Method for the oral/mucosal vaccination by means of recombinant yeasts

Abstract

Recombinant yeast cells are produced which are used for vaccination, among other uses for the oral vaccination by feeding.

Claims

1. A method of vaccination, comprising oral/mucosal administration of a vaccine comprising a recombinant yeast, wherein said recombinant yeast comprises a genomically integrated expression cassette comprising 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 the part of the foreign gene is flanked by the lactose inducible LAC4 promoter and the transcription terminator, which transcription terminator is followed downstream by the KIGAL80-P.

2. The method in accordance with claim 1, wherein the recombinant yeast is Kluyveromyces lactis.

3. The method in accordance with claim 1, in which said recombinant yeast is employed as an expression system.

4. The method in accordance with claim 3, wherein the recombinant yeast allows lactose-inducible expression of the foreign gene or said part of a foreign gene.

5. The method in accordance with claim 1, wherein said recombinant yeast is characterized by absence of any foreign selection markers apart from the foreign gene or said part of a foreign gene.

6. The method in accordance with claim 1, further comprising quantification of the expression of the foreign gene or said part of a foreign gene by expression of an endogenous reporter gene that is a co-regulated Kluyveromyces lactis-intrinsic beta-galactosidase gene (LAC4).

7. The method in accordance with claim 1, wherein the foreign gene or said part of a foreign gene expresses a structure protein from the bovine viral diarrhea virus (BVDV).

8. The method in accordance with claim 1, wherein the vaccine has long storability outside of a cold environment.

9. The method in accordance with claim 1, wherein the recombinant yeast of the vaccine comprises complete yeast cells which provide a specific immunization against the expressed foreign protein or expressed foreign protein part.

10. A method of vaccination comprising inhaled, bronchial, oral or nasal administration of a vaccine, comprising a recombinant yeast comprising complete yeast cells which provide a specific immunization against expressed foreign protein, wherein the recombinant yeast comprises a genomically integrated expression cassette comprising the 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 the part of the foreign gene is flanked by the lactose inducible LAC4 promoter and the transcription terminator, which transcription terminator is followed downstream by the KIGAL80-P.

11. The method in accordance with claim 1 or 10, further comprising subsequently detecting neutralizing antibodies in sera from individuals who were immunized by said vaccine.

12. The method in accordance with claim 1 or 10, further comprising subsequently detecting neutralizing antibodies against a BVDV E2 protein in sera from individuals who were immunized by said vaccine.

13. The method in accordance with claim 1 or 10, further comprising detecting immunization using a challenge with antigen in individuals who were immunized by said vaccine.

14. The method in accordance with claim 13, wherein the antigen is a viral antigen.

15. The method in accordance with claim 14, wherein the viral antigen is BVDV E2.

16. 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 the part of the foreign gene is flanked by the lactose inducible LAC4 promoter and the transcription terminator, which transcription terminator is followed downstream by the KIGAL80-P.

17. The method of vaccination of claim 1, wherein the vaccine comprises a recombinant yeast of the strain Kluyveromyces lactis in which a foreign gene segment of Bovine Viral Diarrhea Virus coding for viral structure protein E2 is inserted into a gene of the strain, whereby, upon oral or mucosal administration of the vaccine to an animal, an immune response to Bovine Viral Diarrhea Virus is induced in the animal.

18. The method in accordance with claim 1, wherein the foreign gene or part of a foreign gene is deliberately integrated into the yeast genome without introduction of an additional marker.

19. The method of claim 1, 10, 16, or 18, wherein said recombinant yeast is characterized by high genetic stability of the expression cassette even during growth of said recombinant yeast under non-selective conditions due to absence of any extended DNA-sequence repeats in the expression cassette.

20. The recombinant yeast as recited in claim 1.

21. The vaccine as recited in claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic illustration relating to exemplary embodiment 1; and

(2) FIG. 2 is a schematic illustration relating to exemplary embodiment 2.

DETAILED DESCRIPTION OF THE INVENTION

(3) In accordance with the invention, a K. lactis strain, preferably VAK367-D4 and variants of this strain, was generated using genetic methods and permits the deliberate integration of foreign genes on the LAC4 locus of the yeast genome without it being necessary to introduce additional DNA sequences (selection marker or the like). The recombinant yeast strains are stable without selection pressure and may be cultivated under fermentation conditions to create high densities. Foreign gene expression may be induced by metered lactose or galactose or, after turning off the KIGAL80 regulator gene, may be constitutively activated. Foreign gene expression may be quantified indirectly using expression of an endogenous reporter gene.

(4) A series of recombinant variants building on the K. lactis VAK367-D4 strain was generated. In general these variants express inducibly significant quantities of a protein, or domains of this protein, or domains of this protein fused with heterogenous protein domains. The related heterogenous protein domains act to deliberately stimulate the immune response (adjuvant purposes) and to deliberately compartmentalize the expressed foreign protein in the yeast cell. In addition to adjuvant effects, compartmentalization of the expressed foreign protein is important for optimizing expression and for formulating the expression product. One of these recombinant K. lactis strains was successfully employed for mucosal/oral vaccination (see exemplary embodiments).

Exemplary Embodiments

(5) 1. Production of the K. lactis VAK367-D4 Strain (Met ura3 lac4::ScURA3).

(6) The initial VAK367 strain for heterologous expression of foreign proteins has the following properties: It permits cultivation to produce high cell density without intracellular proteins being detectably released. This strain is distinguished from many closely related K. lactis strains in this regard. The VAK367 strain was derived from two rounds of mutagenesis of the CBS 2359 strain (Central bureau voor Schimmelcultures, Fungal Biodiversity Centre) and is auxotrophic for the amino acid methionine and the nucleobase uracil. Using genetic methods, the VAK367-D4 strain was derived from the VAK367 strain (deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) in Braunschweig) in that the sequence of +358 to +1181 of the LAC4 gene was replaced with the ScURA3 gene using the plasmid pD4-2. The VAK367-D4 strain now permits the integration of foreign genes at the LAC4 locus without additional markers in that lactose growth is selected. When using a suitable integration vector such as e.g. Klp3 (see below) by homologous recombination, the disruption cassette is replaced such that an intact LAC4 gene is reconstituted and the ScURA3 marker is lost. (FIG. 1)

(7) 2. Production of an Integration Vector that Permits the Inducible Expression of Foreign genes.

(8) Vector: Klp3

(9) The Klp3 vector is an E. coli vector based on YRp7 that cannot replicate autonomically in yeasts because the ARS1 sequence was deleted. Klp3 includes K. lactis sequences that make it possible to integrate at the LAC4 locus using homologous recombination (upstream region of LAC4 and 5 end of the LAC4 reading frame). A DNA segment that includes the TEF1 terminator and the KIGAL80 promotor was inserted between the LAC4 promotor and the transcription start. Thus the LAC4 reading frame is under control of the KIGAL80 promotor that is co-regulated via the KIGal4 transcription factor with the LAC4 promotor (Zenke et al. 1993, Molecular and Cellular Biology, 13:7566-7576). This design makes it possible to track induction of foreign gene expression by measuring the LAC4-coded ?-galactosidase. Klp3 permits the integration of the foreign gene between LAC4 promotor and TEF1 terminator via the unical Sall interface. For integrating the expression cassette, Klp3 is digested with Hpal or EcoRl and transformed into K. lactis VAK367-D4. In doing so the expression cassette is separated form the E. coli vector portion so that the resulting strains do not contain any bacterial sequences.

(10) Plasmid Klp3-E2-1 (9437 bp) (FIG. 2)

(11) The gene segment of the BVDV virus that codes for the viral structure protein E2 was inserted into the Sal interface between LAC4 promotor and TEF1 terminator as Sall-Xhol fragment. The KIGAL80 promotor is downstream and was fused to the 5 end of the LAC4 ORF.

(12) The plasmid was cut with HPal and the larger Hpal fragment was integrated with the E2-ORF chromosomally using homologous recombination. The lac4::URA3 gene locus was replaced and the intact LAC4 gene was reconstituted. Growth on lactose medium was used for selection. The loss of the URA3 gene was confirmed by the uracil auxotrophy. The sequence for the corresponding gene locus was confirmed using DNA sequencing. (Sequence log 1)

(13) 3. Formulation of the E2 Major Antigens of BVDV and CSFV.

(14) A major antigen is characterized by BVDV (bovine viral diarrhea virus), the pathogen of bovine viral diarrhea and mucosal disease (BVD/MD), and also by CSFV (classical swine fever virus), the pathogen of classical swine fever (CSF). This is the envelope (virus-shell integrated) protein E2. Even in the absence of a virus particle, E2 induces a massive humoral immune response, i.e. the formation of effectively virus-neutralizing antibodies. Genetically formulating E2 made it possible to further strengthen the immunogenic potential of the protein and also to produce a cellular immune response. The specific and exclusive immune response to individual protein domains of E2, some of which were genetically formulated, made it possible to discriminate between vaccinated animals and animals infected with the field virus, e.g. by means of the ELISA method.

(15) 4. Design of a K. lactis Strain that Expresses the BVDV E2 Protein VAK367-E2-1, a K. lactis strain, was produced by means of the inventive technology. In this strain, a segment of the BVDV genome (CP7 strain) was integrated into the yeast genome. The corresponding BVDV gene segment included the area that included protein for the E2 and parts of the adjacent E1 and p7-coding regions of the BVDV genome. The E1 and p7 regions include the signal sequences needed for correctly processing the E2 protein (sequence log 2). Correct processing (maturation) of the BVDV E2 protein occurs via signalases.

(16) Expression of E2 in cells of the VAK367-E2-1 K. lactis strain may be established by means of a specifically developed immunofluorescence detection method. An ELISA method specially developed for detecting the BVDV E2 permits detection and quantification of the heterologously expressed antigen. It was possible to use the analog ELISA method for precisely quantifying the antibody titer of immunized animals. Virus neutralization methods and methods for characterizing antibodies and T cells were employed as routine methods.

(17) A novel qRT-PCR method makes it possible to detect and quantify BVDV RNA genomes from serum and cell culture supernatants.

(18) 5. Demonstrating the Effectiveness of K. lactis Strain VAK367-E2-1 in Mucosal/Oral Immunization Studies

(19) Study 1

(20) In animal testing an emulsion of naive K. lactis was applied to a significant number of mice under standard conditions. Various immunization schemes were used.

(21) The main criteria were different quantities of supplied yeasts (3max. 8% portion of daily food intake) and different booster intervals.

(22) Results:

(23) (i) General tolerance for K. lactis was demonstrated: oral administration of the yeast emulsions did not cause any visible changes in the findings for the animals.

(24) (ii) Administration of K. lactis led to a clearly detectable humoral immune response to certain yeast proteins. Using the western blot method it was possible to establish a significant and specific antibody response to yeast proteins in the animals that had been fed with K. lactis compared to control animals. Yeast proteins thus have an immunogenic effect per se. In addition to the proof of principle that an immune response may be produced with the oral application of K. lactis, it was indicated that the oral administration of yeasts in combination with a recombinant antigen may attain an additional adjuvant effect.
Study 2

(25) In other animal studies that used a significant number of mice, oral vaccinations were administered with an optimized immunization scheme (see Study 1) and with recombinant K. lactis of the VAK367-E2-1 strain.

(26) Results:

(27) (i) Using the special ELISA method it was possible to detect the formation of anti-BVDV E2 antibodies in the mice vaccinated with K. lactis strain VAK367-E2-1 compared to control mice (immunized with na?ve K. lactis).

(28) (ii) In a neutralization test with BVDV on bovine culture cells it was possible to detect a neutralizing effect of anti-sera from mice that had been immunized with K. lactis of the VAK367-E2-1 strain, again in comparison to the sera from control mice.

(29) (iii) The immune response could be increased by using adjuvants such as CpG-ODN and QuilA.

(30) (iv) It was possible to attain effective protection using mucosal/oral immunization with the recombinant K. lactis VAK367-E2-1 strain. This could be demonstrated in challenge experiments with recombinant vaccine viruses that express the BVDV E2 protein.

(31) The results of the study prove that in accordance with the invention foreign genes without additional selection markers may be deliberately integrated into them by means of producing a K. lactis strain and variants of this strain. The resulting recombinant descendants of the K. lactis strains in question may be used for expression purposes for the subsequent biochemical representation of a heterologous protein. The cells of the corresponding recombinant K. lactis strains may be employed directly for mucosal/oral or parenteral immunization.