New European type porcine reproductive and respiratory syndrome virus strain

Abstract

The present invention relates to a modified live PRRS vaccine containing a novel PRRSV European type strain of PRRSV, and to methods of using and manufacturing said vaccine.

Description

[0001] This application is a divisional application of the patent application with the application date of February 14, 2012, the priority date of February 17, 2011, the application number of 201280009228.6, and the invention title of "New European Type Porcine Reproductive and Respiratory Syndrome Virus Strain". Technical Field

[0002] The present invention relates to a live attenuated strain of European Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a method for producing said virus strain, a vaccine based on said virus strain, a method for producing said vaccine, and its use for treating pigs. Background Art

[0003] Porcine Reproductive and Respiratory Syndrome (PRRS) is regarded by many as the most important disease currently affecting the worldwide pig industry. In 1987, the syndrome was first described in the United States as the "mystery swine disease" and rapidly spread throughout the world. It causes a severe reduction in fertility, resulting in an increase in mortality due to secondary infections, and leads to a decrease in feed conversion rate and average daily weight gain. Unfortunately, it has proven difficult to control the virus that causes PRRS.

[0004] PRRS virus (PRRSV) is an enveloped single-stranded RNA virus classified in the family Arteriviridae (Cavanaugh, 1997). It causes a widespread porcine disease that was first described in the United States in 1987 as the "mystery swine disease" (Hill, 1990). The disease presents as a respiratory disease in pigs of all age groups, causing death in some of the younger pigs and severe reproductive problems in breeding-age sows.

[0005] PRRSV can be transmitted, and often is, by direct contact between infected pigs and susceptible pigs. It can also be transmitted by air or via semen over very short distances. Once infected, the virus can remain in the blood of adult pigs for about two weeks and in infected pigs for 1 to 2 months or longer. Infected boars can continuously shed the virus in semen for more than 100 days. This long-term viremia significantly increases the likelihood of transmission. In addition, the PRRS virus can cross the placenta during the third trimester of pregnancy, infecting piglets in utero and causing stillbirths or the birth of weak piglets.

[0006] All types and sizes of herds, including those in good or average health or from indoor or outdoor units, can be infected with the PRRS virus. Infected herds may experience severe reproductive failure, as well as increased pneumonia levels and slow growth after weaning. The reproductive period usually lasts two to three months; however, post-weaning problems often become endemic. Reproductive disease is characterized by abortion outbreaks affecting sows and gilts in late pregnancy. Premature farrowing occurs at approximately 109 and 112 days of gestation. The number of stillbirths and weak piglets born increases and causes a significant increase in pre-weaning mortality.

[0007] Respiratory phases have traditionally been seen in farms, especially in continuous flow farms. However, respiratory problems caused by the PRRS virus can also be seen in finishing animals as part of the porcine respiratory disease complex (PRDC). Growth rates are reduced, the percentage of pigs that cannot be sold increases, and post-weaning mortality rises. Diagnostic results indicate a high degree of pneumonia associated with the PRRS virus, as well as a variety of other microorganisms generally regarded as secondary infective agents. Bacterial isolates can particularly include Streptococcus suis, Haemophilus suis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Mycoplasma hyopneumoniae, and Pasteurella multocida. Viral agents commonly involved include swine influenza virus and porcine respiratory coronavirus. Affected pigs rarely respond to high levels of drug treatment, and all-in / all-out systems have been unable to control the disease.

[0008] The PRRSV virus exists in two genotypes called the "American" type and the "European" type, which share approximately 50% sequence homology (Dea S et al., (2000). Arch Virol 145:659-88). These two genotypes can also be distinguished by their immunological properties. Most sequence information on various isolates is based on the structural protein, the envelope protein GP5, which represents only approximately 4% of the viral genome, and little is known about the non-structural proteins (nsp). The isolation of PRRSV and the manufacture of vaccines have been described in a number of publications (WO 92 / 21375, WO 93 / 06211, WO 93 / 03760, WO 93 / 07898, WO 96 / 36356, EP 0 676 467, EP 0 732 340, EP 0 835 930).

[0009] Vaccination is a key method for reducing the burden of PRRS, as pigs recovering from PRRS infection will develop an immune response that will normally protect the pigs from reinfection with the same virus strain. However, the PRRS virus can change (by mutation or recombination); thus, new virus strains may emerge. In such a situation, cross - protection between virus strains may not exist, and new outbreaks may be observed in farms that have previously been infected. Therefore, there is a continuing need for additional vaccines. Summary of the Invention

[0010] The present invention relates to a modified live PRRS vaccine of European genotype, and to new PRRSV strains that can be used to manufacture the vaccine. In particular, the present invention provides modified PRRS virus strains that have been deposited under accession numbers ECACC 11012501 and ECACC 11012502 (each deposited on January 25, 2011) at the European Collection of Cell Cultures (ECACC) in accordance with the provisions of the Budapest Treaty, or any progeny or offspring of one of the above virus strains.

[0011] In a specific embodiment, the present invention describes a European type porcine reproductive and respiratory syndrome virus (PRRSV) that is a virus strain deposited under accession number ECACC 11012501 or accession number ECACC 11012502 at the European Collection of Cell Cultures (ECACC).

[0012] The PRRSV is characterized by attenuation of the virus by passage in cell culture at least 36 times such that when the modified virus is administered to pigs or other mammals susceptible to PRRSV, it does not cause clinical signs of PRRSV disease, but is capable of inducing an immune response that immunizes the mammal against pathogenic forms of PRRSV.

[0013] Also encompassed is a method for preparing a live attenuated PRRSV deposited under accession number ECACC 11012502 at the European Collection of Cell Cultures (ECACC) or a PRRSV attenuated from a parental strain deposited under accession number ECACC 11012501, which comprises adapting a European type PRRSV grown in MA 104 to non - MA 104 mammalian cells.

[0014] Another aspect of the present invention encompasses a vaccine for protecting pigs against PRRSV infection, which comprises a live attenuated PRRSV deposited under accession number ECACC 11012502 at the European Collection of Cell Cultures (ECACC) or a PRRSV attenuated from a parental strain deposited under accession number ECACC 11012501 and a pharmaceutically acceptable carrier. Such a vaccine preferably further comprises one or more attenuated or inactivated pathogens other than PRRSV or antigenic substances thereof. For example, the non-PRRSV pathogens can be selected from Pseudorabies virus, Porcine influenza virus, Porcine parvovirus, Transmissible gastroenteritis virus, Escherichia coli, Erysipelo rhusiopathiae, Bordetella bronchiseptica, Salmonella cholerasuis, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae.

[0015] In other embodiments, the vaccine may further comprise one or more additional European type PRRSV strains selected from the group consisting of the PRRSV strain deposited under accession number Lelystad virus strain (CDI-NL-2.91), or other virus strains such as those deposited under accession numbers ECACC 04102703, ECACC 04102702, ECACC 04102704, CNCM accession number I-1140, CNCM accession number I-1387, CNCM accession number I-1388, ATCC VR 2332, VR 2385, VR 2386, VR 2429, VR 2474, and VR 2402; virus strains under CNCM I-1102, CNCM I-1140, CNCM I-1387, CNCM I-1388, or ECACC V93070108; or may actually be American type strains such as North American PRRS virus pT7P129A; ATCC accession number VR-2332, ATCC accession number VR-2368; ATCC VR-2495; ATCC VR 2385, ATCC VR 2386, ATCC VR 2429, ATCC VR 2474, and ATCC VR 2402.

[0016] The vaccine is expected to comprise a carrier suitable for intradermal or intramuscular application. In some embodiments, the vaccine is in lyophilized form. In a specific embodiment, the vaccine comprises at least about 10 7 virus particles.

[0017] Another aspect of the present invention relates to a method for preparing a live attenuated vaccine for combating PRRS, which comprises mixing a live attenuated PRRSV virus deposited under accession number ECACC 11012502 at the European Collection of Cell Cultures (ECACC) or a PRRSV attenuated from a parental strain deposited under accession number ECACC 11012501 with a pharmaceutically acceptable carrier. In the method, the live attenuated PRRSV preferably may further comprise one or more additional European type PRRSV strains selected from the PRRSV strains deposited under accession numbers ECACC 04102703, ECACC 04102702, ECACC 04102704, CNCM accession number I-1140, CNCM accession number I-1387, and CNCM accession number I-1388.

[0018] In some embodiments, the live attenuated PRRSV may further comprise an adjuvant.

[0019] Also covered is a method of immunizing pigs against porcine reproductive and respiratory syndrome (PRRS), the method comprising the step of administering to a pig a vaccine composition comprising a mixture of a live porcine reproductive and respiratory syndrome virus and a pharmaceutically compatible carrier, the virus comprising PRRS 94881 virus, which has been passaged in cell culture at least 36 times to modify the virus such that when the modified virus is administered to a pig or other mammal susceptible to PRRS, it does not cause clinical signs of PRRS disease but is capable of inducing an immune response that immunizes the mammal against the pathogenic form of PRRS.

[0020] In some embodiments, the method is carried out wherein the pigs have no lung lesions after vaccination. In other embodiments, the pigs have fewer lung lesions after vaccination compared to vaccination with Porcilis vaccine.

[0021] Another aspect of the invention relates to a PRRS virus having a nucleotide sequence that has at least 95% homology to the sequence set forth in SEQ ID NO:1 or SEQ ID NO:10.

[0022] Also covered is a PRRS virus comprising at least one ORF encoding a protein having at least 98% identity to any of the sequences set forth in SEQ ID NO:2 to 9 or SEQ ID NO:11 to SEQ ID NO:18.

[0023] Also covered is a PRRS virus having the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:10 or a nucleotide sequence of a fragment of either SEQ ID NO:1 or SEQ ID NO:2, wherein the fragment encodes an ORF selected from: SEQ ID NO:2, 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:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0024] The present invention further relates to a subunit vaccine for inoculating swine, wherein the vaccine comprises one or more nucleotides encoding an ORF selected from the following: SEQ ID NO:2, 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:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0025] Another aspect of the present invention relates to a subunit vaccine for inoculating swine, wherein the vaccine comprises one or more nucleotides selected from the following: SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; and SEQ ID NO:34.

[0026] Also encompassed is a composition comprising one or more proteins having a sequence selected from the following: SEQ ID NO:2, 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:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0027] Also encompassed is an isolated nucleic acid comprising a sequence selected from the group consisting of: SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34.

[0028] The invention further relates to a recombinant expression vector and / or a vaccine comprising said expression vector, wherein said vector comprises a nucleic acid sequence encoding one or more PRRSV ORFs selected from the group consisting of: SEQ ID NO:2, 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:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18 (the nucleic acid sequence being operably linked to a promoter). In said embodiment, the nucleic acid encoding the ORF is preferably selected from the group consisting of: SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; and SEQ ID NO:34.

[0029] Specifically, the present invention relates to the following items:

[0030] 1. A European type porcine reproductive and respiratory syndrome virus (PRRSV), which is a virus strain deposited at the European Collection of Cell Cultures (ECACC) under accession number ECACC 11012501 or accession number ECACC 11012502.

[0031] 2. A PRRSV as in item 1, wherein the virus is attenuated by passage in cell culture for at least 36 times such that when the modified virus is administered to a pig or other mammal susceptible to PRRSV, it does not cause clinical signs of PRRSV disease but is capable of inducing an immune response that immunizes the mammal against the pathogenic form of PRRSV.

[0032] 3. A method for preparing a live attenuated PRRSV as in item 1, which comprises adapting a European type PRRSV grown in MA 104 to non-MA 104 mammalian cells.

[0033] 4. A vaccine for protecting pigs against PRRSV infection, which comprises a live attenuated PRRSV as in item 1 and a pharmaceutically acceptable carrier.

[0034] 5. The vaccine as in item 4, which further comprises one or more attenuated or inactivated pathogens other than PRRSV or antigenic substances thereof.

[0035] 6. The vaccine as in item 5, wherein the non-PRRSV pathogen is selected from pseudorabies virus, swine influenza virus, porcine parvovirus, transmissible gastroenteritis virus, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Salmonella choleraesuis, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae, and Actinobacillus pleuropneumoniae.

[0036] 7. The vaccine as in item 4, which further comprises one or more other European type PRRSV strains selected from: the PRRSV strain deposited under accession number Lelystad virus strain (Lelystad material (CDI-NL-2.91)), or other virus strains such as those deposited under accession numbers ECACC 04102703, ECACC 04102702, ECACC 04102704, CNCM accession number I-1140, CNCM accession number I-1387, CNCM accession number I-1388, ATCC VR 2332, VR 2385, VR 2386, VR 2429, VR2474, and VR 2402; virus strains under CNCM I-1102, CNCM I-1140, CNCM I-1387, CNCM I-1388, or ECACCV93070108; or those that may actually be American type strains such as North American PRRS virus pT7P129A; ATCC accession number VR-2332, ATCC accession number VR-2368; ATCC VR-2495; ATCC VR 2385, ATCC VR 2386, ATCC VR2429, ATCC VR 2474, and ATCC VR 2402.

[0037] 8. The vaccine according to item 4, which comprises a carrier suitable for intradermal or intramuscular application.

[0038] 9. The vaccine according to item 4, which is in lyophilized form.

[0039] 10. The vaccine according to item 4, wherein the vaccine comprises at least about 10 7 virus particles.

[0040] 11. A method for preparing a live attenuated vaccine for combating PRRS, which comprises mixing the live attenuated PRRSV according to item 1 with a pharmaceutically acceptable carrier.

[0041] 12. The method according to item 11, wherein the live attenuated PRRSV further comprises one or more other European type PRRSV strains selected from the following: PRRSV strains deposited under accession numbers ECACC 04102703, ECACC 04102702, ECACC 04102704, CNCM accession number I-1140, CNCM accession number I-1387 and CNCM accession number I-1388.

[0042] 13. The method according to item 11, wherein the live attenuated PRRSV further comprises an adjuvant.

[0043] 14. A method for immunizing pigs against porcine reproductive and respiratory syndrome (PRRS), the method comprising the step of administering to a pig a vaccine composition, wherein the vaccine composition comprises a mixture of a live porcine reproductive and respiratory syndrome virus and a pharmacologically compatible carrier, the virus comprising the PRRS 94881 virus passaged in cell culture at least 36 times to modify the virus such that when the modified virus is administered to a pig or other mammal susceptible to PRRS, it does not cause clinical signs of PRRS disease but is capable of inducing an immune response that immunizes the mammal against the pathogenic form of PRRS.

[0044] 15. The method according to item 14, wherein the pig does not exhibit lung injury after vaccination.

[0045] 16. The method according to item 14, wherein the pig exhibits less lung injury after vaccination compared to vaccination with Porcilis vaccine.

[0046] 17. A PRRS virus having a nucleotide sequence with at least 95% homology to the sequence described in SEQ ID NO:1 or SEQ ID NO:10.

[0047] 18. A PRRS virus comprising at least one ORF encoding a protein having at least 98% identity to any of the sequences described in SEQ ID NO:2 to 9 or SEQ ID NO:11 to SEQ ID NO:18.

[0048] 19. A PRRS virus having a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:10, or a nucleotide sequence of a fragment of either SEQ ID NO:1 or SEQ ID NO:2, wherein the fragment encodes an ORF selected from the following: SEQ ID NO:2, 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:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0049] 20. A subunit vaccine for inoculating swine, wherein the vaccine comprises one or more nucleotides encoding an ORF selected from the following: SEQ ID NO:2, 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:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0050] 21. A subunit vaccine for inoculating sows, wherein the vaccine comprises one or more nucleotides selected from the following: SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; and SEQ ID NO:34.

[0051] 22. A composition comprising one or more proteins having a sequence selected from the following: SEQ ID NO:2, 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:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0052] 23. An isolated nucleic acid comprising a sequence selected from the following: SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; and SEQ ID NO:34.

[0053] 24. A recombinant expression vector comprising a nucleic acid sequence encoding one or more PRRSV ORFs selected from the following: SEQ ID NO:2, 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:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, said nucleic acid sequence being operably linked to a promoter.

[0054] 25. The recombinant expression vector according to item 24, wherein the nucleic acid encoding the ORF is selected from the following: SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; and SEQ ID NO:34.

[0055] 26. A vaccine comprising the recombinant expression vector according to any one of items 24 or 25. Description of the Drawings

[0056] Figure 1A : Clinical observation results of cough scores in a respiratory challenge model using a European challenge virus strain.

[0057] Figure 1B : Clinical observation results of total clinical scores in a respiratory challenge model using a European challenge virus strain.

[0058] Figure 2 : Rectal temperature measurement in a respiratory challenge model using a European challenge virus strain.

[0059] Figure 3 : Average daily weight gain measurement in a respiratory challenge model using a European challenge virus strain.

[0060] Figure 4 : PRRS viremia as indicated by quantitative PCR in a respiratory challenge model using a European challenge virus strain.

[0061] Figure 5 : PRSS serology as indicated by ELISA in a respiratory challenge model using a European challenge virus strain.

[0062] Figure 6 : Macroscopic examination of lung injury in a respiratory challenge model using a European challenge virus strain.

[0063] Figure 7A -C: Histopathological measurement. Figure 7A Showing average macroscopic lung injury; Figure 7B Showing histopathology of control animals; Figure 7B Showing histopathology of PRRS-infected animals.

[0064] Figure 8: Displays RT - PCT time PCR results depicting the percent viremia in animals vaccinated with the European type PRRS 94881 vaccine.

[0065] Figure 9 : Parallel methods for large - scale production of European type PRRS 94881. Detailed description of the invention

[0066] The present invention provides methods for treating or reducing the severity of porcine reproductive and respiratory syndrome virus (PRRSV) infection and for preventing PRRSV infection. Generally, the methods are for treating or reducing the severity or incidence of porcine reproductive and respiratory syndrome virus (PRRSV) infection. "Treating or reducing severity or incidence" means reducing the severity of the clinical signs, symptoms, and / or pathological signs normally associated with the infection, up to and including preventing any of said signs or symptoms. "Pathological signs" means signs of infection (such as lung lesions) found microscopically or during autopsy.

[0067] The method generally includes the step of administering a therapeutically effective amount of PRRSV antigen to pigs of a specified age or age range. For example, in one aspect of the invention, a therapeutically effective amount of PRRSV antigen can be administered to piglets about three weeks of age or younger, and different therapeutically effective amounts of antigen can be administered to pigs between about 3 and 4 weeks of age. Similarly, yet more different therapeutically effective amounts can be administered to pigs between about four and sixteen weeks of age (or any age within this range, such as five to six weeks of age, nine to fifteen weeks of age, seven to ten weeks of age, etc.) or to pigs over sixteen weeks of age, such as adult sows.

[0068] In a specific embodiment, the present invention relates to an attenuated atypical PRRSV strain and a corresponding modified live vaccine that confers effective immunity against this newly discovered typical PRRSV strain. "Effective immunity" means the ability of the vaccine to prevent porcine PRRSV infection, including atypical PRRSV infection, that can cause substantial clinical signs of disease. It should be understood that vaccinated pigs may be seropositive or seronegative for PRRSV, but the pigs do not show any substantial clinical symptoms.

[0069] In a preferred form, the vaccine of the present invention comprises a live attenuated European type PRRS live virus. The resulting attenuated virus has been shown to be non-toxic and confer effective immunity in challenged control host animal studies. This particular virus strain of European type PRRS is less virulent than others and, therefore, is an attractive choice as a vaccine candidate. The PRRSV 94881 parental strain did not cause severe atypical PRRS disease in pregnant sows nor severe lung damage in piglets. This virus strain was initially isolated from three-week-old piglets with severe respiratory disease in North Rhine Westphalia (Germany). Subsequently, the virus strain was attenuated by serial passage in MA 104 cells. The attenuated strain was deposited on January 25, 2011, by Bioscreen GmbH, Mendelstrasse 11, 48149, Muenster, Germany, at the European Collection of Cell Cultures (ECACC) (Porton Down, Salisbury, Wiltshire, SP4 0JG, Great Britain) and given the accession number 11012502. This attenuated virus is a preferred master seed virus (MSV), which was subsequently passaged and developed into an effective PRRSV vaccine. The virulent parental strain named 94881 was also deposited on January 25, 2011, by Bioscreen GmbH, Mendelstrasse 11, 48149, Muenster, Germany, at the European Collection of Cell Cultures (ECACC) (Porton Down, Salisbury, Wiltshire, SP4 0JG, Great Britain) according to the Budapest Treaty and given the accession number 11012501.

[0070] In certain exemplary embodiments, the modified live virus vaccine was tested in pigs at a dose of 1 ml by intramuscular injection and in sows at a dose of 2 ml and was shown to be effective in generating protective immunity.

[0071] Virus passage to attenuate it was achieved using classical virological methods. In particular, the parental isolate PRRS 94881 was attenuated in vitro by serial passage in MA 104 cells to reach a maximum of 108 passages after the initial isolation. Briefly, the substance was in T-25 cm 2 or T-75 cm 2The cells in the flask were passaged at approximately 1 to 2 passages per week for a total of 108 passages. A confluent MA 104 cell culture with approximately 12 - 30 mL of minimum essential medium (MEM) supplemented with 6% fetal bovine serum (FBS) was inoculated with 100 to 300 μl of the virus. The cultures were incubated at 37 °C and 4 - 6% CO2 in a humidified chamber incubator for 3 - 7 days. Once the cultures reached >25% cytopathic effect (CPE), the flasks were harvested by aspirating the supernatant. A portion of the supernatant was transferred into a new flask and 2 mL of the harvest was aliquoted and stored at -60 °C to -80 °C.

[0072] Those skilled in the art can determine the basic nucleic acid sequence of the attenuated virus deposited under ECACC accession number 11012502 using conventional techniques. Accordingly, the present invention further encompasses the nucleic acid sequences specific to the attenuated PRRSV94481 deposited under ECACC accession number 11012502. Preferably, the present invention further encompasses PRRS virus nucleic acid sequences sharing at least 95% sequence homology with the sequences of SEQ ID NO:1 or SEQ ID NO:10, as the virus may effectively confer immunity to animals after vaccination with the attenuated virus containing the homologous sequence. The sequence shown in SEQ ID NO:1 is the full-length sequence of attenuated PRRS 94881 MSV and has a full-length sequence of 14843 bp. ORFs 1 to 7 of this sequence are annotated as follows:

[0073]

[0074] The sequence shown in SEQ ID NO:10 is the full-length sequence of the 5th passage of the parental PRRSV 94881 strain and has a full-length sequence of 14843 bp. ORFs 1 to 7 of this sequence are annotated as follows:

[0075]

[0076] With the isolation of this new attenuated European type PRRS virus strain, an improved PRRS vaccine can be produced, which contains the latest PRRS strains reflecting the virulent PRRS strains currently found in this field. In particular, the new attenuated European type PRRS virus can be used to prepare a modified live vaccine (MLV). The modified live vaccine is characterized in that it contains live virus that can replicate in pigs but does not cause clinical disease of PRRS. In addition, when administered, it induces an immune response in pigs, which generally provides significant protection against subsequent pathogenic PRRS virus infections. Viruses showing such characteristics are generally referred to as attenuated viruses. In addition, the present invention provides details of the ORF sequences of both the parental and attenuated strains of PRRSV 94881. Accordingly, it is expected that those skilled in the art can adopt the sequences of any one or more of the ORFs shown herein in subunit vaccines.

[0077] As described above, generally, an attenuated virus can be produced from a pathogenic virus isolate by serial passage in a suitable host cell susceptible to infection until the virus exhibits the desired properties (WO 92 / 21375, WO 93 / 06211, WO93 / 03760, WO 93 / 07898, WO 96 / 36356, EP 0 676 467, EP 0 732 340, EP 0 835 930). Alternatively, it can be produced by re-genetic engineering using an infectious clone, usually using a full-length complementary DNA transcript of the viral genome (WO 98 / 18933, EP 1 018 557, WO 03 / 062407, Nielsen et al., J Virol 2003, 77:3702-3711). In a preferred embodiment, the present invention relates to an MLV containing an attenuated PRRS virus of European genotype 94481, which is attenuated from the parental virus deposited under ECACC accession number 11012501. The preferred MLV contains the attenuated virus of the present invention deposited under ECACC accession number 11012502.

[0078] In another aspect, the present invention encompasses the preparation and isolation of progeny or offspring of a PPRS virus deposited on January 25, 2011, at the European Collection of Cell Cultures (ECACC) (Porton Down, Salisbury, Wiltshire, SP4 0JG, Great Britain) under accession numbers ECACC11012502 (attenuated strain of MLV) and 11012501 (parental strain). Accordingly, the present invention extends to PRRS virus strains propagated or bred from the deposited strains in the same or different forms, especially offspring having the basic characteristics of the deposited strains. After continued propagation, the virus strains may acquire mutations, and most of the mutations do not significantly alter the properties of these virus strains.

[0079] The virus strains of the present invention may also be further modified to confer other desired properties on the virus strains. This can be achieved by classical propagation and selection techniques, such as continuous propagation in a suitable host cell to expand the attenuated phenotype. Alternatively, the virus strains can be genetically modified by suitable genetic engineering techniques to introduce directed mutations in the nucleic acid sequences of the genomes of these virus strains. The genomes of PRRSV have been fully or partially sequenced (Conzelmann et al., 1993; Meulenberg et al., 1993a; Murtaugh et al., 1995), and in addition to the RNA-dependent RNA polymerase (ORF 1a and 1b), also encode six structural proteins, including four envelope glycoproteins called GP2 (ORF2), GP3 (ORF3), GP4 (ORF4) and GP5 (ORF5), a non-glycosylated membrane protein M (ORF6) and the nucleocapsid protein N (ORF7) (Meulenberg et al., 1995, 1996; van Nieuwstadt et al., 1996). Immunological characterization and nucleotide sequencing of European and American type PRRSV strains have identified minor antigenic differences within PRRSV strains located in the structural viral proteins (Nelson et al., 1993; Wensvoort et al., 1992; Murtaugh et al., 1995). The PRRS 94881 MSV of the present invention has been compared with the European reference virus strain, Lelystad virus (LV), showing that the nucleotide homology in 8 different viral genes ranges from 85.40% to 95.09%, and the amino acid identity between the two virus strains is from 86.39% to 97.27%. Two deletions in ORF 1a of 94881 MSV compared to LV could be identified.For example, the ORF1a of 94881 MSV has 85.40% nucleotide homology with Riley's virus, resulting in an amino acid identity of 86.39%; the ORF1b of 94881 MSV has 92.12% nucleotide homology with Riley's virus, resulting in an amino acid identity of 97.27%; the ORF2 of 94881 MSV has 91.07% nucleotide homology with Riley's virus, resulting in an amino acid identity of 90.76%; the ORF3 of 94881 MSV has 90.98% nucleotide homology with Riley's virus, resulting in an amino acid identity of 89.43%; the ORF4 of 94881 MSV has 90.58% nucleotide homology with Riley's virus, resulting in an amino acid identity of 87.43%; the ORF5 of 94881 MSV has 90.43% nucleotide homology with Riley's virus, resulting in an amino acid identity of 88.56%; the ORF6 of 94881 MSV has 95.02% nucleotide homology with Riley's virus, resulting in an amino acid identity of 97.11%; the ORF7 of 94881 MSV has 95.09% nucleotide homology with Riley's virus, resulting in an amino acid identity of 92.97%.

[0080] In fact, the PRRS 94881 virus of the present invention can be made into a chimeric virus, in which the backbone of the PRRS virus under the ECACC accession number 11012502 or the parental strain actually deposited under the ECACC accession number 11012501 is modified to replace the endogenous sequences of one or more of ORF 1a, ORF 1b, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6 or ORF7 with the corresponding ORFs from different PRRS virus strains. For example, the different PRRS virus strains can be different European type strains, such as the Lelystad virus strain (Lelystad material (CDI-NL-2.91)), or other strains, such as those deposited under accession numbers ECACC 04102703, ECACC 04102702, ECACC 04102704, CNCM accession number I-1140, CNCM accession number I-1387, CNCM accession number I-1388, ATCC VR 2332, VR 2385, VR 2386, VR 2429, VR 2474 and VR 2402; strains under CNCM I-1102, CNCMI-1140, CNCM I-1387, CNCM I-1388 or ECACC V93070108; or can actually be American type strains, such as North American PRRS virus pT7P129A; ATCC accession number VR-2332, ATCC accession number VR-2368; ATCC VR-2495; ATCCVR 2385, ATCC VR 2386, ATCC VR 2429, ATCC VR 2474 and ATCC VR 2402.

[0081] The recombinant techniques for preparing the modified sequences are well known to those skilled in the art and generally involve constructing a full-length complementary DNA copy (infectious clone) of the viral genome, which can then be modified by DNA recombination and manipulation methods such as site-directed mutagenesis, etc. In this way, for example, the antigenic sites or enzymatic properties of viral proteins can be modified. Infectious clones of PRRS virus strains of European and North American genotypes have been reported in the literature.

[0082] The PRRS virus strains of the present invention suitable for the vaccines of the present invention can be grown and harvested by methods known in the art, for example, by propagating in suitable host cells such as the simian cell line MA-104, Vero cells or porcine alveolar macrophages. PRRSV preferentially grows in alveolar lung macrophages (Wensvoort et al., 1991). Several cell lines such as CL2621 and other cell lines selected from the simian kidney cell line MA-104 (Benfield et al., 1992; Collins et al., 1992; Kim et al., 1993) are also susceptible to virus infection.

[0083] Accordingly, a preferred embodiment of the present invention is a vaccine comprising any PRRS virus of a PRRSV strain deposited under ECACC accession numbers 11012501, 11012501, accession number ECACC 04102703, ECACC 04102702, ECACC 04102704, CNCM accession number I-1140, CNCM accession number I-1387, and CNCM accession number I-1388, and any combination of these strains or their progeny. In a specific embodiment, PRRS virus 94881 is grown by a method in which the virus and host cells are co-inoculated into a bioreactor on the same day, as Figure 9 shown. Other features of the method for producing PRRS virus 94881 may be as described in the U.S. Provisional Application filed concurrently herewith (entitled "A Commercialscale process for production of PRRSV", application number 61 / 444,071). Although this is a method for growing PRRSV 94881, it should be understood that the virus can be propagated according to any known method known to those skilled in the art.

[0084] Preferably, the vaccine of the present invention is a modified live vaccine comprising one or more of these live strains in a suitable carrier, but inactivated virus can also be used to prepare an inactivated vaccine (KV). MLV is typically formulated to allow 10 1 to 10 7 virus particles to be administered per dose, preferably 10 3 to 10 5 particles per dose, more preferably 10 4 to 10 5 particles per dose (4.0 - 5.0 log 10 TCID 50 ). KV can be formulated based on the pre-inactivation titer of 10 3 to 10 10 virus particles per dose. The vaccine can comprise a pharmaceutically acceptable carrier, such as a physiological saline solution.

[0085] Pigs can be infected with PRRSV via the oral and nasal routes. The virus in the lungs is taken up by alveolar macrophages in the lungs and PRRSV replication is completed within 9 hours in these cells. PRRSV travels from the lungs to the pulmonary lymph nodes within 12 hours and to the peripheral lymph nodes, bone marrow, and spleen within 3 days. At these sites, only a few cells stain positive for viral antigen. The virus is present in the blood for at least 21 days and usually much longer. PRRSV antibodies are detected in the blood after 7 days. The combination of virus and antibodies in PRRS-infected pigs indicates that, although antibodies are present, viral infection can persist for a long time, although to a lesser extent. The alveolar cell population in the lungs is different from that of normal SPF lungs for at least 7 weeks.

[0086] The vaccine of the present invention can be provided in the form of a freeze-dried preparation of live virus, which is reconstituted with a solvent to produce an injectable solution. The solvent can be, for example, water, physiological saline, a buffer solution, or a co-solvent. The solvent can contain an adjuvant. The reconstituted vaccine can then be injected into pigs, for example, in the form of an intramuscular or intradermal injection into the neck. For intramuscular injection, a volume of 2 ml can be applied, and for intradermal injection, it is usually 0.2 ml. Thus, in another aspect, the present invention is a vaccine product that contains a freeze-dried composition of the virus and a reconstitution solvent in separate containers, and optionally further contains a leaflet or label containing instructions for use.

[0087] The vaccine of the present invention can not only contain one or more of the above strains, but can also include other components that are active against PRRS or other porcine viral or bacterial diseases, such as porcine circovirus or classical swine fever virus. Thus, the present invention further relates to the vaccine characterized in that it contains at least one other antigen that is active against non-PRRS porcine diseases. For example, the other antigen can include Mycoplasma hyopneumoniae, PCV2, SIV, Haemophilus parasuis, Erysipelothrix rhusiopathiae, Streptococcus suis, Actinobacillus pleuropneumoniae, Leptospira sp., parvovirus, and analogues thereof. In addition, the vaccine can contain certain pharmaceutically or veterinarily acceptable adjuvants. The present invention provides novel vaccine compositions, especially PRRS virus vaccines containing PRRSV94881, which further contain adjuvants that enhance the efficacy of the vaccine, such that when the combination of the adjuvant and the vaccine is administered, a better clinical response / result is seen than when the vaccine is administered alone. For example, the vaccine composition of the present invention can contain a PRRSV94881 virus vaccine and an adjuvant selected from the following: MCP-1, α-tocopherol (for example, α-tocopherol acetate, an exemplary form of which is Diluvac Haemophilus parasuis, Haemophilus sonmus fractions, carbopol, and combinations thereof. In some embodiments, the viral vaccine comprising the PRRS 94881 virus vaccine can be a recombinant subunit vaccine or can be a live attenuated virus vaccine. An exemplary live vaccine present is MLV, and PRRS94881 can be formulated in a manner similar to MLV.

[0088] In addition to the above, the immunogenic compositions of the present invention can also contain other components, provided that the other components do not interfere with the adjuvant or the basic viral vaccine. The other components include, for example, binders, colorants, desiccants, disinfectants, wetting agents, stabilizers, excipients, adhesives, plasticizers, tackifiers, thickeners, attachment materials, ointment bases, keratin removers, alkaline substances, adsorption promoters, fatty acids, fatty acid esters, higher alcohols, surfactants, water, and buffers. Preferred other components include buffers, ointment bases, fatty acids, disinfectants, alkaline substances, or surfactants.

[0089] The content or amount of the adjuvant used in the present invention can vary and can be determined by considering, for example, the nature and dosage form of the PRRS virus vaccine used. The adjuvant can comprise, for example, 1 to 100% by weight. The PRRSV 94881-based composition of the present invention is produced by mixing the adjuvant component (which is a separate component or in the presence of various other components) with the viral vaccine component. The composition can present the viral vaccine and the adjuvant as one formulation, or the adjuvant and the vaccine can be presented in different formulations that can be administered simultaneously or sequentially.

[0090] Thus, the adjuvant component of the immunogenic composition of the present invention can be administered separately from the viral vaccine when administered to an organism. Alternatively, the adjuvant of the present invention can be administered together with the viral vaccine in the form of a single vaccine composition. The viral vaccine can be any viral vaccine. More specific embodiments cover the use of a PRRS virus vaccine comprising PRRSV 94881. In addition, such a vaccine can be combined with other vaccines such as PRRS MLV and / or . This is only an exemplary PRRS virus combination vaccine and other such vaccine combinations can be easily prepared.

[0091] The immunogenic compositions described herein are particularly useful for inducing an antibody response against the PRRS virus. Administration of the vaccine preferably reduces the severity of one or more clinical symptoms, such as lung injury, anorexia, skin discoloration, lethargy, respiratory signs, mummified piglets, coughing, diarrhea, and combinations thereof associated with PRRSV infection.

[0092] Thus, compared to the results obtained by administering the PRRS virus vaccine alone, the composition particularly enhances the clinical outcomes in diseased animals. In a specific embodiment, the enhanced clinical outcome is at least a 50% reduction in the percentage of lung injury compared to animals that did not receive the immunogenic composition in combination with the adjuvant. In other embodiments, the enhanced clinical outcome is at least a 45% reduction in viremia in the animals compared to animals that did not receive the immunogenic composition in combination with the adjuvant.

[0093] Thus, in one aspect, the present invention relates to a modified vaccine, and more particularly to a modified PRRS virus vaccine, wherein the modification comprises mixing an adjuvant selected from MCP-1, Haemophilus somnus fraction, carbopol, and combinations thereof with the viral vaccine. The vaccine composition of the present invention may further comprise a pharmaceutically acceptable carrier.

[0094] The vaccine composition of the present invention can be formulated into, for example, liquid preparations, suspensions, ointments, powders, lotions, water-in-oil emulsions, oil-in-water emulsions, emulsions, creams, poultices, patches, and gels by any method known in the formulation art, and is preferably used as a medicament. Thus, according to another aspect of the present invention, there is provided a pharmaceutical composition comprising the above vaccine composition. The vaccine composition of the present invention can significantly induce antibody production when administered transdermally. Therefore, in another preferred embodiment of the present invention, the vaccine composition can be provided in the form of a transdermal preparation.

[0095] In addition, as described above, the virus and the adjuvant in the present invention can be administered to an organism together in the form of a single vaccine composition, or separately and differently in the form of an adjuvant preparation and the antigenic PRRS virus component of the vaccine, wherein the role of the adjuvant is reflected in that the amount of antibodies produced in the organism in response to the PRRS virus vaccine is significantly increased compared to administering the PRRS virus vaccine alone.

[0096] When the adjuvant and the PRRS virus vaccine are administered to an organism, the clinical outcomes of the animal are enhanced. The effective amount of the adjuvant and the immunologically effective amount of the PRRS virus vaccine can be easily determined by one of ordinary skill in the art considering, for example, the type and nature of the antigenic substance, the species of the organism, age, body weight, disease severity, disease type, administration time, and administration method, and using the amount of antibodies produced in the organism against the antigenic substance as an indicator.

[0097] The PRRS virus vaccine, adjuvant, or a combination thereof can be administered to an organism by any suitable method selected according to, for example, the animal's condition and the nature of the disease. Examples of such methods include intraperitoneal administration, transdermal administration (such as subcutaneous injection, intramuscular injection, intradermal injection, and patches), nasal administration, oral administration, transmucosal administration (such as rectal administration, vaginal administration, and corneal administration). Intramuscular administration is preferred.

[0098] An exemplary therapeutic dose of PRRSV MLV is about two milliliters (2 mL). Those skilled in the art will recognize that the dose can vary based on the breed, size, and other physical factors of the individual, as well as the specific formulation and route of administration of PRRSV MLV. Preferably, PRRSV MLV is administered in a single dose; however, other doses may be applicable. In addition, those skilled in the art will recognize through the present invention that the dose and frequency of administration are affected by the age and physical condition of the individual pig, other considerations common in the industry, and the specific conditions under which PRRSV MLV is administered.

[0099] In certain other embodiments, the vaccine can be a multivalent vaccine comprising two or more PRRS viruses, wherein at least one PRRS virus is the attenuated 94881 virus deposited under ECACC accession number 11012502. The other PRRS viruses can be one or more viruses selected from the following: PRRSV strain Lelystad virus (Lelystad material (CDI-NL-2.91)), or other strains such as those deposited under accession numbers ECACC 04102703, ECACC 04102702, ECACC 04102704, CNCM accession number I-1140, CNCM accession number I-1387, CNCM accession number I-1388, ATCC VR 2332, VR 2385, VR 2386, VR2429, VR 2474, and VR 2402; strains under CNCM I-1102, CNCM I-1140, CNCM I-1387, CNCM I-1388, or ECACCV93070108; or can actually be American type strains such as North American PRRS virus pT7P129A; ATCC accession number VR-2332, ATCC accession number VR-2368; ATCC VR-2495; ATCC VR 2385, ATCC VR 2386, ATCC VR 2429, ATCC VR 2474, and ATCC VR 2402.

[0100] PRRS virus-based vaccines can be used to vaccinate piglets and sows. In one aspect of the present invention, a specific dosing regimen is selected based on the age of the pig and the antigen selected for administration. This will enable pigs of any age to receive the most effective dose. In a preferred method, a therapeutic amount of PRRSV 94881 MLV is administered to pigs or piglets at about two weeks ± 5 days of age. The amount selected will vary depending on the age of the pig. Alternatively, different therapeutic amounts of this MLV are administered to pigs or piglets over about three weeks of age, and this amount also varies as the pigs receiving the administration grow or age. Thus, pigs, gilts or sows at about four weeks, six weeks, eight weeks, ten weeks, twelve weeks, fourteen weeks, sixteen weeks of age will all receive different amounts. The therapeutic dose to be used will be optimized most preferably in the art and is typically determined in clinical studies, where the minimum immunizing dose is determined based on protection against challenge with a virulent heterologous PRRSV in susceptible pigs. Preferably, the PRRSV MLV produced according to the methods described herein is administered intramuscularly; however, other methods of administration known and used in the art, such as intradermal, intranasal, intraretinal, oral, subcutaneous and the like, can be used.

[0101] Those skilled in the art will recognize that the vaccination method can include determining the appropriate timing and dose for vaccinating pigs against PRRSV. The method generally includes the steps of determining at least one variable selected from the age of the pig, health status, degree of innate immunity and degree of active immunity, and adjusting the standard dose concentration to suit these variables. Generally, the degree of innate immunity and the degree of active immunity will be determined by reference to a standard consisting of the average degree from a group of pigs of similar age and health status. In a particularly preferred method, all variables are considered before determining the most preferred dose concentration and dosing time selection.

[0102] In a preferred embodiment, the present invention also relates to isolated nucleic acids encoding specific open reading frames (ORFs) of the attenuated 94881 virus deposited under ECACC Accession No. 11012502 and the parent virulent 94881 virus deposited under ECACC Accession No. 11012501. For example, the complete nucleotide sequence of the attenuated 94881 virus deposited under ECACC Accession No. 11012502 has the sequence of SEQ ID NO: 1, which encodes the ORF1a, ORF1b, ORF2, ORF3, ORF4, ORF5, ORF6, ORF7 protein sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The complete nucleotide sequence of the parental virulent 94881 virus deposited under ECACC accession number 11012501 has the sequence of SEQ ID NO:10, which encodes the ORF1a, ORF1b, ORF2, ORF3, ORF4, ORF5, ORF6, and ORF7 protein sequences of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively.

[0103] The PRRSV 94881 vaccine can be administered in any conventional manner and in some preferred methods the administration is intramuscular. Preferably, the PRRSV vaccine administered after a single dose is administered as Similarly, the benefits of treating PRRSV infection or reducing its severity or morbidity are provided, however, if other antigens or combination or multivalent vaccines are selected, it should be understood that they can be administered in a conventional manner, including one or more booster doses after the initial administration. Those skilled in the art will be able to determine the appropriate dosage based on the selected PRRSV vaccine and the age range of the animal to which the antigen is administered.

[0104] In the specific embodiments presented below, pigs and sows were challenged with a new European origin PRRSV strain capable of reproducibly inducing respiratory disease in piglets. Historically, European origin PRRSV strains have not been able to reproduce respiratory disease in piglet models, and thus respiratory challenge models have relied on infection with non-European type strains. Due to the high genetic diversity, there is a need in Europe for new vaccines based on European type strains. In other embodiments, animals were challenged with a viral strain that causes reproductive failure in the gilts / sows challenge model. It has been found that the efficacy of MLV vaccines based on the attenuated 94881 virus deposited under ECACC accession number 11012502 or any virus prepared from this strain or the parental strain deposited under ECACC accession number 11012501 can be demonstrated using a variety of challenge models, as this strain is also effective in other PRRS virus-induced respiratory or reproductive failure models. Example

[0105] Example 1: Description of the PRRSV Respiratory Challenge Model

[0106] As described above, historically, European origin PRRSV strains have not been able to reproduce respiratory disease in piglet models. Due to the high genetic diversity, there is a need in Europe for new vaccines based on European type strains, and a good model for reproducible respiratory challenge using virulent European origin PRRSV strains is necessary for research. In the following examples, the inventors demonstrate that challenging pigs with a low passage European type challenge strain (4th passage) reliably produces respiratory symptoms.

[0107] In this study, 12 animals were divided into 3 groups, three weeks old at the time of allocation and approximately 10 weeks old at the time of challenge:

[0108] Group 1: Control group

[0109] Group 2: Challenge group (SD 35)

[0110] Group 3: Vaccinated with and then challenged (SD 35).

[0111] The study was conducted for 56 days, and necropsies were performed on 6 animals from each group 10 days after challenge; necropsies on all remaining animals were performed 21 days after challenge. Daily study parameters included: rectal temperature, respiration, and other clinical signs. Other study parameters included: body weight, mortality, viremia, seroconversion, and pathological and histological examination of the lungs.

[0112] On study day -7, the pigs were allocated to the groups. On study day 0, Group 3 was vaccinated with On study day 35, Groups 2 and 3 were challenged with a European type challenge strain. On day 45, 6 animals from each group were euthanized. The remaining animals were euthanized on day 56.

[0113] Figure 1A Shown are the cough measurements for each animal and per week in the form of mean scores. It can be seen that there is an increase in cough after challenge in the single challenge group (Group 2) and the Porcilis group (Group 3). Figure 1B Shown are the total clinical scores obtained from dyspnea, cough, nasal and eye discharges, and behavior. These data show an overall increase in the total clinical scores after challenge in the challenged and Porcilis groups. The rectal temperatures of the animals were monitored before and after challenge and show an increase in rectal temperature after challenge in the challenged and Porcilis groups (SD>35, Group 1-2 p≤0.001; Group 1-3 p≤0.001)( Figure 2 ).

[0114] Measurement of average daily weight ( Figure 3 ) shows that the ADW is significantly lower in the challenged (SD35-44 p≤0.001 and SD35-56 p≤0.01) and Porcilis vaccinated groups (SD35-44 p≤0.05 and SD35-56 p≤0.05) after challenge until the first and until the second necropsy.

[0115] Viremia was monitored using PCR ( Figure 4 ) and ELISA analysis ( Figure 5 ). PCR shown in Group 1: All animals from the control group remained negative. In Group 2: All animals were PRRSV positive after challenge; in Group 3: All animals were PRRSV positive after vaccination. ELISA shows: In Group 1: All animals remained negative; in Group 2: All animals were PRRSV AB positive after challenge; in Group 3: All animals were PRRSV AB positive after vaccination.

[0116] The lungs were also macroscopically examined ( Figure 6 ), where the areas of brownish spots and consolidation in the lungs were evaluated: Compared with the control group, obvious macroscopic changes were observed in the challenged and Porcilis groups (Group 1-2 p≤0.001; Group 1-3 p≤0.05). In the histopathological examination, the data also show the efficacy of vaccination ( Figures 7A to 7C ). The mean lung injury score was significantly higher in the challenged and Porcilis groups compared with the control group (Group 1-2 p≤0.001; Group 1-3 p≤0.001). Microscopic damage was stronger 10 days after infection.

[0117] In summary, compared with the negative control group, after challenge in the challenge control and Porcilis vaccination groups, coughing, total clinical score, and rectal temperature increased, and body weight was significantly lower in the challenge control and Porcilis groups (p<0.05). All animals from the Porcilis group were positive for PRRS virus and antibodies after vaccination. All animals from the challenge control were positive for PRRS virus and antibodies after challenge. Macroscopic and histological analysis of the lungs demonstrated severe macroscopic and microscopic lung lesions in the challenge control and Porcilis groups compared to the negative control group.

[0118] Therefore, this study confirmed that the European challenge strain used induced significant (p<0.05) disease compared to the negative control group: fever, coughing, weight loss, and severe macroscopic and microscopic lung lesions.

[0119] In addition, the European challenge strain has successfully demonstrated the production of a consistent and reproducible PRRSV-specific respiratory disease in a porcine challenge model and is thus suitable as a challenge virus in future efficacy studies. Within the parameters of this study, Porcilis PRRS showed a lack of efficacy against the European challenge strain.

[0120] Example 2: Evaluate the minimum immunizing dose of the attenuated PRRS virus 94881 in susceptible 2-week-old piglets after challenge with a heterologous European type PRRS isolate.

[0121] To evaluate the relative reduction of lung lesions after challenge with a heterologous European type PRRS isolate, a vaccination-challenge study was conducted at three different potency levels by administering a modified live virus (PRRS 94881 MLV) of a European origin isolate of porcine reproductive and respiratory syndrome (PRRS) vaccine to PRRS-susceptible piglets at approximately 14 days of age to evaluate the minimum immunizing dose (MID). Each vaccination group (groups 1-3) and the challenge control group (group 4) included 15 piglets. The negative control group (group 5) included 10 piglets.

[0122] Multiple parameters were monitored in the vaccine groups and the challenge control group, including: viremia after challenge, clinical assessment after vaccination, PRRS serology, viremia after vaccination, clinical observations after challenge, average daily weight gain (ADWG), rectal temperature, and detection of PRRS virus in the lungs. The unchallenged negative control group (group 5) was also included in this study to validate the effectiveness of the study by demonstrating that biosecurity was not breached throughout the study duration.

[0123] The challenge control group and the negative control group were PRRS negative until the day of challenge (D28), and the negative control group remained PRRS negative for the remainder of the study (D38), thus validating the effectiveness of the study.

[0124] The challenge was performed 4 weeks after vaccination. At this time, sera from only 2 animals in the low - potency vaccine group, 1 animal in the medium - potency vaccine group, and 3 animals in the high - potency vaccine group were positive for PRRS qPCR.

[0125] The challenge control group showed significant lung lesions characteristic of PRRS after challenge. After challenge, the low -, medium -, and high - potency vaccine groups had median total lung injury scores of 0.13%, 0.55%, and 0.40%, respectively, while the challenge control group had a median total lung injury score of 33.40%. The median total lung injury scores of the three vaccine - potency groups were significantly lower than those of the challenge control group (p < 0.0001). There was no statistical difference in the total lung injury scores among the vaccine - potency groups (p≥0.1484). The negative control group had a median total lung injury score of 0.00%.

[0126] During days 31, 35, and 38 of the post - challenge period of the study, viremia in the three vaccine - potency groups was significantly less than that in the challenge control group (p≤0.0093). Except at D35, where the high - potency vaccine group showed significantly lower viremia than the medium - potency vaccine group (p = 0.0442), there was no statistical difference in post - challenge viremia among the vaccine - potency groups. Negative - control piglets were viremia - negative at D31, D35, and D38.

[0127] Clinically, during the post - challenge period (days 29 - 38), the severity (p≤0.0082) and frequency (p≤0.0268) of coughing in the three vaccine - potency groups were less severe than those in the challenge control group. After challenge, fever in the challenge control group was more significant than in the three vaccine - potency groups. The ADWG of the three vaccine - potency groups was significantly higher than that of the challenge control group (p≤0.0027).

[0128] Based on the comparison with the challenge control after challenge with a heterologous European - sourced PRRS of variable virulence, the overall lung injury was reduced for all three potency levels, such that the MID of PRRS 94881 MLV as determined in this study was associated with the low - potency vaccine level of 1×10 2.77 TCID 50 / mL. When examining secondary parameters, all three vaccine - potency levels were associated with efficacy and no clear differences were shown among the potency groups.

[0129] General design of the study:

[0130] This was a blinded randomized design study conducted in 70 PRRS - susceptible piglets, 14 - 16 days of age, weaned on day 0 (D0). A description of the treatment groups is shown in Table 2.1 below:

[0131] Table 2.1 Treatment groups

[0132] Group Number of animals on Day 0 Treatment on Day 0 1 15 <![CDATA[IVP No. 1 (PRRS 94881 MLV with an average titer of 1×10 2.77 )]]> 2 15 <![CDATA[IVP No. 2 (PRRS 94881 MLV with an average titer of 1×10 4.42 )]]> 3 15 <![CDATA[IVP No. 3 (PRRS 94881 MLV with an average titer of 1×10 5.84 )]]> 4 15 CP (placebo-matched product without PRRS 94881 MLV) 5 10 CP (placebo-matched product without PRRS 94881 MLV)

[0133] Eighty-three piglets met the study inclusion criteria, and among them, the first 70 were randomly assigned by a biostatistician to one of five groups on D-3. The piglets were assigned to groups 1-4 with 15 piglets per group and 10 piglets to group 5. All 83 piglets were PRRS seronegative.

[0134] The post-vaccination clinical assessment of the piglets was observed from D-1 to D26, and the observations were recorded on a clinical assessment record form.

[0135] Serology: Venous whole blood was collected from the piglets on D0, D7, D14, D21, D28. The collection of the samples was recorded. The blood samples were centrifuged and serum was harvested from each tube, separated and transferred to appropriately labeled tubes. One set of serum samples was kept at 2-8 °C and another set of serum samples was kept at -70 ± 10 °C. The PRRS antibodies in the set of serum samples collected and kept at 2-8 °C on days 0, 7, 14, 21, 28 and 38 were tested. The results were reported as negative (ELISA S / P ratio < 0.4) or positive (ELISA S / P ratio ≥ 0.4).

[0136] PRRS viremia: The PRRSv RNA in the set of serum samples collected and kept at -70 ± 10 °C on days 0, 7, 14, 21, 28, 31, 35 and 38 was tested by qPCR (Appendix 1, Annex 7). The results were reported as n.d. (not detected), positive (European type PRRSv detected but not quantifiable, GE / mL (genome equivalents) = < 3.3 log) or reported value (log GE / mL). For statistical purposes, "not detected" was assigned a value of 0 log GE / mL, and "positive" values were assigned a value of 3.0 log GE / mL.

[0137] Average daily weight gain (ADWG): Each pig was weighed on a calibrated balance and the individual weights were recorded. The average daily weight gain from D0 to D28 and from D28 to D38 was determined.

[0138] Clinical observations after challenge: The clinical signs of disease in the piglets were observed by the study investigator or designee from D27 to D38 and recorded on a clinical observation record form. The observations included respiration, behavior and coughing based on a clinical observation scoring system as shown in Table 2.2 below.

[0139] Table 2.2 Clinical observation scoring system

[0140]

[0141] The total daily clinical observation score for each piglet was determined by summing its daily respiration, behavior and coughing scores.

[0142] Rectal temperature was collected from D27 to D38.

[0143] Total lung injury score: Autopsies were performed on all piglets that died before D38 and the remaining piglets that were euthanized at D38. Any gross lung pathology of each group of lungs was examined and the pathological percentage of each lung lobe was determined. If pathologies of other organs were noted, they were also described and recorded.

[0144] Lung qPCR for PRRSV: For each group of lungs, two samples were retained from the left and right apical lobes, left and right cardiac lobes, left and right diaphragmatic lobes, and the middle lobe. For one group of lung samples, all three samples from the left side were combined in one container; and all three samples from the right side and the middle lobe sample were combined in another container. Each container was filled with a sufficient amount of 10% formaldehyde solution. For the lung samples of other groups, all three left lung samples were combined in one container; and all three samples from the right side and the middle lobe sample were combined in another container.

[0145] Frozen lung tissue samples were kept at -70 ± 10 °C until further analysis. For each piglet, all left lung samples were homogenized and tested as a single combined sample; and all right lung tissues and middle lobe samples were homogenized and tested as a single combined sample. For left and right lung samples, the results were reported as n.d. (not detected), positive (European type PRRSv detected, but not quantifiable, GE / mL (genome equivalent) = < 3.3 log), or the test value (log GE / mL). For the purpose of analyzing each piglet, the average of the qPCR results of the left and right lung samples was recorded. For statistical purposes, "not detected" was designated as a value of 0 log GE / mL, and "positive" values were designated as 3.0 log GE / mL values.

[0146] Results

[0147] Total lung injury score after challenge: An overview of the group minimum, maximum, median, 95% confidence interval, Q range, and mean of the total lung injury score showed that the low, medium, and high - titer vaccine groups had median total lung injury scores of 0.13%, 0.55%, and 0.40%, respectively; while the challenged control group had a median total lung injury score of 33.40%. The median total lung injury scores of the three vaccine - titer groups were significantly lower than that of the challenged control group (p < 0.0001). There was no statistical difference in the total lung injury scores among the vaccine - titer groups (p ≥ 0.1484). The negative control group had a median total lung injury score of 0.00%.

[0148] For one animal (high - potency vaccine group), histological mild suppurative interstitial pneumonia with extensive fibrinopurulent pleurisy was noted. The trachea and alveoli were relatively unremarkable except for scattered neutrophils. Mycoplasma hyopneumoniae, PCV2, PRRSv, and SIV antigens in the lung tissue were IHC negative. The lung lesions were consistent with serositis generally associated with bacterial agents (Appendix 12; accession number 2009030254). Several pure bacterial cultures were isolated from the lung tissue and identified as Bordetella bronchiseptica and coagulase negative Staphylococcus. Although two types of bacteria were isolated from the lung tissue, this piglet was not removed from the total lung injury score analysis of Group 3.

[0149] Two out of 10 negative - control piglets showed minimal lung lesions (No. 1767, 0.55%; No. 1789, 0.61%). These lesions were considered insignificant and did not indicate PRRS. Two out of 10 negative - control piglets showed minimal lung lesions (No. 1767, 0.55%; No. 1789, 0.61%). These lesions were considered insignificant and did not indicate PRRS.

[0150] PRRS viremia after challenge: The individual results of PRRS viremia after challenge (D31 - D38) were tabulated, and it was found that all piglets in the three vaccine - potency groups and the challenge - control group had viremia after challenge. At all three time points after challenge, the viremia in the three vaccine - potency groups was significantly less than that in the challenge - control group (p ≤ 0.0093). Except at D35, where the high - potency vaccine group showed a lower mean viremia than the medium - potency vaccine group (p = 0.0442), there was no statistical difference in viremia between the vaccine - potency groups. The negative - control piglets were viremia - negative at D31, D35, and D38. The area under the curve (AUC) represents the amount and duration of the viral load and is an excellent assessment tool for examining viremia. Significant differences in the AUC were also detected between the three vaccine - potency groups and the challenge - control group for D28 to D38 (p ≤ 0.0162) and D31 to D38 (p < 0.0001). No differences in the AUC for the two time intervals were detected between the vaccine - potency groups (p ≥ 0.3669).

[0151] The group frequencies of viremia - positive piglets from D31 to D28 were also summarized and presented in Table 2.3. Since all vaccine and challenge - control piglets were viremia - positive after challenge, the frequency of viremia - positive piglets in each group was 100% at each time point. Therefore, the frequency of viremia after challenge was not analyzed.

[0152] Table 2.3 Summary of group frequencies of viremia - positive piglets - D31 to D38

[0153]

[0154] *Group 1 = Low - potency PRRS 94881 MLV; Group 2 = Medium - potency PRRS 94881 MLV; Group 3 = High - potency PRRS MLV; Group 4 = Challenge control group; Group 5 = Negative control group

[0155] Lung qPCR results: Inter - group differences in individual lung virus isolation results after challenge were summarized as the frequency (p - value) of qPCR - positive lung sample test results. Lung tissues from piglets in all three vaccine - potency groups and the challenge control group were qPCR - positive for PRRSv after challenge. No significant difference was detected between the vaccine - potency groups and the challenge control group (p = 1.0000). Since all vaccine - potency piglets were qPCR - positive for PRRSv, no tests were performed between the vaccine - potency groups.

[0156] Although no difference in the frequency of qPCR - positive lung tissues was detected between the vaccine - potency groups and the challenge control group, the difference in viral load in the lung tissues was significant. In fact, the low -, medium - and high - potency vaccine groups had median lung qPCR values of 6.88, 6.80 and 6.81 log 10 GE / mL, respectively; while the challenge control group had a median lung qPCR value of 8.13 log 10 GE / mL. The difference between the vaccine - potency groups and the challenge control group was significant (p ≤ 0.0001). Conversely, no difference in median lung qPCR values was detected between the vaccine - potency groups (p ≥ 0.7379).

[0157] Clinical observation scores after challenge: The median maximum clinical score for all five groups was 0 (score 0 represents normal breathing or normal behavior), indicating that abnormal breathing and abnormal behavior were not severe after challenge. In addition, no significant difference in both abnormal breathing and abnormal behavior was detected between the vaccine - potency groups and the challenge control group (p ≥ 0.0996).

[0158] Coughing was noted in all three vaccine - potency groups and the challenge control group, but was more severe in the challenge control group. For the three vaccine - potency groups, each group had a maximum cough score of 1 representing mild or intermittent coughing and a median maximum cough score of 0. Conversely, the challenge control group had a maximum cough score of 2 representing severe or severe recurrent coughing and a median maximum cough score of 1. The coughing severity in the three vaccine - potency groups was significantly less than that in the challenge control group (p ≤ 0.0082). No coughing was noted in the negative control group.

[0159] All three vaccine potency groups had a maximum total score of 1 and a median maximum score of 0. In contrast, the challenge control group had a maximum total score of 4 and a median maximum score of 1. The maximum total clinical scores of the three vaccine groups were significantly lower than those of the challenge control group (p ≤ 0.0047). In addition, the negative control group had a maximum total clinical score of 0 and a median maximum total clinical score of 0.

[0160] The frequency of abnormal breathing or abnormal behavior for at least one day from D29 to D38 was low in all groups. In fact, no abnormal breathing was noted in the low and medium vaccine potency groups from D29 to D38. One out of 15 piglets (7%) in the high vaccine potency group had abnormal breathing, and 3 out of 14 piglets (21%) in the challenge control group had abnormal breathing. No abnormal behavior was noted in any of the vaccine potency groups; while 2 out of 14 challenge control piglets (14%) showed abnormal behavior for at least one day after challenge. No significant difference was observed in the frequency of abnormal breathing or abnormal behavior for at least one day after challenge between the vaccine potency groups and the challenge control group (p ≥ 0.0996). No abnormal breathing or abnormal behavior was noted in the negative control group.

[0161] The coughing frequency in the challenge control group was much higher than that in the three vaccine potency groups. In fact, the frequencies of coughing for at least one day after challenge in the low, medium, and high vaccine potency groups were 14%, 13%, and 27%, respectively. In contrast, the frequency of coughing for at least one day after challenge in the challenge control group was 71%. The coughing frequencies of the three vaccine potency groups were significantly lower than those of the challenge control group (p ≤ 0.0268).

[0162] As indicated by a total clinical score > 0, the frequency of any clinical signs after challenge in the challenge control group was higher than that in the three vaccine potency groups. Similar to the coughing frequency, the frequencies of any clinical signs for at least one day after challenge in the low, medium, and high vaccine potency groups were 14%, 13%, and 33%, respectively; while 79% of the piglets in the challenge control group had at least one clinical sign after challenge. The frequencies of any clinical signs after challenge in the three vaccine potency groups were significantly lower than those of the challenge control group (p ≤ 0.0253). No clinical signs were noted in the negative control group during the same period.

[0163] The average scores of abnormal breathing or abnormal behavior from D29 to D38 were low in all groups. In fact, the average breathing score of the low and medium vaccine potency groups was 0.00 (normal), the average breathing score of the high vaccine potency group was 0.01, and the average breathing score of the challenge control group was 0.03. The average behavior scores of all three vaccine potency groups were 0.00; while the average behavior score of the challenge control group was 0.01. In addition, no significant difference in the average breathing and behavior scores was detected between the vaccine potency groups and the challenge control group (p ≥ 0.0996). The average breathing and behavior scores of the negative control group were 0.00.

[0164] The average cough score of the challenge control group was higher than that of the three vaccine potency groups. In fact, the average cough scores of the low-, medium- and high-potency vaccine groups were 0.01, 0.01 and 0.04, respectively. Conversely, the average cough score of the challenge control group was 0.28. The average cough scores of the three vaccine potency groups were significantly lower than that of the challenge control group (p≤0.0077).

[0165] The average total score in the challenge control group was higher than that of the three vaccine potency groups. Similar to the average cough score, the average total scores after challenge of the low-, medium- and high-potency vaccine groups were 0.01, 0.01 and 0.04, respectively; while the average total score of the challenge control group was 0.32. The average total scores of the three vaccine potency groups were significantly lower than that of the challenge control group (p≤0.0025).

[0166] Rectal temperature after challenge: The maximum group average rectal temperatures of the low-, medium- and high-potency vaccine groups between D29 and D38 were 40.20°C (D33), 40.33°C (D35) and 40.20°C (D37), respectively. The maximum group average rectal temperatures of the challenge control group and the negative control group between D29 and D38 were 40.51°C (D33) and 39.95°C (D33), respectively.

[0167] On D29 (39.47°C vs. 39.90°C), D31 (39.85°C vs. 40.20°C), D35 (39.80°C vs. 40.22°C) and D38 (39.86°C vs. 40.32°C), the rectal temperatures of the low-potency vaccine group were significantly lower than those of the challenge control group (p≤0.0317); while on D30, the rectal temperature of the low-potency vaccine was significantly higher than that of the challenge control group (40.08°C vs. 39.58°C; p = 0.0003). No significant differences were detected between the low-potency vaccine group and the challenge control group on D32 - D34 and D36 - D37 (p≥0.0545).

[0168] On D31 (39.62°C vs. 40.20°C), D33 (40.15°C vs. 40.51°C) and D38 (39.58°C vs. 40.32°C), the rectal temperatures of the medium-potency vaccine group were significantly lower than those of the challenge control group (p≤0.0227). No significant differences were detected between the medium-potency vaccine group and the challenge control group on D29 - D30, D32 and D34 - D37 (p≥0.0580).

[0169] At D33 (40.12 °C vs 40.51 °C), D35 (39.79 °C vs 40.22 °C), and D38 (39.55 °C vs 40.32 °C), the rectal temperatures of the high - titer vaccine group were significantly lower than those of the challenge control group (p ≤ 0.0147); while at D32, the rectal temperature of the high - titer vaccine group was significantly higher than that of the challenge control group (40.31 °C vs 39.90 °C; p = 0.0063). At D29 - D31, D34, and D36 - D37, no significant differences were detected between the high - titer vaccine group and the challenge control group (p ≥ 0.0708).

[0170] The frequency of fever in the three vaccine - titer groups after challenge was lower compared to the challenge control group. The overall frequency of fever was low and similar among the vaccine - titer groups.

[0171] Average daily weight gain (ADWG): The least - squares mean ADWGs of the low, medium, and high - titer vaccine groups from D0 to D28 were 0.4, 0.3, and 0.4 kg / day, respectively. The least - squares mean ADWG of the challenge control group during the same period was 0.3 kg / day. From D0 to D28, the least - squares mean ADWG of the low - titer vaccine group was significantly higher than that of the challenge control group (p = 0.0292); while no significant differences in the least - squares mean ADWG were detected between the vaccine - titer groups (p ≥ 0.1262) or between the vaccine - titer groups and the challenge control group (p ≥ 0.1293). During the same period, the average ADWG of the negative control group was 0.5 kg / day.

[0172] The least - squares mean ADWGs of the low, medium, and high - titer vaccine groups from D28 to D38 were 0.5, 0.5, and 0.4 kg / day, respectively. The least - squares mean ADWG of the challenge control group during the same period was 0.3 kg / day. The weight gain in the three vaccine - titer groups after challenge was significantly more than that of the challenge control group (p ≤ 0.0027). During the same period, the average ADWG of the negative control group was 0.6 kg / day.

[0173] Clinical assessment after vaccination: In the low - potency vaccine group, one piglet (1735) was noted to lose weight from D0 to D10. Additionally, one piglet was noted to start losing weight at D6, which continued for 16 days, showed coughing for 2 days and depression for 9 days, and was euthanized at D21 for animal welfare reasons due to poor health. The total lung injury score for piglet 1727 was 10.8%. Since this value was measured before challenge, it was not included in the post - challenge total lung injury analysis. Additionally, red / purple consolidation areas were noted in the ventral anterior region of the lung, the liver was pale, and multiple red / purple areas were present in the renal pelvis of the kidney. The pathologist noted fatty infiltration of the central lobules in the liver, which is generally seen in cases of negative energy balance and subsequent lipolysis. No other lesions were observed in the liver, kidney, and lung sections. By PCR, the lung tissue was positive for European - type PRRS. No growth was detected for routine bacterial culture.

[0174] In the medium - potency vaccine group, one piglet was excluded from the study at D0 before treatment because of poor health and was replaced with another piglet. Two piglets showed coughing for 1 day and 3 days respectively, starting at D12. Four piglets were noted to lose weight at D2 and / or D3.

[0175] In the high - potency vaccine group, one piglet (1728) showed lameness of one leg or lameness and swelling of one leg from D7 to D26. Starting 18 days after vaccination, one piglet was noted to lose weight for 6 days, showed coughing for 1 day and had rough hair for 4 days. Another piglet was noted to lose weight at D2. Two piglets showed coughing for 2 days and 1 day respectively, starting at D9. One piglet showed diarrhea for 1 day (D14).

[0176] In the challenge control group, six piglets showed periodic coughing, accumulating to 1 to 6 days, starting with the first piglet at D7 and ending with three piglets at D21. Two piglets were noted to lose weight, for 2 days and 11 days respectively, with one piglet starting at D1. The second of these two piglets also showed depression and rough hair for 4 days, weakness of the legs for 1 day, and was found dead at D15. At autopsy of this piglet, no lung injury was noted (lung injury score was 0%), no feed was present in the stomach, and no abdominal fat was present, and the cause of death was diagnosed as starvation. Since this lung injury score was measured before challenge, it was not included in the post - challenge lung injury analysis.

[0177] Group test results (p - values) for any abnormal clinical assessment for at least one day from D1 to D26 are summarized. No significant differences were detected between the vaccine potency groups and the challenge control group (p≥0.0502); no significant differences were detected between the vaccine potency groups either (p≥0.3898). No piglets in the negative control group showed abnormal clinical assessments from D1 to D26.

[0178] PRRS Serology: The PRRS ELISA serological results of individual piglets were summarized. Piglets in the negative control group remained PRRS seronegative throughout the study period. Seroconversion was observed in all three vaccine potency groups up to 14 days post-vaccination; while the challenge control group remained PRRS seronegative until after challenge. At 10 days post-challenge (D38), all piglets in the low and high vaccine potency groups and the challenge control group were PRRS seropositive; while 14 out of 15 piglets in the medium vaccine potency group were PRRS seropositive.

[0179] For between-group differences, the PRRS ELISA positive serological test results (p-values) were determined. At D14, D21, and D28, the frequency of PRRS ELISA positive piglets in the three vaccine potency groups was significantly higher than that in the challenge control group (p < 0.0001). At D38, no significant difference was detected between the three vaccine potency groups and the challenge control group (p = 1.0000 or not tested), and no significant difference was detected between the three vaccine potency groups at any time point (p = 1.0000 or not tested).

[0180] PRRS Viremia after Vaccination: The individual PRRS viremia results after vaccination were determined. For statistical purposes, "not detected" results were assigned a value of 0 log GE / mL, and "positive" values were assigned a value of 3.0 log GE / mL. All groups were PRRS viremia negative at D0. The group viremia (qPCR) (potency (log GE / mL) data from D7 to D28 post-vaccination) was evaluated. Piglets in the three vaccine potency groups reached peak mean viremia at D7, followed by a slow decline in the potency levels of all three groups before challenge (SD28). In contrast, the challenge control group and the negative control group remained viremia negative during the pre-challenge phase of the study. According to the group test results (p-values) of the qPCR results at D7, D14, D21, and D28, it was found that at D7 - D28, the median qPCR values of all three vaccine potency groups were significantly higher than those of the challenge control group (p ≤ 0.0159). The median qPCR of the medium vaccine potency group was significantly higher than that of the low vaccine potency group (p = 0.0193); in addition, no difference in the median qPCR was detected between the vaccine groups before challenge (p ≥ 0.0594).

[0181] Four weeks after vaccination, the viremia frequency was low in the three vaccine potency groups.

[0182] The following conclusions can be drawn based on these study results:

[0183] · Biosecurity was not compromised during the study and it was confirmed that piglets were susceptible to PRRS, confirming the validity and suitability of the study;

[0184] · Substantial PRRS clinical disease was evident in the challenge control group, thus validating this challenge model as a sufficient laboratory tool to evaluate PRRS vaccine efficacy, and more particularly as a MID for PRRS 94881 MLV;

[0185] · All three doses of PRRS 94881 MLV caused a significant reduction in lung lesions, as well as a significant reduction in viremia, viral load in lung tissue, coughing, total clinical observation score, fever, and ADWG after challenge;

[0186] · After challenge with a heterologous European origin PRRS of virulent nature, the overall lung lesions at all three potency levels were relatively reduced compared to the challenge control, thus the MID of PRRS 94881 MLV determined in the study was associated with a low vaccine potency level of 1×10 2.77 TCID 50 / mL.

[0187] Further description of results

[0188] Clinical observations were made daily. Quantitative RT-PCR was performed on samples from blood, oral, fecal, and nasal swabs, as well as bronchoalveolar lavage fluid using PRRSV European type-specific primers.

[0189] Based on these studies, the data showed that the piglets appeared normal and healthy except for a few lame pigs. At necropsy, there were no abnormalities except for slightly enlarged inguinal lymph nodes in 1 - 2 animals. Importantly, no lung lesions were observed in the vaccinated group.

[0190] Figure 8Show the percentage of viremic animals in the sentinel group compared to the group vaccinated with the composition containing the attenuated PRRS virus strain deposited under ECACC accession number 11012502. This figure shows the transmission of the vaccine strain from vaccinated animals to sentinels. At the peak of PRRS viremia (SD21) as detected by quantitative RT-PCR, the viral load in sentinel-infected pigs (average viral load of 3.347 GE / ml) was 78.47% lower than that in vaccinated animals (average viral load of 4.014 GE / ml). In a room where unprocessed sows were mixed with their vaccinated offspring, only 3 out of 8 sows tested positive for PRRSV in the blood by RT-PCR, thus confirming that the exposure of PRRS MLV vaccine to unprocessed adult animals was limited and inefficient. In this study, the vaccine virus 94881 MLV was mainly excreted in feces. In fact, in feces, the virus could be detected from 1 day to 21 days after vaccination. Five days after vaccination, almost 30% of the vaccinated animals excreted the virus in feces. PRRS virus was not detected in nasal secretions, and the PRRS virus was detected via oral secretions in only a few animals (2 out of 56 sampled animals were detected 5 days after vaccination).

[0191] Example 3: Use PRRS as an exemplary material and method for testing vaccine efficacy

[0192] In this study, a selected number (e.g., 14) of healthy pregnant sows from herds confirmed to be PRRSV-negative (by virological and serological tests) were used. The sows were approaching their first or second farrowing and were confirmed pregnant at 94 days of gestation at the time of vaccination / challenge infection. The sows were divided into 3 treatment groups. The first group was treated with 2 ml of a commercial dose of Porcilis 4.0 TCID 50 PRRS (administered intramuscularly) containing at least 10 TM at 94 days of gestation. The challenge control group (Group 2) received 2 ml of cell culture medium containing 10 4.72 TCID 50 of a pathogenic European field isolate (4th passage) via intranasal route. Group 3 was vaccinated intramuscularly with 2 ml of a dose of PRRS MLV containing 10 7.6 TCID 50 of the PRRS MLV containing the attenuated PRRS virus strain deposited under ECACC accession number 11012502 on January 25, 2011, 7 days before insemination, and at 94 days of gestation, challenged with a European field isolate (4th passage) (2 ml of cell culture medium containing 10 4.72 TCID 50 via intranasal route).

[0193] Monitor the animals from Group 1 until 5 days after parturition. Monitor the animals from Group 2 and Group 3 until 28 days after parturition.

[0194] Animal period: One week before vaccination, acclimatize all sows to the animal laboratory. The researchers observe the overall health status of the sows and piglets daily. Each animal that dies or is euthanized undergoes a post-mortem examination and subsequent laboratory analysis.

[0195] Confirm pregnancy by ultrasound examination. Obtain sera from the sows at study days 0, 7, 14 and at parturition for PCR and ELISA studies. Any material associated with abortion undergoes laboratory studies.

[0196] Perform a routine macroscopic pathological study on all stillborn piglets. Collect lung tissue samples from all lung lobes from the stillborn piglets and their mothers. Samples for PCR testing are stored at -70 °C. Collect 2 ml of pre-suckling blood from each piglet on the day of birth. Prepare sera and aliquots are stored at -70 °C. Sera are used to test for viremia to evaluate transplacental infection. All piglets from Group 1 that survive until 5 days of age are euthanized at 5 days of age.

[0197] Clinical and reproductive efficacy parameters: The following criteria are illustrative standards (priority order) for study: the number of live-born piglets per litter, the number of stillborn piglets per litter, the number of mummified piglets per litter and the number of piglets that survive until 5 days or 28 days of age. Determine the number of piglets born with viremia using pre-suckling sera. Study the frequency of PCR-positive blood and tissue samples from sows and / or piglets to evaluate the epidemiology and course of infection.

[0198] Field samples: The field samples studied in this research are taken from routine PRRSV diagnostic methods and consist of blood, sera and various organ materials (mainly lungs and lymph nodes) from different European countries. Samples are stored at -20 °C for a maximum of 3 days, then RNA is prepared, and subsequently the remaining material is transferred to -70 °C for long-term storage. RNA and RT-PCR products are stored at -20 °C.

[0199] Cell culture: MA104 cells (clone CL2621) are grown in MEM (Dulbecco, Germany) supplemented with 10% FCS and antibiotics.

[0200] Harvest porcine alveolar macrophages using the method described by Wensvoort et al. (Wensvoort, G. et al., Vet. Quat. 1991, 13:121 - 130) with the following modifications: Infuse 50 - 100 ml of PBS into each lung lobe and then massage for 3 to 5 minutes. Subsequently, recover the fluid from the lung lobe and pass it through a gauze filter. Repeat this procedure until the lavage fluid is clear. Pool the lavage fluids and centrifuge at 500 g for 15 minutes at room temperature. Wash the centrifuged pellet in PBS and freeze aliquots containing 1×10 7 cells in 50% RPMI 1640 (Biochrom), 40% FCS, and 10% DMSO at - 196°C. For further use, PAM are cultured in RPMI 1640 medium supplemented with 10% FCS and antibiotics.

[0201] Prepare organ material for virus isolation in cell culture: Transfer approximately 0.5 cm 3 of tissue material to 1.8 ml of sterile PBS in a tube containing 1 steel homogenizing ball. Agitate the tube for 10 minutes until the organ material is homogenized. Aggregate cell debris by centrifugation at 450 g and room temperature for 2 minutes. Pass the supernatant through a 0.45 μm pore sterile filter and store at - 70°C. Using a 24 - well microtiter plate, inoculate a semiconfluent cell culture monolayer with 30 μl aliquots.

[0202] RNA isolation: Extract RNA from organ material using the RNeasy Mini kit and from serum, plasma, cell culture supernatant, and vaccine solution using the QTAamp viral RNA Mini kit (both Qiagen) according to the manufacturer's recommendations, preparing using approximately 100 mg of organ material and 140 μl of fluid material respectively. RNA is finally eluted in 65 μl of buffer as recommended by the manufacturer.

[0203] Plaque purification of virus: A confluent monolayer of Ma104 cells that had been inoculated 48 hours previously in a 10 cm cell culture dish was infected with each individual virus diluted serially 10 - fold from 10 -1 to 10 -4 . The cells were incubated with the virus dilutions for 1 hour, then the dilutions were removed and the cells were overlaid with 30 ml of Ma104 medium (Sigma) containing 5% methylcellulose. Plaques were picked 5 to 7 days later and transferred to a Ma104 monolayer in a 24 - well plate. Virus from these plates was harvested at approximately 50% CPE and subjected to further analysis.

[0204] Immunofluorescence assay: Cells were fixed with ice-cold acetone: methanol (1:1) at -20 °C for 15 minutes and then air-dried. After rehydration in PBS, the cells were incubated with a PRRSV-specific monoclonal antibody SDOW17 (Rural Technologies Inc., USA) diluted 1:1000 in PBS for 1 hour. After washing 3 times with PBS, the cells were incubated with a goat anti-mouse FITC-conjugated secondary antibody (Dianova, Hamburg, Germany) (1:150 in PBS) for 1 hour. After a final wash 3 times with PBS, the cells were overlaid with glycerol:PBS solution (1:1) and examined by immunofluorescence microscopy.

[0205] Diagnostic nRT-PCR: Diagnostic RT-nPCR can be performed to examine the presence of PRRSV-European type virus in a sample.

[0206] An exemplary diagnostic RT-nPCR can be performed using the Titan One Tube kit (Roche Molecular Biochemicals) as follows: [5 μl of total RNA preparation, 1* RT-PCR buffer, 0.4 mM dNTP, 20 pmol each of primers PLS and PLR, 5 mM dithiothreitol, 1 mM MgCl2, 2.5 - 5 U RNasin (Promega Ltd), 1 - 2.5 U enzyme mixture, adjusted to a final volume of 25 μl with DEPC-treated distilled water]. The conventional cycling conditions used can be: 45 °C for 1 hour, 94 °C for 2 minutes, and 30 cycles of 94 °C for 30 seconds, 58 °C for 45 seconds, and 68 °C for 45 seconds, with a final extension step of 68 °C for 5 minutes. The nested PCR reaction was performed using Qiagen Taq (Qiagen AG) as follows: [1 μl of RT-PCR product, 1* PCR buffer, 10 μl of Q-solution, 3.5 mM MgCl2, 0.3 mM dNTP, 20 pmol each of EU-7-n-s and EU-7-n-as primers, 2.5 U Taq polymerase, adjusted to a final volume of 50 μl with distilled water]. The cycling conditions were as follows: 7 cycles of 94 °C for 1 minute, 58 °C for 1 minute, and 72 °C for 1 minute, followed by 30 cycles of 94 °C for 1 minute and 70 °C for 1.5 minutes (no annealing step), with a final extension step of 70 °C for 5 minutes.

[0207] Nucleotide sequencing: The nested PCR products can be subjected to nucleotide sequencing, which are generated directly from the PCR reaction using primers containing M13 tags, or from PCR products excised by agarose gel and purified using the JETsorb Gel Extraction kit (Genomed). Sequencing was performed using an automated sequencer LI-COR DNA Analyzer GENE READIR (Performed by LI-COR Inc., Lincoln, Nebr., USA). The nucleotide and deduced amino acid sequences were analyzed using Version 1.1 (LI-COR Inc., Lincoln, Nebr., USA) and the DNASIS.RTM. 2.6 software package (Hitachi Software Genetic Systems Inc., San Francisco, USA).

[0208] Example 4: Determination of the full-length genomic sequence of PRRSV 94881

[0209] This example demonstrates the determination of the full-length genomic nucleotide sequences of the attenuated 94881 strain and its parental 94881 fifth passage. These sequences did not show any undefined nucleotide positions indicating the presence of homologous viral inclusions. Comparison of the 94881 master seed virus with the European reference virus strain, Lelystad virus (LV), showed that nucleotide homology in eight different viral genes was in the range of 85.40% to 95.09%, and amino acid identity between the two virus strains was in the range of 86.39% to 97.27%. Two deletions were identified in ORF 1a of 94881 MSV compared to LV. Comparison between the 94881 master seed virus and its parental fifth passage showed 26 nucleotide exchanges, resulting in a total of 14 amino acid exchanges.

[0210] For the determination of the full-length genomic sequence of the 94881 master seed virus, a total of 1 reverse transcription, 17 external PCRs, and 58 internal PCRs were performed, generating 40 PCR products, which were used for sequencing. In the case of the 94881 fifth passage, 1 reverse transcription, 17 external PCRs, and 67 internal PCRs were also performed, generating 40 PCR products, which were also used for sequencing.

[0211] Overlapping sequence alignment analysis of two virus isolates each containing the complete open reading frames (ORFs) 1a to 7 yielded a full-length sequence of 14,843 nucleotides. In addition, 177 nucleotides of the 5'-untranslated region (5'NTR) and 43 nucleotides of the 3'-untranslated region (3'NTR) could be determined separately. Compared to the European PRRSV reference virus isolate, Lelystad (LV) (GenBank accession number M96262), 44 nucleotides of the 5'NTR and 83 nucleotides of the 3'NTR could not be determined because those regions were used for primer annealing regions.

[0212] Sequencing reactions of the two virus strains produced clear nucleotide sequences without any wobbling or any other indication of mixed sequences. After translation into amino acids, clear amino acid sequences without any suspect amino acids were obtained for sequence comparison of 94881 MSV and LV and with the fifth generation of the parental strain 94881. Nucleotide sequences were aligned and compared between 94881 MSV and Riley-Sta virus, and substantial differences were shown at the nucleotide and amino acid levels. An alignment was also performed between 94881 MSV and its fifth-generation parental strain.

[0213] Sequence comparison with LV yielded nucleotide homologies of 85.40% to 95.09% in eight different viral genes, and amino acid identities between the two virus strains of 86.39% to 97.27%. Compared to LV, two deletions were identified in ORF 1a of 94881 MSV. One deletion of 138 nucleotides was located at positions 2154 to 2292 of LV and resulted in the deletion of 46 amino acids. At positions 2686 to 2688, another triplet was deleted, resulting in the deletion of the amino acid phenylalanine. All alignments of all nucleotide homologies and amino acid identities between LV and the two 94881 strains are shown in Table 4.1.

[0214] Table 4.1: Alignment of nucleotide and amino acid sequence comparisons of 94881 provirus with the European reference virus Riley-Sta virus

[0215]

[0216] NTR: Non-translated region

[0217] * = Only 177 nucleotides were compared between Riley-Sta virus and 94881 MSV. The remaining 44 nucleotides located upstream were not determined.

[0218] ** = Isolate 94881 MSV showed two deletions in ORF 1a, one of 138 nucleotides and one of 3 nucleotides. The complete nucleotide and amino acid sequences of the reference virus LV were used for homology and identity calculations, and the deletions were rated as deviations. The length of the corresponding viral gene of LV was 7191 nucleotides, and the length of the corresponding polyprotein was 2396 amino acids. Calculations of gene homology and amino acid identity refer to the number of 7191 nucleotides and 2396 amino acids, respectively.

[0219] *** = Only 44 nucleotides were compared between Riley-Sta virus and 94881 MSV. The remaining 83 nucleotides located downstream were not determined.

[0220] Sequence comparison of the full-length nucleotide sequences of the 94881 provirus and the 5th generation of the 94881 parental strain showed a total of 26 nucleotide exchanges between the two. The nucleotide exchanges were distributed as follows: 15 in ORF 1a, 4 in ORF 1b, 2 in ORF2, none in ORF 3, 1 in ORF 4, 3 in ORF 5, 1 in ORF 6 and none in ORF 7. These nucleotide exchanges resulted in a total of 14 amino acid exchanges, which were distributed as follows: 8 in polyprotein 1a, 1 in polyprotein 1b, 1 in glycoprotein 2, none in glycoprotein 3, 1 in glycoprotein 4, 2 in glycoprotein 5 and 1 in the matrix protein. Compared with the Lelystad virus, both virus strains showed the same deletion in ORF 1a. The alignment of all nucleotide exchanges and the resulting amino acid exchanges (including their positions in the viral genes and the corresponding proteins) are shown in detail in Table 4.2.

[0221] Example 5 - Cultivation of the Deposited Virus and MSV

[0222] As described above, the parental (low passage) 94881 was deposited with the European Collection of Cell Cultures (ECACC) under accession number ECACC 11012501, and the 94881 provirus (MSV) was deposited with the European Collection of Cell Cultures (ECACC) under accession number ECACC 11012502. The growth and culture conditions for the parental virus and MSV are provided in this example.

[0223] Parental 94881: It is a genotype 1 PRRSV virus and thus a European genotype PRRSV. This virus has a porcine host. The parental virus deposited under 11012501 was stored at a titer of 5.81 Log 10 TCID 50 / mL. The host cells for virus propagation were MA 104 cells. These cells were cultured in Minimum Essential Medium (MEM) with 3.7 g / L sodium bicarbonate and containing 6% irradiated fetal bovine serum at 37 ± 1 °C. The cells were seeded in T-flasks (75 cm 2 ) at a seeding density of 2 × 10 4 to 2 × 10 5 cells / cm 2, and culture for 3 to 7 days until 100% confluence before subculture. To allow virus growth, add the virus to the cells in a T-flask at an MOI of 0.001 - 0.01. Infected cells typically reach a confluence of approximately 80 - 100% 1 - 3 days after cell seeding. After infection, the infected cells are cultured at 37 ± 1 °C for 3 - 10 days and then the virus is harvested. Harvesting is performed when the monolayer shows approximately 80 - 100% cytopathic effect (CPE) 3 - 10 days after infection. Harvest the supernatant of the infected MA104 tissue culture (used medium + PRRSV from cultures with 80 - 100% CPE), which contains the propagated virus. This supernatant can be stored at -70 °C / -80 °C for several months before use. Assess the TCID of the virus by Spearman and Kaerber calculations 50 to determine the log of the sample 10 TCID 50 / mL.

[0224] Vaccine (high passage) 94881 seed virus (MSV): It is a type 1 genotype virus of PRRSV, so it is a European genotype PRRSV. This virus has a porcine host. The MSV preserved under 11012502 is preserved at a titer of 6.43 Log 10 TCID 50 / mL. The host cells used for MSV propagation are MA 104 cells. These cells are cultured at 36 ± 2 °C in minimum essential medium (MEM) with 1.4 g / L sodium bicarbonate and containing 10% irradiated fetal bovine serum. Cells are seeded in T-flasks (75 - 150 cm 2 ) or 850 cm 2 roller bottles at a seeding density of 1 × 10 4 to 1 × 10 5 cells / cm 2 , and culture for 3 to 7 days until 100% confluence before subculture. To allow virus growth, add the virus to the cells in the T-flask or roller bottle at an MOI of 0.001 - 0.01. Infected cells typically reach a confluence of approximately 80 - 100% 1 - 3 days after cell seeding. After infection, the infected cells are cultured at 36 ± 2 °C for 3 - 14 days and then the virus is harvested. Harvesting is performed when the monolayer shows approximately 80 - 100% cytopathic effect (CPE) 3 - 14 days after infection. Harvest the supernatant of the infected MA104 tissue culture (used medium + PRRSV from cultures with 80 - 100% CPE), which contains the propagated virus. This supernatant can be stored at 2 - 8 °C for 5 - 10 days, -70 °C for several months. Assess the TCID of MSV by Reed and Muench calculations 50 to determine the log of the sample10 TCID 50 / mL.

[0225] Table 4.2: Alignment of Nucleotide and Amino Acid Sequences of 94,881 Proviruses and 94,881 Parental Strains

[0226]

[0227] Example 6: PRRS 94881 MLV Gilt MID Study

[0228] Overview

[0229] The objective of this vaccination-challenge study was to evaluate the minimum immunizing dose (MID) of the modified live virus code 19T1.U_ (PRRS 94881 MLV), an isolate of European origin of the porcine reproductive and respiratory syndrome vaccine, in gilts. Approximately 28 days before breeding (day 0; D0), two different potency levels were administered to PRRS-seronegative gilts. At approximately 90 days of gestation (D118), the gilts were challenged with a heterologous European type isolate of PRRSv, and the percentage of total live-born piglets or live-born piglets and piglets weaned at 20 days of age in the gilts was evaluated to determine the MID. At the time of challenge on day 118 (D118), the challenge control group consisted of 8 pregnant gilts (group 1, placebo), the low-potency group consisted of 8 pregnant gilts (group 2, 1×10 2.43 TCID 50 ), the high-potency group consisted of 8 pregnant gilts (group 3, 1×10 3.90 TCID 50 ), and the negative control group consisted of 5 pregnant gilts (group 4, placebo, not challenged).

[0230] Compared to the challenge control group, both the low-potency and high-potency groups resulted in a higher percentage of live piglets per litter at farrowing (P≤0.0455) and a higher percentage and number of live piglets per litter at weaning (P≤0.0203).

[0231] Regarding supportive efficacy parameters, compared to the challenge control group, the high-dose group resulted in a higher percentage and number of healthy piglets per sow at farrowing (P≤0.0211), a lower percentage and number of weak and mummified fetuses (P≤0.0090), a lower percentage of qPCR-positive sows and a lower viral load in sows after challenge at D125, DOF 0, and DOF+13 (P≤0.0155), a lower percentage of qPCR-positive piglets per sow and a lower viral load in DOF 0 piglets (P≤0.0030), a lower percentage of piglets with clinical disease per sow (P<0.0001), and higher piglet body weight and ADWG at DOF+20 (P<0.0013).

[0232] Compared to the challenge control group, the low-dose group resulted in a higher percentage of healthy piglets per sow at farrowing (P = 0.0138), a lower percentage and number of mummified fetuses (P≤0.0190), a lower percentage of qPCR-positive sows and a lower viral load in sows after challenge at D125, D132, DOF 0, and DOF+13 (P≤0.0290), a lower percentage of qPCR-positive piglets per sow at DOF 0 (P = 0.0381), a lower percentage of piglets with clinical disease per sow (P<0.0001), and higher piglet body weight and ADWG at DOF+20 (P<0.0028).

[0233] In summary, the study objective was met and data from this study determined the MID of PRRS 94881 MLV in sows to be 1×10 2.43 TCID 50 / 2 mL. Additionally, this study determined the duration of immunity (DOI) in sows to be approximately 4 months.

[0234] Study objective / aim

[0235] The objective of this vaccination-challenge study was to evaluate the minimum immunizing dose (MID) of a modified live virus code 19T1.U_ (PRRS 94881 MLV) of a European-origin isolate of porcine reproductive and respiratory syndrome vaccine, administered to PRRS-seronegative sows before breeding at two different potency levels (Group 2, low potency; Group 3, high potency), such that after challenging the sows with a heterologous European-type isolate of porcine reproductive and respiratory syndrome virus (PRRSv) at approximately 90 days of gestation, the percentage of live-born piglets and 21-day-old weaned piglets would be higher. The main criterion for meeting this objective was that one or both vaccine groups had to demonstrate a correspondingly higher percentage or number of live-born piglets and 20-day-old (DOF+20) weaned piglets compared to the challenge control group (Group 1).

[0236] Other parameters analyzed between the vaccinated and challenged control groups included gilt clinical assessment after vaccination, gilt PRRS serology, gilt viremia, gilt clinical observations, piglet viremia, total piglets per litter, healthy live piglets per litter, weak live piglets per litter, mummies per litter, stillbirths per litter, crushed / dead piglets per litter, piglet clinical observations, and average daily weight gain of piglets (ADWG). These parameters were analyzed as supportive parameters and not as primary parameters to meet the study objectives.

[0237] Event schedule

[0238] Table 6.1 Event schedule of gilts

[0239]

[0240] *DOF = Day of farrowing

[0241] Table 6.2 Event schedule of piglets

[0242]

[0243] Study design

[0244] Table 6.3 Study design

[0245]

[0246] Blinding criteria

[0247] The study investigators and assignees were unaware of the gilts assigned to groups 1 - 4. To maintain the unawareness of the study investigators and assignees, the IVP and CP were administered to the assigned gilts by non - data collectors on D0. Laboratory staff were unaware of the treatment received by each gilt while performing their respective tasks.

[0248] Materials

[0249] Investigational veterinary product (IVP) and control product (CP)

[0250] Table 6.4 Investigational veterinary product (IVP)

[0251]

[0252] Table 6.6: Control product (CP)

[0253]

[0254] Challenge materials

[0255] Table 6.7 Challenge materials

[0256]

[0257] Extra treatment

[0258] D8 to D21, Matrix TM (6.8 mL; Alternogest; Intervet / Schering Plough Animal Health) was given to each gilt in the feed to synchronize the estrous cycle.

[0259] Oxytocin (VetTek) was given at parturition to assist the gilts in farrowing, rather than to initiate farrowing. At farrowing, all live piglets received 1.0 mL of an iron injection (Phoenix or Durvet) intramuscularly in the right thigh to prevent iron deficiency anemia shortly after birth. Additionally, all live piglets received gentamicin (SparHawk Laboratories Inc) as a prophylactic measure against diarrhea shortly after birth. All treatments were recorded on the Biological & Pharmaceutical Treatment Record form.

[0260] Treatment

[0261] Rationality judgment of drug administration

[0262] Each IVP was given to the allotted gilts at a dose of 2.0 mL to evaluate the MID of PRRS 94881 MLV. The 2.0 mL dose of CP was given to the gilts allotted to Group 1 and Group 4.

[0263] Drug administration plan

[0264] On D0, each IVP or CP was given intramuscularly to the right neck region of each corresponding gilt by a non-research data collector using a sterile 3.0 mL Luer-lock syringe and a sterile 18g × 1-inch (2.54 cm) needle. The drug administration plan is shown in Table 6.8 below.

[0265] Table 6.8 Drug administration plan

[0266] Group Number Treatment Dose / Route Study days 1 28 CP 2.0 mL IM D0 2 28 IVP No. 1 (low potency dose) 2.0 mL IM D0 3 28 IVP No. 2 (high potency dose) 2.0 mL IM D0 4 10 CP 2.0 mL IM D0

[0267] Methods and precautions of research staff

[0268] The staff who administered the IVP, CP, and challenge materials followed safety codes and wore personal protective equipment as outlined for the specific research site.

[0269] Animal information

[0270] Details of research animals

[0271] Table 6.9 Animal information

[0272]

[0273] Inclusion / Exclusion Criteria

[0274] All gilts participating in this study were non-parous gilts negative for PRRS ELISA and were determined to be healthy at the time of vaccination as observed.

[0275] Removal of Gilts after Inclusion

[0276] Five (5) gilts in Group 1 (Nos. 5, 15, 34, 35, and 52), two (2) gilts in Group 2 (Nos. 77 and 94), three (3) gilts in Group 3 (Nos. 2, 25, and 30), and one (1) gilt in Group 4 (No. 20) did not show estrus and thus did not conceive. These gilts were removed from the study on D47.

[0277] Two (2) gilts in Group 1 (Nos. 109 and 110), nine (9) gilts in Group 2 (Nos. 56, 59, 60, 69, 73, 75, 76, 78, and 103), four (4) gilts in Group 3 (Nos. 22, 28, 51, and 53), and one (1) gilt in Group 4 (No. 21) were removed from the study on D89 due to lameness, non-pregnancy, or delayed conception.

[0278] The study protocol stated that if more than 16 pregnant gilts remained in the study in each of Groups 1 - 3 prior to challenge, additional gilts would be randomly selected for removal from the study; thus, 16 pregnant gilts were left in each of Groups 1 - 3. Based on randomization by a statistician, or selected by a non-study personnel, five (5) gilts in Group 1 (Nos. 3, 8, 39, 90, and 101), one (1) gilt in Group 2 (No. 70), and five (5) gilts in Group 3 (Nos. 42, 80, 86, 91, and 105) were removed from the study on D104, reducing the group size of Groups 1 - 3 to 16 gilts.

[0279] Due to space limitations, the study investigator requested that the size of Group 4 be reduced from eight (8) to five (5). A statistician randomly selected three (3) gilts (Nos. 10, 24, and 29) for removal from the study, which were removed on D109.

[0280] Animal Management and Housing

[0281] Animal Housing

[0282] From D-1 until the end of the study, low-IVP-valence gilts were housed in rooms 1 and 2 of Building CB at VRI-Cambridge, and high-IVP-valence gilts were housed in rooms 3 and 4. From D-1 to D85, gilts assigned to the challenge control and negative control groups were housed in individual rooms at VRI-Risdal. At D85, the remaining gilts in the challenge control and negative control groups were moved to VRI-Cambridge. For the remainder of the study, sixteen (16) challenge control gilts were housed in rooms 5-8 of Building CB, and eight (8) negative control gilts were housed in room 12 of Building CA. Since D85, the layout of each room has been the same, with two rows of four farrowing crates each. Each crate housed one gilt and her offspring. Each crate was approximately 5 feet by 7 feet in size, raised off the ground, with a metal bar panel on the sides and a plastic mesh floor. There was no close contact between adjacent crates. The floor of each crate was flushed at least once daily to remove excrement and waste. Each room had independent heating and ventilation to prevent cross-contamination of air between rooms. Each room was cleaned and disinfected before being used for this study. Animal service personnel showered and put on clean clothes before entering each room.

[0283] Isolation of treatment groups was required in this study because it is well known in the scientific community that PRRSv is easily transmitted between pigs via various mechanisms, including aerosolization. This included non-virulent live PRRS vaccines because these biological products contain attenuated virus particles that mimic the characteristics of virulent wild-type PRRS without the ability to cause disease. Appropriate methods were employed to ensure maintenance of biosecurity and that vaccinated animals were not accidentally cross-contaminated with unvaccinated PRRSv-naïve negative control animals.

[0284] Each room in the laboratory had a fan and a heater to promote adequate air circulation and heating. The ventilation system in each room was independent and identical, so air was not shared between rooms. Solid feed was stored in bags and was pest-free. Water was freely available from a well located in the animal laboratory. Gilts were fed commercially prepared drug-free gestation or lactation diets (Heart of Iowa Cooperative, Roland, IA) appropriate for their size, age, and condition.

[0285] As determined by the study investigators, the gilts were in good health and nutritional status before the start of the study. During the study, two gilts were observed to be mildly lame and one gilt was observed to have a swelling in the left neck region. The study investigators considered all of these conditions to be non-specific conditions commonly present in groups of gilts housed in confinement. The study investigators determined that no animals required concurrent treatment during this study.

[0286] All gilts and their piglets allocated to Groups 1 - 3 were disposed of by commercial incineration after euthanasia. Gilts allocated to Group 4 were disposed of by rendering after euthanasia. Piglets in Group 4 were not euthanized and disposed of, but were allocated to another BIVI program. No food from animals participating in this study entered the human food chain.

[0287] Efficacy assessment

[0288] To assess the MID of PRRS 94881 MLV, the low - potency challenge group (Group 2) and the high - potency challenge group (Group 3) were challenged on D118, and the reproductive performance and weaned piglets after challenge were evaluated. The main criteria to meet this objective were: It had to be demonstrated that the percentage or number of live - born piglets and 20 - day - old (DOF + 20) weaned piglets in one or both vaccine groups were statistically higher compared to the challenged control group (Group 1).

[0289] Other parameters analyzed to demonstrate efficacy between the vaccine groups and the challenged control group included the clinical assessment of gilts after vaccination, PRRS serology of gilts, viremia of gilts, clinical observation of gilts after challenge, viremia of piglets at farrowing, total piglets per litter, healthy live piglets per litter, weak live piglets per litter, mummified piglets per litter, stillborn piglets per litter, crushed / dead piglets per litter, clinical observation of piglets, and ADWG of piglets.

[0290] Criteria for a valid test

[0291] The negative control group (Group 4) was not included in any analysis. The negative control group was included in the study to demonstrate that the source of the gilts was PRRS - negative when the other three groups were challenged. In addition, the negative control group had to remain PRRS - negative until the end of the study to exclude the possible introduction of field PRRSv or accidental cross - contamination from the challenged groups.

[0292] PRRS antibody - negative serum samples were required both before purchase and at D0. Serum samples collected from Groups 1 and 4 until the day of challenge had to be free of PRRS antibody, and serum samples collected from Group 4 until the end of the study had to be free of PRRS antibody for the study to be valid.

[0293] Main outcome parameters

[0294] The main efficacy variables for statistical assessment were the live piglets at birth (average number or percentage) per gilt and the live piglets per litter at DOF + 20 (average number and percentage).

[0295] 9.2.1 Percentage of live piglets at birth per gilt

[0296] Litter data were recorded for each gilt during the study period. The date of farrowing (DOF) for each gilt was defined as the date on which the first piglet was born. At farrowing, each piglet was classified into one of the five categories listed in Table 6.10 below. Live piglets at birth were defined as any piglet that was observed and rated as healthy live, weak live, or crushed / dead (death due to crushing was confirmed at necropsy as described below) at birth. Observations were made by the study investigator or designee and recorded on the litter progress record form for each litter.

[0297] Table 6.10 Litter Outcome Categories

[0298]

[0299] Live piglets per litter at DOF + 20

[0300] As outlined in Table 6.11 below, from DOF + 1 to DOF + 20, clinical signs of disease in the piglets were observed. Observations were made by the study investigator or designee and recorded on the clinical observation record form.

[0301] Table 6.11 Clinical Observation Scoring System

[0302]

[0303] The total daily clinical observation score was determined by a statistician summing the respiration, behavior, and cough scores using the SAS program. Any piglet with a clinical score of 0 to 8 at DOF + 20 was rated as surviving at DOF + 20.

[0304] Supportive Parameters

[0305] Other parameters analyzed between the vaccine group and the challenge control group included gilt clinical assessment after vaccination, gilt PRRS serology, gilt viremia, gilt clinical observations, piglet viremia, total piglets per litter, healthy live piglets per litter, weak live piglets per litter, mummified piglets per litter, stillborn piglets per litter, crushed / dead piglets per litter, piglet clinical observations, and average daily weight gain (ADWG) of piglets.

[0306] Daily Gilt Assessment

[0307] All gilts were observed daily by the study investigator or designee once from D - 1 to D21 after vaccination and at least three times a week from D22 to 115 for daily assessment. Observation results were recorded on the daily assessment record form.

[0308] Gilt PRRS Serology

[0309] Before purchase and at D0, D7, D14, D21, D56, D84, D118, D125, D132, DOF 0, DOF+7, DOF+13 and DOF+20, collect whole venous blood from gilts. Immediately after farrowing / abortion, collect blood from gilts that have farrowed / aborted (DOF 0), or collect it up to 8 hours after farrowing / abortion.

[0310] Briefly, collect approximately 10 mL of blood from each gilt into a serum separator tube (SST) of appropriate size. Sample collection is recorded on a sample collection record form. Allow the blood in the SST to clot at room temperature. Blood samples collected on weekdays are transferred to BIVI-Ames on the day of collection. Blood samples collected on weekends are processed by VRI staff on the day of collection. Serum samples from VRI are stored at 2-8 °C. At BIVI-Ames or VRI, the blood samples are briefly centrifuged and the serum is harvested, separated and transferred to appropriate tubes. Each tube is labeled with the gilt's ID number, study number, collection date, study day and sample type. Serum samples from VRI are transferred to BIVI-Ames at the earliest appropriate time. Each shipment includes a complete sample transfer record form. At BIVI-Ames, one set of serum samples is stored at 2-8 °C and other sets of serum samples are stored at -70 ± 10 °C.

[0311] Test the serum samples of gilts stored at 2-8 °C for PRRS antibodies at BIVI-Ames. The results are reported as negative (ELISA S / P ratio < 0.4) or positive (ELISA S / P ratio ≥ 0.4).

[0312] Clinical observations of gilts after challenge

[0313] From D116 to DOF+20, observe the clinical signs of disease in gilts. Observations are made by the study investigator or a designee. Based on the clinical observation scoring system outlined in Table 6.11 above, observe the respiration, behavior and coughing of gilts daily.

[0314] PRRS viremia in piglets

[0315] At DOF 0, DOF+7, DOF+13 and DOF+20, or when a piglet death is detected, collect whole venous blood from piglets. It is preferred to collect pre-colostrum blood from neonatal piglets, but it is not mandatory. If pre-colostrum blood cannot be collected, peripartum blood within 12 hours of farrowing can be collected. Samples collected before the first milking are labeled "peripartum" and are separated from pre-colostrum samples.

[0316] Briefly, approximately 2.0 to 2.5 mL of blood was collected from each live piglet into a serum separator tube (SST) of appropriate size. At least 5.0 mL of blood was collected from each piglet at DOF+20 immediately prior to euthanasia. Blood was collected from each mummified or stillborn fetus, or if blood could not be collected from the stillborn fetus, thoracic or abdominal fluid was collected. Sample collections were recorded on a sample collection record form.

[0317] Average daily gain of piglets

[0318] Individual piglets were weighed by the study investigator or designee at DOF 0 and DOF+20 or on the day of discovery of piglet death. The individual weights at DOF 0 were recorded on the litter progress record form for each litter, and the weights after DOF 0 were recorded on the weight record form.

[0319] Quantitative determination of PRRS virus in lung tissue

[0320] All piglets that died at parturition or were moribund prior to DOF+20 were autopsied by the study investigator. The autopsy results and diagnoses were recorded on an autopsy report form. Two lung samples were collected from each autopsied piglet. One sample was placed in a separate container, and the other sample was placed in an appropriate container filled with a sufficient volume of 10% formalin. Sample collections were recorded on the autopsy report form.

[0321] The container and the formalin container were appropriately labeled with the animal number, study number, sampling date, study day, sample type, and whether the sample was from the left, right, or both sides. The samples in were stored at -70 ± 10 °C and the samples in 10% formalin were stored at room temperature until transferred to BIVI-Ames. The transfer of each sample included a completed specimen transfer record form. At BIVI-Ames, the samples in were stored at -70 ± 10 °C until shipped from BIVI-Ames to Germany, and at BIVI-Ames, the formalin-fixed samples were stored at room temperature.

[0322] After the study ended, the frozen tissue samples in were shipped and tested as described above.

[0323] The formalin-fixed tissue samples were submitted to ISU VDL within one week of collection for embedding in paraffin blocks. The tissue in the paraffin blocks was returned to BIVI and is currently stored at room temperature by BIVI-Ames for possible future testing. The study sponsor and monitor will decide whether to retain or discard these samples after the study report is completed.

[0324] Adverse events

[0325] No adverse events attributable to the IVP were reported during this study. For more information on adverse events, see Section 12.6 Sow evaluation after vaccination.

[0326] Statistical methods

[0327] Experimental unit

[0328] In this study, treatment groups had to be housed in separate rooms to prevent the spread of live PRRSv vaccine to the unvaccinated group. Therefore, the rooms were the experimental units. However, for the purposes of this analysis, possible deviations due to confounding effects of "room" and "treatment" were ignored, and the sows and their corresponding littermates were analyzed as the experimental units.

[0329] Randomization

[0330] Before D0, ninety-four (94) PRRS-seronegative sows from a group of 107 eligible test sows were assigned to one of four groups. Groups 1 - 3 each consisted of 28 sows. Group 4 consisted of 10 sows. For Group 1, before shipment, Nos. 45 and 55 were excluded by the farm manager for health reasons and were replaced by two other sows, Nos. 15 and 18, respectively. For Group 3, before shipment, No. 44 was excluded by the farm manager for health reasons and was replaced by another sow, No. 25.

[0331] Due to space limitations at the time of challenge, Groups 1 - 3 were limited to 16 sows per group, and Group 5 was limited to 5 - 8 sows. For Groups 1 - 3, at D85, 16 sows were randomly selected per group to remain in the study. Since Group 4 consisted of 8 sows at D85, this group was not further reduced by randomization. Subsequently, the study investigator requested that Group 4 be reduced from 8 sows to 5 sows. For Group 4, at D109, 5 sows were randomly selected to remain in the study.

[0332] All randomization procedures were performed by a biostatistician.

[0333] Analysis

[0334] Statistical analysis and data summary were performed by Dr. rer. hort. Martin Vanselow, Biometrie & Statistik, Zum Siemenshof 21, 30539 Hannover, Germany, +49(0)511 606 777650, m.vanselow@t-online.de.

[0335] The main objective of the statistical analysis was to compare two groups of PRRS 94881 MLV vaccinated pigs (Group 2 and Group 3) with the unvaccinated challenged control group (Group 1). All data were entered into SAS for management and evaluation. The data were received from the study sponsor in the form of a validated SAS data set. Prior to the exclusion date, the individual parameters analyzed took into account withdrawals from the study. All data were descriptively summarized (number, minimum, maximum, mean, median, standard deviation, interquartile range or frequency distribution, confidence interval) based on the type of variable. Statistical analysis was performed using SAS software version 8.2 (SAS, 2001, Cary, North Carolina, USA: SAS Institute Inc.).

[0336] Variables for the statistical evaluation of the study:

[0337] Main variables

[0338] Proportion of live piglets at farrowing / abortion (DOF + 0)

[0339] Proportion of live piglets at 20 days of age (DOF + 20)

[0340] Supporting variables

[0341] Clinical evaluation of gilts after vaccination

[0342] PRRS serology in gilts

[0343] Viremia in gilts

[0344] Clinical observations in gilts

[0345] Viremia in piglets

[0346] Reproductive performance

[0347] Clinical observations in piglets

[0348] Average daily weight gain of piglets (ADWG)

[0349] Hypotheses to be tested and assumptions made:

[0350] The non-challenged negative control group (Group 4) was excluded from the statistical tests. The low-titer and high-titer groups (Groups 2 and 3 respectively) were compared with the challenged control group (Group 1). All tests between groups were designed as two-sided tests for differences. In all tests, differences were considered statistically significant only when P ≤ 0.05. Efficacy was demonstrated if the percentage or number of live-born piglets and the percentage or number of weaned piglets at DOF + 20 were significantly higher in one or both of the vaccinated groups compared to the challenged control group.

[0351] Details regarding data manipulation and evaluation:

[0352] Clinical evaluation of gilts after vaccination

[0353] Frequency tables of animals with at least one positive finding between Day 1 and Day 21 of the study and between Day 1 and Day 113 of the study. The differences between the challenged control group and the vaccine group were tested by Fisher's exact test.

[0354] Clinical observations of gilts after challenge

[0355] Frequency tables of animals with at least one positive finding between Day 116 and DOF+20 of the study. The differences between the challenged control group and the vaccine group were tested by Fisher's exact test.

[0356] Serology of gilts

[0357] Frequency tables of "positive" ELISA results at Days 7, 14, 21, 56, 84, 118, 125, 132 (before farrowing) of the study and at DOF+0, DOF+7, DOF+13 and DOF+20. The differences between the challenged control group and the vaccine group were tested by Fisher's exact test.

[0358] Viremia of gilts

[0359] Viremia data were evaluated for each study day (Days 7, 14, 21, 56, 84, 118, 125, 132 before farrowing of the study and DOF+0, DOF+7, DOF+13 and DOF+20). For qualitative evaluation of qPCR data, the results 'not detected' ('n.d.') and 'negative' were classified as 'negative', and the results 'positive' and the measurements were classified as 'positive'. For quantitative evaluation, 'not detected' ('n.d.') and 'negative' were replaced with a log 10 GE / mL value of 0.0, and 'positive' was replaced with 3.0. Quantitative PCR data (PRRS virus load [log 10 GE / mL]) were used for comparison between the challenged control group (Group 1) and Treatment Groups 2 and 3 using the Wilcoxon Mann-Whitney test. Frequency tables of 'positive' qPCR results were generated. The differences between the challenged control group and the vaccine group were tested by Fisher's exact test.

[0360] Reproductive performance

[0361] Determine the absolute frequencies of total, live, healthy, weak, stillborn, dead, and live piglets per gilt at DOF+20 and use as single values for comparison between groups. Calculate the relative frequencies of live, healthy, weak, stillborn, and dead piglets per gilt relative to the total number of piglets at farrowing and use as single values for comparison between groups. Calculate the percentage of live piglets per litter at DOF+20 relative to the number of live piglets at farrowing minus the number of dead and crushed piglets. Test the differences between the challenged control and vaccine groups by the Wilcoxon-Mann-Whitney test.

[0362] Viremia of piglets

[0363] Evaluate viremia data for each study day (DOF+0, DOF+7, DOF+13, and DOF+20) separately. For qualitative evaluation of qPCR data, analyze the results 'not detected' ('n.d.') and 'negative' as 'negative', and analyze the results 'positive' and measured values as 'positive'. Calculate the percentage of 'positive' piglets per litter and use as a single value for comparison between groups by the Wilcoxon-Mann-Whitney test. For quantitative evaluation, replace 'not detected' ('n.d.') and 'negative' with a log 10 GE / mL value of 0.0, and replace 'positive' with 3.0. Calculate the median qPCR value per litter and use as a single value for comparison between groups by the Wilcoxon-Mann-Whitney test. Use individual qPCR data for summary statistics. Only descriptively evaluate the viral load in lung samples.

[0364] Body weight and average daily weight gain of piglets

[0365] Calculate the individual average daily weight gain (ADWG) for the period between DOF+0 and DOF+20. Test the differences between treatment groups by analysis of variance (ANOVA) and subsequent t-tests. The differences between the least-squares means of the groups and the least-squares means with 95% confidence intervals are derived from the ANOVA. Repeat the analysis of DOF+20 and ADWG with the body weight at DOF+0 as a covariate. Descriptively summarize the piglet weight data per gilt.

[0366] Clinical observations of piglets

[0367] Calculate the percentage of piglets per litter with at least one positive finding between study days DOF+1 and DOF+20 and use as a single value for comparison between groups by the Wilcoxon-Mann-Whitney test. Analyze the data with a completely randomized design structure.

[0368] Results

[0369] Gilt reproductive performance

[0370] The average percentages of live piglets per litter (healthy + weak + crushed / dead) at farrowing in the virus-challenged control group, low-titer group, high-titer group, and negative control group were 54.4%, 75.1%, 72.3%, and 93.0%, respectively. Compared with the virus-challenged control group, the percentages of live piglets per litter at farrowing in the low-titer group and high-titer group were significantly higher (P≤0.0455). The average numbers of live piglets per litter at farrowing in the virus-challenged control group, low-titer group, high-titer group, and negative control group were 6.5, 8.3, 8.6, and 10.8, respectively. No significant differences in the numbers of live piglets per litter at farrowing were detected among the groups (P≥0.1039).

[0371] The average percentages of healthy live piglets per litter in the virus-challenged control group, low-titer group, high-titer group, and negative control group were 41.4%, 65.8%, 67.9%, and 93.0%, respectively. Compared with the virus-challenged control group, the percentages of healthy live piglets per litter at farrowing in the low-titer group and high-titer group were significantly higher (P≤0.0138). The average numbers of healthy live piglets per litter at farrowing in the virus-challenged control group, low-titer group, high-titer group, and negative control group were 4.9, 7.2, 8.1, and 10.8, respectively. Compared with the virus-challenged control group, the number of healthy live piglets per litter at farrowing in the high-titer group was significantly higher (P = 0.0211), while no difference was detected in the low-titer group (P = 0.0640).

[0372] The average percentages of weak live piglets per litter at farrowing in the virus-challenged control group, low-titer group, high-titer group, and negative control group were 7.4%, 7.1%, 0.4%, and 0.0%, respectively. The average numbers of weak live piglets per litter at farrowing in the virus-challenged control group, low-titer group, high-titer group, and negative control group were 0.9, 0.8, 0.1, and 0.0, respectively. Compared with the virus-challenged control group, the percentage and number of weak live piglets per litter at farrowing in the high-titer group were significantly lower (P≤0.0090). In contrast, no differences in the percentage or number of weak live piglets at farrowing were detected between the low-titer group and the virus-challenged control group (P≥0.8569).

[0373] The average percentages of mummified piglets per litter at farrowing in the virus-challenged control group, low-titer group, high-titer group, and negative control group were 28.1%, 14.1%, 8.7%, and 0.0%, respectively. The average numbers of mummified piglets per litter at farrowing in the virus-challenged control group, low-titer group, high-titer group, and negative control group were 3.1, 1.6, 0.9, and 0.0, respectively. Compared with the virus-challenged control group, the percentages and numbers of mummified piglets per litter at farrowing in the low-titer group and high-titer group were both significantly lower (P≤0.0190).

[0374] No significant differences in the percentages or numbers of stillborn or dead / crushed piglets per litter at farrowing were detected between the two vaccine-titer groups and the virus-challenged control group (P≥0.1681).

[0375] An overview of the reproductive performance results (percentage of piglets per litter and number of piglets per litter) at DOF in the groups is shown in Tables 6.12 and 6.13 below.

[0376] Table 6.12: Overview of reproductive performance results (percentage of piglets per litter) at DOF in the groups

[0377]

[0378] * Group 1 = challenged control group; Group 2 = low - potency PRRS 94881 MLV group; Group 3 = high - potency PRRS 94881 MLV group; Group 4 = negative control group

[0379] Table 6.13: Overview of reproductive performance results (number of piglets per litter) at DOF in the groups

[0380]

[0381] * Group 1 = challenged control group; Group 2 = low - potency PRRS 94881 MLV group; Group 3 = high - potency PRRS 94881 MLV group; Group 4 = negative control group

[0382] Surviving piglets at DOF + 20

[0383] These scores highlight the number of live piglets at weaning (20 days of age). An overview of the percentage and number of live piglets per litter at DOF + 20 in the groups is shown in Tables 6.12 and 6.13 above.

[0384] The mean percentages of live piglets per litter at weaning (DOF + 20) for the challenged control group, low - potency group, high - potency group, and negative control group were 43.6%, 73.8%, 83.8%, and 100.0% respectively. The mean numbers of live piglets per litter at weaning for the challenged control group, low - potency group, high - potency group, and negative control group were 2.9, 6.2, 6.9, and 10.8 respectively. Compared with the challenged control group, the percentages and numbers of live piglets per litter at weaning (DOF + 20) in the low - potency group and high - potency group were significantly higher (P ≤ 0.0203).

[0385] Gilt qPCR viremia

[0386] At D0, all gilts were qPCR - negative for PRRSv RNA. All challenged control and negative control gilts remained qPCR - negative for PRRSv RNA until and including the day of challenge (D118). The negative control group remained qPCR - negative for the remainder of the study, except for gilt No. 108, which was 'positive' at DOF + 7. By qPCR testing, gilt No. 108 was negative for PRRSv RNA at other time points.

[0387] After vaccination, on D7, 50% and 36% of the low - and high - potency gilts were qPCR - positive for PRRSv RNA, respectively (P ≤ 0.0007). From D14 to D56, only 4% of the low - potency gilts remained qPCR - positive, while during this observation period, up to 4% of the high - potency gilts were intermittently qPCR - positive. From D14 to D56, no difference in the percentage of gilts qPCR - positive for PRRSv RNA was detected between the vaccine group and the challenge control group (P = 1.0000 or not tested). At D84 and D118 (the day of challenge), all vaccinated gilts were qPCR - negative for PRRSv RNA.

[0388] After challenge, on day 125, DOF 0, and DOF + 13, the percentages of gilts qPCR - positive for PRRSv RNA in the low - and high - potency groups were statistically lower compared to the challenge control group (P ≤ 0.0155). At D132, the percentage of gilts qPCR - positive for PRRSv RNA in the low - potency group was significantly lower (P = 0.0290); while no statistical difference was detected between the high - potency group and the challenge control group (P = 0.1556). At DOF + 7 and DOF + 20, no significant difference in the percentage of gilts qPCR - positive for PRRSv RNA was detected between the vaccine group and the challenge control group (P ≥ 0.1719).

[0389] An overview of the percentages of gilts qPCR - positive for PRRSv RNA from D7 to DOF + 20 in the groups is shown in Tables 6.14 and 6.15 below.

[0390] Table 6.14: Overview of the percentages of gilts qPCR - positive for PRRSv RNA from D7 to D132 in the groups

[0391]

[0392] * Group 1 = challenge control group; Group 2 = low - potency PRRS 94881 MLV group; Group 3 = high - potency PRRS94881 MLV group; Group 4 = negative control group; n.a. = not applicable, not tested

[0393] Table 6.15: Overview of the percentages of gilts qPCR - positive for PRRSv RNA from DOF 0 to DOF + 20 in the groups

[0394]

[0395] *Group 1 = challenge control group; Group 2 = low - potency PRRS 94881 MLV group; Group 3 = high - potency PRRS94881 MLV group; Group 4 = negative control group

[0396] At D7, compared with the challenge control group, the median viral loads in both vaccine groups were significantly higher (P ≤ 0.0007). From D14 to D56, no difference in viral load was detected between the vaccine groups and the challenge control group (P = 1.0000). At D84 and D118 (the day of challenge), the viral loads of all vaccinated gilts were 0.

[0397] After challenge, at D125, DOF 0, and DOF + 13, the median viral loads in the low - potency group and the high - potency group were statistically lower compared with the challenge control group (P ≤ 0.0155). At D132, the median viral load in the low - potency group was significantly lower (P = 0.0230); while no statistical difference was detected between the high - potency group and the challenge control group (0.94 and 1.97 log 10 GE / mL; P = 0.1144). At DOF + 7 and DOF + 20, no significant difference in viral load was detected between the vaccine groups and the challenge control group (P ≥ 0.1719).

[0398] An overview of the average gilt qPCR GE / mL results from D7 to DOF + 20 in the groups is shown in Tables 6.16 and 6.17 below.

[0399] Table 6.16: Overview of gilt qPCR results (log 10 GE / mL) from D7 to D132 in the groups

[0400]

[0401] *Group 1 = challenge control group; Group 2 = low - potency PRRS 94881 MLV group; Group 3 = high - potency PRRS94881 MLV group; Group 4 = negative control group

[0402] Table 6.17: Overview of gilt qPCR results (log10GE / mL) from DOF 0 to DOF + 20 in the groups

[0403]

[0404] *Group 1 = challenge control group; Group 2 = low - potency PRRS 94881 MLV group; Group 3 = high - potency PRRS94881 MLV group; Group 4 = negative control group

[0405] Clinical observation scores of gilts after challenge

[0406] From D116 to D0F+20, 25%, 25%, 38% and 60% of the challenged control, low-titer, high-titer and negative control gilts, respectively, showed clinical disease on at least one day from D116 to D0F+20. From D116 to D0F+20, no significant difference in the frequency of gilts positive for clinical disease was detected between the vaccine group and the challenged control group (P≥0.7043).

[0407] An overview of the percentage of gilts positive for clinical disease (clinical observation score > 0) on at least one day from D116 to D0F+20 in the groups is shown in Table 6.18 below.

[0408] Table 6.18: Overview of the percentage of gilts positive for clinical disease (clinical observation score > 0) on at least one day from D116 to D0F+20 in the groups

[0409]

[0410] *Group 1 = challenged control group; Group 2 = low-titer PRRS 94881 MLV group; Group 3 = high-titer PRRS 94881 MLV group; Group 4 = negative control group

[0411] PRRS ELISA serology in gilts

[0412] All gilts were seronegative for PRRS at D0 and D7. All challenged control and negative control gilts remained seronegative for PRRS until and including the day of challenge (D118); while the negative control group remained seronegative for PRRS for the remainder of the study (D0F+20).

[0413] At D14, 18% and 21% of the low-titer and high-titer gilts, respectively, were seropositive for PRRS. The percentage of gilts seropositive for PRRS at D14 in the high-titer group was significantly higher compared to the challenged control group (P = 0.0232), while no difference was detected in the low-titer group (P = 0.0515). At D56, these percentages in the low-titer and high-titer groups reached a high of 65% and 60% in the groups, respectively (P<0.0001). At the day of challenge (D118), 56% and 50% of the low-titer and high-titer gilts, respectively, were seropositive for PRRS (P≤0.0024). At D125, 6%, 88% and 100% of the challenged control, low-titer and high-titer gilts, respectively, were seropositive for PRRS; and the difference between the vaccine group and the challenged control group was significant (P<0.0001). After D125, all remaining challenged control, low-titer and high-titer gilts were seropositive for PRRS for the remainder of the study (not tested).

[0414] An overview of the PRRS ELISA serological results at D14 to DOF + 20 in the groups is shown in Tables 6.19 and 6.20 below.

[0415] Table 6.19: Overview of PRRS ELISA serological results in gilts from D14 to Day 132 in the groups

[0416]

[0417] * Group 1 = challenge control group; Group 2 = low - titer PRRS 94881 MLV group; Group 3 = high - titer PRRS 94881 MLV group; Group 4 = negative control group; n.a. = not applicable, not tested

[0418] Table 6.20: Overview of PRRS ELISA serological results in gilts from DOF 0 to DOF + 20 in the groups

[0419]

[0420] * Group 1 = challenge control group; Group 2 = low - titer PRRS 94881 MLV group; Group 3 = high - titer PRRS 94881 MLV group; Group 4 = negative control group. ** Samples from gilt No. 106 were not tested. n.a. = not applicable, not tested

[0421] Gilts assessment after vaccination

[0422] No abnormal assessments were detected from D1 to 21 in any group, and not tested. From D1 to D113, 4%, 4%, 0%, and 10% of the challenge control, low - titer, high - titer, and negative control gilts, respectively, showed abnormal assessments on at least one day from D1 to D113. No significant difference in abnormal assessments was detected between the vaccine groups and the challenge control group from D1 to D113 (P = 1.0000).

[0423] Individually, No. 109 (challenge control group) showed right hind - leg lameness on D85, No. 12 (low - titer group) showed swelling in the left neck region from D78 to D89, and No. 21 (negative control group) showed lameness from D81 to D83.

[0424] The percentages of gilts in the groups that showed abnormal assessments on at least one day from D1 to D21 and from D1 to D113 are summarized in Table 6.21 below.

[0425] Table 6.21: Overview of abnormal assessments on at least one day from D1 to D113 in the groups

[0426]

[0427] *Group 1 = Challenge control group; Group 2 = Low - potency PRRS 94881 MLV group; Group 3 = High - potency PRRS94881 MLV group; Group 4 = Negative control group

[0428] Clinical observation score of piglets

[0429] The average percentages of piglets per litter that were positive for clinical disease (clinical observation score > 0) on at least one day from DOF + 1 to DOF + 20 in the challenge control group, low - potency group, high - potency group, and negative control group were 91.6%, 32.5%, 33.4%, and 3.2%, respectively. The percentages of piglets per litter that were positive for clinical disease on at least one day from DOF + 1 to DOF + 20 in the low - potency and high - potency groups were significantly lower compared to the challenge control group (p ≤ 0.0001).

[0430] An overview of the percentages of piglets per litter that were positive for clinical disease (clinical observation score > 0) on at least one day from DOF + 1 to DOF + 20 in each group is shown in Table 6.22 below.

[0431] Table 6.22: Overview of the percentages of piglets per litter that were positive for clinical disease (clinical observation score > 0) on at least one day from DOF + 1 to DOF + 20 in each group

[0432]

[0433] *Group 1 = Challenge control group; Group 2 = Low - potency PRRS 94881 MLV group; Group 3 = High - potency PRRS94881 MLV group; Group 4 = Negative control group

[0434] qPCR results of piglet serum / body fluids

[0435] At DOF 0, the average values of piglets per litter in the challenge control group, low - potency group, high - potency group, and negative control group that were qPCR - positive for PRRSv RNA were 86.3%, 58.1%, 55.0%, and 0%, respectively. At DOF 0, the percentages of piglets per litter that were qPCR - positive for PRRSv RNA in the low - potency and high - potency groups were statistically lower compared to the challenge control group (P ≤ 0.0381). At DOF + 7, the percentages of piglets per litter that were qPCR - positive for PRRSv RNA in the low - potency and high - potency groups were again significantly lower compared to the challenge control group (P ≤ 0.0293). At DOF + 13, only the percentage of qPCR - positive piglets per litter in the low - potency group was significantly lower (P = 0.0216); no significant difference was detected in the percentage of qPCR - positive piglets per litter between the high - potency group and the challenge control group (P = 0.0860). At DOF + 20, no significant difference was detected between groups (P ≥ 0.0614).

[0436] An overview of the percentage of serum / body fluid qPCR PRRSv positive piglets per gilt in the groups is shown in Table 6.23 below.

[0437] Table 6.23: Overview of the percentage of serum / body fluid qPCR PRRSv positive piglets per gilt in the groups

[0438]

[0439] * Group 1 = challenge control group; Group 2 = low - potency PRRS 94881 MLV group; Group 3 = high - potency PRRS 94881 MLV group; Group 4 = negative control group

[0440] At DOF 0, compared with the challenge control group, the median qPCR result in the high - potency group was significantly lower (P = 0.0030); while no difference was detected between the low - potency group and the challenge control group (P = 0.0620). At DOF +7, DOF +13 and DOF +20, compared with the challenge control group, the median qPCR values in both vaccine groups were significantly lower (p ≤ 0.0122).

[0441] An overview of the piglet serum / body fluid qPCR GE / mL results per gilt in the groups is shown in Table 6.24 below.

[0442] Table 6.24: Overview of the piglet serum / body fluid qPCR results (log10GE / mL) per gilt in the groups (P - values for differences between groups are based on median qPCR values)

[0443]

[0444] * Group 1 = challenge control group; Group 2 = low - potency PRRS 94881 MLV group; Group 3 = high - potency PRRS 94881 MLV group; Group 4 = negative control group

[0445] Piglet ADWG

[0446] At DOF 0, no difference in LS mean body weight was detected between the groups (P ≥ 0.2972). At DOF +20, with or without factoring the DOF 0 body weight as a covariate in the analysis, the least - squares mean body weights in both vaccine groups were higher compared with the challenge control group (P < 0.0028).

[0447] The average ADWG from DOF 0 to DOF+20 in the challenge control group, low titer group, high titer group and negative control group were 0.1 kg / day, 0.2 kg / day, 0.2 kg / day and 0.2 kg / day, respectively. With or without factoring the DOF 0 body weight as a covariate in the analysis, the ADWG in both vaccine groups were significantly higher than that in the challenge control group (P<0.0028).

[0448] An overview of the body weights of piglets at DOF 0 and DOF+20 and the ADWG (kg / day) from DOF 0 to DOF+20 in the groups are shown in Tables 6.25 and 6.26 below.

[0449] Table 6.25: Overview of the body weights of piglets at DOF 0 and DOF+20 and the ADWG (kg / day) from DOF 0 to DOF+20 in the groups

[0450]

[0451] *Group 1 = challenge control group; Group 2 = low titer PRRS 94881 MLV group; Group 3 = high titer PRRS94881 MLV group; Group 4 = negative control group

[0452] Table 6.26: Overview of the LS average body weight and the ADWG (kg / day) from DOF 0 to DOF+20 in the groups - test results (P values) for differences between groups

[0453]

[0454] *Group 1 = challenge control group; Group 2 = low titer PRRS 94881 MLV group; Group 3 = high titer PRRS94881 MLV group; Group 4 = negative control group. **The body weight at DOF+0 was used as a covariate.

[0455] Autopsy observations and diagnoses of piglets

[0456] Except for 8 fetal pigs, stillborn, mummified or crushed fetal pigs listed at farrowing were confirmed to be correctly classified at autopsy. Although two challenge control fetal pigs were listed as stillborn (40-S1, 66-S1), autopsy results revealed inflated lungs, indicating that they were alive at birth. Although two challenge control fetal pigs were listed as crushed (1-C1, 79-C2), autopsy results showed that the lungs of the two fetal pigs were not inflated, indicating that they did not breathe. Although one low titer fetal pig was listed as stillborn (85-S2), autopsy results revealed inflated lungs, indicating that the piglet was alive at birth. Although three high titer piglets were listed as crushed (36-C1, 36-C2, 65-C1), autopsy results revealed that the lungs of two fetal pigs were not inflated, indicating that they did not breathe. Since the number of fetal pigs not correctly listed at farrowing was small, there was no change in the gilt performance analysis.

[0457] One challenge control piglet 102 - 428 died after blood collection, which was confirmed by autopsy.

[0458] qPCR results of piglet lungs

[0459] Among the fetal pigs and dead piglets examined by autopsy, the mean lung qPCR results of the challenge control group, low - titer group, high - titer group, and negative control group were 4.68, 4.09, 3.55, and 0.0 log 10 GE / mL respectively. Statistical analysis was not performed on these data.

[0460] Overview of lung PRRSv qPCR results (log 10 GE / mL) in each group is shown in Table 6.27 below.

[0461] Table 6.27: Overview of piglet lung PRRSv qPCR results (log 10 GE / mL) in each group

[0462]

[0463] *Group 1 = challenge control group; Group 2 = low - titer PRRS 94881 MLV group; Group 3 = high - titer PRRS94881 MLV group; Group 4 = negative control group

[0464] Discussion / Conclusion

[0465] To achieve the research objectives, at D0, four groups of PRRS - susceptible gilts were included in the study design: the challenge control group (Group 1) receiving the control product; the low - titer vaccine group (IVP No. 1, Group 2) receiving 1×10 2.43 TCID 50 of PRRS 94881 MLV; the high - titer vaccine group (IVP No. 2, Group 3) receiving 1×10 3.90 TCID 50 of PRRS 94881 MLV; and the negative control group (Group 4) also receiving the control product. Each treatment was administered intramuscularly at a dose of 2.0 mL approximately 28 days before insemination (D0).

[0466] To determine the minimum immunizing dose of PRRS 94881 MLV, the two vaccine - titer groups and the challenge control group were challenged at D118 (approximately 90 days of gestation) with a heterologous European - type PRRSv isolate (isolate 190136), and the percentage and number of live piglets per litter at birth (day of farrowing, DOF) and the percentage and number of live piglets per litter at 21 days of age (DOF + 21) were evaluated after challenge.

[0467] Verification of the study (negative control group 4)

[0468] To ensure that the source gilts were free of PRRSv and that no exogenous PRRSv exposure or cross - contamination occurred in the treatment and control groups during the study, a negative control group (Group 4) was included in the study design. The negative control gilts were negative for PRRS antibodies throughout the study. In addition, these gilts and their offspring were also negative for PRRSv viremia (qPCR) at all test time points, except for gilt No. 108 at DOF + 7. Gilt No. 108 was "positive" at DOF + 7, but qPCR negative at all other time points, and its piglets were also negative for PRRSv RNA. This result was considered an error caused by sample contamination rather than PRRSv infection. These results demonstrated that the negative control group remained free of PRRS infection during the study and validated the results of this trial as valid.

[0469] Validation of the PRRSv reproductive challenge model (challenge control group 1)

[0470] A challenge model involving a virulent European - sourced PRRSv strain that induces sufficient and reproducible PRRS clinical disease is needed to fully evaluate the efficacy of PRRS vaccines in a laboratory setting. After inoculation with the European - type PRRS isolate 190136 (1×10 6.30 TCID50 / 6mL), the challenge control group showed only 54.4% live piglets per litter at birth (93.0% in the negative control group), 17.5% and 28.1% stillbirths and mummified fetuses per litter respectively (7.0% and 0.0% in the negative control group), 91.6% of piglets per litter showing clinical disease on at least one day between DOF + 1 and DOF + 20 (3.2% of piglets per litter in the negative control group), an average of 2.9 live piglets per litter at 20 days of age (average of 10.8 in the negative control group) and 86.3% of piglets per litter viremic at birth (0% in the negative control group). These results highlight that severe PRRS - specific clinical disease was induced in the unvaccinated challenge control group of gilts and their offspring, thus validating this challenge model as a clinical experimental tool sufficient to evaluate the efficacy of PRRS vaccines and more particularly the MID of PRRS 94881 MLV.

[0471] Determination of the minimum immunizing dose of PRRS 94881 MLV in gilts (low - potency and high - potency vaccine doses; Groups 2 - 3)

[0472] The determination of the MID of PRRS 94881 MLV in gilts was based on the vaccinated groups that received the lowest - potency vaccine, which had a higher percentage or number of live piglets per litter at birth after challenge and a higher percentage or number of live piglets per litter at 20 days of age compared to the challenge control group.

[0473] The number (percentage or count) of live piglets per litter at farrowing was selected as one of two key criteria for determining the MID of PRRS 94881 MLV. The first key criterion was based on the fact that PRRSv infection in pregnant gilts and sows typically results in mummified and stillborn piglets and a low number of live piglets at farrowing. The number of live piglets per litter at birth was defined as the sum of healthy live piglets, weak live piglets, and crush-dead piglets at farrowing. Piglets classified as crushed or dead were included in the "live" category because autopsy findings confirmed that these piglets were alive at birth and died soon after from trauma. Both the low-titer and high-titer groups showed a significantly higher percentage of live piglets per litter at farrowing (P≤0.0455) compared to the challenge control group, thus meeting this criterion for vaccine efficacy. Although no significant difference in the mean number of live piglets per litter at farrowing was detected between the low-titer and high-titer vaccine groups and the challenge control group (P≥0.1857), the low-titer and high-titer groups showed a significantly higher mean number of live piglets per litter at farrowing (mean of 8.3 and 8.6 piglets per litter, respectively) compared to the challenge control group (mean of 6.5 piglets per litter), thus further demonstrating and supporting the beneficial vaccine treatment effect observed in these animals after challenge.

[0474] The number (percentage or count) of live piglets per litter at 20 days of age was the second criterion for determining the MID of PRRS 94881 MLV because gilt PRRS immunity would affect intrauterine infection of piglets and the transfer of virus from the gilt to live piglets. Piglets infected with PRRS in utero and surviving to birth or infected with virulent PRRS via outflow from the gilt after farrowing typically die from PRRS before weaning. In this study, the challenge control group, low-titer group, high-titer group, and negative control group showed 43.6%, 73.8%, 83.8%, and 100% live piglets per litter at 20 days of age, respectively (P≤0.0203). Similarly, the mean number of live piglets per litter at 20 days of age in the challenge control group, low-titer group, high-titer group, and negative control group was 2.9, 6.2, 6.9, and 10.8, respectively (P≤0.0063). Both the percentage and number of live piglets at weaning were higher in the two vaccine groups (P≤0.0203), thus meeting this criterion of the study objective.

[0475] Further analysis of the litter data revealed more information in support of vaccine efficacy after PRRSv challenge, especially regarding the high-titer group. Compared to the challenged control group, the high-titer group showed a statistically higher percentage and mean number of healthy piglets at birth (P≤0.0211); and also showed a significantly lower percentage and mean number of weak and mummified fetuses (P≤0.0090). These data demonstrate that the high vaccine dose induced protective immunity against virulent and heterologous PRRSv challenge strains. The low-titer group also showed vaccine efficacy at farrowing, as evidenced by a higher percentage of healthy live piglets per litter (P = 0.0138) and a significantly lower percentage and mean number of mummified fetuses (P≤0.0190). In contrast, no differences in the percentage or number of stillborn or crushed / died fetuses at farrowing were detected between the groups (P≥0.1681).

[0476] Seven days post-challenge (D125), compared to the challenged control group, the percentage of gilts positive for PRRSv RNA in the low-titer and high-titer groups was significantly lower as tested by qPCR, and the viral load in both groups was significantly lower (P≤0.0001). These data further demonstrate that both vaccine doses induced immunity in gilts sufficient to significantly reduce viral replication after challenge. Similarly, the percentage of qPCR-positive gilts in the low-titer and high-titer groups was significantly lower at DOF 0 and DOF+13, and the viral load in both groups was lower on these study days (P≤0.0155). The percentage of qPCR-positive gilts in the low-titer group was significantly lower and the viral load was lower at D132 (P≤0.0290); while no statistical differences in the same parameters were detected between the high-titer group and the challenged control group (P≥0.1144). At DOF+7 and DOF+20, no statistical differences in the percentage of qPCR-positive gilts or viral load were detected between the vaccine groups and the challenged control group (P≥0.1719).

[0477] Typically, PRRSv does not induce clinical disease other than abortion in gilts and sows. In this study, 25%, 25%, 38%, and 60% of the challenged control, low-titer, high-titer, and negative control gilts, respectively, showed clinical disease (received a clinical observation score >0) at least one day after challenge. No significant differences in the percentage of gilts with clinical disease on at least one day between D116 and DOF+20 were detected between the vaccine groups and the challenged control group (P≥0.7043). Gilts that showed some form of clinical disease showed it at farrowing rather than immediately after challenge. The high percentage of negative control gilts showing clinical disease (60%), and the fact that clinical disease in all groups in this study was mainly noticed close to farrowing, demonstrate that the clinical disease is not a result of PRRS disease but rather a result of physiological changes related to parturition.

[0478] All gilts in the study were PRRS ELISA seronegative at D0, thus confirming that the test animals met the inclusion criteria for entry into the study. Similarly, all gilts were PRRS ELISA seronegative at D7. Vaccinated gilts began to show PRRS ELISA seropositive results at D14, and the low- and high-dose groups showed peak seroconversion rates of 65% and 60% respectively at D56 (P<0.0001). In contrast, the challenge control group remained PRRS ELISA seronegative until 7 days post-challenge (D125). From D132 until the end of the study, all low-titer, high-titer, and challenge control gilts were PRRS ELISA seropositive. The percentage of viremic gilts post-vaccination in the two vaccine groups reached a peak at D7, as demonstrated by 50% and 36% in the low- and high-titer groups respectively (P≤0.0007). Viremia in the low-titer and high-titer groups rapidly declined to 4% (1 out of 28, No. 64) and 0% (0 out of 28) respectively at D14 (P = 1.0000 or not tested). At D21, viremia remained at 4% in the low-titer group (1 out of 28, No. 56) and the high-titer group (1 out of 28, No. 91). At D56, 1 out of 26 low-titer gilts (4%, No. 89) and 1 out of 25 high-titer gilts (4%, No. 66) were viremic. All gilts were viremia negative at D84 and D118.

[0479] No significant differences (P = 1.0000) were detected in the percentage of gilts with an abnormal clinical assessment on at least one day between D1 and D113 post-vaccination in each group between the two vaccine titer groups and the challenge control group. Individually, only three gilts showed any abnormal assessments during this time period. Two gilts showed lameness (one challenge control gilt and one negative control gilt) and one low-titer gilt showed swelling in the left neck region. No adverse events related to this vaccine were noted as the vaccine was administered in the right neck region.

[0480] The results of PRRS viremia in piglets at DOF provided further insight into the level of protection against cross - placental infection of piglets in gilts. At DOF, for each gilt, an average of 58.1% and 55.0% of the piglets in the low - titer and high - titer groups, respectively, were qPCR - positive. In contrast, an average of 86.3% of the piglets per gilt in the challenge control group were qPCR - positive in serum / body fluids, which was significantly higher than the two vaccine groups (P ≤ 0.0381). When examining the viral load of piglets at DOF 0, the viral load of high - titer piglets was significantly lower compared to challenge - control piglets (P = 0.0030); no difference in viral load was detected between low - titer and challenge - control piglets (P = 0.0620). For each gilt, the percentage of piglets with positive viremia decreased significantly (P ≤ 0.05), indicating a reduction in the vertical transmission of virulent PRRSv from vaccinated gilts to offspring when vaccinated with either dose of European - type PRRS 94881 MLV. Additionally, the median qPCR piglet value per gilt at DOF in the high - titer group was 3.00 log 10 GE / mL; while the median qPCR piglet value in serum / body fluids per gilt in the challenge control group was 6.40 log 10 GE / mL (P = 0.0030). No significant difference in the viral load of piglets at DOF was detected between the low - dose group and the challenge control group (P = 0.0620). This data further demonstrated the efficacy of high - dose PRRS 94881 MLV when administered to gilts and sows.

[0481] The low - titer and high - titer groups showed that the average percentage of piglets with clinical disease (clinical observation score > 0) on at least one day from DOF + 1 to DOF + 20 per litter was 32.5% and 33.4%, respectively. These results were significantly lower than those of the challenge control group, which showed an average of 91.6% of piglets per litter for the same parameter (P ≤ 0.0001), further demonstrating the efficacy of the two doses of the vaccine.

[0482] No significant difference in the average body weight of piglets at DOF 0 was detected between the groups (P ≥ 0.2972); while the body weights at DOF + 20 and the ADWG from DOF 0 to DOF + 20 in the two vaccine groups were significantly higher (P ≤ 0.0028). Again, these results demonstrated the efficacy of the two doses of PRRS 94881 MLV.

[0483] The autopsy results confirmed that almost all fetal pigs were correctly classified at farrowing. Since the number of fetal pigs classified as crushed but actually stillborn and those classified as stillborn but actually crushed at farrowing was very small compared to the total number of correctly classified fetal pigs at farrowing, they were not changed before analyzing the gilt performance data. One challenged control piglet died after blood collection. Since this involved only one piglet compared to the large total number of piglets in the challenged control group, this piglet was not removed from the analysis.

[0484] Lung samples were collected from 141, 79, 75, and 4 dead fetal pigs / piglets in the challenged control group, low titer group, high titer group, and negative control group, respectively. The mean qPCR lung values for the challenged control group, low titer group, high titer group, and negative control group were determined to be 4.68, 4.10, 3.55, and 0.00 log 10 GE / mL. These data were not analyzed because the piglets that survived to 20 days of age were not autopsied, but these results highlight that when gilts were challenged with virulent PRRSv, gilts vaccinated with PRRS 94881 MLV reduced the viral load in the lungs of the piglets.

[0485] In summary, the results of this study demonstrated that compared to the challenged control group, the percentage of live piglets per litter at farrowing in the two vaccine groups was significantly higher (P ≤ 0.0455) and the percentage and number of piglets per litter at weaning were higher (P ≤ 0.0203). Thus, the study objectives were met, and the data from this study determined that the MID of PRRS94881 MLV in gilts was 1×10 2.43 TCID 50 / 2 mL. These results were obtained 118 days after vaccination, and in addition, they determined that the duration of immunity (DOI) in gilts was approximately 4 months.

[0486] When examining the supporting data, the high dose of PRRS 94881 MLV (1×10 3.90 TCID 50 / 2 mL) resulted in a higher percentage and number of healthy piglets per gilt at farrowing (P ≤ 0.0211), a lower percentage and number of weak and shrunken fetal pigs (P ≤ 0.0090), a lower percentage of qPCR-positive gilts at D125, DOF 0, and DOF+13, and a lower viral load in the gilts after challenge (P ≤ 0.0155), a lower percentage of qPCR-positive piglets per gilt at DOF 0 and a lower viral load in the piglets (P ≤ 0.0030), a lower percentage of piglets with clinical disease per gilt (P < 0.0001), and higher piglet body weight and ADWG at DOF+20 (P < 0.0013).

[0487] The low-dose group caused a higher percentage of healthy piglets per sow at farrowing (P = 0.0138), a lower percentage and number of mummified fetuses (P ≤ 0.0190), a lower percentage of qPCR-positive sows and a lower viral load in sows after challenge at D125, D132, DOF 0 and DOF +13 (P ≤ 0.0290), a lower percentage of qPCR-positive piglets per sow at DOF 0 (P = 0.0381), a lower percentage of piglets with clinical disease per sow (P < 0.0001), and higher piglet body weight and ADWG at DOF +20 (P < 0.0028).

[0488] Example 7: Evaluation of the generation of PRRS 94881 MLV immunity after challenge with a heterologous European-type PRRS isolate two weeks after vaccination in susceptible piglets

[0489] The objective of this vaccination-challenge study was to evaluate the onset of immunity (OOI) two weeks after administration of a modified live virus of a candidate vaccine porcine reproductive and respiratory syndrome European-derived isolate 94881 (PRRS 94881 MLV) to susceptible piglets at 14 ± 3 days of age. The primary efficacy criterion for OOI two weeks after vaccination was whether the vaccinated group (Group 1) demonstrated a significant difference in lung lesions after challenge compared to the unvaccinated challenge control group (Group 2) (p ≤ 0.05). Secondary parameters included clinical assessments after vaccination, clinical observations after challenge, rectal temperature, average daily weight gain, assessment of PRRS antibodies and viremia in serum samples, and quantification of PRRS virus in lung samples collected at necropsy.

[0490] Piglets were randomly assigned to Group 1 (receiving 1×10 3.82 TCID 50 / mL of PRRS 94881 MLV - vaccine and challenged; n = 20), Group 2 (receiving placebo vaccine and challenged; n = 20) or Group 3 (receiving placebo vaccine and not challenged; n = 10). Piglets were housed in raised-floor plastic pens (n = 5 pigs / pen). Each treatment group was housed in a different room to avoid mechanical transmission of PRRSv, including aerosolization.

[0491] All animals allocated to this study completed the study. No adverse events were reported during the study. The mean lung injury scores at D24 for PRRS 94881 MLV-vaccinated pigs and challenge controls were 27.4% and 54.8%, respectively. The mean lung injury score for PRRS 94881 MLV-vaccinated pigs was significantly lower than that of the challenge controls (p = 0.0002), and thus met the primary efficacy variable and the OOI was determined to be 2 weeks after a single vaccination. At D14, D17, and D21, the proportion of PRRS 94881 MLV-vaccinated pigs with positive PRRS-antibody titers was significantly higher than that of the challenge controls (p ≤ 0.0012). The mean AUC of viremia for PRRS 94881 MLV-vaccinated pigs from D17 - D24 after challenge was significantly lower than that of the challenge controls (50.72 and 54.61 log 10 GE / mL; p = 0.0039). Compared with 45% lethargy signs in challenge control pigs, PRRS 94881 MLV-vaccinated pigs showed no lethargy signs (0%) after challenge (p = 0.0012). During the post-challenge phase of the study (SD14 - SD24), the weight gain of PRRS 94881 MLV-vaccinated pigs was higher than that of the challenge controls (0.3 and 0.1 kg, respectively; p = 0.0003).

[0492] Significant (p ≤ 0.05) reductions in post-challenge lung injury, clinical signs, viral replication in blood and lung, and improvement in growth performance in vaccinated animals demonstrated the efficacy of the vaccine against virulent PRRSv when challenged 2 weeks after vaccination. Thus, immunity was demonstrated to be generated at least 2 weeks after vaccination with PRRS 94881 MLV.

[0493] Objectives / Purposes of the Study

[0494] The objective of this vaccination-challenge study was to evaluate the onset of immunity (OOI) two weeks after administration of a modified live virus (PRRS 94881 MLV) of a porcine reproductive and respiratory syndrome European-derived isolate 94881 to susceptible piglets at 14 ± 3 days of age. The primary efficacy criterion for meeting the OOI at 2 weeks after vaccination was: whether the vaccinated group (Group 1) demonstrated a significant difference in reduced lung injury after challenge compared to the non-vaccinated challenge control group (Group 2) (p ≤ 0.05).

[0495] Secondary efficacy parameters analyzed between the vaccine and challenge control groups included post-vaccination clinical assessments, PRRS serology, post-challenge PRRS viremia, post-challenge clinical observations, average daily weight gain (ADWG), rectal temperature, and lung PRRSv quantification.

[0496] The study included a negative control group (Group 3) that was not vaccinated or challenged to demonstrate that the source herd was free of PRRSv infection throughout the trial period and that biosecurity was not breached during this trial period.

[0497] Event timeline

[0498] Table 7.1: Event timeline

[0499]

[0500] Study design

[0501] Table 7.2: Study design

[0502]

[0503] Blinding criteria

[0504] The study investigators and designees were unaware of the assigned treatment groups throughout the survival period of the study. To maintain this unawareness, randomization was performed by personnel not involved in pig assessment (i.e., clinical assessment, clinical observation, or necropsy) at D0 and IVP and CP treatments were administered according to the assignment. BIVI laboratory staff were unaware of the treatment received by each pig while performing their respective tasks.

[0505] Materials

[0506] Investigational veterinary product (IVP) and control product (CP)

[0507] Table 7.3: IVP

[0508]

[0509] Table 7.4: CP

[0510]

[0511] Challenge materials

[0512] Table 7.5: Challenge materials

[0513]

[0514] Treatment

[0515] Judgment of dosing rationality

[0516] IVP was administered to the assigned pigs at a dose of 1.0 mL to evaluate the OOI of PRRS 94881 MLV 2 weeks after vaccination. CP was administered as a placebo vaccine at a dose of 1.0 mL to Group 2 and Group 3.

[0517] Dosing regimen

[0518] At D0, IVP or CP was administered intramuscularly to the right neck region of the assigned pigs via a non-research data collector using a sterile 3.0 mL Luer-lock syringe and a sterile 20g x 1 inch (2.54 cm) or 18g x 3 / 4 inch (1.91 cm) needle. The dosing schedule is shown in Table 7.6 below.

[0519] Table 7.6: Dosing Schedule

[0520] Group Number Treatment Dose / Route Study days 1 20 IVP 1.0 mL IM D0 2 20 CP 1.0 mL IM D0 3 10 CP 1.0 mL IM D0

[0521] Animal Information

[0522] Details of the Research Animals

[0523] Table 7.7: Animal Information

[0524]

[0525] Inclusion / Exclusion Criteria

[0526] All piglets participating in this study were PRRS ELISA negative and were determined to be healthy as observed at the time of vaccination.

[0527] Removal Criteria after Inclusion

[0528] Do not remove pigs from the study.

[0529] Animal Management and Caging

[0530] Animal Caging

[0531] During the entire study period, the piglets were caged at Veterinary Resources, Inc. (VRI) (Cambridge, IA). Groups 1, 2, and 3 were caged in the same but separate rooms to ensure biosafety. The piglets were caged in multiple pens (5 piglets / pen) within each room. Group 1 was caged in 4 pens in Room 5, Group 2 was caged in 4 pens in Room 6, and Group 3 was caged in 2 pens in Room 4. The pens consisted of plastic buckets on raised platforms and plastic slatted floors. Each pen contained a plastic 6-hole feeder and a nipple drinker. Each isolation room was constructed the same as the other isolation rooms and met biosafety level 2 (BL2), was filtered through a hepafilter, and was mechanically ventilated under thermostat-regulated temperature control.

[0532] In this study, treatment groups need to be isolated because it is well known in the scientific community that PRRSv is easily transmitted between pigs via various mechanisms, including aerosolization. This includes non - virulent live PRRS vaccines because these biological products contain attenuated virus particles that mimic the characteristics of the virulent wild - type PRRS without the ability to cause disease. Appropriate methods should ensure the maintenance of biosecurity and that vaccinated animals are not accidentally cross - contaminated with unvaccinated PRRSv - untreated negative control animals. Appropriate measures are taken by the testing laboratory staff to thoroughly clean and disinfect each room before use in the study.

[0533] Each room in the laboratory has a fan and a heater to promote adequate air circulation and heating. The ventilation systems of each room are independent and identical, so air is not shared between rooms.

[0534] Solid feed is stored in bags and is pest - free. Water is available ad libitum. According to acceptable livestock husbandry practices in the region, piglets are fed ad libitum a commercial food (Lean Metrics Infant, Purina Mills, St. Louis, MO) supplemented with the drugs tiamulin (35 g / ton) and chlortetracycline (400 g / ton) appropriate for their size, age, and condition.

[0535] As determined by the study investigators, the gilts were in good health and nutritional status before the start of the study.

[0536] During the study, a mild decline in the physical condition of the selected animals was observed, with a rough hair coat appearance, joint swelling, and varying degrees of lameness. The study investigators considered all these conditions to be non - specific conditions commonly present in groups of pigs in confinement. Coughing, sneezing, rapid breathing, dyspnea, and mild to moderate lethargy were also noted in the selected pigs after challenge and were considered typical clinical signs associated with pneumonia, although non - specific etiologically. The study investigators determined that no animal required concomitant treatment during this study.

[0537] All pigs assigned to this study were euthanized at D24 and disposed of by commercial incineration after necropsy. No food from animals participating in this study entered the human food chain.

[0538] Efficacy assessment

[0539] To evaluate the OOI of PRRS 94881 MLV at 2 weeks post-vaccination, Group 1 and Group 2 were challenged at D14 and the lung lesions post-challenge were evaluated. If Group 1 (the minimum immunizing dose of PRRS 94881 MLV) demonstrated a significant reduction in lung lesions post-challenge compared to the challenge control group (Group 2) (p ≤ 0.05), then the OOI at 2 weeks post-vaccination was achieved.

[0540] The secondary efficacy parameters analyzed between the vaccinated group and the challenge control group included clinical assessment post-vaccination, clinical observations post-challenge, rectal temperature, body weight, and average daily weight gain (ADWG), assessment of PRRS antibodies and viremia in serum samples, and quantification of PRRS virus in lung samples collected at necropsy.

[0541] An unchallenged negative control group (Group 3) was included in the study to demonstrate that the source herd was free of PRRS infection and maintained biosecurity throughout the study period.

[0542] Criteria for a valid test

[0543] PRRS antibody-negative serum samples were required both pre-purchase and at D0.

[0544] Serum samples collected from Group 2 and Group 3 until the day of challenge must be free of PRRS antibodies, and serum samples collected from Group 3 until the end of the study must be free of PRRS antibodies for the study to be valid.

[0545] Primary outcome parameters

[0546] The primary efficacy variable for statistical assessment was the total lung lesion score at D24 of the study.

[0547] Total lung lesion score:

[0548] On Day 24 after collecting and recording data and samples, all pigs in the study were euthanized according to VRI SOP PRC1027 (Appendix 1, Annex 8). Each pig was necropsied according to VRI SOP PRC 1028. The thoracic cavity was exposed by a designated person and the heart and lungs were removed. The study investigator examined the lungs of each group, described any macroscopic pathology noted, and determined the pathological percentage of each lung lobe. The observations and data were recorded on the necropsy report recording form. The total lung lesion score for each pig was determined by using the EP rule.

[0549] Supportive parameters

[0550] Other parameters analyzed between Group 1 and Group 2 included clinical assessment post-vaccination, PRRS serology, post-vaccination viremia, clinical observations post-challenge, ADWG, rectal temperature, and post-challenge lung virus quantification. These parameters were analyzed as supportive parameters and not as primary parameters to meet the study objectives.

[0551] Clinical assessment

[0552] On the date outlined in Table 7.1, the clinical assessment of all pigs after vaccination was observed by the study investigator or designee. The observations were recorded on the clinical assessment record form.

[0553] PRRS serology

[0554] On the date outlined in Table 3, whole venous blood was collected. Briefly, approximately 2 - 5 mL of blood was collected from each piglet into a serum separator tube (SST) of appropriate size. The sample collection was recorded on the sample collection record form. Allow the blood in the SST to clot at room temperature. The blood samples were transferred to BIVI - Ames on the day of collection and the sample transfer record form was completed. The blood samples were briefly centrifuged at BIVI - Ames and the serum was harvested, separated and transferred to appropriate tubes. Each tube was labeled with the gilt's ID number, study number, collection date, study day, and sample type. At BIVI - Ames, one set of serum samples was stored at 2 - 8 °C and other sets of serum samples were stored at - 70 ± 10 °C.

[0555] PRRS antibodies in serum samples collected on days 0, 7, 14, 17, 21, and 24 and stored at 2 - 8 °C were tested by BIVI - Ames. The results were reported as negative (ELISA S / P ratio < 0.4) or positive (ELISA S / P ratio ≥ 0.4).

[0556] PRRS viremia

[0557] Other sets of serum samples were collected on days 0, 7, 14, 17, 21, and 24 and stored at - 70 ± 10 °C at BIVI - Ames until the end of the survival period of the study.

[0558] The shipment included the completed sample transfer record form. The serum samples were tested for PRRS vRNA by qPCR at bioScreen. The results were reported as genome equivalents / mL (log GE / mL).

[0559] Clinical observations after challenge

[0560] On the date outlined in Table 7.1, the clinical signs of disease in the piglets were observed. The observations were made by the study investigator or designee and recorded on the clinical observation record form. Based on the clinical observation scoring system outlined in Table 7.8 below, the respiration, behavior, and coughing of the piglets were observed daily.

[0561] Table 7.8: Clinical observation scoring system

[0562]

[0563] Average daily weight gain (ADWG)

[0564] Individual weights were collected on the dates outlined in Table 3. The investigator or designee weighed each pig on a calibrated scale. Results were reported in kg on the weight record form. Average daily gain from D0 to D14 and D14 to D24 was determined.

[0565] Rectal temperature

[0566] Rectal temperatures were collected by the investigator or designee on the dates outlined in Table 6.1. Rectal temperatures were recorded in °C on the clinical observation record form.

[0567] Quantification of PRRS virus in lung tissue

[0568] For each group of lungs, two samples were retained from the left and right cranial, left and right cardiac, left and right diaphragmatic, and middle lobes. Each lung sample was approximately 1 inch (2.54 cm) × 1 inch (2.54 cm). For one set of lung samples, all three samples from the left side were combined in one container; while all three samples from the right side and the middle lobe sample were combined in another container. Each container was filled with a sufficient amount of 10% formaldehyde solution. For the other sets of lung samples, all three left lung samples were combined in one ; while all three samples from the right side and the middle lobe sample were combined in another ; and all containers and were appropriately labeled with the animal number, study number, collection date, study day, sample type, and whether the sample was from the left or right side. The lung samples in [] were stored on dry ice until shipped to BIVI-Ames, while the samples in formaldehyde were stored at room temperature. Sample collection was recorded on the autopsy report record form. The formalin-fixed lung tissue samples and lung samples were transferred to BIVI-Ames. Each shipment included a completed sample transfer record form.

[0569] Shipments included a completed sample transfer record form. The bioScreen tested the lung samples for PRRSv RNA by qPCR (Appendix 1, Attachment 7). The left lung tissue was homogenized and tested. The right lung tissue and middle lobe samples were homogenized and tested. For the left and right lung samples, the results were reported as genome equivalents / mL (log GE / mL).

[0570] Adverse events

[0571] No adverse events were reported during this study.

[0572] Statistical methods

[0573] Experimental unit

[0574] In this study, the treatment groups had to be housed separately to prevent the spread of PRRSv to the unvaccinated groups. Therefore, the rooms were the experimental units. However, for the purposes of this analysis, any potential bias due to confounding effects of "room" and "treatment" was ignored, and the piglets were used as the experimental units.

[0575] Randomization

[0576] Fifty (50) piglets were stratified by body weight (n = 5 piglets / stratum). A random number was assigned to each pig using the random number function in Excel. Within each body weight stratum, the pigs were ranked in ascending order of the assigned random numbers. The treatment groups were then assigned to the pigs in this numerical order: the two lowest random numbers were assigned to Group 1, the next two numbers were assigned to Group 2, and the highest number was assigned to Group 3. Group 1 and Group 2 each contained 20 pigs, and Group 3 contained 10 pigs.

[0577] Analysis

[0578] Statistical analysis and data summarization were performed by Dr. rer. hort. Martin Vanselow, Biometrie & Statistik, Zum Siemenshof 21, 30539 Hannover, Germany, +49(0)511 606 777650, m.vanselow@t-online.de.

[0579] The data were analyzed using a completely randomized design structure. Statistical analysis was performed using SAS software version 8.2 (SAS, Cary, USA / North Carolina, SAS Institute Inc.). All tests for differences were two-sided tests with α = 5%.

[0580] Total lung injury score:

[0581] The total lung injury score was measured on the day of necropsy (D24) as the percentage of lung involvement calculated using the weighted formula recommended in the monograph for porcine enzootic pneumonia vaccine (inactivated). This formula takes into account the relative weights of the seven lung lobes. The percentage of the area of the lung lobe with typical lesions, as assessed, was multiplied by the respective factor for each lung lobe to obtain the total weighted lung injury score. The factors for the individual lung lobes are presented in Table 7.9.

[0582] Table 7.9: Factors for calculating the lung injury score

[0583] Lobe Factor Left parietal lobe 0.05 Left cardiac lobe 0.06 Left diaphragmatic lobe 0.29 Right parietal lobe 0.11 Cardiac lobe 0.10 Right diaphragmatic lobe 0.34 Right accessory / middle lobe 0.05

[0584] The differences between the treatment groups were compared using the Wilcoxon-Mann-Whitney test.

[0585] Clinical assessment after vaccination

[0586] Frequency table of animals with at least one positive finding between D1 and D12. Differences between treatment groups were tested by Fisher's exact test.

[0587] PRRS serology

[0588] Frequency table of animals yielding positive ELISA results. Differences between treatment groups were tested by Fisher's exact test.

[0589] PRRS viremia

[0590] Viremia data were evaluated separately for each day of the study. Additionally, the area under the curve of the individual responses of viral load between D14 and D24 (AUC D14-D24) and between D17 and D24 (AUC D17-D24) was analyzed.

[0591] Quantitative PCR data (PRRS viral load [log 10 GE / mL]) were compared between treatment groups by the Wilcoxon-Mann-Whitney test. Prior to calculation, the analytical result 'not detected' was replaced with a log 10 GE / mL value of 0.0, and 'positive' was replaced with 3.0. The differences between treatment groups were tested using the Wilcoxon-Mann-Whitney test.

[0592] Clinical observations after challenge

[0593] Frequency table of animals with at least one positive finding between D15 and D24. Differences between treatment groups were tested by Fisher's exact test.

[0594] The maximum and mean scores for respiration, behavior, coughing, and all three combined (total) for each animal from D15 to D24 were used for statistical evaluation. Differences between treatment groups were tested by the Wilcoxon-Mann-Whitney test.

[0595] Body weight and average daily gain

[0596] Individual daily gains were calculated for the time periods between D0 and D14 and between D14 and D24. Descriptive statistics were calculated for each day and each time period of the study. Differences between treatment groups were tested using analysis of variance and subsequent t-tests. The least-squares means for the groups and the differences between the least-squares means and their 95% confidence intervals were calculated by analysis of variance.

[0597] Rectal temperature

[0598] Differences in initial temperature data between treatment groups were tested using analysis of variance and subsequent t-tests. The least-squares means for the groups and the differences between the least-squares means and their 95% confidence intervals were calculated by analysis of variance.

[0599] Quantification of PRRS virus in lung tissue

[0600] Quantitative PCR data (PRRS virus load [log 10 GE / mL]) from lungs collected at D24 were compared between treatment groups by the Wilcoxon-Mann-Whitney test. The mean of the left and right lung qPCR results (log 10 GE / mL) was used for evaluation. Prior to calculation, the analytical result of 'not detected' was replaced with a log 10 GE / mL value of 0.0, and 'positive' was replaced with 3.0.

[0601] Frequency table of positive qPCR results. Differences between treatment groups were tested by Fisher's exact test.

[0602] Results

[0603] Total lung injury score

[0604] An overview of the total lung injury scores and associated p-values in the groups is shown in Table 7.10 below.

[0605] Table 7.10: Total lung injury score (%)

[0606]

[0607] 1 Group 1 = PRRS 94881 MLV vaccine of MID, challenged; Group 2 = placebo-treated, challenged; Group 3 = placebo-treated, unchallenged. NI = not included in the statistical analysis.

[0608] The mean total lung injury scores of piglets at D24 in the PRRS 94881 MLV vaccinated group and the challenged control were 27.368% and 54.841%, respectively. The injury score of the PRRS-vaccinated pigs was significantly lower than the mean injury score of the challenged control (p = 0.0002).

[0609] PRRS viremia

[0610] An overview of the qPCR data of PRRSv RNA detected in serum is shown in Table 7.11 below.

[0611] Table 7.11: qPCR of PRRSv RNA detected in serum by day (log 10 GE / mL)

[0612]

[0613] 1Group 1 = PRRS 94881 MLV vaccine of MID, challenged; Group 2 = placebo-treated, challenged; Group 3 = placebo-treated, not challenged. NI = not included in the statistical analysis. AUC = area under the curve; GE / ml per day

[0614] At D0, PRRSv RNA was not detected in the serum of any piglets. The mean values of PRRS 94881 MLV-inoculated pigs at D7 and D14 were 3.17 and 3.30 log 10 GE / mL, respectively. These two-day values were significantly higher than those of the challenged control (p < 0.0001), as the challenged control did not detect PRRSv RNA until D17. On that day, the mean values of PRRS 94881 MLV-inoculated piglets and the challenged control were 6.78 and 8.00 log 10 GE / mL, respectively. The D17 value of the challenged control was significantly higher than that of PRRS 94881 MLV-inoculated piglets (p < 0.0001). At D21 and D24, compared with the mean values of the challenged control at D21 and D24 of 7.88 and 7.34 log 10 GE / mL, the mean values of PRRS 94881 MLV-inoculated pigs on the same days were 7.51 and 7.26 log 10 GE / mL, respectively. There was no significant difference between PRRS94881 MLV-inoculated pigs at D21 or 24 (p ≥ 0.0565). During this study period, PRRSv RNA was not detected in the serum of any negative control pigs.

[0615] There was no difference in AUC 14 - 24 between PRRS 94881 MLV-inoculated pigs and challenged control pigs (65.84 and 66.61, respectively; p = 0.4945). The AUC of PRRS 94881 MLV-inoculated pigs from D17 - D24 was significantly lower than that of the challenged control (50.72 and 54.61, respectively; p = 0.0039).

[0616] Quantification of PRRS virus in lung tissue

[0617] Individual PRRSv qPCR results from lung tissue collected at D24 necropsy are presented in Table 30 of Appendix 1. An overview of PRRSv RNA detected in lung tissue by qPCR data is shown in Table 7.12 presented below, and an overview of the frequency of animals with positive qPCR at necropsy is shown in Table 7.13 below.

[0618] Table 7.12: Lung virus isolation, qPCR at necropsy (D24) (mean log 10 GE / mL)

[0619]

[0620] 1 Group 1 = PRRS 94881 MLV vaccine from MID, challenged; Group 2 = placebo-treated, challenged; Group 3 = placebo-treated, not challenged. NA = not applicable due to lack of variability. NI = not included in the statistical analysis.

[0621] Table 7.13: Frequency of animals with possible PRRSv RNA aPCR in lung tissue collected at necropsy (D24)

[0622]

[0623] 1 Group 1 = PRRS 94881 MLV vaccine from MID, challenged; Group 2 = placebo-treated, challenged; Group 3 = placebo-treated, not challenged. NA = not applicable due to lack of variability. NI = not included in the statistical analysis.

[0624] PRRSv RNA was detected in the lung tissue of all piglets in the PRRS 94881 MLV vaccinated group and all piglets in the challenged control group. There was no difference between these groups. PRRSv RNA was not detected in the lung samples of any negative control piglets.

[0625] Clinical observations after challenge

[0626] The frequency of piglets with at least one positive clinical assessment score during the post-challenge period (D15 - D24) is shown in Table 7.14 below.

[0627] Table 7.14: Frequency of piglets with positive clinical observations after challenge (D15 - D24)

[0628]

[0629] 1 Group 1 = PRRS 94881 MLV vaccine from MID, challenged; Group 2 = placebo-treated, challenged; Group 3 = placebo-treated, not challenged. NI = not included in the statistical analysis.

[0630] Abnormal breathing was observed in the PRRS 94881 MLV vaccinated group (10%) and the challenged control group (30%); however, these values were not significantly different (p = 0.2351).

[0631] Abnormal behavior was observed only in the challenge control group (45%), but not in the PRRS 94881 MLV vaccinated group (0%). The incidence of abnormal behavior in the PRRS 94881 MLV vaccinated group was significantly lower than that in the challenge control group (p = 0.0012).

[0632] Coughing was observed in both the PRRS 94881 MLV vaccinated group (30%) and the challenge control group (55%). These values were not significantly different (p = 0.2003).

[0633] The percentages of piglets with a total clinical score > 0 in the PRRS 94881 MLV vaccinated group and the challenge control group were 30% and 65% respectively. These values were not significantly different (p = 0.0562).

[0634] No clinical signs were observed in the negative control group at any time after challenge.

[0635] An overview of the maximum clinical observation scores during the post - challenge period (D15 to D24) in the groups is shown in Table 7.15 below.

[0636] Table 7.15: Maximum clinical scores after challenge (D15 to D24)

[0637]

[0638] 1 Group 1 = PRRS 94881 MLV vaccine at MID, challenged; Group 2 = placebo - treated, challenged; Group 3 = placebo - treated, not challenged. NI = not included in the statistical analysis.

[0639] After challenge, abnormal breathing was observed in both the PRRS 94881 MLV vaccinated group and the challenge control group, with maximum scores of 1 (wheezing / rapid breathing) and 2 (dyspnea) respectively. There was no significant difference between these respiratory scores (p = 0.1872). The median maximum respiratory score for both groups was 0.

[0640] No abnormal behavior was observed in the PRRS 94881 MLV vaccinated group during the post - challenge period (maximum score = 0). In contrast, the maximum behavior score in the challenge control group was 1 (mild to moderate lethargy; p = 0.0012), although the median score for this group was 0. The maximum score in the PRRS 94881 MLV vaccinated group was significantly lower than the score in the challenge control group (p = 0.0012). The median maximum behavior scores for both groups were 0.

[0641] Coughing was observed in both the PRRS 94881 MLV vaccinated group and the challenge control group after challenge. The maximum scores in the PRRS 94881 MLV vaccinated group and the challenge control group were 1 (gentle or intermittent coughing) and 2 (severe or severe recurrent coughing), respectively, and the median scores were 0 and 1, respectively. There was no significant difference between these groups (p = 0.1129). The median maximum cough scores in the PRRS 94881 MLV vaccinated group and the challenge control group were 0 and 1, respectively.

[0642] The maximum total scores in the PRRS 94881 MLV vaccinated group and the challenge control group were 1 and 4, respectively, and the median total scores were 0 and 1, respectively. The maximum score in the PRRS 94881 MLV vaccinated group was significantly lower than that in the challenge control group (p = 0.0072). The median total scores in the PRRS 94881 MLV vaccinated group and the challenge control group were 0 and 1, respectively.

[0643] No clinical signs were observed from D15 to D24 in the non-challenged negative control group during this study period. The maximum score for each parameter in this group was 0.

[0644] An overview of the mean clinical observation scores in the groups during the post-challenge period (D15 to D24) is shown in Table 7.16 below.

[0645] Table 7.16: Mean clinical scores after challenge from D15 to D24

[0646]

[0647] 1 Group 1 = PRRS 94881 MLV vaccine at MID, challenged; Group 2 = placebo-treated, challenged; Group 3 = placebo-treated, non-challenged. NI = not included in the statistical analysis.

[0648] The mean clinical observation scores followed a pattern similar to that of the maximum clinical scores, where significant differences in the mean behavior score (p = 0.0012) and the mean total score (p = 0.0103) were observed only between the PRRS 94881 MLV vaccinated group and the challenge control group.

[0649] The mean respiratory scores in the PRRS 94881 MLV vaccinated group and the challenge control group were 0.02 and 0.07, respectively. The mean behavior scores in the PRRS 94881 MLV vaccinated group and the challenge control group were 0.00 and 0.12, respectively. The mean cough scores in the PRRS 94881 MLV vaccinated group and the challenge control group were 0.07 and 0.17, respectively. The mean total scores in the PRRS 94881 MLV vaccinated group and the challenge control group were 0.08 and 0.35, respectively.

[0650] During this study period, no clinical signs were observed in the non-challenged negative control group from D15 to D24. The average score for each parameter in this group was 0.

[0651] Body weight and average daily weight gain

[0652] An overview of the body weights at D0, D14, and D24 and the ADWG from D0 to D14 and from D14 to D24 is shown in Table 7.17 below.

[0653] Table 7.17: Body weight and average daily weight gain (kg and kg / d)

[0654]

[0655] 1 Group 1 = PRRS 94881 MLV vaccine from MID, challenged; Group 2 = placebo-treated, challenged; Group 3 = placebo-treated, non-challenged.

[0656] The average body weights of the PRRS 94881 MLV vaccinated group and the challenged control group at D0 were 4.1 and 4.2 kg, respectively. At D14, the average body weights of the PRRS 94881 MLV vaccinated group and the challenged control group were 7.6 and 7.4 kg, respectively. At D24, the average body weights of the PRRS 94881 MLV vaccinated group and the challenged control group were 10.3 and 8.9 kg, respectively. The average daily weight gains of the PRRS 94881 MLV vaccinated group and the challenged control group during the vaccination period (D0 to D14) were 0.25 and 0.23 kg / d, respectively. The ADWG of the PRRS 94881 MLV vaccinated group and the challenged control group during the challenge period (D14 to D24) were 0.26 and 0.15 kg / d, respectively. The ADWG of the negative control group from D0 - D14 and D14 - D24 were 0.23 and 0.34 kg / d, respectively.

[0657] The average body weights of the negative control piglets at D0, D14, and D28 were 4.1, 7.2, and 10.6 kg, respectively.

[0658] An overview of the LS means and statistical analysis of the body weights and ADWG of the PRRS 94881 MLV vaccinated group and the challenged control group is shown in Table 7.18 below.

[0659] Table 7.18: LS mean body weight and daily weight gain (kg)

[0660]

[0661] 1 Group 1 = PRRS 94881 MLV vaccine from MID, challenged; Group 2 = placebo-treated, challenged.

[0662] On day 0, the mean LS body weights of piglets inoculated with PRRS 94881 MLV and the challenge control group were 4.14 and 4.17 kg respectively. The difference was -0.03 kg, with no significant difference (p = 0.8743). At D14, the mean LS body weights of the PRRS 94881 MLV inoculation group and the challenge control group were 7.64 and 7.39 kg respectively. The difference was 0.25 kg, also with no significant difference (p = 0.4297). At D24, the mean LS body weights of the PRRS 94881 MLV inoculation group and the challenge control group were 10.26 and 8.87 respectively. The difference on this day was 1.39 kg, and the body weight of the vaccinated group was significantly higher than that of the challenge control group (p = 0.0063).

[0663] The mean LS ADWG during the vaccination period (D0 to D14) of the PRRS 94881 MLV inoculation group and the challenge control group were 0.25 and 0.23 kg / d respectively. These values had no significant difference (p = 0.1889). The mean LS ADWG during the post-challenge period (D14 to D24) of the PRRS 94881 MLV inoculation group and the challenge control group were 0.26 and 0.15 respectively. The ADWG of the PRRS 94881 MLV inoculation group was significantly higher than that of the challenge control group (p = 0.0003).

[0664] Rectal temperature

[0665] An overview of the rectal temperature is shown in Tables 7.19 and 7.20 below. An overview of the mean LS values and statistical analysis of the rectal temperature of the PRRS 94881 MLV inoculation group and the challenge control group are shown in Tables 7.21 and 7.22 below.

[0666] Table 7.19: Rectal temperature (°C) from day 13 to 22

[0667]

[0668] 1 Group 1 = PRRS 94881 MLV vaccine of MID, challenged; Group 2 = placebo-treated, challenged; Group 3 = placebo-treated, not challenged.

[0669] Table 7.20: Rectal temperature (°C) from day 23 to 24

[0670]

[0671] 1 Group 1 = PRRS 94881 MLV vaccine of MID, challenged; Group 2 = placebo-treated, challenged; Group 3 = placebo-treated, not challenged.

[0672] Table 7.21: Average LS Rectal Temperature (°C) on Days 13 - 20

[0673]

[0674] 1 Group 1 = PRRS 94881 MLV vaccine from MID, challenged; Group 2 = placebo - treated, challenged; Group 3 = placebo - treated, not challenged.

[0675] Table 7.22: Average LS Rectal Temperature (°C) on Days 21 - 24

[0676]

[0677] 1 Group 1 = PRRS 94881 MLV vaccine from MID, challenged; Group 2 = placebo - treated, challenged; Group 3 = placebo - treated, not challenged.

[0678] One day before challenge, the average and LS average rectal temperatures of piglets inoculated with PRRS 94881 MLV were 39.77 °C, and were in the range of 39.69 °C (D15) to 40.68 °C (D16) after challenge. One day before challenge, the average and LS average rectal temperatures of the challenge control were 39.39 °C, and were in the range of 39.77 °C (D16) to 40.61 °C (D20) after challenge. Before challenge (D13 and D14) and at D16 after challenge, the least - squares mean rectal temperature of the challenge control was significantly lower than that of piglets inoculated with PPRRS94881 MLV (p < 0.0001). There were no other significant differences in rectal temperature between PRRS 94881 MLV - inoculated pigs and the challenge control in this study (p ≥ 0.0528). The average and LS average rectal temperatures of the negative control group remained ≤ 39.68 °C throughout the study period.

[0679] Clinical Assessment after Vaccination

[0680] An overview of the percentage of piglets with at least one positive assessment from D1 to D12 is shown in Table 7.23 below.

[0681] Table 7.23: Percentage of Piglets with at Least One Positive Clinical Assessment from D1 - D12

[0682]

[0683] 1 Group 1 = PRRS 94881 MLV vaccine from MID, challenged; Group 2 = placebo - treated, challenged; Group 3 = placebo - treated, not challenged. NI = not included in the statistical analysis.

[0684] During the vaccination period from D-1 to D12, no piglets in the PRRS 94881 MLV vaccination group or the negative control group had any clinically evaluated findings. In the challenge control group, piglet 110 was observed to have an ulcer behind the right front leg, starting from D9. There was no significant difference in this parameter between the PRRS 94881 MLV vaccinated piglets and the challenge control (p = 1.0000).

[0685] PRRS serology

[0686] An overview of the frequencies of piglets positive for PRRS antibody titers is shown in Table 7.24 below.

[0687] Table 7.24: Frequencies of piglets positive for PRRS antibody titers by day

[0688]

[0689] 1 Group 1 = PRRS 94881 MLV vaccine at MID, challenged; Group 2 = placebo-treated, challenged; Group 3 = placebo-treated, not challenged. NA = not applicable, not analyzed. NI = not included in the statistical analysis.

[0690] All piglets in all treatment groups were PRRS antibody negative at D0 and D7. By D14, 85% of the PRRS 94881 MLV vaccinated pigs had positive PRRS antibody titers. At D17, this value increased to 95%, and at D21 and D24 it was 100%. None of the pigs in the challenge control group developed positive PRRS antibody titers until D21 (7 days after challenge), at which time 55% of the pigs had positive titers. By D24, this value increased to 95%. At D14, D17, and D21, the proportion of pigs with positive PRRS antibody titers in the PRRS 94881 MLV vaccinated pigs was significantly higher than that in the challenge control group (p ≤ 0.0012). None of the pigs in the negative control group developed PRRS antibody titers during this study period.

[0691] Discussion / Conclusion

[0692] To achieve the study objectives, at D0, three groups were included in the study design: a vaccine group (Group 1) receiving 1×10 3.82 TCID 50 of PRRS 94881 MLV; a challenge control group (Group 2) receiving the control product; and a negative control group (Group 3) also receiving the control product.

[0693] Twenty (20) healthy, PRRS susceptible and seronegative piglets were inoculated intramuscularly with 1 ml of PRRS 94881 MLV at approximately 14 days of age. Thirty (20 piglets - challenge control group and 10 piglets - negative control group) PRRS susceptible and seronegative piglets were inoculated intramuscularly with 1 ml of control product at approximately 14 days of age.

[0694] To determine whether the development of immunity was achieved at 2 weeks of PRRS 94881 MLV, the vaccine group and the challenge control group were challenged with a heterologous European PRRSv isolate (isolate 205817) 14 days after vaccination and the relative reduction of lung lesions after challenge was evaluated.

[0695] Validation of the study (negative control group 3)

[0696] To ensure that the source piglets were PRRSv-free and that no exogenous PRRSv exposure or cross-contamination occurred in the treatment and control groups during the study, a negative control group (Group 3) was included in the study design. Piglets in the negative control group were PRRSv-negative (viremia; qPCR) and PRRS antibody-negative throughout the study, thus validating this assay.

[0697] Validation of the challenge model (challenge control group 2)

[0698] A challenge model that induces sufficient PRRS clinical disease is needed to fully evaluate the generation of PRRS vaccine immunity in a laboratory setting. After vaccination with European PRRS isolate 205817 by the method described earlier, the challenge control group showed an average rectal temperature of ≥40.50°C on D19, D20, D23 and D24 (≤39.68°C for the negative control group on the same day); the average ADWG was 0.15 kg / day compared to the average ADWG of 0.34 kg / day for the negative control group from D14 to D24, and the abnormal behavior, coughing and median lung lesion scores were 55.2% (0.00% for the negative control group). These results highlight that even if the challenge virus titer is slightly lower than the target dose, severe PRRS-specific clinical disease can be induced in the challenge control group, thus validating this challenge model as a clinical experimental tool sufficient to evaluate the efficacy of PRRS vaccines and more specifically the OOI of PRRS 94881 MLV.

[0699] Determination of immunity to PRRS 94881 MLV (Group 1) for two weeks

[0700] Outcome of immunity (OOI) to PRRS 94881 MLV 2 weeks post-vaccination was determined based on the vaccine group showing a significant (p≤0.05) reduction in lung lesions post-challenge compared to the challenge control group.

[0701] Lung injury was selected as the primary parameter for determining the 2-week OOI because this parameter provides the most clinically relevant and convincing evidence of efficacy when evaluating new vaccines within the PRRS respiratory challenge model in pigs. Lung injury develops as one of the hallmarks of PRRS respiratory disease in pigs. Lung injury is usually accompanied by subsequent manifestation of secondary PRRSv disease characteristics such as clinical signs, fever, reduction in ADWG, etc.

[0702] The PRRS 94881 MLV vaccinated group showed a significant reduction in overall lung injury after challenge, as demonstrated by a median total lung injury score of 27.6% compared to the challenged control group showing a median total lung injury score of 55.2% (p = 0.0002). Therefore, based on the primary parameter of a significant reduction in lung injury after challenge, the 2-week OOI of PRRS 94881 MLV was determined at a dose of 1×10 3.82 TCID 50 . This result was achieved with a vaccine dose slightly lower than the minimum immunizing dose of 1×10 4.5 TCID 50 .

[0703] Viremia after challenge was selected as the most important secondary parameter because it represents the extent and persistence of viral replication that occurs within the host animal after exposure. A significant (p≤0.05) reduction in viremia is consistent with a PRRS vaccine that induces sufficient immunity to limit PRRS pathogenesis within the host. At 3 days post-challenge (D17), the median viremia (qPCR) in the PRRS 94881 MLV vaccinated group was significantly reduced compared to the challenged control group (6.72 GE / mL vs 8.18 GE / mL; p≤0.0001). To further evaluate viremia after challenge between groups, the amount of viral load over a specific duration after challenge was calculated and expressed as the "area under the curve" or AUC. The median AUC value from D17 to D24 in the PRRS 94881 MLV vaccinated group was 49.52 GE / mL per day; while the median AUC value in the challenged control group was 54.35 GE / mL per day. The median AUC value from D17 to D24 in the vaccine group was significantly lower compared to the challenged control group (p = 0.0039). Whether viremia was examined at 3 days post-challenge or during the post-challenge period, PRRS 94881 MLV administered 2 weeks prior to challenge with a virulent heterologous European type PRRS strain significantly (p≤0.05) reduced viremia after challenge inoculation.

[0704] Combined with the reduction in PRRS viremia after challenge, the significant (p≤0.05) reduction in viral load in lung tissue is also significant from the perspective of PRRS vaccine immunity. The reduction in viral load in lung tissue may be associated with reduced viral stability, replication and persistence within the host and, in turn, may reduce the flow of PRRSv to other pigs. In this study, the median lung qPCR result for lung tissue from the PRRS 94881 MLV-vaccinated group was 7.46 log 10 days after challenge (D24). 10 GE / mL, while the median lung qPCR result of the challenge control group was 7.88log 10 The difference between the vaccine group and the challenge control group was significant (p=0.0101), thus further supporting the 2-week OOI.

[0705] The significantly reduced severity and frequency of clinical signs after challenge in piglets also supports the efficacy of the PRRS vaccine and the determination of the 2-week OOI of PRRS94881 MLV. Abnormal breathing of sufficient severity and frequency was not noted in any group after challenge and no differences were detected (p≥0.1394). In contrast, the severity and frequency of coughing were approximately equal between groups and no differences were detected (p≥0.0835). Differences in the severity and frequency of abnormal behavior (lethargy) after challenge were detected between groups. 0 of 20 (0%) and 9 of 20 (45%) piglets in the PRRS 94881 MLV-vaccinated and challenge control groups, respectively, showed abnormal behavior for at least one day after challenge (p=0.0012). Similarly, the PRRS 94881 MLV-vaccinated group showed lower maximum abnormal clinical scores and average abnormal clinical scores after challenge compared to the challenge control group (p=0.0012). When analyzing the maximum and mean scores from D15 to D24, there were significant differences in the total clinical score (the sum of the respiratory, behavioral, and coughing scores) between the groups. Since the abnormal behavior score had an effect on the total score, the maximum total score was significantly lower and the mean total score was lower in the PRRS 94881 MLV-vaccinated group compared with the challenge control group (p≤0.0103). The differences in the severity and frequency of abnormal behaviors between the groups further support OOI 2 weeks after vaccination.

[0706] Before challenge, compared with the challenge control group, the average rectal temperature in the PRRS 94881 MLV vaccinated group was slightly higher at D13 (39.77°C vs 39.39°C; p<0.0001) and D14 (39.76°C vs 39.37°C; p<0.0001). Although significant (p≤0.05) differences were detected between groups before challenge, these differences were not biologically relevant. After challenge, the only day on which significant (p≤0.05) differences in average rectal temperature were detected between groups was D16 (2 days post-challenge). At D16, the average rectal temperatures in the vaccinated and challenge control groups were 40.68°C and 39.77°C, respectively, and the difference between groups was significant (p<0.0001). The average rectal temperature increased above 40°C 4 - 5 days post-challenge and remained above 40°C until the end of the study for both groups.

[0707] The presence of significant abnormal behavior, viremia, lung lesions, and viral load in the lungs caused by PRRS in the challenge control group led to a significant (p≤0.05) difference in ADWG post-challenge between groups. In this study, the average ADWG from D14 to D24 in the vaccinated and challenge control groups was 0.3 kg / day and 0.1 kg / day, respectively, and the difference between groups was significant (p = 0.0003). The significant (p≤0.05) difference in ADWG post-challenge between groups further supported the establishment of OOI 2 weeks after vaccination.

[0708] The post-vaccination parameters examined in this study

[0709] No abnormal clinical assessments related to PRRS 94881 MLV vaccination or the control product were observed in piglets after vaccination at D0. One challenge control piglet showed an ulcer behind the right front leg, starting at D9, which did not seem to be associated with the administration of the control product.

[0710] All piglets were PRRS ELISA seronegative at D0, thus confirming that all piglets met the inclusion criteria of being PRRS negative at the start of the study. Most piglets receiving PRRS 94881 MLV seroconverted to PRRS at D14, and all PRRS vaccinated piglets were seropositive before 7 days (D21) post-challenge. In contrast, the challenge control remained seronegative until 7 days post-challenge, at which time this group started to show PRRS seroconversion. The negative control group remained PRRS seronegative throughout the study.

[0711] At 7 and 14 days post-vaccination, the PRRS 94881 MLV vaccinated group showed average qPCR results of 3.17 and 3.30 log 10 GE / mL, respectively. These results highlighted that within 2 weeks after vaccination, 1×10 3.82 TCID50 The dose of PRRS 94881 MLV induced sufficient replication of MLV, which is generally required to establish protective immunity 2 weeks after vaccination. In contrast, the challenge control group and the negative control group were negative for PRRSv viremia from D0 to D14.

[0712] Conclusion

[0713] The significant (p≤0.05) reduction in lung injury, clinical signs, viral replication in blood and lung, and the improvement in growth performance after challenge supported the establishment of OOI 2 weeks after vaccination of single-dose PRRS 94881 MLV at 1×10 3.82 TCID 50 / mL in 14-day-old piglets.

[0714] Example 8: Evaluation of the duration of immunity of PRRS 94881 MLV after challenge with a heterologous European-type PRRS isolate 26 weeks after vaccination in 2-week-old susceptible pigs

[0715] The objective of this vaccination-challenge study was to evaluate the duration of immunity (DOI) 26 weeks after administration of a modified live virus (PRRS 94881 MLV) of the candidate vaccine porcine reproductive and respiratory syndrome European-derived isolate 94881 to PRRS-seronegative pigs at 14±3 days of age. The primary efficacy criterion for meeting the DOI at 26 weeks after vaccination was a significant reduction (p≤0.05) in the post-challenge lung injury score (overall or histological) in the PRRS 94881 MLV vaccination group (Group 1) compared to the challenge control group (Group 2).

[0716] On Day 0 (D0), 22 pigs assigned to the vaccination group received 1.0 mL IM of PRRS 94881 MLV (1×10 4.27 TCID 50 )(Group 1), 22 pigs assigned to the challenge control group received 1.0 mL IM of a control product (a placebo-matched product without PRRS 94881 MLV, Group 2), and 12 pigs assigned to the negative control group also received 1.0 mL IM of the control product (Group 3). Groups 1 and 2 were challenged with a virulent European-type PRRSV strain on D179 (Day 0 post-challenge {DPC 0}) and the clinical signs, average daily gain, and viremia of the pigs were monitored for 10 days after challenge. The pigs were necropsied on D189 (DPC 10) and the overall and histological lung injury and lung virus load were determined.

[0717] The median overall lung injury scores of PRRS 94881 MLV-vaccinated pigs and challenge controls at D189 (DPC 10) were 0.1% and 13.8%, respectively (p<0.0001). The median histological lung injury scores of PRRS 94881 MLV-vaccinated pigs and challenge controls at DPC 10 were 6.0 and 19.5, respectively (p<0.0001). PRRS 94881 MLV-vaccinated pigs had significantly lower serum virus loads at 3, 7, and 10 days post-challenge compared to challenge controls (p≤0.0001). The area under the curve (AUC) analysis of viremia post-challenge in PRRS 94881 MLV-vaccinated pigs (15.54 and 8.88 log 10 GE / mL per day, respectively) from DPC 0 to DPC 10 and from DPC 3 to DPC 10 was also significantly lower than that of the challenge control group (44.77 and 36.43 log 10 GE / mL per day, respectively, p<0.0001). The median qPCR values of lung tissues collected at necropsy from PRRS 94881 MLV-vaccinated pigs and challenge controls were 3.69 and 6.25 log 10 GE / mL, respectively (p<0.0001). There were no significant differences in clinical signs post-challenge (p≥0.4878).

[0718] The significant reduction (p≤0.05) in overall and histological lung injury, virus load in lung tissues collected at necropsy, and post-challenge viremia in PRRS 94881 MLV-vaccinated pigs compared to challenge controls supported the efficacy of the vaccine against virulent PRRSv at 26 weeks post-vaccination when challenged. The results of this study determined the duration of immunity at 26 weeks post-vaccination in pigs vaccinated with PRRS 94881 MLV at 2 weeks of age. These results were achieved with a vaccine dose of 1×10 4.27 TCID 50 / mL, which was slightly lower than the minimum immunizing dose (1×10 4.5 TCID 50 / mL) of this investigational veterinary product.

[0719] Study objectives

[0720] The objective of this vaccination-challenge study was to evaluate the duration of immunity (DOI) of a modified live virus of a European-origin isolate of porcine reproductive and respiratory syndrome virus 94881, code 19S1.U (PRRS 94881 MLV), administered to PRRS-seronegative pigs at 14 ± 3 days of age against a heterologous European-type PRRS isolate at a virulent challenge 26 weeks post-vaccination. The primary efficacy criterion for a DOI of 26 weeks post-vaccination was a significant reduction (p ≤ 0.05) in the post-challenge lung lesion scores (overall or histological) in the PRRS 94881 MLV vaccinated group (Group 1) compared to the challenge control group (Group 2).

[0721] Secondary efficacy parameters included post-vaccination and post-challenge viremia, post-vaccination clinical assessments, PRRS serology, post-challenge clinical observations, average daily weight gain (ADWG), rectal temperature, and lung PRRSv quantification. Post-challenge viremia was considered the most important secondary parameter as it was an objective and quantifiable parameter. Rectal temperature and clinical observations were then considered supportive parameters in the determination of the DOI. Finally, growth performance, serology, and virus detection in the lung were used as supportive parameters to support the primary parameter in meeting the study objective.

[0722] Study Timeline

[0723] Table 8.1: Study Timeline

[0724]

[0725] Study Design

[0726] This was a blinded vaccination-challenge efficacy study conducted in 56 weaned PRRS-seronegative pigs at 14 ± 3 days of age on Day 0 (D0). An overview of the study is provided in Table 8.2.

[0727] Table 8.2: Study Design

[0728]

[0729] Blinding Criteria

[0730] Study investigators and assignees were unaware of the assigned treatment groups throughout the survival period of the study. To maintain this unawareness, BIVI monitors performed the randomization and the assigned IVP and CP treatments were administered at D0 by individuals not involved in the pig assessments (i.e., clinical assessments, clinical observations, or necropsies). BIVI laboratory staff were unaware of the treatment each pig received while performing their respective tasks.

[0731] Materials

[0732] Investigational Veterinary Product (IVP) and Control Product (CP)

[0733] Table 8.3: IVP

[0734]

[0735] Table 8.4: CP

[0736]

[0737] Challenging material

[0738] Table 8.5: Challenging material

[0739]

[0740] Treatment

[0741] Judgment on rationality of drug administration

[0742] IVP was administered to the allocated pigs at a dose of 1.0 mL to evaluate the DOI of PRRS 94881 MLV at 26 weeks post-vaccination. CP was administered as a placebo vaccine to groups 2 and 3 at a dose of 1.0 mL.

[0743] Drug administration regimen

[0744] At D0, IVP or CP was administered intramuscularly to the right neck region of the allocated pigs by a non-research data collector using a sterile 3.0 mL Luer-lock syringe and a sterile 20g × 1 inch (2.54 cm) needle. The drug administration regimen is shown in Table 8.6 below.

[0745] Table 8.6: Drug administration regimen

[0746] Group Number Treatment Dose / Route Study days 1 22 IVP 1.0 mL IM D0 2 22 CP 1.0 mL IM D0 3 12 CP 1.0 mL IM D0

[0747] Adjunctive treatment

[0748] Since several pigs were found to have died after bacterial infection in the early stage of the study, the investigators and study monitors agreed to administer the following additional adjunctive treatment (section 15.10) to all study animals:

[0749] Day 20: (Vitamin E / Selenium, Intervet / Schering Plough Animal Health (USA)), 0.1 mL was administered intramuscularly into the middle of the right thigh

[0750] Day 21: (Ceftiofur, Pfizer Animal Health, USA), 0.5 mL was administered to the middle of the left thigh

[0751] Day 35: (Ceftiofur, Pfizer Animal Health, USA), 1.0 mL was administered into the middle of the right thigh

[0752] Day 42: (Ceftiofur, Pfizer Animal Health, USA), 1.0 mL was administered into the middle of the left thigh

[0753] Day 47: (Enrofloxacin, Bayer Animal Health, USA), 1.5 mL was administered subcutaneously into the middle of the left neck

[0754] Vitamin E / Selenium was administered to prevent mulberry heart disease, and antibiotic treatment was given to treat / prevent bacterial infections.

[0755] Animal Information

[0756] Details of Animal Studies

[0757] Table 8.7: Animal Information

[0758]

[0759] Inclusion / Exclusion Criteria

[0760] All pigs participating in this study were PRRS ELISA negative (ELISA S / P ratio < 0.4) and were determined to be healthy at the time of vaccination (D0) as determined by observational findings.

[0761] Removal Criteria after Inclusion

[0762] No pigs were removed from the study. Three pigs were found dead before challenge. Further results regarding these three pigs are presented in Section 12.8.

[0763] Animal Management and Housing

[0764] Throughout the study, the pigs were housed at Veterinary Resources, Inc. (VRI) (Cambridge, IA). The pigs were housed in multiple pens (11 or 12 pigs / pen) within each room, where the vaccinated (Group 1) and control animals (Groups 2 and 3) were housed in the same but separate rooms to ensure biosecurity. The PRRS 94881 MLV pigs were housed in room CB8 until D78, then in CC1 until D105, and then in CC3 for the remainder of the study. Throughout the study, the challenge control pigs were housed in room CC2. The negative control pigs were housed in room CB6 until D73, and then in CB7 for the remainder of the study. The animal pens were elevated off the floor with plastic slat flooring and had feeders and cup-type waterers appropriate for the age. Each isolation room was constructed the same as the others and met biosafety level 2 (BL2), was filtered through a hepafilter, and was mechanically ventilated with temperature control regulated by a thermostat.

[0765] Isolation of treatment groups was required in this study because it is well known in the scientific community that PRRSv is easily transmitted between pigs via various mechanisms, including aerosolization. This includes live, non-pathogenic PRRS vaccines because these biological products contain attenuated viral particles that mimic the characteristics of virulent wild-type PRRS without the ability to cause disease. Appropriate methods were employed to ensure biosecurity was maintained and that vaccinated animals were not accidentally cross-contaminated with unvaccinated, PRRSv-naïve negative control animals.

[0766] Appropriate measures were taken by the testing laboratory staff to thoroughly clean and disinfect each room prior to use for the study.

[0767] Each room in the laboratory had fans and heaters to promote adequate air circulation and heating. The ventilation system in each room was independent and identical, so air was not shared between rooms.

[0768] The feed was stored in bags and was pest-free. Feed and water were provided ad libitum. Since arrival, the pigs were fed Lean Metrics Starter Medicated Feed (Purina Mills LLC, St. Louis, MO) until D5, at which time they were switched to Lean Metrics Grower Medicated Feed (Purina Mills LLC, St. Louis, MO). At D64, the pigs were switched to Lean Metrics Complete 85 Feed (Purina Mills LLC, St. Louis, MO), and at D82, they were switched to Lean Metrics Complete CE85, T40 (Purina Mills LLC, St. Louis, MO), and they were fed this feed for the remainder of the study. Throughout the study, the feed provided was appropriate for the size, age, and condition of the pigs according to accepted livestock practices in the area.

[0769] As determined by the study investigators, the gilts were in good health and nutritional condition prior to the start of the study. During the study, other conditions were observed in the selected animals, including emaciation, coughing, swelling, rough hair coats, depression, abscesses, and poor body condition. The study investigators considered all of these conditions to be characteristic of group-housed growing / maturing pigs. These conditions were considered to be transient or insignificant and were not treated.

[0770] Efficacy assessment

[0771] To assess the DOI of PRRS 94881 MLV at 26 weeks post-vaccination, the PRRS 94881 MLV and challenge control groups were challenged at D179 (DPC 0), and the post-challenge lung lesions were evaluated 10 days later (DPC 10). The DOI at 26 weeks post-vaccination was achieved if the post-challenge lung lesions (overall or histological) were significantly reduced (p ≤ 0.05) in the PRRS 94881 MLV group compared to the challenge control group.

[0772] Secondary efficacy parameters analyzed between the vaccine and challenge control groups included post-vaccination and post-challenge viremia, post-challenge clinical observations, post-challenge rectal temperature, post-vaccination clinical assessments, average daily weight gain (ADWG), and PRRS serology. Post-challenge viremia was considered the most important secondary parameter because it is an objective and quantifiable parameter. Next, rectal temperature and clinical observations were considered supportive parameters in the DOI determination process. Finally, growth performance, serology, and virus detection in the lungs were used as supportive parameters to support the primary parameter in meeting the study objectives.

[0773] Criteria for a valid test

[0774] All pigs are required to be PRRS ELISA negative (ELISA S / P ratio < 0.4) at the time of pre-purchase screening and at D0. The challenge control pigs are required to be PRRS antibody negative until the time of challenge, and the negative control group is required to be PRRS antibody negative throughout the study.

[0775] Primary outcome parameters

[0776] The primary efficacy outcome variable is lung injury (overall and histological injury) at study D189 (DPC 10).

[0777] Overall lung injury score:

[0778] At D189, after collecting and recording samples and data, all remaining study pigs are euthanized following VRI SOP PRC1027 (section 15.1). Each pig is necropsied according to VRI SOP PRC 1028 (section 15.1). The chest cavity of each pig is exposed by the designated person and the heart and lungs are removed. The study investigators examine the lungs of each group, describe any macroscopic pathology, and determine the pathological percentage of each lung lobe. The observations and data are recorded on the necropsy report recording form.

[0779] Histological lung injury score

[0780] For the lungs of each group, two samples are retained from the left and right apical lobes, left and right cardiac lobes, left and right diaphragmatic lobes, and the middle lobe. Each lung sample is approximately 1 inch (2.54 cm) × 1 inch (2.54 cm). For one set of lung samples, all three samples from the left side are combined in one container; while all three samples from the right side and the middle lobe sample are combined in another container. Each container is filled with a sufficient amount of 10% formaldehyde solution. For the lung samples of other groups, all three left lung samples are combined in one ; while all three samples from the right side and the middle lobe sample are combined in another ; and all containers and are appropriately labeled with the animal number, study number, collection date, study day, sample type, and whether the sample is from the left or right side. The lung samples in formaldehyde are stored at room temperature, while the lung samples are stored on dry ice until shipped to BIVI-Ames. The sample collection is recorded on the necropsy report recording form. The formalin-fixed lung tissue samples and lung samples are transferred to BIVI-Ames. Each shipment includes a completed sample transfer record form.

[0781] Formalin-fixed lung tissue samples were stored at room temperature by BIVI - Ames until presented by BIVI - Ames to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL). Lung samples were processed and handled by ISU VDL staff within one week of necropsy according to ISU VDL procedures. Each pig produced a single slide containing 7 sections (one section from each of the 7 lung lobes). Each H&E slide was identified with a unique identification code. ISU VDL provided computer records containing the study number, identification code, and associated porcine tissues.

[0782] Once daily, on the day of histopathology reading of the study slides, the ISU VDL pathologist (K. Schwartz) first read the EU PRRS positive and negative control slides. Thereafter, the pathologist read the lung cell overgrowth and hyperplasia, septal infiltration of monocytes, necrotic debris, intra - alveolar accumulation of inflammatory cells, and perivascular accumulation of inflammatory cells on the H&E - stained lung slides. Results were recorded in an Excel spreadsheet. The lung histopathology scoring system is shown in Table 8.8 below.

[0783] Table 8.8: Lung Histopathology Scoring System

[0784]

[0785] After all slides were read, the slides were returned to the sponsor agent and archived at Boehringer Ingelheim Vetmedica, Inc. (St. Joseph, MO) after completion of the final report.

[0786] Secondary Parameters

[0787] Secondary variables included post - vaccination and post - challenge viremia, post - challenge clinical observations, post - challenge rectal temperature, average daily weight gain (ADWG), lung PRRSv quantification, post - vaccination clinical assessments, and PRRS serology.

[0788] Serum PRRS qPCR

[0789] Whole venous blood was collected before purchase and on days 0, 7, 14, 21, 28, 56, 84, 112, 140, 168, 179 (DPC 0), 182 (DPC3), 186 (DPC 7), and 189 (DPC 10). Briefly, approximately 2 - 5 mL of blood was collected from each pig into a serum separator tube (SST) of appropriate size. Sample collection was recorded on a sample collection record form. The blood in the SST was allowed to clot at room temperature. Blood samples were transferred to BIVI - Ames on the day of collection and the sample transfer record form was completed. The blood samples were briefly centrifuged at BIVI - Ames and the serum was harvested, separated, and transferred to appropriate tubes. Each tube was labeled with the pig's ID number, study number, collection date, study day, and sample type. At BIVI - Ames, one set of serum samples was stored at 2 - 8°C and other sets of serum samples were stored at - 70 ± 10°C.

[0790] Clinical observations after challenge

[0791] Clinical signs of disease in pigs from D178 (DPC - 1) to D189 (DPC 10) were observed. The observations were made by the study investigator or designee and recorded on a clinical observation record form. Based on the clinical observation scoring system outlined in Table 8.9 below, the pigs' respiration, behavior, and coughing were observed daily.

[0792] Table 8.9: Clinical observation scoring system

[0793]

[0794] Rectal temperature

[0795] Rectal temperatures from D178 (DPC - 1) to D189 (DPC 10) were collected by the study investigator or designee. The rectal temperatures were recorded in °C on the clinical observation record form.

[0796] Body weight and average daily gain

[0797] Individual body weights were collected on D0, D179 (DPC 0), and D188 (DPC 9). The study investigator or designee weighed each pig on a calibrated balance. The results were recorded in kg on a body weight record form. The average daily gain from D179 (DPC 0) to D188 (DPC9) was determined.

[0798] Lung PRRS qPCR

[0799] The lung tissue samples in BIVI-Ames were stored at -70 ± 10 °C until shipment to the address listed in Section 9.3.1. Shipment included a completed sample transfer record form. The bioScreen tested the lung samples for PRRSv RNA by qPCR (Section 15.1). The left lung tissue was homogenized and tested. The right lung tissue and samples from the middle lung lobe were homogenized and tested. For the left and right lung samples, the results were reported as genome equivalents / mL (log 10 GE / mL). The geometric mean titer of the right and left GE / mL values for each pig was calculated by a statistician using the SAS program.

[0800] Post-vaccination clinical assessment

[0801] The post-vaccination clinical assessment of all pigs was observed by the study investigator or designee. Observations were made daily from D-1 to D21, then at least three times a week from D22 to D177. The observation results were recorded on a clinical assessment record form.

[0802] PRRS serology

[0803] BIVI-Ames tested the serum samples collected before purchase and on days 0, 7, 14, 21, 28, 56, 84, 112, 140, 168, 179 (DPC 0), 182 (DPC 3), 186 (DPC 7), and 189 (DPC 10) for PRRS antibodies (Section 15.1). The results were reported as negative (ELISA S / P ratio < 0.4) or positive (ELISA S / P ratio ≥ 0.4).

[0804] Adverse events

[0805] No adverse events attributable to PRRS 94881 MLV were noted in this study.

[0806] Statistical methods

[0807] Experimental unit

[0808] In this study, the treatment groups had to be housed in separate rooms to prevent the spread of PRRSv to the unvaccinated groups. Therefore, the rooms were the experimental units. However, for the purpose of analysis, the possible deviation resulting from confounding the effects of "room" and "treatment" was ignored, and the pigs were used as the statistical units.

[0809] Randomization

[0810] Fifty-six (56) pigs were randomly assigned to one of three groups. Randomization was performed by BIVI. At the time of shipment, pigs #140 and #143 (challenge control group) and pig #168 (PRRS 94881 MLV group) were selected. From the group of 5 additional pigs meeting the inclusion criteria, pig #178 was randomly selected to replace pig #140, pig #177 was randomly selected to replace 143, and pig #179 was randomly selected to replace pig #168.

[0811] Analysis

[0812] Statistical analysis and data summary were performed by Dr. rer. hort. Martin Vanselow, Biometrie & Statistik, Zum Siemenshof 21, 30539 Hannover, Germany, +49(0)511 606 777 650, m.vanselow@t-online.de. The data were analyzed using a completely randomized design structure. Statistical analysis was performed using SAS software version 8.2 or higher (SAS, 2001, Cary, USA / North Carolina, SAS Institute Inc.). Pigs #179 in the PRRS 94881 MLV group and pigs #124 and #161 in the challenge control group died before challenge and were excluded from the post-challenge analysis. All tests for differences were designed as two-sided tests with α = 5%. The report from the statistician is presented in section 15.9.

[0813] Overall lung injury score

[0814] The overall lung injury score for each pig was calculated by multiplying the pathological percentage of a specific lung lobe by the factor shown in Table 8.10 below. The calculation was performed using a SAS program.

[0815] Table 8.10: Factors for calculating the overall lung injury score

[0816] Lobe Factor Left parietal lobe 0.05 Left cardiac lobe 0.06 Left diaphragmatic lobe 0.29 Right parietal lobe 0.11 Cardiac lobe 0.10 Right diaphragmatic lobe 0.34 Right accessory / middle lobe 0.05

[0817] The Wilcoxon-Mann-Whitney test was used to compare the differences between treatment groups.

[0818] Histological lung injury score

[0819] For each lobe and animal, the individual histological scores of the lung samples were summed. This total score was divided by the number of lobes examined per animal. The result was used as a single value for comparison between treatment groups. The Wilcoxon-Mann-Whitney test was used to test for differences between treatment groups.

[0820] Lung PRRS qPCR

[0821] Quantitative PCR data (PRRS virus load [log 10 GE / mL]) collected from the lungs at D189 were compared between treatment groups by the Wilcoxon-Mann-Whitney test. The mean of the left and right lung qPCR results (log 10 GE / mL) was used for evaluation. Prior to calculation, the analytical result 'not detected' was replaced with a log 10 GE / mL value of 0.0, and 'positive' was replaced with 3.0.

[0822] A frequency table of positive qPCR results was generated. Differences between treatment groups were tested by Fisher's exact test.

[0823] Serum PRRS qPCR

[0824] Viremia data were evaluated separately for each day of the study. Additionally, the area under the individual response curves for virus load between D179 and D189 (AUC 0-10) and between D182 and D189 (AUC 3-10) was analyzed.

[0825] Quantitative PCR data (PRRS virus load [log 10 GE / mL]) were used to compare between treatment groups by the Wilcoxon-Mann-Whitney test. Prior to calculation, the analytical result 'not detected' was replaced with a log 10 GE / mL value of 0.0, and 'positive' was replaced with 3.0. The differences between treatment groups were tested using the Wilcoxon-Mann-Whitney test.

[0826] A frequency table of positive qPCR results was generated. Differences between treatment groups were tested by Fisher's exact test.

[0827] Clinical observations after challenge

[0828] A frequency table of animals with at least one positive finding between D180 and D189 was generated. The total score was the sum of the respiratory score + behavior score + cough score. Calculations were performed using the SAS program. Differences between treatment groups were tested by Fisher's exact test.

[0829] The maximum and mean scores for respiration, behavior, cough, and all three combined (total) for each animal from D180 to D189 were used for statistical evaluation. Differences between treatment groups were tested by the Wilcoxon-Mann-Whitney test.

[0830] Body weight and average daily gain

[0831] Calculate the individual daily weight gain during the period from D179 to D188. For each day and the period of the study, calculate the descriptive statistics. Differences between treatment groups were tested using analysis of variance and subsequent t-tests. The least square means of the groups and the differences between the least square means and their 95% confidence intervals were calculated by analysis of variance.

[0832] Rectal temperature

[0833] Differences between treatment groups regarding the initial temperature data were tested using analysis of variance and subsequent t-tests. The least square means of the groups and the differences between the least square means and their 95% confidence intervals were calculated by analysis of variance.

[0834] Clinical assessment after vaccination

[0835] Generate a frequency table of animals with at least one positive finding between D1 and D21. Differences between treatment groups were tested by Fisher's exact test.

[0836] PRRS serology

[0837] Generate a frequency table of positive ELISA results at each time point. Differences between treatment groups were tested by Fisher's exact test.

[0838] Results

[0839] Overall lung injury score

[0840] The median overall lung injury scores of the PRRS 94881 MLV vaccinated group and the challenge control at D189 (DPC 10) were 0.1% and 13.8%, respectively. The median overall lung injury score of the PRRS vaccinated pigs was significantly lower than that of the challenge control (p < 0.0001). The median overall lung injury score of the negative control group was 0.0%.

[0841] Animal No. 123 (challenge control group) could not be scored for lung injury at D189 due to diffuse pleurisy and adhesions. After autopsy, Moraxella osloensis, Staphylococcus warneri, Staphyloccous hyicus, and Pseudomonas species were isolated from the lung tissue of this pig.

[0842] An overview of the overall lung injury scores and related p-values in the groups is shown in Table 8.11 below.

[0843] Table 8.11: Overview of the overall lung injury scores (%) at D189 in the groups

[0844]

[0845] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. 2 No. 123 could not be scored due to diffuse pleurisy and adhesions caused by bacterial infection. 3 One PRRS 94881 MLV pig and two challenge control pigs died before challenge and were not included in the analysis. NI = not included in the statistical analysis

[0846] Histological lung injury score

[0847] The median histological lung injury scores for the PRRS 94881 MLV vaccinated group and the challenge control were 6.0 and 19.5, respectively. The median histological lung injury score for the PRRS vaccinated group was significantly lower than that of the challenge control (p < 0.0001). The median histological lung injury score for the negative control group was 9.0.

[0848] An overview of the histological lung injury scores and associated p-values in the groups is shown in Table 8.12 below.

[0849] Table 8.12: Overview of histological lung injury scores in the groups

[0850]

[0851] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. 2 One PRRS 94881 MLV pig and two challenge control pigs died before challenge and were not included in the analysis. NI = not included in the statistical analysis

[0852] Lung PRRS qPCR

[0853] The median qPCR lung values from lung tissue for the PRRS 94881 MLV vaccinated pigs and the challenge control were 3.69 and 6.25 log 10 GE / mL, respectively. The median qPCR value for the PRRS 94881 MLV vaccinated pigs was significantly lower than that of the challenge control (p < 0.0001). PRRSv RNA was not detected in any of the lung samples from the negative control pigs.

[0854] An overview of the lung qPCR values and test results (p-values) in the groups is shown in Table 8.13 below.

[0855] Table 8.13: Overview of lung qPCR (mean log 10 GE / mL) values in the groups

[0856]

[0857] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. 2 One PRRS 94881 MLV pig and two challenge control pigs died before challenge and were not included in the analysis. NI = not included in the statistical analysis

[0858] PRRSv RNA was detected in the lung tissues of 90% and 100% of the PRRS 94881 MLV vaccinated pigs and challenge control pigs, respectively. There was no statistical difference between the vaccinated group and the challenge control (p = 0.4878).

[0859] An overview of the frequencies of PRRS qPCR positive lung tissues from pigs at necropsy in the groups is shown in Table 8.14 below.

[0860] Table 8.14: Frequencies of PRRSv qPCR positive lung tissues in the groups

[0861]

[0862] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. 2 One PRRS 94881 MLV pig and two challenge control pigs died before challenge and were not included in the analysis. NI = not included in the statistical analysis

[0863] Serum PRRS qPCR

[0864] At D0, PRRSv RNA was not detected in the serum of any pig. After vaccination, the mean values of the PRRS 94881 MLV vaccinated pigs at D7, D14, D21, D28, D56, D84, D112, D140 and D168 were 3.00, 0, 0, 3.00, 0, 0, 0, 0 and 0 log 10 GE / mL, respectively. At D7, D14, D21 and D28, the values were significantly higher than those of the challenge control (p ≤ 0.0013), because PRRSv RNA was not detected in the challenge control until D182 (DPC 3).

[0865] At D179 (DPC 0), PRRSv RNA was not detected in the serum of any pig. The medians of the challenge control at D182 (DPC 3), D186 (DPC 7) and D189 (DPC 10) were 5.88, 5.30 and 4.24 log 10Compared with 0 GE / mL, after challenge, the median values of PRRS 94881 MLV - inoculated pigs were 4.44, 0, and 0 log 10 GE / mL on the same day. On all days after challenge, the median value of the challenge control was higher than that of the PRRS 94881 MLV group (p ≤ 0.0001).

[0866] During this study period, PRRSv RNA was not detected in the sera from any of the negative - control pigs.

[0867] The median AUC values of PRRS 94881 MLV - inoculated pigs from DPC 0 to DPC 10 and DPC 3 to DPC 10 were 15.54 and 8.88 log 10 GE / mL per day, respectively. In contrast, the median AUC values of the challenge control from DPC 0 to DPC 10 and DPC 3 to DPC 10 were 44.77 and 36.43 log 10 GE / mL per day, respectively. For both periods, the median values of the PRRS MLV group were significantly lower than those of the challenge control (p < 0.0001).

[0868] An overview of the serum PRRS qPCR data is shown in Tables 8.15 and 8.16 below.

[0869] Table 8.15: Overview of serum PRRS qPCR results (log 10 GE / mL) from D0 to D168

[0870]

[0871] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. NI = not included in the statistical analysis

[0872] Table 8.16: Overview of serum PRRS qPCR results (log10GE / mL) from D179 to D189

[0873]

[0874] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. NI = not included in the statistical analysis. AUC = area under the curve; log 10 GE / mL per day

[0875] After vaccination, the proportion of qPCR-positive pigs in the PRRS 94881 MLV group was significantly higher than that in the challenge control group at D7, D14, D21, and D28 (p≤0.0013). At D56, no significant difference in the proportion of qPCR-positive pigs was detected between the groups (p = 0.1069).

[0876] At D182 (DPC 3), 100% of the pigs in the PRRS 94881 MLV and challenge control groups were qPCR-positive (not tested). At D186 (DPC 7) and D189 (DPC 10), the proportion of qPCR-positive pigs in the PRRS MLV group was significantly lower than that in the challenge control group (<0.0001).

[0877] An overview of the proportion of qPCR-positive data in the groups is shown in Tables 8.17 and 8.18 below.

[0878] Table 8.17: Overview of the proportion of post-vaccination serum qPCR-positive results in the groups

[0879]

[0880] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. n.a. = not tested; NI = not included in the statistical analysis

[0881] Table 8.18: Overview of the proportion of post-challenge serum qPCR-positive results in the groups

[0882]

[0883] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. n.a. = not tested; NI = not included in the statistical analysis

[0884] Clinical observations after challenge

[0885] Compared with one challenge control pig (No. 149) showing a score of "1" at D185 (DPC 6), no abnormal respiration was observed in any of the PRRS 94881 MLV-vaccinated pigs after challenge. No difference in the percentage of pigs showing abnormal respiration for at least one day after challenge was detected between the groups (p = 0.4878).

[0886] After challenge, no abnormal behavior or coughing was observed in any of the PRRS 94881 MLV-vaccinated pigs or challenge control pigs.

[0887] The percentages of pigs with a total clinical score > 0 at least one day after challenge in the PRRS 94881 MLV vaccinated group and the challenge control group were 0% and 5%, respectively. These values were not significantly different (p = 0.4878).

[0888] No clinical signs were observed in the negative control group from D179 to D189.

[0889] An overview of the frequencies of pigs in the groups with at least one positive clinical observation score during the post-challenge period is shown in Table 8.19 below.

[0890] Table 8.19: Overview of the frequencies of pigs in the groups with at least one positive clinical observation score after challenge

[0891]

[0892] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. 2 One PRRS 94881 MLV pig and two challenge control pigs died before challenge and were not included in the analysis. NI = not included in the statistical analysis; NA = test not applicable due to lack of variability

[0893] There was no difference in the maximum respiratory score or maximum total score after challenge between the groups (p = 0.4878).

[0894] An overview of the maximum clinical observation scores during the post-challenge period (DPC 1 to DPC 10) in the groups is shown in Table 8.20 below.

[0895] Table 8.20: Overview of the maximum clinical scores after challenge in the groups

[0896]

[0897] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. 2 One PRRS 94881 MLV pig and two challenge control pigs died before challenge and were not included in the analysis. NI = not included in the statistical analysis

[0898] The mean clinical observation scores followed a pattern similar to the percentage of pigs with a positive clinical score. There was no significant difference between the PRRS94881 MLV vaccinated group and the challenge control group (p ≥ 0.4878).

[0899] An overview of the mean clinical observation scores during the post-challenge period (DPC 1 to DPC 10) in the groups is shown in Table 8.21 below.

[0900] Table 8.21: Overview of Mean Clinical Scores after Challenge in Groups

[0901]

[0902] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. 2 One PRRS 94881 MLV pig and two challenge control pigs died before challenge and were not included in the analysis. NI = not included in the statistical analysis

[0903] Body weight and average daily weight gain

[0904] There was no significant difference between groups (p = 0.2389). At D179 (DPC 0), the average body weights and LS mean body weights of the PRRS 94881 MLV group and the challenge control group were 134.6 and 128.2 kg, respectively. The difference was not significant (p = 0.1090). At D188 (DPC 9), the average body weights and LS mean body weights of the PRRS 94881 MLV group and the challenge control group were 138.3 and 130.3 kg, respectively. At D188, the body weight of the vaccinated group was significantly higher than that of the challenge control group (p = 0.0455).

[0905] The LS mean ADWGs of the PRRS 94881 MLV group and the challenge control group during the challenge period (DPC 0 to DPC 9) were 0.4 and 0.2 kg / d, respectively. These values were not significantly different (p = 0.1041).

[0906] The average body weights of the negative control pigs at D0, D179, and D188 were 2.7, 117.2, and 120.0 kg, respectively. The ADWG of the negative control group from D179 to D188 was 0.5 kg / d.

[0907] An overview of the average body weights at D0, D179 (DPC 0), and D188 (DPC 9) and the ADWG from DPC 0 to DPC 9 in the groups is shown in Table 8.22 below. An overview of the LS means and statistical analysis of the body weights and ADWGs of the PRRS 94881 MLV group and the challenge control group is shown in Table 8.23 below.

[0908] Table 8.22: Overview of Body Weight and Average Daily Weight Gain (kg and kg / d) in Groups

[0909]

[0910] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group.

[0911] Table 8.23: Summary of the LS Mean Body Weight and Daily Weight Gain (kg and kg / d) in Groups

[0912]

[0913] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group.

[0914] Rectal temperature

[0915] The mean rectal temperature of the PRRS 94881 MLV group on the day of challenge (D179) was 39.3 °C, and the mean value after challenge ranged from 39.1 °C (D189, DPC 10) to 39.8 °C (D181, DPC 2). The mean rectal temperature of the challenge control group on the day of challenge was 39.1 °C, and the mean value after challenge ranged from 39.1 °C (D183, DPC 4) to 39.9 °C (D182, DPC 3). During the same time period, the mean rectal temperature of the negative control group remained ≤ 39.3 °C.

[0916] A summary of the rectal temperatures in the groups is shown in Table 8.24 below.

[0917] Table 8.24: Summary of the Rectal Temperatures (°C) from D179 (DPC 0) to D189 (DPC 10) in Groups

[0918]

[0919] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. 2 One PRRS 94881 MLV pig and two challenge control pigs died before challenge and were not included in the analysis.

[0920] Compared with the challenge control, the least squares mean rectal temperature of the PRRS 94881 MLV vaccinated pigs was significantly higher at DPC 0 (p = 0.0281), DPC 2 (p = 0.0095), DPC 4 (p = 0.0034), and DPC 5 (p < 0.0001). Compared with the challenge control, the least squares mean rectal temperature of the PRRS 94881 MLV vaccinated pigs was significantly lower at DPC 8 (p = 0.0183) and DPC 10 (p = 0.0001). No significant differences were detected between the groups for the remaining days after challenge (p ≥ 0.0642). A summary of the LS means and statistical analysis of the rectal temperatures in the groups is shown in Table 8.25 below.

[0921] Table 8.25: Overview of the LS mean rectal temperature (°C) of D179 (DPC 0) to D189 (DPC 10) in the groups

[0922]

[0923] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group.

[0924] Among the 21 PRRS 94881 MLV-vaccinated pigs, 3 (14%) and among the 20 challenge control pigs, 5 (25%) had a rectal temperature ≥ 40.5 °C for at least one day after challenge. No difference in the proportion of pigs showing a rectal temperature ≥ 40.5 °C for at least one day after challenge was detected between the groups (p = 0.4537). An overview of the proportion of pigs with fever (≥ 40.5 °C) for at least one day after challenge in the groups is shown in Table 8.26 below.

[0925] Table 8.26: Overview of the proportion of pigs with fever (≥ 40.5 °C) for at least one day after challenge in the groups

[0926]

[0927] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. 2 One PRRS 94881 MLV pig and two challenge control pigs died before challenge and were not included in the analysis. NI = not included in the statistical analysis

[0928] Clinical assessment after vaccination

[0929] Among the 22 PRRS 94881 MLV pigs, 4 (18%), among the 22 challenge control pigs, 8 (36%) and among the 12 negative control pigs, 2 showed an abnormal clinical assessment for at least one day out of D1 to D21. There was no significant difference in this parameter between the groups (p = 0.3102).

[0930] An overview of the percentage of pigs with at least one abnormal clinical assessment out of D1 to D21 in the groups is shown in Table 8.27 below.

[0931] Table 8.27: Overview of the percentage of pigs with at least one abnormal clinical assessment in D1 - D21 in the groups

[0932]

[0933] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. NI = not included in the statistical analysis

[0934] Overall, 7 PRRS 94881 MLV pigs showed an abnormal clinical assessment for at least one day between D1 and D177.

[0935] Pig 121 showed abdominal distension from D61 to D146, scrotal swelling from D147 to D167, abdominal distension on D168, scrotal swelling from D169 - 172, and abdominal distension from 173 to 177. Pig 141 was emaciated from D4 - D10, depressed from D4 - D6, and had rough fur on D5. Pig 144 showed coughing at D26. Pig 146 showed sternal swelling at D82. Pig 147 had weak legs and tremors from D84 - D86 and tremors on D84. Pig 154 was emaciated from D4 - D6. Pig 179 was emaciated from D2 - D5, had rough fur on D5, and was found dead on D6. Thirteen pigs in the challenge control group showed an abnormal clinical assessment for at least one day between D1 and D177: Pig 124 showed tremors and shivering at D20 and was found dead on D21. Pig 134 showed scrotal swelling from D46 - D68, abdominal distension from D69 - D143, umbilical hernia at D144, and abdominal distension from D145 to D177. Pig 137 showed scrotal swelling from D108 - D143. Pig 138 showed scrotal swelling from D115 - D143. Pig 148 showed lameness or leg swelling from D16 - 20 and coughing at D35. Pig 149 was emaciated from D5 - D9 and D12 and had rough fur from D12 - D15. Pig 150 was emaciated from D4 - D9 and D13, had poor body condition from D10 - D12, and was depressed on D11. Pig 161 was emaciated from D4 - D9, had rough fur and central nervous system signs at D9, and was found dead on D10. Pig 167 showed scrotal swelling from D117 - D143. Pig 170 was emaciated from D4 - D7 and was depressed at D7. Pig 172 showed dewclaw ulcers or swelling from D120 - D143. Pig 177 was depressed at D19 and had swelling on the neck from D156 - D159. Pig 178 was depressed on D5, D17 - 20, and D28 - D36, had lameness and / or leg swelling from D15 - D47, was emaciated from D16 - D18, and had leg stiffness from D39 - D47. Six pigs in the negative control group showed an abnormal clinical assessment for at least one day between D1 and D177. Pig 120 showed coughing on D5 - 7 and D12. Pig 126 was emaciated from D2 - 18, was depressed on D4 - D5, D10, and D17 - D19, had rough fur on D5, and had labored breathing from D18 - D22. Pig 132 showed an abscess from D49 - D56. Pig 145 showed scrotal swelling from D37 - D43 and D46 to D74, and scrotal ulcers from D75 - D83 and D85 to D87. Pig 151 showed lameness and / or leg swelling from D78 - D83 and D85. Pig 155 showed an abscess from D69 - D77.

[0936] Three deaths occurred prior to challenge. Pig 179 (PRRS 94881 MLV, D6): Necropsy revealed minimal lesions (emaciation, poor body condition). Laboratory tests showed mild macrophage interstitial pneumonia. Immunohistochemistry was negative for PRRS. Intestinal samples were autolyzed but showed no signs of severe necrosis or severe inflammation. Smooth Escherichia coli and Enterococcus spp (15.9 parts) were isolated. Pig 124 (challenge control, D21): No gross lesions were identified at necropsy. Laboratory tests showed severe suppurative to pyogranulomatous meningoencephalitis and suppurative perivasculitis. Congestion of the lungs and liver was also evident. The diagnosis was Streptococcus suis-associated meningoencephalitis. Pig 161 (challenge control, D10): Necropsy revealed minimal lesions (emaciation, poor body condition). Bordetella bronchiseptica, Streptococcus alpha haemolytic, and Staphylococcus auricularis were isolated from lung tissue.

[0937] PRRS Serology

[0938] All pigs were negative for PRRS ELISA at D0 and D7. By D14, 90% of the PRRS 94881 MLV-vaccinated pigs had a positive PRRS ELISA titer. At D21, this number increased to 95%, and at D28, D56, D84, D112, D140, and D168, this number was 100%, 100%, 100%, 90%, 100%, and 95%, respectively. During the vaccination period of this study, no challenge control pigs developed PRRS antibody titers, and from D14 to D168, the percentage of PRRS 94881 MLV-vaccinated pigs with positive PRRS antibody titers was significantly higher than that of the challenge controls (p < 0.0001).

[0939] During the challenge period of the study, the percentages of PRRS 94881 MLV-vaccinated pigs with positive PRRS ELISA titers at DPC 0, DPC 3, DPC 7, and DPC 10 were 95%, 95%, 100%, and 100%, respectively. In contrast, challenge control pigs did not develop PRRS antibody titers until DPC 7, at which time 30% had titers. At DPC 10, this increased to 80%. Throughout the challenge period of the study, the percentage of animals with positive PRRS antibody titers was higher in PRRS 94881 MLV-vaccinated pigs (p ≤ 0.0478).

[0940] Except for two pigs at D112, pigs in the negative control group were seronegative for PRRS ELISA throughout the study. Pig Nos. 116 and 120 were seropositive for PRRS ELISA at D112.

[0941] An overview of the percentage of pigs in the groups that were positive for PRRS antibody titers before challenge is shown in Table 8.28 below. Data from the challenge portion of the study are presented in Table 8.29 below.

[0942] Table 8.27: Overview of the frequency of pigs in the groups that were positive for PRRS antibody titers from day 0 to day 168 by day

[0943]

[0944] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. NI = not included in the statistical analysis; NA = not applicable, not analyzed

[0945] Table 8.29: Overview of the frequency of pigs in the groups that were positive for PRRS antibody titers from DPC 0 to DPC 10 by day

[0946]

[0947] 1 Group 1 = PRRS 94881 MLV vaccine; Group 2 = challenge control group; Group 3 = negative control group. NI = not included in the statistical analysis

[0948] Discussion / Conclusion

[0949] To achieve the study objectives, twenty-two (22) healthy, PRRS-susceptible, and seronegative pigs were intramuscularly inoculated with 1 ml of PRRS 94881 MLV at approximately 14 days of age. Thirty-four (22 pigs - challenge control group and 12 pigs - negative control group) PRRS-susceptible and seronegative pigs were intramuscularly inoculated with 1 ml of the control product at approximately 14 days of age.

[0950] Validation of the study and challenge model

[0951] During the entire study period, the pigs in the negative control group remained negative for PRRSv (viremia; qPCR). Two pigs in the negative control group (No. 116 and No. 120) had positive ELISA titers at D112, while all other ELISA results in this group were negative. It was considered that viremia was not detected in these pigs overall or in this group; similarly, all other serum samples were ELISA negative, and it was considered that the results of these two pigs at D112 might be false positives due to non-assignable laboratory errors. Therefore, this was a valid study. Irrespective of the establishment of the valid study, in contrast to the median overall injury score of 0.0%, the median histological lung injury score of the negative control group was 9.0 at D189. These data highlight that pigs kept in normal pig-raising conditions for a long time develop minor lung injuries, which are insignificant and not related to specific pathogens.

[0952] After inoculation with the European-type PRRS isolate 205817 by the method described earlier, the challenged control group showed an average ADWG of 0.2 kg / day from DPC0 to DPC 9 (the average ADWG of the negative control group was 0.5 kg / day), a median overall lung injury score of 13.8% (0.0% for the negative control group), a median histological lung injury score of 19.5 (9.0 for the negative control group), and a median of PRRSv RNA detected in the lung tissue of 6.25 log 10 GE / mL (the median for the negative control group was 0.0 log 10 GE / mL). These results highlight that PRRS-specific clinical disease was induced in the challenged control group, thus validating this challenge model as a clinical experimental tool sufficient to evaluate the efficacy of PRRS vaccines and more particularly the 26-week immunity duration of PRRS 94881 MLV.

[0953] Determination of the 26-week immunity duration of PRRS 94881 MLV

[0954] The determination of the DOI of PRRS 94881 MLV at 26 weeks after vaccination was based on the fact that the vaccinated group showed a significant reduction in post-challenge lung injury (overall or histological) compared to the challenged control group (p ≤ 0.05).

[0955] Overall and histological lung injuries were selected as the main parameters for determining the 26-week DOI because this parameter provides the most clinically relevant and convincing evidence of efficacy when evaluating new vaccines within the PRRS respiratory challenge model in pigs. The development of lung injury is one of the hallmarks of PRRS respiratory disease in pigs, and it can be considered the source of all subsequent expressions of secondary PRRSv disease characteristics (such as clinical signs, fever, reduced ADWG, etc.).

[0956] Compared with the challenged control group with a median overall lung injury score of 13.8%, the median overall lung injury score was 0.1% (p < 0.0001) in the PRRS 94881 MLV group, indicating a significant reduction in overall lung injury after challenge in the PRRS 94881 MLV group. Additionally, compared with the challenged control group with a median histological lung injury score of 19.5, the median histological lung injury score was 6.0 (p < 0.0001) in the PRRS 94881 MLV group, indicating a significant decrease in the histological lung injury score in the PRRS 94881 MLV group. Therefore, based on the main parameter of a significant reduction in lung injury after challenge, the DOI of PRRS 94881 MLV was determined to be 26 weeks at a dose of 1×10 4.27 TCID 50 . This result was achieved with a vaccine dose slightly lower than the target minimum immunizing dose of 1×10 4.5 TCID 50 / mL. Although the overall lung injury of one challenged control pig (No. 123) could not be scored due to pleurisy and adhesions caused by bacterial infection, the histological lung injury could be scored. This pig was excluded from the overall lung injury score analysis of the challenged control group without affecting the results of this study.

[0957] Viremia after challenge was selected as the most important secondary parameter because it represents the degree and persistence of viral replication in the host animal after exposure. A significant reduction in viremia (p ≤ 0.05) corresponds to a PRRS vaccine that induces sufficient immunity to limit the pathogenesis of PRRS in the host. At 3, 7, and 10 days after challenge, the PRRS 94881 MLV inoculated group showed a significant reduction in viremia (qPCR) compared with the challenged control group (p < 0.0001). To further evaluate viremia after challenge between groups, the amount of viral load over a specific duration after challenge was calculated and expressed as the "area under the curve" or AUC. The median AUC value of the PRRS 94881 MLV inoculated group from DPC 0 to DPC 10 was 15.54 log 10 GE / mL per day; while the median AUC value of the challenged control group was 44.77 log 10 GE / mL per day (p < 0.0001). Additionally, the median AUC value of the PRRS 94881 MLV inoculated group from DPC 3 to DPC 10 was 8.88 log 10 GE / mL per day; while the median AUC value of the challenged control group during this period was 36.43 log 10 GE / mL per day (p < 0.0001). Whether viremia was examined at specific time points after challenge or over time after challenge, PRRS 94881 MLV administered 26 weeks before challenge with a virulent heterologous European-type PRRS strain significantly (p ≤ 0.05) reduced viremia after challenge inoculation.

[0958] Combined with the reduction in PRRS viremia after challenge, the significant (p≤0.05) reduction in viral load in lung tissue is also significant from the perspective of PRRS vaccine immunity. The reduction in viral load in lung tissue may be associated with reduced viral stability, replication and persistence within the host, and in turn may reduce the flow of PRRSv to other pigs. In this study, the median lung qPCR result for lung tissue from the PRRS94881 MLV group at 10 days post-challenge (DPC 10) was 3.69 log 10 GE / mL, while the median lung qPCR result of the challenge control group was 6.25log 10 The difference between the vaccine group and the challenge control group was significant (p<0.0001), thus further supporting the duration of immunity of 26 weeks.

[0959] The significant reduction in severity and frequency of clinical signs post-challenge in pigs also supports PRRS vaccine efficacy and the determination of a DOI of 26 weeks for PRRS 94881 MLV. Only one pig showed clinical signs post-challenge: Pig 149 (challenge control) had a respiratory score of "1" (panting / rapid breathing) on ​​D185. No pigs in the PRRS 94881 MLV-vaccinated group showed clinical signs during the post-challenge period of this study, and there was no statistical difference between the vaccinated and challenge control groups (p=0.4878 or not tested). The DOI for clinical signs post-challenge was not sufficiently powered in this study.

[0960] After the challenge, fever varied between groups. Compared with the challenge control pigs, the PRRS 94881 MLV-vaccinated pigs showed significantly lower LS mean rectal temperatures on two days (DPC 8 and DPC 10; p≤0.0183), and higher LS mean rectal temperatures on four days (DPC 0, DPC 2, DPC 4 and DPC 5; p≤0.0281). For other days, no significant differences were detected between the groups after the challenge (p≥0.0642). Although statistical differences were detected between the groups after the challenge, these differences were not biologically significant because the mean rectal temperatures of all groups remained ≤39.9°C (challenge control group, D182). No differences in the proportion of pigs with fever for at least one day after the challenge were detected between the groups (p=0.4537).

[0961] In the challenge control group, the presence of significant viremia, lung lesions, and viral load caused by PRRS led to a significant difference in body weight at DPC 9 between the groups (p≤0.05). In this study, the LS mean body weights at DPC 9 for the vaccinated and challenge control groups were 138.3 kg and 130.3 kg, respectively (p = 0.0455). The LS mean ADWG for the vaccinated and challenged groups from DPC 0 to DPC 9 were 0.4 kg / day and 0.2 kg / day, respectively. This difference was not statistically significant (p = 0.1041).

[0962] Post - vaccination parameters examined in this study

[0963] During the vaccination period of this study, three pigs were found dead. Pig 179 (vaccinated with PRRS 94881 MLV) was found dead at D6, associated with infections of Escherichia coli and Enterococcus spp. Pig 161 (challenge control) was found dead at D10, associated with infections of Bordetella bronchiseptica, Streptococcus alpha - hemolyticus, and Staphylococcus auricularis. Pig 124 (challenge control) was found dead at D21, associated with infection of Streptococcus suis causing meningoencephalitis. To control and prevent any more deaths, pigs were treated en masse with injectable vitamins and antibiotics. After treatment, no more deaths occurred. Since deaths occurred in both treatment groups, it is assumed that the IVP itself is not associated with the infections. More likely, the pigs arriving at the research laboratory had these infections. The data from these pigs were included when available. The gross and histological lung injury scores from these pigs were excluded from the lung injury analysis because these pigs died before challenge administration. The loss of 1 PRRS 94881 MLV pig and 2 challenge control pigs over the long period from vaccination to challenge did not affect the study results.

[0964] No abnormal clinical assessments related to PRRS 94881 MLV vaccination or the control product were observed in the pigs after vaccination at D0. Seven pigs in the PRRS 94881 MLV vaccination group had abnormal assessments after vaccination; while 13 pigs in the challenge control group had abnormal assessments. Excluding these three pigs that died due to bacterial infections, these abnormal assessments included emaciation, coughing, swelling, rough fur, depression, abscesses, and poor body condition at various time points, none of which persisted for a long time. In the authors' opinion, these findings are not related to the administration of either experimental product, but are typical findings in pigs growing / maturing over a long period in group - housed conditions.

[0965] All pigs were PRRS ELISA seronegative at D0, thus confirming that all pigs met the inclusion criteria of PRRS seronegativity at the time of entry into the study. Most pigs (90%) receiving PRRS 94881 MLV seroconverted for PRRS at D14, and all PRRS-vaccinated pigs were seropositive at D28. In contrast, the challenge control pigs remained seronegative until 7 days post-challenge, at which time this group began to show PRRS seroconversion. As previously reported, 2 negative control pigs were PRRS ELISA seropositive at D112, which was considered an incidental finding, likely due to non-assignable laboratory error.

[0966] At 7, 14, 21, and 28 days post-vaccination, the PRRS 94881 MLV-vaccinated groups showed median qPCR results of 3.00, 0, 0, and 3.00 log 10 GE / mL, respectively. These results highlight that within 4 weeks post-vaccination, a 1×10 4.27 TCID 50 dose of PRRS 94881 MLV induced sufficient replication of the MLV, which is generally required to establish protective immunity 4 weeks post-vaccination. In contrast, the challenge control group was negative for PRRS viremia until 3 days post-challenge.

[0967] Conclusions

[0968] Compared to the challenge control group, the significant reduction (p≤0.05) in overall and histological lung lesions at necropsy, viral load in lung tissue at necropsy, and post-challenge viremia in the PRRS 94881 MLV group demonstrated the efficacy of the vaccine against virulent PRRSv when vaccinated at 2 weeks of age and challenged 26 weeks post-vaccination. Therefore, the results of this study demonstrated the duration of immunity 26 weeks after vaccination with PRRS 94881 MLV. These results were achieved with a vaccine dose of 1×10 4.27 TCID 50 / mL, which was slightly lower than the minimum immunizing dose (1×10 4.5 TCID 50 / mL).

[0969] The sequences of the PRRSV 94881 attenuated strain and the parental strain are as follows:

[0970] SEQ ID NO:1: Full-length nucleotide sequence of the PRRS prototype virus of 94881

[0971]

[0972]

[0973]

[0974]

[0975]

[0976] SEQ ID NO:2: ORF 1a of 94881 MSV encoded by the sequence between nucleotides 178..7227 in SEQ ID NO:1

[0977]

[0978] VQTAPQQGFDPKSEAPVGTVVIGGITYNRHLVKGKEVLVPKPDNCLEAARLSLEQALAGMGQTCDLTATEVEKLKRIISQLQGLTTEQALNC

[0979] SEQ ID NO:3: ORF 1B of 94881MSV encoded by the sequence between nucleotides 7209...11600 in SEQ ID NO:1

[0980]

[0981] SEQ ID NO:4: ORF 2 of 94881MSV encoded by the sequence between nucleotides 11611..12360 in SEQ ID NO:1

[0982] MQWVYCGVKSVSCSWMPSLSSLLVWLTLSSFSPYCLGSLLQAGYWSSFSEWFAPRFSVRALPFTLPNYRRSYEGLLPNCRPDVPQFAVKHPLGILWHMRVSHLIDEMVSRRIYRTMEHSGQAAWKQVVSEATLTKLSRLDVVTHFQHLAAVEADSCRFLSSRLAMLKNLAVGNVSLEYNTTLDRVELIFPTPGTRPKLTDFRQWLISVHASIFSSVASSVTLFTVLWLRIPALRYVFGFHWPTATHHSN

[0983] SEQ ID NO:5: ORF 3 of 94881MSV encoded by the sequence between nucleotides 12219..13016 in SEQ ID NO:1

[0984] MAYQRARFHLLLCGFVCYLVHSALASNSSSTLCFWFPLAHGNTSFELTINYTICKPCPTSQAAQQRLEPGRNVWCKIGHDRCEERDHDELSMSIPSGYDNLKLEGYYAWLAFLSFSYAAQFHPELFGIGNVSRVFVDKRHQFICAEHDGQNSTISARHNISASYAVYYHHQIDGGNWFHLEWLRPFFSSWLVLNISWFLRRSPASPASRRIYQILRPTRPRLPVSWSFRTSIVSNLTGPQQRKVPLPSGGRPNVVKPSAFPSTSR

[0985] SEQ ID NO:6: ORF 4 of 94881MSV encoded by the sequence between nucleotides 12761..13312 in SEQ ID NO:1

[0986] MAATILFLLAGAQHLMVSEAFACKPCFSTHLSDIKTNTTAAAGFMVLQNINCFQSHRASTAQGTTPLRRSSQCREAVGIPQYITITANVTDESYLYNADLLMLSACLFYASEMSEKGFKVIFGNISGVVSACVNFTDYVAHVTQHTQQHHLVIDHIRLLHFLTPSTMRWATTIACLLAILLAV

[0987] SEQ ID NO:7: The ORF 5 of 95881MSV encoded by the sequence between nucleotides 13309..13914 in SEQ ID NO:1

[0988] MKCSCKLGHFLTPHSCFWWLFLLCTGLSWSFVDGNDDSSTSQYIYNLTICELNGTEWLSGHFDWAVETFVLYPVATHIISLGFLTTSHFLDALGLGAVSATGFIGERYVLSSMYGVCAFAAFVCFVIRAAKNCMACRYARTRFTNFIVDDRGRIHRWKSSIVVEKLGKAEVGGDLVNIKHVVLEGVKAQPLTRTSAEQWEA

[0989] SEQ ID NO:8: The ORF 6 of 94881MSV encoded by the sequence between nucleotides 13902..14423 in SEQ ID NO:1

[0990] MGSLDDFCNDPTAAQKLVLAFSITYTPIMIYALKVSRGRLLGLLHILIFLNCSFTFGYMTYVHFQSTNRVAFTLGAVVALLWGVYSLTESWKFITSRCRLCCLGRRYILAPAHHVESAAGLHSIPASGNRAYAVRKPGLTSVNGTLVPGLRSLVLGGKRAVKRGVVNLVKYGR

[0991] SEQ ID NO:9: The ORF 7 of 94881MSV encoded by the sequence between nucleotides 14413..14799 in SEQ ID NO:1

[0992] MAGKNQSQKKRRNAAPMGKGQPVNQLCQLLGTMIKSQRQQSRGGQAKKKKPEKPHFPLAAEDDIRHHLTQAERSLCLQSIQTAFNQGAGTASLSSSGKVSFQVEFMLPVAHTVRLIRVTSTSASQGAN

[0993] SEQ ID NO:10: Full-length nucleotide sequence of parental PRRS strain 94881

[0994]

[0995]

[0996]

[0997]

[0998]

[0999] SEQ ID NO:11: ORF 1a of parental PRRSV strain 94881 encoded by the sequence between nucleotides 178..7227 in SEQ ID NO:10

[1000]

[1001] SEQ ID NO: 12: ORF 1B of parental PRRSV strain 94881 encoded by the sequence between nucleotides 7209..11600 in SEQ ID NO: 10

[1002]

[1003] SEQ ID NO:13: ORF 2 of parental PRRSV strain 94881 encoded by the sequence between nucleotides 11611..12360 in SEQ ID NO:10

[1004] MQWVHCGVKSVSCSWMPSLSSLLVWLTLSSFSPYCLGSLLQAGYWSSFSEWFAPRFSVRALPFTLPNYRRSYEGLLPNCRPDVPQFAVKHPLGILWHMRVSHLIDEMVSRRIYRTMEHSGQAAWKQVVSEATLTKLSRLDVVTHFQHLAAVEADSCRFLSSRLAMLKNLAVGNVSLEYNTTLDRVELIFPTPGTRPKLTDFRQWLISVHASIFSSVASSVTLFTVLWLRIPALRYVFGFHWPTATHHSN

[1005] SEQ ID NO:14: ORF 3 of parental PRRSV strain 94881 encoded by the sequence between nucleotides 12219..13016 in SEQ ID NO:10

[1006] MAYQRARFHLLLCGFVCYLVHSALASNSSSTLCFWFPLAHGNTSFELTINYTICKPCPTSQAAQQRLEPGRNVWCKIGHDRCEERDHDELSMSIPSGYDNLKLEGYYAWLAFLSFSYAAQFHPELFGIGNVSRVFVDKRHQFICAEHDGQNSTISARHNISASYAVYYHHQIDGGNWFHLEWLRPFFSSWLVLNISWFLRRSPASPASRRIYQILRPTRPRLPVSWSFRTSIVSNLTGPQQRKVPLPSGGRPNVVKPSAFPSTSR

[1007] SEQ ID NO:15: ORF 4 of parental PRRSV strain 94881 encoded by the sequence between nucleotides 12761..13312 in SEQ ID NO:10

[1008] MAATILFLLAGAQHLMVSEAFACKPCFSTHLSDIKTNTTAAAGFMVLQNINCFQSHRASTAQGTTPLRRSSQCREAVGIPQYITITANVTDESYLYNADLLMLSACLFYASEMSEKGFKVIFGNISGVVSACVNFTDYVAHVTQHTQQHHLVIDHIRLLHFLTPSTMRWATTIACLFAILLAV

[1009] SEQ ID NO:16: The ORF 5 of the parental PRRSV strain 94881 encoded by the sequence between nucleotides 13309..13914 in SEQ ID NO:10

[1010] MKCSCKLGHFLTPHSCFWWLFLLCTGLSWSFVDGNDNSSTSQYIYNLTICELNGTEWLSGHFDWAVETFVLYPVATHIISLGFLTTSHFLDALGLGAVSATGFIGERYVLSSMYGVCAFAALVCFVIRAAKNCMACRYARTRFTNFIVDDRGRIHRWKSSIVVEKLGKAEVGGDLVNIKHVVLEGVKAQPLTRTSAEQWEA

[1011] SEQ ID NO:17: The ORF 6 of the parental PRRSV strain 94881 encoded by the sequence between nucleotides 13902..14423 in SEQ ID NO:10

[1012] MGSLDDFCNDPTAAQKLVLAFSITYTPIMIYALKVSRGRLLGLLHILIFLNCSFTFGYMTYVHFQSTNRVALTLGAVVALLWGVYSLTESWKFITSRCRLCCLGRRYILAPAHHVESAAGLHSIPASGNRAYAVRKPGLTSVNGTLVPGLRSLVLGGKRAVKRGVVNLVKYGR

[1013] SEQ ID NO:18: The ORF 7 of the parental PRRSV strain 94881 encoded by the sequence between nucleotides 14413..14799 in SEQ ID NO:10

[1014] MAGKNQSQKKRRNAAPMGKGQPVNQLCQLLGTMIKSQRQQSRGGQAKKKKPEKPHFPLAAEDDIRHHLTQAERSLCLQSIQTAFNQGAGTASLSSSGKVSFQVEFMLPVAHTVRLIRVTSTSASQGAN

[1015] SEQ ID NO:19: Nucleotides encoding attenuated PRRSV 94881 ORF1A

[1016]

[1017]

[1018]

[1019] SEQ ID NO:20: Nucleotides encoding attenuated PRRSV 94881 ORF1B

[1020]

[1021]

[1022] SEQ ID NO:21: Nucleotides encoding attenuated PRRSV 94881 ORF2

[1023]

[1024] SEQ ID NO:22: Nucleotides encoding attenuated PRRSV 94881 ORF3

[1025]

[1026] SEQ ID NO:23: Nucleotides encoding attenuated PRRSV 94881 ORF4

[1027]

[1028] SEQ ID NO:24: Nucleotides encoding attenuated PRRSV 94881 ORF5

[1029]

[1030] SEQ ID NO:25: Nucleotides encoding attenuated PRRSV 94881 ORF6

[1031]

[1032] SEQ ID NO:26: Nucleotides encoding the attenuated PRRSV 94881 ORF7

[1033]

[1034] SEQ ID NO:27: Nucleotides encoding the parental PRRSV 94881 ORF1a

[1035]

[1036]

[1037]

[1038] SEQ ID NO:28: Nucleotides encoding the parental PRRSV 94881 ORF1b

[1039]

[1040]

[1041] SEQ ID NO:29: Nucleotides encoding the parental PRRSV 94881 ORF2

[1042]

[1043] SEQ ID NO:30: Nucleotides encoding the parental PRRSV 94881 ORF3

[1044]

[1045] SEQ ID NO:31: Nucleotides encoding the parental PRRSV 94881 ORF4

[1046]

[1047] SEQ ID NO:32: Nucleotides encoding the parental PRRSV 94881 ORF5

[1048]

[1049] SEQ ID NO:33: Nucleotides encoding the parental PRRSV 94881 ORF6

[1050]

[1051] SEQ ID NO:34: Nucleotides encoding the parental PRRSV 94881 ORF7

[1052]

Claims

1. A composition comprising a European type porcine reproductive and respiratory syndrome virus (PRRSV), said PRRSV being the virus strain deposited under accession number ECACC 11012502 at the European Collection of Cell Cultures (ECACC).

2. The composition of claim 1, wherein said PRRSV, when administered to pigs or other mammals susceptible to PRRSV, does not cause clinical signs of PRRSV disease but is capable of inducing an immune response that immunizes the mammal against pathogenic forms of PRRSV.

3. The composition according to claim 1 or 2, which is in lyophilized form.

4. The composition according to claim 1 or 2, wherein the composition is formulated to permit administration of 10 1 to 10 7 viral particles per dose, or wherein the composition comprises at least 10 7 viral particles.

5. The composition according to claim 1 or 2, further comprising one or more attenuated or inactivated non-PRRSV pathogens or their antigenic materials.

6. The composition of claim 5, wherein said non-PRRSV pathogens are selected from pseudorabies virus, swine influenza virus, porcine parvovirus, transmissible gastroenteritis virus, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Salmonella choleraesuis, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae, and Actinobacillus pleuropneumoniae.

7. The composition according to claim 1 or 2, further comprising an adjuvant.

8. The composition according to claim 7, wherein the adjuvant is selected from the group consisting of: MCP-1, α-tocopherol, Haemophilus somnus Haemophilus sonmus ) fraction, carbopol, and combinations thereof.

9. The composition of claim 8, wherein said α-tocopherol is α-tocopherol acetate.

10. Use of a composition according to any one of claims 1 to 9 in the manufacture of a medicament for: - treating porcine reproductive and respiratory syndrome virus (PRRSV) infection or reducing its severity, - preventing PRRSV infection, - inducing an antibody response against PRRS virus, and / or - reducing the severity of one or more clinical symptoms associated with PRRSV infection, wherein said one or more clinical symptoms are selected from the group consisting of lung lesions, anorexia, skin discoloration, lethargy, respiratory signs, mummified piglets, coughing, diarrhea, and combinations thereof.

11. Use of a composition according to any one of claims 7 to 9 in the manufacture of a medicament for: - reducing the percentage of lung lesions by at least 50% when compared to animals not receiving an immunogenic composition combined with said adjuvant, - reducing viremia in animals by at least 45% when compared to animals not receiving an immunogenic composition combined with said adjuvant.

12. Use of a composition according to any one of claims 1 to 9 in the manufacture of a medicament for administering a therapeutically effective amount of PRRSV antigen to: - piglets 3 weeks of age or younger, - pigs between 3 and 4 weeks of age, - pigs between 4 and 16 weeks of age, or - pigs older than 16 weeks.

13. The use of claim 12, wherein the pigs are between 5 and 6 weeks of age or between 9 and 15 weeks of age.

14. The use of claim 12, wherein the pigs are between 7 and 10 weeks of age.

15. The use of claim 12, wherein the pigs older than 16 weeks are gilts or adult sows.

16. The composition according to claim 1 or 2, wherein the composition is administered in the following manner: - By intramuscular injection at a dose of 1 ml for pigs and 2 ml for sows, and / or - As a single dose.

17. A vaccine product comprising the lyophilized composition according to any one of claims 1 to 9 in a separate container and a solvent for reconstitution.

18. The vaccine product according to claim 17, further comprising instructions or a label which includes instructions for use.

19. The vaccine product of claim 17 or 18, wherein - The solvent is selected from the group consisting of water, physiological saline, or buffer, or co-solvent, and / or - The solvent contains an adjuvant.

20. A composition comprising a protein consisting of the sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:

9.

21. An isolated nucleic acid comprising a nucleic acid sequence encoding a protein consisting of the sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, wherein the nucleic acid sequence is SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO:

26.

22. A recombinant expression vector comprising a nucleic acid sequence encoding the PRRSV ORF of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 operably linked to a promoter.

23. The recombinant expression vector of claim 22, wherein the nucleic acid sequence encoding the ORF is SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26.

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