A yeast Kluyveromyces lactis-based host / vector system optimized for the production of monovalent and multivalent subunit prophylactic vaccines

Abstract

The present invention relates to a recombinant Kluyveromyces lactis (K. lactis) yeast strain capable of highly efficient expression of one or more heterologous proteins and suitable for use as a vaccine to elicit a protective immune response against pathogens. The present invention particularly provides a K. lactis strain for targeted cloning of nucleic acids encoding heterologous antigens into the yeast genome of the K. lactis strain, characterized in that the K. lactis strain contains an integrated expression cassette for the heterologous antigen at the KlURA3-20 locus (KLLA0E22771g) and / or KlMET5-1 locus (KLLA0B03938g) instead of or in addition to the KlLAC4 locus. The present invention also relates to an integrative expression vector and a method for producing the K. lactis strain of the present invention, as well as its use as a vaccine. [Selection diagram] None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to recombinant Kluyveromyces lactis (K. lactis) yeast strains suitable for highly efficient expression of one or more heterologous proteins and for use as vaccines for generating protective immune responses against pathogens. The invention particularly relates to K. lactis strains for targeted cloning of nucleic acids encoding heterologous antigens into the yeast genome, characterized in that the expression cassette for the heterologous antigen is integrated into the KlURA3-20 locus (KLLA0E22771g) and / or the KlMET5-1 locus (KLLA0B03938g) instead of or in addition to the KlLAC4 locus. The invention also relates to integrative expression vectors and methods for producing the K. lactis strains of the invention, as well as their use as vaccines. [Background technology]

[0002] BACKGROUND OF THE INVENTION Vaccines are used to protect against disease (prophylactic vaccines) or treat existing disease (immunotherapeutic vaccines). Over the past century or so, preventive vaccination programs have significantly contributed to the reduction of infectious diseases. However, immunotherapeutic vaccines, for example, against persistent viral, bacterial, or parasitic infections or oncogenic diseases, have only been developed and used for about 20 years. The goal of vaccination is to induce a cellular (i.e., essentially T cell and NK cell-mediated) and / or humoral (i.e., essentially B cell / antibody-mediated) immune response, as well as immunological memory against antigenic components of pathogens or malignant (tumorigenic) cells.

[0003] Classical vaccines contain whole pathogens in attenuated (inactivated) or killed form, including their genetic material, i.e., nucleic acids in the form of DNA or RNA. Producing these vaccines usually requires special safety measures and / or the use of infectious organisms and / or cell cultures. Furthermore, such vaccines often require complex storage and transportation, often involving the use of cold chains. Furthermore, the use of classical vaccines carries the risk of material from the production process (e.g., from test animals or cell cultures) causing adverse effects in vaccinated individuals or the risk of undesirable reactivation of pathogens. Diagnostic problems also exist. For example, when vaccinating useful animals with a whole pathogen, vaccinated animals cannot be distinguished from naturally infected animals, which means that early warning systems based on detecting new infections are useless. Therefore, so-called "subunit vaccines," which vaccinate with only specific components of the pathogen, have been developed. A prerequisite for their use is that the "major antigen" of the pathogen in question is known. A major antigen is typically a surface component of a pathogen that can be recognized by the immune system, such as a viral coat protein or a viral capsid protein. The major antigen can also induce humoral and / or cellular immune responses and immunological memory in a host against the virus, even in the absence of intact virus particles. In "subunit vaccination," additional components of the pathogen are lost, allowing vaccinated individuals to be distinguished from naturally infected individuals by differential diagnosis (DIVA [Distinguishing Infection from Vaccinated Animals]). Therefore, these vaccines are also referred to as "subunit marker vaccines." The drawbacks of many subunit vaccines are their often complex manufacturing process and often poor immunogenicity. While the pathogen itself can be efficiently cultivated (with the limitations described above), its major antigen must be genetically engineered and purified by costly and usually inefficient means. Therefore, the resulting subunit vaccines are biological materials with a short shelf life and must often be stored and transported under refrigeration.For these reasons, the majority of commercially available animal vaccines are still based on the classical principle of using whole pathogens.

[0004] For example, infectious bursal disease (IBD), a widespread poultry disease, is caused by infectious bursal disease virus (IBDV), a non-enveloped virus with a double-stranded segmented RNA genome belonging to the Birnaviridae family. Most vaccines against IBD are based on attenuated or inactivated viruses. However, a problem arises: while highly attenuated, non-inactivated "live" and inactivated viruses also provide protection against IBD viruses of average pathogenicity, this is not the case for highly virulent IBD virus strains (vvIBDV). Until recently, several highly virulent attenuated viruses (intermediate hot strains) were protective against vvIBDV, but these vaccine strains were associated with side effects in the form of potential immunosuppression due to temporary damage to B cells in the bursa of Fabricius, a lymphoid organ (Rautenschlein et al., 2005). However, even these intermediate hot strain vaccines do not provide complete protection against recently discovered vvIBDV strains (Negash et al., 2012; Kasanga et al., 2007). Furthermore, a problem with vaccination with highly attenuated live viruses is that maternal antibodies inhibit viral replication and therefore prevent the induction of an immune response. Therefore, effective vaccination with these vaccines is only possible within three weeks of hatching (Kumar et al., 2000; Rautenschlein et al., 2005).

[0005] For example, influenza A virus is one of the most important viral pathogens worldwide (Short et al., 2015; Silva et al., 2012). Influenza viruses belong to the Orthomyxoviridae family and are enveloped viruses with a single-stranded, segmented RNA genome. Like most RNA viruses, influenza viruses are also subject to a high mutation rate. In particular, reassortment of viral RNA segments generates viral progeny with new genetic and biological properties (Short et al., 2015). Due to rapid evolution, a problem that arises, especially in the case of vaccination against influenza viruses, is that existing vaccines cannot "catch up" with newly emerging viral variants. Therefore, attempts have already been made for a long time to develop vaccines that offer cross-protection and therefore long-term protection against different influenza variants (Steel et al., 2010; Krammer and Palese, 2013; Kirchenbaum and Ross, 2014; Berthoud et al., 2011).

[0006] Bovine viral diarrhea virus (BVDV) is a widespread pathogen in ungulates. BVDV belongs to the genus Pestivirus in the family Flaviviridae. The single-stranded RNA genome of this virus is also subject to a high mutation rate. Furthermore, in pregnant animals, the fetus may become infected, and persistently infected (PI) animals may be born due to immune tolerance. These PI animals may further spread the virus and, in the case of 100% viral mutation, may die from so-called mucosal disease. Again, attempts have already been made for a long time to develop vaccines that offer cross-protection and long-term protection against different BVD virus variants (Ridpath (2015)).

[0007] An effective subunit vaccine could address or solve these problems. In most cases, the subunits are protein components of the pathogen, which can be produced in a variety of host cells by genetic engineering. In addition to the enterobacterium Escherichia coli, mammalian or insect cells that can be grown in cell culture, plant cells, and various fungi have been established as host systems for heterologous protein expression. Microbial systems such as bacteria and fungi can be cultivated on a large scale, particularly cost-effectively.

[0008] Yeast cells of the yeast genera Saccharomyces, Pichia, and Kluyveromyces have been routinely used for decades to express heterologous proteins. In contrast to bacteria, yeast cells have the advantage of being eukaryotic, i.e., similar in many respects to animal cells, and eukaryotic proteins, i.e., proteins that must be formed and / or functional in animal cells, can be produced cost-effectively in yeast in their native or substantially native form (Bathurst (1994); Gellissen and Hollenberg (1997)). Yeast was initially used only for the production of heterologous proteins, and after expression, the proteins were purified from the yeast cells and used as subunit vaccines. Attempts to administer yeast itself or yeast cell fractions as vaccines have only recently been made. Such "yeast-based vaccines" are yeast particles that contain immunologically effective components of a pathogen (antigen) and, after administration (e.g., subcutaneous, intramuscular, or oral / mucosal), are capable of inducing a specific immune response in the host organism against the antigen and also against the pathogen from which the antigen was derived. What is desired is the induction of immunological "memory" in the vaccinated organism, which will prevent the proliferation and / or spread of the corresponding pathogen upon subsequent infection ("challenge") and / or reduce the pathological consequences of the infection. As already mentioned above, antigens are usually structural proteins of the pathogen, and the nucleic acid sequences encoding them (antigen-encoding genes) are introduced into yeast cells using genetic engineering methods, allowing the expression of one or more of these structural proteins. The recombinant yeast thus produced, in live form (yeast cells), dead and dried (yeast particles) or in powder form after cell disruption, and in homogenized form (yeast lysate), constitute yeast-based vaccines. After administration of this vaccine, the antigen is recognized by the immune system and elicits humoral and / or cellular immune defenses.

[0009] Yeast-based vaccination is well known to those skilled in the art. A range of U.S. patent applications and patents, such as U.S. Patent Application Publication No. 20090304741A1, U.S. Patent No. 5,830,463A, U.S. Patent No. 7,465,454B2, and U.S. Patent Application Publication No. 20070166323A1, describe the use of S. cerevisiae strains containing at least one recombinant antigen in immunotherapy. These yeasts have been shown to be effective in stimulating immune responses, particularly cell-mediated immune responses.

[0010] International Publication No. 2006044923 discloses a yeast (S. cerevisiae) that recombinantly expresses various proteins of the hepatitis C virus (HCV) and is capable of eliciting an immune response, particularly a T cell response, against the HCV proteins, which is intended to be used as a vaccine against chronic hepatitis C.

[0011] WO 2007092792 describes the possible use of recombinant S. cerevisiae yeast against influenza virus infection, including the use of a combination of different yeast strains, the administration of which results in the induction of T cells, i.e., a cellular immune response.

[0012] WO 20101054649 and WO 2013107436 describe the use of strains of the species Kluyveromyces lactis containing antigens identified to generate a protective humoral immune response after oral / mucosal or subcutaneous administration of whole killed yeast cells. The latter patents include applications in which recombinant K. lactis strains derived from the starting strain VAK367-D4 have been successfully used for vaccination.

[0013] The possibility of using recombinant K. lactis yeast for vaccination is known to those skilled in the art (Arnold et al. (2012), WO 20101054649 and WO 2013107436). In an example application, we demonstrated that subcutaneous administration of the yeast K. lactis, intracellularly expressing the VP2 capsid protein of infectious bursal disease virus (IBDV) via an expression cassette controlled by the LAC4 promoter, elicits a humoral immune response and provides effective protection against viral infection. While this has been demonstrated against IBD viruses of average virulence, it has not been possible to demonstrate this against highly virulent IBDV (vvIBDV). Previous data have shown that increasing the intracellular concentration of viral antigens enhances the efficacy of yeast vaccines (Arnold et al. (2012)). The technical modification to achieve increased antigen concentration involves introducing additional copies of the transcriptional activator gene KlGAL4-1 (also known as LAC9-1) into IBDV-VP2-expressing strains (deposit strains DSM25406 and DSM25407) by integrating the pLI-1 plasmid (Krijger et al. (2012) and WO 2013107436). Thus, until now, the generation of such K. lactis vaccine strains has been based on two genetic interventions: first, the integration of a heterologous gene encoding an antigen; and second, the integration of the KlGAL4-1 gene. However, in current implementations, the latter always results in the integration of tandem repeats of the plasmid, which not only leads to cytotoxic effects due to strong overexpression of the activator (Breunig 1989), but also to variations in the copy number of the KlGAL4-1 and ScURA3 genes in the vaccine strains generated in this way.

[0014] The strategy of achieving heterologous gene expression via the unmodified LAC4 promoter, as described in the application examples (Arnold et al. (2012), WO 20101054649 and WO 2013107436), has the secondary effect that minimal expression of the heterologous gene occurs even under non-inducing conditions, i.e., when the promoter is somewhat open. This effect becomes more pronounced again with increasing KlGAL4-1 gene dosage. Therefore, in the case of heterologously expressed proteins that have a cytopathic effect (CPE) on yeast cells, biomass formation during cultivation, for example, during fed-batch fermentation processes, may be severely limited. In these cases, it is necessary to find alternative ways to minimize gene expression under non-inducing conditions.

[0015] Various subunit vaccines are effective and efficient only when multiple, rather than single, pathogen subunits are used in vaccination. Furthermore, the use of multiple antigen subunits in vaccination greatly increases cross-protection against different variants of the pathogen. Co-expression of the same or different antigens can also be used to increase antigen concentration in yeast cells or to produce vaccines that protect against different pathogens.

[0016] These strains are generally auxotrophic and often grow less well than prototrophic strains on complete media. Therefore, rapid and feasible conversion of auxotrophic yeast strains to the prototrophic form can improve growth characteristics. Summary of the Invention [Problem to be solved by the invention]

[0017] Description of the Invention Therefore, an object of the present invention is to provide a novel K. lactis vaccine strain that overcomes the drawbacks of the prior art. Specifically, a recombinant K. lactis strain is provided that contains a limited number of copies of the KIGAL4-1 gene integrated into a specific site within the genome. Furthermore, a strain is provided that allows for little or no expression of heterologous proteins under non-inducing conditions, allows for the expression of multiple copies of an antigen or multiple antigens in yeast, and is more suitable for cultivation and can be used more effectively for prophylactic vaccination against pathogens. At the same time, the heterologous gene encoding the immunomodulatory active protein (antigen) must be integrated into a specific site within the K. lactis genome. Resistance genes should not be used as selection markers when selecting target clones with integrated heterologous genes. Furthermore, prototrophic strains should be generated from auxotrophic strains in the simplest possible manner. This would also enable simplified fermentation of yeast vaccine strains produced in unsupplemented synthetic media. [Means for solving the problem]

[0018] The above objective has been achieved by providing a modular system comprising novel vectors and novel genetically modified K. lactis yeast variants, allowing the generation of vaccine strains optimized for protein antigen specificity. Building block-type exchange of DNA elements between vectors allows efficient cloning of regions encoding common heterologous antigens into the yeast genome, separate from the heterologous gene to be expressed. As a result of genomic integration of the targeted, relevant heterologous gene, the yeast strain remains stable and genetically intact for many generations. Thanks to these properties, the fermentation process proceeds reproducibly under non-selective conditions and can be standardized. The optimization of the K. lactis yeast according to the present invention aims to control the protein production rate so that it is as high as possible and below the threshold at which the cytopathic effect of the antigen severely interferes with the efficient fermentation process. This can be achieved by one or a combination of several genetic manipulations, namely: (i) an increase in lactose-inducible transcriptional activator concentrations; (ii) targeted modification of the LAC4 promoter, and / or (iii) stepwise escalation of gene dosage of heterologous genes encoding antigens; This was achieved by

[0019] Furthermore, optimization of the K. lactis yeast of the present invention includes: (iv) establishing multiple, novel integration sites for heterologous gene-encoding cassettes in the yeast genome to simultaneously express multiple antigens; It consists of:

[0020] In a preferred embodiment, the object of the present invention is achieved by providing a K. lactis strain for targeted cloning of heterologous gene-encoding nucleic acids into the yeast genome of said K. lactis strain, characterized in that said K. lactis strain comprises an expression cassette for said heterologous antigen integrated into the KlURA3-20 locus (KLLA0E22771g) and / or the KlMET5-1 locus (KLLA0B03938g) instead of or in addition to the KlLAC4 locus. A particularly preferred case is when the K. lactis strain comprises an expression cassette for said heterologous antigen integrated into the KlURA3-20 locus (KLLA0E22771g) and / or the KlMET5-1 locus (KLLA0B03938g) in addition to the KlLAC4 locus. A particularly preferred case is when the K. lactis strain has expression cassettes for the heterologous antigens integrated into the KlURA3-20 locus (KLLA0E22771g) and the KlMET5-1 locus (KLLA0B03938g) in addition to the KlLAC4 locus. Such modified K. lactis strains have the advantage that the genes for expressing heterologous genes are integrated into special, defined loci within the K. lactis genome, and the copy number of the heterologous genes is controllable. Furthermore, the K. lactis strain allows the integration of different genes for the expression of different heterologous antigens into defined loci within the K. lactis genome.

[0021] "Heterologous antigen" or "heterologous protein" in the context of the present invention means all peptides, polypeptides and proteins suitable for generating an immune response, preferably a protective immune response, in humans or animals against pathogens or oncogenically altered cells. Heterologous proteins may be derived from any kind of pathogen or tumor, for which antigens have been characterized that are capable of inducing a protective immune response, preferably a protective immune response, alone.

[0022] In a preferred embodiment, the heterologous protein is derived from a pathogen (virus, bacterium, parasite) for which an antigen has been characterized that is capable of inducing a protective immune response, preferably a protective humoral immune response, alone.

[0023] For example, they are illustrated below. (heterologous proteins derived from parasites) American hookworm; Duodenal hookworm (Ancylostoma duodenalis): ASP protein, hemoglobin-degrading protease, Leishmania: gp63, 46kD promastigote antigen, LACK, Plasmodium (malaria parasites): CSP proteins, CSA-1, CSA-3, EXP1, SSP2, STARP, SALSA, MSP1, MSP2, MSP3, AMA-1, GLURP, Pfs25, Pfs28, Pvs25, Pvs28, Pfs48 / 45, Pfs230, Schistosoma (Schistosomes): TP1, Sm23, ShGSTs 26 and 28, paramyosin, parasite myosin, Sm14.

[0024] (bacterial heterologous protein) Mycobacterium tuberculosis: Ag85A, Hsp65, R8307, 19kD, 45kD, 10.4, Heliobacter pylori: VacA, LagA, NAP, hsp, urease, catalase, Group A Streptococcus: M, SCPA peptidase, exotoxins SPEA and SPEC, fibronectin-binding protein, Streptococcal pneumonia: PspA, PsaA, BHV3, BHV4, Salmonella typhimurium: Vi antigen, Shigella: LPS, Vibrio cholerae:CTB, E. coli ETEC: LT, LT-ST, CTB, Yersinia pestis: F1, V.

[0025] (heterologous proteins derived from tumor cells / tumors (tumor-associated antigens, TAAs)) CEA, 5T4, MUC1, MART1, HER-2.

[0026] (Heterologous proteins derived from viruses are particularly preferred.) Caliciviridae (Norwalk, HEV): NV 60kD; HEV ORF2; Reoviridae (Rota): VP7, VP4, Retroviridae (HIV): Gag, Pol, Nef, Env, gp160, gp120, gp140, gp41, Flaviviridae (Flavivirus genus: WNV, Dengue, YF, TBE, JEV): preM-Env, NS3, NS4, NS5, Flaviviridae (Pestiviruses BVDV, CSFV, BDV; Hepacivirus HCV): E1, E2, E RNS (Pesti), C, NS3, NS4, NS5, Hepadnaviridae (HBV): HBS antigen, Paramyxoviridae (subfamily Paramyxovirinae: PIV-1, PIV-2, mumps, Sendai, PIV-2, PIV-4, Morbilli): M, HN, N, F, Paramyxoviridae (Pneumovirinae: RSV): F, G, SH, M, Rhabdoviridae (rabies): G, Herpesviridae (EBV, HSV2): gp350 / 220 (EBV), gB2, gD2 (HSV), Coronaviridae (SARS): CoV, N, M, S, Orthomyxoviridae (influenza A, B): HA, NA, M1, M2, NP, Papillomaviridae: L2, E6, E7.

[0027] In a further embodiment of the invention, the modified K. lactis strain comprises an expression cassette comprising a K. lactis LAC4-12 promoter (P LAC4-12 ) or a variant of said promoter, the ORF of the antigen to be expressed, and the AgTEF1 terminator. LAC4-12 Expression of heterologous genes under promoter control has the advantage that after integration into the LAC4 and / or KlURA3 and / or KlMET5 loci they are almost equally strongly induced by lactose.

[0028] As mentioned above, there is a positive correlation between antigen concentration in the vaccine strain and the immunogenic effect of the yeast vaccine in the target organism. To prevent CPE in the event of excessively strong overexpression, for example, due to the integration of an additional KlGAL4 gene, the vector system can be alternatively modified to rapidly and efficiently link multiple gene copies in series and introduce this expression cassette into one of three loci in a single step (see Example 5 and Figure 7A).

[0029] Thus, as a further advantageous result of the present invention, the modified K. lactis strain comprises multiple copies of a nucleic acid sequence encoding a heterologous antigen at the KlLAC4 locus, or the KlURA3-20 locus, or the KlMET5-1 locus, which copies are inserted via a tandem or multiple expression cassette, each of which is driven by the LAC4-12 promoter (P LAC4-12) or a variant of the promoter and multiple copies of the antigen-encoding region (gene) flanked by the AgTEF1 terminator. Duplication of antigen gene copies in this manner, one at each locus, can significantly increase their expression.

[0030] In a preferred embodiment of the invention, the gene for the heterologous antigen IBDV-VP2 is present in the form of a tandem expression cassette at the KlLAC4 locus of the K. lactis strain. This K. lactis strain has the advantage that the heterologous antigen IBDV-VP2 is expressed in increased amounts compared to strains that have a single copy of the gene encoding the heterologous antigen IBDV-VP2. Particularly preferred according to this embodiment of the invention is strain VAK1118 (DSM32701), which has the gene for the heterologous antigen IBDV-VP2 in the form of a tandem expression cassette at the KlLAC4 locus.

[0031] Even more preferred is a K. lactis strain of the invention in which one or more copies of nucleic acids encoding different heterologous antigens are inserted into the KlLAC4 locus, the KlURA3-20 locus, and / or the KlMET5-1 locus via a single expression cassette, a tandem expression cassette, or multiple expression cassettes, such that, firstly, different heterologous antigens can be expressed in the yeast cell, and secondly, the different heterologous antigens can be expressed at different concentrations. Particularly preferred in this embodiment are K. lactis strains in which nucleic acid sequences encoding the heterologous antigens influenza A HA (A / Puerto Rico / 8 / 1934(H1N1)) and influenza A M1 (A / Puerto Rico / 8 / 1934(H1N1)) are inserted into the KlLAC4 and KlURA3-20 loci of the K. lactis strain and expressed therein. This particularly preferred embodiment of the invention is strain VAK1283 (DSM32697) in which nucleic acid sequences encoding the heterologous antigens influenza A HA (A / Puerto Rico / 8 / 1934 (H1N1)) and influenza A M1 (A / Puerto Rico / 8 / 1934 (H1N1)) have been inserted into the KlLAC4 and KlURA3-20 loci of the K. lactis strain.

[0032] As mentioned above, it is known that increasing the dosage of the KlGAL4 gene can increase antigen production (Krijger et al., 2012, and International Publication No. 2013107436). The drawbacks of achieving this by integrating the pLI-1 plasmid expressing KlGAL4 in a two-step process were discussed above. This drawback has been overcome by the present invention by providing a stable starting strain for integrating heterologous genes, including a second copy of the KlGAL4 gene. All derivative strains have the same genetic background, ensuring that exactly one additional copy of the KlGAL4 gene is present in the strain. This reduces the cytotoxicity observed with the expression of multiple copies and reduces the multiple steps required for vaccine strain production to just one. Furthermore, genetic stability is improved because the reversible integration / excision of the plasmid is omitted. Such strains can be generated, for example, as described in Example 1.

[0033] Thus, a further advantageous embodiment of the present invention provides a K. lactis strain that contains, in addition to the native KIGAL4 gene of the genome, a second ectopic copy of the KIGAL4 gene. In this strain, expression of the KIGAL4 transcriptional activator can be increased up to two-fold, and expression of heterologous genes inserted into the KlLAC4 locus, KlURA3-20 locus, and / or KlMET5-1 locus can be increased in a specified manner by the LAC4-12 promoter or variants of this promoter described below. In conventional practice, KlGAL4-encoding plasmids are transiently introduced into cells at multiple, uncontrolled copy numbers. As a result, heterologous antigens are often expressed at high concentrations, resulting in cytotoxic effects. In the case of the K. lactis strain of this embodiment of the present invention, cytotoxic effects can be reduced or avoided with high efficiency. Furthermore, future loci developed for the same purpose (insertion of an LAC4-regulated expression cassette) can also be controlled in this way. It has been found to be advantageous when the ectopic copy of the KIGAL4 gene is flanked by the KIGAL4 promoter and KIGAL4 terminator and integrated at locus KLLA0E13795g (Klavt3::KlGAL4-1, SEQ ID NO: 1) in the K. lactis strain. Particularly preferred in this embodiment of the invention is strain VAK1111 (DSM 32696) which possesses these properties.

[0034] In a further preferred embodiment, the present invention provides a K. lactis strain in which the nucleic acid sequence encoding the heterologous antigen IBDV VP2 is present at the locus KlLAC4. Particularly preferred according to this embodiment of the present invention is strain VAK1171 (DSM32699). This strain additionally contains a second ectopic copy of the KIGAL4 gene, in which the nucleic acid sequence encoding the heterologous antigen IBDV VP2 is also present. This strain shows increased expression of the heterologous antigen IBDV-VP2 compared to a strain without an additional ectopic copy of the KIGAL4 gene.

[0035] The production of heterologous proteins in microorganisms can be problematic if it leads to cytopathic effects (CPE). The present invention therefore provides a method for separating the antigen-producing phase from the biomass-accumulating phase. Thanks to the inducible LAC4 promoter, this is partly possible, for example, by a fed-batch fermentation process, but also by the promoter P LAC4-12 This approach is also hindered by the fact that KlGAL4 is not completely inactivated (i.e., remains somewhat open) under non-inducing conditions. For antigens with very strong CPE, the result is a decrease in growth rate and induction of a cellular stress response, which adversely affects antigen production. This problem is exacerbated by doubling the KlGAL4 gene dosage and / or increasing the number of antigen-encoding sequences (see below).

[0036] Therefore, a further advantageous development of the K. lactis strain of the present invention is a K. lactis strain having a modified promoter structure of the LAC4-12 promoter, which expresses little or no heterologous protein under non-inducing conditions. The modified structure of the LAC4-12 promoter is, in particular, the promoter P between positions 1065 and 1540. LAC4-12 The basic control region (BCR) of the LAC4-12-LR2’ ; SEQ ID NO: 2) is deleted (see also Example 2). As already mentioned above, this embodiment of the invention has the advantage over previous implementations that the cytotoxic effects previously caused by excessively strong expression of heterologous genes are highly efficiently reduced and avoided. Preferred in this embodiment are K. lactis strains in which the nucleic acid sequence encoding the heterologous antigen influenza A HA (A / Puerto Rico / 8 / 1934 (H1N1)) is located at the K1LAC4 locus. Particularly preferred according to this embodiment of the invention is strain VAK1243 (DSM32702). Said strain comprises an LR2 deletion in the LAC4-12 promoter.

[0037] The K. lactis strain also has a modified promoter structure of the LAC4-12 promoter, allowing for the regulation of heterologous protein expression. The promoter has a variable number of binding sites for the activator KlGal4 ("upstream activation sequences" 1, 2, and 4, 5), with one, two, three, or four KlGal4 binding sites. This allows for the expression of different heterologous proteins at different concentrations (quality assurance by design) in a single yeast cell. The shortened promoter variants are particularly important for the modularization of the system, e.g., for the expression of multiple proteins in optimal stoichiometry within the same strain, e.g., for the formation of highly immunogenic virus-like particles (VLPs). A preferred embodiment of the invention is when a nucleic acid sequence encoding the heterologous antigen IBDV VP2 is inserted into the KlLAC4 locus of the K. lactis strain. Particularly preferred according to this embodiment of the invention is strain VAK1131 (DSM32700). The strain contains a deletion of LR2 and a deletion of upstream activating sequences 4 and 5 within the LAC4-12 promoter.

[0038] One of the objectives of the present invention was to provide a K. lactis strain that is more suitable for cultivation. This problem was solved by restoring the gene function of the Kllac4, Klura3-20, and Klmet5-1 alleles in the K. lactis strain of the present invention. The resulting K. lactis strain is prototrophic (Example 6, Figure 8). This simplifies the fermentation of vaccine strains, facilitating the establishment of production processes and making them more cost-effective. According to this embodiment of the present invention, preferred is a K. lactis strain in which nucleic acid sequences encoding the heterologous antigens BVDV E2 ectodomain (type 1, CP7), BVDV E2 ectodomain (type 2, New York 93), and BVDV Npro-NS3 (type 1, CP7) have been inserted into the KlLAC4, KlURA3-20, and KlMet5-1 loci of the K. lactis strain. According to this embodiment of the present invention, the strain VAK1400 (DSM32698) is particularly preferred. The strain is prototrophic.

[0039] In a particularly preferred embodiment, the present invention provides a K. lactis strain selected from the following strains: VAK952 (accession number: DSM32705), VAK1111 (accession number: DSM32696), VAK1118 (accession number: DSM32701), VAK1131 (accession number: DSM32700), VAK1171 (accession number: DSM32699), VAK1243 (accession number: DSM32702), VAK1283 (accession number: DSM32697), VAK1395 (accession number: DSM32706), VAK1400 (accession number: DSM32698).

[0040] The strains were deposited in accordance with the Budapest Treaty on November 24, 2017 or December 1, 2017 (DSM 32705, DSM 32706) with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures, DSMZ, Inhoffenstrasse 7B, 38124 Braunschweig, Germany) under the above numbers.

[0041] In a further aspect, the present invention provides an integrative expression vector, thanks to which the K. lactis strain of the invention can be produced.

[0042] In a preferred embodiment, the present invention provides the integrative expression vectors KIpURA3 (SEQ ID NO: 3) and KIpMET5 (SEQ ID NO: 4). These vectors utilize the LAC4-12 promoter (P LAC4-12) or a variant of said promoter, which contains the ORF of the antigen to be expressed, as well as the AgTEF1 terminator sequence and, similarly, targeting sequences that allow targeted functional restoration of the Klura3-20 and Klmet5-1 alleles after integration. The antigen-encoding sequence is cloned between the promoter and terminator sequences of the expression cassette at a specified restriction site. This vector allows stable integration of heterologous gene expression cassettes into the K. lactis genome without markers and without the use of antibiotic resistance. Thus, the strength of this vector system is that multiple heterologous genes can be easily exchanged between different vectors and that the promoters and terminators of the expression cassettes can be exchanged with others. The expression cassette is LAC4-12 It consists of a promoter and an AgTEF1 terminator with a heterologous gene between them. The heterologous gene is exchangeable at the restriction sites AscI and NotI. LAC4-12 Promoters can be exchanged between the SmaI and AscI restriction sites in both vectors, and terminators can be exchanged between NotI and BoxI (or MluI) in KIpURA3 and NotI and Ecl136II (or SacI) in KIpMET5. Another expression cassette is cloned between the SmaI and BoxI (or MluI) restriction sites in KIpURA3 and between the SmaI and Ecl136II (or SacI) restriction sites in KIpMET5. Using designated restriction enzymes, expression cassettes can also be exchanged between the KIpMET5 and KIpURA3 vectors, or additional expression cassettes can be introduced. An improvement over the KIp3 and KIp3-MCS vectors (WO 20101054649) is that selection is performed under non-inducing conditions (no lactose), resulting in high transformation rates even for proteins with CPE and reducing the possibility of many transformants even with low heterologous gene expression. See also Examples 3.1 and 3.2.

[0043] In a particularly preferred embodiment of the present invention, KIpMET5-P LAC4-12 -Et, KIpMET5-P LAC4-12-LR2 -Et, KIpMET5-PLAC4 -Et, KIpMET5-P LAC4-LR2 From, and KIpURA3-P LAC4-12 -Et, KIpURA3-P LAC4-12-LR2 -Et, KIpURA3-P LAC4 -Et and KIpURA3-P LAC4-LR2 Integrative expression vectors selected from (SEQ ID NO: 3, 4 in combination with SEQ ID NO: 5, 6, 7 or 8) are provided.

[0044] Vector KIpURA3-P LAC4-12 -Et, KIpURA3-P LAC4-12-LR2 -Et, KIpURA3-P LAC4 -Et and KIpURA3-P LAC4-LR2 are variants of the vector KIpURA3-Et, in each case a nucleic acid sequence encoding the Etx.B-HA protein is inserted. LAC4-12 -Et, KIpURA3-PLAC 4-12-LR2 -Et, KIpURA3-P LAC4 -Et and KIpURA3-P LAC4-LR2 has a different promoter compared to the vector KIpURA3-Et.

[0045] Vector KIpMET5-P LAC4-12 -Et, KIpMET5-P LAC4-12-LR2 -Et, KIpMET5-P LAC4 -Et, KIpMET5-P LAC4-LR2 are variants of the vector KIpMET5, in each case a nucleic acid sequence encoding the Etx.B-HA protein is inserted. LAC4-12 -Et, KIpMET5-P LAC4-12-LR2 -Et, KIpMET5-P LAC4 -Et, KIpMET5-P LAC4-LR2 has a different promoter compared to the vector KipMET5.

[0046] In a further aspect, the present invention provides a method for producing a K. lactis strain of the invention, comprising the steps of: (i) inserting a nucleic acid sequence encoding an antigen of interest into a KIpURA3 vector or a KIpMET5 vector; (ii) transforming a K. lactis culture with the modified and pre-enzymatically digested vector construct; (iii) selecting transformed K. lactis cells using solid medium lacking uracil and / or methionine; and (iv) optionally restoring prototrophy; The method includes:

[0047] In one embodiment of the method of the present invention, gene sequences for multiple antigens can be ectopically inserted and expressed in a controlled manner simultaneously. It is preferred that different gene sequences encoding antigens of different variants of a single pathogen be ectopically inserted and expressed in a controlled manner. It is also preferred that different gene sequences encoding antigens of different pathogens be ectopically inserted and expressed in a controlled manner.

[0048] In a further aspect, the present invention provides pharmaceutical or veterinary compositions for parenteral, enteral, intramuscular, mucosal or oral administration comprising a K. lactis strain of the invention, optionally in combination with conventional vehicles and / or excipients. In particular, the present invention provides pharmaceutical or veterinary compositions suitable for vaccination.

[0049] Preferably, the pharmaceutical or veterinary pharmaceutical composition comprises at least one physiologically compatible vehicle, diluent, adjuvant and / or excipient. The K. lactis strain of the present invention may be contained in a pharmaceutically acceptable vehicle, e.g., a conventional medium such as a saline medium or a buffer solution, as a pharmaceutical composition for injection. Such a medium may also contain conventional pharmaceutical substances, e.g., pharmaceutically acceptable salts for setting the osmotic pressure, buffers, preservatives, etc. Preferred media include physiological saline and human serum. A particularly preferred medium is PBS-buffered saline.

[0050] Further suitable pharmaceutically acceptable vehicles are known to those skilled in the art, for example from Remington's Practice of Pharmacy, 13th Edition and J. of Pharmaceutical Science & Technology, Vol. 52, No. 5, Sept-Oct, pages 238-311.

[0051] A further aspect of the invention provides the use of the recombinant K. lactis yeast of the invention for vaccination, e.g., to generate protective immunization, particularly protective immunization against pathogens.

[0052] Methods relating to the generation of protective immunization include, for example, the following steps: a) culturing and growing the recombinant yeast of the invention; b) harvesting and inactivating the yeast; c) administering the recombinant yeast according to a specific immunization scheme; d) determining the titer of antibodies formed; and / or e) Detecting immunization.

[0053] Cultivation and propagation of the recombinant yeast of the present invention can be achieved using any conventionally available method. Particularly preferred are methods that result in high cell yields in a cost-effective manner. These include fermentation methods, especially high cell density fermentation methods. Performing fermentation using a fed-batch fermentation protocol has been found to be particularly advantageous.

[0054] In a preferred embodiment, protective (prophylactic) immunization is achieved by administering recombinant yeast orally / mucosally, intramuscularly or subcutaneously.

[0055] Recombinant yeast cells should be used in the inactivated / killed state in the method of the present invention. For this purpose, the yeast is dried after cultivation and expression of the heterologous gene and then inactivated. Inactivation can be carried out using any conventionally available method. Particularly suitable for use in the method of the present invention are heat inactivation (e.g., heat inactivation at 90°C for 2 hours) or gamma irradiation (e.g., 25 kGy or 50 kGy).

[0056] The present invention also provides a vaccination method comprising administering a K. lactis strain of the present invention to a subject, e.g., an animal or a human, preferably an animal, in an amount sufficient to elicit an immune response, preferably a protective immune response, in the subject against one or more heterologous antigens.

[0057] A particular advantage of using the K. lactis strains of the invention is that they can elicit a protective immune response against one pathogen alone, after one application / immunization ("single inoculation"), or after two applications / immunizations ("prime-boost"). A further advantage of using the K. lactis strains of the invention has been discovered to be that they can elicit cross-protective immune responses against different variants of one pathogen, after one application / immunization ("single inoculation"), or after two applications / immunizations ("prime-boost"). Even when the K. lactis strains of the invention harbor and express different heterologous genes against antigens of different pathogens, they can still elicit a protective immune response after one application / immunization ("single inoculation") or two applications / immunizations ("prime-boost"). [Effects of the Invention]

[0058] (Summary of Advantages of the Invention) The above improvements within the K. lactis platform have yielded a number of advantages. a) Great simplification (ready-to-use toolboxes / kits) and high reproducibility have been achieved in the construction of yeast-based "subunit vaccine" strains, which can now be produced within a specific short timeframe. b) Yeast vaccines can contain one or more antigens, which can be flexibly customized and produced in different amounts. c) Furthermore, efficient fermentation of prototrophic yeasts became possible. d) It allows for precise inducibility of recombinant protein production, the latter being particularly important for proteins that may cause CPE. e) The desired stable integration of the heterologous gene into the genome and the associated genetic stability of the strain offers the advantage that the production process proceeds reproducibly, which is particularly important for GMP production. f) The protection of yeast vaccines is improved by increasing recombinant antigen production, achieved as a result of increasing the heterologous gene copies and / or increasing KlGAL4 concentrations. g) Furthermore, the administered vaccine dose can be reduced due to the increased recombinant antigen production achieved as a result of increased heterologous gene copies and / or elevated KlGAL4 concentrations, making yeast production more cost-effective and improving vaccine suitability for vaccinated recipients. h) Polyvalent yeast vaccines can be used in a cross-protective or polyvalent manner to protect against different varieties of the same pathogen or against different pathogens. No further post-treatment of yeast used as a vaccine is required other than inactivation and mixing with an appropriate adjuvant and / or an appropriate liquid volume. [Brief explanation of the drawings]

[0059] The invention is explained more particularly below on the basis of drawings and exemplary embodiments.

[0060] [Figure 1]Figure 1 shows the characterization of the newly generated K. lactis background strain carrying two KlGAL4 copies. The presence of a second ectopic KlGAL4 copy at the identified integration site was confirmed and the effect of the integration on yeast growth was analyzed. A: Diagram of the integration site of the ectopic KlGAL4 copy. The integration site is indicated and the gene name is also labeled. B: Yeast strains with an additional integrated ectopic KlGAL4 gene at the KlAVT3 locus (VAK1110) and without an additional integration (VAK367) were analyzed using primers [ka] Agarose gel of PCR-amplified fragments using the . The predicted fragment sizes for each are shown to the right of the diagram. C: Titration test with 10-fold serial dilutions (starting OD1) in glucose (YPD) or lactose (YPLac). Incubations were performed at 30°C and 37°C, respectively. Growth was compared for a yeast strain with a KlGAL4 copy at the native locus (VAK1139), a yeast strain with a KlGAL4 copy at an ectopic locus but lacking KlGAL4 at the native locus (VAK1110), a yeast strain with no KlGAL4 copies (ΔKlgal4; VAK964), or a yeast strain with two KlGAL4 copies (VAK1168). It was shown that only the specific integration of the additional KlGAL4 gene resulted in a growth defect with little productivity. This defect was visible only under inducing conditions at 37°C. Even more pronounced was the growth defect upon complete deletion of KlGAL4.

[0061] [Figure 2]Figure 2 shows a Western blot analysis of proteins from a K. lactis strain producing IBDV-VP2 and harboring an additional ectopic KlGAL4 copy. The effect of the additional KlGAL4 copy on LAC4-12 promoter-dependent recombinant protein production was analyzed by Western blot. The test strain used was a yeast strain carrying an IBDV-VP2 expression cassette, and this yeast strain was compared with other IBDV-VP2 yeast strains. The presence (+) or absence (-) of the ectopic KlGAL4 copy and the tandem IBDV-VP2 expression cassette (see below) are indicated at the top. In strain VAK911, an ectopic copy was introduced by linearization of the plasmid pLI-1 with BstEII (Krijger et al. 2012 and WO 2013107436), and in strain VAK1130, an ectopic KlGAL4 copy resides at the KlAVT3 locus (see Figure 1). Yeast strain VAK367 was included as a wild-type control without heterologous genes. Yeast strains were pre-cultured in YPD and then grown in YPLac for 15 h. Twenty micrograms of protein extract from each yeast strain was analyzed by SDS-PAGE. Immunoblotting was performed using anti-IBDV rabbit serum (1:8000) and goat-derived HRP-conjugated anti-rabbit antibody (1:10000). Multimeric (agg.) and monomeric (mon.) IBDV-VP2 are indicated by arrows on the right, and nonspecific bands are indicated by asterisks. It was shown that ectopic expression of an additional KlGAL4 gene, similar to the presence of a tandem expression cassette, strongly increased heterologous antigen concentrations (see also below).

[0062] [Figure 3]Figure 3 shows the effect of LR2 deletion within the LAC4-12 promoter on uninduced recombinant protein production and yeast growth on glucose. The unmodified LAC4-12 promoter also exhibits basal expression of the GOI (gene of interest) under uninducing conditions. This is particularly problematic in the case of cytotoxic heterologous antigens. These experiments tested whether a deletion within the BC region of the LAC4-12 promoter (LR2 deletion) could reduce or completely suppress recombinant protein production under uninducing conditions. A: Diagram of the LAC4-12 promoter (PLAC4-12). The basic control region (BCR), LR2 deletion, and four KlGal4-binding sites (upstream activation sequences: U1, U2, U4, U5), as well as the nucleic acid sequence encoding the heterologous gene (GOI), are also shown. B: Western blot analysis of IBDV-VP2 yeast strains with and without LR2 deletion (VAK1131) and those without (VAK1130) after cultivation under non-inducing conditions (YPD 3% EtOH). VAK1111 was used as a wild-type control without a heterologous gene. For each yeast strain, 50 μg of protein extract was loaded onto a 12% SDS gel. Immunoblotting was performed using anti-IBDV rabbit serum (1:5000) and goat-derived HRP-conjugated anti-rabbit antibody (1:10,000). The loaded control KlNop1 was detected using mouse anti-Nop1 antibody (1:5000) and goat-derived HRP-conjugated anti-mouse antibody (1:10,000). C: Titration test with 10-fold serial dilutions (starting at OD1) in YPD, YPD with 0.5% glucose, and YPLac. Incubations were performed at 30°C and 37°C, respectively. Growth of yeast strains containing an influenza A HA heterologous gene at the LAC4 locus with (VAK1243) and without (VAK952) the LR2 deletion was compared. Yeast strain VAK367 was used as a wild-type control without the heterologous gene. It was shown that LR2 deletion suppressed the expression of undesired basic heterologous proteins. Furthermore, it was shown that LR2 deletion improved the growth of yeast strains expressing a cytotoxic protein (influenza hemagglutinin, HA) under both non-inducing and inducing conditions.This is especially evident at 37°C.

[0063] [Figure 4] Figure 4 shows Kip vectors that can be used to integrate protein expression cassettes into different loci in the K. lactis genome. While the use of the LAC4 locus (KIp3 vector system) has been previously described (WO 20101054649 and WO 2013107436), the use of the KlURA3 and KlMET5 loci is novel. A: Diagram of different Kip vectors, each with an integration site in the genome. B and C: Expression cassettes and flanking ends in the newly described KipURA3 (B) and KipMET5 (C) vectors. Different DNA sequence segments and associated restriction sites are indicated. GOI: heterologous gene (gene of interest). D: Western blot analysis of heterologous protein expression in yeast strains constructed with Kip vectors (A, B, and C). Here, the heterologous gene is Etx.B-HA. Yeast "housekeeping" KlNop1 protein (KLLA0C04389g) was detected as a loading control. Yeast strains were pre-cultured in YPD (+U) and then grown in YPLac (+U) for 4 hours. For each yeast strain, 30 μg of protein extract was loaded onto a 12% SDS-PAGE. Immunoblotting was performed using monoclonal mouse anti-HA (1:5000) and anti-KlNop1 (1:5000; Santa Cruz, TX, USA) antibodies and also a goat-derived HRP-conjugated anti-mouse antibody (1:10000; Jackson ImmunoResearch, PA, USA). It has been shown that both the KlURA3 and KlMET5 loci, as well as the LAC4 locus (WO 20101054649 and WO 2013107436), can be used for heterologous gene expression.

[0064] [Figure 5]Figure 5 shows the production of different recombinant proteins in the same yeast strain. The yeast strain (VAK1234) was constructed using the KIpURA3 and KIp3-MCS vectors. Western blot analysis of proteins from a tandem IBDV VP2-expressing yeast strain (see below) was performed using the KIpURA3 vector to introduce an additional expression cassette carrying Etx.B-HA as a heterologous gene (VAK1234). Controls used were a yeast strain carrying an expression cassette carrying Etx.B-HA at the LAC4 locus (VAK899) or at the KlURA3 locus (VAK1235), or a yeast strain carrying only the tandem IBDV-VP2 expression cassette at the LAC4 locus (VAK1171). The yeast strains were pre-cultured in YPD and then cultured in YPLac for 6 hours. For each yeast strain, 30 μg of protein extract was loaded onto a 12% SDS-PAGE gel. Protein detection by immunoblotting was performed using mouse anti-HA antibody (1:5000; Santa Cruz, TX, USA) and goat HRP-conjugated anti-mouse antibody (1:10,000) for Etx.B-HA, and rabbit anti-IBDV antiserum (1:5000; Granzow et al., 1997) and goat HRP-conjugated anti-rabbit antibody (1:10,000; Jackson ImmunoResearch, PA, USA) for IBDV-VP2. Both heterologous proteins were expressed in the same yeast cells. Surprisingly, the expression level of one antigen was not limited by the coexpression of another antigen. This is evident from a comparison of expression levels in monovalent and bivalent strains (see also Figure 12).

[0065] [Figure 6]Figure 6 shows differentially induced LAC4-12 promoter variants for expression cassettes in KIp vectors. The expression cassettes in KIp vectors were provided with different variants of the LAC4-12 promoter. The effect of the promoter variants on the strength of protein synthesis induction was tested based on the analysis of yeast strains containing the corresponding expression cassettes with Etx.B-HA as the heterologous gene. A: Schematic diagram of the promoter variants, the related KIpURA3 vector with Etx.B-HA as the heterologous gene, and the yeast strains constructed from them. BCR: Binding regions for the transcriptional activators KlCat8 and KlSip4 (transcriptional activators under non-inducing conditions); U1, U2, U4, U5: Binding regions for the transcriptional activator KlGal4 (upstream activation sequence). B: Western blot analysis for the characterization of the LAC4-12 promoter variants (A) in yeast strains constructed using the KIpURA3 vector. Yeast strains were pre-cultured in YPD and then grown in YPLac for 4 hours. For each yeast strain, 30 μg of protein extract was loaded onto a 12% SDS-PAGE. Immunoblotting was performed using monoclonal mouse anti-HA (1:5000) and anti-Nop1 (1:5000) antibodies and also a goat-derived HRP-conjugated anti-mouse antibody (1:10000). It was shown that the expression rate of heterologous genes varies depending on the nature of the promoter used.

[0066] [Figure 7]Figure 7 shows the effect of doubling the heterologous gene copy number using a tandem expression cassette on recombinant protein production. The effect of increasing the heterologous gene copy number using a tandem expression cassette on recombinant protein production (IBDV-VP2) was tested. A: Schematic diagram of the tandem expression cassette. The DNA segment and relevant restriction sites are indicated. GOI: heterologous gene (gene of interest). B: The tandem construct from (A) for random integration using the ScURA3 selection marker is shown. C: Western blot analysis comparing IBDV-VP2 protein production in a yeast strain (VAK1118) harboring a tandem expression cassette (A) and a yeast strain (VAK910) harboring an expression cassette containing only one heterologous gene copy. Yeast strains were pre-cultured in YPD and then cultured in YPLac for 3 or 6 hours. For each yeast strain, 60 μg of protein extract was loaded onto a 12% SDS-PAGE. Immunoblotting was performed using anti-IBDV rabbit serum (1:10,000) and goat-derived HRP-conjugated anti-rabbit antibody (1:10,000). Multimeric (agg.) and monomeric (mon.) IBDV-VP2 are indicated by arrows on the right, and nonspecific bands are indicated by asterisks. D: Western analysis of yeast strains containing randomly integrated tandem IBDV-VP2 expression cassettes (B) compared with a Kip3-MCS-constructed yeast strain (VAK910) containing a single expression cassette and a derived yeast strain containing an additional KlGAL4-1 copy (pLI-1). Yeast strains were precultured in YPD and then grown in YPLac for 8 hours. Immunoblotting was performed as described in (b) below. It was shown that the use of tandem expression cassettes significantly increased the expression rate of heterologous proteins.

[0067] [Figure 8]Figure 8 shows gene fragments for restoring gene function of the Klura3-20 and Klmet5-1 (A) alleles. The loci and gene fragments amplified using the specified primers for KlURA3 (A) and KlMET5 (B) are shown schematically. Mutations in the Klura3-20 (A) and Klmet5-1 (B) alleles that were reconstructed by homologous recombination using these gene fragments are indicated by an asterisk below the gene. Restriction sites for excision of the subcloned fragments are drawn in. This diagram shows a strategy for generating yeast strains that express prototrophic heterologous genes at the URA3 or MET5 locus.

[0068] [Figure 9]Figure 9, combined with Tables 1 and 2, shows the protective (prophylactic) immunization of chickens against vvIBDV in a classical prime-boost vaccination scheme. In two experiments (A and B), groups of at least 16 SPF chickens were subcutaneously vaccinated with lyophilized and heat-inactivated yeast cells of the genetically optimized tandem IBDV-VP2 K. lactis yeast strain VAK1127 in a prime-boost regimen. The first vaccination was administered two weeks after hatch (prime), followed by a second vaccination two weeks later (boost). Two weeks after the boost, a viral challenge with a highly virulent vvIBDV strain (89163 / 7.3) was administered. One group of subjects, which served as infection control, was mock-treated with PBS or adjuvant alone. In experiment 1 (A), wild-type yeast (VAK367) was also administered as a control. At least seven chickens per group, and at least five in Experiment 2 (B), served as controls without virus challenge. Serum was collected immediately before the first dose, immediately before and after challenge, and at other 10-day intervals. Seroconversion intensity was determined by ELISA (ProFLOK IBD Plus, Synbiotics). Converted titers are shown according to the kit instructions. A: Experiment 2 was performed identically to Experiment 1 (A). The mean ELISA titers of 12 animals are shown with standard deviations. Both experiments demonstrate the development of strong anti-IBDV VP2 antibody titers in VAK1127-vaccinated animals. The associated table summarizes the protection results of vaccinated animals against challenge with vvIBDV. Complete protection against virus infection was achievable in both vaccination experiments.

[0069] [Figure 10]Figure 10 shows the effect of genetic modification for restoring prototrophy on the recombinant protein production and immunogenicity of tandem IBDV-VP2 yeast strains. The auxotrophic tandem IBDV-VP2 yeast strain VAK1127 and its derived prototrophic yeast strain VAK1171 were compared in terms of recombinant protein production and immunogenicity efficiency. A: Western blot analysis to confirm IBDV-VP2 content in freshly harvested yeast material. Yeast strains were pre-cultured in YPD and then cultured in YPLac for 8 hours. For each yeast strain, 40 μg of protein extract was loaded onto a 12% SDS-PAGE gel. Immunoblotting was performed using anti-IBDV rabbit antiserum (1:10,000) and goat-derived HRP-conjugated anti-rabbit antibody (1:10,000). Aggregated (agg.) and monomeric (mon.) IBDV-VP2 are indicated by arrows on the right, and nonspecific bands are indicated by asterisks. B: Western blot analysis to confirm IBDV-VP2 content in lyophilized, heat-inactivated yeast material, which was subsequently used in immunization studies in BALB / c mice (C). Yeast strains were pre-cultured in YPD followed by 15 h in YPLac. For each yeast strain, 10 μg of protein extract was loaded onto 12% SDS-PAGE, while immunoblotting was performed as in (A) above, showing the corresponding bands. C: Immunogenicity test of the two yeast strains VAK1127 and VAK1171 in immunization experiments in BALB / c mice. Groups of five mice were each vaccinated subcutaneously three times with 0.1 mg (dry weight) of the yeast material analyzed above (B). The wild-type strain (VAK367) without antigen was used as a control. The first dose was administered using CFA (Complete Freund's Adjuvant) as the adjuvant, followed by two more doses at two-week intervals using IFA (Incomplete Freund's Adjuvant) as the adjuvant. One week after the third dose, mice were euthanized and bled. Serum was analyzed by IBDV-VP2 ELISA (IDEXX). Absorbance at 650 nm, which correlates with anti-IBDV-VP2 antibody titers, is shown with standard error.A monoclonal anti-IBDV-VP2 antibody (positive, mab64) was used as a positive control for ELISA, and sample buffer (negative 1) or a nonspecific antibody (negative 2) were used as controls. It was shown that the heterologous protein expression levels of both strains were similar and showed immunogenic potential.

[0070] [Figure 11] Figure 11, in conjunction with Table 3, shows the protective (prophylactic) immunization of SPF chickens against vvIBDV by a single subcutaneous administration of genetically optimized IBDV-VP2 vaccine yeast. Groups of at least 18 SPF chickens were vaccinated subcutaneously once at 2 weeks post-hatch with 10 mg of heat-inactivated cells of the genetically optimized tandem IBDV-VP2 K. lactis yeast strain VAK1171. Controls used were animals vaccinated with 10 mg of PBS or VAK367. Animals were vaccinated twice, at 2 and 4 weeks post-hatch. All animals were challenged with vvIBDV 6 weeks post-hatch. Sera were analyzed by ELISA (ProFLOK IBD Plus, Synbiotics) as described above. Confirmed antibody titers are shown. Individual points represent individual antibody titers of 12 chickens analyzed per group, and bars represent the mean with standard deviation. In the control cases, antibody titers were only observed in surviving chickens after challenge, demonstrating that only a "single dose" vaccination with the yeast subunit vaccine VAK1171 achieves complete protection against subsequent exposure to vvIBDV.

[0071] [Figure 12]Figure 12 shows the characterization of strains VAK952 and VAK1283. (A) Yeast strains VAK952 (monovalent HA) and VAK1283 (bivalent HA, M1) were preincubated in shake flasks with YPD and then induced in YPL for 6 hours. The optical density at 600 nm was measured, and 30 OD units of the culture were harvested. The pellet was disrupted with glass beads, and the soluble protein fraction (LF) and insoluble protein fraction (P, pellet) were examined by immunoblotting. The primary antibody used was α-HA1 or α-M1, and the secondary antibody used was α-Mouse-IR-Dye800CW. Signals were detected using an infrared imaging system (LI-COR Biosciences). (B, C) Yeast strains were preincubated in shake flasks with YPD and then induced in YPL for 24 hours. At designated time points, the optical density of the yeast culture was determined, and 30 OD units were harvested. (B) VAK1283 pellets were disrupted with glass beads and analyzed by immunoblotting. (C) Optical density measurements of VAK952 and VAK1283 were combined into growth curves as a function of time and averaged from at least two independent experiments. (D) For the dot test, yeast strains were grown on YPD-containing nutrient agar plates at 30°C for 48 hours. Starting at 1 OD unit, the yeast was serially diluted and then spotted onto YPD- or YPL-containing nutrient agar plates. The plates were grown at 30°C for 48 hours and then photographed. Ponceau S: staining of total yeast protein for each fraction and loading control. It is shown that VAK952 (monovalent HA) and VAK1283 (bivalent HA, M1) express equivalent amounts of HA protein. It was further shown that VAK1283 and VAK952 exhibited comparable growth characteristics, with VAK1283 being slightly superior.

[0072] [Figure 13]Figure 13 shows antibody titers in the serum of BALB / c mice after immunization with VAK952 (monovalent HA) and VAK1283 (bivalent HA, M1), both before and after challenge. Both yeast strains were preincubated in YPD in shake flasks and then induced in YPL for 12 hours (VAK952) or 6 hours (VAK1283). Cultures were then harvested, lyophilized, and the yeast material inactivated at 90°C for 2 hours. For immunization, 9-week-old female BALB / c mice were subcutaneously vaccinated twice (prime-boost) or once (single inoculation) with 2 mg of yeast (VAK952, VAK1283) or 1 mg of VAK1283, or with PBS (no adjuvant), at 3-week intervals. The adjuvant used was AddaVax. Three or six weeks after the final dose, animals were intranasally infected with 5xMLD50 of influenza A / PR / 8 / 34 (H1N1) virus. The infection control used was a mock-infected animal (mock) administered only intranasally with virus-free PBS. Three or six weeks after the final dose and during the challenge infection, animal serum was collected and tested for neutralizing antibodies (nAb) by VNT. nAb titer 50: the serum dilution that reduces the number of plaques by 50% compared to the virus-free control. It is determined as the log2 of the corresponding serum dilution. For logarithmic plots, a titer of log2(2) = 1 was considered to represent a serum sample with no detectable antibodies. mAb: test system control (α-H1(H37-66)). It was shown that both immunization schemes resulted in significant induction of neutralizing antibodies (Ab). Furthermore, it is clear that the neutralizing anti-HA antibody titers obtained in the primer-boost and single-shot vaccination experiments are not significantly different from those obtained with VAK952 and VAK1283.

[0073] [Figure 14]Figure 14 shows challenge with influenza A / PR / 8 / 34 (H1N1) after immunization with VAK952 (monovalent HA) and VAK1283 (bivalent HA, M1). Three or six weeks after the final dose (see Figure 13 for the immunization scheme), BALB / c mice were intranasally infected with 5x MLD50 of influenza A / PR / 8 / 34 (H1N1) virus. The control animals were mock-infected by intranasal administration of virus-free PBS alone (mock). Animals were then examined multiple times daily for survival (A), weight (B), and clinical signs (C) over a 14-day period. Clinical signs were scored on a scale of 0 to 4 (0: no abnormalities; 1: slightly unkempt coat; 2: unkempt coat and reduced activity; 3: unkempt coat and 15% weight loss; 4: unkempt coat and >20% weight loss). It was shown that prime-boost immunization with VAK952 did not provide optimal protection against viral challenge, while VAK1283 provided optimal protection. A single-dose scheme with both vaccines produced optimal protection with a 2 mg vaccine dose. Even when administered at 1 mg, the protection achieved by VAK1283 was similar to that achieved with a 2 mg VAK952 dose in a prime-boost regimen. [Example]

[0074] (Experimental Embodiment) Example 1: Generation of a host strain with two KlGAL4 gene copies stably integrated into unlinked loci A second copy of the KlGAL4 gene was inserted into a different locus (ectopically) without a selectable marker. Sequencing allowed us to localize the insertion site within the KlAVT3 gene (KLLA0E13795g) (Klavt3::KlGAL4-1, SEQ ID NO: 1) (Figure 1). The resulting strain was designated VAK1111. Independent meiotic segregation of the two KlGAL4 copies on chromosomes E (ectopic copy) and D (original copy) was confirmed by crossover experiments. Furthermore, the same experiments established that the copy number of the KlGAL4-1 gene in the genome is exactly two. VAK1111, like VAK367-D4, was used for targeted integration of expression cassettes into the LAC4 locus by introducing a one-step lac4::ScURA3 disruption, allowing for markerless integration of a heterologous gene of interest between the LAC4 promoter and the LAC4 reading frame under selection for lactose growth using Kip vector technology (Krijger et al., 2012). The resulting strain, VAK1123, differs from VAK367-D4 only in that it contains a second, ectopic copy of the KlGAL4 gene.

[0075] Example 1.1: Improved productivity of yeast vaccine strains with an additionally integrated KlGAL4 gene In one exemplary embodiment, IBDV-oVP2 T2S The gene (Arnold et al. (2012)) was inserted into the LAC4 locus of strain VAK1123 (resulting strain VAK1130). Compared to a strain (VAK910) with the same genetic background but only one KlGAL4 copy, an increased production of IBDV-VP2 could be established. For comparison, a strain with only one KlGAL4 gene but two CDS VP2 IBDV The strain VAK1118 (see below) carrying the copy is additionally shown (Figure 2).

[0076] Example 2: Peptides with reduced basal activity for optimizing expression of cytopathic antigens LAC4-12LR2’ promoter) The production of heterologous proteins in microorganisms is problematic when this leads to cytopathic effects (CPE). The challenge faced is therefore to find a way to separate the antigen production phase from the biomass accumulation phase. Thanks to the inducible LAC4 promoter, this is partly possible by fed-batch fermentation processes, but under non-inducing conditions the promoter P LAC4-12 The overall inhibition of KlGAL4 expression is hindered by the lack of complete inactivation of the promoter region between -1065 and -1540. In the case of antigens with very strong CPE, a decrease in growth rate and induction of a cellular stress response occur, adversely affecting antigen production. This problem is exacerbated by doubling the KlGAL4 gene dosage and / or increasing the number of antigen-encoding sequences (see below). The solution is to insert the promoter region between -1065 and -1540 into the promoter region. LAC4-12 The deletion of the basic control region (BCR) (Mehlgarten et al. (2015)) in Figure 3A (LR2 deletion; P LAC4-12-LR2’ ; SEQ ID NO: 2). The deletion was introduced into the native LAC4 locus of the genome of the starting strains VAK367 (one KlGAL4 copy) and VAK1111 (two KlGAL4 copies), along with the lac4::ScURA3 disruption. The resulting strains VAK1109 and VAK1124 are suitable for the expression of antigens with CPE. The promoter P LAC4-12LR2’ was also inserted into the integrating vectors KIpURA3-Et and KIpMET5-Et (see below).

[0077] Example 2.1: Inhibition of basal (uninduced) expression of antigens by modified promoters After integration of a single tandem IBDV-VP2 expression cassette into VAK1124 (the resulting yeast strain: VAK1131; see below and Figure 7 for an explanation of the term "tandem expression cassette"), we were able to show that the LR2 deletion within the LAC4-12 promoter led to a strong reduction in VP2 protein production under non-inducing conditions (Figure 3B). Using strains expressing the influenza A antigen hemagglutinin (VAK952 without the LR2 deletion within the promoter and VAK1243 with the LR2 deletion within the promoter), we were able to show that the LR2 deletion resulted in the suppression of the cytopathic effect of influenza A HA antigen and improved growth under non-inducing conditions (Figure 3C).

[0078] Example 3: A versatile vector system for targeted integration of multiple expression cassettes into the K. lactis genome. Like VAK367-D4 (Krijger et al. (2012), WO 20101054649), the yeast strain VAK367 forms the genetic background for all K. lactis strains described herein. This strain background requires uracil and methionine (uracil and methionine auxotrophy) due to mutations in two genes, KlURA3 (KLLA0E22771g) and KlMET5 (KLLA0B03938g), alleles designated Klura3-20 (base pair deletion at position +345) and Klmet5-1 (G2555A and A3682T), which are loss-of-function gene variants.

[0079] These mutant alleles were used to utilize additional loci for targeted integration, separate from the integration site LAC4 (Krijger et al., 2012) already developed in KIp3 / KIp3-MCS, and thereby generate multivalent vaccine strains (Figure 4A). Selection can be achieved by restoring gene function of these mutated genes without the need for additional insertion of a selection marker. For this purpose, novel integrative vectors were constructed. Within these vectors, expression cassettes (in each case under the control of the LAC4-12 promoter or its variants) are flanked by gene segments that allow upstream integration of the KlURA3 gene and downstream integration of the KlMET5 gene by homologous recombination, while simultaneously restoring the wild-type sequences of these genes. Further loci can be developed similarly by mutagenesis of integration sites and auxotrophic selection for alternative growth substances.

[0080] Example 3.1: Vectors KIpURA3 and KIpMET5 for targeted integration of expression cassettes (with an inducible LAC4-12 promoter) into the KlURA3 (KLLA0E22771g) and / or KlMET5 (KLLA0B03938g) loci of K. lactis strains carrying the Klura3-20 and / or Klmet5-1 alleles The integrative expression vectors KIpURA3 (SEQ ID NO: 3) and KIpMET5 (SEQ ID NO: 4) were constructed with appropriate gene fragments (KlMET5 / KlURA3 target sequences) that allow targeted restoration of the function of the Klura3-20 and Klmet5-1 alleles, respectively.

[0081] The KIpMET5 expression vector is driven by the LAC4-12 promoter (P LAC4-12 or a variant thereof), a nucleic acid sequence encoding the antigen to be expressed, and an expression cassette consisting of the AgTEF1 terminator, which is adjacent downstream to the native KlMET5 fragment of the genome with the introduced ScCYC1 terminator, and adjacent upstream to the KlAIM18 promoter with the downstream KlAIM18 gene. The KIpURA3 expression vector is driven by the LAC4-12 promoter (P LAC4-12 or a variant thereof), a nucleic acid sequence encoding the antigen to be expressed, and an expression cassette consisting of the AgTEF1 terminator, which is flanked downstream of KLLAOE22749g with its associated promoter and upstream of the KlURA3 promoter with the downstream KlURA3 fragment (Figure 4B, C).

[0082] In either case, the antigen-encoding sequence is cloned between the promoter and terminator via the AscI and NotI restriction sites. The entire expression cassette is separated from the KIpURA3 vector backbone by Eco91I or KpnI restriction of the resulting plasmid, and the entire expression cassette is separated from the KIpMET5 vector backbone by HindIII or BoxI restriction of the resulting plasmid, and the restricted material is transformed into a K. lactis host strain harboring the Klura3-30 and / or Klmet5-1 alleles. The heterologous gene-containing expression cassette thus integrated into KlURA3-20 or KlMET5-1 thus corresponds to that which can be integrated into LAC4 in VAK367-D4 by the KIp3-MCS vector (WO 20101054649). Verification of uracil and / or methionine prototrophy of transformants was performed by standard colony PCR using primers MAB6 and VK211 for KIpMET5 transformants and primers MAB6 and VK71 for KIpURA3 transformants. Integration of the expression cassette into the precise target site between KlURA3 or KlMET5 and the respective flanking gene yielded products of 1652 bp in size for KIpMET5 transformants and 1307 bp in size for KIpURA3 transformants. No indication was found that the insertion impaired the functionality of the flanking genes.

[0083] Primer: [ka]

[0084] Example 3.2: Expression of heterologous antigens after integration of the coding gene cassette into the KlURA3 or KlMET5 locus P LAC4-12 Under promoter control, heterologous genes were induced by lactose with almost equal potency after integration into the LAC4, KlURA3, and KlMET5 loci. The heat-labile, nontoxic enterotoxin subunit B (Etx.B) and C-terminal (HA)3 epitope (Etx.B-HA) from Escherichia coli (E. coli) were used as test proteins for vector system evaluation. The coding sequences were cloned into vectors KipMET5, KipURA3, and Kip3-MCS and integrated into the KlMET5 (VAK1251), KlURA3 (VAK1235), and LAC4 (VAK899) loci (Figure 4D). As shown by Western blotting, the concentrations of Etx.B-HA protein in all three strains were very similar (Figure 4D). Therefore, we were unable to establish a positional effect on the amount of recombinant protein production depending on the integration site of the expression cassette within the genome.

[0085] Example 3.3: Co-expression of two heterologous antigens in the same yeast cell A novel vector system allows for the expression of P LAC4-12 The possibility of producing different heterologous proteins under the control of promoters was demonstrated by the Etx.B-HA expression cassette at the KlURA3 locus and two VP2 promoters present in tandem at the LAC4 locus. IBDV This was demonstrated by constructing a yeast strain containing an expression cassette with two copies of Etx.B-HA or VP2 (VAK1234; Figure 5; see below and Figure 7 for a description of the tandem cassette). Compared with yeast strains (VAK1235 or VAK1171) each containing only one expression cassette in the genome, the expression cassettes for Etx.B-HA or VP2 in VAK1234 were significantly higher than those for VAK1235 or VAK1171. IBDV No decrease in protein concentration was observed.

[0086] Example 4: LAC4 promoter variants for regulating recombinant protein synthesis under similar induction conditions The immunogenic effect of an antigen is often based on the assembly of multiple proteins in non-stoichiometric ratios. To make this possible in yeast-based vaccines, P3000, which can be differentially induced by lactose or galactose, is used. LAC4-12LR2’ Promoter variants were constructed (Figure 6A). They were characterized by the number of binding sites for the activator KlGal4 (U1, U2, U4, and U5; Godecke et al., 1991) and the presence or absence of the basic regulatory region BCR. In addition to the construct shown in Figure 3A inserted into the KIpURA3 vector, it was also possible to construct promoter variants with increased promoter strength by inserting additional binding sites. The result was a lactose-inducible synthetic promoter that can be used to extend this vector system and achieve different protein production or gene expression rates under the same induction conditions.

[0087] Example 4.1: Expression of heterologous antigens under the control of different LAC4 promoter variants Expression of Etx.B-HA under the control of four LAC4-12 promoter variants. Four LAC4 promoter variants were tested, differing in the number of binding sites for the transcriptional activator KlGal4 and in the presence or absence of a regulatory region for basal expression under non-inducible conditions (basal control region BCR; Figure 6A; SEQ ID NO: 14). Using these promoter variants, KIpURA3-Et vector variants KIpURA3-PL412-Et, KIpURA3-PL412LR2-Et, KIpURA3-PL4-Et, and KIpURA3-PL4LR2 were constructed, and Etx.B-HA protein was inserted as a test GOI. As previously mentioned, the AscI and NotI restriction sites allow insertion of alternative GOIs. The expression cassette was integrated into the KlURA3 locus, and Etx.B-HA protein levels were quantified by Western blotting (Figure 6B). It was shown that under identical induction conditions (4 hours in lactose-containing complete medium), the longest promoter variant P, which contains the complete intergenic region between the LAC4 and LAC12 genes and contains four KlGal4-binding sites (U1, U2, U4, U5) (Godecke et al., 1991), was expressed. LAC4-12The highest protein concentration was obtained by deleting the BCR (-1540 to -1065) when only the two proximal U1 and U2 binding sites (-1064 to -10) of LAC4 were present.

[0088] Example 5: Increased antigen production by increasing the copy number of the antigen-encoding gene Therefore, the vector system was modified to rapidly and efficiently combine multiple gene copies sequentially and to introduce this expression cassette into three loci in a single step (Figure 7A). To generate tandem expression cassettes that can integrate at the LAC4 locus, three PCR-amplified fragments were fused in one step (in-fusion cloning) with the desired KIP3(-MCS)-GOI template: (1) and (2) P LAC4-LR2 and T TEF (1) an expression cassette containing the LAC4 target sequence (primers: VK30 and VK31, and VK32 and VK33), and (2) an expression cassette containing the LAC4 target sequence (VK34 and VK35). After restriction (treatment) using, for example, HpaI, the tandem expression cassette can be integrated into the lac4::URA3 locus as described (Figure 7). After successful integration of the expression cassette, the first heterologous gene copy is inserted into the P LAC4-12 or P LAC4-12-LR2 The second gene copy is regulated by either P LAC4-LR2Alternatively, inserting a selectable marker between two expression cassettes at the SmiI, MluI, or PmeI restriction sites and removing the LAC4 target sequence with KpnI creates a tandem cassette that integrates non-directionally into the genome via NHEJ. When the expression cassettes are excised using MreI and AvaI, compatible ends can be ligated together to create longer, multiple expression cassettes. Repeated restriction with MreI and AvaI enriches the ligation mix for fragments containing expression cassettes arranged in tandem (head-to-tail). These fragments are transformed and integrated non-directionally under marker selection conditions.

[0089] Primer: [ka]

[0090] Example 5.1: Successful Use of a Multi-Copy Strategy This strategy involves the use of IBDV-VP2 as an antigen and two IBDV-VP2-encoding sequences (CDS-VP2 IBDV This was confirmed by using an expression cassette derived from KIp3 containing tandem oVP2. T2S , SEQ ID NO: 21) (Figure 7A) is a tandem IBDV-VP2 expression cassette in T2S VP2 from (Arnold et al., (2012)) IBDV (CDS-VP2 IBDV The CDS-VP2 consists of two LAC4 promoter-controlled coding sequences for the first and second copies. The promoter sequence consists of the LAC4 promoter region from -1123 to -10 for the first copy and from -1099 to -10 for the second copy. IBDV is flanked at the 3' end by the AgTEF1 terminator. Plasmid KIP3-Tandem-oVP2 T2SThe vector was digested with HpaI and the restricted material was transformed into strain VAK367-D4. The resulting yeast strain, VAK1118, contains the tandem expression cassette integrated into the LAC4 locus. Western blotting revealed higher IBDV-VP2 protein concentrations in this strain compared to a single-copy strain in the same genetic background (Figure 7B). The tandem expression cassette was highly genetically stable; after 78 generations of growth in inducible medium (YNB + lactose), none of 100 colonies tested by PCR showed any genetic alterations of the expression cassette (data not shown).

[0091] Example 6: Tools for generating prototrophy in K. lactis strains for simple fermentation on synthetic and complete media Studies have shown that uracil-auxotrophic yeast strains grow more poorly in complete medium than uracil-prototrophic strains, an effect that is partially neutralized by the addition of uracil. To simplify the fermentation of vaccine strains, facilitate the establishment of production processes, make them more cost-effective, and avoid growth effects due to insufficient incorporation of methionine and / or uracil, a rapid and reproducible method for neutralizing these auxotrophies required for strain construction must be discovered. To reconstruct KlURA3 from Klura3-20, a DNA fragment was generated by PCR using primers VK67 and VK69 and the wild-type KlURA3 gene as a template (Figure 8A). To restore the Klmet5-1 allele, a PCR fragment was similarly generated using primers VK74 and VK75 and the wild-type allele KlMET5 as a template (Figure 8B). Transformation of the PCR fragments (individually or together) into the corresponding mutant strains and selection on methionine- and / or uracil-free media results in highly efficient reconstitution of the wild-type allele. This process was carried out, inter alia, to generate strains VAK1171 and VAK1400 (see above).

[0092] Primer [ka]

[0093] Example 7: Protective immunization with optimized inactivated vaccine yeast The modifications and optimization of the K. lactis vaccine platform carried out in Examples 1-5 were validated in various vaccination studies.

[0094] Example 7.1: Immunogenicity of the optimized K. lactis platform using the example of an IBDV-VP2 yeast strain (VAK1127) The VAK1127 strain contains a tandem IBDV-VP2 expression cassette (SEQ ID NO: 21), two KlGAL4 copies, and an LR2 deletion within the LAC4 promoter. To characterize the immunogenicity of the yeast strain, immunization experiments were performed in the target organism, chickens. In challenge experiments, complete protection of SPF chickens against the highly virulent (vv) IBDV strain 89163 / 7.3 (AFSSA, Ploufragan, France), well characterized by Eterradossi and colleagues (1997), was achieved (Tables 1 and 2). To this end, in two independent experiments, 1 mg of lyophilized, heat-inactivated (2 h, 90°C) yeast (VAK1127) was administered subcutaneously (prime-boost) twice (Figures 9A and B) with incomplete Freund's adjuvant (IFA). Administration was performed 2 and 4 weeks after hatch, and virus challenge was performed 6 weeks after hatch. After 19 days, high titers of anti-IBDV-VP2 antibodies were already measurable in VAK1127-vaccinated animals. In controls, anti-IBDV-VP2 antibody titers developed only after vvIBDV challenge (Figure 9). In both experiments, complete protection (0% morbidity, 0% mortality) was observed in VAK1127-vaccinated animals against challenge with vvIBDV (Tables 1 and 2). In these experiments, protection against vvIBDV could be observed using a subunit vaccine in a classical primer-boost vaccination strategy.

[0095] The immunogenicity of vaccine yeast is not affected by genetic reversion to the antigen-carrying prototrophic yeast strain. This could be demonstrated in mouse vaccination experiments using auxotrophic or prototrophic forms of the IBDV-VP2 yeast strain (Figure 10C). The yeast strain VAK1127 (auxotrophic) was made prototrophic in two steps using a PCR fragment to generate VAK1171, as previously described (Example 6; Figure 8). Both strain types showed no significant differences in the expression level of the recombinant protein (Figures 10A and 10B). Mice were subcutaneously vaccinated three times at 2-week intervals with 0.1 mg of heat-inactivated yeast together with IFA. No differences in the intensity of seroconversion between the auxotrophic IBDV-VP2 strain (VAK1127) and its prototrophic progeny (VAK1171) could be established (Figure 10C).

[0096] Example 7.2: Complete protection by vaccination in a "single dose" scheme "Single-dose" vaccination, i.e., vaccination with a single vaccine administration, is typically ineffective due to the lack of immunogenicity of subunit vaccines. However, antibody titer data obtained using the VAK1127 strain optimized with a prime / boost strategy (Figure 9) suggest that protection may also be achieved with a single-dose approach. This was confirmed by single-dose vaccination with the prototrophic yeast strain VAK1171 (Figure 11; Table 3). To achieve this goal, a single dose of yeast was administered at a targeted high dose (10 mg), followed by challenge at 4-week intervals. With VAK1171, complete protection against vvIBDV (0% morbidity, 0% mortality) was indeed achievable using a "single-dose" approach (Table 3). This result can be attributed to the high protective antibody titers achieved approximately 20 days after vaccination (Figure 11). The fact that a single-dose vaccination scheme protects against vvIBDV with high protection indicates the strong immunogenic potential of the vaccine used and provides excellent validation of the optimized vaccine platform.

[0097] Example 7.3: Improved Protection of Bivalent Yeast Vaccines Compared to Monovalent Yeast Vaccines When Used Against Influenza A Virus Infection Three different vaccine strains were constructed for vaccination against influenza A virus. First, VAK952 (DSM32705) was constructed, which expresses the major antigen, HA (hemagglutinin) gene, of the influenza A strain (Puerto Rico / 8 / 1934; PR8 / 34). In VAK952, the gene is integrated into the LAC4 locus of the genome, as described by Krijger et al. (2012) and Arnold et al. (2012). Second, VAK1283 (DSM32697) was constructed. In this strain, in addition to the HA gene from PR8 / 34 at the LAC4 locus, the M1 gene is also integrated into the URA3 locus. The M1 gene encodes a more important influenza A antigen that is significantly more conserved than the HA. Previously published reports have shown that combining both antigens can enhance the immunogenicity of vaccines against influenza A and achieve cross-protection against different influenza viruses. To verify this aspect with a bivalent yeast vaccine, we further constructed strains (VAK1395; DSM32706) that also contained the M1 gene at the URA3 locus, but in which the HA gene from PR8 / 34 was replaced with the HA gene from influenza virus (California / 4 / 2009). Equivalent expression of HA and additional expression of M1 were confirmed in each strain. Furthermore, these strains showed comparable growth, with VAK1283 showing slight superiority over VAK952 (Figure 12). Vaccination studies in a mouse model, using a prime-boost scheme with different yeast concentrations and a single-dose scheme in each case, showed that VAK952 and VAK1283 elicited comparable virus-neutralizing antibody titers (Figure 13). However, challenge experiments revealed that the bivalent VAK1283 vaccine provided maximal protection in both the prime-boost and single-dose schemes, which differed from the monovalent VAK952 vaccine. Furthermore, in a single-vaccination experiment with half the yeast material used, vaccine VAK1283 achieved a similar protective effect as VAK952 in a prime-boost approach (Figure 14 and Table 3).In experiments using VAK1395 as a vaccine, it was also possible to establish protection against influenza PR8 / 34. Thus, cross-protection against different influenza variants was achieved using a bivalent yeast vaccine.

[0098] [Table 1]

[0099] [Table 2]

[0100] [Table 3]

[0101] (Notes for Table 1) (a) Two-week-old chickens were subcutaneously vaccinated with 1 mg yeast (or PBS) and IFA as an adjuvant. Two weeks after vaccination, they were boosted in the same manner. After another two weeks, a virus challenge test was performed. 4 EID vvIBDV (highly virulent 89163 / 7.3) was used via the oculonasal route. Inactivated whole yeast strain VAK1127 was used as the vaccine yeast, and groups vaccinated with PBS and IFA alone served as infection controls. Groups receiving antigen-free wild-type yeast (VAK367) served as controls for yeast effects alone.

[0102] (b) Histopathological assessment of capsular lesions was performed using a scale of 0 to 4: 0: no lesion; 1: 5-25% of follicles affected; 2: 26-50% of follicles affected; 3: 51-75% of follicles affected; 76-100% capsular damage (loss of structure).

[0103] (c) The mean bursa weight to body mass index (bu / bod) was calculated using the formula: (bursa weight / body mass) x 1000. The unexposed control group consisted of at least 7 chickens, and the exposed group consisted of 10 chickens. The standard deviation is also shown.

[0104] (d) Morbidity is expressed as the number of affected chickens per total number of chickens in the flock, with the percentage of affected chickens shown in brackets.

[0105] (e) Mortality is expressed as the number of dead chickens per total number of chickens in the flock, with the percentage of dead chickens shown in brackets.

[0106] (Notes for Table 2) (a) Two-week-old chickens were subcutaneously vaccinated with 1 mg yeast (or PBS) and IFA as an adjuvant. Two weeks after vaccination, they were boosted in the same manner. After another two weeks, a virus challenge test was performed. 4 Vaccination was performed via the oculonasal route using EID vvIBDV (highly virulent 89163 / 7.3). Inactivated strain VAK1127 whole yeast was used as vaccine yeast, and groups vaccinated with PBS and IFA only served as infected controls.

[0107] (b) Histopathological assessment of capsular lesions was performed using a scale of 0 to 4: 0: no lesion; 1: 5-25% of follicles affected; 2: 26-50% of follicles affected; 3: 51-75% of follicles affected; 76-100% capsular damage (loss of structure).

[0108] (c) The mean bursa weight to body mass index (bu / bod) was calculated using the formula: (bursa weight / body mass) x 1000. The unexposed control group consisted of at least 5 chickens, and the exposed group consisted of 9 chickens. The standard deviation is also shown.

[0109] (d) Morbidity is expressed as the number of affected chickens per total number of chickens in the flock, with the percentage of affected chickens shown in brackets.

[0110] (e) Mortality is expressed as the number of dead chickens per total number of chickens in the flock, with the percentage of dead chickens shown in brackets.

[0111] (Notes for Table 3) (a) Two-week-old chickens were subcutaneously vaccinated with 10 mg yeast (or PBS) and IFA as an adjuvant. After 4 weeks, a virus challenge test was performed. 4 EID vvIBDV (highly virulent 89163 / 7.3) was used via the oculonasal route. Inactivated whole yeast strain VAK1171 was used as a single yeast vaccine. The infected controls used were a group vaccinated with PBS and MF59 alone, and a group vaccinated with wild-type yeast and MF59 two weeks after the first vaccination, both of which received a boost containing the same amount of yeast or PBS.

[0112] (b) Histopathological assessment of capsular lesions was performed using a scale of 0 to 4: 0: no lesion; 1: 5-25% of follicles affected; 2: 26-50% of follicles affected; 3: 51-75% of follicles affected; 76-100% capsular damage (loss of structure).

[0113] (c) The mean bursa weight to body mass index (bu / bod) was calculated using the formula: (bursa weight / body mass) x 1000. Each group consisted of at least 9 chickens. The standard deviation is also shown.

[0114] (d) Morbidity is expressed as the number of affected chickens per total number of chickens in the flock, with the percentage of affected chickens shown in brackets.

[0115] (e) Mortality is expressed as the number of dead chickens per total number of chickens in the flock, with the percentage of dead chickens shown in brackets.

[0116] (array) This patent application includes the following sequences as part of its description:

[0117] [Table 4] The present application provides the following aspects of the invention. (Aspect 1) A Kluyveromyces lactis (K. lactis) strain for targeted cloning of a nucleic acid encoding a heterologous antigen into the yeast genome of said K. lactis strain, characterized in that said K. lactis strain has an expression cassette for said heterologous antigen integrated into the KlURA3-20 locus (KLLA0E22771g) and / or the KlMET5-1 locus (KLLA0B03938g) instead of or in addition to the KlLAC4 locus. (Aspect 2) The expression cassette contains the K. lactis LAC4-12 promoter (P LAC4-12 2. The K. lactis strain according to claim 1, characterized in that it comprises the promoter LAC12 or a variant thereof which also comprises the intergenic region between LAC12 and LAC4, the region encoding the antigen, and the AgTEF1 terminator. (Aspect 3) 3. A K. lactis strain according to aspect 1 or 2, characterized in that multiple copies of a nucleic acid encoding a heterologous antigen are inserted into the KlLAC4 locus, or the KlURA3-20 locus, or the KlMET5-1 locus of the resulting K. lactis strain via a tandem or multi-expression cassette. (Aspect 4) 4. The K. lactis strain according to any one of aspects 1 to 3, characterized in that the gene for the heterologous antigen IBDV VP2 is present in the form of a tandem expression cassette at the KlLAC4 locus of said K. lactis strain. (Aspect 5) 4. The K. lactis strain according to any one of aspects 1 to 3, characterized in that one or more copies of nucleic acids encoding different heterologous antigens are inserted into the KlLAC4 locus, and / or the KlURA3-20 locus, and / or the KlMET5-1 locus via a single expression cassette, a tandem expression cassette or multiple expression cassettes. (Aspect 6) 6. The K. lactis strain according to any one of aspects 1 to 3 and 5, wherein genes encoding the heterologous antigens influenza A HA (A / Puerto Rico / 8 / 1934(H1N1)) and influenza A M1 (A / Puerto Rico / 8 / 1934(H1N1)) are inserted into and expressed at the KlLAC4 and KlURA3-20 loci of said K. lactis strain. (Aspect 7) 7. The K. lactis strain according to any one of aspects 1 to 6, wherein the K. lactis strain additionally comprises, in addition to the KIGAL4 gene native to the genome, a second ectopic copy of the KIGAL4 gene. (Aspect 8) 8. The K. lactis strain of embodiment 7, wherein the ectopic copy of the KIGAL4 gene is flanked by a KIGAL4 promoter and a KIGAL4 terminator and is integrated at the locus KLLA0E13795g (Klavt3::KlGAL4-1, SEQ ID NO: 1) in said K. lactis strain. (Aspect 9) 9. The K. lactis strain according to any one of aspects 1 to 8, wherein the gene for the heterologous antigen IBDV VP2 is present at the K1LAC4 locus of said K. lactis strain. (Aspect 10) 10. The K. lactis strain according to any one of aspects 1 to 9, wherein the K. lactis strain has a promoter structure that is a modified LAC4-12 promoter, which expresses little or no heterologous proteins under non-inducing conditions, and wherein the promoter P LAC4-12 The basic control region (BCR) of the LAC4-12-LR2’ ;SEQ ID NO: 2) has been deleted. (Aspect 11) 11. The K. lactis strain according to aspect 10, characterized in that the gene for the heterologous antigen influenza A HA (A / Puerto Rico / 8 / 1934 (H1N1)) is present in the K1LAC4 locus of said K. lactis strain. (Aspect 12) 12. The K. lactis strain according to any one of aspects 1 to 11, having a promoter structure which is a modified version of the LAC4-12 promoter, allowing for regulation of heterologous protein expression, characterized in that the promoter has a variable number of binding sites for the activator KlGal4 ("upstream activation sequences" 1, 2 and 4, 5), with 1, 2, 3 or 4 KlGal4 binding sites being present. (Aspect 13) 13. The K. lactis strain according to any one of aspects 1 to 12, characterized in that the gene for the heterologous antigen IBDV VP2 is inserted at the KlLAC4 locus of said K. lactis strain. (Aspect 14) 14. The K. lactis strain according to any one of aspects 1 to 13, wherein the gene function of the alleles Kllac4, Klura3-20 and Klmet5-1 is restored, and wherein the K. lactis strain is prototrophic. (Aspect 15) 15. The K. lactis strain according to any one of aspects 1 to 14, characterized in that the genes for the heterologous antigens BVDV E2 ectodomain (type 1, CP7), BVDV E2 ectodomain (type 2, New York 93) and BVDV Npro-NS3 (type 1, CP7) are inserted into the loci KlLAC4, KlURA3-20, and KlMet5-1 of said K. lactis strain. (Aspect 16) A K. lactis strain selected from the following strains: VAK952 (accession number: DSM32705), VAK1111 (accession number: DSM32696), VAK1118 (accession number: DSM32701), VAK1131 (accession number: DSM32700), VAK1171 (accession number: DSM32699), VAK1243 (accession number: DSM32702), VAK1283 (accession number: DSM32697), VAK1395 (accession number: DSM32706), and VAK1400 (accession number: DSM32698). (Aspect 17) An integrative expression vector selected from KIpURA3 (SEQ ID NO: 3) or KIpMET5 (SEQ ID NO: 4). (Aspect 18) KIpMET5-P L4-12 -Et, KIpMET5-P L4-12-LR2 -Et, KIpMET5-P L4 -Et, KIpMET5-P L4-LR2 -Et and KIpURA3-P L4-12 -Et, KIpURA3-P L4-12-LR2 -Et, KIpURA3-P L4 -Et and KIpURA3-P L4-LR2 -Et (SEQ ID NO: 3, 4 in combination with SEQ ID NO: 5, 6, 7 or 8). (Aspect 19) A method for producing the K. lactis strain according to any one of aspects 1 to 16, comprising the steps of: (i) inserting the gene sequence of the antigen of interest into the KIpURA3 vector and / or the KIpMET5 vector; (ii) transforming a K. lactis culture with the modified and pre-enzymatically digested vector construct; (iii) selecting transformed K. lactis cells using solid medium lacking uracil and / or methionine; and (iv) optionally restoring prototrophy; The method comprising: (Aspect 20) 20. The method of embodiment 19, wherein the gene sequences of the multiple antigens are ectopically inserted simultaneously and expressed in a regulated manner. (Aspect 21) 21. The method of embodiment 20, wherein different gene sequences encoding antigens of different variants of a single pathogen are ectopically inserted and expressed in a controlled manner. (Aspect 22) 21. The method of embodiment 20, wherein different gene sequences encoding antigens of different pathogens are ectopically inserted and expressed in a regulated manner. (Aspect 23) A pharmaceutical composition comprising the K. lactis strain according to any one of aspects 1 to 16. (Aspect 24) 17. The K. lactis strain according to any one of embodiments 1 to 16 for use in vaccination. (Aspect 25) 17. The K. lactis strain according to any one of embodiments 1 to 16 for use in prophylactic vaccination. (Aspect 26) A method of vaccination comprising administering to a subject the K. lactis strain according to any one of aspects 1 to 16, in an amount sufficient to raise in the subject a protective immune response against one or more heterologous antigens. (Aspect 27) A K. lactis strain according to embodiment 24 or 25, or a method according to embodiment 26, wherein the K. lactis strain is administered subcutaneously, intramuscularly or orally / mucosally. (Aspect 28) A K. lactis strain according to aspect 24 or 25, or a method according to aspect 26 or 27, characterized in that the K. lactis strain elicits a protective immune response against a pathogen with one application / immunization ("single inoculation") or two applications / immunizations ("prime-boost"). (Aspect 29) A K. lactis strain according to aspect 24 or 25, or a method according to aspect 26 or 27, characterized in that the K. lactis strain induces a cross-protective immune response against different variants of a single pathogen with a single use / immunization ("single inoculation") or with two applications / immunizations ("prime-boost"). (Aspect 30) A K. lactis strain according to aspect 24 or 25, or a method according to aspect 26 or 27, characterized in that the K. lactis strain elicits a protective immune response against multiple different pathogens with a single use / immunization ("single inoculation") or with two applications / immunizations ("prime-boost"). [Advanced Technology Documents]

Non-licensed literature

[0118] (References)

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Claims

1. A Kluyveromyces lactis (K. lactis) strain for targeted cloning of a nucleic acid encoding a heterologous antigen into the yeast genome of the K. lactis strain, characterized in that the K. lactis strain has an expression cassette for the heterologous antigen integrated into the KlURA3-20 locus (KLLA0E22771g) and / or the KlMET5-1 locus (KLLA0B03938g) in addition to the KlLAC4 locus, wherein the K. lactis expression cassette has a variant of the LAC4-12 promoter (P LAC4-12 ; SEQ ID NO: 5) comprising the intergenic region between LAC12 and LAC4, the antigen-encoding region, and the AgTEF1 terminator, and wherein the LAC4-12 promoter (P LAC4-12 ; SEQ ID NO: 5) variants are The LAC4-12 promoter (P LAC4-12 ; SEQ ID NO: 5) has a modified promoter structure that allows little or no expression of heterologous proteins under non-inducing conditions, and the basic control region (BCR) (LR2 deletion; P LAC4-12-LR2’ the LAC4-12 promoter (P LAC4-12 ; SEQ ID NO: 5), wherein the LAC4-12 promoter (P LAC4-12 ; SEQ ID NO: 2) has been deleted; and 1. The LAC4-12 promoter (P LAC4-12 ; SEQ ID NO: 5) has a modified promoter structure that allows for the regulation of heterologous protein expression, characterized in that the number of binding sites for the promoter activator KlGal4 ("upstream activation sequences" 1, 2 and 4, 5) is variable, with 1, 2, 3 or 4 KlGal4 binding sites present. is selected from and multiple copies of nucleic acids encoding heterologous antigens are inserted into the KlLAC4 locus, or the KlURA3-20 locus, or the KlMET5-1 locus of the resulting K. lactis strain via tandem or multiple expression cassettes, or one or more copies of nucleic acids encoding different heterologous antigens are inserted into the KlLAC4 locus, and / or the KlURA3-20 locus, and / or the KlMET5-1 locus via single, tandem or multiple expression cassettes, and wherein the gene functions of the Kllac4, Klura3-20 and Klmet5-1 alleles are restored, and the K. lactis strain is prototrophic.

2. 2. The K. lactis strain according to claim 1, characterized in that the gene for the heterologous antigen IBDV VP2 is present in the form of a tandem expression cassette at the KlLAC4 locus of said K. lactis strain.

3. A K. lactis strain according to any one of claims 1 and 2, characterized in that genes encoding the heterologous antigens influenza A HA (A / Puerto Rico / 8 / 1934 (H1N1)) and influenza A M1 (A / Puerto Rico / 8 / 1934 (H1N1)) are inserted into and expressed at the KlLAC4 and KlURA3-20 loci of the K. lactis strain.

4. A K. lactis strain according to any one of claims 1 to 3, characterized in that the K. lactis strain contains, in addition to the genomic KlGAL4 gene, a second ectopic copy of the KlGAL4 gene.

5. The K. lactis strain according to claim 4, characterized in that the ectopic copy of the KlGAL4 gene is flanked by the KlGAL4 promoter and KlGAL4 terminator and is integrated into the locus KLLA0E13795g (Klavt3::KlGAL4-1, SEQ ID NO: 1) in the K. lactis strain.

6. A K. lactis strain according to any one of claims 1 to 5, characterized in that the gene for the heterologous antigen IBDV VP2 is located at the locus KlLAC4 of said K. lactis strain.

7. 2. The K. lactis strain according to claim 1, characterized in that the gene for the heterologous antigen influenza A HA (A / Puerto Rico / 8 / 1934 (H1N1)) is present in the KlLAC4 locus of said K. lactis strain.

8. A K. lactis strain according to any one of claims 1 to 7, characterized in that the gene for the heterologous antigen IBDV VP2 is inserted at the KlLAC4 locus of said K. lactis strain.

9. A K. lactis strain according to any one of claims 1 to 8, characterized in that the gene function of the alleles Kllac4, Klura3-20 and Klmet5-1 is restored and said K. lactis strain is prototrophic.

10. A K. lactis strain according to any one of claims 1 to 9, characterized in that the genes for the heterologous antigens BVDV E2 ectodomain (type 1, CP7), BVDV E2 ectodomain (type 2, New York 93) and BVDV Npro-NS3 (type 1, CP7) are inserted into the loci KlLAC4, KlURA3-20 and KlMet5-1 of said K. lactis strain.

11. A K. lactis strain selected from the group consisting of: VAK1283 (accession number: DSM32697), VAK1395 (accession number: DSM32706), and VAK1400 (accession number: DSM32698).

12. A method for producing a K. lactis strain according to any one of claims 1 to 10, comprising the steps of: (i) inserting the gene sequence of the antigen of interest into the KlpURA3 vector and / or the KlpMET5 vector in addition to the Klp3-MCS vector; (ii) transforming a K. lactis culture with the modified and pre-enzymatically digested vector construct; (iii) selecting the transformed K. lactis cells using solid medium lacking uracil and / or methionine; The method comprising:

13. 13. The method of claim 12, further comprising the step of (iv) restoring prototrophy.

14. 14. The method according to claim 12 or 13, characterized in that the gene sequences of multiple antigens are ectopically inserted simultaneously and expressed in a controlled manner.

15. 14. The method of claim 13, wherein different gene sequences encoding antigens of different variants of one pathogen are ectopically inserted and expressed in a controlled manner.

16. 14. The method of claim 13, wherein different gene sequences encoding antigens of different pathogens are ectopically inserted and expressed in a controlled manner.

17. A pharmaceutical composition comprising a K. lactis strain according to any one of claims 1 to 11.

18. 18. The pharmaceutical composition according to claim 17 for use in vaccination.

19. 19. The pharmaceutical composition according to claim 18, wherein the K. lactis strain is administered subcutaneously, intramuscularly, or orally / mucosally.

20. 19. The pharmaceutical composition of claim 18, wherein the K. lactis strain induces a protective immune response against a pathogen with one application / immunization ("single inoculation") or two applications / immunizations ("prime-boost").

21. 19. The pharmaceutical composition of claim 18, wherein the K. lactis strain induces a cross-protective immune response against different variants of a single pathogen with a single use / immunization ("single inoculation") or with two applications / immunizations ("prime-boost").

22. 19. The pharmaceutical composition of claim 18, wherein the K. lactis strain induces a protective immune response against different pathogens with a single use / immunization ("single inoculation") or with two applications / immunizations ("prime-boost").

23. 18. The pharmaceutical composition according to claim 17, for use in prophylactic humoral vaccination.

24. 24. The pharmaceutical composition according to claim 23, wherein the K. lactis strain is administered subcutaneously, intramuscularly, or orally / mucosally.

25. 24. The pharmaceutical composition of claim 23, wherein the K. lactis strain induces a protective immune response against the pathogen with one application / immunization ("single inoculation") or two applications / immunizations ("prime-boost").

26. 24. The pharmaceutical composition of claim 23, wherein the K. lactis strain induces a cross-protective immune response against different variants of a pathogen with a single use / immunization ("single inoculation") or with two applications / immunizations ("prime-boost").

27. 24. The pharmaceutical composition of claim 23, wherein the K. lactis strain induces a protective immune response against different pathogens with a single use / immunization ("single inoculation") or with two applications / immunizations ("prime-boost").

28. A pharmaceutical composition for vaccination comprising a K. lactis strain described in any one of claims 1 to 11, said pharmaceutical composition being used to administer the K. lactis strain to a subject in an amount sufficient to induce a protective immune response in the subject against one or more heterologous antigens.

29. 29. The pharmaceutical composition according to claim 28, wherein the K. lactis strain is administered subcutaneously, intramuscularly, or orally / mucosally.

30. 30. The pharmaceutical composition of claim 28 or 29, wherein the K. lactis strain induces a protective immune response against a pathogen with one application / immunization ("single inoculation") or two applications / immunizations ("prime-boost").

31. 30. A pharmaceutical composition according to claim 28 or 29, characterized in that the K. lactis strain induces a cross-protective immune response against different variants of one pathogen with a single use / immunization ("single inoculation") or with two applications / immunizations ("prime-boost").

32. 30. The pharmaceutical composition of claim 28 or 29, wherein the K. lactis strain induces a protective immune response against different pathogens with a single use / immunization ("single inoculation") or with two applications / immunizations ("prime-boost").

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