EBV gB Cysteine Mutations for Prefusion Vaccine Antigen Stability
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Solution Overview
Problem
The development of an effective vaccine against Epstein-Barr virus (EBV) has been hindered by the inability to select appropriate antigen targets, lack of a suitable vaccine platform, and inadequate evaluation systems, leading to a need for a prophylactic vaccine that provides protection against EBV-associated diseases.
Innovation Solution
Engineered polypeptides of EBV glycoprotein B (gB) with specific amino acid substitutions to stabilize the prefusion conformation through disulfide bond formation, which are used in immunogenic compositions to induce an immune response.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type EBV gB is used as the antigen target, then the virus can naturally induce infection, but the antigen conformation is unstable and cannot provide reliable protective immunity
Solution Approach 1:
The patent applies preliminary action by introducing cysteine mutations at specific positions (e.g., C51, C68, C141, C295, C551) in the gB protein before vaccination to pre-establish disulfide bonds that will stabilize the prefusion conformation. This preliminary structural modification ensures the antigen maintains its protective conformation during storage and delivery, resolving the instability issue without compromising immunogenicity.
Solution Approach 2:
The patent changes the structural parameters of the gB protein by introducing specific cysteine mutations that alter the protein's conformational stability. These mutations create disulfide bonds that lock the protein in its prefusion state, changing the physical-chemical parameters of the antigen to achieve both stability and immunogenicity simultaneously.
2Reliability
If disulfide bonds are introduced to stabilize prefusion conformation, then antigen stability improves, but protein structure complexity increases
Solution Approach 1:
The patent applies local quality by introducing disulfide bonds at specific localized positions within the gB protein structure rather than uniformly throughout. The cysteine mutations are placed at specific locations (C51, C68, C141, C295, C551) where they provide maximum stabilizing effect on the prefusion conformation with minimal impact on overall protein complexity. This targeted approach resolves the contradiction by stabilizing only the critical regions needed for immunogenicity.
3Ease of manufacture
If conventional EBV vaccine platforms are used, then existing infrastructure can be utilized, but no protective immunity has been achieved
Solution Approach 1:
The patent applies universality by designing a stabilized gB antigen that can be utilized across multiple vaccine platforms (e.g., subunit vaccines, viral vector vaccines, mRNA vaccines). The disulfide-stabilized prefusion gB serves as a universal antigen target that can be adapted to different delivery systems, maintaining ease of manufacture while achieving protective immunity that was previously unattainable with conventional approaches.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The engineered EBV gB variants stabilize the prefusion conformation, potentially enhancing the development of a vaccine by providing a stable antigen target and improving immune response induction.
Implementation Method 1
engineered variants of the glycoprotein B (gB) of Epstein-Barr virus (EBV)... wherein the modifications comprise substituting one or more pairs of amino acid residues with one or more pairs of sulfhydryl-containing amino acid residues... one or more disulfide bonds are formed in a prefusion conformation upon protein expression
Data Source
AI summary
We have generated a 3D model of the glycoprotein B (gB) of Epstein-Barr virus (EBV) to design candidate stabilizing mutations that increase the stability of the prefusion state essential for an effective EBV gB based vaccine. Provided herein are engineered polypeptides derived from the EBV gB, which include an altered EBV gB ectodomain that has modifications relative to the native EBV gB ectodomain that stabilize a prefusion conformation of the polypeptides. In various aspects, the modifications are amino acid substitutions to generate pairs of cysteine amino acid residues, preferably positioned to connect different domains of the poly peptide or different copies of the polypeptide in a trimeric or multimeric conformation via formation of disulfide bonds during protein expression. In additional aspects, the modifications and/or the engineered polypeptides do not contain pairs of cysteine amino acid residues that may form disulfide bonds in a postfusion conformation.