Unlike muscle, skin is the first line of defense against most pathogen and is therefore, very rich in immune cells and molecules. Skin specifically contains Langerhans cells, which are known to help in generating a robust immune response. Many researchers have also demonstrated that a smaller vaccine dose when delivered to the skin as compared to the muscle can produce a comparable immune response, suggesting that skin immunization can lead to dose reduction by as much as a factor of ten. This is especially relevant in the current context of shortage of influenza vaccine, where dose reduction will be very beneficial. Microneedles make targeting the skin layer for immunization more straight forward, and therefore can be expected to result in a safer, simpler, painless and highly-effective vaccination method.
Microneedles can be used in a variety of ways to deliver vaccines/drugs into the skin, for example by coating the vaccine/drug onto solid microneedles which then dissolves in the aqueous environment of the skin upon penetration. The goal of this study was to study the immune response in mice upon vaccination using vaccine-coated microneedles.
EXPERIMENTAL METHODS Two different antigen models, namely a protein-based vaccine, and a DNA based vaccine were studied. The model antigen for protein-based vaccine was ovalbumin (OVA), while for the DNA based vaccine two different disease models were studied: a DNA encoding influenza hemagglutinin (HA) gene (DNA-HA group), and a DNA encoding the hepatitis-C non-structural gene - NS3/4A (NS3/4A-DNA group). The respective antigens were coated onto microneedles and inserted into Balb/C mice or C57Bl/6 for vaccine delivery. A boost dose was also done at 4 weeks time following the first immunization for the OVA and DNA-HA group. Quantitative ELISA was used to quantify the antibody levels upon immunization. To assess the ability of mice to generate cytotoxic T-cells, an in vivo tumor challenge assay was done.
RESULTS AND DISCUSSION For the OVA immunized mice, the dose of immunization was 30 µg followed by a boost at 4 weeks. The ovalbumin-specific IgG titers increased by 2-fold between immunizations and 22-fold relative to pre-immunization levels. It is noteworthy that ovalbumin, which by other vaccination approaches requires the use of adjuvant to elicit specific antibodies, was able to induce substantial IgG titers by microneedle immunization without an adjuvant.
In the influenza model, mice were immunized with 3.5 µg of DNA-HA per mouse both for prime and boost immunizations. The antibody binding titers for the DNA-HA group increased by 6-fold between immunizations and 37-fold relative to pre-immunization levels. As for the hemagglutination-inhibition (HI) titers, they increased almost 5-fold between priming and boosting in the DNA-HA group and 19-fold relative to pre-immunization levels. It is also noteworthy that significant titers of IgG and HI antibodies were produced against influenza hemagglutinin. The HI titers achieved are expected to be sufficiently high to provide protection against viral exposure. Viral challenge studies are currently underway to test this expectation.
In the case of hepatitis-C vaccination, mouse immunization with NS3/4A-DNA was done with 16 µg of DNA. Since a boost immunization was not done, the antibody titers were not found to increase much. This has also been observed in other modes of immunization (our previous gene gun immunization) using the same antigen. However, since previous experiments using gene gun immunization had shown development of cytotoxic T-cells, we checked for similar in vivo cytotoxic T-cell generation in microneedle immunized mice using a tumor challenge assay. Microneedle-immunized mice were challenged with tumor cells expressing the NS3/4A protein on their surface. If the mice had developed immunity to the NS3/4A upon microneedle immunization, they should reject the tumor cells, while in the case of naïve animals, the tumor should grow. The size of the tumor was found to be 3 ± 2.1 mm in the case of microneedle immunized mice as compared to 12 ± 1.3 mm in the naïve mice at day 15. These results show that even with just a single dose of microneedle-based immunization, the mice had developed cytotoxic T-cells specific to NS3/4A, a hepatitis-C antigen. These results were repeatable even with a lower dose of 3.2 µg of NS3/4A-DNA per mouse. Experiments are in progress to perform prime and boost immunizations.
CONCLUSION Microneedles were successfully coated with ovalbumin, influenza DNA-HA vaccine and hepatitis-C NS3/4A-DNA vaccine in a controllable dose of 0.3 to 3 µg per microneedle. Mouse immunization using these antigens resulted in specific antibody and cytotoxic-T cell responses without the use of adjuvants. These results show that coated microneedle-based immunization has the potential to be used as a safe and painless (from our other studies) mass immunization method.
ACKNOWLEDGEMENTS The study was funded in part by the National Institute of Health (USA) and in part by the Swedish Research Council and the European Community.