Health & Medical Vaccinations

Microneedle and Mucosal Delivery of Influenza Vaccines

Microneedle and Mucosal Delivery of Influenza Vaccines

Skin Vaccination Using Microneedles

Concept of Skin Delivery Using Various Forms of Microneedles

The initial designs of microneedles were arrays of microneedles that spanned several hundred microns (Figure 2). Coated or dissolvable microneedles, or microneedles alone can be used either to pierce or make microscopic holes in the skin's outer layer, the straturm corneum. Compounds delivered to the skin using microneedles include bovine serum albumin (as a model protein antigen), oligonucleotides or plasmid DNA vaccines, latex particles of viral dimensions, recombinant bacterial proteins, and live attenuated virus vaccines.

Microneedles are fabricated to create micron-scale needles. Solid microneedle vaccines were demonstrated to be successfully used for coating with protein antigens in a dry formulation. These coated vaccines can dissolve from microneedles within the skin on a time scale of seconds or minutes (Figure 3). This concept of solid microneedle vaccines was applied to various model compounds including proteins such as ovalbumin.

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Figure 3.

Histology images of insertion and delivery of fluorescence by a coated microneedle into guinea pig skin. (A) White light and (B) fluorescence images (scale bar: 200 µm).

In contrast to solid-coated microneedles, dissolvable polymer microneedles with encapsulated drugs or vaccines have also been developed. Dissolvable microneedles composed of a biocompatible and mechanically robust material have been fabricated from polymers such as polylactic-co-glycolic acid, dissolving sugar or polysaccharide. Dissolving microneedles in the skin leaves behind no sharp or hazardous waste. This concept of dissolvable polymer microneedles has been demonstrated for the delivery of insulin and other model compounds. In a recent study, the concept of dissolving polymer microneedles was demonstrated to deliver encapsulated inactivated influenza virus to the skin of mice. Immunized mice with dissolving microneedle influenza vaccines showed effective lung viral control and cellular recall responses after challenge, suggesting that dissolving microneedles can provide a simpler and safer vaccination method.

Intradermal Influenza Vaccination Using Hollow Microneedles

In contrast to the solid microneedles mentioned above, hollow microneedles are composed of a hollow fluid conduit through the body of the individual needle, a vaccine reservoir and fluid delivery system such as a syringe, pump or other pressure-generating device. One or more hollow needles that are used to allow passage of a liquid formulation into the skin are generally in the range of 1.0–1.5 mm in length (30–34 gauge). Intradermal delivery using hollow microneedles has been demonstrated for a variety of compounds and vaccines in animals and in humans. Intradermal influenza vaccines were first licensed (Table 1) and marketed under various tradenames through the commercially available hollow microneedle system, BD Soluvia™ (Becton Dickinson, NJ, USA). This licensed intradermal influenza vaccine is based on a single 1.5-mm stainless steel microneedle in a glass prefilled syringe with a self-deploying safety shield after delivery, and has been reported in several studies.

The microinjection system using seasonal trivalent influenza vaccine at a lower dose in adult volunteers induced humoral immune responses (hemagglutination inhibition titers) comparable to those by the standard intramuscular route. That is, 9 or 15 µg of intradermal dose of trivalent seasonal influenza vaccines was comparably immunogenic to the 15 µg standard intramuscular dose for all strains. In the elderly volunteers, intradermal immunization with 15 µg of trivalent inactivated vaccine using the BD Soluvia microinjection system is likely to be more immunogenic as compared with the intramuscular delivery route or noninferior to conventional intramuscular vaccination. In a multicenter, randomized study including 1107 healthy volunteers of more than 60 years of age, intradermal trivalent inactivated influenza vaccines containing 15 or 21 µg of hemagglutinin per strain were administered using a microinjection system and the strain-specific hemagglutination inhibition geometric mean titers were determined in comparison with standard intramuscular immunization. Seroprotection and seroconversion rates were significantly higher in each intradermal vaccine. Although the mechanisms in enhancing the immunogenicity of intradermal vaccines are not well understood, dermal DCs might be involved in stimulating cellular immune responses in the elderly. Also, in 112 healthy young children aged 3–18 years, intradermal influenza vaccination at a fifth of a standard dose was reported to elicit comparable immunogenicity to full-dose intramuscular vaccination.

In a study of skin thickness (epidermis–dermis) to have an insight into the potential site of intradermal vaccine delivery, it was reported that the suprascapular had 2.54 mm, the deltoid 2.02 mm and the waist 1.91 mm of mean skin thickness. Therefore, microneedles with 1.5 mm length would be assumed to deliver the antigen into the dermal layer. The skin sites of deltoid or suprascapular are therefore deemed to be appropriate in the body. The microneedle injection system appears to be easy to perform, reliable and consistent in administering the given dose. The systemic reactogenicity (e.g., muscle pain) of the intradermal route is significantly less but minor transient reactions (redness) at the injection sites are more frequently observed. The acceptability of the intradermal route using a microinjection system has a high, up to over 95%, satisfaction rate, with most responding that the new intradermal vaccination was less painful and quickly administered. It is thus expected that intradermal vaccines will help increase seasonal influenza vaccination rates in adults. Therefore, clinical trials provide sufficient evidence that delivering vaccines to the dermal layer of skin can be a promising approach for vaccination alternative to intramuscular delivery.

Influenza Vaccination in the Skin of Mice Using Solid Microneedles

Intradermal injection of influenza vaccines using needles and syringes or microinjection systems has been widely demonstrated in humans. However, its general application may have some limitations due to discomfort associated with needle sticks (in cases of using hypodermic needles and syringes) and/or cost of microinjection devices. As a new influenza vaccination, solid microneedles coated with inactivated influenza vaccines have been demonstrated in mice. In this study, an array of five microneedles 750 µm in length (Figure 2) was coated with 3.3 µg total protein of inactivated H1N1 influenza virus vaccines at a concentration of 5 mg of vaccine protein per ml. Using three sets of five-needle arrays, a total of 10 µg of influenza vaccine was successfully delivered to the mouse skin (Figure 3). Microneedle vaccinated mice showed comparable vaccine-specific antibody responses and protection as intramuscular vaccination. A similarly comparable immunogenicity was observed in another microneedle skin vaccination with 3–10 µg of inactivated H3N2 influenza virus vaccine used as intramuscular immunization. Relatively high doses of influenza vaccines and multiple arrays of microneedles were required, probably due to the unstabilized dry microneedle formulation.

Stable microneedle formulations were developed to improve the efficacy of microneedle influenza vaccines. A trehalose (disaccharide) known to stabilize biomolecules during drying was included in the microneedle coating formulation as a stabilizer (Table 1). As a result, microneedles coated with as low as 0.4 µg of inactivated influenza (A/PR8, H1N1) virus vaccine induced protective immunity, which was superior to the intramuscular immunization with the same vaccine. These findings suggest that vaccination in the skin of mice using a microneedle patch improves protective immunity and simplifies the delivery of influenza vaccines. This approach has been proved to be applicable to other influenza vaccines such as influenza H1N1, H5N1 virus-like particle vaccines and 2009 H1N1 inactivated influenza virus vaccines.

Immunological Responses After Influenza Vaccination Using Solid Microneedles

A mouse model provides a valuable tool in understanding the detailed host immune responses after delivering vaccines to the skin, which is difficult in humans. With the development of solid microneedle influenza vaccines, we could investigate host immune responses in detail after microneedle vaccination in the skin of mice. Influenza vaccine-coated solid microneedle delivery to the skin of mice could induce virus neutralizing antibody and hemagglutination inhibition responses at comparable (or higher) levels to the intramuscular route. Enhanced lung virus clearance and fewer inflammatory cytokines were observed in mice that received microneedle vaccination after challenge infection, indicating improved protective efficacy of microneedle vaccination. In addition, microneedle vaccination showed significantly higher levels of virus-specific recall IgG antibody responses after challenge, suggesting rapid host anamnestic immune responses on exposure to challenge virus. Intramuscular immunization showed decreases in levels of virus-specific antibodies at this early time after challenge infection, which might be related to delayed virus clearance.

Regarding cellular immune responses, microneedle vaccination induced higher levels of IFN-γ and IL-4 cytokine-secreting splenocytes as compared with intramuscular immunization upon MHC2 peptide stimulation, indicating enhanced MHC2-associated CD4 T-helper cell responses, which might be especially important to provide protection in the elderly. Finally, microneedle vaccination in the skin was demonstrated to increase trafficking of DCs to regional lymph nodes, which plays a role in contributing to improved protective immunity. Thus, microneedle vaccination would provide an excellent research tool in studying detailed immune responses after delivery of vaccine antigens to the skin.

Stability of Solid Microneedle Vaccines & Their Immunogenicity

We found that the simple process of microneedle coating and drying significantly decreased the stability of influenza microneedle vaccines, as indicated by the loss of hemagglutination activity, probably due to the phase change from liquid to solid formulation. Unstabilized vaccine yielded much weaker protective immune responses. Immunization using unstabilized vaccine induced IgG1 antibody as a dominant isotype whereas stabilized vaccines maintaining hemagglutination activity shifted the pattern of antibodies to IgG2a antibodies implicating the T-helper Type 1 (Th1) immune responses. Also, a much higher dose (10 µg) of unstable microneedle vaccines was required to induce protection. Importantly, the addition of trehalose to the microneedle coating formulation was required to retain hemagglutination activity after microneedle coating of influenza vaccines. Microneedle vaccines of a low dose of 0.4 µg inactivated virus with a trehalose stabilizer in the coating formulation conferred significantly enhanced protection, indicating a positive correlation between the stabilization of coated influenza vaccines and the efficacy of protection. However, it was found that trehalose sugar did not have an adjuvant effect on enhancing immunogenicity. Intact influenza virus vaccines (not exposed to microneedle coating) with and without the addition of trehalose showed similar antibody responses after intramuscular immunization, indicating a critical role of trehalose as a stabilizing agent in retaining hemagglutination activity of influenza vaccines during the coating of microneedles and drying process but not as an immune adjuvant.

Influenza vaccines provide a useful system that utilizes an easy assay of hemagglutination activity to investigate the effects of vaccine integrity and stability on its immune responses after skin vaccination. Solid microneedle vaccine studies of stabilized and unstabilized influenza vaccines provide evidence that the functional integrity of hemagglutinin in the influenza vaccine may have a significant impact on the types and qualities of host protective immune responses to influenza vaccination. Thus, it is speculated that retaining the receptor-binding functional activity of hemagglutinin protein antigens in influenza vaccines is important for the effective induction of protective immune responses. Some lymphoid DCs are more likely to induce Th1 type immune responses whereas non-receptor-mediated uptake of vaccines via macrophage cells may induce Th2 type responses affecting the pattern of antibody isotypes. Among the range of immunologic data, the recall immune responses with microneedle vaccination to the skin were significantly stronger than intramuscular immunization. After taking up transdermally delivered antigens, skin-derived DCs are known to migrate to the systemic and mucosal compartments, which may be involved in rapid recall immune responses after microneedle vaccination in the skin. Therefore, detailed immunologic study provides deeper explanations for potential improved protective efficacies by vaccine delivery to the skin. This correlation between the functional integrity of hemagglutinin and protective immunity against influenza is further supported by other influenza vaccines such as H1N1 and H5N1 influenza virus-like particle vaccines.

Coating Formulations & Stability of Solid Microneedle Vaccines

The stability of microneedle vaccines seems to be related to the composition of coating solution that is composed of 1% carboxymethylcellulose (CMC) sodium salt, detergent Lutrol F-68 NF, and with or without 15% (w/v) d-(+)-trehalose dihydrate stabilizer. It would be informative to better understand the possible roles of microneedle coating formulations. Sugar combinations of trehalose, mannitol, dextran and arginine glutamate were developed as a stabilizing formulation for a dry powder form of influenza vaccines. Some carbohydrate compounds such as trehalose, sucrose, glucose, inulin and dextran were shown to prevent damages caused by drying or freezing of biomolecules. Among the different carbohydrate stabilizers tested, trehalose was found to be the most effective for stabilizing influenza microneedle vaccines. Addition of trehalose retained 50–80% hemagglutination activity for all three major strains of seasonal influenza A H1N1 and H3N2, and influenza B viruses. In the case of a spray freeze-drying process, HEPES-buffered saline provided good stability.

Drying influenza vaccines in the coating solution caused more damage to hemagglutination activity than drying in phosphate-buffered saline, indicating the potential effects of components in microneedle coating solution. In the absence of trehalose, hemagglutination activity of vaccines almost disappeared independent of CMC concentrations. Although removing CMC from the coating formulation allowed us to retain 100% hemagglutination activity after drying the vaccines in the presence of trehalose, CMC was nonetheless required to produce thick coatings onto microneedles. Without CMC, the mass of virus coated on microneedles was significantly reduced by more than an order of magnitude. The presence of detergent Lutrol F-68 NF (0.5%) that is also needed for effective coating onto metal microneedles did not show significant effects on stability of microneedle vaccines. Therefore, trehalose and CMC are important excipients in the coating solution for microneedle vaccine formulations.

Long-term Stability of Solid Microneedle Vaccines

Cold-chain delivery and storage is required during an influenza vaccination campaign as temperature is an important factor for determining the efficacy of influenza vaccines. In liquid formulation of influenza vaccines, storage at 4°C is needed for maintaining the stability of vaccines as measured by hemagglutination activity and storage at 25°C resulted in a significant loss of vaccine activity. By contrast, microneedle vaccines showed a similar pattern of stability kinetics at 4 and 25°C. Within a day storage of influenza microneedle vaccines, there was an approximately 30% loss in hemagglutination activity at both 4 and 25°C and 40% loss at 37°C. After 7 days of storage, a low flat point of approximately 30% activity was maintained up to 28 days, monitored at 25°C storage. Most importantly, 100% protective immunity was observed with microneedle vaccines stored at 25°C for 28 days. Therefore, solid microneedle vaccines can be developed as an alternative to cold-chain influenza vaccines (Table 1).

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