Dataset Information

Identification of the HERV-K gag antigen in prostate cancer by SEREX using autologous patient serum and its immunogenicity.

ABSTRACT: The prostate cancer HERV-K gag-related NGO-Pr-54 antigen was identified by SEREX analysis using autologous patient serum. NGO-Pr-54 mRNA was observed to be faintly expressed in normal prostate and strongly expressed in a variety of cancers, including ovarian cancer (5/8), prostate cancer (6/9), and leukemia (5/14). A phage plaque assay showed that a strong reaction was constantly observed with clone ZH042 in which the 5' end of NGO-Pr-54 is deleted, suggesting that it contained the sequence coding for the protein product. A TI-35 mAb was produced using a recombinant protein (438 aa) deduced from the sequence of ZH042. Transfection of clone ZH042 into 293T cells resulted in the production of an approximately 50-kDa molecule visualized by Western blotting. Natural production of the molecule was confirmed in a SK-MEL-23 melanoma cell line. An indirect immunofluorescence assay showed that NGO-Pr-54 protein was expressed on the cell surface as well as in the cytoplasm. Cell surface expression was confirmed by flow cytometry using the TI-35 mAb. The antibody response against NGO-Pr-54 was observed in patients with bladder (5.1%), liver (4.1%), lung (3.4%), ovarian (5.6%), and prostate (4.2%) cancer, as well as with malignant melanoma (13.2%).

SUBMITTER: Ishida T

PROVIDER: S-EPMC2935773 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Identification of the HERV-K gag antigen in prostate cancer by SEREX using autologous patient serum and its immunogenicity.

Ishida Toshiaki T Obata Yuichi Y Ohara Nobuya N Matsushita Hirokazu H Sato Shuichiro S Uenaka Akiko A Saika Takashi T Miyamura Takako T Chayama Kosuke K Nakamura Yurika Y Wada Hisashi H Yamashita Toshiharu T Morishima Tsuneo T Old Lloyd J LJ Nakayama Eiichi E

Cancer immunity 20081113

The prostate cancer HERV-K gag-related NGO-Pr-54 antigen was identified by SEREX analysis using autologous patient serum. NGO-Pr-54 mRNA was observed to be faintly expressed in normal prostate and strongly expressed in a variety of cancers, including ovarian cancer (5/8), prostate cancer (6/9), and leukemia (5/14). A phage plaque assay showed that a strong reaction was constantly observed with clone ZH042 in which the 5' end of NGO-Pr-54 is deleted, suggesting that it contained the sequence codi ...[more]

PMID: 19006261

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Project description:BACKGROUND: Esophageal squamous cell carcinoma (SCC) represents one of the most malignant tumors. To improve the poor prognosis, it is necessary to diagnose esophageal SCC at early stages using new tumor markers. SEREX (serological identification of antigens by recombinant cDNA expression cloning) is suitable for large-scale screening of tumor antigens and has been applied for various types of human tumors. METHODS: Tumor markers of esophageal squamous cell carcinoma (SCC) were screened by SEREX method. The presence of serum anti-makorin 1 (MKRN1) antibodies (s-MKRN1-Abs) was examined by Western blotting using bacterially expressed MKRN1 protein. The expression levels of MKRN1 mRNA in tissues were examined by RT-PCR. The biological activity of MKRN1 was examined by transfection of ras-NIH3T3 mouse fibroblasts with MKRN1 cDNA. Major ubiquitinated proteins in MKRN1-transfected cells were identified by immunoprecipitation with anti-ubiquitin antibody followed by mass spectrometry. RESULTS: MKRN1 was identified as a novel SEREX antigen of esophageal SCC. Although a total of 18 (25%) of 73 patients with esophageal SCC had s-MKRN1-Abs, none of the 43 healthy donors had a detectable level of s-MKRN1-Abs. There was no correlation between the presence of s-MKRN1-Abs and clinicopathological variables other than histological grading. Well-differentiated tumors were associated significantly with the presence of s-MKRN1-Abs in the patients. The mRNA levels of MKRN1 were frequently higher in esophageal SCC tissues than in the peripheral normal esophageal mucosa. Stable transfection of ras-NIH3T3 cells with MKRN1 cDNA induced prominent morphological changes such as enlargement of the cell body and spreading. Ubiquitination of 80- and 82-kDa proteins were clearly observed in MKRN1-transfected cells but not in the parental cells, which were identified as L-FILIP (filamin A interacting protein 1). CONCLUSION: MKRN1 is a novel SEREX antigen of esophageal SCC, and s-NKRN1-Abs can be a candidate of diagnostic markers of esophageal SCC with high specificity. It is plausible that MKRN1 is involved in carcinogenesis of the well-differentiated type of tumors possibly via ubiquitination of L-FILIP.

Project description:BackgroundHIV Gag-specific CD4+ and CD8+ T-cell responses are important for HIV immune control. Pulsing overlapping Gag peptides on autologous lymphocytes (OPAL) has proven immunogenic and effective in reducing viral loads in multiple pigtail macaque studies, warranting clinical evaluation.MethodologyWe performed a phase I, single centre, placebo-controlled, double-blinded and dose-escalating study to evaluate the safety and preliminary immunogenicity of a novel therapeutic vaccine approach 'OPAL-HIV-Gag(c)'. This vaccine is comprised of 120 15mer peptides, overlapping by 11 amino acids, spanning the HIV Gag C clade sequence proteome, pulsed on white blood cells enriched from whole blood using a closed system, followed by intravenous reinfusion. Patients with undetectable HIV viral loads (<50 copies/ml plasma) on HAART received four administrations at week 0, 4, 8 and 12, and were followed up for 12 weeks post-treatment. Twenty-three people were enrolled in four groups: 12 mg (n?=?6), 24 mg (n?=?7), 48 mg (n?=?2) or matching placebo (n?=?8) with 18 immunologically evaluable. T-cell immunogenicity was assessed by IFN? ELIspot and intracellular cytokine staining (ICS).ResultsThe OPAL-HIV-Gag(c) peptides were antigenic in vitro in 17/17 subjects. After vaccination with OPAL-HIV-Gag(c), 1/6 subjects at 12 mg and 1/6 subjects at 24 mg dose groups had a 2- and 3-fold increase in ELIspot magnitudes from baseline, respectively, of Gag-specific CD8+ T-cells at week 14, compared to 0/6 subjects in the placebo group. No Gag-specific CD4+ T-cell responses or overall change in Rev, Nef, Tat and CMV specific responses were detected. Marked, transient and self-limiting lymphopenia was observed immediately post-vaccination (4 hours) in OPAL-HIV-Gag(c) but not in placebo recipients, with median fall from 1.72 to 0.67 million lymphocytes/mL for active groups (P<0.001), compared to post-placebo from 1.70 to 1.56 lymphocytes/ml (P?=?0.16).Conclusion/significanceDespite strong immunogenicity observed in several Macaca nemestrina studies using this approach, OPAL-HIV-Gag(c) was not significantly immunogenic in humans and improved methods of generating high-frequency Gag-specific T-cell responses are required.Name of registryClinicalTrials.gov, Registry number: NCT01123915, URL trial registry database: http://www.clinicaltrials.gov/ct2/results?term=OPAL-HIV-1001&Search=Search.

Project description:BACKGROUND: The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. RESULTS: By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and proline-rich peptides, QP1 and QP2, spanning 23 and 19 amino acids, respectively. CONCLUSIONS: Expression of reconstituted sequences of original HERV elements is an important tool for studying fundamental aspects of the biology of these ancient viruses. The analysis of HERV-K(HML-2) Gag processing and the nature of the mature Gag proteins presented here will facilitate further studies of the discrete functions of these proteins and of their potential impact on the human host.

Dataset Information

Identification of the HERV-K gag antigen in prostate cancer by SEREX using autologous patient serum and its immunogenicity.

Publications

Identification of the HERV-K gag antigen in prostate cancer by SEREX using autologous patient serum and its immunogenicity.

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