Stabilization of influenza vaccine enhances protection by microneedle delivery in the mouse skin

Abstract

Background: Simple and effective vaccine administration is particularly important for annually recommended influenza vaccination. We hypothesized that vaccine delivery to the skin using a patch containing vaccine-coated microneedles could be an attractive approach to improve influenza vaccination compliance and efficacy.

Methodology/principal findings: Solid microneedle arrays coated with inactivated influenza vaccine were prepared for simple vaccine delivery to the skin. However, the stability of the influenza vaccine, as measured by hemagglutination activity, was found to be significantly damaged during microneedle coating. The addition of trehalose to the microneedle coating formulation retained hemagglutination activity, indicating stabilization of the coated influenza vaccine. For both intramuscular and microneedle skin immunization, delivery of un-stabilized vaccine yielded weaker protective immune responses including viral neutralizing antibodies, protective efficacies, and recall immune responses to influenza virus. Immunization using un-stabilized vaccine also shifted the pattern of antibody isotypes compared to the stabilized vaccine. Importantly, a single microneedle-based vaccination using stabilized influenza vaccine was found to be superior to intramuscular immunization in controlling virus replication as well as in inducing rapid recall immune responses post challenge.

Conclusions/significance: The functional integrity of hemagglutinin is associated with inducing improved protective immunity against influenza. Simple microneedle influenza vaccination in the skin produced superior protection compared to conventional intramuscular immunization. This approach is likely to be applicable to other vaccines too.

Figure 1. Coating of influenza vaccines on microneedles.

(A) (i) Comparison of a microneedle array and hypodermic needle (23 gauge). (ii) The circled part of the microneedle array in (i) is shown as a bright-field micrograph with 5 microneedles coated with inactivated influenza virus. (B) Experimental design. Microneedles were coated with inactivated influenza A/PR8 with or without trehalose coating formulation. Some vaccine coated on microneedles with or without trehalose was dissolved in PBS for intramuscular injections. (C) HA activities (% of unprocessed control) were determined after reconstituting from microneedles coated with or without trehalose formulation.

Figure 2. Influenza A/PR8 specific IgG responses.

Mice (n = 12 per group) were immunized with microneedles coated with 0.4 µg of inactivated A/PR8 vaccine or by intramuscular injection with 0.4 µg of A/PR8 vaccine reconstituted from coated microneedles. Blood samples were collected at weeks 1, 2 and 4 after a single vaccination. Virus-specific antibody responses measured by ELISA are expressed as the endpoint titers. MN, microneedle vaccine without trehalose; MN+T, microneedle vaccine with trehalose; IM, intramuscular injection of reconstituted A/PR8 vaccine without trehalose; IM+T, intramuscular injection of reconstituted A/PR8 vaccine from trehalose-formulated microneedles; Mock, microneedles with trehalose coating buffer only (without A/PR8 vaccine). Statistical significances among groups compared are as follows: p<0.005 between MN+T and MN or IM at weeks 1, 2, and 4. p<0.05 between IM+T and IM or MN at weeks 1, 2, and 4. p<0.05 between MN+T and IM+T at week 4.

Figure 3. Effects of trehalose stabilized vaccine on antibody isotype responses.

(A) MN group, (B) MN+T group, (C) IM group, and (D) IM+T group. Kinetics of A/PR8 virus specific isotype antibody responses (IgG1, IgG2a, IgG2b) were determined at weeks 1, 2 and 4 after a single vaccination. Results are expressed as averages of optical density readings at 450 nm (OD₄₅₀) with 100-fold diluted serum samples in each group of mice (n = 12). (E) Ratios of IgG2a/IgG1 based on optical density readings with week-4 samples. Groups are described in the legend of Fig. 2.

Figure 4. Trehalose stabilized vaccine enhances functional antibody responses.

Serum neutralizing titers (A) and hemagglutination inhibition (HAI) titers (B) were determined at week 4 after a single vaccination (n = 12). Neutralizing activities were expressed as the percentage of plaque reduction compared to a naïve serum control. Significant differences were found among groups of mice: For neutralizing titers at the 540 dilution for plaque reduction, p<0.005 between MN+T and MN or IM. At the serum dilution 270, p<0.05 between IM+T and IM or MN. For HAI titers, p<0.001 between MN+T and MN or IM, and p<0.05 between IM+T and IM or MN.

Figure 5. Trehalose stabilization of vaccine improves protective efficacy.

At week 5 after a single vaccination, mice (n = 12) were challenged with a lethal dose (A/PR8 virus, 20× LD50) and were monitored daily to record body weight changes (A) and survival rates (B). Groups of mice are described as in the legend of Fig. 2.

Figure 6. Trehalose-stabilized microneedle vaccine improves control of lung viral replication.

Lungs from individual mice in each group (n = 6 out of 12 mice per group) were collected on day 4 post-challenge, weighed, and extracted in media. Slight variations in lung tissue weight recovered from individual mice were adjusted (0.25 g lung tissue/ml). Virus titers (plaque forming units, pfu) in log10 (A), or IFN- γ (B) and IL-6 (C) cytokines in nano-grams (ng) per gram (g) lung tissue are expressed as geometric mean values. IFN-γ and IL-6 in lung extracts were determined by ELISA. Groups of mice are described in the legend of Fig. 2. Naïve is an uninfected mouse control.

Figure 7. Higher recall immune responses induced by trehalose stabilized microneedle vaccine.

Lung, serum, and spleen samples were collected from individual mice in each group (n = 6) at day 4 post challenge. (A) Virus specific IgG1 and IgG2a antibodies in lungs (ng/g tissue: ng antibodies per g lung tissue). (B) Virus specific IgG1 and IgG2a antibodies in sera (ng/ml: ng antibodies per ml sera). (C) IFN- γ secreting splenocytes (spots/1×10⁶ spleen cells) after stimulation with hemagglutinin-specific MHC I and II peptides. (D) Neutralizing activities in lung. Serial dilutions of lung samples were incubated with infectious influenza viruses and percentiles of plaque forming units were determined. Titers/g tissue: dilution factors per g lung tissue from the lung extracts (0.25 g/ml). Groups of mice are the same as in Fig. 5. Significant differences were found among the groups: For lung IgG2a antibody (A), p<0.001 between MN+T and other groups; for lung IgG1 (A), p<0.001 between MN+T and IM or IM+T. For serum IgG2a antibody (B), p<0.001 between MN+T and other groups; for IgG1 (B), p<0.005 between MN and IM or IM+T, p<0.05 between MN+T and IM or IM+T. For spleen IFN-γ secreting cells (C), p<0.05 between MN+T and other groups. For lung neutralizing titers, p<0.05 between MN+T and other groups at 15× dilution.