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

Martin Luther University Halle Wittenberg • Verovaccines GmbH

Abstract

The invention relates to 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 relates to a recombinant Kluyveromyces lactis vaccine platform with targeted genomic integration of foreign antigen-encoding expression cassettes into defined K. lactis genome loci. The K. lactis strain has integrated expression cassettes for foreign antigens at one or more of the KlURA3-20 locus, the KlMET5-1 locus, and/or the KlLAC4 locus, providing a defined, targeted one-shot genomic cloning and expression framework using alternative loci for foreign antigen-encoding nucleic acids.

A modular integrative, marker-free vector system generates the described K. lactis strains and integrates foreign antigen-encoding expression cassettes without antibiotic resistance. The platform supports stable defined integration and multi-antigen and multivalent vaccine design, including multiple expression cassettes and tandem or multi-expression cassette configurations.

The platform includes inducibility control through LAC4-12 promoter variants and promoter structure modifications to reduce basal expression. The LAC4-12 promoter variants include constructs such as LR2 basal control region deletion and altered KlGal4 binding-site counts, and the platform supports fold-controlled antigen expression via promoter activator context and additional antigen gene copies, including an ectopic second KIGAL4 copy at a defined locus/sequence ID.

The document also frames the platform for restoring auxotrophy to generate prototrophic strains for improved fermentation and production. Vaccine outcomes described include reproducible GMP-relevant production and multivalent or cross-protection protection, including one-shot or prime-boost efficacy.

Claims Coverage

The provided independent claim set includes two independent claims: one directed to a K. lactis strain with integrated, locus-specific foreign antigen expression cassettes at specified genomic loci, and a second directed to a defined group of integrative expression vector variants. Across these independent claims, the inventive subject matter is centered on targeted integration into KlURA3-20, KlMET5-1 and/or KlLAC4 loci and use of a specified integrative vector family with defined PL4 and LR2-Et variant combinations.

Overall, the independent claim coverage focuses on K. lactis strains with integrated foreign antigen-encoding expression cassettes placed into specified loci and on integrative expression vector variants from a defined set that include PL4-12 and LR2/E t variants in combination with KIpURA3 or KIpMET5 backbone selections.

Stated Advantages

Documented Applications