Evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions

<p>Abstract</p> <p>Background</p> <p>Porcine reproductive and respiratory syndrome virus (PRRSV) causes chronic, economically devastating disease in pigs of all ages. Frequent mutations in the viral genome result in viruses with immune escape mutants. Irrespective of regular vaccination, control of PRRSV remains a challenge to swine farmers. In PRRSV-infected pigs, innate cytokine IFN-α is inhibited and the adaptive arm of the immunity is delayed. To elucidate both cellular and innate cytokine responses at very early stages of PRRSV infection, seven weeks old pigs maintained on a commercial pig farm were infected and analyzed.</p> <p>Results</p> <p>One pig in a pen containing 25 pigs was PRRSV infected and responses from this pig and one penmate were assessed two days later. All the infected and a few of the contact neighbor pigs were viremic. At day 2 post-infection, approximately 50% of viremic pigs had greater than 50% reduction in NK cell-mediated cytotoxicity, and nearly a 1-fold increase in IFN-α production was detected in blood of a few pigs. Enhanced secretion of IL-4 (in ~90%), IL-12 (in ~40%), and IL-10 (in ~20%) (but not IFN-γ) in PRRSV infected pigs was observed. In addition, reduced frequency of myeloid cells, CD4^-CD8⁺T cells, and CD4⁺CD8⁺T cells and upregulated frequency of lymphocytes bearing natural T regulatory cell phenotype were detected in viremic pigs. Interestingly, all viremic contact pigs also had comparable immune cell modulations.</p> <p>Conclusion</p> <p>Replicating PRRSV in both infected and contact pigs was found to be responsible for rapid modulation in NK cell-meditated cytotoxicity and alteration in the production of important immune cytokines. PRRSV-induced immunological changes observed simultaneously at both cellular and cytokine levels early post-infection appear to be responsible for the delay in generation of adaptive immunity. As the study was performed in pigs maintained under commercial environmental conditions, this study has practical implications in design of protective vaccines.</p

Mutations in a Highly Conserved Motif of nsp1? Protein Attenuate the Innate Immune Suppression Function of Porcine Reproductive and Respiratory Syndrome Virus

Citation: Li Y, Shyu D-L, Shang P, Bai J, Ouyang K, Dhakal S, Hiremath J, Binjawadagi B, Renukaradhya GJ, Fang Y. 2016. Mutations in a highly conserved motif of nsp1? protein attenuate the innate immune suppression function of porcine reproductive and respiratory syndrome virus. J Virol 90:3584–3599. doi:10.1128/JVI.03069-15.Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1? (nsp1?) is a multifunctional viral protein, which is involved in suppressing the host innate immune response and activating a unique ?2/?1 programmed ribosomal frameshifting (PRF) signal for the expression of frameshifting products. In this study, site-directed mutagenesis analysis showed that the R128A or R129A mutation introduced into a highly conserved motif (123GKYLQRRLQ131) reduced the ability of nsp1? to suppress interferon beta (IFN-?) activation and also impaired nsp1?'s function as a PRF transactivator. Three recombinant viruses, vR128A, vR129A, and vRR129AA, carrying single or double mutations in the GKYLQRRLQ motif were characterized. In comparison to the wild-type (WT) virus, vR128A and vR129A showed slightly reduced growth abilities, while the vRR129AA mutant had a significantly reduced growth ability in infected cells. Consistent with the attenuated growth phenotype in vitro, pigs infected with nsp1? mutants had lower levels of viremia than did WT virus-infected pigs. Compared to the WT virus in infected cells, all three mutated viruses stimulated high levels of IFN-? expression and exhibited a reduced ability to suppress the mRNA expression of selected interferon-stimulated genes (ISGs). In pigs infected with nsp1? mutants, IFN-? production was increased in the lungs at early time points postinfection, which was correlated with increased innate NK cell function. Furthermore, the augmented innate response was consistent with the increased production of IFN-? in pigs infected with mutated viruses. These data demonstrate that residues R128 and R129 are critical for nsp1? function and that modifying these key residues in the GKYLQRRLQ motif attenuates virus growth ability and improves the innate and adaptive immune responses in infected animals

Biodegradable nanoparticle-entrapped vaccine induces cross-protective immune response against a virulent heterologous respiratory viral infection in pigs.

Biodegradable nanoparticle-based vaccine development research is unexplored in large animals and humans. In this study, we illustrated the efficacy of nanoparticle-entrapped UV-killed virus vaccine against an economically important respiratory viral disease of pigs called porcine reproductive and respiratory syndrome virus (PRRSV). We entrapped PLGA [poly (lactide-co-glycolides)] nanoparticles with killed PRRSV antigens (Nano-KAg) and detected its phagocytosis by pig alveolar macrophages. Single doses of Nano-KAg vaccine administered intranasally to pigs upregulated innate and PRRSV specific adaptive responses. In a virulent heterologous PRRSV challenge study, Nano-KAg vaccine significantly reduced the lung pathology and viremia, and the viral load in the lungs. Immunologically, enhanced innate and adaptive immune cell population and associated cytokines with decreased secretion of immunosuppressive mediators were observed at both mucosal sites and blood. In summary, we demonstrated the benefits of intranasal delivery of nanoparticle-based viral vaccine in eliciting cross-protective immune response in pigs, a potential large animal model

Reduced lung pathology and viral load in Nano-KAg vaccinated MN184 challenged pigs.

<p>Pigs were unvaccinated or vaccinated as indicated and challenged with PRRSV MN184 and euthanized at DPC 15. (A) A representative pig lung H&E picture from indicted pig group. (B) A representative lung immunohistochemistry (IHC) picture from indicted pig group showing PRRSV N antigen positive cells (asterisk). (C) Gross lung lesions were graded based on percent lung area affected and severity of inflammatory pathology. (D) PRRSV N antigen positive cells in IHC were counted in 10 random fields from each pig. The PRRSV titer in fluorescence foci units at indicated DPC in (E) serum and (F) in the lungs was determined by immunofluorescence assay. Each bar represents average values from three pigs ± SEM. Asterisk represents the statistical significant difference (p<0.05) between K-Ag and Nano-KAg received pig groups, and φ represents the statistical significant difference (p<0.05) between unvaccinated and Nano-KAg received pig groups. A similar trend in results was obtained in an independent second trial performed using same number of animals.</p

Nano-KAg elicited enhanced innate and suppressed regulatory response in a pre-challenge study.

<p>Pigs were unvaccinated or vaccinated as indicated. MNC were immunostained to analyze the frequency of immune cells: (A) NK cells, (B) Dendritic cells, (C) γδ T cells, (D) Th/memory cells, (E) CD8⁺ T cells in lung MNC; and (H) γδ T cells and (I) Dendritic cells in PBMC. Harvested culture supernatants from restimulated MNC were analyzed for cytokines: (F) IL-6 and (G) IL-10 in lung MNC; (J) IL-10 in PBMC; (K) IFN-α in serum by ELISA. Each bar represents the average cytokine amounts from three pigs ± SEM. Asterisk represents the statistical significant difference (p<0.05) between Nano-KAg and K-Ag received pig groups.</p

Frequency of immune cells in PBMC and TBLN of Nano-KAg vaccinated pigs.

<p>Pigs were unvaccinated or vaccinated with either K-Ag or Nano-KAg once intranasally and challenged with PRRSV strain MN184 and euthanized at DPC 15. Different immune cell subsets present in PBMC and TBLN MNC were enumerated by flow cytometry. ^a CD172⁺ cells were gated to enumerate CD11c and SLAII expression and the percent of DCs rich fraction (CD172⁺CD11c⁺SLAII⁺) is shown.^b CD3⁻ cells were gated to enumerate CD4 and CD8α expression and the percent NK cell rich fraction (CD3⁻CD4⁻ CD8α⁺) is shown. ^c CD25⁺ cells were gated to enumerate CD4 and Foxp3 expression and the percent of CD4⁺CD25⁺Foxp3⁺ cell is shown. Each number is an average percent of immune cells from three pigs +/− SEM. Asterisk represents the statistical significant difference (p<0.05) between Nano-KAg and K-Ag received pig groups.</p

Enhanced PRRSV specific IgA and neutralizing antibody response in Nano-KAg vaccinated virus challenged pigs.

<p>Pigs were unvaccinated or vaccinated, challenged and euthanized as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051794#pone-0051794-g003" target="_blank">Fig 3</a> legend. Anti-PRRSV IgA and IgG antibody response in the (A&B) lungs, (D&E) serum, and (G&H) nasal swabs, was determined by ELISA. PRRSV neutralizing antibody response in (C) lungs and (F) serum was determined by immunofluorescence assay. Each bar represents average optical density value or VN titer from three pigs ± SEM. Asterisk represents the statistical significant difference (p<0.05) between K-Ag and Nano-KAg received pig groups, and φ represents the statistical significant difference (p<0.05) between unvaccinated and Nano-KAg received pig groups. A similar trend in results was obtained in an independent second trial performed using same number of animals.</p