Delivery of heterologous antigens through the mucosal surface by recombinant Salmonella vectors is a powerful strategy for inducing both mucosal and systemic immune responses. After oral vaccination, the Salmonella bacteria invade the mucosal surfaces and spread through the mesenteric lymph nodes to distal sites, such as spleen and liver [16, 17]. This normally results in the induction of both mucosal and systemic cellular and humoral immune responses [18–20]. In the current study, we investigated the immunogenicity of a recombinant HIV-1 Subtype C Gag-expressing attenuated live Salmonella enterica serovarTyphimurium vaccine vector in mice after oral immunization. The HIV-1 gag gene was codon-optimized to reflect codons commonly used by Salmonella bacteria. The presence of rare codons in foreign genes may affect mRNA and plasmid stability and, in some cases, protein synthesis and bacterial growth [21–24]. In this study, we found that the expression of the HIV-1 Gag in Salmonella was improved when the gene was codon-optimized confirming published data . However, other studies have shown that the level of antigen expression was decreased in Salmonella when the gene was codon-optimized . Codon-optimization of genes for expression in recombinant Salmonella vaccine vectors has been reported to have an impact on the nature, breadth and magnitude of the immune responses induced after vaccination. It has been shown that antigen-specific immune responses against a Salmonella-based vaccine that expressed human papillomavirus type 16 L1 improved after codon-optimization . The expression of measles virus (MV) epitopes in a Salmonella vaccine vector has been shown to be enhanced by codon-optimization . Oral vaccination of MV-susceptible mice with the recombinant Salmonella vector induced MV-specific serum antibodies and CD4+ T cell response . Codon-optimization of HIV-1 gag for expression in Salmonella resulted in enhanced mucosal immunity in vaccinated mice .
Our recombinant HIV-1 Gag-expressing Salmonella vaccine induced specific Th1 and Th2 cytokine responses in the spleen. This indicates that the recombinant bacteria successfully delivered the heterologous HIV-1 antigen to the systemic immune system after oral vaccination. Live Salmonella can be taken up by antigen-presenting cells by the process of phagocytosis and the bacteria are able to reside and replicate in the phagosomes. The Salmonella antigens are, therefore, presented to MHC class II-restricted CD4+ T cells [28–30]. This elicits antigen-specific Th1 and/or Th2 cytokine responses [31–34]. The two types of responses (CD4+ Th1 and Th2) induced by the recombinant Salmonella vector in this study are crucial for vaccines that are required to induce both cell-mediated and antibody responses for protection against infection by a number of pathogens. CD4+ Th1 cytokines such as IFN-γ and TNF-α provide protective immunity against intracellular pathogens such as viruses. They promote CD8+ T cell responses and B cell class-switching to IgG2a [35, 36]. In contrast, CD4+ Th2 cytokines such as IL-4 and IL-5 promote B cell class switching to neutralizing antibodies such as IgG1 and they further regulate the magnitude of Th1 cytokine responses [36–38]. In this study, we also tested for the induction of Gag-specific CD8+ T cells in vaccinated mice. No specific CD8+ T cells were detectable by ELISPOT and CBA assays (results not shown). This was not unexpected; the secretion of antigens from the Salmonella bacteria has been shown to result in induction of antigen-specific CD8+ T cell responses in vaccinated mice [39–41]. Our HIV-1 Gag antigen was expressed inside the bacterial cytoplasm as inclusion bodies and was therefore unlikely to induce potent antigen-specific CD8+ T cell responses.
The nature of immune responses found in this study has relevance to the field of HIV-1 vaccinology. In HIV-1 infection, CD4+ Th1 and Th2 cells play regulatory roles in controlling infection and replication [42, 43]. HIV-infected long-term non-progressors have been found to have strong CD4+ T cell responses to HIV-1 antigens such as Gag . Recent studies have also suggested that HIV-specific CD4+ Th1 cell that produce INF-γ and IL-2 are important in long-term reduction of HIV viremia [45, 46]. Other studies have shown that the loss of CD8+ T cell responses in HIV-1 infection could be reversed by vaccine-induced CD4+ Th cell responses . Although CD4+ Th cells provide immunological help to CD8+ T cells and B cells, they can also play a more direct role in antiviral activity . CD4+ T cells, like CD8+ T cells, have cytolytic activities against HIV-infected cells and can provide protective immune responses [49–52]. The cytokines, such as IFN-γ and TNF-α, secreted by activated CD4+ T cells have direct antiviral activities . HIV-1 vaccines should, therefore, provoke both specific CD8+ and CD4+ T cell responses, so as to maximize the chance of preventing or controlling infection.
The development of an HIV-1 vaccine that elicits protective humoral immune responses is still a challenge to the scientific community. Such antibody responses should be able to neutralize many strains of the virus if they are to be useful. Although HIV-1 Gag is not a target for neutralizing antibodies, antibodies to Gag may play a role in other responses such as antibody-mediated cellular cytotoxicity and complement-mediated lysis of HIV-1 virions . In the current study, we investigated whether Salmonella that express codon-optimized Gag could induce HIV-1 specific antibodies. Gag-specific IgG responses were induced in vaccinated mice. Further characterization of the anti-HIV-1 Gag antibody responses induced after secondary vaccinations with aroC+Gag showed the presence of both IgG1 and IgG2a subclasses. This result was supported by the finding that aroC+Gag induced Gag-specific CD4+ Th1 and Th2 cytokines. These cytokines produced by the Th1 and Th2 cells were most probably responsible for the induction of heavy-chain isotype switching to both IgG2a and IgG1 respectively . In summary, our results highlight the potential of using recombinant Salmonella as a vector for HIV-1 antigens. A Salmonella-delivered HIV-1 vaccine would be convenient for mass-vaccinations and inexpensive to produce. This would be advantageous for developing countries, where the HIV/AIDS is most prevalent, and the pandemic urgently needs to be brought under control.