C3d Enhancement Of The Immunogenicity Of The Swine Influenza HA Expressed By A DNA Vaccine

Swine influenza is a common respiratory disease in pigs, and it is an acute, feverish and infectious disease. Currently, three main subtypes of influenza virus are circulating in different swine populations throughout the world: H1N1, H3N2, and H1N2. Swine is the only animal which can be infected by either avian or human original influenza viruses, and can be considered as the intermediate host for the process of genetic reassortments between viruses of different hosts. This can lead to the generation of new strains of influenza, some of which may be transmitted to other species including humans. As such, the development of a highly effective influenza virus vaccine for pigs would be of benefit to both veterinary and human health.Currently, Bivalent, killed vaccines are now commercially available but antigenic variation in the hemagglutinin (HA) protein of influenza viruses passaged in eggs can reduce the efficacy of this vaccine in eliciting the desired protective immune responses. Furthermore, larger dose injection and addition of adjuvant increased cost. Therefore, it is important to development new vaccines that are capable of inducing virus-specific neutralizing antibody plus cell mediated immunity, which provides superior protection against the acute influenza diseases. Furthermore, the new vaccines should provide protection against several main subtypes of swine influenza virus.DNA vaccines can induce protective cellular and humoral immune responses and have therefore been used during the last decade to develop vaccines against a variety of different pathogens. Several studies indicated that DNA vaccines expressed HA can elicit an immune response and confers protection against an influenza virus challenge. However, developments of DNA vaccines were limited because of low protein expression level in vivo and weak immune response elicited especially in larger animals. Therefore, various tactics have been used to increase the potency of DNA vaccines. For example, several studies have convincingly demonstrated that conjugation of target antigens to multiple copies of C3d can dramatically enhance the antibody responses to the target antigens. Utilizing DNA as a priming vaccination and a different modality for boosting (e.g., live viral vectors) has induced remarkable levels of cellular immunity.This study evaluated the effects of C3d on the immunogenicity of the swine influenza HA expressed by a DNA vaccine. Plasmid encoding soluble HA, complete HA or soluble fused form of HA were constructed from H3N2 subtype of swine influenza virus. Mice of Four groups were respectively injected with plasmid pCA-sHA, pCA-tmHA, pCA-sHA/mC3d3, pCA-sHA/sC3d3 and empty vector pCAGGS. The immune response was monitored by an enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI) assays, and virus neutralization tests. Analysis of antibody titers indicated that immunizations with pCA-sHA/mC3d3 or pCA-sHA/sC3d3 had higher titers of anti-HA antibodies and stronger antibody affinity compared to serum from mice vaccinated with pCA-sHA or pCA-tmHA. Furthermore, C3d fusion increased Th2-biased immune response by inducing IL-4 production. Splenocytes from mice vaccinated with pCA-sHA/mC3d3 or pCA-sHA/sC3d3 produced about three-fold more IL-4 than did splenocytes from mice vaccinated with pCA-sHA or pCA-tmHA. Seven days post-challenge with homologous virus (H3N2), no virus was isolated from the mice immunized with HA-expressing plasmids. However, 10 days post-challenge with heterologous virus (H1N1), only mice immunized with pCA-sHA/mC3d3 or pCA-sHA/sC3d3 had no virus or microscopic lesions in the kidneys and cerebrum. These results demonstrated that C3d gene fused to truncated HA gene significantly enhanced the humoral immunity of the soluble HA expressed by DNA vaccine. Furthermore, effects of molecule adjuvant wine C3d were evaluated in mice model.In order to investigate the immunogenicity and protective efficacy of a heterologous prime-boost immunization against swine influenza virus, mice were first primed with naked DNA (pCA-sHA, pCA-tmHA, pCA-sHA/mC3d3 and pCA-sHA/sC3d3, respectively) and then boosted with same plasmid or recombinant pseudorabies virus expressing HA (rPRV-HA). The mice immunized with the prime-boost regime developed a high level of specific antibody against HA and cytokine production in the splenocytes when compared with the mice immunized single rPRV-HA. After plasmid DNA immunization, mice boosted with rPRV-HA produced similar antibody level and higher cytokine production in the splenocytes than that boosted with same plasmid DNA. Four weeks after the booster immunization with rPRV-HA or plasmid DNA, the mice were challenged with homologous virus (H3N2). All groups immunized with DNA or rPRV-HA were protected to a great extent. Especially, prime-boost immunization with DNA followed by rPRV-HA improved protective immunity against homologous virus (H3N2).In conclusion, C3d enhanced antibody responses to hemagglutinin and protective immunity against SIV of different subtypes. The results implied the plasmid DNA expressing sHA/C3d3 fusion proteins may represent an effective candidate vaccine against swine influenza virus. Furthermore, this study also indicated that heterologous prime-boost immunization approach might be useful against SIV infection.