Method for the oral/mucosal vaccination by means of recombinant yeasts
Inventors
Breunig, Karin • Behrens, Sven-Erik
Assignees
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Publication Number
US-9885052-B2
Publication Date
2018-02-06
Expiration Date
Abstract
Recombinant yeast cells are produced which are used for vaccination, among other uses for the oral vaccination by feeding.
Core Innovation
The invention relates to oral/mucosal vaccination using complete, non-pathogenic recombinant yeasts with GRAS status, specifically recombinant Kluyveromyces lactis yeasts, as immunogens. The recombinant yeasts enable direct use of yeast cells for vaccination and provide immunization against expressed foreign proteins.
A genomically integrated expression cassette drives lactose-inducible expression of a foreign gene or a part of a foreign gene expressing an immunogenic protein. The cassette includes a lactose inducible LAC4 promoter from Kluyveromyces lactis, a transcription terminator, and a GAL80 promoter from K. lactis (KIGAL80-P) that drives transcription of a LAC4 coding sequence.
The foreign gene or part of it is flanked by the lactose inducible LAC4 promoter and the transcription terminator, and the transcription terminator is followed downstream by KIGAL80-P. The document further describes indirect quantification of foreign gene expression via an endogenous LAC4 beta-galactosidase reporter linked to LAC4 coding sequences.
The document provides examples including a platform strain (VAK367-D4) and recombinant yeast (VAK367-E2-1) expressing BVDV E2, and demonstrates immunogenicity and protective efficacy in mouse studies. These studies include induction of anti-BVDV E2 antibodies and neutralizing antibodies, assessment by ELISA and neutralization tests, and enhancement of immune responses by adjuvants including CpG-ODN and QuilA, followed by challenge experiments.
Claims Coverage
The provided independent claims are directed to three inventive coverage points: oral/mucosal vaccination using a recombinant yeast with a genomically integrated promoter/terminator/KIGAL80-P cassette architecture that drives LAC4-coding transcription of an expression cassette; vaccination via inhaled, bronchial, oral or nasal routes using complete yeast cells with the same core cassette architecture; and oral/mucosal vaccination using a recombinant yeast derived from Kluyveromyces lactis VAK367-D4 where lac4::URA3 is replaced by the cassette via homologous recombination.
Oral/mucosal vaccination using lactose inducible LAC4 promoter and KIGAL80-P cassette flanking a foreign immunogenic gene
A method of vaccination comprising oral/mucosal administration of a vaccine comprising a recombinant yeast, where the recombinant yeast comprises a genomically integrated expression cassette including a lactose inducible LAC4 promoter from Kluyveromyces lactis, a foreign gene or a part of a foreign gene expressing an immunogenic protein, a transcription terminator and GAL80 promoter from K. lactis (KIGAL80-P) which drives transcription of a LAC4 coding sequence, wherein the foreign gene or part is flanked by the lactose inducible LAC4 promoter and the transcription terminator, and the transcription terminator is followed downstream by KIGAL80-P.
Route-flexible vaccination using complete yeast cells with genomically integrated lactose inducible LAC4 promoter and KIGAL80-P expression cassette
A method of vaccination comprising inhaled, bronchial, oral or nasal administration of a vaccine comprising a recombinant yeast comprising complete yeast cells that provide a specific immunization against expressed foreign protein, wherein the recombinant yeast comprises a genomically integrated expression cassette comprising a lactose inducible LAC4 promoter from Kluyveromyces lactis, a foreign gene or a part of a foreign gene expressing an immunogenic protein, a transcription terminator and GAL80 promoter from K. lactis (KIGAL80-P) which drives transcription of a LAC4 coding sequence, wherein the foreign gene or part is flanked by the lactose inducible LAC4 promoter and the transcription terminator, and the transcription terminator is followed downstream by KIGAL80-P.
Oral/mucosal vaccination using VAK367-D4-derived yeast replacing genomic lac4::URA3 with lactose inducible LAC4/KIGAL80-P cassette by homologous recombination
A method of vaccination comprising oral/mucosal administration of a vaccine comprising a recombinant yeast derived from the strain Kluyveromyces lactis VAK367-D4 by homologous recombination resulting in replacement of the genomic lac4::URA3 locus with an expression cassette comprising a foreign gene or a part of a foreign gene expressing an immunogenic protein, lactose inducible LAC4 promoter from said strain, a transcription terminator, and GAL80 promoter from K. lactis (KIGAL80-P) which drives transcription of a LAC4 coding sequence, wherein the foreign gene or part is flanked by the lactose inducible LAC4 promoter and the transcription terminator, and the transcription terminator is followed downstream by the KIGAL80-P.
Across the independent claims, the core inventive concept is vaccination with recombinant Kluyveromyces lactis yeasts that contain a genomically integrated, lactose inducible LAC4 promoter/terminator arrangement flanking a foreign gene or part and followed downstream by KIGAL80-P to drive transcription of LAC4 coding sequences; administration routes are oral/mucosal, inhaled, bronchial, oral or nasal, and oral/mucosal for VAK367-D4-derived lac4::URA3 replacement via homologous recombination.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Oral/mucosal vaccination using recombinant yeast vaccine.
Inhaled, bronchial, oral or nasal administration vaccination using complete yeast cells providing specific immunization against expressed foreign protein.
Mouse studies with oral tolerance and induction of anti-BVDV E2 antibody and neutralizing antibody responses following vaccination with recombinant yeast expressing BVDV E2, including adjuvant enhancement using CpG-ODN and QuilA, followed by challenge experiments.
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