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Optimizing peptide matrices for identifying T-cell antigens.

ABSTRACT: Mapping T-cell epitopes for a pathogen or vaccine requires a complex method for screening hundreds to thousands of peptides with a limited amount of donor sample. We describe an optimized deconvolution process by which peptides are pooled in a matrix format to minimize the number of tests required to identify peptide epitopes. Four peptide pool matrices were constructed to deconvolute the HIV-specific T-cell response in three HIV-infected individuals. ELISpot assays were used to map peptide antigens. Many HIV peptides were mapped in all three individuals. However, there were several challenges and limitations associated with the deconvolution process. Peptides that induced low-frequency responses or were masked by peptide competition within a given pool were not identified, because they did not meet the threshold criteria for a positive response. Also, amino acid sequence variation limited the ability of this method to map autologous HIV peptides. Alternative analysis strategies and revisions to the original matrix optimizations are presented that address ways to increase peptide identification. This optimized deconvolution method allows for efficient mapping of T-cell peptide epitopes. It is rapid, powerful, efficient, and unrestricted by HLA type.

SUBMITTER: Precopio ML

PROVIDER: S-EPMC2586828 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Optimizing peptide matrices for identifying T-cell antigens.

Precopio Melissa L ML Butterfield Tiffany R TR Casazza Joseph P JP Little Susan J SJ Richman Douglas D DD Koup Richard A RA Roederer Mario M

Cytometry. Part A : the journal of the International Society for Analytical Cytology 20081101 11

Mapping T-cell epitopes for a pathogen or vaccine requires a complex method for screening hundreds to thousands of peptides with a limited amount of donor sample. We describe an optimized deconvolution process by which peptides are pooled in a matrix format to minimize the number of tests required to identify peptide epitopes. Four peptide pool matrices were constructed to deconvolute the HIV-specific T-cell response in three HIV-infected individuals. ELISpot assays were used to map peptide anti ...[more]

PMID: 18781655

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Project description:BACKGROUND:Nasopharyngeal carcinoma (NPC) is a solid tumor of the head and neck. Multimodal therapy is highly effective when NPC is detected early. However, due to the location of the tumor and the absence of clinical signs, early detection is difficult, making a biomarker for the early detection of NPC a priority. The dysregulation of small non-coding RNAs (miRNAs) during carcinogenesis is the focus of much current biomarker research. Herein, we examine several miRNA discovery methods using two sample matrices to identify circulating miRNAs (c-miRNAs) associated with NPC. METHODS:We tested two miRNA discovery workflows on two sample sources for miRNAs associated with NPC. In the first workflow, we assumed that NPC tumor tissue would be enriched for miRNAs, so we compared miRNA expression in FFPE from NPC cases and controls using microarray and RNA-Seq technologies. Candidate miRNAs from both technologies were verified by qPCR in FFPE and sera from an independent NPC sample set. In a second workflow, we directly interrogated NPC case and control sera by RNA-Seq for c-miRNAs associated with NPC, with candidate c-miRNAs verified by qPCR in the sera from the same independent NPC sample set. RESULTS:Both microarray and RNA-Seq narrowed the miRNA signature to 1-5% of the known mature human miRNAs. Moreover, these two methods produced similar results when applied to the same sample type (FFPE), with RNA-Seq additionally indicating "unknown" miRNAs associated with NPC. However, we found different miRNA profiles in NPC sera compared to FFPE using RNA-Seq, with the few overlapping miRNAs found to be significantly up-regulated in FFPE significantly down-regulated in sera (and vice versa). Despite the different miRNA profiles found in FFPE and sera, both profiles strongly associated with NPC, providing two potential sources for biomarker signatures for NPC. CONCLUSIONS:We determined that the direct interrogation of sera by RNA-Seq was the most informative method for identifying a c-miRNA signature associated with NPC. We also showed that there are different miRNA expression profiles associated with NPC for tumor tissue and sera. These results reflect on the methods and meaning of miRNA biomarkers for NPC in tissue and peripheral blood.

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Optimizing peptide matrices for identifying T-cell antigens.

Publications

Optimizing peptide matrices for identifying T-cell antigens.

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