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Vaccination with Ad5 vectors expands Ad5-specific CD8 T cells without altering memory phenotype or functionality.

Hutnick NA, Carnathan DG, Dubey SA, Cox KS, Kierstead L, Makadonas G, Ratcliffe SJ, Lasaro MO, Robertson MN, Casimiro DR, Ertl HC, Betts MR - PLoS ONE (2010)

Bottom Line: Adenoviral (Ad) vaccine vectors represent both a vehicle to present a novel antigen to the immune system as well as restimulation of immune responses against the Ad vector itself.Peripheral blood Ad5-specific CD8(+) T-cell numbers expanded after Ad5-HIV vaccination in all subjects, but differential expansion kinetics were noted in some baseline Ad5-neutralizing antibody (Ad5 nAb) seronegative subjects compared to baseline Ad5 nAb seropositive subjects.These data indicate that repeat Ad5-vector administration in humans expands Ad5-specific CD8(+) T-cells without overtly affecting their functional capacity or phenotypic properties.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Center for AIDS Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: Adenoviral (Ad) vaccine vectors represent both a vehicle to present a novel antigen to the immune system as well as restimulation of immune responses against the Ad vector itself. To what degree Ad-specific CD8(+) T cells are restimulated by Ad vector vaccination is unclear, although such knowledge would be important as vector-specific CD8(+) T cell expansion could potentially further limit Ad vaccine efficacy beyond Ad-specific neutralizing antibody alone.

Methodology/principal findings: Here we addressed this issue by measuring human Adenovirus serotype 5 (Ad5)-specific CD8(+) T cells in recipients of the Merck Ad5 HIV-1 vaccine vector before, during, and after vaccination by multicolor flow cytometry. Ad5-specific CD8(+) T-cells were detectable in 95% of subjects prior to vaccination, and displayed primarily an effector-type functional profile and phenotype. Peripheral blood Ad5-specific CD8(+) T-cell numbers expanded after Ad5-HIV vaccination in all subjects, but differential expansion kinetics were noted in some baseline Ad5-neutralizing antibody (Ad5 nAb) seronegative subjects compared to baseline Ad5 nAb seropositive subjects. However, in neither group did vaccination alter polyfunctionality, mucosal targeting marker expression, or memory phenotype of Ad5-specific CD8(+) T-cells.

Conclusions: These data indicate that repeat Ad5-vector administration in humans expands Ad5-specific CD8(+) T-cells without overtly affecting their functional capacity or phenotypic properties. This is a secondary analysis of samples collected during the 016 trial. Results of the Merck 016 trial safety and immunogenicity have been previously published in the journal of clinical infectious diseases [1].

Trial registration: ClinicalTrials.gov NCT00849680[http://www.clinicaltrials.gov/show/NCT00849680].

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Baseline CD8+ T-cell responses.Forty total subjects with a range of Ad5 nAb titers were analyzed. Five seronegative (Ad5 nAb titer ≤18, gray symbols) and five seropositive subjects (Ad5 nAb titer >18, black symbols and white boxes) received Merck Ad5 gag/pol/nef as described in Methods. Each circle represents a subject with lines representing the mean. CD8+ T-cell responses were measured by flow cytometry following whole Ad5 vector stimulation. A) Gating strategy for measuring Ad5-specific T cells by intracellular cytokine staining. At least 100,000 PBMCs were collected on a modified LSR II. Singlets were selected with a FSC-A and FSC-H, followed by a lymphocytes gate, dead cell exclusion, and exclusion of contaminating CD14+ monocytes and CD19+ B-cells. CD3+ T-cells were selected and then CD8+ cells by CD8+CD4−. Central memory, effector memory and effector CD8+ T cells were selected before gating on each cytokine. Because cells can store perforin and these appear perforin+, Ad5-specific CD8+perforin+ T cells must also be positive for another function to be considered as a responding event. B) Total Ad-specific CD8+ response. The total Ad-specific CD8+ response was computed by summing cells making at least IL-2, IFN-γ, MIP1α, or TNFα as measured by flow cytometry. C) There was no difference in the magnitude of the Ad-specific CD8+ T-cell response between serogroups at baseline. D) There was no correlation between the magnitude of Ad-specific CD8+ T-cell responses and nAb titer at baseline. E) There is a correlation between the magnitude of Ad-specific CD4+ and Ad-specific CD8+ T-cell responses at baseline.
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pone-0014385-g001: Baseline CD8+ T-cell responses.Forty total subjects with a range of Ad5 nAb titers were analyzed. Five seronegative (Ad5 nAb titer ≤18, gray symbols) and five seropositive subjects (Ad5 nAb titer >18, black symbols and white boxes) received Merck Ad5 gag/pol/nef as described in Methods. Each circle represents a subject with lines representing the mean. CD8+ T-cell responses were measured by flow cytometry following whole Ad5 vector stimulation. A) Gating strategy for measuring Ad5-specific T cells by intracellular cytokine staining. At least 100,000 PBMCs were collected on a modified LSR II. Singlets were selected with a FSC-A and FSC-H, followed by a lymphocytes gate, dead cell exclusion, and exclusion of contaminating CD14+ monocytes and CD19+ B-cells. CD3+ T-cells were selected and then CD8+ cells by CD8+CD4−. Central memory, effector memory and effector CD8+ T cells were selected before gating on each cytokine. Because cells can store perforin and these appear perforin+, Ad5-specific CD8+perforin+ T cells must also be positive for another function to be considered as a responding event. B) Total Ad-specific CD8+ response. The total Ad-specific CD8+ response was computed by summing cells making at least IL-2, IFN-γ, MIP1α, or TNFα as measured by flow cytometry. C) There was no difference in the magnitude of the Ad-specific CD8+ T-cell response between serogroups at baseline. D) There was no correlation between the magnitude of Ad-specific CD8+ T-cell responses and nAb titer at baseline. E) There is a correlation between the magnitude of Ad-specific CD4+ and Ad-specific CD8+ T-cell responses at baseline.

Mentions: To assess the total magnitude, functionality, and phenotypes of Ad5-specific CD8+ T-cells, we stimulated human PBMCs with Ad5 vector and measured CD8+ T-cell responses by polychromatic flow cytometry (Fig. 1A). We detected Ad5-specific CD8+ T-cell responses in 95% of all donors at the week 0 baseline (Fig. 1B). There was no difference in frequencies of Ad5-specific CD8+ T-cells between baseline seropositive and seronegative subjects (Fig. 1C, p>0.05). Likewise there was no correlation between frequencies of Ad5-specific CD8+ T-cells and the Ad5 nAb titer at baseline (Fig. 1D, p>0.05). There was however a positive correlation between frequencies of baseline Ad5-specific CD4+ and CD8+ T-cells (Fig. 1E).


Vaccination with Ad5 vectors expands Ad5-specific CD8 T cells without altering memory phenotype or functionality.

Hutnick NA, Carnathan DG, Dubey SA, Cox KS, Kierstead L, Makadonas G, Ratcliffe SJ, Lasaro MO, Robertson MN, Casimiro DR, Ertl HC, Betts MR - PLoS ONE (2010)

Baseline CD8+ T-cell responses.Forty total subjects with a range of Ad5 nAb titers were analyzed. Five seronegative (Ad5 nAb titer ≤18, gray symbols) and five seropositive subjects (Ad5 nAb titer >18, black symbols and white boxes) received Merck Ad5 gag/pol/nef as described in Methods. Each circle represents a subject with lines representing the mean. CD8+ T-cell responses were measured by flow cytometry following whole Ad5 vector stimulation. A) Gating strategy for measuring Ad5-specific T cells by intracellular cytokine staining. At least 100,000 PBMCs were collected on a modified LSR II. Singlets were selected with a FSC-A and FSC-H, followed by a lymphocytes gate, dead cell exclusion, and exclusion of contaminating CD14+ monocytes and CD19+ B-cells. CD3+ T-cells were selected and then CD8+ cells by CD8+CD4−. Central memory, effector memory and effector CD8+ T cells were selected before gating on each cytokine. Because cells can store perforin and these appear perforin+, Ad5-specific CD8+perforin+ T cells must also be positive for another function to be considered as a responding event. B) Total Ad-specific CD8+ response. The total Ad-specific CD8+ response was computed by summing cells making at least IL-2, IFN-γ, MIP1α, or TNFα as measured by flow cytometry. C) There was no difference in the magnitude of the Ad-specific CD8+ T-cell response between serogroups at baseline. D) There was no correlation between the magnitude of Ad-specific CD8+ T-cell responses and nAb titer at baseline. E) There is a correlation between the magnitude of Ad-specific CD4+ and Ad-specific CD8+ T-cell responses at baseline.
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pone-0014385-g001: Baseline CD8+ T-cell responses.Forty total subjects with a range of Ad5 nAb titers were analyzed. Five seronegative (Ad5 nAb titer ≤18, gray symbols) and five seropositive subjects (Ad5 nAb titer >18, black symbols and white boxes) received Merck Ad5 gag/pol/nef as described in Methods. Each circle represents a subject with lines representing the mean. CD8+ T-cell responses were measured by flow cytometry following whole Ad5 vector stimulation. A) Gating strategy for measuring Ad5-specific T cells by intracellular cytokine staining. At least 100,000 PBMCs were collected on a modified LSR II. Singlets were selected with a FSC-A and FSC-H, followed by a lymphocytes gate, dead cell exclusion, and exclusion of contaminating CD14+ monocytes and CD19+ B-cells. CD3+ T-cells were selected and then CD8+ cells by CD8+CD4−. Central memory, effector memory and effector CD8+ T cells were selected before gating on each cytokine. Because cells can store perforin and these appear perforin+, Ad5-specific CD8+perforin+ T cells must also be positive for another function to be considered as a responding event. B) Total Ad-specific CD8+ response. The total Ad-specific CD8+ response was computed by summing cells making at least IL-2, IFN-γ, MIP1α, or TNFα as measured by flow cytometry. C) There was no difference in the magnitude of the Ad-specific CD8+ T-cell response between serogroups at baseline. D) There was no correlation between the magnitude of Ad-specific CD8+ T-cell responses and nAb titer at baseline. E) There is a correlation between the magnitude of Ad-specific CD4+ and Ad-specific CD8+ T-cell responses at baseline.
Mentions: To assess the total magnitude, functionality, and phenotypes of Ad5-specific CD8+ T-cells, we stimulated human PBMCs with Ad5 vector and measured CD8+ T-cell responses by polychromatic flow cytometry (Fig. 1A). We detected Ad5-specific CD8+ T-cell responses in 95% of all donors at the week 0 baseline (Fig. 1B). There was no difference in frequencies of Ad5-specific CD8+ T-cells between baseline seropositive and seronegative subjects (Fig. 1C, p>0.05). Likewise there was no correlation between frequencies of Ad5-specific CD8+ T-cells and the Ad5 nAb titer at baseline (Fig. 1D, p>0.05). There was however a positive correlation between frequencies of baseline Ad5-specific CD4+ and CD8+ T-cells (Fig. 1E).

Bottom Line: Adenoviral (Ad) vaccine vectors represent both a vehicle to present a novel antigen to the immune system as well as restimulation of immune responses against the Ad vector itself.Peripheral blood Ad5-specific CD8(+) T-cell numbers expanded after Ad5-HIV vaccination in all subjects, but differential expansion kinetics were noted in some baseline Ad5-neutralizing antibody (Ad5 nAb) seronegative subjects compared to baseline Ad5 nAb seropositive subjects.These data indicate that repeat Ad5-vector administration in humans expands Ad5-specific CD8(+) T-cells without overtly affecting their functional capacity or phenotypic properties.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Center for AIDS Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: Adenoviral (Ad) vaccine vectors represent both a vehicle to present a novel antigen to the immune system as well as restimulation of immune responses against the Ad vector itself. To what degree Ad-specific CD8(+) T cells are restimulated by Ad vector vaccination is unclear, although such knowledge would be important as vector-specific CD8(+) T cell expansion could potentially further limit Ad vaccine efficacy beyond Ad-specific neutralizing antibody alone.

Methodology/principal findings: Here we addressed this issue by measuring human Adenovirus serotype 5 (Ad5)-specific CD8(+) T cells in recipients of the Merck Ad5 HIV-1 vaccine vector before, during, and after vaccination by multicolor flow cytometry. Ad5-specific CD8(+) T-cells were detectable in 95% of subjects prior to vaccination, and displayed primarily an effector-type functional profile and phenotype. Peripheral blood Ad5-specific CD8(+) T-cell numbers expanded after Ad5-HIV vaccination in all subjects, but differential expansion kinetics were noted in some baseline Ad5-neutralizing antibody (Ad5 nAb) seronegative subjects compared to baseline Ad5 nAb seropositive subjects. However, in neither group did vaccination alter polyfunctionality, mucosal targeting marker expression, or memory phenotype of Ad5-specific CD8(+) T-cells.

Conclusions: These data indicate that repeat Ad5-vector administration in humans expands Ad5-specific CD8(+) T-cells without overtly affecting their functional capacity or phenotypic properties. This is a secondary analysis of samples collected during the 016 trial. Results of the Merck 016 trial safety and immunogenicity have been previously published in the journal of clinical infectious diseases [1].

Trial registration: ClinicalTrials.gov NCT00849680[http://www.clinicaltrials.gov/show/NCT00849680].

Show MeSH
Related in: MedlinePlus