C. Andreoni

Enhanced protective response and immuno-adjuvant effects of porcine GM-CSF on DNA vaccination of pigs against Aujeszky's disease virus.

Abstract This study was conducted to investigate whether the co-delivery of DNA encoding porcine cytokines would enhance a protective immune response in pigs to a Pseudorabies virus (PRV; or Aujeszky’s disease virus) DNA vaccine. Aujeszky’s disease in pigs results in respiratory and nervous symptoms with important economic losses. To evaluate cytokine effects, eukaryotic expression vectors were constructed for porcine GM-CSF, IL-2 and IFN-γ. cDNA for each of these cytokines was inserted under the control of a CMV promoter in the pcDNA3 plasmid and cytokine expression was confirmed after DNA transfection in various mammalian cell cultures by bioassays (GM-CSF and IL2) and ELISA (IFN-γ). Pigs were vaccinated by single intramuscular injection with plasmid DNA encoding PRV gB and gD along with various combinations of cytokine plasmid constructs. Pig serum was tested for the production of antibody by isotype specific anti-PRV ELISA. Pigs were then challenged with the highly virulent PRV strain NIA3 on day 21 after vaccination. The survival and growth rate of pigs were monitored for seven days after the viral challenge. The co-administration of GM-CSF plasmid increased the immune response induced by gB and gD PRV DNA vaccine. This immune response was characterized by an earlier appearance of anti-PRV IgG2, a significantly enhanced anti-PRV IgG1 and IgG2 antibody response, a significantly decreased and shortened viral excretion in nasal swabs and an improved protection to the viral challenge. In contrast, the co-administration of porcine IL-2 or IFN-γ had no adjuvant effects. Our results thus demonstrate for the first time that the application of porcine GM-CSF gene in a DNA vaccine formulation can exert immuno-adjuvant and protective effects with single vaccination in the natural host pig against Aujeszky’s disease.

A West Nile virus (WNV) recombinant canarypox virus vaccine elicits WNV-specific neutralizing antibodies and cell-mediated immune responses in the horse

Successful vaccination against West Nile virus (WNV) requires induction of both neutralizing antibodies and cell-mediated immune responses. In this study, we have assessed the ability of a recombinant ALVAC-WNV vaccine (RECOMBITEK WNV) to elicit neutralizing antibodies and virus-specific cell-mediated immune responses in horses. In addition, we examined whether prior exposure to ALVAC-WNV vaccine would inhibit B and cell-mediated immune responses against the transgene product upon subsequent booster immunizations with the same vaccine. The results demonstrated that the recombinant ALVAC-WNV vaccine induced neutralizing antibodies and prM/E insert-specific IFN-gamma(+) producing cells against WNV in vaccinated horses. Prior exposure to ALVAC-WNV vaccine did not impair the ability of horses to respond to two subsequent booster injections with the same vaccine, although anti-vector-specific antibody and cell-mediated immune responses were induced in vaccinated horses. This report describes, for the first time, the induction of antigen-specific cell-mediated responses following vaccination with an ALVAC virus recombinant vaccine encoding WNV antigens. Moreover, we showed that both WNV-specific IFN-gamma producing cells and anti-WNV neutralizing antibody responses, are not inhibited by subsequent vaccinations with the same vector vaccine.

DNA vaccination of neonate piglets in the face of maternal immunity induces humoral memory and protection against a virulent pseudorabies virus challenge

DNA vaccination represents a unique opportunity to overcome the limitations of conventional vaccine strategy in early life in the face of maternal-derived immunity. We used the model of pseudorabies virus (PRV) infection in pigs to further explore the potential of DNA vaccination in piglets born to sows repeatedly vaccinated with a PRV inactivated vaccine. A single immunisation of 8-week-old piglets with a DNA vaccine expressing secreted forms of PRV gB, gC, and gD, triggered an active serological response, confirming that DNA vaccination can over-ride significant residual maternal-derived immunity. A clear anamnestic response was evidenced when a secondary DNA vaccination was performed at 11 weeks of age, suggesting that DNA vaccination, performed in the face of passive immunity, elicited a strong humoral memory. We subsequently explored the potential of DNA vaccination in neonate piglets (5-6 days of age) in the face of very high titres of maternal antibodies and demonstrated that very high titres of passive antibodies selectively inhibited serological responses but not the establishment of potent memory responses. Finally, we demonstrated that DNA vaccination provided protection against an infectious PRV challenge at the end of the fattening period (i.e. at approximately 5 months of age). Collectively, our results pave the way for a new flexible vaccination program, which could ensure uninterrupted protection of fattening pigs over their entire economical life under field conditions.

Quantitative analysis of the antigen-specific IFNγ+ T cell-mediated immune response in conventional outbred pigs: kinetics and duration of the DNA-induced IFNγ+ CD8+ T cell response

It is now well established that antigen-specific CD8(+) T cells play a major role in vaccine-induced immunity against intracellular pathogens and tumor cells. The detection of these immune cells in outbred animals has been hampered mainly by the need to generate individual autologous antigen-presenting cells (APCs) due to the high degree of polymorphism of the major histocompatibility complex (MHC) Class I loci. We used individually derived immature porcine dendritic cells infected with a pox-based recombinant viral vector to ex vivo stimulate PBMCs from vaccinated conventional pigs. The frequencies of antigen-specific T cells was determined by the number of IFNgamma-secreting cells in a quantitative enzyme-linked immune spot (ELISPOT) assay. Using this approach we were able to rank different pseudorabies virus (PRV) vaccines strategies for their ability to prime viral-specific IFNgamma(+) T cells. Plasmid DNA has recently emerged as a promising tool with multiple applications in the field of infectious diseases, allergy and cancer. We showed for the first time in this study that DNA immunization induced a long-lived antigen-specific IFNgamma(+) T cells response in conventional pigs. Additional studies allowed us to show that these virus-specific IFNgamma(+) responding cells detected in this ELISPOT assay were MHC-restricted and comprised in the CD8alpha(bright) pig T cell subset. These new data confirm the usefulness of DNA vaccines to control diseases requiring cellular immunity in pigs.

Intradermal vaccination with un-adjuvanted sub-unit vaccines triggers skin innate immunity and confers protective respiratory immunity in domestic swine.

Intradermal (ID) vaccination constitutes a promising approach to induce anti-infectious immunity. This route of immunization has mostly been studied with influenza split-virion vaccines. However, the efficacy of ID vaccination for sub-unit vaccines in relation to underlying skin innate immunity remains to be explored for wider application in humans. Relevant animal models that more closely mimic human skin immunity than the widely used mouse models are therefore necessary. Here, we show in domestic swine, which shares striking anatomic and functional properties with human skin, that a single ID delivery of pseudorabies virus (PRV) glycoproteins without added adjuvant is sufficient to trigger adaptive cellular and humoral immune responses, and to confer protection from a lethal respiratory infection with PRV. Analysis of early events at the skin injection site revealed up-regulation of pro-inflammatory cytokine and chemokine genes, recruitment of neutrophils and monocytes and accumulation of inflammatory DC. We further show that the sustained induction of pro-inflammatory cytokine genes results from the combined effects of skin puncture, liquid injection in the dermis and viral antigens. These data highlight that immune protection against respiratory infection can be induced by ID vaccination with a subunit vaccine and reveal that adjuvant requirements are circumvented by the mechanical and antigenic stress caused by ID injection, which triggers innate immunity and mobilization of inflammatory DC at the immunization site. ID vaccination with sub-unit vaccines may thus represent a safe and efficient solution for protection against respiratory infections in swine and possibly also in humans, given the similarity of skin structure and function in both species.