Vaccination against porcine reproductive and respiratory syndrome virus (PRRSV) and exposure to natual infection by a heterologous field isolate: immune efficiency and neuroendocrine response in conventional pigs

The functional characterization and quantification of the anti-viral immune response are particularly relevant with regards both to the study of the mechanisms mounted by the immune system to cope with infections and to the employment of new strategies for improving vaccination efficacy and immune protection. In this view, also the determination of systemic neuroendocrine modulation can be informative on the progression of infection since a bi-directional communication exists between the immune system and neuroendocrine axes. Hormones such as cortisol and growth hormone (GH) can act as immunomodulatory mediators, thus influencing the immune response to the pathogen and the severity of the associated disease. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) represents one of the most important and deleterious viruses affecting the swine population. However, the interaction between PRRSV and the swine immune system is highly complex and still poorly understood, because of its ability of evading and dampening several immune responses and since different outcomes are observed upon vaccination and/or infection. Moreover, genetically and antigenically divergent viral strains make the development of efficacious vaccines even more difficult. In this thesis, before considering immune reactivity to PRRSV vaccination and infection, different immunological techniques and approaches of analysis were evaluated and optimized in order to properly measure anti-viral immune responses, with particular attention to cellular immunity (flow cytometry and ELISpot assay). Moreover, since much attention has been given to vaccine formulations and administration systems, an innovative needle-less intradermal (ID) vaccine delivery system (I.D.A.L.®) was assessed in comparison with the more conventional intramuscular (IM) route. The efficiency of needle-less intradermal vaccination was evaluated in a model of intense viral immune stimulation [Aujeszky’s Disease Virus (ADV)] in order to transfer such application to the more complex virus-to-host interaction observed upon PRRSV vaccination and infection. It was demonstrated that ID vaccination is able to elicit an intense immune response, strictly comparable to IM vaccination, both in terms of humoral (VN antibodies) and cellular (IFN-γ secreting cells, IFN-γ productivity per cell, IFN-γ responsiveness categories) immunity. Flow cytometric data showed a positive modulation of peripheral innate (NK) and adaptive (CD8β+) cytotoxic cells as well as of CD4-(CD8+)CD25+ activated and regulatory CD4+CD25+ T cells in ID-vaccinated animals. Also IFN-γ gene expression in PBMC was up-regulated in both groups.The qualitative and quantitative characterization of the immune response against PRRSV was then determined after vaccination (Lelystad-like vaccine strain) and subsequent natural infection by exposure to a heterologous field strain (Italian cluster virus, 84% homology) and associated with the degree of cross-protection and reduction of the disease. Besides immunological and clinical evaluations, also cytokine and hormonal activation were assessed. Compared to controls, vaccinated pigs (IM and ID groups) showed a reduction of the respiratory signs by 72% and 79% and of the overall clinical signs by 68% and 72%, respectively. Clinical protection was associated with marked activation of the cell-mediated immune response. The highest levels of PRRSV-specific IFN-γ secreting cells (mean levels and responsive animals identified by cut-off analysis) were associated with the increase of peripheral NK cells, γ/δ T lymphocytes and cytotoxic CD4-CD8+high T lymphocytes. Evidence of European vaccine-induced clinical protection against natural exposure to a genetically diverse PRRSV isolate (Italian cluster) was demonstrated by the statistically significant reduction of clinical signs in terms of incidence, duration and severity as well as by a more efficient cell-mediated immune response in vaccinated pigs as compared to unvaccinated controls. The early and prompt increase after PRRSV infection and the subsequent decrease to basal levels of IL-1β, IL-6, TNF-α, MCP-1 and IFN-γ gene expression in PBMC of vaccinated animals, associated with temporally regulated endocrine modifications (cortisol and GH) and lower IL-10 expression levels, are a clear indication of both a more efficient responsiveness of peripheral innate immune cells (i.e. monocyte, NK cells) in the early phase of infection and a more efficient control of inflammation in a later phase. Furthermore, the degree of clinical protection was not affected by the route of vaccine administration, since intradermally vaccinated pigs using a needle-less injector did not show any significant difference in terms of clinical signs in comparison with intramuscularly vaccinated pigs. Overall, the results confirm that the ability of a PRRSV strain to induce a strong immune response is more important than the genetic similarity between vaccine and field strains in order to induce clinical protection.