Immune responses in neonatal and adult pigs following Toll-like receptor agonist adjuvanted vaccination

Young piglets are very susceptible to infections and therefore vaccination early after birth can help to decrease the disease incidence on swine farms. However, vaccination during the neonatal period is a challenge, because vaccination of newborn pigs is in general less successful compared to adult pigs. The innate and adaptive immune system are still developing during early-life and this results in qualitative and quantitative differences between the immune responses of newborn or neonatal pigs (< 1-week-old) and adult or immunocompetent (> 8-week-old) pigs after vaccination. Research has indicated that effective immuno-stimulators (adjuvants) in adult vaccines may not fulfil the requirements to stimulate the neonatal immune system effectively. Therefore, age-group-adapted vaccine formulations may be necessary to enhance the success of vaccination at an early age. The aim of this thesis was to investigate immune responses in neonatal and adult pigs after Toll-like receptor (TLR) stimulation to increase our understanding of the porcine (neonatal) immune system and the immune responses after vaccination in pigs of different age-groups. In addition to conventional intramuscular (i.m.) vaccination, skin vaccination was used as a delivery route, because skin has the potential to increase vaccine efficacy due to its abundance of antigen presenting cells (APCs). Porcine reproductive and respiratory syndrome virus (PRRSV) was selected as vaccine-antigen, because this is an endemic and important disease in the swine farming. In addition, to date no effective inactivated PRRSV-vaccine has been developed yet.

In chapter 2, we studied dendritic cells (DCs) and showed that neonatal porcine DCs derived from blood were composed of the same DC subsets and DC proportions as their adult counterparts, namely: plasmacytoid DCs (pDC), and two types of conventional DCs (cDC1 and cDC2). After TLR1/2 or TLR9 stimulation peripheral blood mononuclear cells (PBMCs) and DCs were activated in both neonatal and adult pigs in an age-dependent way. The results of chapter 2 indicated that TLR1/2 agonists (TLR1/2a) and TLR9a could be promising adjuvant candidates in porcine vaccines for both age-classes.

In chapter 3, neonatal and adult pigs were vaccinated in the skin using dissolving microneedle (DMN)-patches as the delivery system. The DMN-patches contained three different individual TLRa-vaccine formulations in combination with inactivated PRRSV (iPRRSV)-antigen. In this study, we investigated the early immune responses in skin and draining lymph nodes 24h after skin vaccination. The DMN-patches containing TLR7/8a induced the most pronounced immune and/or inflammatory response in both age-groups. In neonatal pigs a higher number of genes related to early immune responses were upregulated after TLR7/8a vaccination compared to the adult pigs in both the skin and draining LN, while no age-dependent difference for the local influx of immune cells and the number of APCs in the skin after TLR7/8a vaccination was found. Less striking local vaccine immune responses were noticed also after TLR1/2a and TLR9a application. These results suggested that neonatal and adult pigs could be effectively vaccinated in the skin, especially with TLR7/8a containing vaccines. However, age-dependent immune responses after skin vaccination could be expected.

In the adult PRRSV vaccine-study (chapter 4) we investigated if the specific individual TLRa, which have shown in vitro potential (chapter 2) and induced early immune responses after skin vaccination (chapter 3), generated a protective immune response after vaccination in combination with iPRRSV-antigen. To study this, we administered the TLRa containing vaccines both i.m. and into the skin with the same DMN-patches as used in chapter 3. Pigs received a prime vaccination followed by a booster vaccination four weeks later. To measure vaccine efficacy the pigs were infected with PRRSV three weeks after the booster vaccination. In adult pigs TLR1/2a, TLR7/8a or TLR9a containing PRRSV-vaccines did not induce a detectable PRRSV-specific immune response, independent of the administration route. However, the i.m. administered TLR9a vaccine showed reduction of PRRSV shedding after infection compared to the non-vaccinated animals without detectable specific immune responses. In contrast, iPRRSV-antigen combined with the reference oil-in water (O/W) emulsion Montanide™ ISA28 (ISA28) induced an antigen-specific humoral immune response after booster vaccination combined with reduced shedding of PRRSV. A local skin immune response was observed after skin application of TLR7/8a. This skin reaction was comparable to that observed in adult pigs in chapter and was not observed in the other TLRa skin vaccines. These results indicated that the individual TLRa were less effective than the O/W reference adjuvant ISA28 .

We considered it unlikely that the same TLRa containing vaccine would be more effective when administered in neonatal pigs. Therefore, in chapter 5 we decided to use different adjuvant formulations for the neonatal PRRSV-vaccine-study. In this study a mixture of the previously used TLRa (TLR1/2a+TLR7/8a+TLR9a) was combined with a squalene based O/W emulsion (SWE) and this vaccine formulation (SWE+TLRa) was administered i.m and into the skin with DMN-patches using a study design comparable to the adult PRRSV-study. The reference O/W adjuvant ISA28 was used in both the neonatal and adult PRRSV-vaccine study. In this neonatal study, only ISA28 induced a specific humoral immune response, while a specific cellular immune response was generated in the SWE group. This suggests that prime vaccination in neonates induced a specific immune response after booster vaccination, dependent on the O/W emulsion formulation, but not dependent on the presence of the TLRa or delivery route. In contrast to the adult pigs none of the vaccines in neonatal pigs with or without TLRa were able to reduce the viral shedding after PRRSV challenge. This study demonstrated that the O/W adjuvant ISA28 was less effective in neonatal pigs compared to adult pigs and that the TLRa in an adjuvant-mixture did not enhance the immune response in a PRRSV vaccine

This thesis showed age-dependent differential immune responses after in vitro  and in vivo TLR stimulation and also with O/W adjuvanted PRRSV vaccination. This suggests that neonatal vaccines require a specific adjuvant choice. Further, I conclude that traditional O/W formulations induced a better detectable immune response than when the TLRa were used as adjuvant in combination with the iPRRSV-antigen. Also, we showed that skin vaccination with TLRa did not overcome the weak immunogenic potential of the iPRRSV-antigen. Therefore, the administered vaccine formulations with TLRa were less successful as expected and it can be anticipated that formulating an effective iPRRSV vaccine for (newborn) pigs will continue to be a challenge.