EA046085B1 - NEW EHV INSERT SITE UL-43 - Google Patents

Description

List of sequences

The invention contains a sequence listing in accordance with 37 C.F.R. 1.821 - 1.825. The sequence listing accompanying the invention is hereby incorporated by reference in its entirety.

Prerequisites for creating the invention

A. The technical field to which the invention relates.

The present invention relates to the field of (vector) vaccines, and in particular to the new EHV insertion site UL43. The present invention further relates to related expression cassettes and vectors that are useful for expressing genes of interest, particularly antigen coding sequences. The viral vectors of the present invention are useful for producing an immunogenic composition or vaccine.

B. Background and Description of the Prior Art.

The equine pathogen equine alphaherpesvirus type 1 (mare abortion virus, EHV-1) belongs to the genus Varicellovirus in the subfamily Alphaherpesvirinae in the family Herpesviridae in the order Herpesvirales. It is a large, enveloped virus with a double-stranded DNA genome of approximately 150,000 base pairs. Other important members of the subgenus Varicellovirus are human alphaherpesvirus type 3 (varicella zoster virus), Aujeszky's disease virus 1 (pseudorabies virus), bovine alphaherpesvirus type 1 (infectious bronchitis virus), and equine alphaherpesvirus type 4 (equine rhinopneumonia virus, EHV-4). (http://www.ictvonline.org/virustaxonomy.asp Virus Taxonomy: 2015 Release EC 47, London, UK, July 2015; Email ratification 2016 (MSL #30)). EHV-1 and EHV-4 are endemic and affect horses worldwide. While EHV-4 causes predominantly mild upper respiratory tract infection, EHV-1 can cause systemic infection with a range of illnesses ranging from respiratory symptoms to abortion and fatal myeloencephalopathy depending on the strain and immunological status of the host. There are currently two licensed modified live vaccines (MLV) against EHV-1 in the US and Europe, Rhinomune® (Boehringer Ingelheim) and Prevaccinol® (MSD), respectively. Both contain a classically attenuated EHV-1 RacH strain that has been passivated 256 times in porcine epithelial cells for attenuation (Ma et al. 2013). The mechanism of attenuation was studied at the molecular level. Osterrieder et al. (1996) showed that RacH lacks two genomic copies of orf67 (IR6) and that restoration of one copy is sufficient to restore virulence. In addition, RacH carries a 1283 deletion that removes more than 90% of the coding sequence of orf1(UL56), which encodes an immunosuppressive viral protein. Other mutations may also have an effect on attenuation, but have not been studied in detail to date. All this makes RacH an extremely safe vaccine strain, since restoration of virulence by passivation in vaccinated animals is extremely unlikely, if not impossible.

Two variants of the E. coli bacterial artificial chromosome (BAC) anchoring the complete genome of the RacH vaccine strain of equine alphaherpesvirus type 1 (EHV-1): pRacH and pRacH-SE are known as platforms for vector vaccine development. The BAC pRacH-SE was generated from pRacH, with the BAC initially cloned in the laboratory of Klaus Osterrieder, FU Berlin. pRacH has a deletion of orf71 (US5), encoding glycoprotein II (gpII; Wellington et al., 1996). BAC vector sequences and a GFP expression cassette were introduced into this location. However, in order to recover unmodified EHV-1 RacH from pRacH, it must be cotransfected using a plasmid containing the complete orf71 (US5) plus flanking regions, thus, during viral replication, parts of the BAC vector sequence and the GFP expression cassette are replaced by orf71 (US5) by homologous recombination in such a way that the original RacH genome is restored. pRacH has been modified in the present invention such that the BAC vector sequences/GFP expression cassette become self-truncating (SE) when transfected in cell cultures (Tischer et al., 2007). This improved BAC was designated pRacH-SE. pRacH and pRacH-SE can both serve as platforms for vector vaccine development, with the only difference being that pRacH-SE significantly facilitates the recovery of orf71 (US5) -repaired virus.

EHV-1 RacH vector vaccines have been shown to induce immunity in several mammalian species, including pigs, cattle and dogs (Rosas et al. 2007, Rosas et al. 2008, Trapp et al. 2005, Said et al. . 2013). Genes encoding antigenic proteins of pathogens can be expressed using recombinant EHV-1 RacH. The EHV-1-RacH genome is manipulated in its BAC form in E. coli and adapted to express additional proteins, usually by insertion of transgene expression cassettes (Tischer et al., 2010). When pRacH-SE DNA is transfected into cultured permissive cells, EHV-1 replication is initiated by cellular transcription factors. Viral DNA polymerase activity results in the deletion of all BAC vector-associated sequences and restoration of the EHV-1 RacH genome to its original state. A pathogenic virus is created that is indistinguishable from RacH.

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If pRacH-SE is manipulated in E. coli, for example by insertion of transgene expression cassettes, then the virus recovered from transfection in permissive cells will carry the modification and will express the additional gene. Recombinant EHV-1 RacH can be used as a vector vaccine.

Wild-type EHV-1 strains have three open reading frames (orfs), called orf1 (UL56), orf 2, and orf3, at one end of a long, unique segment of their genome (sequence coordinates 1298-3614; Fig. 1). Orf1 (UL56) and orf3 are sequentially assembled on the same DNA strand, while orf2 is encoded by the complementary strand. The RacH vaccine strain has a 1283 bp deletion in this region involving orf 1 and orf 2, indicating that these genes are nonessential for viral replication. For this reason, this site serves as the transgene insertion site. This insertion site is called ORF1/3 (UL56).

However, the size and number of transgenes that can be inserted into the ORF1/3 (UL56) insertion site are usually limited. Thus, in order to expand the capabilities of the EHV vector, there is an unmet need for new and alternative ways of insertion and expression of transgenes from the EHV vector, especially the recombinant EHV-1 RacH vector.

Summary of the invention

In order to enhance the capabilities of the EHV vector, the present invention provides new and alternative routes for the insertion and expression of transgenes from the EHV vector backbone.

The present invention relates to a new, alternative insertion site for the UL43 transgene that can be used to insert a transgene sequence and express a transgene protein from an EHV vector, particularly recombinant EHV-1 RacH.

The new UL43 insertion site in the EHV vector is characterized by partial deletion, truncation, substitution, modification, or the like. relative to UL43 (ORF17). It is believed that deletion of complete UL43 will be detrimental to viral replication and thus vaccine production and efficacy because complete deletion of UL43 will affect the UL44 promoter encoding glycoprotein C. New UL43 insertion site and/or insertion (expression cassette) into UL43 functionally defined such that UL44 remains functional or intact.

In a specific aspect, the UL43 insertion site spans the deletion of approximately the 870 portion of (SEQ ID NO: 21) within UL43 for RacH (SEQ ID NO: 18) or 70, 80, 85, 90, 95, 99% of its homologous sequence. The deleted portion of the RacH genomic sequence is shown at SEQ ID NO: 21 (no nucleotide numbers available because the complete RacH genomic sequence is not known). In another specific aspect, the UL43 insertion site spans the theoretical 870 deletion within UL43 (nt 23021 to 24226, reverse complementary) for wild-type EHV-1 strain V592 (Genbank accession number AY464052.1) (SEQ ID NO: 23). The deleted portion corresponds to the wild-type V592 genomic sequence (Genbank accession number AY464052.1), nucleotides 23353 and 24226, with reverse complementarity (SEQ ID NO: 24).

In the present invention, the flanking regions direct recombination of an expression cassette comprising a sequence or gene of interest, preferably an antigen coding sequence, in the EHV genome. These flanking regions are naturally present in EHV. The Up UL43 flanking region (226 bp, SEQ ID NO: 19) and the Up UL44 flanking region (305 bp, SEQ ID NO: 20) are selected for classical homologous recombination for all transfer vectors/plasmids used for the UL43 site. In wild-type EHV-1 strain V592 (Genbank accession number AY464052.1), the corresponding sequences are located at nucleotides 24227-24452 with reverse complementarity (flanking region up UL43, SEQ ID NO: 26) and 23049-23354 with reverse complementarity (flanking region up UL44, SEQ ID NO: 27).

Plasmid/vector maps in Figs. 21 for transfer plasmid pU-mC70-BGH (SEQ ID NO: 37), in FIG. 22 for transfer vector pU70-p455-71K71 (SEQ ID NO: 28), and FIG. 3 for transfer plasmid pU70-p455-H3-71K71 (SEQ ID NO: 29) are examples of vectors containing an expression cassette including a new ORF70 insertion site. Plasmid/vector maps in Figs. 23 for transfer vector pU-1-3-p430-BGHKBGH (SEQ ID NO: 30), and FIG. 4 for transfer plasmid pU1-3-p430-H1av-BGHKBGH (SEQ ID NO: 31) are examples of vectors containing an expression cassette including an ORF1/3 (UL56) insertion site.

Plasmid/vector maps in Figs. 24 for the transfer vector pUUL43-p422-18K18 (SEQ ID NO: 34), in FIG. 14 for transfer plasmid pUUL43-422-mC-18K18 (SeQ ID NO: 35), and FIG. 16 for transfer plasmid pUUL43-422-H1pdm-18K18 (SEQ ID NO: 36) are examples of vectors containing an expression cassette including a new UL43 insertion site.

The present invention further provides an EHV vector expressing two different transgenes from a single vector backbone without linking the two transgenes using RNA virus-derived functions (2a peptides, IRES sites) under the control of a single promoter.

The present invention further provides an EHV vector expressing two or three different transgenes from a single vector backbone without linking the two or three transgenes using RNA virus-derived functions (2a peptides, IRES sites) under the control of one

- 2 046085 promoters.

The present invention further provides an equine alphaherpesvirus (EHV) vector, preferably EHV-1, RacH or RacH-SE, comprising a first sequence or gene of interest inserted into a new UL43 insertion site, and a second sequence or gene of interest inserted into a well-known insertion site, such as ORF1/3 (UL56) or another ORF70 insertion site (US4). In addition, the present invention further relates to vectors based on other herpesviruses, in particular alphaherpesviruses, in particular Varicelloviruses, including equine alphaherpesvirus type 3 (EHV-3), equine alphaherpesvirus type 4 (EHV-4), equine alphaherpesvirus type 8 (EHV -8), equine alphaherpesvirus type 9 (EHV-9), bovine alphaherpesvirus type 1 (BHV-1), bovine alphaherpesvirus type 5 (BHV-5), canine alphaherpesvirus type 1, and feline alphaherpesvirus type 1.

The present invention further provides an equine alphaherpesvirus (EHV) vector, preferably EHV-1, RacH or RacH-SE, comprising a first sequence or gene of interest inserted into a new UL43 insertion site, and a second sequence or gene of interest inserted into another ORF70 (US4) insertion site, and a third sequence or gene of interest inserted into a well known insertion site , such as ORF1/3 (UL56). In addition, the present invention further relates to vectors based on other herpesviruses, in particular alphaherpesviruses, in particular Varicelloviruses, including equine alphaherpesvirus type 3 (EHV-3), equine alphaherpesvirus type 4 (EHV-4), equine alphaherpesvirus type 8 (EHV -8), equine alphaherpesvirus type 9 (EHV9), bovine alphaherpesvirus type 1 (BHV-1), bovine alphaherpesvirus type 5 (BHV-5), canine alphaherpesvirus type 1, and feline alphaherpesvirus type 1.

The present invention further relates to mammalian host cells comprising such vectors and methods for creating vector vaccines using such host cells, as well as immunogenic compositions and vaccines comprising the equine alphaherpesvirus (EHV) vector of the present invention.

The present invention further relates to a promoter sequence comprising p422 (SEQ ID NO: 5) or a complementary nucleotide sequence thereof or a functional fragment thereof or complementary nucleotide sequences thereof, wherein said promoter sequence results in the expression of a nucleotide sequence of interest, preferably a gene of interest, more preferably antigen coding sequence. The present invention also provides functional promoter sequence fragments having sequence identity and/or homology of 70, 80, 85%, preferably 90, 91, 92, 93, 94%, more preferably 95, 96, 97, 98, 99, 99.9%.

Thus, the solution to the above-described technical problem is achieved by the description and embodiments described in the claims, and the invention in its various aspects is implemented in accordance with the claims.

These properties provide the opportunity to create recombinant EHV vector vaccines, preferably EHV-1 RacH, expressing at least one antigen from the first described UL43 insertion site or at least two different antigens in parallel with similar potency from the first described UL43 insertion site and another site insertion like ORF1/3 (UL56) and/or another insertion site ORF70 (US4). If the vaccine target consists of two different pathogens, then the use of a new UL43 insertion site in parallel with a well-known ORF1/3-like insertion site (UL56) and/or another ORF70 insertion site (US4) can reduce the cost of goods significantly and represents an obvious advantage over a vector expressing only one antigenic component.

These properties provide the opportunity to create recombinant EHV vector vaccines, preferably EHV-1 RacH, expressing at least one antigen from the first described UL43 insertion site, or at least two different antigens in parallel with similar potency from the first described UL43 insertion site and another an ORF1/3-like insertion site (UL56), or another ORF70 insertion site (US4), or at least three different antigens in parallel from the first described UL43 insertion site and another ORF70 insertion site (US4), and another ORF1-like insertion site /3 (UL56). If the vaccine target consists of two different pathogens, then the use of a new UL43 insertion site in parallel with a well-known ORF1/3-like insertion site (UL56) or another ORF70 insertion site (US4) can reduce the cost of goods significantly and represents a clear advantage in compared to a vector expressing only one antigenic component. If the vaccine target consists of three different pathogens, then using the new UL43 insertion site in parallel with the well-known ORF1/3-like insertion site (UL56) and another ORF70 insertion site (US4) can reduce the cost of goods significantly and represents a clear advantage in compared to a vector expressing only one or two antigenic components.

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Detailed Description of the Invention

The present invention solves the problems inherent in the prior art and provides a significant improvement over the prior art.

In general, the present invention provides an expression cassette comprising (i) at least one exogenous nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence, wherein said nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence, operably linked to the promoter sequence, and (ii) at least one upstream UL43 flanking region selected from the group consisting of: SEQ ID NO: 19 and at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, SEQ ID NO: 26 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99% of its homologous and/or identical sequence, and (iii) at least one upstream UL44 flanking region selected from the group consisting of: SEQ ID NO: 20 and at 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, SEQ ID NO: 27 and at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence.

The present invention further provides equine herpesvirus (EHV), specifically equine alphaherpesvirus such as EHV-1, EHV-3, EHV-4, EHV-8 and EHV-9, more specifically equine alphaherpesvirus type 1 (EHV-1) vector, most specifically a RacH strain comprising the expression cassette of the present invention.

The present invention provides an equine alphaherpesvirus (EHV) vector, preferably EHV-1 or RacH strain, comprising an expression cassette of the present invention.

Furthermore, the present invention relates to equine herpesvirus (EHV), specifically equine alphaherpesvirus such as EHV-1, EHV-3, EHV-4, EHV-8 and EHV-9, more specifically equine alphaherpesvirus type 1 (EHV-1) vector , most specifically a RacH strain comprising (i) no at least one exogenous nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence, wherein said nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence, is operably linked to promoter sequence, and (ii) at least one upstream of UL43 flanking region selected from the group consisting of: SEQ ID NO: 19 and at 70, 80, 85, 90, 91, 92, 93, 94, 95 , 96, 97, 98, 99% of its homologous and/or identical sequence, SEQ ID NO: 26 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% its homologous and/or identical sequence, and (iii) at least one upstream UL44 flanking region selected from the group consisting of: SEQ ID NO: 20 and at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, SEQ ID NO: 27 and at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 , 98, 99% of its homologous and/or identical sequence.

The experimental data provided by the present invention successfully show that a new insertion site has been provided within the EHV vector that can be used for insertion and expression of antigens. In addition, the creation of a new insertion site now allows for the insertion and expression of antigens from different insertion sites and the expression of more than one antigen, respectively.

The present invention further relates to equine herpesvirus (EHV), specifically equine alphaherpesvirus such as EHV-1, EHV-3, EHV-4, EHV-8 and EHV-9, more specifically equine alphaherpesvirus type 1 (EHV-1) vector, most specifically a RacH strain comprising a nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence inserted into UL43.

The present invention further relates to equine herpesvirus (EHV), specifically equine alphaherpesvirus such as EHV-1, EHV-3, EHV-4, EHV-8 and EHV-9, more specifically equine alphaherpesvirus type 1 (EHV-1) vector, most specifically a RacH strain comprising a first nucleotide sequence or gene of interest, preferably an antigen coding sequence inserted into UL43, and a second nucleotide sequence or gene of interest, preferably another antigen coding sequence inserted into a second insertion site, preferably UL56 (orf1/3) or US4 (orf70). In a specific aspect of the EHV vector of the present invention, at least two genes of interest are operably linked to regulatory sequences, preferably promoter sequences.

In a specific aspect of the vector of the present invention, the insertion in UL43 is characterized by a partial deletion, truncation, substitution, modification, or the like. in UL43, with UL44 remaining functional.

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In another specific aspect of the vector of the present invention, the insertion in UL43 is characterized by a deletion of approximately 870 parts within UL43 for RacH (SEQ ID NO: 21) or 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence.

In another specific aspect of the vector of the present invention, the insertion in UL43 is characterized by a deletion of approximately 870 portions within UL43 for RacH (SEQ ID NO: 21) or a deletion thereof of 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence in any other strain.

In a further specific aspect of the vector of the present invention, the insertion in UL43 is characterized by a deletion of approximately 870 portions within UL43 for wild-type EHV-1 strain V592 (Genbank accession number AY464052.1), the deleted portion being located in the wild-type V592 genomic sequence between nucleotides 23353 and 24226 (SEQ ID NO: 24), or 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homologous and/or identical sequence.

In another specific aspect of the vector of the present invention, the EHV vector, specifically the EHV-1 vector, includes (i) at least one upstream UL43 flanking region selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 26 , and (ii) at least one upstream of UL44 flanking region selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 27.

In a further specific aspect of the vector or expression cassette of the present invention, said nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence, is not naturally occurring and/or is recombinant.

In another specific aspect of the vector or expression cassette of the present invention, said nucleotide sequence of interest is recombinant and/or heterologous and/or exogenous.

In a further specific aspect of the vector or expression cassette of the present invention, said antigen coding sequence relates to a pathogen infecting an animal, such as a food animal such as a pig, poultry or cattle, or companion animals such as cats, dogs or horses.

In a further specific aspect of the vector or expression cassette of the present invention, said vector or expression cassette further includes at least one additional regulatory sequence, such as a stop codon or a polyadenylation sequence.

In another specific aspect of the vector or expression cassette of the present invention, said vector or expression cassette further includes additional regulatory sequences, such as a stop codon and/or a polyadenylation sequence.

In a further specific aspect of the vector or expression cassette of the present invention, said vector or expression cassette further includes at least one additional nucleotide sequence of interest, preferably another gene of interest, more preferably an antigen coding sequence. In one aspect, at least one additional nucleotide sequence of interest, preferably another gene of interest, more preferably an antigen coding sequence, is inserted into the same UL43 insertion site, for example, by IRES/2a peptide(s). In another aspect, said vector or expression cassette includes at least one additional nucleotide sequence of interest, preferably another gene of interest, more preferably an antigen coding sequence, inserted into another insertion site, preferably in UL56 and/or US4.

In a further aspect of the vector or expression cassette of the present invention, at least one additional nucleotide sequence of interest, preferably another gene of interest, more preferably an antigen coding sequence, is inserted into UL56. The UL56 (ORF1/3) insertion site has been described in the prior art.

In a further aspect of the vector or expression cassette of the present invention, at least one additional nucleotide sequence of interest, preferably another gene of interest, more preferably an antigen coding sequence, is inserted into US4 (ORF70).

An alternative US4 transgene insertion site (ORF70) can be used to insert a transgene sequence and express a transgene protein from an EHV vector, particularly recombinant EHV-1 or EHV-1 RacH.

The US4 (ORF70) insertion site in the EHV vector is characterized by partial deletion, truncation, substitution, modification, or the like. relative to US4 (ORF70). It is believed that deletion of complete US4 (ORF70) will be detrimental to viral replication and thus vaccine production and efficacy, since complete deletion of US4 (ORF70) will affect the promoter of US5 (ORF71),

- 5 046085 encoding gpII. The US4 (ORF70) insertion site and/or insertion (expression cassette) into US4 (ORF70) is functionally defined such that US5 (ORF71) remains functional or intact.

In a specific aspect, the US4 (ORF70) insertion site spans the deletion of approximately 801 bp within US4 (ORF70) for RacH (SEQ ID NO: 17) or by 70%, 80%, 85%, 90%, 95%, 99% its homologous sequence. The deleted portion of the RacH genomic sequence is shown at SEQ ID NO: 17 (no nucleotide numbers available because the complete RacH genomic sequence is not known). In another specific aspect, the ORF70 insertion site spans the theoretical 801 deletion within ORF70 for EHV-1 wild-type strain ab4 (Genbank accession number AY665713.1, SEQ ID NO: 16). The deleted portion is located on the wild-type ab4 genomic sequence (Genbank accession number AY665713.1) between nucleotides 127681 and 128482 (SEQ ID NO: 16).

In the present invention, the flanking regions direct recombination of an expression cassette comprising a sequence or gene of interest, preferably an antigen coding sequence, into the EHV genome. These flanking regions are naturally present in EHV. The Up70 flanking region (417 bp, SEQ ID NO: 9) and the Up71 flanking region (431 bp, SEQ ID NO: 10) are selected for classical homologous recombination for all transfer vectors/plasmids used for the orf70 site. In the EHV-1 wild-type ab4 strain (Genbank accession number AY665713.1), the corresponding sequences are located at nucleotides 127264 - 127680 (flanking region up orf70, SEQ ID NO: 11) and 128483 - 128913 (flanking region up orf71, SEQ ID NO: 12). For RED recombination, the flanking regions are truncated due to cleavage with the restriction enzyme XbaI. These truncated flanking regions are identical to 3'283 of 417 of the classical flanking region (Up70 flanking region, SEQ ID NO: 9) and 5'144 of 431 of the classical flanking region (Up71 flanking region, SEQ ID NO: 10) which are described above. These truncated flanking regions are named Up70 flanking region (283 bp), included as SEQ ID NO: 13, and Up71 flanking region (144 bp), included as SEQ ID NO: 14. These different flanking regions define the same ORF70 insertion site . Flanking regions are always used in pairs, one left flanking region, such as SEQ ID NO: 9, 11, 13, and one right flanking region, such as SEQ ID NO: 10, 12, 14.

In a further aspect of the vector of the present invention, the vector further includes (i) at least one exogenous nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence, wherein said nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence, is functionally associated with a promoter sequence, and (ii) at least one US4 left (ORF70) flanking region selected from the group consisting of: SEQ ID NO: 9 and at 70, 80, 85, 90, 91, 92, 93, 94 , 95, 96, 97, 98, 99% of its homologous and/or identical sequence, SEQ ID NO: 11 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homologous and/or identical to the sequence, and SEQ ID NO: 13 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homologous and/or identical to it sequence, and (iii) at least one right US4 (ORF70) flanking region selected from the group consisting of: SEQ ID NO: 10 and at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, SEQ ID NO: 12 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, and SEQ ID NO: 14 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homologous and/or identical sequence.

Furthermore, the present invention relates to an equine herpesvirus (EHV), specifically an equine alphaherpesvirus type 1 (EHV-1) vector, most specifically a RacH strain, comprising (i) a first exogenous nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen-coding sequence, wherein said nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence, is operably linked to a promoter sequence, and at least one upstream UL43 flanking region selected from the group consisting of: SEQ ID NO: 19 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, SEQ ID NO: 26 and at 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, and at least one upstream UL44 flanking region selected from the group consisting of: SEQ ID NO: 20 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homologous and/or identical to the sequence, SEQ ID NO: 27 and 70, 80, 85, 90 , 91, 92, 93, 94, 95, 96, 97, 98, 99% homologous and/or identical sequence thereof, and (ii) a second exogenous nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence,

- 6 046085 wherein said nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence, is operably linked to a promoter sequence, and at least one left US4 (ORF70) flanking region selected from the group consisting of: SEQ ID NO: 9 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, SEQ ID NO: 11 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homologous and/or identical to the sequence, and SEQ ID NO: 13 and at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, and at least one right US4 (ORF70) flanking region selected from the group consisting of: SEQ ID NO: 10 and at 70, 80 , 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, SEQ ID NO: 12 and at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, and SEQ ID NO: 14 and at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence.

In a further aspect of the vector of the present invention, the insertion vector into US4 (ORF70) is characterized by a deletion of approximately 801 bp within US4 (ORF70) for RacH (SEQ ID NO: 17) or at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence.

In a further aspect of the vector of the present invention, the vector includes at least one flanking region selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homologous and/or identical sequence to any of these sequences.

In a further aspect of the vector of the present invention, the vector includes (i) at least one left US4 (ORF70) flanking region selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, and (ii) at least one US4 right flanking region selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14.

In a further aspect of the vector or expression cassette of the present invention, a second additional nucleotide sequence of interest, preferably another gene of interest, more preferably an antigen coding sequence, is inserted into US4, and a third additional nucleotide sequence of interest, preferably another gene of interest, more preferably an antigen coding sequence, inserted into UL56.

In a specific aspect of the vector of the present invention, the gene of interest is operably linked to a regulatory sequence, preferably a promoter sequence.

In a further aspect of the vector or expression cassette of the present invention, the gene of interest is operably linked to a regulatory sequence, preferably a promoter sequence, or an EHV vector as described herein, wherein at least two genes of interest are operably linked to regulatory sequences, preferably promoter sequences.

In a further aspect of the vector or expression cassette of the present invention, the promoter sequence(s) operably linked to one or two or more sequences or genes of interest are selected from the group consisting of the following: SV40 large T, HCMV and MCMV immediate early gene 1, human elongation factor alpha promoter, baculovirus polyhedrin promoter, functional fragment from 4pgG600 (SEQ ID NO: 1), preferably said functional fragment is p430 (SEQ ID NO: 3), functional fragment from complementary nucleotide sequence of 4pgG600 (SEQ ID NO: 1), a functional fragment from 4pMCP600 (SEQ ID NO: 2), preferably said functional fragment is p455 (SEQ ID NO: 4), a functional fragment from the complementary nucleotide sequence of 4pMCP600 (SEQ ID NO: 2), or p422 (SEQ ID NO: 5) or a functional fragment thereof or complementary nucleotide sequences thereof.

In a further aspect of the vector or expression cassette of the present invention, the promoter sequence operably linked to the at least one gene of interest is p422 (SEQ ID NO: 5) or a functional fragment thereof or complementary nucleotide sequences thereof.

In a specific aspect of the vector or expression cassette of the present invention, at least two genes of interest are operably linked to regulatory sequences, preferably promoter sequences.

In a further specific aspect of the vector or expression cassette of the present invention, the promoter sequences operably linked to the at least two genes of interest are different.

In another specific aspect of the vector or expression cassette of the present invention

- 7 046085 a promoter sequence operably linked to at least one gene of interest is p422 (SEQ ID NO: 5) or a functional fragment or derivative thereof or complementary nucleotide sequences thereof, and a promoter sequence operably linked to another gene of interest genome, is p430 (SEQ ID NO: 3) or a functional fragment or derivative thereof or complementary nucleotide sequences thereof, and a promoter sequence operably linked to another gene of interest is p455 (SEQ ID NO: 4) or a functional one thereof fragment or derivative thereof or their complementary nucleotide sequences.

In a further specific aspect of the vector or expression cassette of the present invention, the polyadenylation sequence is BGHpA, 71pA (SEQ ID NO: 6), or 18pA (SEQ ID NO: 7).

In a further specific aspect of the vector or expression cassette of the present invention, the EHV vector or expression cassette is recombinant.

In a further specific aspect of the vector or expression cassette of the present invention, said sequences or exogenous nucleotide sequence of interest or gene of interest are an antigen coding sequence.

In a further specific aspect of the vector or expression cassette of the present invention, the antigen coding sequence belongs to a pathogen selected from the list of: Schmallenberg virus, influenza A virus, porcine respiratory and reproductive syndrome virus, porcine circovirus, classical swine fever virus, African swine fever virus, hepatitis virus E, bovine diarrhea virus, rabies virus, feline morbillivirus, tetanus bacillus, mycobacterium tuberculosis, causative agent of actinobacillus pleuropneumonia.

In a further specific aspect of the vector or expression cassette of the present invention, the antigen coding sequence is a hemagglutinin coding sequence.

In a further specific aspect of the vector or expression cassette of the present invention, the sequence encoding the influenza hemagglutinin antigen is derived from swine influenza virus type A.

The four most common influenza A strains in Europe are the H1N2, H3N2 and H1N1 subtypes (avian H1N1 and pandemic H1N1). Thus, there is a need for vaccines that are highly effective against the H1N2, H3N2 and H1N1 subtypes (avian H1N1 and pandemic H1N1) and thus provide very broad protection against these field strains of swine IAV.

In addition, it is preferable to have a multivalent vaccine available since multivalent vaccines are generally more cost effective and time effective than monovalent vaccines.

The EHV vector vaccine disclosed herein, while not a modified live vaccine (MLV), provides maximum safety against porcine IAVs because no live IAVs are created or administered to the animals, thereby preventing potential reversion to virulence of the vaccine(s) strain(s) and genetic recombination or reassortment with field strains of pigs or humans. Moreover, unlike killed vaccines (the current standard), a vector vaccine is expected to not only induce neutralizing antibodies against porcine IAV, but also potently stimulate cellular immunity against porcine IAV through both MHC class I and II pathways. Thus, there is a need for vector-based SIAV vaccines. In addition, a vector vaccine expressing IAV hemagglutinins provides the ability to differentiate between infected and vaccinated animals (DIVA), since other influenza virus proteins that induce antibodies are not part of the vector vaccine. Thus, vaccination will not induce any antibodies specific for NP (nucleoprotein) or N (neuraminidase), both of which are structural proteins of the virus and are contained in standard killed vaccines.

In a further specific aspect of the vector or expression cassette of the present invention, the exogenous antigen coding sequence is a hemagglutinin coding sequence, and the influenza hemagglutinin subtype is selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H13, H15, H16, H17 and H18.

In a further specific aspect of the vector or expression cassette of the present invention, the exogenous antigen coding sequence is a hemagglutinin coding sequence, and the influenza hemagglutinin antigen coding sequence is selected from the group of strains consisting of A/swine/Italy/116114/2010(H1N2), A/ swine/Italy/7680/2001(H3N2), A/swine/Gent/132/2005(H1N1), A/swine/Italy/4675/2003(H1N2),

A/swine/Italy/259543/2003(H1N2), A/swine/Denmark/13772-1/2003(H1N1),

A/swine/England/MD0040352R/2009(H1N1), A/swine/Hungary/13509/2007(H3N2),

- 8 046085

A/swine/Italy/13962/95(H3N2), A/swine/Cotes d'Armor/1121/00(H1N1), A/Swine/Colorado/ 1/77, A/Swine/Colorado/23619/99, A /Swine/Cote d'Armor/3633/84, A/Swine/England/ 195852/92, A/Swine/Finistere/2899/82, A/Swine/Hong Kong/10/98, A/Swine/Hong Kong/ 9/98, A/Swine/Hong Kong/81/78, A/Swine/Illinois/100084/01, A/Swine/Illinois/100085A/01, A/Swine/Illinois/21587/99,

A/Swine/Indiana/1726/88, A/Swine/Indiana/9K035/99, A/Swine/Indiana/P1243 9/00, A/Swine/Iowa/30, A/Swine/Iowa/15/30, A /Swine/Iowa/533/99, A/Swine/Iowa/569/99, A/Swine/Iowa/3421/90, A/Swine/Iowa/8548-1/98, A/Swine/Iowa/930/01 , A/Swine/Iowa/17672/88, A/Swine/Italy/1513-1/98,

A/Swine/Italy/1523/98, A/Swine/Korea/CY02/02, A/Swine/Minnesota/55551/00, A/Swine/Minnesota/593/99, A/Swine/Minnesota/9088-2/ 98, A/Swine/Nebraska/1/92, A/Swine/Nebraska/209/98,

A/Swine/Netherlands/12/85, A/Swine/North Carolina/16497/99, A/Swine/North Carolina/35922/98, A/Swine/North Carolina/93523/01, A/Swine/North Carolina/ 98225/01, A/Swine/Oedenrode/7C/96, A/Swine/Ohio/891/01, A/Swine/Oklahoma/18717/99, A/Swine/Oklahoma/18089/99, A/Swine/Ontario/ 019111/99, A/Swine/Ontario/01911-2/99, A/Swine/Ontario/41848/97, A/Swine/Ontario/97, A/Swine/Quebec/192/81, A/Swine/Quebec/ 192/91, A/Swine/Quebec/5393/91, A/Swine/Taiwan/7310/70, A/Swine/Tennessee/24/77, A/Swine/Texas/4199-2/98, A/Swine/ Wisconsin/125/97, A/Swine/Wisconsin/136/97, A/Swine/Wisconsin/I63/97, A/Swine/Wisconsin/164/97, A/Swine/Wisconsin/166/97, A/Swine/ Wisconsin/168/97

A/Swine/Wisconsin/235/97, A/Swine/Wisconsin/238/97, A/Swine/Wisconsin/457/985

A/Swine/Wisconsin/458/98, A/Swine/Wisconsin/464/98 and A/Swine/Wisconsin/14094/99.

In a further specific aspect of the vector or expression cassette of the present invention, the exogenous antigen coding sequence is a hemagglutinin coding sequence, and the influenza hemagglutinin antigen coding sequence is selected from the group of strains consisting of A/swine/Italy/116114/2010(H1N2), A/ swine/Italy/7680/2001(H3N2), A/swine/Gent/132/2005(H1N1) and A/swine/Italy/4675/2003(H1N2).

In a further specific aspect of the vector or expression cassette of the present invention, the exogenous antigen coding sequence is a hemagglutinin coding sequence, and the influenza hemagglutinin subtype is H1 and/or H3.

In a further specific aspect of the vector or expression cassette of the present invention, the antigen coding sequence is a hemagglutinin coding sequence, and the influenza hemagglutinin antigen coding sequence includes a nucleic acid sequence encoding an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47.

In a further specific aspect of the vector or expression cassette of the present invention, the EHV vector or expression cassette does not include NP (nucleoprotein) or N (neuraminidase) sequences encoding influenza antigen.

H1pdm in UL43 with p422.

In a further specific aspect of the vector or expression cassette of the present invention, a p422 promoter sequence (SEQ ID NO: 5), or a functional fragment or functional derivative thereof, or any complementary nucleotide sequence thereof, is operably linked to a nucleic acid sequence encoding an amino acid sequence having at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as indicated in SEQ ID NO: 44 (H1pdm).

In a further specific aspect of the vector or expression cassette of the present invention, the EHV vector includes two or more sequences encoding influenza hemagglutinin antigens.

H1av in UL56 with p430.

In a further specific aspect of the vector or expression cassette of the present invention, the additional sequence encoding the influenza hemagglutinin antigen is a nucleic acid sequence encoding an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98% or at least 99% identity to the amino acid sequence as indicated in SEQ ID NO: 46 (H1av).

In a further specific aspect of the vector or expression cassette of the present invention, said additional sequence encoding a hemagglutinin antigen

- 9 046085 influenza, inserted into UL56.

In a further specific aspect of the vector or expression cassette of the present invention, the influenza hemagglutinin antigen coding sequence as described herein is operably linked to the p430 promoter sequence (SEQ ID NO: 3) or a functional fragment or functional derivative thereof or complementary nucleotide sequences thereof.

H3 to US4 with p455.

In a further specific aspect of the vector or expression cassette of the present invention, the additional sequence encoding influenza hemagglutinin antigens is a nucleic acid sequence encoding an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98% or at least 99% identity to the amino acid sequence as indicated in SEQ ID NO: 45 (H3).

In an additional specific aspect of the vector or expression cassette of the present invention, said additional sequence encoding influenza hemagglutinin antigens is inserted into US4.

In a further specific aspect of the vector or expression cassette of the present invention, the influenza hemagglutinin antigen coding sequence as described herein is operably linked to the p455 promoter sequence (SEQ ID NO: 4) or a functional fragment or functional derivative thereof or complementary nucleotide sequences thereof.

Combinations.

Furthermore, the present invention relates to an equine herpesvirus (EHV) comprising a p422 promoter sequence (SEQ ID NO: 5), or a functional fragment or functional derivative thereof, or any complementary nucleotide sequence thereof operably linked to a nucleic acid sequence encoding an amino acid sequence, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 44 (H1pdm) , inserted into UL43, and additionally a p430 promoter sequence (SEQ ID NO: 3), or a functional fragment or a functional derivative thereof, or any complementary nucleotide sequence thereof operably linked to a nucleic acid sequence encoding an amino acid sequence having at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, according at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as defined in SEQ ID NO: 46 (H1av), inserted into UL56.

Furthermore, the present invention relates to an equine herpesvirus (EHV) comprising a p422 promoter sequence (SEQ ID NO: 5), or a functional fragment or functional derivative thereof, or any complementary nucleotide sequence thereof operably linked to a nucleic acid sequence encoding an amino acid sequence, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 44 (H1pdm) , inserted into UL43, and additionally a p455 promoter sequence (SEQ ID NO: 4), or a functional fragment or a functional derivative thereof, or any complementary nucleotide sequence thereof operably linked to a nucleic acid sequence encoding an amino acid sequence having at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, according at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as defined in SEQ ID NO: 45 (H3) inserted in US4.

Furthermore, the present invention relates to an equine herpesvirus (EHV) comprising a p422 promoter sequence (SEQ ID NO: 5), or a functional fragment or functional derivative thereof, or any complementary nucleotide sequence thereof operably linked to a nucleic acid sequence encoding the amino acid sequence

- 10 046085 capacity having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence as specified in SEQ ID NO : 44 (H1pdm) inserted into UL43, and additionally the p430 promoter sequence (SEQ ID NO: 3), or a functional fragment or functional derivative thereof, or any complementary nucleotide sequence thereof operably linked to a nucleic acid sequence encoding an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as defined in SEQ ID NO: 46 (H1av), inserted into UL56, and additionally a p455 promoter sequence (SEQ ID NO: 4), or a functional fragment or a functional derivative thereof, or any complementary nucleotide sequence thereof operably linked to a nucleic acid sequence encoding an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as specified in SEQ ID NO: 45 (H3) inserted in US4.

In another aspect of the vector of the present invention, the EHV vector is selected from the group consisting of EHV-1, EHV-3, EHV-4, EHV-8 and EHV-9.

In another aspect of the vector of the present invention, the EHV vector is EHV-1 or EHV-4.

In another aspect of the vector of the present invention, the EHV vector is EHV-1, preferably RacH.

The present invention further relates to a vector or equine herpesvirus (EHV) comprising:

a) a first nucleotide sequence of interest, preferably a gene of interest, such as an antigen coding sequence, in UL43, where

b) said first nucleotide sequence of interest is optionally operably linked to a regulatory nucleic acid sequence/promoter sequence, preferably p455, p430 or p422,

c) said first nucleotide sequence of interest is optionally operably linked to an (additional) regulatory nucleic acid sequence, for example a polyadenylation sequence, preferably BGHpA, 71pA (SEQ ID NO: 6) or 18pA (SEQ ID NO: 7).

In a specific aspect, the vector or EHV further includes

a) a second nucleotide sequence of interest, preferably a gene of interest such as an antigen coding sequence, at a second insertion site, preferably UL56 or US4, wherein

b) said second nucleotide sequence of interest is optionally operably linked to a regulatory nucleic acid sequence/promoter sequence, preferably p455, p430 or p422,

c) said second nucleotide sequence of interest is optionally operably linked to an (additional) regulatory nucleic acid sequence, for example a polyadenylation sequence, preferably BGHpA, 71pA (SEQ ID NO: 6), or 18pA (SEQ ID NO: 7).

In a specific aspect, the vector or EHV further includes

a) a third nucleotide sequence of interest, preferably a gene of interest such as an antigen coding sequence, at a third insertion site, preferably UL56 or US4, wherein

b) said third nucleotide sequence of interest is optionally operably linked to a regulatory nucleic acid sequence/promoter sequence, preferably p455, p430 or p422,

c) said third nucleotide sequence of interest is optionally operably linked to a regulatory nucleic acid sequence, for example a polyadenylation sequence, preferably 71pA, BGHpA, or18pA.

The present invention further provides a plasmid including flanking regions for homologous recombination or RED-mediated recombination (both described above) at a specific target site in a viral vector genome, preferably at the orf70 (US4) site from an EHV vector, specifically EHV-1, more specifically RacH vector such as transfer plasmid pU-mC70

- 11 046085

BGH (SEQ ID NO: 37) and/or transfer vector pU70-p455-71K71 (SEQ ID NO: 28), and/or transfer plasmid pU70-p455-H3-71K71 (SEQ ID NO: 29), and/or transfer plasmid pU70-p455-H1pdm-71K71 (SEQ ID NO: 32).

The present invention further provides a plasmid including flanking regions for homologous recombination or RED-mediated recombination (both described above) at a specific target site in a viral vector genome, preferably at the UL43 site from an EHV vector, specifically EHV-1, more specifically a RacH vector, such as transfer plasmid pUUL43-42218K18 (SEQ ID NO: 34) or pUUL43-422-H1pdm-18K18 (SEQ ID NO: 36).

The present invention further provides a plasmid including flanking regions for homologous recombination or RED-mediated recombination (both described above) at a specific target site in a viral vector genome, preferably at the orf1/3 (UL56) site from an EHV vector, specifically EHV-1, more specifically a RacH vector, and a nucleic acid regulatory sequence, preferably a promoter, preferably p430, such as the transfer vector pU-1-3-p430-BGHKBGH (SEQ ID NO: 30) and/or the transfer plasmid pU1-3-p430-H1av-BGHKBGH (SEQ ID NO: 31), and/or transfer plasmid pU1-3-p430-H1hu-BGHKBGH (SEQ ID NO: 33).

The present invention further provides a plasmid including flanking regions for homologous recombination or RED-mediated recombination (both described above) at a specific target site in a viral vector genome, preferably at the UL43 site from an EHV vector, specifically EHV-1, more specifically a RacH vector, and a regulatory nucleic acid sequence, preferably a promoter, preferably p422, such as transfer vector pUUL43-422-18K18 (SEQ ID: 34) or transfer plasmid pUUL43-422-mC-18K18 (SEQ ID NO: 35), or pUUL43-422-H1pdm -18K18 (SEQ ID NO: 36).

The present invention further provides a plasmid including flanking regions for homologous recombination or RED-mediated recombination (both described above) at a specific target site in a viral vector genome, preferably at the orf70 (US4) site from an EHV vector, specifically EHV-1, more specifically RacH vector, and a regulatory nucleic acid sequence, preferably a promoter, preferably p455, such as transfer vector pU70-p455-71K71 (SEQ ID NO: 28) and/or transfer plasmid pU70-p455-H3-71K71 (SEQ ID NO: 29), and/or transfer plasmid pU70-p455-H1pdm-71K71 (SEQ ID nO: 32).

The present invention further provides a plasmid including flanking regions for homologous recombination or RED-mediated recombination (both described above) at a specific target site in a viral vector genome, preferably at the UL56 (orf1/3) site from an EHV vector, specifically EHV-1, more a specifically RacH vector, and a nucleic acid regulatory sequence, preferably a promoter, preferably p430, such as the transfer vector pU-1-3-p430-BGHKBGH (SEQ ID NO: 30) and/or the transfer plasmid pU1-3-p430-H1av-BGHKBGH (SEQ ID NO: 31), and/or transfer plasmid pU1-3-p430-H1hu-BGHKBGH (SEQ ID NO: 33).

The present invention further provides a plasmid including flanking regions for homologous recombination or RED-mediated recombination (both described above) at a specific target site in a viral vector genome, preferably at the UL56 (orf1/3) site from an EHV vector, specifically EHV-1, more a specifically RacH vector, and a nucleic acid regulatory sequence, preferably a promoter, preferably p430, and a second nucleic acid regulatory sequence, preferably a polyadenylation sequence, preferably a BGH polyadenylation sequence, such as the transfer vector pU-1-3-p430BGHKBGH (SEQ ID NO: 30) and/or transfer plasmid pU1-3-p430-H1av-BGHKBGH (SEQ ID NO: 31), and/or transfer plasmid pU1-3-p430-H1hu-BGHKBGH (SEQ ID NO: 33).

The present invention further provides a plasmid including flanking regions for homologous recombination or RED-mediated recombination (both described above) at a specific target site in a viral vector genome, preferably at the US4 (orf70) site from an EHV vector, specifically EHV-1, more specifically RacH vector, and a regulatory nucleic acid sequence, preferably a promoter, preferably p455, and a second regulatory nucleic acid sequence, preferably a polyadenylation sequence, preferably a 71pA polyadenylation sequence, such as transfer vector pU70-p45571K71 (SEQ ID NO: 28) and/or transfer plasmid pU70 -p455-H3-71K71 (SEQ ID NO: 29), and/or transfer plasmid pU70-p455-H1pdm-71K71 (SEQ ID NO: 32).

The present invention further provides a plasmid including flanking regions for homologous recombination or RED-mediated recombination (both described above) at a specific target site in a viral vector genome, preferably at the UL43 site from an EHV vector, specifically EHV-1, more specifically a RacH vector, and a regulatory nucleic acid sequence, preferably a promoter, preferably p422, and a second regulatory nucleic acid sequence, preferably a polyadenylation sequence, preferably an 18pA polyadenylation sequence (SEQ ID NO: 7), such as transfer vector pUUL43-422-18K18 (SEQ ID NO: 34) and/or transfer plasmid pUUL43-422-H1pdm-18K18 (SEQ ID

- 12 046085

NO: 36), and/or transfer plasmid pUUL43-422-mC-18K18 (SEQ ID NO: 35).

The present invention further relates to a method for producing a vector in accordance with the present invention, comprising:

a) inserting a first nucleotide sequence of interest, preferably a gene of interest such as an antigen coding sequence, into UL43,

b) optionally operably linking said first nucleotide sequence of interest to a regulatory nucleic acid sequence/promoter sequence, preferably p455, p430 or p422.

c) optionally operably linking said first nucleotide sequence of interest to an (additional) regulatory nucleic acid sequence, for example a polyadenylation sequence, preferably BGHpA, 71pA (SEQ ID NO: 6) or 18pA (SEQ ID NO: 7).

In a specific aspect, the method also includes

a) inserting a second nucleotide sequence of interest, preferably a gene of interest such as an antigen coding sequence, into a second insertion site, preferably UL56 or US4,

b) optionally operably linking said second nucleotide sequence of interest to a regulatory nucleic acid sequence/promoter sequence, preferably p455, p430 or p422,

c) optionally operably linking said second nucleotide sequence of interest to a regulatory nucleic acid sequence, for example a polyadenylation sequence, preferably BGHpA, 71pA (SEQ ID NO: 6) or 18pA (SEQ ID NO: 7).

In a specific aspect, the method also includes

a) inserting a third nucleotide sequence of interest, preferably a gene of interest such as an antigen coding sequence, into a third insertion site, preferably UL56 or US4,

b) optionally operably linking said third nucleotide sequence of interest to a regulatory nucleic acid sequence/promoter sequence, preferably p455, p430 or p422.

c) optionally operably linking said third nucleotide sequence of interest to a regulatory nucleic acid sequence, for example a polyadenylation sequence, preferably BGHpA, 71pA (SEQ ID NO: 6), or 18pA (SEQ ID NO: 7).

The present invention further provides a kit consisting of a vector in accordance with the present invention, optional transfection reagent(s), and an instruction leaflet.

The present invention also relates to a mammalian host cell characterized in that it includes a vector in accordance with the present invention.

The present invention further relates to a method for obtaining a host cell, which is characterized by the following steps:

a) infecting a mammalian host cell in accordance with the present invention with a vector in accordance with the present invention,

b) culturing the infected cells under suitable conditions,

c) optionally collecting said host cells.

The present invention further relates to the use of UL43 in an equine herpesvirus (EHV) vector, specifically in equine alphaherpesviruses such as EHV-1, EHV-3, EHV-4, EHV-8 and EHV-9, more specifically in equine alphaherpesvirus type 1 vector ( EHV-1), most specifically in RacH, as an insertion site in said equine herpesvirus (EHV) vector, wherein said insertion site supports/facilitates the expression of a nucleotide sequence of interest, preferably a gene of interest, such as an antigen coding sequence, wherein said UL43 insertion site comprises a partial deletion, truncation, substitution, modification, or the like. in UL43, and UL44 remains functional.

The invention further relates to the use of a vector in accordance with the present invention or a mammalian host cell in accordance with the present invention for the preparation of an immunogenic composition or vaccine.

The invention further relates to an immunogenic composition comprising

a) a vector according to the present invention, and/or

b) a polypeptide expressed by a vector in accordance with the present invention, such as a virus, modified live virus, virus-like particle (VLP) or the like, and

c) optionally a pharmaceutically or veterinary acceptable carrier or excipient, said carrier preferably being suitable for oral, intradermal, intradermal,

- 13 046085 for intramuscular or intranasal administration, wherein said immunogenic composition preferably includes a virus. In a specific aspect, said virus is a pathogenic virus.

The invention further relates to a vaccine or pharmaceutical composition comprising a). vector in accordance with the present invention, and/or

b) a polypeptide expressed by a vector in accordance with the present invention, such as a modified live virus, virus-like particle (VLP) or the like, and

c) a pharmaceutically or veterinary acceptable carrier or excipient, said carrier being preferably suitable for oral, intradermal, intramuscular or intranasal administration,

d) optionally said vaccine further includes an adjuvant.

Preferably, the vaccine includes an EHV vector as described in the present invention. Preferably, the immunogenic composition includes a pharmaceutically or veterinarily acceptable carrier or excipient.

In one aspect of the present invention, said pharmaceutically acceptable carrier is a cell culture medium or a physiological resuspension buffer.

In one aspect of the present invention, said resuspension buffer is phosphate buffered saline.

In a specific aspect, said immunogenic composition or vaccine or pharmaceutical composition comprises a vector or expression cassette of the present invention, wherein said antigen-coding sequence relates to a pathogen that infects pigs. In a further specific aspect, said pathogen is swine influenza A virus (IAV). In a further specific aspect, said antigen is a hemagglutinin (HA) antigen, in particular, said hemagglutinin antigen is derived from influenza A virus. For example, influenza A virus is influenza A virus (A/swine/Italy/116114/2010(H1N2) ), influenza A virus (A/swine/Italy/7680/2001(H3N2)), influenza A virus (A/swine/Gent/132/2005(H1N1)), and/or influenza A virus (A/swine/Italy /4675/2003(H1N2)). In a further specific aspect, said antigen includes or consists of a sequence encoded by SEQ ID NO selected from the group consisting of: SEQ ID NOs: 44, 45, 46, and 47. In another specific aspect, said antigen includes or consists of a sequence encoded by amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence as specified in SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47.

The invention further relates to a vaccine or DIVA vaccine comprising one or more EHV vectors as described in the present invention.

The present invention further relates to a promoter sequence comprising p422 (SEQ ID NO: 5) or a complementary nucleotide sequence thereof or a functional fragment thereof or complementary nucleotide sequences thereof, wherein said promoter sequence results in the expression of a nucleotide sequence of interest, preferably a gene of interest, more preferably antigen coding sequence.

The present invention also provides an expression cassette comprising a p422 promoter sequence (SEQ ID NO: 5) or a complementary nucleotide sequence thereof or a functional fragment and a complementary nucleotide sequence thereof, wherein the promoter sequence is operably linked to a sequence of interest, preferably a gene of interest, such as an antigen-encoding gene a sequence, more preferably a heterologous and/or exogenous sequence of interest, a gene of interest or an antigen-coding sequence of interest, wherein said promoter sequence leads to the expression of a nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen-coding sequence, wherein said promoter sequence is preferably a heterologous promoter sequence, more preferably an exogenous promoter sequence.

The present invention also provides a vector including a promoter sequence or expression cassette as described in the present invention.

In a further specific aspect of the promoter or expression cassette or vector of the present invention, the functional promoter sequence fragment has a sequence identity and/or homology of 70, 80, 85%, preferably 90, 91, 92, 93, 94%,

- 14 046085 more preferably 95, 96, 97, 98, 99, 99.9% relative to the p422 sequence (SEQ ID NO: 5).

In a further specific aspect of the promoter or expression cassette or vector of the present invention, said functional promoter sequence fragment is 100 nucleotides in length, preferably 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400 nucleotides, most preferably 410 or 420 nucleotides, or the functional promoter sequence fragment is 100 to 422 nucleotides, 200 to 422 nucleotides, 300 to 422 nucleotides, or 350 to 422 nucleotides in length.

In a further specific aspect of the expression cassette or vector of the present invention, said expression cassette or vector includes one or more additional regulatory sequences, such as a stop codon, a polyadenylation signal, or a regulatory element like an IRES and/or 2a peptide.

In a further specific aspect of the expression cassette or vector of the present invention, the expression cassette or vector further includes a polyadenylation sequence, preferably BGHpA, 71pA (SEQ ID NO: 6), or 18pA (SEQ ID NO: 7).

In a further specific aspect of the vector of the present invention, said vector is a recombinant and/or heterologous and/or exogenous vector.

In a further specific aspect of the vector of the present invention, said vector is a viral vector, preferably selected from the group consisting of Herpesviridae such as equine alphaherpesvirus type 1 (EHV-1), equine alphaherpesvirus type 4 (EHV-4) and other Varicellovirus like PrV (pseudorabies virus) and BHV-1 (bovine herpesvirus type 1), Adenoviridae (AdV) such as CAdV (canine adenovirus), adeno-associated viruses, Baculoviridae, Lentiviridae such as retroviruses, and Poxviridae.

In a further specific aspect of the vector of the present invention, said vector is a member of the family Herpesviridae, preferably the genus Alphaherpesvirinae, more preferably the subgenus Varicellovirus, most preferably said vector is equine alphaherpesvirus type 1 (EHV-1).

DIVA.

The main advantage of an effective DIVA vaccine is that it provides the ability to identify food animals (preferably pigs) that are acutely infected or have been infected for some time (at least approximately 3 weeks) before sampling in the vaccinated animal population, and thus makes it possible to control the spread or re-infection of a pathogen (preferably the swine flu virus) in an animal population. Therefore, this provides some confidence that the vaccinated pig population is not infected with swine influenza A virus based on laboratory results.

The marker vaccine allows rapid and effective administration and provides the ability to distinguish between animals infected with the field virus (associated with the disease) and vaccinated animals.

The immunogenic composition or DIVA vaccine of the present invention does not contain any antigen coding sequence, influenza N antigen coding sequence (neuraminidase) and/or influenza NP antigen coding sequence (nucleoprotein).

In contrast, following infection of animals with wild type swine influenza virus type A, or vaccination with a modified live vaccine, or vaccination with an inactivated whole virus vaccine, or in the case of animals in which residual maternal antibodies are present, such infected/vaccinated animals produce/have specific antibodies against N (neuraminidase) and/or NP (nucleoprotein). However, in animals vaccinated with the immunogenic composition according to the present invention, such specific antibodies against N (neuraminidase) and/or NP (nucleoprotein) cannot be detected.

Using exemplary immunoassays and/or genomic analytical tests, animals vaccinated only with the immunogenic composition of the present invention can be distinguished from animals that have been infected with wild-type swine influenza virus or vaccinated with a modified live vaccine, or vaccinated with an inactivated whole virus vaccine, or that have residual maternal antibodies, due to the fact that animals vaccinated only with the immunogenic composition of the present invention do not have any specific antibodies against N (neuraminidase) and/or NP (nucleoprotein) and any specific sequences of swine influenza virus type A encoding N (neuraminidase ) and/or NP (nucleoprotein), respectively.

The present invention provides a method for differentiating food animals infected with swine influenza virus type A from food animals vaccinated with an immunogenic composition or DIVA vaccine as described in the present invention, comprising

- 15 046085

a) obtaining a sample from an animal raised for food purposes, and

b) carrying out the analysis of the specified sample using an immunoassay and/or a genomic analytical test.

In one aspect of the present invention, the immunoassay includes testing whether the sample contains antibodies that specifically recognize the N protein (neuraminidase) or the NP protein (nucleoprotein) of swine influenza.

In one aspect of the present invention, an animal raised for food purposes is infected with swine influenza virus type A if antibodies that specifically recognize the swine influenza N protein (neuraminidase) or NP protein (nucleoprotein) of swine influenza have been detected.

In one aspect of the present invention, the genomic analytical test includes testing whether the sample contains specific swine influenza virus type A sequences encoding N (neuraminidase) and/or NP (nucleoprotein).

In one aspect of the present invention, a food animal is infected with swine influenza virus type A if specific swine influenza virus type A sequences encoding N (neuraminidase) and/or NP (nucleoprotein) have been detected.

In one aspect of the present invention, the immunoassay is an EIA (enzyme-linked immunosorbent assay) or ELISA (enzyme-linked immunosorbent assay), or the genomic analytical test is PCR (polymerase chain reaction), RT-PCR (reverse transcriptase polymerase chain reaction) or PCR (polymerase chain reaction). reaction) in real time.

In one aspect of the present invention, the food animal is a pig.

In one aspect of the present invention, the sample is a serum sample.

Preferably, an antibody specific for wild-type SIAV N (neuraminidase) and/or NP (nucleoprotein) is used to detect SIAV antigen in the respiratory tract of a pig that is suspected of being infected with SIAV or that has been vaccinated with a vaccine in accordance with the invention. In such a case, only a sample from an infected pig or a pig vaccinated with a modified live vaccine or vaccinated with an inactivated whole virus vaccine, or having residual maternal antibodies, will test positive with the indicated N (neuraminidase) and/or NP (nucleoprotein) specific antibody. In contrast, a sample from a pig vaccinated with the vaccine of the present invention will not test with the indicated N (neuraminidase) and/or NP (nucleoprotein) specific antibody due to the absence of such antigens (hemagglutinin only) in the vaccine of the present invention.

However, the N (neuraminidase) and/or NP (nucleoprotein) epitope is evolutionarily conserved and specific for SIAV and is a target for neutralizing antibodies.

Thus, the test could, for example, contain wells with the wild-type SIAV N (neuraminidase) and/or NP (nucleoprotein) epitope cross-linked to microwell assay plates. Said cross-linking is preferably carried out through an anchor protein such as, for example, poly-L-lysine. Expression systems for producing wild-type N (neuraminidase) and/or NP (nucleoprotein) epitopes are well known to one skilled in the art. Alternatively, said N (neuraminidase) and/or NP (nucleoprotein) epitopes can be chemically synthesized.

Animals vaccinated only with the vaccine of the present invention do not increase the level of antibodies against the wild-type N (neuraminidase) and/or NP (nucleoprotein) epitope. However, in such animals the level of antibodies against the HA epitope (hemagglutinin) is increased in accordance with the present invention. As a consequence, antibodies do not bind to a well coated with the wild-type N (neuraminidase) and/or NP (nucleoprotein) epitope. In contrast, if the well has been coated with an HA epitope in accordance with the present invention, the antibodies bind to said substituted HA epitope.

In one aspect of the present invention, the ELISA is an indirect ELISA, a sandwich ELISA, a competitive ELISA, or a blocking ELISA.

However, various ELISA techniques are well known to one skilled in the art. ELISA methods have been described illustratively by Wensvoort G. et al., 1988 (Vet. Microbiol. 17(2): 129-140), Robiolo B. et al., 2010 (J. Virol. Methods. 166(1-2): 21-27) and Colijn, E.O. et al., 1997 (Vet. Microbiology 59: 15-25).

Preferably, the test for differentiating an animal that is infected with field SIAV or vaccinated with a modified live vaccine, or vaccinated with an inactivated whole virus vaccine, or that has residual maternal antibodies, and an animal that is vaccinated only with the vaccine of the present invention is carried out by isolating RNA from respiratory cells and reverse transcriptase followed by cDNA amplification. Using specific primers for N (neuraminidase) and/or NP (nucleoprotein) PCR can be performed. In this case, if there is a positive PCR signal, the pig is infected with wild type SIAV.

- 16 046085

However, if it is not possible to amplify a specific N (neuraminidase) and/or NP (nucleoprotein) sequence, the animal is vaccinated with the vaccine of the present invention.

In this case, real-time techniques can be used using primers and/or probes that recognize N (neuraminidase) and/or NP (nucleoprotein), and/or specific HA (hemagglutinin). However, such methods are well known in the art.

Primers and/or probes based on real-time techniques that recognize either N (neuraminidase) and/or NP (nucleoprotein) and/or a specific HA (hemagglutinin) can be used. However, such methods are well known to those skilled in the art.

In another aspect of the present invention, the genomic analytical test is a PCR (polymerase chain reaction), RT-PCR (reverse transcriptase polymerase chain reaction) or real-time PCR (polymerase chain reaction).

The invention further relates to a method for producing an immunogenic composition or vaccine for reducing the frequency or severity of one or more clinical symptoms associated with or caused by an infection, comprising the following steps:

a) infecting a mammalian host cell in accordance with the present invention with a vector in accordance with the present invention,

b) culturing the infected cells under suitable conditions,

c) collection of infected cell cultures,

d) optionally purifying the collected infected cell cultures from step c),

e) optionally mixing said harvested infected cell culture with a pharmaceutically acceptable carrier.

Medical use:

The invention further relates to a method of immunizing an animal, comprising administering to such animal an immunogenic composition, vaccine or DIVA vaccine as described in the present invention.

The invention further provides a method of reducing or preventing clinical symptoms caused by a pathogen in an animal in need thereof, which comprises administering to the animal a therapeutically effective amount of an immunogenic composition, vaccine or DIVA vaccine as described herein.

The invention further provides a method of reducing or preventing clinical symptoms caused by swine influenza virus in an animal in need thereof, which includes administering to the animal a therapeutically effective amount of an immunogenic composition, vaccine or DIVA vaccine as described in the present invention.

In a further specific aspect of the medical use or method of the present invention, said animal is a pig, piglet or sow, poultry, cattle, horse, dog or cat.

In a further specific aspect of the medical use or method of the present invention, the immunogenic composition, vaccine or DIVA vaccine is administered once.

It should be understood that a single dose is administered only once. Preferably, the single dose has a total volume of between about 0.2 ml and 2.5 ml, more preferably between about 0.2 ml and 2.0 ml, even more preferably between about 0.2 ml and 1.75 ml, even more preferably between about 0.2 ml and 1.5 ml, even more preferably between about 0.4 ml and 1.25 ml, even more preferably between about 0.4 ml and 1.0 ml, with a single dose of 0.5 ml or 1.0 ml being most preferred. The most preferred single dose has a total volume of 0.5 ml, 1 ml, 1.5 ml or 2 ml.

Preferably, the immunogenic composition or DIVA vaccine is administered to the piglets before they reach three weeks of age. Preferably, the immunogenic composition or DIVA vaccine is administered to each of the piglets between 1 day of life and 21 days of life, more preferably between 1 day of life and 10 days of life, even more preferably between 1 day of life and 9 days of life, even more preferably between 1 day of life and 8 days of life, even more preferably between 1 day of life and 7 days of life, even more preferably between 1 day of life and 6 days of life, even more preferably between 1 day of life and 5 days of life, even more preferably between 1 day of life and 4 days of life, even more preferably between 1 day of life and 3 days of life, even more preferably on 1 or 2 days of life, and most preferably on 1 day of life.

In a further specific aspect of the medical use or method of the present invention, the immunogenic composition, vaccine or DIVA vaccine is administered to the animal during the first three weeks of life, during the first two weeks of life, during the first week of life, or during the first day of life.

In a further specific aspect of the medical use or method of the present invention, the immunogenic composition, vaccine or DIVA vaccine is administered in two doses.

However, the immunogenic composition can be administered to the animal in two or more doses, with the first dose administered before the second (booster) dose. Preferably, the first dose is administered within

- 17 046085 the first two weeks of life, more preferably during the first week of life and even more preferably during the first day of life. Preferably, the second dose is administered at least 15 days after the first dose. More preferably, the second dose is administered between 15 days and 40 days after the first dose. Even more preferably, the second dose is administered at least 17 days after the first dose. Even more preferably, the second dose is administered between the 17th day and 30th day after the first dose. Even more preferably, the second dose is administered at least 19 days after the first dose. Even more preferably, the second dose is administered between day 19 and day 25 after the first dose. Most preferably, the second dose is administered at least 21 days after the first dose. In a preferred aspect of a double-dose regimen, both first and second doses of the immunogenic composition are administered in equal amounts. Preferably, each dose is within the preferred amounts stated above, with the most preferred dose being 1 ml for both the first and second dose. In addition to the first and second dosing schedule, an alternative embodiment includes additional subsequent doses. For example, in these aspects, a third, fourth, or fifth dose may be administered. Preferably, in such a regimen followed by additional doses, the third, fourth and fifth dose are administered in the same amount as the first dose, with time intervals between doses corresponding to the time intervals between the first and second doses mentioned above.

In a further specific aspect of the medical use or method of the present invention, the immunogenic composition, vaccine or DIVA vaccine is administered to the animal for the first time during the first week of life and a second time during the second, third or fourth week of life.

The immunogenic composition or DIVA vaccine is preferably administered locally or systemically. Typically, a suitable route of administration used is oral or parenteral, such as intranasal, intravenous, intramuscular, intraperitoneal, subcutaneous, as well as inhalation route. However, depending on the nature and mechanism of action of the compound, the immunogenic composition or DIVA vaccine can also be administered by other routes. However, most preferably, the immunogenic composition or DIVA vaccine is administered intramuscularly or intranasally.

In a further specific aspect of the medical use or method of the present invention, said immunogenic composition, vaccine or DIVA vaccine is administered intramuscularly or intranasally.

In a further specific aspect of the medical use or method of the present invention, said immunogenic composition or DIVA vaccine comprises an EHV vector at a tissue culture semi-infectious dose ( _TCID50 ) of 1x104 to 1x109, preferably 1x104 to ^1x108 TCID50, even more preferably from 1x104 to 1x10 ⁷ TCID50

In a further specific aspect of the medical use or method of the present invention, the immunogenic composition, vaccine or DIVA vaccine comprises an EHV vector at a dose of 1x104 to ^1x107 TCID50.

In a further specific aspect of the medical use or method of the present invention, said method results in an improvement in an efficacy parameter selected from the group consisting of: decreased weight loss, decreased rectal temperature, decreased clinical symptoms, increased induction of (neutralizing) antibodies, or combinations thereof, compared with an animal from a non-immunized control group of the same species.

In a specific aspect of the medical use of the present invention described above, or the method of immunizing an animal described above, said antigen-coding sequence relates to a pathogen infecting pigs. In a further specific aspect, said pathogen is swine influenza A virus (IAV). In a further specific aspect, said antigen is a hemagglutinin (HA) antigen, in particular, said hemagglutinin antigen is derived from influenza A virus. For example, influenza A virus is influenza A virus (A/swine/Italy/116114/2010(H1N2) ), influenza A virus (A/swine/Italy/7680/2001(H3N2)), influenza A virus (A/swine/Gent/132/2005(H1N1)), and/or influenza A virus (A/swine/Italy /4675/2003(H1N2)). In a further specific aspect, said antigen includes or consists of a sequence encoded by SEQ ID NO selected from the group consisting of: SEQ ID NOs: 44, 45, 46, and 47. In another specific aspect, said antigen includes or consists of a sequence encoded by amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence as set forth in SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47.

The invention also relates to a kit for immunizing an animal, preferably a food animal such as a pig, poultry or cattle, or companion animals such as cats, dogs or horses, against a disease associated

- 18 046085 with and/or reduction in the frequency or severity of one or more clinical symptoms associated with or caused by a pathogen in an animal, including:

a) a dispenser capable of administering the vaccine to the specified animal; And

b) an immunogenic composition, vaccine or DIVA vaccine according to the present invention, and

c) optionally an instruction leaflet.

Definitions

Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one skilled in the art to which this invention relates at the time of filing. The meaning and scope of the terms must be clear; however, in the event of any underlying ambiguity, the definitions provided in this invention supersede any dictionary or external definition. In addition, unless the context otherwise requires, singular terms shall include sets, and plural terms shall include singular variants. In this case, the use of the conjunction or means and/or unless otherwise indicated. Moreover, the use of the term including, as well as other forms such as includes and included, is not limiting. All patents and publications mentioned herein are incorporated herein by reference.

In carrying out the present invention, unless otherwise indicated, conventional techniques of virology, molecular biology, microbiology, recombinant DNA technology, protein chemistry and immunology, which are known to specialists in the art, will be used. Such techniques are described in detail in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, volumes I, II and III, second edition (1989); DNA Cloning, volumes I and II (ed. D. N. Glover, 1985); Oligonucleotide Synthesis (ed. M. J. Gait, 1984); Nucleic Acid Hybridization (ed. B. D. Hames & S. J. Higgins, 1984); Animal Cell Culture (ed. R. K. Freshney, 1986); Immobilized Cells and Enzymes (IRL, ed., 1986); Perbal, B., A Practical Guide to Molecular Cloning (1984); series, Methods In Enzymology (eds. S. Colowick and N. Kaplan, Academic Press, Inc.); Protein purification methods - a practical approach (eds. E.L.V. Harris and S. Angal, IRL Press at Oxford University Press); and Handbook of Experimental Immunology, volumes I-IV (eds. D. M. Weir and C. C. Blackwell, 1986, Blackwell Scientific Publications).

Before describing the present invention in more detail, it should be noted that the present invention is not limited to specific DNA, polypeptide sequences or process parameters, which, of course, may vary. It should also be understood that the terminology used in this invention is intended only for the purpose of describing specific embodiments of the invention, and is not intended to be limiting. It should also be noted that, as used in this specification and the accompanying claims, the singular forms include references to the plural unless the content clearly indicates otherwise. Thus, for example, a reference to an antigen includes a mixture of two or more antigens, a reference to an excipient includes mixtures of two or more excipients, and the like.

Molecular biological definitions.

The term vector, as it is known in the art, refers to a polynucleotide construct, typically a plasmid or bacterial artificial chromosome, used to transfer genetic material into a host cell. Vectors may be, for example, bacteria, viruses, phages, bacterial artificial chromosomes, cosmids or plasmids. A vector as used in this invention may consist of or contain either DNA or RNA. In some embodiments, the vector consists of DNA. In some embodiments, the vector is a pathogenic virus. Such a viral vector contains a viral genome that is processed in such a way that it carries a foreign gene that does not participate in the replication of the viral vector either in cell culture or in the host animal. In accordance with specific aspects of the present invention, the vector can be used in various aspects, such as transfer of genetic material only, for transfection of host cells or organisms, for use as vaccines, for example, DNA vaccines, or for gene expression purposes. Gene expression is a term that describes protein biosynthesis in a cell, controlled by a specific polynucleotide sequence called a gene. In a specific aspect, the vector may be an expression vector, which is a vector capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is in a suitable environment.

Vectors and methods for producing and/or using expression vectors (or recombinants) may be the same or similar to those described, in particular, in the following documents: US patent Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807, 4,722,848, 5,942,235 , 5,364,773, 5,762,938, 5,770,212, 5,942,235, 382,425, PCT publications WO 94/16716, WO 96/39491, WO 95/30018; Paoletti, Applications of pox virus vectors to vaccination: An update, PNAS USA 93: 11349-11353, October 1996; Moss, Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety PNAS USA 93: 11341-11348, October 1996; Smith et al., US Patent No. 4,745,051 (recombinant baculovirus); Richardson, C. D. (ed.), Methods in Molecular Biology 39, Baculovirus

- 19 046085

Expression Protocols (1995 Humana Press Inc.); Smith et al., Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector, Molecular and Cellular Biology, December, 1983, vol. 3, no. 12, cc. 2156-2165; Pennock et al., Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus Vector, Molecular and Cellular Biology, March 1984, vol. 4, no. 3, p. 406; EP AO 370 573; US Patent Application No. 920,197, filed October 16, 1986; EP Patent Publication No. 265785; US Patent No. 4,769,331 (recombinant herpesvirus); Roizman, The function of herpes simplex virus genes: A primer for genetic engineering of novel vectors, PNAS USA 93:11307-11312, October 1996; Andreansky et al., The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors, PNAS USA 93: 11313-11318, October 1996; Robertson et al., Epstein-Barr virus vectors for gene delivery to B lymphocytes, PNAS USA 93: 11334-11340, October 1996; Frolov et al., Alphavirus-based Expression Vectors: Strategies and applications, PNAS USA93: 11371-11377, October 1996; Kitson et al., J. Virol. 65, 3068-3075, 1991; US Patent Nos. 5,591,439, 5,552,143; WO 98/00166; accepted applications for US patent ser. Nos. 08/675,556 and 08/675,566, both filed July 3, 1996 (recombinant adenovirus); Grunhaus et al., 1992, Adenovirus as cloning vectors, Seminars in Virology (vol. 3) cc. 237-52, 1993; Ballay et al. EMBO Journal, vol. 4, p. 3861-65, Graham, Tibtech 8, 85-87, April, 1990; Prevec et al., J. Gen. Virol. 70, 42434; PCT WO 91/11525; Feigner et al. (1994), J. Biol. Chem. 269, 2550-2561, Science, 259: 1745-49, 1993; and McClements et al., Immunization with DNA vaccines, encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease, PNAS USA 93: 11414-11420, October 1996; and US Patent Nos. 5,591,639, 5,589,466 and 5,580,859, as well as WO 90/11092, WO 93/19183, WO 94/21797, WO 95/11307, WO 95/20660; Tang et al., Nature, and Furth et al., Analytical Biochemistry, relating to DNA Expression Vectors. See also WO 98/33510; Ju et al., Diabetologia, 41: 736-739, 1998 (lentiviral expression system); Sanford et al., US Patent No. 4,945,050; Fischbach et al. (Intracel); WO 90/01543; Robinson et al., Seminars in Immunology vol. 9, cc. 271-283 (1997), (DNA vector systems); Szoka et al., US Patent No. 4,394,448 (method of inserting DNA into living cells); McCormick et al., US Patent No. 5,677,178 (use of cytopathic viruses); and US Patent No. 5,928,913 (gene delivery vectors); as well as other documents cited in the present invention.

The term viral vector describes a genetically modified virus that can be manipulated using recombinant DNA techniques such that its entry into a host cell results in a specific biological activity, such as expression of a transgene carried by the vector. In a specific aspect, the transgene is an antigen. The viral vector may be able to replicate or unable to replicate in the target cell, tissue or organism.

The creation of a viral vector can be accomplished using any suitable genetic engineering techniques well known in the art, including, but not limited to, standard restriction endonuclease digestion, ligation, transformation, plasmid purification, DNA sequencing, cell culture transfection techniques, e.g. as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (1989)) or K. Maramorosch and H. Koprowski (Methods in Virology, Volume VIII, Academic Press Inc. London, UK (2014)).

The viral vector may include sequences from the genome of any known organism. The sequences may be inserted in their native form or may be modified in any way to obtain the desired activity. For example, the sequences may include insertions, deletions, or substitutions.

The viral vector may include coding regions for two or more proteins of interest. For example, the viral vector may include a coding region for a first protein of interest and a coding region for a second protein of interest. The first protein of interest and the second protein of interest may be the same or different. In some embodiments, the viral vector may include coding region(s) for a third or fourth protein of interest. The third or fourth protein of interest may be the same or different. The total length of two or more proteins of interest encoded by a single viral vector may vary. For example, the total length of two or more proteins may be at least about 200 amino acids. At least about 250 amino acids, at least about 300 amino acids, at least about 350 amino acids, at least about 400 amino acids, at least about 450 amino acids, at least about 500 amino acids, at least about 550 amino acids, according to at least about 600 amino acids, at least about 650 amino acids, at least about 700 amino acids, at least about 750 amino acids, at least about 800 amino acids, or more.

Preferred viral vectors include herpesvirus vectors such as those derived from EHV-1 or EHV-4 or other Varicellovirus like PrV (pseudorabies virus) or

- 20 046085

BHV-1 (bovine herpesvirus type 1).

In accordance with specific aspects of the present invention, the term viral vector or alternatively viral construct refers to a recombinant viral construct derived from a virus that is selected from the Herpesviridae families, such as EHV-1, EHV-4. Preferred viral vectors include herpesvirus vectors, such as those derived from EHV-1 or EHV-4.

The terms viral vector and viral construct may be used interchangeably.

The term construct, as used in the present invention, refers to a recombinant nucleic acid, such as a plasmid, BAC or recombinant virus, which has been created artificially.

The term plasmid refers to cytoplasmic DNA that replicates independently of the bacterial chromosome in the bacterial host cell. In a specific aspect of the present invention, the term plasmid and/or transfer plasmid refers to an element of recombinant DNA technology used to construct, for example, an expression cassette for insertion into a viral vector. In another specific aspect, the term plasmid can be used to indicate a plasmid used for DNA vaccination purposes.

As used herein, the terms nucleic acid and polynucleotide are used interchangeably and refer to any nucleic acid.

The term nucleic acid, nucleic acid sequence, nucleotide sequence, polynucleotide, polynucleotide sequence, RNA sequence or DNA sequence, as used herein, refers to an oligonucleotide, nucleotide or polynucleotide and fragments and parts thereof, and DNA or RNA of genomic or synthetic origin. , which can be single-stranded or double-stranded and represent a sense or antisense strand. The sequence may be a non-coding sequence, a coding sequence, or a mixture of both. The nucleic acid sequences of the present invention can be obtained using standard techniques well known to one skilled in the art.

The terms nucleic acid and polynucleotide also specifically include nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

The terms regulatory nucleic acid, regulatory element and expression control element are used interchangeably and refer to nucleic acid molecules that can influence the expression of an operably linked coding sequence in a particular host organism. These terms are used in a broader sense to cover all elements that promote or regulate transcription, including promoters, promoter sequences, core elements required for the basic interaction of RNA polymerase and transcription factors, upstream elements, enhancers, and response elements. Exemplary regulatory elements in prokaryotes include promoters, operator sequences, and ribosome binding sites. Regulatory elements that are used in eukaryotic cells may include, but are not limited to, sequences that control transcription and translation, such as promoters, enhancers, splicing signals, polyadenylation signals, terminators, protein decay signals, internal ribosome entry sites (IRES) , picornavirus 2A sequences, and the like, which provide and/or regulate expression of the coding sequence and/or production of the encoded polypeptide in the host cell.

The internal ribosome entry site or IRES describes a sequence that functionally facilitates the initiation of translation independent of the 5' IRES gene and allows the translation of two cistrons (open reading frames) from a single transcript in an animal cell. The IRES provides an independent ribosome entry site for translation of the open reading frame, located immediately downstream of it. Unlike bacterial mRNA, which can be polycistronic, i.e., encode several different polypeptides that are translated sequentially from the mRNA, most animal cell mRNAs are monocistronic and encode the synthesis of only one polypeptide. With a polycistronic transcript in a eukaryotic cell, translation will initiate from the 5'most translation initiation site, terminating at the first stop codon, and the transcript will be released from the ribosome, resulting in translation of only the first encoded polypeptide into mRNA. In a eukaryotic cell, a polycistronic transcript having an IRES operably linked to a second or subsequent open reading frame in the transcript enables sequential translation of the downstream open reading frame to produce two or more polypeptides encoded by the same transcript. IRES can be of different lengths and from different sources, for example, encephalomyocarditis virus (EMCV), picornaviruses (eg foot-and-mouth disease virus, FMDV or poliovirus (PV) or hepatitis C virus (HCV). Various IRES sequences and their use in vector constructs have been described and they are well known in the art. Cody

- 21 046085 The downstream directing sequence is functionally linked to the 3rd end of the IRES at any distance that does not adversely affect the expression of the downstream gene. The optimal or tolerable distance between an IRES and the start of a downstream gene can be easily determined by varying the distance and measuring expression as a function of the distance.

The term 2a or 2a peptide refers to short oligopeptide sequences, described as 2a and ^-like, that serve as linkers capable of mediating cotranslational cleavage between proteins through a process defined as ribosomal escape. Such 2a and '2a-like' sequences (from Picornaviridae and other viruses or cellular sequences) can be used to link multiple gene sequences into a single gene, allowing them to be co-expressed in the same cell (see Luke and Ryan, 2013).

As used in the present invention, the term promoter or promoter sequence means a nucleotide sequence that permits binding of RNA polymerase and directs transcription of a gene. Typically, the promoter is located in the 5' non-coding region of the gene, proximal to the transcription initiation site of the gene. Sequence elements within promoters that function to initiate transcription are often characterized by consensus nucleotide sequences. Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses and animals such as mammals (including horses, pigs, cattle, poultry, dogs, cats and humans), birds or insects. A promoter can be inducible, repressible and/or constitutive. Inducible promoters initiate increased levels of transcription from DNA under their control in response to a specific change in culture conditions, such as a change in temperature (Ptashne, 2014). Examples of promoters well known to one skilled in the art include, for example, SV40 large T, HCMV and MCMV immediate early gene 1, human elongation factor alpha promoter, baculovirus polyhedrin promoter.

As used herein, in the context of the present invention, the term promoter refers in particular to a functional fragment, for example, a truncation of 4pgG600 (SEQ ID NO: 1) or its complementary nucleotide sequence, where the sequence identity is preferably (at least) 72% relative to the entire length (or higher). Furthermore, as used herein, in the context of the present invention, the term promoter refers in particular to a functional fragment, for example, a truncation of 4pMCP600 (SEQ ID NO: 2) or its complementary nucleotide sequence, where the sequence identity is preferably (at least ) 78% relative to the entire length (or higher). Furthermore, as used herein, in the context of the present invention, the term promoter refers particularly to p422 (SEQ ID NO: 5) or a functional fragment thereof or complementary nucleotide sequences thereof. Most preferably, the promoter is p430 (SEQ ID NO: 3), p455 (SEQ ID NO: 4) or p422 (SEQ ID NO: 5). As further used herein, in the context of the present invention, the term promoter refers specifically to a functional derivative of p430 (SEQ ID NO: 3) or p455 (SEQ ID NO: 4) or 4pgG600 (SEQ ID NO: 1) or 4pMCP600 ( SEQ ID NO: 2) having, for example, a small substitution, mutation or inversion such that the sequence identity is 70, 80, 85, 90, 95, 99% identity or homology.

The terms 4pgG430, p430, gG 430 and 430 are used synonymously and interchangeably throughout the specification, figures, sequence listing, etc. The terms 4pMCP455, p455, MCP 455 and 455 are used synonymously and interchangeably throughout the specification, figures, sequence listing, etc. The terms p422 and 422 are used synonymously and interchangeably throughout the specification, figures, sequence listing, etc.

The term enhancer means a polynucleotide sequence that, in cis-localization, influences the activity of a promoter and thereby stimulates transcription of a gene or coding sequence operably linked to that promoter. Unlike promoters, the influence of enhancers is independent of position and orientation and, therefore, they can be located upstream or downstream of a transcription unit, within an intron, or even within a coding region. The enhancer can be located either in close proximity to the transcription unit or at a considerable distance from the promoter. It also seems possible to have physical and functional overlap with the promoter. One skilled in the art has information regarding various enhancers from various sources (in particular, they are deposited in data banks such as GenBank, for example, SV40 enhancers, CMV enhancers, polyoma enhancers, adenovirus enhancers), which are available as independent elements or elements, cloned within polynucleotide sequences (eg, deposited in the ATCC or from commercial and individual sources). Various promoter sequences also contain enhancer sequences, such as the commonly used CMV promoter. The human CMV enhancer is one of the most potent enhancers identified

- 22 046085 new to date. One example of an inducible enhancer is the metallothionein enhancer, which can be stimulated by glucocorticoids or heavy metals.

The term complementary nucleotide sequences describes one strand of two paired strands of polynucleotides such as DNA or RNA. The nucleotide sequence of the complementary strand is a mirror image of the nucleotide sequence of its paired strand in such a way that for each adenosine it contains thymine (or uracil for RNA), for each guanine it contains cytosine, and vice versa. The complementary nucleotide sequence, for example, 5'-GCATAC3' is 3'-CGTATG-5', or for RNA 3'-CGUAUG-5'.

The terms gene, gene of interest, as used in this invention, have the same meaning and refer to a polynucleotide sequence of any length that encodes a product of interest. The gene may further include regulatory sequences located upstream (5' non-coding or untranslated sequences) and downstream (3' non-coding or untranslated sequences) of the coding sequence. The selected sequence may be full length or a truncated, fused, or tagged sequence, and may be a cDNA, genomic DNA, or DNA fragment. In general terms, it is understood that genomic DNA encoding a polypeptide or RNA may include non-coding regions (i.e., introns) that are spliced from mature messenger RNA (mRNA) and therefore not present in the cDNA encoding the same polypeptide or RNA. It may be a native sequence, i.e. naturally occurring form(s), or may be mutated, or include sequences originating from different sources, or otherwise modified as desired. These modifications include codon optimizations to optimize the frequency of codon usage in the selected host cell or tagging. In addition, they may include deletions or additions of cis-acting sites such as (cryptic) splice donors, acceptor sites and branch points, polyadenylation signals, TATA boxes, chi sites, ribosome entry sites, repeat sequences, secondary structures (e.g. loop -stem), binding sites for transcription factors or other regulatory factors, restriction enzyme sites, etc. to present only some, but not limiting, examples. The selected sequence may encode a secreted, cytoplasmic, nuclear, membrane-bound or cell surface polypeptide.

The term nucleotide sequence of interest, as used herein, is a more general term than gene of interest because it does not necessarily include a gene, but may include elements or portions of a gene or other genetic information, such as ori (origin of replication). The nucleotide sequence of interest may be any DNA or RNA sequence, whether or not it includes a coding sequence.

An open reading frame or ORF refers to a stretch of nucleic acid sequence, either DNA or RNA, that includes a translation start signal or start codon, such as ATG or AUG, and a stop codon, and can potentially be translated into a polypeptide sequence.

The term UL (long unique) is an abbreviation to describe a long unique segment of EHV, preferably the EHV-1 genome.

The term US (short unique) is an abbreviation to describe a short unique segment of EHV, preferably the EHV-1 genome.

The term transcription describes the biosynthesis of mRNA in a cell.

The term expression, as used in this invention, refers to the transcription and/or translation of a nucleic acid sequence in a host cell. In accordance with specific aspects of the present invention, the term expression refers to the transcription and/or translation of a heterologous and/or exogenous nucleic acid sequence in a host cell. The level of expression of a desired product in a host cell can be determined based on either the amount of the corresponding RNA or mRNA that is present in the cell or the amount of the desired polypeptide encoded by the selected sequence. For example, the mRNA transcribed from a selected sequence can be quantified by Northern blot hybridization, RNA ribonuclease protection, in situ hybridization with cellular RNA, or by RT qPCR (reverse transcription followed by quantitative PCR). Proteins expressed from a selected sequence can be quantified using various methods, for example, by ELISA, Western blotting, radioimmunoassays, by immunoprecipitation, by testing the biological activity of the protein, or by protein immunostaining followed by FACS analysis.

The term expression cassette or transcription unit or expression unit defines a region in a vector, construct or polynucleotide sequence that contains one or more transcribed genes, where nucleotide sequences encoding the transcribed gene(s), as well as polynucleotide sequences containing a regulator

- 23 046085 elements contained in the expression cassette are functionally linked to each other. They are transcribed from a promoter and transcription is terminated by at least one polyadenylation signal. In one specific aspect, they are transcribed from a single promoter. As a result of this, different genes are at least transcriptionally related. More than one protein or product can be transcribed and expressed from each transcription unit (multicistronic transcription unit). Each transcription unit will include regulatory elements necessary for the transcription and translation of any of the selected sequences that are contained in that unit. And each transcription unit may contain the same or different regulatory elements. For example, each transcription unit may contain the same terminator, an IRES element, or introns may be used to operably link genes within the transcription unit. The vector or polynucleotide sequence may contain more than one transcription unit.

The term increased expression, increased titer or productivity, or improved expression or productivity means increased expression, synthesis or secretion of a heterologous and/or exogenous sequence introduced into a host cell, e.g., a gene encoding a therapeutic protein, by comparison with a suitable control, e.g. , encoded by the cDNA relative to the protein encoded by the intron-containing gene. An increased titer or productivity is observed if a cell in accordance with the invention is cultured in accordance with the method in accordance with the invention described in the present invention, and if this cell has at least 1.2-fold, 1.5-fold, 2-fold, 3-fold , a fourfold or fivefold increase in specific productivity or titer. There is also increased titer or productivity if a cell in accordance with the invention is cultured in accordance with the method in accordance with the invention described in the present invention, and if this cell has at least 1.2-fold or at least 1.5-fold or at least twofold or at least threefold increase in specific productivity or titer. There is also, in particular, an increased titer or productivity if the cell in accordance with the invention is cultured in accordance with the method in accordance with the invention described in the present invention, and if this cell has at least 1.2-fold - five-fold, preferably 1 .5-fold - five-fold, more preferably two-fold - five-fold, especially preferably three-fold - five-fold increase in specific productivity or titer. Increased expression may also indicate that more cells actually express the gene/sequence of interest. For example, increased expression may mean that the novel promoters of the present invention are active for a longer period of time during the viral replication cycle compared to other promoters.

Increased expression, titer or productivity can be obtained by using a heterologous vector in accordance with the invention. This can be combined with other approaches such as FACS-assisted selection of recombinant host cells that contain, as an additional selectable marker, one or more fluorescent proteins (eg GFP) or a cell surface marker. It is also possible to use other methods for obtaining increased expression, and a combination of various methods, based on, for example, the use of cis-active elements to manipulate chromatin structure (for example, LCR, UCOE, EASE, insulators, S/MAR, STAR elements), the use of (artificial) transcription factors, treatment of cells with natural or synthetic agents to activate the expression of endogenous or heterologous and/or exogenous genes, improving the stability (half-life) of mRNA or protein, improving the initiation of translation of mRNA, increasing gene dosage through the use of episomal plasmids (based on the use of viral sequences in as origins of replication, for example SV40, polyoma, adenovirus, EBV or BPV), the use of amplification sequences or in vitro amplification systems based on DNA concatemers.

The study to measure increased expression is LC-MS/MS based protein determinations such as multiple reaction monitoring (MRM); antibody-based detection methods such as Western blotting, dot blotting or immunodiffusion, and flow cytometry; and measuring biological activity through hemagglutination assays.

Promoter activity is measured indirectly by quantifying the mRNA transcribed under the control of the corresponding promoter. mRNA is quantified by RT qPCR against an endogenous standard.

The term viral titer is a measure of infectious units per volume of viral preparation. The viral titer is the end point of a biological procedure and is defined as the dilution at which a certain proportion of tests performed in parallel is effective (Reed and Muench, 1938). Specifically, the tissue culture semi-infective dose per milliliter (TSI ₅₀ /ml) provides a dilution of the viral preparation such that 50% of the number of cell cultures inoculated in parallel with such dilution are infected.

Transcription regulatory elements usually include a promoter located upstream

- 24 046085 transcription sequence of the expressed gene, transcription initiation and termination sites, and polyadenylation signal.

The term transcription start site refers to the nucleic acid in the construct corresponding to the first nucleic acid incorporated into the primary transcript, i.e. precursor to mRNA. The transcription start site may overlap with promoter sequences.

A stop codon or terminator or polyadenylation signal or polyA or transcription termination site or transcription termination element is a signal sequence that causes cleavage at a specific site at the 3' end of a eukaryotic mRNA and post-transcriptional incorporation of a sequence of approximately 100 to 200 adenine nucleotides (polyA tail ) at the cleaved 3' end, and thus causes termination of transcription carried out by RNA polymerase. The polyadenylation signal includes the AATAAA sequence approximately 10-30 nucleotides upstream of the restriction site and a sequence located downstream of the restriction site. Various polyadenylation elements are known, such as tk polyA, SV40 late and early polyA, BGH polyA (described, for example, in US Pat. No. 5,122,458) or hamster growth hormone polyA (WO 2010010107).

Translation regulatory elements include a translation initiation site (AUG), a stop codon, and a polyA signal for each individual polypeptide to be expressed. Some constructs may include an internal ribosome entry site (IRES). In order to optimize expression, it may be desirable to remove, add or modify 5' and/or 3' untranslated regions of the expressed nucleic acid sequence to eliminate any potential additional inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, or at the level of transcription or translation. Consensus ribosome binding sites (Kozak sequence) can be inserted immediately upstream of the start codon to enhance translation and thus expression. Increased A/U content around this ribosome binding site promotes more efficient ribosome binding.

By definition, each polynucleotide sequence or gene inserted into a host cell, and the corresponding protein or RNA so encoded, is designated as exogenous, exogenous sequence, exogenous gene, exogenous coding sequence, with respect to the host cell, if it ( it) has origins from different (viral) species. Accordingly, the EHV-4 based promoters of the present invention are exogenous to the EHV-1 viral vector. As used herein, with respect to a sequence or gene of interest, such as an antigen, the term exogenous means that said sequence or gene of interest is specifically identified as an antigen , is expressed outside the environment of its natural species. Accordingly, H3, H1av or H1pdm antigens from porcine IAV are examples of exogenous antigens to the EHV-1 vector. Therefore, any sequence having origin from a pathogen other than EHV-1 is an exogenous sequence of interest or a gene or antigen in accordance with a specific aspect of the present invention.

By definition, each polynucleotide sequence or gene inserted into a host cell and the corresponding protein or RNA so encoded is referred to as a heterologous, heterologous sequence, heterologous gene, heterologous coding sequence, transgene, or heterologous protein with respect to the host cell. This applies even if the introduced sequence or introduced gene is identical to the endogenous sequence or endogenous gene of the host cell. For example, an EHV-4 promoter sequence introduced into an EHV-4 viral vector at a different site or in a modified form than in a wild-type EHV-4 virus is by definition a heterologous sequence. As used herein, with respect to a sequence or gene of interest, such as an antigen, the term heterologous means that said sequence or gene of interest, specifically the antigen, is expressed outside of environment of its natural subspecies. Accordingly, any non-EHV-1 specific sequence or gene of interest, such as an antigen, e.g., antigen from any equine alphaherpesvirus other than EHV-1, e.g., EHV-3 , EHV-8, is therefore a heterologous sequence of interest or gene or antigen in accordance with a specific aspect of the present invention.

The term non-naturally occurring means any sequence or gene of interest, such as an antigen, that does not occur naturally in that environment, such as a hybrid sequence or sequence of interest or a gene, such as an antigen, from different species, or a sequence or gene of interest, such as an antigen, that is not a natural product due to artificial mutation, in

- 25 046085 insertions, deletions, etc.

The term recombinant is used interchangeably with the terms non-naturally occurring, heterologous and exogenous throughout the present invention. Thus, a recombinant protein is a protein expressed from either a heterologous or exogenous polynucleotide sequence. The term recombinant, as used in relation to a virus, means a virus produced by artificial manipulation of the viral genome. A virus comprising a heterologous or exogenous sequence, such as an exogenous antigen coding sequence, is a recombinant virus. The term recombinant virus and the term unnatural virus are used interchangeably.

Thus, the term heterologous vector means a vector that includes a heterologous or exogenous polynucleotide sequence. The term recombinant vector means a vector that includes a heterologous or recombinant polynucleotide sequence.

As used in the present invention, the term operably linked is used to describe the relationship between regulatory elements and a gene or coding region thereof. Typically, gene expression is placed under the control of one or more regulatory elements, such as, but not limited to, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. When a gene or coding region is stated to be operably linked to or operably linked to or operably associated with regulatory elements, it means that the gene or coding region is controlled by or is influenced by the regulatory element. For example, a promoter is operably linked to a coding sequence if the promoter influences the transcription or expression of the coding sequence.

In addition, within the scope of the present description, the terms operably linked, operably linked or operably linked mean that two or more nucleic acid sequences or sequence elements are arranged in such a way as to allow them to function properly. For example, a promoter/enhancer or terminator is operably linked to a coding gene sequence if it is capable of controlling or modulating transcription of the associated gene sequence in cis. In general, but not necessarily, DNA sequences that are operably linked are contiguous and, if necessary to join two polypeptide coding regions or in the case of signal peptide secretion, contiguous and in frame. However, although an operably linked promoter is typically located upstream of transcription or an operably linked terminator is typically located downstream of a coding sequence, it is not always adjacent to them. Enhancers do not need to be contiguous because they increase transcription of the coding sequence. To do this, they can be located upstream of the transcription or along the transcription of the coding sequence, and even at some distance. A polyadenylation site is operably linked to a coding sequence if it is located at the 3' end of the coding sequence such that transcription occurs through the coding sequence to the polyadenylation signal. Linking is accomplished using recombinant methods well known in the art, for example, by ligation at suitable restriction sites or blunt ends, or by using PCR techniques with overlapping primers. Synthetic oligonucleotide linkers or adapters can be used in accordance with conventional practice if suitable restriction sites are not present.

Accordingly, the term functional fragment or functional derivative of a promoter sequence means that the fragment or derivative still has promoter activity. Functional assays to assess promoter activity are well known to one of ordinary skill in the art (Bustin 2000, Nolan et al. 2006). An exemplary embodiment of such a functional study involves, for example, a promoter kinetics experiment. Cells infected with vector viruses carrying expression cassettes, where a promoter or fragment thereof drives transcription of a reporter transgene, are incubated for various periods of time. Total RNA was obtained from samples collected at various times after infection. After the contaminating DNA is destroyed by DNase I digestion, the RNA is reverse transcribed. One replicate sample was processed with the addition of reverse transcriptase (RT), and a second replicate was processed without the addition of RT to demonstrate successful removal of contaminating DNA from the RNA preparation. The resulting cDNA is purified and used as a template in conventional PCR. Only samples processed with the addition of RT will produce a PCR product. These cDNAs can then be used for quantitative PCR with primers for the reporter transgene and in parallel with primers for the viral vector core gene (internal standard gene), the transcription of which provides the internal standard for infection and replication efficiency. Reporter qPCR values are normalized between different constructs and time postinfection using internal qPCR values.

- 26 046085 standard gene. This provides the opportunity to interpret the activities of various promoters and their fragments.

Sequence homology, as used in the present invention, refers to a method for determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned, and gaps are introduced if necessary. However, unlike sequence identity, conservative amino acid substitutions are counted as matches when determining sequence homology.

In other words, to obtain a comparable polypeptide or polynucleotide having 95% sequence homology with the comparison sequence, 85%, preferably 90, 91, 92, 93, 94%, even more preferably 95, 96, 97, 98, 99, 99.9% amino acids residues or nucleotides in the comparison sequence must match or include a conservative substitution for another amino acid or nucleotide. Alternatively, the amount of amino acids or nucleotides is up to 15%, preferably up to 10, 9, 8, 7, 6%, even more preferably up to 5, 4, 3, 2, 1, 0.1% of the total amino acid residues or nucleotides, not including conservative substitutions in the comparison sequence can be inserted into the comparison sequence. Preferably, the homologous sequence is at least 50, even more preferably 100, even more preferably 250, even more preferably 500 nucleotides in length.

The term sequence identity, as it is known in the art, refers to the relationship of two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence that is compared with the reference sequence. Sequence identity is determined by comparing a given sequence to a reference sequence after optimally aligning the sequences to obtain the highest degree of sequence similarity, as determined by matches between stripes of such sequences. In such an alignment, sequence identity is established based on positional comparisons, eg, sequences are identical at a particular position if, at that position, the nucleotides or amino acid residues are identical. The total number of such identical positions is then divided by the total number of nucleotides or residues in the reference sequence, yielding % sequence identity. Sequence identity can be easily calculated using known methods, including, but not limited to, those described in Computational Molecular Biology, ed. Lesk, A. N., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, ed. Smith, D.W., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, ed. Griffin, A.M. and Griffin, H. G., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, ed. Gribskov, M. and Devereux, J., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods for determining sequence identity are created to obtain the greatest match between the test sequences. Methods for determining sequence identity are codified in publicly available computer programs that determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG software package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F., et al. ., J. Molec. Biol., 215:403410 (1990) The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, S. F. et al. ., J. Molec. Biol., 215:403-410 (1990), the details of which are incorporated herein by reference.) These programs optimally align sequences using default gap opening penalties to obtain the highest level of sequence identity between a given and comparative sequences. By way of illustration, a polynucleotide having a nucleotide sequence having at least, for example, 85%, preferably 90, 91, 92, 93, 94%, even more preferably 95, 96, 97, 98, 99, 99.9% sequence identity to the compared nucleotide sequence, it is understood that the nucleotide sequence of a given polynucleotide is identical to the comparison sequence except that a given polynucleotide sequence may include up to 15, preferably up to 10, even more preferably up to 5 point mutations for every 100 nucleotides of the nucleotide sequence comparison. In other words, in a polynucleotide having a nucleotide sequence having at least 85%, preferably 90, 91, 92, 93, 94%, even more preferably 95, 96, 97, 98, 99, 99.9% identity with respect to the nucleotide sequence comparison, up to 15%, preferably 10, 9, 8, 7, 6%, even more preferably 5, 4, 3, 2, 1, 0.1% of the nucleotides in the comparison sequence may be deleted or replaced by another nucleotide, or a number of nucleotides up to 15%, preferably 10, 9, 8, 7, 6%, even more preferably 5, 4, 3, 2, 1, 0.1% of all nucleotides in the reference sequence can be inserted into the reference sequence. These comparison sequence mutations can occur at the 5' or 3' terminal

- 27 046085 positions of the reference nucleotide sequence or at any other place between these terminal positions, scattered either individually between nucleotides in the reference sequence or in one or more contiguous groups in the comparison sequence. Likewise, a polypeptide having a given amino acid sequence having at least, for example, 85%, preferably 90, 91, 92, 93, 94%, even more preferably 95, 96, 97, 98, 99% sequence identity to the amino acid sequence comparison, it is understood that a given amino acid sequence of a polypeptide is identical to the comparison sequence except that a given polypeptide sequence may include up to 15, preferably up to 10, 9, 8, 7, 6, even more preferably up to 5, 4, 3, 2, 1 amino acid changes for every 100 amino acids of the comparison amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90, 91, 92, 93, 94%, even more preferably 95, 96, 97, 98, 99% sequence identity with the reference amino acid sequence, up to 15%, preferably up to 10, 9, 8, 7%, even more preferably up to 5, 4, 3, 2, 1% of the amino acid residues in the reference sequence may be deleted or replaced with another amino acid, or up to 15 amino acids %, preferably up to 10, 9, 8, 7%, even more preferably up to 5, 4, 3, 2, 1% of the total number of amino acid residues in the comparison sequence can be inserted into the comparison sequence. These reference sequence changes may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between these terminal positions, scattered either individually between residues in the reference sequence or in one or more contiguous groups in the reference sequence. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity.

The terms sequence identity or percent identity are used interchangeably herein. For purposes of the present invention, the present invention specifies that to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). The amino acid or nucleotide residues at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e. % identity = number of identical positions / total number of positions (i.e. overlapping positions) x 100). Preferably, the two sequences are of the same length.

Sequence comparisons can be made for the full lengths of the two sequences being compared or for a fragment of two sequences. Typically, the comparison is made for the full length of the two sequences being compared. However, sequence identity can be determined for a region of, for example, twenty, fifty, one hundred or more contiguous amino acid residues.

One skilled in the art will be aware that several different computer programs are available for determining homology between two sequences. For example, comparing sequences and determining the percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percentage identity between two amino acid or nucleic acid sequences is determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)), which was included in the GAP program in the software package Accelrys GCG collateral (available at http://www.accelrys.com/products/gcg/), using either a Blosum 62 matrix or a PAM250 matrix, and a gap penalty of 16, 14, 12, 10, 8, 6, or 4 and a penalty length of 1, 2, 3, 4, 5, or 6. One skilled in the art will appreciate that all of these different parameters will produce slightly different results, but the overall percent identity of the two sequences will not change significantly when used various algorithms.

The protein or nucleic acid sequences of the present invention may further be used as a search sequence to search public databases, for example, to identify other members of families or related sequences. Such searches can be performed using the programs BLASTN and BLASTP (version 2.0) from Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST searches for proteins can be performed using the BLASTP program, score=50, word length=3 to obtain amino

- 28 046085 acid sequences homologous to protein molecules according to the invention. To obtain gap alignments for comparisons, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17): 3389-3402. When using the BLAST and Gapped BLAST programs, you can use the default parameters of the corresponding programs (for example, BLASTP and BLASTN). See the National Center for Biotechnology Information home page at http://www.ncbi.nlm.nih.gov/.

Definitions of EHV-1 and EHV-4/recombinant vector technology.

The term equidae or equine or odd-toed ungulate refers to or refers to the equidae family (Equidae), which includes horses, donkeys and zebras, preferably equines. In addition, the term equidae or equine or equid also covers hybrids of members of the family Equidae (e.g. mules, hinnies, etc.).

Herpes virus or herpes virus vector belongs to the species in the family Herpesviridae in the order Herpesvirales.

The term equine herpes virus vector or equine herpes virus or EHV refers to a member of the Herpesviridae family that infects horses. To date, eight different species of equine herpesviruses have been identified, five of them belonging to the subfamily Alphaherpesvirinae (EHV-1, EHV-3, EHV-4, EHV-8 and EHV-9) and three of them belonging to Gammaherpesvirinae. (http://www.ictvonline.org/virustaxonomy.asp Virus Taxonomy: 2015 Release EC 47, London, UK, July 2015; Email ratification 2016 (MSL #30)).

The term EHV-1 refers to equine alphaherpesvirus type 1, a member of the subgenus Varicellovirus in the genus Alphaherpesvirinae in the family Herpesviridae. A non-limiting reference sequence for EHV-1 could be, for example, wild-type EHV-1 strain ab4 (Genbank accession number AY665713.1) or RacH (Hubert 1996).

The term EHV-4 refers to equine alphaherpesvirus type 4, a member of the subgenus Varicellovirus in the genus Alphaherpesvirinae in the family Herpesviridae.

The term inserted into ORF70 means that the DNA fragment has been inserted into the genomic DNA at a location encoding the equine alphaherpesvirus type 1 open reading frame 70. In a specific aspect of the present invention, the insertion results in the deletion of 801 5' base pairs of ORF70, leaving the remaining 423 at the 3' end intact but abolishing expression of the orf70 gene product glycoprotein G. G glycoprotein of several alphaherpesviruses, including EHV-1, has been shown to secreted from infected cells and acts as an immunomodulatory protein by binding proinflammatory cytokines. Abolition of its expression in the viral vector will increase the immunogenicity of the viral infection compared with wild-type EHV-1 expressing intact glycoprotein G.

The term inserted into UL43 means that a DNA fragment has been inserted into the genomic DNA at a location encoding the equine alphaherpesvirus type 1 open reading frame UL43 (ORF17). In a specific aspect of the present invention, the insertion results in the deletion of 870 5' base pairs of UL43, leaving the remaining 336 at the 3' end intact but abolishing expression of the UL43 product of the pUL43 gene. pUL43 from EHV-1 has been shown to act as an immunomodulatory protein together with pUL56 by influencing MHC-I presentation.

The term inserted into ORF1/3 means that a DNA fragment is inserted into the viral genome at a position where, by random deletion during passivation during the attenuation procedure of the EHV-1 vaccine strain, RacH fragment 1283, comprising 90% of ORF1 and complete ORF2, was lost. This insertion site was chosen because it is believed that there is a very low likelihood that expression of the transgene from this location will interfere with viral replication.

Vaccine definitions.

An immunogenic or immunological composition refers to a composition of a substance that includes at least one antigen or immunogenic portion thereof that induces a cell- or antibody-mediated immunological response to the composition in the host.

The term antigen as used in this invention is well known in the art and includes substances that are immunogenic, i.e. immunogens, as well as substances that induce immunological unresponsiveness or anergy, i.e. lack of reactions through the body's defense reactions to foreign substances. As used in the present invention, the term antigen means full-length proteins, as well as peptide fragments thereof containing or including an epitope.

The term food animal means animals that are used for human consumption such as pig, cattle, poultry, fish and the like, preferably food animal means pig and cattle, most preferably a pig.

An immunogenic composition as used in the present invention may refer to a polypeptide or protein, such as, for example, a viral surface protein, that induces an immunological response as described in the present invention. The term immunogenic fragment or immu

- 29 046085 gene part refers to a fragment or truncated and/or substituted form of a protein or polypeptide that includes one or more epitopes and thus causes the immunological response described in the present invention. In general, such truncated and/or substituted forms or fragments will comprise at least six consecutive amino acids from the full-length protein. Such fragments can be identified using any of a number of techniques that are used for epitope mapping well known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, vol. 66 (Glenn E. Morris, ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes can be determined by simultaneously synthesizing large numbers of peptides on solid supports, identifying peptides corresponding to parts of the protein molecule, and reacting the peptides with antibodies while the peptides still remain attached to the supports. Such techniques are known and described in the art, see, for example, US Pat. No. 4,708,871; Geysen et al (1984) Proc. Natl. Acad. Sci. USA 81:39984002; and Geysen et al. (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes are easily identified by determining the spatial conformation of amino acids, for example, by X-ray crystallography and two-dimensional nuclear magnetic resonance. See Epitope Mapping Protocols as described above. Synthetic antigens are also included in this definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically produced antigens. See, for example, Bergmann et al. (1993) Eur. J. Immunol. 23:2777-2781; Bergmann et al (1996), J. Immunol. 157:3242-3249; Suhrbier, A. (1997), Immunol, and Cell Biol. 75:402-408; and Gardner et al., (1998) 12th World AIDS Conference, Geneva, Switzerland, July 28, 1998 (The disclosure and contents of which are incorporated herein by reference.)

The term immunization refers to active immunization by administering an immunogenic composition to the food animal to be immunized, thereby eliciting an immunological response to the antigen included in such immunogenic composition.

The term needing or in need, as used herein, means that the administration/treatment is associated with a boost or improvement in health status or clinical symptoms, or any other beneficial medical effect on the health status of animals that receive the immunogenic composition in accordance with the present invention. invention.

The term vaccine as used herein refers to a pharmaceutical composition comprising at least one immunologically active component that induces an immunological response in an animal, and optionally, but not necessarily, one or more additional components that enhance the immunological activity of the active component. The vaccine may additionally include other components that are typical of pharmaceutical compositions. In contrast, the immunologically active vaccine component may comprise complete viral particles either in their original form or as attenuated particles in a so-called modified live vaccine (MLV) or particles inactivated by suitable methods in a so-called killed vaccine (KV). In another form, the immunologically active vaccine component may include suitable elements of organisms (subunit vaccines), these elements being created either by destruction of the whole particle or by growing cultures containing such particles and optionally subsequent purification steps resulting in the desired vaccine(s). structure(s), or by synthetic processes, including suitable manipulation by use of a suitable system based on, for example, bacteria, viruses, mammals or other species, plus optional subsequent isolation and purification procedures, or by induction of synthetic processes in an animal in need of a vaccine , by direct incorporation of genetic material using suitable pharmaceutical compositions (polynucleotide vaccination). The vaccine may include one or more than one of the elements described above. As used in specific aspects of the present invention, a vaccine refers to a live vaccine or live virus, also referred to as a recombinant vaccine. In another specific aspect of the present invention, the vaccine refers to an inactivated or killed virus, including virus-like particles (VLPs). Thus, the vaccine may be a subunit vaccine or a killed (KV) or inactivated vaccine.

The term multiplicity of infection (M.O.I.) describes how many infectious units, such as TCID50, of a viral preparation are used per cell to infect cultured cells. For example, M.O.I. 0.01 means that one infectious unit is inoculated for every 100 cells in the culture vessel.

The term DNA vaccination or polynucleotide vaccination refers to the direct inoculation of genetic material using suitable pharmaceutical compositions.

Various physical and chemical inactivation methods are known in the art. The term inactivated refers to a previously virulent or non-virulent virus or bacterium that has been irradiated (ultraviolet (UV), x-rays, electron beam or gamma rays), heated or chemically treated to inactivate or kill such virus

- 30 046085 or bacteria while maintaining its immunogenicity. Suitable inactivating agents include beta-propiolactone, binary or beta- or acetyl-ethylenimine, glutaraldehyde, ozone and formalin (formaldehyde).

In the case of inactivation with formaldehyde or formaldehyde, formaldehyde is typically mixed with water and methyl alcohol to create formaldehyde. The addition of methyl alcohol prevents degradation or cross-reaction during the activation process. In one embodiment, approximately 0.1 to 1% of a 37% formaldehyde solution is used to inactivate the virus or bacteria. It is very important to adjust the amount of formaldehyde to ensure that the material is inactivated, but not so much that side effects from using a high dose occur.

More preferably, the term inactivated in the context of a virus means that the virus is unable to replicate in vivo or in vitro and, correspondingly, the term inactivated in the context of a bacterium means that the bacterium is unable to reproduce under in vivo or in vitro conditions. For example, the term inactivated may refer to a virus that has been propagated in vitro and has then been inactivated using chemical or physical means such that it is no longer capable of replication. In another example, the term inactivated may refer to a bacterium, a bacterium that has been propagated and then inactivated using chemical or physical means, resulting in a suspension of bacteria, fragments or components of the bacterium, such as a bacterin, which can be used as vaccine component.

As used in the present invention, the terms inactivated, killed or KV are used interchangeably.

The term live vaccine refers to a vaccine comprising either a living organism or a replication competent virus or viral vector.

A pharmaceutical composition essentially consists of one or more components capable of modifying the physiological, eg immunological, functions of the organism into which it is administered or other organisms living in or on the said organism. The term includes, but is not limited to, antibiotics or antiparasitics, as well as other constituents that are typically used to achieve certain other objectives such as, but not limited to, processability, sterility, stability, ability to administer the composition via enteral or parenteral routes, such as oral, intranasal, intravenous, intramuscular, subcutaneous, intradermal or other suitable route, tolerability after administration, or controlled release properties. One non-limiting example of such a pharmaceutical composition, presented for demonstration purposes only, can be prepared as follows: the cell culture supernatant, which is an infected cell culture, is mixed with a stabilizer (for example, spermidine and/or bovine serum albumin (BSA)) and then the mixture is lyophilized or dehydrated by other methods.Prior to vaccination, the mixture is then reconstituted in aqueous (eg, saline, phosphate buffered saline (PBS)) or non-aqueous solutions (eg, oil emulsion, aluminum adjuvant).

As used herein, the term pharmaceutically or veterinarily acceptable carrier includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption retarding agents, and the like. In some preferred embodiments, and particularly those involving lyophilized immunogenic compositions, stabilizing agents for use in the present invention include lyophilization or freeze-drying stabilizers.

In some embodiments, the immunogenic composition of the present invention contains an adjuvant. Adjuvants as used in the present invention may include aluminum hydroxide and aluminum phosphate, saponins, e.g. Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL) , water-in-oil emulsion, oil-in-water, water-in-oil-in-water. The emulsion can be based, in particular, on light petroleum jelly (in accordance with the European Pharmacopoeia); isoprenoid oil such as squalane or squalene; oils obtained from the oligomerization of alkenes, in particular isobutene or decene; esters of acids or alcohols containing a linear alkyl group, more preferably vegetable oil, ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form an emulsion. Emulsifiers are preferably non-ionic surfactants, in particular esters of sorbitan, mannitol (eg anhydromannite oleate), glycol, polyglycerol, propylene glycol and oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene block copolymers , in particular, Pluronic products, especially L121. See Hunter et al., The Theory and

- 31 046085

Practical Application of Adjuvants (ed. Stewart-Tull, D.E.S.), JohnWiley and Sons, NY, pp. 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997). Exemplary adjuvants are SPT emulsion, described on page 147 of Vaccine Design, The Subunit and Adjuvant Approach, ed. M. Powell and M. Newman, Plenum Press, 1995, and MF59 emulsion, described on page 183 of the same source.

Another example of an adjuvant is a compound selected from polymers of acrylic or methacrylic acid and copolymers of maleic anhydride and an alkenyl derivative. Preferred adjuvant compounds are polymers of acrylic or methacrylic acid which are cross-linked, in particular by polyalkenyl sugar esters or polyalcohols. These compounds are known under the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art may also refer to US Patent No. 2,909,462, which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably no more than 8, with at least three hydrogen atoms hydroxyl groups are replaced by unsaturated aliphatic radicals containing at least 2 carbon atoms. Preferred radicals are those containing from 2 to 4 carbon atoms, for example vinyls, allyls and other ethylenically unsaturated groups. Unsaturated radicals themselves may contain other substituents, such as methyl. Products sold under the name CARBOPOL®; (BF Goodrich, Ohio, USA) are particularly suitable. They are cross-linked with allyl sucrose or allyl pentaerythritol. Among them, Carbopol 974P, 934P and 971P can be mentioned. The most preferred is the use of CARBOPOL® 971P. Among the copolymers of maleic anhydride and an alkenyl derivative, mention should be made of EMA copolymers (Monsanto), which are copolymers of maleic anhydride and ethylene. Dissolution of these polymers in water results in an acidic solution, which must be neutralized, preferably to a physiological pH, in order to obtain an adjuvant solution into which the immunogenic or immunological vaccine composition itself will be included.

Additional suitable adjuvants include, but are not limited to, RIBI adjuvant system (Ribi Inc.), block copolymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid amine adjuvant , heat labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide, either natural or recombinant cytokines or analogs thereof or stimulators of endogenous cytokine release, among many others.

It is expected that the adjuvant may be added in an amount of from about 100 μg to about 10 mg per dose, preferably in an amount from about 100 μg to about 10 mg per dose, more preferably in an amount from about 500 μg to about 5 mg per dose, further more preferably in an amount of from about 750 μg to about 2.5 mg per dose, and most preferably in an amount from about 1 mg per dose. Alternatively, the adjuvant may be present at a concentration of about 0.01 to 50%, preferably at a concentration of about 2 to 30%, more preferably at a concentration of about 5 to 25%, even more preferably at a concentration of about 7 to 22%, and most preferably in a concentration of 10 to 20% of the volume of the final product.

Diluents may include water, saline, dextrose, ethanol, glycerin, and the like. Isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali metal salts with ethylenediaminetetraacetic acid, among others.

Isolated means altered by the hand of man from its natural state, i.e., if it occurs in nature, then it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living organism is not isolated, but the same polynucleotide or polypeptide, when separated from coexisting materials in its natural state, is isolated as that term is used herein. .

Attenuation means reducing the virulence of a pathogen. In the present invention, attenuation is synonymous with avirulence. In the present invention, an attenuated virus is a virus in which the virulence has been reduced such that it does not cause clinical symptoms of infection but is capable of inducing an immune response in the target animal, but may also mean that clinical symptoms are reduced in frequency or severity in animals infected with an attenuated virus, particularly the EHV-1 RacH viral vector, as stated, compared with a control group of animals infected with a non-attenuated virus or pathogen and not receiving the attenuated virus. In this context, the term reduced/reduced means a reduction of at least 10%, preferably 25%, even more preferably 50%, even more preferably 60%, even more preferably 70%, even more preferably 80%, even more preferably 90% and most preferably

- 32 046085 100% positive compared to the control group as defined above. Thus, an attenuated, avirulent pathogen, such as, for example, the inventive attenuated viral vector, especially the inventive EHV-1 (preferably RacH) viral vector, is suitable for creating a modified live vaccine (MLV) or a modified live immunogenic composition.

The term treatment and/or prevention refers to reducing the incidence of infection (particularly infection with swine influenza virus type A) in a herd or reducing the severity of clinical symptoms caused by or associated with a particular infection (particularly infection with swine influenza virus type A). Thus, the term treatment and/or prevention also refers to reducing the number of animals in a herd that become infected with a pathogen (specifically swine influenza virus type A = reducing the frequency of a particular infection with swine influenza virus type A) or reducing the severity of clinical symptoms usually associated with or caused by infection (in particular, infection with swine influenza virus type A) in a group of animals where the animals receive an effective amount of an immunogenic composition as provided in this invention, compared to a group of animals where the animals do not receive such an immunogenic composition.

Treatment and/or prophylaxis generally involves administering an effective amount of an immunogenic composition of the present invention to an animal or herd of animals that needs it or that would benefit from such treatment/prophylaxis. The term "treatment" refers to the administration of an effective amount of an immunogenic composition once to an animal or at least some of the animals of a herd that has already been infected with such a pathogen (particularly swine influenza virus type A), and wherein such animals will already exhibit some of the clinical symptoms caused by or associated with infection by such a pathogen (particularly swine influenza virus type A). The term prophylaxis refers to the administration of a given composition to an animal prior to any infection of such animal with a pathogen (in particular, swine influenza virus type A) or, at least, in the event that such animal or none of the animals in the group of animals exhibits any or clinical symptoms caused by or associated with infection with such a pathogen (in particular, swine influenza virus type A). The terms prophylaxis and prevention are used interchangeably in the present invention.

The term clinical signs, as used in this invention, refers to symptoms of infection of an animal with a pathogen (specifically, swine influenza virus type A). Clinical symptoms of infection depend on the pathogen chosen. Examples of such clinical symptoms include, but are not limited to, respiratory dysfunction, otitis media, roughening of the coat, low-grade fever, depression, and decreased appetite. However, clinical symptoms also include, but are not limited to, clinical symptoms that are directly observed in a living animal. Examples of clinical symptoms that are directly observed in a living animal include nasal and eye discharge, lethargy, coughing, wheezing, throbbing, fever, weight loss, dehydration, limping, emaciation, pale skin, frailty, etc.

As used herein, effective dose means, but is not limited to, the amount of antigen that produces or is capable of producing an immune response that results in a reduction in clinical symptoms in an animal to which the antigen is administered.

As used herein, the term effective amount means, in the context of a composition, an amount of an immunogenic composition capable of inducing an immune response that reduces the frequency or severity of infection, or the incidence of disease in an animal. Specifically, the effective amount refers to colony-forming units (CFU) per dose. Alternatively, in the context of therapy, the term effective amount refers to an amount of therapy that is sufficient to reduce or ameliorate the severity or duration of a disease or disorder, or one or more symptoms thereof, prevent the development of the disease or disorder, cause regression of the disease or disorder, prevent the relapse, development, onset or progression of one or more symptoms associated with a disease or disorder, or enhance or improve the prevention or treatment of another therapy or therapeutic agent.

Immune response or immunological response means, but is not limited to, the development of a cellular and/or antibody-mediated immune response to the (immunogenic) composition or vaccine of interest. Typically, an immune or immunological response includes, but is not limited to, one or more of the following effects: production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells specifically directed to an antigen or antigens included in the compositions or vaccines of interest. Preferably, the host will demonstrate either a therapeutic or a protective immunological (immunological memory) response such that resistance to a new infection will be enhanced and/or the clinical severity of the disease will be reduced. Such protection will manifest itself either less

- 33 046085 by the number of symptoms, the severity of symptoms, or the absence of one or more symptoms associated with the infected pathogen, a delay in the onset of viremia, a decrease in viral persistence, a decrease in the overall viral load, and/or a decrease in viral excretion.

Protection from disease, protective immunity, functional immunity, reduction of clinical symptoms, induction/production of neutralizing antibodies and/or serological transformations, and similar phrases, mean a partial or complete response against a disease or condition that is induced by administration of one or more therapeutic compositions in accordance with the invention, or a combination thereof, resulting in less harmful effects than would be expected in a non-immunized subject who has been exposed to disease or infection. That is, the severity of the harmful effects of infection is reduced in the vaccinated subject. Infection may be reduced, delayed, or possibly completely prevented in a vaccinated subject. Moreover, when complete prevention of infection is meant, this is specifically indicated. If complete prevention is not specified, the term includes partial prevention.

In the present invention, reducing the incidence of disease and/or the severity of clinical symptoms or reducing clinical symptoms means, but is not limited to, reducing the number of infected subjects in a group, reducing or eliminating the number of subjects who exhibit clinical symptoms of infection, or reducing the severity of any clinical symptoms. that are present in one or more subjects, compared with wild-type infection. For example, any reduction in pathogen load, pathogen shedding, reduction in pathogen transmission, or reduction in any clinical sign of symptomatic malaria infection should be mentioned. Preferably, these clinical symptoms are reduced in one or more subjects receiving the therapeutic composition of the present invention by at least 10% compared to subjects not receiving the composition and who are infected. More preferably, clinical symptoms are reduced in subjects receiving the composition of the present invention by at least 20%, preferably by at least 30%, more preferably by at least 40%, and even more preferably by at least 50%.

The term enhanced protection as used herein means, but is not limited to, a statistically significant reduction in one or more clinical symptoms that are associated with infection with an infectious agent in a group of vaccinated subjects versus an unvaccinated control group of subjects. The term statistically significant reduction in clinical symptoms means, but is not limited to, the incidence of at least one clinical symptom in a group of vaccinated subjects that is at least 10%, preferably 20%, more preferably 30%, even more preferably 50%, and even more preferably 70% lower than in the unvaccinated control group after challenge with the infectious agent.

Long-term protection refers to improved effectiveness that lasts for at least 3 weeks, but more preferably at least 3 months, even more preferably at least 6 months. In the case of cattle, it is most preferable for long-term protection to be maintained until the average age at which the animals are sold for meat.

The term reduction of virus-induced viremia means, but is not limited to, reducing the amount of virus entering the animal's circulatory system where the level of viremia, i.e. the number of copies of viral DNA or RNA per ml of blood serum, or the number of plaque-forming colonies per deciliter of blood serum, is reduced in the serum of animals receiving the composition of the present invention by at least 50% compared to animals that did not receive the composition and may become infected. More preferably, the level of viremia is reduced in animals receiving the composition of the present invention by at least 90%, preferably by at least 99.9%, more preferably by at least 99.99%, and even more preferably by at least 99.999%.

The term pathogen is well known to one skilled in the art. However, the term pathogen includes bacteria and viruses. The term pathogen includes pathogens such as Schmallenberg virus, influenza A virus, porcine respiratory and reproductive syndrome virus, porcine circovirus, classical swine fever virus, African swine fever virus, hepatitis E virus, bovine diarrhea virus, rabies virus, feline morbillivirus , tetanus bacillus, mycobacterium tuberculosis, causative agent of actinobacillus pleuropneumonia

The term food animal means animals that are used for human consumption, such as pig, cattle, poultry, fish and the like, preferably pig.

As used in the present invention, the term viremia is specifically understood as a condition in which viral particles reproduce and/or circulate in the circulatory system

- 34 046085 animal, in particular a mammal, bird or insect.

Safety refers to the absence of adverse effects in the vaccinated animal following vaccination, including, but not limited to: the potential for the virus-based vaccine to become virulent, clinically significant adverse effects such as persistent, systemic disease or unacceptable inflammation at the site of vaccine administration.

The terms vaccination or vaccinating or variants thereof, as used in this invention, mean, but are not limited to, a process that involves the administration of an immunogenic composition in accordance with the invention, which, when administered to an animal, causes or can cause - directly or indirectly - immune response from the specified animal.

Mortality, in the context of the present invention, refers to death caused by infection, and includes the situation where the infection is so severe that the animal is euthanized to prevent suffering and ensure a humane end to its life.

Compositions.

The subject to which the composition is administered is preferably an animal, including, but not limited to, cattle, horses, sheep, pigs, poultry (eg chickens), goats, cats, dogs, hamsters, mice and rats, most preferably the mammal is a pig.

The compositions in accordance with the invention include an effective immunizing amount of one or more immunogenic compositions and a physiologically acceptable carrier. Vaccines include an effective immunizing amount of one or more immunogenic compositions and a physiologically acceptable carrier. The composition must correspond to the method of administration.

The immunogenic composition, if desired, may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The immunogenic composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The oral formulation may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc., having pharmaceutical grade purity.

Methods of treatment.

Preferred routes of administration include, but are not limited to, intranasal, oral, intradermal and intramuscular. Administration in drinking water is desirable, most preferably in a single dose. One skilled in the art will appreciate that the compositions of the invention can also be administered in one, two or more doses, as well as using other routes of administration. For example, such other routes include subcutaneous, intradermal, intraperitoneal, intradermal administration, and depending on the desired duration and effectiveness of treatment, the compositions in accordance with the invention may be administered one or more times, as well as periodically, for example on a daily basis for several days , weeks or months, and at varying dosages, such as from approximately 10 ³ to 10 ⁸ TCID50 (see virus titer above). In a specific aspect of the present invention, the dosage is approximately 10 ³ to 10 ⁸ TCID50, particularly for a live virus/live vaccine.

The compositions, if desired, may be presented in a package or dosage device, which may contain one or more unit dosage forms containing the active ingredient. The packaging may, for example, comprise metal foil or plastic film such as a blister pack. The package or dosing device may be accompanied by instructions for administration, preferably administration to a mammal, especially a pig. Such container(s) may be accompanied by information in a form prescribed by the government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, which information reflects agency approval for manufacture, use, or sale for administration to humans.

Antigen definitions.

The term swine influenza virus is known to one skilled in the art. The term swine flu virus refers to influenza virus type A or type C from the orthomyxovirus family that causes swine flu. While orthomyxovirus has three groups: type A, type B and type C, only influenza viruses type A and type C infect pigs. Preferably, the swine influenza virus is a swine influenza virus type A. Subtypes of the swine influenza virus include H1N1, H1N2, H3N2 and H3N1. H9N2 and H5N1 can also be found in pigs. Preferably, the swine influenza virus is an influenza virus that has been isolated from pigs.

The terms HA or H, NA or N and NP are known to one skilled in the art. However, in general, influenza A viruses are divided into 17 H (hemagglutinin) and 10 N (neuraminidase) subtypes, which can give rise to many different combinations (referred to as H1N1, H1N2....H2N1, H2N2....H5N1 , H5N2.... and so on). H (hemagglutinin) and N (neuraminidase) are surface glycoproteins in influenza A viruses such as SIAV. In addition, N is the main antigenic target of neutralizing antibodies. In addition, NP (nucleoprotein) forms

- 35 046085 nucleocapsid.

DIVA Definitions.

The term DIVA (Differentiation Between Infected and Vaccinated Animals) refers to a vaccine that can be used to differentiate a vaccinated animal from a naturally infected animal.

The term sample refers to a sample of body fluid, a sample of separated cells, or a sample of tissue or organ. Body fluid samples can be obtained using well known techniques and preferably include blood, plasma, serum or urine samples, more preferably blood, plasma or serum samples. Tissue or organ samples can be obtained from any tissue or organ, for example by biopsy. Isolated cells can be obtained from body fluids or tissues or organs using separation techniques such as centrifugation or cell sorting.

The term obtained may include an isolation and/or purification step known to one skilled in the art, preferably using precipitation, columns, etc.

The term immunoassays and genomic analytical tests is the basis for differentiating animals vaccinated with the immunogenic composition in accordance with the present invention from animals infected with natural (disease associated) swine influenza virus. Examples of immunoassays include any enzyme-linked immunoassay or immunochemical detection method such as ELISA (enzyme-linked immunosorbent assay), EIA (enzyme-linked immunosorbent assay), RIA (radio immunoassay), enzyme-linked immunosorbent assays, fluorescent antibody test (FAT), electrochemiluminescent immunochemical sandwich assays (ECLIA), dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) or enzyme-linked immunoassays, immunofluorescence test (IFT), immunohistochemical staining method, Western blot analysis, or any other suitable method available to those skilled in the art. Depending on the assay used, antigens or antibodies may be labeled with an enzyme, fluorophore, or radioisotope. See, for example, Coligan et al. Current Protocols in Immunology, John Wiley & Sons Inc., New York, N.Y. (1994); and Frye et al., Oncogen 4: 1153-1157, 1987.

The term genomic analytical test refers to a genomic analytical method based on polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR (r-PCR) or real-time reverse transcription PCR (rRT). -PCR), Templex-PCR, nucleic acid sequence-based amplification (NASBA), and isothermal amplification methods using polymerases and specific oligonucleotides as primers. The above amplification methods are well known in the art.

Items.

The following items are described in the present invention: The invention provides the following items.

1. An expression cassette comprising (i) at least one exogenous nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence, wherein said nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence is operably linked to a promoter sequence , and (ii) at least one upstream UL43 flanking region selected from the group consisting of: SEQ ID NO: 19 and at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96 , 97, 98, 99% homologous and/or identical to the sequence, SEQ ID NO: 26 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homologous and/or identical sequence, and (iii) at least one upstream UL44 flanking region selected from the group consisting of: SEQ ID NO: 20 and at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, SEQ ID NO: 27 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99% of its homologous and/or identical sequence.

2. An equine alphaherpesvirus (EHV) vector comprising the expression cassette of claim 1.

3. An equine alphaherpesvirus (EHV) vector comprising (i) at least one exogenous nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence, wherein said nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence, operably linked to the promoter sequence, and (ii) at least one upstream UL43 flanking region selected from the group consisting of: SEQ ID NO: 19 and at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, SEQ ID NO: 26 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99% of its homologous and/or identical sequence, and

- 36 046085 (iii) at least one upstream UL44 flanking region selected from the group consisting of: SEQ ID NO: 20 and at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence, SEQ ID NO: 27 and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence.

4. An equine alphaherpesvirus (EHV) vector comprising a nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence, inserted into UL43.

5. An equine alphaherpesvirus (EHV) vector comprising a first nucleotide sequence or gene of interest, preferably an antigen coding sequence inserted into UL43, and a second nucleotide sequence or gene of interest, preferably another antigen coding sequence, inserted into a second insertion site, preferably UL56 or US4.

6. The EHV vector according to any one of paragraphs 2-5, wherein the insertion in UL43 is characterized by a partial deletion, truncation, substitution, modification, or the like. in UL43, with UL44 remaining functional.

7. The EHV vector of any one of claims 2-6, wherein the UL43 insertion is characterized by a deletion of approximately bp 870 within UL43 for RacH (SEQ ID NO: 21) or at 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of its homologous and/or identical sequence.

8. The EHV vector according to any one of paragraphs 2-7, wherein the EHV vector includes at least one flanking region selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27, and 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homologous and/or sequence identical to any of these sequences.

9. The EHV vector according to any one of claims 2-8, wherein the EHV vector includes (i) at least one upstream UL43 flanking region selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 26, and (ii) at least one upstream of UL44 flanking region selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 27.

10. The EHV vector of any one of claims 2 to 9 or the expression cassette of claim 1, wherein said nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen coding sequence is not naturally occurring and/or is recombinant.

11. The EHV vector of any one of claims 2 to 10 or the expression cassette of claim 1 or 10, wherein said nucleotide sequence of interest is recombinant and/or heterologous and/or exogenous.

12. The EHV vector according to any of paragraphs 2-11 or the expression cassette according to any of paragraphs 1 or 10-11, wherein said antigen-coding sequence relates to a pathogen infecting an animal, such as an animal raised for food purposes, such as a pig, poultry or cattle or companion animals such as cats, dogs or horses.

13. An EHV vector according to any of paragraphs 2-12 or an expression cassette according to any of paragraphs 1 or 10-12, also including additional regulatory sequences, such as a stop codon or a polyadenylation sequence.

14. The EHV vector according to any one of claims 2-13, further comprising at least one additional nucleotide sequence of interest, preferably another gene of interest, more preferably an antigen coding sequence, optionally inserted into another insertion site, such as UL56 and/or US4.

15. The EHV vector according to any one of claims 2-14, wherein at least one additional nucleotide sequence of interest, preferably another gene of interest, more preferably an antigen coding sequence, is inserted into UL56.

16. The EHV vector according to any one of claims 2-15, wherein at least one additional nucleotide sequence of interest, preferably another gene of interest, more preferably an antigen coding sequence, is inserted into US4.

17. The EHV vector according to any one of claims 2-14, wherein a second additional nucleotide sequence of interest, preferably another gene of interest, more preferably an antigen coding sequence, is inserted into US4 and wherein a third additional nucleotide sequence of interest, preferably another gene of interest, more preferably the antigen coding sequence inserted into UL56.

18. The EHV vector of any one of claims 2-17, wherein the gene of interest is operably linked to a regulatory sequence, preferably a promoter sequence, or the EHV vector of claims 5-17, wherein at least two genes of interest are operably linked to regulatory sequences, preferably promoter sequences sequences.

19. The EHV vector according to any of paragraphs 2-18 or the expression cassette according to any of paragraphs 1 or 10-13, where the promoter sequence(s) is operably linked to one or

- 37 046085 two or more sequences or genes of interest selected from the group consisting of the following: SV40 large T, HCMV and MCMV immediate early gene 1, human elongation factor alpha promoter, baculovirus polyhedrin promoter, functional fragment from 4pgG600 (SEQ ID NO : 1), preferably said functional fragment is p430 (SEQ ID NO: 3), a functional fragment from the complementary nucleotide sequence of 4pgG600 (SEQ ID NO: 1), a functional fragment from 4pMCP600 (SEQ ID NO: 2), preferably said functional fragment is p455 (SEQ ID NO: 4), a functional fragment from the complementary nucleotide sequence of 4pMCP600 (SEQ ID NO: 2), or p422 (SEQ ID NO: 5) or a functional fragment thereof, or complementary nucleotide sequences thereof.

20. The EHV vector of any one of claims 2-19 or the expression cassette of any of claims 1 or 10-13 or 19, wherein the promoter sequence operably linked to the at least one gene of interest is p422 (SEQ ID NO: 5 ) or a functional fragment thereof or their complementary nucleotide sequences.

21. The EHV vector according to any one of claims 5-20, wherein the promoter sequences operably linked to the at least two genes of interest are different.

22. The EHV vector according to any of paragraphs 2-21 or the expression cassette according to any of paragraphs 1 or 10-13 or 19-20, where the EHV vector or expression cassette is recombinant.

23. The EHV vector of any of claims 2-22 or the expression cassette of any of claims 1 or 10-13 or 19-20 or 22, wherein said sequences or an exogenous nucleotide sequence of interest or a gene of interest is an antigen-coding sequence.

24. EHV vector according to any of paragraphs 2-23 or an expression cassette according to any of paragraphs 1 or 10-13 or 19, 20 or 22, 23, where the antigen-coding sequence belongs to a pathogen selected from the list: Schmallenberg virus, influenza virus type A , porcine respiratory and reproductive syndrome virus, swine circovirus, classical swine fever virus, African swine fever virus, hepatitis E virus, bovine diarrhea virus, rabies virus, feline morbillivirus, tetanus bacillus, mycobacterium tuberculosis, causative agent of actinobacillus pleuropneumonia.

25. The EHV vector according to any of paragraphs 2-24 or the expression cassette according to any of paragraphs 1 or 10-13 or 19, 20 or 22-24, where the antigen coding sequence is a hemagglutinin coding sequence.

26. The EHV vector or expression cassette of claim 25, wherein the sequence encoding the influenza hemagglutinin antigen is derived from swine influenza virus type A.

27. The EHV vector or expression cassette of claim 25 or 26, wherein the exogenous antigen coding sequence is a hemagglutinin coding sequence and the influenza hemagglutinin subtype is selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and H18.

28. The EHV vector or expression cassette according to any one of claims 25-27, wherein the exogenous antigen coding sequence is a hemagglutinin coding sequence and the influenza hemagglutinin antigen coding sequence is selected from the group of strains consisting of

A/swine/Gent/132/2005(H1N1),

A/swine/Denmark/13772-1/2003(H1N1),

A/swine/Italy/116114/2010(H1N2), A/swine/Italy/7680/2001(H3N2),

A/swine/Italy/4675/2003(H1N2), A/swine/Italy/259543/2003(H1N2),

A/swine/England/MD0040352R/2009(H1N1),

A/swine/Hungary/13509/2007(H3N2), A/swine/Italy/13962/95(H3N2), A/swine/Cotes d'Armor/1121/00(H1N1), A/Swine/Colorado/ 1/ 77, A/Swine/Colorado/23619/99, A/Swine/Cote d'Armor/3633/84, A/Swine/England/ 195852/92, A/Swine/Finistere/2899/82, A/Swine/Hong Kong/10/98, A/Swine/Hong Kong/9/98, A/Swine/Hong Kong/81/78, A/Swine/Illinois/100084/01, A/Swine/Illinois/100085A/01, A/ Swine/Illinois/21587/99,

A/Swine/Indiana/1726/88, A/Swine/Iowa/15/30, A/Swine/Iowa/8548-1/98,

A/Swine/Indiana/9K035/99, A/Swine/Indiana/P12439/00, A/Swine/Iowa/30,

A/Swine/Iowa/533/99, A/Swine/Iowa/930/01,

A/Swine/Iowa/569/99, A/Swine/Iowa/17672/88,

A/Swine/Italy/1523/98, A/Swine/Korea/CY02/02, A/Swine/Minnesota/55551/00,

A/Swine/Minnesota/9088-2/98, A/Swine/Nebraska/1/92,

A/Swine/Netherlands/12/85, A/Swine/North Carolina/16497/99,

A/Swine/Iowa/3421/90,

A/Swine/Italy/1513-1/98, A/Swine/Minnesota/593/99, A/Swine/Nebraska/209/98,

A/Swine/North Carolina/35922/98,

A/Swine/North Carolina/93523/01, A/Swine/North Carolina/98225/01, A/Swine/Oedenrode/7C/96, A/Swine/Ohio/891/01, A/Swine/Oklahoma/18717/ 99, A/Swine/Oklahoma/18089/99, A/Swine/Ontario/019111/99, A/Swine/Ontario/01911-2/99, A/Swine/Ontario/41848/97, A/Swine/Ontario/ 97, A/Swine/Quebec/192/81, A/Swine/Quebec/192/91, A/Swine/Quebec/5393/91, A/Swine/Taiwan/7310/70, A/Swine/Tennessee/24/ 77,

A/Swine/Texas/4199-2/98,

A/Swine/Wisconsin/163/97,

A/Swine/Wisconsin/168/97,

A/Swine/Wisconsin/457/985

A/Swine/W isconsin/125/97,

A/Swine/Wisconsin/164/97,

A/Swine/Wisconsin/235/97,

A/Swine/Wisconsin/458/98,

A/Swine/W isconsin/136/97, A/Swine/W isconsin/166/97, A/Swine/Wisconsin/238/97,

A/Swine/Wisconsin/464/98

- 38 046085

A/Swine/Wisconsin/14094/99.

29. The EHV vector or expression cassette according to any one of claims 25 to 28, where the exogenous antigen coding sequence is a hemagglutinin coding sequence and the influenza hemagglutinin antigen coding sequence is selected from the group of strains consisting of A/swine/Italy/116114/2010 (H1N2), A/swine/Italy/7680/2001(H3N2),

A/swine/Gent/132/2005(H1N1) and A/swine/Italy/4675/2003(H1N2).

30. The EHV vector or expression cassette of any one of claims 25 to 29, wherein the exogenous antigen coding sequence is a hemagglutinin coding sequence and the influenza hemagglutinin subtype is H1 and/or H3.

31. The EHV vector or expression cassette according to any one of claims 25-30, wherein the antigen coding sequence is a hemagglutinin coding sequence, and the influenza hemagglutinin antigen coding sequence includes a nucleic acid sequence encoding an amino acid sequence having at least 70% at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47.

32. The EHV vector or expression cassette according to any one of claims 25-31, wherein the EHV vector or expression cassette does not include NP (nucleoprotein) or N (neuraminidase) sequences encoding an influenza antigen.

33. The EHV vector or expression cassette according to any one of claims 25 to 32, wherein the p422 promoter sequence (SEQ ID NO: 5), or a functional fragment or functional derivative thereof, or any complementary nucleotide sequence thereof is operably linked to a nucleic acid sequence encoding an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% , at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 44 ( H1pdm).

34. The EHV vector of claim 2-33, wherein the EHV vector includes two or more sequences encoding influenza hemagglutinin antigens.

35. The EHV vector of claim 34, wherein the additional sequence encoding the influenza hemagglutinin antigen is a nucleic acid sequence encoding an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence as indicated in SEQ ID NO: 46 (H1av).

36. The EHV vector of claim 35, wherein said additional sequence encoding the influenza hemagglutinin antigen is inserted into UL56.

37. The EHV vector of claim 35 or 36, wherein the sequence encoding the influenza hemagglutinin antigen of claim 35 is operably linked to the p430 promoter sequence (SEQ ID NO: 3) or a functional fragment or functional derivative thereof or complementary nucleotide sequences thereof.

38. The EHV vector of claim 34-37, wherein the additional sequence encoding influenza hemagglutinin antigens is a nucleic acid sequence encoding an amino acid sequence having at least 70%, at least 75%, at least 80%, at least at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% identity to the amino acid sequence as indicated in SEQ ID NO: 45 (H3).

39. The EHV vector of claim 38, wherein said additional sequence encoding influenza hemagglutinin antigens is inserted into US4.

40. The EHV vector of claim 38 or 39, wherein the sequence encoding the influenza hemagglutinin antigens of claim 38 is operably linked to the p455 promoter sequence (SEQ ID NO: 4) or a functional fragment or functional derivative thereof or complementary nucleotide sequences thereof.

41. The EHV vector as claimed in any one of claims 2-40, wherein the EHV vector is selected from the group consisting of EHV-1, EHV-3, EHV-4, EHV-8 and EHV-9.

42. The EHV vector according to any one of paragraphs 2-41, where the EHV vector is EHV-1 or EHV-4.

43. The EHV vector according to any one of paragraphs 2-42, where the EHV vector is EHV-1, preferably

- 39 046085 specifically RacH.

44. A mammalian host cell, characterized in that it includes a vector in accordance with paragraphs 2-43.

45. Use of a vector in accordance with paragraphs 2-43 or a mammalian host cell in accordance with paragraph 44 to obtain an immunogenic composition or vaccine.

46. Immunogenic composition, including

a) vector in accordance with paragraphs 2-43, and/or

b) a polypeptide expressed by a vector in accordance with paragraphs 2-43, such as a virus, modified live virus, virus-like particle (VLP) or the like, and

c) optionally a pharmaceutically or veterinarily acceptable carrier or excipient, wherein said carrier is preferably suitable for oral, intradermal, intramuscular or intranasal administration, preferably said immunogenic composition comprising a virus, such as a pathogenic virus.

47. A vaccine or pharmaceutical composition comprising

a) vector in accordance with paragraphs 2-43, and/or

b) a polypeptide expressed by a vector in accordance with paragraphs 2-43, such as a modified live virus, virus-like particle (VLP) or the like, and

c) a pharmaceutically or veterinary acceptable carrier or excipient, wherein said carrier is preferably suitable for oral, intradermal, intramuscular or intranasal administration, optionally said vaccine further comprising an adjuvant.

48. A vaccine or DIVA vaccine comprising one or more EHV vectors according to any of paragraphs 2-43.

49. A method of producing an immunogenic composition or vaccine to reduce the frequency or severity of one or more clinical symptoms associated with or caused by an infection, comprising the following steps:

a) infection of a mammalian host cell in accordance with paragraph 41 using a vector in accordance with paragraphs 2 to 43,

b) culturing the infected cells under suitable conditions,

c) collection of infected cell cultures,

d) optionally purifying the harvested infected cell culture from step c) and optionally mixing said harvested infected cell culture with a pharmaceutically acceptable carrier.

50. An immunogenic composition, vaccine or DIVA vaccine in accordance with any of paragraphs 46 to 48 for use in a method of immunizing an animal, comprising administering to said animal said immunogenic composition, vaccine or DIVA vaccine.

51. An immunogenic composition, vaccine or DIVA vaccine in accordance with any of paragraphs 46 to 48 for use in a method of reducing or preventing clinical symptoms caused by a pathogen in an animal in need thereof, which includes administering to the animal a therapeutically effective amount of said immunogenic composition, vaccine or DIVA vaccines.

52. An immunogenic composition, vaccine or DIVA vaccine according to any one of paragraphs 46 to 48 for use in a method of reducing or preventing clinical symptoms caused by a swine influenza virus in an animal in need thereof, which includes administering to the animal a therapeutically effective amount of said immunogenic composition, vaccines or DIVA vaccines.

53. A method of immunizing an animal, including administering to such an animal an immunogenic composition, vaccine or DIVA vaccine according to any one of paragraphs 46-48.

54. A method of reducing or preventing clinical symptoms caused by a pathogen in an animal in need thereof, which comprises administering to the animal a therapeutically effective amount of an immunogenic composition, vaccine or DIVA vaccine in accordance with any of paragraphs 46-48.

55. A method of reducing or preventing clinical symptoms caused by swine influenza virus in an animal in need thereof, which comprises administering to the animal a therapeutically effective amount of an immunogenic composition, vaccine or DIVA vaccine in accordance with any of paragraphs 46-48.

56. The method or use of any one of paragraphs 50-55, where the animal is a pig, piglet or sow, poultry, cattle, horse, dog or cat.

57. The method or use of any one of claims 50-56, wherein the immunogenic composition, vaccine or DIVA vaccine is administered once.

58. The method or use according to any one of paragraphs 50-57, where the immunogenic composition, vaccine or DIVA vaccine is administered to the animal during the first six weeks of life, during the first two weeks

- 40 046085 days of life, during the first week of life or during the first day of life.

59. The method or use of any one of claims 50-58, wherein the immunogenic composition, vaccine or DIVA vaccine is administered in two doses.

60. The method or use of claim 59, wherein the immunogenic composition, vaccine or DIVA vaccine is administered to the animal for the first time during the first week of life and a second time during the second, third or fourth week of life.

61. The method or use of any one of claims 50-60, wherein said immunogenic composition, vaccine or DIVA vaccine is administered intramuscularly or intranasally.

62. The method or use of any one of claims 50-61, wherein the immunogenic composition, vaccine or DIVA vaccine includes from 1x 10 ⁴ to 1x 10 ⁷ TCID50 EHV vector.

63. The method or use according to any of paragraphs 50 - 62, where the specified method leads to an improvement in an effectiveness parameter selected from the group consisting of: decreased weight loss, decreased rectal temperature, decreased clinical symptoms, increased induction of (neutralizing) antibodies, or their combinations, compared with an animal from a non-immunized control group of the same species.

64. A kit for immunizing an animal, preferably an animal raised for food purposes such as a pig, poultry or cattle, or companion animals such as cats, dogs or horses, against a disease associated with and/or reducing the incidence or the severity of one or more clinical symptoms associated with or caused by the pathogen in the animal, including:

a) a dispenser capable of administering the vaccine to the specified animal; And

b) an immunogenic composition in accordance with paragraph 46, a vaccine in accordance with paragraph 47 or a DIVA vaccine in accordance with paragraph 48, and

c) optionally an instruction leaflet.

65. A promoter sequence comprising p422 (SEQ ID NO: 5) or a complementary nucleotide sequence thereof or a functional fragment thereof or complementary nucleotide sequences thereof, wherein said promoter sequence results in the expression of a nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen-coding sequence .

66. An expression cassette comprising a p422 promoter sequence (SEQ ID NO: 5) or a complementary nucleotide sequence or functional fragment thereof and complementary nucleotide sequences thereof, wherein the promoter sequence is operably linked to a sequence of interest, preferably a gene of interest, such as an antigen-encoding one a sequence, more preferably a heterologous and/or exogenous sequence of interest, a gene of interest or an antigen-coding sequence of interest, wherein said promoter sequence leads to the expression of a nucleotide sequence of interest, preferably a gene of interest, more preferably an antigen-coding sequence, wherein said promoter sequence is preferably a heterologous promoter sequence, more preferably an exogenous promoter sequence.

67. A vector comprising the promoter sequence or expression cassette of claim 65 or 66.

68. The promoter or expression cassette or vector according to any one of paragraphs 65-67, wherein the functional fragment of the promoter sequence has sequence identity and/or homology of 70, 80, 85%, preferably 90, 91, 92, 93, 94%, more preferably 95, 96, 97, 98, 99, 99.9% relative to the p422 sequence (SEQ ID NO: 5).

69. The promoter or expression cassette or vector according to any of paragraphs 65 to 68, where the specified functional fragment of the promoter sequence has a length of 100 nucleotides, preferably 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400 nucleotides, most preferably 410 or 420 nucleotides, or wherein the functional promoter sequence fragment is 100 to 422 nucleotides, 200 to 422 nucleotides, 300 to 422 nucleotides, or 350 to 422 nucleotides in length.

70. The expression cassette or vector of any one of claims 66-69, wherein the expression cassette or vector further comprises a polyadenylation sequence, preferably BGHpA, 71pA (SEQ ID NO: 6), or 18pA (SEQ ID NO: 7).

71. The expression cassette or vector according to any one of paragraphs 66-70, wherein said expression cassette or vector includes one or more additional regulatory sequences, such as a stop codon, a polyadenylation signal, or a regulatory element like an IRES and/or 2a peptide.

- 41 046085

72. The vector according to any one of paragraphs 67-71, where the specified vector is a recombinant and/or heterologous and/or exogenous vector.

73. The vector according to any one of claims 67-72, wherein said vector is a viral vector, preferably selected from the group consisting of Herpes viridae, such as equine alphaherpesvirus type 1 (EHV-1), equine alphaherpesvirus type 4 (EHV-4 ) and other Varicelloviruses like PrV (pseudorabies virus) and BHV-1 (bovine herpesvirus type 1), Adenoviridae (AdV) such as CAdV (canine adenovirus), adeno-associated viruses, Baculoviridae, Lentiviridae, such as retroviruses, and Poxviridae.

74. The vector according to any one of claims 67-73, wherein said vector is a member of the family Herpesviridae, preferably the genus Alphaherpesvirinae, more preferably the subgenus Varicellovirus, most preferably said vector is equine alphaherpesvirus type 1 (EHV-1).

Sequence overview.

The following sequences are set out in detail and are thus disclosed in the present invention.

Promoter and polyA sequences.

(SEQ ID NO: 1) 600 DNA fragment 4pgG600.

(SEQ ID NO: 2) 600 DNA fragment 4pMCP600:

(SEQ ID NO: 3) Specifically, the 600b promoters were truncated to 430b for 4pgG, new name: p430.

(SEQ ID NO: 4) and up to 449 for 4pMCP, new name: p455.

(SEQ ID NO: 5) Sequence of p422 promoter.

(SEQ ID NO: 6) Sequence 71pA, polyadenylation sequence.

(SEQ ID NO: 7) Sequence 18pA, polyadenylation sequence.

Sequences of insertion sites.

(SEQ ID NO: 8) US4 (orf70) RacH sequence.

(SEQ ID NO: 9) Up70 flanking region (417 bp).

(SEQ ID NO: 10) Up71 flanking region (431 bp).

(SEQ ID NO: 11) 127264-127680 (flanking region up orf70).

(SEQ ID NO: 12) 128484-128913 (flanking region up orf71).

(SEQ ID NO: 13) Up70 flanking region (283 bp) = identical to 3' 283 bp of the classic 417 bp flanking region.

(SEQ ID NO: 14) Up71 flanking region (144bp) = identical to 5' 144bp of the classic 431bp flanking region.

(SEQ ID NO: 15) Sequence of US4 (orf70) wt - strain ab4 nt 127681-128916.

(SEQ ID NO: 16) Deleted portion of orf70 (US4) in the wild-type ab4 genomic sequence (Genbank accession number AY665713.1): nt 127681-128482.

(SEQ ID NO: 17) Deleted portion of orf70 (US4) in the RacH genomic sequence (no nt numbers available because the complete genomic sequence is unknown).

(SEQ ID NO: 18) UL43 RacH sequence.

(SEQ ID NO: 19) Sequence of the upstream recombination region of Up UL43.

(SEQ ID NO: 20) Sequence of the recombination region downstream of Up UL44.

(SEQ ID NO: 21) Sequence of the deleted portion of UL43 in RacH.

(SEQ ID NO: 22) Sequence of the conserved 3' end of UL43 in RacH.

(SEQ ID NO: 23) UL43 sequence in wt EHV-1 V592 (nt 23021-24226, reverse complementary).

(SEQ ID NO: 24) Deleted portion (870 bp) of UL43 in wt EHV-1 V592 (nt 23353-24226, reverse complementary).

(SEQ ID NO: 25) Retained part of UL43 reading frame in wt EHV-1

V592 (nt 23021-23354, reverse complementary).

(SEQ ID NO: 26) Upstream sequence of the corresponding Up UL43 recombination region in wt EHV-1 V592 (nt 24227-24452, with reverse complementarity).

(SEQ ID NO: 27) Downstream sequence of the corresponding Up recombination region of UL44 in wt EHV-1 V592 (nt 23049-23354, with reverse complementarity).

Plasmid sequences.

(SEQ ID NO: 28) Nucleotide sequence of transfer vector pU70-p455-71K71.

(SEQ ID NO: 29) Nucleotide sequence of transfer plasmid pU70-p455-H3-71K71.

(SEQ ID NO: 30) Nucleotide sequence of transfer vector pU-1-3-p430-BGHKBGH.

(SEQ ID NO: 31) Nucleotide sequence of transfer plasmid pU1-3-p430-H1avBGHKBGH.

(SEQ ID NO: 32) Nucleotide sequence of transfer plasmid pU70-p455-H1pdm- 42 046085

71K71.

(SEQ ID NO: 33) Nucleotide sequence of transfer plasmid pU1-3-p430-H1huBGHKBGH.

(SEQ ID NO: 34) Nucleotide sequence of transfer vector pUUL43-p422-18K18.

(SEQ ID NO: 35) Nucleotide sequence of transfer plasmid pUUL43-p422-mC18K18.

(SEQ ID NO: 36) Nucleotide sequence of transfer plasmid pUUL43-p422-H1pdm18K18.

(SEQ ID NO: 37) Nucleotide sequence of transfer plasmid pUmC70.

Primer sequences.

For the insertion site orf70/US4.

(SEQ ID NO: 38) Forward primer AGGCTCGTGCGCGGATACATCG.

(SEQ ID NO: 39) Reverse primer TTCGGGGCTGTTAGACTCCTCC.

For the insertion site orf1/3/UL56.

(SEQ ID NO: 40) Forward primer CCAACTCGCCGCCATGAGACCC.

(SEQ ID NO: 41) Reverse primer AGCGCGCCCCGTACCCAGTGGG.

For insertion site UL43.

(SEQ ID NO: 42) Forward primer CGACGCGCGTCGGAGG.

(SEQ ID NO: 43) Reverse primer GTTATAAACATACCATGCACC.

Amino acid sequences of influenza A virus hemagglutinins.

(SEQ ID NO: 44) hemagglutinin [influenza A virus (A/swine/Italy/116114/2010(H1N2))]; GenBank: ADR01746.1 H1pdm.

(SEQ ID NO: 45) hemagglutinin [influenza A virus (A/swine/Italy/7680/2001(H3N2))]; GenBank: ABS50302.2 H3.

(SEQ ID NO: 46) hemagglutinin [influenza A virus (A/swine/Gent/132/2005(H1N1))]; GenBank: AFR76623.1 H1av.

(SEQ ID NO: 47) hemagglutinin [influenza A virus (A/swine/Italy/4675/2003(H1N2))]; GenBank: ADK98476.1* H1hu.

Brief description of the figures

The following figures form part of the present description and are included to further demonstrate certain aspects of the present invention. The invention may be better understood with reference to one or more of these figures in combination with a detailed description of the specific embodiments presented in the present invention.

Fig. 1. Schematic illustration of comparison of UL56 (orf1/3) regions of EHV-1 wild-type (wt) strain ab4 and the attenuated EHV-1 vaccine strain RacH. orf1, orf2, orf3 = first three open reading frames in the EHV-1 genome, orf1 has a homologue in other alphaherpesviruses designated UL56 Flank A, Flank B = recombination sites for insertion of a transgene expression cassette at the orf1/3 (UL56) site (prior art ).

Fig. 2. Schematic representation of the US4 (orf70) insertion site.

UL = long unique segment,

US = short unique segment,

IR = internal inverted repeat,

TR = terminal inverted repeat, gG = glycoprotein G, pA = polyadenylation sequence at the end of the coding sequence, gpII = glycoprotein II, orf = open reading frame, orf69, orf70, orf71 = US3, US4, US5 (open reading frames relevant for orf70 /US4 insertion site),

Aorf1/3 = orf1/3 (UL56) insertion site (prior art), by = base pairs.

Fig. 3. Plasmid map of the transfer plasmid pU-p455-H3-71K71.

H3 = open reading frame encoding influenza A virus hemagglutinin H3,

71pA = new polyA sequence as described in the disclosure, EM P2016-022,

I-SceI = restriction site for restriction endonuclease I-SceI, aph promoter = prokaryotic kanamycin-resistant gene promoter,

Kana = kanamycin resistance gene,

3' end of ORF70 = recombination region located downstream of the insertion site,

ORI = plasmid origin of replication

AP _r = ampicillin-resistant plasmid gene, upstream orf70 = recombination site located

- 43 046085 upstream of the insertion site, p455 = new p455 promoter, by = base pairs.

Fig. 4. Plasmid map of the transfer vector pU1-3-p430-H1av-BGHKBGH.

H1av = open reading frame encoding influenza A virus H1av hemagglutinin,

BGHpA = polyA sequence of the bovine growth hormone gene,

I-SceI = restriction site for restriction endonuclease I-SceI, aph promoter = prokaryotic kanamycin-resistant gene promoter, Kana = kanamycin-resistant gene,

Flank A = recombination site located upstream of the insertion site, ORI = origin of plasmid replication,

AP _r = ampicillin resistance gene plasmid,

Flank B = recombination site located downstream of the insertion site, p430 = new p430 promoter, by = base pairs.

Fig. 5. Schematic illustration of the rEHV-1 RacH-SE-70-p455-H3 genome with an extended US4 (orf70) insertion site.

orf69/US3: open reading frame number 69 (US3) upstream of the insertion site in orf70 (US4), p455: a new promoter described in this invention,

H3: influenza virus hemagglutinin transgene,

71pA: new polyadenylation sequence,

Aorf70 (US4): a residue from orf70 (US4) containing the promoter for orf71 (US5), which encodes the structural viral glycoprotein II (gpII), at = base pairs.

Fig. 6. Schematic illustration of the rEHV-1 RacH-SE-1/3-p430-H1av genome with the extended UL56 (orf1/3) insertion site.

p430: new promoter described in this invention,

H1av: influenza virus hemagglutinin transgene,

BGHpA: bovine growth hormone polyadenylation sequence,

Δorf1/UL56: residue from orf1 (UL56),

Orf3: EHV-1 open reading frame orf3 (no homolog in other Alphaherpesviridae), by = base pairs.

Fig. 7. Schematic illustration of the rEHV-1 RacH-SE-1/3-p430-H1av-70-p455-H3 genome (rEHV-1RacH-SE B) with two extended insertion sites.

p430: new promoter described in this invention,

H1av: influenza virus hemagglutinin transgene,

BGHpA: bovine growth hormone polyadenylation sequence,

Δorf1/UL56: residue from orf1 (UL56),

Orf3: EHV-1 open reading frame orf3 (no homolog in other Alphaherpesviridae), orf69/US3: open reading frame number 69 (US3) upstream of the insertion site in orf70 (US4), p455: new promoter described in this invention,

H3: influenza virus hemagglutinin transgene,

71pA: new polyadenylation sequence,

Aorf70 (US4): a residue from orf70 (US4) containing the promoter for orf71 (US5), which encodes the structural viral glycoprotein II (gpII), at = base pairs.

Fig. 8. Plasmid map of the transfer plasmid pU1/3-p430-H1hu-BGHKBGH.

p430 = new p430 promoter,

H1hu = open reading frame encoding influenza A virus H1hu hemagglutinin, BGHpA = bovine growth hormone gene polyA sequence, I-SceI = restriction site for restriction endonuclease I-SceI, aph promoter = prokaryotic kanamycin-resistant gene promoter, Kana = kanamycin-resistant gene gene,

Flank A = recombination site located upstream of the insertion site, ORI = plasmid origin of replication,

Flank B = recombination site located downstream of the insertion site, I-Ceu = homing endonuclease to release fragment for RED recombination, by = base pairs.

Fig. 9. Plasmid map of the transfer plasmid pU70-p455-H1pdm-71K71.

- 44 046085 upstream orf 70 = recombination sequence located upstream of the insertion site, p455 = new promoter described in this invention,

H1pdm = influenza virus H1pdm hemagglutinin transgene,

71pA = new polyadenylation sequence,

3' end orf70 = recombination sequence located downstream of the insertion site, aph promoter = prokaryotic kanamycin-resistant gene promoter,

Kana = kanamycin resistance gene, by = base pairs,

ScaI, EcoRI, SalI, NotI, KpnI, BamHI, XbaI = restriction endonuclease restriction sites.

Fig. 10. Schematic illustration of the rEHV-1 RacH-SE-70-p455-H1pdm genome with an extended US4 (orf70) insertion site.

orf69/US3 = open reading frame number 69 (US3) upstream of the insertion site in orf70 (US4), p455 = new promoter described in this invention,

H1pdm = influenza virus H1pdm hemagglutinin transgene,

71pA = new polyadenylation sequence,

Aornf7() (US4): a residue from orf70 (US4) containing the promoter for orf71 (US5), which encodes the structural viral glycoprotein II (gpII), at = base pairs.

Fig. 11. Schematic illustration of the rEHV-1 RacH-SE-1/3-p430-H1hu genome with the extended UL56 (orf1/3) insertion site.

p430 = new promoter described in this invention,

H1hu = influenza virus H1hu hemagglutinin transgene,

BGHpA = bovine growth hormone polyadenylation sequence,

Aorf1/UL56 = residue from orf1 (UL56),

Orf3 = EHV-1 open reading frame of orf3 (no homolog in other Alphaherpesviridae), by = base pairs.

Fig. 12. Schematic illustration of the genome of rEHV-1 RacH-SE-1/3-p430-H1hu-70-p455-H1pdm (virus D) with an extended insertion site.

p430 = new promoter described in this invention,

H1hu = influenza virus H1hu hemagglutinin transgene,

BGHpA = bovine growth hormone polyadenylation sequence,

Aorn/UL56 = residue from orf1 (UL56),

Orf3 = EHV-1 open reading frame of orf3 (no homologue in other Alphaherpesviridae), orf69/US3 = open reading frame number 69 (US3) upstream of the insertion site in orf70 (US4), p455 = new promoter described in this invention,

H1pdm = influenza virus H1pdm hemagglutinin transgene,

71pA = new polyadenylation sequence,

Aorf70 (US4) = residue from orf70 (US4) containing the promoter for orf71 (US5), which encodes structural viral glycoprotein II (gpII), at = base pairs.

Fig. 13. Schematic illustration of the construction of a new insertion site for the transgene UL43 UL44, UL43, UL42 open reading frames in the insertion site, 18pA: new polyadenylation site, 422 promoter: new p422 promoter, by: base pairs.

Fig. 14. Plasmid map of the transfer plasmid pUUL43-422-mC-18K18.

UpUL43 = viral genomic DNA sequence flanking insertion site upstream,

UpUL44 = viral genomic DNA sequence flanking the insertion site located downstream of transcription,

422 promoter = promoter that controls transgene expression, mC = transgene (mChermy autofluorescent protein),

18pA = new polyadenylation sequence,

I-Sce1 = restriction site for I-Sce1, promoter aph = prokaryotic promoter driving kanamycin-resistant gene expression,

Kana = kanamycin-resistant orf,

- 45 046085

P(BLA) = prokaryotic promoter that drives ampicillin-resistant gene expression,

AP(R) = ampicillin resistance gene,

ORI = plasmid origin of replication,

P(LAC) = prokaryotic promoter of lacZ, encoding beta-galactosidase,

I-Ceu = recognition site for homing endonuclease I-Ceu.

Fig. 15. Schematic illustration of the rEHV-1 RacH-SE-UL43-422-mC genome with the extended UL43 insertion site.

UL = Unique long segment of the EHV genome,

US = Unique short segment of the EHV genome,

IRS and TRS = Inner and tail repeat sections, inserting a short unique segment into the frame,

UL44, UL43, UL42 = open reading frames at the insertion site,

AUL43 = remainder of UL43,

18pA = new polyadenylation site, p422 = new p422 promoter, by = base pairs.

Fig. 16. Plasmid map of the transfer plasmid pUUL43-422-H1pdm-18K18.

UpUL43 = viral genomic DNA sequence flanking the upstream insertion site,

UpUL44 = viral genomic DNA sequence flanking the insertion site located downstream of transcription,

422 promoter = promoter that controls the expression of the transgene,

H1pdm = transgene (influenza A hemagglutinin H1pdm),

18pA = new polyadenylation sequence,

I-Sce1 = restriction site for I-Sce1, promoter aph = prokaryotic promoter driving kanamycin-resistant gene expression,

Kana = kanamycin-resistant orf,

P(BLA) = prokaryotic promoter that drives ampicillin-resistant gene expression,

AP(R) = ampicillin resistance gene,

ORI = plasmid origin of replication,

P(LAC) = prokaryotic promoter of lacZ, encoding beta-galactosidase,

I-Ceu = recognition site for homing endonuclease I-Ceu.

Fig. 17. Schematic illustration of the rEHV-1 RacH-SE-UL43-422-H1pdm genome with the extended UL43 insertion site.

UL = Unique long segment of the EHV genome,

US = Unique short segment of the EHV genome,

IRS and TRS = Inner and tail repeat sections, inserting a short unique segment into the frame,

UL44, UL43, UL42 = open reading frames at the insertion site,

AUL43 = remainder of UL43,

18pA = new polyadenylation site,

H1pdm = transgene (influenza A hemagglutinin H1pdm), p422 = new p422 promoter, by = base pairs.

Fig. 18. Schematic illustration of the rEHV-1 genome RacH-SE-1/3-p430-H1av-UL43-422-H1pdm-70p455-H3 with an extended insertion site.

Δorf1/UL56: residue from UL56 at the boundary of the expression cassette, p430: new p430 promoter,

BGHpA: bovine growth hormone polyadenylation site,

H1av, H3, H1pdm: transgenes (influenza A hemagglutinins),

Aorf70/US4: residue from US4 at the border of the expression cassette, orf69 (US3) and orf71 (US5) open reading frames in the US4 insertion site,

71pA: new polyadenylation sequence,

UL44, UL43, UL42 open reading frames in the UL43 insertion site,

18pA: new polyadenylation site, p422: new p422 promoter, by: base pairs.

Fig. 19. Western blotting.

- 46 046085

Quadruplicate blots incubated with four different antibodies, a: blot incubated with a proprietary monoclonal antibody against influenza H1av HA, b: blot incubated with a commercial rabbit antiserum specific for influenza H3 HA, c: blot incubated with a proprietary monoclonal antibody anti-HA H1pdm influenza, d: blot incubated with proprietary monoclonal antibody against EHV-1 gpII,

M = molecular weight marker (kDa = kilodalton, 250, 150, 100, 75, 50, 37, 25, 20)

Virus name abbreviation Insertion sites used Expressed transgenes rEHV-1 RacH-SE-70-p455-H3 US4-H3 US4 nz rEHV-1 RacH-SE-l/3-p430Hlav UL56-Hlav UL56 Hlav rEHV-1 RacH-SE-70-p455Hlpdm US4-Hlpdm US4 Hlpdm rEHV-1 RacH-SE-l/3-p430Hlhu UL56-Hlhu UL56 Hlhu rEHV-1 RacH-SE-l/3-p430- Hlav-70-455-H3 IN US4 and UL56 NC and Hlav rEHV-1 RacH-SE-l/3-p430- Hlhu-70-455-Hlpdm D US4 and UL56 Hlpdm and Hlhu rEHV-1 RacH-SE-UL43- UL43-Hlpdm UL43 Hlpdm Hlpdm rEHV-1 RacH-SE-l/3-p430Hlav-UL43-422-Hlpdm70455-H3 E US4 and UL56 and UL43 NC, Hlav, and Hlpdm rEHV-1 RacH-SE S.E. none none

Fig. 20. Results of tests for neutralization of influenza A virus in mouse sera.

Virus neutralization tests were performed in duplicate or triplicate depending on the amounts of mouse sera available. Neutralization titers were normalized to 100 TCID50 and shown as reciprocal neutralization abilities. *Error bars indicate standard deviation.

Fig. 21. Plasmid map of transfer plasmid pUmC70 3' end of orf69 = part of orf69(US3) contained in the transfer vector.

up70 = recombination sequence located upstream of the insertion site, mCherry = transgene (mCherry autofluorescent protein),

BGHpA = bovine growth hormone polyadenylation sequence, up71 = recombination sequence located downstream of the insertion site,

3' end of orf70 = residue from orf70 (US4), located downstream of the insert, at = base pairs,

Seal, EcoRI, Sall, NotI, Kpnl, BamHI, Xbal = restriction endonuclease restriction sites.

Fig. 22. Plasmid map of transfer vector pU70-p455-71K71.

Up70 = viral genomic DNA sequence flanking the upstream insertion site,

Up71 = viral genomic DNA sequence flanking the insertion site located downstream of transcription,

4pMCP455 = promoter driving transgene expression,

EHV-4orf71pApA = new 71pA polyadenylation sequence,

I-Sce1 = restriction site for I-Sce1, promoter aph = prokaryotic promoter driving kanamycin-resistant gene expression,

Kana = kanamycin-resistant orf,

P(BLA) = prokaryotic promoter that drives ampicillin-resistant gene expression,

- 47 046085

AP(R) = ampicillin resistance gene,

ORI = plasmid origin of replication,

P(LAC) = prokaryotic promoter of lacZ, encoding beta-galactosidase,

I-Ceu = recognition site for homing endonuclease I-Ceu,

KpnI, NotI, XbaI = restriction endonuclease restriction sites, by = base pairs.

Fig. 23. Plasmid map of the transfer vector pU1/3-p430-BGHKBGH.

Flank A = viral genomic DNA sequence flanking the insertion site located upstream of transcription.

Flank B = viral genomic DNA sequence flanking the insertion site located downstream of transcription,

4pgG430 = promoter driving transgene expression,

BGHpA = bovine growth hormone gene polyadenylation sequence,

I-Sce1 = restriction site for I-Sce1, promoter aph = prokaryotic promoter driving kanamycin-resistant gene expression,

Kana = kanamycin-resistant orf,

I-Ceu = recognition site for homing endonuclease I-Ceu,

KpnI, NotI = restriction endonuclease sites, by = base pairs.

Fig. 24. Plasmid map of transfer plasmid pUUL43-422-H1pdm-18K18.

UpUL43 = viral genomic DNA sequence flanking the upstream insertion site,

UpUL44 = viral genomic DNA sequence flanking the insertion site located downstream of transcription, p422 = promoter driving transgene expression,

18pA = new polyadenylation sequence,

I-Sce1 = restriction site for I-Sce1, promoter aph = prokaryotic promoter driving kanamycin-resistant gene expression,

Kana = kanamycin-resistant orf,

P(BLA) = prokaryotic promoter that drives ampicillin-resistant gene expression,

AP(R) = ampicillin resistance gene,

ORI = plasmid origin of replication,

P(LAC) = prokaryotic promoter of lacZ, encoding beta-galactosidase,

I-Ceu = recognition site for homing endonuclease I-Ceu by = base pairs.

Examples

The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art will appreciate that the methods disclosed in the examples represent methods disclosed by the inventors for the normal practice of the present invention, and thus may be considered preferred methods for its practice. However, it will be apparent to those skilled in the art in light of the present disclosure that many changes can be made to the specific embodiments disclosed and still achieve a similar or similar result without departing from the spirit and scope of the present invention.

Example 1.

Creation of a new ORF70/US4 insertion site.

In order to increase the capabilities of the EHV-1 vector, the inventors discovered a way to express two different transgenes from a single vector backbone without linking the two transgenes using RNA virus-derived functions under the control of a single promoter. The inventors hypothesized that the herpes virus genome would be tolerant to the use of two independent transgene insertion sites in parallel. To determine whether EHV-1 ORF70/US4 was a suitable transgene insertion site, 801 bp at the 5' end of orf70/US4 (bp 1236) was replaced with an expression cassette encoding the autofluorescent mCherry protein (Shaner et al. 2004) using the classical homologous recombination. A map of plasmid pU-mC70-BGH is shown in Fig. 21 (SEQUENCE ID NO: 37). The DNA fragment used for homologous recombination was excised from pU-mC70-BGH using XbaI. The gel-purified fragment was cotransfected with viral genomic DNA from EHV-1 RacH into RK13 cells. Efficient release of the recombinant vector virus and efficient replication in cultured cells were demonstrated using live fluorescence and virus titrations (not shown). Deletion of two-thirds of orf70/US4 had the added benefit of expressing glycoprotein G,

- 48 046085 coded orf70/US4, has been cancelled. Glycoprotein G from EHV-1 has been shown to be a nonstructural, secreted chemokine-binding protein that antagonizes the host immune response (Drummer et al., 1998; Bryant et al., 2003). Since the vector vaccine is intended to stimulate an immune response to the vaccine, removal of this particular immunosuppressive function of the viral vector should further improve the platform of functioning of the EHV-1 RacH-SE viral vector.

Example 2.

Application of a new ORF70/US4 insertion site with the p455 promoter in a recombinant EHV-1 vaccine vector and construction of a recombinant virus.

p455 promoter.

For the first animal experiment, influenza H3 subtype hemagglutinin from swine influenza A virus (A/swine/Italy/7680/2001(H3N2), GenBank accession number: ABS50302.2) was used. Its coding sequence was synthesized and subcloned into the transfer vector pU70-p455-71K71 (SEQ ID NO: 28), creating the transfer plasmid pU70-p455-H3-71K71, placing H3 under the control of the new p455 promoter and the new 71pA polyadenylation signal, and inserting in frame cassette with recombination sites for insertion into orf70 (Fig. 3, SEQ ID NO: 29).

By en-passant mutagenesis using the RED recombination system (Tischer et al. 2006), the p455-H3-71 expression cassette was inserted into orf70/US4 from pRacH-SE to generate pRacH-SE70p455-H3.

PK/WRL cells were transfected with pRacH-SE70-p455-H3, and the recombinant virus rEHV1 RacH-SE70-p455-H3 (Fig. 5) was released and plaque purified twice. The correct insertion of the expression cassette was confirmed by sequencing the product using high-fidelity PCR with corrective exonuclease activity of the insertion site. Transgene expression in infected cells was analyzed by indirect immunofluorescence assay.

Reconstitution of orf71, encoding EHV-1 gpII, was confirmed by IFA (not shown) and Western blotting (Fig. 19) using monoclonal antibody Ai2G7 (owned by BI). The appearance of H3 trimers on the plasma membrane of infected cells was analyzed by a hemadsorption reaction using chicken erythrocytes (not shown). Peak titers, measured as _TIR50 /ml in PK/WRL cells, were in the same range as those of the parental rEHV-1 RacH-SE virus, indicating that transgene expression does not have a negative effect on viral replication (not shown). This was confirmed by passivation of rEHV-1 RacH-SE70-p455H3 in PK/WRL cells up to passage 20 (P20) after release. At passages P5, P10, P15, and P20, the virus was characterized by titration, sequencing, and Western blotting, at PP10 and P20 additionally by IFA, and the HA expression and genetic stability of the insert that encodes HA, along with the promoter and polyA sequences, were confirmed.

By dual immunofluorescence assay (dIFA) of viral plaques in cells infected with P20, using anti-H3 monoclonal antibody and horse anti-EHV serum, it was confirmed that virtually all EHV-1-induced plaques also express H3 (not shown). All tests confirmed the stability of recombinant EHV-1 RacH-SE-70-p455-H3.

Example 3.

Application of a new p430 promoter in a recombinant EHV-1 vaccine vector and construction of a recombinant virus.

p430 promoter.

The newly identified p430 promoter was used to drive the expression of another hemagglutinin from the H1N1 influenza virus ((A/swine/Gent/132/2005(H1N1), GenBank accession number: AFR76623.1). Because the hemagglutinin gene in this virus isolate was of avian IAV origin , then it was designated as H1av.H1av was synthesized and subcloned into the transfer vector pU1/3-p430-BGHKBGH (SEQ ID NO: 30) for the orf1/3/UL56 insertion site to obtain pU1/3-p430H1av-BGH_K_BGH (Fig. 4, SEQ ID NO: 31) H1av expression was controlled by the p430 promoter and the bovine growth hormone (BGH) polyA signal.

By en-passant mutagenesis using the RED recombination system (Tischer et al. 2006), the p430-H1av-BGH expression cassette was inserted into orf1/3/UL56 from pRacH-SE to generate pRacHSE1/3-p430-H1av.

PK/WRL cells were transfected with pRacH-SE1/3-p430-H1av, and the recombinant rEHV-1 virus RacH-SE1/3-p430-H1av (Fig. 6) was released and plaque purified twice. The correct insertion of the expression cassette was confirmed by sequencing the product using high-fidelity PCR with corrective exonuclease activity of the insertion site. Transgene expression in infected cells was analyzed by indirect immunofluorescence assay (IFA) and Western blotting using monoclonal and polyclonal antibodies (Fig. 19). The specific detection of a broad band migrating at 75 kDa by antibody PA-34929 is consistent with the expected appearance of a recombinant HA glycoprotein as predicted from its sequence. Reconstitution of orf71/US5 encoding EHV-1 gpII was confirmed by IFA and Western blotting using monoclonal antibody Ai2G7 (owned by BI) (FIG. 19). Peak titers, measured as TIR50/ml in PK/WRL cells, were in the same range as those of the parental RacH-SE virus, indicating that transgene expression does not negatively affect viral replication (not shown) .

To test whether the expressed recombinant hemagglutinins would be processed and transported as expected, VERO cells were infected with rEHV-1 RacH-SE-1/3-p430-H1av, rEHV-1 RacH-SE-70-p455- H3, rEHV-1 RacH-SE (parental) at M.O.I. 0.01, or left uninfected. After 24 hours p.i. live infected and uninfected cells were incubated with a suspension of chicken red blood cells in PBS, washed with PBS, and stained with fluorescent Hoechst 33342 to stain nuclei. Since avian red blood cells contain cell nuclei, they can be stained with Hoechst33342 and are detected as miniature blue spots using fluorescence microscopy. Compared to cells that were infected with rEHV-1 RacH-SE, which do not express hemagglutinin, chicken erythrocyte adsorption was significantly increased for cells infected with either rEHV-1 RacH-SE-1/3-p430H1av or rEHV- 1 RacH-SE-70-p455-H3 (not shown). Based on this, it can be concluded that hemagglutinins were translated, processed and transported to the plasma membrane of cells infected with the vector virus in the same way as if they were produced by authentic infection with influenza virus.

The distinct hemadsorption phenotype of infected cells is confirmed by the results of Western blots and latency assays, which show efficient expression of transgenic proteins and suggest the formation of functional HA trimers on the cell surface of cells infected with the EHV-1 vector.

Example 4.

Application of the new ORF70 insertion site and ORF1/3(UL56) insertion site in a recombinant EHV-1 vaccine vector in parallel.

To demonstrate that the two new promoters can be used in parallel, recombinant EHV-1 RacH was generated expressing two different hemagglutinins from two different influenza A virus subtypes.

The specificity and lack of cross-reactivity of the polyclonal commercially available anti-H3 antibody (PA5-34930) and the proprietary monoclonal antibodies to H1av and H1pdm is evident from Western blots of infected cells, as shown in FIG. 19. Identical samples were subjected to quadruplicate SDS-PAGE and transferred to nylon membranes before incubation with four different antibodies.

The open reading frame encoding the influenza A virus hemagglutinin (A/swine/Gent/132/2005(H1N1)) was synthesized and cloned into the transfer vector pU1-3-p430-BGHKBGH (SEQ ID NO: 30), which provided pU1-3- p430-H1av-BGHKBGH (Fig. 4, SEQ ID NO: 31). Using the recombinant BAC pRacH-SE-70-p455-H3 as a starting point, the p430-H1av-BGH expression cassette, which was assembled into pU1/3-p430-H1av-BGHKBGH (Fig. 4, SEQ ID NO: 31), was inserted into orf1/3/UL56 insertion site by two-step RED recombination to generate pRacH-SE-1/3-p430-H1av-70-p455-H3. PK/WRL cells were transfected with pRacH-SE1/3-p430-H1av-70-p455-H3, and the recombinant rEHV-1 virus RacH-SE1/3-p430-H1av-70-p455-H3 (Fig. 7) was released and cleared plaques twice.

The short name for this recombinant virus is rEHV-1 RacH-SEB. The correctness of the insertion of the expression cassette was confirmed by sequencing the high-precision PCR products with corrective exonuclease activity of the insertion sites along with the flanking sequences. Transgene expression in infected cells was analyzed by indirect immunofluorescence assay (IFA, not shown) and Western blotting using monoclonal and polyclonal antibodies (Fig. 19). Reconstitution of orf71/US5, encoding EHV-1 gpII, was confirmed by IFA (not shown) and Western blotting using monoclonal antibody Ai2G7 (owned by BI) (Fig. 19).

As shown in FIG. 19, both H3 and H1av transgenes were expressed in parallel in cell cultures infected with the double recombinant rEHV-1 insert RacH-SE-1/3-p430-H1av-70p455-H3 (B). Transgene expression was stable and did not impair viral titers tested to passage 11 in AI-ST A1 cells (BI proprietary porcine testis cell line, Table 3).

Two novel promoters p430 and p455 have been shown to be functional in the context of rEHV1-RacH-SE replication in cell cultures. Levels of activity during the viral replication cycle appeared to be very similar, as inferred from comparable signal intensities in Western blots specific for individual transgenes. These properties provide the opportunity to create recombinant vector vaccines based on EHV-1 RacH or other vector platforms that express two different antigens in parallel with similar efficacy. If the vaccine target consists of two different pathogens, then the use of two new promoters at two insertion sites in combination with two polyadenylation sequences can reduce the cost of goods significantly and represents a clear advantage over vector expression

- 50 046085 that contain only one antigenic component.

Example 5.

Creation. In vitro characterization and in vivo testing of a bivalent influenza a ha vaccine based on the ehv-1 vector.

As described below, in the described invention, two of the four porcine IAV hemagglutinin (HA) antigens described above, derived from the H3N2 and H1N1 sub-/serotypes of avian/porcine IAV, are expressed by a single recombinant EHV-1 vector virus. This new bivalent porcine IAV vaccine provides DIVA signature, for example, by detecting antibodies against porcine IAV NP or NA proteins in animals that are infected with field strains of porcine IAV, but not in animals vaccinated only with the vaccine described in this invention, since they express only the HA proteins of porcine IAV.

A novel bivalent swine IAV vaccine was characterized in vitro and tested in vivo for its ability to induce neutralizing antibodies to influenza A virus in mice.

To test whether the expressed recombinant hemagglutinins would be processed and transported as expected, VERO cells were infected with rEHV-1 RacH-SE-1/3-p430-H1av, rEHV-1 RacH-SE-70-p455- H3, rEHV-1 RacH-SE (parental) at M.O.I. 0.01, or left uninfected. After 24 hours p.i. live infected and uninfected cells were incubated with a suspension of chicken red blood cells in PBS, washed with PBS, and stained with fluorescent Hoechst 33342 to stain nuclei. Since avian red blood cells contain cell nuclei, they can be stained with Hoechst33342 and are detected as miniature blue spots using fluorescence microscopy. Compared to cells that were infected with rEHV-1 RacH-SE, which do not express hemagglutinin, chicken erythrocyte adsorption was significantly increased for cells infected with either rEHV-1 RacH-SE-1/3-p430H1av or rEHV- 1 RacH-SE-70-p455-H3 (not shown). Based on this, it can be concluded that the hemagglutinins were translated, processed and transported to the plasma membrane of cells infected with the vector virus in the same way as if they were produced by authentic replication of the influenza virus.

The hemadsorption phenotype of the infected cells is confirmed by the results of Western blots (Fig. 19) and immunofluorescence assays (not shown), which show efficient expression of the transgenic proteins and suggest the formation of functional HA trimers on the cell surface of cells infected with the EHV-1 vector.

An improved EHV-1 vector with two insertion sites and two new promoters was shown to express two influenza virus hemagglutinins in parallel. The subcellular localization determined by IFA and the mobility in SDS-PAGE determined by Western blotting (Fig. 19) were consistent with authentic uncleaved hemagglutinins expressed in cells infected with influenza A virus known from the literature.

The genetic and phenotypic stability of recombinant rEHV-1 was demonstrated by passivation in cell culture with viral titers determined every 5 passages. The sequences of the insertion sites were confirmed every ten passages, as was the expression of the transgene by Western blotting (not shown). Expression fidelity was assessed by double IFA of plaques under the top methylcellulose ball, counting plaques stained with anti-EHV and transgene-specific antibodies (not shown).

Example 6.

Induction of neutralizing antibody responses against two antigens in mice vaccinated with the bivalent rEHV-1 RacH vector vaccine.

rEHV-1 RacH SE B (rEHV-1 RacH-SE-1/3-p430-H1av-70-p455-H3 see Fig. 7) was used to immunize Balb/c mice to demonstrate that the expressed transgenes are immunogenic in species other than pig, and that neutralizing antibodies were raised against one of the two antigens by intranasal administration.

In more detail, three groups of five Balb/c mice per group, 3–5 weeks of age, were inoculated intranasally on study days 0 and 21 with either 40 μl rEHV-1 RacH SE B (rEHV-1 RacH-SE-1/3- 430H1av-7-455-H3, group 1), either 40 μl of empty vector (rEHV-1 RacH-SE, group 2, vector control) or 40 μl of tissue culture medium (group 3, negative control), respectively. For groups 1 and 2, infectious dosages of recombinant EHV-1 were 1x 10 ^L 5 TCID50/40 μl, respectively. Blood samples were collected from mice on study days 0 (before ^1st inoculation), 7, 14, 21 (before ^2nd inoculation), 28, and 35. Serum was prepared from the blood samples and stored frozen at -80°C.

Immunofluorescence assay for detection of antibodies to vector virus.

AI-ST cells were infected at a multiplicity of infection (MOI) of 0.001 with rEHV-1 RacH-SE1212, and virus was released from the empty BAC vector pRacH-SE1.2. After 24 hours p.i. Clear plaques were observed, and cells were processed for indirect immunofluorescence analysis (IFA). Sera were tested from all three groups of the final blood draw (obtained 14 days after the second vaccine

- 51 046085 tion), diluted 1:50 in PBS. Serum from an EHV-1 vaccinated horse was used as a positive control at a dilution of 1:500. The secondary antibodies used were commercially available FITC-conjugated rabbit anti-mouse IgG for mouse sera and Cy5-conjugated goat anti-horse IgG for horse sera, and were used at a dilution of 1:200. Antibody binding was assessed by fluorescence microscopy. All vaccinated mice developed antibodies that reacted in IFA with rEHV-1 RacH-SE-infected cells. Uninfected cells did not bind to any of the sera tested. Sera from negative control mice showed no specific binding to either infected or uninfected cells. The data is summarized in the table below.

Table 4

Fluorescence microscopy IFA results for antibodies to EHV-1________

Treatment Mouse number Ident. experiment number Not divorced Uninfected cells Infected cells Group 3 (Negative Control) 1 1 1:50 neg. neg. 2 2 1:50 neg. neg. 3 3 1:50 neg. neg. 4 4 1:50 neg. neg. 5 5 1:50 neg. neg. Group 2 (Blank Vector) 1 6 1:50 neg. positive 2 7 1:50 neg. positive 3 8 1:50 neg. positive 4 9 1:50 neg. positive 5 10 1:50 neg. positive Group 1 (rEHV1 RacH SE B) 1 eleven 1:50 neg. positive 2 12 1:50 neg. positive 3 13 1:50 neg. positive 4 14 1:50 neg. positive 5 15 1:50 neg. positive Control antibody Specific to Horse serum EHV-1 22 1:500 neg. positive Secondary antibodies Specific to FITC-goat anti- MOUSE 23 1:200 neg. neg. Su5 goat anti- horse 24 1:200 neg. neg.

From this it can be concluded that inoculation of rEHV-1 into the nostrils of mice leads to infection and replication of the virus, such that the immune systems of the mice were stimulated to produce antibodies to EHV-1.

Virus Neutralization Tests (VNT).

- 52 046085

To demonstrate the induction of protective immunity against expressed transgenes derived from either influenza A virus (IAV) (A/swine/Italy/7680/2001(H3N2)) or (A/swine/Gent/132/2005(H1N1) ), mouse sera were tested for neutralization activity against the respective viruses (Allwinn et al. 2010; Trombetta et al. 2014). The IAV used for neutralization tests were isolates obtained from pigs in Germany since 2014, specifically A/swine/Germany/AR452/2014 (H3N2) and A/swine/Germany/AR1181/2014 (H1N1) . Because they are heterologous to the strains from which the study vaccines are derived, any neutralization of these viruses by mouse sera would indicate broad and effective induction of protective immunity by rEHV-1 vaccination. As a negative control serum, pig serum was used that was demonstrated to be negative for influenza virus antibodies.

Influenza A virus neutralization tests.

MDCK cells for virus neutralization and back titration in 96-well plates were incubated for two days at 37°C/5%CO ₂ before use. IAV-matched stocks of H3N2 and H1avN1 were thawed on ice and diluted in MEM containing gentamicin and 2x trypsin (MEM/Genta/2x trypsin).

Test sera were obtained from the last blood draw of group 1 (rEHV-1 RacH SE B), group 2 (empty vector), positive control (serum from a pig vaccinated with inactivated multivalent IAV vaccine), and negative control.

Sera were heat inactivated and, in two and three independent experiments, respectively, serially diluted 1:2, starting from 1:16 up to 1:4096. IAV was diluted to approximately 100 TCID50/reaction mixture for the neutralization reaction. Reaction mixtures for the neutralization reaction were incubated for 2 hours at 37°C, 5% CO ₂ . Back titration of the virus used was carried out in quadruple repetition. Growth medium was removed and MDCK cells were washed with medium containing gentamicin and trypsin before adding neutralization reaction mixtures or back titration dilutions of viruses. VNTs and titration plates were incubated at 37°C/5% CO ₂ for 1 hour after the addition of neutralization reaction mixtures or virus dilutions to MDCK cells, respectively. After this, the inoculum was removed and fresh medium containing gentamicin and trypsin was applied to the cells. After five days p.i. monitored and documented CPE. The actual virus titer used in the assay was calculated as TIR50/ml according to Reed and Munch, and the dilutions at which the test sera prevented the induction of typical influenza virus CPE were recorded, see tables below.

- 53 046085

H1avN1 influenza virus VNT results

Table 5

HlavNl VNT#1 VNT#2 VNT#3 Average value of neutralization ability 146 TCID50/well Ability 32 TCID50/well Ability 181 TCID50/well Ability SD (standard deviation) MOUSE Reciprocal neutralizing dilution Reciprocal neutralizing dilution Reciprocal neutralizing dilution rEHV-1 RacH SE B- 1 32 4672 128 4096 32 5792 4853 862 rEHV-1 RacH SE B- 2 16 2336 64 2048 neg. 2192 204 rEHV-1 RacH SE B- 3 32 4672 128 4096 16 2896 3888 906 rEHV-1 RacH SE B- 4 128 18688 512 16384 64 11584 15552 3624 rEHV-1 RacH SE B- 5 32 4672 256 8192 16 2896 5253 2695 Empty and about. n.d. neg. n.d. neg. n.d. n.d. n.d. vector-1 Blank vector-2 and about. n.d. neg. n.d. neg. n.d. N.d. n.d. Blank vector-3 AND ABOUT. n.d. neg. n.d. neg. n.d. N.d. n.d. Blank vector-4 neg. n.d. neg. n.d. neg. n.d. N.d. n.d. Blank vector-5 But. n.d. neg. n.d. neg. n.d. N.d. n.d. Posit, control, pork whey 32 n.d. But. n.d. But. n.d. N.d. n.d.

- 54 046085

Table 6

H3N2 influenza virus VNT results

H3N2 VNT#1 VNT#2 VNT#3 Average value of neutralization ability 16 TCID50/well ability 24 TCID50/well ability 15 TCID50/well ability SD (standard deviation) MOUSE Reciprocal neutralizing dilution Reciprocal neutralizing dilution Reciprocal neutralizing dilution rEHV-1 RacH SE B - 1 4096 65536 1024 24576 2048 30720 40277 22089 rEHV-1 RacH SE B - 2 1024 16384 512 12288 128 1920 10197 7455 rEHV-1 RacH SE B - 3 1024 16384 512 12288 256 3840 10837 6397 rEHV-1 RacH SE B - 4 256 4096 256 6144 64 960 3733 2611 rEHV-1 RacH SE B - 5 256 4096 128 3072 64 960 2709 1599 Blank vector-1 neg. n.d. neg. n.d. neg. n.d. n.d. n.d. Blank vector-2 neg. n.d. neg. N.d. neg. N.d. n.d. N.d. Blank vector-3 neg. N.d. neg. N.d. neg. N.d. n.d. N.d.

In order to compare the results of independent tests, the neutralizing capacity was calculated by multiplying the reciprocal dilution of the serum by the corresponding titer that was neutralized by it. The average of the three tests was then divided by 100 to reflect neutralization of 100 TCID50 (Tables 4, 5, and 6). The data is summarized and presented graphically in Fig. 20.

All mice vaccinated with rEHV-1 RacH SE B developed neutralizing antibodies against the corresponding IAV heterologous strains of the H3N2 and H1avN1 subtypes. Thus, double intranasal administration of rEHV-1 RacH-SE, which express IAV hemagglutinins from the orf70 insertion site under the control of the p455 promoter (H3) and in parallel from the orf1/3 insertion site under the control of the p430 promoter (H1av), successfully stimulated a protective immune response in BALB/c mice.

It can be concluded that the rEHV-1 RacH-SE vector can be used to express two different transgenes in parallel to stimulate an immune response after intranasal vaccination.

Western blotting.

1. Infection: Three wells of each confluent AI-ST cell monolayer in 6-well plates were infected at M.O.I with approximately 1 recombinant virus by directly adding 10 μl of thawed virus stocks to the growth medium. Three wells were left uninfected. Infected and uninfected cells were incubated for two days and then processed for Western blotting. The viruses used for infection are summarized in the table below (Table 2).

table 2

Viruses tested by Western blot

Virus name abbreviation Insertion sites used Expressed transgenes rEHV-1 RacH-SE-70-p455-H3 NZ US4 NZ rEHV-1 RacH-SE-l/3-p430Hlav av UL56 Hlav rEHV-1 RacH-SE-70-p455Hlpdm 4p US4 Hlpdm rEHV-1 RacH-SE-l/3-p430Hlhu hu UL56 Hlhu

- 55 046085

rEHV-1 RacH-SE-l/3-p430- Hlav-70-455-H3 IN US4 and UL56 NC and Hlav rEHV-1 RacH-SE-l/3-p430- Hlhu-70-455-Hlpdm D US4 and UL56 Hlpdm and Hlhu rEHV-1 RacH-SE-UL43Hlpdm 43p UL43 Hlpdm rEHV-1 RacH-SE-l/3-p430Hlav-UL43-422-Hlpdm70455-H3 E US4 and UL56 and UL43 NC, Hlav, and Hlpdm rEHV-1 RacH-SE S.E. absent absent

2. Preparation of lysates: RIPA buffer supplemented with protease inhibitor cocktail (RIPA+PI) was prepared as follows: 0.7 ml 10x RIPA lysis buffer Millipore Cat. No. 20-188 was added to 6.3 ml H ₂ O, Fisher Scientific Cat. No. BP2470-1, and 1 tablet of Complete™ Mini Protease inhibitor cocktail (Roche cat. no. 11 836 153 001) were dissolved in 7 ml of 1xRIPA buffer. Uninfected controls were scraped into the medium, and suspensions from triplicate wells were pooled in 15-ml centrifuge tubes and placed on ice. Infected cells were washed into the medium, and suspensions from triplicate wells were pooled in 15-ml centrifuge tubes and placed on ice. Cells were pelleted by centrifugation at 1000xg 4°C for 5 min. Supernatants were carefully aspirated and cell pellets were resuspended in RIPA +PI (uninfected cells in 300 μl, infected cells in 150 μl). The suspensions were incubated on ice for 30 minutes and shaken every 10 minutes. The suspensions were transferred to 1.5 ml microcentrifuge tubes and the undissolved material was pelleted by centrifugation at 15,000 rpm, 4°C, for 10 min in a microcentrifuge. Clear supernatants were transferred to new 1.5 ml microcentrifuge tubes and stored at -80°C until use.

3. SDS-PAGE and transfer to nylon membranes: Materials: BioRad Criterion TGX Stain Free Precast Gels, 4-20%, 26 well, cat. No. 567-8095: Bio Rad Precision Plus Dual Color Marker, cat. No. 161-0374; Bio Rad Precision Plus All Blue Marker, cat. No. 161-0373; Bio Rad Trans Blot Turbo, transfer kit, Midi format cat. No. 170-4159; Bio Rad 4x Laemmli Sample Buffer (cat. no. 161-0747) (Bio Rad Laboratories GmbH, Heidemannstrasse 164, D-80939 Munchen); TGS running buffer (Sambrook et al.), blocking solution 1: 5% FBS in PBST (Sambrook et al.); PBST.

Samples were prepared without adding a reducing agent. Samples were thawed on ice and mixed with 1 volume of 4x Lammli buffer, boiled for 6 minutes at 96°C, and kept at RT until loading on the gel. The gel was run for 30 min at 230 mA and then collected for electroblotting using a BioRad Trans Blot Turbo system. Transfer was set to 2.5 A 25 B for 10 minutes. The membrane was washed in sterile distilled H ₂ O and incubated with 25 ml blocking solution of 5% FBS in PBST for 30 minutes at 4°C.

Incubation and detection of antibodies.

Materials: Immun-Star WesternC Chemiluminescent Kit (Bio Rad Laboratories GmbH, Heidemannstrasse 164, D-80939 Munchen) Cat. No. 170-5070

Primary Antibodies: See legend to Fig. 19 a-d.

Secondary antibody: peroxidase-conjugated goat anti-mouse, (Jackson Immune Research #115-035-146) 1:5000.

All incubations were carried out in sufficient volume with constant stirring. Antibodies were diluted in 5%FBS/TBST. Primary antibodies were incubated overnight at 4°C. The antibody solution was removed and the blots were washed three times with TBST for 5-10 minutes. The diluted secondary antibody was incubated with the blots for 1 h at RT, removed, and the blots were washed three times with TBST for 5–10 min. The blots were placed on a clear plastic protective sheet. Peroxide and lumino/enhancer solutions 1 ml + 1 ml were mixed (total 2 ml for each blot), pipetted onto the blots and incubated for 3-5 minutes. The membranes were then placed into a ChemiDocXRS imaging system (Bio Rad Laboratories GmbH, Heidemannstrasse 164, D-80939 Munchen) and the signals were recorded using Image Lab software.

Virus titration.

AI-ST cells were seeded in 96-well plates (Corning Incorporated -Life Sciences, One Becton Circle, Durham, NC 27712, USA; REF 353072) at 2x104 cells/well in MEM supplemented with 10% FBS one day before infection. Virus stocks were quickly thawed and placed on ice. Ten serial dilutions of 1:10 in MEM were prepared in a volume of 1.2 ml per dilution. Dilutions of viruses in an amount of 100 μl/well were added to the cells, 8 wells in one vertical row per dilution. Vertical rows 11 and 12 of each plate served as control medium by adding 100 μl/well of MEM. Titrations were performed in triplicate and cells were incubated for 5 days at

- 56 046085

37°C/5%CO2. Cell cultures were analyzed microscopically and wells in which EHV-1 RacH typical CPE was observed were recorded. Titers were calculated as TSGO50/ml according to the method of Reed and Muench (1938).

Example 7.

Creating a new UL43 insertion site.

Using the EHV vector platform as described in the previous examples, only two antigens can be expressed in parallel in their authentic forms. A mixture of two vector vaccines will increase the cost of the product and may also result in unbalanced transgene expression if replication efficiency varies between different recombinant viruses, which is likely. Although there are ways to link two antigens at a single insertion site, either using an internal ribosome entry site (IRES) or a picornavirus 2a (2a) peptide, such techniques are not sufficient for this purpose. If two transgenes are linked by the 2a peptide, which triggers ribosomal slip, resulting in the synthesis of a discrete translation product located downstream of transcription (Donnelly et al., 2001), the 2a peptide structurally modifies the first of the expressed proteins, which will result in 19 amino acid residues of 2a peptide added to the C-terminus. One amino acid residue, proline, will be added to the N-terminus of the second protein (Ryan et al., 1994). Since this one extra amino acid will be cleaved off by the HA signal peptide, it will most likely have no consequences. However, the 19 amino acid tail on the first HA may interfere with trimerization and prevent sufficient efficiency. To find a solution to overcome the described obstacles, the inventors installed a third transgene expression site in pRacH-SE.

Use of a unified nomenclature for alphaherpesviruses.

With the availability of the first genomic sequences of various alphaherpesviruses, in-silico identified open reading frames (orfs) were numbered for each virus individually according to their position in the respective genomes. It was later discovered that most alphaherpesvirus genes were homologues present in different species. To facilitate comparison of data, it is now common practice to assign genes and gene products the designation of their homologs in the human herpes virus type 1 genome. Accordingly, we have modified the old EHV orfs designations to match the new nomenclature, as indicated in Table 1. 1.

Table 1

EHV orf Unified nomenclature Gene product orfl UL56 pUL56 orf2 (absent) orf2 protein orf3 (absent) orf3 protein orfl 6 UL44 Glycoprotein C orfl 7 UL43 pUL43 orfl 8 UL42 DNA polymerase processivity factor orf70 US4 Glycoprotein G orf71 US5 Glycoprotein II (or Glycoprotein J)

However, to design the insertion site, care must be taken not to disrupt the putative promoter and polyA signals located upstream and downstream of the UL42 gene, encoding the DNA polymerase processivity factor, and the UL44 gene, encoding glycoprotein C.

Construction of the new UL43 insertion site is illustrated in FIG. 13.

Thus, 870 bp (SEQ ID NO: 21) of the 5' end of UL43 (SEQ ID NO: 18) was replaced with an expression cassette encoding autofluorescent mCherry protein by RED recombination of BAC pRacH-SE. The open reading frame (orf) for mCherry was placed under the control of the putative promoter (p422, SEQ ID NO: 5) and polyA sequence (SEQ ID NO: 7) of EHV-4 UL18 encoding capsid triplex subunit 2.

The 18pA polyadenylation sequence (SEQ ID NO: 7) was introduced into a transfer vector for RED recombination upstream and downstream of the kanamycin-resistant expression cassette (Kana) to perform dual functions: 1. During the second step of en-passant mutagenesis (2- x stage Red-mediated strategy) it serves as a homologous site for the Kana, 2 deletion. It acts as a polyadenylation signal for the transgene. A map of the pUUL43-422-mC-18K18 transfer vector is shown in FIG. 14.

The pUUL43-422-mC-18K18 fragment (Fig. 14; SEQ ID NO: 35), covering the flanking regions for recombination in the viral genome, the expression cassette and the kanamycin resistance cassette, was excised from the transfer vector using restriction homing endonuclease I-CeuI And

- 57 046085 was purified by agarose gel electrophoresis. The purified DNA fragment was then inserted into the BAC pRacH-SE using en-passant RED recombination (Tischer et al. 2006). After sequence integrity was confirmed, recombinant EHV-1 RacH-SE-UL43-422-mCherry (Fig. 15) was released following transfection of permissive cell cultures and plaque purified. Fluorescent mCherry protein expression by fluorescence microscopy showed that the new expression cassette at the new insertion site was functional (not shown).

To test the functionality of the third insertion site as a vector vaccine, influenza subtype H1pdm hemagglutinin (SEQ ID NO: 44) from swine influenza A virus ((A/swine/Italy/116114/2010 (H1N2), GenBank accession number: ADR01746) was inserted to the new pRacH-SE site.

For this purpose, the orf encoding mCherry was excised from the transfer vector pUUL43-422-mC18K18 (Fig. 14; SEQ ID NO: 35) and the orf encoding H1pdm was inserted in its place. The resulting transfer plasmid was named pUUL43-422-H1pdm-18K18 (FIG. 16, SEQ ID NO: 36). The pUUL43-422-H1pdm-18K18 fragment, covering the flanking regions for recombination in the viral genome, the expression cassette and the kanamycin resistance cassette, was excised from the transfer vector using restriction homing endonuclease I-CeuI and purified by agarose gel electrophoresis. The purified DNA fragment was then inserted into the BAC pRacH-SE using en-passant RED recombination (Tischer et al. 2006). After sequence integrity was confirmed, recombinant EHV-1 RacH-SE-UL43-422-H1pdm (Fig. 17) was released following transfection of permissive cell cultures and plaque purified.

The same procedure was used to generate recombinant EHV-1 RacH-SE from rEHV1 RacH-SE-B (rEHV-1 RacH-SE-orf1/3-p430-H1av-70-p455-H3, Fig. 7). The generated recombinant containing the triple insert was named rEHV-1 RacH-SE-UL56-430-H1av-UL43-422-H1pdm-US4-455-H3 (abbreviated rEHV-1 RacH-SE-E, Fig. 18).

A schematic representation of the rEHV-1 RacH-SE-UL56-430-H1av-UL43422-H1pdm-US4-455-H3 triple insert genome (abbreviated rEHV-1-E) is depicted in FIG. 18. While the name of the precursor construct uses the original EHV-orf nomenclature, the new triple-insert virus name is based on the uniform alphaherpesvirus nomenclature, where genes are named according to their homologs in human herpes virus type 1.

Recombinant plaque-purified viruses were characterized by sequencing of insertion site regions (not shown), Western blotting (Fig. 19), and virus titration (Table 3).

A double-insert recombinant EHV-1, rEHV-1 RacH-SE-UL56-430-H1hu-US4-455-H1pdm (abbreviated rEHV-1 RacH-SE-D, Fig. 12) was used to compare the expression strength of the transgenes. In addition, the studies included a rEHV-1 RacH-SE with a single insert, rEHV-1 RacH-SE-orf70p455-H1pdm (Fig. 10), which expresses the IAV HA H1pdm from a novel orf70/US4 expression site under the control of the p455 promoter.

To evaluate the expression strength of the novel recombinant EHV-1 RacH-SE-UL43-422-H1pdm and EHV-1 RacH-SE-E in comparison with the other two rEHV-1 RacH-SE expressing H1pdm, Western blotting was performed. In addition, all single-insert rEHV-1 RacH-SEs expressing different IAV HAs and double-insert rEHV-1 RacH-SEs B and D, respectively, were included. For the list of viruses used, see table. 2.

table 2

List of viruses analyzed using Western blotting (Fig. 19)

Long title Abbreviated Transgenes rEHV-1 RacH-SE-UL56-430-Hlav-US4-455-H3 IN Hlav H3 rEHV-1 RacH-SE-UL56-430-Hlhu-US4-455- D HlhuHlpdm rEHV-1 RacH-SE-UL56-430-Hlav-UL43-422- E Hlav Hlpdm H3 rEHV-1 RacH-SE-UL5 6-43 0-H lav av Hlav rEHV-1 RacH-SE-UL56-430-Hlhu hu Hlhu rEHV-1 RacH-SE-US4-455-H3 H3 H3 rEHV-1 RacH-SE-US4-455-Hlpdm 4p Hlpdm rEHV-1 RacH-SE-UL43-Hlpdm 43p Hlpdm rEHV-1 RacH-SE S.E. absent

Three proprietary monospecific monoclonal antibodies directed against hemagglutinins H1av or H1pdm, or EHV-1 glycoprotein II, and a commercial polyclonal antibody against H3 were used. This method allowed for a semi-quantitative assessment of the amount

- 58 046085 transgenes expressed in cells infected with the various recombinant viruses tested. As a cell culture control, cells were left uninfected, and rEHV-1 RacH-SE, which was rescued and plaque purified from the BAC empty vector backbone (SE), was used as a background viral control. AI-ST cell cultures infected with recombinant EHV-1 B, D, E, SE, av, hu, H3, 4p, 43p (see Table 2), or remaining uninfected, were harvested after 30 hours p.i. and treated with SDS-PAGE under reducing conditions. After electrophoresis, proteins were electrotransferred to nylon membranes and incubated with monoclonal antibodies to either HA H1av, H1pdm, EHV-1 glycoprotein II, or a commercial rabbit polyclonal antibody to HA H3. Western blotting (Fig. 19d) confirmed successful infection and replication of all nine viruses. The amounts of gpII expressed in B, D, av, hu, H3, 4p, 43p, and SE-infected cells are similar, indicating comparable replication efficiency. The amount of gpII in E-infected cells is slightly reduced compared to others. Western blotting methods (Fig. 19a and 19b) confirm the expression of hemagglutinins H1av and H3, respectively, by the new recombinant E. Compared to B, the amounts appear to be comparable. In contrast, the amount of hemagglutinin H1pdm expressed by the novel recombinant EHV-1 RacH-SE-E and -43p (Western blot (Fig. 19c)) appears to be significantly reduced compared to D and 4p, where an identical protein is expressed in US4 ( orf70) insertion site under the control of promoter 455.

To assess whether expression of the three hemagglutinins in parallel would impair the efficiency of viral replication, rEHV-1 RacH-SE-B, -D and -E were passivated in AI-ST cells until the eleventh passage, and titers were determined in parallel in triplicate (Table 3). . All titers were in a comparable range, indicating that the third transgene expression cassette had no obvious effect on the fitness of the virus under cell culture conditions.

Table 3

Comparison of virus titers at passage 11

Virus ID Passage No. TCU50/ml Standard deviation rEHV-lRacH-SE-B eleven 2.01E+08 1.09E+07 rEHV-lRacH-SE-D eleven 1.76E+08 8.59E+07 rEHV-lRacH-SE-E eleven 1.67E+08 8.88E+07

Overall, recombinant EHV-1 was shown to express three different influenza A hemagglutinins from three different expression sites in parallel. While expression from the UL56 (orf1/3) and US4 (orf70) insertion sites under the control of the 430 and 455 promoters, respectively, was comparable in strength, expression from the new UL43 site under the control of the new 422 promoter was weaker. Also, in recombinant EHV-1 RacH-SE, expressing only the hemagglutinin H1pdm from a new insertion site in UL43 under the control of the p422 promoter, the amount was reduced compared to the same protein expressed from the US4 (orf70) insertion site under the control of the p455 promoter. Thus, the new expression system provides an option to address the need for less robust expression of a third transgene in addition to those expressed from the UL56 site and the US4 site. Lower expression from the UL43 site is an advantage when the expressed proteins are known to have toxic effects in cell cultures when present in large quantities. In addition, a combination of strong and weak expression sites can be used if a specific ratio of proteins is needed for a particular purpose, for example, to produce virus-like particles consisting of different viral structural proteins in specific ratios. In addition, weaker expression of the transgene may be desirable if the expressed protein tends to destabilize the recombinant vector virus.

The improved EHV-1 BAC vector pRacH-SE can be used as a platform for the development of vector vaccines against various pathogens of mammalian species, including horses, dogs and pigs (Trapp et al. 2005, Rosas et al. 2007a, 2007b, 2008). Three different transgenes can be expressed in parallel by the improved vector virus in their authentic form. The three different antigens may represent three serotypes of the same pathogen or may come from different pathogens of the species for which the vaccine is intended. In addition, a vector vaccine based on the improved EHV-1 vector pRacH-SE expressing equine pathogen antigens has the potential to be quadrivalent as it will also vaccinate against EHV-1 infection.

Information about the EHV-1 improved BAC vector pRacH-SE has not been published or presented outside of BI.

All compositions and methods disclosed and claimed in this invention can be prepared and carried out without undue experimentation in light of the present description. Although the compositions and methods in

- 59 046085 in accordance with this invention have been described in the context of preferred embodiments, it will be apparent to those skilled in the art that changes may be made to the compositions and methods, as well as to the steps or sequence of steps of the method described herein, without departing from the concept , spirit and scope of the present invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein to achieve the same or similar results. All such such substitutions and modifications apparent to those skilled in the art are intended to be within the spirit, scope and concept of the invention as defined in the appended claims.

Links

The following references, to the extent that they provide illustrative methodological or other details additional to those set forth herein, are expressly incorporated herein by reference.

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13. C.T. Rosas, V.K. Tischer, G. A. Perkins, W. Wagner, L.B. Goodman, N. Osterrieder Live-attenuated recombinant equine herpesvirus type 1 (EHV-1) induces a neutralizing antibody response against West Nile virus (WNV) Virus Research, 125 2007b, cc. 69-78.

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Sequence listing <110> Boehringer Ingelheim Vetmedica GmbH <120> New EHV insertion site UL43 <130> P01-3304 <160> 47 <170> PatentIn version 3.5

- 62 046085 <210>1 <211>600 <212> DNA <213> Equine herpesvirus type 4 <400>1 gcagactttg gagcagcaca atttccggtt gtggacccca tggaccttgg tttggctggt60 accgtggaaa ctaacgctcc ggaagttttg gccagagcaa aatacaattc gaaggtagac120 atatggagcg ccggaatagt tctgtttgaa atgctcgcat atccatcaac tctatttgag180 gacccgccga gtaccccaca agagtatgta aaaagctgtc attctcaact actgagaata240 atatcaaagc taaagataaa ccctgaggag tttccacggg aaccagagtc taggctcgtg300 cgcggataca tcgaatacgc cagcctagag cgtaagccac atacgcgcta tccttgcttc360 cagcgcgtga acctacacat tgacggggaa tttttgatcc ataaaatgct agcgt tcaat420 gctgcgatgc gcccatccgc agaagagttg ttgtcctacc caatgtttat gaatctgtag480 gatgactaac agatttgggg tggagacggc gtgggcgata ctgtataaag ttgtactact540 taccagccca gtcagtgtgc tgtagtgcca ccacctgtaa agctgtgata agctgcagtt600 <210>2 <211>600 <212> DNA <213 > Equine herpesvirus type 4 <400>2 agctggggga gtttgtacta tagtgtatta catgcggctt gcaataactg cctggtttat60 gtttcgcaac attcaagcag acatgctacc gctaaacact ttgcaacaat tttttattgg120 gtgtttggcc tttggtagaa ctgtcgcgtt tt tggtggta gcatatacta ccttatttat180 acgctccgag ctgtttttca gcatgctagc acccaacgcc gagcgagagt atataactcc240 catcattgcc cacaagctta tgccacttat tagcgtccgc tctgccgttt gcttagtcat300 aatatctacc gccgtttacg cagcagacgc tatctgcgac acaattggat ttgcgatacc360 gcgcatgtgg atgtgtattt taatgagatc aacctccatg aagcgtaact aggggggcctc420 ccactgaggc actaccggct tagcagctga ctaacacagt ataaaacgtg agaagaaatc480 agtctcatgc gccattagcg ctaggctagt tagcgtggag gaccggagcg ctaccgccag540 cagtttcatc cgcctggtta cgggtttgtt aacacctacc ggtgtttt ac cgctaccata600 <210>3 <211>430 <212> DNA <213> Equine herpesvirus type 4 <400>3 tctatttgag gacccgccga gtaccccaca agagtatgta aaaagctgtc attctcaact60

- 63 046085 actgagaata atatcaaagc taaagataaa ccctgaggag tttccacggg aaccagagtc taggctcgtg cgcggataca tcgaatacgc cagcctagag cgtaagccac atacgcgcta tccttgcttc cagcgcgtga acctacacat tgacggggaa tttttgatcc ataa aatgct agcgttcaat gctgcgatgc gcccatccgc agaagagttg ttgtcctacc caatgtttat gaatctgtag gatgactaac agatttgggg tggagacggc gtgggcgata ctgtataaag ttgtactact taccagccca gtcagtgtgc tgtagtgcca ccacctgtaa agctgtgata agctgcagtt <210>4 <211>449 <212 > DNA <213> Equine herpesvirus type 4 <400>4 ttggtggtag catatactac cttatttata cgctccgagc tgtttttcag catgctagca cccaacgccg agcgagagta tataactccc atcattgccc acaagcttat gccacttatt agcgtccgct ctgccgtttg cttagtcata atatctaccg ccg tttacgc agcagacgct atctgcgaca caattggatt tgcgataccg cgcatgtgga tgtgtatttt aatgagatca acctccatga agcgtaacta gggggcctcc cactgaggca ctaccggctt agcagctgac taacacagta taaaacgtga gaagaaatca gtctcatgcg ccattagcgc taggctag tt agcgtggagg accggagcgc taccgccagc agtttcatcc gcctggttac gggtttgtta acacctaccg gtgttttacc gctaccata <210>5 <211>422 <212> DNA <213> Equine herpesvirus type 4 <400>5 ccatggatcg tcagacttgg tggaagaccc atgttcgttg tttcaagaag cgtatcctct actcagctcc actgatacag cattgctacg cacgcctcac gttggcgaaa ttggcgcaga tgaaggacat tttgcccagt acctaattcg cgacgaatcc cccctgaaag gctgttttcc acgaatttag gttgtgcccg cctacaactt ttcacttgca aactcaataa aacgcacagt ttgtatattc agttgtcagt ttgctctact cgagcgtcgg cgctttgtct agccctctta gtgggtattg ttaccggctg gggttttatt ggcgttgtta ttggggagat tttagttgat agaaag cata ccgaggtttt gggggtgtcg cttaatttcg gtgtctgtaa acgtaaaaag ag

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422 <210> 6 <211> 125

- 64 046085 <212> DNA <213> Artificial sequence <220>

<223> Polyadenylation sequence 71pA <400>6 aataaacgcg gtatgtctac cttcaagcct atgatgaacg gatgtttggt gtttgcggct60 attataacgc tcttgagttt tatgctatct ctgggaacat gcgaaaatta caggcgtgtg120 gttcg125 <210 >7 <211>123 <212> DNA <213> Artificial sequence <220>

<223> Polyadenylation sequence 18pA <400>7 ataaaaactg agactgttat attcatttca gtgtgtttaa taagaattgt gaacataact60 tattctatat ctcattgcgt ggaaagactg gaaaacgcat tggtggtagg tggaaggctc120 gcc123 <210>8 <211>1 235 <212> DNA <213> Equine herpesvirus type 1 <400> 8

atgttgactg tcttagcagc tctgagtctg ctcagcttgc ttacgagcgc aaccggacgg 60 ctcgccccag atgaactctg ttatgccgaa ccccgcagaa ctggcagccc accaaacacc 120 cagcccgaac gccacccgt aatatttgag cccccaacaa ttgcgattaa agctgaatcc 180 aagggttgtg agctaatttt attagatcca cccatagatg taagctatcg cagagagat 240 aaggtgaatg cgtccattgc ttggtttttt gactttggcg cttgccggat gcccatcgca 300 tacagagt attacggttg tattggcaat gctgttccct ccccagagac ttgtgatgcg 360 tactcatta cccttattag gaccgaggt atcgtggagt ttaccatcgt aaacatgagc 420 ctcctgtttc agcctggaat atacgatagt ggcaatttta tctacagcgt tctcctggac 480 taccacatat ttacaggacg tgtaacgttg gaagtggaaa aggacakaaa ctatccctgt 540 ggcatgattc atggactcac tgcttacgga aacatcaacg tagatgaaac catggacaac 600 gccagcccac acccgcgtgc cgtggggtgc tttcccgagc ccatcgacaa cgaagcgtgg 660 gcaaacgtta cattactga attggggata ccagacccaa actcatttct cgatgacgag 720 ggtgattacc cgaatatatc agactgtcac tcgtggggagt catacaccta cccaaatacg 780 ctgaggcagg ccacaggacc ccagaccctg ttggtgggtg cggttggact cagaatcttg 840

- 65 046085 gcgcaggcat aatttagaga caggaggagt acacacacgg atgcctgctt tttgtgtatt atagaaatta ggaagtttgt cccacgtacc ctaacagccc gggtgcgtcg ggctctaatt aaggtcaaaa ccagcgactt cggtgacgaa ctcaagtgct cgaagttgcc a acggcatcc ggactcggca ctgtcctctc gagtacgttg acatacgaca gcagagtcgt cacctgcgaa aggactgtga catgtgccat ggaatttttc cttaa ccatccgcgc ctctagaaaa gcgtcaacag cagtcagctc gatagggttg gcgcgcgcaa agaagcaaag ccaatcgaca cgatgacagt aaaactgtgt atagtttaca aatgtaaaac

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1235 <210> 9 <211> 417 <212> DNA <213> Equine herpesvirus type 1 <400> 9

ctccgagtac cccagaggag tatgtgaaaa gctgccactc gcaactactg aagataattt caacgctcaa gataaatccg gagggagtttc ctcgagaccc cgggtcgagg ctcgtgcgcg gatacatcga gtattctaga ctcgagcgca agccctacac gcgctacccc tgctttcaac gcgtcaacct gcacattgac ggggagtttc tggttcacaa gatgctagcg ttcaatgccg cgatgcgccc atcggccgag gagctgctgt cataccaat gtttgctcaa ctttaggatg actaacctgt ttctgggagg agacagcgtg ggcgacggtg tataaagttg gtctgctttc aagccctgcc actgcgctac agtgccacca actgtaaagc ggtagtaagc tgcagtg

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417 <210> 10 <211> 431 <212> DNA <213> Equine herpesvirus type 1 <400> 10 gaccctgttg tgacgaaaca aagtgctgca agttgcccac cggcatccag actcggcaca gtcctctcgg gtgggtgcgg tacgacacca gagtcgtctc ctgcgaagcg g actgtgaca tgtgccatga aatttttcgc ttggactcag tccgcgcaga tagaaaacca tcaacagcga gtcagctcaa tagggttgat gcgcgcaaaa aatcttggcg agcaaagaat atcgacacag tgacagtaca aactgtgtat agtttacatt tgtaaaacat caggcatgga ttagagaccc gaggagtcta cacacggggg gcctgcttgg tgtgtattaa agaaattacc agtttgtcgg acgtaccctc acagccccga gtgcgtcgaa ctctaattgg ggtcaaaact agcgacttga gtacgttgct t

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431 <210> 11 <211> 417 <212> DNA

- 66 046085 <213> Equine herpesvirus type 1 <400> 11

ctccgagtac cccagaggag tatgtgaaaa gctgccactc gcaactactg aagataattt caacgctcaa gataaatccg gagggagtttc ctcgagaccc cgggtcgagg ctcgtgcgcg gatacatcga gtattctaga ctcgagcgca agccctacac gcgctacccc tgctttcaac gcgtcaacct gcacattgac ggggagtttc tggttcacaa gatgctagcg ttcaatgccg cgatgcgccc atcggccgag gagctgctgt cataccaat gtttgcacaa ctttaggatg actaacctgt ttctgggagg agacagcgtg ggcgacggtg tataaagttg gtctgctttc aagccctgcc actgcgctac agtgccacca actgtaaagc ggtagtaagc tgcagtg

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417 <210> 12 <211> 530 <212> DNA <213> Equine herpesvirus type 1 <400> 12 gaccctgttg tgacgaaaca aagtgctgca agttgcccac cggcatccag actcggcaca gtcctctcgg gtacgttgct aactgagtgt gtgggtgcgg tacgacacca gag tcgtctc ctgcgaagcg gactgtgaca tgtgccatga aatttttcgc taacacctgt ggttttaaaa ttggactcag tccgcgcaga tagaaaacca tcaacagcga gtcagctcaa tagggttgat gcgcgcaaaa caaataaaag atactaaatc aatcttggcg agcaaagaat atcgacacag tgacagtaca aactgtgtat agtttacatt tgtaaaacat tttcaaatca gcggcaattc caggcatgga ttagagaccc gaggagtcta cacacggggg gcctgcttgg tgtgtattaa agaaattacc aaaacattgt cggaaatagc ag tttgtcgg acgtaccctc acagccccga gtgcgtcgaa ctctaattgg ggtcaaaact agcgacttga tgtctgtaat

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283

DNA

Equine herpesvirus type 1 <210>

<211>

<212>

<213>

<400>

tctagactcg attgacgggg gccgaggagc gggaggagac cgctacagtg agcgcaagcc agtttctggt tgctgtcata agcgtgggcg ccaccaactg ctacacgcgc tcacaagatg cccaatgttt acggtgtata taaagcggta tacccctgct ctagcgttca gctcaacttt aagtt ggtct gtaagctgca ttcaacgcgt atgccgcgat aggatgacta gctttcaagc gtg caacctgcac gcgcccatcg acctgtttct cctgccactg

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283 <210> 14 <211> 144 <212> DNA

- 67 046085 <213> Equine herpesvirus type 1 <400>14 gaccctgttg gtgggtgcgg ttggactcag aatcttggcg caggcatgga agtttgtcgg tgacgaaaca tacgacacca tccgcgcaga agcaaagaat ttagagaccc acgtaccctc aagtgctgca gagtcgtctc taga

120

144 <210>15 <211>1236 <212> DNA <213> Equine herpesvirus type 1 <400>15 atgttgactg tcttagcagc cctgagtctg ctcagcttgc ttacgagcgc aaccggacgg60 ctcgccccag atgaactctg ttatgccgaa ccccgcagaa ctggcagccc acca aacacc120 cagcccgaac gcccacccgt aatatttgag cccccaacaa ttgcgattaa agctgaatcc180 aagggttgtg agctaatttt attagatcca cccatagatg taagctatcg cagagaagat240 aaggtgaatg cgtccattgc ttggtttttt gactttggcg cttgccggat gcccatcgca300 tacagagagt attacggttg tattggcaat gctgttccct ccccagagac ttgtgatgcg360 tactcattta cccttattag gaccgaggt atcgtggagt ttaccatcgt aaacatgagc420 ctcctgttt c agcctggaat atacgatagt ggcaatttta tctacagcgt tctcctggac480 taccacatat ttacaggacg tgtaacgttg gaagtggaaa aggacacaaa ctatccctgt540 ggcatgattc atggactcac tgcttacgga aacatcaacg tagatgaaac catggacaac600 gccagcccac acccg cgtgc cgtggggtgc tttcccgagc ccatcgacaa cgaagcgtgg660 gcaaacgtta catttactga attggggata ccagacccaa actcatttct cgatgacgag720 ggtgattacc cgaatatatc agactgtcac tcgtgggagt catacaccta cccaaatacg780 ctgaggcagg ccacaggacc ccagaccctg ttggtgggtg cggttggact cagaatcttg840 gcgcaggcat ggaagtttgt cggtgacgaa acatacgaca ccatccgcgc agaagcaaag900 aatttagaga cccacgtacc ctcaagtgct gcagagtcgt ctctaga aaa ccaatcgaca960 caggaggagt ctaacagccc cgaagttgcc cacctgcgaa gcgtcaacag cgatgacagt1020 acacacacgg ggggtgcgtc gaacggcatc caggactgtg acagtcagct caaaactgtg1080 tatgcctgct tggctctaat tggactcggc acatgtgcca t gatagggtt gatagtttac1140 atttgtgtat taaggtcaaa actgtcctct cggaattttt cgcgcgcgca aaatgtaaaa1200 catagaaatt accagcgact tgagtacgtt gcttaa1236 <210 >16 <211>801 <212> DNA <213> Equine herpesvirus type 1

- 68 046085 <400>16 atgttgactg tcttagcagc cctgagtctg ctcagcttgc ttacgagcgc aaccggacgg60 ctcgccccag atgaactctg ttatgccgaa ccccgcagaa ctggcagccc accaaacacc120 cagcccgaac gcccacccgt aatatttgag ccccca acaa ttgcgattaa agctgaatcc180 aagggttgtg agctaatttt attagatcca cccatagatg taagctatcg cagagaagat240 aaggtgaatg cgtccattgc ttggtttttt gactttggcg cttgccggat gcccatcgca300 tacagagagt attacggttg tattggcaat gct gttccct ccccagagac ttgtgatgcg360 tactcattta cccttattag gaccgagggt atcgtggagt ttaccatcgt aaacatgagc420 ctcctgtttc agcctggaat atacgatagt ggcaatttta tctacagcgt tctcctggac480 taccacatat ttacaggacg tgtaacgttg gaagtggaaa aggacacaaa ctatccctgt540 ggcatgattc atggactcac tgcttacgga aacatcaacg tagatgaaac catggacaac600 gccagcccac acccg cgtgc cgtggggtgc tttcccgagc ccatcgacaa cgaagcgtgg660 gcaaacgtta catttactga attggggata ccagacccaa actcatttct cgatgacgag720 ggtgattacc cgaatatatc agactgtcac tcgtgggagt catacaccta cccaaatacg780 ctgaggcagg ccacagg acc c801 <210>17 <211>803 <212> DNA <213> Equine herpesvirus type 1 <400>17 atgttgactg tcttagcagc tctgagtctg ctcagcttgc ttacgagcgc aaccggacgg60 ctcgccccag atgaactctg ttatgccgaa ccccgcagaa ctggcagccc accaaacacc120 cagcccgaac gcccacccgt aatatttgag c ccccaacaa ttgcgattaa agctgaatcc180 aagggttgtg agctaatttt attagatcca cccatagatg taagctatcg cagagaagat240 aaggtgaatg cgtccattgc ttggtttttt gactttggcg cttgccggat gcccatcgca300 tacagagagt attacggttg tattggcaat gctgttccct ccccagagac ttgtgatgcg360 tactcattta cccttattag gaccgaggt atcgtggagt ttaccatcgt aaacatgagc420 ctcctgtttc agcctggaat atacgatagt ggcaatttta tctaagcgt tctcctggac480 taccacatat ttacaggacg tgtaacgttg gaagtggaaa aggacacaaa ctatccctgt540 ggcatgattc atggactcac tg cttacgga aacatcaacg tagatgaaac catggacaac600 gccagcccac acccgcgtgc cgtggggtgc tttcccgagc ccatcgacaa cgaagcgtgg660 gcaaacgtta catttactga attggggata ccagacccaa actcatttct cgatgacgag720 ggtgattacc cgaatatatc agactgt cac tcgtgggagt catacaccta cccaaatacg780

- 69 046085 ctgaggcagg ccacaggacc cca

803 <210>18 <211>1206 <212> DNA <213> Equine herpesvirus type 1 <400>18 atgatgtacc agccagatag agagcccggt gaagactcgt gtctggtgct aagctcatct60 tcggtccaac gctgcaccgg ctcccagagg ggctgcatgc catgtacctg ggcggcctcc1 20 aaagctttcg ttggaatcgg actacaagct tgcgtcctca cttcatcgat cttacacatt180 gacctgctaa cccggaactc aacatgtctg attctgatga tcatctcgat gtatgtgttg240 agcctgatcc gcgtacccat atctaagatg gaaactatag taaccgtatg tcgatcgata300 caggcgctgg ccactctagt ggcagccagt gtctgggtcg cgggatctgc agtcaaaaag360 gaacatttac ttatagttgt taccgtttgc attttgtttg tgtttatagc cggaactcaa420 atttcc ctat tttacgtcat atgctcagcc aatggaacgg ggactcactt tagagcgagc480 ctattggcta ttatcggtgg atgtgtgcta ggggtttccg taaagctcgt tgagctgaaa540 gatgtaccca tagggatagg gatagctatc gctattatag cttcctgtca agactttggg600 ctt gctcttc gagacacatg ccactatcga atcggacggt atgcgtgcat gcgcaccttt660 acggaccttg gccggggtat taactacaga tgggtgacgg acgttgaagc cgtccccaag720 atcgaagaag tcgcggaaga aaaggtttcg ctgttcaagt ttttcaagga gatgccgggg780 gtgattttct ccccagcggt cggaactcac gcaaccccca taatatggat cgtcctacgc840 ttggtctacg gaatttccaa cgtgtggcaa accccggcgt atgttgtctt ctgtctgact900 gttggacacg tctctgcgat gctgctggag cagcttgtca tcagagtaaa ctacacggca960 gaggcgagtt ccggcatcca ctccacggcc cacgctgtct gcatggtgct tgccgccttt1020 gggtacggcg tggccggtcc cctctcgctc gcatttactg tatccggggg tatactgggg1080 gcgctatacc ttcgcaagcg cgca acgggc gcgcgccgcc tggcggcaac tcacatttcg1140 aggtggctta ttgtttgtgt atatgttgcc gccggtttgt gttatgcaac tataatcaca1200 cattaa1206 <210>19 < 211>226 <212> DNA <213> Equine herpesvirus type 1 <400>19 gtataccatg cttgctatac tgaaaataaa aacgcatatt gtaaacgaca gacgcggaaa60 tgtttattgc ttagtttcac tatttggtta aaactattta cacttgtaga aacacgccca120 ctaagtattt gt tttatgac taatacctgg tgcataaaac catcctcttg ggtccctgta180

- 70 046085 cctcaaactc tccaaaggtt ggcttgctac atcaaggtta tcaatc <210>20 <211>305 <212> DNA <213> Equine herpesvirus type 1 <400>20 accccggcgt atgttgtctt ctgtctgact gttggacacg tctctgcgat gctgctggag cagcttgtca tcagagtaaa ctacacggca gaggcgagtt ccggcatcca ctccacggcc cacgctgtct gcatggtgct tgccgccttt gggtacggcg tggccggtcc cctctcgctc gcatttactg tatccggggg tatactgggg gcgctatacc ttcgcaagcg cgcaacgggc gcgcgccgcc tggcggcaac tcacatttcg aggtggctta ttgtttgtgt atatgttgcc gccgg <210>21 <211>870 <212> DNA <213> Equine herpesvirus type 1 <400>21 atgatg tacc agccagatag agagcccggt gaagactcgt gtctggtgct aagctcatct tcggtccaac gctgcaccgg ctcccagagg ggctgcatgc catgtacctg ggcggcctcc aaagctttcg ttggaatcgg actacaagct tgcgtcctca cttcatcgat cttacacatt gacctgctaa cccggaactc aacatgtctg attctgatga tcatctcgat gtatgtgttg agcctgatcc gcgtacccat atctaagatg gaaactatag taaccgtatg tcgatcgata caggcgctgg ccactctagt ggcagccagt gtctgg gtcg cgggatctgc agtcaaaaag gaacatttac ttatagttgt taccgtttgc attttgtttg tgtttatagc cggaactcaa atttccctat tttacgtcat atgctcagcc aatggaacgg ggactcactt tagagcgagc ctattggcta ttatcggtgg atgtgtgcta ggggtt tccg taaagctcgt tgagctgaaa gatgtaccca tagggatagg gatagctatc gctattatag cttcctgtca agactttggg cttgctcttc gagacacatg ccactatcga atcggacggt atgcgtgcat gcgcaccttt acggaccttg gccggggtat taactacaga tgggtgacgg acgttgaagc cgtccccaag atcgaagaag tcgcggaaga aaaggtttcg ctgttcaagt ttttcaagga gatgccgggg gtgattttct ccccagcggt cggaactcac gcaaccccca taatatggat cgtcctac gc ttggtctacg gaatttccaa cgtgtggcaa <210> 22 <211> 336 <212> DNA

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840

870

- 71 046085 <213> Equine herpesvirus type 1 <400>22 accccggcgt atgttgtctt ctgtctgact gttggacacg tctctgcgat gctgctggag60 cagcttgtca tcagagtaaa ctacacggca gaggcgagtt ccggcatcca ctccacggcc120 cacgctgtct gcatggtgct tgccgccttt gggtacggcg tggccggtcc cctctcgctc180 gcatttactg tatccggggg tatactgggg gcgctatacc ttcgcaagcg cgcaacgggc240 gcgcgccgcc tggcggcaac tcacatttcg aggtggctta ttgtttgtgt atatgttgcc300 gccggtttgt gttatgcaac tataatcaca cattaa336 <210 >23 <211>1206 <212> DNA <213> Equine herpesvirus type 1 <400>23 atgatgtacc agccagatag agagcccggt gaagactcgt gtctggtgct aagctcatct60 tcggtccaac gctgcaccgg ctcccagagg ggctgcatgc catgtacctg ggcggcctcc120 aaagcttt cg ttggaatcgg actacaagct tgcgtcctca cttcatcgat cttacacatt180 gacctgctaa cccggaactc aacatgtctg attctgatga tcatctcgat gtatgtgttg240 agcctgatcc gcgtacccat atctaagatg gaaactatag taaccgtatg tcgatcgata300 caggcgctgg ccactctagt ggcagccagt gtctgggtcg cgggatctgc agtcaaaaag360 gaacatttac ttatagttgt taccgtttgc attttgtttg tgtttatagc cggaactcaa420 atttccctat tttacgtcat atgctcagcc aatgga acgg ggactcactt tagagcgagc480 ctattggcta ttatcggtgg atgtgtgcta ggggtttccg taaagctcgt tgagctgaaa540 gatgtaccca tagggatagg gatagctatc gctattatag cttcctgtca agactttggg600 cttgctcttc gagacacatg ccactatcga atcgg acggt atgcgtgcat gcgcaccttt660 acggaccttg gccggggtat taactacaga tgggtgacgg acgttgaagc cgtccccaag720 atcgaagaag tcgcggaaga aaaggtttcg ctgttcaagt ttttcaagga gatgccgggg780 gtgattttct ccccagcggt cggaactcac gcaaccccca taatatggat cgtcctacgc840 ttggtctacg gaatttccaa cgtgtggcaa accccggcgt atgttgtctt ctgtctgact900 gttggacacg tctctgcgat gctgctggag cagcttgtca tcagagtaaa ctacacggca960 gaggcgagtt ccggcattca ctccacggcc cacgctgtct gcatggtgct tgccgccttt1020 gggtacggcg tggccgctcc cctctcgctc gcatttactg tatccggggg tatactgggg1080 gcgctatacc ttcgcaagcg cgcaacgggc gcgcgccgcc tggcggcaac tcacatt tcg1140 aggtggctta ttgtttgtgt atatgttgcc gccggtttgt gttatgcaac tataatcaca1200 cattaa1206

- 72 046085 <210>24 <211>870 <212> DNA <213> Equine herpesvirus type 1 <400> 24

atgatgtacc agccagatag agagcccggt gaagactcgt gtctggtgct aagctcatct 60 tcggtccaac gctgcaccgg ctcccagagg ggctgcatgc catgtacctg ggcggcctcc 120 aaagctttcg ttggaatcgg actacaagct tgcgtcctca cttcatcgat cttacacatt 180 gacctgctaa cccggaactc aacatgtctg attctgatga tcatctcgat gtatgtgttg 240 agcctgatcc gcgtacccat atctaagatg gaaactatag taaccgtatg tcgatcgata 300 caggcgctgg ccactctagt ggcagccagt gtctgggtcg cgggatctgc agtcaaaaag 360 gaacatttac ttatagttgt taccgtttgc attttgtttg tgtttatagc cggaactcaa 420 atttccctat tttacgtcat atgctcagcc aatggaacgg ggactcactt tagagcgagc 480 ctattggcta ttatcggtgg atgtgtgcta ggggtttccg taaagctcgt tgagctgaaa 540 gatgtaccca tagggatagg gatagctatc gctattatatag cttcctgtca agactttgggg 600 cttgctcttc gagacacatg ccactatcga atcggacggt atgcgtgcat gcgcaccttt 660 acggaccttg gccggggtat taactacaga tgggtgacgg acgttgaagc cgtccccaag 720 atcgaagaag tcgcggaaga aaaggtttcg ctgttcaagt ttttcaagga gatgccgggg 780 gtgattttct ccccagcggt cggaactcac gcaacccca taatatggat cgtcctacgc 840 ttggtctacg gaatttccaa cgtgtggcaa 870

<210>25 <211>336 <212> DNA <213> Equine herpesvirus type 1 <400> 25

accccggcgt atgttgtctt ctgtctgact gttggacacg tctctgcgat gctgctggag 60 cagcttgtca tcagagtaaa ctacacggca gaggcgagtt ccggcattca ctccacggcc 120 cacgctgtct gcatggtgct tgccgccttt gggtacggcg tggccgctcc cctctcgctc 180 gcatttactg tatccgggggg tatactgggg gcgctatacc ttcgcaagcg cgcaacggggc 240 gcgcgccgcc tggcggcaac tcacatttcg aggtggctta ttgtttgtgt atatgttgcc 300 gccggtttgt gttatgcaac tataatcaca cattaa 336

<210>26 <211>226 <212> DNA <213> Equine herpesvirus type 1

- 73 046085 <400>26 gtataccatg cttgctatac tgaaaataaa aacgcatatt gtaaacgaca gacgcggaaa60 tgtttattgc ttagtttcac tatttggtta aaactattta cacttgtaga aacacgccca120 ctaagtattt gttttatgac taatacctgg tgcata aaac catcctcttg ggtccctgta180 cctcaaactc tccaaaggtt ggcttgctac atcaaggtta tcaatc226 <210>27 <211>305 <212> DNA <213> Equine herpesvirus type 1 < 400>27 accccggcgt atgttgtctt ctgtctgact gttggacacg tctctgcgat gctgctggag60 cagcttgtca tcagagtaaa ctacacggca gaggcgagtt ccggcattca ctccacggcc120 cacgctgtct gcatggtgct tgccgccttt gggtacggcg tgg ccgctcc cctctcgctc180 gcatttactg tatccggggg tatactgggg gcgctatacc ttcgcaagcg cgcaacgggc240 gcgcgccgcc tggcggcaac tcacatttcg aggtggctta ttgtttgtgt atatgttgcc300 gccgg305 <210>28 <211>5191 < 212> DNA <213> Artificial sequence <220>

<223> Nucleotide sequence of transfer vector pU70-p455-71K71 <400> 28

caataaacgc ggtatgtcta ccttcaagcc tatgatgaac ggatgtttgg tgtttgcggc 60 tattataacg ctcttgagtt ttatgctatc tctgggaaca tgcgaaaatt acaggcgtgt 120 ggttcgggat cctagggata acaggtaat cgatttattc aacaaagcca cgttgtgtct 180 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg 240 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct 300 tgctcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatggggct 360 cgcgataatg tcggggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg 420 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgatg 480 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt 540 actcctgatg atgcatggtt actaccact gcgatccccg ggaaaacagc attccaggta 600 ttagagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc 660 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc 720 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag 780

- 74 046085

cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataagctttt gccattctca 840 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg 900 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt 960 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gcttttttcaa 1020 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag 1080 tttttctaaa aaaacgcgg tatgtctacc ttcaagccta tgatgaacgg atgtttggtg 1140 tttgcggcta ttataacgct cttgagtttt atgctatctc tgggaacatg cgaaaattac 1200 aggcgtgtgg ttcgggatcc gaccctgttg gtgggtgcgg ttggactcag aatcttggcg 1260 caggcatgga agtttgtcgg tgacgaaaca tacgacacca tccgcgcaga agcaaagaat 1320 ttagagacc acgtaccctc aagtgctgca gagtcgtctc tagaaaacca atcgacacag 1380 gaggagtcta acagccccga agttgcccac ctgcgaagcg tcaacagcga tgacagtaca 1440 cacacgggggg gtgcgtcgaa cggcatccag gactgtgaca gtcagctcaa aactgtgtat 1500 gcctgcttgg ctctaattgg actcggcaca tgtgccatga tagggttgat agttacatt 1560 tgtgtattaa ggtcaaaact gtcctctcgg aatttttcgc gcgcgcaaaa tgtaaaacat 1620 agaaattacc agcgacttga gtacgttgct taagcttggc gtaatcatgg tcatagctgt 1680 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 1740 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 1800 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 1860 cggggagagg cggtttgcgt attggggcgct cttccgcttc ctcgctcact gactcgctgc 1920 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 1980 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 2040 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 2100 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 2160 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 2220 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 2280 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 2340 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 2400 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 2460 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 2520 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 2580 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 2640

- 75 046085

gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 2700 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 2760 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 2820 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 2880 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 2940 catctggccc cagtgctgca atgataccgc gagaccacg ctcaccggct ccagatttat 3000 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 3060 cctccacatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 3120 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 3180 tggcttcatt cagctccggt tccaacgat caaggcgagt tacatgatcc cccatgttgt 3240 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 3300 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 3360 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 3420 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 3480 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 3540 tgttgagatc cagttcgatg taaccactc gtgcacccaa ctgatcttca gcatctttta 3600 ctttcaccag cgtttctgggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 3660 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 3720 ttttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 3780 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 3840 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt 3900 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc 3960 tgtaagcgga tgccggggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt 4020 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc 4080 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat 4140 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc 4200 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt 4260 cacgacgttg taaaacgacg gccagtgaat tcctccgagt accccagagg agtatgtgaa 4320 aagctgccac tcgcaactac tgaagataat ttcaacgctc aagataaatc cggagaggagtt 4380 tcctcgagac cccggggtcga ggctcgtgcg cggatacatc gagtattcta gactcgagcg 4440 caagccctac acgcgctacc cctgctttca acgcgtcaac ctgcacattg acggggagtt 4500 tctggttcac aagatgctag cgttcaatgc cgcgatgcgc ccacggccg aggagctgct 4560

- 76 046085

gtcataccca atgtttgctc aactttagga tgactaacct gtttctggga ggagacagcg 4620 tggggcgacgg tgtataaagt tggtctgctt tcaagccctg ccactgcgct acagtgccac 4680 caactgtaaa gcggtagtaa gctgcagtgg tcgactggtg gtagcatata ctaccttatt 4740 tatacgctcc gagctgtttt tcagcatgct agcaccaac gccgagcgag agtatataac 4800 tcccatcatt gccccacaagc ttatgccact tattagcgtc cgctctgccg tttgcttagt 4860 cataatact accgccgttt acgcagcaga cgctatctgc gacacaattg gatttgcgat 4920 accgcgcatg tggatgtgta ttttaatgag atcaacctcc atgaagcgta actagggggc 4980 ctccactga ggcactaccg gcttagcagc tgactaacac agtataaaac gtgagaagaa 5040 atcagtctca tgcgcatta gcgctaggct agttagcgtg gaggaccgga gcgctaccgc 5100 cagcagtttc atccgcctgg ttacgggttt gttaacacct accggtgttt taccgctacc 5160 ataggatccg atccatggggc ggccgcggta c 5191

<210> 29 <211> 6892 <212> DNA <213> Artificial sequence <220>

<223> Nucleotide sequence of transfer plasmid pU70-p455-H371K71 <400> 29

caataaacgc ggtatgtcta ccttcaagcc tatgatgaac ggatgtttgg tgtttgcggc 60 tattataacg ctcttgagtt ttatgctatc tctgggaaca tgcgaaaatt acaggcgtgt 120 ggttcgggat cctagggata acaggtaat cgatttattc aacaaagcca cgttgtgtct 180 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg 240 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct 300 tgctcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatggggct 360 cgcgataatg tcggggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg 420 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgatg 480 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt 540 actcctgatg atgcatggtt actaccact gcgatccccg ggaaaacagc attccaggta 600 ttagagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc 660 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc 720 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag 780 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataagctttt gccattctca 840 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg 900

- 77 046085

aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt 960 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gcttttttcaa 1020 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag 1080 tttttctaaa aaaacgcgg tatgtctacc ttcaagccta tgatgaacgg atgtttggtg 1140 tttgcggcta ttataacgct cttgagtttt atgctatctc tgggaacatg cgaaaattac 1200 aggcgtgtgg ttcgggatcc gaccctgttg gtgggtgcgg ttggactcag aatcttggcg 1260 caggcatgga agtttgtcgg tgacgaaaca tacgacacca tccgcgcaga agcaaagaat 1320 ttagagacc acgtaccctc aagtgctgca gagtcgtctc tagaaaacca atcgacacag 1380 gaggagtcta acagccccga agttgcccac ctgcgaagcg tcaacagcga tgacagtaca 1440 cacacgggggg gtgcgtcgaa cggcatccag gactgtgaca gtcagctcaa aactgtgtat 1500 gcctgcttgg ctctaattgg actcggcaca tgtgccatga tagggttgat agttacatt 1560 tgtgtattaa ggtcaaaact gtcctctcgg aatttttcgc gcgcgcaaaa tgtaaaacat 1620 agaaattacc agcgacttga gtacgttgct taagcttggc gtaatcatgg tcatagctgt 1680 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 1740 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 1800 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 1860 cggggagagg cggtttgcgt attggggcgct cttccgcttc ctcgctcact gactcgctgc 1920 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 1980 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 2040 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 2100 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 2160 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 2220 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 2280 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 2340 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 2400 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 2460 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 2520 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 2580 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 2640 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 2700 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 2760

- 78 046085 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt2820 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc2880 gttcatccat agttgcctga ctccccgtcg t gtagataac tacgatacgg gagggcttac2940 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat3000 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg3060 cctccatcca gtctattaat tgttgccggg aagctaga gt aagtagttcg ccagttaata3120 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta3180 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt3240 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag3300 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa3360 gatgcttttc tgtgactggt gagtactcaa ccaag tcatt ctgagaatag tgtatgcggc3420 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt3480 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc3540 tgttgagatc cagttcgatg ta acccactc gtgcacccaa ctgatcttca gcatctttta3600 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa3660 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca3720 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac3780 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta3840 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt390 0 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc3960 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt4020 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc4080 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat4140 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc4200 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt4260 cacgacgttg taaaacgacg gccagtgaat tcctccgagt accccagagg agtatgtgaa4320 aagctgccac tcgcaactac tgaagataat ttcaacgctc aagataaatc cggaggagtt4380 tcctcgagac cccggg tcga ggctcgtgcg cggatacatc gagtattcta gactcgagcg4440 caagccctac acgcgctacc cctgctttca acgcgtcaac ctgcacattg acggggagtt4500 tctggttcac aagatgctag cgttcaatgc cgcgatgcgc ccatcggccg aggagctgct4560 gtcat accca atgtttgctc aactttagga tgactaacct gtttctggga ggagacagcg4620 tgggcgacgg tgtataaagt tggtctgctt tcaagccctg ccactgcgct acagtgccac4680

- 79 046085 caactgtaaa gcggtagtaa gctgcagtgg tcgactggtg gtagcatata ctaccttatt4740 tatacgctcc gagctgtttt tcagcatgct agcacccaac gccgagcgag agtatataac4800 tcccatcatt gcccacaagc ttatgccact tattagcgtc cgctctgcc g tttgcttagt4860 cataatatct accgccgttt acgcagcaga cgctatctgc gacacaattg gatttgcgat4920 accgcgcatg tggatgtgta ttttaatgag atcaacctcc atgaagcgta actagggggc4980 ctcccactga ggcactaccg gcttagcagc tgactaacac agtataa aac gtgagaagaa5040 atcagtctca tgcgccatta gcgctaggct agttagcgtg gaggaccgga gcgctaccgc5100 cagcagtttc atccgcctgg ttacgggttt gttaacacct accggtgttt taccgctacc5160 ataggatccg atccatgggc ggccgcatga agaccgtgat cgccctgagt tacatcttct5220 gcctggtgtt tgggcaggac ctccctggta aaggcaacaa cacggccacg ctgtgccttg5280 ggcaccacgc cgtgccgaac ggcacccttg tgaaaactat taccgacgat cagatcgagg5340 tgaccaacgc caccgaactg gttcagaatt ttagcatggg caaaatttgc aataacccgc5400 accgcattct ggacggggcc aactgcacgc tgatcgattc attgctgggt gatccccact5460 gcgatggctt tcaaaacgaa aagtgggact tgttcatcga ac gcagcaag gcattcagca5520 actgctaccc atacgacgtg cccgaataca ccagcctgcg aagcctgatc gcgagctctg5580 ggaccctgga gttcaccaat gagaacttca attggaccgg agtgacccaa aacggtggct5640 ccagcgcctg taaaagggga cccaataaca gcttctttag caagttgaat tggctttaca5700 agagcggcaa tacttacccg atgttgaatg tgaccatgcc caacagtgac gactttgata5760 aactgtacat atggggcgtg caccatccca gcacggaccg cgaacagata aacctgtacg5820 tgcaggccag cggga agata atcgtgagca ccaagcgcag ccagcagacc atcattccca5880 acattggcag ccgaccgtgg gtgcgcggtc tgagctcccg catcagcata tactggacca5940 ttgtcaagcc gggagacatc ctgatcatca actctaatgg caatcttatc gccccacgcg6000 gctacttcaa gatgcagacc ggcaaaagca gtgtgatgag gagcgacgcc cccatcgaca6060 cctgcaatag cgaatgcatc acccccaatg gcagcatccc caacgacaag cctttccaga6120 acgtgaataa gatcacctac ggcgcgtgcc ccaagtacat caagcagaac accctgaagc6180 tggccaccgg catgcgcaac atccccgagc gacagacacg gggcattttt ggcgcaatcg6240 cagggttcat tgagaatggc tgggagggaa tggttaacgg ctggtacggc ttccgccatc6300 agaactctga aggaatcggc caagctgcgg atct gaagtc cacgcaagca gccatcaacc6360 agatcaacgg caagcttaac cgcgtgattg aaaagacgaa cgagaaattc caccaaatag6420 agaaagaatt cagcgaggtg gagggccgca tccaagacct cgagcgctac gtggaggaca6480 ccaagatcga cctgtggagc tacaatgccg agctcct ggt cgccttggaa aaccaacaca6540

- 80 046085

ccattgacct gaccgacagc gagatgaata aactcttcga gaagacccgg aagcaactcc 6600 gagagaacgc cgaagacatg ggtaatggggt gttttaagat ctaccacaag tgcgacaata 6660 gctgcatgga gagcatccga aacggaacct acgaccacaa cgagtaccgc gatgaggcag 6720 ttaataaccg cttccaaatc aaaagcgtgg aactgaagag tggctataag gactggatac 6780 tgtggatcag ctttgccata agctgcttcc tgctgtgcgc cgtttggttg ggtttcatca 6840 tgtggggcctg tcaaaagggc aatattcgct gtaacatctg catttgaggt ac 6892

<210> 30 <211> 4977 <212> DNA <213> Artificial sequence <220>

<223> Nucleotide sequence of transfer vector pU-1-3-p430BGHKBGH <400> 30

cctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 60 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 120 ctgagtagt gtcattctat tctggggggt ggggtggggc aggacagcaa ggggggat 180 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggatcc tagggataac 240 agggtaatcg atttattcaa caaagccacg ttgtgtctca aaatctctga tgttacattg 300 cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata 360 caaggggtgt tatgagccat attcaacggg aaacgtcttg ctcgaggccg cgattaaatt 420 ccaacatgga tgctgattta tatgggtata aatggggctcg cgataatgtc gggcaatcag 480 gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg 540 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg 600 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac 660 tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag 720 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt 780 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga 840 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac 900 aagtctggaa agaaatgcat aagcttttgc cattctcacc ggattcagtc gtcactcatg 960 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg 1020 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg 1080 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg 1140 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaacca tggctgtgcc 1200

- 81 046085

ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 1260 tgccactccc actgtccttt cctaataaaa tgaggaatt gcatcgcatt gtctgagtag 1320 gtgtcattct attctgggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 1380 caatagcagg catgctgggg atgcggtggg ctctatggat ccgaccctcc ccggggctaa 1440 aaagctgcgt cttcacgccc gaggcgctta ttgccactg ggtacggggc gcgcttttat 1500 atgtgtaacg tcccaccggt gtgacgcacg tactacggtt gttctaaata gctgtccccg 1560 tgattgcctc ggctgcacac atcgcctagg tttccgccgt gcctggtgtc gagggcccac 1620 ccctgtaacc aacatcgatg ggggcctgct gctccttcgc taccttagga ccgttatagt 1680 tacgtcaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 1740 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 1800 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 1860 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 1920 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 1980 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 2040 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 2100 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 2160 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 2220 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 2280 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 2340 tcgctccaag ctggggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 2400 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 2460 cactggtaac aggattagca gagcgaggta tgtagggcggt gctacagagt tcttgaagtg 2520 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 2580 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 2640 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 2700 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 2760 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 2820 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 2880 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 2940 cgtcgtgtag ataactacga tacggggaggg cttaccatct ggccccagtg ctgcaatgat 3000 accgcgagac ccacgctcac cggctccaga ttttatcagca aaaaccagc cagccggaag 3060 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 3120

- 82 046085

ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 3180 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 3240 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 3300 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 3360 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 3420 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 3480 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 3540 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 3600 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctggggtgagc 3660 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 3720 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 3780 cggatacata tttgaatgta ttttagaaaaa taaacaaata ggggttccgc gcacatttcc 3840 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa 3900 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg 3960 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 4020 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc 4080 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt 4140 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg 4200 gcgatcggtg cggggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag 4260 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag 4320 tgaattcgac gtaactataa cggtcctaag gtagcgaatt tttcattgg gcccctccct 4380 tttggctctg ggtatttagc ttccctccca cttctcattc cactttctcc acctgcacct 4440 tttccatctc ctctccaact cgccgccatg agacccgagg gagtttcgcg gggccgcgcc 4500 tcctctgtct ccatctccaa ctagtgtcga cctctatttg aggacccgcc gagtacccca 4560 caagagtatg taaaaagctg tcattctcaa ctactgagaa taatatcaaa gctaaagata 4620 aaccctgagg agtttccacg ggaaccagag tctaggctcg tgcgcggata catcgaatac 4680 gccagcctag agcgtaagcc acatacgcgc tatccttgct tccagcgcgt gaacctacac 4740 attgacgggg aatttttgat cataaaatg ctagcgttca atgctgcgat gcgcccatcc 4800 gcagaagagt tgttgtccta cccaatgttt atgaatctgt aggatgacta acagatttgg 4860 ggtggagacg gcgtggggcga tactgtataa agttgtacta cttaccagcc cagtcagtgt 4920 gctgtagtgc caccacctgt aaagctgtga taagctgcag ttgcggccgc cggggtac 4977

- 83 046085 <210> 31 <211> 6678 <212> DNA <213> Artificial sequence <220>

<223> Nucleotide sequence of transfer plasmid pU1-3-p430H1av-BGHKBGH <400> 31

cctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 60 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 120 ctgagtagt gtcattctat tctggggggt ggggtggggc aggacagcaa ggggggat 180 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggatcc tagggataac 240 agggtaatcg atttattcaa caaagccacg ttgtgtctca aaatctctga tgttacattg 300 cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata 360 caaggggtgt tatgagccat attcaacggg aaacgtcttg ctcgaggccg cgattaaatt 420 ccaacatgga tgctgattta tatgggtata aatggggctcg cgataatgtc gggcaatcag 480 gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg 540 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg 600 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac 660 tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag 720 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt 780 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga 840 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac 900 aagtctggaa agaaatgcat aagcttttgc cattctcacc ggattcagtc gtcactcatg 960 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg 1020 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg 1080 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg 1140 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaacca tggctgtgcc 1200 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 1260 tgccactccc actgtccttt cctaataaaa tgaggaatt gcatcgcatt gtctgagtag 1320 gtgtcattct attctgggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 1380 caatagcagg catgctgggg atgcggtggg ctctatggat ccgaccctcc ccggggctaa 1440 aaagctgcgt cttcacgccc gaggcgctta ttgccactg ggtacggggc gcgcttttat 1500 atgtgtaacg tcccaccggt gtgacgcacg tactacggtt gttctaaata gctgtccccg 1560

- 84 046085 tgattgcctc ggctgcacac atcgcctagg tttccgccgt gcctggtgtc gagggcccac1620 ccctgtaacc aacatcgatg ggggcctgct gctccttcgc taccttagga ccgttatagt1680 tacgtcaagc ttggcgtaat catggtcata gctgt ttcct gtgtgaaatt gttatccgct1740 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg1800 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct1860 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg1920 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc1980 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg2040 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct2100 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca2160 gaggtggcga aacccgacag gactataaag ataccagg cg tttccccctg gaagctccct2220 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc2280 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt2340 tcgctccaag ctggggct gtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc2400 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc2460 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg2520 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc2580 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag2640 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaaga aga2700 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat2760 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag2820 ttttaaatca atctaaagta tatatgagta aacttggtct gacagtt acc aatgcttaat2880 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc2940 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat3000 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag3060 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg3120 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc3180 tacaggcatc gtgg tgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca3240 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg3300 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc33 60 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta3420 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc3480

- 85 046085

aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 3540 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 3600 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctggggtgagc 3660 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 3720 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 3780 cggatacata tttgaatgta ttttagaaaaa taaacaaata ggggttccgc gcacatttcc 3840 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa 3900 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg 3960 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 4020 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc 4080 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt 4140 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg 4200 gcgatcggtg cggggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag 4260 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag 4320 tgaattcgac gtaactataa cggtcctaag gtagcgaatt tttcattgg gcccctccct 4380 tttggctctg ggtatttagc ttccctccca cttctcattc cactttctcc acctgcacct 4440 tttccatctc ctctccaact cgccgccatg agacccgagg gagtttcgcg gggccgcgcc 4500 tcctctgtct ccatctccaa ctagtgtcga cctctatttg aggacccgcc gagtacccca 4560 caagagtatg taaaaagctg tcattctcaa ctactgagaa taatatcaaa gctaaagata 4620 aaccctgagg agtttccacg ggaaccagag tctaggctcg tgcgcggata catcgaatac 4680 gccagcctag agcgtaagcc acatacgcgc tatccttgct tccagcgcgt gaacctacac 4740 attgacgggg aatttttgat cataaaatg ctagcgttca atgctgcgat gcgcccatcc 4800 gcagaagagt tgttgtccta cccaatgttt atgaatctgt aggatgacta acagatttgg 4860 ggtggagacg gcgtggggcga tactgtataa agttgtacta cttaccagcc cagtcagtgt 4920 gctgtagtgc caccacctgt aaagctgtga taagctgcag ttgcggccgc cgatggaggc 4980 aaaattgttc gtgctgttct gcgccttcac tgctctgaag gcagacacca tctgcgtggg 5040 ttaccacgcc aataattcca ccgacacggt ggataccatc ctggagaaga acgtgaccgt 5100 gactcattcc gtgaacctct tggagaactc acacaatggt aaattgtgca gccttaacgg 5160 caaagccccg ctgcaattgg ggaattgtaa cgtggccgga tggatactgg ggaaccccga 5220 gtgcgacctt ctcctgaccg ccaacagttg gtcctacatc attgagacga gcaacagcaa 5280 gaatggcgcc tgctatcctg ggggagttcgc tgactacgag gagctgcgcg agcagttgtc 5340

- 86 046085

tacagtcagc agcttcgaaa gattcgagat cttcccaaag gccactagct ggcccaacca 5400 cgatactacc aagggcacta cagtgagttg cagccacagc ggtgccaata gcttctaccg 5460 caacctgctg tggatcgtga agaagggtaa cagctacccc aagctgagca aatcttacac 5520 aaacaacaaa ggcaaagagg tgttggttat ctggggcgtg catcatcccc caaccgactc 5580 cgatcagcaa accctgtacc agaacaacca cacctacgtg agcgtcggta gctctaagta 5640 ttaccagcgc ttcacccccg aaatcgtcgc acgaccgaag gtgagagggc aggccggggag 5700 aatgaactac tactggacc tgctggatca aggcgacact attaccttcg aggctaccgg 5760 caacttgatc gccccgtggc acgcgttcgc cctcaataaa ggatctaata gcggcataat 5820 gatgagtgat gcccacgtgc ataactgcac cacgaagtgc cagacccctc acggcgcact 5880 gaaaagcaat ctgccctttc agaatgtgca ccccatcacc atcggcgagt gccccaagta 5940 tgttaaaagc actcagctcc gcatggccac cggactgcgc aacatcccga gcatccaatc 6000 ccgcggactg ttcggcgcaa tcgcgggctt tataggggc ggctggaccg gcatgatcga 6060 cggctggtac ggctaccacc atcaaaatga gcaaggttcc ggctacgccg cagaccagaa 6120 gagcaccaa atagcaatcg atggcatctc caacaaggtg aacagcgtga tcgaaaagat 6180 gaacatccag ttcacaagcg tggggaagga gttcaataac ctggaaaagc gcatcgagaa 6240 tctgaacaag aaggttgacg atgggttcct cgatgtctgg acctataacg ccgagctcct 6300 gatactgctt gagaacgagc gcaccctgga cttccacgac ttcaacgtga aaaacctgta 6360 cgaaaaggtc aagtcacagt tgcgaaacaa tgcgaagggag ataggcaacg gctgcttcga 6420 gttctatcac aagtgtgaca acgagtgcat gggagagcgtc aagaacggca cttacaacta 6480 cccgcgctac tctgaggaga gtaagctcaa ccgcgaagag attgacggcg tgaaactgga 6540 aagcgttggt gtccatcaga tcctggccat ctacagcacc gtggctagct ctctggttct 6600 gttggtgagc ctggggcgcta taagcttttg gatgtgttct aatggggagcc tgcagtgccg 6660 catctgcatc tgaggtac 6678

<210> 32 <211> 6892 <212> DNA <213> Artificial sequence <220>

<223> Nucleotide sequence of transfer plasmid pU70-p455H1pdm-71K71 <400> 32

caataaacgc ggtatgtcta ccttcaagcc tatgatgaac ggatgtttgg tgtttgcggc 60 tattataacg ctcttgagtt ttatgctatc tctgggaaca tgcgaaaatt acaggcgtgt 120 ggttcgggat cctagggata acaggtaat cgatttattc aacaaagcca cgttgtgtct 180

- 87 046085 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg240 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct300 tgctcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct360 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg420 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg480 gtcagactaa actggctgac ggaatttatg cctcttcc ga ccatcaagca ttttatccgt540 actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta600 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc660 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc720 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag780 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataagctttt gccattctca840 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg900 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt960 gccatcctat ggaactgcct cggtgagtt t tctccttcat tacagaaacg gctttttcaa1020 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag1080 tttttctaaa ataaacgcgg tatgtctacc ttcaagccta tgatgaacgg atgtttggtg1140 tttgcggcta ttataacgct cttgagtttt atgctatctc tgggaacatg cgaaaattac1200 aggcgtgtgg ttcgggatcc gaccctgttg gtgggtgcgg ttggactcag aatcttggcg1260 caggcatgga agtttgtcgg tgacgaaaca tacgacacca tccgcgcaga agcaaagaat1 320 ttagagaccc acgtaccctc aagtgctgca gagtcgtctc tagaaaacca atcgacacag1380 gaggagtcta acagccccga agttgcccac ctgcgaagcg tcaacagcga tgacagtaca1440 cacacggggg gtgcgtcgaa cggcatccag gactgtgaca gtcagctcaa aactg tgtat1500 gcctgcttgg ctctaattgg actcggcaca tgtgccatga tagggttgat agtttacatt1560 tgtgtattaa ggtcaaaact gtcctctcgg aatttttcgc gcgcgcaaaa tgtaaaacat1620 agaaattacc agcgacttga gtacgttgct taagcttggc gtaatcatgg tcatagctgt1680 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa1740 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac1800 tgcccgcttt ccagt cggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg1860 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc1920 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat198 0 cccagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca2040 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc2100

- 88 046085 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc2160 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg2220 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta2280 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg2340 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac2400 acgacttatc gccact ggca gcagccactg gtaacaggat tagcagagcg aggtatgtag2460 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat2520 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat2580 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc2640 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt2700 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct2760 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatat gagtaaactt2820 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc2880 gttcatccat agttgcct ga ctccccgtcg tgtagataac tacgatacgg gagggcttac2940 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat3000 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg3060 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata3120 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta3180 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgt tgt3240 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag3300 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa3360 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctg agaatag tgtatgcggc3420 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt3480 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc3540 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta3600 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa3660 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca3720 tttatcaggg ttattg tctc atgagcggat acatatttga atgtatttag aaaaataaac3780 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta3840 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt3900 tcggt gatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc3960

- 89 046085 tgtaagcgga tgccggggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt4020 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc4080 ggtgtgaaat accgcacaga tgcgtaagga gaa aataccg catcaggcgc cattcgccat4140 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc4200 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt4260 cacgacgttg taa aacgacg gccagtgaat tcctccgagt accccagagg agtatgtgaa4320 aagctgccac tcgcaactac tgaagataat ttcaacgctc aagataaatc cggaggagtt4380 tcctcgagac cccgggtcga ggctcgtgcg cggatacatc gagtattcta gactcgagcg4440 caagccctac acgcgctacc cctgctttca acgcgtcaac ctgcacattg acggggagtt4500 tctggttcac aagatgctag cgttcaatgc cgcgatgcgc ccatcggccg aggagctgct4560 gtcataccca atgtttgctc aactttagga tgactaacct g tttctggga ggagacagcg4620 tgggcgacgg tgtataaagt tggtctgctt tcaagccctg ccactgcgct acagtgccac4680 caactgtaaa gcggtagtaa gctgcagtgg tcgactggtg gtagcatata ctaccttatt4740 tatacgctcc gagctgtttt tcagcatgct agcacc caac gccgagcgag agtatataac4800 tcccatcatt gcccacaagc ttatgccact tattagcgtc cgctctgccg tttgcttagt4860 cataatatct accgccgttt acgcagcaga cgctatctgc gacacaattg gatttgcgat4920 accgcgcatg tggatgtgta ttttaatgag atcaacctcc atgaagcgta actagggggc4980 ctcccactga ggcactaccg gcttagcagc tgactaacac agtataaaac gtgagaagaa5040 atcagtctca tgcgccatta gcgctaggct agttagcgtg gaggaccgga gcgctaccgc5100 cagcag tttc atccgcctgg ttacggggttt gttaacacct accggtgttt taccgctacc5160 ataggatccg atccatgggc ggccgcatga aggcgatcct ggttgtgctg ctgtacacct5220 ttgccaccgc caacgccgat acgctgtgca tcggctatca cgccaacaac agcaccgaca5 280 cagtggatac cgttttggag aaaaatgtga cggtgacgca cagtgtgaat ctgttggagg5340 acaaacataa cggcaagctg tgtaagctga gaggtgtggc tccccttcac ctgggcaagt5400 gtaacatcgc cggctggatt cttggaaacc ccgagtgcga gagcctgagc accgcaagct5460 cctggagcta catcgttgaa accagttcat ccgacaatgg cacctgctac cccggagact5520 tcatcgacta cgaggagctg cgcgaacaac ttagcagcgt ctcctcattc gagcgcttcg5580 agatcttccc gaagaccagc a gctggccca accacgacag caataaggga gtgacagccg5640 cctgtcccca cgccggtgcc aagagcttct ataagaatct gatttggctg gtgaaaaagg5700 ggaactccta tccaaagctg tccaagtctt atatcaatga taagggcaag gaggtgctgg5760 tgttgtgggg aattcaccac ccaagca cca gcgccgatca gcagagcctc taccagaacg5820 cggatgctta tgtgttcgtc ggtacgtcac gctacagcaa gaagttcaag cccgagattg5880

- 90 046085

ccatccgccc gaaagtgcgg gaccaagaag gccgcatgaa ctactactgg accctggtgg 5940 aacctggcga caagatcacg tttgaggcta ccggcaacct ggtcgttccc cgctacgcct 6000 tcgccatgga gcgcaaagcg ggctctggca tcataatcag cgacacacct gtgcacgact 6060 gcaacactac ctgccagacc cccaagggcg ccataacac tagcctccct ttccagaaca 6120 ttcaccccat caccataggc aaatgcccca agtatgttaa gagcactaag ctgcggctcg 6180 ccactggcct gagaaacatc ccgagcatcc agtctcgcgg tctgttcgga gccattgccg 6240 gatttatcga aggaggctgg accggtatgg tggacggctg gtatggctac caccatcaaa 6300 atgagcaggg cagcggctat gccgctgats ttaaaagtac ccaaaatgca atcgacgaaa 6360 tcactaacaa ggttaacagc gtgattgaaa agatgaatac ccagtttact gcggtgggca 6420 aagagtttaa ccacctggaa aaacgcatcg aaaacctgaa caaaaaagtg gacgacggct 6480 tcctggatat ctggacatac aatgcagaac tgcttgtgtt gcttgagaac gagcgcactc 6540 tggactacca cgatagcaac gtcaaaaatc tctatgaaaa ggtgcgcagc cagctcaaaa 6600 acaatgcgaa agaaatcggg aatggctgct tcgaattcta ccacaagtgc gacaacacgt 6660 gcatggagag cgttaagaac ggacgtatg attatcctaa gtatagcgaa gaagccaagc 6720 tgaatcgcga agagatcgac ggagtgaaac tggaatccac ccgcatatat cagatactgg 6780 ccattacag cacagttgcg agcagcctgg tcctgatcgt gagcctggggt gctatatcat 6840 tctggatgtg cagcaacggc tctctccagt gccgcatctg tatctgaggt ac 6892

<210> 33 <211> 6670 <212> DNA <213> Artificial sequence <220>

<223> Nucleotide sequence of transfer plasmid pU1-3-p430H1hu-BGHKBGH <400> 33

cctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 60 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 120 ctgagtagt gtcattctat tctggggggt ggggtggggc aggacagcaa ggggggat 180 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggatcc tagggataac 240 agggtaatcg atttattcaa caaagccacg ttgtgtctca aaatctctga tgttacattg 300 cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata 360 caaggggtgt tatgagccat attcaacggg aaacgtcttg ctcgaggccg cgattaaatt 420 ccaacatgga tgctgattta tatgggtata aatggggctcg cgataatgtc gggcaatcag 480 gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg 540

- 91 046085 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg600 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac660 tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat cctgatt cag720 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt780 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga840 ataacggttt ggttgatgcg agtgatttt g atgacgagcg taatggctgg cctgttgaac900 aagtctggaa agaaatgcat aagcttttgc cattctcacc ggattcagtc gtcactcatg960 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg1020 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg1080 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg1140 atatgaataa attgcagttt catttgatgc tcgatgag tt tttctaacca tggctgtgcc1200 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg1260 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag1320 gtgtcattct attctggggg g tggggtggg gcaggacagc aagggggagg attgggaaga1380 caatagcagg catgctgggg atgcggtggg ctctatggat ccgaccctcc ccggggctaa1440 aaagctgcgt cttcacgccc gaggcgctta ttgcccactg ggtacggggc gcgcttttat1500 atgtgtaacg tcccaccggt gtgacgcacg tactacggtt gttctaaata gctgtccccg1560 tgattgcctc ggctgcacac atcgcctagg tttccgccgt gcctggtgtc gagggcccac1620 ccctgtaacc aacatcgatg ggggcctgct gctccttcgc taccttagga ccgttatagt 1680 tacgtcaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct1740 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg1800 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttcca gt cgggaaacct1860 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg1920 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc1980 ggtatcagct cactcaaagg cggtaatac g gttatccaca gaatcagggg ataacgcagg2040 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct2100 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca2160 gaggtggcga aaccc gacag gactataaag ataccaggcg tttccccctg gaagctccct2220 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc2280 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt234 0 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc2400

- 92 046085 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 2460 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 2520 gtggcctaac tacggctaca ctagaaggac agtatttggt atctg cgctc tgctgaagcc 2580 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 2640 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 2700 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 2760 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 2820 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 2880 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 2940 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 3000 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 3060 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 3120 ccgggaagct agagtaagta gttcgcca gt taatagtttg cgcaacgttg ttgccattgc 3180 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 3240 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 3300 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 3360 actgcataat tctcttactg tcatgccatc cgtaagatgc ttt tctgtga ctggtgagta 3420 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 3480 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 3540 ttcttcgggg cgaaaactct caaggat ctt accgctgttg agatccagtt cgatgtaacc 3600 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 3660 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 3720 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 3780 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 3840 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa 3900 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg 3960 acacatgcag ctcccggaga cggtcacagc ttg tctgtaa gcggatgccg ggagcagaca 4020 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc 4080 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt 4140 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg 4200 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag 4260 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag 4320

- 93 046085

tgaattcgac gtaactataa cggtcctaag gtagcgaatt tttcattgg gcccctccct 4380 tttggctctg ggtatttagc ttccctccca cttctcattc cactttctcc acctgcacct 4440 tttccatctc ctctccaact cgccgccatg agacccgagg gagtttcgcg gggccgcgcc 4500 tcctctgtct ccatctccaa ctagtgtcga cctctatttg aggacccgcc gagtacccca 4560 caagagtatg taaaaagctg tcattctcaa ctactgagaa taatatcaaa gctaaagata 4620 aaccctgagg agtttccacg ggaaccagag tctaggctcg tgcgcggata catcgaatac 4680 gccagcctag agcgtaagcc acatacgcgc tatccttgct tccagcgcgt gaacctacac 4740 attgacgggg aatttttgat cataaaatg ctagcgttca atgctgcgat gcgcccatcc 4800 gcagaagagt tgttgtccta cccaatgttt atgaatctgt aggatgacta acagatttgg 4860 ggtggagacg gcgtggggcga tactgtataa agttgtacta cttaccagcc cagtcagtgt 4920 gctgtagtgc caccacctgt aaagctgtga taagctgcag ttgcggccgc atgaaggcca 4980 agcttcttat actgtggtgc gccctcagcg ctaccgacgc tgacaccatt tgcatcggtt 5040 accacgccaa caacagcacc gataccgttg acaccgtgct ggaaaagaac gtgaccgtta 5100 cccacagtgt taatctgctg gaggacaacc acaacggaaa gttgtgtaag ttgaagggcg 5160 tcgcacccct gcagctggga aagtgcagca tagcaggctg gattctcggt aacccggagt 5220 gcgagagcct gtttagcaaa aagtcttggt catacatcgc ggaaacccca aacgccgaaa 5280 atggtatttg ctaccccggc tacttcagcg actacgaaga gctccgcgag cagttgagca 5340 gcgttagtag ctttgaacgc ttcgagatct tccccaaaga aagctcctgg ccaaagcact 5400 ccacggagc caccgccagc tgttccaagc aggggccgcag cagcttctac cgcaacctcc 5460 tgtggctcac cgaaaaaaac ggttcttacc ccaaccttag caagagctac gtcaacgaca 5520 aggagagaga ggtgcttgtt ctgtggggcg ttcatcaccc ctctaacatc gaggaccaaa 5580 gagccatata tcgaaaggag accgcctacg tgagcgttat gagtagtctg tacaacaggc 5640 gcttcacccc cgagatcgcc aagcgcccca agatcaggaa ccaggagggc aggatcaact 5700 attattggac ccttctggaa cccaaggaca ctatcatctt cgaggccaac gggaacctca 5760 tcgccccgtg gtacgccttc gccctgtcac gcggctttga gagcgggatc atcgtgagca 5820 acgcctctat ggatgagtgt gacgccaagt gccaaacccc gcagggcgcg atcaatagca 5880 gtcttcatt tcagaatgtg cacccggtga ccattgggga gtgcccgaag tatgtgaaga 5940 gtaccaagct gaagatggcc acggggcctgc gcaacatccc cagcattcag acacggggtc 6000 tgttcggcgc tatcgccggc ttcattgaag gcggttggac tggcatgatt gatggctggt 6060 atggctatca ccatcagaac gaacaaggaa gcggctacgc ggcagatcag aagtcaaccc 6120 agaacgctat aaacggcata actaacaagg tgaacagcgt cattgacaaa atgaatacc 6180

- 94 046085 agtttaccgc cgtgggcaaa gagttcaata agcttgaaaa acgcatggaa aaccttaaca6240 aaaaggtgga cgacggcttc ctggacatct ggacctacaa tgccgaactg ttggtgctcc6300 ttgagaatga gcggacgctt gatttccacg acagcaac gt gaagtcactc tacgagaagg6360 ttaagggaca gcttaaaaac aacgctaaag aaatcggcaa cggctgtttc gagttctacc6420 acaagtgcaa caacgagtgc atggatagcg tgaaaaatgg gacctatgac tacccccgct6480 actcagagga gagcaagctc aaccgcgaga a gatcgacgg cgtggagctg aaaagcatgg6540 gcgtgtacca gatactggcc atatacagca ctgtcgccag cagccttgtg ttgcttgtga6600 gccttggagc gaccagtttt tggatgtgct ccaatggcag cttgcagtgc cgcatctgca6660 tctgaggtac6670 <210>34 <211>4916 <212> DNA <213> Artificial sequence <220>

<223> Nucleotide sequence of transfer vector pUUL43-p42218K18 <400> 34

aattctaact ataacggtcc taaggtagcg aagtatacca tgcttgctat actgaaaata 60 aaaacgcata ttgtaaacga cagacgcgga aatgtttatt gcttagtttc actatttggt 120 taaaactatt tacacttgta gaaacacgcc cactaagtat ttgttttatg actaatacct 180 ggtgcataaa accatcctct tgggtccctg tacctcaaac tctccaaagg ttggcttgct 240 acatcaaggt tatcaatccc atggatcgtc agacttggtg gaagacccat gttcgttgtt 300 tcaagaagcg tatcctctac tcagctccac tgatacagca ttgctacgca cgcctcacgt 360 tggcgaaatt ggcgcagatg aaggacattt tgccagtac ctaattcgcg acgaatcccc 420 cctgaaaggc tgttttccac gaatttagt tgtgcccgcc tacaactttt cacttgcaaa 480 ctcaataaaa cgcacagttt gtatattcag ttgtcagttt gctctactcg agcgtcggcg 540 ctttgtctag ccctcttagt gggtattgtt accggctggg gttttattgg cgttgttatt 600 ggggagattt tagttgatag aaagcatacc gaggttttgg gggtgtcgct taatttcggt 660 gtctgtaaac gtaaaaagag ggcgcgcctc agcggccgca ggtaccaata aaaactgaga 720 ctgttattatt catttcagtg tgtttaataa gaattgtgaa cataacttat tctatatctc 780 attgcgtgga aagactggaa aacgcattgg tggtaggtgg aaggctcgcc ggatcctagg 840 gataacaggg taatcgattt attcaacaaa gccacgttgt gtctcaaaat ctctgatgtt 900 acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca 960 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctcg aggccgcgat 1020

- 95 046085 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc1080 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga1140 aacatggcaa aggtagcgtt gccaatgatg ttacagat ga gatggtcaga ctaaactggc1200 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat1260 ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg1320 attcaggtga aaatattgtt gatgcgctgg cag tgttcct gcgccggttg cattcgattc1380 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac1440 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg1500 ttgaacaagt ctggaaagaa atgcataagc ttttgccatt ctcaccggat tcagtcgtca1560 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta1620 ttgatgttgg acgagtcgga atcgcagacc gataccag ga tcttgccatc ctatggaact1680 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata1740 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taaaataaac1800 gtcgacaata aaaactgaga ctgtt atatt catttcagtg tgtttaataa gaattgtgaa1860 cataacttat tctatatctc attgcgtgga aagactggaa aacgcattgg tggtaggtgg1920 aaggctcgcc tctagaaccc cggcgtatgt tgtcttctgt ctgactgttg gacacgtctc1980 tgcgatgctg ctggagcagc ttgtcatcag agtaaactac acggcagagg cgagttccgg2040 catccactcc acggcccacg ctgtctgcat ggtgcttgcc gcctttgggt acggcgtggc2100 cggtcccctc tcgctcgcat ttactgtatc cgggggtata ctgggggcgc tataccttcg21 60 caagcgcgca acgggcgcgc gccgcctggc ggcaactcac atttcgaggt ggcttattgt2220 ttgtgtatat gttgccgccg gttcgctacc ttaggaccgt tatagttact gcaggcatgc2280 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgtt atc cgctcacaat2340 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag2400 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg2460 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc2520 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc2580 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa2640 catgtgagca aa aggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt2700 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg2760 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 2820 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag2880 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc2940

- 96 046085

caagctggggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 3000 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 3060 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 3120 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 3180 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 3240 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 3300 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 3360 catgatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 3420 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 3480 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 3540 gtagataact acgatacgggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 3600 agaccaccgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 3660 gcgcagaagt ggtcctgcaa ctttatccgc ctccacatccag tctattaatt gttgccggga 3720 agctagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 3780 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 3840 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 3900 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 3960 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 4020 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 4080 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 4140 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aaccactcg 4200 tgcaccaac tgatcttcag catcttttac tttcaccagc gtttctggggt gagcaaaaac 4260 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 4320 actcttcctt tttcaatatt attgaagcat ttatcaggt tattgtctca tgagcggata 4380 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 4440 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg 4500 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat 4560 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccggggagca gacaagcccg 4620 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga 4680 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaagggag 4740 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc 4800

- 97 046085 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt4860 aagttgggta acgccaggt tttcccagtc acgacgttgt aaaacgacgg ccagtg4916 <210>35 <211>5626 <212> DNA <213> Artificial sequence <220>

<223> Nucleotide sequence of transfer plasmid pUUL43-p422-mC18K18 <400> 35

aattctaact ataacggtcc taaggtagcg aagtatacca tgcttgctat actgaaaata 60 aaaacgcata ttgtaaacga cagacgcgga aatgtttatt gcttagtttc actatttggt 120 taaaactatt tacacttgta gaaacacgcc cactaagtat ttgttttatg actaatacct 180 ggtgcataaa accatcctct tgggtccctg tacctcaaac tctccaaagg ttggcttgct 240 acatcaaggt tatcaatccc atggatcgtc agacttggtg gaagacccat gttcgttgtt 300 tcaagaagcg tatcctctac tcagctccac tgatacagca ttgctacgca cgcctcacgt 360 tggcgaaatt ggcgcagatg aaggacattt tgccagtac ctaattcgcg acgaatcccc 420 cctgaaaggc tgttttccac gaatttagt tgtgcccgcc tacaactttt cacttgcaaa 480 ctcaataaaa cgcacagttt gtatattcag ttgtcagttt gctctactcg agcgtcggcg 540 ctttgtctag ccctcttagt gggtattgtt accggctggg gttttattgg cgttgttatt 600 ggggagattt tagttgatag aaagcatacc gaggttttgg gggtgtcgct taatttcggt 660 gtctgtaaac gtaaaaagag ggcgcgcctc agcggccgca tggtgagcaa gggcgaggag 720 gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg 780 aacggccacg agttcgagat cgagggcgag ggcgaggggcc gcccctacga gggcacccag 840 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc 900 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac 960 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc 1020 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg 1080 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatggggc 1140 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag 1200 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag 1260 gccaagaagc ccgtgcagct gccccggcgcc tacaacgtca acatcaagtt ggacatcacc 1320 tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgagg ccgccactcc 1380 accggcggca tggacgagct gtacaagtaa ggtaccaata aaaactgaga ctgttattatt 1440

- 98 046085 catttcagtg tgtttaataa gaattgtgaa cataacttat tctatatctc attgcgtgga1500 aagactggaa aacgcattgg tggtaggtgg aaggctcgcc ggatcctagg gataacaggg1560 taatcgattt attcaacaaa gccacgttgt gtctcaaaat ctctgatgtt acattgcaca1620 agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag1680 gggtgttatg agccatattc aacgggaaac gtcttgctcg aggccgcgat taaattccaa1740 catggatgct gatttatatg ggtataaatg ggctcgc gat aatgtcgggc aatcaggtgc1800 gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa1860 aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt1920 tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac1980 cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga2040 aaatattgtt gatgcgctgg cagtgttcct gcgccggt tg cattcgattc ctgtttgtaa2100 ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa2160 cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt2220 ctggaaagaa at gcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga2280 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg2340 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga2400 gt tttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat2460 gaataaattg cagtttcatt tgatgctcga tgagtttttc taaaataaac gtcgacaata2520 aaaactgaga ctgttatatt catttcagtg tgtttaataa gaattgtgaa cataacttat2580 tctatatctc attgcgtgga aagactggaa aacgcattgg tggtaggtgg aaggctcgcc2640 tctagaaccc cggcgtatgt tgtcttctgt ctgactgttg gacacgtctc tgcgatgctg2700 ctggagcagc ttgtcatcag agtaaactac acggcagagg cgagttccgg catcc actcc2760 acggcccacg ctgtctgcat ggtgcttgcc gcctttgggt acggcgtggc cggtcccctc2820 tcgctcgcat ttactgtatc cggggggtata ctgggggcgc tataccttcg caagcgcgca2880 acgggcgcgc gccgcctggc ggcaactcac atttcgaggt ggcttattgt ttgtgtatat2940 gttgccgccg gttcgctacc ttaggaccgt tatagttact gcaggcatgc aagcttggcg3000 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac3060 atacgagccg gaagcataa a gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca3120 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat3180 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc32 40 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca3300 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca3360

- 99 046085

aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 3420 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 3480 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 3540 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 3600 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctggggc 3660 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 3720 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 3780 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 3840 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 3900 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 3960 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 4020 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgatta 4080 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 4140 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 4200 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 4260 acgatacgggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agaccaccgc 4320 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 4380 ggtcctgcaa ctttatccgc ctccacatccag tctattaatt gttgccggga agctagta 4440 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 4500 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 4560 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 4620 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 4680 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 4740 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 4800 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 4860 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aaccactcg tgcaccaac 4920 tgatcttcag catcttttac tttcaccagc gtttctggggt gagcaaaaac aggaaggcaa 4980 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 5040 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 5100 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 5160 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 5220

- 100 046085

ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg 5280 gagacggtca cagcttgtct gtaagcggat gccggggagca gacaagcccg tcagggcgcg 5340 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta 5400 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaagggag aaaataccgc 5460 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc 5520 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta 5580 acgccaggt tttcccagtc acgacgttgt aaaacgacgg ccagtg 5626

<210> 36 <211> 6616 <212> DNA <213> Artificial sequence <220>

<223> Nucleotide sequence of transfer plasmid pUUL43-p422H1pdm-18K18 <400> 36

aattctaact ataacggtcc taaggtagcg aagtatacca tgcttgctat actgaaaata 60 aaaacgcata ttgtaaacga cagacgcgga aatgtttatt gcttagtttc actatttggt 120 taaaactatt tacacttgta gaaacacgcc cactaagtat ttgttttatg actaatacct 180 ggtgcataaa accatcctct tgggtccctg tacctcaaac tctccaaagg ttggcttgct 240 acatcaaggt tatcaatccc atggatcgtc agacttggtg gaagacccat gttcgttgtt 300 tcaagaagcg tatcctctac tcagctccac tgatacagca ttgctacgca cgcctcacgt 360 tggcgaaatt ggcgcagatg aaggacattt tgccagtac ctaattcgcg acgaatcccc 420 cctgaaaggc tgttttccac gaatttagt tgtgcccgcc tacaactttt cacttgcaaa 480 ctcaataaaa cgcacagttt gtatattcag ttgtcagttt gctctactcg agcgtcggcg 540 ctttgtctag ccctcttagt gggtattgtt accggctggg gttttattgg cgttgttatt 600 ggggagattt tagttgatag aaagcatacc gaggttttgg gggtgtcgct taatttcggt 660 gtctgtaaac gtaaaaagag ggcgcgcctc agcggccgca tgaaggcgat cctggttgtg 720 ctgctgtaca cctttgccac cgccaacgcc gatacgctgt gcatcggcta tcacgccaac 780 aacagcaccg acacagtgga taccgttttg gagaaaaatg tgacggtgac gcacagtgtg 840 aatctgttgg aggacaaaca taacggcaag ctgtgtaagc tgagaggtgt ggctcccctt 900 cacctggggca agtgtaacat cgccggctgg attcttggaa accccgagtg cgagagcctg 960 agcaccgcaa gctcctggag ctacatcgtt gaaaccagtt catccgacaa tggcacctgc 1020 taccccggag acttcatcga ctacgaggag ctgcgcgaac aacttagcag cgtctcctca 1080 ttcgagcgct tcgagatctt cccgaagacc agcagctggc ccaaccacga cagcaataag 1140

- 101 046085

ggagtgacag ccgcctgtcc ccacgccggt gccaagagct tctataagaa tctgatttgg 1200 ctggtgaaaa aggggaactc ctatccaaag ctgtccaagt cttatatcaa tgataagggc 1260 aaggaggtgc tggtgttgtg gggaattcac cacccaagca ccagcgccga tcagcagagc 1320 ctctaccaga acgcggatgc ttatgtgttc gtcggtacgt cacgctacag caagaagttc 1380 aagcccgaga ttgccatccg cccgaaagtg cgggaccaag aaggccgcat gaactactac 1440 tggaccctgg tggaacctgg cgacaagatc acgtttgagg ctaccggcaa cctggtcgtt 1500 ccccgctacg ccttcgccat ggagcgcaaa gcggggctctg gcatcataat cagcgacaca 1560 cctgtgcacg actgcaacac tacctgccag acccccaagg gcgccattaa cactagcctc 1620 cctttccaga acattcaccc catcaccata ggcaaatgcc ccaagtatgt taagagcact 1680 aagctgcggc tcgccactgg cctgagaaac atcccgagca tccagtctcg cggtctgttc 1740 ggagccatg ccggatttat cgaaggaggc tggaccggta tggtggacgg ctggtatggc 1800 taccaccatc aaaatgagca gggcagcggc tatgccgctg atcttaaaag tacccaaaat 1860 gcaatcgacg aaatcactaa caaggttaac agcgtgattg aaaagatgaa tacccagttt 1920 actgcggtgg gcaaagagtt taaccacctg gaaaaacgca tcgaaaacct gaacaaaaaa 1980 gtggacgacg gcttcctgga tatctggaca tacaatgcag aactgcttgt gttgcttgag 2040 aacgagcgca ctctggacta ccacgatagc aacgtcaaaa atctctatga aaaggtgcgc 2100 agccagctca aaaacaatgc gaaagaaatc gggaatggct gcttcgaatt ctaccacaag 2160 tgcgacaaca cgtgcatgga gagcgttaag aacgggacgt atgattatcc taagtatagc 2220 gaagaagcca agctgaatcg cgaagatc gacggagtga aactggaatc cacccgcata 2280 tatcagatac tggccatta cagcacagtt gcgagcagcc tggtcctgat cgtgagcctg 2340 ggtgctatat cattctggat gtgcagcaac ggctctctcc agtgccgcat ctgtatctga 2400 ggtaccaata aaaactgaga ctgttattatt catttcagtg tgtttaataa gaattgtgaa 2460 cataacttat tctatatctc attgcgtgga aagactggaa aacgcattgg tggtaggtgg 2520 aaggctcgcc ggatcctagg gataacaggg taatcgattt attcaacaaa gccacgttgt 2580 gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa 2640 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac 2700 gtcttgctcg aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg 2760 ggctcgcgat aatgtcggggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga 2820 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga 2880 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccata agcattttat 2940 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca 3000 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct 3060

- 102 046085 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg3120 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga3180 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataagc ttttgccatt3240 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga3300 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga3360 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt3420 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga3480 tgagtttttc taaaataaac gtcgacaata aaaactgaga ctgttatatt catttcagtg3540 tgtttaataa gaattgtgaa cataacttat tctatatctc attgcgtgga aagactggaa3600 aacgcattgg tggtaggtgg aaggctcgcc tctagaaccc cggcgtatgt tgtcttctgt3660 ctgactgttg gacacgtctc tgcgatgctg ctgg agcagc ttgtcatcag agtaaactac3720 acggcagagg cgagttccgg catccactcc acggcccacg ctgtctgcat ggtgcttgcc3780 gcctttgggt acggcgtggc cggtcccctc tcgctcgcat ttactgtatc cgggggtata3840 ctgggggcgc tataccttcg caag cgcgca acgggcgcgc gccgcctggc ggcaactcac3900 atttcgaggt ggcttattgt ttgtgtatat gttgccgccg gttcgctacc ttaggaccgt3960 tatagttact gcaggcatgc aagcttggcg taatcatggt catagctgtt tcctgtgtga4020 aattgtt atc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc4080 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc4140 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc42 00 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt4260 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca4320 ggggataacg caggaaagaa catgtgagca aaacccagc aaaa ggccag gaaccgtaaa4380 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat4440 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc4500 cctggaagct ccctcgtgcg ctctcct gtt ccgaccctgc cgcttaccgg atacctgtcc4560 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt4620 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac4680 cgctgcgcct t atccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg4740 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca4800 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc4860 gctctgctga a gccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa4920

- 103 046085 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa4980 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac5040 tcacgttaag ggattttggt catgagatta t caaaaagga tcttcaccta gatcctttta5100 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt5160 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata5220 gttgcctgac tccccgt cgt gtagataact acgatacggg agggcttacc atctggcccc5280 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac5340 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag5400 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac5460 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc5520 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg5580 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc5640 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct5700 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc5760 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc5820 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc5880 agttc gatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc5940 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca6000 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcaggt6060 t attgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt6120 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca6180 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac 6240 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat6300 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg6360 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata6420 ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc6480 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg6540 ggatgtgctg ca aggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt 6600 aaaacgacgg ccagtg6616 <210>37 <211>4435 <212> DNA <213> Artificial sequence

- 104 046085 <220>

<223> Nucleotide sequence of transfer plasmid pUmC70 <400>37 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcaggcgcg tcagcgggtg120 ttggcgg gtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cctccgagta ccccagagga420 gtatgtgaaa agctgccact cgcaactact gaagataatt tcaacgctca agataaatcc480 ggaggagttt cctcgagacc ccgggtcgag gctcgtgcgc ggatacatcg agtattctag540 actcgagcgc aagcccta ca cgcgctaccc ctgctttcaa cgcgtcaacc tgcacattga600 cggggagttt ctggttcaca agatgctagc gttcaatgcc gcgatgcgcc catcggccga660 ggagctgctg tcatacccaa tgtttgctca actttaggat gactaacctg tttctgggag720 gagacagcgt g ggcgacggt gtataaagtt ggtctgcttt caagccctgc cactgcgcta780 cagtgccacc aactgtaaag cggtagtaag ctgcagtggt cgacatggtg agcaagggcg840 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct900 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca960 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc1020 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg1080 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg1140 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca1200 aggtgaagct gcgcggcacc aacttcccct ccgac ggccc cgtaatgcag aagaagacca1260 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga1320 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct1380 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca1440 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc1500 actccaccgg cggcatggac gagctgtaca agtaactgtg ccttctagtt gccagccatc156 0 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct1620 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg1680 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatag ca ggcatgctgg1740

- 105 046085

ggatgcggtg ggctctatgg atccgaccct gttggtgggt gcggttggac tcagaatctt 1800 ggcgcaggca tggaagtttg tcggtgacga aacatacgac accatccgcg cagaagcaaa 1860 gaatttagag acccacgtac cctcaagtgc tgcagagtcg tctctagaaa accaatcgac 1920 acaggaggag tctaacagcc ccgaagttgc ccacctgcga agcgtcaaca gcgatgacag 1980 tacacacacg ggggggtgcgt cgaacggcat ccaggactgt gacagtcagc tcaaaactgt 2040 gtatgcctgc ttggctctaa ttggactcgg cacatgtgcc atgatagggt tgatagttta 2100 catttgtgta ttaaggtcaa aactgtcctc tcggaatttt tcgcgcgcgc aaaatgtaaa 2160 acatagaaat taccagcgac ttgagtacgt tgcttaagct tggcgtaatc atggtcatag 2220 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 2280 aaaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 2340 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 2400 cgcgcgggga gagggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 2460 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 2520 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 2580 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 2640 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 2700 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 2760 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 2820 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tggggctgtgt gcacgaaccc 2880 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 2940 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 3000 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 3060 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 3120 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 3180 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacgggg gtctgacgct 3240 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 3300 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 3360 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 3420 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acggggagggc 3480 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 3540 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 3600 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 3660

- 106 046085

aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 3720 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 3780 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 3840 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 3900 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 3960 cggcgaccga gttgctcttg cccggcgtca atacggata ataccgcgcc acatagcaga 4020 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 4080 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatch ttcagcatct 4140 tttacttttca ccagcgtttc tggggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 4200 ggaataaggg cgacacggaa atgttgaata ctcatactct tccttttttca atattattga 4260 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 4320 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc 4380 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc 4435

<210> 38 <211> 22 <212> DNA <213> Artificial sequence <220>

<223> Forward primer <400>38 aggctcgtgc gcggatacat cg <210>39 <211>22 <212> DNA <213> Artificial sequence <220>

<223> Reverse primer <400>39 ttcggggctg ttagactcct cc <210>40 <211>22 <212> DNA <213> Artificial sequence <220>

<223> Direct primer <400> 40 ccaactcgcc gccatgagac cc

- 107 046085 <210> 41 <211> 22 <212> DNA <213> Artificial sequence <220>

<223> Reverse primer <400> 41 agcgcgcccc gtacccagtg gg <210> 42 <211> 16 <212> DNA <213> Artificial sequence <220>

<223> Forward primer <400> 42 cgacgcgcgt cggagg <210> 43 <211> 21 <212> DNA <213> Artificial sequence <220>

<223> Reverse primer <400> 43 gttataaaca taccatgcac c <210>44 <211>566 <212> PRT <213> swine flu virus <400>44

Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn

5 1015

Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30

Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45

Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val 50 55 60

Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly 65 70 75 80

- 108 046085

Asn

Val

Ile

Glu

Ser 145

Phe

Lys

Leu

Tyr

Lys 225

Glu

Ile

Ala

Val

Thr 305

Pro

Pro Glu Cys Glu Ser 85

Glu Thr Ser Ser Ser

100

Asp Tyr Glu Glu Leu

115

Arg Phe Glu Ile Phe 130

Asn Lys Gly Val Thr

150

Tyr Lys Asn Leu Ile

165

Leu Ser Lys Ser Tyr 180

Trp Gly Ile His His 195

Gln Asn Ala Asp Ala 210

Lys Phe Lys Pro Glu

230

Gly Arg Met Asn Tyr

245

Thr Phe Glu Ala Thr

260

Met Glu Arg Lys Ala

275

His Asp Cys Asn Thr 290

Ser Leu Pro Phe Gln

310

Lys Tyr Val Lys Ser

325

Leu Ser Thr Ala Ser

Ser Trp Ser Tyr Ile 95

Asp Asn Gly Thr Cys 105

Arg Glu Gln Leu Ser

120

Pro Lys Thr Ser Ser 135

Ala Ala Cys Pro His

155

Trp Leu Val Lys Lys

170

Ile Asn Asp Lys Gly

185

Pro Ser Thr Ser Ala

200

Tyr Val Phe Val Gly 215

Ile Ala Ile Arg Pro

235

Tyr Trp Thr Leu Val

250

Gly Asn Leu Val Val 265

Gly Ser Gly Ile Ile

280

Thr Cys Gln Thr Pro 295

Asn Ile His Pro Ile

315

Thr Lys Leu Arg Leu

330

Tyr Pro Gly Asp Phe 110

Ser Val Ser Ser Phe

125

Trp Pro Asn His Asp 140

Ala Gly Ala Lys Ser

160

Gly Asn Ser Tyr Pro

175

Lys Glu Val Leu Val

190

Asp Gln Gln Ser Leu

205

Thr Ser Arg Tyr Ser 220

Lys Val Arg Asp Gln

240

Glu Pro Gly Asp Lys

255

Pro Arg Tyr Ala Phe 270

Ile Ser Asp Thr Pro

285

Lys Gly Ala Ile Asn 300

Thr Ile Gly Lys Cys

320

Ala Thr Gly Leu Arg

335

- 109 046085

Asn Ile Pro Ser Ile Gln Ser Arg Gly

340 345

Phe Ile Glu Gly Gly Trp Thr Gly Met

355 360

His His Gln Asn Glu Gln Gly Ser Gly

370 375

Thr Gln Asn Ala Ile Asp Glu Ile Thr 385 390

Glu Lys Met Asn Thr Gln Phe Thr Ala

405

Leu Glu Lys Arg Ile Glu Asn Leu Asn

420 425

Leu Asp Ile Trp Thr Tyr Asn Ala Glu

435 440

Glu Arg Thr Leu Asp Tyr His Asp Ser

450 455

Lys Val Arg Ser Gln Leu Lys Asn Asn

465 470

Cys Phe Glu Phe Tyr His Lys Cys Asp

485

Lys Asn Gly Thr Tyr Asp Tyr Pro Lys

500 505

Asn Arg Glu Glu Ile Asp Gly Val Lys

515 520

Gln Ile Leu Ala Ile Tyr Ser Thr Val

530 535

Val Ser Leu Gly Ala Ile Ser Phe Trp 545 550

Phe Gly Ala Ile Ala Gly

350

Asp Gly Trp Tyr Gly Tyr

365

Ala Ala Asp Leu Lys Ser

380

Lys Val Asn Ser Val Ile

395 400

Gly Lys Glu Phe Asn His

415

Lys Val Asp Asp Gly Phe

430

Leu Val Leu Leu Glu Asn

445

Val Lys Asn Leu Tyr Glu

460

Lys Glu Ile Gly Asn Gly

475 480

Thr Cys Met Glu Ser Val

495

Ser Glu Glu Ala Lys Leu

510

Glu Ser Thr Arg Ile Tyr

525

Ser Ser Leu Val Leu Ile

540

Cys Ser Asn Gly Ser Leu

555 560

Gln Cys Arg Ile Cys Ile

565 <210> 45

- 110 046085 <211>566 <212> PRT <213> swine virus <400>45

Met Lys Thr Val Ile 15 flu

Ala Leu Ser Tyr Ile 10

Phe Cys Leu Val Phe

Gln Asp Leu Pro Gly

Lys Gly Asn Asn Thr 25

Ala Thr Leu Cys Leu 30

His His Ala Val Pro

Asn Gly Thr Leu Val 40

Lys Thr Ile Thr Asp 45

Gln Ile Glu Val Thr 50

Asn Ala Thr Glu Leu 55

Val Gln Asn Phe Ser

Gly Lys Ile Cys Asn 65

Asn Pro His Arg Ile 70

Leu Asp Gly Ala Asn 75

Thr Leu Ile Asp Ser

Leu Leu Gly Asp Pro

His Cys Asp Gly Phe

Asn Glu Lys Trp Asp

100

Leu Phe Ile Glu Arg

105

Ser Lys Ala Phe Ser

110

Cys Tyr Pro Tyr Asp

115

Val Pro Glu Tyr Thr

120

Ser Leu Arg Ser Leu

125

Ala Ser Ser Gly Thr

130

Leu Glu Phe Thr Asn

135

Glu Asn Phe Asn Trp 140

Gly Val Thr Gln Asn 145

Gly Gly Ser Ser Ala 150

Cys Lys Arg Gly Pro 155

Asn Ser Phe Phe Ser

165

Lys Leu Asn Trp Leu

170

Tyr Lys Ser Gly Asn

175

Tyr Pro Met Leu Asn

180

Val Thr Met Pro Asn

185

Ser Asp Asp Phe Asp

190

Leu Tyr Ile Trp Gly

195

Val His His Pro Ser

200

Thr Asp Arg Glu Gln 205

Asn Leu Tyr Val Gln

210

Ala Ser Gly Lys Ile 215

Ile Val Ser Thr Lys

220

Ser Gln Gln Thr Ile

Ile Pro Asn Ile Gly

Ser Arg Pro Trp Val

Gly

Gly

Asp

Met

Cys 80

Gln

Asn

Ile

Thr

Asn 160

Thr

Lys

Ile

Arg

Arg

- 111 046085

225

230

235

Gly Leu Ser Ser Arg

245

Ile Ser Ile Tyr Trp

250

Thr Ile Val Lys Pro

255

Asp Ile Leu Ile Ile

260

Asn Ser Asn Gly Asn

265

Leu Ile Ala Pro Arg

270

Tyr Phe Lys Met Gln

275

Thr Gly Lys Ser Ser 280

Val Met Arg Ser Asp

285

Pro Ile Asp Thr Cys

290

Asn Ser Glu Cys Ile 295

Thr Pro Asn Gly Ser 300

Pro Asn Asp Lys Pro 305

Phe Gln Asn Val Asn 310

Lys Ile Thr Tyr Gly 315

Cys Pro Lys Tyr Ile

325

Lys Gln Asn Thr Leu

330

Lys Leu Ala Thr Gly

335

Arg Asn Ile Pro Glu

340

Arg Gln Thr Arg Gly

345

Ile Phe Gly Ala Ile

350

Gly Phe Ile Glu Asn

355

Gly Trp Glu Gly Met 360

Val Asn Gly Trp Tyr

365

Phe Arg His Gln Asn

370

Ser Glu Gly Ile Gly

375

Gln Ala Ala Asp Leu 380

Ser Thr Gln Ala Ala 385

Ile Asn Gln Ile Asn 390

Gly Lys Leu Asn Arg 395

Ile Glu Lys Thr Asn

405

Glu Lys Phe His Gln

410

Ile Glu Lys Glu Phe

415

Glu Val Glu Gly Arg

420

Ile Gln Asp Leu Glu

425

Arg Tyr Val Glu Asp

430

Lys Ile Asp Leu Trp

435

Ser Tyr Asn Ala Glu

440

Leu Leu Val Ala Leu

445

Asn Gln His Thr Ile

450

Asp Leu Thr Asp Ser 455

Glu Met Asn Lys Leu

460

Glu Lys Thr Arg Lys 465

Gln Leu Arg Glu Asn 470

Ala Glu Asp Met Gly 475

240

Gly

Gly

Ala

Ile

Ala 320

Met

Ala

Gly

Lys

Val 400

Ser

Thr

Glu

Phe

Asn 480

- 112 046085

Gly Cys Phe Lys Ile Tyr His Lys Cys 485

Ile Arg Asn Gly Thr Tyr Asp His Asn

500 505

Asn Asn Arg Phe Gln Ile Lys Ser Val

515 520

Asp Trp Ile Leu Trp Ile Ser Phe Ala

530 535

Ala Val Trp Leu Gly Phe Ile Met Trp

545 550

Arg Cys Asn Ile Cys Ile

565 <210>46 <211>566 <212> PRT <213> swine flu virus <400>46

Met Glu Ala Lys Leu Phe Val Leu Phe 15

Ala Asp Thr Ile Cys Val Gly Tyr His

25

Val Asp Thr Ile Leu Glu Lys Asn Val

40

Leu Leu Glu Asn Ser His Asn Gly Lys

55

Ala Pro Leu Gln Leu Gly Asn Cys Asn 65 70

Asn Pro Glu Cys Asp Leu Leu Leu Thr 85

Ile Glu Thr Ser Asn Ser Lys Asn Gly

100 105

Ala Asp Tyr Glu Glu Leu Arg Glu Gln

115 120

Asn Ser Cys Met Glu Ser

495

Tyr Arg Asp Glu Ala Val

510

Leu Lys Ser Gly Tyr Lys

525

Ser Cys Phe Leu Leu Cys

540

Cys Gln Lys Gly Asn Ile

555 560

Ala Phe Thr Ala Leu Lys 15

Asn Asn Ser Thr Asp Thr 30

Val Thr His Ser Val Asn 45

Cys Ser Leu Asn Gly Lys 60

Ala Gly Trp Ile Leu Gly 75 80

Asn Ser Trp Ser Tyr Ile

Cys Tyr Pro Gly Glu Phe

110

Ser Thr Val Ser Ser Phe

125

- 113 046085

Glu

Thr 145

Phe

Lys

Ile

Tyr

Gln 225

Ala

Ile

Ala

Val

Ser 305

Pro

Asn

Phe

His

Arg Phe Glu Ile Phe 130

Thr Lys Gly Thr Thr

150

Tyr Arg Asn Leu Leu

165

Leu Ser Lys Ser Tyr 180

Trp Gly Val His 195

Gln Asn Asn His Thr 210

Arg Phe Thr Pro Glu

230

Gly Arg Met Asn Tyr

245

Thr Phe Glu Ala Thr

260

Leu Asn Lys Gly Ser

275

His Asn Cys Thr Thr 290

Asn Leu Pro Phe Gln

310

Lys Tyr Val Lys Ser

325

Ile Pro Ser Ile Gln

340

Ile Glu Gly Gly Trp

355

His Gln Asn Glu Gln 370

Pro Lys Ala Thr Ser 135

Val Ser Cys Ser His

155

Trp Ile Val Lys Lys

170

Thr Asn Asn Lys Gly 185

Pro Pro Thr Asp Ser

200

Tyr Val Ser Val Gly 215

Ile Val Ala Arg Pro

235

Tyr Trp Thr Leu Leu

250

Gly Asn Leu Ile Ala 265

Asn Ser Gly Ile Met 280

Lys Cys Gln Thr Pro 295

Asn Val His Pro Ile

315

Thr Gln Leu Arg Met

330

Ser Arg Gly Leu Phe

345

Thr Gly Met Ile Asp 360

Gly Ser Gly Tyr Ala 375

Trp Pro Asn His Asp 140

Ser Gly Ala Asn Ser

160

Gly Asn Ser Tyr Pro

175

Lys Glu Val Leu Val

190

Asp Gln Gln Thr Leu

205

Ser Ser Lys Tyr Tyr 220

Lys Val Arg Gly Gln

240

Asp Gln Gly Asp Thr

255

Pro Trp His Ala Phe 270

Met Ser Asp Ala His

285

His Gly Ala Leu Lys 300

Thr Ile Gly Glu Cys

320

Ala Thr Gly Leu Arg

335

Gly Ala Ile Ala Gly

350

Gly Trp Tyr Gly Tyr

365

Ala Asp Gln Lys Ser 380

- 114 046085

Thr Gln Ile Ala Ile Asp Gly Ile

385 390

Ser Asn Lys Val Asn

395

Ser Val Ile

400

Glu Lys Met Asn Ile Gln Phe Thr Ser Val Gly Lys Glu

405 410

Phe Asn Asn

415

Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp

420 425

Asp Gly Phe 430

Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Ile Leu

435 440 445

Leu Glu Asn

Glu Arg Thr Leu Asp Phe His Asp Phe Asn Val Lys Asn

450 455 460

Leu Tyr Glu

Lys Val Lys Ser Gln Leu Arg Asn Asn Ala Lys Glu Ile

465 470 475

Gly Asn Gly

480

Cys Phe Glu Phe

Tyr His Lys Cys Asp Asn Glu Cys Met 485 490

Glu Ser Val

495

Lys Asn Gly Thr Tyr Asn Tyr Pro Arg Tyr Ser Glu Glu

500 505

Ser Lys Leu 510

Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Val

515 520 525

Gly Val His

Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu

530 535 540

Val Leu Leu

Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn

545 550 555

Gly Ser Leu

560

Gln Cys Arg Ile Cys Ile

565 <210> 47 <211> 564 <212> PRT <213> swine flu virus <220>

<221> other_character <222> (531) .. (531) <223> Xaa can be any naturally occurring amino acid <400> 47

Met Lys Ala Lys Leu Leu Ile Leu Trp Cys Ala Leu Ser

Ala Thr Asp

- 115 046085

Ala

Val

Leu

Ala 65

Asn

Ala

Ser

Glu

Ile 145

Arg

Ser

Gly

Lys

Phe 225

Arg

Asp Thr Ile Cys Ile 20

Asp Thr Val Leu Glu 35

Leu Glu Asp Asn His 50

Pro Leu Gln Leu Gly

Pro Glu Cys Glu Ser 85

Glu Thr Pro Asn Ala

100

Asp Tyr Glu Glu Leu 115

Arg Phe Glu Ile Phe 130

Gly Ala Thr Ala Ser

150

Asn Leu Leu Trp Leu

165

Lys Ser Tyr Val Asn

180

Val His His Pro Ser

195

Glu Thr Ala Tyr Val 210

Thr Pro Glu Ile Ala

230

Ile Asn Tyr Tyr Trp

245

Gly Tyr His Ala Asn 25

Lys Asn Val Thr Val 40

Asn Gly Lys Leu Cys 55

Lys Cys Ser Ile Ala 75

Leu Phe Ser Lys Lys

Glu Asn Gly Ile Cys 105

Arg Glu Gln Leu Ser

120

Pro Lys Glu Ser Ser 135

Cys Ser Lys Gln Gly

155

Thr Glu Lys Asn Gly

170

Asp Lys Glu Arg Glu 185

Asn Ile Glu Asp Gln

200

Ser Val Met Ser Ser 215

Lys Arg Pro Lys Ile

235

Thr Leu Leu Glu Pro

250

Asn Ser Thr Asp Thr 30

Thr His Ser Val Asn

Lys Leu Lys Gly Val 60

Gly Trp Ile Leu Gly

Ser Trp Ser Tyr Ile 95

Tyr Pro Gly Tyr Phe 110

Ser Val Ser Ser Phe

125

Trp Pro Lys His Ser 140

Arg Ser Ser Phe Tyr

160

Ser Tyr Pro Asn Leu

175

Val Leu Val Leu Trp

190

Arg Ala Ile Tyr Arg 205

Leu Tyr Asn Arg Arg 220

Arg Asn Gln Glu Gly

240

Lys Asp Thr Ile Ile

255

- 116 046085

Phe

Ser

Glu

Leu 305

Tyr

Pro

Glu

Gln

Asn 385

Met

Lys

Ile

Thr

Lys 465

Glu

Gly

Glu Ala Asn Gly Asn 260

Arg Gly Phe Glu Ser

275

Cys Asp Ala Lys Cys 290

Pro Phe Gln Asn Val

310

Val Lys Ser Thr Lys

325

Ser Ile Gln Thr Arg 340

Gly Gly Trp Thr Gly

355

Asn Glu Gln Gly Ser 370

Ala Ile Asn Gly Ile

390

Asn Thr Gln Phe Thr

405

Arg Met Glu Asn Leu

420

Trp Thr Tyr Asn Ala

435

Leu Asp Phe His Asp 450

Gly Gln Leu Lys Asn

470

Phe Tyr His Lys Cys

485

Thr Tyr Asp Tyr Pro 500

Leu Ile Ala Pro Trp 265

Gly Ile Ile Val Ser

280

Gln Thr Pro Gln Gly 295

His Pro Val Thr Ile

315

Leu Lys Met Ala Thr

330

Gly Leu Phe Gly Ala 345

Met Ile Asp Gly Trp

360

Gly Tyr Ala Ala Asp 375

Thr Asn Lys Val Asn

395

Ala Val Gly Lys Glu

410

Asn Lys Lys Val Asp 425

Glu Leu Leu Val Leu

440

Ser Asn Val Lys Ser 455

Asn Ala Lys Glu Ile

475

Asn Asn Glu Cys Met

490

Arg Tyr Ser Glu Glu

505

Tyr Ala Phe Ala Leu 270

Asn Ala Ser Met Asp

285

Ala Ile Asn Ser Ser 300

Gly Glu Cys Pro Lys

320

Gly Leu Arg Asn Ile

335

Ile Ala Gly Phe Ile 350

Tyr Gly Tyr His His

365

Gln Lys Ser Thr Gln 380

Ser Val Ile Asp Lys

400

Phe Asn Lys Leu Glu

415

Asp Gly Phe Leu Asp 430

Leu Glu Asn Glu Arg

445

Leu Tyr Glu Lys Val 460

Gly Asn Gly Cys Phe

480

Asp Ser Val Lys Asn

495

Ser Lys Leu Asn Arg 510

- 117 046085

Glu Lys Ile Asp Gly Val Glu Leu Lys Ser Met Gly Val Tyr Gln Ile

515 520525

Leu Ala Xaa Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser

530 535540

Leu Gly Ala Thr Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys

545 550 555560

Arg Ile Cys Ile

Claims (13)

1. An equine alphaherpesvirus (EHV) RacH vector comprising an expression cassette at UL43, wherein the expression cassette comprises: (i) at least one exogenous antigen coding sequence operably linked to a promoter sequence, and (ii) at least one upstream UL43 flanking region selected from the group consisting of: a sequence that is at least 90% homologous to and /or identical to SEQ ID NO: 19, a sequence at least 90% homologous and/or identical to SEQ ID NO: 26, and (iii) at least one upstream UL44 flanking region selected from the group consisting of : a sequence at least 90% homologous and/or identical to SEQ ID NO: 20, and a sequence at least 90% homologous and/or identical to SEQ ID NO: 27. 2. The equine alphaherpesvirus (EHV) RacH vector according to claim 1, comprising at least one additional antigen-coding sequence inserted into UL56 and/or US4. 3. The equine alphaherpesvirus (EHV) RacH vector of claim 1, wherein the insertion in UL43 results in a partial deletion, truncation or substitution in UL43, with UL44 remaining functional. 4. The EHV vector of claim 1, wherein the insertion in UL43 results in a deletion of approximately the 870 bp portion within UL43 for RacH (SEQ ID NO: 21) or at least 90% of the deletion of SEQ ID NO: 21. 5. The EHV vector according to claim 1, wherein the EHV vector includes at least one flanking region selected from the group consisting of a sequence at least 90% identical and/or homologous to SEQ ID NO: 19, SEQ ID NO: 20 , SEQ ID NO: 26 and SEQ ID NO: 27. 6. The EHV vector of claim 1, wherein said antigen-coding sequence relates to an infectious animal pathogen selected from the group consisting of pig, poultry, cattle, cat, dog or horse. 7. The EHV vector of claim 1, wherein the antigen coding sequence is operably linked to a promoter sequence selected from the group consisting of SV40 large T, HCMV and MCMV immediate early gene 1, human elongation factor alpha promoter, baculovirus polyhedrin promoter, 4pgG600 (SEQ ID NO: 1), p430 (SEQ ID NO: 3), nucleotide sequence complementary to 4pgG600 (SEQ ID NO: 1), 4pMCP600 (SEQ ID NO: 2), p455 (SEQ ID NO: 4), nucleotide sequence complementary 4pMCP600 (SEQ ID NO: 2) or p422 (SEQ ID NO: 5). 8. The EHV vector according to claim 1, where the antigen-coding sequence is obtained from a pathogen selected from the group consisting of Schmallenberg virus, influenza virus type A, porcine respiratory and reproductive syndrome virus, porcine circovirus, classical swine fever virus, African swine fever virus, hepatitis E virus, bovine diarrhea virus, rabies virus, feline morbillivirus, tetanus bacillus, mycobacterium tuberculosis, causative agent of actinobacillus pleuropneumonia. 9. The EHV vector of claim 1, wherein the antigen coding sequence is a hemagglutinin coding sequence, or where the antigen coding sequence is a hemagglutinin influenza antigen coding sequence derived from swine influenza virus type A. 10. The EHV vector according to claim 9, wherein the antigen coding sequence is a hemagglutinin coding sequence and a hemagglutinin anti-coding sequence - 118 046085 influenza gene, includes a nucleic acid sequence encoding an amino acid sequence at least 90% identical to the amino acid sequence presented in SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47 . 11. An immunogenic composition comprising an EHV-1 vector according to any one of claims 1 to 10. 12. A method of immunizing a pig animal, including administering to such an animal an immunogenic composition according to claim 11, where the antigen-coding sequence encodes at least one influenza A antigen. 13. A method of reducing or preventing clinical symptoms caused by a pathogen in a swine animal, which includes administering to the animal a therapeutically effective amount of an immunogenic composition according to claim 11, wherein the antigen coding sequence encodes at least one influenza A antigen.