In an attempt to improve H5N1 vaccine delivery approaches, we have determined the immunogenicity and protective efficacy elicited by microneedle administration of an H5 VLP antigen to mouse skin. Our results indicate that prime-boost microneedle vaccination in the skin with low doses of H5 VLPs can induce long-lasting humoral and cellular immune responses, as well as protective immunity. Higher levels of antibody-secreting cell responses in both spleen and bone marrow were found to be induced by the microneedle vaccination than by traditional intramuscular immunization when determined at 8 months postvaccination. More importantly, we found that microneedle delivery of H5 VLP vaccines to viable human skin can mobilize CD207+ human LCs. In addition, microneedle vaccination has logistic advantages, such as potential self-administration and avoiding the use of hypodermic needles and syringes. Therefore, the results in this study have significant implications in demonstrating the feasibility of microneedle vaccination for humans.
We showed that an H5 VLP vaccine delivered via a surface coating on microneedles induces protective immunity at least comparable to that induced by intramuscular immunization. VLPs alone would not cross the skin barrier at a meaningful level, as we have shown that the immune responses without microneedles were minimal even with over 200-fold-higher doses (100 μg) of inactivated whole virus applied to the skin (39
). The VLP doses used for microneedle vaccination were 0.4 μg total protein containing approximately 40 ng of HA (19
). The immunogenicity of egg-derived inactivated subunit H5N1 vaccines administered without adjuvants in preclinical and clinical studies was far less satisfactory than that of seasonal influenza vaccines (14
). We found that a single dose of microneedle vaccination with H5 VLPs induced low levels of antibody responses, which is consistent with results showing low immunogenicity of H5 vaccines in previous studies. High doses and/or multiple injections of H5 vaccines were needed to obtain satisfactory seroconversion rates. In humans, two 90-μg HA doses of baculovirus-expressed or inactivated subunit vaccines produced in eggs were needed to induce antibody responses that were expected to be protective in 54 to 58% of individuals vaccinated (5
). A prime-boost immunization regimen of adjuvanted inactivated H5N1 whole virus or a split vaccine containing 3 μg HA has been applied to induce protective immunity or to improve protective efficacy in mice (9
). Our results demonstrated that significant levels of H5-specific antibodies were induced after prime-boost vaccination with an even lower dose of H5 VLPs via either microneedle skin delivery or the intramuscular route. Therefore, our study suggests that VLPs are an effective immunogen for inducing protective immunity against H5N1 influenza virus via microneedle vaccination in the skin.
The microneedles used in this study were designed to deliver coated vaccines in a dry form to the epidermis and dermis of the skin, as previously described (21
). H5 VLPs delivered to the skin via microneedles induced effective humoral and cellular immune responses after boost immunization, including IgG1 and IgG2a isotype antibodies and cytokine (IFN-γ and IL-4)-secreting splenocytes, which were observed to be significantly higher in the microneedle group than in the intramuscular group. Microneedle immunization also induced more balanced immune responses than the intramuscular route, including both T helper type 1 (Th1) and Th2, as shown by increases in both IgG1 and IgG2a antibody levels after boost vaccination. In contrast, intramuscular immunization with influenza VLPs induced IgG2a antibody as a dominant isotype, probably due to preferential activation and differentiation of conventional B2 cells to secrete IgG2a isotype antibody by VLP immunization (52
). It was previously reported that epidermal gene gun delivery of DNA vaccines induced IgG1 as a dominant antibody, as well as IFN-γ- and IL-4-secreting T-cell responses (10
), whereas the same DNA vaccines delivered by intramuscular injection induced a Th1 pattern of immune responses, including IgG2a (10
). Therefore, the route of vaccination can influence the pattern of immune responses, as well as protective efficacy.
It was demonstrated that aggregates of inactivated split influenza vaccines can influence the Th1/Th2 immune responses by increasing the Th2-type cytokine-producing cells (IL-4 and IL-5), but not by changes in the ratios of antibody isotypes IgG1 and IgG2a (2
). Microneedle vaccination with inactivated influenza H1N1 viral vaccines (A/PR/8/34) without trehalose stabilization induced high IgG1 and low IgG2a antibody levels compared to trehalose-stabilized microneedle vaccination, which was partially attributed to the formation of particle aggregates (21
). A Th2-type immune response was implicated as being undesirable in humans (2
). Thus, it is important to improve the Th1-type immune responses after microneedle vaccination with H5 VLPs. Although trehalose stabilization was shown to prevent the formation of influenza vaccine aggregates and to induce higher levels of IgG2a antibodies (21
), the low immunogenicity of H5 vaccines (A/VN/1203/04) might also have contributed to the types of immune responses. Microneedle coating with H5 VLPs needs further investigation to improve Th1-type immune responses.
A recent study demonstrated that simian-human immunodeficiency VLPs trafficked to and were associated with local lymph nodes for longer times after intradermal immunization on the abdominal site than after intramuscular injection of mice and resulted in enhanced immune responses (8
). Also, we demonstrated that microneedle delivery on the dorsal skin can load a model compound into dendritic cells, more effectively draining to the local lymph nodes than intramuscular injection, using a fluorescent dye as a model compound (22
). These studies indicate that different anatomical sites of the skin did not significantly affect immune responses in a mouse model, but it remains to be determined in humans. Our complementary experiments investigated the effects on LCs after delivery of VLPs from coated microneedles using a previously validated excised-human-skin model (28
). Importantly, this study demonstrated that the numbers of human CD207+
LCs in a given epidermal plane were significantly lower after administration of influenza H5 VLP vaccine to the skin using microneedles. Tissue sections further confirmed changes in the LC distribution pattern following VLP exposure, suggesting their mobilization in the human skin environment. VLP vaccines were shown to effectively activate dendritic cells, enhancing the cell surface activation markers CD80 and CD86 (38
). Therefore, effective uptake and stimulation of antigen-presenting cells, such as LCs, by delivery of particulate VLP vaccines to the skin is likely to be a mechanism for inducing long-lasting immune responses, as evidenced by high levels of antibody-secreting B-cell responses and IFN-γ cytokine-producing T cells after skin vaccination.
Vaccine delivery using coated microneedles is a new platform for vaccination. The vaccines are delivered in a dry formulation, and delivery does not involve hypodermic needles and syringes. The patch-based format of microneedles should simplify vaccination and may enable self-administration. Production of VLP vaccines does not require the handling of live influenza viruses during vaccine manufacture and does not rely on problematic egg supplies. Therefore, microneedle vaccination with VLPs provides an attractive approach to improve both vaccination efficacy and coverage for seasonal and pandemic viruses that warrants further studies.