Recombinant Virus Heterologous Antigen Insertion E-NS1 Intergenic Region
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Solution Overview
Problem
Current methods for developing flavivirus vaccines that express heterologous antigens face challenges in maintaining viral structure, replication, and immunogenicity, particularly in integrating foreign sequences into the viral genome without compromising attenuation or inducing appropriate immune responses.
Innovation Solution
The method involves modifying heterologous sequences to be compatible with the 5' end of the NS1 gene and 3' end of the E protein stem and anchor domains of the vector virus, inserting them into the intergenic region between the E and NS1 genes, ensuring stable integration and proper processing, and maintaining immunogenic properties to induce an appropriate immune response.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If heterologous sequences are inserted into the viral genome to express foreign antigens, then the vaccine can induce immune response to multiple pathogens, but the viral structure and replication may be compromised
Solution Approach 1:
The patent divides the viral genome into specific regions for insertion, using the intergenic region between E and NS1 genes as a designated segment for heterologous antigen insertion. This segmentation allows foreign sequences to be inserted without disrupting the structural integrity of essential viral genes, thereby maintaining viral replication while enabling multi-pathogen immunity.
Solution Approach 2:
The patent uses the intergenic region between E and NS1 genes as an intermediary site for heterologous antigen insertion. This region serves as a mediator that allows foreign sequences to be integrated without interfering with the function of adjacent viral genes, thus preserving viral structure and replication capability while enabling expression of heterologous antigens.
2Adaptability or versatility
If foreign sequences are integrated into the viral genome, then heterologous antigens can be expressed, but the virus may lose its attenuated phenotype
Solution Approach 1:
The patent applies local quality by restricting heterologous antigen insertion to a specific region (intergenic region between E and NS1 genes) rather than allowing insertion throughout the genome. This localized approach ensures that the attenuated phenotype is preserved in essential viral regions while enabling heterologous antigen expression in a non-critical region.
Solution Approach 2:
The patent performs preliminary characterization of the intergenic region between E and NS1 genes to identify it as a suitable insertion site that maintains viral attenuation. By pre-establishing this safe harbor region through prior research and selection, the patent ensures that subsequent heterologous insertions will not compromise the attenuated phenotype while enabling antigen expression.
3Stability of the object's composition
If heterologous sequences are inserted into the E-NS1 intergenic region, then stable integration and proper processing are ensured, but the integration process becomes complex
Solution Approach 1:
The patent establishes the intergenic region between E and NS1 genes as a universal insertion site that can accommodate various heterologous antigens from different pathogens. This multi-functional region serves as a standardized platform for integrating diverse foreign sequences, ensuring stable integration and proper processing regardless of the specific antigen being inserted, thereby simplifying the overall process despite the initial complexity.
Data Source
AI summary
The purpose of the present invention is the production of recombinant virus through the cloning and expression of sequences of nucleotides encoding whole or part of heterologous proteins, through the following method: (a) modification of the heterologous nucleotides sequences in such way that when cloned and expressed in a vector virus, the 5′ region nucleotides present in the 5′ edge of the gene NS1 of this vector virus or of other virus or equivalent functional sequences, and in its 3′ region, the correspondent genome region in the whole or part of the domains of the stem and anchor of the protein E of the vector virus or equivalent functional sequences, and not comprising the structure and the replication of the mentioned vector virus; (b) insertion of the modified heterologous sequences in (a) in the intergenic region at the structural protein E level and of the nonstructural NS1 vector virus; (c) obtaining the non pathogenic recombinant virus with immunologic properties, having the heterologous sequences integrated in the viral genome according to the insertion described in (b) and, then expressing the heterologous antigen in such way that it can induce an appropriate immune response. The present invention is also addressed to vaccine compositions to immunize against the Flavivirus and/or other pathogens.