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Long-term central and effector SHIV-specific memory T cell responses elicited after a single immunization with a novel lentivector DNA vaccine.

Arrode-Brusés G, Moussa M, Baccard-Longere M, Villinger F, Chebloune Y - PLoS ONE (2014)

Bottom Line: Prevention of HIV acquisition and replication requires long lasting and effective immunity.During the primary expansion phase, immediate effector cells as well as increasing numbers of proliferating cells with limited effector functions were detected which expressed markers of effector (EM) and central (CM) memory phenotypes.Strong PHPC responses comprising vaccine-specific CM and EM T cells that readily expanded and acquired immediate effector functions were detected at 40/47 weeks PI.

View Article: PubMed Central - PubMed

Affiliation: INRA, ANRS, Université Joseph Fourier, PAVAL Lab./Nanobio 2, UJF Grenoble, Grenoble, France.

ABSTRACT
Prevention of HIV acquisition and replication requires long lasting and effective immunity. Given the state of HIV vaccine development, innovative vectors and immunization strategies are urgently needed to generate safe and efficacious HIV vaccines. Here, we developed a novel lentivirus-based DNA vector that does not integrate in the host genome and undergoes a single-cycle of replication. Viral proteins are constitutively expressed under the control of Tat-independent LTR promoter from goat lentivirus. We immunized six macaques once only with CAL-SHIV-IN- DNA using combined intramuscular and intradermal injections plus electroporation. Antigen-specific T cell responses were monitored for 47 weeks post-immunization (PI). PBMCs were assessed directly ex vivo or after 6 and 12 days of in vitro culture using antigenic and/or homeostatic proliferation. IFN-γ ELISPOT was used to measure immediate cytokine secretion from antigen specific effector cells and from memory precursors with high proliferative capacity (PHPC). The memory phenotype and functions (proliferation, cytokine expression, lytic content) of specific T cells were tested using multiparametric FACS-based assays. All immunized macaques developed lasting peripheral CD8+ and CD4+ T cell responses mainly against Gag and Nef antigens. During the primary expansion phase, immediate effector cells as well as increasing numbers of proliferating cells with limited effector functions were detected which expressed markers of effector (EM) and central (CM) memory phenotypes. These responses contracted but then reemerged later in absence of antigen boost. Strong PHPC responses comprising vaccine-specific CM and EM T cells that readily expanded and acquired immediate effector functions were detected at 40/47 weeks PI. Altogether, our study demonstrated that a single immunization with a replication-limited DNA vaccine elicited persistent vaccine-specific CM and EM CD8+ and CD4+ T cells with immediate and readily inducible effector functions, in the absence of ongoing antigen expression.

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Polyfunctional SHIV-specific CD8+ T cell recall responses.At various indicated time points pre- and post-immunization PBMCs from vaccinated animals were labeled with CFSE and cultured in presence of specific pools of SHIV peptides Gag, Env, TRN, Pol or medium without peptide for 5 days. On day 5, cells were harvested and restimulated overnight with the same cocktail of peptides (designated as Gag(5d) Gag(16 h)) in the presence of costimulatory Abs and Brefeldin A. Cells were then surface-stained with EMA, CD3, CD8, CD4 mAbs, permeabilized and stained with IFN-γ, IL-2 and Granzyme B mAbs. For flow cytometry analysis, we initially gated on live lymphocytes (low FSC/SSC, EMA-, CD3+), bright CD8+ T cell populations (colored in orange). Antigen-specific T cells were identified by their capacity to proliferate, secrete cytokines and contain lytic molecules (black dots). A) Results for two representative animals (BX72 and BX78) were displayed. The proportion of cells producing IFN-γ (contour plot; upper number) and proliferating (CFSE dilution, contour plot, lower number) in response to specific antigens is indicated in each plot. Frequencies for antigen-specific responses are reported as the percent of cytokine-secreting and proliferating CD8+ T cells after the subtraction of backgrounds obtained with cells cultured for 5 days with medium only and restimulated for 6 h with relevant peptide pools. B) Summary of the frequencies of proliferating (CFSE low) CD8+ T cells detected against each indicated antigen (Gag (blue), Env (red), TRN (green), Pol (purple)) in each immunized animal (BX72, 73, 78, 80, 83, 84) at various weeks post-immunization. Of note, results obtained between W3 and W8 for BX83 were excluded due to non-specific T cell hyperactivation (indicated by an interrupted x-axis).
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pone-0110883-g002: Polyfunctional SHIV-specific CD8+ T cell recall responses.At various indicated time points pre- and post-immunization PBMCs from vaccinated animals were labeled with CFSE and cultured in presence of specific pools of SHIV peptides Gag, Env, TRN, Pol or medium without peptide for 5 days. On day 5, cells were harvested and restimulated overnight with the same cocktail of peptides (designated as Gag(5d) Gag(16 h)) in the presence of costimulatory Abs and Brefeldin A. Cells were then surface-stained with EMA, CD3, CD8, CD4 mAbs, permeabilized and stained with IFN-γ, IL-2 and Granzyme B mAbs. For flow cytometry analysis, we initially gated on live lymphocytes (low FSC/SSC, EMA-, CD3+), bright CD8+ T cell populations (colored in orange). Antigen-specific T cells were identified by their capacity to proliferate, secrete cytokines and contain lytic molecules (black dots). A) Results for two representative animals (BX72 and BX78) were displayed. The proportion of cells producing IFN-γ (contour plot; upper number) and proliferating (CFSE dilution, contour plot, lower number) in response to specific antigens is indicated in each plot. Frequencies for antigen-specific responses are reported as the percent of cytokine-secreting and proliferating CD8+ T cells after the subtraction of backgrounds obtained with cells cultured for 5 days with medium only and restimulated for 6 h with relevant peptide pools. B) Summary of the frequencies of proliferating (CFSE low) CD8+ T cells detected against each indicated antigen (Gag (blue), Env (red), TRN (green), Pol (purple)) in each immunized animal (BX72, 73, 78, 80, 83, 84) at various weeks post-immunization. Of note, results obtained between W3 and W8 for BX83 were excluded due to non-specific T cell hyperactivation (indicated by an interrupted x-axis).

Mentions: We used the same assay to longitudinally examine the T cell responses from PBMCs in the macaques immunized in the current study. Results from two representative macaques (BX72 and 78) are shown in Figure 2A. During the first 4 weeks PI, Gag-specific CD8+ T cells that were detected have moderate capacity of proliferation (<3 divisions), no immediate effector functions (IFN-γ-, IL-2-) but contained the lytic Granzyme B (>80% positive cells). By week 6 PI, cells with higher proliferative capacity (>3 divisions) together with cells producing IFN-γ have developed. However, the proportions of IFN-γ producing cells always represented minor fractions of antigen-specific proliferative T cells and they never exceeded 1% of total CD8+ T cell populations at any of the time points or animals tested. After this primary peak, Gag-specific proliferative responses decreased and contracted to levels below detection for several weeks until they started to re-emerge (Figure 2A). These secondary T cell responses appeared at later time points, in the absence of booster immunization, and their profile showed similar properties to those found in the primary peak. These Gag specific CD8+ T responses were composed of cells with low and high proliferative capacity with limited immediate IFN-γ responses but containing Granzyme B in more than 50% of the responding cells.


Long-term central and effector SHIV-specific memory T cell responses elicited after a single immunization with a novel lentivector DNA vaccine.

Arrode-Brusés G, Moussa M, Baccard-Longere M, Villinger F, Chebloune Y - PLoS ONE (2014)

Polyfunctional SHIV-specific CD8+ T cell recall responses.At various indicated time points pre- and post-immunization PBMCs from vaccinated animals were labeled with CFSE and cultured in presence of specific pools of SHIV peptides Gag, Env, TRN, Pol or medium without peptide for 5 days. On day 5, cells were harvested and restimulated overnight with the same cocktail of peptides (designated as Gag(5d) Gag(16 h)) in the presence of costimulatory Abs and Brefeldin A. Cells were then surface-stained with EMA, CD3, CD8, CD4 mAbs, permeabilized and stained with IFN-γ, IL-2 and Granzyme B mAbs. For flow cytometry analysis, we initially gated on live lymphocytes (low FSC/SSC, EMA-, CD3+), bright CD8+ T cell populations (colored in orange). Antigen-specific T cells were identified by their capacity to proliferate, secrete cytokines and contain lytic molecules (black dots). A) Results for two representative animals (BX72 and BX78) were displayed. The proportion of cells producing IFN-γ (contour plot; upper number) and proliferating (CFSE dilution, contour plot, lower number) in response to specific antigens is indicated in each plot. Frequencies for antigen-specific responses are reported as the percent of cytokine-secreting and proliferating CD8+ T cells after the subtraction of backgrounds obtained with cells cultured for 5 days with medium only and restimulated for 6 h with relevant peptide pools. B) Summary of the frequencies of proliferating (CFSE low) CD8+ T cells detected against each indicated antigen (Gag (blue), Env (red), TRN (green), Pol (purple)) in each immunized animal (BX72, 73, 78, 80, 83, 84) at various weeks post-immunization. Of note, results obtained between W3 and W8 for BX83 were excluded due to non-specific T cell hyperactivation (indicated by an interrupted x-axis).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4206452&req=5

pone-0110883-g002: Polyfunctional SHIV-specific CD8+ T cell recall responses.At various indicated time points pre- and post-immunization PBMCs from vaccinated animals were labeled with CFSE and cultured in presence of specific pools of SHIV peptides Gag, Env, TRN, Pol or medium without peptide for 5 days. On day 5, cells were harvested and restimulated overnight with the same cocktail of peptides (designated as Gag(5d) Gag(16 h)) in the presence of costimulatory Abs and Brefeldin A. Cells were then surface-stained with EMA, CD3, CD8, CD4 mAbs, permeabilized and stained with IFN-γ, IL-2 and Granzyme B mAbs. For flow cytometry analysis, we initially gated on live lymphocytes (low FSC/SSC, EMA-, CD3+), bright CD8+ T cell populations (colored in orange). Antigen-specific T cells were identified by their capacity to proliferate, secrete cytokines and contain lytic molecules (black dots). A) Results for two representative animals (BX72 and BX78) were displayed. The proportion of cells producing IFN-γ (contour plot; upper number) and proliferating (CFSE dilution, contour plot, lower number) in response to specific antigens is indicated in each plot. Frequencies for antigen-specific responses are reported as the percent of cytokine-secreting and proliferating CD8+ T cells after the subtraction of backgrounds obtained with cells cultured for 5 days with medium only and restimulated for 6 h with relevant peptide pools. B) Summary of the frequencies of proliferating (CFSE low) CD8+ T cells detected against each indicated antigen (Gag (blue), Env (red), TRN (green), Pol (purple)) in each immunized animal (BX72, 73, 78, 80, 83, 84) at various weeks post-immunization. Of note, results obtained between W3 and W8 for BX83 were excluded due to non-specific T cell hyperactivation (indicated by an interrupted x-axis).
Mentions: We used the same assay to longitudinally examine the T cell responses from PBMCs in the macaques immunized in the current study. Results from two representative macaques (BX72 and 78) are shown in Figure 2A. During the first 4 weeks PI, Gag-specific CD8+ T cells that were detected have moderate capacity of proliferation (<3 divisions), no immediate effector functions (IFN-γ-, IL-2-) but contained the lytic Granzyme B (>80% positive cells). By week 6 PI, cells with higher proliferative capacity (>3 divisions) together with cells producing IFN-γ have developed. However, the proportions of IFN-γ producing cells always represented minor fractions of antigen-specific proliferative T cells and they never exceeded 1% of total CD8+ T cell populations at any of the time points or animals tested. After this primary peak, Gag-specific proliferative responses decreased and contracted to levels below detection for several weeks until they started to re-emerge (Figure 2A). These secondary T cell responses appeared at later time points, in the absence of booster immunization, and their profile showed similar properties to those found in the primary peak. These Gag specific CD8+ T responses were composed of cells with low and high proliferative capacity with limited immediate IFN-γ responses but containing Granzyme B in more than 50% of the responding cells.

Bottom Line: Prevention of HIV acquisition and replication requires long lasting and effective immunity.During the primary expansion phase, immediate effector cells as well as increasing numbers of proliferating cells with limited effector functions were detected which expressed markers of effector (EM) and central (CM) memory phenotypes.Strong PHPC responses comprising vaccine-specific CM and EM T cells that readily expanded and acquired immediate effector functions were detected at 40/47 weeks PI.

View Article: PubMed Central - PubMed

Affiliation: INRA, ANRS, Université Joseph Fourier, PAVAL Lab./Nanobio 2, UJF Grenoble, Grenoble, France.

ABSTRACT
Prevention of HIV acquisition and replication requires long lasting and effective immunity. Given the state of HIV vaccine development, innovative vectors and immunization strategies are urgently needed to generate safe and efficacious HIV vaccines. Here, we developed a novel lentivirus-based DNA vector that does not integrate in the host genome and undergoes a single-cycle of replication. Viral proteins are constitutively expressed under the control of Tat-independent LTR promoter from goat lentivirus. We immunized six macaques once only with CAL-SHIV-IN- DNA using combined intramuscular and intradermal injections plus electroporation. Antigen-specific T cell responses were monitored for 47 weeks post-immunization (PI). PBMCs were assessed directly ex vivo or after 6 and 12 days of in vitro culture using antigenic and/or homeostatic proliferation. IFN-γ ELISPOT was used to measure immediate cytokine secretion from antigen specific effector cells and from memory precursors with high proliferative capacity (PHPC). The memory phenotype and functions (proliferation, cytokine expression, lytic content) of specific T cells were tested using multiparametric FACS-based assays. All immunized macaques developed lasting peripheral CD8+ and CD4+ T cell responses mainly against Gag and Nef antigens. During the primary expansion phase, immediate effector cells as well as increasing numbers of proliferating cells with limited effector functions were detected which expressed markers of effector (EM) and central (CM) memory phenotypes. These responses contracted but then reemerged later in absence of antigen boost. Strong PHPC responses comprising vaccine-specific CM and EM T cells that readily expanded and acquired immediate effector functions were detected at 40/47 weeks PI. Altogether, our study demonstrated that a single immunization with a replication-limited DNA vaccine elicited persistent vaccine-specific CM and EM CD8+ and CD4+ T cells with immediate and readily inducible effector functions, in the absence of ongoing antigen expression.

Show MeSH
Related in: MedlinePlus