One of the yawning information gaps highlighted by the Merck HIV vaccine trial is the absence of data regarding the impact of vaccination on adenovirus-specific T cell immune responses. Although it was logical for researchers to focus on the HIV-specific T cell responses induced by the vaccine, in retrospect it was an oversight to not pay attention to the effect of the vector on adenovirus-specific T cells, particularly CD4 T cells which are potential targets for HIV infection.
The literature on adenovirus-specific T cell immunity is relatively sparse, but the published data indicates:
- Adenovirus-specific T cell responses are detectable in the majority of individuals studied
- Responses are biased toward CD4 T cells with an effector memory phenotype
- Adenovirus-specific CD4 T cell responses are generally of a high magnitude but wane with age
- Adenovirus-specific CD4 T cells recognize epitopes that are conserved across adenovirus serotypes, including epitopes that are present even in Ad5 vectors with multiple gene deletions
Taken together, the published data certainly suggests that studies of the impact of Merck’s Ad5 vector on adenovirus-specific CD4 T cells should be a priority in the ongoing effort to understand the outcome of the Merck HIV vaccine trial.
J. Virol. 2008 Jan;82(1):546-54. Epub 2007 Oct 17.
Identification of hexon-specific CD4 and CD8 T-cell epitopes for vaccine and immunotherapy.
Leen AM, Christin A, Khalil M, Weiss H, Gee AP, Brenner MK, Heslop HE, Rooney CM, Bollard CM.
Center for Cell and Gene Therapy, Baylor College of Medicine, 6621 Fannin Street, MC 3-3320, Houston, TX 77030, USA.
Adenoviral infections in the immunocompromised host are associated with significant morbidity and mortality. Although the adoptive transfer of adenovirus-specific T cells may prevent and treat such infections, the T-cell immune response to the multiplicity of adenovirus serotypes and subspecies that infect humans has not been well characterized, impeding the development of such approaches. We have, therefore, analyzed the specificities of T-cell responses to the viral capsid hexon antigen, since this structure is highly conserved in human pathogens. We screened 25 human cytotoxic T-cell lines with adenovirus specificity to extensively characterize their responses to adenoviral hexon and to identify a panel of novel CD4(+) and CD8(+) T-cell epitopes. Using a peptide library spanning the entire sequence of the hexon protein, we confirmed the responsiveness of these cytotoxic T-cell lines to seven peptides described previously and also identified 33 new CD4- or CD8-restricted hexon epitopes. Importantly, the majority of these epitopes were shared among different adenovirus subspecies, suggesting that T cells with such specificities could recognize and be protective against multiple serotypes, simplifying the task of effective adoptive transfer or vaccine-based immunotherapy for treating infection by this virus.
J Gen Virol. 2007 Sep;88(Pt 9):2417-25.
Onion D, Crompton LJ, Milligan DW, Moss PA, Lee SP, Mautner V.
CRUK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK.
Adenovirus is a significant pathogen in immunocompromised patients and is widely utilized as a gene delivery vector, so a detailed understanding of the human immune response to adenovirus infection is critical. This study characterized the adenovirus-specific CD4(+) T-cell response of healthy donors by incubation with whole virus or with individual hexon and fiber proteins. Adenovirus-specific CD4(+) T cells averaged 0.26 % of the CD4(+) T-cell pool and were detectable in all donors. T cells recognizing the highly conserved hexon protein accounted for 0.09 %, whereas no response was observed against the fiber protein. A panel of hexon-specific CD4(+) T-cell clones was generated and shown to lyse targets infected with adenovirus from different serotypes and species. Three CD4 T-cell epitopes are described, which map to highly conserved regions of the hexon protein.
Br J Haematol. 2007 Jan;136(1):117-26. Epub 2006 Nov 8.
Capture and generation of adenovirus specific T cells for adoptive immunotherapy.
Chatziandreou I, Gilmour KC, McNicol AM, Costabile M, Sinclair J, Cubitt D, Campbell JD, Kinnon C, Qasim W, Gaspar HB.
Molecular Immunology Unit, Institute of Child Health, University College London, London, UK.
Adenoviral infections represent a major cause of morbidity and mortality following haematopoietic stem cell transplantation. Current anti-viral agents are virostatic and it is evident that elimination of adenovirus (ADV) infection is only achieved by recovery of cellular immunity. Using an interferon-gamma (IFN-gamma) secretion and capture assay to isolate ADV-specific T cells, followed by a 2 week expansion and restimulation protocol, we generated ADV T cells that may be used for cellular immunotherapy. In contrast to virus-specific T cells for cytomegalovirus or Epstein-Barr virus, the ADV response was dominated by CD4(+) T cells and the majority of captured cells exhibited an effector/memory immunophenotype. Highly specific antigen responses were demonstrated by intracellular IFN-gamma expression and cytotoxicity assays when the expanded cells underwent restimulation with ADV-pulsed target cells. Although T cells were initially generated in response to ADV species C, the expanded populations also showed strong activity against ADV species B, suggesting cross-reactivity across ADV species; a finding that has important clinical consequences in the paediatric setting, where the majority of infections are caused by ADV type B and C. The protocols can be readily translated to generate ADV-specific T cells suitable for clinical use and offer an effective immunotherapeutic strategy to control ADV infection.
Int Immunol. 2006 Nov;18(11):1521-9. Epub 2006 Sep 5.
Haveman LM, Bierings M, Legger E, Klein MR, de Jager W, Otten HG, Albani S, Kuis W, Sette A, Prakken BJ.
Department of Pediatric Immunology and Hematology, Wilhelmina Children's Hospital, University Medical Center, KC.03.063.0, Lundlaan 6, Postbus 85090, 3508 AB, Utrecht, The Netherlands.
Adenovirus can cause fatal infections in the immunocompromised host. To date, no effective anti-viral therapy is available. Adoptive therapy with adenovirus-specific T cells could be a promising treatment, but requires the identification of such T cells. Aim of this study was to identify conserved adenoviral T cell epitopes recognized in a majority of healthy individuals. By using a computer algorithm designed to predict pan-HLA-DR-binding T cell epitopes, we selected 19 peptides of adenovirus serotype 5. PBMCs from 26 healthy subjects were isolated and incubated with these peptides to test epitope-specific T cell proliferation. Six epitopes derived from E1B protein, hexon protein (two epitopes), DNA polymerase, E3A glycoprotein and fiber protein induced a proliferative T cell response in the majority of healthy controls. In vitro MHC binding assays confirmed the potential capacity of the adenovirus epitopes to bind multiple MHC alleles. The cytokine and chemokine profile induced by these epitopes was determined with a multiplex immunoassay and revealed a predominant pro-inflammatory pattern. Based on the broad recognition and the induced cytokine and chemokine profile, the detected epitopes can be regarded as potential candidates to select adenovirus-specific T cells for immune intervention in the immunocompromised host.
J Virol. 2003 Jun;77(11):6562-6. (free access to full text)
Heemskerk B, Veltrop-Duits LA, van Vreeswijk T, ten Dam MM, Heidt S, Toes RE, van Tol MJ, Schilham MW.
Department of Pediatrics, Leiden University Medical Center, The Netherlands.
Adenovirus (Ad)-specific T-cell responses in healthy adult donors were investigated. Ad5, inactivated by methylene blue plus visible light, induced proliferation and gamma interferon (IFN-gamma) production in peripheral blood mononuclear cells of the majority of donors. Responding T cells were CD4(+) and produced IFN-gamma upon restimulation with infectious Ad5 and Ads of different subgroups. T-cell clones showed distinct cross-reactivity patterns recognizing Ad serotypes from either one subgroup (C), two subgroups (B and C), or three subgroups (A, B, and C). This cross-reactivity of Ad-specific T cells has relevance both for Ad-based gene therapy protocols, as well as for the feasibility of T-cell-mediated adoptive immunotherapy in recipients of an allogeneic stem cell transplantation.
Hum Gene Ther. 2002 Jul 1;13(10):1167-78.
The adenovirus capsid protein hexon contains a highly conserved human CD4+ T-cell epitope.
Olive M, Eisenlohr L, Flomenberg N, Hsu S, Flomenberg P.
Center of Human Virology and Department of Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA.
The immunogenicity of adenovirus vectors remains a major obstacle to their safe and efficacious use for gene therapy. In order to identify T-cell epitopes directly from adenoviruses, four viral protein sequences were screened for the well-characterized 9-mer HLA-A2 binding motif. Peripheral blood mononuclear cells (PBMC) from healthy adults were tested for responses to 17 selected viral peptides using a short-term interferon-gamma ELISPOT assay. Memory T-cell responses were identified to a single peptide derived from the major capsid protein hexon in 5 of 6 HLA-A2-positive donors. Unexpectedly, responses to this hexon peptide were also detected in 4 of 6 HLA-A2-negative donors, and responder cells were identified as CD4(+) T cells by immunomagnetic depletion experiments. A longer 15-mer peptide, H910-924, was identified as the optimal CD4(+) T-cell epitope. This hexon epitope induces strong proliferative T-cell responses that can be blocked by a monoclonal antibody against HLA-DR, and molecular HLA typing of donors suggests that the peptide response is restricted by multiple HLA-DR alleles. Additionally, quantitative analysis of responses to H910-924 and whole adenovirus reveals that the frequency of circulating CD4(+) T cells specific for this single hexon epitope (mean = 61 per 10(6) PBMC) represents up to one third of the total adenovirus-specific T-cell response. Finally, comparison of hexon sequences from over 20 different human adenovirus serotypes indicates that H910-924 is highly conserved. In most individuals, therefore, T-cell responses to this hexon epitope will be induced by all adenovirus vectors, including "gutted" vectors packaged with capsid proteins and vectors based on different serotypes.
J Infect Dis. 2002 May 15;185(10):1379-87. Epub 2002 Apr 30. (free access to full text)
Age-related decrease in adenovirus-specific T cell responses.
Sester M, Sester U, Alarcon Salvador S, Heine G, Lipfert S, Girndt M, Gärtner B, Köhler H.
Medical Department IV, Institute of Medical Microbiology and Hygiene, University of the Saarland, Homburg, Germany.
Infections with persistent viruses, such as cytomegalovirus (CMV) or adenovirus, are not, in general, clinically apparent but may cause serious complications in the immunocompromised host. As has been shown for CMV, the cellular arm of the immune response is essential in controlling viral replication. However, cellular immunity toward adenoviruses has not been well characterized in humans. The aim of the present study was the quantitative and functional analysis of adenovirus-specific T cell responses from 171 healthy individuals and 59 long-term renal transplant recipients by use of flow-cytometric, as well as standard proliferation and enzyme-linked immunosorbant, assays. Adenovirus-specific immunity is dominated by CD4 T cells with memory/effector phenotype. Of interest, the frequency of adenovirus-specific T cells decreases significantly with age. This age-related decline indicates the eventual elimination of adenoviruses within a lifetime that may explain the well-known clinical observation of a predominant incidence of adenoviral complications in children and young adults, compared with older adults, after transplantation.
Viral Immunol. 2001;14(4):403-13.
Quantitative analysis of adenovirus-specific CD4+ T-cell responses from healthy adults.
Olive M, Eisenlohr LC, Flomenberg P.
Department of Medicine, Center for Human Virology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Although nearly all adults are seropositive for adenoviruses, little is known about the cellular immune responses to these ubiquitous pathogens. We have previously identified adenovirus-specific proliferative T-cell responses in peripheral blood mononuclear cells (PBMC) from healthy adults. In this study, memory T-cell responses to adenovirus were further evaluated in healthy adult donors using a short term, quantitative enzyme-linked immunospot assay (ELISPOT) assay. Adenovirus antigen induced specific secretion of interferon-gamma (IFN-gamma) from PBMC within 12 hours of incubation. PBMC from 20 of 22 healthy donors (90.9%) expressed IFN-y in response to adenovirus. Responder cells were identified as CD4+ T cells by immunomagnetic depletion methods. Interleukin-4 (IL-4) secretion was not detected, consistent with a TH1 response. There was a 10-fold variation in the frequencies of adenovirus-specific CD4+ T cells between donors (range, 34 to 294; median, 122 per million PBMC). Adenovirus-specific T cell frequencies remained stable over periods up to 2 years among individual donors, but there was an inverse correlation between frequency and donor age. These quantitative data suggest that most adults retain adenovirus-specific cellular memory after childhood exposure. This assay may be useful for the evaluation of adenovirus-specific CD4+ T-cell responses in patients treated with adenovirus gene therapy vectors and the identification of major T-cell epitopes.
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