Biomedical subjects

David D Eckels

Publications and source records attributed to David D Eckels.

7 recordsLinked to original sources

PubMed2005

Interactions between helper T-cell epitopes of hepatitis C virus.

The premise of this work is that within a given hepatitis C virus (HCV) protein there exists an array of Th1 and Th2 epitopes, each of which can provide synergistic (positive or negative) effects upon other epitopes by intramolecular, cytokine-mediated immunoregulation of helper T-cell responses. To address this question, we constructed minigene plasmids pHCVTh1, pHCVTh1X3 and pHCVThR, and HCV NS3 full-length plasmid pHCVNS3. 293T cells were transfected with these plasmids and cell lysates from the transfected cells were used to stimulate PBMC from a patient with chronic HCV infection. IL-2 and IFN-gamma in the supernatant of the cultured PBMC were tested and proliferation of the PBMC was measured. The results demonstrate that interactions exist among helper T-cell epitopes; the synergistic effects of suppressive Th2 epitopes upon Th1 epitopes will inhibit the responses induced by Th1 epitopes, which may contribute to chronic infection by HCV; synergistic effects among Th1 epitopes induce higher levels of IFN-gamma, which may suggest a new strategy for HCV vaccine development. Further, stimulation of an HCV NS3 specific clone with cell lysates from 293T cells transfected with different constructs shows that the HCV NS3 clone could respond to all suggesting that the epitope-specific suppression may be due to an imbalance of Type 1 and Type 2 cytokines or regulatory T-cells.

Amino Acid Sequence↗

PubMed2005

Recognition of endogenously synthesized HLA-DR4 restricted HCV epitopes presented by autologous EBV transformed B-lymphoblastoid cell line.

Hepatitis C virus (HCV) causes non-A, non-B hepatitis and infects an estimated 170 million people worldwide. The treatment for HCV infection is often unsuccessful with high costs and many side-effects. There is a great need for alternative therapies including preventive and therapeutic vaccination for HCV infection. The experiments in this study were carried out to elucidate whether endogenously expressed antigen can be presented to helper T-cells restricted by class II molecules and to determine whether responses to plasmid-derived antigen resemble those that we have reported for recombinant antigens or synthetic peptides. To address these issues, a multi-epitope minigene was expressed in 293T-cells and Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells (BLCL). The transfected BLCLs were employed as APCs to stimulate epitope-specific T-cell hybridomas (THC). The results demonstrated that the endogenously expressed minigene antigens could be processed and presented to T-cell hybridomas by HLA matched BLCL. Five out of seven incorporated epitopes were recognized. Blockade of HLA DR could abolish the release of IL-2, which demonstrated that the endogenously expressed minigene antigens were presented by MHC class II molecules. The presentation of endogenously expressed antigens was much more efficient than that of exogenous antigens, at least in the present study. The findings obtained here have important significance for the development of an HCV DNA vaccine.

Animals↗

PubMed2003

Modulation of the peripheral T-Cell response by CD4 mutants of hepatitis C virus: transition from a Th1 to a Th2 response.

A disturbing feature of hepatitis C virus (HCV) is its long-term persistence in roughly 85% of those infected. Escape mutants may play a major role in HCV persistence. Our previous studies have identified a human leukocyte antigen DRB1*15 (HLA-DRB1*15) restricted Th1 epitope in the HCV NS3 protein, NS3(358-375), and escape variants of this epitope that may emerge under immune selection. Such variants attenuate or fail to stimulate T-cell proliferation. Here we provide data from peripheral blood mononuclear cells derived from four HLA-DRB1*15 patients chronically infected with HCV, and report that naturally occurring single amino acid substitutions in the Th1 epitope NS3(358-375) fail to stimulate proliferation, which is accompanied by a shift in cytokine secretion patterns from one characteristic of a Th1 antiviral responses to a Th2 form. Further, in one patient, we demonstrate that HCV variant peptides can effectively inhibit host polyclonal peripheral T-cell proliferation. We speculate that this phenomenon may be a factor in chronic HCV infection.

CD4-Positive T-Lymphocytes↗

PubMed2003

Cryptic infection and autoimmunity.

Infection as an environmental factor in autoimmunity has long been recognized. Numerous examples can be found in which pathogens express antigens that cross-react with host antigens or induce local inflammatory responses that can lead to autoimmune responses through a very complex set of circumstances. Borrowing from the relationship between chronic infection with hepatitis C virus and autoimmune hepatitis as an example, we consider the possibility that infection with an unknown virus having specific tissue tropism could lead to a perceived autoimmune process. We raise the question whether such should be considered for Type 1 diabetes.

Animals↗

PubMed2002

Sequence variation in the gene encoding the nonstructural 3 protein of hepatitis C virus: evidence for immune selection.

To determine whether the persistent nature of hepatitis C infection is related to the emergence of antigenic variants driven by immune selection, we examined the sequence heterogeneity in a portion of the hepatitis C virus (HCV) nonstructural 3 (NS3) gene of a patient infected over the course of more than 2 years. By PCR amplification, cloning, and sequencing, we observed several variable and conserved regions in the NS3 segment of the HCV genome. All variable regions had higher ratios of nonsynonymous/synonymous mutations and encompassed immunodominant epitopes, and their locations were not essential to maintain the known function of HCV RNA helicase. In contrast, the regions that are critical for HCV RNA helicase activity were found to be conserved with lower heterogeneity or lower ratios of nonsynonymous/synonymous mutations, and none except one of these regions was encoded within immunodominant epitopes. Our results are consistent with immune selection of viral variants at the epitope and molecular levels that may enable HCV to evade host defenses over time. Plotting the relatedness of sequence variants revealed a star topology suggesting that a wild-type HCV sequence is maintained, unlike HIV.

Amino Acid Sequence↗

PubMed2002

Functionally distinct helper T-cell epitopes of HCV and their role in modulation of NS3-specific, CD8+/tetramer positive CTL.

Hepatitis C virus specific (HCV-specific) CD8+ cytotoxic T cells play a critical role in viral clearance. Low HCV-specific cytotoxic T lymphocyte (CTL) responses in chronic HCV infection may favor the persistence of virus, whereas stimulation and expansion of HCV-specific CTL activity may assist elimination of HCV infection. Helper T cells control the intensity of CD8+ T-cell responses and helper T-cell responses are known to be compromised in chronic carriers of HCV. In this study, we wanted to ascertain if strengthening the Th response could increase the intensity of CTL activity against HCV target antigens. We selected a synthetic CTL peptide NS3(1073-1081)), two Th1 epitopes, peptide NS3(358-375) and NS5B(155-172), and one Th2 epitope, peptide NS3(505-521). By using the four peptides alone or in combinations, we stimulated peripheral blood cells isolated from a chronic hepatitis C patient in vitro and then analyzed CD8 T cells specific for the NS3(1073-1081) CTL epitope in A2 tetramer staining and cytotoxicity assays. The results demonstrated that CTL responses could be augmented by helper T-cell epitopes NS3(358-375) and NS5B(155-172). Th2 epitope NS3(505-521) inhibited augmentation of CTL activity by Th1 epitopes. This inhibitory effect could be overcome by combining the two Th1 epitopes NS3(358-375) and NS5B(155-172) together with NS3(505-521). Under such conditions, CTL frequency was restored, but cytotoxic activity remained low suggesting that the help provided under these cultures was sufficient to drive proliferation of CTL, but not sufficient to drive differentiation into mature killer cells. These results may provide some insights into compromised CTL activity in HCV viral persistence.

Amino Acid Sequence↗

PubMed2002

Epitopes of the NS3 protein of hepatitis C virus: recognition in HLA-DR4 transgenic mice.

Hepatitis C virus (HCV) infects more than 180 million of the world's population and causes a persistent infection that over decades can result in cirrhosis and hepatocellular carcinoma. Treatment is only partially effective and control is likely only with the development of effective vaccines. Currently, only chimpanzees can be infected with HCV and alternative animal and tissue culture models are badly needed. We have used mice transgenic for HLA-DR and human CD4 to analyse the specificity of murine responses to the HCV NS3 antigen in an effort to determine whether the epitopes recognized correspond to those recognized by human T cells. Indeed, determinants mapped in transgenic mice overlap with those in a patient exposed to HCV through infection. This result provides hope that such an animal model may be a useful tool with which to analyse particular aspects of immune responses to HCV in vivo.

Animals↗