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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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Vaccine-specific PHPC T cell responses at late time points post-immunization.A) PBMCs were isolated from blood samples taken between W40 and W47 PI for all animals (W28 to 35 PI for BX73). A portion of cells from each animal was used for ELISPOT assay to detect IFN-γ producing cells in response to medium, Gag and TRN pools of peptides (day 1). The other portion was cultured for 11 days in the presence or absence of relevant peptide pools, in medium supplemented on day 3 with recombinant simian IL-2 only and on day 7 with recombinant simian IL-2, IL-15 and IL-7 cytokines. On day 11, cells were collected, counted and used for ELISPOT assay using the same conditions as day 1 ELISPOT, to detect IFN-γ producing cells in response to Gag and TRN peptides (day 12). This assay is referred as PHPC assay for memory precursors with high proliferative capacity. Numbers of IFN-γ producing cells obtained per million of PBMCs against Gag + TRN at day 1 and day 12 are indicated in the y-axis. The number of median responses obtained among all 6 animals are represented (n = 457 spots/106 at day 1 and n = 11628 spots/106 at day 12). B) Memory phenotyping of Gag and TRN-specific T cells for animal BX72 at W40 PI. Day 1 and day 11 cultured PBMCs, as described in section A, were restimulated for 16 h in the presence or absence of Gag or TRN 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. Cells were gated on live lymphocytes (low FSC/SSC, EMA-, CD3+), bright CD8+ T cell populations (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 simplicity, the percentage of total IFN-γ+ or TNF-α+ or IL-2+ antigen specific responses (black dots) obtained are indicated after the subtraction of background obtained with cells cultured with medium only. All black dots were superimposed to the total CD8+ or CD4+ T cell population and plotted against EM and CM memory markers (CD28 and CD95). C) The same procedure as described in B was performed on day 11-cultured PBMCs that have been isolated at W47 PI (W35 PI for BX73) from all six animals. The histograms represent the percentage of total IFN-γ+ (green) or TNF-α+ (red) or IL-2+ (blue) antigen-specific T cell responses obtained against Gag and TRN (indicated by G and M respectively in the histogram) within CD8+ (left histogram) and CD4+ (right histogram) T cells. Individual dot plot analysis for cytokine and memory phenotype shown in the Figure 4C, are displayed in supplemental Figure S3 and S4.
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pone-0110883-g004: Vaccine-specific PHPC T cell responses at late time points post-immunization.A) PBMCs were isolated from blood samples taken between W40 and W47 PI for all animals (W28 to 35 PI for BX73). A portion of cells from each animal was used for ELISPOT assay to detect IFN-γ producing cells in response to medium, Gag and TRN pools of peptides (day 1). The other portion was cultured for 11 days in the presence or absence of relevant peptide pools, in medium supplemented on day 3 with recombinant simian IL-2 only and on day 7 with recombinant simian IL-2, IL-15 and IL-7 cytokines. On day 11, cells were collected, counted and used for ELISPOT assay using the same conditions as day 1 ELISPOT, to detect IFN-γ producing cells in response to Gag and TRN peptides (day 12). This assay is referred as PHPC assay for memory precursors with high proliferative capacity. Numbers of IFN-γ producing cells obtained per million of PBMCs against Gag + TRN at day 1 and day 12 are indicated in the y-axis. The number of median responses obtained among all 6 animals are represented (n = 457 spots/106 at day 1 and n = 11628 spots/106 at day 12). B) Memory phenotyping of Gag and TRN-specific T cells for animal BX72 at W40 PI. Day 1 and day 11 cultured PBMCs, as described in section A, were restimulated for 16 h in the presence or absence of Gag or TRN 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. Cells were gated on live lymphocytes (low FSC/SSC, EMA-, CD3+), bright CD8+ T cell populations (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 simplicity, the percentage of total IFN-γ+ or TNF-α+ or IL-2+ antigen specific responses (black dots) obtained are indicated after the subtraction of background obtained with cells cultured with medium only. All black dots were superimposed to the total CD8+ or CD4+ T cell population and plotted against EM and CM memory markers (CD28 and CD95). C) The same procedure as described in B was performed on day 11-cultured PBMCs that have been isolated at W47 PI (W35 PI for BX73) from all six animals. The histograms represent the percentage of total IFN-γ+ (green) or TNF-α+ (red) or IL-2+ (blue) antigen-specific T cell responses obtained against Gag and TRN (indicated by G and M respectively in the histogram) within CD8+ (left histogram) and CD4+ (right histogram) T cells. Individual dot plot analysis for cytokine and memory phenotype shown in the Figure 4C, are displayed in supplemental Figure S3 and S4.

Mentions: The difference in IFN-γ responses detected by the 18 h ELISPOT assay and the 6-day polyfunctional recall assay, clearly indicated that these two types of assays identify different subsets of antigen-specific T cells. While the IFN-γ ELISPOT assay is thought to identify effector T cells with low proliferative capacity, another peptide-based cultured IFN-γ ELISPOT assay has been used to quantify antigen-specific memory T cells precursors with high proliferative capacity (PHPC assay) [38]. This assay is thought to assess central memory T cells that require more time to expand and give rise to cells with immediate effector functions. Importantly, in untreated HIV seropositive individuals, strong PHPC responses correlated with low viremia and high CD4+ counts [42]. The memory phenotype analysis performed on day 1 stimulated PBMCs (Figure 1C) clearly indicated the presence of antigen-specific central memory cells within the cytokine secreting population. Antigen-specific CM T cells were also found within the population of cells that proliferate only in our 6 day culture assay (data not shown). We developed and performed a slightly modified PHPC assay to examine peripheral blood samples of vaccinated animals collected at W40 to W47 PI time points (W28 to W35 PI for BX73). Briefly, PBMCs were expanded for 11 days in culture supplemented or not with antigen on day 1 followed with recombinant simian IL-2 only (to force short life effectors to die) on day 3 and then with a cocktail of recombinant simian IL-2, IL-15 and IL-7 cytokines on day 7 (to promote expansion of memory CD8+ and CD4+ T cells). On day 11, cells were collected, counted and tested in the IFN-γ ELISPOT assay and polychromatic flow cytometry assay. The results shown in Figure 4A clearly illustrated that the number of SHIV-specific IFN-γ immediate effector cells could be greatly expanded by culturing the cells for 11 days in presence of antigenic and homeostatic signals. Among all six vaccinated animals, we measured and calculated a mean of Gag+TRN response of 457 spots/106 PBMCs (range 57–1248, n = 6) for the day 1 ELISPOT assay and a mean of Gag+TRN response of 11628 spots/106 PBMCs (range 5387–19007, n = 6) for the day 12 ELISPOT assay. This represented an average 60 fold increase of expansion. Each of the six macaques also had positive PHPC responses against Gag GW9 and/or Nef RM9 antigen (Figure S2B). We next assessed the phenotype and function of the Gag and TRN-specific T cells at day 1 and day 12 similar to the experiment depicted in Figure 1C. PBMCs, fresh or cultured for 11 days, were stimulated for 16 h with Gag or TRN pools of peptides or with medium only, in presence of Brefeldin A. Cells were then surface-stained with anti-CD3, CD4, CD8, CD28 and CD95 mAbs in the presence of EMA (for dead cell exclusion). After a permeabilization step, intracellular staining was performed with anti-IFN-γ, TNF-α and IL-2 mAbs. A representative example is illustrated in Figure 4B for animal BX72 on day 1 and day 12 starting with PBMCs collected at W40 PI. For the day 1 assay, this analysis identified Gag specific-CD8+ T cells capable of secreting IFN-γ and/or TNF-α as well as TRN-specific CD4+ T cells capable of secreting IFN-γ and/or IL-2 with a proportion of single or multiple cytokine secreting cells around 0.01% of total live CD8+ or CD4+ T cells. The vast majority of vaccine-induced T cells expressed EM markers for CD8+ T cells and CM markers for CD4+ T cells. At day 12, Gag and TRN cytokine secreting-specific T cells had greatly expanded. Gag specific-CD8+ T cells capable of secreting mostly IFN-γ (2.0%) and TNF-α (0.1%) were found as well as TRN-specific CD4+ T cells capable of secreting IFN-γ (0.4%), IL-2 (0.05%) and TNF-α (0.2%). When plotted against memory markers, expanded Gag-specific CD8+ T cells were distributed within EM and CM T cell populations, while expanded TRN-specific CD4+ T cells mostly remained within the CM T cell population. These results indicate that within the CD8+ T cell compartment, CM specific T cells have expanded to maintain and enrich their initial pool size as well as to promote the massive development of secondary immediate effector EM type of 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)

Vaccine-specific PHPC T cell responses at late time points post-immunization.A) PBMCs were isolated from blood samples taken between W40 and W47 PI for all animals (W28 to 35 PI for BX73). A portion of cells from each animal was used for ELISPOT assay to detect IFN-γ producing cells in response to medium, Gag and TRN pools of peptides (day 1). The other portion was cultured for 11 days in the presence or absence of relevant peptide pools, in medium supplemented on day 3 with recombinant simian IL-2 only and on day 7 with recombinant simian IL-2, IL-15 and IL-7 cytokines. On day 11, cells were collected, counted and used for ELISPOT assay using the same conditions as day 1 ELISPOT, to detect IFN-γ producing cells in response to Gag and TRN peptides (day 12). This assay is referred as PHPC assay for memory precursors with high proliferative capacity. Numbers of IFN-γ producing cells obtained per million of PBMCs against Gag + TRN at day 1 and day 12 are indicated in the y-axis. The number of median responses obtained among all 6 animals are represented (n = 457 spots/106 at day 1 and n = 11628 spots/106 at day 12). B) Memory phenotyping of Gag and TRN-specific T cells for animal BX72 at W40 PI. Day 1 and day 11 cultured PBMCs, as described in section A, were restimulated for 16 h in the presence or absence of Gag or TRN 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. Cells were gated on live lymphocytes (low FSC/SSC, EMA-, CD3+), bright CD8+ T cell populations (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 simplicity, the percentage of total IFN-γ+ or TNF-α+ or IL-2+ antigen specific responses (black dots) obtained are indicated after the subtraction of background obtained with cells cultured with medium only. All black dots were superimposed to the total CD8+ or CD4+ T cell population and plotted against EM and CM memory markers (CD28 and CD95). C) The same procedure as described in B was performed on day 11-cultured PBMCs that have been isolated at W47 PI (W35 PI for BX73) from all six animals. The histograms represent the percentage of total IFN-γ+ (green) or TNF-α+ (red) or IL-2+ (blue) antigen-specific T cell responses obtained against Gag and TRN (indicated by G and M respectively in the histogram) within CD8+ (left histogram) and CD4+ (right histogram) T cells. Individual dot plot analysis for cytokine and memory phenotype shown in the Figure 4C, are displayed in supplemental Figure S3 and S4.
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Related In: Results  -  Collection

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

pone-0110883-g004: Vaccine-specific PHPC T cell responses at late time points post-immunization.A) PBMCs were isolated from blood samples taken between W40 and W47 PI for all animals (W28 to 35 PI for BX73). A portion of cells from each animal was used for ELISPOT assay to detect IFN-γ producing cells in response to medium, Gag and TRN pools of peptides (day 1). The other portion was cultured for 11 days in the presence or absence of relevant peptide pools, in medium supplemented on day 3 with recombinant simian IL-2 only and on day 7 with recombinant simian IL-2, IL-15 and IL-7 cytokines. On day 11, cells were collected, counted and used for ELISPOT assay using the same conditions as day 1 ELISPOT, to detect IFN-γ producing cells in response to Gag and TRN peptides (day 12). This assay is referred as PHPC assay for memory precursors with high proliferative capacity. Numbers of IFN-γ producing cells obtained per million of PBMCs against Gag + TRN at day 1 and day 12 are indicated in the y-axis. The number of median responses obtained among all 6 animals are represented (n = 457 spots/106 at day 1 and n = 11628 spots/106 at day 12). B) Memory phenotyping of Gag and TRN-specific T cells for animal BX72 at W40 PI. Day 1 and day 11 cultured PBMCs, as described in section A, were restimulated for 16 h in the presence or absence of Gag or TRN 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. Cells were gated on live lymphocytes (low FSC/SSC, EMA-, CD3+), bright CD8+ T cell populations (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 simplicity, the percentage of total IFN-γ+ or TNF-α+ or IL-2+ antigen specific responses (black dots) obtained are indicated after the subtraction of background obtained with cells cultured with medium only. All black dots were superimposed to the total CD8+ or CD4+ T cell population and plotted against EM and CM memory markers (CD28 and CD95). C) The same procedure as described in B was performed on day 11-cultured PBMCs that have been isolated at W47 PI (W35 PI for BX73) from all six animals. The histograms represent the percentage of total IFN-γ+ (green) or TNF-α+ (red) or IL-2+ (blue) antigen-specific T cell responses obtained against Gag and TRN (indicated by G and M respectively in the histogram) within CD8+ (left histogram) and CD4+ (right histogram) T cells. Individual dot plot analysis for cytokine and memory phenotype shown in the Figure 4C, are displayed in supplemental Figure S3 and S4.
Mentions: The difference in IFN-γ responses detected by the 18 h ELISPOT assay and the 6-day polyfunctional recall assay, clearly indicated that these two types of assays identify different subsets of antigen-specific T cells. While the IFN-γ ELISPOT assay is thought to identify effector T cells with low proliferative capacity, another peptide-based cultured IFN-γ ELISPOT assay has been used to quantify antigen-specific memory T cells precursors with high proliferative capacity (PHPC assay) [38]. This assay is thought to assess central memory T cells that require more time to expand and give rise to cells with immediate effector functions. Importantly, in untreated HIV seropositive individuals, strong PHPC responses correlated with low viremia and high CD4+ counts [42]. The memory phenotype analysis performed on day 1 stimulated PBMCs (Figure 1C) clearly indicated the presence of antigen-specific central memory cells within the cytokine secreting population. Antigen-specific CM T cells were also found within the population of cells that proliferate only in our 6 day culture assay (data not shown). We developed and performed a slightly modified PHPC assay to examine peripheral blood samples of vaccinated animals collected at W40 to W47 PI time points (W28 to W35 PI for BX73). Briefly, PBMCs were expanded for 11 days in culture supplemented or not with antigen on day 1 followed with recombinant simian IL-2 only (to force short life effectors to die) on day 3 and then with a cocktail of recombinant simian IL-2, IL-15 and IL-7 cytokines on day 7 (to promote expansion of memory CD8+ and CD4+ T cells). On day 11, cells were collected, counted and tested in the IFN-γ ELISPOT assay and polychromatic flow cytometry assay. The results shown in Figure 4A clearly illustrated that the number of SHIV-specific IFN-γ immediate effector cells could be greatly expanded by culturing the cells for 11 days in presence of antigenic and homeostatic signals. Among all six vaccinated animals, we measured and calculated a mean of Gag+TRN response of 457 spots/106 PBMCs (range 57–1248, n = 6) for the day 1 ELISPOT assay and a mean of Gag+TRN response of 11628 spots/106 PBMCs (range 5387–19007, n = 6) for the day 12 ELISPOT assay. This represented an average 60 fold increase of expansion. Each of the six macaques also had positive PHPC responses against Gag GW9 and/or Nef RM9 antigen (Figure S2B). We next assessed the phenotype and function of the Gag and TRN-specific T cells at day 1 and day 12 similar to the experiment depicted in Figure 1C. PBMCs, fresh or cultured for 11 days, were stimulated for 16 h with Gag or TRN pools of peptides or with medium only, in presence of Brefeldin A. Cells were then surface-stained with anti-CD3, CD4, CD8, CD28 and CD95 mAbs in the presence of EMA (for dead cell exclusion). After a permeabilization step, intracellular staining was performed with anti-IFN-γ, TNF-α and IL-2 mAbs. A representative example is illustrated in Figure 4B for animal BX72 on day 1 and day 12 starting with PBMCs collected at W40 PI. For the day 1 assay, this analysis identified Gag specific-CD8+ T cells capable of secreting IFN-γ and/or TNF-α as well as TRN-specific CD4+ T cells capable of secreting IFN-γ and/or IL-2 with a proportion of single or multiple cytokine secreting cells around 0.01% of total live CD8+ or CD4+ T cells. The vast majority of vaccine-induced T cells expressed EM markers for CD8+ T cells and CM markers for CD4+ T cells. At day 12, Gag and TRN cytokine secreting-specific T cells had greatly expanded. Gag specific-CD8+ T cells capable of secreting mostly IFN-γ (2.0%) and TNF-α (0.1%) were found as well as TRN-specific CD4+ T cells capable of secreting IFN-γ (0.4%), IL-2 (0.05%) and TNF-α (0.2%). When plotted against memory markers, expanded Gag-specific CD8+ T cells were distributed within EM and CM T cell populations, while expanded TRN-specific CD4+ T cells mostly remained within the CM T cell population. These results indicate that within the CD8+ T cell compartment, CM specific T cells have expanded to maintain and enrich their initial pool size as well as to promote the massive development of secondary immediate effector EM type of 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