Early immune responses in skin and lymph node after skin delivery of Toll-like receptor agonists in neonatal and adult pigs.

The skin is potentially an important vaccine delivery route facilitated by a high number of resident antigen presenting cells (APCs), which are known to be stimulated by different Toll-like receptor agonists (TLRa). In this study, neonatal and adult pigs were vaccinated in the skin using dissolving microneedle patches to investigate the immuno-stimulatory potential of different TLRa and possible age-dependent differences early after vaccination. These patches contained TLR1/2a (Pam3Cys), TLR7/8a (R848) or TLR9a (CpG ODN) combined with inactivated porcine reproductive and respiratory syndrome virus (PRRSV) or with an oil-in-water stable emulsion. Vaccinated skin and draining lymph nodes were analysed for immune response genes using microfluidic high-throughput qPCR to evaluate the early immune response and activation of APCs. Skin pathology and immunohistochemistry were used to evaluate the local immune responses and APCs in the vaccinated skin, respectively. In both neonatal and adult pigs, skin vaccination with TLR7/8a induced the most prominent early inflammatory and immune cell responses, particularly in the skin. Skin histopathology and immunohistochemistry of APCs showed comparable results for neonatal and adult pigs after vaccination with the different TLRa vaccines. However, in vaccinated neonatal pigs in the skin and draining lymph node more immune response related genes were upregulated compared to adult pigs. We showed that both neonatal and adult skin could be stimulated to develop an immune response, particularly after TLR7/8a vaccination, with age-dependent differences in regulation of immune genes. Therefore, age-dependent differences in local early immune responses should be considered when developing skin vaccines.

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.

Electroporation of a nanoparticle-associated DNA vaccine induces higher inflammation and immunity compared to its delivery with microneedle patches in pigs.

DNA vaccination is an attractive technology, based on its well-established manufacturing process, safety profile, adaptability to rapidly combat pandemic pathogens, and stability at ambient temperature; however an optimal delivery method of DNA remains to be determined. As pigs are a relevant model for humans, we comparatively evaluated the efficiency of vaccine DNA delivery in vivo to pigs using dissolvable microneedle patches, intradermal inoculation with needle (ID), surface electroporation (EP), with DNA associated or not to cationic poly-lactic-co-glycolic acid nanoparticles (NPs). We used a luciferase encoding plasmid (pLuc) as a reporter and vaccine plasmids encoding antigens from the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a clinically-significant swine arterivirus. Patches were successful at inducing luciferase expression in skin although at lower level than EP. EP induced the cutaneaous recruitment of granulocytes, of MHC2posCD172Apos myeloid cells and type 1 conventional dendritic cells, in association with local production of IL-1ß, IL-8 and IL-17; these local responses were more limited with ID and undetectable with patches. The addition of NP to EP especially promoted the recruitment of the MHC2posCD172Apos CD163int and CD163neg myeloid subsets. Notably we obtained the strongest and broadest IFNγ T-cell response against a panel of PRRSV antigens with DNA + NPs delivered by EP, whereas patches and ID were ineffective. The anti-PRRSV IgG responses were the highest with EP administration independently of NPs, mild with ID, and undetectable with patches. These results contrast with the immunogenicity and efficacy previously induced in mice with patches. This study concludes that successful DNA vaccine administration in skin can be achieved in pigs with electroporation and patches, but only the former induces local inflammation, humoral and cellular immunity, with the highest potency when NPs were used. This finding shows the importance of evaluating the delivery and immunogenicity of DNA vaccines beyond the mouse model in a preclinical model relevant to human such as pig and reveals that EP with DNA combined to NP induces strong immunogenicity.