Humoral and cell-mediated immunity to Porcine Circovirus type 2 (PCV2) vaccination and natural infection

Porcine circovirus type 2 (PCV2) has been identified as the main causative agent of the postweaning multisystemic wasting syndrome (PMWS), one of the major swine diseases worldwide that is commonly referred, together with other relevant porcine diseases related to PCV2, as belonging to the porcine circovirus associated diseases (PCVD). The most important strategy to prevent and control PCV2 associated diseases, apart from management procedures and control of coinfections, is the vaccination of piglets or sows and gilts. Nowadays there are three commercial PCV2 vaccines available; even if their efficacy in reducing the viremia burden and viral-induced specific lymphoid lesions has been proved, the mechanisms by which they are able to elicit protective immunity have not been thoroughly clarified. Besides the development of humoral immunity that is generally characterised by the detection of total anti-PCV2 and virus-neutralizing antibodies, the mechanisms that allow the adaptive cell-mediated immune response to control PCV2 infection and the related diseases have not been clearly elucidated, particularly under field conditions. The present Thesis investigated the efficacy of a one-dose porcine circovirus 2 (PCV2) subunit vaccine based on the PCV2 Cap protein expressed in a baculovirus system in two different farms (farm1 and 2) at which a history of porcine circovirus-associated disease (PCVD) was present. Morbidity, mortality, average daily weight gain, carcass weight, PCV2 load in serum and vaccine immunogenicity, in terms of PCV2-specific antibodies, PCV2-specific IFN-? secreting cell frequencies and mRNA expression profiles of relevant pro-inflammatory and immune cytokines, were assessed. Serology to potential coinfections due to porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae (M. hyo.) was also carried out. A double-blind, randomised, and controlled field trial was performed distributing 818 piglets in two treatment groups. At inclusion (weaning at 21±3 days of age), 408 animals received a 2-ml intramuscular dose of Porcilis PCV® (vaccinated group) suspended in a tocopherol-based adjuvant (Diluvac Forte®). Controls (410 piglets) received 2 ml of the same adjuvant alone intramuscularly. Weights were recorded at inclusion and at 12 and 26 weeks of age, and the average daily weight gain (ADWG) was calculated. The carcass weights of the pigs from farm 2 were recorded at slaughter (274 day-old pigs). All dead animals (died or culled) underwent autopsy to classify them as PMWS-affected or not. At each farm, blood samples were collected for serologic and cellular studies aimed at investigating the humoral (ELISA determination of PCV2-antibody titres in serum) and cell-mediated (ELISpot assay for the measurement of the PCV2-specific IFN-? secreting cell frequencies in PBMC) immune response of pigs. The analyses of the present Thesis showed that vaccination with a single dose of a PCV2 Cap vaccine had beneficial effects against the PCVD, and especially PMWS. The vaccination reduced the mortality rate and morbidity, PCV2 viremia and viral load, and improved productive performances (e.g. ADWG: +70 g/day between 12 and 26 weeks of age when viremia and the specific disease occurred) as well as carcass weight at slaughter age (+4.5 kg). These effects were associated with virologic and clinical protection derived from the immunogenicity of the vaccine measured as activation of both humoral and a cellular immune responses. In this regard, ELISA quantification of PCV2-specific antibodies showed seroconversion (with exception of pigs with a titre of maternally derived antibodies >8 log2) and long lasting protective immunity in all vaccinated pigs. Furthermore, the increased frequency of IFN-? secreting cells that was detected by the ELISpot assay during the post-vaccination period demonstrated the capability of a single dose of the PCV2 Cap-based vaccine to induce a virus-specific cell-mediated immune response. During the post-exposure period, vaccinated animals rapidly and efficiently counteracted virus spread since both humoral and cell-mediated immunity were associated with absent or low viremia and less severe clinical signs. In addition, in order to obtain more thorough information about the mechanisms of cellular immune reactivity, the evaluation of expression patterns of relevant pro-inflammatory (IL-8, TNF-?, IL-1?) and immune (IFN-?, IL-10) cytokines was carried out. Cytokine modulation and course of viremia were assessed in 10 PCV2-vaccinated and 20 non-vaccinated pigs from farm 1. These analyses were performed by reverse transcriptional-quantitative PCR (RT-qPCR) before the onset of PCV2 viremia (16 weeks of age), upon PCV2 infection and after the onset of PMWS clinical signs (19 and 22 weeks of age, respectively). The cytokine response was evaluated with regards to evident clinical signs related to PMWS and course of viremia, grouping the animals into three groups: 1) vaccinated (PCV2-vac) pigs; 2) unvaccinated spontaneously infected/non-PMWS-affected (Ctrl) pigs; 3) unvaccinated spontaneously infected/PMWS-affected (Ctrl-PMWS+) pigs. Moreover, in order to establish an association between cytokine expression and viremia burden, each of the above mentionated groups was analysed dividing the animals in three different subgroups based on viremia: non-viremic pigs (NV), pigs with viremia <106 (V<106) and pigs with viremia ?106 (V?106) viral genome copy number / ml of serum. Higher IL-8, TNF-? and IFN-? levels were detected in the PCV2-vac group, testifying a more efficient immune responsiveness, especially when compared to the Ctrl-PMWS+ group. In Ctrl-PMWS+ pigs, lower IFN-? at 19 weeks of age was associated with high IL-10 at 19 weeks of age and low levels of pro-inflammatory cytokines at 22 weeks of age, namely IL-8 and TNF-?, a condition likely correlated with the onset of the disease. Contrarily, at 19 weeks of age, PCV2-vac and Ctrl pigs showed lower IL-10 expression, together with higher IFN-? levels than the Ctrl-PMWS+ animals. At 22 weeks of age, vaccinated animals maintained higher levels of the pro-inflammatory cytokines. These evidences support that the outcome of PMWS could be associated with a reduction of the innate/pro-inflammatory response. Overall, the results show a different cytokine modulation in vaccinated and unvaccinated-infected pigs also developing PMWS. Vaccinated pigs coped with infection showing low or absent viremia burden, absence of PMWS disease and stronger inflammatory response and cellular IFN-?-related reactivity.