Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although much effort has been expended on evaluating recombinant proteins and synthetic peptides as immunogens, they have generally proved incapable of inducing an efficient cytotoxic T-cell (CTL) response. Filamentous bacteriophage fd can display multiple copies of foreign peptides in the N-terminal region of its major coat protein pVIII, 2,700 copies of which make up the virus capsid. Here we show that fd virions displaying peptide RT2 (ILKEPVHGV), corresponding to residues 309-317 of the reverse transcriptase (RTase) of HIV-1, are able to prime a CTL response specific for this HIV-1 epitope in human cell lines. Successful priming also requires a T-helper epitope, pep23 (KDSWTVNDIQKLVGK), corresponding to residues 249-263 of HIV-1 RTase. Supplying this by displaying it on either the same or a separate bacteriophage virion led to activation of antigen-specific CD4+ T cells. Likewise, HLA-A2 transgenic mice immunized with bacteriophage virions displaying peptide RT2 were shown to mount an effective, specific anti-HIV-RT2 CTL response. This unexpected ability to elicit a designated cytolytic T-cell response, in addition to a B-cell response, has important implications for access to the class I major histocompatibility complex (MHC) loading compartment and the development of recombinant vaccines.
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PMID:Phage display of peptide epitopes from HIV-1 elicits strong cytolytic responses. 1093 58

Vigorous HIV-1-specific CD8(+) cytotoxic T lymphocyte (CTL) responses play an important role in the control of HIV-1 replication and the induction of a strong, broadly cross-reactive CTL response remains an important goal of HIV vaccine development. It is known that the display of high levels of class I MHC-viral peptide complexes at the cell surface of target cells is necessary to elicit a strong CTL response. We now report two strategies to enhance the presentation of defined HIV-1 epitope-specific CTL target structures, by incorporating subdominant HIV-1 reverse transcriptase (RT) CTL epitope sequences into the human class I MHC molecule HLA-A2. We show that either incorporation of HIV-1 CTL epitopes into the signal sequence of HLA or tethering of epitopes to the HLA-A2 heavy chain provide simple ways to create effective CTL target structures that can be recognized and lysed by human HLA-A2-restricted RT-specific CD8(+) CTL. Moreover, cells expressing these epitope-containing HLA-A2 constructs stimulated the generation of primary epitope-specific CTL in vitro. These strategies offer new options in the design of plasmid DNA-based vaccines or immunotherapeutics for the induction of CTL responses against subdominant HIV-1 epitopes.
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PMID:Creating HIV-1 reverse transcriptase cytotoxic T lymphocyte target structures by HLA-A2 heavy chain modifications. 1096 24

The functional status of circulating human immunodeficiency (HIV)-specific CD8 T cells in chronically infected subjects was evaluated. By flow cytometry, only 5 of 7 subjects had detectable CD8 T cells that produced IFN-gamma after stimulation with HIV-infected primary CD4 T cells. In 2 subjects, the frequency of IFN-gamma-producing cells increased 4-fold when IL-2 was added to the culture medium; in another subject, IFN-gamma-producing cells could be detected only after IL-2 was added. IFN-gamma-producing cells ranged from 0.4% to 3% of CD8 T cells. Major histocompatibility complex-peptide tetramer staining, which identifies antigen-specific T cells irrespective of function, was used to evaluate the proportion of HIV-specific CD8 T cells that may be nonfunctional in vivo. CD8 T cells binding to tetramers complexed to HIV gag epitope SLYNTVATL and reverse transcriptase epitope YTAFTIPSI were identified in 9 of 15 and 5 of 12 HLA-A2-expressing seropositive subjects at frequencies of 0.1% to 1.1% and 0.1 to 0.7%, respectively. Freshly isolated tetramer-positive cells expressed a mixed pattern of memory and effector markers. On average, IFN-gamma was produced by less than 25% of tetramer-positive CD8 T cells after stimulation with the relevant gag or reverse transcriptase peptide. In all subjects tested, freshly isolated CD8 T cells were not cytolytic against peptide-pulsed B lymphoblastoid cell line or primary HIV-infected CD4 T-cell targets. Exposure to IL-2 enhanced the cytotoxicity of CD8 T cells against primary HIV-infected CD4 targets in 2 of 2 subjects tested. These results suggest that a significant proportion of HIV-specific CD8 T cells may be functionally compromised in vivo and that some function can be restored by exposure to IL-2.
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PMID:Impaired function of circulating HIV-specific CD8(+) T cells in chronic human immunodeficiency virus infection. 1104 89

An autologous melanoma cell line selected for loss of expression of the immunodominant MART-1 and gp100 antigens was initially used to carry out a mixed lymphocyte tumor culture (MLTC) in a patient who expressed the human leukocyte antigen (HLA)-AI and HLA-A2 class I major histocompatibility complex alleles. Ten clones identified from this MLTC seemed to recognize melanoma in an HLA-A1-restricted manner but failed to recognize a panel of previously described melanoma antigens. The screening of an autologous melanoma cDNA library with one HLA-Al-restricted melanoma-reactive T-cell clone resulted in the isolation of a cDNA clone called AIM-2 (antigen isolated from immunoselected melanoma-2). The AIM-2 transcript seemed to have retained an intronic sequence based on its alignment with genomic sequences as well as expressed sequence tags. This transcript was not readily detected after Northern blot analysis of melanoma mRNA, indicating that only low levels of this product may be expressed in tumor cells. Quantitative reverse transcriptase-polymerase chain reaction analysis, however, demonstrated a correlation between T-cell recognition and expression in HLA-A1-expressing tumor cell lines. A peptide that was encoded within a short open reading frame of 23 amino acids and conformed to the HLA-A1 binding motif RSDSGQQARY was found to represent the T-cell epitope. The AIM-2-reactive T-cell clone recognized a number of neuroectodermal tumors as well as breast, ovarian, and colon carcinomas that expressed HLA-A1, indicating that this represents a widely expressed tumor antigen. Thus, AIM-2 may represent a potential target for the development of vaccines in patients bearing tumors of a variety of histologies.
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PMID:Melanoma-Reactive CD8+ T cells recognize a novel tumor antigen expressed in a wide variety of tumor types. 1156 34

The presentation of endogenously synthesized peptides in association with HLA class I molecules allows the activation of CD8(+) lymphocytes. Tumor cells often fail to present antigenic peptides resulting in the immune escape of metastasizing cells. The aim of this study was to elucidate possible molecular mechanisms leading to reduced antigen presentation in melanoma. Melanoma cell short-time cultures were genotypically and phenotypically HLA-typed by sequence-specific primer polymerase chain reaction and complement-mediated microlymphocytotoxicity assays, respectively. Flow cytometric analysis of HLA-A2 and HLA-A3 allospecificities were performed to confirm typing results. Transcriptional levels of classical HLA-A, HLA-B genes and nonclassical HLA-G genes were detected using quantitative real-time reverse transcriptase polymerase chain reaction (LightCycler). We found loss or downregulation of HLA proteins in 18% (for HLA-A) and 53% (for HLA-B) of all tested metastases. Genomic analysis, however, revealed the presence of the corresponding HLA class I gene in six out of seven cases. On the level of gene transcription we observed a differential regulation of HLA-A, HLA-B, and HLA-G mRNA expression. There was no correlation between classical and nonclassical HLA gene transcription, but the transcriptional levels of classical HLA corresponded to the protein expression levels. Furthermore, an overall reduced amount of HLA class I gene transcription was observed in melanoma metastases during disease progression in three individuals. We postulate that there is a transcriptional regulation of HLA class I gene expression in melanoma cells. These data suggest that treatment approaches aimed at activating specific cytotoxic T lymphocytes are most successful in early disease.
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PMID:Decreased intraindividual HLA class I expression is due to reduced transcription in advanced melanoma and does not correlate with HLA-G expression. 1188 14

Immunization with wild-type sequence (wt) p53 epitopes represents a novel therapeutic strategy for cancer patients with tumors accumulating mutant p53. To evaluate usefulness of p53-derived peptides as future cancer vaccines, frequencies of wt p53(264-272) peptide-specific CD8+ T cells were determined in the peripheral circulation of patients with squamous cell carcinoma of the head and neck (SCCHN). T cells of 30 HLA-A2.1+ patients and 31 HLA-A2.1+ healthy individuals were evaluated by multicolor flow cytometry analysis using peptide-HLA-A2.1 complexes (tetramers). T cells specific for an influenza matrix peptide (a model recall antigen) or an HIV reverse transcriptase peptide (a model novel antigen) were studied in parallel. Patients with SCCHN had a significantly higher mean frequency of CD8+ T cells specific for wt p53(264-272) than normal donors (P = 0.0041). Surprisingly, the frequency of epitope-specific T cells in the circulation of patients did not correlate with p53 accumulation in the tumor. In patients whose tumors had normal p53 expression or had p53 gene mutations preventing presentation of this epitope, high frequencies of wt p53(264-272)-specific CD8+ T cells were found, of which many were memory T cells. In contrast, patients whose tumors accumulated p53 had low frequencies of wt p53(264-272)-specific CD8+ T cells, which predominantly had a naive phenotype and were unable to proliferate ex vivo in response to the epitope, as reported by us previously (T. K. Hoffmann, J. Immunol., 165: 5938-5944, 2000). This seemingly contradictory relationship between the high frequency of epitope-specific T cells and wt p53 expression in the tumor suggests that other factors may contribute to the observed anti-p53 responses. Human papillomavirus-16 E6/E7 expression is common in SCCHN, and E6 is known to promote presentation of wt p53 epitopes. Although human papillomavirus-16 E6/E7 expression was detected in 46% of the tumors, it did not correlate with the frequency of wt p53(264-272)-specific CD8+ T cells or with p53 expression in the tumor. These findings emphasize the complexity of interactions between the tumor and the host immune system, and, thus, have particularly important implications for future p53-based immunization strategies.
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PMID:Frequencies of tetramer+ T cells specific for the wild-type sequence p53(264-272) peptide in the circulation of patients with head and neck cancer. 1206 99

The icosahedral protein scaffold (1.5MDa) generated by self-assembly of the catalytic domains of the dihydrolipoyl acetyltransferase core of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus has been engineered to display 60 copies of one or more peptide epitopes on a single molecule (E2DISP). An E2DISP scaffold displaying pep23, a 15-residue B- and T-helper epitope from the reverse transcriptase of HIV-1, was able to induce a pep23-specific T-helper response in cell lines in vitro. The same scaffold displaying both pep23 and peptide RT2, a nine-residue CTL epitope from HIV-1 reverse transcriptase, was able to prime an RT2-specific CD8(+) T-cell response in human cell lines in vitro and in HLA-A2 transgenic mice in vivo. This was accompanied by a humoral antibody response specific for E2DISP-presented epitopes. Thus, the icosahedral acetyltransferase core constitutes a simple and flexible scaffold for multiple epitope display with access to both cellular and humoral immune response pathways.
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PMID:Induction of specific T-helper and cytolytic responses to epitopes displayed on a virus-like protein scaffold derived from the pyruvate dehydrogenase multienzyme complex. 1261 47

HIV epitopes may have developed to be poor immunogens. As a counterapproach HIV vaccine strategy, we used epitope enhancement of a conserved HIV reverse transcriptase (RT) epitope for induction of antiviral protection in HLA-A2-transgenic mice mediated by human HLA-A2-restricted CTLs. We designed two epitope-enhanced peptides based on affinity for HLA-A2, one substituted in anchor residues (RT-2L9V) and the other also with tyrosine at position 1 (RT-1Y2L9V), and examined the balance between HLA binding and T cell recognition. CTL lines and bulk cultures in two HLA-A2-transgenic mouse strains showed that RT-2L9V was more effective in inducing CTL reactive with wild-type Ag than RT-1Y2L9V, despite the higher affinity of the latter, because the 1Y substitution unexpectedly altered T cell recognition. Accordingly, RT-2L9V afforded the greatest protection in vivo against a surrogate virus expressing HIV-1 RT mediated by HLA-A2-restricted CTL in a mouse in which all CTL are restricted to only the human HLA molecule. Such antiviral protection has not been previously achieved with an HLA epitope-enhanced vaccine. These findings define a critical balance between MHC affinity and receptor cross-reactivity required for effective epitope enhancement and also demonstrate construction and efficacy of such a component of a new generation vaccine.
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PMID:Epitope-enhanced conserved HIV-1 peptide protects HLA-A2-transgenic mice against virus expressing HIV-1 antigen. 1292 5

Fibromodulin (FMOD) was shown to be highly overexpressed in chronic lymphocytic leukemia (CLL) cells compared with normal B lymphocytes by gene expression profiling. Therefore FMOD might serve as potential tumor-associated antigen (TAA) in CLL, enabling expansion of FMOD-specific T cells. In CLL samples derived from 16 different patients, high expression of FMOD by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was detectable in contrast to normal B lymphocytes. We used unpulsed native CLL cells and CD40 ligand (CD40L)-stimulated CLL cells as antigen-presenting cells (APCs) to expand autologous T cells from 13 patients. The number of T cells during 4 weeks of in vitro culture increased 2- to 3.5-fold and the number of T cells recognizing FMOD peptides bound to HLA-A2 dimers increased 10-fold. The expanded T cells also were able to secrete interferon-gamma (IFN-gamma) upon recognition of the antigen demonstrated by IFN-gamma ELISPOT assays. T cells not only recognized HLA-A2-binding FMOD peptides presented by transporter-associated with antigen-processing (TAP)-deficient T2 cells, but also FMOD overexpressing autologous CLL cells in an HLA-A2-restricted manner. In summary, FMOD was shown for the first time to be naturally processed and presented as TAA in primary CLL cells, enabling the expansion of autologous tumor-specific T cells.
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PMID:Fibromodulin as a novel tumor-associated antigen (TAA) in chronic lymphocytic leukemia (CLL), which allows expansion of specific CD8+ autologous T lymphocytes. 1547 55

We have generated a codon-optimized hGagp17p24-Polp51 plasmid DNA expressing the human immunodeficiency virus type 1 (HIV-1) Gag-Pol fusion protein that consists of clusters of highly conserved cytotoxic T lymphocyte (CTL) epitopes presented by multiple MHC class I alleles. In the hGagp17p24-Polp51 construct, the ribosomal frameshift site had been deleted together with the potentially immunosuppressive Gag nucleocapsid (p15) as well as Pol protease (p10) and integrase (p31). Analyses of the magnitude and breadth of cellular responses demonstrated that immunization of HLA-A2/K(b) transgenic mice with the hGagp17p24-Polp51 construct induced 2- to 5-fold higher CD8+ T-cell responses to Gag p17-, p24-, and Pol reverse transcriptase (RT)-specific CTL epitopes than the full-length hGag-PolDeltaFsDeltaPr counterpart. The increases were correlated with higher protection against challenge with recombinant vaccinia viruses (rVVs) expressing gag and pol gene products. Consistent with the profile of Gag- and Pol-specific CD8+ T cell responses, an elevated level of type 1 cytokine production was noted in p24- and RT-stimulated splenocyte cultures established from hGagp17p24-Polp51-immunized mice compared to responses induced with the hGag-PolDeltaFsDeltaPr vaccine. Sera of mice immunized with the hGagp17p24-Polp51 vaccine also exhibited an increased titer of p24- and RT-specific IgG2 antibody responses. The results from our studies provide insights into approaches for boosting the breadth of Gag- and Pol-specific immune responses.
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PMID:Clustered epitopes within the Gag-Pol fusion protein DNA vaccine enhance immune responses and protection against challenge with recombinant vaccinia viruses expressing HIV-1 Gag and Pol antigens. 1568 Apr 12


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