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]

Defending the genome from the enemy within:mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline

Adenoviruses in Lymphocytes of the Human Gastro-Intestinal Tract

Objective: Persistent adenoviral shedding in stools is known to occur past convalescence following acute adenoviral infections. We wished to establish the frequency with which adenoviruses may colonize the gut in normal human subjects. Methods: The presence of adenoviral DNA in intestinal specimens obtained at surgery or autopsy was tested using a nested PCR method. The amplified adenoviral DNA sequences were compared to each other and to known adenoviral species. Lamina propria lymphocytes (LPLs) were isolated from the specimens and the adenoviral copy numbers in the CD4+ and CD8+ fractions were determined by quantitative PCR. Adenoviral gene expression was tested by amplification of adenoviral mRNA. Results: Intestinal tissue from 21 of 58 donors and LPLs from 21 of 24 donors were positive for the presence of adenoviral DNA. The majority of the sequences could be assigned to adenoviral species E, although species B and C sequences were also common. Multiple sequences were often present in the same sample. Forty-one non-identical sequences were identified from 39 different tissue donors. Quantitative PCR for adenoviral DNA in CD4+ and CD8+ fractions of LPLs showed adenoviral DNA to be present in both cell types and ranged from a few hundred to several million copies per million cells on average. Active adenoviral gene expression as evidenced by the presence of adenoviral messenger RNA in intestinal lymphocytes was demonstrated in 9 of the 11 donors tested

Pre-Existing Adenovirus Immunity Modifies a Complex Mixed Th1 and Th2 Cytokine Response to an Ad5/HIV-1 Vaccine Candidate in Humans

The results of the recent Step Study highlight a need to clarify the effects of pre-existing natural immunity to a vaccine vector on vaccine-induced T-cell responses. To investigate this interaction, we examined the relationship between pre-existing Ad5 immunity and T-cell cytokine response profiles in healthy, HIV-uninfected recipients of MRKAd5 HIV-1 gag vaccine (HVTN 050, ClinicalTrials.gov #NCT00849732). Participants were grouped by baseline Ad5 neutralizing antibody titer as either Ad5-seronegative (titer ≤18; n = 36) or Ad5-seropositive (titer >200; n = 34). Samples from vaccine recipients were analyzed for immune responses to either HIV-1 Gag peptide pools or Ad5 empty vector using an ex vivo assay that measures thirty cytokines in the absence of long-term culture. The overall profiles of cytokine responses to Gag and Ad5 had similar combinations of induced Th1- and Th2-type cytokines, including IFN-γ, IL-2, TNF-α, IP-10, IL-13, and IL-10, although the Ad5-specific responses were uniformly higher than the Gag-specific responses (p<0.0001 for 9 out of 11 significantly expressed analytes). At the peak response time point, PBMC from Ad5-seronegative vaccinees secreted significantly more IP-10 in response to Gag (p = 0.008), and significantly more IP-10 (p = 0.0009), IL-2 (p = 0.006) and IL-10 (p = 0.05) in response to Ad5 empty vector than PBMC from Ad5-seropositive vaccinees. Additionally, similar responses to the Ad5 vector prior to vaccination were observed in almost all subjects, regardless of Ad5 neutralizing antibody status, and the levels of secreted IFN-γ, IL-10, IL-1Ra and GM-CSF were blunted following vaccination. The cytokine response profile of Gag-specific T cells mirrored the Ad5-specific response present in all subjects before vaccination, and included a number of Th1- and Th2-associated cytokines not routinely assessed in current vaccine trials, such as IP-10, IL-10, IL-13, and GM-CSF. Together, these results suggest that vector-specific humoral responses may reduce vaccine-induced T-cell responses by previously undetected mechanisms

Reconstitution of huPBL-NSG Mice with Donor-Matched Dendritic Cells Enables Antigen-Specific T-cell Activation

Humanized mouse models provide a unique opportunity to study human immune cells in vivo, but traditional models have been limited to the evaluation of non-specific T-cell interactions due to the absence of antigen-presenting cells. In this study, immunodeficient NOD/SCID/IL2r-γnull (NSG) mice were engrafted with human peripheral blood lymphocytes alone or in combination with donor-matched monocyte-derived dendritic cells (DC) to determine whether antigen-specific T-cell activation could be reconstituted. Over a period of 3 weeks, transferred peripheral blood lymphocytes reconstituted the spleen and peripheral blood of recipient mice with predominantly human CD45-positive lymphocytes. Animals exhibited a relatively normal CD4/CD8 ratio (average 1.63:1) as well as reconstitution of CD3/CD56 (averaging 17.8%) and CD20 subsets (averaging 4.0%). Animals reconstituted with donor-matched CD11c+ DC also demonstrated a CD11c+ population within their spleen, representing 0.27% to 0.43% of the recovered human cells with concurrent expression of HLA-DR, CD40, and CD86. When immunized with adenovirus, either as free replication-incompetent vector (AdV) or as vector-transduced DC (DC/AdV), there was activation and expansion of AdV-specific T-cells, an increase in Th1 cytokines in serum, and skewing of T-cells toward an effector/memory phenotype. T-cells recovered from animals challenged with AdV in vivo proliferated and secreted a Th1-profile of cytokines in response to DC/AdV challenge in vitro. Our results suggest that engrafting NSG mice with a combination of lymphocytes and donor-matched DC can reconstitute antigen responsiveness and allow the in vivo assessment of human immune response to viruses, vaccines, and other immune challenges

Comparison of Human Memory CD8 T Cell Responses to Adenoviral Early and Late Proteins in Peripheral Blood and Lymphoid Tissue

Treatment of invasive adenovirus (Ad) disease in hematopoietic stem cell transplant (SCT) recipients with capsid protein hexon-specific donor T cells is under investigation. We propose that cytotoxic T cells (CTLs) targeted to the late protein hexon may be inefficient in vivo because the early Ad protein E3-19K downregulates HLA class I antigens in infected cells. In this study, CD8+ T cells targeted to highly conserved HLA A2-restricted epitopes from the early regulatory protein DNA polymerase (P-977) and late protein hexon (H-892) were compared in peripheral blood (PB) and tonsils of naturally infected adults. In tonsils, epitope-specific pentamers detected a significantly higher frequency of P-977+CD8+ T cells compared to H-892+CD8+ T cells; this trend was reversed in PB. Tonsil epitope-specific CD8+ T cells expressed IFN-γ and IL-2 but not perforin or TNF-α, whereas PB T cells were positive for IFN-γ, TNF-α, and perforin. Tonsil epitope-specific T cells expressed lymphoid homing marker CCR7 and exhibited lower levels of the activation marker CD25 but higher proliferative potential than PB T cells. Finally, in parallel with the kinetics of mRNA expression, P-977-specific CTLs lysed targets as early as 8 hrs post infection. In contrast, H-892-specific CTLs did not kill unless infected fibroblasts were pretreated with IFN-γ to up regulate HLA class I antigens, and cytotoxicity was delayed until 16–24 hours. These data show that, in contrast to hexon CTLs, central memory type DNA polymerase CTLs dominate the lymphoid compartment and kill fibroblasts earlier after infection without requiring exogenous IFN-γ. Thus, use of CTLs targeted to both early and late Ad proteins may improve the efficacy of immunotherapy for life-threatening Ad disease in SCT recipients

Decreased Pre-existing Ad5 Capsid and Ad35 Neutralizing Antibodies Increase HIV-1 Infection Risk in the Step Trial Independent of Vaccination

<div><h3>Background</h3><p>The Step trial raised the possibility that uncircumcised men with pre-existing Ad5 neutralizing antibodies carried an increased risk of HIV infection after vaccination. Thus, understanding Ad seropositivity in humans is important to the development of an AIDS vaccine. Here, we analyze the impact of different Ad5-specific neutralizing antibodies on immune function and clinical outcome.</p> <h3>Methods and Findings</h3><p>Ad seropositivity in the Step trial volunteers was analyzed using chimeric rAd5/35 vectors to characterize their specificity for Ad5 fiber and non-fiber external (capsid) proteins. Immune responses and HIV seropositivity were correlated with the specificity of Ad5-neutralizing antibodies. Neutralizing antibodies induced by the vaccine in Ad5 seronegative subjects were directed preferentially to Ad5 capsid proteins, although some fiber-neutralizing antibodies could be detected. Pre-vaccination Ad5 serostatus did not affect the capsid-directed response after three vaccinations. In contrast, anti-fiber antibody titers were significantly higher in volunteers who were Ad5 seropositive prior to vaccination. Those Ad5 seropositive subjects who generated anti-capsid responses showed a marked reduction in vaccine-induced CD8 responses. Unexpectedly, anti-vector immunity differed qualitatively in Ad5 seropositive participants who became HIV-1 infected compared to uninfected case controls; Ad5 seropositive participants who later acquired HIV had lower neutralizing antibodies to capsid. Moreover, Ad35 seropositivity was decreased in HIV-infected subjects compared with uninfected case controls, while seroprevalence for other serotypes including Ad14, Ad28 and Ad41 was similar in both groups.</p> <h3>Conclusions</h3><p>Together, these findings suggest that the case subjects were less immunologically responsive prior to infection. Subjects infected during the Step trial had qualitative differences in immunity that increased their risk of HIV-1 infection independent of vaccination.</p> </div

Effects of supervised exercise on cancer-related fatigue in breast cancer survivors: a systematic review and meta-analysis

Background: Cancer-related fatigue (CRF) is the most common and distressing symptom in breast cancer survivors. Approximately 40% to 80% of cancer patients undergoing active treatment suffer from CRF. Exercise improves overall quality of life and CRF; however, the specific effects of the training modalities are not well understood.Methods: This study aimed to determine the pooled effects of supervised exercise interventions on CRF in breast cancer survivors. We searched PubMed/MEDLINE, EMBASE, Scopus, CENTRAL and CINAHL databases between December 2013 and January 2014 without language restrictions. Risk of bias and methodological quality were evaluated using the PEDro score. Pooled effects were calculated with a random-effects model according to the DerSimonian and Laird method. Heterogeneity was evaluated with the I2 test.Results: Nine high-quality studies (n = 1156) were finally included. Supervised aerobic exercise was statistically more effective than conventional care in improving CRF among breast cancer survivors (SMD = −0.51, 95%CI −0.81 to −0.21), with high statistical heterogeneity (P = 0.001; I2 = 75%). Similar effects were found for resistance training on CRF (SMD = −0.41, 95%CI −0.76 to −0.05; P = 0.02; I2 = 64%). Meta-regression analysis revealed that exercise volume parameters are closely related with the effect estimates on CRF. Egger’s test suggested moderate evidence of publication bias (P = 0.04).Conclusions: Supervised exercise reduces CRF and must be implemented in breast cancer rehabilitation settings. High-volume exercises are safe and effective in improving CRF and overall quality of life in women with breast cancer. Further research is encouraged.The authors would like to acknowledge Universidad Santo Tomás, Bogotá for the financial support to the GICAEDS Group (Project: Práctica del autoexamen de seno y los conocimientos, factores de riesgo y estilos de vida relacionados al cáncer de mama en mujeres jóvenes de la USTA – Number: 4110060001-008)

Vaccines against Tuberculosis: Where Are We and Where Do We Need to Go?

In this review we discuss recent progress in the development, testing, and clinical evaluation of new vaccines against tuberculosis (TB). Over the last 20 years, tremendous progress has been made in TB vaccine research and development: from a pipeline virtually empty of new TB candidate vaccines in the early 1990s, to an era in which a dozen novel TB vaccine candidates have been and are being evaluated in human clinical trials. In addition, innovative approaches are being pursued to further improve existing vaccines, as well as discover new ones. Thus, there is good reason for optimism in the field of TB vaccines that it will be possible to develop better vaccines than BCG, which is still the only vaccine available against TB