Evaluation of the sublingual route for administration of influenza vaccines in a murine model

Influenza is a one of the most important respiratory viruses, and it infects millions of people every year. Due to mutational changes in the virus genome (antigenic drift), influenza cause occasional pandemics. Pandemic influenza strains arise when gene segments from two or more influenza viruses re-assort (antigenic shift), leading to a novel virus. Vaccination is still the most effective way of preventing influenza, and the recent H1N1 pandemic emphasised the need for effective pandemic vaccines that can induce rapid protection in an immunologically naïve population. Mucosally administered vaccines are an attractive approach for delivery of influenza vaccines since they are needle-free and have the ability to induce mucosal immune responses. Intranasal (IN) vaccination against influenza has been used for decades, however, an inactivated intranasal influenza vaccine was recently associated with Bell's Palsy (facial nerve paralysis). Sublingual (SL) vaccination (application under the tongue) can be a novel alternative for mucosal administration of influenza vaccines. Avian influenza subtypes have previously shown to be poorly immunogenic in man, thus an effective adjuvant is needed to boost the vaccine effect. In this study we have vaccinated BALB/c mice IM, IM or SL with two doses, three weeks apart, of a virosomal H5N1 influenza vaccine (2 μg of HA) alone or in combination with 7.5 μg of the novel mucosal adjuvant c-di-GMP. Serum, saliva and nasal wash samples were analysed for influenza specific antibodies using the ELISA and HI antibodies were detected in serum and saliva using the HI assay. Splenocytes were used in the memory B cell ELISPOT, and stimulated in vitro before the cytokine profiles were measured by multiplex bead assay. In addition, influenza stimulated splenocytes were fixed and stained intracellularly for cytokines, and the frequency of cytokine producing cells was determined using multiparametric flow cytometry. The intramuscular, the intranasal and the sublingual routes all induced strong immune responses both in the humoral and the cellular immune assays when the virosomes were combined with c-di-GMP adjuvant. The non-adjuvanted vaccine induced lower immune responses as compared to the adjuvanted vaccine, irrespective of administration route. After the first vaccine dose, IM administration of the adjuvanted vaccine showed the highest IgG antibody response. In contrast, after the second vaccine dose, the IN adjuvanted group showed the highest responses in all assays. A strong local humoral immune response together with systemic IgG and IgA antibodies was elicited in both the sublingual and the intranasal adjuvanted groups. Interestingly, the IN adjuvanted group showed a dominant Th1 profile, whereas the SL adjuvanted group showed a more balanced Th2/Th1 profile. In addition, high levels of IL-17 (a Th17 cytokine) were produced in both the mucosal administered vaccines groups. The frequency of multifunctional CD4+ T cells was highest in the IN adjuvanted group, but also SL vaccination of virosomes combined with c-di-GMP induced high frequencies of multifunctional T cells. This is the first study to report that SL vaccination with H5N1 virosomes induces both humoral and cellular immune responses. These results demonstrate that the SL route is a promising way of administering influenza vaccines; we therefore suggest further investigation of influenza vaccines administered sublingually.