Protection against influenza virus by sublingual immunization with subunit vaccine and development of mucosal adjuvant derived from cholera toxin

Abstract

Influenza virus causes seasonal epidemics and global pandemics, leading to severe morbidity and mortality worldwide. Influenza pandemic in 2009 and infection with highly pathogenic avian influenza viruses in humans suggest a need for the development of effective vaccines against various influenza viruses. It is likely that mucosal vaccination is more effective in providing protection against various influenza viruses than parenteral route. Despite its advantage, most of currently available influenza vaccines are immunized via parenteral routes. Actually, it is difficult to induce proper immune response to antigens (Ags) such as protein-based vaccines at mucosal surfaces. Thus, the development of mucosal adjuvant would contribute to improve Ag-specific immune responses via mucosal vaccination. In this study, recombinant hemagglutinin protein 1 (HA1) and M2 protein (3M2eC) with three tandem copies of the M2 ectodomain (M2e) were expressed in Escherichia coli and protective efficacy of the recombinant proteins between sublingual (s.l.) and parenteral vaccinations in mice against influenza viral infection compared. In addition, in order to develop a safe and effective mucosal adjuvant, a new protein (TCTA1T) was generated by fusion of HIV Tat protein transduction domain (PTD) at both N and C termini of A1 subunit (CTA1) of cholera toxin (CT) without B subunit. The results showed that s.l. immunization of 3M2eC with CT was more efficient for inducing protection in mice against homologous and heterologous influenza viral challenges than parenteral routes, although levels of M2e-specific serum antibodies (Abs) induced by parenteral route were higher than that in s.l. group. On the other hand, s.l. immunization induced higher levels of M2-specific IgG and IgA Abs in the lung tissue than those in parenteral routes. The results indicate that the protection induced by M2 protein is associated with Ab response in lung tissue rather than that in serum. In contrast to M2 protein, HA1-specific serum IgG Ab titer induced by s.l. immunization was comparable to those in mice by intramuscular (i.m.) vaccination. Mice immunized by either s.l. or i.m. route showed significant neutralizing Ab (nAb) responses in serum. Furthermore, both s.l. and i.m. immunizations with the HA protein induced complete protection against infection with pandemic influenza virus in mice. These results indicate that nAbs to HA1 in the serum are important for the protection against influenza virus. Finally, TCTA1T was compared for the safety and adjuvanticity as mucosal adjuvant with CT in mice. The results showed that TCTA1T in combination with OVA induced Ag-specific systemic and mucosal immune responses which were comparable to those induced by CT after intranasal (i.n.) immunization. In addition, TCTA1T induced significant CTL response in mice. Furthermore, i.n. immunization of influenza M2 protein with TCTA1T provided complete protection against influenza virus challenge. Importantly, on the contrary to CT, TCTA1T was non-toxic and non-immunogenic. These results indicate that TCTA1T could be an effective mucosal adjuvant for enhancing immune responses to Ags when immunized mucosally. Taken together, these studies suggest that s.l. administration of influenza recombinant vaccine together with TCTA1T as a mucosal adjuvant may be a promising approach to prevent epidemic and pandemic influenza viral infection.