K. Lactis Vaccine Host Strain with Multi-Locus Antigen Integration
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Solution Overview
Problem
Existing vaccines, particularly subunit vaccines, face challenges such as complex production processes, inadequate immunogenicity, and limited effectiveness against virulent strains, requiring improved yeast-based vaccine strains with controlled antigen expression and multiple integration sites for enhanced cross-protection.
Innovation Solution
Development of recombinant Kluyveromyces lactis strains with controlled KIGAL4-1 gene copy number, optimized LAC4 promoter, and multiple integration sites at KIURA3-20 and KIMET5-1 loci for stable expression of multiple antigens, allowing efficient fermentation and standardized production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple antigens are expressed in yeast vaccine strains, then cross-protection against different pathogens is enhanced, but production complexity and fermentation variability increase
Solution Approach 1:
The patent divides the yeast genome into multiple independent integration loci (KILAC4, KIURA3-20, KIMET5-1), allowing separate insertion of expression cassettes for different antigens. This segmentation enables controlled multivalent vaccine production while maintaining independent regulation of each antigen expression, thereby reducing fermentation variability despite expressing multiple antigens.
Solution Approach 2:
The patent creates a universal yeast host system with standardized integration loci and promoter elements (LAC4, GAL1, etc.) that can accommodate multiple different antigen-encoding cassettes. This multi-functional platform allows the same yeast strain to be adapted for producing vaccines against different pathogens by simply changing the inserted antigen genes, without redesigning the entire production system.
2Reliability
If antigen expression is increased to improve immunogenicity, then protective efficacy is enhanced, but cytopathic effects and fermentation stability deteriorate
Solution Approach 1:
The patent employs different promoter elements with varying strengths (LAC4, GAL1, PGK1, etc.) that can be selectively assigned to different antigen expression cassettes based on the specific immunogenic requirements of each antigen. This local optimization allows high expression for highly immunogenic antigens while using moderate promoters for other antigens, preventing cytopathic effects and maintaining fermentation stability.
Solution Approach 2:
The patent introduces inducible promoters (LAC4 induced by galactose, GAL1 induced by glucose depletion) that allow dynamic control of antigen expression timing and level. Expression can be upregulated during specific fermentation phases to achieve high immunogenicity while downregulating during growth phases to maintain fermentation stability, creating a temporal separation between growth and production phases.
3Stability of the object's composition
If integrated expression cassettes are added to yeast genome, then antigen expression stability is improved, but genetic manipulation complexity increases
Solution Approach 1:
The patent pre-establishes three validated integration loci (KILAC4, KIURA3-20, KIMET5-1) with characterized properties and compatible selection markers in the yeast genome before antigen cassette insertion. This preliminary preparation provides ready-to-use integration sites with known stability and expression characteristics, eliminating the need for de novo locus characterization for each new vaccine development project and simplifying the overall genetic manipulation process.
Solution Approach 2:
The patent uses identical or highly similar expression cassette designs (promoter-antigen-terminator structure) that can be repeatedly copied and inserted into different loci. The standardized cassette architecture with universal restriction sites and selection markers allows rapid cloning and integration of multiple antigens using the same molecular biology protocols, reducing the complexity of genetic manipulation despite multiple integrations.
Data Source
AI summary
Described herein are recombinant Kluyveromyces lactis (K. lactis) yeasts which are capable of the highly efficient expression of one or more foreign proteins and are suitable for use as a vaccine for generating a protective immune response against pathogens. The invention provides in particular K. lactis strains for the targeted cloning of foreign antigen-coding nucleic acids into the yeast genome of the K. lactis strain, which is characterized in that the K. lactis strain has integrated expression cassettes for foreign antigens as an alternative or in addition to the KILAC4 locus on the KIURA3-20 locus (KLLA0E22771g) and/or on the KIMET5-1 locus (KLLA0B03938g). The invention further relates to integrative expression vectors and to methods for producing the K. lactis strains of the invention as well as to the use thereof as vaccines.