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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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SHIV-specific CD4+ T cell recall responses.PBMCs were processed and labeled using the procedure described in the legend of Figure 2. A) Flow cytometry data analyses were performed by gating on low FSC/SSC, EMA-, CD3+ and CD4+ T cell populations (colored in blue). Representative results obtained for macaques BX73, 80 and 83 at the pre-immunization time point and at the time of maximal immune response (IR) for Env antigen are displayed. Frequencies for Env-specific responses are reported as the percent of proliferating CD4+ T cells after the subtraction of backgrounds obtained with cells cultured for 5 days with medium alone and restimulated for 6 h with relevant peptides pools. B) Summary of the frequency of proliferating (CFSE low) CD4+ 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) following 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-g003: SHIV-specific CD4+ T cell recall responses.PBMCs were processed and labeled using the procedure described in the legend of Figure 2. A) Flow cytometry data analyses were performed by gating on low FSC/SSC, EMA-, CD3+ and CD4+ T cell populations (colored in blue). Representative results obtained for macaques BX73, 80 and 83 at the pre-immunization time point and at the time of maximal immune response (IR) for Env antigen are displayed. Frequencies for Env-specific responses are reported as the percent of proliferating CD4+ T cells after the subtraction of backgrounds obtained with cells cultured for 5 days with medium alone and restimulated for 6 h with relevant peptides pools. B) Summary of the frequency of proliferating (CFSE low) CD4+ 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) following 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: In our polyfunctional recall assay we included cell surface staining for CD4+ T cells to examine vaccine-specific CD4+ T cell responses. Examples of data obtained with the pre-immune samples and those showing maximal proliferative responses against Env antigens with samples from 3 animals (BX80, 73, 83) are shown in Figure 3A. Similar to vaccine-specific CD8+ T cell responses, CD4+ T cells showed sustained proliferative ability (>3 divisions) but no immediate effector functions (IFN-γ-, IL-2-) in the 6 days culture assay. Interestingly, similar to the data obtained in our previous lentiviral-based DNA vaccine study, the vaccine-specific CD4+ T cell responses progressed through the same dynamic as the one observed with CD8+ T cell responses. Indeed, primary peaks of expansion were observed between W2 and W6 PI, followed by a contraction phase between W8 and W20 PI, and then late re-emergence peaks (W22 to W47 PI), in the absence of booster immunization, as summarized in Figure 3B. Vaccine-specific CD4+ T cells were directed against all tested antigens. The maximal proportion of total antigen responses measured across the longitudinal analysis in all animals remained below 5% of total CD4+ T cells, except for 3 animals (BX73, 78 and 83) that reached levels above 7%, mostly due to a high Env response, at single distinct time-points (W4 for BX73, W26 for BX78 and W47 for BX83). Maximal responses measured in all animals at various time points PI ranged from 0.3% to 2.9% and from 0.8% to 2.2% of total live CD4+ T cells for Gag and TRN respectively. For Pol and Env, these responses varied from 0.3% to 1.6% and from 0.3% to 7.7% respectively (Table S2). Overall, these results indicate that in parallel to vaccine-specific CD8+ T cell responses, broad and long-lasting vaccine specific CD4+ T cell responses have been induced in all vaccinated animals, and may contribute to the longevity of vaccine-specific T and B cell responses.


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)

SHIV-specific CD4+ T cell recall responses.PBMCs were processed and labeled using the procedure described in the legend of Figure 2. A) Flow cytometry data analyses were performed by gating on low FSC/SSC, EMA-, CD3+ and CD4+ T cell populations (colored in blue). Representative results obtained for macaques BX73, 80 and 83 at the pre-immunization time point and at the time of maximal immune response (IR) for Env antigen are displayed. Frequencies for Env-specific responses are reported as the percent of proliferating CD4+ T cells after the subtraction of backgrounds obtained with cells cultured for 5 days with medium alone and restimulated for 6 h with relevant peptides pools. B) Summary of the frequency of proliferating (CFSE low) CD4+ 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) following 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

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

pone-0110883-g003: SHIV-specific CD4+ T cell recall responses.PBMCs were processed and labeled using the procedure described in the legend of Figure 2. A) Flow cytometry data analyses were performed by gating on low FSC/SSC, EMA-, CD3+ and CD4+ T cell populations (colored in blue). Representative results obtained for macaques BX73, 80 and 83 at the pre-immunization time point and at the time of maximal immune response (IR) for Env antigen are displayed. Frequencies for Env-specific responses are reported as the percent of proliferating CD4+ T cells after the subtraction of backgrounds obtained with cells cultured for 5 days with medium alone and restimulated for 6 h with relevant peptides pools. B) Summary of the frequency of proliferating (CFSE low) CD4+ 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) following 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: In our polyfunctional recall assay we included cell surface staining for CD4+ T cells to examine vaccine-specific CD4+ T cell responses. Examples of data obtained with the pre-immune samples and those showing maximal proliferative responses against Env antigens with samples from 3 animals (BX80, 73, 83) are shown in Figure 3A. Similar to vaccine-specific CD8+ T cell responses, CD4+ T cells showed sustained proliferative ability (>3 divisions) but no immediate effector functions (IFN-γ-, IL-2-) in the 6 days culture assay. Interestingly, similar to the data obtained in our previous lentiviral-based DNA vaccine study, the vaccine-specific CD4+ T cell responses progressed through the same dynamic as the one observed with CD8+ T cell responses. Indeed, primary peaks of expansion were observed between W2 and W6 PI, followed by a contraction phase between W8 and W20 PI, and then late re-emergence peaks (W22 to W47 PI), in the absence of booster immunization, as summarized in Figure 3B. Vaccine-specific CD4+ T cells were directed against all tested antigens. The maximal proportion of total antigen responses measured across the longitudinal analysis in all animals remained below 5% of total CD4+ T cells, except for 3 animals (BX73, 78 and 83) that reached levels above 7%, mostly due to a high Env response, at single distinct time-points (W4 for BX73, W26 for BX78 and W47 for BX83). Maximal responses measured in all animals at various time points PI ranged from 0.3% to 2.9% and from 0.8% to 2.2% of total live CD4+ T cells for Gag and TRN respectively. For Pol and Env, these responses varied from 0.3% to 1.6% and from 0.3% to 7.7% respectively (Table S2). Overall, these results indicate that in parallel to vaccine-specific CD8+ T cell responses, broad and long-lasting vaccine specific CD4+ T cell responses have been induced in all vaccinated animals, and may contribute to the longevity of vaccine-specific T and B cell responses.

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