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Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important global swine pathogen. PRRSV infects porcine dendritic cells (DCs), but the effects of the interactions with DCs are largely unknown. Current research focuses on the production and regulation of interferons and selected inflammatory cytokines in DCs, which may play key roles in immune modulation. In addition, PRRSV also downregulates swine leukocyte antigen class I (SLA-I), SLA-II, and CD80/86 costimulatory molecules in DCs. In this study, we aim to evaluate the PRRSV immunomodulatory effects on monocyte-derived DCs (MoDCs) through interactions with porcine DC-SIGN (pDC-SIGN) receptor. We demonstrated that blocking the PRRSV and pDC-SIGN interactions in MoDCs with recombinant hICAM-3 did not affect the regulatory effects of PRRSV on SLA-I, SLA-II, or CD80/86 molecules. The hICAM-3 did not affect the morphological changes on MoDCs associated with their activation and maturation after PRRSV infection, and did not impair the virus infectivity in these cells either. The mRNA levels of tumor necrosis factor alpha (TNF-α), IL-12p35, IL-1β, and IL-6 were upregulated after hICAM-3 treatment or PRRSV infection, but in the presence of the blockage of pDC-SIGN in MoDCs with hICAM-3, PRRSV did not modulate the expression of these genes. However, in the presence of an anti-pDC-SIGN monoclonal antibody (mAb), we showed that PRRSV infection significantly reduced the mRNA expression levels of TNF-α and IL-1α, but enhanced the expression of IL-12p35 in MoDCs. Both hICAM-3-Fc and pDC-SIGN mAb treatments did not modulate proinflammatory cytokine protein levels in the culture supernatants of PRRSV-infected MoDCs. The results indicate that blocking the PRRSV-pDC-SIGN interactions by recombinant hICAM-3-Fc did not significantly affect virus infectivity, DC maturation, and proinflammatory cytokine gene expression in infected MoDCs. However, blocking the PRRSV-pDC-SIGN interactions on MoDCs with an anti-pDC-SIGN mAb revealed differential regulatory effects on specific proinflammatory gene expressions in those cells. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) is arguably the most economically-important global swine pathogen. Here we demonstrated that PRRSV down-regulates Swine Leukocyte Antigen class I (SLA-I) expression in porcine alveolar macrophages, PK15-CD163 cells and monocyte-derived dendritic cells. To identify the viral protein(s) involved in SLA-I down-regulation, we tested all 22 PRRSV structural and non-structural proteins and identified that Nsp1α and Nsp2TF, and GP3 significantly down-regulated SLA-I expression with Nsp2TF showing the greatest effect. We further generated a panel of mutant viruses in which the Nsp2TF protein synthesis was abolished, and found that the two mutants with disrupted −2 ribosomal frameshifting elements and additional stop codons in the TF domain were unable to down-regulate SLA-I expression. Additionally we demonstrated that the last 68 amino acids of TF domain in Nsp2TF are critical for this function. Collectively, the results indicate a novel function of Nsp2TF in negative modulation of SLA-I expression. [ABSTRACT FROM AUTHOR]

Immunogenicity of protein subunit vaccines may be dramatically improved by targeting them through antibodies specific to c-type lectin receptors (CLRs) of dendritic cells in mice, cattle, and primates. This novel vaccine development approach has not yet been explored in pigs or other species largely due to the lack of key reagents. In this study, we demonstrate that porcine reproductive and respiratory syndrome virus (PRRSV) antigen was targeted efficiently to dendritic cells through antibodies specific to a porcine CLR molecule DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) in pigs. A recombinant PRRSV antigen (shGP45M) was constructed by fusing secretory-competent subunits of GP4, GP5 and M proteins derived from genetically-shuffled strains of PRRSV. In vaccinated pigs, when the PRRSV shGP45M antigen was delivered through a recombinant mouse-porcine chimeric antibody specific to the porcine DC-SIGN (pDC-SIGN) neck domain, porcine dendritic cells rapidly internalized them in vitro and induced higher numbers of antigen-specific interferon-γ producing CD4T cells compared to the pigs receiving non-targeted PRRSV shGP45M antigen. The pDC-SIGN targeting of recombinant antigen subunits may serve as an alternative or complementary strategy to existing vaccines to improve protective immunity against PRRSV by inducing efficient T cell responses. [ABSTRACT FROM AUTHOR]