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

Combadière B, Vogt A, Mahé B, Costagliola D, Hadam S, Bonduelle O, Sterry W, Staszewski S, Schaefer H, van der Werf S, Katlama C, Autran B, Blume-Peytavi U - PLoS ONE (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institut National de Santé et de Recherche Médicale, INSERM U945, Paris, France. behazine.combadiere@upmc.fr

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.

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Related in: MedlinePlus

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.
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pone-0010818-g002: 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.

Mentions: The epidermal route of immunization described in our study is intended to target mostly epidermal LCs [36]. These APCs have been shown to be more potent in inducing CD8 cells in in vitro studies [20]–[23]. In addition, effector CD4 and CD8 cells both provide cellular immune responses and can secrete multiple cytokines that reflect the quality of the effector-cell compartment of the immune responses. Multiparametric flow cytometry assays were performed to determine the relative importance of the subpopulations of influenza-specific CD3+CD4+ and CD3+CD8+ T cells that produce IL-2, IFN-γ, and TNF-α. Overlapping peptides were designed for three major influenza antigens included in the seasonal vaccine: i) H3, a recent strain not contained in seasonal influenza vaccines in Europe over the past five years, ii) H1, repeatedly present in influenza seasonal vaccines over the past five years, and iii) NP. We further analyzed IL-2, IFN-γ, and TNF-α production by CD4 and CD8 cells after ex vivo stimulation of T cells with overlapping 20-mer peptides of H3, H1, and NP. Because of the high variability of baseline influenza-specific T cells in healthy individuals, we measured the course of influenza-specific T cell responses from D0. Strikingly, we found that the frequencies of H3-, H1-, and NP-specific CD8 cells producing cytokines (IL-2, IFN-γ, and TNF-α) were significantly higher after TC than after IM vaccination (H3 p = 0.0164, H1 p = 0.031, NP p = 0.007), mainly because the levels of CD8 responses after IM administration was extremely low (Figure 1, upper panels). Influenza-specific CD4 responses were similar for both routes (H3 p = 0.719, H1 p = 0.408, NP p = 0.299). In addition, we found a higher proportion of positive responders after TC compared with IM immunization, as depicted in Figure 1 (upper panels) (χ2 test, H3 p = 0.02, H1 p = 0.035, NP p = 0.035) for influenza-specific CD8 but not CD4 responses (Fig 2, lower panels). Note the very high frequencies of CD8+cytokine+ cells against H3 proteins after TC vaccination. Overall, we observed preferential induction of CD8 responses against all three protein compounds included in the inactivated influenza vaccine when administered by the TC but not the IM route.


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

Combadière B, Vogt A, Mahé B, Costagliola D, Hadam S, Bonduelle O, Sterry W, Staszewski S, Schaefer H, van der Werf S, Katlama C, Autran B, Blume-Peytavi U - PLoS ONE (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2877091&req=5

pone-0010818-g002: 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.
Mentions: The epidermal route of immunization described in our study is intended to target mostly epidermal LCs [36]. These APCs have been shown to be more potent in inducing CD8 cells in in vitro studies [20]–[23]. In addition, effector CD4 and CD8 cells both provide cellular immune responses and can secrete multiple cytokines that reflect the quality of the effector-cell compartment of the immune responses. Multiparametric flow cytometry assays were performed to determine the relative importance of the subpopulations of influenza-specific CD3+CD4+ and CD3+CD8+ T cells that produce IL-2, IFN-γ, and TNF-α. Overlapping peptides were designed for three major influenza antigens included in the seasonal vaccine: i) H3, a recent strain not contained in seasonal influenza vaccines in Europe over the past five years, ii) H1, repeatedly present in influenza seasonal vaccines over the past five years, and iii) NP. We further analyzed IL-2, IFN-γ, and TNF-α production by CD4 and CD8 cells after ex vivo stimulation of T cells with overlapping 20-mer peptides of H3, H1, and NP. Because of the high variability of baseline influenza-specific T cells in healthy individuals, we measured the course of influenza-specific T cell responses from D0. Strikingly, we found that the frequencies of H3-, H1-, and NP-specific CD8 cells producing cytokines (IL-2, IFN-γ, and TNF-α) were significantly higher after TC than after IM vaccination (H3 p = 0.0164, H1 p = 0.031, NP p = 0.007), mainly because the levels of CD8 responses after IM administration was extremely low (Figure 1, upper panels). Influenza-specific CD4 responses were similar for both routes (H3 p = 0.719, H1 p = 0.408, NP p = 0.299). In addition, we found a higher proportion of positive responders after TC compared with IM immunization, as depicted in Figure 1 (upper panels) (χ2 test, H3 p = 0.02, H1 p = 0.035, NP p = 0.035) for influenza-specific CD8 but not CD4 responses (Fig 2, lower panels). Note the very high frequencies of CD8+cytokine+ cells against H3 proteins after TC vaccination. Overall, we observed preferential induction of CD8 responses against all three protein compounds included in the inactivated influenza vaccine when administered by the TC but not the IM route.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institut National de Santé et de Recherche Médicale, INSERM U945, Paris, France. behazine.combadiere@upmc.fr

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.

Show MeSH
Related in: MedlinePlus