Adenovirus Specific T-Cell Responses in Humans Following Natural Infection and Vaccination

The adenovirus (Ad) vector is an attractive candidate for vaccines designed to elicit cellular immunity as studies in animals and humans have proven Ad vectors are capable of inducing large transgene-specific T-cell responses. However, given that natural infection by Ad is prevalent globally, pre-existing Ad immunity is a major impediment to the use of recombinant Ad-based vaccines. Though the prevalence of pre-existing neutralizing antibodies has been well characterized, there is a lack of information on the functionality and phenotype of Ad-specific T-cell responses among heterogeneous human cohorts. The lack of protection and possible increased risk of HIV infection in the Merck Ad5 HIV vaccine STEP trial further highlights the need to understand vector-specific immunity in order to produce safe, effective Ad-based vaccines. We aimed to characterize Ad-specific T-cell responses in humans following natural infection and vaccination. Ad-specific T-cell responses were measured by stimulating peripheral blood mononuclear cells (PBMCs) with whole Ad vector or overlapping Ad hexon peptide pools. PBMCs were obtained from 17 healthy adults to study natural infection and longitudinally from 40 participants in Merck phase I Ad5 HIV vaccine studies, 10 of which were enrolled in the STEP trial precursor study using the same vector, dosing, and schedule used for the STEP study. T-cell phenotype and functionality were measured by polychromatic flow cytometry. We found that both CD4+ and CD8+ Ad5-specific T-cells were universally present in subjects independent of their serostatus. Ad5-specific CD8+ T-cells exhibited an effector phenotype and produced the effector functions MIP1α and perforin whereas Ad5-specific CD4+ T-cells had an effector memory phenotype producing IL-2, IFN-γ and TNFα. Ad5-specific T-cells recognized both conserved and variable hexon epitopes making them highly cross-reactive with chimpanzee serotypes. Upon Ad5-based vaccination, Ad5-specific CD4+ T-cells were only transiently expanded and there were no differences in activation or mucosal homing marker expression between Ad5-seronegative and Ad5-seropositive subjects. These data suggest the increased risk of HIV infection observed in the STEP trial was not a result of Ad5-specific CD4+ T-cells. Ad5-specific CD8+ T-cells were also transiently expanded by Ad5-based vaccination, however, there were no changes in functionality. Together, these data suggest though pre-existing Ad-specific T-cells may reduce vaccine efficacy, they should not represent a safety concern for the use of Ad-based vaccines

Vaccination with Ad5 Vectors Expands Ad5-Specific CD8+ T Cells without Altering Memory Phenotype or Functionality

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

Exercise and lymphocyte activation following chemotherapy for breast cancer.

PURPOSE: To determine whether exercise training would increase lymphocyte activation in patients with breast cancer following chemotherapy. Activation was determined by the presence of CD4(+)CD69(+) T-helper lymphocytes, mitogen-induced proliferation, and levels of cytokines produced by mitogen-stimulated lymphocytes and in the patients\u27 plasma. METHODS: Patients with breast cancer (N = 28) who participated in a 6-month exercise program were compared with patients (N = 21) who did not exercise. Following chemotherapy, and 3 and 6 months later, patients underwent fitness evaluations and had blood drawn. The exercise program consisted of resistance training and aerobic activity at 60-75% functional capacity three times a week with a personal trainer. Immunochemistry and flow cytometry were used to measure the number of CD4(+)CD69(+) blood lymphocytes. Whole blood was stimulated with concanavalin A (ConA), phytohemagglutin (PHA), or pokeweed mitogen (PWM) to determine proliferation potential. Enzyme-linked immunosorbent assays (ELISA) were used to determine the concentration of interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) in the culture medium of mitogen-stimulated lymphocytes as well as the plasma concentrations of IL-6, soluble IL-6 receptor, soluble gp130, and IFN-gamma. Analysis of groups across time was done using the Wilcoxon signed rank test, and comparisons of groups were done using the Mann-Whitney U test. RESULTS: The exercising patients showed increases in maximal oxygen uptake and upper body strength. This group also showed a greater percentage of CD4(+)CD69(+) cells and a greater level of tritiated thymidine incorporation (DNA synthesis) when stimulated with ConA, PHA, and PWM at the end of the intervention. Plasma and mitogen-stimulated IL-6 and IFN-gamma production were similar in both groups. CONCLUSION: Exercise may improve immune function by increasing lymphocyte activation in patients with breast cancer following treatment

Adenovirus-specific immunity after immunization with an Ad5 HIV-1 vaccine candidate in humans

The immunologic basis for the potential enhanced HIV-1 acquisition in adenovirus serotype 5 (Ad5)-seropositive individuals who received the Merck recombinant Ad5 HIV-1 vaccine in the STEP study remains unclear. Here we show that baseline Ad5-specific neutralizing antibodies are not correlated with Ad5-specific T lymphocyte responses and that Ad5-seropositive subjects do not develop higher vector-specific cellular immune responses as compared with Ad5-seronegative subjects after vaccination. These findings challenge the hypothesis that activated Ad5-specific T lymphocytes were the cause of the potential enhanced HIV-1 susceptibility in the STEP stud

AAV-1–mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells

In a clinical trial for adeno-associated virus serotype 1 (AAV-1)–mediated gene transfer to muscle for lipoprotein lipase (LPL) deficiency, 1 subject from the high-dose cohort experienced a transient increase in the muscle enzyme creatine phosphokinase (CPK) 4 weeks after gene transfer. Simultaneously, after an initial downward trend consistent with expression of LPL, plasma triglyceride levels returned to baseline. We characterized B- and T-cell responses to the vector and the transgene product in the subjects enrolled in this study. IFN-γ enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining assays performed on peripheral blood mononuclear cells (PBMCs) from the subject who experienced the CPK elevation showed the activation of capsid-specific CD4+ and CD8+ T cells. Four of 8 subjects had detectable T-cell responses to capsid with dose-dependent kinetics of appearance. Subjects with detectable T-cell responses to capsid also had higher anti–AAV-1 IgG3 antibody titer. No subject developed B- or T-cell responses to the LPL transgene product. These findings suggest that T-cell responses directed to the AAV-1 capsid are dose-dependent. Whether they also limit the duration of expression of the transgene at higher doses is unclear, and will require additional analyses at later time points