EHV insertion site UL43

Abstract

The present invention relates to the field of (vector) vaccines, and especially to the novel EHV insertion site UL43. The present invention further concerns related expression cassettes and vectors, which are suitable to express genes of interest, especially antigen encoding sequences. The viral vectors of the present invention are useful for producing an immunogenic composition or vaccine.

Claims

1. An Equid Alphaherpesvirus (EHV) RacH vector comprising an expression cassette into UL43, wherein the expression cassette comprises (i) at least one exogenous antigen encoding sequence operably linked to a promoter sequence, and (ii) at least one upstream UL43 flanking region selected from the group consisting of: sequences having at least 90% sequence identity and/or homology to SEQ ID NO:19, sequences having at least 90% sequence identity to SEQ ID NO:26, and (iii) at least one upstream UL44 flanking region selected from the group consisting of: sequences having at least 90% sequence identity and/or homology to SEQ ID NO:20, sequences having at least 90% sequence identity to SEQ ID NO:27.

2. The Equid Alphaherpesvirus (EHV) RacH vector of claim 1 comprising at least one further antigen encoding sequence, inserted into UL56 and/or US4.

3. The EHV RacH vector of claim 1, whereby the insertion into UL43 results in a deletion, truncation, or substitution in UL43, whereby UL44 remains functional.

4. The EHV RacH vector of claim 1, whereby the insertion into UL43 results in the deletion of an approximately 870 bp portion within UL43 for RacH (SEQ ID NO:21) or at least 90% of SEQ ID NO:21 is deleted.

5. The EHV RacH vector of claim 1, whereby the EHV vector comprises at least one flanking region selected from the group consisting of: sequences having at least 90% sequence identity and/or homology to SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:26, and SEQ ID NO:27.

6. The EHV RacH vector of claim 1, wherein said antigen encoding sequence relates to a pathogen infecting an animal selected from the group consisting of swine, poultry, cattle, cats, dogs, and horses.

7. The EHV RacH vector of claim 1, wherein the antigen encoding 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), the complementary nucleotide sequence of 4pgG600 (SEQ ID No. 1), 4pMCP600 (SEQ ID No. 2), p455 (SEQ ID NO:4), the complementary nucleotide sequence of 4pMCP600 (SEQ ID No. 2) or p422 (SEQ ID NO:5.

8. The EHV RacH vector of claim 1, wherein the antigen encoding sequence is from a pathogen selected from the group consisting of: 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 Viral Diarrhea Virus, Rabies Virus, Feline Morbillivirus, Clostridium tetani, Mycobacterium tuberculosis, Actinobacillus Pleuropneumoniae.

9. The EHV RacH vector of claim 1, wherein the antigen encoding sequence is a hemagglutinin encoding sequence or whereby the antigen encoding sequence is a hemagglutinin influenza antigen encoding sequence from a Swine influenza A virus.

10. The EHV RacH vector of claim 9, wherein the antigen encoding sequence is a hemagglutinin encoding sequence and the hemagglutinin influenza antigen encoding sequence comprises a nucleic acid sequence encoding an amino acid sequence with at least, at least 90% 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.

11. An immunogenic composition comprising the EHV RacH vector of claim 1.

12. A method for immunizing a porcine animal comprising administering to such animal an immunogenic composition of claim 11, wherein the antigen encoding sequence encodes for at least one influenza antigen.

13. A method for reducing or preventing clinical signs caused by a pathogen in a porcine, the method comprising administering to the animal a therapeutically effective amount of an immunogenic composition according to claim 11, wherein the antigen encoding sequence encodes for at least one infulenza antigen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

(2) FIG. 1. Schematic illustration comparing the UL56 (orf1/3) regions of wild-type (wt) EHV-1 strain ab4 and attenuated vaccine strain EHV-1 RacH. orf1, orf2, orf3=first three open reading frames in the EHV-1 genome, orf1 has a homolog in other alphaherpesviruses designated UL56

(3) Flank A, Flank B=recombination regions for insertion of transgene expression cassette into the orf1/3 (UL56) site (prior art)

(4) FIG. 2. Schematic drawing of the US4 (orf70) insertion site

(5) UL=long unique segment

(6) US=short unique segment

(7) IR=inner inverted repeat

(8) TR=terminal inverted repeat

(9) gG=glycoprotein G

(10) pA=polyadenylation sequence at the termination of a coding sequence

(11) gpII=glycoprotein II

(12) orf=open reading frame orf69, orf70, orf71=US3, US4, US5 (open reading frames relevant for the orf70/US4 insertion site)

(13) Δorf1/3=orf1/3 (UL56) insertion site (prior art)

(14) bp=base pairs

(15) FIG. 3. Plasmid map of transfer plasmid pU-p455-H3-71K71 H3=open reading frame encoding for Influenza A virus hemagglutinin H3

(16) 71 pA=new polyA sequence as described in invention disclosure EM P2016-022

(17) I-SceI=cleavage site for the restriction endonuclease I-SceI

(18) promoter aph=prokaryotic Kanamycin resistance gene promoter

(19) Kana=Kanamycine resistance gene

(20) 3′ end ORF70=recombination region downstream of insertion site

(21) ORI=origin of replication of the plasmid

(22) AP.sub.r=Ampicillin resistence gene of the plasmid

(23) upstream orf70=recombination region upstream of insertion site

(24) p455=new promoter p455

(25) bp=base pairs

(26) FIG. 4. Plasmid map of transfer vector pU1-3-p430-H1av-BGHKBGH H1av=open reading frame encoding for Influenza A virus hemagglutinin H1av

(27) BGHpA=polyA sequence of the bovine growth hormone gene

(28) I-SceI=cleavage site for the restriction endonuclease I-SceI

(29) promoter aph=prokaryotic Kanamycin resistance gene promoter

(30) Kana=Kanamycine resistance gene

(31) Flank A=recombination region upstream of insertion site

(32) ORI=origin of replication of the plasmid

(33) AP.sub.r=Ampicillin resistence gene of the plasmid

(34) Flank B=recombination region downstream of insertion site

(35) p430=new promoter p430

(36) bp=base pairs

(37) FIG. 5. Schematic illustration of the genome of rEHV-1 RacH-SE-70-p455-H3 with the US4 (orf70) insertion region enlarged orf69/US3: open reading frame number 69(US3) upstream of the insertion site in orf70 (US4)

(38) p455: new promoter described herein

(39) H3: transgene Influenza Virus hemagglutinin

(40) 71 pA: new polyadenylation sequence

(41) Δorf70 (US4): remainder of orf70 (US4) containing the promoter for orf71 (US5), which encodes the structural viral glycoprotein II (gpII)

(42) bp=base pairs

(43) FIG. 6. Schematic illustration of the genome of rEHV-1 RacH-SE-1/3-p430-H1av with the UL56 (orf1/3) insertion region enlarged.

(44) p430: new promoter described herein

(45) H1av: transgene Influenza Virus hemagglutinin

(46) BGHpA: bovine growth hormone polyadenylation sequence

(47) Δorf1/UL56: remainder of orf1 (UL56)

(48) Orf3: EHV-1 open reading frame orf3 (no homolog in other alphaherpesviridae)

(49) bp=base pairs

(50) FIG. 7. Schematic illustration of the genome of rEHV-1 RacH-SE-1/3-p430-H1av-70-p455-H3 (rEHV-1-RacH-SE B) with the two insertion regions enlarged.

(51) p430: new promoter described herein

(52) H1av: transgene Influenza Virus hemagglutinin

(53) BGHpA: bovine growth hormone polyadenylation sequence

(54) Δorf1/UL56: remainder of orf1 (UL56)

(55) Orf3: EHV-1 open reading frame orf3 (no homolog in other alphaherpesviridae)

(56) orf69/US3: open reading frame number 69(US3) upstream of the insertion site in orf70 (US4)

(57) p455: new promoter described herein

(58) H3: transgene Influenza Virus hemagglutinin

(59) 71 pA: new polyadenylation sequence

(60) Δorf70 (US4): remainder of orf70 (US4) containing the promoter for orf71 (US5), which encodes the structural viral glycoprotein II (gpII)

(61) bp=base pairs

(62) FIG. 8. Plasmid map of transfer plasmid pU1/3-p430-H1hu-BGHKBGH p430=new promoter p430

(63) H1hu=open reading frame encoding for Influenza A virus hemagglutinin H1hu

(64) BGHpA=polyA sequence of the bovine growth hormone gene

(65) I-SceI=cleavage site for the restriction endonuclease I-SceI

(66) promoter aph=prokaryotic Kanamycin resistance gene promoter

(67) Kana=Kanamycine resistance gene

(68) Flank A=recombination region upstream of insertion site

(69) ORI=origin of replication of the plasmid

(70) Flank B=recombination region downstream of insertion site

(71) I-Ceu=homing endonuclease for release of fragment for RED recombination

(72) bp=base pairs

(73) FIG. 9. Plasmid map of transfer plasmid pU70-p455-H1pdm-71K71 upstream orf 70=recombination sequence upstream of insertion site

(74) p455=new promoter described herein

(75) H1pdm=transgene Influenza Virus hemagglutinin H1pdm

(76) 71 pA=new polyadenylation sequence

(77) 3′ end orf70=recombination sequence downstream of insertion site

(78) promoter aph=prokaryotic Kanamycin resistance gene promoter

(79) Kana=Kanamycine resistance gene

(80) bp=base pairs

(81) ScaI, EcoRI, SalI, NotI, KpnI, BamHI, XbaI=restriction endonuclease cleavage sites

(82) FIG. 10. Schematic illustration of the genome of rEHV-1 RacH-SE-70-p455-H1pdm with the US4 (orf70) insertion region enlarged.

(83) orf69/US3=open reading frame number 69 (US3) upstream of the insertion site in orf70 (US4)

(84) p455=new promoter described herein

(85) H1pdm=transgene Influenza Virus hemagglutinin H1pdm

(86) 71 pA=new polyadenylation sequence

(87) Δorf70 (US4): remainder of orf70 (US4) containing the promoter for orf71 (US5), which encodes the structural viral glycoprotein II (gpII)

(88) bp=base pairs

(89) FIG. 11. Schematic illustration of the genome of rEHV-1 RacH-SE-1/3-p430-H1hu with the UL56 (orf1/3) insertion region enlarged.

(90) p430=new promoter described herein

(91) H1hu=transgene Influenza Virus hemagglutinin H1hu

(92) BGHpA=bovine growth hormone polyadenylation sequence

(93) Δorf1/UL56=remainder of orf1 (UL56)

(94) Orf3=EHV-1 open reading frame orf3 (no homolog in other alphaherpesviridae)

(95) bp=base pairs

(96) FIG. 12. Schematic illustration of the genome of rEHV-1 RacH-SE-1/3-p430-H1hu-70-p455-H1pdm (virus D) with the insertion regions enlarged.

(97) p430=new promoter described herein

(98) H1hu=transgene Influenza Virus hemagglutinin H1hu

(99) BGHpA=bovine growth hormone polyadenylation sequence

(100) Δorf1/UL56=remainder of orf1 (UL56)

(101) Orf3=EHV-1 open reading frame orf3 (no homolog in other alphaherpesviridae)

(102) orf69/US3=open reading frame number 69(US3) upstream of the insertion site in orf70 (US4)

(103) p455=new promoter described herein

(104) H1pdm=transgene Influenza Virus hemagglutinin H1pdm

(105) 71 pA=new polyadenylation sequence

(106) Δorf70 (US4)=remainder of orf70 (US4) containing the promoter for orf71 (US5), which encodes

(107) the structural viral glycoprotein II (gpII)

(108) bp=base pairs

(109) FIG. 13. Schematic illustration of the construction of the new transgene insertion site UL43

(110) UL44, UL43, UL42 open reading frames in the insertion region

(111) 18 pA: new polyadenylation site

(112) 422 promoter: new p422 promoter

(113) bp: basepairs

(114) FIG. 14. Plasmid map of transfer plasmid pUUL43-422-mC-18K18

(115) UpUL43=viral genomic DNA sequence flanking the insertion site upstream

(116) UpUL44=viral genomic DNA sequence flanking the insertion site downstream

(117) 422promoter=promoter driving expression of transgene

(118) mC=transgene (autofluorescent protein mCherry)

(119) 18 pA=new polyadenylation sequence

(120) I-Sce1=cleavage site for I-Sce1

(121) promoter aph=prokaryotic promoter driving expression of Kanamycin-resistence gene

(122) Kana=Kanamycine resistance orf

(123) P(BLA)=prokaryotic promoter driving expression of Ampicillin-resistence gene

(124) AP(R)=Ampicillin-resistance gene

(125) ORI=plasmid origin of replication

(126) P(LAC)=prokaryotic promoter of lacZ encoding Betagalactosidase

(127) I-Ceu=recognition site of the homing endocuclease I-Ceu

(128) FIG. 15. Schematic illustration of the genome of rEHV-1 RacH-SE-UL43-422-mC with the UL43 insertion region enlarged

(129) UL=Unique long segment of the EHV genome

(130) US=Unique short segment of the EHV genome

(131) IRS and TRS=Inner and terminal repeat regions framing the unique short segment

(132) UL44, UL43, UL42=open reading frames in the insertion region

(133) ΔUL43=remainder of UL43

(134) 18 pA=new polyadenylation site

(135) p422=new p422 promoter

(136) bp=basepairs

(137) FIG. 16. Plasmid map of transfer plasmid pUUL43-422-H1pdm-18K18

(138) UpUL43=viral genomic DNA sequence flanking the insertion site upstream

(139) UpUL44=viral genomic DNA sequence flanking the insertion site downstream

(140) 422promoter=promoter driving expression of transgene

(141) H1pdm=transgene (Influenza A hemagglutinin H1pdm)

(142) 18 pA=new polyadenylation sequence

(143) I-Sce1=cleavage site for I-Sce1

(144) promoter aph=prokaryotic promoter driving expression of Kanamycin-resistence gene

(145) Kana=Kanamycine resistance orf

(146) P(BLA)=prokaryotic promoter driving expression of Ampicillin-resistence gene

(147) AP(R)=Ampicillin-resistance gene

(148) ORI=plasmid origin of replication

(149) P(LAC)=prokaryotic promoter of lacZ encoding Betagalactosidase

(150) I-Ceu=recognition site of the homing endocuclease I-Ceu

(151) FIG. 17. Schematic illustration of the genome of rEHV-1 RacH-SE-UL43-422-H1pdm with the UL43 insertion region enlarged

(152) UL=Unique long segment of the EHV genome

(153) US=Unique short segment of the EHV genome

(154) IRS and TRS=Inner and terminal repeat regions framing the unique short segment

(155) UL44, UL43, UL42=open reading frames in the insertion region

(156) ΔUL43=remainder of UL43

(157) 18 pA=new polyadenylation site

(158) H1pdm=transgene (Influenza A hemagglutinin H1pdm)

(159) p422=new p422 promoter

(160) bp=basepairs

(161) FIG. 18. Schematic illustration of the genome of rEHV-1 RacH-SE-1/3-p430-H1av-UL43-422-H1pdm-70-p455-H3 with insertion regions enlarged

(162) Δorf1/UL56: remainder of UL56 at the boundary of the expression cassette

(163) p430: new promoter p430

(164) BGHpA: bovine growth hormone polyadenylation site

(165) H1av, H3, H1pdm: transgenes (Influenza A hemagglutinins)

(166) Δorf70/US4: remainder of US4 at the boundary of the expression cassette

(167) orf69 (US3) and orf71 (US5) open reading frames in the US4 insertion region

(168) 71 pA: new polyadenylation sequence

(169) UL44, UL43, UL42 open reading frames in the UL43 insertion region

(170) 18 pA: new polyadenylation site

(171) p422: new p422 promoter

(172) bp: basepairs

(173) FIG. 19. Western blot

(174) Quadruplicate blots incubated with four different antibodies

(175) a: Blot incubated with a proprietary monoclonal antibody against Influenza HA H1av

(176) b: Blot incubated with a commercial rabbit antiserum specific for Influenza HA H3

(177) c: Blot incubated with a proprietary monoclonal antibody against Influenza HA H1pdm

(178) d: Blot incubated with a proprietary monoclonal antibody against EHV-1 gpII

(179) TABLE-US-00004 M = molecular weight marker (kDa = kilodalton, 250, 150, 100, 75, 50, 37, 25, 20) Virus name Abbreviation Used insertion sites Expressed transgenes rEHV-1 RacH-SE-70-p455-H3 US4-H3 US4 H3 rEHV-1 RacH-SE-1/3-p430- UL56-H1av UL56 H1av H1av rEHV-1 RacH-SE-70-p455- US4-H1pdm US4 H1pdm H1pdm rEHV-1 RacH-SE-1/3-p430- UL56-H1hu UL56 H1hu H1hu rEHV-1 RacH-SE-1/3-p430- B US4 and UL56 H3 and H1av H1av-70-455-H3 rEHV-1 RacH-SE-1/3-p430- D US4 and UL56 H1pdm and H1hu H1hu-70-455-H1pdm rEHV-1 RacH-SE-UL43- UL43-H1pdm UL43 H1pdm H1pdm rEHV-1 RacH-SE-1/3-p430- E US4 and UL56 and H3, H1av, and H1av-UL43-422-H1pdm70- UL43 H1pdm 455-H3 rEHV-1 RacH-SE SE none none

(180) FIG. 20: Results of Influenza A virus neutralization tests of mice sera. Virus neutralization tests were done in duplicate or triplicate depending on the available amounts of mouses sera. Neutralization titres were normalized to 100 TCID50 and shown as reciprocal neutralization capacities. *Error bars indicate standard deviation.

(181) FIG. 21: Plasmid map of transfer plasmid pUmC70

(182) 3′ end orf69=portion of orf69(US3) contained in the transfer vector

(183) up70=recombination sequence upstream of insertion site

(184) mCherry=transgene (autofluorescent protein mCherry)

(185) BGHpA=bovine growth hormone polyadenylation sequence

(186) up71=recombination sequence downstream of insertion site

(187) 3′ end orf70=remainder of orf70 (US4) downstream of insert

(188) bp=base pairs

(189) ScaI, EcoRI, SalI, NotI, KpnI, BamHI, XbaI=restriction endonuclease cleavage sites

(190) FIG. 22: Plasmid map of transfer vector pU70-p455-71K71

(191) Up70=viral genomic DNA sequence flanking the insertion site upstream

(192) Up71=viral genomic DNA sequence flanking the insertion site downstream

(193) 4pMCP455=promoter driving expression of transgene

(194) EHV-4orf71pApA=new polyadenylation sequence 71 pA

(195) I-Sce1=cleavage site for I-Sce1

(196) promoter aph=prokaryotic promoter driving expression of Kanamycin-resistence gene

(197) Kana=Kanamycine resistance orf

(198) P(BLA)=prokaryotic promoter driving expression of Ampicillin-resistence gene

(199) AP(R)=Ampicillin-resistance gene

(200) ORI=plasmid origin of replication

(201) P(LAC)=prokaryotic promoter of lacZ encoding Betagalactosidase

(202) I-Ceu=recognition site of the homing endocuclease I-Ceu

(203) KpnI, NotI, XbaI=restriction endonuclease cleavage sites

(204) bp=base pairs

(205) FIG. 23: Plasmid map of transfer vector pU1/3-p430-BGHKBGH

(206) Flank A=viral genomic DNA sequence flanking the insertion site upstream

(207) Flank B=viral genomic DNA sequence flanking the insertion site downstream

(208) 4pgG430=promoter driving expression of transgene

(209) BGHpA=polyadenylation sequence of the bovine growth hormone gene

(210) I-Sce1=cleavage site for I-Sce1

(211) promoter aph=prokaryotic promoter driving expression of Kanamycin-resistence gene

(212) Kana=Kanamycine resistance orf

(213) I-Ceu=recognition site of the homing endocuclease I-Ceu

(214) KpnI, NotI=restriction endonuclease cleavage sites

(215) bp=base pairs

(216) FIG. 24: Plasmid map of transfer plasmid pUUL43-422-H1pdm-18K18

(217) UpUL43=viral genomic DNA sequence flanking the insertion site upstream

(218) UpUL44=viral genomic DNA sequence flanking the insertion site downstream

(219) p422=promoter driving expression of transgene

(220) 18 pA=new polyadenylation sequence

(221) I-Sce1=cleavage site for I-Sce1

(222) promoter aph=prokaryotic promoter driving expression of Kanamycin-resistence gene

(223) Kana=Kanamycine resistance orf

(224) P(BLA)=prokaryotic promoter driving expression of Ampicillin-resistence gene

(225) AP(R)=Ampicillin-resistance gene

(226) ORI=plasmid origin of replication

(227) P(LAC)=prokaryotic promoter of lacZ encoding Betagalactosidase

(228) I-Ceu=recognition site of the homing endocuclease I-Ceu

(229) bp=base pairs

EXAMPLES

(230) The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Establishment of the New Insertion Site ORF70/US4

(231) In order to augment the capabilities of the EHV-1 vector the inventors sought to find a way to express two different transgenes from one vector backbone without coupling two transgenes by RNA-virus-derived functions under control of one promoter. The inventors hypothesized that the herpesvirus genome would tolerate the use of two independent transgene insertion sites in parallel. To determine whether the EHV-1 ORF70/US4 was a suitable transgene insertion site, 801 basepairs of the 5′ end of orf70/US4 (1236 bp) were replaced with an expression cassette coding for the autofluorescent mCherry protein (Shaner et al. 2004) by classical homologous recombination. A map of the plasmid pU-mC70-BGH is in FIG. 21 (SEQUENCE ID NO:37). The DNA fragment used for homologous recombination was excised from pU-mC70-BGH with XbaI. The gel-purified fragment was co-transfected with viral genomic DNA of EHV-1 RacH into RK13 cells. Efficient rescue of recombinant vector virus and efficient replication in cultured cells were shown by live fluorescence and virus titrations (not shown). Deletion of two thirds of orf70/US4 had the additional benefit that expression of glycoprotein G encoded by orf70/US4 was abolished. Glycoprotein G of EHV-1 was shown to be a non-structural, secreted chemokine binding protein counter-acting the host's immune response (Drummer et al., 1998; Bryant et al., 2003). Since a vector vaccine is intended to stimulate the vaccinee's immune response, removal of this particular immunosuppressive function of the viral vector might additionally improve performance of the viral vector platform EHV-1 RacH-SE.

Example 2

Use of the New ORF70/US4 Insertion Site with p455 Promoter in Recombinant EHV-1 Vector Vaccines and Construction of a Recombinant Virus

(232) The p455 promoter:

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

(234) By en-passant mutagenesis using the RED recombination system (Tischer et al. 2006) the expression cassette p455-H3-71 was inserted in orf70/US4 of pRacH-SE to generate pRacH-SE70-p455-H3.

(235) PK/WRL cells were transfected with pRacH-SE70-p455-H3, recombinant virus rEHV-1 RacH-SE70-p455-H3 (FIG. 5) was rescued and plaque-purified twice. Correct insertion of the expression cassette was verified by sequencing of a high-fidelity PCR product of the insertion region. Expression of the transgene in infected cells was analyzed by indirect immunofluorescence assay.

(236) Restoration of orf71 encoding EHV-1 gpII was confirmed by IFA (not shown) and Western blot (FIG. 19) using a monoclonal antibody Ai2G7 (owned by BI). Appearance of trimers of H3 on the plasma membrane of infected cells was assayed by a hemadsorption test using chicken erythrozytes (not shown). Peak titers determined as TCID.sub.50/ml in PK/WRL cells were in the same range as titers of the parental virus rEHV-1 RacH-SE which indicates that transgene expression had no detrimental effect on viral replication (not shown). This was confirmed by passaging of rEHV-1 RacH-SE70-p455-H3 in PK/WRL cells up to passage 20 (P20) after rescue. At P5, P10, P15, and P20 the virus was characterized by titration, sequencing, and Western blot, at P10 and P20 additionally by IFA, and HA expression and genetic stability of the HA encoding insert along with the promoter and polyA sequences were confirmed.

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

Example 3

Use of the New p430 Promoter in Recombinant EHV-1 Vector Vaccines and Construction of a Recombinant Virus

(238) The p430 promoter:

(239) The newly identified p430 promoter was used to drive expression of another Influenza hemagglutinin from an H1N1 virus ((A/swine/Gent/132/2005(H1N1), GenBank accession NO:AFR76623.1). Since the hemagglutinin gene in this virus isolate originated from an avian IAV it will be referred to as H1av. H1av was synthesized and subcloned in a transfer vector pU1/3-p430-BGHKBGH (SEQ ID NO:30) for the orf1/3/UL56 insertion region to generate pU1/3-p430-H1av-BGH_K_BGH (FIG. 4, SEQ ID NO:31). Expression of H1av was placed under control of the p430 promoter and the bovine growth hormone (BGH) polyA signal.

(240) By en-passant mutagenesis using the RED recombination system (Tischer et al. 2006) the expression cassette p430-H1av-BGH was inserted in orf1/3/UL56 of pRacH-SE to generate pRacH-SE1/3-p430-H1av.

(241) PK/WRL cells were transfected with pRacH-SE1/3-p430-H1av, recombinant virus rEHV-1 RacH-SE1/3-p430-H1av (FIG. 6) was rescued and plaque-purified twice. Correct insertion of the expression cassette was verified by sequencing of a high-fidelity PCR product of the insertion region. Expression of the transgene in infected cells was analyzed by indirect immunofluorescence assay (IFA) and Western blot using monoclonal and polyclonal antibodies (FIG. 19). Specific detection of a broad band migrating at 75 kDa by antibody PA-34929 is in concordance with the expected appearance of the recombinant HA glycoprotein as predicted from its sequence.

(242) Restoration of orf71/US5 encoding EHV-1 gpII was confirmed by IFA and Western blot using a monoclonal antibody Ai2G7 (owned by BI), (FIG. 19). Peak titers determined as TCID50/ml in PK/WRL cells were in the same range as titers of the parental virus RacH-SE which indicates that transgene expression had no detrimental effect on viral replication (not shown).

(243) In order to test whether the expressed recombinant hemagglutinins were 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 (parent) at an m.o.i. of 0.01, or left uninfected. 24 h p.i. live infected and uninfected cells were incubated with a suspension of chicken erythrocytes in PBS, washed with PBS and stained with the fluorescent Hoechst 33342 nuclear stain. Since erythrocytes of birds contain cell nuclei they can be stained with Hoechst33342 and appear as tiny blue specks by fluorescence microscopy, Compared with cells that were infected with rEHV-1 RacH-SE that does not express hemagglutinin, adsorption of chicken erythrocytes was significantly increased on cells infected with either rEHV-1 RacH-SE-1/3-p430-H1av or rEHV-1 RacH-SE-70-p455-H3 (not shown). From this it can be concluded that the hemagglutinins were translated, processed and transported to the plasma membrane of vector virus infected cells in a manner as if they were produced by authentic influenza virus infection.

(244) The clear phenotype of hemadsorption of infected cells supports the findings of the Western blots and immunofluorescence assays showing efficient expression of the transgenic proteins and suggesting formation of functional HA trimers on the cell surface of EHV-1 vector infected cells.

Example 4

Use of the New ORF70 Insertion Site and the ORF1/3(UL56) Insertion Site in Recombinant EHV-1 Vector Vaccines in Parallel

(245) To show that the two new promoters can be used in parallel a recombinant EHV-1 RacH was generated expressing two different hemagglutinins of two different Influenza A virus subtypes.

(246) Specificity and lack of cross-reactivity of the polyclonal commercial antibody to H3 (PA5-34930) and the proprietary monoclonal antibodies to H1av and H1pdm is obvious from the Western blots of infected cells as shown in FIG. 19. Identical samples were run in quadruplicate SDS-PAGE and transferred to nylon membranes before incubation with four different antibodies.

(247) The open reading frame encoding the hemagglutinin of Influenza A virus (A/swine/Gent/132/2005(H1N1)) was synthesized and cloned into the transfer vector pU1-3-p430-BGHKBGH (SEQ ID NO:30) resulting in pU1-3-p430-H1av-BGHKBGH (FIG. 4, SEQ ID NO:31). Starting with the recombinant BAC pRacH-SE-70-p455-H3, the expression cassette p430-H1av-BGH as assembled in pU1/3-p430-H1av-BGHKBGH (FIG. 4, SEQ ID NO:31) was inserted into the orf1/3/UL56 insertion site by two-step RED recombination to generate pRacH-SE-1/3-p430-Hiav-70-p455-H3. PK/WRL cells were transfected with pRacH-SE1/3-p430-Hav-70-p455-H3, and recombinant virus rEHV-1 RacH-SE1/3-p430-H1av-70-p455-H3 (FIG. 7) was rescued and plaque-purified twice.

(248) The short designation for this recombinant virus is rEHV-1 RacH-SE_B. Correct insertion of the expression cassette was verified by sequencing of high-fidelity PCR products of the insertion regions together with flanking sequences. Expression of the transgenes in infected cells was analyzed by indirect immunofluorescence assay (IFA, not shown) and Western blot using monoclonal and polyclonal antibodies (FIG. 19). Restoration of orf7l/US5 encoding EHV-1 gpII was confirmed by IFA (not shown) and Western blot using a monoclonal antibody Ai2G7 (owned by BI), (FIG. 19).

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

(250) The two new promoters p430 and p455 were shown to be functional in the context of rEHV1-RacH-SE replication in cell cultures. Activity levels during the viral replication cycle appear to be very similar as deduced from comparable intensities of signals in Western blots specific for the individual transgenes. These properties allow creation of recombinant vector vaccines based on EHV-1 RacH or other vector platforms expressing two different antigens in parallel with similar efficiency. If a vaccine target consists of two different pathogens application of the two new promoters in two insertion sites combined with two polyadenylation sequences can reduce cost of goods significantly and represents a clear advantage over a vector expressing only one antigenic component.

Example 5

Generation, In Vitro Characterization and In Vivo Testing of A Bivalent EHV-1 Vectored Influenza a Virus Vaccine

(251) As described below, in the described invention two of the four above-described Swine IAV hemagglutinin (HA) antigens derived from H3N2 and H1N1 avian Swine IAV sub-/serotypes are expressed by one recombinant EHV-1 vector virus. This new bivalent vaccine against swine IAV provides a DIVA feature, e.g. by detection of antibodies against Swine IAV proteins NP or NA in animals that were infected by Swine IAV field strains but not in animals only vaccinated with the vaccine described here since it only expresses the Swine IAV HA proteins.

(252) The new bivalent Swine IAV vaccine was characterized in vitro and tested in vivo for its ability to induce Influenza A virus neutralizing antibodies in mice.

(253) In order to test whether the expressed recombinant hemagglutinins were 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 (parent) at an m.o.i. of 0.01, or left uninfected. 24 h p.i. live infected and uninfected cells were incubated with a suspension of chicken erythrocytes in PBS, washed with PBS and stained with the fluorescent Hoechst 33342 nuclear stain. Since erythrocytes of birds contain cell nuclei they can be stained with Hoechst33342 and appear as tiny blue specks by fluorescence microscopy, compared with cells that were infected with rEHV-1 RacH-SE that does not express hemagglutinin, adsorption of chicken erythrocytes was significantly increased on cells infected with either rEHV-1 RacH-SE-1/3-p430-H1av or rEHV-1 RacH-SE-70-p455-H3 (not shown). From this it can be concluded that the hemagglutinins were translated, processed and transported to the plasma membrane of vector virus infected cells in a manner as if they were produced by authentic influenza virus replication. The phenotype of hemadsorption of infected cells supports the findings of the Western blots (FIG. 19) and immunofluorescence assays (not shown) showing efficient expression of the transgenic proteins and suggesting formation of functional HA trimers on the cell surface of EHV-1 vector infected cells.

(254) The enhanced EHV-1 vector with two insertion sites and two new promoters was shown to express two Influenza virus hemagglutinins in parallel. Subcellular localization as determined by IFA and mobility in SDS-PAGE as determined by Western blot (FIG. 19) corresponded to authentic uncleaved hemagglutinins expressed in Influenza A virus infected cells known from the literature.

(255) Genetic and phenotypic stabilities of the recombinant rEHV-1 were shown by passaging in cell culture, determining viral titres every 5 passages. Sequences of the insertion regions were confirmed every ten passages as well as transgene expression by Western blot (not shown). Expression fidelity was assessed by double IFA of plaques under methocel-overlay, counting plaques stained with anti-EHV-antibodies and transgene-specific antibodies (not shown).

Example 6

Induction of a Neutralizing Antibody Response Against Two Antigens in Mice Vaccinated with a Bivalent rEHV-1 Rach Vector Vaccine

(256) The rEHV-1 RacH SE B (rEHV-1 RacH-SE-1/3-p430-H1av-70-p455-H3 see FIG. 7) was used for immunization of Balb/c mice in order to demonstrate that the expressed transgenes are immunogenic in another species than swine and that neutralizing antibodies are induced against either one of the two antigens by intranasal application.

(257) In detail, three groups of five Balb/c mice per group, 3-5 weeks of age, were intranasally inoculated on study days 0 and 21 either with 40 μl of rEHV-1 RacH SE B (rEHV-1 RacH-SE-1/3-430-H1av-7-455-H3, group 1), or 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 recombinant EHV-1 dosages were 1×10{circumflex over ( )}TCID50/40 μl, respectively. Mice were bled on study days 0 (before 1.sup.st inoculation), 7, 14, 21 (before 2.sup.nd inoculation), 28, and 35. Serum was prepared from the blood samples and stored frozen at −80° C.

(258) Immunofluorescence Assay for Detection of Antibodies Against the Vector Virus

(259) AI-ST cells were infected at amultiplicity of infection (MOI) of 0.001 with rEHV-1 RacH-SE1212, a virus rescued from the empty vector BAC pRacH-SE1.2. 24 hours p.i. distinctive plaques were observed and cells were processed for indirect immunofluorescence assay (IFA). Sera of all three groups of the final bleeds (obtained 14 days after the second vaccination) diluted 1:50 in PBS were tested. As positive control serum from an EHV-1 vaccinated horse was used in a dilution of 1:500. Secondary antibodies were commercially available FITC-conjugated rabbit anti-mouse IgG for the mice sera and Cy5-conjugated goat-anti horse IgG for the horse serum and used at 1:200 dilution. Antibody binding was evaluated by fluorescence microscopy. All vaccinated mice had developed antibodies reactive in IFA with rEHV-1 RacH-SE-infected cells. Uninfected cells were not bound by any of the tested sera. Sera from the negative control group of mice did not show any specific binding neither to infected nor to uninfected cells. Data are summarized in the table below.

(260) TABLE-US-00005 TABLE 4 Fluorescence microscopy results of IFA for anti-EHV-1 antibodies Mouse ID in Uninfected Infected Treatment number experiment dilution cells cells Group 3 1 1 1:50 neg neg (Negative control) 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 1 6 1:50 neg pos (Empty vector) 2 7 1:50 neg pos 3 8 1:50 neg pos 4 9 1:50 neg pos 5 10 1:50 neg pos Group 1 1 11 1:50 neg pos (rEHV-1 RacH SE B) 2 12 1:50 neg pos 3 13 1:50 neg pos 4 14 1:50 neg pos 5 15 1:50 neg pos Control antibody Specific for Horse serum EHV-1 22 1:500 neg pos Secondary antibodies Specific for FITC-goat mouse 23 1:200 neg neg anti- Cy5 goat horse 24 1:200 neg neg anti-

(261) From this it can be concluded that inoculation of the rEHV-1 into the nostrils of the mice resulted in infection and viral replication, so that the mice immune systems were stimulated to produce anti-EHV-1 antibodies.

(262) Virus Neutralization Tests (VNT)

(263) In order to show induction of protective immunity against the expressed transgenes originating either from Influenza A virus (IAV) (A/swine/Italy/7680/2001(H3N2)) or (A/swine/Gent/132/2005(H1N1)) the mice sera were tested for neutralizing activity against the respective viruses (Allwinn et al. 2010; Trombetta et al. 2014). IAV used for neutralization tests were isolates from pigs in Germany from 2014, specifically A/swine/Germany/AR452/2014 (H3N2) and A/swine/Germany/AR1181/2014 (H1N1). As these are heterologous from the strains the vaccine targets were derived from, any neutralization of these viruses by the mouse sera will be indicative of broad and efficient induction of protective immunity by the rEHV-1 vaccination. As a negative control serum, a serum from a pig which had been shown to be negative for Influenza virus antibodies was used.

(264) Influenza a Virus Neutralization Tests:

(265) MDCK cells for virus neutralization as well as back-titration in 96-well plates were incubated for two days at 37° C./5% CO.sub.2 prior to use. The respective IAV stocks H3N2 and H1avN1 were thawed on ice and diluted in MEM containing Gentamycin and the double concentration of trypsin (MEM/Genta/2× trypsin).

(266) Sera tested were from the final bleeds of group 1 (rEHV-1 RacH SE B), group 2 (empty vector), a positive control (serum from a pig vaccinated with inactivated multivalent IAV vaccine, and a negative control.

(267) Sera were heat inactivated and in two and three independent tests, respectively, serially 1:2 diluted starting at 1:16 up to 1:4096. IAV was diluted to approximately 100 TCID50/neutralization reaction. Neutralization reactions were incubated for 2 hours at 37° C., 5% CO.sub.2. Back-titration of used virus was done in quadruplicate. Growth medium was removed and MDCK-cells were washed with medium containing Gentamycin and trypsin before adding the neutralization reactions or the virus dilutions of the back-titrations. VNT and titration plates were incubated at 37° C./5% CO.sub.2 for 1 h after addition of neutralization reaction or virus dilutions to the MDCK-cells, respectively. Thereafter inocula were removed and cells were overlaid with fresh medium containing Gentamycin and trypsin. Five days p.i. CPE was monitored and documented. Actually used virus titre in the test was calculated as TCID50/ml according to Reed and Münch and dilutions at which the tested sera prevented induction of Influenza virus-typical CPE were reported, see tables below.

(268) TABLE-US-00006 TABLE 5 Results Influenza H1avN1 VNT H1avN1 VNT#1 VNT#2 VNT#3 146 32 181 TCID50/ TCID50/ TCID50/ well well well Reciprocal Reciprocal Reciprocal Average SD neutralizing neutralizing neutralizing neutralizing (standard mouse dilution capacity dilution capacity dilution capacity capacity deviation) rEHV-1 32 4672 128 4096 32 5792 4853 862 RacH SE B-1 rEHV-1 16 2336 64 2048 neg 2192 204 RacH SE B-2 rEHV-1 32 4672 128 4096 16 2896 3888 906 RacH SE B-3 rEHV-1 128 18688 512 16384 64 11584 15552 3624 RacH SE B-4 rEHV-1 32 4672 256 8192 16 2896 5253 2695 RacH SE B-5 Empty n.d. n/a neg n/a neg n/a n/a n/a vector-1 Empty n.d. n/a neg n/a neg n/a n/a n/a vector-2 Empty n.d. n/a neg n/a neg n/a n/a n/a vector-3 Empty neg n/a neg n/a neg n/a n/a n/a vector-4 Empty n.d. n/a neg n/a neg n/a n/a n/a vector-5 Pos 32 n/a n.d n/a n.d n/a n/a n/a control pig serum

(269) TABLE-US-00007 TABLE 6 Results Influenza H3N2 VNT H3N2 VNT#1 VNT#2 VNT#3 16 24 15 TCID50/ TCID50/ TCID50/ well well well Reciprocal Reciprocal Reciprocal Average SD neutralizing neutralizing neutralizing neutralizing (standard mouse dilution capacity dilution capacity dilution capacity capacity deviation) rEHV-1 4096 65536 1024 24576 2048 30720 40277 22089 RacH SE B-1 rEHV-1 1024 16384 512 12288 128 1920 10197 7455 RacH SE B-2 rEHV-1 1024 16384 512 12288 256 3840 10837 6397 RacH SE B-3 rEHV-1 256 4096 256 6144 64 960 3733 2611 RacH SE B-4 rEHV-1 256 4096 128 3072 64 960 2709 1599 RacH SE B-5 Empty neg n/a neg n/a neg n/a n/a n/a vector-1 Empty neg n/a neg n/a neg n/a n/a n/a vector-2 Empty neg n/a neg n/a neg n/a n/a n/a vector-3

(270) In order to compare results of independent tests neutralizing capacity was calculated by multiplication of the reciprocal serum dilution and the respective titre that was neutralized by it. Averages of three tests were then divided by 100 to reflect neutralization of 100 TCID50 (Tables 4, 5, and 6). Data are summarized and shown graphically in FIG. 20.

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

(272) It can be concluded that the vector rEHV-1 RacH-SE can be used for parallel expression of two different transgenes to stimulate immune response after intranasal vaccination.

(273) Western Blot

(274) 1. Infection: Three wells each of confluent monolayers of AI-ST cells in 6-well plates were infected at an M.O.I. of approximately 1 with recombinant viruses 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 blot. Viruses used for infection are summarized in the table below (Table 2)

(275) TABLE-US-00008 TABLE 2 Viruses tested in Western blot Virus name Abbreviation Used insertion sites Expressed transgenes rEHV-1 RacH-SE-70-p455-H3 H3 US4 H3 rEHV-1 RacH-SE-1/3-p430- av UL56 H1av H1av rEHV-1 RacH-SE-70-p455- 4p US4 H1pdm H1pdm rEHV-1 RacH-SE-1/3-p430- hu UL56 H1hu H1hu rEHV-1 RacH-SE-1/3-p430- B US4 and UL56 H3 and H1av H1av-70-455-H3 rEHV-1 RacH-SE-1/3-p430- D US4 and UL56 H1pdm and H1hu-70-455-H1pdm H1hu rEHV-1 RacH-SE-UL43-H1pdm 43p UL43 H1pdm rEHV-1 RacH-SE-1/3-p430- E US4 and UL56 and H3, H1av, and H1av-UL43-422-H1pdm70- UL43 H1pdm 455-H3 rEHV-1 RacH-SE SE none none

(276) 2. Preparation of lysates: RIPA buffer supplemented with protease inhibitor cocktail (RIPA+PI) was prepared as follows: 0.7 ml 10×RIPA lysis buffer Millipore Cat #20-188 were added to 6.3 ml H.sub.2O, Fisher Scientific Cat #BP2470-1, and 1 tablet Complete™ Mini Protease inhibitor cocktail (Roche cat #11 836 153 001) was dissolved in 7 ml 1×RIPA buffer. Uninfected controls were scraped into the medium and suspensions from the three replicate wells were pooled in 15 ml centrifuge tubes and placed on ice. Infected cells were rinsed off in the medium and the suspensions from the three replicate wells were pooled in 15 ml centrifuge tubes and placed on ice. Cells were sedimented by centrifugation at 1000×g 4° C. for 5 min. Supernatants were carefully aspirated and the cell pellets were resuspended in RIPA+PI (Uninfected cells in 300 μl, infected cells in 150 μl). Suspensions were incubated on ice for 30 min and vortexed every 10 min. Suspensions were transferred to 1.5 ml microfuge tubes and undissolved material was sedimented by centrifugation at 15000 rpm, 4° C., for 10 min in a microcentrifuge. Clear supernatants were transferred to new 1.5 ml microfuge tubes and stored at −80° C. until use.

(277) 3. SDS-PAGE and transfer on nylon membranes: Materials: BioRad Criterion TGX Stain Free Precast Gels, 4-20%, 26 well Cat #_567-8095; Bio Rad Precision Plus Dual Colour Marker, Cat #161-0374; Bio Rad Precision Plus All Blue Marker, Cat #161-0373; Bio Rad Trans Blot Turbo transfer kit, Midi format Cat #170-4159; Bio Rad 4× Laemmli Sample Buffer (Cat no. 161-0747) (Bio Rad Laboratories GmbH, Heidemannstrasse 164, D-80939 Mnchen); TGS Running buffer (Sambrook et al.), Blocking Solution 1: 5% FBS in PBST (Sambrook et al.); PBST. Samples were prepared without addition of a reducing agent. Samples were thawed on ice and mixed with 1 volume of 4× Lämmli buffer, boiled for 6 min at 96° C., and kept at RT until loading of the gel. Gel was run for 30 min at 230 mA and then assembled for electrotransfer using the BioRad Trans Blot Turbo system. Transfer was set to 2.5 A 25 V 10 min. Membrane was rinsed in sterile distilled H.sub.2O and incubated with 25 mL Blocking Solution 5% FBS in PBST for 30 min at 4° C.

(278) Antibody Incubation and Detection

(279) Materials: Immun-Star WesternC Chemiluminecent Kit (Bio Rad Laboratories GmbH,

(280) Heidemannstrasse 164, D-80939 München) Cat #170-5070

(281) Primary Antibodies see figure legend 19 a to d.

(282) Secondary Antibody: Peroxidase conjugated Goat anti-mouse, (Jackson Immune Research #115-035-146) 1:5000.

(283) All incubations were done in sufficient volume under constant agitation. Antibodies were diluted in 5% FBS/TBST. Primary antibodies were incubated over night at 4° C. Antibody solution was removed and blots were washed three times with TBST for 5-10 min. Diluted secondary antibody was incubated with the blots for 1 h at RT, removed and blots were washed three times with TBST for 5-10 min. Blots were placed on a clear plastic sheet protector. Peroxide and Lumino/Enhancer solutions were mixed 1 ml+1 ml (2 ml total for each blot), pipetted on the blots and incubated for 3 to 5 min. Thereafter the membranes were placed in the ChemiDocXRS imaging system (Bio Rad Laboratories GmbH, Heidemannstrasse 164, D-80939 München) and signals were recorded using Image Lab software.

(284) Virus Titrations

(285) AI-ST cells were seeded in 96-well plates (Corning Incorporated—Life Sciences, One Becton Circle, Durham, N.C. 27712, USA; REF 353072) at 2×10.sup.4 cells/well in MEM supplemented with 10% FBS one day before infection. Virus stocks were quickly thawed and placed on ice. Ten serial 1:10 dilutions were prepared in MEM in 1.2 ml volume per dilution. 100 μl/well of the virus dilutions were added to the cells, 8 wells in one vertical row per dilution. Vertical rows 11 and 12 of each plate served as medium control by addition of 100 l/well MEM. Titrations were done in triplicate and cells were incubated for 5 days at 37° C./5% CO.sub.2. Cell cultures were inspected microscopically and wells where EHV-1 RacH typical CPE was observed were recorded. Titres were calculated as TCID50/ml according to the method by Reed and Muench (1938).

Example 7

Establishment of the New UL43 Insertion Site

(286) Using the EHV-vector platform as described in the previous examples only two antigens can be expressed in parallel in their authentic forms. A blend of two vector vaccines would increase cost of goods and might also result in biased expression of transgenes, if replication efficiency varies between the different recombinant viruses, which is not unlikely. Although there are ways to couple two antigens in one insertion site either by an internal ribosome entry site (IRES) or by a picornavirus 2a peptide (2a) these techniques are not sufficient for the task. If two transgenes are coupled by a 2a peptide, which triggers a ribosomal skip which results in the synthesis of a discrete downstream translation product (Donnelly et al., 2001) the 2a peptide will structurally alter the first one of the expressed proteins, which will have 19 amino acid residues from the 2a peptide added to its C-terminus. One amino acid residue, a proline, will be added to the N-terminus of the second protein (Ryan et al., 1994). Since this one additional amino acid will be cleaved off with the signal peptide of HA, it is very likely not of any consequence. Still, the 19 amino acid tail on the first HA might interfere with trimerization and prevent sufficient efficacy. To find a solution to overcome the described hurdles the inventors established a third transgene expression site in pRacH-SE.

(287) Use of the unified Alphaherpesvirus nomenclature

(288) With the availability of the first genomic sequences of the various alphaherpesviruses, the in-silico identified open reading frames (orfs) were numbered for each virus individually according to their positions in the respective genomes. Later it was found that the majority of the alphaherpesvirus genes were homologs present in the different species. In order to facilitate comparison of data it is now a common practice to assign genes and gene products the designation of their homologs in the genome of human herpesvirus-1. Accordingly, we have changed the old designations of the EHV orfs according to the new nomenclature as listed in table 1.

(289) TABLE-US-00009 TABLE 1 EHV orf Unified nomenclature Gene product orf1  UL56 pUL56 orf2  (none) orf2 protein orf3  (none) orf3 protein orf16 UL44 Glycoprotein C orf17 UL43 pUL43 orf18 UL42 DNA polymerase processivity factor orf70 US4  Glycoprotein G orf71 US5  Glycoprotein II (or glycoprotein J)

(290) For the construction of the insertion site though, care had to be taken not to destroy the putative promoter and poly A signals of the upstream and downstream genes UL42 encoding for a DNA polymerase processivity factor and UL44 encoding for glycoprotein C.

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

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

(293) The 18 pA polyadenylation sequence (SEQ ID NO:7) was introduced in the transfer vector for RED recombination upstream and downstream of a Kanamycin-resistence expression cassette (Kana) to fulfill a dual function: 1. During the second step of the en-passant-mutagenesis (2nd RED) it serves as the homologous region for deletion of Kana, 2. It functions as polyadenylation signal for the transgene. For a map of the transfer vector pUUL43-422-mC-18K18 see FIG. 14.

(294) A fragment of pUUL43-422-mC-18K18 (FIG. 14; SEQ ID NO:35) encompassing the flanking regions for recombination in the viral genome, the expression cassette, and the Kanamycin-resistance cassette was cut out of the transfer vector using the homing restriction endonuclease I-CeuI and purified by agarose-gel-electrophoresis. The purified DNA fragment was then inserted in the BAC pRacH-SE by en-passant RED recombination (Tischer et al. 2006). After sequence integrity was confirmed, recombinant EHV-1 RacH-SE-UL43-422-mCherry (FIG. 15) was rescued after transfection of permissive cell cultures and plaque purified. Expression of the fluorescent mCherry protein as investigated by fluorescence microscopy showed that the new expression cassette in the new insertion site was functional (not shown).

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

(296) To this end, the orf encoding for mCherry was cut out of the transfer vector pUUL43-422-mC-18K18 (FIG. 14; SEQ ID NO:35) and the orf encoding H1pdm was inserted instead. The resulting transfer plasmid was named accordingly pUUL43-422-H1pdm-18K18 (FIG. 16, SEQ ID NO:36). A fragment of pUUL43-422-H1pdm-18K18 encompassing the flanking regions for recombination in the viral genome, the expression cassette, and the Kanamycin-resistance cassette was cut out of the transfer vector using the homing restriction endonuclease I-CeuI and purified by agarose-gel-electrophoresis. The purified DNA fragment was then inserted in the BAC pRacH-SE by en-passant RED recombination (Tischer et al. 2006). After sequence integrity was confirmed, recombinant EHV-1 RacH-SE-UL43-422-H1pdm (FIG. 17) was rescued after transfection of permissive cell cultures and plaque purified.

(297) The same procedure was used to generate a recombinant EHV-1 RacH-SE based on rEHV-1 RacH-SE-B (rEHV-1 RacH-SE-orf1/3-p430-Hiav-70-p455-H3, FIG. 7). The generated triple-insert recombinant was named rEHV-1 RacH-SE-UL56-430-H1av-UL43-422-H1pdm-US4-455-H3 (abbreviated rEHV-1 RacH-SE-E, FIG. 18).

(298) A schematic drawing of the genome of the triple-insert rEHV-1 RacH-SE-UL56-430-H1av-UL43-422-H1pdm-US4-455-H3 (abbreviated as rEHV-1-E) is depicted in FIG. 18. While the name of the predecessor construct uses the original EHV-orf nomenclature, the new triple insert virus name is based on the unified nomenclature of Alphaherpesviruses, where genes are named according to their homologs in Human Herpesvirus 1.

(299) Recombinant plaque-purified viruses were characterized by sequencing the insertion site regions (not shown), Western blots (FIG. 19) and virus titrations (Table 3).

(300) The dual-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 expression strength of the transgenes. In addition a single-insert rEHV-1 RacH-SE, rEHV-1 RacH-SE-orf70-p455-H1pdm (FIG. 10), which expresses the IAV HA H1pdm from the new orf70/US4 expression site under control of the p455 promoter was included in the tests.

(301) In order to assess expression strength of the new recombinant EHV-1 RacH-SE-UL43-422-H1pdm and EHV-1 RacH-SE-E in comparison with the two other rEHV-1 RacH-SE expressing H1pdm Western blot analysis was performed. In addition, all single-insert rEHV-1 RacH-SE expressing different IAV HA and the two dual-insert rEHV-1 RacH-SE B and D, respectively, were included. For a list of the used viruses see table 2.

(302) TABLE-US-00010 TABLE 2 List of viruses analyzed by Western blot (FIG. 19) Long name Abbreviation transgenes rEHV-1 RacH-SE-UL56-430-H1av- B H1av H3 US4-455-H3 rEHV-1 RacH-SE-UL56-430-H1hu- D H1hu H1pdm US4-455-H1pdm rEHV-1 RacH-SE-UL56-430-H1av-UL43- E H1av H1pdm H3 422-H1pdm-US4-455-H3 rEHV-1 RacH-SE-UL56-430-H1av av H1av rEHV-1 RacH-SE-UL56-430-H1hu hu H1hu rEHV-1 RacH-SE-US4-455-H3 H3 H3 rEHV-1 RacH-SE-US4-455-H1pdm 4p H1pdm rEHV-1 RacH-SE-UL43-H1pdm 43p H1pdm rEHV-1 RacH-SE SE none

(303) Three proprietary monospecific monoclonal antibodies directed against hemagglutinins H1av or H1pdm, or against the EHV-1 glycoprotein II and a commercial polyclonal anti-H3 antibody were used. The method allowed for a semi-quantitative assessment of the amounts of transgenes expressed in cells infected with the different tested recombinant viruses. As cell culture control cells were left uninfected and as background virus control a rEHV-1 RacH-SE was used that had been rescued and plaque purified from an “empty” vector backbone BAC (SE). AI-ST cell cultures infected with the recombinant EHV-1 B, D, E, SE, av, hu, H3, 4p, 43p (see table 2), or left uninfected were collected 30 h p.i. and processed for SDS-PAGE under reducing conditions. After electrophoresis, proteins were electro-transferred to nylon membranes and incubated with monoclonal antibodies to either HA H1av, H1pdm or the EHV-1 glycoprotein II or a commercial rabbit polyclonal antibody to HA H3. The Western blot (FIG. 19d) confirms successful infection and replication of all nine viruses. Quantities of gpII expressed in B, D, av, hu, H3, 4p, 43p and SE-infected cells appear similar, which is indicative of comparable replication efficiency. The gpII amount in E-infected cells is slightly reduced compared to the others. Western blots (FIGS. 19a and 19b) confirm expression of the hemagglutinins H1av and H3, respectively, by the new recombinant E. In comparison to B, the quantities appear comparable. In contrast, the amount of hemagglutinin H1pdm expressed by the new recombinant EHV-1 RacH-SE-E and -43p (Western blot (FIG. 19c)) appears greatly reduced when compared to D and 4p, where the identical protein is expressed in the US4 (orf70) insertion site under control of the 455 promoter.

(304) To assess whether expression of three hemagglutinins in parallel would impair viral replication efficiency, rEHV-1 RacH-SE-B, -D, and -E were passaged in AI-ST cells until passage eleven and titres were determined in parallel as triplicates (Table 3). All titres were in a comparable range indicating that the third transgene expression cassette had no obvious negative impact on viral fitness under cell culture conditions.

(305) TABLE-US-00011 TABLE 3 Comparison of viral titres at passage 11 Virus ID Passage no. TCID50/ml Standard deviation rEHV-1RacH-SE-B 11 2.01E+08 1.09E+07 rEHV-1RacH-SE-D 11 1.76E+08 8.59E+07 rEHV-1RacH-SE-E 11 1.67E+08 8.88E+07

(306) Taken together it was shown that a recombinant EHV-1 expressed three different Influenza A hemagglutinins from three different expression sites in parallel. While expression from the UL56 (orf1/3) and the US4 (orf70) insertions sites under control of the 430 and 455 promoters, respectively, was of comparable strength, expression from the new site UL43 under control of the new 422 promoter was weaker. Also in a recombinant EHV-1 RacH-SE expressing only hemagglutinin H1pdm in from the new insertion site in UL43 under control of the p422 promoter, the amount appeared reduced as compared to the same protein expressed from the US4 (orf70) insertion site under control of the p455 promoter. Thus, the new expression system presents itself as an option if the goal demands less strong expression of a third transgene in addition to the ones being expressed from the UL56 site and the US4 site. A lower expression from the UL43 site is advantageous when expressed proteins are known to exert toxic effects in cell cultures when present in high amounts. Furthermore, combination of strong and weak expression sites can be used if a certain ratio of proteins is needed for a purpose, e.g. for the formation of virus like particles consisting of different viral structural proteins at specific ratios. In addition, a weaker transgene expression might be desirable if the expressed protein has a tendency to destabilize the recombinant vector virus.

(307) The enhanced EHV-1 vector BAC pRacH-SE can be used as a platform for the generation of vector vaccines against diverse 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 enhanced vector virus in their authentic form. Three different antigens may represent three serotypes of one pathogen or originate from different pathogens of the species the vaccine is designed for. In addition, a vector vaccine generated on the basis of the enhanced EHV-1 vector pRacH-SE expressing antigens of horse pathogens has the putative potential to be tetravalent, since it would also vaccinate against EHV-1 infection.

(308) Information on the enhanced EHV-1 vector BAC pRacH-SE has not been published or presented outside of BI.

(309) All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the following claims.

REFERENCES

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