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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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Evaluation and characterization of immediate effector T cell responses induced by a single immunization with the CAL-SHIV-IN− DNA vaccine.A) PBMCs isolated from blood samples taken at the indicated weeks post-immunization (PI) were used for ELISPOT assay to detect IFN-γ producing cells in response to the pools of SIV Gag (indicated in blue), HIV Env (indicated in red), HIV Tat+Rev+ SIV Nef (TRN, indicated in green) and SIV Pol (indicated in purple) peptides. Numbers of IFN-γ producing cells obtained per million of PBMCs against all Ags tested are indicated in the y-axis. B) Mean and range of IFN-γ ELISPOT responses to Gag and TRN pools across 22 (for BX72, 78, 80, 83, 84) and 14 (for BX73) time-points collected. C) Memory phenotyping of Gag-specific T cells at W8 PI. PBMCs were restimulated for 16 h in presence or absence of Gag pool of peptides in medium containing Brefeldin A. Cells were then surface stained with EMA, CD3, CD8, CD4, CD28, CD95 mAbs, permeabilized and stained with IFN-γ, IL-2 and TNF-α mAbs. For analysis, cells were initially gated on live lymphocytes (low FSC/SSC, EMA-, CD3+), CD8 bright T cells (orange color) and CD4+ T cell populations (blue color). Antigen-specific T cells were identified by their capacity to secrete one or more cytokines (black dots). For each indicated animal (BX72, 78, 80, 84), we displayed the maximal response obtained at this time point within CD8+ or CD4+ T cell populations. For simplicity, the percentage of total responses (total black dots) obtained through all quadrants (IFN-γ+, IFN-γ+ &TNF-α+, IFN-γ+&IL-2+ and IL-2+) are indicated after the subtraction of background obtained with cells cultured with medium only. For memory T cell subset determinations, all black dots were superimposed to the total CD8+ or CD4+ T cell populations and plotted against memory markers (CD28 and CD95). The naïve population was defined as CD28+ CD95−, effector memory (EM) as CD28− CD95+ and central memory (CM) as CD28+ CD95+. D) Sequential evaluation of SIV Gag p55 specific antibody responses in immunized macaques (“homemade kit”). E) Sequential evaluation of HIV Env gp160 specific antibody responses in immunized macaques (Biorad, France).
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pone-0110883-g001: Evaluation and characterization of immediate effector T cell responses induced by a single immunization with the CAL-SHIV-IN− DNA vaccine.A) PBMCs isolated from blood samples taken at the indicated weeks post-immunization (PI) were used for ELISPOT assay to detect IFN-γ producing cells in response to the pools of SIV Gag (indicated in blue), HIV Env (indicated in red), HIV Tat+Rev+ SIV Nef (TRN, indicated in green) and SIV Pol (indicated in purple) peptides. Numbers of IFN-γ producing cells obtained per million of PBMCs against all Ags tested are indicated in the y-axis. B) Mean and range of IFN-γ ELISPOT responses to Gag and TRN pools across 22 (for BX72, 78, 80, 83, 84) and 14 (for BX73) time-points collected. C) Memory phenotyping of Gag-specific T cells at W8 PI. PBMCs were restimulated for 16 h in presence or absence of Gag pool of peptides in medium containing Brefeldin A. Cells were then surface stained with EMA, CD3, CD8, CD4, CD28, CD95 mAbs, permeabilized and stained with IFN-γ, IL-2 and TNF-α mAbs. For analysis, cells were initially gated on live lymphocytes (low FSC/SSC, EMA-, CD3+), CD8 bright T cells (orange color) and CD4+ T cell populations (blue color). Antigen-specific T cells were identified by their capacity to secrete one or more cytokines (black dots). For each indicated animal (BX72, 78, 80, 84), we displayed the maximal response obtained at this time point within CD8+ or CD4+ T cell populations. For simplicity, the percentage of total responses (total black dots) obtained through all quadrants (IFN-γ+, IFN-γ+ &TNF-α+, IFN-γ+&IL-2+ and IL-2+) are indicated after the subtraction of background obtained with cells cultured with medium only. For memory T cell subset determinations, all black dots were superimposed to the total CD8+ or CD4+ T cell populations and plotted against memory markers (CD28 and CD95). The naïve population was defined as CD28+ CD95−, effector memory (EM) as CD28− CD95+ and central memory (CM) as CD28+ CD95+. D) Sequential evaluation of SIV Gag p55 specific antibody responses in immunized macaques (“homemade kit”). E) Sequential evaluation of HIV Env gp160 specific antibody responses in immunized macaques (Biorad, France).

Mentions: We immunized six cynomolgus macaques (BX72, 73, 78, 80, 83, 84) with each animal delivered a single immunization with the CAL-SHIV-IN− DNA vaccine consisting of combined IM and ID-EP (intradermal plus electroporation) administrations as described in material and methods. The vaccine-induced IFN-γ responses were measured longitudinally against Gag-, Pol-, Env- and Tat+Rev+Nef in PBMCs isolated from fresh blood samples collected across a year post-immunization (PI). Samples were analyzed weekly during the first 4 weeks, every other week up to W32 PI and then every 4 weeks up to W47 PI. Monkey BX73 was vaccinated 3 months after the other animals and sampled every other week from W2 to W20 PI and then every 4 weeks up to W35 PI. Data from this longitudinal examination reported in Figure 1A showed that all six animals immunized once with a total of 5 mg of DNA vaccine developed circulating antigen-specific IFN-γ producing T cell responses that persisted until the end of follow-up (W47 PI). While individual variations were noted, most primary expansion for total antigen responses peaked during the first three months (W2 to 12) PI. Interestingly, secondary peaks of expansion occurred, in the absence of any antigen boost, at multiple late time points PI. In five of six animals, maximal peak responses observed during the primary expansion phase or at reemergence time points ranged between 2000 to 3000 spots/million of PBMCs (106). The sixth animal, BX73 mounted lower peak responses at about 1000 spots/106. The magnitude and timing of the responses varied between macaques, however at the level of individual antigens, responses against Gag and TRN were detected in all animals at most time points with mean responses and ranges shown in Figure 1B. For Env and Pol antigens, the calculated overall average responses reached in all 6 vaccinated animals were generally lower (85 spots/106 for Env and 51 spots/106 for Pol), commensurate with the relative lower levels of production of these proteins. Thus, all vaccinated macaques developed broad IFN-γ responses directed against all administered antigens with dominant responses against Gag and TRN antigens.


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)

Evaluation and characterization of immediate effector T cell responses induced by a single immunization with the CAL-SHIV-IN− DNA vaccine.A) PBMCs isolated from blood samples taken at the indicated weeks post-immunization (PI) were used for ELISPOT assay to detect IFN-γ producing cells in response to the pools of SIV Gag (indicated in blue), HIV Env (indicated in red), HIV Tat+Rev+ SIV Nef (TRN, indicated in green) and SIV Pol (indicated in purple) peptides. Numbers of IFN-γ producing cells obtained per million of PBMCs against all Ags tested are indicated in the y-axis. B) Mean and range of IFN-γ ELISPOT responses to Gag and TRN pools across 22 (for BX72, 78, 80, 83, 84) and 14 (for BX73) time-points collected. C) Memory phenotyping of Gag-specific T cells at W8 PI. PBMCs were restimulated for 16 h in presence or absence of Gag pool of peptides in medium containing Brefeldin A. Cells were then surface stained with EMA, CD3, CD8, CD4, CD28, CD95 mAbs, permeabilized and stained with IFN-γ, IL-2 and TNF-α mAbs. For analysis, cells were initially gated on live lymphocytes (low FSC/SSC, EMA-, CD3+), CD8 bright T cells (orange color) and CD4+ T cell populations (blue color). Antigen-specific T cells were identified by their capacity to secrete one or more cytokines (black dots). For each indicated animal (BX72, 78, 80, 84), we displayed the maximal response obtained at this time point within CD8+ or CD4+ T cell populations. For simplicity, the percentage of total responses (total black dots) obtained through all quadrants (IFN-γ+, IFN-γ+ &TNF-α+, IFN-γ+&IL-2+ and IL-2+) are indicated after the subtraction of background obtained with cells cultured with medium only. For memory T cell subset determinations, all black dots were superimposed to the total CD8+ or CD4+ T cell populations and plotted against memory markers (CD28 and CD95). The naïve population was defined as CD28+ CD95−, effector memory (EM) as CD28− CD95+ and central memory (CM) as CD28+ CD95+. D) Sequential evaluation of SIV Gag p55 specific antibody responses in immunized macaques (“homemade kit”). E) Sequential evaluation of HIV Env gp160 specific antibody responses in immunized macaques (Biorad, France).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110883-g001: Evaluation and characterization of immediate effector T cell responses induced by a single immunization with the CAL-SHIV-IN− DNA vaccine.A) PBMCs isolated from blood samples taken at the indicated weeks post-immunization (PI) were used for ELISPOT assay to detect IFN-γ producing cells in response to the pools of SIV Gag (indicated in blue), HIV Env (indicated in red), HIV Tat+Rev+ SIV Nef (TRN, indicated in green) and SIV Pol (indicated in purple) peptides. Numbers of IFN-γ producing cells obtained per million of PBMCs against all Ags tested are indicated in the y-axis. B) Mean and range of IFN-γ ELISPOT responses to Gag and TRN pools across 22 (for BX72, 78, 80, 83, 84) and 14 (for BX73) time-points collected. C) Memory phenotyping of Gag-specific T cells at W8 PI. PBMCs were restimulated for 16 h in presence or absence of Gag pool of peptides in medium containing Brefeldin A. Cells were then surface stained with EMA, CD3, CD8, CD4, CD28, CD95 mAbs, permeabilized and stained with IFN-γ, IL-2 and TNF-α mAbs. For analysis, cells were initially gated on live lymphocytes (low FSC/SSC, EMA-, CD3+), CD8 bright T cells (orange color) and CD4+ T cell populations (blue color). Antigen-specific T cells were identified by their capacity to secrete one or more cytokines (black dots). For each indicated animal (BX72, 78, 80, 84), we displayed the maximal response obtained at this time point within CD8+ or CD4+ T cell populations. For simplicity, the percentage of total responses (total black dots) obtained through all quadrants (IFN-γ+, IFN-γ+ &TNF-α+, IFN-γ+&IL-2+ and IL-2+) are indicated after the subtraction of background obtained with cells cultured with medium only. For memory T cell subset determinations, all black dots were superimposed to the total CD8+ or CD4+ T cell populations and plotted against memory markers (CD28 and CD95). The naïve population was defined as CD28+ CD95−, effector memory (EM) as CD28− CD95+ and central memory (CM) as CD28+ CD95+. D) Sequential evaluation of SIV Gag p55 specific antibody responses in immunized macaques (“homemade kit”). E) Sequential evaluation of HIV Env gp160 specific antibody responses in immunized macaques (Biorad, France).
Mentions: We immunized six cynomolgus macaques (BX72, 73, 78, 80, 83, 84) with each animal delivered a single immunization with the CAL-SHIV-IN− DNA vaccine consisting of combined IM and ID-EP (intradermal plus electroporation) administrations as described in material and methods. The vaccine-induced IFN-γ responses were measured longitudinally against Gag-, Pol-, Env- and Tat+Rev+Nef in PBMCs isolated from fresh blood samples collected across a year post-immunization (PI). Samples were analyzed weekly during the first 4 weeks, every other week up to W32 PI and then every 4 weeks up to W47 PI. Monkey BX73 was vaccinated 3 months after the other animals and sampled every other week from W2 to W20 PI and then every 4 weeks up to W35 PI. Data from this longitudinal examination reported in Figure 1A showed that all six animals immunized once with a total of 5 mg of DNA vaccine developed circulating antigen-specific IFN-γ producing T cell responses that persisted until the end of follow-up (W47 PI). While individual variations were noted, most primary expansion for total antigen responses peaked during the first three months (W2 to 12) PI. Interestingly, secondary peaks of expansion occurred, in the absence of any antigen boost, at multiple late time points PI. In five of six animals, maximal peak responses observed during the primary expansion phase or at reemergence time points ranged between 2000 to 3000 spots/million of PBMCs (106). The sixth animal, BX73 mounted lower peak responses at about 1000 spots/106. The magnitude and timing of the responses varied between macaques, however at the level of individual antigens, responses against Gag and TRN were detected in all animals at most time points with mean responses and ranges shown in Figure 1B. For Env and Pol antigens, the calculated overall average responses reached in all 6 vaccinated animals were generally lower (85 spots/106 for Env and 51 spots/106 for Pol), commensurate with the relative lower levels of production of these proteins. Thus, all vaccinated macaques developed broad IFN-γ responses directed against all administered antigens with dominant responses against Gag and TRN antigens.

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