title: Lymphocyte activation as cytokine gene expression and secretion is related to the porcine reproductive and respiratory syndrome virus (PRRSV) isolate after in vitro homologous and heterologous recall of peripheral blood mononuclear cells (PBMC) from pigs vaccinated and exposed to natural infection.
abstract: The present study evaluated the lymphocyte activation in PRRSV-vaccinated pigs subsequently exposed to natural infection by in vitro stimulation of peripheral blood mononuclear cells (PBMC) with homologous vaccine and two heterologous PRRSV isolates. The responsiveness was assessed by determining IFN-γ secreting cells by ELISpot assay, lymphocyte CD8 phenotype by intracellular staining/flow cytometry, cytokine gene expression by real-time quantitative PCR and cytokine secretion by ELISA. Conventional pigs were weaned at 28 days of age and inoculated intramuscularly (IM) or needle-less intradermally (ID) with a modified-live PRRSV vaccine suspended in adjuvant, while control pigs were injected with adjuvant alone (ADJ). Blood samples were collected at vaccination, 35 days post-vaccination and after 35 days post-exposure to natural infection by a heterologous field strain. Thirty-five days post-vaccination, PRRSV vaccine induced a low but significant virus-specific IFN-γ secreting cell response upon stimulation with both the vaccine strain and the two isolates in vaccinated pigs. Conversely, after 35 days post-exposure, only the vaccine strain and the BS/114/S isolate triggered this response. Intracellular staining showed that PRRSV-specific immune cells reacting upon vaccine strain and BS/114/S stimulation were mostly CD8(+) IFN-γ producing cells whereas the stimulation with BS/55 isolate induced an IFN-γ production associated to the CD8(-)IFN-γ(+) phenotype. At 35 days post-vaccination, PBMC from vaccinated pigs showed lower IL-10 expression and release, and higher TNF-α gene expression upon stimulation with both the vaccine and viral isolates. After infection, both cytokines were not differently modulated in different groups. Immune parameters give evidence that IFN-γ secreting cells in the peripheral blood can be elicited upon PRRSV infection although vaccination itself does not stimulate high levels of these reactive cells. Moreover, the cross-reactivity against divergent PRRS viruses can show a different intensity and be differently associated with cytotoxic CD8(+)IFN-γ(+) as well as CD8(-)IFN-γ(+) cells. Overall, the obtained data confirmed that the immune activation against PRRSV is not dependent on the genetic divergence of the virus. Especially after infection, a different immune reactivity was evident upon stimulation with the different isolates in terms of frequency and CD8 phenotype of PRRSV-specific IFN-γ producing cells. The modulation of cytokines in vaccinated pigs appeared to be more dependent on vaccination or infection conditions than on stimulation by different isolates, and the changes of IL-10 more relevant than those of TNF-α at gene and protein levels. Moreover, under the conditions of this study, the PRRSV vaccine administered via the intradermal route by a needle-less device was confirmed to induce an immune response comparable or in some cases higher than the intramuscular route.
authors.affiliation: Department of Veterinary Sciences, University of Parma, Via del Taglio, 10 - 43126 Parma, Italy. luca.ferrari@unipr.it
authors.forename: Luca
authors.initials: L
authors.lastname: Ferrari
authors.forename: Paolo
authors.initials: P
authors.lastname: Martelli
authors.forename: Roberta
authors.initials: R
authors.lastname: Saleri
authors.forename: Elena
authors.initials: E
authors.lastname: De Angelis
authors.forename: Valeria
authors.initials: V
authors.lastname: Cavalli
authors.forename: Marcello
authors.initials: M
authors.lastname: Bresaola
authors.forename: Michele
authors.initials: M
authors.lastname: Benetti
authors.forename: Paolo
authors.initials: P
authors.lastname: Borghetti
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood; D000914:Antibodies, Viral / Q000097:blood; D000918:Antibody Specificity; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D003429:Cross Reactions; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics*; D015870:Gene Expression; D007111:Immunity, Cellular; D007277:Injections, Jet; D007371:Interferon-gamma / Q000096:biosynthesis; D016753:Interleukin-10 / Q000096:biosynthesis / Q000235:genetics; D008213:Lymphocyte Activation; D019318:Porcine Reproductive and Respiratory Syndrome / Q000276:immunology / Q000517:prevention & control; D019316:Porcine respiratory and reproductive syndrome virus / Q000276:immunology*; D034421:Sus scrofa / Q000235:genetics / Q000276:immunology* / Q000821:virology*; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics; D014611:Vaccination / Q000379:methods / Q000662:veterinary; D014765:Viral Vaccines / Q000008:administration & dosage / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D016207:Cytokines; D014409:Tumor Necrosis Factor-alpha; D014765:Viral Vaccines; D016753:Interleukin-10; D007371:Interferon-gamma