IM T3 6 PRRSV Wetzel Clone 9 p48 2 mL/IM C 5 Control (Sterile PBS Placebo) 2 mL/IM
Randomization Method

(169) For randomization, a roster of all animals included in the study was prepared by the study investigator prior to study initiation. A computer random number generator (Microsoft Excel) was used to assign each animal a unique random number. Animals were sorted into weight blocks (experimental blocks). Within experimental blocks, the animal with the lowest random number was assigned to the first treatment group. Assignment continued by increasing random number until all animals had been assigned to a treatment group. See Attachment 7 for group assignments.

(170) Methods of Blinding

(171) The study was blinded as to eliminate bias in laboratory sample testing. The person conducting the laboratory testing of study samples was not the same person administering treatment or making daily observations. See Table 7 for inclusion criteria.

(172) TABLE-US-00007 TABLE 7 Inclusion Criteria a) Species: Porcine b) Breed: Commercial cross c) Age Requirements: 5 weeks 5 days of age d) Weight Range: Uniform for age e) Sex: Both female and neutered males f) Number: 23 g) Source of test animal and Commercial Class A Dealer; ownership: Boehringer Ingelheim Vetmedica, Inc. h) Special Requirements: Cesarean delivered and colostrum deprived (CD/CD) i) Serological Status: Seronegative to PRRSV (S/P < 0.400)
Exclusion Policy

(173) No animals were excluded from the study prior to completion of the animal phase.

(174) Identification of Test Animals

(175) All test animals were identified by duplicated ear tags not associated with treatment group.

(176) Animal Management and Housing

(177) Animals were housed in the facilities at Veterinary Resources, Inc. according to their standard operating procedures.

(178) Animal Care

(179) The animals were deemed to be in good health and nutritional status before the study was initiated. The investigator conducted a health examination prior to the randomization procedure.

(180) Due to the susceptibility of CD/CD animals, all piglets received one dose of Pfizer EXCEDE on six days prior to inoculation (D-6) as per label instructions. Additionally, piglets were fed PURINA Mills Senior Medicated feed from D-6 through D0. From D1 through the end of the study, piglets received PURINA Mills Senior feed.

(181) Management of Food and Water

(182) Feed rations were appropriate for the age, condition, and species of test animal according to facility standard operating procedure. Water was provided ad libitum throughout the study.

(183) Investigational veterinary products (IVP)

(184) The PRRSV clones were prepared by infection of AK MA-104 cells in modified Minimum Essential Media (mMEM) with 2% FBS. The cultures were allowed to incubate at 37 C. until CPE was 80% at which time they were placed at 70 C. On the day of inoculation (D0), flasks were removed from the freezer and allowed to thaw at room temperature. Each clones' suspension was then transferred to a plastic, stopper-topped vaccine bottle and labeled with T1, T2, or T3 (see Table 1). They were then delivered directly on ice to the study site. All clone preparations were tested and quantified by the TCID.sub.50 assay pre- and post-inoculation. For the control Group C, 1PBS was used as a placebo. The pigs were administered 2.0 mL IM of the appropriate IVP or placebo on D0. Any remaining IVP's were returned to BIVI-Ames after inoculation. See Tables 8, 9, and 10 for details of the PRRSV clone IVP's.

(185) TABLE-US-00008 TABLE 8 Investigational Veterinary Products, PRRSV Isolate Y-6 Generic Product PRRSV Isolate Y-6 Name: Manufacturer: Boehringer Ingelheim Vetmedica, Inc. Lot or 250-071-MRC6P81 Serial Number: Expiration Date: N/A Storage 70 C. Requirements Testing On the day of challenge, a TCID.sub.50 assay was performed pre- and post-challenge resulting in respective titers of 6.23 and 6.66 with an overall average of 6.45 log.sub.10 TCID.sub.50/mL. Test Article The required doses of IVP were Transfer transferred by the Study Monitor to the study site just prior to inoculation and kept on ice. Test Article Any unused IVP was disposed of by autoclaving. Retention: Applied Dose: 2.0 mL of the PRRSV Isolate 6 was administered IM on Day 0 to each pig in Group T1.

(186) TABLE-US-00009 TABLE 9 Investigational Veterinary Products, PRRSV Wetzel Clone 3 p51 Generic Product PRRSV Wetzel Clone 3 p51 Name: Manufacturer: Boehringer Ingelheim Vetmedica, Inc. Lot or 250-096-WC3P51 Serial Number: Expiration Date: N/A Storage 70 C Requirements Testing On the day of challenge, a TCID.sub.50 assay was performed pre- and post-challenge resulting in respective titers of 6.29 and 6.72 with an overall average of 6.51 log.sub.10 TCID.sub.50/mL. Test Article The required doses of IVP were transferred Transfer by the Study Monitor to the study site just prior to inoculation and kept on ice. Test Article Any unused IVP was disposed of by autoclaving. Retention: Applied Dose: 2.0 mL of the PRRSV Wetzel Clone 3 p51 was administered IM on Day 0 to each pig in Group T2.

(187) TABLE-US-00010 TABLE 10 Investigational Veterinary Products, PRRSV Wetzel Clone 9 p48 Generic Product PRRSV Wetzel Clone 9 p48 Name Manufacturer Boehringer Ingelheim Vetmedica, Inc. Lot or 250-046-WC9P48 Serial Number Expiration Date N/A Storage 70 C. Requirements Testing On the day of challenge, a TCID.sub.50 assay was performed pre- and post-challenge resulting in respective titers of 6.98 and 6.91 with an overall average of 6.95 log.sub.10 TCID.sub.50/mL. Test Article The required doses of IVP were transferred Transfer by the Study Monitor to the study site just prior to inoculation and kept on ice. Test Article Any unused IVP was disposed of by autoclaving. Retention Applied Dose 2.0 mL of the PRRSV Wetzel Clone 9 p48 was administered IM on Day 0 to each pig in Group T3.

(188) Carcasses of all euthanized study pigs were disposed of by rendering. No test animals were removed from the study after inoculation or prior to necropsy. After all of the doses of the Investigational Veterinary Products were administered, the remaining IVP's were returned to the Study Monitor for appropriate disposal.

(189) Evaluation of CLONE Safety

(190) The safety of the clones was evaluated based on seroconversion to PRRSV, viremia and presence/severity of PRRSV-associated lung lesions. A single occurrence of viremia at any time point following inoculation classified a pig as positive for viremia. Percentage pathology for each animal's lungs was calculated according to the procedure described in section 12.3.1. Results from each Test Group were compared to those of the Control Group. Pigs were also assessed for the isolation of PRRSV in serum and lung lavage fluid, clinical observations/scores, and mortality.

(191) No more than 10 mL of venous whole blood was collected via the anterior vena cava from each pig using a sterile 18-20g1 inch (2.54 cm) to 1.5 inch (3.81 cm)1-1 VACCUTAINER needle, a VACCUTAINER needle holder and 13 mL serum separator tubes (SST) by the Study the Investigator or a designee. Blood was collected prior to inoculation and on Day 7 and 14. Each blood tube was labeled with the animal's ID number, the study number, the date of collection, the study day and the sample type.

(192) Serum samples were tested for PRRSV viremia by PCR and for PRRSV antibodies using a commercially available testIDEXX PRRS ELISA. Serum samples were also tested for the presence of PRRSV by virus isolation (VI). Every animal was PRRS sero-negative (ELISA S/P ratio of <0.4) prior to vaccination indicating a valid study. Virus isolation results were reported as positive or negative.

(193) Clinical Observations

(194) Pigs were observed for overall health and clinical signs associated with PRRS disease by the Investigator or designee daily from Day 1 to Day 14. Specifically, pigs were examined each day for respiration, behavior, and cough with each category rated 0 for normal and 1 for abnormal (See Table 11). A total daily score for each pig was the summation of its Respiratory Score, Behavior Score and Cough Score for that particular day. No deaths occurred during the study.

(195) Respiration, Behavior, Cough, and Observation Scores were scored as follows: Respiration Score: 0=Normal Respiration, 1=Abnormal Respiration; Behavior Score: 0=Normal Behavior, 1=Abnormal Behavior; Cough Score: 0=No Coughing Noted, 1=Cough Present.

(196) On the day of necropsy, after data and samples were collected, each pig was euthanized and necropsied by the Investigator or designee. Following euthanasia, the Investigator or designee removed the lungs and trachea intact, with a clamp placed across the trachea to prevent blood cross contamination. A general description of lung pathology was observed and the percentage of pathology in each lung lobe was recorded. Total % lung pathology was determined for each pig by summation of percent lung pathology for each lung lobe.

(197) After the lungs were evaluated for pathology, a lung lavage sample was collected from each set of lungs by the Investigator or designee. Each sample was labelled with the animal number, study number, sample type, date of collection and study day. No lung tissue samples were collected for this study.

(198) Results:

(199) ELISA results were negative (S/P<0.400) for all pigs on Day 0, indicating a lack of maternal antibodies present at the time of inoculation. Day 0 averages for group T1, T2, T3, and C are 0.070, 0.042, 0.037, and 0.032, respectively. All pigs remained negative on Day 7 with averages for group T1, T2, T3, and C at 0.069, 0.038, 0.023, and 0.053, respectively. On Day 14, there were four positive pigs (4/6, 66.7%) in Group Ti, five positive pigs (5/6, 83.3%) in Group T2, and one positive pig (1/6, 16.7%) in Group T3. However, Group C remained all negative (0/5, 0.0%). Averages for Day 14 were 1.122, 1.024, 0.247, and 0.045 for Group T1, T2, T3, and C, respectively. ELISA data can be seen in Table 11.

(200) TABLE-US-00011 TABLE 11 PRRSV IDEXX ELISA Sample to Positive Ratios Pig ID Group D0 D7 D14 9 T1 0.013 0.079 0.305 23 0.002 0.024 2.007* 31 0.007 0.045 1.626* 32 0.385 0.241 0.083 33 0.018 0.009 1.333* 36 0.002 0.016 1.380* Average S/P 0.070 0.069 1.122 # of Positive 0/6 (0.0%) 0/6 (0.0%) 4/6 (66.7%) Pigs (%) 60 T2 0.049 0.049 0.187 61 0.016 0.002 0.447* 62 0.005 0.040 2.490* 65 0.131 0.089 1.281* 76 0.022 0.011 1.020* 80 0.029 0.040 0.720* Average S/P 0.042 0.038 1.024 # of Positive 0/6 (0.0%) 0/6 (0.0%) 5/6 (83.3%) Pigs (%) 70 T3 0.018 0.029 0.031 81 0.163 0.054 0.246 88 0.007 0.022 0.107 90 0.016 0.005 0.241 95 0.017 0.016 0.014 97 0.002 0.011 0.869* Average S/P 0.037 0.023 0.247 # of Positive 0/6 (0.0%) 0/6 (0.0%) 1/6 (16.7%) Pigs (%) 57 C 0.022 0.033 0.040 84 0.020 0.033 0.052 91 0.025 0.047 0.045 92 0.061 0.074 0.059 94 0.032 0.080 0.028 Average S/P 0.032 0.053 0.045 # of Positive 0/5 (0.0%) 0/5 (0.0%) 0/5 (0.0%) Pigs (%) *Positive Sample = S/P > 0.400

(201) All pigs were PCR negative on Day 0 indicating a lack of exposure to PRRSV at the time of inoculation. On Day 7, all pigs in Group T1 (6/6, 100%) and T2 (6/6, 100%) were positive, while Group T3 (0/6, 0.0%) and C (0/5, 0.0%) had all negative pigs. The number of positive pigs per group for Day 14 was T1 6/6 (100%), T2 6/6 (100%), T3 2/6 (33.3%), and C 0/5 (0.0%). The lack of positive pigs throughout the study in the control Group C indicates that there was no environmental exposure to PRRSV. PCR totals can be seen in Table 12.

(202) TABLE-US-00012 TABLE 12 NAPRRSV Multiplex PCR Results Pig ID Group D0 D7 D14 9 T1 Neg Pos Pos 23 Neg Pos Pos 31 Neg Pos Pos 32 Neg Pos Pos 33 Neg Pos Pos 36 Neg Pos Pos # of Positive 0/6 (0.0%) 6/6 (100%) 6/6 (100%) Pigs (%) 60 T2 Neg Pos Pos 61 Neg Pos Pos 62 Neg Pos Pos 65 Neg Pos Pos 76 Neg Pos Pos 80 Neg Pos Pos # of Positive 0/6 (0.0%) 6/6 (100%) 6/6 (100%) Pigs (%) 70 T3 Neg Neg Neg 81 Neg Neg Neg 88 Neg Neg Pos 90 Neg Neg Neg 95 Neg Neg Neg 97 Neg Neg Pos # of Positive 0/6 (0.0%) 0/6 (0.0%) 2/6 (33.3%) Pigs (%) 57 C Neg Neg Neg 84 Neg Neg Neg 91 Neg Neg Neg 92 Neg Neg Neg 94 Neg Neg Neg # of Positive 0/5 (0.0%) 0/5 (0.0%) 0/5 (0.0%) Pigs (%) *Neg = negative, Pos = positive

(203) Due to the fact that Group T3 did not show any PCR positive pigs until Day 14, sequencing was performed to verify that pigs #88 and 97 were infected with Wetzel Clone 9. ORF 5 sequences were compared between the PRRSV isolated from the above listed pigs and the three PRRSV clones used in this study. Results from pigs 88 and 97 were homologous with Wetzel clone 9. Sequence results can be seen in FIG. 5.

(204) Virus isolation on serum samples was similar to PCR results. All pigs were negative by CPE and FA on Day 0. Positive pigs per group on Day 7 were 6/6 (100%), 6/6 (100%), 2/6 (33.3%), and 0/5 (0.0%) for Group T1, T2, T3, and C, respectively, for both CPE and FA. Positive pigs per group on Day 14 were 5/6 (83.3%), 2/6 (33.3%), 5/6 (83.3%), and 0/5 (0.0%) for Group T1, T2, T3, and C, respectively, for both CPE and FA. Again, control Group C remained negative for the entire study showing lack of exposure to PRRSV. See Table for Virus Isolation-Serum results. Virus isolation was also performed on bronchial-alveolar lavage samples taken at the time of necropsy. Results show 2/6 (33.3%), 3/6 (50.0%), 1/6 (16.7%), and 0/5 (0.0%) positive pigs for Group T1, T2, T3, and C, respectively. See Table 14 for Virus Isolation-Lavage results.

(205) TABLE-US-00013 TABLE 13 Virus Isolation-Serum Pig D0 D7 D14 ID Group CPE/FA CPE/FA CPE/FA 9 T1 Neg/Neg Pos/Pos Pos/Pos 23 Neg/Neg Pos/Pos Pos/Pos 31 Neg/Neg Pos/Pos Pos/Pos 32 Neg/Neg Pos/Pos Pos/Pos 33 Neg/Neg Pos/Pos Pos/Pos 36 Neg/Neg Pos/Pos Neg/Neg # of Positive 0/6 (0.0%) 6/6 (100%) 5/6 (83.3%) Pigs (%) 60 T2 Neg/Neg Pos/Pos Neg/Neg 61 Neg/Neg Pos/Pos Pos/Pos 62 Neg/Neg Pos/Pos Pos/Pos 65 Neg/Neg Pos/Pos Neg/Neg 76 Neg/Neg Pos/Pos Neg/Neg 80 Neg/Neg Pos/Pos Neg/Neg # of Positive 0/6 (0.0%) 6/6 (100%) 2/6 (33.3%) Pigs (%) 70 T3 Neg/Neg Neg/Neg Pos/Pos 81 Neg/Neg Neg/Neg Neg/Neg 88 Neg/Neg Pos/Pos Pos/Pos 90 Neg/Neg Neg/Neg Pos/Pos 95 Neg/Neg Neg/Neg Pos/Pos 97 Neg/Neg Pos/Pos Pos/Pos # of Positive 0/6 (0.0%) 2/6 (33.3%) 5/6 (83.3%) Pigs (%) 57 C Neg/Neg Neg/Neg Neg/Neg 84 Neg/Neg Neg/Neg Neg/Neg 91 Neg/Neg Neg/Neg Neg/Neg 92 Neg/Neg Neg/Neg Neg/Neg 94 Neg/Neg Neg/Neg Neg/Neg # of Positive 0/5 (0.0%) 0/5 (0.0%) 0/5 (0.0%) Pigs (%) *Neg = negative, Pos = positive; CPE = cytopathic effect, FA = fluorescent antibody

(206) TABLE-US-00014 TABLE 14 Virus Isolation-Lavages Pig Lavage ID Group CPE/FA 9 T1 Pos/Pos 23 Neg/Neg 31 Neg/Neg 32 Pos/Pos 33 Neg/Neg 36 Neg/Neg # of Positive 2/6 (33.3%) Pigs (%) 60 T2 Pos/Pos 61 Neg/Neg 62 Pos/Pos 65 Neg/Neg 76 Neg/Neg 80 Pos/Pos # of Positive 3/6 (50.0%) Pigs (%) 70 T3 Pos/Pos 81 Neg/Neg 88 Neg/Neg 90 Neg/Neg 95 Neg/Neg 97 Neg/Neg # of Positive 1/6 (16.7%) Pigs (%) 57 C Neg/Neg 84 Neg/Neg 91 Neg/Neg 92 Neg/Neg 94 Neg/Neg # of Positive 0/5 (0.0%) Pigs (%) *Neg = negative, Pos = positive; CPE = cytopathic effect, FA = fluorescent antibody

(207) Clinical Observations

(208) All pigs had a clinical score of 0, meaning there were no observed abnormal clinical respiratory signs, abnormal behavior, or cough. See Table 15 for Observation Scores.

(209) TABLE-US-00015 TABLE 15 Clinical Observation Scores Pig Total ID Group D-1 D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 D14 Score 9 T1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 23 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 31 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 32 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 33 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 36 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Average 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Score 60 T2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 61 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 62 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 65 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 76 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 80 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Average 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Score 70 T3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 81 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 88 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 90 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 95 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 97 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Average 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Score 57 C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 84 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 91 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 92 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 94 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Average 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Score

(210) All pigs had a % lung lobe pathology score of 0, meaning no lesions were seen on any of the lung lobes upon gross pathological examination. See Table 16 for Lung Lesion Scores and Table 17 for IVP Titration.

(211) TABLE-US-00016 TABLE 16 Lung Lesion Scores Total Pig ID Group RA RC RD LA LC LD Int Score 9 T1 0 0 0 0 0 0 0 0 23 0 0 0 0 0 0 0 0 31 0 0 0 0 0 0 0 0 32 0 0 0 0 0 0 0 0 33 0 0 0 0 0 0 0 0 36 0 0 0 0 0 0 0 0 Average Score 0 0 0 0 0 0 0 0 60 T2 0 0 0 0 0 0 0 0 61 0 0 0 0 0 0 0 0 62 0 0 0 0 0 0 0 0 65 0 0 0 0 0 0 0 0 76 0 0 0 0 0 0 0 0 80 0 0 0 0 0 0 0 0 Average Score 0 0 0 0 0 0 0 0 70 0 0 0 0 0 0 0 0 0 81 0 0 0 0 0 0 0 0 0 88 0 0 0 0 0 0 0 0 0 90 0 0 0 0 0 0 0 0 0 95 0 0 0 0 0 0 0 0 0 97 0 0 0 0 0 0 0 0 0 Average Score 0 0 0 0 0 0 0 0 57 0 0 0 0 0 0 0 0 0 84 0 0 0 0 0 0 0 0 0 91 0 0 0 0 0 0 0 0 0 92 0 0 0 0 0 0 0 0 0 94 0 0 0 0 0 0 0 0 0 Average Score 0 0 0 0 0 0 0 0 *RA = right apical lobe, RC = right cardiac lobe, RD = right diaphragmatic lobe, LA = left apical lobe, LC = left cardiac lobe, LD = left diaphragmatic lobe, Int = intermediate lobe

(212) TABLE-US-00017 TABLE 17 IVP Titration Results Pre-Inoculation Titers PRRSV Clone 1 2 3 4 5 Ave.sup.1 Isolate X-6 6.25 6.25 6.40 6.50 5.75 6.23 Wetzel Clone 3 p51 5.70 6.16 6.50 6.88 6.22 6.29 Wetzel Clone 9 p48 7.00 6.60 7.16 7.16 7.00 6.98 Post-Inoculation Titers 1 2 3 4 5 Ave.sup.2 Isolate X-6 6.50 6.60 6.75 6.69 6.76 6.66 Wetzel Clone 3 p51 7.36 6.84 6.45 6.36 6.60 6.72 Wetzel Clone 9 p48 6.70 6.75 6.60 7.50 7.00 6.91 Average Total (Ave.sup.1 + Ave.sup.2) Isolate X-6 6.45 Wetzel Clone 3 p51 6.51 Wetzel Clone 9 p48 6.95 *Titer expressed as Log.sub.10 TCID.sub.50/mL

CONCLUSION

(213) The objective of this study was to evaluate the safety of the PRRSV clones Isolate

(214) Y-6, Wetzel Clone 3 p51, and Wetzel Clone 9 p48 in 5-week old piglets to determine their viability as vaccine candidates. Although no lung lesions were visible 14 days post-inoculation, for two clones (Isolate Y-6 and Wetzel Clone 3 p51) PRRS viremia was present by Day 7. Two piglets inoculated with the third clone (Wetzel Clone 9 p48) tested PCR positive at Day 14. Additionally, no PRRSV antibodies were detected in any of the IVP piglets until Day 14, one week after the presence of viremia. Despite the fact that piglets developed viremia by Day 14, the data indicates that the PRRSV clones used in this study caused no lung lesions and/or clinical signs of PRRSV infection. These clones are deemed to be safe in 5-week old piglets. Future studies should include vaccination-challenge models to determine these clones' abilities to reduce lung lesions after virulent challenge with PRRSV.

Example 3

Efficacy of Attenuated PRRS Virus (Wetzel Attenuated) in a Respiratory Challenge Model

(215) The objective of this study was to evaluate the efficacy of INGELVAC PRRS MLV and an experimental vaccine against a recent NA-PRRSV challenge isolate in a respiratory challenge model. Evaluations included mortality, clinical scores (respiratory distress, behavior, cough, etc.), viremia, lung lesions, serology, virus isolation, average daily gain, and rectal temperature.

(216) The objective of this study was to evaluate the efficacy of INGELVAC PRRS MLV and an experimental vaccine against a recent NA-PRRSV challenge isolate in a respiratory challenge model. Evaluations included mortality, clinical scores (respiratory distress, behavior, cough, etc.), viremia, lung lesions, serology, virus isolation, average daily gain, and rectal temperature. Schedule of events is provided in Table 18.

(217) TABLE-US-00018 TABLE 18 SCHEDULE OF EVENTS Day Event Samples Testing 2 or Transportation to study site None None before Health examination None None 0 Sample collection (all) Blood (SST) ELISA, PCR, VI Weight (all) None None Vaccination Retention Titration (TCID.sub.50) (V1, V2, C1) 7, 14, 22 Sample collection (all) Blood (SST) ELISA, PCR, VI 22 Necropsy strict controls % lung pathology, VI, Histopathology (if lavage; lung; other necessary) tissues if necessary 22 Sample collection (all) Blood (SST) ELISA, PCR, VI Weight (all) & move V1 and C1 None None to the same room, separate pens Challenge Retention Titration (TCID.sub.50) (V1, V2, C1) 29 Sample collection (all) Blood (SST) ELISA, PCR, VI 21-36 Clinical observations, temp (all) None None 36 Sample collection (all) Blood (SST) ELISA, PCR, VI Weight (all) None None Necropsy (all) % lung pathology, VI, Histopathology (if lavage; lung; other necessary) tissues if necessary

(218) Sixty-five PRRS-susceptible study pigs were individually identified and examined by the Investigator and were deemed healthy for enrollment. Pigs were randomly divided into four groups as shown in Table 19.

(219) TABLE-US-00019 TABLE 19 Treatment Groups Room Day 22- # of Room Vx Challenge Challenge Group Pigs phase Day 0-Vaccine Phase Isolate 1 20 CC2 Placebo (Diluent) CC2 1-4-4 2 20 CA5 INGELVAC CC2 1-4-4 PRRS MLV 3 20 CC1 W96117 #9 p42 CC1 1-4-4 SC 5 CC2 NA NA NA

(220) On Day 00n Day 0 (D0) of the study, Groups 2 and 3 were vaccinated with INGELVAC PRRS MLV per label instructions or 2.0 mL of the experimental vaccine, 1M, respectively. Groups 1 received an equivalent dose of a placebo in the same manner. Group SC served as strict controls and received no treatment. Pigs were vaccinated at >3 weeks of age. On D22, Groups 1, 2, and 3 were challenged with the 1-4-4 isolate. Only two rooms were available during the challenge period. Group 1 and 2 were housed together and Group 3 remained in its original room. Challenge route and dose was 1.0 ml intranasal (0.5 mL/nostril). The challenge virus was titrated for potency on the day of challenge, results indicated a delivered dose of 5.13 log/dose. Fourteen days following challenge (D36), all pigs were euthanized and necropsied. The lungs and trachea were removed and a general description of lung pathology was observed. The percentage of pathology in each lung lobe was recorded. After scoring, lung lavages were collected. Individual lung lavage fluid was tested for presence of PRRSV by PCR.

(221) Rectal temperatures were taken and animal observations were performed from one day prior to challenge (D21) through the day of necropsy (D36); observations were for mortality, respiratory distress, behavior, and cough. Serum was collected prior to vaccination on D0, and at D7, 14, 21, D28, and D35. Serum samples were individually tested for PRRSV antibodies by IDEXX ELISA and viremia by PCR, qPCR and VI. In addition, fixed lung tissue was collected and retained for histopathological testing, if necessary. Animals were weighed at the beginning of the study (Day 0), at challenge (Day 22) and just prior to necropsy (Day 36).Sixty-five, mixed breed, mixed sexed, individually identified pigs were used in the study that were PRRSV antibody and viremia negative prior to allotment in the study. Animals were housed in a BSL2 facility in accordance with the appropriate SOP(s). To eliminate the possibility of shedding across groups, vaccinates were housed separately from controls for the duration of the study, see Table 1. Also, Group SC remained with non-vaccinated Group 1 until the day of challenge and was necropsied on Day 22.

(222) Animal space was appropriate for the size and number of pigs in accordance with acceptable animal husbandry practices for these regions. Feed rations were appropriate for the age, condition, and species of test animal according to facility standard operating procedure. Water was provided ad libitum throughout the study. No extra-protocol biologicals or pharmaceuticals were administered to the pigs during the study.

(223) Vaccine, Placebo, and Challenge Virus

(224) See Table 20, 21, 22, and 23 for vaccine, placebo, and challenge virus that were used in this study.

(225) TABLE-US-00020 TABLE 20 Vaccine, INGELVAC PRRS MLV Vaccine: INGELVAC PRRS MLV, reconstituted with Diluent. Formulation: Released serial formulated according to Outline of Production. Manufacturer: Boehringer Ingelheim Vetmedica, Inc. Lot/Serial Lot 245-C29; Diluent Item #180460 Number: Storage: Lyophilized: 2-8 C. Reconstituted: On ice and protected from light Testing: Potency titration results: 6.0 log/dose (5.71 Log.sub.10 TCID.sub.50/mL) Dose/Route: 2.0 mL/intramuscular (IM) injection; Groups 2

(226) TABLE-US-00021 TABLE 21 Experimental Vaccine Vaccine: W96117#9 p42 Formulation: Frozen cell culture preparation. The virus was propagated twice in AKMA-104 cells p78 from Wetzel#9 p41 Lot#262-132. T-75 flasks of cells were used and inoculated with 200 ul of virus. The flasks were incubated at 37 C. with CO2 for seven days, then frozen. The two sets were thawed, combined and harvested. (N288-029 through N288-039) Manufacturer: Boehringer Ingelheim Vetmedica, Inc. Lot/Serial Lot #N288-038 Number: Storage: Frozen, thaw in lukewarm water bath; hold on ice during use Testing: Potency titration results: 6.6 log/dose (6.29 Log.sub.10 TCID.sub.50/mL) Dose/Route: 2.0 mL/intramuscular (IM) injection; Group 3

(227) TABLE-US-00022 TABLE 22 Placebo, Diluent Only Placebo: Diluent, Sterile water for injection Formulation: Formulated according to Outline of Production. Manufacturer: Boehringer Ingelheim Vetmedica, Inc. Lot/Serial Item #180460 Number: Storage: Room temperature. Testing: After vaccination, any remaining diluent was frozen at <40 C. and checked for sterility. Dose/Route: 2.0 mL/intramuscular (IM) injection; Groups 1

(228) TABLE-US-00023 TABLE 23 Challenge Virus, North American PRRSV (NA-PRRSV) Challenge Isolates Virus: NA-PRRSV, 109866 (144 or 1-4-4) Formulation: Isolate 109866 (144) was used (200 ul) to inoculate a T-75 flask of AKMA-104 cells, incubated at 37 C. with CO2 for five days, and was then frozen. The material was thawed and aliquoted to individual vials. The necessary number of vials of tissue culture harvest was removed from the freezer, thawed, combined, transferred to vaccine bottles and put on ice. Manufacturer: Boehringer Ingelheim Vetmedica, Inc. Lot/Serial Number: Lot N191-154, preparation N288-136 Storage: Long term: frozen 70 10 C. Frozen, thaw in lukewarm water bath; put on ice during use. Testing: Potency testing: 5.13 log/dose Dose/Route: 1.0 mL/intranasal (IN) at 0.5 mL/nostril; Groups 1, 2, 3.
Assessment of Effectiveness

(229) Vaccine efficacy was assessed based on reduction of PRRSV viremia and percentage of total PRRSV-associated lung lesions. A single occurrence of viremia at any time point following vaccination classified a pig as positive for viremia. Percentage pathology for each animal's lungs was calculated according to the procedure described in the protocol. Pigs were also assessed for the isolation of PRRSV in serum and lung lavage fluid, clinical observations/scores, serology, mortality, average daily gain (ADG), and rectal temperature.

(230) Results

(231) A general description of lung pathology and the percentage of pathology in each lung lobe were recorded on the Necropsy Report Record. Total percent lung pathology was determined for each pig by summation of percent lung pathology for each lung lobe. Individual lung lesion scores are included in the appendix. The summary statistics are listed in the Table 24. The lung scores were analyzed using SAS in the mixed procedure using Type 3 Tests and the difference of least squared means; treatment and room were confounded due to housing constraints. There were statistically significant lower lung scores in the both vaccinated groups when compared to the challenge control group. No histopathology was submitted.

(232) TABLE-US-00024 TABLE 24 Lung Score summary statistics with a scatter plot of individual values. p-val vs Back- diluent % Lung transformed group Scores Average Min Max StdDev LSM for LSM Diluent 52.9 11.3 100.0 25.1 54.7 INGELVAC 37.4 0.6 90.5 28.1 33.9 0.0452 PRRS MLV W96117#9 35.5 0.1 72.0 22.1 32.7 0.0181 p42

(233) After the lungs were evaluated for pathology, a lung lavage sample was collected from each set of lungs by the Investigator or designee. Lung lavage samples were tested for PRRSV by PCR, qPCR and VI. Based on the qPCR all of the BAL samples were positive for the challenge virus. There were no statistical differences of quantity of virus detected in the BALs.

(234) Blood was collected on D0, D7, 14, 22, D28, and D36. Serum was harvested from each tube and transferred to cryovials for retention and to HMC for testing. Serum samples at BIVI-Ames were held at 2-8 C. until testing and 7010 C. for retention.

(235) Serum samples were for PRRSV antibodies using a commercial available test (IDEXX PRRS ELISA). ELISA results were recorded as Sample-to-Positive (S/P) ratios. An S/P ratio of 0.4 is considered positive. Results are shown in FIG. 6.

(236) Viremia for PRRSV as tested by PCR and qPCR. Results were reported as positive or negative which used generic primers for any PRRS virus. A single occurrence of viremia at any time point following vaccination classified a pig as positive for viremia. Relative copy numbers of PRRS virus was determined by qPCR testing that used primers specifically designed to detect the challenge 144 PRRS virus without detecting either vaccine virus. The quantitative data were analyzed using SAS in the mixed procedure using Type 3 Tests and the difference of least squared means; treatment and room were confounded due to housing constraints. On day 29 the W96117#9 p42 vaccinate groups had significantly less virus in the serum than the challenge control group (p-value=<0.0001). On day 36, the group vaccinated with INGELVAC PRRSMLV has significantly less viremia than the challenge control group (p-value=0.0089). The results are shown in FIG. 7.

(237) Clinical Observations

(238) Pigs were observed for overall health and clinical signs associated with PRRS disease by the Investigator or designee daily from one day prior to challenge (D27) to the day of necropsy (D36). Specifically, pigs were examined each day for respiration, behavior, and cough with each category rated 0 for normal and 1 for abnormal. The results are shown in FIG. 8.

(239) Rectal temperatures were taken daily, from D22 through D36. The data were analyzed using JMP8.0 student's t test. When comparing the temperature response of the group vaccinated with the W96117#9 p42 vaccine, the average temperatures were significantly higher on days 22-25 when compared to the challenge controls and significantly lower on days 26, 33-35. When comparing the group vaccinated with, there were significantly higher average temperatures than the challenge control pigs on days 24 and 29; the average temperatures were significantly lower on days 22, 33-35. In this study one pig in the challenge control group was euthanized on day 35 due to morbidity. The results are shown in FIG. 9.

(240) Animals were weighed on Day 0, 22 and 36. The total average weight gain during the vaccination period and during the challenge period was calculated. The weight gain data were analyzed using SAS in the mixed procedure using Type 3 Tests and the difference of least squared means; treatment and room were confounded due to housing constraints. The group vaccinated with INGELVAC PRRS MLV had statistically lower gain during the vaccination period (p-value=<0.0001) and statistically higher gain during the challenge period (p-value=0.0039) when compared to the challenge control group. The W96117#9 p42 group has significantly higher gain than the challenge control group during the challenge period (p-value=<0.0001). The results are shown in FIG. 10.

(241) Discussion:

(242) The objective of this study was to evaluate the efficacy of INGELVAC PRRS MLV and an experimental vaccine against a recent NA-PRRSV challenge isolate in a respiratory challenge model. The challenge isolate was highly virulent and created very high (up to 100% lung lesion score indicative of pneumonia) lung lesions and noticeable clinical disease, including weight loss and mortality.

(243) Two vaccines were evaluated for efficacy in the presence of the virulent challenge. Typically, it is recommended to vaccinate pigs four weeks prior to the anticipated period of challenge. However, in this study, the pigs were challenged at 22 days post-vaccination. Both vaccines resulted in a statistically significant reduction of lung lesions with improved weight gain during the post-challenge period and reduction in viremia post-challenge.

(244) Through the course of this study a specific semi-quantitative PCR was developed for the detection of the 144 challenge virus, specifically. With this tool, the serum viremia data could be specific for the challenge virus and not detect the vaccine virus. It was demonstrated that there was no detectable vaccine virus in the unvaccinated group during the challenge period after being moved into the room with a vaccinated animal.

Example 4

Nucleotide and Protein Sequences Encoded Thereby of the Wetzel Pre-Attenuated (p3 Wetzel (Nucleotide Sequence of SEQ ID NO:1)) and Wetzel Attenuated ((Nucleotide Sequence of SEQ ID NO:2))

(245) Nucleotide Sequence

(246) The goal of this study was to provide PRRSV full genome sequence for PRRSV isolates pre-attenuated Wetzel strain (p3) and post-attenuated vaccine Wetzel strain (p41). Genomic sequencing of pre- and post-attenuated vaccine strains can provide critical information to help understand the biological determinates of viral fitness and thresholds required for viral reversion. Note that although the PRRSV virus is a single-stranded RNA virus, DNA equivalents (wherein T is substituted for the U in the actual RNA viral sequence) are disclosed herein for consistency with NCBI GenBank usage.

(247) Genomic Sequencing

(248) A viral metagenomic approach was employed in which tissue culture materials for each of the PRRS viruses were subjected to viral particle protected nucleic acid extraction and 454-sequencing of randomly primed amplicons. In total, 6 strains were attempted: 1-4-4 (data not shown), passage 3 Wetzel (p3), p10 Wetzel, p20 Wetzel, p30 Wetzel, p40 Wetzel, and vaccine strain p41 Wetzel. Of the 7 attempted, sufficient sequencing was achieved for complete/near-complete genome analysis of 6 strains, in which p20 Wetzel failed. Table 25 indicates the number of sequences and average coverage depth for each of the isolates. A high percentage of PRRSV sequences were obtained and no other viruses were detected in any of the samples. Genomic sequences are listed below for p3 Wetzel and p41 Wetzel as sequence IDs land 2, respectively.

(249) TABLE-US-00025 TABLE 25 Summary of Sequencing % of Genome Average Sequence Avg complete/ Seq. Total # of align to coverage near- Strain Length Sequences PRRSV depth complete 1-4-4 225 27299 90% 375X Near- challenge complete p3 Wetzel 216 40182 90% 520X Complete p10 Wetzel 205 10214 87% 123X Near- complete p20 Wetzel 202 32 100% N/A N/A p30 Wetzel 216 11655 88% 149X Near- complete p40 Wetzel 219 7472 93% 113X Partial p41 Wetzel 207 48806 94% 636X Complete
Components of Attenuation

(250) Detailed analysis of those positions which were modified or polymorphic at each passage tested during the attenuation was conducted; based on the sequencing data, no large scale deletions were identified in the vaccine strain. Summarized in Table 26, 33 point mutations were identified between p3 and p41, the majority (19/33) were either silent or polymorphic.

(251) TABLE-US-00026 TABLE 26 Summary of p3 vs p41 changes Nuc. Polymorphic/ Amino acid Ref. Position Gene Change Substitution change 931 ORF1a C > T Sub. Silent 936 ORF1a G > GT Poly. R252R/L 938 ORF1a G > CG Poly. V253L/V 1147 ORF1a T > C Sub. Silent 2047 ORF1a T > C Sub. Silent 2137 ORF1a C > T Sub. Silent 2916 ORF1a A > G Sub. Y912C 3011 ORF1a C > T Sub. Silent 3151 ORF1a T > CT Poly. Silent 3450 ORF1a C > T Sub. A1090V 3896 ORF1a T > C Sub. Silent 5626 ORF1a C > T Sub. Silent 5713 ORF1a C > T Sub. Silent 5797 ORF1a C > T Sub. Silent 6449 ORF1a A > T Sub. T2090S 6769 ORF1a T > A Sub. Silent 6947 ORF1a A > C Sub. K2256Q 7345 ORF1b A > G Sub. T16A 7602 ORF1b C > T Sub. Silent 8364 ORF1b T > A Sub. Silent 8379 ORF1b C > T Sub. Silent 9957 ORF1b G > A Sub. Silent 10361 ORF1b A > G Sub. D1021G 10894 ORF1b C > T Sub. R1199W 11976 ORF2a A > G Sub. K102R 12693 ORF3 C > T Sub. Silent 12721 ORF3 T > C Sub. F143L 12722 ORF3 T > GT Poly. F143F/C 12968 ORF3 C > T Sub. A225V 13398 ORF5 A > T Sub. K4N 13876 ORF5 G > A Sub. G164R 13964 ORF5 C > T Sub. S193L 14098 ORF6 T > C Sub. L42S
Protein Sequences:

(252) From Wang et al. (22) (internal references deleted; expressly incorporated by reference herein): PRRSV is a small, enveloped, single positive-stranded RNA virus including a genome of about 15 kb, encoding nine ORFs. The PRRSV genome is comprised of two polymerase genes, ORF1a and 1b, and seven structural genes, ORF2a, 2b, 3, 4, 5, 6, and 7. ORF1a and ORF1b constitutes approximately 75% of the viral genome, and are characterized by a process of ribosomal frame shifting translated into a large polyprotein; which by self-cleavage gives rise to the non-structural proteins (NSPs) including the RNA-dependent RNA polymerase. Open reading frames 2a, 3, 4 and 5 all encode glycosylated proteins, designated GP2a, GP3, GP4, and GPS, respectively. The newly defined ORF2b encodes the smallest protein of the virus particle designated GP2b. ORF7 encodes the non-glycosylated nucleocapsid protein (N), constituting 20-40% of the protein content of the virion. ORF6 encodes the likewise non-glycosylated matrix protein (M).

(253) Polypeptides encoded by the nucleotide sequences of SEQ ID NOs: 1 and 2 were identified, and are set forth as follows:

(254) TABLE-US-00027 p3WetzelproteinsequencesencodedbySEQIDNO:1 Protein/ SeqID NO SEQUENCE ORF1a/ MSGILDRCTCTPNARVFVADGQVYCTRCLSARTLLPLNLQVSELGVLGLFYRPEEPL SEQID RWTLPRAFPTVECSPAGACWLSAIFPIARMTSGNLNFQQRIVRVAAEIYRVGQLTPTV NO:3 LKNLQVYERGCRWYPIVGPVPGVAVYANSLHVSDKPFPGATHVLTNLPLPQRPKPE DFCPFECAMAVVYDIGHDAVMYVAKERVSWAPRGGEKGKFETVPEELRLVAEQLF TSFPPHHVVDMSKFIFTAPDCGASMRVERQYGCLPAGTVPDGNCWWSLFSSLPPEV QCREIRRATQFGYQTKHGVAGKYLQRRLQANGLRAVVDSNGPIVIQYFSVKESWIR HVKLAGEPCYPGFEDLLRIRVEPNTLPLSDKGDKVFRFGGHKWYGAGKRARRSRAG AATTVAGHASPVRETQQAKKHEAASANKAELLERYSPPAEGNCGWHCISAIVNRM VNSKFETALPEKVRPPEDWATDEDLVNTIQILRLPAALDRNGVCASAKYVLKLEGEH WTVSVTPGMPPSLLPLECVQGCCEHKGSLRSPDAVEVSGFDPASLDRLAGVMHLPSS AIPAALAELSGDPDRPVSPATTAWTVSQFYARHSGGEHPDQKYLRKIISLCEVIESCC CSQNKINLVTPEEVKTKIDQYLSGAASLEECLARLEKARPPSVLDTSFDWDVVLPGV GAAVRAAKLPLANQCHAPVTVVTQRPSLKFQPRKAESVKSLPESRPLPAPRRKIRSR CGSLTSLDGNFPDSWEDLAGGPFHFPTLPEPTTRPGEPVPVPAPRKTVPRLVSSLIVSV PVPAPRRGIRQAEGMNLVAVTPACQDELLDLSESSQAEHGAPSLALPRSEDALAVGR REAEEVLSEISGMPDDIRLVPVSSSSSLSSVEITRPKYSAQAIIDSGGPCCGHLQEVKEK YLNVMREACDATKLDDPATREWLSRMWDRVDMLTWRNTSIFQAPFTLADKFKLLP KMILETPPPYPCGFVMMPRTPAPSVGAESDLTVGSVATEDVPRILGKVQGVCKTTVH EPLAPFADGPTDGQPAREPRTQTPPAGTGGVGLVLDSEGSPELTDSPPPNGTDASGG GPLYTVKKKAERCFDQLSRRVFDIVSHLPVFFSRLFKSDGHYAPGDWGFAAFTLLCL FLCYSYPAFGVVPLLGVFSGSSRRVRMGVFGCWLAFAVSLFKPAPDPVGTACEFDSP ECRDILHSFELLQPWDPVRSLVVGPVGLGLAILGRLLGGARYVWLFLLRLGIVSDCIL AGAYVLSQGRCKKCWGSCIRTAPSEVAFNVFPFTRATRSSLIDLCDRFCAPKGMDPIF LATGWRGCWAGQSPIEQPTEKPIAFAQLDEKKITAKTVVAQPYDPNQAVKCLRVLQ AGGAMVAEAIPKVVKVSAIPFRAPFFPVGVKVDPECRVVVDSDTFTTALRSGYSTTN LILGVGDFAQLNGLKIRQISKPSGGGPHIMAALHVACSMALHMLVGIYVTTVGSCGS GTNDPWCTNPFAVPVYGPGSLCTSRLCISQHGLTLPLTALVAGFGIQEVALVVLIFVS IGGMAHRLSCKADVLCILLAIASYVWLPLTWLLCVFPCWLRWFSLHPLTVTWLVFF LISVNMPSGVLALVLLISLWLLGRYTNVAGLVTPYDIHHYTNGPRGVAALATAPDGT YLAAVRRAALTGRTMLFTPSQLGSLLEGAFRTQKPTLNTVNVVGSSMGSGGVFTID GKIKCVTAAHILTGNSARVSGVGFNQMLDFDVKGDFAIADCPDWQGVAPKSQFCED GWTGRAYWLTSSGVEPGVIGRGFAFCFTACGDSGSPVITEAGELVGVHTGSNKQGG GIVTRPSGQFCNVTPTKLSELSEFFAGPRVPLGDVKVGNHIIKDTNEVPSDLCALLAA KPELEGGLSTVQLLCVFFLLWRMMGHAWTPLVAVGFFVLNEILPAVLVRSVFSFGM FALSWFTPWSAQILMIRLLTAALNRNRWSLAFYSLGALTGFAADLAINQGHSLHVA MNFSTYAFLPRAMAVTSPVPTIACGVVHLLAIVLYLFKYRSLHTVLVGDGAFSAAFF LRYFAEGKLREGVSQSCGMNHESLTGAIAIRLDDEDLDFLIKLTDFKCFVSASNMRN AAGQFIEAAYAKALRVELAQLVQVDKVRGVLAKLEAFADTATPQLSPGDIVVALGH TPVGSIFDLKVGSTKHTLQAIETRVLAGSRMTVARVVDPTPAPPPVPVPIPLPPKVLE NGPRAWEDEDRLNKKRRRKMEAVGIYVMDGKKYQKFWDKNSGDVFYEEVHDNT DAWECLRTDDPADLDPEKGTLCGHLTIENRPYHVYASPSGRKFLVPANPESGKAQW EAARLSIEQALGMMNVDGELTAKEVEKLKRIIDKLQGLTKEQCLNY ORF1b/ GAVFKLLAASGLTRCGRGGLVITETAVKIVRFHSRTFTLGPVNLKVASEVELKDAVE SEQID HNQHPIARPVDGGVVLLRSAVPSLIDVLISGADASPQLLAHHGPGNTGIDGTLWDFE NO:4 SVATKEEVTLSAQIIQACGIRRGDAPEIGLPYKLHPVRGNPERVKGVLKNTRFGDIPY KTPSDTGSPVHAAACLTPNATPVTDGRSVLATTMPSGFELYVPTIPASVLDYLDSRPD CPKQLTEHGCEDAALRDLSKYDLSTQGFVLPGVLRLVRKYLFAHVGKCPPVHRPST YPAKNSMAGINGNRFPTKDIQSIPEIDVLCAQAVRENWQTVTPCTLKKQYCGKKKT RTILGTNNFIALAHRAALSGVTQGFMKKAFNSPIALGKNKFKELQTPVLGRCLEADL ASCDRSTPAIVRWFAAHLLYELACAEEHLPSYVLNCCHDLLVTQSGAVTKRGGLSS GDPITSVSNTIYSLVIYAQHMVLSYFKSGHPHGLLFLQDQLKFEDMLKVQPLIVYSDD LVLYAESPTMPNYHWWVEHLNLMLGFQTDPKKTTITDSPSFLGCRIMNGCQLVPNR DRILAALAYHMKANNVSEYYASAAAILMDSCACLEYDPEWFEELVVGMAQCARKD GYSFPGPPFFLSMWEKLRSNYEGKRSRVCGYCGASAPYATSCGLDVCVYHTHFHQH CPVIIWCGHPAGSGSCDDCKSPTGKDTNPLDEVLKQVPYKPPRTVLMHVEQGLTPLD PGRYQTRRGLVAVRRGIRGNEVDLPDGDYASTALLPTCKEINMVAVASNVLRSRFII GPPGAGKTHWLLQQVQDGDVIYTPTHQTMLDMIKALGTCRFNVPAGTTLQFPAPSR TGPWVRILAGGWCPGKNSFLDEAAYCNHLDVLRLLSKTTLTCLGDFKQLHPVGFDS HCYVFDIMPQTQLKTIWRFGQNICDAIQPDYRDKLMSMVNTTRVTYVEKPVKYGQV LTPYHRDREDGAITIDSSQGATFDVVTLHLPTKDSLNKQRALVAITRARHAIFVYDPY RQLQSLFDLPAKSTPVNLAVHRDGQLIVLDRNNKECTVAQALGNGDKFRATDKRV VDSLRAICADLEGSSSPLPKVAHNLGFYFSPDLTQFAKLPIELAPHWPVVTTQNNEN WPDRLVASLRPIHKYSRACIGAGYMVGPSVFLGTPGVVSYYLTKFVKGEAQVLPET VFSTGRIEVDCREYLDDREREVAASLPHAFIGDVKGTTVGGCHHVTSKYLPRFLPKE SVAVVGVSSPGKAAKAVCTLTDVYLPDLEAYLHPVTQSKCWKMMLDFKEVRLMV WKDKTAYFQLEGRHFTWYQLASFASYIRVPVNSTVYLDPCMGPALCNRKVVGSPH WGADLAVTPYDYGARKILSSAYHGEMPPGYKILACAEFSLDDPVRYKHTWGFESDT AYLYEFTGNGEDWEDYNDAFRARQKGKIYKATATSLKFHFPPGHIVEPTLGLN GP2/ MRWEPHRAFLTKLVNFLLMPSRSSWCLLLISSYFWPFCSASPSPVGWWSFASDWFSP SEQID RYSVRALPFTLSNYRRSYEAYLSQCQVDIPAWGTKHPLGMIWHHKVSTLIDEMVSR NO:5 RMYRTMEQAGQAAWKQVVTEATLSRISSLDVVAHFQHLAAIEAETCKYLASRLPM LHNLRLTGSNVTIVYNSSLDRVFAVFPTSSSRPKLHDFRQWLIAVHSSIFSSVAASCTL FVVLWLRLPIIRTVFGFRWLGAIFLSSSQ GP3/ MANSCAFLHILLCCGFLYPFRRTVVAASNNTYCFWFPLVRGNFSFELTVNYTVCPPC SEQID LTRQAASEIYEPSRSLWCRIGQDRCTESDHDELGFLVPPGLSNEGHLISVYAWLAFLS NO:6 FSYTSQFHPEIFGIGNVSEVYVDIKHQFICAVHDGQNTTLPRHDNITAVYQTYYQHQV DGGNWFHLEWLRPFFSSWLVLNVSWFLRRSPASRVSVRVFQTSKPTPPQLQALLSSK TSAVLGMATRPLRRLAKAANAVRR GP4/ MAASLLFFLVGFECFMVSQAFACKPCFSSSLSDIKTNTTAAAGFAVLQDISCLRHGDS SEQID SSEATRKSRQCRTAIGTPVYITVTANVTDENYLHSSDLLMLSSCLFYASEMSEKGFK NO:7 VIFGNVSGIVAVCVNFTSYVQHVKEFTRRSLVVDHVRLLHFMTPETMRWATVLACL FAILLAI GP5/ MLGKCLTAGCCSQLLFLWCIVPFCFVALVNANNSSSSHLQLIYNLTICELNGTDWLN SEQID RKFDWAVETFVIFPVLTHIVSYGALTTSHFLDTVGLVTVSTAGYYHRRYVLSSIYAV NO:8 CALAALICFAIRLTKNCMSWRYSCTRYTNFLLDTKGKLYRWRSPVIIEKGGKIEVNG HLIDLKRVVLDGSAATPVTKVSAEQWGRP M/ MGTSLDDFCNDSTAPQKVLLAFSITYTPIMIYALKVSRGRLLGLLHLLIFLNCAFTFG SEQID YMTFAHFRSTNKVALTMGAVVALLWGVYSAIETWRFITSRCRLCLLGRRYILAPAH NO:9 HVESVAGFHPITASDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGRRAVKRGVVNLV KYAK N/SEQ MPNNNGKQQKRKKGDGQPVNQLCQMLGRIIAQQNQPRGKGPGKRNKKKSPEKPHF IDNO:10 PLATEDDVRHHFTPSERQLCLSSIQTAFNQGAGTCTLSDSGRVSYAVEFSLPTHHTVR LIRATTSPSA

(255) p41 attenuated Wetzel protein sequences encoded by SEQ ID NO:2 which differ from the p3 sequences

(256) TABLE-US-00028 Protein/ SeqID SEQUENCE NO (changesfromp3boldedandunderlined) ORF1a/ MSGILDRCTCTPNARVFVADGQVYCTRCLSARTLLPLNLQVSELGVLGLFYR SEQID PEEPLRWTLPRAFPTVECSPAGACWLSAIFPIARMTSGNLNFQQRIVRVAAEI NO:11 YRVGQLTPTVLKNLQVYERGCRWYPIVGPVPGVAVYANSLHVSDKPFPGAT HVLTNLPLPQRPKPEDFCPFECAMAVVYDIGHDAVMYVAKERVSWAPRGGE KGKFETVPEELRLVAEQLFTSFPPHHVVDMSKFIFTAPDCGASMRVERQYGC LPAGTVPDGNCWWSLFSSLPPEVQCREIRRATQFGYQTKHGVAGKYLQRRL QANGLRAVVDSNGPIVIQYFSVKESWIRHVKLAGEPCYPGFEDLLRIRVEPNT LPLSDKGDKVFRFGGHKWYGAGKRARRSRAGAATTVAGHASPVRETQQAK KHEAASANKAELLERYSPPAEGNCGWHCISAIVNRMVNSKFETALPEKVRPP EDWATDEDLVNTIQILRLPAALDRNGVCASAKYVLKLEGEHWTVSVTPGMP PSLLPLECVQGCCEHKGSLRSPDAVEVSGFDPASLDRLAGVMHLPSSAIPAAL AELSGDPDRPVSPATTAWTVSQFYARHSGGEHPDQKYLRKIISLCEVIESCCC SQNKINLVTPEEVKTKIDQYLSGAASLEECLARLEKARPPSVLDTSFDWDVV LPGVGAAVRAAKLPLANQCHAPVTVVTQRPSLKFQPRKAESVKSLPESRPLP APRRKIRSRCGSLTSLDGNFPDSWEDLAGGPFHFPTLPEPTTRPGEPVPVPAPR KTVPRLVSSLIVSVPVPAPRRGIRQAEGMNLVAVTPACQDELLDLSESSQAEH GAPSLALPRSEDALAVGRREAEEVLSEISGMPDDIRLVPVSSSSSLSSVEITRPK YSAQAIIDSGGPCCGHLQEVKEKCLNVMREACDATKLDDPATREWLSRMW DRVDMLTWRNTSIFQAPFTLADKFKLLPKMILETPPPYPCGFVMMPRTPAPS VGAESDLTVGSVATEDVPRILGKVQGVCKTTVHEPLAPFADGPTDGQPAREP RTQTPPAGTGGVGLVLDSEGSPELTDSPPPNGTDASGGGPLYTVKKKVERCF DQLSRRVFDIVSHLPVFFSRLFKSDGHYAPGDWGFAAFTLLCLFLCYSYPAFG VVPLLGVFSGSSRRVRMGVFGCWLAFAVSLFKPAPDPVGTACEFDSPECRDI LHSFELLQPWDPVRSLVVGPVGLGLAILGRLLGGARYVWLFLLRLGIVSDCIL AGAYVLSQGRCKKCWGSCIRTAPSEVAFNVFPFTRATRSSLIDLCDRFCAPK GMDPIFLATGWRGCWAGQSPIEQPTEKPIAFAQLDEKKITAKTVVAQPYDPN QAVKCLRVLQAGGAMVAEAIPKVVKVSAIPFRAPFFPVGVKVDPECRVVVD SDTFTTALRSGYSTTNLILGVGDFAQLNGLKIRQISKPSGGGPHIMAALHVAC SMALHMLVGIYVTTVGSCGSGTNDPWCTNPFAVPVYGPGSLCTSRLCISQHG LTLPLTALVAGFGIQEVALVVLIFVSIGGMAHRLSCKADVLCILLAIASYVWL PLTWLLCVFPCWLRWFSLHPLTVTWLVFFLISVNMPSGVLALVLLISLWLLG RYTNVAGLVTPYDIHHYTNGPRGVAALATAPDGTYLAAVRRAALTGRTMLF TPSQLGSLLEGAFRTQKPTLNTVNVVGSSMGSGGVFTIDGKIKCVTAAHILTG NSARVSGVGFNQMLDFDVKGDFAIADCPDWQGVAPKSQFCEDGWTGRAY WLTSSGVEPGVIGRGFAFCFTACGDSGSPVITEAGELVGVHTGSNKQGGGIV TRPSGQFCNVTPTKLSELSEFFAGPRVPLGDVKVGNHIIKDTNEVPSDLCALL AAKPELEGGLSTVQLLCVFFLLWRMMGHAWTPLVAVGFFVLNEILPAVLVR SVFSFGMFALSWFTPWSAQILMIRLLTAALNRNRWSLAFYSLGALTGFAADL AINQGHSLHVAMNFSTYAFLPRAMAVTSPVPTIACGVVHLLAIVLYLFKYRS LHTVLVGDGAFSAAFFLRYFAEGKLREGVSQSCGMNHESLTGAIAIRLDDED LDFLIKLSDFKCFVSASNMRNAAGQFIEAAYAKALRVELAQLVQVDKVRGV LAKLEAFADTATPQLSPGDIVVALGHTPVGSIFDLKVGSTKHTLQAIETRVLA GSRMTVARVVDPTPAPPPVPVPIPLPPKVLENGPRAWEDEDRLNKKRRRKME AVGIYVMDGKKYQKFWDQNSGDVFYEEVHDNTDAWECLRTDDPADLDPE KGTLCGHLTIENRPYHVYASPSGRKFLVPANPESGKAQWEAARLSIEQALGM MNVDGELTAKEVEKLKRIIDKLQGLTKEQCLNY ORF1b/ GAVFKLLAASGLTRCGRGGLVITETAVKIVRFHSRTFTLGPVNLKVASEVEL SEQID KDAVEHNQHPIARPVDGGVVLLRSAVPSLIDVLISGADASPQLLAHHGPGNT NO:12 GIDGTLWDFESVATKEEVTLSAQIIQACGIRRGDAPEIGLPYKLHPVRGNPER VKGVLKNTRFGDIPYKTPSDTGSPVHAAACLTPNATPVTDGRSVLATTMPSG FELYVPTIPASVLDYLDSRPDCPKQLTEHGCEDAALRDLSKYDLSTQGFVLPG VLRLVRKYLFAHVGKCPPVHRPSTYPAKNSMAGINGNRFPTKDIQSIPEIDVL CAQAVRENWQTVTPCTLKKQYCGKKKTRTILGTNNFIALAHRAALSGVTQG FMKKAFNSPIALGKNKFKELQTPVLGRCLEADLASCDRSTPAIVRWFAAHLL YELACAEEHLPSYVLNCCHDLLVTQSGAVTKRGGLSSGDPITSVSNTIYSLVI YAQHMVLSYFKSGHPHGLLFLQDQLKFEDMLKVQPLIVYSDDLVLYAESPT MPNYHWWVEHLNLMLGFQTDPKKTTITDSPSFLGCRIMNGCQLVPNRDRIL AALAYHMKANNVSEYYASAAAILMDSCACLEYDPEWFEELVVGMAQCAR KDGYSFPGPPFFLSMWEKLRSNYEGKRSRVCGYCGASAPYATSCGLDVCVY HTHFHQHCPVIIWCGHPAGSGSCDDCKSPTGKDTNPLDEVLKQVPYKPPRTV LMHVEQGLTPLDPGRYQTRRGLVAVRRGIRGNEVDLPDGDYASTALLPTCK EINMVAVASNVLRSRFIIGPPGAGKTHWLLQQVQDGDVIYTPTHQTMLDMIK ALGTCRFNVPAGTTLQFPAPSRTGPWVRILAGGWCPGKNSFLDEAAYCNHL DVLRLLSKTTLTCLGDFKQLHPVGFDSHCYVFDIMPQTQLKTIWRFGQNICD AIQPDYRDKLMSMVNTTRVTYVEKPVKYGQVLTPYHRDREDGAITIDSSQG ATFDVVTLHLPTKDSLNKQRALVAITRARHAIFVYDPYRQLQSLFGLPAKSTP VNLAVHRDGQLIVLDRNNKECTVAQALGNGDKFRATDKRVVDSLRAICADL EGSSSPLPKVAHNLGFYFSPDLTQFAKLPIELAPHWPVVTTQNNENWPDRLV ASLRPIHKYSRACIGAGYMVGPSVFLGTPGVVSYYLTKFVKGEAQVLPETVF STGRIEVDCREYLDDWEREVAASLPHAFIGDVKGTTVGGCHHVTSKYLPRFL PKESVAVVGVSSPGKAAKAVCTLTDVYLPDLEAYLHPVTQSKCWKMMLDF KEVRLMVWKDKTAYFQLEGRHFTWYQLASFASYIRVPVNSTVYLDPCMGP ALCNRKVVGSPHWGADLAVTPYDYGARKILSSAYHGEMPPGYKILACAEFS LDDPVRYKHTWGFESDTAYLYEFTGNGEDWEDYNDAFRARQKGKIYKATA TSLKFHFPPGHIVEPTLGLN GP2/ MRWEPHRAFLTKLVNFLLMPSRSSWCLLLISSYFWPFCSASPSPVGWWSFAS SEQID DWFSPRYSVRALPFTLSNYRRSYEAYLSQCQVDIPAWGTKHPLGMIWHHRV NO:13 STLIDEMVSRRMYRTMEQAGQAAWKQVVTEATLSRISSLDVVAHFQHLAAI EAETCKYLASRLPMLHNLRLTGSNVTIVYNSSLDRVFAVFPTSSSRPKLHDR QWLIAVHSSIFSSVAASCTLFVVLWLRLPIIRTVFGFRWLGAIFLSSSQ GP3/ MANSCAFLHILLCCGFLYPFRRTVVAASNNTYCFWFPLVRGNFSFELTVNYT SEQID VCPPCLTRQAASEIYEPSRSLWCRIGQDRCTESDHDELGFLVPPGLSNEGHLIS NO:14 VYAWLAFLSFSYTSQFHPEIFGIGNVSEVYVDIKHQLICAVHDGQNTTLPRHD NITAVYQTYYQHQVDGGNWFHLEWLRPFFSSWLVLNVSWFLRRSPASRVSV RVFQTSKPTPPQLQVLLSSKTSAVLGMATRPLRRLAKAANAVRR GP4 Aminoacidsequenceisidenticaltop3GP4(SEQIDNO:7) GP5/ MLGNCLTAGCCSQLLFLWCIVPFCFVALVNANNSSSSHLQLIYNLTICELNGT SEQID DWLNRKFDWAVETFVIFPVLTHIVSYGALTTSHFLDTVGLVTVSTAGYYHRR NO:15 YVLSSIYAVCALAALICFAIRLTKNCMSWRYSCTRYTNFLLDTKGKLYRWRS PVIIEKRGKIEVNGHLIDLKRVVLDGSAATPVTKVLAEQWGRP M Aminoacidsequenceisidenticaltop3M(SEQIDNO:9) N Aminoacidsequenceisidenticaltop3N(SEQIDNO:10)

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(258) 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.