Yeast kluyveromyces lactis based host / vector system optimized in order to produce monovalent and multivalent subunit prophylactic vaccine

Abstract

To provide a recombinant Kluyveromyces lactis (K.lactis) yeast that is capable of highly efficiently expressing one or more heterogenous protein, and is appropriate to be used as a vaccine which causes defensive immune response to a pathogen.SOLUTION: There is provided a K. lactis strain for performing target cloning of nucleic acid that encodes heterologous antigen to a yeast genome of a K. lactis strain, and has an expression cassette integrated for the heterologous antigen in a KlURA3-20 gene locus (KLLA0E22771g) and / or a KlMET5-1 gene locus (KLLA0B03938g) instead of or additionally to a KlLAC4 gene locus. Further, an integrated expression vector, and a method for producing K. lactis strain of the present invention, and use thereof as a vaccine are provided.SELECTED DRAWING: None

Description

Technical Field

[0001] (Field of the Invention) The present invention is suitable for highly efficiently expressing one or more heterologous proteins and is suitable for use as a vaccine for generating a protective immune response against pathogens. It relates to recombinant Kluyveromyces lactis (K. lactis) yeast. In particular, the present invention relates to a Kluyveromyces strain for targeted cloning of nucleic acids encoding heterologous antigens into the yeast genome of a K. lactis strain, which has an expression cassette incorporated for the heterologous antigen instead of or in addition to the KlLAC4 locus, and / or at the KlURA3-20 locus (KLLA0E22771g), and / or at the KlMET5-1 locus (KLLA0B03938g). The present invention further relates to an integrative expression vector, and a method for generating the K. lactis strain of the present invention, and its use as a vaccine.

Background Art

[0002] (Background of the Invention)[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0003] Classical vaccines contain attenuated inactivated or killed whole pathogens, as well as their genetic material, i.e., nucleic acids in the form of DNA or RNA. In order to produce such classical vaccines usually requires special safety measures and / or the use of infectious organisms and / or cell cultures. Furthermore, such vaccines often require storage and transportation involving complex cold chains. Moreover, the use of classical vaccines is associated with the risk that substances from the production process (e.g., from test animals or cell cultures) may cause adverse effects in vaccinated individuals or the risk of unwanted reactivation of pathogens. Similar problems also exist in the diagnostic aspect. For example, when vaccinating useful animals with whole pathogens, vaccinated animals cannot be distinguished from naturally infected animals, which means that early warning systems based on the detection of new infections are not useful. Therefore, so-called "subunit vaccines" that vaccinate only specific components of pathogens have been developed. An essential condition for their use is that the "major antigen" of the pathogen in question is known. The major antigen is usually a surface component of the pathogen that can be recognized by the immune system, such as the capsid protein of a virus or the viral coat protein. The major antigen can also induce humoral and / or cellular immune responses and immunological memory against the virus in the host even when complete virus particles are absent. In "subunit vaccination", further components of the pathogen are lost, so that vaccinated individuals can be identified by differential diagnosis (DIVA), i.e., distinguish infections from vaccinated animals from natural infections For example, when vaccinating useful animals with whole pathogens, vaccinated animals cannot be distinguished from naturally infected animals, which means that early warning systems based on the detection of new infections are not useful. Therefore, so-called "subunit vaccines" that vaccinate only specific components of pathogens have been developed. An essential condition for their use is that the "major antigen" of the pathogen in question is known. The major antigen is usually a surface component of the pathogen that can be recognized by the immune system, such as the capsid protein of a virus or the viral coat protein. The major antigen can also induce humoral and / or cellular immune responses and immunological memory against the virus in the host even when complete virus particles are absent. In "subunit vaccination", further components of the pathogen are lost, so that vaccinated individuals can be identified by differential diagnosis (DIVA), i.e., distinguish infections from vaccinated animals from natural infections For example, when vaccinating useful animals with whole pathogens, vaccinated animals cannot be distinguished from naturally infected animals, which means that early warning systems based on the detection of new infections are not useful. Therefore, so-called "subunit vaccines" that vaccinate only specific components of pathogens have been developed. An essential condition for their use is that the "major antigen" of the pathogen in question is known. The major antigen is usually a surface component of the pathogen that can be recognized by the immune system, such as the capsid protein of a virus or the viral coat protein. The major antigen can also induce humoral and / or cellular immune responses and immunological memory against the virus in the host even when complete virus particles are absent. In "subunit vaccination", further components of the pathogen are lost, so that vaccinated individuals can be identified by differential diagnosis (DIVA), i.e., distinguish infections from vaccinated animals from natural infections It can be distinguished from other individuals, and is therefore also called a "subunit marker vaccine." The drawbacks of subunit vaccines are that the manufacturing process is often complex and often... The immunogenicity is insufficient, and the pathogen itself can be cultured efficiently (with the limitations described above). It is possible, but the main antigens are obtained through genetic engineering using costly and usually inefficient methods. It needs to be prepared and purified by complex means. Therefore, the sub-yeast obtained in this way Nit vaccines have a short shelf life and often need to be stored and transported under refrigeration. It is not a biological material. For these reasons, most mass-produced vaccines for useful animals are This is still based on the classical principle of using complete pathogens.

[0004] For example, infectious bursal disease (IBD), a widespread poultry disease, is a contagious disease. It is caused by the bursal disease virus (IBDV), which belongs to the family Birnaviridae. It is a non-enveloped virus with a segmented RNA genome. Most vaccines are based on weakened or inactivated viruses. However, the problem that arises here is the highly weakened, inactivated "live virus" and Inactivated viruses also provide protection against IBD viruses with average pathogenicity, This is not true for highly virulent IBD virus strains (vvIBDV). Therefore, several highly virulent, attenuated viruses (intermediate hot strains) are being protected against vvIBDV. Although it was a target, these vaccine strains temporarily reach the bursa of Fabricius and B cells of lymphoid organs. This can lead to side effects in the form of immunosuppression due to physical damage (Rautenschlein et al. (2 005)). However, even the aforementioned intermediate hot strain vaccine is not effective against the recently discovered vvIBDV strain. Complete defense is not possible (Negash et al. (2012), Kasanga et al. (2007)). Furthermore, it is highly attenuated. The problem with vaccination using live viruses is that maternal antibodies can help the virus The goal is to inhibit replication and thus prevent the induction of an immune response. Therefore, these vaccines Effective vaccination is only possible within 3 weeks after hatching (Kumar et al. (2000), Rautenschle). in et al. (2005).

[0005] For example, influenza A virus is one of the most important viral pathogens in the world. (Short et al. (2015), Silva et al. (2012)). Influenza viruses belong to the Orthomyxoviridae family. They belong to a group that possesses envelope-type RNA, which has single-stranded segmented RNA as its genome. It is a virus. Like most RNA viruses, the influenza virus is also highly It is affected by the mutation rate. In particular, the reassembly of viral RNA segments is related to new genetic and It produces viral offspring with biological characteristics (Short et al. (2015)). For rapid evolution, The problem that arises in the case of vaccination against the influenza virus is that existing vaccines However, this cannot "keep up" with newly emerging virus variants. Therefore, cross-protection Develop a vaccine that demonstrates efficacy and, consequently, long-term protection against different influenza strains. Attempts have already been made for a long time (Steel et al. (2010), Krammer and Palese (2013), Kirch Enbaum and Ross (2014), Berthoud et al. (2011).

[0006] Bovine viral diarrhea virus (BVDV) is a pathogen that is widespread in even-toed ungulates. DV is a species of pestivirus belonging to the Flaviviridae family. This virus has a single-stranded RNA gene. Nomu is similarly affected by a high mutation rate. Furthermore, in pregnant animals, the fetus becomes infected and immune... In some cases, animals with persistent infection (PI) may be born due to disease tolerance. These PI animals may then be further infected with the virus. It spreads, and in the case of 100% viral mutation, it can lead to death from so-called mucosal diseases. Here too, we have developed a vaccine that shows cross-protection and long-term protection against different BVD virus variants. Attempts to do so have already been made for a long time (Ridpath (2015)).

[0007] Effective subunit vaccines can address or solve these problems in most cases. In combination, subunits are protein components of pathogens, and through genetic engineering, they can be modified in various ways. It can be produced within host cells. In addition to the intestinal bacterium Escherichia coli, mammalian cells that can proliferate in cell culture media can also produce it. Alternatively, insect cells, plant cells, and various fungi have been established as host systems for heterologous protein expression. Microbial systems such as bacteria and fungi can be cultured on a large scale, and in particular, with good cost-effectiveness. can.

[0008] Yeast cells of the yeast genus Saccharomyces, Pichia, and Cluyveromyces contain heterologous proteins. It has been routinely used for decades to express quality. In contrast to bacteria, yeast cells It has the advantage of being a eukaryote, that is, the advantage of being similar to animal cells in many ways, and eukaryotes Proteins, that is, proteins that are formed and / or must be functional within animal cells. The quality is that it can be produced in a natural or substantially natural form within yeast at good cost-effectiveness (Bathurst( 1994), Gellissen and Hollenberg (1997). Yeast was initially used only for the production of heterologous proteins. After use and expression, the protein is purified from yeast cells and used as a subunit vaccine. Attempts to administer yeast itself or yeast cell fractions as a vaccine have only recently begun. This is being done. Such "yeast-based vaccines" are immunologically effective against pathogens (antigens). It contains effective ingredients, and after administration (e.g., subcutaneously, intramuscularly, or orally / through mucous membranes), This elicits a specific immune response to the antigen, and also to the pathogen from which the antigen originates. These are yeast particles that can induce [a reaction]. What is needed is [a reaction] within a vaccinated organism. This is the induction of immunological "memory," which allows the body to respond to the next infection ("challenge"). To prevent the increase and / or spread of pathogens and / or reduce the pathological effects of infection. And so, as already mentioned above, antigens are usually structural proteins of pathogens, and The nucleic acid sequence that codes for the antigen (the gene that codes for the antigen) is synthesized in yeast cells using genetic technology. It is introduced into cells, enabling the expression of one or more of these structural proteins. It is thus produced. This refers to the living form (yeast cells), the dead and dried form (yeast particles), or the powder after cell destruction. Recombinant yeast in its final form and post-homogenized form (yeast lysate) is yeast-based It is a vaccine. After administration of this vaccine, the antigen is recognized by the immune system, and in humors and / Alternatively, it triggers cellular immune defense.

[0009] Yeast-based vaccines are known to those skilled in the art through prior art. License applications and patents, such as U.S. Patent Application Publication No. 20090304741A1 and U.S. Patent No. 5830463A. U.S. Patent No. 7,465,454,522 and U.S. Patent Application Publication No. 2007,0166,323,A1 describe immunotherapy. and a strain of Saccharomyces cerevisiae containing at least one recombinant antigen Uses are described. These yeasts stimulate immune responses, particularly cell-mediated immune responses. It has been shown to be effective for that purpose.

[0010] International Publication Number 2006044923 contains genes for various proteins of the hepatitis C virus (HCV). It is recombinantly expressed and triggers an immune response against the HCV protein, particularly a T cell response. A yeast (S. cerevisiae) capable of producing [something] has been disclosed, and it is used in vaccines for chronic hepatitis C. It is intended to be used as a tool.

[0011] International Publication Number 2007092792 describes recombinant S.ce for influenza virus infection. The possible uses of the revisiae yeast are described, and its use involves various combinations of yeast strains. Its use includes the induction of T cells, i.e., a cellular immune response, upon administration.

[0012] International Publication Numbers 20101054649 and 2013107436 contain information on all dead yeast cells. To induce a protective humoral immune response after oral / mucosal or subcutaneous administration of the body The use of the Kluiveromyces lactis strain containing the antigen is described. The listed patents include a recombinant K. lactis strain derived from the starting strain VAK367-D4, which is used as a vaccine. This includes examples of applications where vaccination was effectively used.

[0013] The possibility of using recombinant Kluiveromyces lactis yeast for vaccination is , and are known to those skilled in the art through prior art (Arnold et al. (2012), International Publication No. 20101054649 and (and International Publication No. 2013107436). In the application example, the development controlled by the LAC4 promoter The VP2 capsule of infectious bursal disease virus (IBDV) is transmitted within the cell via the current cassette. Subcutaneous administration of the yeast K. lactis, which expresses the sidoprotein, triggers a humoral immune response. And we were able to demonstrate that it provides effective protection against viral infection. While it was possible to demonstrate this for sexually transmitted IBD viruses, it was not possible for highly virulent IBDV (vvIBDV) It had been impossible to demonstrate this against ) until now. Previous data showed that the viral antigen It has been shown that increasing intracellular concentrations enhances the effectiveness of yeast vaccines (Arn Old et al. (2012). Technical modifications to achieve increased antigen concentration involve transcriptional activator genes. Additional copies of the child KlGAL4-1 (also known as LAC9-1) were added to IBDV-VP2 expressing strains (deposited strains DSM25406 and DSM25407). The method involves introducing it by incorporating the pLI-1 plasmid (Krijger et al. (2012) and international (Publication number 2013107436). Therefore, the generation of such K. lactis vaccine strains until now has been It was based on two gene interventions. First, the integration of a heterologous gene encoding an antigen. Secondly, it is based on the integration of the KlGAL4-1 gene. However, the implementation to date In this form, the latter always results in the incorporation of tandem repeats of plasmids, and as a result, In addition to the cytotoxic effects of strong overexpression of tivators (Breunig 1989), this method can also be used to produce This also causes variations in the copy number of the KlGAL4-1 and ScURA3 genes in the resulting vaccine strain.

[0014] Strategies for achieving heterologous gene expression via an unmodified LAC4 promoter are described in the application examples above. As stated (Arnold et al. (2012), International Publication No. 20101054649 and International Publication No. 2013107436) (Note), the minimum expression of heterologous genes is achieved under non-inducible conditions, i.e., the promoter is somewhat open. It has a secondary effect that occurs even under certain conditions. Increased KlGAL4-1 gene dosage. Then, this effect becomes even more pronounced. Therefore, in heterologous expression, against yeast cells In the case of proteins that have cytopathic effects (CPE), during culture, for example, in a fed-batch fermentation process... Biomass formation in these areas may be severely restricted, especially in uninducible cases. We need to find another way to minimize gene expression under these conditions.

[0015] Various subunit vaccines contain multiple subunits of the pathogen, rather than just one. It is effective and beneficial only when used for vaccination. Furthermore, multiple antigens can be detected through vaccination. Using subunits significantly increases cross-protection against different variants of pathogens. Co-expression of the same or different antigens also increases the antigen concentration in yeast cells, or differs from that expression. It can be used to produce vaccines that provide protection against pathogens.

[0016] The aforementioned strains are generally trophic strains and often grow more than prototrophic strains in complete media. It is inferior in terms of performance. Therefore, if a nutrient-requiring yeast strain is converted to a prototrophic form quickly and efficiently, Reproductive characteristics can be improved. [Overview of the project] [Problems that the invention aims to solve]

[0017] (Description of the present invention) Therefore, the objective of the present invention is to provide a novel K. lactiform that can overcome the shortcomings of the prior art. The objective is to provide a vaccine strain. In particular, the strain provided is one that is incorporated into a specific region within the genome. This is a recombinant K. lactis strain containing a limited number of copies of the KIGAL4-1 gene. Furthermore, what is provided is that, under non-inducible conditions, it expresses little or no heterologous protein. This makes it possible to express multiple copies of an antigen or multiple antigens within yeast. Furthermore, it is more suitable for cultivation and can be used more effectively for preventive vaccination against pathogens. It is a viable strain. At the same time, it has heterologous genes that encode immunomodulatoryly active proteins (antigens). This needs to be integrated into a specific site in the K. lactis genome. (Incorporation of heterologous genes) When selecting a target clone that possesses resistance genes, use resistance genes as selection markers. It should not be done. Furthermore, the prototrophic strain is created from the trophication-requiring strain in the simplest and most feasible way. It should be released. This is also yeast produced in a synthetic medium without (nutrient) supplementation. This will allow for simplified fermentation of vaccine strains. [Means for solving the problem]

[0018] The aforementioned objective is to develop a novel vector and a novel genetically modified yeast K. lactis. This enables the creation of vaccine strains that include variants and are optimized for the specificity of protein antigens. This was achieved by providing a regular system. The structure of DNA elements between vectors is block-type. Through exchange, the yeast genome can efficiently clone the normal, heterologous antigen-coding regions. The expression was achieved separately from the heterologous gene that was to be expressed. As a result of gene genome integration, yeast strains are stable across a very large number of generations, and genes It was clearly visible in its original state. Thanks to these characteristics, the fermentation process is non-selective. The process proceeds reproducibly under the specified conditions and is standardizable. Optimization of K. lactis yeast according to the present invention. The goal is to maximize the protein production rate and efficiently utilize the cytopathic effect of the antigen through fermentation. The goal is to control it so that it remains below a threshold that severely disrupts the process. Genetic manipulation of a single gene, or a combination of multiple gene manipulations, i.e., (i) Increase the concentration of lactose-inducible transcription activators, (ii) Modifications targeted by the LAC4 promoter, and / or (iii) Gradual increase in the gene dose of a heterologous gene encoding an antigen, This was achieved by [the method / method].

[0019] Furthermore, the optimization of K. lactis yeast according to the present invention is (iv) In order to express multiple antigens simultaneously, heterogeneous gene encodings in the yeast genome Establishing multiple novel integration sites for the set, It consists of.

[0020] In a preferred embodiment, the objective of the present invention is to introduce heterologous genes into the yeast genome of the K. lactis strain. The lactis strain for targeted cloning of child encoding nucleic acids, and KlLA Instead of or in addition to the C4 locus, the KlURA3-20 locus (KLLA0E22771g) and / or if The KlMET5-1 gene locus (KLLA0B03938g) contains an expression cassette incorporated for the heterologous antigen. This was achieved by providing the K. lactis strain characterized by having [specific feature]. If preferred, the K. lactis strain has the KlURA3-20 locus in addition to the KlLAC4 locus. The heterogeneous gene incorporated into KLLA0E22771g) and / or KlMET5-1 locus (KLLA0B03938g) This is when it has an expression cassette for the antigen. Particularly very preferred is the K. lactate In addition to the KlLAC4 locus, the strain also possesses the KlURA3-20 locus (KLLA0E22771g) and the KlMET5-1 locus. In the case where an expression cassette for the heterologous antigen is incorporated into the gene locus (KLLA0B03938g) Yes. In this modified K. lactis strain, the genes for expressing heterologous genes are It is integrated into a specific, identified locus within the K. lactis genome, and is heterogeneous. The copy number has the advantage of being controllable. Furthermore, the K. lactis strain is also K. lactis geno A set of different genes for the expression of multiple different heterologous antigens at a specific locus within the molecule. Allows for insertion.

[0021] In the context of this invention, "heterogeneous antigen" or "heterogeneous protein" refers to a disease in humans or animals. To induce an immune response against the pathogen or cancerous degenerated cells, preferably a protective immune response. This refers to all peptides, polypeptides, and proteins suitable for [the purpose]. Heterogeneous proteins are, They may originate from any kind of pathogen or tumor, and for them alone, provide defensive immunity. Antigens capable of inducing an epidemic response, preferably a protective immune response, were characterized.

[0022] In a preferred embodiment, the heterologous protein is derived from a pathogen (virus, bacteria, parasite). Therefore, for these reasons, a protective immune response, preferably a protective humoral immune response, occurs on its own. Antigens capable of inducing this reaction were characterized.

[0023] For example, these are illustrated below. (Parasite-derived heterologous proteins) American hookworm; duodenal hookworm (Anthococcus fuscoguttatus): ASP protein, hemoglobin-degrading protein Arze, Leishmania genus: gp63, 46kD preflagellar antigen, LACK, Plasmodium genus (malaria parasites): CSP protein, CSA-1, CSA-3, EXP1, SSP2, STARP , SALSA, MSP1, MSP2, MSP3, AMA-1, GLURP, Pfs25, Pfs28, Pvs25, Pvs28, Pfs48 / 45, P fs230, Cystosoma genus (blood flukes): TP1, Sm23, ShGSTs26 and 28, paramyosin, parasitic myo Shin, Sm14.

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

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

[0026] (Viral-derived heterologous proteins 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, NS 4. NS5, Flaviviridae (Pestivirus genus BVDV, CSFV, BDV; Hepacivirus genus HCV): E1, E2, E RNS (Pesti), C, NS3, NS4, NS5, Hepadnaviridae (HBV): HBS antigen, Paramyxoviridae (Paramyxovirinae: PIV-1, PIV-2, mumps, sen Dye, PIV-2, PIV-4, Morbilli): M, HN, N, F, Paramyxoviridae (Pneumovirinae: RSV): F, G, SH, M, Rhabdoviridae (rabies): G, Herpesvirus family (EBV, HSV2): gp350 / 220 (EBV), gB2, gD2 (HSV), Coronavirus family (SARS): CoV, N, M, S Orthomyxoviridae (Influenza A, B): HA, NA, M1, M2, NP, Papillomaviridae: L2, E6, E7

[0027] In embodiments of the present invention, the modified K. lactis strain further has the expression cassette K. lactis Ktis LAC4-12 promoter (P LAC4-12 ) or a variant of the promoter, or the antigen to be expressed. The embodiment is characterized by having an ORF and an AgTEF1 terminator. LAC4-12 The expression of heterologous genes under promoter control is LAC4 and / or KlURA3 and / or After being incorporated into the KlMET5 gene locus, it is almost equally strongly induced by lactose. It has the advantage of being able to do so.

[0028] As described above, the antigen concentration within the vaccine strain and the immunogenicity of the yeast vaccine in the target organism There is a positive correlation between the fruits. For example, this occurs due to the incorporation of additional KlGAL4 genes. To prevent CPE in cases of excessively strong overexpression, the vector system is modified as an alternative. By modifying and rapidly and efficiently ligating multiple gene copies in sequence, this expression cassette is created in 3 It can be introduced into one of the gene loci in a single step (see Example 5 and Figure 7A).

[0029] Therefore, as a further advantageous outcome of the present invention, the modified K. lactis strain has the KlLAC4 gene The locus, or the KlURA3-20 locus, or the KlMET5-1 locus, encodes a heterologous antigen. It contains multiple copies of the nucleic acid sequence, and these copies are expressed in a tandem expression cassette or a multiexpression cassette. The expression cassette is inserted via a set. Each expression cassette contains the LAC4-12 promoter (P L AC4-12 ) or a variant of the promoter and adjacent to the AgTEF1 terminator, the antigen It includes multiple copies of the region (gene) to be copied. Replication can significantly increase the expression of each gene through one of its loci. This is the result.

[0030] In a preferred embodiment of the present invention, the gene for the heterologous antigen IBDV-VP2 is expressed in a tandem expression cassette. It is present at the K1LAC4 locus of the K. lactis strain in this form. The K. lactis strain contains the heterologous antigen IB. Compared to strains with a single copy of the gene encoding DV-VP2, heterologous antigen IBDV-VP2 It has the advantage of being expressed in increased amounts. Particularly preferred according to this embodiment of the present invention is A strain that possesses the gene for the heterologous antigen IBDV-VP2 in the form of a tandem expression cassette at the KlLAC4 locus. This is VAK1118 (DSM32701).

[0031] Even more preferably, one or more copies of nucleic acids encoding different heterologous antigens are present in the present invention. The KlLAC4 locus and / or the KlURA3-20 locus of the K. lactis strain, and / or Alternatively, the KlMET5-1 gene locus can be used with a single expression cassette, a tandem expression cassette, or a multi-expression cassette. This is the case when it is inserted via the current cassette. As a result, within the yeast cell, first Furthermore, it is possible to express different heterologous antigens, and secondly, the said different heterologous antigens can be expressed in different concentrations. It is possible to express it at a certain degree. In this embodiment, a heterologous antigen influenza Influenza A HA (A / Puerto Rico / 8 / 1934(H1N1)) and Influenza A M1 (A / Puerto Rico / 8 / 193 The nucleic acid sequence encoding 4(H1N1) is found at the KlLAC4 and KlURA3-20 loci of the K. lactis strain. This is an inserted and expressed K. lactis strain. This particularly preferred embodiment of the present invention is different Influenza A HA (A / Puerto Rico / 8 / 1934(H1N1)) and influenza A M1 (A / Puerto Rico) antigens The nucleic acid sequences encoding elrico / 8 / 1934(H1N1) are KlLAC4 and KlURA3 of the K. lactis strain. This is the VAK1283 (DSM32697) strain, which has an insertion at the -20 locus.

[0032] As mentioned above, it is known that increasing the dose of the KlGAL4 gene can increase antigen production (K Rijger et al. (2012, and International Publication No. 2013107436). A pLI-1 plasmid expressing KlGAL4 The drawbacks of achieving this through a two-step process are described above. The point is that, according to the present invention, for incorporating heterologous genes including a second copy of the KlGAL4 gene, This was overcome by providing a fixed starting strain. All derived strains have the same genetic background. Furthermore, it is certain that exactly one additional KlGAL4 gene copy is present in the strain. This reduces the cytotoxicity observed in cases of multiple copy expression, and the vaccine strain Reducing the multiple steps involved in production to just one. Furthermore, reversible plasmid integration. Because the excision process is omitted, gene stability is improved. Such strains are, for example, implemented It can be manufactured as shown in Example 1.

[0033] Therefore, a more advantageous embodiment of the present invention is provided in which the K. lactis strain is the same as In addition to the original KIGAL4 gene in the genome, it also contains a second ectopic copy of the KIGAL4 gene. This is the K. lactis strain. Within this strain, the expression of KIGAL4 transcription activator is increased by up to twofold. This can be done to the KlLAC4 locus and / or the KlURA3-20 locus and / or KlM The expression of a heterologous gene inserted into the ET5-1 locus is controlled by the LAC4-12 promoter or the promoter -The following variants can be increased in a specified manner. In conventional practices, Kl Plasmid encoding GAL4 transiently enters cells, resulting in multiple uncontrolled copies. They were introduced in large numbers. As a result, heterologous antigens are often expressed at high concentrations, leading to cytotoxic effects. In the case of the K. lactis strain of this embodiment of the present invention, the cytotoxic effect is achieved with high efficiency. It can be reduced or avoided. Furthermore, for the same purpose (insertion of LAC4 control expression cassette) The gene loci that will be developed in the future can also be controlled by this method. The ectopic copy of the gene is adjacent to the KIGAL4 promoter and KIGAL4 terminator, and the K. It is incorporated at the KLLA0E13795g (Klavt3::KlGAL4-1, SEQ ID NO: 1) locus within the Lactis strain. In some cases, these characteristics have been found to be advantageous. In this embodiment of the present invention, these characteristics are particularly preferred. This is the stock VAK1111 (DSM32696) that possesses [this characteristic].

[0034] In a more preferred embodiment, the present invention relates to a nucleic acid sequence encoding the heterologous antigen IBDV VP2. The present invention provides a K. lactis strain located at the K1LAC4 gene locus. Particularly preferred is strain VAK1171 (DSM32699). The strain is further characterized by the following characteristics of the KIGAL4 gene. It contains two ectopic copies, both of which similarly contain nucleic acid sequences encoding the heterologous antigen IBDV VP2. This strain exhibits heterologous antigen IBDV-V compared to strains without additional ectopic copies of the KIGAL4 gene. It shows elevated expression of P2.

[0035] The production of heterologous proteins within microorganisms is problematic when it leads to cytopathic effects (CPE). Therefore, the present invention provides a method for separating the antigen-producing phase from the biomass-accumulating phase. This is made possible, for example, in a fed-batch fermentation process, thanks to the inducible LAC4 promoter which is partially possible, but promoter P LAC4-12 is not completely inactivated (clo se down) under non-inducing conditions (i.e., is somewhat open), so this method is also inhibited . In the case of an antigen with a very strong CPE, what results is a decrease in the growth rate and the induction of a cellular stress response, which has an adverse effect on antigen production. This problem is exacerbated by doubling the KlGAL4 gene dosage and / or increasing the number of sequences encoding the antigen (see below).

[0036] Therefore, a further advantageous development of the K. lactis strain of the present invention is a promoter structure modified from the LAC4-12 promoter that expresses little or no heterologous protein under non-inducing conditions, in a K. lactis strain. The modified structure of the LAC4-12 promoter is characterized in particular by the absence of the basic control region (BCR) of promoter P between positions 1065 and 1540 (LR2 deletion; P LAC4-12 ; SEQ ID NO: 2) (see also Example 2, likewise). As already mentioned above, this embodiment of the present invention has the advantage that the cytotoxic effects that were previously caused by the overly strong expression of heterologous genes can be highly efficiently reduced and avoided LAC4-12-LR2 ’ . Preferably, in this embodiment, the nucleic acid sequence encoding the heterologous antigen influenza A HA (A / Puerto Rico / 8 / 1934 (H1N1)) is present at the KlLAC4 locus in the K. lactis strain . Particularly preferably, according to this embodiment of the present invention, it is strain VAK1243 (DSM32702). The above-mentioned strain contains an LR2 deletion in the LAC4-12 promoter. ​

[0037] The K. lactis strain also enables the regulation of heterologous protein expression, as described above. It has a motor-modified promoter structure, and the promoter's activator KlGal4( The number of binding sites for the "upstream activation sequence" 1, 2 and 4, 5) varies, with 1, 2, 3 or 4 KlGal 4 binding sites may be present. In this way, different concentrations (designed product) can be found within one yeast cell. (Quality Assurance) It is possible to express multiple different heterologous proteins. Shortened Promo The Tah variant, in particular, forms highly immunogenic virus-like particles (VLPs). For example, to express multiple proteins in the optimal stoichiometric ratio within the same strain, cis This is important for modularization of the system. A preferred embodiment of the present invention is the aforementioned heterogeneous The nucleic acid sequence encoding the original IBDV VP2 was inserted into the KlLAC4 locus of the K. lactis strain. In this case, VAK1131 (DSM32700) is particularly preferred according to this embodiment of the present invention. The aforementioned strain has LR2 deficiency and deficiencies of upstream activation sequences 4 and 5 within the LAC4-12 promoter. Includes.

[0038] Part of the objective of this invention was to provide a K. lactis strain that is more suitable for cultivation. In the K. lactis strain of the invention, the gene functions of the alleles Kllac4, Klura3-20, and Klmet5-1 are reversed. This problem was solved by restoration. The resulting K-lactis strain is prototrophic. (Example 6, Figure 8). Therefore, the fermentation of the vaccine strain is simplified, and the establishment of the production process is facilitated. This is more cost-effective. According to this embodiment of the present invention, preferably the heterogeneous anti The original BVDV E2 external domain (Type 1, CP7), BVDV E2 external domain (Type 2, New York 93), and B The nucleic acid sequence encoding VDV Npro-NS3 (type 1, CP7) is the K1LAC4 locus of the K. lactis strain. This is a K. lactis strain inserted into URA3-20 and KlMet5-1. This embodiment of the present invention According to the study, 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 aforementioned shares were issued on November 24, 2017, or December 1, 2017, in accordance with the Budapest Convention (DSM32705, DSM32706), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Biological and Cellular Collection Institute, DSMZ, Inhoffenstrasse 7B, 38124 Braunschweig, D. It was deposited with Italy under the aforementioned number.

[0041] In a further embodiment, the present invention provides an embedded expression vector for K. lactis The strain becomes capable of production thanks to this vector.

[0042] In a preferred embodiment, the present invention relates to the embedded expression vector KIpURA3 (SEQ ID NO: 3) and K IpMET5 (SEQ ID NO: 4) is provided. These vectors are (as described above, K. lactis) (For stocks) LAC4-12 promoter (P LAC4-12 ) or a variant of the promoter, which expresses The ORF of the antigen to be incorporated, as well as the AgTEF1 terminator sequence, and similarly, the Klura3- after integration. The antigen has target sequences that enable the restoration of targeted function in the 20 and Klmet5-1 alleles. The sequence encoding the promoter sequence of the expression cassette at the identified restriction site and Cloning occurs between the terminator sequences. The vector enables heterologous gene expression. The cassette stably analyzes the K. lactis genome without markers or antibiotic resistance. It is incorporated internally. Therefore, the strength of this vector system is that multiple heterologous genes are different The ability to easily exchange between vectors, and the promoter and terminator of the expression cassette The key feature is that the ter is interchangeable with other things. The expression cassette is P LAC4-12 promoter It consists of the AgTEF1 terminator and heterogeneous genes between them. The heterogeneous genes are controlled It can be replaced with limited-part AscI and NotI. LAC4-12 The promoter is within both of the aforementioned vectors. The restricted areas SmaI and AscI are replaceable, and the terminator is NotI and Box within KIpURA3. It is interchangeable with I (or MluI), as well as with NotI and Ecl136II (or SacI) in KIpMET5. The expression cassette is between the restriction sites SmaI and BoxI (or MluI) within KIpURA3, and within SmaI within KIpMET5. It is cloned between Ecl136II (or SacI). Using a specified restriction enzyme, the expression cassette The set is also exchanged between the KIpMET5 and KIpURA3 vectors, or an additional expression cassette. Improvements to KIp3 and KIp3-MCS vectors (International Publication Number 20101054649) will be introduced. The point is that selection was performed under non-inducing conditions (without lactose), which resulted in the selection of proteins accompanied by CPE. Even in such cases, a high transformation rate is achieved, and a large number of transformants can be produced even with low heterologous gene expression. Suppresses the possibility. 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-P LAC4 -Et, KIpMET5-P LAC4-LR2 From, as well as KIpURA3-P LAC4-12 -Et, KIpURA3-P L AC4-12-LR2 -Et, KIpURA3-P LAC4 -Et and KIPURA3-P LAC4-LR2 From (Sequence number: 5, 6, 7 or 8 and A combination of sequence numbers 3 and 4) is provided as an embedded expression vector.

[0044] Vector KIpURA3-P LAC4-12 -Et, KIpURA3-P LAC4-12-LR2 -Et, KIpURA3-P LAC4 -Et and KIpU RA3-P LAC4-LR2 This is a variant of vector KIpURA3-Et, and in each case, Etx.B-HA A nucleic acid sequence encoding a protein is inserted. Vector KIpURA3-P LAC4-12 -Et, KIpURA3-P LAC 4-12-LR2 -Et, KIpURA3-P LAC4 -Et and KIPURA3-P LAC4-LR2 This is compared to the vector KIpURA3-Et. The promoter is different.

[0045] Vector KIpMET5-P LAC4-12 -Et, KIpMET5-P LAC4-12-LR2 -Et, KIpMET5-P LAC4 -Et, KIpMET 5-P LAC4-LR2 This is a variant of the vector KIpMET5, and in each case, the Etx.B-HA protein A nucleic acid sequence encoding quality is inserted. Vector KIpMET5-P LAC4-12 -Et, KIpMET5-P LAC4-1 2-LR2 -Et, KIpMET5-P LAC4 -Et, KIpMET5-P LAC4-LR2 Compared to Vector KIpMET5, the promotion The engine is different.

[0046] In a further embodiment, the present invention relates to a method for producing the K. lactis strain of the present invention, wherein the following Step: (i) Insert the nucleic acid sequence encoding the target antigen into the KIpURA3 vector or KIpMET5 vector. Steps to enter, (ii) A K. lactis culture is transformed with a modified and pre-enzymatically digested vector construct. Steps to transform quality (iii) Using a solid medium that does not contain uracil and / or methionine, transform the K. larvae The step of selecting cutis cells, and (iv) Optional step to restore prototrophicity, The present invention provides the method including the above.

[0047] In one embodiment of the method of the present invention, the gene sequences of multiple antigens are ectopically and simultaneously inserted. It can be expressed under controlled conditions. It codes for antigens of different variants of a single pathogen. It is preferable that different gene sequences are ectopically inserted and regulated in their expression. , different gene sequences encoding antigens of multiple different pathogens are ectopically inserted, It is preferable that the expression is controlled.

[0048] In a further embodiment, the present invention relates to the present invention for extraintestinal, enteral, intramuscular, mucosal, or oral administration. We provide a pharmaceutical or veterinary drug composition containing the K. lactis strain, and the composition is optionally adapted to It may be combined with vehicles and / or excipients for use in vaccination. In particular, the present invention is for use in vaccination To provide a suitable pharmaceutical composition or veterinary pharmaceutical composition.

[0049] Preferably, the pharmaceutical composition or veterinary pharmaceutical composition contains at least one physiologically compatible The present invention comprises a suitable vehicle, diluent, adjuvant and / or excipient. This is a pharmaceutical composition for injection, contained in a pharmacocompatible vehicle, for example, in a physiological saline medium or a slow-release pharmacokinetic. It may be contained in conventional media such as a cinder. Such media may also, for example, have an osmotic pressure. It may include conventional pharmaceutical substances such as pharmaceutically acceptable salts, buffers, and preservatives for determining suitability. Preferred media include physiological saline and human serum. Particularly preferred media is PBS. It is a saline solution.

[0050] Furthermore, a more appropriate pharmacopoeia vehicle is, for example, Remington's Practice of Pharmacy. , 13th edition and J. of Pharmaceutical Science & Technology, Vol.52, No.5, Sept-Oct, p This is known to those skilled in the art by ages 238-311.

[0051] Furthermore, in aspects of the present invention, recombinant K. lactis yeast of the present invention is used, for example, for defensive immunization. Use for vaccination, particularly to induce protective immunization against pathogens. To provide.

[0052] Methods involved in the development of protective immunization include, for example, the following steps: a) A step of culturing and growing recombinant yeast of the present invention, b) Steps to harvest and inactivate the yeast, c) A step of administering recombinant yeast according to a specific immunization scheme, d) A step of determining the titer of the formed antibody, and / or e) A step to detect immunization.

[0053] The culture and propagation of recombinant yeast of the present invention can be achieved using any conventionally available method. This is possible. Particularly preferred is a cost-effective method that yields high cell yields. These include fermentation methods, particularly high-cell-density fermentation methods. Using ru to carry out fermentation proved to be particularly advantageous.

[0054] In a preferred embodiment, protective (preventive) immunization is achieved by recombinant yeast administered orally / through mucosa. This is achieved by administration via intramuscular or subcutaneous injection.

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

[0056] The present invention also applies the K. lactis strain of the present invention to animals or humans, preferably. To induce an immune response, preferably a protective immune response, against one or more heterologous antigens within the animal. The present invention provides a vaccination method that includes administering a sufficient amount to induce the vaccine.

[0057] A special advantage of using the K. lactis strain of the present invention is the protective immune response against pathogen 1. After one application / immunization ("single dose"), or two applications / immunizations ("Prime Boost") ") Afterwards, it is caused by itself. Further benefits of using the K. lactis strain of the present invention The discovery of this as a point indicates a cross-protective immune response against multiple different variants of a single pathogen. After one application / immunization ("single dose"), or two applications / immunizations ("Prime Boost") This can be caused later. The K. lactis strain of the present invention is an anti-pathogen of multiple different pathogens. Even when expressing different heterologous genes against the original organism, a single protective immune response is applied. / Immunization ("single dose") or two doses / Immunization ("prime boost") It is even possible to do that. [Effects of the Invention]

[0058] (Summary of the advantages of the present invention) The aforementioned improvements within the K. Ractis platform resulted in numerous advantages. a) Excellent simplification (immediate) in the construction of yeast-based "subunit vaccine" strains. The availability of toolboxes / kits and high reproducibility have become possible. They are now specific It can be produced in a short amount of time. b) Yeast vaccines can contain one or more antigens. They can be flexibly customized and different It can be produced in a certain quantity. c) Furthermore, efficient fermentation of prototrophic yeast became possible. d) Strict induction of recombinant protein production became possible. The latter causes CPE or This is especially important for proteins that may be affected. e) The objective is the stable integration of heterologous genes into the genome, and the related genes of the strain. Stability offers the advantage of ensuring that the production process proceeds reproducibly. This is for GMP production. This is especially important. f) The protective effect of the yeast vaccine is due to an increase in heterologous gene copies and / or an increase in KlGAL4 concentration. The result is improved by increased recombinant antigen production. g) Furthermore, the administered vaccine dose is used to increase heterologous gene copies and / or KlGAL4 concentration. This can be reduced by the increase in recombinant antigen production achieved as a result of the increase. Maternal production becomes more cost-effective, and vaccine suitability for vaccinated individuals improves. h) Polyvalent yeast vaccines are effective against multiple different variants of the same pathogen, or against multiple different diseases. It can be used in a cross-protective or multivalent protective manner for the prevention of the active ingredient. Inactivation, and Anything other than mixing with an appropriate adjuvant and / or an appropriate amount of liquid is not permitted to be used as a vaccine. No further processing of the yeast is necessary. [Brief explanation of the drawing]

[0059] The present invention is described more specifically below based on the drawings and exemplary embodiments. It can be done.

[0060] [Figure 1] Figure 1 shows the characteristics of a newly generated K. lactis background strain containing two KlGAL4 copies. The presence of a second ectopic KlGAL4 copy at the identified integration site was confirmed, and the effect of integration on yeast growth was analyzed. A: Diagram of the integration site of the ectopic KlGAL4 copy. The integration site is shown, and the gene name is also displayed. B: Yeast strains with an additionally integrated ectopic KlGAL4 gene at the KlAVT3 locus (VAK1110) and without additional integration (VAK367) were marked with primers. [ka] Agarose gel of PCR-amplified fragments using [a specific method]. The predicted fragment size for each is [a specific method]. It is shown on the right side of the eargram. C: Droplet test with glucose (YPD) or lactose (YPLac) using a 10-fold serial dilution (start-OD1). Incubation was carried out at 30°C and 37°C, respectively. KlGAL4 copies were added to the natural locus. The yeast strain (VAK1139) possesses four copies of KlGAL at an ectopic locus, and the native locus of KlGAL Yeast strain 4 is missing (VAK1110), yeast strain lacking KlGAL4 copy (ΔKlgal4;VAK964), Alternatively, the growth of a yeast strain (VAK1168) with two KlGAL4 copies was compared. What was shown was further... Only the identified incorporation of the KlGAL4 gene results in a growth defect with very little productivity. The aforementioned defect is only visible under induction conditions at 37°C. It is more clearly visible in KlGAL4. This is a growth defect that occurs when a certain component is completely absent.

[0061] [Figure 2]Figure 2 shows the Western blot analysis of K. lactis strain proteins with additional ectopic KlGAL4 copies producing IBDV-VP2. The effect of the additional KlGAL4 copies on LAC4-12 promoter-dependent recombinant protein production was analyzed by Western blotting. The test strain used was a yeast strain with an IBDV-VP2 expression cassette, and this yeast strain was compared with other IBDV-VP2 yeast strains. The presence (+) or absence (-) of ectopic KlGAL4 copies and tandem IBDV-VP2 expression cassettes (see below) is shown in the upper panel. Ectopic copies were introduced into strain VAK911 by linearization of plasmid pLI-1 using BstEII (Krijger et al. 2012 and International Publication No. 2013107436), and ectopic KlGAL4 copies are present at the KlAVT3 locus in strain VAK1130 (see Figure 1). Yeast strain VAK367 was included as a wild-type control without heterologous genes. The yeast strains were pre-cultured in YPD and then cultured in YPLac for 15 hours. Protein extracts were analyzed at a rate of 20 μg for each yeast strain using 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 to the right, and non-specific bands are indicated by asterisks. It was shown that ectopic expression of additional KlGAL4 genes, as well as the presence of tandem expression cassettes, leads to a strong increase in heterologous antigen concentration (see also below).

[0062] [Figure 3]Figure 3 shows the effect of LR2 deficiency in the LAC4-12 promoter on uninducible recombinant protein production and glucose-induced yeast growth. The unmodified LAC4-12 promoter also exhibits basal expression of GOIs (Gene of Interest) under uninducible conditions. This is particularly problematic in the case of cytotoxic heterologous antigens. These experiments tested whether deficiency in the BC region of the LAC4-12 promoter (LR2 deficiency) reduces or completely suppresses recombinant protein production under uninducible conditions. A: Diagram of the LAC4-12 promoter (LAC4-12). The basal regulatory region (BCR), LR2 deficiency, four KlGal4-binding sites (upstream activation sequences: U1, U2, U4, U5), and nucleic acid sequences encoding heterologous genes (GOIs) are also shown. B: Western blotting of IBDV-VP2 yeast strains with and without LR2 deficiency (VAK1131 and VAK1130) after culturing under non-inducible conditions (YP3%EtOH). VAK1111 was used as a wild-type control without heterologous genes. 50 μg of protein extract was loaded onto a 12% SDS gel for each yeast strain. Immunoblotting was performed using anti-IBDV rabbit serum (1:5000) and goat-derived HRP-conjugated anti-rabbit antibody (1:10000). Loaded control KlNop1 was detected using mouse anti-Nop1 antibody (1:5000) and goat-derived HRP-conjugated anti-mouse antibody (1:10000). C: Droplet test using YPD, 0.5% glucose-containing YPD, and YPLac with 10-fold serial dilutions (start-OD1). Incubation was performed at 30°C and 37°C, respectively. The growth of yeast strains carrying heterologous influenza A HA genes at the LAC4 locus was compared between those with and without LR2 deficiency (VAK1243) and those without (VAK952). Yeast strain VAK367 was used as a wild-type control without heterologous genes. It was shown that LR2 deficiency suppressed the expression of undesirable basic heterologous proteins. Furthermore, it was shown that LR2 deficiency improved the growth of yeast strains expressing cytotoxic proteins (influenza hemagglutinin, HA) under both non-inducible and induced conditions.This is particularly evident at 37°C.

[0063] [Figure 4] Figure 4 shows KIp vectors that can be used to integrate protein expression cassettes into different loci of the K. lactis genome. The use of the LAC4 locus (KIp3 vector system) has been previously described (International Publication Nos. 20101054649 and 2013107436), but the use of the KlURA3 and KlMET5 loci is novel. A: Diagrams of different KIp vectors, each with an integration site in the genome. B and C: Expression cassettes and both adjacent ends in the KIpURA3 (B) and KIpMET5 (C) vectors newly described herein. Different DNA sequence segments and associated restriction sites are shown. GOI: Heterogene (target gene). D: Western blot analysis of heterogeneous protein expression in yeast strains constructed by KIp vectors (A, B, and C). Here, the heterogene is Etx.B-HA. The yeast "housekeeping" KlNop1 protein (KLLA0C04389g) was detected as a loading control. Yeast strains were pre-cultured in YPD(+U) and then cultured in YPLac(+U) for 4 hours. 30 μg of protein extract was loaded onto 12% SDS-PAGE for each yeast strain. Immunoblotting was performed using monoclonal mouse anti-HA (1:5000) and anti-KlNop1 (1:5000; Santa Cruz, TX, USA) antibodies, as well as goat-derived HRP-conjugated anti-mouse antibody (1:10000; Jackson ImmunoResearch, PA, USA). The results showed that both the KlURA3 and KlMET5 loci, as well as the LAC4 locus (International Publication Nos. 20101054649 and 2013107436), can be used for heterologous gene expression.

[0064] [Figure 5]Figure 5 shows the production of different recombinant proteins within the same yeast strain. The aforementioned yeast strain (VAK1234) was constructed using KIpURA3 and KIp3-MCS vectors. Western blot analysis of proteins from tandem IBDV VP2-expressing yeast strains (see below) was performed. The yeast strain (VAK1234) had an additional expression cassette containing Etx.B-HA as a heterologous gene introduced using the KIpURA3 vector. The controls used were yeast strains with an expression cassette containing Etx.B-HA in the LAC4 locus (VAK899), or yeast strains with it at the KIpURA3 locus (VAK1235), or yeast strains with 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 12% SDS-PAGE. Immunoblotting was performed for Etx.B-HA using mouse anti-HA antibody (1:5000; Santa Cruz, TX, USA) and goat-derived HRP-conjugated anti-mouse antibody (1:10000), and for IBDV-VP2 using rabbit anti-IBDV antiserum (1:5000; Granzow et al. (1997)) and goat-derived HRP-conjugated anti-rabbit antibody (1:10000; Jackson ImmunoResearch, PA, USA). It was shown that both heterologous proteins were expressed within the same yeast cells. Surprisingly, the expression level of one antigen was not limited by the co-expression of the other antigen. This is evident from the comparison of expression levels in monovalent and bivalent strains (see also Figure 12).

[0065] [Figure 6]Figure 6 shows differently induced LAC4-12 promoter variants for expression cassettes within the KIp vector. KIp vector expression cassettes 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 analysis of yeast strains containing the corresponding expression cassettes with Etx.B-HA as the heterogene. A: Schematic diagram of the promoter variant, the associated KIpURA3 vector with Etx.B-HA as the heterogene, and the yeast strains constructed from them. BCR: Binding regions for transcriptional activators KlCat8 and KlSip4, transcriptional activators under non-inducible conditions; U1, U2, U4, U5: Binding regions for transcriptional activator KlGal4 (upstream activation sequence). B: Western blot analysis for characterization of the LAC4-12 promoter variant (A) in the yeast strain constructed using the KIpURA3 vector. Yeast strains were pre-cultured in YPD and then cultured in YPLac for 4 hours. For each yeast strain, 30 μg of protein extract was loaded onto 12% SDS-PAGE. Immunoblotting was performed using monoclonal mouse anti-HA (1:5000) and anti-Nop1 (1:5000) antibodies, as well as goat-derived HRP-conjugated anti-mouse antibody (1:10000). The results showed that the expression rates of heterologous genes varied depending on the properties 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. DNA segments and associated restriction sites are shown. GOI: Heterologous gene (target gene). B: Tandem construct from (A) for random incorporation using a ScURA3 selection marker is shown. C: Western blot analysis comparing IBDV-VP2 protein production in yeast strain (VAK1118) (A) with a tandem expression cassette and in yeast strain (VAK910) with 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. 60 μg of protein extract was loaded onto 12% SDS-PAGE for each yeast strain. Immunoblotting was performed using anti-IBDV rabbit serum (1:10000) 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. D: Western blotting comparing yeast strains (B) with randomly incorporated tandem IBDV-VP2 expression cassettes with KIp3-MCS-constructed yeast strains (VAK910) with one expression cassette and yeast strains derived therefrom that having additional KlGAL4-1 copies (pLI-1). Yeast strains were pre-cultured in YPD and then cultured in YPLac for 8 hours. Immunoblotting was performed as described in (b) below. It was shown that the use of tandem expression cassettes significantly increases the expression rate of heterologous proteins.

[0067] [Figure 8]Figure 8 shows gene fragments for restoring the gene function of the alleles Klura3-20 and Klmet5-1(A). The gene loci and gene fragments amplified using identified primers for KlURA3(A) and KlMET5(B) are schematically shown. Mutations in the alleles Klura3-20(A) and Klmet5-1(B), reconstructed by homologous recombination using these gene fragments, are indicated by asterisks below the genes. A restriction site, the site for excising the subcloned fragment, is drawn in. This diagram illustrates a strategy for creating yeast strains expressing prototrophic heterologous genes at the URA3 or MET5 locus.

[0068] [Figure 9]Figure 9, in combination with Tables 1 and 2, shows protective (preventive) immunization of chickens against vvIBDV in a classic prime-boost vaccination scheme. In two experiments (A and B), at least 16 SPF chickens were subcutaneously inoculated with freeze-dried and heat-inactivated yeast cells of the genetically optimized tandem IBDV-VP2 K. lactis yeast strain VAK1127 using the prime-boost method. The first vaccination was performed two weeks after hatching (prime), and the second vaccination (boost) was performed two weeks later. Two weeks after the boost, a viral challenge was performed with the vvIBDV strain (highly virulent 89163 / 7.3). One control group, designated as an infection control, underwent simulated treatment with either PBS alone or adjuvant alone. In Experiment 1(A), wild-type yeast (VAK367) was also administered as a control. At least 7 chickens per group, and at least 5 chickens in Experiment 2(B), were used as controls without viral challenge. Serum was collected immediately before the first dose, immediately before and after the challenge, and at other 10-day intervals. Seroconversion (antibody seroconversion) intensity was determined by ELISA (ProFLOK IBD Plus, Synbiotics). The converted titer is shown according to the kit information. A: Experiment 2 was conducted in the same manner as Experiment 1(A). The mean ELISA titer of 12 animals is shown along with the standard deviation. Both experiments showed that the titer of anti-IBDV VP2 antibody was stronger in animals vaccinated with VAK1127. The related table summarizes the results of protection against vvIBDV challenge in vaccinated animals. Complete protection against viral infection was achievable in both vaccination experiments.

[0069] [Figure 10]Figure 10 shows the effect of genetic modification for restoring prototrophicity on recombinant protein production and immunogenicity of tandem IBDV-VP2 yeast strains. The trophic-requiring tandem IBDV-VP2 yeast strain VAK1127 and the derived prototrophic yeast strain VAK1171 were compared in terms of the efficiency of recombinant protein production and immunogenicity. A: Western blot analysis to confirm the IBDV-VP2 content in newly collected yeast material. Yeast strains were pre-cultured in YPD and then cultured in YPLac for 8 hours. 40 μg of protein extract was loaded onto 12% SDS-PAGE for each yeast strain. Immunoblotting was performed using anti-IBDV rabbit antiserum (1:10000) and goat-derived HRP-conjugated anti-rabbit antibody (1:10000). Agglutinative (agg.) and monomeric (mon.) IBDV-VP2 are indicated by arrows on the right, and non-specific bands are indicated by asterisks. B: Western blot analysis to confirm the IBDV-VP2 content in freeze-dried and heat-inactivated yeast material, which was subsequently used in immunization studies in BALB / c mice (C). Yeast strains were cultured in YPLac for 15 hours after preliminary culture in YPD. 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 band. C: Immunogenicity testing of two yeast strains, VAK1127 and VAK1171, in immunization experiments in BALB / c mice. Each of five mouse groups was subcutaneously inoculated three times with 0.1 mg (dry weight) of the yeast material analyzed in (B) above. The control used was the antigen-free wild-type strain (VAK367). The initial dose was administered using CFA (complete Freund's adjuvant) as an adjuvant, followed by two more doses at two-week intervals using IFA (incomplete Freund's adjuvant) as an adjuvant. One week after the third dose, mice were euthanized and blood was collected. Serum was analyzed by IBDV-VP2 ELISA (IDEXX). Absorption at 650 nm, which correlates with anti-IBDV-VP2 antibody titer, is shown along with the 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) was used as a control. The results showed that heterologous protein expression levels in both strains were similar and indicated potential immunogenicity.

[0070] [Figure 11] Figure 11, in combination with Table 3, shows protective (preventive) immunization against vvIBDV in SPF chickens by a single subcutaneous administration of genetically optimized IBDV-VP2 vaccine yeast. At least 18 SPF chickens were subcutaneously vaccinated 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. The control group used consisted of animals vaccinated with 10 mg of PBS or VAK367. The animals were vaccinated twice, at 2 and 4 weeks post-hatch. All animals underwent vvIBDV challenge at 6 weeks post-hatch. Serum was analyzed by ELISA (ProFLOK IBD Plus, Synbiotics) as described above. The confirmed antibody titers are shown. Each dot represents the individual antibody titer of 12 chickens analyzed in each group, and the bars represent the mean value along with the standard deviation. In the control group, only the antibody titers of surviving chickens were confirmed after the challenge. It was shown that only a single dose of the yeast subunit vaccine VAK1171 provided complete protection against subsequent vvIBDV exposure.

[0071] [Figure 12]Figure 12 shows the characterization of strains VAK952 and VAK1283. (A) Yeast strains VAK952 (monovalent HA) and VAK1283 (divalent HA, M1) were pre-incubated in YPD in a shaking flask and then induced in YPL for 6 hours. Optical density was measured at 600 nm, 30 OD units of the culture were taken, the pellet was broken using 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 generated using an infrared imaging system (LI-COR Biosciences). (B, C) Yeast strains were pre-incubated in YPD in a shaking flask and then induced in YPL for 24 hours. (B) The optical density of the yeast culture was determined at a predetermined time, and 30 OD units were collected. (C) The VAK1283 pellet was disrupted using glass beads and analyzed by immunoblotting. (D) The measured optical densities of VAK952 and VAK1283 were combined as growth curves as a function of time and averaged from at least two independent experiments. (E) In the dot test, the yeast strains were cultured on YPD-containing nutrient agar plates at 30°C for 48 hours. Starting with 1 OD unit, the yeast was serially diluted and then dropped onto YPD-containing or YPL-containing nutrient agar plates. The plates were cultured at 30°C for 48 hours, and then photographs were taken. Ponceau S: Staining of all yeast proteins in each fraction and supported control. It is shown that VAK952 (monovalent HA) and VAK1283 (divalent HA, M1) express HA protein in equivalent amounts. Furthermore, it was shown that VAK1283 and VAK952 exhibited similar growth characteristics, but VAK1283 was slightly superior.

[0072] [Figure 13]Figure 13 shows antibody titers in the serum of BALB / c mice before and after exposure infection, following immunization with VAK952 (monovalent HA) and VAK1283 (bivalent HA, M1). Both yeast strains were pre-incubated in YPD in a shaking flask, then induced in YPL for 12 hours (VAK952) or 6 hours (VAK1283). The cultures were then harvested, lyophilized, and the yeast material was 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 PBS (unadjuvant), at 3-week intervals. The adjuvant used was AddaVax. Three or six weeks after the final dose, animals were intranasally infected with 5xMLD50 influenza A / PR / 8 / 34(H1N1) virus. The infection control used was a simulated infection of animals administered intranasally with virus-free PBS only. Three or six weeks after the final dose, and during exposure infection, serum was collected from the animals and tested for neutralizing antibodies (nAb) by VNT. nAb titer 50: Serum dilution that reduces plaque count by 50% compared to the virus-free control. Identified as log2 of the corresponding serum dilution. Due to the logarithmic plot, a titer of log2(2)=1 was considered to be a serum sample without detectable antibody. 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 vaccination experiment and the single-dose vaccination experiment were not significantly different for VAK952 and VAK1283.

[0073] [Figure 14]Figure 14 shows exposure to 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 5xMLD50 influenza A / PR / 8 / 34 (H1N1) virus. The infection control used was a simulated animal (simulation) that had been intranasally administered only virus-free PBS. Subsequently, the survival rate (A), weight (B), and clinical symptoms (C) of the animals were examined multiple times daily for 14 days. For clinical symptoms, a score of 0-4 was determined, averaged across each group (0: no abnormalities, 1: slightly poor coat, 2: poor coat, decreased activity, 3: poor coat, 15% weight loss, 4: poor coat, over 20% weight loss). The study demonstrated that the prime-boost immunization method with VAK952 did not provide optimal protection against viral exposure, while VAK1283 did. Single-dose schemes with both vaccines yielded optimal protection with a 2 mg dose. Even with a 1 mg dose, the protection achieved by VAK1283 was similar to that achieved with a 2 mg dose of VAK952 using the prime-boost method. [Examples]

[0074] (Experimental embodiment) (Example 1: Two KlGAL4 genes stably incorporated into multiple gene loci that are not bound to each other) (Creation of host strains with child copies) Without a selection marker, a second KlGAL4 gene copy was inserted (ectopically) into a different gene locus. Sequencing was used to determine the insertion site within the KlAVT3 gene (KLLA0E13795g). It is possible to do this (Klavt3::KlGAL4-1, Sequence ID: 1) (Figure 1). The resulting strain was named VAK1111. I attached it. Two KlGAL4 copies located on chromosome E (ectopic copy) and D (original genome copy). The independent meiotic separation of each was confirmed by crossover experiments. Furthermore, in the same experiment, this It was established that the KlGAL4-1 gene copy number in the genome is exactly 2. VAK1111 was identified as VAK367. -Similar to D4, one step for use in targeting expression cassettes to the LAC4 locus. We introduced a processable lac4::ScURA3 disruption and, under the selectivity for lactose proliferation, without a marker. Using KIp vector technology, the target heterologous gene is converted to an LAC4 promoter and an LAC4 leaflet. It was possible to incorporate it into the ding frame (Krijger et al. (2012)). The resulting strain VAK1123 is It differs from VAK367-D4 only in that it has a second ectopic KlGAL4 gene copy.

[0075] (Example 1.1: Improved live yeast vaccine strain having the additionally incorporated KlGAL4 gene) (Productivity) In one exemplary embodiment, IBDV-oVP2 T2S (Arnold et al. (2012)) The gene is LAC of strain VAK1123 Insertion occurred at 4 gene loci (the resulting strain is VAK1130). It possesses only one KlGAL4 copy. Compared to a genetically homogeneous strain (VAK910), it was possible to establish increased production of IBDV-VP2. It worked. For comparison, there is only one KlGAL4 gene, but there are two CDSVP2 IBDV Share V, which has a copy AK1118 (see below) is also shown (Figure 2).

[0076] (Example 2: To optimize the expression of antigens with cytopathic effects, the basic activity was reduced.) P LAC4-12LR2’ promoter) The production of heterologous proteins within microorganisms can lead to cytopathic effects (CPE). There is a problem. Therefore, the challenge we face is to separate the antigen production phase from the biomass accumulation phase. The goal is to find a way to separate them. Thanks to the inducible LAC4 promoter, this can be achieved through fluid culture. Partially possible through the fermentation process, but under non-inducible conditions, promoter P LAC4-12 The completion Because it is not completely inactivated, the overall effect is inhibited. An antigen with very strong CPE (Critical Prescription Emission). In this case, a decrease in proliferation rate and induction of a cellular stress response occur, negatively impacting antigen production. Boss. This problem involves doubling the dose of the KlGAL4 gene and / or increasing the number of sequences encoding the antigen. It worsens with the addition (see below). The solution is promoter P between -1065 and -1540. LAC4-12 ( This involves deleting the basic control region (BCR) (Mehlgarten et al. (2015)) as shown in Figure 3A (LR2 deletion). ;P LAC4-12-LR2’ (Sequence ID: 2). The aforementioned deletions are due to the starting strain VAK367 (one KlGAL4 copy) and VA K1111 (two copies of KlGAL4) is introduced into the original LAC4 locus of the genome, along with disruption of lac4::ScURA3. The obtained strains VAK1109 and VAK1124 are suitable for expressing antigens with CPE. Motor P LAC4-12LR2’ It is also inserted into the embedded vectors KIpURA3-Et and KIpMET5-Et. (See below.)

[0077] (Example 2.1: Inhibition of basic (uninduced) antigen expression by a modified promoter) One tandem IBDV-VP2 expression cassette was incorporated into VAK1124 (Yeast strain created: VAK 1131; For an explanation of the term "tandem expression cassette," see below and Figure 7. Afterwards, LAC4-12 promo This shows that LR2 deficiency in the receptor leads to a sharp decrease in VP2 protein production under non-inducible conditions. This was possible (Figure 3B). A strain expressing the influenza A antigen hemagglutinin (red blood cell agglutinin) ( VAK952, which does not have LR2 deficiency in its promoter, and VAK1243, which has LR2 deficiency in its promoter) As a result of LR2 deficiency, the cytopathic effect of influenza A HA antigen is suppressed, under non-inducible conditions. We were able to demonstrate that proliferation was improved (Figure 3C).

[0078] (Example 3: Multi-purpose use for targeted integration of multiple expression cassettes into the K. lactis genome) (Vector System) As with VAK367-D4 (Krijger et al. (2012), International Publication No. 20101054649), yeast strain VAK Strain 367 forms the genetic background for all K. lactis strains described herein. The background is that it requires uracil and methionine (uracil and methionine nutrition (Requirement) This is due to mutations in two genes, KlURA3 (KLLA0E22771g) and KlMET5 (KLLA0B03938g). Alternates known as Klura3-20 (base pair deletion at position +345) and Klmet5-1 (G2555A and A3682T) This is because there is a gene (this allele is a functionally deficient variant).

[0079] These mutant alleles are already developed in KIp3 / KIp3-MCS, specifically in the integration site LAC4(Kr In addition to ijger et al. (2012), to use multiple further gene loci for target integration, This was then used to create a multivalent vaccine strain (Figure 4A). The selection was based on these. By restoring the gene function of the mutated gene, without the additional insertion of a selection marker. This can be achieved. For this purpose, a novel embedded vector was created. The vector Within the expression cassette (in each case, the LAC4-12 promoter or its variants) (Below) Homologous recombination leads to upstream integration of the KlURA3 gene and downstream integration of the KlMET5 gene. It is adjacent to the gene segment that enables integration, and at the same time, the wild type of those genes Recover the array. By selecting the integration site through mutagenesis and the nutritional requirements for alternative growth substances This means that multiple more gene loci can be developed in the same way.

[0080] (Example 3.1: KlURA of a K. lactis strain having the Klura3-20 and / or Klmet5-1 alleles) Expression cassettes for the 3(KLLA0E22771g) and / or KlMET5(KLLA0B03938g) gene loci (inducible Vectors KipURA3 and KipMET5 (with LAC4-12 promoter) for target incorporation. The embedded expression vectors KIpURA3 (SEQ ID NO: 3) and KIpMET5 (SEQ ID NO: 4) were introduced into Klura3-20. And appropriate gene fragments that enable the targeted restoration of the function of each Klmet5-1 allele. It was constructed using the (KlMET5 / KlURA3 target sequence).

[0081] The KIpMET5 expression vector uses the LAC4-12 promoter (P LAC4-12 or its variants), the expressed antibody It comprises an expression cassette consisting of a nucleic acid sequence encoding the progenitor and an AgTEF1 terminator, It is located downstream and adjacent to the original KlMET5 fragment of the genome that has the introduced ScCYC1 terminator, It is located upstream of the KlAIM18 promoter, which contains the downstream KlAIM18 gene. The KIpURA3 expression vector uses the LAC4-12 promoter (P LAC4-12or its variants), the expressed antibody It comprises an expression cassette consisting of a nucleic acid sequence encoding the progenitor and an AgTEF1 terminator, It is located downstream of KLLAOE22749g, which has the associated promoter, and has a downstream KlURA3 fragment. It is located upstream of the KlURA3 promoter (Figure 4B, C).

[0082] In either case, the sequence encoding the antigen is promoted via the AscI and NotI restriction sites. -It is cloned between the terminator and the plasmid Eco91I or KpnI. Due to limitations, the entire expression cassette was separated from the KIpURA3 vector backbone, and also prepared. Due to HindIII or BoxI restriction of the plasmid, the entire expression cassette becomes a KIpMET5 vector Klura3-30 and / or Klmet5-1 alleles were isolated from the Klura bone, and the restriction material had Klura3-30 and / or Klmet5-1 alleles. It is transformed into a Lactis host strain. In this way, it is incorporated into KlURA3-20 or KlMET5-1. Therefore, the heterologous gene-containing expression cassette is used by the KIp3-MCS vector to express LAC4 within VAK367-D4. It corresponds to the embeddable version (International Publication Number 20101054649) itself. Confirmation of nutritional and / or methionine prototrophic transformants is performed on KIpMET5 transformants. For the KIpURA3 transformants, primers MAB6 and VK211 were used. The expression is then performed using a standard method by colony PCR. The expression cassette is KlURA3 or KlMET5 and By incorporating it into the precise target site between each adjacent gene, a size of 1652 bp is achieved. We obtain KIpMET5 transformants and KIpURA3 transformants of size 1307 bp. This insertion No sign of impaired functionality of adjacent genes was confirmed.

[0083] Primer:

Chemical formula

[0084] (Example 3.2: Expression of heterologous antigen after integration of the coding gene cassette into the KlURA3 or KlMET5 locus) (Expression of heterologous antigen) P LAC4-12 Under the control of the promoter, the heterologous gene is integrated into the LAC4, KlURA3 and KlMET5 loci and then induced almost equally strongly by lactose. The heat-labile and non-toxic enterotoxin subunit B (Etx.B) and the C-terminal (HA )3 epitope (Etx.B-HA) derived 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 loci KlMET5 (VAK1251), KlURA3 (VAK1235) and LAC4 (VAK899) (Figure 4D). As shown by Western blot analysis, the concentrations of Etx.B-HA protein in all three strains were very similar (Figure 4D). Therefore, depending on the integration site of the expression cassette in the genome, a positional effect on the amount of recombinant }protein production could not be established. <00009{22> (Example 3.3: Co-expression of two heterologous antigens in the same yeast cell) With the new vector system, the possibility of producing different LAC4-12 heterologous proteins under the control of the P promoter in the same yeast strain was investigated by introducing an Etx.B-HA expression cassette into the KlURA3 locus and ​​​Two VP2s exist as a tandem at the LAC4 locus. IBDV It has an expression cassette that has copies. This was demonstrated by constructing the yeast strain (VAK1234; Figure 5, Tandem cassette explanation below and See Figure 7). Each yeast strain has only one expression cassette in its genome (VAK1235 or Compared to VAK1171, Etx.B-HA or VP2 in the case of VAK1234 IBDV The decrease in protein concentration is It could not be confirmed.

[0086] (Example 4: LAC4 promoter for regulating recombinant protein synthesis under similar induction conditions) (Variant) The immunogenic effect of an antigen is often due to the assembly of multiple proteins in non-stoichiometric ratios (assemblin). Based on g). To make this possible with yeast-based vaccines, lactose or galactose P can be induced differently depending on the method. LAC4-12LR2’ A variant of the promoter was created (Figure 6A). They found that the number of binding sites for the activator KlGal4 (U1, U2, U4, U5; Godecke et al. (1991)) and the basic Characterized by the presence or absence of the control region BCR. As shown in Figure 3A, inserted into the KIpURA3 vector. In addition to the structure, the promoter strength has been increased by the insertion of further bonding sites. —It was also possible to create variants. The result was that this vector system could be extended. It is a lactose-inducible synthetic promoter that produces different proteins under the same induction conditions. Alternatively, it may be possible to achieve a specific gene expression rate.

[0087] (Example 4.1: Expression of heterologous antigens under the control of various LAC4 promoter variants) Expression of Etx.B-HA under the control of four LAC4-12 promoter variants. The expression tested was transcriptional activity The number of binding sites for tivator KlGal4 differs, and the regulatory region of basic expression under non-inducible conditions (bas These are four LAC4 promoter variants that differ in the presence or absence of the control region BCR (Figure 6A; Sequence ID: 14). Using the aforementioned promoter variant, the KIpURA3-Et vector variant KIpURA3-PL412-Et, KIpURA3- PL412LR2-Et, KIpURA3-PL4-Et, and KIpURA3-PL4LR2 were prepared, and each was converted into Etx.B-HA protein. Quality was inserted as the test GOI. As mentioned above, the insertion of alternative GOIs was limited to the AscI and NotI. This is possible. The expression cassette is inserted into the KlURA3 gene locus, and the protein concentration of Etx.B-HA is adjusted. Quantitative analysis was performed using Western blotting (Figure 6B). The results showed that under the same induction conditions, (lact (In complete medium containing -4 hours), the LAC4 and LAC12 genes contain a complete intergenetic region, and 4 The longest promoter variant P, which includes KlGal4-binding sites (U1, U2, U4, U5) (Godecke et al. (1991)). LAC 4-12 The best protein concentration is obtained from the two U1 and U2 proximal to LAC4. If only the binding site (-1064 to -10) is present, the additional deletion of the BCR (-1540 to -1065) is similar. It has a protein-reducing effect under induction conditions.

[0088] (Example 5: Increased antigen production due to increased copy number of the gene encoding the antigen) Therefore, the aforementioned vector system rapidly and efficiently merges multiple gene copies in sequence. To combine them, and to introduce this expression cassette into one of the three gene loci in a single step. , modified (Figure 7A). To create a tandem expression cassette that can be integrated at the LAC4 locus, three PCR-amplified fragments were fused in one step using any KIp3(-MCS)-GOI template of interest (in-fu sion cloning): (1 and 2) P LAC4-LR2 and T TEF (primers: VK30 and VK31, and, VK32 and VK33), and (3) an expression cassette containing the LAC4 target sequence (VK34 and VK35)). For example, after digestion with 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 regulated by either P or P LAC4-12 depending on the starting strain, and the second gene LAC4-12-LR2 copy is regulated by P . Alternatively, if a selection marker between the two expression cassettes is inserted at the restriction sites SmiI, MluI or PmeI and the LAC4 target sequence is removed with KpnI, a tandem LAC4-LR2 cassette that is integrated randomly into the genome via NHEJ is created when the LAC4 target sequence is removed via KpnI . When the expression cassette is excised using MreI and AvaI, compatible ends are ligated, thereby allowing the creation of longer, multiple expression cassettes . By repeating the digestion using MreI and AvaI, fragments where the expression cassettes are arranged in tandem (head-to-tail) are enriched in the ligation mix. They are transformed and integrated randomly under marker selection conditions. Thereby, it is possible to create longer, multiple expression cassettes. By repeating the restriction using MreI and AvaI, fragments where the expression cassettes are arranged in tandem (head-to-tail) are enriched in the ligation mix. They are transformed and integrated randomly under marker selection conditions. They are transformed and integrated randomly under marker selection conditions.

[0089] Primers:

Chemical formula

[0090] (Example 5.1: Successful use of the multicopy strategy) This strategy involves using IBDV-VP2 as an antigen and two sequences encoding IBDV-VP2 (CDS-VP2). IBD V This was confirmed by using an expression cassette derived from KIp3 containing ) in tandem. Kutar (plasmid KIp3-tandem-oVP2) T2S Tandem IBDV-VP2 expression case within Sequence ID: 21) The kit (Figure 7A) is KIp3-MCS-oVP2 T2S (From Arnold et al., (2012)) VP2 IBDV (CDS-VP2 IBDV ) It consists of two LAC4 promoter-controlled coding sequences. The promoter sequence is initially From the LAC4 promoter region -1123 to -10 for the first copy, and from -1099 to -10 for the second copy. Both CDS-VP2 IBDV It is adjacent to the AgTEF1 terminator at its 3' end. Plasmid KIp3-Tandem-oVP2 T2S The restricted material was cut using HpaI and transformed into strain VAK367-D4. The yeast strain VAK1118 produced in this way was incorporated into the LAC4 gene locus in a tandem gene. Includes expression cassette. As shown by Western blotting, the genetic background is homogeneous, 1 Compared to strains containing only one copy, the aforementioned strain exhibits a higher IBDV-VP2 protein concentration. (Figure 7B). The tandem expression cassette is genetically highly stable and does not involve an inducible medium (YNB+). After growing for more than 78 generations in lactose, 100 colonies were tested by PCR, and the expression cassette was... No genetic changes were found (data not shown).

[0091] (Example 6: Producing prototrophicity of K. lactis strain for simple fermentation in synthetic and complete media) (A tool for outputting) In the studies conducted, uracil-dependent yeast strains were found to be uracil prototrophic strains in complete culture media. The growth is poorer than that, which is partially neutralized by the addition of uracil. It was revealed that this was the effect. In order to simplify the fermentation of the vaccine strain, the production process To promote the establishment of methionine and / or uranium, and to make them more cost-effective. To avoid the proliferation effect caused by insufficient uptake of sil, what needs to be discovered is eye This method aims to quickly and reproducibly neutralize the nutritional requirements necessary for constructing the target strain. To reconstruct Klura3 from Klura3-20, primers VK67 and VK69, as well as Temp Using the wild-type KlURA3 gene as the reference, DNA fragments were constructed by PCR (Figure 8A). To repair the met5-1 allele, primers VK74 and VK75, and a template are used. PCR fragments were similarly prepared using the wild-type allele KlMET5 (Figure 8B). The mutant strains were transformed (individually or together) and methionine-free and / or uracil-free. When selected in the culture medium, wild-type alleles are reconstituted with high efficiency. This process is This was done to produce strains VAK1171 and VAK1400 (see above).

[0092] Primer [ka]

[0093] (Example 7: Protective immunization with optimized and inactivated vaccine yeast) The modifications and optimizations of the K. lactis vaccine platform performed in Examples 1-5 were: This has been verified in various vaccine studies.

[0094] (Example 7.1: Optimized K. lactis using the IBDV-VP2 yeast strain (VAK1127)) (Immunogenicity of the platform) The VAK1127 strain has a tandem IBDV-VP2 expression cassette (SEQ ID NO: 21), two KlGAL4 copies and L Includes LR2 deletion within the AC4 promoter. For characterizing the immunogenicity of yeast strains, in target organisms... An immunization experiment was conducted on a chicken. In the challenge experiment, Eterradossi and colleagues (1997) A highly virulent (vv)IBDV strain 89163 / 7.3 (AFSSA, Ploufragan), well characterized by ) Complete protection of SPF chickens against (France) was achieved (Tables 1 and 2). Therefore, in two independent experiments, 1 mg of freeze-dried and heat-inactivated (2 hours, 90°C) was used. Yeast (VAK1127) was administered subcutaneously twice (Figures 9A and B) with incomplete Freund's adjuvant (IFA) at ℃. Administered (Prime Boost). Administration was performed at 2 weeks and 4 weeks after hatching, and viral exposure (C) The challenge was performed at 6 weeks after hatching. 19 days later, animals vaccinated with VAK1127 showed anti-IBDV- High titers of VP2 antibodies were already measurable. In the control group, the titer of anti-IBDV-VP2 antibody was This occurred only after vvIBDV challenge (Figure 9). In both experiments, the patient was vaccinated with VAK1127. Complete protection (0% morbidity, 0% mortality) was observed in animals against challenge with vvIBDV. Tables 1 and 2). In these experiments, subunits were found using the classical primer-boost vaccination method. Using the cot vaccine, we were able to observe protection against vvIBDV.

[0095] The immunogenicity of vaccine yeast is under the influence of genetic reverse mutations into prototrophic yeast strains that possess the antigen. It does not affect the mouse. This is because the trophicity type or prototrophic type of the IBDV-VP2 yeast strain affects the mouse. This was demonstrated in a vaccination experiment (Figure 10C). The yeast strain VAK1127 (nutrient requirement) was used as described above. In (Example 6; Figure 8), the prototrophic stage was created in two steps using PCR fragments to produce VAK1171. The two strains did not show significant differences in recombinant protein expression levels (Figure 10A and B). Mice were subcutaneously vaccinated with 0.1 mg of heat-inactivated yeast together with IFA three times at two-week intervals. The cellophane between the trophic IBDV-VP2 strain (VAK1127) and its prototrophic offspring (VAK1171) was found. We were unable to establish any difference in strength between the versions (Figure 10C).

[0096] (Example 7.2: Complete protection by vaccination using a "single-dose" scheme) "Single-dose" vaccination, that is, vaccination with a single dose of vaccine, is usually, Subunit vaccines are ineffective because they lack immunogenicity. However, prime / boost Study data on antibody titers obtained using the VAK1127 strain optimized by the STR method (Figure 9) are single-stage This demonstrates the possibility of protection being obtained through an inoculation approach. This involves the prototrophic yeast strain VAK1171. This was confirmed by administering the vaccine as a single dose (Figure 11; Table 3). To achieve this, administer yeast once at the target high dose (10 mg), followed by 4 weeks. The challenge was conducted at intervals. With VAK1171, complete protection against vvIBDV was achieved (morbidity 0%, death). A mortality rate of 0% was found to be achievable using a single dose (Table 3). These results can be attributed to the high protective antibody titers achieved approximately 20 days after vaccination. (Figure 11). A single-dose vaccination scheme provides high protection against vvIBDV. This fact suggests the potential for the potent immunogenicity of the vaccine used, and the optimization of the vaccine. It provides excellent validation of the platform.

[0097] (Example 7.3: Monovalent yeast vaccine when used against influenza A virus infection) (Improved protection with bivalent yeast vaccine compared to other vaccines) Three different vaccines are used to administer the influenza A virus vaccine. A strain was created. First, the major antigen of influenza A strain (Puerto Rico / 8 / 1934; PR8 / 34) was used. We created VAK952 (DSM32705), which expresses the HA (hemagglutinin) gene. In VAK952, As described by Krijger et al. (2012) and Arnold et al. (2012), the gene is located at the LAC4 locus of the genome. It is incorporated into. Secondly, VAK1283 (DSM32697) was created. Here, the LAC4 gene In addition to the HA gene from the PR8 / 34 locus, the M1 gene is further integrated into the URA3 locus. The M1 gene is clearly more conserved than the HA gene and contains the more important influenza A antigen. It is being reported that when both antigens are combined, influenza To enhance the immunogenicity of the vaccine against influenza A, and against different influenza viruses. It has been shown that cross-protection can be achieved. This aspect will be verified with a bivalent yeast vaccine. Therefore, the URA3 locus also contains the M1 gene, and the HA gene from PR8 / 34 is influenza The HA gene of the virus (California / 4 / 2009) has been replaced in the strain (VAK1395;DSM3270) 6) was further prepared. Equivalent expression of HA and additional expression of M1 were confirmed in each strain. Furthermore, these strains showed comparable growth, with VAK1283 being slightly superior to VAK952. This was done (Figure 12). In a mouse model, prime boost schemes and single-batch tests were performed with different yeast concentrations. In the vaccination studies where the vaccination scheme was used in each case, VAK952 and VAK1283 It was shown that each of these induces equivalent titers of virus-neutralizing antibodies (Figure 13). However, In the challenge experiment, the bivalent VAK1283 vaccine showed a prime boost scheme and a single-dose scheme. It has become clear that this monovalent VAK952 vaccine provides maximum protection in both the genital and malformations. This is different from the case. Furthermore, in a single-dose inoculation experiment using half of the yeast material used, the vaccine VAK1283 Using this, a similar defensive effect to that of VAK952 in the prime boost approach was achieved. (Figure 14 and Table 3). In experiments using VAK1395 as a vaccine, it was effective against influenza PR8 / 34. It was also possible to establish a defense against cross-breeding against different influenza variants. Protection was achieved using a bivalent yeast vaccine.

[0098] [Table 1]

[0099] [Table 2]

[0100] [Table 3]

[0101] (Note for Table 1) (a) Administer 1 mg of yeast (or PBS) and IFA as an adjuvant subcutaneously to chickens two weeks after hatching. I was vaccinated. Two weeks after vaccination, I was boosted again. Two weeks later, I received the virus. Exposure test, 10 4 The drug was administered via the ophthalmosin route using EID vvIBDV (highly toxic 89163 / 7.3). The entire inactivated strain VAK1127 yeast was used as the vaccine yeast, and the vaccine was administered using only PBS and IFA. The group inoculated with tin was used as an infection control. The group was treated with wild-type yeast (VAK367) that did not contain the antigen. The group administered ) was used as a control for the yeast effect alone.

[0102] (b) Histopathological evaluation of cystic lesions was performed using a scale of 0 to 4. 0: No lesion. 1: Filtration 1: 5-25% of the follicles were affected. 2: 26-50% of the follicles were affected. 3: 51-75% of the follicles were affected. It was affected. 76-100% sac damage (loss of structure).

[0103] (c) The average of the bag weight versus body weight index (bu / bod) is calculated using the formula: (bag weight / body weight) × 1000 The calculation was performed. The unexposed control group consisted of at least 7 chickens, while the exposed group consisted of 10 or It will also display the standard deviation.

[0104] (d) The incidence rate is expressed as the number of affected chickens per total number of chickens in the flock. Percentages are shown in parentheses.

[0105] (e) Mortality rate is expressed as the number of dead chickens per total number of chickens in the flock. Percentages are shown in parentheses.

[0106] (Notes for Table 2) (a) Administer 1 mg of yeast (or PBS) and IFA as an adjuvant subcutaneously to chickens two weeks after hatching. I was vaccinated. Two weeks after vaccination, I was boosted again. Two weeks later, I received the virus. Exposure test, 10 4 The drug was administered via the ophthalmosin route using EID vvIBDV (highly toxic 89163 / 7.3). The entire inactivated strain VAK1127 yeast was used as the vaccine yeast, and the vaccine was administered using only PBS and IFA. The group that received the chlorine vaccine was used as an infection control group.

[0107] (b) Histopathological evaluation of cystic lesions was performed using a scale of 0 to 4. 0: No lesion. 1: Filtration 1: 5-25% of the follicles were affected. 2: 26-50% of the follicles were affected. 3: 51-75% of the follicles were affected. It was affected. 76-100% sac damage (loss of structure).

[0108] (c) The average of the bag weight versus body weight index (bu / bod) is calculated using the formula: (bag weight / body weight) × 1000 The calculations were performed. The unexposed control group consisted of at least 5 chickens, while the exposed group consisted of 9 chickens. Yes, it will. The standard deviation will also be displayed.

[0109] (d) The incidence rate is expressed as the number of affected chickens per total number of chickens in the flock. Percentages are shown in parentheses.

[0110] (e) Mortality rate is expressed as the number of dead chickens per total number of chickens in the flock. Percentages are shown in parentheses.

[0111] (Notes for Table 3) (a) Administer 10 mg of yeast (or PBS) and IFA as an adjuvant subcutaneously to chickens two weeks after hatching. I was vaccinated. I will undergo a viral exposure test four weeks later. 4 EID vvIBDV (highly toxic 89163 / 7.3) was used, and the procedure was carried out via the oculonasal route. The entire inactivated strain VAK1171 yeast was administered once as a yeast vaccine. It was used as a vaccine. The infection control used was primarily PBS and MF59, and the vaccine was administered only to the vaccine. In the seeded group, the second group was inoculated with wild-type yeast and MF59 two weeks after the initial vaccination. Both groups received the same amount of boost containing yeast or PBS.

[0112] (b) Histopathological evaluation of cystic lesions was performed using a scale of 0 to 4. 0: No lesion. 1: Filtration 1: 5-25% of the follicles were affected. 2: 26-50% of the follicles were affected. 3: 51-75% of the follicles were affected. It was affected. 76-100% sac damage (loss of structure).

[0113] (c) The average of the bag weight versus body weight index (bu / bod) is calculated using the formula: (bag weight / body weight) × 1000 The calculations were performed. Each group consists of at least 9 chickens. The standard deviation is also displayed.

[0114] (d) The incidence rate is expressed as the number of affected chickens per total number of chickens in the flock. Percentages are shown in parentheses.

[0115] (e) Mortality rate is expressed as the number of dead chickens per total number of chickens in the flock. Percentages are shown in parentheses.

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

[0117] [Table 4] [Advanced Technology Documents]

Non-licensed literature

[0118] (References)

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Claims

[Claim 1] The novel products, methods and processes substantially as herein described.

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