Porcine reproductive and respiratory syndrome vaccine virus

Abstract

The present invention relates to a modified live porcine reproductive and respiratory syndrome virus. The virus is genetically analyzed and selected based on phylogenetic grouping for modification by repeated passage in tissue culture. The ability of the modified live virus to provide protective immunity against heterologous viruses is evaluated. The modified live virus can be used in a vaccine, particularly a vaccine that can treat infections in pigs caused by a variety of heterologous viruses.

Description

Technical Field

[0001] The present invention relates to a modified live porcine reproductive and respiratory syndrome virus. The modified live virus can be used in vaccines, particularly vaccines that provide protection against heterologous viruses. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), initially described in Europe as an enigmatic swine disease, has now spread worldwide. PRRS causes late-term abortions, stillbirths, and infertility in breeding sows of reproductive age, as well as respiratory diseases, reduced growth performance, and even death in breeding pigs and growing / finishing pigs in breeding farms. PRRS causes significant economic losses.

[0003] Symptoms of PRRS virus infection in adult swine animals include, but are not limited to, reduced appetite, lethargy, and fever. Pregnant sows may give birth to piglets prematurely, abort the fetuses, or deliver mummified or stillborn piglets, and up to 10% of pregnant sows may die from PRRS virus infection. Infected piglets have a high pre-weaning mortality rate, are usually weak, and may have edema around the eyes. PRRS virus infection in weaned breeding pigs or growing / finishing pigs can cause (but is not limited to) growth retardation, respiratory distress, dyspnea or tachypnea, red skin spots, and a rough coat.

[0004] The PRRS virus is an enveloped virus with a linear positive-strand RNA genome of approximately 15 kb, and the virus has been classified into the family Arteriviridae. To date, at least eleven open reading frames have been identified in the genome. The PRRS virus is divided into two genotypes. The European genotype - type 1 PRRS virus (PRRSV-1) - is exemplified by the Lelystad strain, while the North American PRRS virus type 2 (PRRSV-2) is exemplified by the strain VR-2332.

[0005] These two genotypes may have as little as approximately 60% sequence identity in their genomes, and even within a genotype, individual strains may vary by up to approximately 20% in their genomic identity. This variability complicates the development of vaccines that effectively treat and / or prevent PRRS. Modified live virus (MLV) variants of the PRRS virus can confer immunity against PRRS virus challenge, but the vaccine is most effective when the challenge is with a PRRS virus that is genetically homologous to the MLV. MLV vaccines are less effective against heterologous virus challenges. In addition, MLV has shown some reversion to virulence, such that the vaccine virus causes disease in vaccinated animals. Vaccines containing inactivated (i.e., killed) PRRS virus have a better safety profile but limited efficacy against heterologous challenges.

[0006] Since current PRRS vaccines have not shown sufficient safety and efficacy to reduce the economic impact of PRRS virus infection, new and improved vaccines are needed. Preferably, these vaccines will be both safe and effective. If the vaccine contains attenuated MLV, these attenuated MLV should not show a reversion to virulence in order to be considered safe for use in the field. For example, by adapting the PRRS strain to grow in tissue culture cells for at least 60 passages, at least 70 passages, at least 80 passages, or preferably at least 85 passages, the MLV should not show a reversion to virulence. To be effective, the vaccine virus strain should be able to elicit protective immunity against a range of phylogenetically diverse wild-type PRRS strains in swine animals. Preferably, the new PRRS vaccine virus strain will be able to elicit protective immunity against at least three phylogenetically distinct wild-type PRRS strains in swine animals. Summary of the Invention

[0007] The present invention provides a modified live porcine reproductive and respiratory syndrome vaccine virus strain, wherein the consensus complementary DNA sequence of the PRRS strain is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Preferably, the modified live strain may have a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. More preferably, the modified live strain may also have a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. As will be understood by those of ordinary skill in the art, due to the high mutation rate of the PRRS virus, the modified live PRRS strain may contain multiple subpopulations, each with a homologous but not identical genome.

[0008] The present invention provides modified live porcine reproductive and respiratory syndrome (PRRS) virus strains, wherein the PRRS virus strains are DE 14-3073, ES 13-49, IT 14-32 or PL 14-02 strains. The PRRS virus strains should preferably be passaged in tissue culture cells at least 60 times, or more preferably 70 times, or even more preferably 80 times. Most preferably, the PRRS virus strains should be passaged 85 times in tissue culture cells. This passage in tissue culture cells can be used to attenuate the modified live PRRS virus strains. When the attenuated PRRS virus strains are administered to swine animals, these strains may cause subclinical but not clinical disease. The modified live PRRS virus strains passaged at least 80 times have a lower probability of reverting to wild-type virulence. Most preferably, the modified live PRRS virus strains passaged 85 times have a lower probability of reverting to wild-type virulence.

[0009] The present invention provides an immunogenic composition comprising a modified live PRRS virus strain having a consensus complementary DNA sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Preferably, the modified live strain can have a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. More preferably, the modified live strain can further have a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. As will be understood by those of ordinary skill in the art, due to the high mutation rate of the PRRS virus, the modified live PRRS strains may contain multiple subpopulations, each having a homologous but not identical genome.

[0010] The present invention provides an immunogenic composition comprising a modified live PRRS virus strain, wherein the PRRS virus strain is a DE 14-3073 strain, an ES 13-49 strain, an IT 14-32 strain, or a PL 14-02 strain. The DE 14-3073 strain or the ES 13-49 strain or the IT 14-32 strain or the PL 14-02 strain can be passaged in tissue culture cells at least 80 times, or preferably even 85 times. Most preferably, the immunogenic composition comprises at least one pharmaceutically acceptable excipient. The immunogenic composition can further comprise additional antigens from different viruses or from bacterial strains or from parasites.

[0011] The present invention provides an immunogenic composition comprising a modified live PRRS virus strain, wherein the consensus complementary DNA sequence of said PRRS strain is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Preferably, the modified live strain can have a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Most preferably, the modified live strain can further have a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The vaccine can further comprise an adjuvant. The vaccine can further comprise a pharmaceutically acceptable excipient, stabilizer, solubilizer, or diluent. The vaccine can comprise additional antigens from different viruses or from bacterial strains or from parasites.

[0012] The present invention provides a vaccine for preventing porcine reproductive and respiratory syndrome in swine animals. Since PRRS is caused by the PRRS virus, the present invention provides a vaccine for preventing PRRS virus infection. The present invention also provides a vaccine for use in swine animals to alleviate the symptoms caused by PRRS virus infection. The infection may be from a wild-type lethal strain of the PRRS virus. The symptoms can be, but are not limited to, reduced appetite, lethargy, fever, premature birth, abortion, stillbirth, edema, growth retardation, coughing, respiratory distress, dyspnea or tachypnea, red skin spots, rough hair coat, lung lesions, virus shedding, and death. The present invention provides a vaccine for preventing PRRS in swine animals. Preferably, the vaccine comprises a modified live PRRS strain having a consensus complementary DNA sequence that is at least 90%, at least 95%, or at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Most preferably, the vaccine comprises a modified live PRRS strain that is the DE 14-3073 strain, the ES 13-49 strain, the IT 14-32 strain, or the PL 14-02 strain. The vaccine can further comprise a pharmaceutically acceptable excipient. The vaccine can further comprise an adjuvant. The vaccine can comprise additional antigens from different viruses or from bacterial strains or from parasites.

[0013] The present invention provides a method for preventing porcine reproductive and respiratory syndrome (PRRS) symptoms in swine animals, comprising administering to the swine animal an immunogenic composition comprising a modified live PRRS virus strain. The present invention also provides a method for preventing porcine reproductive and respiratory syndrome in swine animals, comprising administering to the swine animal an immunogenic composition comprising a modified live PRRS virus strain. Preferably, the modified live PRRS virus strain for use in the method will have a consensus complementary DNA sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. More preferably, the modified live PRRS virus strain for use in the method will have a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Most preferably, the modified live PRRS virus strain for use in the method will have a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The immunogenic composition may further comprise a pharmaceutically acceptable excipient. The immunogenic composition may comprise additional antigens from different viruses or from bacteria or from parasites.

[0014] The present invention provides a method for preventing porcine reproductive and respiratory syndrome in swine animals, comprising administering to the swine animal an immunogenic composition comprising a modified live porcine reproductive and respiratory syndrome (PRRS) virus strain, wherein the PRRS virus strain is the DE 14-3073 strain, the ES 13-49 strain, the IT 14-32 strain, or the PL 14-02 strain, and can be passaged in tissue culture cells at least 80 times, or preferably even 85 times, for use in the method. The immunogenic composition may further comprise a pharmaceutically acceptable excipient. The immunogenic composition may comprise additional antigens from different viruses or from bacterial strains or from parasites.

[0015] The present invention provides a method for preventing symptoms caused by PRRS virus infection in swine animals, said method comprising administering to said swine animal an immunogenic composition comprising a modified live porcine reproductive and respiratory syndrome (PRRS) virus strain, wherein the consensus complementary DNA sequence of said PRRS strain is preferably at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. More preferably, the modified live strain for use in said method may also have a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. Most preferably, the modified live strain for use in said method may also have a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. The immunogenic composition may further comprise a pharmaceutically acceptable excipient. The immunogenic composition may also comprise additional antigens from a different virus or from a bacterial strain or from a parasite.

[0016] The present invention provides a method for alleviating symptoms caused by PRRS virus infection in swine animals, said method comprising administering to said swine animal an immunogenic composition comprising a modified live porcine reproductive and respiratory syndrome (PRRS) virus strain, wherein the PRRS virus strain is the DE 14-3073 strain, the ES 13-49 strain, the IT 14-32 strain or the PL 14-02 strain, which can be passaged in tissue culture cells at least 80 times, or preferably even 85 times, for use in said method. The immunogenic composition may further comprise a pharmaceutically acceptable excipient. The immunogenic composition may also comprise additional antigens from a different virus or from a bacterial strain or from a parasite. The PRRS virus infection may be an infection caused by a virulent PRRS virus heterologous to the modified live PRRS virus strain in the immunogenic composition. Two PRRS virus strains are considered heterologous if the genomic consensus sequence of each virus strain maps to a different phylogenetic group. Two PRRS virus strains are considered heterologous if the complementary DNA consensus sequence of each virus strain maps to a different phylogenetic group.

[0017] The present invention provides the use of a modified live PRRS virus strain in the preparation of a medicament for preventing or alleviating PRRS symptoms, wherein the modified live PRRS virus comprises a consensus complementary DNA sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Preferably, the modified live strain may also have a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. More preferably, the modified live strain may also have a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0018] The present invention provides the use of a modified live PRRS virus strain comprising the DE 14-3073 strain, ES 13-49 strain, IT 14-32 strain, or PL 14-02 strain in the preparation of a medicament for preventing or alleviating PRRS symptoms. The modified live PRRS virus strain should be passaged in tissue culture cells at least 80 times, or preferably even 85 times. This passage in tissue culture cells can be used to appropriately attenuate the modified live PRRS virus strain. When the attenuated PRRS virus strain is administered to swine animals, these strains may cause subclinical but not clinical disease. The modified live PRRS virus strain passaged at least 80 times has a lower probability of reverting to wild-type virulence.

[0019] The present invention provides the use of an immunogenic composition comprising a modified live PRRS virus strain in the preparation of a medicament for preventing PRRS virus infection, wherein the modified live PRRS virus strain comprises a consensus complementary DNA sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The modified live strain for such use may also have a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The modified live strain for such use may also have a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0020] The present invention provides the use of an immunogenic composition comprising a modified live PRRS virus strain in the preparation of a medicament for preventing PRRS virus infection, wherein the modified live PRRS virus strain comprises the DE 14-3073 strain, the ES13-49 strain, the IT 14-32 strain or the PL 14-02 strain. The PRRS virus strain should be passaged in tissue culture cells at least 80 times, or preferably even 85 times. This passage in tissue culture cells can be used to appropriately attenuate the modified live PRRS virus strain. When the attenuated PRRS virus strains are administered to swine animals, these strains may cause subclinical but not clinical disease. The modified live PRRS virus strains passaged at least 80 times have a low probability of reverting to wild-type virulence. The modified live PRRS virus strains passaged 85 times have a low probability of reverting to wild-type virulence.

[0021] The present invention provides the use of an immunogenic composition comprising a modified live PRRS virus strain in the preparation of a medicament for protecting swine animals against PRRS virus infection, wherein the modified live PRRS virus strain comprises a consensus complementary DNA sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The modified live strain for such use may also have a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The modified live strain for such use may also have a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0022] The present invention provides the use of an immunogenic composition comprising a modified live PRRS virus strain in the preparation of a medicament for protecting swine animals against PRRS virus infection, wherein the modified live PRRS virus strain comprises the DE 14-3073 strain, the ES 13-49 strain, the IT 14-32 strain or the PL 14-02 strain. The PRRS virus strain should be passaged in tissue culture cells at least 80 times, or preferably even 85 times. This passage in tissue culture cells can be used to appropriately attenuate the modified live PRRS virus strain. When the attenuated PRRS virus strains are administered to swine animals, these strains may cause subclinical but not clinical disease. The modified live PRRS virus strains passaged at least 80 times have a low probability of reverting to wild-type virulence. The modified live PRRS virus strains passaged 85 times have a low probability of reverting to wild-type virulence. Description of the Drawings

[0023] Figure 1 .Phylogenetic analysis of PRRSV-1 DE 14-3073 strain, ES 13-49 strain, IT 14-32 strain or PL 14-02 strain, commercially available vaccines, and isolates selected based on the ORF5 nucleotide sequence constructed by the proximity ligation method. Detailed Description of the Invention

[0024] Unless otherwise specified, as used in the following discussion, the terms "a" or "an" shall be understood to cover one or more.

[0025] As used herein, the term "virus" may refer to a species of virus or, interchangeably, may encompass an individual infectious unit that contains nucleic acid and protein. The individual infectious unit is also referred to as a "virion" or "virus particle", and the latter two terms are synonyms.

[0026] As used herein, a "strain" or "isolate" of a virus refers to a collection of genetically homologous virions. Two viruses are considered "homologous" if they map to the same phylogenetic branch. Two viruses are considered "heterologous" if they map to different phylogenetic branches. Since the PRRS virus has a high mutation rate, it should be understood that a single PRRS strain contains individual virions with related but variable genetic sequences. Thus, subpopulations of strains exist within each PRRS strain, and the genetic sequence of a PRRS strain is a consensus sequence such that the genetic sequences of individual members of a PRRS strain may not be identical to the consensus sequence of that strain. A "consensus" sequence is a nucleic acid sequence in which each nucleic acid residue at a given position is present in >51% of the polynucleotides of a PRRS virus strain or isolate.

[0027] "Percent identity" can be determined by calculating the number of identical nucleotides or amino acids at the same positions in a nucleic acid or protein. Calculation of percent identity includes determining the best alignment between two or more sequences. The alignment can take into account insertions and deletions (i.e., "gaps") in each of the sequences being tested (such as but not limited to in non-coding regions of nucleic acids), as well as truncations or extensions of polypeptide sequences. Computer programs and algorithms such as the Basic Local Alignment Search Tool (BLAST) can be used to determine percent identity. BLAST is one of a number of resources provided by the National Center for Biotechnology Information in the United States. Because the genetic code is degenerate and more than one codon can encode a given amino acid, the coding regions of nucleic acids are considered identical if they encode the same polypeptide. Thus, percent identity can also be calculated based on the polypeptide encoded by the nucleic acid. Percent identity can be calculated based on the full-length consensus genomic sequence or a portion of the genomic sequence, such as for example but not limited to an individual open reading frame (ORF).

[0028] As used herein, the term "modified live virus" applies to any individual virus particle (i.e., "virion") or population of virus particles whose genetic sequence has been altered from the genetic sequence of a naturally occurring wild-type virus but still elicits protective immunity against the wild-type virus. Alterations include but are not limited to genetic mutations such as insertions and deletions of nucleotides and transitions and transversions that change one nucleotide to another. Alterations can be achieved by adapting a wild-type virus to replicate in a tissue culture system and continuing to passage the virus in the tissue culture system, whereby the virus accumulates genetic mutations. Alterations can also be accomplished using molecular techniques. Attenuated viruses form a subset of modified live viruses.

[0029] As used herein, the term "attenuated" or "attenuation" means that the ability of a virus to cause or exacerbate clinical disease has been reduced or eliminated. An attenuated virus can still infect host cells in vitro or in vivo, and the infection can result in subclinical effects in the host organism, but the infection does not result in one or more symptoms of clinical disease.

[0030] In contrast, as used herein, an "inactivated" virus is a virus that can no longer replicate in a host cell. An inactivated virus is considered to be a killed or dead virus. Inactivation can be accomplished by a variety of methods, including but not limited to chemical alteration of viral proteins, chemical or physical alteration of the structure of the virion, or chemical or physical alteration of viral nucleic acids.

[0031] "Antigen" is any molecule that can be specifically detected by the immune system of an organism. Typically, viral antigens are viral proteins encoded by or derived from the viral genome. The presence of viral antigens can be specifically detected by surface antigen receptors of host T lymphocytes and host B lymphocytes, as well as by antibody molecules synthesized by host cells.

[0032] "Immunogenicity" refers to the ability of an antigen to elicit an immune response, which includes antigen-specific responses and non-antigen-specific responses or innate immune responses. "Protective immunity" is such an immune response that can mitigate or prevent clinical symptoms when an immunized animal is challenged with or exposed to a pathogenic virus strain. As will be understood by those skilled in the art, protective immunity can decline over time or with increasing age of the immunized animal. As used herein, protective immunity should be effective for at least four months, but preferably at least six months, from the date of the most recent vaccination. Protective immunity can be induced by a single-dose vaccine. A second dose or additional doses can be used to increase or prolong the protective immune response. For example, enhancing the protective immune response in breeding sows may result in elevated levels of maternal antibodies in piglets.

[0033] In contrast to an antigen, an "adjuvant" is a non-specific stimulator of the immune response. Adjuvants can stimulate the innate immune response by binding to and activating pattern recognition receptors (PRRs). Such stimulators of PRRs can be, for example, viral or bacterial nucleic acids, lipids from bacteria or parasites, or bacterial proteins or toxins, or any artificially constructed mimics of such molecules. Adjuvants also include, but are not limited to: inorganic compounds that aggregate antigens to facilitate recognition by B lymphocytes or uptake by phagocytes, such as alum, aluminum hydroxide, aluminum phosphate, hydroxyapatite, or ammonium sulfate; oils; and detergents. Adjuvants can also be host mediators of immune signaling, such as, but not limited to, cytokines, lymphokines, chemokines, interferons, anaphylatoxins, growth factors, differentiation factors, and adhesion molecules.

[0034] As used herein, an "immunogenic composition" is a composition that elicits an immune response when administered to an animal. An immunogenic composition comprises at least one antigen and at least one pharmaceutically acceptable excipient. The antigen can be a live or inactivated whole virus, bacterium, or other pathogen. The antigen can also be an antigen molecule isolated, purified, or partially purified from a virus, bacterium, or other pathogen. The antigen can be a polypeptide, polysaccharide, nucleic acid, or lipid.

[0035] As used herein, "vaccine" is such an immunogenic composition that, when administered to an animal, confers protection, resistance, prevention, or alleviation of the symptoms of a disease, wherein said symptoms are caused by a pathogenic organism such as a virus. A PRRS vaccine can include, but is not limited to, a live or inactivated viral antigen or whole virion in combination with at least one pharmaceutically acceptable excipient.

[0036] As used herein, the terms "treating", "treat", or "treatment" include, but are not limited to, inhibiting, slowing, stopping, alleviating, improving, or reversing the progression or severity of existing symptoms, disorders, conditions, or diseases. Treatment can be applied or administered therapeutically.

[0037] As used herein, the terms "preventing", "prevent", or "prevention" include, but are not limited to, reducing, decreasing, or improving the risk of symptoms, disorders, conditions, or diseases, as well as protecting an animal from symptoms, disorders, conditions, or diseases. Prevention can be applied or administered prophylactically.

[0038] As used herein, "administered to an animal" includes, but is not limited to, administration by skin, subcutaneous, intramuscular, mucosal, submucosal, transdermal, oral, or intranasal routes. Administration can include injection or topical application.

[0039] The term "pharmaceutically acceptable excipient" refers to those excipients commonly used in the preparation of veterinary and pharmaceutical compositions and should be pure and non-toxic in the amounts used. In certain embodiments, the pharmaceutical composition may contain excipients for modifying, maintaining or preserving, for example, the pH, osmotic pressure, viscosity, clarity, isotonicity, sterility, stability, adsorption or permeability of the composition. Some examples of acceptable excipients can be found, for example, in "Remington's Pharmaceutical Sciences and the Handbook of Pharmaceutical Excipients", 18th edition, (edited by A.R. Genrmo), 1990, Mack Publishing Company and subsequent editions, and "Remington: The Science and Practice of Pharmacy", edited by Lloyd V. Allen, Pharmaceutical Press, London, UK, 22nd edition, 2012, and include diluents, vehicles, carriers, stabilizers, preservatives, solvents, suspending agents, emulsifying agents, antimicrobial agents, antioxidants, buffers, chelating agents, complexing agents, carbohydrates, proteins, diluents and / or pharmaceutical adjuvants. In certain embodiments, the major vehicle or carrier in the pharmaceutical composition can be essentially aqueous or non-aqueous. For example, suitable vehicles or carriers can be water for injection, saline solutions and other materials common in compositions for parenteral administration.

[0040] As used herein, the term "porcine animal" refers to swine, any animal belonging to the genus Sus within the even-toed ungulate family Suidae.

[0041] The following experimental examples illustrate the modified live PRRS virus. The following experimental examples also illustrate immunogenic compositions comprising the modified live PRRS virus. The following experimental examples also illustrate the use of the modified live PRRS virus to prevent or alleviate PRRS symptoms in porcine animals. It should be understood that other embodiments and uses will be apparent to those skilled in the art and the present invention is not limited to these specific illustrative examples or preferred embodiments.

[0042] Example 1

[0043] The purpose of this study was to identify potential strains for vaccine development. A total of 36 field isolates of PRRS type 1 were evaluated for their potential as vaccines. The following is the preparation of pre-master seed virus (Pre-MSV) of four type 1 (European) porcine reproductive and respiratory syndrome virus (PRRSV-1) strains: DE 14-3073, ES 13-49, IT 14-32, and PL 14-02.

[0044] The initial isolation of PRRSV-1 strains was performed using sera and lung tissues sourced from pig herds diagnosed with positive PRRSV test results in Europe during the period from 2013 to 2014. The pig herds were not vaccinated against PRRSV but presented clinical symptoms characteristic of PRRSV infection, including reproductive failure in pregnant gilts and sows (i.e., late abortions, premature farrowings, weak-born and stillborn piglets, and / or increased pre-weaning mortality) and / or growth retardation and respiratory disease problems in young pigs.

[0045] Virus isolation was carried out in primary cultures of porcine alveolar macrophages (PAM). To obtain PAM cultures, 3-week-old piglets were used as donors. Briefly, after humane euthanasia of the piglets, lungs were obtained under aseptic conditions and then rinsed with phosphate-buffered saline to recover PAM cells. The obtained cell suspension was centrifuged at 800×g for 15 minutes at 4°C, and the supernatant was discarded. The precipitated cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) and washed twice using DMEM as a diluent after the above centrifugation conditions.

[0046] In DMEM supplemented with 10% fetal bovine serum (FBS) and an antibiotic-antifungal solution (100 units / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B), the cells were counted at a final concentration of 3×10 6 cells / mL and inoculated in different supports (i.e., cell culture flasks or plates of different sizes). The cells were cultured at 37°C in an atmosphere containing 5% CO 2 2.

[0047] Clinical samples were processed according to different procedures depending on their nature. Thus, serum samples were filtered through a 0.22-μm sterile syringe filter and kept at -80°C until used for virus isolation. On the other hand, tissue samples including tonsils and lungs were homogenized 1:10 using DMEM as a diluent. The homogenate was clarified by centrifugation at 2500×g for 15 minutes, and the supernatant was filtered through a 0.22-μm sterile syringe filter and kept at -80°C until used for virus isolation.

[0048] To infect PAM cultures, the medium was removed and clinical samples (i.e., processed serum samples or processed tissue samples) were added in different amounts depending on the support used. After adsorption at 37 °C for 1.5 hours, the cultures were washed and fresh DMEM supplemented for cell maintenance was added. The cytopathic effect (CPE) of the cultures was observed daily. When CPE was observed, the cultures were harvested. After 3 cycles of freezing and thawing, cell debris was removed by centrifugation at 2500 × g for 15 minutes at 4 °C, and the supernatant was frozen and stored at -80 °C. The presence of porcine reproductive and respiratory syndrome virus (PRRSV) in the cultures was confirmed by reverse transcription and polymerase chain reaction (RT-PCR).

[0049] When no CPE was observed, the cultures were considered negative and the original clinical samples (i.e., serum samples or tissue samples) were used for bioassays and inoculated into 3-week-old piglets housed in isolation. For this purpose, the clinical samples were filtered and injected into the pigs by the intramuscular route. After one week, blood samples were taken from the exposed pigs to confirm viremia by RT-PCR. Pigs confirmed to be viremic were euthanized, and blood, tonsil, and lung samples were collected at necropsy and used as inoculum for a second virus isolation attempt in PAM cultures according to the method described above.

[0050] The growth of PRRSV isolates in the MARC-145 cell line was attempted only for isolates that grew well in PAM and could produce seed stock solutions. For this purpose, MARC-145 cells were inoculated at a concentration of 5 x 10 5 cells per flask in 25 cm 2 cell culture flasks and maintained at 37 °C in an atmosphere containing 5% CO 2 in DMEM supplemented with 10% FBS and an antibiotic-antifungal solution (100 units / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B). The confluent pre-cultures were infected with the supernatant of positive PAM cultures according to the same protocol described for the infection of PAM cultures. After adsorption, the cells were washed and cultured in DMEM medium supplemented with 5% FBS and the aforementioned antibiotic-antifungal solution.

[0051] After primary isolation, the virus was amplified and a primary virus stock solution (with a volume of at least 100 mL) was produced for each isolate in PAM cultures. For this purpose, PAM cells were seeded at a concentration of 3 x 10 2 cells per mL in 75 cm 6 flasks in DMEM containing 5% CO 2Cultivate for 24 hours in an atmosphere of 50 and discard the medium. Wash the cells with fresh DMEM and add the virus inoculum. After adsorption at 37 °C for 1.5 hours, wash the culture and add fresh DMEM supplemented for cell maintenance. Observe the CPE of the culture daily. When most of the cells in the culture are killed, harvest the culture. After 3 cycles of freezing and thawing, remove cell debris by centrifugation at 2500 x g for 15 minutes at 4 °C, and freeze the supernatant containing the virus and store it at -80 °C. Calculate the virus titer according to the method of Reed and Muench (1938) and express it as log TCID

[0052] Use the same procedure for MARC-145 cell cultures. Inoculate MARC-145 cell cultures as described previously for initial isolation and maintain them at 37 °C in an atmosphere containing 5% CO 2 . When the CPE is obvious (i.e., affecting approximately 70 - 80% of the monolayer), or after 5 days of culture (if no CPE is observed), subject the culture to 3 cycles of freezing and thawing, clarify by centrifugation at 2500 x g for 15 minutes, and use the supernatant to inoculate fresh MARC-145 cell cultures. Store the remaining supernatant from each passage at -80 °C. Perform a total of 60 passages for each PRRSV isolate in the MARC-145 cell line.

[0053] At passage 30 and passage 50, clone the virus stock by plaque purification according to the standard method. Briefly, when the culture is pre-confluent with each virus stock serially diluted from 10 1 to 10 6 , inoculate a 6-well plate previously seeded with MARC-145. After adsorption for 1.5 hours, remove the inoculum from each well and cover the cells with fresh DMEM medium supplemented with 5% FBS, the above antibiotic - antifungal solution, and 1% low melting point agarose. After 2 - 4 days of culture, select and pick individual plaques under a phase contrast microscope according to the isolate. Based on their complete isolation in the monolayer, select at least five plaques for each virus in each round of purification to ensure the clonal nature of the selected virus. The selected plaques are used as the inoculum for the next round of purification. Repeat this procedure three times to ensure that the virus progeny obtained is derived from a single virus.

[0054] Example 2

[0055] The aim of this study was to further characterize the PRRS-1 isolate. Use a set of previously designed primers to amplify the portions of ORF1 encoding nsp2 and ORF 2 to 7 by RT-PCR. For this purpose, use A viral RNA mini kit (Qiagen, USA) was used to obtain total RNA from all virus stocks. For reverse transcription and polymerase chain reaction (RT-PCR), 15 μL of total RNA was used as a template. According to the manufacturer's instructions, a commercial one-step RT-PCR kit (SuperScript III One-Step RT-PCR PLATINUM Invitrogen, USA) was used for the reaction. The RT-PCR products were purified using a commercial kit ( Purification Gel Kit, Qiagen, USA) according to the manufacturer's instructions. Using the same primer pair for RT-PCR, the samples were amplified by asymmetric PCR with fluorescent terminators, and the products were analyzed by electrophoresis on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, USA) to determine the individual sequences of the two DNA strands of each PCR product. At least two different RT-PCR products were sequenced to verify that no errors occurred during DNA amplification and that the sequences obtained were correct. The sequences obtained were manually corrected, error-cleared, and aligned using ClustalOmega software. The sequences obtained were compared with the genotype 1 PRRSV prototype Lelystad virus and the vaccine strains PRRS (MSD Animal Health) (DV strain), PRRS (Laboratorios Hipra) (VP-046BIS strain) and INGELVAC EU (Boehringer Ingelheim) (strain 94881). In addition, they have been compared with the genotype 2 prototype strain VR-2332, which is the strain of the INGELVAC MLV vaccine (Boehringer Ingelheim) and is also commercially available in Europe.

[0056] The nucleotide similarity between the obtained PRRSV isolates and the similarity between each field isolate and the available vaccine strains at the time of isolation were calculated to confirm that the isolates were not related to each other and that they were not derivatives of commercially available vaccines at the time of clinical sample collection. In addition, a phylogenetic tree was constructed using the neighbor-joining method, and the US genotype prototype VR-2332 was included as an outgroup to determine the subtype to which the European isolates belong. To evaluate the statistical reliability of the phylogenetic tree, bootstrap values were calculated (random number seed: 123; 1,000 replicates). All phylogenetic analyses were performed using MEGA5.0 software.

[0057] PRRSV-1 was passaged 10 times (to P70) in MARC-145 cells in growth medium supplemented with 2% fetal bovine serum (FBS; catalog numbers 12003C and 12007C from Sigma and catalog number 04 - 4000DJ from Gibco) and 50 μg gentamicin / mL (catalog number 15750, Life Technologies) and was further attenuated by an additional passage of more than 15 times (to P85) in the same growth medium supplemented with 2% FBS without gentamicin. The identity of the 85th passage (P85) PRRSV-1 was confirmed by indirect immunofluorescence assay (IFA) using PRRSV-specific monoclonal antibodies, and P85 PRRSV-1 was considered the Pre-Master Seed Virus (Pre-MSV).

[0058] The following procedure was used to determine the titer of PRRSV-1. MARC-145 cells were seeded at a density of 0.75 to 1.5 x 10 cells in 100 μL of growth medium (medium supplemented with 5% FBS and 50 μg / mL gentamicin 4 I) into 96-well plates. The cells were incubated in a 37 ± 2 °C and 5 ± 1% CO 2 incubator for 48 - 72 hours until the cells were more than 95% confluent. On the day of titration, all the medium was removed from the 96-well plates and replaced with 100 μL of fresh growth medium.

[0059] Using a diluent ( I medium, 50 μg / mL gentamicin), ten-fold serial dilutions of PRRSV-1 were prepared and transferred, along with a negative control consisting of the diluent alone and a positive control with a known titer, to the corresponding wells on the plates prepared as above. The titration plates were incubated in a 37 ± 2 °C and 5 ± 1% CO 2 incubator for 4 days. At the end of the incubation period, the presence of virus-induced cytopathic effect (CPE) in each sample well of each plate was observed using an inverted microscope. The 50% tissue culture infective dose (TCID 50 ) was calculated using the Reed-Muench method, and the titer was recorded as log 10 TCID 50 / mL. The titer of P85 PRRSV-1 ranged from 8.1 - 8.5 log 10 TCID 50 / mL.

[0060] Example 3

[0061] The aim of this study was to determine the sequence of Pre-MSV. To determine the genomic sequence of each virus isolate, the virus grown in MARC-145 cells was concentrated and purified by ultracentrifugation on a sucrose cushion, and RNA was extracted using the MINELUTE Virus Spin Kit (Qiagen) and TRIzol LS (Invitrogen). The whole-genome sequence was determined by multiple runs of next-generation sequencing (NGS) using the platform and / or the NEXTSEQ500 system located at Bioreliance (Rockville, MD) and ACGT (Wheeling, IL). Variable and ambiguous sequences and gaps were fixed and confirmed by Sanger dideoxy sequencing, and a consensus whole-genome sequence was generated. The nucleotide sequences were aligned using the neighbor-joining tree nucleotide alignment tool in Geneious 10.1.3 software and compared with selected known PRRSV-1 and commercial vaccine viruses.( Figure 1 ).

[0062] The PRRSV-1 MLV strain labeled Pre-MSV is deposited under conditions that will ensure that access to the culture during the pendency of this patent application is limited to those having a need to know in accordance with 37 C.F.R. § 1.14 and 35 U.S.C. § 122, the Commissioner of Patents and Trademarks. The deposit will be available as required by the patent laws of the countries / regions in which the family or its progeny of the subject application is filed. However, it should be understood that the availability of the deposit does not constitute a license to practice the subject invention in derogation of the patent rights granted by government action. The subject culture deposit will be stored and made available to the public in accordance with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., for at least five years after the most recent request for a sample of the deposit and, in any case, for at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent that may issue disclosing the deposited culture, the deposit will be carefully preserved as necessary to maintain its viability and freedom from contamination. The depositor acknowledges an obligation to replace the deposit if, due to the conditions of the deposit, the depositor is unable to provide a sample upon request. Upon the grant of a patent disclosing the deposit of the subject culture, all restrictions on access by the public to the deposit of the subject culture will be irrevocably removed. On March 7, 2019, in accordance with the terms of the Budapest Treaty, the deposit of PRRSV-1 Pre-MSV was incorporated by the repository into the permanent collection of the American Type Culture Collection Patent Depository Center, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA, where the strain was assigned the deposit numbers PTA-125490 (DE14-3073); PTA-125489 (ES 13-49); PTA-125488 (IT 14-32); PTA-125487 (PL 14-02).

[0063] The safety and efficacy of immunogenic compositions and vaccines of modified live porcine reproductive and respiratory syndrome virus can be determined by methods well known in the art, including dose response, onset of immunity, duration of immunity, and shedding and transmission of porcine reproductive and respiratory syndrome virus. The lack of reversion to virulence of any Pre-MSV can also be readily determined.

[0064] The cDNA consensus sequences of four PRRS virus isolates of the 85th passage (P85) are deposited in the GenBank genetic sequence database, an annotated collection of all publicly available nucleic acid sequences. The GenBank database is maintained by the National Center for Biotechnology Information (NCBI), part of the National Institutes of Health (NIH). GenBank is part of the International Nucleotide Sequence Database Collaboration.

[0065] The consensus cDNA sequence of the PRRS strain DE14 - 3073 of P85 has been designated as GenBank accession number MK024324 (SEQ.ID.NO:1). The consensus cDNA sequence named SEQ.ID.NO:1 is:

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] The consensus cDNA sequence of the PRRS strain ES13 - 49 of P85 has been designated as GenBank accession number MK024325 (SEQ.ID.NO:2). The consensus cDNA sequence named SEQ.ID.NO:2 is:

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] The consensus cDNA sequence of the PRRS strain IT 14 - 32 of P85 has been designated as GenBank accession number MK024326 (SEQ.ID.NO:3). The consensus cDNA sequence named SEQ.ID.NO:3 is:

[0078]

[0079]

[0080]

[0081]

[0082] The cDNA consensus sequence of the PRRS strain PL 14-02 of P85 has been designated as GenBank accession number MK024327 (SEQ.ID.NO:4). The cDNA consensus sequence designated as SEQ.ID.NO:4 is:

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Those skilled in the art will recognize that the length of the polyadenylation tail (if any) of each genomic consensus sequence may differ from the sequences reported above.

[0089]

[0090]

Claims

1. A modified live porcine reproductive and respiratory syndrome (PRRS) virus strain, wherein the consensus complementary DNA sequence of said PRRS strain is represented by SEQ ID NO:

2.

2. The modified live PRRS virus strain according to claim 1, wherein said PRRS strain is the ES 13-49 strain deposited in the permanent depository of the American Type Culture Collection Patent Depository Center under the accession number PTA-125489.

3. The modified live PRRS strain according to claim 1 or 2, wherein said PRRS virus strain is passaged in tissue culture cells at least 85 times.

4. Use of a modified live porcine reproductive and respiratory syndrome (PRRS) virus strain according to any one of claims 1 to 3 in the preparation of a medicament for the treatment of PRRS.

5. An immunogenic composition comprising a modified live PRRS virus strain and a pharmaceutically acceptable excipient, stabilizer, solubilizer or diluent, said modified live PRRS virus strain having a consensus complementary DNA sequence represented by SEQ ID NO:

2.

6. The immunogenic composition according to claim 5, wherein said modified live PRRS virus strain is the ES13-49 strain deposited in the permanent depository of the American Type Culture Collection Patent Depository Center under the accession number PTA-125489.

7. The immunogenic composition according to claim 5 or 6, further comprising an adjuvant.

8. A vaccine comprising a modified live PRRS virus strain, wherein said PRRS virus strain is the ES13-49 strain deposited in the permanent depository of the American Type Culture Collection Patent Depository Center under the accession number PTA-125489.

9. A vaccine comprising a modified live PRRS virus strain according to any one of claims 1 to 3 for the treatment or prevention of symptoms of porcine reproductive and respiratory syndrome in swine animals.

10. Use of an immunogenic composition in the preparation of a medicament for the treatment or prevention of symptoms of type 2 porcine reproductive and respiratory syndrome (PRRS) virus, said immunogenic composition comprising a modified live PRRS virus strain, said modified live PRRS virus strain having a consensus complementary DNA sequence represented by SEQ ID NO:

2.

11. The use according to claim 10, wherein the symptoms of said PRRS are caused by PRRS virus infection in swine animals.

12. Use of a vaccine in the preparation of a medicament for the treatment or prevention of symptoms of type 2 porcine reproductive and respiratory syndrome (PRRS) virus in swine animals, said vaccine comprising a modified live PRRS virus strain, wherein said PRRS virus strain is the ES13-49 strain deposited in the permanent depository of the American Type Culture Collection Patent Depository Center under the accession number PTA-125489.

13. The use according to claim 12, wherein the symptoms of said PRRS are caused by PRRS virus infection in swine animals.

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