Safety, tolerability, and immunogenicity of influenza vaccination with a high-density microarray patch: Results from a randomized, controlled phase I clinical trial

Abstract

Background: The Vaxxas high-density microarray patch (HD-MAP) consists of a high density of microprojections coated with vaccine for delivery into the skin. Microarray patches (MAPs) offer the possibility of improved vaccine thermostability as well as the potential to be safer, more acceptable, easier to use, and more cost-effective for the administration of vaccines than injection by needle and syringe (N&S). Here, we report a phase I trial using the Vaxxas HD-MAP to deliver a monovalent influenza vaccine that was to the best of our knowledge the first clinical trial to evaluate the safety, tolerability, and immunogenicity of lower doses of influenza vaccine delivered by MAPs.

Methods and findings: HD-MAPs were coated with a monovalent, split inactivated influenza virus vaccine containing A/Singapore/GP1908/2015 H1N1 haemagglutinin (HA). Between February 2018 and March 2018, 60 healthy adults (age 18-35 years) in Melbourne, Australia were enrolled into part A of the study and vaccinated with either: HD-MAPs delivering 15 μg of A/Singapore/GP1908/2015 H1N1 HA antigen (A-Sing) to the volar forearm (FA); uncoated HD-MAPs; intramuscular (IM) injection of commercially available quadrivalent influenza vaccine (QIV) containing A/Singapore/GP1908/2015 H1N1 HA (15 μg/dose); or IM injection of H1N1 HA antigen (15 μg/dose). After 22 days' follow-up and assessment of the safety data, a further 150 healthy adults were enrolled and randomly assigned to 1 of 9 treatment groups. Participants (20 per group) were vaccinated with HD-MAPs delivering doses of 15, 10, 5, 2.5, or 0 μg of HA to the FA or 15 μg HA to the upper arm (UA), or IM injection of QIV. The primary objectives of the study were safety and tolerability. Secondary objectives were to assess the immunogenicity of the influenza vaccine delivered by HD-MAP. Primary and secondary objectives were assessed for up to 60 days post-vaccination. Clinical staff and participants were blind as to which HD-MAP treatment was administered and to administration of IM-QIV-15 or IM-A/Sing-15. All laboratory investigators were blind to treatment and participant allocation. Two further groups in part B (5 participants per group), not included in the main safety and immunological analysis, received HD-MAPs delivering 15 μg HA or uncoated HD-MAPs applied to the forearm. Biopsies were taken on days 1 and 4 for analysis of the cellular composition from the HD-MAP application sites. The vaccine coated onto HD-MAPs was antigenically stable when stored at 40°C for at least 12 months. HD-MAP vaccination was safe and well tolerated; any systemic or local adverse events (AEs) were mild or moderate. Observed systemic AEs were mostly headache or myalgia, and local AEs were application-site reactions, usually erythema. HD-MAP administration of 2.5 μg HA induced haemagglutination inhibition (HAI) and microneutralisation (MN) titres that were not significantly different to those induced by 15 μg HA injected IM (IM-QIV-15). HD-MAP delivery resulted in enhanced humoral responses compared with IM injection with higher HAI geometric mean titres (GMTs) at day 8 in the MAP-UA-15 (GMT 242.5, 95% CI 133.2-441.5), MAP-FA-15 (GMT 218.6, 95% CI 111.9-427.0), and MAP-FA-10 (GMT 437.1, 95% CI 254.3-751.3) groups compared with IM-QIV-15 (GMT 82.8, 95% CI 42.4-161.8), p = 0.02, p = 0.04, p < 0.001 for MAP-UA-15, MAP-FA-15, and MAP-FA-10, respectively. Higher titres were also observed at day 22 in the MAP-FA-10 (GMT 485.0, 95% CI 301.5-780.2, p = 0.001) and MAP-UA-15 (367.6, 95% CI 197.9-682.7, p = 0.02) groups compared with the IM-QIV-15 group (GMT 139.3, 95% CI 79.3-244.5). Results from a panel of exploratory immunoassays (antibody-dependent cellular cytotoxicity, CD4+ T-cell cytokine production, memory B cell (MBC) activation, and recognition of non-vaccine strains) indicated that, overall, Vaxxas HD-MAP delivery induced immune responses that were similar to, or higher than, those induced by IM injection of QIV. The small group sizes and use of a monovalent influenza vaccine were limitations of the study.

Conclusions: Influenza vaccine coated onto the HD-MAP was stable stored at temperatures up to 40°C. Vaccination using the HD-MAP was safe and well tolerated and resulted in immune responses that were similar to or significantly enhanced compared with IM injection. Using the HD-MAP, a 2.5 μg dose (1/6 of the standard dose) induced HAI and MN titres similar to those induced by 15 μg HA injected IM.

Trial registration: Australian New Zealand Clinical Trials Registry (ANZCTR.org.au), trial ID 108 ACTRN12618000112268/U1111-1207-3550.

Fig 1. Trial profile.

Randomisation and flow of participants in parts A and B of the study. Participants in part A were vaccinated with either A/Singapore/GP1908/2015 H1N1 delivered by HD-MAP (A-MAP-FA-15), IM injection of Afluria quadrivalent vaccine (A-IM-QIV-15), uncoated HD-MAP (A-MAP-FA-0), or A/Singapore/GP1908/2015 H1N1 monovalent pooled harvest injected IM (IM-SIN-15). Participants in part B were vaccinated with A/Singapore/GP1908/2015 H1N1 at 15, 10, 5, or 2.5 μg HA/dose delivered by HD-MAPs applied to the volar forearm (MAP-FA-15, MAP-FA-10, MAP-FA-5, MAP-FA-2.5), uncoated HD-MAPs (MAP-FA-0), A/Singapore/GP1908/2015 H1N1 at 15 μg HA/dose delivered by HD-MAP applied to the upper arm (MAP-UA-15), or injected IM with Afluria quadrivalent vaccine (IM-QIV-15). HA, haemagglutinin; HD-MAP, high-density microarray patch; IM, intramuscular.

Fig 2. Coating and stability of A/Sing HA on HD-MAPs.

The left-hand panel shows a scanning electron micrograph of HD-MAPs coated with A/Sing MPH. The coated vaccine is visible on the tips and top half of the projections. The graphs show stability of A/Sing HA on HD-MAPs coated with either 5 μg or 15 μg HA (in A/Sing MPH) indicated by the dotted line. Antigen-coated HD-MAPs were stored at 2 to 8°C, 25°C or 40°C at 60% ± 5% RH for 1, 3, 6, or 12 months. At each time point, antigen was eluted from the HD-MAP and the HA content determined by enzyme immunoassay. A/Sing, A/Singapore/GP1908/2015 H1N1; HA, haemagglutinin; HD-MAP, high-density microarray patch; RH, relative humidity.

Fig 3. Representative images of skin reactions over time at HD-MAP application sites (single participant (S316/B0139), MAP-FA-10 group).

Photographs show 3 HD-MAPs applied to adjacent sites on the forearm; site 1 (nearest the elbow crease) is the uncoated patch, and sites 2 and 3 are applications of the A/Sing coated HD-MAPs delivering 5 μg HA into the skin per patch. Timepoints at 1 h and 24 h are not shown in the image sequence. Photo credit Nucleus Network Pty Ltd. A/Sing, A/Singapore/GP1908/2015 H1N1; FA, forearm; HA, haemagglutinin; HD-MAP, high-density microarray patch; MAP, microarray patch.

Fig 4. Time course of injection site reaction resolution.

(A) SII, scale 0 to 8. (B) visibility (i.e., colouration at the application site), scale 0 to 3. Symbols represent mean scores ± 95% confidence intervals. Day, study day (HD-MAP applied on day 1); HD-MAP, high-density microarray patch; Min, minutes after HD-MAP application; SII, skin irritation index.

Fig 5. Self-reported pain scores using a visual analogue scale from 0 (no pain) to 10 (worst pain imaginable).

Symbols represent mean scores ± 95% confidence intervals. Day, study day (HD-MAP applied on day 1); HD-MAP, high-density microarray patch; Min, minutes after HD-MAP application.

Fig 6. HAI titres for participants in part B at study days 1 (prevaccination), 4, 8, 22, and 61.

Participants were vaccinated with A/Singapore/GP1908/2015 H1N1 at 15, 10, 5, or 2.5 μg HA/dose delivered by HD-MAPs applied to the volar forearm (MAP-FA-15, MAP-FA-10, MAP-FA-5, MAP-FA-2.5); uncoated HD-MAPs (MAP-FA-0); A/Singapore/GP1908/2015 H1N1 at 15 μg HA/dose delivered by HD-MAP applied to the upper arm (MAP-UA-15); or injected IM as a component of the Afluria quadrivalent vaccine (IM-QIV-15). Symbols represent the GMTs and the error bars show the 95% confidence intervals. GMT, geometric mean titre; HAI, HA inhibition; HD-MAP, high-density microarray patch; IM, intramuscular.

Fig 7. HA-specific FcR-binding antibodies.

Antibodies specific for A/Singapore/GP1908/2015 monovalent purified harvest that engage with dimeric, soluble recombinant FcγRIII were measured by ELISA. (A) Midpoint ELISA titres. (B) Fold-change in midpoint titres, day 22 versus day 1. Symbols represent individual responses before (D1) and after (D22) immunisation, in which horizontal lines indicate the mean response (A); columns with error bars represent the median with interquartile ranges (B). ELISA, enzyme-linked immunosorbent assay; FcR, Fc receptor; HA, haemagglutinin.

Fig 8. MBC frequencies pre- and postvaccination.

The frequencies of HA-specific MBC were assessed in cryopreserved PBMC samples by flow cytometry. Samples were gated for live, CD19+, IgD negative B cells, and specificity determined based upon binding to A/Michigan/2015 probes alone or in combination with A/New Caledonia/1999 or a stabilised H1N1 stem probes. (A and B) A/Michigan/2015 H1N1; (C and D) A/New Caledonia/1999 and A/Michigan/2015 H1N1 cross-reactive MBC; (E and F) H1 stem. Results are expressed as the frequency of probe-binding cells at days 1 and 22 (A, C, and E) with symbols representing individual responses before (D1) and after (D22) immunisation, and horizontal lines indicating the mean response; fold-change at day 22 compared with baseline (B, D, and F). Columns represent the median fold change; error bars represent the median with interquartile ranges. HA, haemagglutinin; MBC, memory B cell; PBMC, peripheral blood mononuclear cell.

Fig 9

Frequency of CD4⁺ cells producing IL-2, TNF-α, or IFN-γ following stimulation of PBMC with (A) overlapping peptides spanning A/Sing HA (5 μg/ml) or (B) A/Sing MPH (20 μg/ml). Cells were labelled with Live/Dead Aqua for viability; CD3, CD4, and CD8; permeabilised; and subsequently labelled with anti-IFN-γ, anti-TNF-α, and anti-IL-2. Approximately 500,000 events were acquired using a Becton Dickinson LSR Fortessa X20 and data analysed using SPICE software. Day 1 versus day 22 comparisons were made using the Wilcoxon rank sum test: **p < 0.01, *p < 0.05. HA, haemagglutinin; IFN, interferon; IL, interleukinMPH, monovalent purified harvest; PBMC, peripheral blood mononuclear cell; TNF, tumour necrosis factor.