Method for predicting the effectiveness of a live attenuated vaccine against porcine reproductive and respiratory syndrome virus (PRRSV)

Abstract

A method is provided for inducing heterologous immunogenicity against heterologous porcine reproductive and respiratory syndrome virus (PRRSV) strains and enabling the evaluation of innate and adaptive immunity. In other aspects, a method is provided for determining the effectiveness of a vaccine against PRRSV. In still other aspects, a method is provided for predicting the effectiveness of a vaccine against PRRSV in pigs suspected of being infected with PRRSV.

Description

Technical Field

[0001] The present disclosure generally relates to methods for inducing heterologous immunogenicity against heterologous porcine reproductive and respiratory syndrome virus (PRRSV) strains and enabling the evaluation of innate and adaptive immunity. In other aspects, methods for determining the efficacy of a vaccine against PRRSV are provided. In still other aspects, methods for predicting the efficacy of a vaccine against PRRSV in pigs suspected of being infected with PRRSV are provided.

Background Art

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) remains one of the most economically important animal pathogens. This virus causes reproductive disorders and respiratory diseases and is a major contributor to porcine respiratory disease complex (PRDC). See Lunney et al., "Porcine Reproductive and Respiratory Syndrome Virus (PRRSV): Pathogenesis and Interaction with the Immune System", (2016) Ann. Rev Anim Biosci, 4:129-154. Just the respiratory diseases alone result in an annual production loss of approximately 7.4% and losses exceeding $664 million annually. See Valdes-Donoso et al., "Production Losses from an Endemic Animal Disease: Porcine Reproductive and Respiratory Syndrome (PRRS) in Selected Midwest US Sow Farms", (2018) Front Vet Sci, 5, 102: doi:10.3389 / fvets.2018.00102.

[0003] In addition to its immunosuppressive capabilities, the high mutation rate of PRRSV can evade the host immunity obtained by either infection or vaccination. Loving et al., "Innate and adaptive immunity against Porcine Reproductive and Respiratory Syndrome Virus", (2015) Vet Immunol Immunopathol 167: 1-14; Geldhof et al., "Comparison of the efficacy of autogenous inactivated Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) vaccines with that of commercial vaccines against homologous and heterologous challenges", (2012) BMC Vet Res, 8, 182: doi:10.1186 / 1746-6148-8-182; Kvisgaard et al., "Challenge of Naive and Vaccinated Pigs with a Vaccine-Derived Recombinant Porcine Reproductive and Respiratory Syndrome Virus 1 Strain (Horsens Strain)", (2021) Vaccines (Basel), 9(5): doi:10.See 3390 / vaccines9050417; Murtaugh et al., "Immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (PRRS)", (2011) Vaccine, 29(46): 8192-8204; Rowland et al., "Alternative strategies for the control and elimination of PRRS", (2017) Vet Microbiol 209: 1-4; Shi et al., "Molecular epidemiology of PRRSV: a phylogenetic perspective", (2010) Virus Res 154(1-2): 7-17; Zhou et al., "Efficacy evaluation of three modified-live virus vaccines against a strain of porcine reproductive and respiratory syndrome virus NADC30-like", (2017) Vet Microbiol 207: 108-116.

[0004] These mutations have led to a large number of strains. PRRSV is divided into two species: type 1, i.e., PRRSV-1, which is mainly identified in Europe, and type 2, i.e., PRRSV-2, which is prevalent in North America. Based on the open reading frame (ORF), PRRSV-2 is further divided into nine lineages, and there are numerous PRRSV strains. This high diversity poses a major challenge to PRRSV vaccines. Vaccines are required to defend against the continuous evolution of various PRRSV strains existing in the swine industry. The currently most prevalent lineages of PRRSV-2 are lineage 1, lineage 5, lineage 8, and lineage 9. See Brar et al., "Genomic evolution of porcine reproductive and respiratory syndrome virus (PRRSV) isolates revealed by deep sequencing", (2014) PLoS One 9(4): e88807, doi:10.1371 / journal.pone.0088807.

Prior Art Documents

Non-Patent Documents

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Summary of the Invention

Problems to be Solved by the Invention

[0006] However, based on the high morbidity rates of these strains, there is a need for a PRRSV vaccine that the industry is confident can provide broad cross-reactivity against various PRRSV strains.

[0007] The present disclosure addresses this need.

Means for Solving the Problems

[0008] The inventors have discovered a method for inducing heterologous immunogenicity against heterologous porcine reproductive and respiratory syndrome virus (PRRSV) strains and enabling the evaluation of innate and adaptive immunity. In other aspects, a method for determining the efficacy of a vaccine against PRRSV is provided. In still other aspects, a method for predicting the efficacy of a vaccine against PRRSV in pigs suspected of being infected with PRRSV is provided.

[0009] In one embodiment, a method is provided for inducing heterologous immunogenicity against heterologous porcine reproductive and respiratory syndrome virus (PRRSV) strains and enabling the evaluation of innate and adaptive immunity. This method first includes the step of administering an effective amount of a live attenuated PRRSV vaccine or a control injection to a pig. On approximately the 28th day after vaccine administration, the pig is challenged by intranasal inoculation with a certain amount of a live PRRSV of a known strain. In the pig, body temperature and body weight are measured immediately before administration of the live attenuated PRRSV vaccine, immediately before challenge by intranasal inoculation with a known PRRSV strain, and at least on the 7th and 14th days after challenge. Blood samples from the pig are taken immediately before administration of the live attenuated PRRSV vaccine, immediately before challenge by intranasal inoculation with a known PRRSV strain, and at least on the 7th and 14th days after challenge. Next, various correlates of protection (CoP) are measured and determined from each blood sample. The measurement items include the CD4 T cell response, the presence of strain-specific neutralizing antibodies, the presence of CD4 cells, CD8 cells, and TCR-γδ cells, IFN-γ levels, and the levels of PRRSV-specific immunoglobulin A (IgA) and immunoglobulin G (IgG). All measurement values from the vaccinated pigs are compared with the measurement values taken from the pigs injected with the control.

[0010] In any embodiment, the known porcine reproductive and respiratory syndrome virus (PRRSV) strain is selected from a PRRSV type 1 (PRRSV-1) virus strain and a PRRSV type 2 (PRRSV-2) virus strain. In other embodiments, the known porcine reproductive and respiratory syndrome virus (PRRSV) strain is a PRRSV type 2 (PRRSV-2) virus strain selected from the group consisting of NADC30 and NC174 (lineage 1), VR2332 (lineage 5), and NADC20 (lineage 8).

[0011] In any embodiment, the strain-specific neutralizing antibody is one or more of an anti-NADC30 neutralizing antibody, an anti-VR2332 neutralizing antibody, and an anti-NADC20 neutralizing antibody.

[0012] In yet other embodiments, the step of administering an effective amount of a live attenuated PRRSV vaccine to a pig, followed by the step of challenging the pig by intranasal inoculation with a defined amount of a known live PRRSV strain at least 28 days after vaccine administration, induces a protective immune correlate (CoP) characterized by, compared to control injection, i) an increase in T cell activation as demonstrated by the differentiation of CD4 T cells and CD8 cells, ii) an increase in the amount of PRRSV-specific immunoglobulin G (IgG) levels, iii) the production of serum neutralizing antibodies, and iv) an increase in serum IFN-γ levels.

[0013] In any of the embodiments, the method includes isolating, storing, and banking peripheral blood mononuclear cells (PBMCs) obtained from a blood sample of a pig administered an effective amount of a live attenuated PRRSV vaccine.

[0014] Another embodiment of the invention provides a method for determining the efficacy of a vaccine against porcine reproductive and respiratory syndrome virus (PRRSV), comprising: i) administering to a pig an effective amount of a live attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine; ii) challenging the pig by intranasal inoculation with a defined amount of a known live PRRSV strain at least 28 days after vaccine administration; iii) measuring the body temperature and weight of the pig immediately before administration of the live attenuated PRRSV vaccine, immediately before challenge by intranasal inoculation with the known PRRSV strain, and at least on days 7 and 14 after challenge; iv) collecting blood samples and nasal swabs from the pig immediately before administration of the live attenuated PRRSV vaccine, immediately before challenge by intranasal inoculation with the known PRRSV strain, and at least on days 7 and 14 after challenge; v) evaluating the pathology of the lungs and lymph nodes of the pig at necropsy; vi) collecting bronchoalveolar lavage samples at necropsy; vii) measuring the amount of virus, the amount of PRRSV-specific immunoglobulin A and immunoglobulin G present in each blood sample, nasal swab, and bronchoalveolar lavage sample; and viii) comparing all measurements to measurements taken from pigs injected with a control.

[0015] In some embodiments, the steps of administering an effective amount of a live attenuated PRRSV vaccine to a pig, followed by challenging the pig by intranasal inoculation with a defined amount of a known live PRRSV strain at least 28 days after vaccine administration, can induce, when the measured values are compared to the measured values taken from pigs injected with a control, i) little or no pathology in the lungs and lymph nodes of the pigs at necropsy; ii) a decrease in the amount of PRRSV virus in samples taken from blood, nasal swabs, and bronchoalveolar lavage at necropsy; and iii) an increase in the amounts of PRRSV-specific immunoglobulin A and immunoglobulin G.

[0016] In yet another embodiment of the invention, there are provided steps of: i) isolating PRRSV from a blood sample or nasal swab sample taken from a pig suspected of being infected with PRRSV; ii) loading, with a sample of PRRSV isolated from a pig suspected of being infected with PRRSV and peripheral blood mononuclear cells (PBMCs) previously obtained from a blood sample from a pig administered an effective amount of a live attenuated PRRSV vaccine, the PBMCs being isolated, stored, and banked, where the pigs are further challenged by intranasal inoculation with a known live PRRSV strain at least 28 days after vaccine administration, and where the CD4 T cell and CD8 T cell responses have been previously obtained as differentiations of CD4 T cells and CD8 cells; iii) measuring a correlate of protection (CoP) as the CD4 T cell and CD8 T cell responses in the PBMCs after loading with PRRSV from a pig suspected of being infected with PRRSV; and iv) comparing the CD4 T cell and CD8 T cell responses of step iii) to the CD4 T cell and CD8 T cell responses previously obtained from the isolated, stored, and banked PBMC samples of step ii), to provide a method for predicting the effectiveness of a vaccine against PRRSV in pigs suspected of being infected with porcine reproductive and respiratory syndrome virus (PRRSV).

[0017] These and other embodiments and features of the present disclosure will become more apparent by reference to the following description, the accompanying drawings, and the claims. Further, it should be understood that the features of the various embodiments described herein are not mutually exclusive and may exist in various combinations and permutations.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0019] Throughout this disclosure, various quantities such as amounts, sizes, dimensions, ratios, etc. are presented in range format. It should be understood that the description of a quantity in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Thus, a description of a range should, unless the context clearly indicates otherwise, be considered to specifically disclose all possible sub-ranges, as well as all individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual values within that range, such as 1.1, 2, 2.3, 4.62, 5, 5.9, etc. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in a smaller range and are included within the scope of the disclosure, subject to the limitation that they are specifically excluded within the described range. If one or both of the limiting values are included in the described range, then, unless the context clearly indicates otherwise, the range excluding one or both of those included limiting values is also included in the disclosure.

[0020] The terms used in this specification are for the sole purpose of describing particular embodiments and are not intended to limit any embodiments. As used in this specification, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Further, the terms "includes", "comprises", "including", and / or "comprising", when used in this specification, specify the presence of the described features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used in this specification, the term "and / or" includes any and all combinations of one or more of the associated listed items. Further, note that items included in a list in the form of "at least one of A, B, and C" can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C). Similarly, items listed in the form of "at least one of A, B, or C" can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C).

[0021] Unless specifically stated or apparent from the context, as used in this specification, the term "about" in reference to a numerical value or range of numerical values means the recited numerical value and numerical values within + / - 10% thereof, or for values listed as a range, 10% below the listed lower limit and 10% above the listed upper limit.

[0022] As used in this specification, the term "subject" or "patient" refers to a mammal, more particularly a pig or swine.

[0023] Embodiments and features of the present disclosure will become more apparent by referring to the following specification, the accompanying drawings, and the claims. Further, it should be understood that the features of the various embodiments described herein are not mutually exclusive and may exist in various combinations and permutations.

[0024] The present disclosure is based on the finding that, despite the availability of porcine reproductive and respiratory syndrome virus (PRRSV) vaccines in North America for approximately 30 years, there is a significant hurdle in that they must provide cross-protection against a very diverse range of PRRSV strains. This cross-protection, i.e., the effectiveness of heterologous vaccines, depends largely on heterologous immunogenicity, which is the ability of a vaccine to induce a strong immune response against various strains.

[0025] The inventors investigated the vaccine efficacy and immunogenicity of live attenuated virus (MLV) against four different heterologous type 2 porcine reproductive and respiratory syndrome virus (PRRSV-2) strains as a model for testing the vaccine efficacy and immunogenicity of MLV against any suspect PRRSV strain. In the present disclosure, pigs were divided into several groups. Half were mock vaccinated (i.e., using phosphate-buffered saline), and the other half were vaccinated with the PRSSV live attenuated vaccine, Prevacent® PRRS MLV vaccine (Elanco, Inc.). Four weeks after vaccination, each group was challenged with mock or one of four PRRSV-2 strains: NC174 or NADC30 (both genotype 1), VR2332 (genotype 5), or NADC20 (genotype 8). Before and after challenge, lung pathology, viral load in nasal swabs and serum, anti-PRRSV IgA / G, neutralizing antibodies, and PRRSV-2 strain-specific T cell responses were evaluated. At necropsy, lungs were collected and viral load, pathology, and IgA levels in bronchoalveolar lavage (BAL) were evaluated. Lung pathology was induced only by NC174, NADC20, and NADC30, and of these, vaccination reduced the pathology of the NADC20 and NADC30 strains. All pigs became viremic, and during challenge with NADC20, NADC30, and VR2332, vaccinated pigs had reduced viremia. Regarding vaccine immunogenicity, vaccination induced a strong systemic IgG response, and serum IgG levels increased after challenge with all strains. Furthermore, vaccination increased the number of animals with neutralizing antibodies against three of the four challenge strains, namely NADC20, NADC30, and VR2332. Heterologous T cell responses were also improved by vaccination. Vaccination not only increased the induction of heterologous effector / memory CD4 T cells but also improved heterologous CD4 and CD8 proliferation and / or IFN-γ responses against all strains. Importantly, correlation analysis revealed that serum IgG levels (non-PRRSV strain-specific) and PRRSV strain-specific CD4 T cell responses are the best immunological correlates of protection.Overall, Prevacent® induces varying degrees of efficacy and immunogenicity against different heterologous strains of three different PRRSV-2 lineages and serves as a model for testing vaccine efficacy and immunogenicity of live attenuated viruses (MLVs) against any suspected strain of PRRSV.

[0026] Vaccine immunogenicity One aspect of the present disclosure includes the analysis and validation of vaccine immunogenicity assays, including both humoral and T cell immune responses following immunization with a live attenuated PRRSV vaccine, to develop methods and tests for determining the efficacy of a live attenuated PRRSV vaccine against unknown or suspected strains of the PRRSV virus.

[0027] Neutralizing antibodies (nAbs) play an important role in both viral clearance and protection against reinfection. See Lopez et al., "Role of neutralizing antibodies in PRRSV protective immunity," (2004) Vet Immunol Immunopathol 102(3): 155-163. However, several studies have noted a delayed induction of nAbs before clearing viremia. See, for example, Lunney et al., 2016; Butler et al., "Antibody Repertoire Development in Swine," (2017) Ann. Rev Anim Biosci 5: 255-279; Pileri et al., "Review on the transmission porcine reproductive and respiratory syndrome virus between pigs and farms and impact on vaccination," (2016) Vet Res 47(1): 108. These recent studies highlighted that clearance of PRRSV viremia can occur before the presence of nAbs.

[0028] Therefore, the development of nAbs is important for the defense against PRRSV, but other factors also seem to play a related role. The reduction or elimination of viremia in the absence of nAbs is partially explained by cell-mediated immune responses, including T cell responses such as IFN-γ production by CD4, CD8, and TCR-γδ T cells. See Chae et al., "Commercial PRRS Modified-Live Virus Vaccines", (2021) Vaccines (Basel) 9(2): doi:10.3390 / vaccines9020185; Kick et al., "The T-Cell Response to Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)", (2019) Viruses, 11(9): doi:10.3390 / v11090796; Nan et al., "Improved Vaccine against PRRSV: Current Progress and Future Perspective", (2017) Front Microbiol 8: 1635. Based on this central role of T cells in the control of PRRSV, the inventors herein provide a detailed analysis of the PRRSV strain-specific proliferation and IFN-γ responses of CD4, CD8, and TCR-γδ T cells. Responsive CD4 T cells were further analyzed for their differentiation from CD8α- naive to CD8α+ antigen-experienced memory / effector cells. This differentiation allows for the distinction between the primary and secondary responses of CD4 T cells.

[0029] Overview of the plan To achieve a detailed study of the immunogenicity and efficacy of the heterologous vaccine, 60 pigs were divided into 10 groups, with 5 pigs immunized with the commercially available live attenuated PRRSV vaccine Prevacent® PRRS MLV vaccine (Elanco, Greenfield, Indiana) and 5 pigs mock-inoculated (phosphate-buffered saline). Four weeks after vaccination, the pigs were challenged with one of the above PRRSV-2 strains or mock-inoculated. Virus shedding and viremia, as well as the induced immune response, were followed for 2 weeks. The pigs were then euthanized, and the virus load in bronchoalveolar lavage (BAL), gross and histological pathology of the lungs, and the size of the inguinal lymph nodes were further evaluated. Analysis of the immune response included humoral and T cell immune responses. The humoral response was examined not only by quantifying mucosal IgA in nasal swabs and bronchoalveolar lavage (BAL), but also by measuring the levels of serum IgG and nAbs. The systemic T cell response was analyzed in detail, including the proliferation and IFN-γ responses of CD4, CD8, and TCR-γδ T cells, as well as CD4 T cell differentiation. Furthermore, these immune parameters were investigated for their correlations, i.e., the immune correlations with the vaccine efficacy parameters examined, i.e., lung pathology, virus shedding, and viremia.

[0030] Prevacent® was found to induce various levels of heterologous immunity, including a strong IgA response in BAL, induction of a strong systemic IgG response, and increased prevalence of anti-NADC30, anti-VR2332, and anti-NADC20 nAbs. Furthermore, immunization with Prevacent® also promoted i) CD4 T cell differentiation, ii) proliferation of CD4, CD8, and TCR-γδ cells, and iii) enhanced IFN-γ response after challenge.

[0031] From the correlation analysis between vaccine efficacy and immunogenicity parameters, two important immune correlates of protection - systemic IgG levels and CD4 T cell responses - were revealed. However, in contrast to ELISA, which is used to quantify systemic IgG levels, in vitro restimulation assays followed by multicolor flow cytometry determine the CD4 response specific to the challenge strain. As a conclusion, for the first time, the inventors have identified the systemic CD4 T cell response as an immune correlate of protection (CoP) specific to the strain against PRRSV-2.

[0032] Therefore, the inventors have discovered that the immune correlate of protection (CoP) can strongly facilitate vaccine development and can be used to predict vaccine efficacy against newly emerging PRRSV-2 strains.

[0033] Research plan The research plan of the present disclosure is shown in FIG. 1. Sixty 4-week-old weaned piglets from a PRRSV-2 negative farm (North Carolina State University Swine Education Unit, Raleigh, North Carolina, USA) were transferred to the BSL-2 Laboratory Animal Research - LAR facility of the College of Veterinary Medicine, North Carolina State University (Raleigh, North Carolina, USA). These 60 weaned piglets were randomly divided into 10 groups using the GraphPad online randomization tool. Five groups were mock-inoculated intramuscularly (IM) with phosphate-buffered saline (PBS), and five groups were inoculated with Prevacent® as recommended by the manufacturer. Twenty-eight days after vaccination, the piglets were challenged nasally (500 μL / nostril, 1 mL total) using a Nasal Mist Intranasal Mucosal Atomization Device (Mountainside Medical Equipment, Marcy, New York).

[0034] Each of the mock-vaccinated group and the MLV-vaccinated group was challenged with either NC174 (lineage 1A), NADC30 (lineage 1C), VR2332 (lineage 5), or NADC20 (lineage 8) at a dose of 106 TCID50 / mL. Mock-challenged pigs were challenged with either 1% bovine serum albumin (BSA) in PBS (3 / 6 pigs) or Opti-MEMTM (3 / 6 pigs) because these were the two suspension media used for the different virus strains. Pigs were clinically monitored daily. Blood was collected on days -28, 0, 7, and 14 days post-challenge (dpc) to isolate serum and / or peripheral blood mononuclear cells (PBMC). Body weight and rectal temperature were recorded weekly. For ease of handling, necropsies were performed over a two-day period on days 15 and 16 dpc. Pigs were euthanized using a lethal injection, and the lungs were excised.

[0035] First, the gross pathology of the lungs was evaluated and photographed for documentation. The lungs were then filled with 50 mL of PBS and gently massaged to collect bronchoalveolar lavage (BAL) to evaluate downstream lung virus load and local humoral and cellular immune responses. Subsequently, tissue samples were taken to quantify the pathology and characterize infiltrating lung tissue T cells. The inguinal lymph nodes were also taken and weighed as a clinical indicator of PRRSV exposure. See Rossow et al., "Pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs", (1995) Vet Pathol 32(4): 361-373. This experimental procedure was approved by the North Carolina State University Institutional Animal Care and Use Committee (IACUC) ID# 17-166A (November 29, 2017).

[0036] PRRSV strain NC174, NADC20, and NADC30 were provided by Elanco. VR2332 was manufactured in-house and titrated in MA-104 cells. Serum pools from mock-vaccinated challenged pigs at 7 dpc were sent to ISU VDL for ORF5 sequencing. Sequence analysis confirmed the correct identity of the challenged strain (data not shown, d.n.s).

[0037] Bronchoalveolar lavage, nasal swab, and blood processing A 0.4 mL aliquot of BAL was added to 0.6 mL of TriReagent (Ambion, Austin, Texas, USA), mixed, and stored at -80 °C for downstream qPCR-based quantification of PRRSV. The remaining BAL was centrifuged at 400 g for 10 min at 4 °C to pellet BAL immune cells. The cell pellet was collected, counted, and used for the analysis of local T cell immune responses. The supernatant was aliquoted and stored at -80 °C for downstream analysis of humoral immune responses by IgA ELISA. Nasal swabs were rotated within each nostril and placed into tubes filled with 1 mL of PBS. After collection, the swabs were vortexed and rotated in a circular motion while pressing against the wall of the tube before removing the swabs from the tubes. The PBS from these nasal swabs was aliquoted and stored at -80 °C for quantification of downstream PRRSV and antibodies. Whole blood for serum isolation was collected into SST tubes (BD Bioscience, San Jose, California, USA) and incubated upright for 30 min. After incubation, the blood was centrifuged at 2,000 g for 20 min at 23 °C. Serum was collected, aliquoted, and stored at -80 °C. Whole blood for peripheral blood mononuclear cell (PBMC) isolation was collected into Hepann tubes (BD Bioscience). Isolation of PBMCs was performed by density centrifugation using Sepmate tubes (StemCell, Vancouver, Canada) and Ficoll-Paque (GE Healthcare, Uppsala, Sweden). After isolation, PBMCs were used fresh for in vitro restimulation to examine PRRSV strain-specific T cell immune responses.

[0038] Viremia and viral load

[0039] Isolated sera and nasal swabs were sent to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL), Ames, IA, USA, to quantify PRRSV using either PRRSV universal or “Elanco Prevacent®-like” specific reverse transcription (RT)-quantitative PCR. Results were reported as cycle threshold (Ct) values (“Elanco Prevacent®-like” RT-qPCR) or genome copies / mL (universal RT-qPCR).

[0040] Serum anti-PRRSV IgG and anti-PRRSV IgA Isolated sera and nasal swabs were sent to ISU VDL. Serum IgG levels were determined by PRRSV X3 enzyme-linked immunosorbent assay (ELISA, IDEXX, Westbrook, ME, USA). PRRSV oral fluid IgA ELISA was used to determine IgA in nasal swabs.

[0041] Neutralizing antibodies Serum samples at 0 dpc and 14 dpc were sent to the South Dakota State University Animal Research and Diagnostic Laboratory (SDSU ARDL). Neutralizing antibodies were measured by the fluorescent focus neutralization (FFN) test. See Valdes-Donoso et al. (2018). Titers greater than 1:4 were considered positive. Isolated sera were tested against their respective homologous challenge strains. Both mock-challenged groups were tested against all four virus strains.

[0042] Gross pathology, histology, and lymph node mass of the lung At necropsy, the lungs and inguinal lymph nodes were collected. Photographs of the dorsal and ventral sides of the lungs were taken. The lung lobes were scored by a blinded veterinarian. For histopathological evaluation, tissue was collected from seven lung lobes: left apical, left ventricle, left diaphragm (caudal), right apical, right ventricle, right diaphragm (caudal), and intermediate (accessory). Tissue samples were fixed in formaldehyde / Zn fixative (Electron Microscopy Sciences, Hatfield, PA) for 24 h and then transferred to 70% ethanol. Tissue processing, hematoxylin and eosin (H&E) staining, and slide preparation were performed by the Histology Laboratory at North Carolina (NC) State University. Histopathology was evaluated by a blinded pathologist as previously described by Halbur et al. (1995). Briefly, scores were recorded as (0) normal, (1) slight change, (2) mild, (3) moderate, or (4) severe. For a general assessment of immune activation, both inguinal lymph nodes were collected at euthanasia and weighed in grams.

[0043] Proliferation of PRRSV load strain-specific T cells To measure the proliferation of PRRSV-specific T cell subsets, freshly isolated PBMCs were stained using the CellTrace™ Violet cell Proliferation Kit (Invitrogen) according to the manufacturer's instructions. The stained cells were seeded at 200,000 cells / well in a 96-well round-bottom plate (Sarstedt, Nuembrecht, Germany). The cells were stimulated for 72 hours with medium (mock), NC174, NADC20, NADC30, or VR2332 (MOI = 0.1); or stimulated with concanavalin A (ConA, 2.5 μg / mL, Alfa Aesar) as a positive control. Cells from eight replicates were pooled and stained for flow cytometry analysis according to Table 1. Flow cytometry data were acquired on a Cytoflex using CytExpert software (Beckman Coulter). Data analysis was performed using FlowJo version 10.5.3 (FLOWJO LLC) with gates based on relevant FMO controls.

[0044]

Table 1

[0045] IFN-γ production of PRRSV load strain-specific T cells PBMCs were seeded at 500,000 cells / well and left overnight. The next day, the cells were stimulated with either medium (mock), NC174, NADC20, NADC30, or VR2332 (MOI = 0.1). Phorbol 12-myristate 13-acetate (PMA, 5 ng / mL, Alfa Aesar, Ward Hill, Massachusetts, USA) / ionomycin (500 ng / mL, AdipoGen, San Diego, California, USA) was used as a positive control. The plates were cultured for 18 hours. Monensin (5 μg / mL, Alfa Aesar) was added during the last 4 hours of culture. Then, eight replicates were pooled and stained according to Table 1 for flow cytometry analysis. Data were acquired on a Cytoflex using CytExpert software (Beckman Coulter). Data analysis was performed using FlowJo version 10.5.3 with gates based on FMO controls.

[0046] Statistical analysis Statistical analysis was performed using GraphPad Prism 9.1.1 (GraphPad Software, San Diego, California). All qPCR data were log-transformed prior to statistical analysis. Depending on the dataset, statistical significance was analyzed by two-way ANOVA or two-sided independent Student's t-test. Multiple comparisons were performed using Tukey's or Sidak's multiple comparison tests.

[0047] Further reference is made to the following experimental examples, which are described for the purpose of illustrating various embodiments of the invention and are not intended to limit the present disclosure in any way. This example, together with the methods described herein, represents the presently preferred embodiments and is provided by way of illustration only and is not intended to limit the scope of the invention. Modifications and other uses that are encompassed within the spirit of the present disclosure as defined by the claims will be apparent to those skilled in the art.

Example

[0048] (Example 1) Efficacy of heterologous vaccines The efficacy of Prevacent® heterologous vaccines was determined in three ways: i) clinical signs including rectal temperature, ii) evaluation of the amount of PRRSV in nasal swabs and sera at 0, 7, and 14 dpc (Figure 2); iii) evaluation of the virus amount and gross and histopathological pathology of the lungs at necropsy (14 dpc, Figure 3).

[0049] Weight gain, clinical signs, and virus amount in sera, BAL, and nasal swabs Figures 2A - C show the efficacy of Prevacent® heterologous vaccines. Rectal temperature (Figure 2A), viremia (Figure 2B), and virus amount in nasal swabs (Figure 2C) were determined on days 0, 7, and 14 after challenge with mock (gray), or PRRSV strains 1 - 4 - 4 (NADC30, dark blue), NC174 (red), VR2332 (green), or 1 - 4 - 2 (NADC20, light blue). The line graph in Figure 2A shows the mean and standard deviation of rectal temperature [°C]. Viremia (Figure 2B) and virus shedding (Figure 2C) were quantified by PRRSV - specific RT - qPCR in sera and nasal swabs, respectively (genome copies / mL [log10]). Black bars indicate the median. Additionally, individual data points are shown for mock - vaccinated animals (white diamonds) and MLV - vaccinated animals (filled squares). Data were analyzed using Sidak multiple - comparison test two - way ANOVA. Each vaccinated group was compared at each time point to its respective PRRSV type 2 - challenged mock - vaccinated group. **** p < 0.0001. *** p < 0.001,[[]] ** p < 0.01,[[]] * p < 0.05.

[0050] No significant differences in weight gain were observed between the groups. Clinical symptoms were mild. Clinical symptoms included lethargy and dyspnea at approximately 7 - 14 dpc. Only two differences were observed between the mock - inoculated group and the Prevacent® vaccine - inoculated group: i) In the NC174 - challenged group, lethargy in Prevacent® vaccine - inoculated pigs was slightly reduced; ii) In contrast to Prevacent® vaccine - inoculated pigs, the mock group showed prominent clinical symptoms upon challenge with NADC20: Mock pigs showed symptoms of dyspnea, strong lethargy, and anorexia from 7 - 14 dpc (data not shown, d.n.s.). Rectal temperatures varied between the groups (Figure 2A): A temperature increase was shown at 7 dpc only in pigs challenged with NC174 and NADC20. In the NC174 - challenged group, the body temperature of Prevacent® vaccine - inoculated pigs was even higher than that of mock - vaccinated pigs. Body temperatures upon challenge with NADC20 were comparable between mock - vaccinated and Prevacent® vaccine - inoculated pigs. Overall, under the conditions of this study, most clinical symptoms were mild and included lethargy, anorexia, respiratory disease, and a slight increase in body temperature in NC174 and NADC20. The most prominent protective effect of Prevacent® was the reduction of respiratory disease, anorexia, and lethargy after NADC20 challenge.

[0051] Due to the limited clinical symptoms, quantification of the virus amount in nasal swabs and sera was performed to more appropriately evaluate the efficacy of the heterologous vaccines (Figure 2B, 2C). Analysis of the pre - challenge virus amount at 0 dpc showed that all animals vaccinated with Prevacent® had similar PRRSV virus copy numbers. This confirmed that not only was the Prevacent® vaccination successful, but it was also homogeneous (Figure 2B). Challenge with various PRRSV strains induced viremia, which peaked at 7 dpc. At that time, challenge with VR2332 caused mild to moderate viremia in the mock - vaccinated group, and the median genome copy number was 10 6.2In contrast, the challenge with NC174, NADC30, and NADC20 induced strong viremia in mock-vaccinated pigs. The median genome copy number / mL was 10 9.0 , 10 8.6 , and 10 9.2 , respectively. At 14 dpc, viremia decreased by approximately 1–2 logs. For VR2332 and NADC30, vaccination with Prevacent® significantly reduced viremia at both time points. For NADC30, viremia decreased at 14 dpc (Figure 2B). Notably, all mock-vaccinated pigs had persistent viremia during VR2332 challenge, whereas 4 / 6 Prevacent®-vaccinated pigs were able to eliminate this PRRSV strain by 14 dpc.

[0052] In addition to the universal PRRSV RT-qPCR, Prevacent®-specific RT-qPCR analysis was performed on sera at 7 dpc and 14 dpc. The purpose of this analysis was to provide insights into the contribution of the Prevacent® vaccine strain and challenge strains to the total amount of PRRSV in the sera. From this analysis, it was revealed that i) at 14 dpc, Prevacent® was not detected (d.n.s.); ii) at 7 dpc, it had disappeared from the sera or was present only at very low levels (Ct ≥ 31). Interestingly, most animals (4 / 6) in the mock and VR2332-challenge groups still had detectable levels of the Prevacent® vaccine strain in their sera, whereas all or 5 / 6 animals in the NADC30, NC174, and NADC20 groups had eliminated the Prevacent® vaccine strain. These data indicate that pigs challenged with PRRSV strains that induce high viremia eliminated the Prevacent® strain more rapidly (Figure 8, and d.n.s.).

[0053] Figure 8 shows the quantification of the Prevacent® vaccine strain in serum. The prevalence of the Prevacent® vaccine strain was quantified by Prevacent®-specific RT-qPCR. This table shows the Ct values of mock, NADC20, NC174, VR2332, and NADC20-challenged animals on day 7 post-challenge.

[0054] In addition to viremia, viral load was also evaluated by nasal swab to assess viral shedding. Importantly, the Prevacent® vaccine strain was not detected in nasal swabs at the time points of analysis at 0, 7, and 14 dpc (28, 35, 42 dpv; d.n.s., respectively). In VR2332-challenged pigs, the viral load in nasal swabs was low (less than 10 4 genome copies / ml in 4 / 12 animals) or completely absent (8 / 12 animals; Figure 2C). In contrast, challenge with other PRRSV strains led to a significant increase in the viral load in nasal swabs of all inoculated animals (including the range of mean genome copies; Figure 2C). Similar to viremia, the viral load in nasal swabs peaked at 7 dpc. Prevacent® vaccination reduced the viral load in nasal swabs at 7 dpc for NC174, NADC20, and NADC30. At 14 dpc, Prevacent® vaccination significantly reduced and completely eliminated the viral load in nasal swabs of NADC20- and NADC30-challenged pigs.

[0055] Figures 3A - 3C show the efficacy of the heterologous vaccine against the viral load in the lungs, lesions, and the size of the inguinal lymph nodes. (Figure 3A) The viral load in the lungs was evaluated by PRRSV - specific RT - qPCR (genome copies / mL [log10]) in bronchoalveolar lavage (BAL). Notably, PRRSV was not detected in the BAL of one mock - vaccinated / NADC20 - challenged animal, but this pig showed the second - highest lung pathology score in combination with the highest PRRSV load in the nasal swab at 14 dpc. Based on this discrepancy, it was excluded from this analysis. (Figures 3B and C) On day 14 post - challenge (dpc), the gross and histological pathology of all seven lung lobes were evaluated by a veterinarian who was blinded. (Figure 3B) shows the percentage of lung lesions for each individual pig. (Figure 3C) shows the histopathological scores of all seven lung lobes according to the scoring guidelines of Halbur et al. See Halbur et al., "Comparison of the pathogenicity of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus", (1995) Vet Pathol. 32(6): 648 - 660. Each vaccinated group was compared to its respective PRRSV type 2 - challenged non - vaccinated group using a two - sided independent t - test. The black bars represent the median. Additionally, individual data points are shown for mock - vaccinated animals (white diamonds) and MLV - vaccinated animals (filled squares). Each vaccinated group was compared to its respective PRRSV type 2 - challenged non - vaccinated group. Data comparisons were performed using a two - sided independent t - test. **** p < 0.0001. *** p < 0.001, ** p < 0.01, * p < 0.05.

[0056] At necropsy, virus loads were further quantified by universal PRRSV-specific qPCR in BAL (Figure 3A). All sham-challenged pigs were PRRSV-2 negative in BAL. Sham-vaccinated pigs showed approximately 10 8 genome copies per mL of BAL, showing the highest median virus load among all challenged groups. Vaccination with Prevacent® reduced these virus loads to 10 6.5 in NC174, 10 3.7 in NADC30, and 10 5.3 in NADC20, significantly reducing them to these values, and VR2332 was eliminated from the BAL of 4 / 6 pigs. In conclusion, Prevacent® was not present in nasal swabs 4 - 6 weeks post-vaccination and was either absent or present at low levels in sera at these time points.

[0057] Viremia was significantly reduced by Prevacent® vaccination during VR2332 challenge. Both viremia and virus shedding were restricted in the NADC20 and NADC30 challenge groups. Also, Prevacent® significantly reduced the BAL virus loads of NADC20, NADC30, and VR2332 (Figure 3A).

[0058] Vaccine efficacy in tissues - virus load, lung lesions, and lymph node size At necropsy, virus loads were evaluated by PRRSV-specific qPCR in BAL (Figure 3A). All sham-challenged animals were negative. Sham-vaccinated animals in the NADC30, NC174, and NADC20 groups had virus loads of approximately 10 8 genome copies / mL. In contrast, sham-inoculated and VR2332-vaccinated animals had median virus loads approximately 1,000-fold lower, at approximately 10 5It was. Vaccination with Prevacent® vaccine was able to reduce the median viral load of all challenged strains. This reduction was significant in NADC30, VR2332, and NADC20. Notably, the BAL of 4 / 6 Prevacent®-vaccinated animals and VR2332-challenged animals was PRRSV-2 negative.

[0059] Macroscopic pathological changes in the lungs were hardly present in both the mock-challenged groups, minimally present in the VR2332-challenged group, but clearly present in NC174-, NADC20-, and NADC30-challenged pigs (Figure 3B). Prevacent® vaccination reduced the macroscopic pathology of the lungs in challenge with two of the three strains that induced pathology, NADC30 and NADC20. As seen in macroscopic pathology, the median of histopathological changes in mock-vaccinated pigs was also highest in the NADC30, NC174, and NADC20 groups.

[0060] However, histopathological changes were also present in the mock and VR2332-challenged groups (Figure 3C). When comparing the mock-vaccinated group and the Prevacent®-vaccinated group, only one difference was revealed by histopathological analysis. Prevacent®-vaccinated animals had a significantly lower histopathological score compared to mock-vaccinated animals.

[0061] In addition to lung pathology, the size of lymph nodes was also evaluated because an increase in lymph node size is often associated with inflammation. Both the mock and VR2332 groups maintained an almost healthy mass at necropsy (median was approximately 2 grams). PRRSV-2 challenge enlarged the lymph nodes of NC174, NADC20, and NADC30. Prevacent® was able to significantly reduce the size of NADC30 lymph nodes by 1 gram (median mass from 4.7 grams to 3.6 grams).

[0062] In conclusion, Prevacent® reduced the amount of BAL virus in NADC30, VR2332, and NADC20. VR2332 induced minimal lung pathology, while Prevacent® not only reduced the gross and / or histological pathology of the lungs, but also reduced the median mass of the inguinal lymph nodes numerically in NADC30 and in NADC20.

[0063] (Examples 2 - 6) Immunogenicity and Correlates of Protection (CoP) of Heterologous Vaccines Overview In addition to vaccine efficacy, the immunogenicity of heterologous vaccines was examined for both humoral and T cell immune responses, and the correlates of protection (CoP) were determined. The humoral immune response was examined by quantifying the local anti - PRRSV IgA levels in nasal swabs and BAL, and serum anti - PRRSV IgG and nAb levels (Figures 4A - 4D). To examine the T cell response, PBMCs were isolated and restimulated in vitro with each PRRSV challenge strain, and analyzed by polychromatic flow cytometry for three major read - out parameters: i) proliferation of CD4, CD8, and TCR - γδ T cells (Figures 5A - 5D), ii) IFN - y production (Figures 6A - 6D), and iii) differentiation of CD4 T cells into memory / effector cells (Figures 7A - 7B).

[0064] (Example 2) Humoral Immune Response The local humoral immune response was examined by quantifying the anti - PRRSV IgA levels in BAL at necropsy (Figure 4A), and the anti - PRRSV IgA levels in nasal swabs at 0 dpc and 14 dpc (Figure 4B).

[0065] Figures 4A-4D show the immunogenicity of the heterologous vaccines as a humoral immune response. (Figure 4A) Immunoglobulin A in bronchoalveolar lavage (BAL), (Figure 4B) nasal swabs, and (Figure 4C) serum IgG levels were evaluated by PRRSV X3 ELISA on days 0 and 14 post-challenge (dpc). IgA and IgG ELISA S / P ratios were compared within their respective challenge groups of mock (gray), 1-4-4 (NADC30, blue), NC174 (red), VR2332 (green), 1-4-2 (NADC20, light blue). Black bars represent the median. Additionally, individual data points are shown for mock-vaccinated animals (white diamonds) and MLV-vaccinated animals (filled squares). Data were statistically analyzed using two-way ANOVA with time and vaccination as the two parameters and Tukey's multiple comparison test. **** p < 0.0001. *** p < 0.001, ** p < 0.01, * p < 0.05. (Figure 4D) Neutralizing antibody (NA) titers determined by the FFN test against each challenge strain at 0 dpc and 14 dpc. Since no animals showed FFN titers at 0 dpc, only 14 dpc data are shown. Titers ≥ 1:4 were considered positive. Titers for each individual PRRSV-challenged pig are shown. Positive NA titers are highlighted in blue (NADC30), red (NC174, not detected), green (VR2332), and light blue (NADC20).

[0066] It should be noted that, in contrast to the samples of undiluted nasal swabs, the BAL samples were diluted 1:200 prior to analysis. At 2 weeks post-challenge, the majority of BAL samples from mock-vaccinated pigs were negative for PRRSV-specific IgA. The BAL of vaccinated pigs and VR2332-challenged pigs were also negative, but Prevacent® vaccination induced a strong IgA response against NADC30, NC174, and NADC20. The S / P ratios of 4 / 6 of the Prevacent®-vaccinated pigs in the NADC30 and NC174 pigs and all Prevacent®-vaccinated pigs in the NADC20-challenged group exceeded 0.4. As a result, Prevacent increased the lung PRRSV-specific IgA levels (numerical) of NC174 and significantly increased them in NADC30 and NADC20.

[0067] The IgA levels in nasal swabs were significantly low (Figure 3B). Except for some outliers, the IgA levels of mock-, NADC30-, and VR2332-challenged pigs maintained an S / P ratio of less than 0.4. However, challenge with NC174 and NADC20 was observable and induced a mainly significant local IgA response. However, there was no difference between each mock-vaccinated group and Prevacent®-vaccinated group.

[0068] The systemic humoral immune response was evaluated in two ways: the levels of anti-PRRSV IgG and strain-specific nAbs in serum (Figure 4C, Figure 4D). At 4 weeks post-vaccination, i.e., 0 dpc, all vaccinated animals had high positive IgG levels, S / P 1.3 - 2.1, and the control animals were not positive. By 14 dpc, infection with each of the four PRRSV strains also induced anti-PRRSV serum IgG in mock-vaccinated animals. However, all vaccinated animals had significantly higher serum IgG levels than their respective mock-vaccinated groups (Figure 4C).

[0069] The neutralizing antibody (nAb) titers specific to the challenged strain were determined by the FFN test at 0 dpc and 14 dpc (Figure 4D). nAbs were not detected at 0 dpc (d.n.s.). At 14 dpc, neither the mock-challenged (d.n.s.) group nor the NC174-challenged group generated nAbs against the challenged strain. However, in the NADC20-, NADC30-, and VR2332-challenged pigs, mainly low-titer serum nAbs were induced by 14 dpc. Among six pigs per group, only 1 to 2 mock-vaccinated animals developed serum nAb titers. In contrast, 3 / 6, 5 / 6, and 6 / 6 pigs in the Prevacent®-vaccinated groups developed nAbs against the VR2332-, NADC20-, and NADC30-challenged strains, respectively (Figure 4D).

[0070] These data indicate that Prevacent® vaccination induced strong local IgA responses in the BAL against NADC30, NC174 (numerical), and NADC20. It also induced a systemic humoral immune response, with high serum IgG titers in all groups and a high frequency of nAb-positive animals against VR2332, NADC30, and NADC20 after challenge.

[0071] (Example 3) Proliferation of T cell subsets In addition to the humoral immune response, the cellular immune response is very important for protection against PRRSV and can provide a model for evaluating the correlates of protection (CoP). To better understand the immunogenicity of MLV-induced heterologous vaccines, the PRRSV strain-specific proliferation (Figures 5A - 5D) and IFN-γ (Figures 6A - 6D) responses of CD4, CD8, and TCR-γδ T cells were examined.

[0072] Figures 5A to 5D show the immunogenicity of the heterologous vaccine as the proliferation of CD4, CD8, and TCR-γδ T cells. (Figure 5A) shows the gating hierarchy for evaluating the heterologous proliferation responses of T cell subsets to each PRRSV type 2 challenge strain. A Live / dead discrimination dye was included to exclude dead cells. Live cells were used to identify live lymphocytes through the FSC / SSC lymphocyte gate. From the live lymphocytes, doublets were excluded using the singlet FSC width (FSC-W) / FSC area (FSC-A) gate. These single live lymphocytes were gated for T cells (FSC-A / CD3) and further used to discriminate between TCR-αβ cells and TCR-γδ T cells. TCR-αβ T cells were further divided into CD4 T cells and CD8 T cells based on the CD4 / CD8α expression profile. The proliferation of CD4, CD8, and TCR-γδ T cells was discriminated by a violet proliferation dye. Two examples show the representative staining patterns of control animals (upper right figure) and highly responsive animals (lower right figure). (Figures 5B to 5D) show the proliferation responses of CD4 (Figure 5B), CD8 (Figure 5C), TCR-γδ T cells (Figure 5D) according to mock (gray), NC174 (red), NADC20 (light blue), NADC30 (navy), VR2332 (green) of the challenge groups. The black bars represent the median. Additionally, individual data points are shown for mock-vaccinated animals (white diamonds) and MLV-vaccinated animals (filled squares). The data were statistically analyzed using two-way ANOVA with time and vaccination as two parameters and Tukey's multiple comparison test. *** p < 0.0001. *** p < 0.001, ** p < 0.01, * p < 0.05.

[0073] The proliferative responses of CD4, CD8, and TCR-γδ T cells were analyzed after in vitro restimulation with each challenge PRRSV-2 strain (MOI 0.1) according to the multicolor flow cytometry gating hierarchy shown in Figure 5A. At -28 dpc, CD4 T cells showed very limited background proliferation (Figure 5B). Four weeks later (0 dpc), in contrast to mock-vaccinated pigs, CD4 T cells in Prevacent®-vaccinated pigs began to exhibit a heterologous proliferative response. This response was moderate with NADC20, but was clearly observable with NADC30 and NC174 and was significant for the mock-challenge group and the VR2332-challenge group. At 14 dpc, the proliferative CD4 T cell response also increased significantly in Prevacent®-vaccinated animals in the NC174 and NADC20 groups. In contrast to CD4 T cells, CD8 T cells mainly showed a low proliferative response (Figure 5C). However, they showed a similar pattern: i) generally, the proliferative response increased over time; ii) at 0 dpc, proliferation mainly increased in the mock and VR2332 groups; and, iii) after challenge, Prevacent® was able to promote the proliferation of the NC174 group. The proliferative TCR-γδ response showed high within-group variability. When comparing the mock and Prevacent® groups, the only clear and significant effect of Prevacent was an increase in TCR-γδ at 14 dpc against the NADC20 strain (Figure 5D).

[0074] In summary, these data indicate that the effect on TCR-δγ proliferation was limited to NADC20, but Prevacent® vaccination increased the proliferation of CD4 T cells and CD8 T cells against three heterologous PRRSV-2 strains, VR2332 (before challenge), NC174 (after challenge), and NADC20 (only CD4 T cells, after challenge).

[0075] (Example 4) IFN-γ Production by T Cell Subsets In addition to the systemic proliferative response, the immunogenicity of the heterologous vaccine was also evaluated by examining IFN-γ, perhaps the most relevant antiviral T cell cytokine (Figures 6A-6D).

[0076] Figures 6A-6D show the heterologous vaccine immunogenicity as IFN-γ production by CD4, CD8, and TCR-γδ T cells. (Figure 6A) Gating hierarchy for evaluating the heterologous IFN-γ responses of T cell subsets to each PRRSV type 2 challenge strain. The gating hierarchy mainly follows the proliferation analysis shown in Figure 5. However, instead of gating on proliferating cells, IFN-γ was analyzed in the FSC-A / IFN-γ plot. The IFN-γ gate was set using an appropriate FMO control (upper right figure). (Figures 6B-6D) show the IFN-γ responses of CD4 (Figure 6B), CD8 (Figure 6C), and TCR-γδ T cells (Figure 6D) according to the challenge groups (mock (gray), 1-4-4 (NADC30, dark blue), NC174 (red), VR2332 (green), and 1-4-2 (NADC20, light blue)). The black bars represent the median. Additionally, individual data points are shown for mock-vaccinated animals (white diamonds) and MLV-vaccinated animals (filled squares). Data were statistically analyzed using two-way ANOVA with time and vaccination as two parameters and Tukey's multiple comparison test. **** p < 0.0001. *** p < 0.001, ** p < 0.01, * p < 0.05.

[0077] The gating hierarchy used to selectively analyze IFN-γ production in CD4, CD8, and TCR-γδ T cells is the same as that for proliferation analysis and is shown in Figure 6A. IFN-γ production before challenge (-28 dpc and 0 dpc) was low in all T cell subsets, and there were no significant differences between each vaccination group (Figures 6B–6D). In contrast, at 14 dpc, significant IFN-γ production was present in most of the PRRSV-2 challenged groups. For CD4 T cells, this post-challenge response was significantly increased by Prevacent® vaccination against NC174, NADC20, and NADC30 (Figure 6B). In CD8 T cells, Prevacent® significantly enhanced the IFN-γ response against NC174 and NADC20 (Figure 6C). Also, in TCR-γδ T cells, Prevacent® vaccination increased IFN-γ production against NADC20 (Figure 6D). Therefore, Prevacent® vaccination was able to enhance the IFN-γ response after heterologous challenge against NADC30 (in CD4 T cells), NC174 (in CD4 and CD8 T cells), and NADC20 (in all T cell subsets).

[0078] (Example 5) Differentiation of IFN-γ-producing CD4 T cells Heterologous IFN-γ production showed the strongest response after challenge, but T cell differentiation analysis of IFN-γ-producing T cells revealed significant differences before challenge (Figures 7A–7B).

[0079] Figures 7A–7B show the immunogenicity of heterologous vaccines as the differentiation of IFN-γ-producing CD4 T cells. (Figure 7A) shows the gating hierarchy for evaluating the differentiation of IFN-γ-producing CD4 T cells. After gating IFN-γ+ CD4 T cells as described in Figure 6, their differentiation was analyzed via the CD4 / CD8α expression profile, naive (CCR7+CD8α-), central memory (T CM CCR7+CD8α+), effector memory (T EM, CCR7-CD8α+) CD4 T cells were distinguished (upper right figure). Most of the CD8α+ IFN-γ-producing CD4 T cells belonged to the T CM subset (data not shown), so in downstream analysis, both T CM and T EM were grouped into the "memory / effector" subset. (Figure 7B) shows the frequencies of these memory / effector cells within IFN-γ-producing CD4 T cells according to their respective challenge groups: mock (gray), 1-4-4 (NADC30, dark blue), NC174 (red), VR2332 (green), and 1-4-2 (NADC20, light blue). The black bars represent the median. Additionally, individual data points are shown for mock-vaccinated animals (white diamonds) and MLV-vaccinated animals (filled squares). The data were statistically analyzed using two-way ANOVA with time and vaccination as the two parameters and Tukey's multiple comparison test. **** p < 0.0001. *** p < 0.001, ** p < 0.01, * p < 0.05.

[0080] Once again, multi-color flow cytometry with a sophisticated gating hierarchy was used to evaluate the differentiation of IFN-γ-producing CD4 T cells into CCR7+CD8α-naïve, CCR7+CD8α+ central memory (T CM ) and CCR7-, CD8α+ effector memory (T EM ) CD4 T cells (Figure 7A). Most of the CD8α + CD4 T cells that produce IFN-γ belonged to the CCR 7+ T CM subset (data not shown), so the antigen-experienced T CM subset and T EMThe subsets were grouped into one "memory / effector" subset (Figure 7B). Before vaccination, i.e., at -28 dpc, most of the IFN-γ was produced by naive CD4 T cells - median: 10 - 40% memory / effector CD4 T cells. At 0 dpc, in the mock-vaccinated group, IFN-γ was still mainly produced by naive CD4 T cells - median 0 - 15% memory / effector CD4 T cells.

[0081] In contrast, in the Prevacent®-vaccinated group, IFN-γ was mainly produced by memory / effector CD4 T cells, with a median of approximately 50% ->90%. This difference in the pre-challenge differentiation of IFN-γ-producing CD4 T cells was significant in all groups and for all PRRSV-2 challenge strains. At 14 dpc, the frequency of memory / effector CD4 T cells increased in the mock-vaccinated group. However, at least numerically, the Prevacent®-vaccinated group still had the highest median frequency of memory / effector cells among all PRRSV-2 challenged groups (Figure 7B). In conclusion, this CD4 differentiation analysis revealed an important immune mechanism in the immunogenicity of heterologous vaccines. Prevacent® did not increase the CD4 IFN-γ response pre-challenge but had already promoted the pre-challenge differentiation of these CD4 T cells against all PRRSV-2 strains analyzed - NC174, NADC20, NADC30, and VR2332.

[0082] (Example 6) Correlates of protection (CoP) The above data demonstrate that Prevacent® has shown the immunogenicity and efficacy of various heterologous vaccines. An important parameter that has been little analyzed for PRRSV is the correlate of protection (CoP). See Plotkin et al., "Nomenclature for immune correlates of protection after vaccination", (2012) Clin Infect Dis 54(11): pp. 1615-1617. These correlations can not only facilitate vaccine development but also enable the prediction of the efficacy of vaccines against newly emerging PRRSV strains.

[0083] To provide insights into potential CoPs for heterologous PRRSV strains, the inventors performed a correlation analysis between the pre-challenge immune parameters analyzed (0 dpc) and three post-challenge (14 dpc) parameters related to protection: lung pathology, virus shedding, and viremia (Table 2).

[0084] [Table 2A]

[0085] [Table 2B]

[0086] Table 2 shows the R values (-1 to +1) of the correlations between various immune parameters (e.g., proliferation, IFN-γ production, differentiation into CD4 memory cells) at 0 dpc and three protection evaluations (gross pathology (A), shedding (B), viremia (C)) at 14 dpc. Italicized numbers represent non-significant correlations, while bold numbers highlight significant correlations (p < 0.05). A negative correlation (R = 0 > -1) indicates that an increase in immune parameters before challenge correlates with a reduction in lesions or viral load. Neither the systemic CD8 nor the TCR-γδ responses were significantly correlated with protection. Only CD8 proliferation showed a negative correlation with NADC30 shedding. The TCR-γδ IFN-γ response showed a significant positive correlation with NADC20 shedding.

[0087] In contrast, except for the gross pathology of NC174, the CD4 T cell responses showed negative correlations with all protection parameters analyzed. The most robust and significant CD4 correlations were observed with the NADC20 strain and / or the NADC30 strain. The CD4 IFN-γ response and differentiation into memory / effector cells showed a significant negative correlation with NADC20-induced pulmonary gross pathology. CD4 T cell proliferation showed a significant negative correlation with NADC20 shedding and viremia. Also, all CD4 parameters (proliferation, IFN-γ, and differentiation) correlated with both NADC30 shedding and viremia. Regarding the humoral immune response, the IgA levels in nasal swabs (local IgA) showed both positive and negative correlations with protection, while the systemic IgG levels correlated well with most protection parameters. The systemic IgG levels showed a significant negative correlation with NADC20-induced gross pathology, NADC30-induced shedding, and viremia induced by NADC30, VR2332, and NADC20. These data indicate that while only T cell responses were analyzed strain-dependently, both systemic IgG levels and CD4 T cell responses are candidates serving as important CoPs for PRRSV.

[0088] Conclusion The present disclosure combined Prevacent® vaccination with subsequent in vivo challenge with four different PRRSV strains and extensive ex vivo and in vitro analyses of lung pathology, viral loads in various tissues, and humoral and adaptive immune responses. Detailed analysis of heterologous humoral and T cell immune responses appropriately explains the immunogenicity of Prevacent®. Initially (0 dpc), Prevacent® induced early T cell activation and differentiation, as shown by increased proliferative responses of CD8 T cells and predominantly CD4 T cells. Furthermore, Prevacent® induced the differentiation of heterologous CD4 T cells into memory / effector cells. Downstream, this early T cell activation and differentiation not only provided B cell help that drove serum IgG levels (0 dpc and 14 dpc) and the frequency of nAb-positive animals (14 dpc), but also stimulated increased IFN-γ production (14 dpc) after challenge of vaccinated pigs. Induction of this combination of T cell and humoral immune responses induced partial protection against at least three of the four PRRSV strains, NADC30 (lineage 1), VR2332 (lineage 5), and NADC20 (lineage 8). From the CoP analysis included, serum IgG levels and CD4 T cell responses (proliferation, differentiation, and IFN-γ production) were shown to be the best systemic CoP. However, only the CD4 T cell response can be reliably used as CoP against specific PRRSV strains.

[0089] As will be appreciated from the description herein, a wide variety of aspects and embodiments are contemplated by the present disclosure, examples of which include, but are not limited to, the aspects and embodiments listed below.

[0090] After inoculating pigs with an effective amount of a live attenuated PRRSV vaccine, the pigs are challenged by intranasal inoculation with a certain amount of live PRRSV of a known strain at least 28 days after vaccine administration, thereby inducing heterologous immunogenicity against heterologous strains of porcine reproductive and respiratory syndrome virus (PRRSV) and enabling the evaluation of innate and adaptive immunity. In pigs, various measurements are taken including body temperature and body weight immediately before administration of the live attenuated PRRSV vaccine, immediately before challenge by intranasal inoculation with a known PRRSV strain, and at least 7 and 14 days after challenge. Further, blood samples are taken from the pigs immediately before administration of the live attenuated PRRSV vaccine, immediately before challenge by intranasal inoculation with a known PRRSV strain, and at least 7 and 14 days after challenge. Measured in each blood sample are the CD4 T cell response, the presence of strain-specific neutralizing antibodies, the presence of CD4, CD8, and TCR-γδ cells, IFN-γ levels, and the amounts of PRRSV-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) levels.

[0091] In this method, the steps of administering an effective amount of a live attenuated PRRSV vaccine to pigs, followed by challenging the pigs by intranasal inoculation with a known live PRRSV strain at least 28 days after vaccine administration, induce i) an increase in T cell activation demonstrated by the differentiation of CD4 T cells and CD8 cells, ii) an increase in the amount of PRRSV-specific immunoglobulin G (IgG) levels, iii) the production of serum neutralizing antibodies, and iv) an increase in serum IFN-γ levels.

[0092] This method further includes isolating, storing, and banking peripheral blood mononuclear cells (PBMCs) obtained from blood samples of pigs administered an effective amount of a live attenuated PRRSV vaccine.

[0093] Another method of disclosure includes: i) administering to a pig an effective amount of a live attenuated vaccine against porcine reproductive and respiratory syndrome virus (PRRSV); ii) challenging the pig by intranasal inoculation with a certain amount of a known live PRRSV strain at least 28 days after vaccine administration; iii) measuring the body temperature and body weight of the pig immediately before administration of the live attenuated PRRSV vaccine, immediately before challenge by intranasal inoculation with the known PRRSV strain, and on at least the 7th and 14th days after challenge; iv) collecting blood samples and nasal swabs from the pig immediately before administration of the live attenuated PRRSV vaccine, immediately before challenge by intranasal inoculation with the known PRRSV strain, and on at least the 7th and 14th days after challenge; v) evaluating the pathology of the lungs and lymph nodes of the pig at necropsy; vi) collecting bronchoalveolar lavage samples at necropsy; and vii) measuring the amount of virus, the amount of PRRSV-specific immunoglobulin A and immunoglobulin G present in each blood sample, nasal swab, and bronchoalveolar lavage sample. A method for determining the effectiveness of a vaccine against porcine reproductive and respiratory syndrome virus (PRRSV).

[0094] In this method, the step of administering to a pig an effective amount of a live attenuated PRRSV vaccine and the step of challenging the pig by intranasal inoculation with a certain amount of a known live PRRSV strain at least 28 days after vaccine administration induce: i) little or no lesions in the lungs and lymph nodes of the pig at necropsy; ii) a decrease in the amount of PRRSV virus in samples obtained from blood, nasal swabs, and bronchoalveolar lavage at necropsy; and iii) an increase in the amount of PRRSV-specific immunoglobulin A and immunoglobulin G.

[0095] Another method of the disclosure includes: i) isolating PRRSV from a blood sample or nasal swab sample taken from a pig suspected of being infected with PRRSV; ii) isolating, storing, and banking peripheral blood mononuclear cells (PBMCs) obtained in advance from a blood sample of a pig suspected of being infected with PRRSV or from a pig administered an effective amount of a live attenuated PRRSV vaccine, wherein the pig is further challenged by intranasal inoculation with a known live PRRSV strain at least 28 days after vaccination, and wherein those PBMCs are isolated, stored, and banked, and wherein the responses of CD4 T cells and CD8 T cells have been obtained in advance as the differentiation of CD4 T cells and CD8 cells; iii) measuring the correlates of protection (CoP) as the CD4 T cell and CD8 T cell responses in the PBMCs after loading with PRRSV from a pig suspected of being infected with PRRSV; and iv) comparing the CD4 T cell and CD8 T cell responses of step iii) with the CD4 T cell and CD8 T cell responses obtained in advance from the isolated, stored, and banked PBMC samples of step ii). A method for predicting the efficacy of a vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) in a pig suspected of being infected with PRRSV.

[0096] In any of the disclosed methods, porcine reproductive and respiratory syndrome virus (PRRSV) infection can be caused by infection with any PRRSV strain.

[0097] Although embodiments of the present disclosure are described herein, it will be understood by those skilled in the art that such embodiments are provided for illustrative purposes only. Numerous variations, modifications, and alternatives will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. i) administering to the pig an effective amount of a live attenuated vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) or a control injection; ii) challenging the pig by intranasal inoculation with a defined amount of a known live PRRSV strain at least 28 days after vaccine administration; iii) measuring the body temperature and body weight in the pig immediately before administration of the live attenuated PRRSV vaccine, immediately before challenge by intranasal inoculation with the known PRRSV strain, and at least on days 7 and 14 after challenge; iv) collecting blood samples from the pig immediately before administration of the live attenuated PRRSV vaccine, immediately before challenge by intranasal inoculation with the known PRRSV strain, and at least on days 7 and 14 after challenge; v) measuring in each blood sample the amount of CD4 T cell response, presence of strain-specific neutralizing antibodies, presence of CD4, CD8, and TCR-γδ cells, IFN-γ levels, and PRRSV-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) levels; vi) comparing all measurements with measurements taken from pigs injected with the control A method for inducing heterologous immunogenicity against a heterologous porcine reproductive and respiratory syndrome virus (PRRSV) strain and enabling the evaluation of innate and adaptive immunity, comprising the above steps.

2. The method according to claim 1, wherein the known porcine reproductive and respiratory syndrome virus (PRRSV) strain is selected from a PRRSV type 1 (PRRSV-1) virus strain and a PRRSV type 2 (PRRSV-2) virus strain.

3. The method according to claim 2, wherein the known porcine reproductive and respiratory syndrome virus (PRRSV) strain is a PRRSV type 2 (PRRSV-2) virus strain selected from the group consisting of NADC30 and NC174 (lineage 1), VR2332 (lineage 5), and NADC20 (lineage 8).

4. The method according to claim 1, wherein the strain-specific neutralizing antibody is one or more of an anti-NADC30 neutralizing antibody, an anti-VR2332 neutralizing antibody, and an anti-NADC20 neutralizing antibody.

5. The step of administering to the pig an effective amount of a live attenuated PRRSV vaccine, followed by challenging the pig by intranasal inoculation with a defined amount of a known live PRRSV strain at least 28 days after vaccine administration, compared to injection of the control, results in i) an increase in T cell activation demonstrated by the differentiation of CD4 T cells and CD8 cells; ii) an increase in the amount of PRRSV-specific immunoglobulin G (IgG) levels; iii) the production of serum neutralizing antibodies; and iv) induction of an increase in serum IFN-γ levels The method according to claim 1, which induces the above.

6. The method according to claim 1, further comprising the step of isolating, storing, and banking peripheral blood mononuclear cells (PBMCs) collected from a blood sample of a pig administered with an effective amount of a live attenuated PRRSV vaccine.

7. i) administering an effective amount of a live attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine to a pig; ii) challenging the pig by intranasal inoculation with a certain amount of a known live PRRSV strain at least 28 days after vaccine administration; iii) measuring the body temperature and body weight of the pig immediately before administration of the live attenuated PRRSV vaccine, immediately before challenge by intranasal inoculation with the known PRRSV strain, and at least on days 7 and 14 after challenge; iv) collecting blood samples and nasal swabs from the pig immediately before administration of the live attenuated PRRSV vaccine, immediately before challenge by intranasal inoculation with the known PRRSV strain, and at least on days 7 and 14 after challenge; v) evaluating the pathology of the lungs and lymph nodes of the pig at necropsy; vi) collecting bronchoalveolar lavage samples at necropsy; vii) measuring the amount of virus present, the amounts of PRRSV-specific immunoglobulin A and immunoglobulin G in each blood sample, nasal swab, and bronchoalveolar lavage sample; viii) comparing all measured values with the measured values obtained from pigs injected with a control A method for determining the effectiveness of a vaccine against porcine reproductive and respiratory syndrome virus (PRRSV), comprising the above steps.

8. The method according to claim 6, wherein the known porcine reproductive and respiratory syndrome virus (PRRSV) strain is selected from a PRRSV type 1 (PRRSV-1) virus strain and a PRRSV type 2 (PRRSV-2) virus strain.

9. The method according to claim 7, wherein the known porcine reproductive and respiratory syndrome virus (PRRSV) strain is a PRRSV type 2 (PRRSV-2) virus strain selected from the group consisting of NADC30 and NC174 (lineage 1), VR2332 (lineage 5), and NADC20 (lineage 8).

10. When the step of administering an effective amount of a live attenuated PRRSV vaccine to a pig, followed by the step of challenging the pig by intranasal inoculation with a certain amount of a known live PRRSV strain at least 28 days after vaccine administration, and comparing the measured values with the measured values obtained from pigs injected with a control i) at autopsy, there are few or no lesions in the lungs and lymph nodes of the pig; ii) at autopsy, the amount of PRRSV virus in samples taken from blood, nasal swabs, and bronchoalveolar lavage is decreased; iii) the amounts of PRRSV-specific immunoglobulin A and immunoglobulin G are increased The method according to claim 6, which induces the above.

11. i) isolating PRRSV from a blood sample or a nasal swab sample collected from a pig suspected of being infected with porcine reproductive and respiratory syndrome virus (PRRSV); ii) isolating, storing, and banking peripheral blood mononuclear cells (PBMCs) obtained in advance from a blood sample of a pig administered with an effective amount of a live attenuated PRRSV vaccine and loading them with the PBMCs isolated, stored, and banked from a blood sample of a pig suspected of being infected with PRRSV, wherein the pig is further loaded by intranasal inoculation with a certain amount of a known live PRRSV strain at least on the 28th day after vaccine administration, and wherein their PBMCs are isolated, stored, and banked, and wherein the responses of CD4 T cells and CD8 T cells have been obtained in advance as the differentiation of CD4 T cells and CD8 cells; iii) measuring the CD4 T cell and CD8 T cell responses in PBMCs after loading them with PRRSV from a pig suspected of being infected with PRRSV; iv) comparing the CD4 T cell and CD8 T cell responses in step iii) with the CD4 T cell and CD8 T cell responses obtained in advance from the isolated, stored, and banked PBMC samples in step ii) A method for predicting the effectiveness of a vaccine against PRRSV in a pig suspected of being infected with porcine reproductive and respiratory syndrome virus (PRRSV), comprising the above steps.

12. The method according to claim 11, wherein the known porcine reproductive and respiratory syndrome virus (PRRSV) infection is caused by infection with a strain selected from the group consisting of PRRSV type 1 (PRRSV-1) virus strains and PRRSV type 2 (PRRSV-2) virus strains.

13. The method according to claim 12, wherein the porcine reproductive and respiratory syndrome virus (PRRSV) infection is caused by infection with a PRRSV type 2 (PRRSV-2) virus strain selected from the group consisting of NADC30 and NC174 (lineage 1), VR2332 (lineage 5), and NADC20 (lineage 8).

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