Optimized host/vector system for producing protective mono-and multivalent subunit vaccines on the basis of the yeast Kluyveromyces lactis
Inventors
Hührlimann, Hans Caspar • Behrens, Martina • Gebauer, Mandy • Breunig, Karin • Behrens, Sven-Erik
Assignees
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Publication Number
US-12473562-B2
Publication Date
2025-11-18
Expiration Date
Abstract
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.
Core Innovation
The invention describes an optimized recombinant Kluyveromyces lactis vaccine platform in which expression cassettes for foreign antigens are integrated at defined genomic loci. The integration targets the KIURA3-20 locus, the KIMET5-1 locus, and/or the KILAC4 locus in specified combinations, enabling single-locus and multi-locus configurations. The approach supports stable subunit/yeast vaccines using foreign antigen expression within Kluyveromyces lactis.
A key aspect of the platform is improved inducibility of foreign protein expression from a lactose-inducible LAC4-12 promoter system. The disclosed promoter includes modified variants intended to reduce basal, noninduced expression, including a promoter variant with an LR2 deletion and a basal control region deletion, together with variable numbers of KIGal4 activator binding sites. This inducible design is described in the context of avoiding cytopathic effects and enabling controlled antigen expression.
The platform also specifies genetic control over antigen expression and modular, marker-free targeted integration. It includes controlled antigen copy number through tandem and/or multi-expression cassettes and additional integration sites enabled by marker-free selection, including restoring URA3/MET5 function for prototrophy. Integrative vectors KIpURA3 and KIpMET5 are used to modularly insert antigen ORFs together with promoter/terminator components for locus-specific genomic integration.
Claims Coverage
The provided independent claims are clm-00001 and clm-00019. Across these claims, the main inventive features cover locus-specific integration of foreign antigen expression cassettes in K. lactis, and production using specified integrative vectors and uracil/methionine selection with optional prototrophy restoration.
Locus-specific foreign antigen expression cassettes for vaccination
Administering to a subject an effective amount of a K. lactis strain sufficient for triggering a protective immune response against one or more foreign antigens, wherein the K. lactis strain comprises expression cassettes for foreign antigens integrated at KIURA3-20, KIMET5-1, and/or KILAC4 in combinations of the loci.
Vaccination using specified K. lactis strain variants and antigen insert configurations
The vaccination method of the K. lactis strain is refined by specifying foreign antigens and/or by selecting the K. lactis strain from a group of named VAK/DSM entries, and the foreign antigen expression cassettes correspond to the defined integration loci including KIURA3-20, KIMET5-1, and KILAC4.
Producing K. lactis strains with foreign antigen expression cassettes at defined loci using KIpURA3 and KIpMET5
A method for producing a K. lactis strain comprising expression cassettes for foreign antigens integrated at KIURA3-20, KIMET5-1, and/or KILAC4 in combinations of the loci, comprising inserting a desired antigen gene sequence into the KIpURA3 vector and/or KIpMET5 vector, transforming a K. lactis culture with the modified and previously enzymatically digested vector constructs, and selecting transformed K. lactis cells on a solid medium not containing uracil and/or methionine with optional restoration of prototrophy.
Taken together, the claim coverage focuses on vaccination by administering K. lactis strains that carry foreign antigen expression cassettes integrated into specified genomic loci and on producing such K. lactis strains by using KIpURA3 and/or KIpMET5 vector constructs, selecting on uracil/methionine-omitting solid medium, with optional prototrophy restoration.
Stated Advantages
Stable subunit/yeast vaccine operation via foreign antigen expression in Kluyveromyces lactis.
Improved inducibility by reducing basal, noninduced expression using a modified LAC4-12 promoter system.
Avoidance of cytopathic effects through stringent inducibility.
Reproducible GMP-compatible fermentation.
Dose reduction and cross-/multivalent protection after single or double immunization.
Cross-protection and demonstrated protection outcomes in challenge studies.
Documented Applications
Protective immunization against very virulent infectious bursal disease virus (vvIBDV) in chickens, including prime-boost and one-shot complete protection.
Bivalent influenza yeast vaccines (HA+M1) protection and challenge studies, including cross-protection against variants, with comparisons to monovalent vaccines.
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