CMV VLP Antigen Insertion and T Helper Epitope Replacement
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Solution Overview
Problem
Current vaccine platforms, such as those using Cucumber Mosaic Virus (CMV) virus-like particles, face challenges in inducing consistent and strong immune responses, particularly in inducing good T helper cell responses and accommodating long antigenic peptides without affecting the viral structure, leading to variable antibody responses and issues like non-responsiveness in individuals.
Innovation Solution
Modified virus-like particles of CMV are developed by inserting antigenic polypeptides of varying lengths into specific positions of CMV polypeptides, replacing the N-terminal region with a T helper cell epitope, and using amino acid linkers to facilitate assembly, allowing for the incorporation of antigenic peptides up to 200 amino acids in length, enhancing immunogenicity and structural integrity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If antigenic polypeptides are inserted into CMV polypeptides to create chimeric forms, then the ability to present different antigens on VLP surfaces is improved, but the structural integrity and assembly capability of the virus-like particles deteriorates
Solution Approach 1:
The CMV coat protein is divided into functional domains: the N-terminal region (residues 1-83) is replaced with T helper cell epitopes, the middle region (residues 84-220) retains the native CMV sequence for structural integrity, and antigenic polypeptides are inserted at specific positions (e.g., between residues 84-85 or 131-132). This segmentation allows different regions to fulfill different functions while maintaining overall VLP stability
Solution Approach 2:
Different regions of the chimeric CMV polypeptide are assigned different qualities: the N-terminal region provides immunogenicity through T helper cell epitopes, the central region provides structural stability through native CMV sequences, and specific insertion sites provide antigen presentation capability. This local differentiation resolves the contradiction between versatility and structural integrity
2Reliability
If long antigenic polypeptides (up to 200 amino acids) are incorporated into CMV VLPs, then the immunogenicity and vaccine efficacy are improved, but the difficulty of maintaining proper viral structure and assembly increases
Solution Approach 1:
The design of chimeric CMV polypeptides is performed in silico before expression, optimizing the insertion positions of long antigenic polypeptides (up to 200 amino acids) within the CMV coat protein framework. This preliminary design ensures that the native structural elements are preserved and assembly signals are maintained, allowing long antigens to be incorporated without compromising VLP formation
Solution Approach 2:
The chimeric CMV polypeptide acts as a composite material combining native CMV structural elements with foreign antigenic sequences. The native CMV regions (particularly residues 84-220) provide the structural framework and self-assembly capability, while the inserted antigenic polypeptides (up to 200 amino acids) provide immunogenicity, creating a hybrid structure that maintains both functionality
3Reliability
If the N-terminal region of CMV polypeptide is replaced with T helper cell epitopes, then the induction of T helper cell responses is improved, but the protein-RNA interaction stability deteriorates
Solution Approach 1:
The CMV coat protein is segmented into functional regions: the N-terminal region (residues 1-83) containing the R-domain for RNA binding is replaced with T helper cell epitopes to enhance immunogenicity, while the central region (residues 84-220) retains native sequences that maintain structural integrity and protein-RNA interactions. This segmentation allows independent optimization of immunogenicity and stability
Solution Approach 2:
The central region of the CMV coat protein (residues 84-220) acts as an intermediary that bridges the N-terminal T helper cell epitopes and the C-terminal structural domains. This intermediary region maintains the structural framework and protein-RNA interactions while allowing the N-terminal region to provide enhanced immunogenicity through T helper cell epitope incorporation
Data Source
AI summary
The present invention relates to a modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprising at least one fusion protein, wherein said at least one fusion protein comprises, or preferably consists of b) a chimeric CMV polypeptide, wherein said chimeric CMV polypeptide comprises, or preferably consists of (iii) a CMV polypeptide, wherein said CMV polypeptide comprises, or preferably consists of, a coat protein of CMV, wherein preferably said coat protein of CMV comprises, or preferably consists of, SEQ ID NO:62; or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90%, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% with SEQ ID NO:62; and (iv) an antigenic polypeptide, wherein said antigenic polypeptide is inserted into said CMV polypeptide, wherein said insertion of said antigenic polypeptide is between amino acid residues of said CMV polypeptide corresponding to amino acid residues of position 84 and position 85 of SEQ ID NO:62; and (iii) a T helper cell epitope, wherein said T helper cell epitope replaces a N-terminal region of said CMV polypeptide, and wherein preferably said N-terminal region of said CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO:62.