HCMV Deletion Mutants for T-Cell Epitope Identification
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Solution Overview
Problem
Current methods for identifying T-cell specificities for human cytomegalovirus (HCMV) are hindered by the virus's large protein coding capacity and immune evasion strategies, limiting the effectiveness of treatments and vaccines for HCMV-associated pathologies.
Innovation Solution
The use of HCMV deletion mutant viruses lacking HLA class I immunoevasins allows for the direct identification of naturally presented HLA ligands by mass spectrometry, revealing a broad panel of T-cell epitopes that can be used in vaccines and therapies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If HCMV deletion mutant viruses lacking HLA class I immunoevasins are used, then the identification of HLA ligands and T-cell epitopes is improved, but the complexity of virus cultivation and experimental procedures increases
Solution Approach 1:
The patent extracts and removes the HLA class I immunoevasin genes (US2, US6, US11, US3) from the HCMV genome to create deletion mutant viruses. This extraction eliminates the immunoevasion mechanisms that normally prevent HLA ligand presentation, thereby enabling direct identification of T-cell epitopes through mass spectrometry without the interference of viral countermeasures.
Solution Approach 2:
The patent performs preliminary genetic modification of HCMV by deleting immunoevasin genes before conducting the actual epitope identification experiment. This preliminary action ensures that when the virus infects antigen-presenting cells, HLA molecules can naturally present viral peptides without being disrupted by immunoevasins, thus preparing the system for successful mass spectrometry analysis.
2Loss of information
If HCMV deletion mutant viruses lacking HLA class I immunoevasins are used, then the identification of T-cell specificities is improved, but the time and resources required for virus cultivation and analysis increase
Solution Approach 1:
The patent replaces traditional mechanical and biochemical methods of epitope identification (such as peptide library screening and overlapping peptide assays) with mass spectrometry technology. This substitution enables direct, high-throughput identification of naturally presented HLA ligands from infected cells, significantly reducing the time and resources required while providing comprehensive T-cell specificity data.
Solution Approach 2:
The patent uses mass spectrometry to directly detect and identify the actual peptide sequences presented by HLA molecules on the surface of infected cells. This copying approach captures the authentic viral epitopes without requiring synthetic peptide libraries or indirect prediction methods, thereby obtaining complete and accurate T-cell specificity information more efficiently.
3Ease of manufacture
If traditional methods using overlapping peptides or prediction algorithms are used, then the identification process is simpler, but the accuracy and comprehensiveness of identified epitopes deteriorates
Solution Approach 1:
The patent replaces indirect prediction algorithms and overlapping peptide screening methods with direct mass spectrometry detection. This substitution eliminates the need for computational predictions and synthetic peptide libraries, directly identifying the actual viral peptides naturally presented by HLA molecules, thereby achieving both high accuracy and comprehensive epitope coverage.
Solution Approach 2:
The patent directly copies and sequences the authentic viral peptide fragments that are naturally processed and presented by HLA molecules during HCMV infection. This direct copying approach captures the true immunogenic epitopes without relying on prediction algorithms or incomplete overlapping peptide coverage, ensuring high accuracy and comprehensiveness in epitope identification.
Data Source
AI summary
The present invention relates to relates to T cell epitope peptides, proteins, nucleic acids and cells for use in immunother-apeutic methods. In particular, the present invention relates to the immunotherapy of viral infection. The present invention specifically relates to virus-associated T-cell peptide epitopes, alone or in combination with other virus-associated peptides that can serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-viral immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.