Immune responses induced by inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in neonatal pigs using different adjuvants.

Vaccination of neonatal pigs could be supportive to prevent porcine reproductive and respiratory syndrome virus (PRRSV), which is an important porcine pathogen causing worldwide welfare and health problems in pigs of different age classes. However, neonatal immunity substantially differs to adults, thus different vaccines may be required in neonateal pigs. We examined if the immunogenicity and efficacy of inactivated PRRSV (iPRRSV) vaccines in neonatal pigs could be improved with adjuvants containing oil-in water (O/W) emulsions with or without Toll-like receptor (TLR) agonists and by altering the delivery route from intramuscular (i.m.) to the skin. Three-day-old PRRSV-naïve piglets (n = 54, divided in 6 groups) received a prime vaccination and a booster vaccination four weeks later. The vaccine formulations consisted of different O/W emulsions (Montanide™ ISA28RVG (ISA28)), a squalene in water emulsion (SWE) for i.m. or a Stable Emulsion (SE) with squalene for skin vaccination) and/or a mixture of TLR1/2, 7/8 and 9 agonists (TLRa) combined with iPRRSV strain 07V063. These vaccines were delivered either i.m. (ISA28, SWE, TLRa or SWE + TLRa) or into the skin (skiSE + TLRa) with dissolving microneedle (DMN)-patches. All animals received a challenge with homologous PRRSV three weeks after booster vaccination. Specific antibodies, IFN-γ production and viremia were measured at several time-points after vaccination and/or challenge, while lung pathology was studied at necropsy. After booster vaccination, only ISA28 induced a specific antibody response while a specific T-cell IFN-γ response was generated in the SWE group, that was lower for ISA28, and absent in the other groups. This suggests that prime vaccination in neonates induced a specific immune response after booster vaccination, dependent on the emulsion formulation, but not dependent on the presence of the TLRa or delivery route. Despite the measured immune responses none of the vaccines showed any efficacy. Further research focused on the early immune response in draining lymph nodes is needed to elucidate the potential of TLR agonists in vaccines for neonatal pigs.

Toll-like receptor agonists as adjuvants for inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine.

Toll-like receptor (TLR) agonists can effectively stimulate antigen-presenting cells (APCs) and are anticipated to be promising adjuvants in combination with inactivated vaccines. In this study, the adjuvant potential of three different TLR-agonists were compared with an oil-in-water (O/W) adjuvant in combination with inactivated porcine reproductive and respiratory syndrome virus (iPRRSV) applied by different administration routes: intramuscular (i.m.) or into the skin using dissolving microneedle (DMN) patches. Pigs received a prime vaccination followed by a booster vaccination four weeks later. TLR1/2 (Pam3Cys), TLR7/8 (R848) or TLR9 (CpG ODN) agonists were used as adjuvant in combination with iPRRSV strain 07V063. O/W adjuvant (Montanide™) was used as reference control adjuvant and one group received a placebo vaccination containing diluent only. All animals received a homologous challenge with PRRSV three weeks after the booster vaccination. Antibody and IFN-γ production, serum cytokines and viremia were measured at several time-points after vaccination and/or challenge, and lung pathology at necropsy. Our results indicate that a TLR 1/2, 7/8 or 9 agonist as adjuvant with iPRRSV does not induce a detectable PRRSV-specific immune response, independent of the administration route. However, the i.m. TLR9 agonist group showed reduction of viremia upon challenge compared to the non-vaccinated animals, supported by a non-antigen-specific IFN-γ level after booster vaccination and an anamnestic antibody response after challenge. Montanide™-adjuvanted iPRRSV induced antigen-specific immunity after booster combined with reduction of vireamia. Skin application of TLR7/8 agonist, but not the other agonists, induced a local skin reaction. Further research is needed to explore the potential of TLR agonists as adjuvants for inactivated porcine vaccines with a preference for TLR9 agonists.

Characterization of immune responses following homologous reinfection of pigs with European subtype 1 and 3 porcine reproductive and respiratory syndrome virus strains that differ in virulence.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry worldwide. Vaccination results often in limited protection. Understanding host immune responses elicited by different PRRSV strains could help to develop more efficacious vaccines. In the current study we characterized immunological responses and viral kinetics in pigs after primo infection and homologous challenge of the highly virulent European subtype 3 strain Lena, and the moderate to low virulent subtype 1 strain LV. Eighteen pigs were infected per strain, and 18 non-infected pigs served as control. Post mortem analysis was performed at days 7, 46 and 60 p.i. At day 46, pigs were challenged with the homologous strain. After the first inoculation, pigs infected with Lena developed fever and clinical symptoms, while this was not observed in pigs infected with LV. Virus titres in serum were about 100-fold higher in pigs infected with Lena than in pigs infected with LV. An inflammatory response was observed in pigs after primo infection with Lena with significantly higher levels of IL-12, IL-1ß and TNF-α in the bronchoalveolar lavage. IFN-γ ELISPOT assay showed comparable responses between Lena and LV. Neutralizing antibodies were detected earlier in serum of pigs infected with Lena than in pigs infected with LV. After the challenge, a boost in antibody levels in both groups was observed. Challenge infection resulted in both groups in complete protection and sterile immunity, with no viraemia, clinical symptoms or viral RNA in tissues. In conclusion, although there were clear differences in immunological, clinical and virological responses to the primo infection, there were no differences observed in protection against homologous challenge.

Lung pathogenicity of European genotype 3 strain porcine reproductive and respiratory syndrome virus (PRRSV) differs from that of subtype 1 strains.

Porcine reproductive and respiratory syndrome (PRRS) is difficult to control due to a high mutation rate of the PRRS virus (PRRSV) and the emergence of virulent strains. The objective of this study was to analyse early and late pathological responses in the respiratory tract after infection with the European PRRSV subtype 3 strain Lena in comparison to two European PRRSV subtype 1 strains: Belgium A and Lelystad-Ter Huurne (LV). For each virus strain, groups of twelve pigs were inoculated, and four pigs per group were euthanized at days 3, 7 and 35 post-infection (p.i.) for consecutive examination. Infection with strain Lena resulted in a more severe disease than with the subtype 1 strains, an inflammatory response within the first week of infection with expression of IL-1α in the lung and lymph node, and an influx of neutrophils and monocytes in bronchoalveolar lavage fluid (BALF). Infection with strain Belgium A or LV resulted in mild or no pathology within the first week of infection, but inflammatory cell influx in the lung interstititium was increased at the end of the experiment at day 35 p.i. At five weeks p.i., all strains induced a higher percentage of cytotoxic T cells and higher levels of IFN-γ producing cells in BALF. This might have contributed to clearance of virus. In general, subtype 3 strain Lena induced a stronger early inflammatory response which led to more severe clinical disease and pathology. On the other hand, this may have supported an enhanced or faster clearance of virus in tissues, compared to subtype 1 strains.

Phenotypic modulation and cytokine profiles of antigen presenting cells by European subtype 1 and 3 porcine reproductive and respiratory syndrome virus strains in vitro and in vivo.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes continuous problems in the pig industry, due to high costs of outbreaks and reduced welfare of diseased pigs. The severity of infection is, partly, dependent on the virus strain. Recently isolated Eastern-European subtype 3 strains are more pathogenic than the widespread subtype 1 strains. There is, however, almost no information available about the mechanisms involved in the pathogenicity of these subtype 3 strains. The objective of the present study was to characterize the in vitro and in vivo response of two European subtype 1 strains, Belgium A and Lelystad-Ter Huurne (LV), and a virulent subtype 3 strain, Lena, in bone marrow-derived dendritic cells (BM-DC) (in vitro) and alveolar macrophages (in vitro and in vivo). It was shown that infection with the Lena strain resulted in a higher apoptosis of cells in vitro and a higher level of infectivity in vitro and in vivo than the other virus strains. Furthermore, infection with Lena resulted in a small downregulation of the immunologically relevant cell surface molecules SLA-I, SLA-II and CD80/86 in vitro, and SLA-II in vivo. In spite of these differences, in vitro cytokine responses did not differ significantly between strains, except for the absence of IL-10 production by Lena in BM-DC. The higher infectivity, apoptosis and downregulation of the cell surface molecules, may have contributed to the increased pathogenicity of Lena, and have dampened specific immune responses. This could explain the delayed and decreased adaptive immune responses observed after infections with this strain.

Comparative analysis of immune responses following experimental infection of pigs with European porcine reproductive and respiratory syndrome virus strains of differing virulence.

Porcine reproductive and respiratory syndrome virus (PRRSV) is difficult to control due to a high mutation rate and the emergence of virulent strains. The objective of this study was to analyze the immunological and pathological responses after infection with the European subtype 3 strain Lena in comparison to subtype 1 strains Belgium A and Lelystad-Ter Huurne (LV). Sixteen pigs were inoculated per strain, and sixteen pigs with PBS. At days 7 and 21 post-inoculation (p.i.), four pigs per group were immunized with an Aujeszky disease vaccine (ADV) to study the immune competence after PRRSV infection. Infection with the Lena strain resulted in fever and clinical signs. This was not observed in the Belgium A or LV-infected pigs. Infection with the Lena strain resulted in high virus titers in serum, low numbers of IFN-γ secreting cells, a change in leukocyte populations and a delayed antibody response to immunization with ADV. Levels of IL-1ß, IFN-α, IL-10, IL-12, TNF-α and IFN-γ mRNA of the Lena-infected pigs were increased during the first week of infection. For pigs infected with the Belgium A or LV strain, the effects of infection on these parameters were less pronounced, although for the Belgium A-infected pigs, the level of the analyzed cytokines, except for TNF-α, and leukocyte populations were comparable to the Lena-infected pigs. These results suggest that while the outcome of infection for the three strains was comparable, with mostly clearance of viremia at day 33 p.i, differences in immune responses were observed, perhaps contributing to their virulence.