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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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AdHu5 hexon quantitative real-time PCR.RNA isolated from non-infected cells (dashed bar) and from cells infected with AdC7rab.gp (white bar), Ad5 wt (black bar) or Ad5rab.gp (gray bar) were reverse transcribed and normalized to equal amount of gapdH mRNA copies. Ad5 hexon mRNA copies were quantified by real-time PCR using specific primers. No Ad5 hexon specific mRNA was detected in non-infected cells or cells infected with AdC7rab.gp. Ad5 hexon mRNA levels were assessed in triplicates. Averages (Ad5 wt and Ad5rab.gp) and p values from two-tail student's t test are shown on top of the bars.
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pone-0014385-g003: AdHu5 hexon quantitative real-time PCR.RNA isolated from non-infected cells (dashed bar) and from cells infected with AdC7rab.gp (white bar), Ad5 wt (black bar) or Ad5rab.gp (gray bar) were reverse transcribed and normalized to equal amount of gapdH mRNA copies. Ad5 hexon mRNA copies were quantified by real-time PCR using specific primers. No Ad5 hexon specific mRNA was detected in non-infected cells or cells infected with AdC7rab.gp. Ad5 hexon mRNA levels were assessed in triplicates. Averages (Ad5 wt and Ad5rab.gp) and p values from two-tail student's t test are shown on top of the bars.

Mentions: E1-deleted Ad5 vectors are replication-defective as E1 gene products are needed to initiate transcription of the other viral genes. Nevertheless, cellular factors can in part compensate for loss of E1 and E1-deleted Ad vectors can thus be transcribed and even proliferate in certain cell types [25], [26]. To test if the E1-deleted Ad5 vector were able to transcribe structural late genes, and thus be recognized by CD8+ T cells, CHO cells stably transfected with a vector expressing the coxsackie adenovirus receptor (CAR) were infected with 1000 vp per cell of an E1-deleted Ad5 vector, 1000 vp/cell of wild-type Ad5 virus as a positive control or 1000 vp of an E1-deleted chimpanzee-derived Ad vector (AdC7) as a negative control. RNA was isolated 48 hours later and the amount of mRNA encoding the viral hexon was determined using a quantitative real-time PCR with primers designed to distinguish hexon transcripts of AdHu5 from those of AdC7. Results clearly showed that hexon was being transcribed although at levels that were at least 500 fold below those achieved by wild-type virus (Figure 3). A reduction in vector dose resulted in reduced levels of transcripts (data not shown). Cells infected with the AdC7 vector or non-infected cells were negative for Ad5 hexon transcripts.


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)

AdHu5 hexon quantitative real-time PCR.RNA isolated from non-infected cells (dashed bar) and from cells infected with AdC7rab.gp (white bar), Ad5 wt (black bar) or Ad5rab.gp (gray bar) were reverse transcribed and normalized to equal amount of gapdH mRNA copies. Ad5 hexon mRNA copies were quantified by real-time PCR using specific primers. No Ad5 hexon specific mRNA was detected in non-infected cells or cells infected with AdC7rab.gp. Ad5 hexon mRNA levels were assessed in triplicates. Averages (Ad5 wt and Ad5rab.gp) and p values from two-tail student's t test are shown on top of the bars.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008674&req=5

pone-0014385-g003: AdHu5 hexon quantitative real-time PCR.RNA isolated from non-infected cells (dashed bar) and from cells infected with AdC7rab.gp (white bar), Ad5 wt (black bar) or Ad5rab.gp (gray bar) were reverse transcribed and normalized to equal amount of gapdH mRNA copies. Ad5 hexon mRNA copies were quantified by real-time PCR using specific primers. No Ad5 hexon specific mRNA was detected in non-infected cells or cells infected with AdC7rab.gp. Ad5 hexon mRNA levels were assessed in triplicates. Averages (Ad5 wt and Ad5rab.gp) and p values from two-tail student's t test are shown on top of the bars.
Mentions: E1-deleted Ad5 vectors are replication-defective as E1 gene products are needed to initiate transcription of the other viral genes. Nevertheless, cellular factors can in part compensate for loss of E1 and E1-deleted Ad vectors can thus be transcribed and even proliferate in certain cell types [25], [26]. To test if the E1-deleted Ad5 vector were able to transcribe structural late genes, and thus be recognized by CD8+ T cells, CHO cells stably transfected with a vector expressing the coxsackie adenovirus receptor (CAR) were infected with 1000 vp per cell of an E1-deleted Ad5 vector, 1000 vp/cell of wild-type Ad5 virus as a positive control or 1000 vp of an E1-deleted chimpanzee-derived Ad vector (AdC7) as a negative control. RNA was isolated 48 hours later and the amount of mRNA encoding the viral hexon was determined using a quantitative real-time PCR with primers designed to distinguish hexon transcripts of AdHu5 from those of AdC7. Results clearly showed that hexon was being transcribed although at levels that were at least 500 fold below those achieved by wild-type virus (Figure 3). A reduction in vector dose resulted in reduced levels of transcripts (data not shown). Cells infected with the AdC7 vector or non-infected cells were negative for Ad5 hexon transcripts.

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