Method for predicting efficacy of modified live vaccines of porcine reproductive and respiratory syndrome virus (PRRSV)

Abstract

The present invention provides methods for eliciting heterologous immunogenicity against heterologous porcine reproductive and respiratory syndrome virus (PRRSV) strains to allow for assessment of innate immunity and adaptive immunity. In other aspects, methods are provided for determining the efficacy of a vaccine against PRRSV. In still other aspects, methods are provided for predicting the efficacy of a vaccine against PRRSV in a pig suspected of being infected with PRRSV.

Description

Technical Field

[0001] The present disclosure generally relates to methods for eliciting heterologous immunogenicity against heterologous porcine reproductive and respiratory syndrome virus (PRRSV) strains to allow for the assessment 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 having PRRSV infection are provided. Background Art

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) remains the most economically important animal pathogen. This virus causes reproductive disorders and respiratory diseases and is an important cause of porcine respiratory disease syndrome (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: pp. 129-54. Respiratory diseases alone result in an annual production decline of approximately 7.4%, equivalent to an annual loss of more than $664 million. 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 capacity, the high mutation rate of PRRSV also enables it to escape immunity provided by the host through infection or vaccination. See Loving et al., "Innate and adaptive immunity against Porcine Reproductive and Respiratory Syndrome Virus," (2015) Vet Immunol Immunopathol 167: pp. 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: 10.3390 / vaccines9050417; Murtaugh et al., "Immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (PRRS)," (2011) Vaccine, 29(46): pp.8192-204; Rowland et al., "Alternative strategies for the control and elimination of PRRS,” (2017) VetMicrobiol 209: pp.1-4; Shi et al., "Molecular epidemiology of PRRSV: a phylogeneticperspective," (2010) Virus Res 154(1-2): pp.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:pp.108-116. .

[0004] These mutations have resulted in a plethora of strains: PRRSV can be divided into two types, type 1 or PRRSV-1, found primarily in Europe, and type 2 or PRRSV-2, prevalent in North America. Based on open reading frames (ORFs), PRRSV-2 is further divided into nine lineages of numerous PRRSV strains. This high diversity presents a huge challenge for PRRSV vaccines: they need to protect against the various evolving PRRSV strains present in the swine industry. Currently the most prevalent PRRSV-2 lineages are lineages 1, 5, 8, and 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.

[0005] However, based on the high prevalence of these strains, the industry needs a PRRSV vaccine that is confidently able to provide broad cross-reactivity against a variety of PRRSV strains.

[0006] The present disclosure satisfies this need. Summary of the invention

[0007] The inventors have discovered methods for eliciting heterologous immunogenicity against heterologous porcine reproductive and respiratory syndrome virus (PRRSV) strains to allow for the assessment 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 having PRRSV infection are provided.

[0008] In one embodiment, a method for eliciting heterologous immunogenicity against heterologous porcine reproductive and respiratory syndrome virus (PRRSV) strains is provided to allow for the assessment of innate and adaptive immunity. The method comprises first administering an effective amount of a modified live PRRSV vaccine or a control injection to a pig. About 28 days after the vaccine is administered, the pig is challenged by intranasal inoculation of a certain amount of a live known PRRSV strain. Immediately before administering the modified live PRRSV vaccine, immediately before attacking with a known PRRSV strain by intranasal inoculation, and at least 7 days and 14 days after the attack, the body temperature and weight of the pig are measured. Immediately before administering the modified live PRRSV vaccine, immediately before attacking with a known PRRSV strain by intranasal inoculation, and at least 7 days and 14 days after the attack, a blood sample is obtained from the pig. Then, various immune protection correlations (CoPs) from each blood sample are measured and determined. Measurements included CD4 T cell responses, the presence of strain-specific neutralizing antibodies, the presence of CD4, CD8 and TCR-γδ cells, IFN-γ levels, and the amount of PRRSV-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) levels. All measurements from vaccinated pigs were compared to those obtained from control injected pigs.

[0009] In any embodiment, the known porcine reproductive and respiratory syndrome virus (PRRSV) strain is selected from type 1 PRRSV (PRRSV-1) and type 2 PRRSV (PRRSV-2) strains. In other embodiments, the known porcine reproductive and respiratory syndrome virus (PRRSV) strain is a type 2 PRRSV (PRRSV-2) strain selected from NADC30 and NC174 (lineage 1), VR2332 (lineage 5) and NADC20 (lineage 8).

[0010] In any embodiment, the strain-specific neutralizing antibodies are one or more of anti-NADC30, anti-VR2332, and anti-NADC20 neutralizing antibodies.

[0011] In yet other embodiments, administration of an effective amount of a modified live PRRSV vaccine to pigs, followed by challenge of the pigs by intranasal inoculation with a live known PRRSV strain at least 28 days after vaccine administration, induces a correlation of immune protection (CoP) characterized by: i) increased T cell activation, as evidenced by CD4 T and CD8 cell differentiation; ii) increased levels of PRRSV-specific immunoglobulin G (IgG); iii) production of serum neutralizing antibodies; and, iv) increased serum IFN-γ levels, compared to control injections.

[0012] In any embodiment, the method comprises the isolation, storage and banking of peripheral blood mononuclear cells (PBMCs) obtained from a blood sample of a pig administered an effective amount of a modified live PRRSV vaccine.

[0013] Another embodiment of the present invention provides a method for determining the efficacy of a vaccine against porcine reproductive and respiratory syndrome virus (PRRSV), comprising: i) administering an effective amount of a modified live PRRSV vaccine to a pig; ii) challenging the pig by intranasal inoculation of a live known PRRSV strain at least 28 days after the administration of the vaccine; iii) measuring the body temperature and weight of the pig immediately before administration of the modified live PRRSV vaccine, immediately before challenge by intranasal inoculation of a known PRRSV strain, and at least 7 days and 14 days after challenge; iv) measuring the body temperature and weight of the pig immediately before administration of the modified live PRRSV vaccine, immediately before challenge by intranasal inoculation of a known PRRSV strain, and at least 7 days and 14 days after challenge; iv) measuring the body temperature and weight of the pig immediately before administration of the modified live PRRSV vaccine, immediately before challenge by intranasal inoculation of a known PRRSV strain, and at least 7 days and 14 days after challenge. Obtain blood samples, nasal swabs from pigs before modified live PRRSV vaccine, immediately before challenge by intranasal inoculation with a known PRRSV strain, and at least 7 and 14 days after challenge; v) after necropsy, evaluate the pigs for lung and lymph node pathology; vi) after necropsy, obtain bronchoalveolar lavage samples; vii) measure the amount of virus, PRRSV-specific immunoglobulin A and immunoglobulin G present in each blood sample, nasal swab and bronchoalveolar lavage sample; and, viii) compare all measurements with those obtained from control injected pigs.

[0014] In some embodiments, administration of an effective amount of a modified live PRRSV vaccine to pigs, followed by challenge of the pigs by intranasal inoculation with a live known PRRSV strain at least 28 days after vaccine administration, can induce: i) little or no lung and lymph node pathology in the pigs after necropsy; ii) a decrease in the amount of PRRSV virus in samples obtained from blood, nasal swabs, and bronchoalveolar lavage after necropsy; and iii) an increase in the amount of PRRSV-specific immunoglobulin A and immunoglobulin G, as compared to measurements obtained from control injected pigs.

[0015] Yet another embodiment of the present invention provides a method for predicting the efficacy of a vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) in a pig suspected of having PRRSV infection, comprising: i) isolating PRRSV from a blood or nasal swab sample obtained from a pig suspected of having PRRSV infection; ii) challenging with PRRSV from the pig suspected of having PRRSV infection, isolating, storing and warehousing a sample of peripheral blood mononuclear cells (PBMCs) previously obtained from a blood sample from a pig administered with an effective amount of a modified live PRRSV vaccine, wherein at least 28 days after vaccine administration, the pig is further challenged by intranasal inoculation with a certain amount of a live known PRRSV strain, and isolating, storing and warehousing its PBMCs, wherein CD4 T and CD8 T cell responses differentiated as CD4 T and CD8 cells are previously obtained; iii) measuring the correlation of immune protection (CoP) as CD4 T and CD8 T cell responses in PBMCs after challenging with PRRSV from the pig suspected of having PRRSV infection; and iv) separating the CD4 T and CD8 T cell responses differentiated as CD4 T and CD8 T cells in step iii) from the pig suspected of having PRRSV infection. The T cell responses were compared to the CD4 T and CD8 T cell responses previously obtained from the isolated, stored and banked PBMC samples in step ii).

[0016] These and other embodiments and features of the present disclosure will become more apparent by reference to the following description, drawings and claims.In addition, it should be understood that the features of the various embodiments described herein are not mutually exclusive and can exist in various combinations and arrangements. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The design of an in vivo animal study is described. Four-week-old weaned pigs were divided into ten groups: five mock (MOCK, phosphate-buffered saline) groups and five Vaccination groups. Pigs were vaccinated at -28 days post-challenge (dpc). On the day of challenge (0 dpc), each of the five groups received one of five intranasal inoculations: MOCK, NADC30, NC174, VR2332, or NADC20. At 14 dpc, pigs were sacrificed to assess lung pathology. As shown in the timeline, blood and nasal swabs were collected throughout the study to assess viral load and humoral and T cell immune responses.

[0018] Figure 2A-C Depicted Heterologous vaccine efficacy. Rectal temperature (Figure 2A), viremia (Figure 2B), and viral load in nasal swabs (Figure 2C) were determined 0, 7, and 14 days (dpc) after challenge with MOCK (grey) 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 viral shedding (Figure 2C) (genome copies / mL [log10]) were quantified in serum and nasal swabs, respectively, by PRRSV-specific RT-qPCR. The black bars represent the median values; in addition, individual data points for MOCK-vaccinated animals (open diamonds) and MLV-vaccinated animals (filled squares) are shown. Use Data were analyzed by two-way ANOVA with multiple comparisons. Within each time point, each vaccination group was compared with its respective MOCK vaccination group challenged with type 2 PRRSV. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

[0019] Fig. 3A-C depicts the efficacy of heterologous vaccines on lung viral load, pathology and inguinal lymph node size.(Fig. 3A) Pulmonary viral load was assessed in bronchoalveolar lavage fluid (BAL) by PRRSV-specific RT-QPCR (genome copy number / mL [log10]).It is noteworthy that, although PRRSV was not detected in the BAL of an animal receiving MOCK-inoculation / NADC20-attack, the pig had the second highest lung pathology score, together with the highest PRRSV load in 14dpc nasal swabs.Based on this difference, it was excluded from this analysis.(Fig. 3B and C) At 14 days (dpc) after attack, the lung gross and histopathology of all seven lobes were assessed by veterinarians blindly.(Fig. 3B) The lung lesion percentage of each individual pig is depicted.(Fig. 3C) Histopathological scores of all seven lobes are shown, which follow 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): pp. 648-60. Each vaccinated group was compared with its corresponding unvaccinated group challenged with type 2 PRRSV using a two-tailed unpaired t-test. The black bar represents the median value; in addition, individual data points for MOCK-vaccinated animals (open diamonds) and MLV-vaccinated animals (solid squares) are shown. Each vaccinated group was compared with its respective unvaccinated group challenged with type 2 PRRSV. Data were compared using a two-tailed unpaired t-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

[0020] Figure 4A-DDepict heterologous vaccine immunogenicity as humoral immune response. Immunoglobulin A of (Fig. 4A) bronchoalveolar lavage fluid (BAL), (Fig. 4B) nasal swabs and IgG levels of serum (Fig. 4C) at 0 and 14 days (dpc) post-challenge were assessed via PRRSV X3 ELISA. IgA and IgG ELISA S / P ratios were compared within their challenge groups: MOCK (grey), 1-4-4 (NADC30, dark blue), NC174 (red), VR2332 (green) and 1-4-2 (NADC20, light blue). Black bars represent median values; in addition, individual data points for MOCK-vaccinated animals (hollow diamonds) and MLV-vaccinated animals (solid squares) are also shown. Data were statistically analyzed using a two-factor ANOVA with time and inoculation as two parameters and Tukey's multiple comparison test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. (Fig. 4D) Neutralizing antibody (NA) titers against the corresponding challenge strains at 0 and 14 dpc were determined via the FFN test. Since no animals showed FFN titers at 0 dpc, only data for 14 dpc are shown. Titers ≥1:4 were considered positive. Titers for each individual pig challenged with PRRSV are shown. Positive NA titers are highlighted in blue (NADC30), red (NC174, not detected), green (VR2332) and light blue (NADC20).

[0021] Figure 5A-DHeterologous vaccine immunogenicity as CD4, CD8 and TCR-γδT cell proliferation is shown.(Fig. 5A) shows a gating hierarchy, which is used to assess the heterologous proliferation response of T cell subsets to corresponding type 2 PRRSV attack strains.Includes distinguishing live / dead dyes to exclude dead cells.Live cells are used to identify live lymphocytes via FSC / SSC lymphocyte gating.Use FSC width (FSC-W) / FSC area (FSC-A) gating to single cells (singlet), exclude doublets (doublet) from live lymphocytes.These single live lymphocytes are used to gate T cells (FSC-A / CD3), and further distinguish TCR-αβ and TCR-γδT cells.TCR-αβT cells are further divided into CD4 and CD8 T cells via their CD4 / CD8α expression profiles.The proliferation of CD4, CD8 and TCR-γδT cells is identified via purple proliferation dye. Two examples show representative staining patterns of control animals (upper right) and high-responding animals (lower right). (Figure 5B-D) Proliferative responses of CD4 (Figure 5B), CD8 (Figure 5C), and TCR-γδT cells (Figure 5D) are shown according to their challenge groups (MOCK (gray), NC174 (red), NADC20 (light blue), NADC30 (dark blue), and VR2332 (green)). The black bars represent the median values; in addition, individual data points for MOCK-vaccinated animals (open diamonds) and MLV-vaccinated animals (solid squares) are shown. Data were statistically analyzed using a 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.

[0022] Figure 6A-DHeterologous vaccine immunogenicity as IFN-γ production of CD4, CD8, and TCR-γδ T cells is shown. (FIG. 6A) Gating hierarchy used to assess heterologous IFN-γ responses of T cell subsets to the corresponding type 2 PRRSV challenge strains. The gating hierarchy largely follows the proliferation analysis shown in FIG5. However, instead of gating on proliferating cells, IFN-γ analysis was performed in the FSC-A / IFN-γ plot. IFN-γ gating was set using the appropriate FMO controls (top right). (FIG. 6B-D) IFN-γ responses of CD4 (FIG. 6B), CD8 (FIG. 6C), and TCR-γδ T cells (FIG. 6D) are shown according to their challenge groups (MOCK (grey), 1-4-4 (NADC30, dark blue), NC174 (red), VR2332 (green), and 1-4-2 (NADC20, light blue)). The black bar represents the median value; in addition, individual data points for MOCK-vaccinated animals (open diamonds) and MLV-vaccinated animals (filled squares) are shown. Data were statistically analyzed using a 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.

[0023] Figure 7A-B The immunogenicity of heterologous vaccines as differentiation of IFN-γ-producing CD4 T cells is depicted. (FIG. 7A) shows the gating hierarchy used to assess the differentiation of IFN-γ-producing CD4 T cells. After gating on IFN-γ+CD4 T cells as described in FIG. 6, their differentiation was analyzed via their CD4 / CD8α expression profile to distinguish between naive (CCR7+CD8α-), central memory (T CM CCR7+CD8α+) ​​and effector memory type (T EM , CCR7-CD8α+)CD4 T cells (upper right). Since the vast majority of CD4 T cells producing CD8α+IFN-γ belong to T CM subset (data not shown), so T CM and T EMThe two are combined into the "memory / effector" subset. (Figure 7B) The frequency of these memory / effector cells within IFN-γ-producing CD4 T cells is shown according to their challenge group (MOCK (gray), 1-4-4 (NADC30, dark blue), NC174 (red), VR2332 (green), and 1-4-2 (NADC20, light blue)). The black bar represents the median value; in addition, individual data points are shown for MOCK-vaccinated animals (open diamonds) and MLV-vaccinated animals (filled squares). Data were statistically analyzed using a 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.

[0024] Figure 8 Shows Quantification of vaccine strains in serum. Specific RT-qPCR quantification Prevalence of vaccine strains. The table shows the Ct values ​​for animals challenged with MOCK, NADC20, NC174, VR2332 and NADC20 at 7 days post-challenge. DETAILED DESCRIPTION OF THE INVENTION

[0026] Throughout the present disclosure, various quantities, such as amount, size, dimensions, ratio, etc., are all displayed in range format. It should be understood that describing quantity in range format is only for convenience and simplicity, and should not be interpreted as a strict limitation of the scope of any embodiment. Therefore, unless the context clearly stipulates otherwise, the description of the scope should be deemed to have clearly disclosed all possible sub-ranges and all single values ​​in the range. For example, the description of the scope (for example, from 1 to 6) should be deemed to have clearly disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single values ​​in the range, such as 1.1, 2, 2.3, 4.62, 5 and 5.9. Regardless of the width of the scope, this applies. The upper and lower limits of these intermediate ranges can be independently included in a smaller range, and are also included in the present disclosure, subject to any clearly excluded limit in the scope. When the scope includes one or two limits, the scope excluding one or two of these included limits is also included in the present disclosure, unless the context clearly stipulates otherwise.

[0027] The terms used herein are only for the purpose of describing specific embodiments and are not intended to limit any embodiment. As used herein, the singular forms "a", "an", and "the" are also intended to include plural forms unless the context clearly indicates otherwise. It should be further understood that the terms "include", "comprise", "including", and / or "comprising", when used in this specification, specify the presence of the features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more related listed items. Additionally, it should be understood that the items included in the list of "at least one of A, B, and C" form can represent (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C). Similarly, items listed in the format “at least one of A, B, or C” may mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C).

[0028] Unless otherwise specified or obvious from the context, as used herein, the term "about" in reference to a number or numerical range should be understood to refer to the stated number and + / - 10% thereof, or, for a listed range value, 10% below the listed lower limit and 10% above the listed upper limit.

[0029] As used herein, the term "subject" or "patient" refers to a mammal, more specifically a pig or swine.

[0030] By referring to the following description, drawings and claims, embodiments and features of the present disclosure will become more apparent.In addition, it should be understood that the features of the various embodiments described herein are not mutually exclusive and can exist in various combinations and arrangements.

[0031] The present disclosure is based on the knowledge that, although porcine reproductive and respiratory syndrome virus (PRRSV) vaccines have been available in North America for nearly 30 years, they face a significant obstacle: they must provide cross-protection against highly diverse PRRSV strains. This cross-protection, or heterologous vaccine efficacy, relies heavily on the ability of the vaccine to induce a strong immune response against a variety of strains, i.e., heterologous immunogenicity.

[0032] The inventors studied the vaccine efficacy and immunogenicity of modified live virus (MLV) against four heterologous type 2 PRRSV (PRRSV-2) strains as a model for testing the vaccine efficacy and immunogenicity of modified live virus (MLV) against any suspected PRRSV strain. In the present disclosure, pigs were divided into several groups. Half were vaccinated with MOCK (i.e., phosphate buffered saline) and the other half were vaccinated with modified live PRRSV vaccine: PRRS MLV vaccine (Elanco, Inc.). Four weeks after vaccination, each group was challenged with MOCK or four PRRSV-2 strains (NC174 or NADC30 (all pedigree 1), VR2332 (pedigree 5) or NADC20 (pedigree 8)). Before and after the challenge, lung pathology, viral load in both nasal swabs and serum, anti-PRRSVIgA / G, neutralizing antibodies, and PRRSV-2 strain-specific T cell responses were evaluated. At autopsy, lungs were collected to assess viral load, pathology, and IgA levels in BAL. Lung pathology was induced only by NC174, NADC20, and NADC30; among them, vaccination did reduce the pathology of NADC20 and NADC30 strains. All pigs developed viremia, and the vaccinated pigs had reduced viremia after being challenged with NADC20, NADC30, and VR2332. Regarding vaccine immunogenicity, vaccination induced a strong systemic IgG response and increased post-challenge serum IgG levels for all strains. In addition, vaccination increased the number of animals with neutralizing antibodies against three of the four challenge strains (NADC20, NADC30, and VR2332). Vaccination also improved heterologous T cell responses: 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 analyses showed that (non-PRRSV strain-specific) serum IgG levels and PRRSV strain-specific CD4 T cell responses were the best correlates of immune protection. In summary, Different degrees of efficacy and immunogenicity were elicited against different heterologous strains from three different PRRSV-2 lineages, thus serving as a model for testing modified live virus (MLV) vaccine efficacy and immunogenicity against any suspected PRRSV strain.

[0033] Vaccine immunogenicity

[0034] One aspect of the present disclosure includes analysis and validation of vaccine immunogenicity, including both humoral and T cell immune responses following immunization with a modified live PRRSV vaccine, in order to develop methods and tests for determining the efficacy of a modified live PRRSV vaccine against unknown or suspected strains of PRRSV virus strains.

[0035] Neutralizing antibodies (nAbs) play a key role in both viral clearance and protection against repeated infection. See Lopez et al., "Role of neutralizing antibodies in PRRSV protective immunity," (2004) Vet Immunol Immunopathol 102(3): pp.155-163. However, some studies have noted that the induction of nAbs is delayed before the clearance of viremia. See, for example, Lunney et al., 2016; Butler et al., "Antibody Repertoire Development in Swine," (2017) Ann. Rev Anim Biosci 5: pp.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): p.108. These existing studies emphasize that clearance of PRRSV viremia can occur before the appearance of nAbs.

[0036] Thus, although the development of nAbs is important for protection against PRRSV, other factors also appear to play relevant roles. In the absence of nAbs, the reduction of viremia or even viral clearance is partly 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: pp. 1635. Based on this central role of T cells in controlling PRRSV, the inventors herein provide a detailed analysis of the PRRSV strain-specific proliferation and IFN-γ response of CD4, CD8, and TCR-γδ T cells. Additionally, the reactive CD4 Differentiation of T cells from CD8α naive cells to antigen-experienced CD8α+ memory / effector cells. This differentiation enables the differentiation of primary and secondary responses of these CD4 T cells.

[0037] Design Overview

[0038] To complete a detailed study of the immunogenicity and efficacy of heterologous vaccines, 60 pigs were divided into 10 groups: 5 groups were treated with a commercially available modified live PRRSV vaccine PRRS MLV vaccine (Elanco, Greenfield, IN) was used for immunization, and 5 groups were vaccinated with MOCK (phosphate-buffered saline). Four weeks after vaccination, pigs were challenged with one of the above-mentioned PRRSV-2 strains or vaccinated with MOCK. Viral shedding and viremia, as well as the induced immune response, were followed for two weeks; then, pigs were sacrificed for additional evaluation of viral load in bronchoalveolar lavage (BAL), lung macroscopic and histopathology, and inguinal lymph node size. Immune response analysis included humoral and T-cell immune responses: the humoral response was studied not only by quantifying mucosal IgA in nasal swabs and bronchoalveolar lavage (BAL), but also by determining serum IgG and nAb levels; systemic T-cell responses were analyzed in detail, including proliferation of CD4, CD8, and TCR-γδ T cells and IFN-γ responses and CD4 T cell differentiation. In addition, the correlation of these immune parameters was studied, that is, the correlation of immunity with the studied vaccine efficacy parameters (lung pathology, viral shedding, and viremia).

[0039] Discover Various levels of heterologous immunity were induced, including strong IgA responses in BAL, strong systemic IgG responses, and increased prevalence of nAbs against NADC30, -VR2332, and -NADC20. Immunization also promoted i) CD4 T cell differentiation, ii) CD4, CD8 and TCR-γδ cell proliferation, and iii) a stronger post-challenge IFN-γ response.

[0040] Correlation analysis between vaccine efficacy and immunogenicity parameters revealed two important correlates of immune protection: systemic IgG levels and CD4 T cell responses. However, in contrast to ELISA, which was used to quantify systemic IgG levels, specific CD4 responses to the challenge strain were determined by an in vitro restimulation method followed by multicolor flow cytometry. Conclusively, the inventors identified systemic CD4 T cell responses as a strain-specific correlate of immune protection (CoP) for PRRSV-2 for the first time.

[0041] Therefore, the present inventors have found that the Correlation of Protection (CoP) can powerfully facilitate vaccine development and can be used to predict vaccine efficacy against emerging PRRSV-2 strains.

[0042] Study Design

[0043] The research design of this disclosure is as follows Figure 1As shown. Sixty four-week-old weaned pigs from a PRRSV-2 negative farm (NCState University Swine Education Unit, Raleigh, NC, USA) were brought to the BSL-2 Laboratory Animal Research Unit (LAR Site) of the College of Veterinary Medicine at North Carolina State University (NC State University, College of Veterinary Medicine, Raleigh, NC, USA). These 60 weaned pigs were randomly divided into ten groups using the GraphPad online randomization tool. Five groups were inoculated intramuscularly (IM) with MOCK using phosphate buffered saline (PBS), and five groups were inoculated with the manufacturer's recommended Vaccination. Twenty-eight days after vaccination, pigs were challenged intranasally (500 μL / nostril; 1 mL total) using a nasal spray intramucosal atomization device (Mountainside Medical Equipment, Marcy, NY).

[0044] Each MOCK and MLV inoculated group was challenged with 106TCID50 / mL dose of NC174 (lineage 1A), NADC30 (lineage 1C), VR2332 (lineage 5) or NADC20 (lineage 8). Pigs challenged with MOCK were challenged with 1% bovine serum albumin (BSA) (3 / 6 pigs) or Opti-MEMTM (3 / 6 pigs) in PBS, which are two suspension media for different virus strains. Pigs were clinically monitored every day. Blood was collected to obtain serum and / or isolate peripheral blood mononuclear cells (PBMC) at -28 days, 0 days, 7 days and 14 days (dpc) after the attack. Body weight and rectal temperature were recorded weekly. For ease of handling, autopsies were performed over two days: 15 and 16dpc. Pigs were euthanized using a lethal injection and lungs were collected.

[0045] First, the lungs were evaluated for gross pathology and photographed. Then, the lungs were filled with 50 mL PBS, gently massaged and bronchoalveolar lavage fluid (BAL) was collected for downstream evaluation of lung viral load and local humoral and cellular immune responses. Subsequently, tissue samples were collected for histopathological quantification and characterization of T cells infiltrating lung tissue. Inguinal lymph nodes were also collected and weighed as clinical indicators of PRRSV exposure. See Rossow et al., "Pathogenesis of porcinereproductive and respiratory syndrome virus infection in gnotobiotic pigs," (1995) Vet Pathol 32 (4): pp. 361-73. The experimental procedures have been approved by the Institutional Animal Care and Use Committee (IACUC) ID#17-166A of North Carolina State University (November 29, 2017).

[0046] PRRSV strains

[0047] NC174, NADC20 and NADC30 were provided by Elanco. VR2332 was produced in-house and titrated on MA-104 cells. Pools of serum from challenged pigs receiving MOCK vaccination at 7 dpc were sent to ISU VDL for ORF5 sequencing: Sequence analysis confirmed the correct identity of the challenge strain (data not shown, dns).

[0048] Processing of bronchoalveolar lavage fluid, nasal swabs, and blood

[0049] 0.4mL BAL sample was added to 0.6mL TriReagent (Ambion, Austin, TX, USA), mixed and stored at -80°C for downstream PRRSV quantification via qPCR. The remaining BAL was centrifuged at 400g and 4°C for 10 minutes to precipitate BAL immune cells. Cell pellets were collected, counted and used to analyze local T cell immune responses. The supernatant was aliquoted and stored at -80°C for downstream analysis of humoral immune responses via IgA ELISA. Nasal swabs were rotated in each nostril and placed in a test tube containing 1mL PBS. After collection, the swabs were vortexed and then pressed against the tube wall to rotate in a circular motion, and then the swabs were removed from the tube. PBS from these nasal swabs was aliquoted and stored at -80°C for downstream PRRSV and antibody quantification. Whole blood for serum separation was collected in SST tubes (BD Bioscience, San Jose, CA, USA) and incubated upright for 30 minutes. After incubation, the blood was centrifuged at 2,000 g for 20 minutes at 23°C. Serum was collected and stored in aliquots at -80°C. Whole blood for peripheral blood mononuclear cell (PBMC) separation was collected in heparin tubes (BD Bioscience). PBMC separation was performed by density centrifugation using Sepmate tubes (StemCell, Vancouver, Canada) and Ficoll-Paque (GE Healthcare, Uppsala, Sweden). After separation, fresh PBMCs were used for in vitro restimulation to study PRRSV strain-specific T cell immune responses.

[0050] Viremia and viral load

[0051] Separated serum and nasal swabs were shipped to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) (Ames, IA, USA) for use with PRRSV universal or “Elanco-like” " specific reverse transcription (RT) quantitative PCR was used to quantify PRRSV. The results were expressed as Ct values ​​("similar to Elanco ” RT-qPCR) or genome copies / mL (universal RT-qPCR) are given.

[0052] Serum anti-PRRSV IgG and anti-PRRSV IgA

[0053] Separate serum and nasal swabs were shipped to the ISU VDL. Serum IgG levels were determined using the PRRSV X3 enzyme-linked immunosorbent assay (ELISA, IDEXX, Westbrook, ME, USA). The PRRSV oral fluid IgA ELISA was used to determine IgA in nasal swabs.

[0054] Neutralizing antibodies

[0055] Serum samples at 0 and 14 dpc were shipped to South Dakota State University Animal Research and Diagnostic Laboratory (SDSUARDL). Neutralizing antibodies were measured by fluorescence focus neutralization (FFN) test. See Valdes-Donoso et al. (2018). Titers > 1:4 were considered positive. The isolated sera were tested against the corresponding homologous challenge strains. Both MOCK challenge groups were tested against all four virus strains.

[0056] Lung gross pathology, histology, and lymph node weights

[0057] At necropsy, lungs and inguinal lymph nodes were collected. Photographs of the dorsal and ventral sides of the lungs were taken. Lung lobes were scored blindly by a veterinarian. For histopathological evaluation, tissues from seven lobes of the lung were extracted—left apex, left lobe, left diaphragm (caudal), right apex, right lobe, right diaphragm (caudal), and intermediate (appendageal). Tissue samples were fixed in formaldehyde / Zn fixative (Electron Microscopy Sciences, Hatfield, PA) for twenty-four hours; then, they were transferred to 70% ethanol. Tissue processing, hematoxylin and eosin (H&E) staining, and slide preparation were performed by the Histology Laboratory at North Carolina 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) slightly altered, (2) mild, (3) moderate, or (4) severe. For a general assessment of immune activation, bilateral inguinal lymph nodes were collected at sacrifice and weighed in grams.

[0058] Proliferation of PRRSV challenge strain-specific T cells

[0059] In order to measure the proliferation of PRRSV-specific T cell subsets, freshly isolated PBMCs were stained with CellTraceTM Violet cell proliferation kit (Invitrogen) according to the manufacturer's instructions. The stained cells were seeded in 96-well round-bottom plates (Sarstedt, Nümbrecht, Germany) at 200,000 cells / well. Cells were stimulated with culture medium (MOCK), NC174, NADC20, NADC30 or VR2332 (MOI of 0.1) for 72 hours; Concanavalin A (ConA, 2.5 μg / mL, Alfa Aesar) was used as a positive control. Cells from eight replicates were merged and stained for flow cytometry analysis according to Table 1. Flow cytometry data were obtained on Cytoflex using CytExpert software (Beckman Coulter). Data analysis was performed using FlowJo version 10.5.3 (FLOWJO LLC), where gates were set according to the relevant FMO controls.

[0060] Table 1: Flow cytometry staining panel

[0061]

[0062] While both panels include CD3, CD4, CD8α, TCR-γδ, CRR7, and live / dead staining, IFN-γ staining (*) is included only in the IFN-γ analysis, and proliferation ( # ) staining was included only in the proliferation analysis.

[0063] PRRSV-challenging strain-specific IFN-γ production in T cells

[0064] PBMCs were plated at 500,000 cells / well and allowed to stand overnight. The next day, cells were stimulated with culture medium (MOCK), NC174, NADC20, NADC30 or VR2332 (MOI 0.1); phorbol 12-myristate 13-acetate (PMA, 5ng / mL, AlfaAesar, Ward Hill, MA, USA) / ionomycin (500ng / mL, AdipoGen, San Diego, CA, USA) was used as a positive control. The culture plate was cultured for 18 hours; monensin (5 μg / mL, AlfaAesar) was added in the last 4 hours of culture. Then, 8 replicates were merged and dyed according to Table 1 to perform flow cytometry analysis. Data were acquired on Cytoflex using CytExpert software (Beckman Coulter). Data analysis was performed using FlowJo version 10.5.3, where gates were set according to FMO controls.

[0065] Statistical analysis

[0066] Statistical analysis was performed using GraphPad Prism 9.1.1 (GraphPad Software, San Diego, CA). All qPCR data were log-transformed prior to statistical analysis. Statistical significance was analyzed by two-way ANOVA or two-tailed unpaired Student's t-test, depending on the data set. Multiple comparisons were performed using multiple comparison tests.

[0067] Further reference is made to the following experimental examples, which are provided for the purpose of illustrating various embodiments of the present invention, and are not intended to limit the present disclosure in any way. The present examples and the methods described herein are current representatives of preferred embodiments, which are provided only as examples, and are not intended to limit the scope of the present invention. Those skilled in the art will appreciate its changes and other uses within the spirit of the present disclosure defined by the scope of the claims. Example

[0068] Example 1: Heterologous vaccine efficacy

[0069] The heterologous vaccine efficacy was determined in three ways: i) clinical symptoms, including rectal temperature; ii) assessment of PRRSV load in nasal swabs and serum at 0, 7 and 14 dpc (Figure 2); and iii) assessment of viral load and lung gross and histopathology at necropsy (14 dpc, Figure 3).

[0070] Weight gain, clinical symptoms, and viral load in serum, BAL, and nasal swabs

[0071] Figure 2A-C Depicted Heterologous vaccine efficacy. Rectal temperature (Figure 2A), viremia (Figure 2B), and viral load in nasal swabs (Figure 2C) were determined 0, 7, and 14 days (dpc) after challenge with MOCK (grey) 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 viral shedding (Figure 2C) (genome copies / mL [log10]) were quantified in serum and nasal swabs, respectively, by PRRSV-specific RT-qPCR. The black bars represent the median values; in addition, individual data points for MOCK-vaccinated animals (open diamonds) and MLV-vaccinated animals (filled squares) are shown. Use Data were analyzed by two-way ANOVA with multiple comparisons. Within each time point, each vaccination group was compared with its respective MOCK vaccination group challenged with type 2 PRRSV. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

[0072] There were no relevant differences in weight gain between the groups. Clinical signs were mild; including lethargy and respiratory distress at approximately 7-14 dpc. Only two differences were noted between the vaccinated groups: i) in the NC174 challenge group, Vaccinated pigs showed slightly lower levels of lethargy; and ii) Compared with the pigs challenged with NC174, the MOCK group showed obvious clinical signs after challenge with NADC20: MOCK pigs showed signs of respiratory distress, severe lethargy and anorexia at 7-14 dpc (data not shown, dns). Rectal temperatures differed between the groups (Figure 2A): only pigs challenged with NC174 and NADC20 showed elevated body temperature at 7 dpc. The body temperature of pigs vaccinated with MOCK was even higher than that of their counterparts vaccinated with MOCK. Overall, under the current study conditions, clinical signs of NC174 and NADC20 were mostly mild and included lethargy, anorexia, respiratory disease, and some elevated body temperatures. The most prominent protective aspects were the reduction in respiratory disease, anorexia, and lethargy following NADC20 challenge.

[0073] Due to limited clinical symptoms, viral loads in nasal swabs and serum were quantified to better evaluate heterologous vaccine efficacy (Figure 2B, 2C). Analysis of pre-challenge viral loads at 0 dpc showed that all vaccinated The number of PRRSV copies in animals is similar: This confirms Vaccination was not only successful, but also homogeneous (Figure 2B). Challenge with different PRRSV strains induced viremia, which peaked at 7dpc. At this time, VR2332 challenge resulted in mild to moderate viremia in the MOCK-vaccinated group, with a median genome copy number of 10^6.2. In contrast, challenges with NC174, NADC30, and NADC20 induced strong viremia in pigs vaccinated with MOCK: the median genome copy number / mL was 10^9.0, 10^8.6, and 10^9.2, respectively. At 14dpc, viremia was reduced by about 1-2 logs. For VR2332 and NADC30, Vaccination significantly reduced viremia at both time points; for NADC30, it reduced viremia at 14 dpc (Figure 2B). Of note, while all pigs vaccinated with MOCK remained viremic after VR2332 challenge, 4 / 6 pigs vaccinated with Pigs can clear this PRRSV strain at 14dpc.

[0074] In addition to the PRRSV universal RT-qPCR, sera from 7dpc and 14dpc were also tested. Specific RT-qPCR analysis: The purpose of this analysis is to provide The analysis showed that i) at 14 dpc, no PRRSV was detected. (dns); and ii) at 7 dpc, it was either cleared from the serum or present only at very low levels (Ct≥31). Interestingly, although the majority of animals (4 / 6) in the MOCK and VR2332 challenge groups still had detectable levels of vaccine strains, but all or 5 / 6 animals in the NADC30, NC174, and NADC20 groups cleared These data suggest that pigs challenged with a PRRSV strain that induces high viremia clear the strain ( Figure 8 and dns).

[0075] Figure 8 Shows serum Quantification of vaccine strains. Specific RT-qPCR quantification Prevalence of vaccine strains. The table shows the Ct values ​​for animals challenged with MOCK, NADC20, NC174, VR2332 and NADC20 at 7 days post-challenge.

[0076] In addition to viremia, viral load in nasal swabs was assessed to evaluate viral shedding. Importantly, no nasal swabs were detected at the time points analyzed (0, 7, and 14 dpc (28, 35, and 42 dpv; DNs, respectively). Vaccine strains. In pigs challenged with VR2332, viral loads in nasal swabs were low (<10^4 genome copies / ml in 4 / 12 pigs) or completely absent (8 / 12 pigs; Figure 2C). In contrast, challenge with other PRRSV strains resulted in significantly higher viral loads in nasal swabs of all vaccinated animals (including the mean genome copy number range; Figure 2C). As viremia, viral loads in nasal swabs peaked at 7dpc. For NC174, NADC20, and NADC30, Vaccination resulted in a numerical reduction in viral load in nasal swabs at 7 dpc. Vaccination significantly reduced and completely eliminated viral loads in nasal swabs of NADC20- and NADC30-challenged pigs.

[0077] Fig. 3A-C depicts the efficacy of heterologous vaccines on lung viral load, pathology and inguinal lymph node size. (Fig. 3A) Pulmonary viral load was assessed in bronchoalveolar lavage fluid (BAL) by PRRSV-specific RT-qPCR (genome copy number / mL [log10]). It is worth noting that, although PRRSV was not detected in the BAL of an animal receiving MOCK-inoculation / NADC20-attack, the pig had the second highest lung pathology score, together with the highest PRRSV load in 14dpc nasal swabs. Based on this difference, it was excluded from this analysis. (Fig. 3B and C) At 14 days (dpc) after the attack, the lungs of all seven lobes were evaluated by blind veterinarians. (Fig. 3B) The percentage of lung lesions for each individual pig is depicted. (Fig. 3C) Histopathological scores of all seven lobes are shown, which follow 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): pp. 648-660. Each vaccinated group was compared with its corresponding unvaccinated group challenged with type 2 PRRSV using a two-tailed unpaired t-test. The black bar represents the median value; in addition, individual data points for MOCK-vaccinated animals (open diamonds) and MLV-vaccinated animals (solid squares) are shown. Each vaccinated group was compared with its respective unvaccinated group challenged with type 2 PRRSV. Data were compared using a two-tailed unpaired t-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

[0078] At necropsy, viral load in BAL was additionally quantified via universal PRRSV-specific qPCR (Figure 3A). All pigs challenged with MOCK were negative for PRRSV-2 in BAL. MOCK-vaccinated pigs showed a genome copy number of ~10^8 per ml of BAL, the highest median viral load in all challenge groups. Vaccination reduced these viral loads numerically to 10^6.5 for NC174, and significantly to 10^3.7 for NADC30, 10^5.3 for NADC20, and even eliminated VR2332 in the BAL of 4 / 6 pigs. In summary, there was no presence of VR2332 in nasal swabs 4-6 weeks after vaccination. And at these time points, it was absent or present at low levels in serum.

[0079] Vaccination significantly reduced viremia after VR2332 challenge; it limited viremia and viral shedding in the NADC20 and NADC30 challenged groups; and The BAL viral loads of NADC20, NADC30, and VR2332 were significantly reduced (Figure 3A).

[0080] Vaccine efficacy in tissues - viral load, lung pathology, and lymph node size

[0081] At necropsy, viral load in BAL was assessed via PRRSV-specific qPCR (Fig. 3A). All animals challenged with MOCK were negative. The viral load of animals inoculated with MOCK from NADC30, NC174, and NADC20 groups was ~10^8 genome copies / mL. In contrast, animals challenged with MOCK-inoculation and VR2332-had a median viral load of ~10^5, about 1000 times lower. Vaccination numerically reduced the median viral load for all challenge strains. This reduction became significant for NADC30, VR2332, and NADC20. Notably, 4 / 6 recipients BAL of -vaccinated and VR2332-challenged animals were negative for PRRSV-2.

[0082] Gross lung pathology was essentially absent in both MOCK-challenged groups and minimal in the VR2332-challenged group; however, it was clearly present in pigs challenged with NC174, NADC20, and NADC30 (Figure 3B). Vaccination reduced the gross lung pathology after challenge with two of the three pathology-inducing strains (NADC30 and NADC20). As can be seen in gross pathology, the median histopathological changes in MOCK-vaccinated pigs were also the highest in the NADC30, NC174, and NADC20 groups.

[0083] However, histopathological changes were also present in the MOCK and VR2332 challenge groups (Figure 3C). vaccinated group, histopathological analysis revealed only one difference: compared with MOCK-vaccinated animals, Histopathology scores were significantly lower in vaccinated animals.

[0084] In addition to lung pathology, lymph node size was also assessed, as increases in lymph node size are often associated with inflammation. Both the MOCK and VR2332 groups maintained approximately healthy weights at necropsy (median weight of ~2 grams). PRRSV-2 challenge resulted in enlarged lymph nodes in the NC174, NADC20, and NADC30 groups. was able to significantly reduce lymph node size by one gram in the NADC30 group (median weight decreased from 4.7 grams to 3.6 grams).

[0085] decisively, The BAL viral loads of NADC30, VR2332, and NADC20 were reduced. Although VR2332 induced only minimal lung pathology, Not only was lung macroscopic and / or histopathology reduced, but median inguinal lymph node weights were also reduced for NADC30 and NADC20 (numerically).

[0086] Example 2-6: Heterologous vaccine immunogenicity and correlation of immune protection (CoP)

[0087] Overview

[0088] In addition to vaccine efficacy, heterologous vaccine immunogenicity was investigated with respect to both humoral and T cell immune responses to determine the correlate of immune protection (CoP). Humoral immune responses were investigated by quantifying local anti-PRRSV IgA levels in nasal swabs and BAL and serum anti-PRRSV IgG and nAb levels ( Figure 4A-D To investigate T cell responses, PBMCs were isolated, restimulated in vitro with the corresponding PRRSV challenge strains, and analyzed by multicolor flow cytometry for three main readout parameters: i) proliferation of CD4, CD8 and TCR-γδ T cells ( Figure 5A-D ), and ii) IFN-γ production ( Figure 6A-D ), and iii) differentiation of CD4 T cells into memory / effector cells ( Figure 7A-B ).

[0089] Example 2: Humoral Immune Response

[0090] Local humoral immune responses were investigated by quantifying anti-PRRSV IgA levels in BAL at necropsy ( FIG. 4A ) and in nasal swabs at 0 and 14 dpc ( FIG. 4B ).

[0091] Figure 4A-D Heterologous vaccine immunogenicity as a humoral immune response is depicted. Immunoglobulin A (FIG. 4A) bronchoalveolar lavage fluid (BAL), (FIG. 4B) nasal swabs and serum IgG levels (FIG. 4C) were assessed at 0 and 14 days post-challenge (dpc) via PRRSV X3 ELISA. IgA and IgG ELISA S / P ratios were compared within their challenge groups: MOCK (grey), 1-4-4 (NADC30, dark blue), NC174 (red), VR2332 (green) and 1-4-2 (NADC20, light blue). The black bars represent median values; in addition, individual data points for MOCK-vaccinated animals (open diamonds) and MLV-vaccinated animals (solid squares) are also shown. Data were statistically analyzed using a 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. (Fig. 4D) Neutralizing antibody (NA) titers against the corresponding challenge strains at 0 and 14 dpc were determined via the FFN test. Since no animals showed FFN titers at 0 dpc, only data for 14 dpc are shown. Titers ≥ 1:4 were considered positive. Titers for each individual pig challenged with PRRSV are shown. Positive NA titers are highlighted in blue (NADC30), red (NC174, not detected), green (VR2332) and light blue (NADC20).

[0092] Of note, BAL samples were diluted 1:200 prior to analysis compared to undiluted nasal swab samples. At two weeks post-challenge, most BAL samples from MOCK-vaccinated pigs were negative for PRRSV-specific IgA. Although BAL from vaccinated and VR2332-challenged pigs were also negative, Vaccination induced strong IgA responses to NADC30, NC174, and NADC20: 4 / 6 NADC30 and NC174 pigs All the pigs in the vaccinated group and the NADC20 challenge group The S / P ratios of the vaccinated pigs were all >0.4. Thus, Prevacent increased the lung PRRSV-specific IgA levels of NC174 (numerically) and significantly increased the lung PRRSV-specific IgA levels of NADC30 and NADC20.

[0093] IgA levels in nasal swabs were significantly lower (Figure 3B): With the exception of some outliers, IgA levels in pigs challenged with MOCK, NADC30, and VR2332 remained below an S / P ratio of 0.4. However, challenge with NC174 and NADC20 induced observable and mostly significant local IgA responses. There were no differences between vaccination groups.

[0094] The whole body humoral immune response is evaluated in two ways as follows: anti-PRRSV IgG in serum and challenge strain-specific nAb levels (Fig. 4C, 4D). At four weeks after vaccination, i.e., 0dpc, each vaccinated animal (but no control animal) had a high positive IgG level, i.e., S / P was 1.3-2.1. By 14dpc, each of the four PRRSV strains infected also induced anti-PRRSV serum IgG in animals vaccinated with MOCK; However, all vaccinated animals had serum IgG levels significantly higher than their respective MOCK vaccination groups (Fig. 4C).

[0095] At 0 and 14 dpc, challenge strain-specific serum nAb titers were determined by FFN testing (Figure 4D). No nAb(dns) was detected at 0 dpc. At 14 dpc, neither the MOCK(dns) nor the NC174 challenge groups produced nAbs against the challenge strain. However, by 14 dpc, NADC20, NADC30, and VR2332 challenges induced mainly low titers of serum nAbs: among the six pigs in each group, only 1-2 pigs produced serum nAb titers in the MOCK-vaccinated animals; in contrast, In the vaccinated groups, 3 / 6, 5 / 6, and 6 / 6 pigs produced nAbs against the VR2332, NADC20, and NADC30 challenge strains, respectively ( FIG. 4D ).

[0096] These data prove that Vaccination induced strong local IgA responses in BAL to NADC30, NC174 (numerically) and NADC20; it also induced systemic humoral immune responses with high serum IgG titers in all groups and a higher frequency of post-challenge nAb-positive animals to VR2332, NADC30 and NADC20.

[0097] Example 3: Proliferation of T cell subsets

[0098] In addition to humoral responses, cellular immune responses are critical for protection against PRRSV and can provide a model for assessing the correlate of protection (CoP). To provide a more detailed understanding of the immunogenicity of MLV-induced heterologous vaccines, the PRRSV strain-specific proliferation of CD4, CD8, and TCR-γδ T cells was investigated. Figure 5A-D ) and IFN-γ( Figure 6A-D )reaction.

[0099] Figure 5A-D Heterologous vaccine immunogenicity as CD4, CD8 and TCR-γδT cell proliferation is shown. (Fig. 5A) shows a gating hierarchy, which is used to evaluate the heterologous proliferation response of T cell subsets to corresponding type 2 PRRSV attack strains. Including distinguishing live / dead dyes to exclude dead cells. Live lymphocytes are identified via FSC / SSC lymphocyte gating using live cells. Double cells are excluded from live lymphocytes using FSC width (FSC-W) / FSC area (FSC-A) gating to single cells. T cells (FSC-A / CD3) are gated using these single live lymphocytes, and TCR-αβ and TCR-γδT cells are further distinguished. TCR-αβ T cells are further divided into CD4 and CD8 T cells via their CD4 / CD8α expression profiles. The proliferation of CD4, CD8 and TCR-γδT cells is identified via purple proliferation dye. Two examples show representative staining patterns of control animals (upper right figure) and highly responsive animals (lower right figure). (Fig. 5B-D) Proliferative responses of CD4 (Fig. 5B), CD8 (Fig. 5C), and TCR-γδ T cells (Fig. 5D) are shown according to their challenge groups (MOCK (grey), NC174 (red), NADC20 (light blue), NADC30 (dark blue), and VR2332 (green)). Black bars represent median values; in addition, individual data points are shown for MOCK-vaccinated animals (open diamonds) versus 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.

[0100] After restimulation with the corresponding PRRSV-2 challenge strain (MOI 0.1) in vitro, the proliferation response of CD4, CD8 and TCR-γδ T cells was analyzed by multicolor flow cytometry, and the gate hierarchy is shown in Figure 5. At -28dpc, CD4 T cells showed very limited background proliferation (Figure 5B). Four weeks later (0dpc) and compared with MOCK-vaccinated pigs, the proliferation of CD4 T cells in the MOCK-vaccinated pigs was significantly higher than that in the MOCK-vaccinated pigs. CD4 T cells of pigs in the 14th day of the experiment began to generate allogeneic proliferative responses. This response was moderate for NADC20, clearly visible for NADC30 and NC174, and significant for MOCK and VR2332 challenge groups. The proliferative CD4 T cell response of vaccinated animals was also significantly increased. Compared with CD4 T cells, CD8 T cells mostly showed a lower proliferative response (Figure 5C). However, they showed similar patterns: i) overall, the proliferative response increased over time; ii) at 0dpc, mainly MOCK and VR2332 groups experienced increased proliferation; and iii) after challenge, The proliferation was enhanced in the NC174 group. The proliferative TCR-γδ response showed higher intra-group variability. In the control group, the only clear and significant effect of Prevacent was the increase of TCR-γδ against the NADC20 strain at 14 dpc ( FIG5D ).

[0101] Overall, these data suggest that while the effect on TCR-γδ proliferation is limited to NADC20, Vaccination increased the proliferation of CD4 and CD8 T cells against three heterologous PRRSV-2 strains: VR2332 (pre-challenge), NC174 (post-challenge), and NADC20 (CD4 T cells only, post-challenge).

[0102] Example 4: IFN-γ production by T cell subsets

[0103] In addition to systemic proliferative responses, heterologous vaccine immunogenicity was evaluated by studying IFN-γ, which is considered to be the most relevant antiviral T cell cytokine ( Figure 6A-D ).

[0104] Figure 6A-DHeterologous vaccine immunogenicity is shown as IFN-γ production of CD4, CD8, and TCR-γδ T cells. (Fig. 6A) The gating hierarchy was used to assess heterologous IFN-γ responses of T cell subsets to the corresponding type 2 PRRSV challenge strains. The gating hierarchy largely follows the proliferation analysis shown in Fig. 5. However, instead of gating on proliferating cells, IFN-γ analysis was performed in the FSC-A / IFN-γ plot. IFN-γ gating was set using the appropriate FMO controls (top right). (Fig. 6B-D) IFN-γ responses of CD4 (Fig. 6B), CD8 (Fig. 6C), and TCR-γδ T cells (Fig. 6D) are shown according to their challenge group (MOCK (grey), 1-4-4 (NADC30, dark blue), NC174 (red), VR2332 (green), and 1-4-2 (NADC20, light blue)). The black bar represents the median value; in addition, individual data points for MOCK-vaccinated animals (open diamonds) and MLV-vaccinated animals (filled squares) are shown. Data were statistically analyzed using a 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.

[0105] The gating hierarchy for selective analysis of IFN-γ production by CD4, CD8, and TCR-γδ T cells was similar to that for proliferation analysis and is shown in Figure 6A. Before challenge (-28 and 0 dpc), IFN-γ production was low in all T cell subsets and there were no significant differences between the corresponding vaccination groups (Figure 6B-D). In contrast, at 14 dpc, significant IFN-γ production was observed in most PRRSV-2 challenged groups. For CD4 T cells, IFN-γ production was significantly increased by targeting NC174, NADC20, and NADC30. In CD8 T cells, The IFN-γ response to NC174 and NADC20 was significantly enhanced (Figure 6C); and for TCR-γδ T cells, Vaccination resulted in increased IFN-γ production against NADC20 ( FIG6D ). Vaccination enhanced IFN-γ responses after heterologous challenge against NADC30 (in CD4 T cells), NC174 (in CD4 and CD8 T cells), and NADC20 (in all T cell subsets).

[0106] Example 5: Differentiation of IFN-γ-producing CD4 T cells

[0107] Although heterologous IFN-γ production showed the strongest response after challenge, T cell differentiation analysis of IFN-γ producing T cells revealed significant differences before challenge ( Figure 7A-B ).

[0108] Figure 7A-B The immunogenicity of heterologous vaccines as differentiation of IFN-γ-producing CD4 T cells is depicted. (FIG. 7A) shows the gating hierarchy used to assess the differentiation of IFN-γ-producing CD4 T cells. After gating on IFN-γ+CD4 T cells as described in FIG. 6, their differentiation was analyzed via their CD4 / CD8α expression profile to distinguish between naive (CCR7+CD8α-), central memory (T CM CCR7+CD8α+) ​​and effector memory type (T EM , CCR7-CD8α+)CD4 T cells (upper right). Since the vast majority of CD4 T cells producing CD8α+IFN-γ belong to T CM subset (data not shown), so T CM and T EM The two are combined into the "memory / effector" subset. (Figure 7B) The frequency of these memory / effector cells within IFN-γ-producing CD4 T cells is shown according to their challenge group (MOCK (gray), 1-4-4 (NADC30, dark blue), NC174 (red), VR2332 (green), and 1-4-2 (NADC20, light blue)). The black bar represents the median value; in addition, individual data points are shown for MOCK-vaccinated animals (open diamonds) and MLV-vaccinated animals (filled squares). Data were statistically analyzed using a 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.

[0109] Again, multicolor flow cytometry with a complex gating hierarchy was used to assess the expression of IFN-γ-producing CD4 T cells to CCR7 + CD8α - Initial, CCR7 + CD8α + Central memory type (T CM ) and CCR7 - , CD8α + Effect memory type (T EM ) Differentiation of CD4 T cells (Figure 7A). + CD4 T cells belong to CCR7+T CM subset (data not shown), and therefore, will experience the antigen's TCM and T EM The subsets were combined into a "memory / effector" subset (Figure 7B). Before vaccination, i.e., -28dpc, most IFN-γ was produced by naive CD4 T cells: a median of 10-40% of memory / effector CD4 T cells. At 0dpc, IFN-γ in the MOCK-vaccinated group was still mainly produced by naive CD4 T cells: a median of 0-15% of memory / effector CD4 T cells.

[0110] In contrast, In the vaccinated groups, IFN-γ was produced primarily by memory / effector CD4 T cells: median values ​​ranged from ∼50% to >90%. Differences in the differentiation of IFN-γ-producing CD4 T cells prior to challenge were significant for all groups and 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 median frequency of memory / effector cells in the vaccinated group was still higher in all PRRSV-2 challenged groups (Figure 7B). In summary, this CD4 differentiation analysis revealed important immune mechanisms in the immunogenicity of heterologous vaccines: although Rather than increasing CD4 IFN-γ responses before challenge, it already promoted pre-challenge differentiation of these CD4 T cells against each of the PRRSV-2 strains analyzed (NC174, NADC20, NADC30, and VR2332).

[0111] Example 6: Correlation of Immune Protection (CoP)

[0112] The above data confirms Different degrees of heterologous vaccine immunogenicity and efficacy are shown. For PRRSV, an important parameter that has hardly been analyzed is the correlation of immune 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 facilitate vaccine development and predict vaccine efficacy against emerging PRRSV strains.

[0113] To provide insight into the potential CoP of heterologous PRRSV strains, the inventors performed correlation analyses between the analyzed pre-challenge immune parameters (0 dpc) and three post-challenge (14 dpc) parameters associated with protection (lung pathology, viral shedding and viremia) (Table 2).

[0114] Table 2(A),(B),(C): Correlation of immune protection.

[0115] A)

[0116]

[0117]

[0118] B)

[0119]

[0120] C)

[0121]

[0122] Table 2 lists the R values ​​(-1 to +1) for the correlations between various immune parameters at 0 dpc (e.g., proliferation, IFN-γ production, and differentiation into CD4 memory cells) and three protective measures at 14 dpc (gross pathology (A), shedding (B), and viremia (C)). Italic numbers represent non-significant correlations, while bold numbers emphasize significant correlations (p<0.05). Negative correlations (R=0>-1) indicate that increases in pre-challenge immune parameters are associated with decreases in pathology or viral load. Neither systemic CD8 nor TCR-γδ responses were significantly correlated with protection: only CD8 proliferation was negatively correlated with NADC30 shedding; the IFN-γ response of TCR-γδ was even significantly positively correlated with NADC20 shedding.

[0123] In contrast, CD4 T cell responses were negatively correlated with all protection parameters analyzed, except for NC174 gross pathology. The strongest and most significant CD4 correlations were observed for NADC20 and / or NADC30 strains: CD4 IFN-γ responses and differentiation into memory / effector cells were significantly negatively correlated with NADC20-induced lung gross pathology; CD4 T cell proliferation was significantly negatively correlated with NADC20 shedding and viremia; and all CD4 parameters (proliferation, IFN-γ, and differentiation) were correlated with both NADC30 shedding and viremia. Regarding humoral immune responses, while IgA levels in nasal swabs (local IgA) were both positively and negatively correlated with protection, systemic IgG levels were well correlated with most protection parameters: systemic IgG levels were significantly negatively correlated with NADC20-induced gross pathology, NADC30-induced shedding, and NADC30, VR2332, and NADC20-induced viremia. These data suggest that both systemic IgG levels and CD4 T cell responses are candidates to serve as important CoPs for PRRSV, although T cell responses were only analyzed in a strain-dependent manner.

[0124] in conclusion

[0125] This disclosure will Vaccination followed by in vivo challenge with four heterologous PRRSV strains was combined with extensive ex vivo and in vivo analysis of lung pathology, viral loads in various tissues, and humoral and adaptive immune responses. In-depth analysis of heterologous humoral and T cell immune responses well explained Immunogenicity: Early stage (0dpc), Induces early T cell activation and differentiation, manifested by increased proliferation of CD8 but mainly CD4 T cells. Induce heterologous CD4 T cells to differentiate into memory / effector cells. Downstream, this early T cell activation and differentiation not only leads to B cell help drive serum IgG levels (at 0 and 14dpc) and the frequency of nAb-positive animals (14dpc), but also prompts vaccinated pigs to increase the production of IFN-γ after challenge (14dpc). The induction of this combined T cell and humoral immune response induces at least partial protection against at least three of the four PRRSV strains: NADC30 (lineage 1), VR2332 (lineage 5) and NADC20 (lineage 8). The included CoP analysis revealed that serum IgG levels and CD4 T cell responses (proliferation, differentiation and IFN-γ production) are the best systemic CoPs; however, only CD4 T cell responses can be reliably used as CoPs against specific PRRSV strains.

[0126] It can be understood from the description herein that the present disclosure contemplates a variety of aspects and embodiments, examples of which include but are not limited to the aspects and embodiments listed below:

[0127] A method for eliciting heterologous immunogenicity against heterologous porcine reproductive and respiratory syndrome virus (PRRSV) strains, thereby allowing the evaluation of innate and adaptive immunity after: inoculating pigs with an effective amount of a modified live PRRSV vaccine, and then challenging the pigs by intranasal inoculation of a certain amount of live known PRRSV strains at least 28 days after vaccine administration. Various measurements of the pigs, including body temperature and body weight, are obtained immediately before the administration of the modified live PRRSV vaccine, immediately before the challenge of the known PRRSV strain by intranasal inoculation, and at least 7 days and 14 days after the challenge. In addition, blood samples are obtained from the pigs immediately before the administration of the modified live PRRSV vaccine, immediately before the challenge of the known PRRSV strain by intranasal inoculation, and at least 7 days and 14 days after the challenge. The amount of CD4 T cell responses, the presence of strain-specific neutralizing antibodies, the presence of CD4, CD8 and TCR-γδ cells, IFN-γ levels, and PRRSV-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) levels are measured in each blood sample.

[0128] In this method, an effective amount of a modified live PRRSV vaccine is administered to pigs, and then at least 28 days after vaccine administration, the pigs are challenged by intranasal inoculation with a live known PRRSV strain that induces: i) increased T cell activation, as evidenced by CD4 T and CD8 cell differentiation; ii) increased levels of PRRSV-specific immunoglobulin G (IgG); iii) production of serum neutralizing antibodies; and iv) increased serum IFN-γ levels.

[0129] The method also includes the isolation, storage and warehousing of peripheral blood mononuclear cells (PBMCs) obtained from a blood sample of a pig administered an effective amount of a modified live PRRSV vaccine.

[0130] Another method disclosed is a method for determining the efficacy of a vaccine against porcine reproductive and respiratory syndrome virus (PRRSV), comprising: i) administering an effective amount of a modified live PRRSV vaccine to a pig; ii) challenging the pig by intranasal inoculation of a live known PRRSV strain at least 28 days after vaccine administration; iii) measuring the efficacy of the modified live PRRSV vaccine immediately before administration, immediately before challenge by intranasal inoculation of a known PRRSV strain, and at least 7 and 14 days after challenge. The invention relates to the present invention to: measuring the body temperature and body weight of the pigs; iv) obtaining blood samples, nasal swabs from the pigs immediately before administration of the modified live PRRSV vaccine, immediately before challenge by intranasal inoculation of a known PRRSV strain, and at least 7 days and 14 days after challenge; v) evaluating the lung and lymph node pathology of the pigs after necropsy; vi) obtaining bronchoalveolar lavage samples after necropsy; vii) measuring the amount of virus, PRRSV-specific immunoglobulin A and immunoglobulin G present in each blood sample, nasal swab and bronchoalveolar lavage sample.

[0131] In this method, an effective amount of a modified live PRRSV vaccine is administered to pigs, and at least 28 days after vaccine administration, the pigs are challenged by intranasal inoculation with a live known PRRSV strain, inducing: i) little or no lung and lymph node pathology in the pigs after necropsy; ii) a decrease in the amount of PRRSV virus in samples obtained from blood, nasal swabs, and bronchoalveolar lavage after necropsy; and iii) an increase in the amount of PRRSV-specific immunoglobulin A and immunoglobulin G.

[0132] Another method disclosed is a method for predicting the efficacy of a vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) in pigs suspected of having PRRSV infection, comprising: i) isolating PRRSV from a blood or nasal swab sample obtained from a pig suspected of having PRRSV infection; ii) challenging with PRRSV from the pig suspected of having PRRSV infection, isolating, storing and warehousing a sample of peripheral blood mononuclear cells (PBMCs) previously obtained from a blood sample from a pig administered with an effective amount of a modified live PRRSV vaccine, wherein at least 28 days after vaccine administration, the pig is further challenged by intranasal inoculation with a certain amount of a live known PRRSV strain, and isolating, storing and warehousing its PBMCs, wherein CD4 T and CD8 T cell responses differentiated as CD4 T and CD8 T cells are previously obtained; iii) measuring the CD4 T and CD8 T cell responses in the PBMCs after challenging with PRRSV from the pig suspected of having PRRSV infection; and iv) separating the CD4 T and CD8 T cell responses in step iii) from the PBMCs; The T cell responses were compared to the CD4 T and CD8 T cell responses previously obtained from the isolated, stored and banked PBMC samples in step ii).

[0133] In any of the disclosed methods, the porcine reproductive and respiratory syndrome virus (PRRSV) infection can be caused by infection with any strain of PRRSV.

[0134] Although embodiments of the present disclosure have been described herein, it will be appreciated by those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art will now appreciate that many variations, changes, and substitutions do not depart from the present invention. It will be appreciated that in the practice of the present invention, various alternatives to the embodiments of the present invention described herein may be employed. It is intended that the scope of the present invention be defined by the following claims, and that methods and structures within the scope of these claims and their equivalents are encompassed thereby.

Claims

1. A method for eliciting heterologous immunogenicity against a heterologous porcine reproductive and respiratory syndrome virus (PRRSV) strain to allow evaluation of innate and adaptive immunity, comprising: i) administering an effective amount of a modified live PRRSV vaccine or a control injection to a pig; ii) challenging pigs by intranasal inoculation with a live known PRSSV strain at least 28 days after vaccine administration; iii) measuring the body temperature and body weight of the pigs immediately prior to administration of the modified live PRRSV vaccine, immediately prior to challenge by intranasal inoculation with a known PRRSV strain, and at least 7 and 14 days after challenge; iv) obtaining blood samples from the pigs immediately prior to administration of the modified live PRRSV vaccine, immediately prior to challenge by intranasal inoculation with a known PRRSV strain, and at least 7 and 14 days after challenge; v) measuring the amount of 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 levels of PRRSV-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) in each blood sample; and vi) All measurements were compared with those obtained from control injected pigs.

2. The method of claim 1, wherein the known porcine reproductive and respiratory syndrome virus (PRRSV) strain is selected from the group consisting of PRRSV type 1 (PRRSV-1) and PRRSV type 2 (PRRSV-2) strains.

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

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

5. The method of claim 1, wherein said administering to the pigs an effective amount of a modified live PRRSV vaccine, followed by challenging the pigs by intranasal inoculation with an amount of a live known PRRSV strain at least 28 days after administration of the vaccine, induces: i) increased T cell activation, as evidenced by CD4 T and CD8 cell differentiation; ii) an increase in the level of PRRSV-specific immunoglobulin G (IgG); iii) production of serum neutralizing antibodies; iv) Increased serum IFN-γ levels.

6. The method of claim 1, further comprising the isolation, storage and warehousing of peripheral blood mononuclear cells (PBMCs) obtained from a blood sample of a pig administered an effective amount of a modified live PRRSV vaccine.

7. A method for determining the efficacy of a vaccine against porcine reproductive and respiratory syndrome virus (PRRSV), comprising: i) administering an effective amount of a modified live PRRSV vaccine to a pig; ii) challenging the pigs by intranasal inoculation with a live known PRSSV strain at least 28 days after vaccine administration; iii) measuring the body temperature and body weight of the pigs immediately prior to administration of the modified live PRRSV vaccine, immediately prior to challenge by intranasal inoculation with a known PRRSV strain, and at least 7 and 14 days after challenge; iv) obtaining blood samples, nasal swabs from the pigs immediately prior to administration of the modified live PRRSV vaccine, immediately prior to challenge by intranasal inoculation with a known PRRSV strain, and at least 7 and 14 days after challenge; v) evaluating the pigs for lung and lymph node pathology following necropsy; vi) obtaining bronchoalveolar lavage samples after autopsy; vii) measuring the amount of virus, PRRSV-specific immunoglobulin A and immunoglobulin G present in each blood sample, nasal swab, and bronchoalveolar lavage sample; and, viii) All measurements were compared to those obtained from control injected pigs.

8. The method of claim 6, wherein the known porcine reproductive and respiratory syndrome virus (PRRSV) strain is selected from the group consisting of PRRSV type 1 (PRRSV-1) and PRRSV type 2 (PRRSV-2) strains.

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

10. The method of claim 6, wherein said administering to pigs an effective amount of a modified live PRRSV vaccine followed by challenging said pigs by intranasal inoculation with an amount of a live known PRRSV strain at least 28 days after vaccine administration induces: i) upon necropsy, the pigs had little or no lung and lymph node pathology; ii) a reduction in PRRSV viral load in samples obtained from blood, nasal swabs, and bronchoalveolar lavage after necropsy; and, iii) The amount of PRRSV-specific immunoglobulin A and immunoglobulin G increased.

11. A method for predicting the efficacy of a vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) in pigs suspected of having PRRSV infection, comprising: i) isolating PRRSV from a blood or nasal swab sample obtained from the pig suspected of having PRRSV infection; ii) challenging with PRRSV from the pig suspected of having PRRSV infection, isolating, storing and warehousing a sample of peripheral blood mononuclear cells (PBMC) previously obtained from a blood sample from a pig administered with an effective amount of a modified live PRRSV vaccine, wherein at least 28 days after vaccine administration, the pig is further challenged by intranasal inoculation with a certain amount of a live known PRRSV strain, and isolating, storing and warehousing its PBMC, wherein CD4 T and CD8 T cell responses differentiated as CD4 T and CD8 cells are previously obtained; iii) measuring CD4 T and CD8 T cell responses in PBMCs after challenge with PRRSV from said pigs suspected of having PRRSV infection; as well as iv) comparing the CD4 T and CD8 T cell responses of step iii) with the CD4 T and CD8 T cell responses previously obtained from the isolated, stored and banked PBMC sample of step ii).

12. The method of claim 11, wherein the 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) and PRRSV type 2 (PRRSV-2) strains.

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

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