Preferential amplification of CD8 effector-T cells after transcutaneous application of an inactivated influenza vaccine: a randomized phase I trial

Abstract

Background: Current conventional vaccination approaches do not induce potent CD8 T-cell responses for fighting mostly variable viral diseases such as influenza, avian influenza viruses or HIV. Following our recent study on vaccine penetration by targeting of vaccine to human hair follicular ducts surrounded by Langerhans cells, we tested in the first randomized Phase-Ia trial based on hair follicle penetration (namely transcutaneous route) the induction of virus-specific CD8 T cell responses.

Methods and findings: We chose the inactivated influenza vaccine - a conventional licensed tetanus/influenza (TETAGRIP) vaccine - to compare the safety and immunogenicity of transcutaneous (TC) versus IM immunization in two randomized controlled, multi-center Phase I trials including 24 healthy-volunteers and 12 HIV-infected patients. Vaccination was performed by application of inactivated influenza vaccine according to a standard protocol allowing the opening of the hair duct for the TC route or needle-injection for the IM route. We demonstrated that the safety of the two routes was similar. We showed the superiority of TC application, but not the IM route, to induce a significant increase in influenza-specific CD8 cytokine-producing cells in healthy-volunteers and in HIV-infected patients. However, these routes did not differ significantly for the induction of influenza-specific CD4 responses, and neutralizing antibodies were induced only by the IM route. The CD8 cell response is thus the major immune response observed after TC vaccination.

Conclusions: This Phase Ia clinical trial (Manon05) testing an anti-influenza vaccine demonstrated that vaccines designed for antibody induction by the IM route, generate vaccine-specific CD8 T cells when administered transcutaneously. These results underline the necessity of adapting vaccination strategies to control complex infectious diseases when CD8 cellular responses are crucial. Our work opens up a key area for the development of preventive and therapeutic vaccines for diseases in which CD8 cells play a crucial role.

Trial registration: Clinicaltrials.gov NCT00261001.

Figure 1. Differential induction of CD4 and CD8 T cell responses after TC vaccine application compared to IM immunization in healthy individuals.

Intracytoplasmic cytokine staining (ICS) of influenza-specific effector CD4 and CD8 responses was performed on frozen PBMC samples from vaccinated individuals: 10/12 from the TC group and 7–9/12 from IM group with 90% cell viability after thawing. Three million cells were stimulated with the overlapping peptide covering H3, H1, and NP for 12 hours at 37°C. Brefeldin A was added 4 h before harvesting. ICS was performed by flow-cytometric assays on CD3+CD4+ (left panels) and CD3+CD8+ T cells (Right panels). At least 1,000,000 live events according to forward and side scatter parameters were accumulated and analyzed (M&M section). The expression of IFN-γ, TNF-α, and/or IL-2 (triple+double+single cytokine positive cells) by influenza-specific T cells was analyzed with the Boolean gating function of FlowJo software. Results are shown as percentages of cytokine-producing T cells (Δ Day 28- Day 0) after subtracting the unstimulated cell background. Mann-Whitney test was used to compare continuous variables between the groups. Significance was set at p<0.05. Responders are determined when (Δ Day 28-Day 0) were >0. The χ2 test was used to define categorical variables between TC and IM groups.

Figure 2. Flow cytometric representation of influenza-specific T cell response at day 28 post-vaccination by TC and IM routes.

A, B) Representative flow cytometric analysis of cytokine-producing influenza-specific effector CD4 and CD8 responses. Experiments were performed on frozen PBMCs from individuals vaccinated by TC and IM routes as described in figure 1. Results are shown for a representative healthy individual with a TC route (A) and an IM route (B) vaccination at day 28. C, D) Pie chart analyses of single (white), double (gray) and triple (black)-cytokine positive cells for CD4 (C) and CD8 (D) effector cells specific for the indicated influenza protein. The expression of IFN-γ, TNF-α, and/or IL-2 (triple+double+single cytokine positive cells) by influenza-specific T cells was analyzed with the Boolean gating function of FlowJo software. NA: not applicable.

Figure 3. Differential induction of CD4 and CD8 T cell responses after TC and IM vaccine application in HIV-infected volunteers.

Intracytoplasmic cytokine staining (ICS) of influenza-specific effector CD4 and CD8 responses was performed on frozen PBMCs from vaccinated individuals. Experiments were performed on 12 HIV-infected individuals for whom cell sample viability was at least 90% after thawing. Three million cells were stimulated with the overlapping peptide covering H3, H1, and NP for 12 hours at 37°C. Brefeldin A (5 µg/ml) was added 4 h before harvesting. ICS was performed for IFN-γ by flow-cytometric assay on CD3+CD4+ (left panels) and CD3+CD8+ T cells (Right panels). At least 1,000,000 live events according to forward and side scatter parameters were accumulated and analyzed (M&M section). The expression of IFN-γ, TNF-α, and/or IL-2 (triple+double+single cytokine positive cells) by influenza-specific T cells was analyzed with the Boolean gating function of FlowJo software. Results are shown as percentages of cytokine-producing T cells (Δ Day 28-Day0) after subtracting the unstimulated cell background. Mann-Whitney tests were used to compare continuous variables between the groups. Significance was set at p<0.05.