Herpesvirus of turkeys vectored vaccine against avian influenza in poultry

ES 2 635 019 T3

DESCRIPTION

Turkey Herpesvirus Vector Vaccine Against Avian Influenza In Poultry

The present application applies to the field of veterinary vaccines, in particular of vaccines for poultry against avian influenza. The vaccine is based on a recombinant viral vector expressing the hemagglutinin protein of an avian influenza virus, in which the vector is turkey herpesvirus (HVT) and the hemagglutinin gene is driven by a glycoprotein B gene promoter of a mammalian herpesvirus. A vaccine comprising this HVT + HA vector can be used to induce a protective immune response against avian influenza in poultry, and to reduce the spread of VGA. The invention also relates to methods, uses and vaccines involving the HVT + HA vector.

Turkey herpesvirus (HVT) was described around 1970 as a herpesvirus that infects turkeys and has antigenic elements in common with Marek's disease virus (VEM). While VEM is highly pathogenic for chickens, HVT is non-pathogenic for chickens and could be used for effective vaccination against infection and disease caused by VEM (Okazaki et al., 1970, Avian Diseases, vol. 14, p. 413-429). Since then, vaccinating chickens against EMV using HVT has become part of the conventional vaccination program for billions of chickens produced worldwide each year. Very helpful in this regard was the finding that HVT, unlike VEM, can be purified from the host cells in which it was produced, for example by ultrasound, and can be marketed as a stable, lyophilized vaccine.

HVT replicates in avian lymphocytes, particularly peripheral blood lymphocytes (PBL), therefore it is a systemic virus. It induces a long-lasting immune response, which is primarily directed at the cellular immune system, not the humoral one.

HVT vaccines can be given to chickens at a young age, which is a combined result of the apathogenic nature of HVT, as well as its relative insensitivity to maternally derived antibodies against EMV or HVT. Consequently, HVT vaccines can be inoculated into chicks on the day of egg hatching (day one), or even before hatching, while they are still in the egg. The latter approach, in-egg vaccination, is typically applied on day 18 of embryonic development (ED), which is approximately 3 days before hatching.

HVT is currently classified in the alphaherpesvirinae subfamily, and is also known as: meleagid herpesvirus 1, turkey herpesvirus, or Marek's disease virus serotype 3.

The HVT virion has all the elements of a typical herpesvirus, and is approximately 160 nm in size in its enveloped form. Within the capsid it comprises a large linear double-stranded DNA genome. The complete viral genome sequence of approximately 159 kb has been known since 2001 (Genbank reference number AF291866).

However, long before this, the HVT genome had been studied and manipulated, particularly its apathogenic properties have led to research on the use of HVT as a viral vector for expression and delivery of various proteins to a host chicken that has been inoculated. with the recombinant HVT. Examples are the expression of genes encoding antigens from other poultry pathogens such as: infectious bursal disease virus (IBV) (Darteil et al., 1995, Virology, vol. 211, p. 481-490) and Newcastle disease virus (VEN) (Sondermeijer et al., 1993, Vaccine, vol. 11, p. 349-358). However, the expression of a parasitic antigen has also been described (Cronenberg et al., 1999, Acta Virol., Vol. 43, p. 192-197), or of a cytokine, to manipulate the immune response of chicken (document WO 2009 / 156,367; Tarpey et al., 2007, Vaccine, vol. 25, p. 85298535).

Many locations for insertion of the heterologous gene into the HVT genome at suitable non-essential loci have been investigated, eg, in the unique short region of the HVT genome (EP 431,668); or in the single long region (EP 794,257).

Several methods have been described for inserting heterologous nucleic acids into HVT: use of homologous recombination (Sondermeijer et al., Mentioned above), regeneration of cosmids (US 5,961,982) or bacmids (bacterial artificial chromosomes) (Baigent et al., 2006, J. of Gen. Virol., Vol. 87, p. 769-776).

For large-scale production, HVT is commonly produced in vitro, in chick embryo fibroblast cell (FEP) cultures. These are primary cells prepared by trypsinization of chicken embryos. FEPs are seeded in monolayers and infected with HVT. It then replicates in these fibroblast cells, even though HVT in vivo replicates in lymphoid cells.

Several commercial vaccine products are currently available that comprise an HVT vector that expresses a heterologous antigen. For example: the F antigen of VEN: Innovax®-ND-SB (MSD Animal Health) and Vectormune® HVT-VEN (Ceva); the VP2 antigen of IBV: Vaxxitek® HVT + IBD (Merial) and Vectormune® HVT-IBD

ES 2,635,019 T3 (Ceva); or infectious laryngotracheitis virus antigens: Innovax®-ILT (MSD Animal Health).

The application of such HVT vector vaccines to poultry will generate an immune response against the expressed heterologous gene, as well as against HVT / VEM. Due to the virulence of VEM field strains have increased over time, a typical vaccination against VEM now incorporates an additional MVD vaccine component in addition to the HVT virus or vector, such as a VEM vaccine strain. serotype 1 or 2, for example a VEM Rispens or VEM SB1 strain respectively.

The influenza virus (VG) is an orthomyxovirus that is infectious to many host species. From the influenza particle itself, it is not entirely possible to determine what type of host has been infected, or will become infected in the future. Therefore, in practice, an influenza virus that can infect and replicate in a certain species is usually indicated as belonging to that species, although cross-infections with other species occur regularly, for example: from waterfowl to chickens; from chickens to pigs, to cats or to humans; from humans to horses, etc. Consequently, avian influenza virus (AGV) refers to the virus that can infect birds. VGA can cause the disease: avian flu (GA), which is also known as “avian plague” or “bird flu” and is a notifiable disease in many countries. Depending on the infectious VGA pathotype and the immune status of infected birds, the disease can range from a subclinical to a mild respiratory to a highly lethal outcome.

Avian influenza in commercial poultry is routinely counteracted by vaccination in areas of the world where VGA is endemic, for example in Asia and the Middle East. In other areas, such as Europe and North America, vaccination is regulated by the government and is only allowed in cases of outbreaks, and in combination with quarantine and eradication measures.

VGA viruses of the so-called highly pathogenic (PA) type are of particular concern, as they present significant zoonotic risks of spreading from birds to other species, including humans. The VGA AP possesses an HA protein that contains a number of basic amino acids at the cleavage site of the HA1 and HA2 parts of the HA protein. The presence of these basic amino acids means that the activation of the HA protein by cleavage can be carried out by a protease that also appears in organs other than the respiratory tract where low pathogenic VGAs replicate. This results in the most systemic viremia and severity of AP VGA infection.

A type A influenza virion, such as VGA, comprises a genome consisting of negative polarity single stranded DNA, divided into 8 segments, encoding 10 proteins. The most relevant viral proteins for immunological purposes are hemagglutinin (HA) and neuraminidase (N). HA is the main antigen, which can induce a protective humoral immune response. VGAs are classified by the serotype variant of their HA and N proteins: H1-H16 and N1-N9 have been described to date. VGA APs are always H5 or H7 subtype.

Even though a flu particle is not limited to infection of a specific species, there does appear to be a prevalence of certain VG serotypes in certain species: VG serotypes H1 and H3 in pigs; H3 and H7 in horses; H3 in dogs; H5 in cats; H7 and H9 in turkeys; and H5, H7 and H9 in chickens.

Because an immune response against influenza is serotype specific, influenza vaccines generally match the immune subtype of VG circulating in the field. Commercial GA vaccines comprise complete inactivated VGA in an emulsion with oil as adjuvant, or a live attenuated VGA vaccine strain.

However, changes occur in the VG field virus over time, known as "genetic drift." In practice, a VG strain that differs from existing strains by more than 90% in the amino acid sequence identity of its HA protein will be designated as a new antigenic class, and will obtain a new "clade" number. This phenomenon can confront a target population with a VG that has more or less changed its immunological profile since the last infection or vaccination. This can make existing vaccines, even when they are of the correct subtype, less effective over time, thus requiring an update of the vaccine virus. Among other reasons, influenza vaccines based on recombinant DNA techniques have been developed to facilitate such updating. For example a VG-HA subunit vaccine that is expressed by the baculovirus expression vector system. By means of routine molecular biological techniques, the expressed HA H5 gene can be exchanged for a more recent one, when required.

Similarly, vector vaccines for GA have been developed that express an HA protein in the context of a living carrier microorganism. Examples of such vectors are viruses such as: infectious laryngotracheitis virus (VLTI) (Lüschow et al., 2001, Vaccine, vol. 19, p. 4249-59); rinderpest virus (Walsh et al., 2000, J. Virol., vol. 74, p. 10165-10175); Vesicular stomatitis virus (Roberts et al., 1998, J. Virol., vol. 247, p. 47044711); fowl pox virus (Swayne et al., 2000, Vaccine, vol. 18, p. 1088-1095); adenovirus (Toro et al., 2010, Avian Diseases, vol. 54, p. 224-231) and VEN (Veits et al., 2006, PNAS USA, vol. 103, p. 8197-8202).

Of these, the HAV-H5 Trovac® vaccine based on the fowl pox vector (Merial), is available in the

ES 2 635 019 T3 market.

A GA vaccine for poultry is of course intended to protect the vaccinated animal against symptoms of avian influenza, and against reinfection in the future. However, for a viral disease with zoonotic and pandemic potential such as VGA, the ability of the vaccine to reduce the spread of wild-type virus in the environment, for example to other herds, migratory or indigenous wild birds, or other animals is almost equally relevant. animal species. Reduction of viral spread can be obtained by inducing a highly effective immune response in the vaccinated bird.

GA vaccines that are subunit or vector vaccines have the advantage that they can be applied in a DIVA approach: differentiation of infected and vaccinated animals, also known as: 'marker vaccines'. This applies because recombinant vaccines only induce antibodies against the expressed viral protein, not for other viral proteins as would appear in the case of infection with a whole virus. DIVA is important for countries or economic sectors that want to maintain and certify VGA-free status, for example for export purposes.

Current vaccines that are based on complete inactivated VGA in an adjuvanted oil emulsion do not allow for distinction by DIVA. In the worst case, poultry vaccinated with such vaccines will carry a broad spectrum of antibodies against VGA, but if these are not completely protective, the birds could still carry live infectious VGA, although it would not be observed.

A live recombinant viral vector for the expression and delivery of a heterologous antigen must be able to overcome several biological stresses on its stability and efficacy: firstly, the ability to generate offspring after transfection. This indicates that the recombinant virus is viable. Next, the ability to replicate in vitro in a host cell line for many cycles while maintaining the expression of the heterologous gene. This indicates that the recombinant was not attenuated by the insert, and the insert is stably replicated and expressed. Then replication and expression in vivo. This indicates that the recombinant can overcome significant selection pressure in a living animal, such as that exhibited by the immune system. In general, the loss of expression of the foreign gene favors faster replication in the animal; Such "escape mutants" have acquired mutations, or significant deletions in the foreign gene, and outgrow intact vectors. Finally, replication in the animal needs to be able to generate such an efficient immune response that the inoculated animal is protected.

Especially important with respect to in vivo efficacy is the behavior of the viral vector vaccine in animals that already possess antibodies; against the vector and / or the heterologous gene that it expresses. For young animals these antibodies are derived mainly from their mothers that have been extensively vaccinated against common pathogens; hence their designation as maternally derived antibodies (ADM). Such antibodies can alter the replication of the vector and / or the expression of the foreign gene, because they can stimulate the immune system of the animals for the elimination (not intended) of the vaccine from the vector.

Recombinant viral vector constructs of an HVT vector with a VGA-HA gene insert have been described: the company CEVA has announced a VECTORMUNE HVT-A1 product on a website (http://www.ceva.com/en/ Responsibility / Contributions), but details are not yet available.

Lan et al. (2009, Acta Microbiologica Sinica, vol. 49, p. 78-84) describe an HVT vector with a VGA-HA H5 gene insert, generated using an improved bacmid recombination technique. From a translation of this article (which is in Chinese) as well as a corresponding article on recombination technology used for VEM (Cui et al., 2009, J. of Virol. Meth., Vol. 156, p. 66-72), it is clear that Lan et al. they constructed their recombinant HVT by inserting an expression cassette into the HVT Us2 gene; the expression cassette contained a VGA H5 HA gene under the control of the human cytomegalovirus immediate early gene promoter (lE-hCMV). The cassette contains additional items necessary for the cloning and selection process. The article by Lan et al. it only describes the cloning and rescue of a HVT + H5 recombinant; no animal tests are indicated, nor any efficacy or stability data from in vitro or in vivo tests.

Alternatively, Zhou et al. (2010, Vaccine, vol. 28, p. 3990-3996) mentions the use of a gB promoter for the expression of VP2 of VBI, from the Us10 locus of MDV1. Notably this element is briefly mentioned in the abstract, but the remainder of the article describes the construction and use of an MDV1 vector expressing lacZ and VP2 driven by the hCMV-IE promoter from the Us2 locus.

Sonoda et al. (2000, J. of Virol., Vol. 74, p. 3217-3226) describe the use of an MDV1 gB gene promoter to drive the expression of a VEN F gene, from the MDV1 Us10 locus.

Takekoshi et al. (1998, Tokai J. Exp. Clin. Med., Vol. 23, p. 39-44) describe the use of a gB gene promoter from hCMV for the expression of heterologous genes in hCMV.

US2008 / 0241188 describes the use of the CMV IE gene promoter to drive an HA gene from

ES 2 635 019 T3

VGA in an HVT vector.

WO2007 / 022151 describes the use of a hCMV early gene promoter to drive a VGA HA gene in a human adenovirus vector.

WO01 / 05988 describes the use of the mCMV IE gene promoter and the SV40 promoter to drive avian leukosis virus genes in an HVT vector.

Sonoda et al. (J. of Virol., Vol. 74, p. 3217) describe the use of the MDV1 gB gene promoter to drive the VEN F gene in an MDV1 vector.

WO2010 / 119112 describes (in examples 23-25) the use of a CMV IE gene promoter to drive the expression of a VGA H5-type HA gene in the context of an HVT vector.

It is an objective of the present invention to generate a GA vaccine based on an HVT vector; the vector vaccine should induce effective immune protection against VGA infection and disease in poultry.

The main requirement of such an immunologically and economically feasible vector vaccine product is that it be stable, both in vector replication and in the expression of the inserted heterologous gene. This combination allows for the extensive in vitro replication cycles that are necessary for large scale production, as well as continued expression and presentation to the host immune system of the inserted foreign gene, when the vector vaccine is replicated in an inoculated host animal. In addition, the stability will allow the vector vaccine to meet the very high standards of safety and biological stability that a recombinant virus that is going to be introduced to the field must meet, in order to obtain a marketing authorization from national government authorities.

The inventors were surprised to discover that promoters that had been used in the prior art to drive the expression of heterologous genes in HVT could not be used for the expression of a VGA HA vector in the context of an HVT vector.

Several promoters were tested: a Rous sarcoma virus long terminal repeat promoter (RSV LTR) (as described in EP 431,668: derived pRSVcat (Gorman et al., 1982, PNAS USA, vol. 79, p 6777-6781)); and a hCMV IE gene promoter (derived from pI17: Cox et al., 2002, Scand. J. Immunol., vol. 55, p. 14-23), to drive the expression of a VGA HA H5 gene , at the Us10 locus of the HVT genome. The vector with the hCMV IE promoter produced plaques after transfection, however these could not amplify for several cycles; the HVT vector with LTR promoter produced plaques that could be amplified, however these only showed very weak HA expression, and when tested in animals as recombinant virus HVP142, they did not provide a significant protective effect in a period of 2-3 ( see Examples).

In this situation, it was totally unexpected that a mammalian herpesvirus gB gene promoter, which had not been previously described for driving the expression of heterologous genes in HVT, nor for the expression of a VGA HA gene, could be used. to construct an HVT vector vaccine expressing a VGA HA gene insert, which advantageously exhibited stability in vector replication and immunological efficacy in expression of foreign genes.

Without wishing to be bound by theory, the inventors speculate that the gB gene promoter from a mammalian herpesvirus, when used for expression of a VGA HA gene in the context of an HVT vector, provides the right balance between the strength of expression of the heterologous gene and the stress this places on the replicative capacity of the recombinant HVT.

Therefore, the invention relates to an HVT vector comprising a heterologous nucleic acid comprising a nucleotide sequence encoding a VGA HA protein, characterized in that said nucleotide sequence is operably linked to a glycoprotein B gene promoter ( gB) from a mammalian herpesvirus.

The HVT vector according to the invention is stable in replication, and provides a sustained expression of the inserted VGA HA gene, both in vitro and in vivo. The HVT + HA vector when used in a poultry vaccine induced a strong immune response that could protect birds against disease caused by a severe VGA challenge infection, and could significantly reduce the spread of the challenge virus To the environment.

A "vector" for the invention is a living recombinant carrier microorganism, herein an HVT. A "heterologous nucleic acid" for the invention is a nucleic acid that did not appear in the parental HVT that was

ES 2 635 019 T3 used to generate the recombinant HVT vector according to the invention.

A "protein" for the invention is a molecular chain of amino acids. The protein can be modified, if required, in vivo or in vitro, by, for example, glycosylation, amidation, carboxylation, phosphorylation, pegylation, or changes in spatial folding. A protein can be of biological or synthetic origin. The protein can be a native or mature protein, a pre or proprotein, or a functional fragment of a protein. Among others, immunologically active peptides, oligopeptides and polypeptides are included within the definition of protein.

It is well known that a "promoter" is a functional region in the genome of an organism that directs the transcription of a downstream coding region. A promoter is therefore a DNA fragment that is located upstream, that is to say towards the 5 'side, of an open reading frame, usually a gene.

As is well known, a promoter initiates mRNA synthesis of the gene it controls, starting from the "transcription initiation site" (SIT). The mRNA produced is in turn translated into protein starting from the gene's start codon, which is the first ATG sequence in the open reading frame (the first AUG in the mRNA). Typically the SIT is located 30-40 nucleotides upstream from the start codon. A SIT can be determined by sequencing the 5 'end of the mRNA of a gene, for example by the RACE technique.

A promoter does not have a specific length, however in general promoters it is comprised at a distance of 1,000 nucleotides upstream from the A position of the start codon, which is generally indicated as A + 1; most promoters are between -500 and A + 1, usually between nucleotides -250 and A + 1.

Furthermore, promoters do not have a fixed nucleotide sequence, but do contain several conserved, recognizable sequence elements; These elements are involved in the binding to transcription factors, and the targeting of RNA polymerase, but also in the regulation of the time, duration, conditions, and level of transcription thereafter. In this way the promoter is sensitive to signals from regulatory elements such as enhancers, or to DNA binding factors such as drugs, hormones, metabolites, etc. A well known conserved promoter element is the TATA box, typically located within 50 nucleotides upstream of the SIT, usually about 30 nt upstream of the SIT. Other examples of conserved promoter elements are the CAAT box, typically at about 75 nt upstream of the SIT, and the GC box, typically at about 90 nt upstream of the SIT.

The location and size of a promoter can be conveniently determined using standard assays, such as the expression of a marker gene by subcloned larger or smaller sections of a suspected promoter. In a similar way, by testing the expression of a marker gene (detecting RNA or protein production), the relative strength of different promoters can be determined and compared.

In practice a promoter can simply be selected by subcloning the region between two consecutive genes, for example from the poly A signal of the upstream gene to the SIT of the downstream gene, followed by cloning the area where appropriate.

Because a promoter is adjacent to the gene it controls expression of in the native context, knowledge of the location of a gene, or the initiation of transcription of its mRNA, inherently reveals the position of its adjoining promoter. This also applies in the invention, where the "mammalian herpesvirus gB gene promoter" refers to the promoter that drives the expression of a herpesvirus gB gene, and is located immediately upstream of that gB gene. The gB protein in normal herpesvirus replication is involved in cell entry and cell propagation. Because the gB gene is such a well-documented and clearly recognizable gene, and because the genomes of many herpesviridae have been sequenced (in whole or in part), one of ordinary skill in the art can easily identify and obtain such a promoter by techniques. routine.

A review of herpesvirus gB proteins was presented in Perreira (1994, Infect. Agents Dis., Vol. 3, p. 9-28). The promoter of the HSV1 gB gene was studied in detail in Pederson et al. (1992, J. of Virol., Vol. 66, p. 6226-6232). None of these, however, describe or suggest the use of a herpesvirus gB promoter to drive heterologous gene expression, neither in HVT nor in any other expression vector system.

For the invention, it is necessary that the gB gene promoter of a mammalian herpesvirus is capable of driving the expression of the HA gene. This is usually indicated as either that the promoter is "operably linked" to the gene or that the gene is "under the control of the" promoter. This usually means that in the final HVT vector construct the gB gene promoter and the HA gene are connected on the same DNA, in effective proximity, and with no signals or sequences between them that would interfere with efficient transcription and translation.

In vector constructs of the invention, the HA gene provides the start codon. Furthermore, the vector constructs prepared were as clean as possible, indicating that except for some restriction enzyme sites, there were no substantial foreign elements in the recombinant vector construct such as an expression cassette with heterologous elements required for cloning. or selection of

ES 2 635 019 T3 recombinants.

Although not strictly necessary, in a preferred embodiment the HA gene is constructed to contain a downstream polyA signal, for example from SV40. Such a signal can provide more complete transcription termination and polyadenylation of the transcript for translation.

The generation of the HVT + HA vector construct can be accomplished by well-known molecular biological techniques, involving cloning, transfection, recombination, selection, and amplification.

A "mammalian herpesvirus" for the invention refers to a herpesvirus that habitually infects and replicates in a mammalian species. Preferably these are from the taxonomic subfamily of Alphaherpesvirinae. For example: human herpesvirus 1 (herpes simplex virus 1), bovine herpesvirus 1, feline herpesvirus 1, equine herpesvirus 1 (EHV), or pseudorabies virus (PRV, also known as suid herpesvirus 1).

The gB gene promoters of such mammalian herpesviruses are usefully used for the invention.

Therefore, in a preferred embodiment the gB gene promoter from a mammalian herpesvirus for the invention is from VPR or HVE.

HVT vectors comprising these gB gene promoters have been shown to be sufficiently stable both in vitro and in vivo, and when used in a poultry vaccine were immunologically very effective in protecting poultry from GA and reduction of VGA propagation.

Such promoters can be conveniently obtained from the prior art, such as from Genbank, for example for:

- VPR, from Genbank reference no .: BK001744, region 20139 - 19596 (the gB VPR gene is UI 27 or gii), or

- HVE of Genbank reference no .: AY665713, region 60709-61570 (the gB gene of HVE1 is ORF 33).

In addition, Genbank reference no. Pfam00606 conveniently represents a group of herpesvirus gB proteins.

The HVP311 vector construction as described in the examples contained the HVE gB gene promoter (SEQ ID NO: 1) and demonstrated in vitro and in vivo stability. When used as a vaccine, this construct showed good immune protection and reduced virus spread, see Examples.

To improve the efficiency of the gB gene promoter from a mammalian herpesvirus for the invention even further, while maintaining its stability, the promoter was adapted. The adaptation was an elongation of the promoter sequence, so that now it did not end before A + 1, but extended downstream of A + 1 of the start codon of the gB gene, to the coding region of the gB gene that normally it is translated into protein.

One result was that the extended promoter now comprised one or more ATG codons, specifically the original start codon and possibly other triplets encoding methionine. Said ATG codons, in this position downstream of the TATA box in the promoter could be interpreted by the cellular transcription machinery as a start codon, which leads to unwanted premature initiation of translation. Therefore the ATG codons downstream of the TATA box of the gB gene promoter, which were now comprised in the extended promoter sequence, were mutated to render such ATGs non-functional as a potential start codon. This allowed the gB promoter for the invention to incorporate nucleotides that spanned the native gB start codon and extend into the translated region of the gB gene, however these additional nucleotides are not being translated but are acting as the leader sequence. extended.

Consequently, promoter sequences containing nucleotides from the coding region of gB downstream of the original A + 1 were constructed.

Therefore, in a more preferred embodiment the gB gene promoter from a mammalian herpesvirus comprises nucleotide sequences from the translated region of said gB gene, wherein any ATG nucleotide sequence had been changed.

The "change" of the ATG nucleotide sequence in the extended promoter for the invention is preferably carried out by mutation. The ATG nucleotide sequence can in principle be changed to any other triplet, provided this does not reduce the stability of replication, or the expression of the vector construct. Preferably the change is by a single nucleotide, preferably from ATG to TTG.

The number of nucleotides downstream of ATG that are comprised in an extended gB promoter for the invention is at least 10, preferably at least 20, 30, 50, 75 or 100, in that order of preference. In the

In practice, the number of nucleotides downstream of A + 1 that are incorporated into the extended promoter for the invention can conveniently be taken as the sequence of A + 1 up to, but not including, the next ATG codon downstream. In this case, it is only necessary to change one ATG sequence (that of the start codon) by mutation.

The HVP310 vector construction as described in the examples contains a VPR gB gene promoter extended 129 nt beyond A + 1. The only ATG sequence comprised in the extended sequence was the original start codon, this was changed to TTG by mutation. This vector showed similar in vitro efficacy and stability to the maladaptive HVE gB gene promoter, however with greatly improved in vivo efficacy, see Examples.

The extended VPR gB gene promoter is as presented in: SEQ ID NO: 2.

Therefore in a further preferred embodiment of the gB gene promoter from a mammalian herpesvirus according to the invention, the promoter has a nucleotide sequence as in SEQ ID NO: 2, or its equivalent.

The HA gene that is comprised in an HVT vector according to the invention, can in principle be any HA gene of avian influenza virus, therefore in principle of any VGA, and of any serotype H1-H16, or similar HA genes described in the future.

For optimal efficacy of the poultry vaccine against GA based on the HVT vector of the invention, the inserted HA gene is preferably a highly pathogenic type HA gene (AP), therefore comprising the amino acids basic at the HA1-HA2 cleavage site. Expression of an HA AP gene provides the possibility to effectively vaccinate poultry against infection with an AP-type VGA, and reduce further spread to the environment.

Therefore, in a further preferred embodiment of the HVT vector according to the invention, the nucleotide sequence encoding a VGA HA protein was derived from a VGA AP.

It is well known in the art that VGA is classified as AP, and many sequences are publicly available. Furthermore, HA AP genes in the invention can be readily obtained from field isolates of VGA AP, from different host species, using routine molecular biology techniques such as RT-PCR.

Preferably, the AP-type HA for the invention is obtained from a VGA AP.

NB: Working with live VGA AP isolates will require laboratory facilities of an appropriate containment level.

To further improve the efficacy of a poultry vaccine comprising the HVT vector according to the invention, the HA gene comprised in this vector was subjected to codon optimization. The codon optimization process is well known in the art, and involves the adaptation of a nucleotide sequence that encodes a protein to encode the same amino acids as the original coding sequence, albeit with other nucleotides, that is, the mutations are essentially silent. . This improves the level at which the coding sequence is expressed in a context that differs from the origin of the expressed gene. For example when a certain gene is expressed in the new context of a recombinant expression system, the adapted codon usage is then adjusted to the codon preference of the new system. In practice this will mean that although most amino acids will remain the same, the coding nucleotide sequence may differ considerably (up to 25% identity) from the original sequence.

For the invention, the coding sequence of the VGA HA gene cDNA used in the invention was optimized for expression in a eukaryotic viral vector, such as HVT.

Examples of AP-type VGA HA gene sequences, the codons of which have been optimized for the invention: SEQ ID NO: 3 and 3, of the HA H5 and H7 genes respectively, and the corresponding encoded HA proteins in SEQ ID NO : 4 and 6. As is well known, HA proteins that are 90% amino acid sequence identity of these genes are commonly considered to be of the same antigenic class.

Therefore, in a more preferred embodiment of the nucleotide sequence encoding the VGA HA protein for the HVT vector according to the invention, the encoded VGA HA protein has at least 90% amino acid sequence identity with the amino acid sequence as in SEQ ID NO 4 or 6. Even more preferably 95, 96, 97, 98, 99 or 100%, in that order of preference.

In a further preferred embodiment, the nucleotide sequence encoding the VGA HA protein of the invention has a nucleotide sequence that has at least 90% nucleotide sequence identity with the nucleotide sequence as in SEQ ID NO: 3 or 5, even more preferably 95, 96, 97, 98, 99, or 100%, in that order of preference.

ES 2 635 019 T3

The most preferred HVT vector according to the invention comprises a heterologous nucleic acid comprising a VPR extended gB gene promoter (e.g. SEQ ID NO: 2) and a codon optimized VGA HA gene of a type H5 (eg SEQ ID NO: 3), whereby this heterologous nucleic acid is inserted into the HVT genome at the Us2 locus.

An example of such a HVT + HA vector virus is represented by the HVP310 vector construct (see Examples), which provided the highest measured immunization efficiency and reduction in viral spread.

The nucleotide sequence of a heterologous nucleic acid that can be used to assemble said recombinant HVT + HA vector virus by routine techniques is as presented in SEQ ID NO: 7. The sequence can conveniently be incorporated into a conventional carrier plasmid as is. commercially available pUC series. The resulting plasmid is then commonly referred to as a "transfer vector", and is suitable for use in transfection protocols.

As described, the HVT + HA vector construction according to the invention can be generated by standard techniques well known in the art. Central to these techniques is the integration into the HVT genome of a heterologous nucleic acid comprising a mammalian herpesvirus gB gene promoter and a VGA HA gene, both in accordance with the invention.

Therefore a further aspect of the invention relates to a method for the preparation of the HVT vector according to the invention, which comprises the integration into the genome of an HVT of a heterologous nucleic acid comprising a nucleotide sequence that encodes a VGA HA protein, wherein said nucleotide sequence is operably linked to a mammalian herpesvirus gB gene promoter.

The advantageous use of the HVT + HA vector according to the invention is in a vaccine for poultry against GA; protect birds and their environment from VGA infection and disease.

Therefore, in a further aspect the invention relates to the HVT vector according to the invention, or to the HVT vector as can be obtained by the method of the invention, for use in the vaccination of poultry against GA.

Said use in vaccination according to the invention is advantageously carried out using a vaccine composition comprising the HVT vector according to the invention.

Therefore, in a further aspect the invention relates to the HVT vector according to the invention, or to the HVT vector as can be obtained by the method of the invention, for use in a vaccine against GA in poultry. Said use of the vector according to the invention is carried out in a vaccine for poultry.

Therefore in a further aspect the invention relates to a vaccine against GA in poultry, comprising the HVT vector according to the invention, or as can be obtained by the method of the invention, and a pharmaceutically acceptable carrier.

It is well known that a vaccine is a composition comprising an immunologically active compound in a pharmaceutically acceptable carrier. The "immunologically active compound" or "antigen" is a molecule that is recognized by the target's immune system and induces an immune response. The response can originate from the innate or acquired immune systems, and can be cellular and / or humoral. For the present invention, the antigen is a protein.

In general, a vaccine induces an immune response that helps prevent, alleviate, reduce sensitivity to, or treat a disease or disorder resulting from infection with a microorganism. Protection is achieved as a result of administering at least one antigen derived from that microorganism. This will cause the target animal to show a reduction in the number, or intensity of clinical signs elicited by the microorganism. This may be the result of a reduced invasion, colonization or infection rate by the microorganism, leading to a reduction in the number or severity of lesions and effects that are caused by the microorganism or by the response of the target to it.

The "pharmaceutically acceptable carrier" is intended to aid in the effective administration of a compound, without causing (serious) adverse effects to the health of the animal to which it is administered. Said vehicle can be for example sterile water or a sterile physiological saline solution. In a more complex form the carrier can be, for example, a buffer, which can comprise additional additives, such as stabilizers or preservatives. For example, details and examples are described in well-known manuals such as: Remington: the science and practice of pharmacy (2000, Lippincot, United States, ISBN: 683306472) and Veterinary vaccinology (P. Pastoret et al. Ed., 1997 , Elsevier, Amsterdam, ISBN 0444819681).

The vaccine according to the invention is prepared from live HVT + HA virus vector particles of

ES 2 635 019 T3 according to the invention by methods described herein, which are easily applicable by a person of ordinary skill in the art. For example, the HVT + HA vector according to the invention is constructed by transfection and recombination and the desired recombinant HVT vector is selected as described herein. The HVT vector viruses are then industrially produced in larger or smaller volumes. Although production in host animals is possible, proliferation in in vitro cultures, for example in FEP, is preferred. After collecting a suspension comprising the virus, either whole cells or an ultrasound cell product, this suspension is formulated into a vaccine and the final product is packaged. After extensive testing regarding quality, quantity and sterility these vaccine products are published for sale.

General techniques and considerations that apply to vaccinology are well known in this field and are described for example in government regulations (pharmacopoeia) and in manuals such as: Veterinary vaccinology and Remington (both mentioned above).

The HVT + HA vector vaccine according to the present invention can in principle be provided to target poultry by different routes of application, and at different points in their lifetime, provided that the inoculated HVT + HA vector virus can establish a protective infection.

However, since an infection with VGA can be established already at a very young age, it is advantageous to apply the vaccine according to the invention as soon as possible. Therefore, the vaccine according to the invention is preferably applied on the day of hatching (day 1) or in the egg, for example at 18 days ED. Furthermore, the application is preferably by a mass vaccination method. This provides the earliest possible protection, while minimizing labor cost.

Well-known methods for such early-age mass application routes are: by coarse spray on day 1, or by automatic injection into the egg. Equipment suitable for industrial scale application is available on the market.

Therefore, in a further preferred embodiment, the vaccine according to the invention can be applied in the egg.

Different routes of inoculation are known in the egg, such as in the yolk sac, the embryo or the allantoic fluid cavity; these can be optimized as required. Inoculation is preferably in the allantoic fluid cavity.

Alternatively, when the vaccine according to the invention is to be combined with an additional antigenic component, parenteral application may be required, for example, by injection into or through the skin: for example, intramuscular, intraperitoneal, subcutaneous, etc.

Formulations of the vaccine according to the invention are for example a suspension, solution, dispersion or emulsion.

When applied by spray vaccination, the size of the droplets used is important; in general a coarse spray (droplet size of more than 50 µm) is applied, which is in effect an oral, nasal and / or ocular application.

Depending on the route of application of the vaccine according to the invention, it may be necessary to adapt the vaccine composition. This is within the capabilities of one of ordinary skill in the art, and generally involves adjusting the efficacy or safety of the vaccine. This can be done by adapting the dose, amount, frequency, route of the vaccine, using the vaccine in another form or formulation or by adapting the other constituents of the vaccine (for example a stabilizer or an adjuvant).

For example, to be suitable for egg application, the vaccine composition is required to be very mild, so as not to reduce the hatchability of the eggs. Some reduction in hatchability may be acceptable, for example by 10%, more preferably 5, 4, 3, 2, 1 or 0% in that order of preference.

In general the safety of the vaccine according to the invention is provided by the use as the parental HVT virus for the vector construction according to the invention, an established safe HVT vaccine strain, such as an HVT strain PB1 or BC126. These are generally available and are known to be suitable for inoculation into the egg. Incorporation of a heterologous nucleic acid probably does not increase its virulence or pathogenicity (on the contrary), and a return to virulence is not applicable.

The exact amount of HVT vector virus according to the invention in a vaccine dose is not as critical as it would be for an inactivated emulsion type vaccine, because the HVT vector virus will replicate and therefore multiply in the host. up to a level of viremia that is biologically sustainable. It is only necessary that the dose of vaccine is sufficient to generate said producing infection. Higher inoculum dose hardly shortens

ES 2 635 019 T3 the time it takes to reach optimal viremia in the host; Very high doses are not effective because the viremia they establish cannot be greater than the natural optimum, in addition, such a very high inoculum dose is not attractive for economic reasons.

A preferred inoculum dose is therefore between 1X10⁰ and 1X10⁶ HVT vector virus plaque-forming units (pfu) per animal dose, more preferably between 1X10¹ and 1x⁵ pfu / dose, even more preferably between 1x10² and 1X10⁴ pfu / dose; more preferably between 500 and 5,000 pfu / dose.

Determination of the immunologically effective amount of the vaccine according to the invention is within the scope of the person skilled in the art, for example by monitoring the immune response after vaccination, or after a challenge infection, for example by re-isolating the pathogen. or monitoring the targets for clinical signs of disease, or serological parameters, and comparing these with responses seen in unvaccinated animals.

The dose schedule for applying the vaccine according to the invention to a target organism can be in one or multiple doses, which can be given at the same time or sequentially, in a manner compatible with the vaccine formulation, and in such an amount that is immunologically effective.

The vaccine according to the invention can be used for both prophylactic and therapeutic treatment, and therefore interferes with the establishment and / or with the progression of an infection or its clinical symptoms of disease.

The vaccine according to the invention can act effectively as a prime vaccination, which can then be followed and amplified by a booster vaccination, for example with a classical whole inactivated virus, virus with adjuvant.

The protocol for the administration of the vaccine according to the invention is ideally integrated into existing vaccination programs of other vaccines.

Preferably the vaccine according to the invention is applied only once, at the time of hatching, or in the egg.

The volume per animal dose of the HVT + HA vector vaccine according to the invention can be optimized according to the intended route of application: inoculation into the egg is usually applied with a volume of between 0.05 and 0.5 ml / egg, and parenteral injection is usually carried out with a volume of between 0.1 and 1 ml / bird.

Determination and optimization of the dosage volume are within the capabilities of the skilled person.

It is highly effective to formulate the vaccine according to the invention as a combination vaccine, since in this way multiple immunological agents can be administered at the same time, providing reduction of time and labor costs, as well as reduction of discomfort for the target animals. vaccinated. A combination vaccine comprises in addition to the vaccine according to the invention, another antigenic compound. In principle this can be any living or dead microorganism or subunit product, as long as this does not reduce the replication stability or the expression of the HVT + HA vector construct. The additional immunoactive component (s) may be an antigen, an immune-enhancing substance, a cytokine, and / or a vaccine.

Alternatively, the vaccine according to the invention can itself be added to a vaccine.

Therefore, in a further preferred embodiment, the vaccine according to the invention is characterized in that the vaccine comprises one or more additional immunoactive components.

In a more preferred embodiment the vaccine according to the invention is a combination vaccine, comprising at least one additional antigen from a microorganism that is pathogenic for poultry.

Preferably the additional antigen from a microorganism that is pathogenic for poultry is selected from the groups consisting of:

- Viruses: infectious bronchitis virus, Newcastle disease virus, adenovirus, egg drop syndrome virus, infectious bursal disease virus (i.e. gumborovirus), chicken anemia virus, avian encephalomyelitis virus, smallpox virus avian, turkey rhinotracheitis virus, duck plague virus (duck viral enteritis), pigeon pox virus, VEM, avian leukosis virus, ITLV, avian pneumovirus and reovirus;

- bacteria: Escherichia coli, Salmonella spec., Ornitobacterium rhinotracheale, Haemophilis paragallinarum, Pasteurella multocida, Erysipelothrix rhusiopathiae, Erysipelas spec., Mycoplasma spec. and Clostridium spec .;

- parasites: Eimeria spec .; Y

- fungi: for example Aspergillus spec.

ES 2 635 019 T3

VEM, VLTI, VBI and VEN are more preferred.

The preferred target poultry animals for application of the vaccine according to the invention are chickens. Said chickens can be layers, breeders, combination breeds or parental lines of any of said chicken breeds.

The age, weight, sex, immune status and other parameters of the poultry to be vaccinated are not critical, although it is obviously favorable to vaccinate healthy targets, and vaccinate as soon as possible to avoid any field infection.

The vaccine according to the invention is usefully used in a DIVA approach, as a "marker vaccine". A marker vaccine is known as a vaccine that allows differentiation between vaccinated and infected subjects in the field. This can conveniently be detected by a serological assay such as an ELISA or immunofluorescence assay.

Therefore, in a preferred embodiment, the vaccine according to the invention is a marker vaccine.

As described, there are several ways in which the vaccine according to the invention can be composed and formulated, depending on the desired route of application, antigenic combination, etc.

Therefore, in a further aspect the invention relates to the use of the HVT vector according to the invention or the HVT vector as can be obtained by the method of the invention, for the manufacture of a vaccine against GA in poultry .

Alternatively, in a further aspect the invention relates to a method for the preparation of the vaccine according to the invention, the method comprising mixing the HVT vector according to the invention or with the HVT vector as can be obtained by the method of the invention and a pharmaceutically acceptable carrier.

Due to the advantageous properties of HVT, the vaccine manufactured according to the use or the method of the invention can be presented in different forms, in particular in cell-associated or cell-free form. To obtain the cell-associated form, the HVT + HA vector virus is harvested together with its host cells in which it was produced, eg FEP. In the cell-free form, the host production cells are sonicated in a stabilizer solution, and the cell-free HVT is collected as supernatant of the sonication product.

The vaccine according to the invention can be manufactured to contain one or more components that aid the viability and quality of the HVT vector according to the invention, thereby promoting productive replication and the establishment of a protective infection in poultry. Diana.

Therefore, in a preferred embodiment, the vaccine manufactured according to the use or method of the invention comprises a stabilizer.

Stabilizers are compounds that stabilize the quantity and quality of the HVT vector according to the invention during storage, handling and inoculation, such as by injection or ingestion. In general these are large, high molecular weight molecules, such as lipids, carbohydrates or proteins; for example milk powder, gelatin, serum albumin, sorbitol, trehalose, spermidine, dextran or polyvinylpyrrolidone.

Preservatives such as thimerosal, merthiolate, phenolic compounds or gentamicin can also be added.

In a preferred embodiment, the compounds used for the manufacture of the vaccine composition according to the invention are serum-free (ie without animal serum); protein-free (no animal protein, but may contain other animal-derived components); free of animal compounds (SCA; containing no component derived from an animal); or even "chemically defined", in that order of preference.

It is clear that mixing other compounds such as carriers, diluents, emulsions and the like with vaccines according to the invention is also within the scope of the invention. Such additives are described in well known manuals such as: "Remington" and "Veterinary Vaccinology" (both mentioned above).

For reasons of stability or economy, a vaccine according to the invention can be manufactured in lyophilized form. In general this will allow prolonged storage at temperatures above zero ° C, for example at 4 ° C. Procedures for lyophilization are known to those skilled in the art, and equipment for lyophilization at different scales is commercially available.

Therefore, in a further preferred embodiment, the vaccine manufactured according to the use or the method of the invention is in lyophilized form.

ES 2 635 019 T3

To reconstitute a lyophilized vaccine composition, it is usually suspended in a physiologically acceptable diluent. Said diluent can be, for example, as simple as sterile water, or a physiological saline solution, for example phosphate buffered saline (PBS); alternatively the diluent may contain an adjuvant compound, such as tocopherol, as described in EP 382,271. In a more complex form the lyophilized vaccine can be suspended in an emulsion for example as described in EP 1,140,152.

As described, the vaccine according to the invention can be advantageously applied to poultry by a vaccination method such as by spraying, inoculation or egg application.

Therefore, in a further aspect the invention relates to a method of vaccinating poultry against avian influenza, comprising the step of inoculating said poultry with a vaccine according to the invention.

Examples

1. Assembling vector constructions

1.1. HVP142

The HVP142 HVT vector viruses carry as a heterologous insert a VGA H5 gene, driven by the RSV LTR promoter. The transfection cassette was inserted into the US10 locus of the HVT PB1 strain, using the homologous recombination technique. The H5 gene was obtained from a 1998 VGA H5N2 isolate.

The methods for transfection, recombination, selection and amplification were essentially as described in Sondermeijer et al., 2003 (mentioned above) and EP 431,668.

The antiserum used for selection of HA expression plaques was a polyclonal chicken antiserum against a VGA strain of the H5N6 type.

1.2. HVP310

The HVP310 vector viruses comprised a codon optimized H5 gene (SEQ ID NO: 3), which was driven by a VPR gB gene promoter that had been extended downstream of the ATG start codon of the gB gene (SEQ ID NO: 2). The heterologous construct was inserted into the Us2 locus of the HVT genome of strain FC126, using a cosmid clone regeneration technique. The total expression construct was as depicted in SEQ ID NO: 7.

The H5 gene originated from an H5N1 AP isolate taken from a 2005 Asian cat. This had been corrected by codon usage optimization for expression in a viral expression vector system.

The methods for transfection, recombination, selection and amplification were essentially as described in US 5,961,982. Transfected FEP cells were seeded after recombination in 10 cm tissue culture plates; after about a week the plaques became clearly visible. The plates were stained with Evans blue contrast dye, and the plates could be selected directly from the seed plates. Recombinant HVT vector virus DNA was routinely checked for recombination correctness and insertion of the HA gene and promoter by restriction enzyme analysis.

Expression of the integrated HA gene was performed by immunofluorescence assays in microtiter plates, using H5N6 chicken polyclonal antiserum.

1.3. HVP311

The HVP311 vector viruses comprised a codon optimized H5 gene, which was driven by an HVE gB gene promoter (SEQ ID NO: 1). The construction, recombination and selection was similar to that of the HVP310 virus. Furthermore, the same codon optimized HA H5 gene insert was used.

1.4. In vitro stability tests

To determine in vitro stability, recombinant HVT vector viruses HVP142, 310 and 311 were passaged at least 15 times in FEP monolayers. After a plate was selected, it was amplified for 15 cycles.

Finally, the 10 cm plates were inoculated and after incubation, stained with chicken H5N6 antiserum for an immunofluorescence assay (EIF). The number of plaques showing positive immunofluorescence was counted per total number of plaques. All recombinants tested were found to be completely stable in in vitro cultures as 100% of the plates fluoresced positively. This meant that first the hA insert had replicated correctly through the more than 15 passages of cell culture, and

ES 2 635 019 T3 second, that the HA gene was still intact and was being expressed correctly.

two. Animal test in chickens SPE

2.1. Preparation of the animal test

The animal experiment was set up to determine the efficacy of HVT + HA recombinants after specific pathogen-free (SPE) vaccination of one-day-old broilers. The protective efficacy was evaluated by exposure-infection with an HPAI H5N1 virus at two or three weeks after vaccination (bv). Chicks were observed daily for clinical signs of avian influenza infection or mortality. In addition, tracheal and cloacal swabs were collected to assess virus excretion by PCR challenge.

Groups of 10 SPE broilers were placed in negative pressure isolators in the high containment facilities of the central veterinary institute (Lelystad, NL). Blood samples were taken weekly during the course of the test.

The vaccines tested were the recombinant HVT vector viruses HVP142, 310 and 311, together with a conventional H5-type inactivated emulsion vaccine, and a mock vaccinated group that received only PBS. Recombinant HVT vaccines had been prepared as cell-associated preparations at approximately 5x10⁵ pfu / ml, which were stored in liquid nitrogen until use.

Chickens were placed, individually tagged, and vaccinated; HVT was administered intramuscularly, with 0.2 ml / dose at 2000 pfu / chick.

After two to three weeks the chicks were exposed to 10^6,0 DIH50 per chick of exposure virus VGA H5N1 AP (H5N1 Pavo / Pavo / 01/05 Clado 2.2), with 0.1 ml through the nasal route and 0.1 ml through the intratracheal route.

After challenge the chickens were observed daily for GA signals. Clinical scores were classified ranging from 0 to 3 (none - severe) for typical GA symptoms such as depression, oronasal discharge, respiratory distress, neurological signs, diarrhea, etc. The seriously ill chicks were euthanized. Dead chicks were histopathologically tested for cause of death.

Serum samples from before exposure and from 14 days after exposure were determined by hemagglutination inhibition (HI) assay using mainly the HPAI type H5 challenge virus.

For evaluation of spread of the challenge virus, swabs were taken from the trachea and cloaca of each chicken at 2, 3, 4, 7 and 14 days after challenge. The swabs were individually examined by Q-PCR on the VGA matrix protein gene, to compare if, and how much (Ct value) of challenge virus was shed by vaccinated and control chickens.

2.2. Results

The results of the tests in chickens SPE are presented in Tables 1-3.

With regard to "protection of clinical signs" as presented in Table 2, only animals that did not show any clinical signs of GA were scored as protected.

In Table 3, the "positive in viral reisolation" indicates from which animals it was possible to reisolate the virus; only if an animal was positive for two consecutive days was it listed positive for virus reisolation. As none of the cloaca swab specimens were positive, only tracheal swab results are presented.

Table 1: HI titers before exposure, in SPE vaccinated im at one day of age

<td rowspan="2">Vaccine</td><td rowspan="2">no. of animals</td><td colspan="2">HI (log2, ag. H5N1 AP)</td>

<td>Exp. At 2 weeks pv</td><td>Exp. At 3 weeks pv</td>

<td>HVP142</td><td> 20</td><td> &lt;4</td><td> &lt;4</td>

<td>HVP310</td><td> 20</td><td> 5,9</td><td> 8,6</td>

<td>HVP311</td><td> 20</td><td> 4,2</td><td> 8,1</td>

<td>H5 inac</td><td> 20</td><td> &lt;4 *)</td><td> &lt;4 *)</td>

<td>thinner</td><td> 10</td><td> &lt;4</td><td> &lt;4</td>

ES 2 635 019 T3

*) When tested in an HI assay with another H5-type antigen, there was clear evidence of seroconversion, with HI titers of 6.7 and 8.6, at 2 and 3 weeks pv respectively.

Table 2: Protection against clinical signs of GA, in SPE, vaccinated im at one day of age, after lethal challenge (&lt;48 h) with VGA AP H5N1.

<td rowspan="2">Vaccine</td><td rowspan="2">no. of animals</td><td colspan="2">Protection against clinical signs</td>

<td>Exp. At 2 weeks pv</td><td>Exp. At 3 weeks pv</td>

<td>HVP142</td><td> 20</td><td> 0/10</td><td> 1/10</td>

<td>HVP310</td><td> 19</td><td> 10/10</td><td> 9/9</td>

<td>HVP311</td><td> 20</td><td> 10/10</td><td> 10/10</td>

<td>H5 inac</td><td> 20</td><td> 3/10</td><td> 8/10</td>

<td>thinner</td><td> 10</td><td> 0/5</td><td> 0/5</td>

Table 3: Protection against virus re-isolation, in SPE, vaccinated im at one day of age, after lethal challenge (&lt;48 h) with VGA HSN1 AP.

<td rowspan="2">Vaccine</td><td rowspan="2">no. of animals</td><td colspan="2">Positive in virus reisolation (trachea)</td>

<td>Exp. At 2 weeks pv</td><td>Exp. At 3 weeks pv</td>

<td>HVP142</td><td> 20</td><td> 10/10</td><td> 10/10</td>

<td>HVP310</td><td> 20</td><td> 6/10</td><td> 1/10</td>

<td>HVP311</td><td> 20</td><td> 6/10</td><td> 2/10</td>

<td>H5 inac</td><td> 20</td><td> 10/10</td><td> 10/10</td>

<td>diluent *)</td><td> 0</td><td> --</td><td> --</td>

*) Animal swabs could not be taken in the diluent group as all died within 48 hours after exposure

3. Animal testing in chickens MDA +

3.1. Animal test preparation

The setup of the MDA + broiler animal test was generally the same as for the SPE chicken test except that no HVP142 vector vaccine was included. The MDA + broilers were derived from parents that had been vaccinated twice with a standard inactivated H5N2 emulsion vaccine; the chicks had starting H5 IH titers between 5 and 6.

3.2. Results

The results of the tests on MDA + chickens are presented in Tables 4-6.

For Tables 5 and 6 the same observations apply as for Tables 2 and 3 above.

Table 4: IH titers on the day of exposure, in MDA + vaccinated im at one day of age

<td rowspan="2">Vaccine</td><td rowspan="2">no. of animals</td><td colspan="2">HI (log2, ag. H5N1 AP)</td>

<td>Exp. At 2 weeks pv</td><td>Exp. At 3 weeks pv</td>

<td>HVP310</td><td> 20</td><td> &lt;4</td><td> 5,4</td>

<td>HVP311</td><td> 20</td><td> &lt;4</td><td> 4,4</td>

<td>H5 inac</td><td> 20</td><td> &lt;4</td><td> &lt;4</td>

<td>thinner</td><td> 10</td><td> &lt;4</td><td> &lt;4</td>

ES 2 635 019 T3

Table 5: protection against clinical signs of GA in MDA +, vaccinated im at one day of age, after lethal challenge (&lt;120 h) with VGA H5N1 AP.

<td rowspan="2">Vaccine</td><td rowspan="2">no. of animals</td><td colspan="2">Protection against clinical signs</td>

<td>Exp. At 2 weeks pv</td><td>Exp. At 3 weeks pv</td>

<td>HVP310</td><td> 20</td><td> 1/10</td><td> 9/10</td>

<td>HVP311</td><td> 19</td><td> 0/10</td><td> 4/9</td>

<td>H5 inac</td><td> 18</td><td> 0/9</td><td> 0/9</td>

<td>thinner</td><td> 20</td><td> 0/10</td><td> 0/10</td>

Table 6: protection against virus re-isolation, in MDA +, vaccinated im at one day of age, after lethal exposure (&lt;120 h), with VGA H5N1 AP.

<td rowspan="2">Vaccine</td><td rowspan="2">no. of animals</td><td colspan="2">Positive in virus reisolation (trachea)</td>

<td>Exp. At 2 weeks pv</td><td>Exp. At 3 weeks pv</td>

<td>HVP310</td><td> 20</td><td> 10/10</td><td> 7/10</td>

<td>HVP311</td><td> 19</td><td> 10/10</td><td> 9/9</td>

<td>H5 inac</td><td> 18</td><td> 9/9</td><td> 9/9</td>

<td>thinner</td><td> 20</td><td> 10/10</td><td> 10/10</td>

3.3. Quantification by Q-PCR

In the animal test in which MDA + chickens were challenged, virus reisolation samples were obtained by taking swabs from the trachea on day 2 and 3 post challenge. Nucleic acids were then extracted, and real-time RT-PCR assays were performed as described in Maas et al. (2007, Emerging Infectious Diseases, vol. 13, p. 1219-1221). Threshold values ​​(Ct) were expressed in relative copy numbers and compared with the value measured in birds that were not vaccinated (control) or vaccinated with an emulsion vaccine. The copy number corresponding to the lowest Ct value in this group was arbitrarily set at 1000.

Figure 1 presents the results: an approximately 250-fold reduction in challenge virus replication with the HVP310 strain.

Four. Conclusions from the results of animal tests

4.1. General:

- HVP142 lacked efficacy in SPE trial and was not included in MDA + trial.

- The HVP310 and 311 vector viruses replicated well, in both SPE and MDA + chicks, indicating their stable, viable constitution. The expression of the inserted HA gene was equally stable and efficient, as demonstrated by the highly efficient immune response that was generated.

- The applied challenge infection turned out to be extremely heavy, considering that all the controls and many of the vaccines with conventional emulsion vaccine died. However, this allowed HVT + HA vector vaccines to demonstrate their protective capabilities under the most stringent conditions.

4.2. SPE Assay:

- The clinical protection induced in SPE chicks was very spectacular: the SPE chicks vaccinated with HVP310 and 311 were completely protected against each and every one of the clinical signs of GA, as early as 2 weeks after vaccination, while the Emulsion provided only partial protection, and unvaccinated chicks died within 48 hours.

- The SPE chicks were also fully protected from the spread of the challenge virus, as demonstrated by the virus re-isolation results; reduction of virus isolation of 80 and 90% was achieved for HVP 311 and 310 respectively, while no reduction of virus spread could be achieved by the emulsion vaccine.

- The efficacy of the HVP310 and 311 vectors in SPE chicks therefore differed only minimally.

ES 2 635 019 T3

4.3. MDA + Assay:

- Protection of MDA + chicks from clinical signs of GA after exposure was much better at 3 weeks pv than at 2 weeks pv HVP 310 was able to protect 90% of MDA + chicks from the appearance of no clinical signs; HVP311 achieved only 45% protection, while the emulsion vaccine did not provide protection. All unvaccinated MDA + chicks died within 120 hours.

- Under the harsh conditions of the trial, the HVP310 vector vaccine was still able to reduce the viral spread in MDA + chicks by 30% at 3 weeks after vaccination, while no reduction in the spread of the virus could be achieved by the HVP311 vaccines or emulsion.

- The reduction in viral suppression induced by the HVP310 vaccine vector, relative to the emulsion vaccinated and the control vaccinated birds, was a factor of 250 on day 2 post challenge.

5. Reisolated Vaccine Virus Stability Test:

Vector vaccines HVP310 and HVP311 will be re-isolated from chickens at 2 and 3 weeks after vaccination. The virus will be seeded in 10 cm FEP plates and allowed to infect. After 5-7 days, the plates will be stained by EIF with chicken H5N6 antiserum, as described. The number of plaques versus the number of fluorescent positive plaques will indicate whether all viruses still contain and express the inserted HA gene.

6. Safety of use for egg vaccination:

To seal the safety for use in egg of the HVT vector vaccine HVP310 and 311, these will be used in the egg.

Three days before the start of the experiment (t = -3 days), three groups of 40 18-day-old embryonated chicken eggs will be inoculated with the vector vaccines HVP310 and 311, as follows:

before vaccination the eggs shall be candled. The blunt end of 18-day-old embryonated eggs will be disinfected with 70% ethanol. A hole will be drilled in the eggshell using an egg drill. The eggs will be vaccinated by inserting a needle (1 ml Becton &amp; Dickinson Plastipak® syringes and 0.6x25, 23G, Microlance® needles) vertically into the egg and injecting 0.05 ml of the vaccines. Later the holes will be sealed with glue and the eggs will be placed in incubators, under appropriate conditions.

The eggs will then hatch in three incubators in animal facilities. After hatching, 25 chickens per group will be banded and placed in group 1 to 3 (t = 1 day), and housed in three isolates respectively, and observed for another week.

The resulting numbers and health of the hatched chicks will be monitored to determine if any effect on hatchability or health occurs from inoculation of the HVT HVP310 and 311 vector vaccine into the egg.

7. Difference in the properties of gB gene promoters derived from avian or mammalian herpesviruses, when used in an HVT vector

When different promoters were tested for their suitability to drive the expression of a heterologous gene in the context of an HVT viral vector, the MDV1 gB gene promoter was shown to be ineffective in HVT. On the other hand, the equine herpesvirus (HVE) gB gene promoter was operative in HVT.

The constructs used for this purpose were assembled essentially as described in Example 1, and comprised a gene from an Eimeria tenella parasite, the Etsc2 gene. This gene encodes an antigen of approximately 37 kDa, which is the homologue of the Easc2 antigen of Eimeria acervulina which is described for example in EP 775,746. Transfer vector constructs were prepared containing the Etsc2 gene under the control of the gB gene promoter from HVE1 (in transfer vector construct pVEC102), or from MDV1 (construct pVEC103).

Recombinant HVTs were generated by transfection and homologous recombination, and seeded into FEP monolayers as described. Recombinant HVT plates were selected and assayed for Etsc2 antigen expression by immunofluorescence assay in 96-well plates with FEP cell monolayers. Two plates were tested from both constructions, and each plate was tested in duplicate. A rabbit anti Etsc2 antiserum was used as the primary antibody, followed by a secondary antibody conjugated to FITC. This initial scan revealed weakly positive fluorescence for pVEC102 recombinants, but no fluorescence for pVEC103 recombinants.

The four plates were then amplified, and the IFA was repeated. This time all plaques from pVEC102 (using the HVE gB gene promoter) were clearly positive for Etsc2 antigen expression; however,