WO2010128338A2

WO2010128338A2 - Vaccines

Info

Publication number: WO2010128338A2
Application number: PCT/GB2010/050753
Authority: WO
Inventors: Ian Pardoe; John Burden
Original assignee: Henderson Morley Plc
Priority date: 2009-05-08
Filing date: 2010-05-10
Publication date: 2010-11-11
Also published as: WO2010128338A3; GB0907935D0

Abstract

The use of a Herpes Simplex Virus (HSV) or HSV Virus Like Particle (VLP) to express one or more non-HSV antigens, wherein said non HSV antigens comprises a cancer antigen, a Human Cytomegalovirus (HCMV) polyepitope or an Epstein-Barr Virus (EBV) polyepitope.

Description

Vaccines

The present invention relates to improved vaccine preparations suitable for the prevention and treatment of conditions such as cancer, and infections caused by, e.g. herpesviridae, including human Cytomegalovirus (HCMV) and Epstein-Barr Virus (EBV). The invention also relates to improved methods of preparing such vaccines.

Human Cytomegalovirus is a widespread herpes virus infecting at least 50% of adults in developed countries. The consequences of infection with HCMV are most serious in immunocompromised individuals, notably transplant patients (where it is the most important pathogen) and HIV infected individuals, and in pregnant women (because of the morbidity and mortality threat to unborn babies with an immature immune system). Immunocompromised individuals infected with HCMV may be prone to liver failure, cytomegalovirus retinitis and cytomegalovirus colitis. New born babies who have acquired congenital HCMV infection may exhibit birth defects such as hearing loss, vision impairment and mental retardation.

Epstein-Barr virus is even more widespread than HCMV, occurring in 95% of adults in the USA. Infection is often acquired in early childhood or during adolescence and in adolescence can result in infectious mononucleosis, causing fatigue, malaise, headaches, chills, puffy eyes, and loss of appetite. Symptoms may progress to fever, sore throat, swollen lymph nodes, difficulty in swallowing due to tonsillitis, minor aches and pains, and bleeding gums as well as jaundice or a rash.

While antiviral compounds may be used in the management of HCMV infections, treatment of EBV infection is currently limited to management of the symptoms of mononucleosis. An alternative therapeutic approach for both viral diseases is the development of a vaccine to prevent infection or to treat infected individuals. Potential antigens from both viruses have been identified and initial results using adenovirus delivery vectors for HCMV antigens have shown promise. PCT patent application WO 03/000720 (the entire disclosure of which is herein incorporated by reference) describes CTL epitope peptides and polypeptides from 14 distinct antigens of HCMV that are restricted through HLA alleles that are common. WO 96/03144 (the entire disclosure of which is herein incorporated by reference) describes a polyepitope protein of EBV containing nine different epitopes restricted by different HLA alleles, and a vaccinia virus based vaccine composition containing the polyepitope protein.

The use of more than one CTL epitope in the preparation of a vaccine for HCMV or EBV is expected to provide a wider range of protection in a broader ethnic spectrum, by including epitopes restricted by different HLA alleles. Epitopes can be selected from conserved regions of the viral proteins reducing the likelihood of escape mutants. CTL and T-helper epitopes can be combined in a single coding sequence and may further be covalently linked to virus neutralizing antigens. Vaccines based on such polyepitopes are expected to be safe as the potentical pathogenic or oncogenic effects of viral proteins may be reduced or eliminated. Moreover, a polyepitope vaccine allows the inclusion of multiple epitopes from different genes and even from different pathogens (to provide, for example, a combined EBV and CMV vaccine).

Zhong et al. (PLoS ONE, September 2008, 3(9) e3256) describes a chimeric vaccine based on a replication deficient adenovirus which encodes 46 HCMV CTL epitopes from 8 different HCMV antigens, restricted through multiple HLA Class I and Class II alleles, as a polyepitope. Additionally, the polyepitope was covalently linked to a truncated HCMV- encoded gB antigen as a fusion protein. The fusion protein vaccine preparation was shown to induce pluripotent cellular and humoral immunity in vivo and to recall and expand HCMV-specific CD8+ and CD4+ cells. One disadvantage with adenovirus based vaccines relates to the risk of decreases immunogenicity to the vaccine antigens in individuals with preexisting immunity to the adenovirus vector. A need therefore remains for different and improved delivery vectors for CMV, EBV and other antigens.

Summary of the Invention

In one aspect the present invention provides the use of Human Herpes Virus (HHV), e.g. Herpes Simplex Virus (HSV), particles and virus like particles (VLPs) as vectors for the delivery of vaccine antigens.

Preferred VLPs are the HSV light particles (L-particles) and pre-viral DNA replication envelope particles (PREPS). However the use of HSV heavy particles (H-particles), usually attenuated or otherwise inactivated, is also envisaged, preferably in combination with VLPs. Other useful HSV particles and VLPs include genetically disabled viruses (e.g. in which a gene essential for replication has been deleted), and the disabled infectious single cycle (DISC) virus. HSV virus and VLP preparations may be used in purified form, or may be used as unpurified extracts. Preparations for use as a vaccine are preferably sterilized by UV or gamma irradiation, or by formalin or heat treatment.

Nucleic acid encoding vaccine antigens may be cloned into a recombinant HSV genome so as to be expressed in the envelope, the tegument or both the envelope and the tegument of HSV virus particles or VLPs. In a preferred embodiment, the recombined HSV genome is constructed to express the vaccine antigen or antigens as one or more fusion proteins with envelope or tegument proteins.

Suitable tegument proteins for fusion to vaccine antigens include UL41, UL46 and VP22. Suitable envelope proteins for fusion to vaccine antigens include gD.

In some embodiments, the vaccine antigen is a cancer antigen. In other embodiments the vaccine antigen is a peptide or protein containing at least two CTL epitopes, optionally from at least two different vaccine antigen proteins. Such peptides or proteins are referred to herein as polyepitopes.

The polyepitope may contain any combination of vaccine CTL epitopes from any vaccine antigen protein, as may be determined and selected by the skilled person.

In some embodiments the separate epitopes of the polyepitope will include epitopes that are restricted by different HLA alleles in order to expand the range of individuals in which an immune response may be effected.

In some embodiments the vaccine antigens will further include at least one B cell epitope. Often, the vaccine antigens will include at least one B cell epitope and at least one polyepitope. In some embodiments the vaccine antigens will include at least one B cell epitope and more than one polyepitope. In some embodiments the vaccine antigens will include more than one B cell epitope and more than one polyepitope.

In some embodiments the vaccine target is HCMV. The vaccine antigen may be a polyepitope of HCMV. Preferably, the polyepitope comprises CTL epitopes from more than one protein of HCMV. In some instances the polyepitope comprises at least 8 CTL epitopes from HCMV proteins. In other embodiments the polyepitope comprises at least 46 CTL epitopes from HCMV proteins. A polyepitope may, however, include any number of CTL epitopes from any number of different HCMV proteins as desired by the skilled person.

HCMV epitopes for use in a vaccine delivery system of the current invention may also include one or more B cell epitopes, and in some embodiments may include the gB surface glycoprotein from HCMV. The B cell epitope may be expressed as a fusion with the one or more polyepitopes, fused to an envelope or tegument protein of the HSV particle or VLP, or may be expressed separately fused to an envelope or tegument protein, with the polyepitope expressed as a fusion to a different envelope or tegument protein.

In another embodiment the vaccine target is EBV. The vaccine antigen may be a polyepitope of EBV comprising more than one CTL epitope of

EBV. Preferably, the polyepitope comprises CTL epitopes from more than one protein of EBV. In some instances the polyepitope comprises CTL epitopes from at least 9 different EBV proteins. A polyepitope may, however, include any number of CTL epitopes from any number different proteins as desired by the skilled person.

The EBV polyepitope is preferably inserted into multiple tegument and/or envelope proteins. In some embodiments the EBV polyepitope is inserted into UL46 or another tegument protein. Preferably at least two further tegument proteins are engineered to carry the EBV polyepitope as a fusion. In some embodiments the HSV particle or VLP carries the EBV polyepitope as a fusion with at least one protein of the tegument and as a fusion with at least one protein of the envelope.

A further aspect of the invention is an HSV particle or Virus Like Particle (VLP) which expresses a non HSV antigen, for example a cancer antigen or a HCMV or EBV polyepitope. The antigen or polyepitope may be expressed as a fusion with an HSV tegument or envelope protein. In some embodiments the Virus Like Particle may be an L-particle or a PREP. In some embodiments in which the antigen is an HCMV polyepitope, the HSV particle or VLP may further express a B cell epitope of HCMV. In some embodiments the HSV particle or VLP may express a polyepitope from HCMV and a polyepitope from EBV, and optionally further a B cell epitope from HCMV. In embodiments in which a B cell epitope from HCMV is expressed, the B cell epitope may be expressed as a fusion with the HCMV polyepitope, or may be expressed separately from the HCMV polyepitope. A yet further aspect of the invention is an HSV particle or VLP which expresses an immuno modulator such as LIGHT, a member of the TNF superfamily.

HSV particle or Virus Like Particles of the invention may be used to raise an immune response in a human or non-human mammal to one or more of the non HSV antigens or epitopes that are expressed. Where an immune response is immunoprotective, the HSV particles or VLPs of the invention may be suitable for use as a vaccine. Accordingly, in a further aspect, the invention provides the use of an HSV particle or Virus Like Particle which expresses a non HSV antigen in the manufacture of a vaccine for the treatment or prevention of HCMV or EBV infection. Preferably the non HSV antigen is a cancer antigen or a HCMV or EBV polyepitope. In a further aspect, the invention provides an HSV particle or Virus Like Particle which expresses a non HSV antigen, e.g. a cancer antigen or a HCMV or EBV polyepitope, for use as a vaccine.

A further aspect of the invention provides a method for selecting for a recombinant HSV genome encoding a fusion between a tegument protein or an envelope protein, and a polyepitope or other epitope. Selection for recombinant HSV particles may be based upon the use of a antibiotic, e.g. Zeocin, resistance selectable marker gene, or the use of an enhanced green fluorescent protein (eGFP) gene. The invention therefore provides a "toolkit" vector suitable for the insertion of heterologous DNA sequences into a desired location in a HSV gene as a functional fusion protein, together with a marker gene. The toolkit vector is subsequently used for recombination with HSV and the marker gene enables selection of recombinant HSV particles containing the polyepitope or epitope of interest.

In an alternative embodiment of this aspect of the invention, positive selection of recombinant HSV viruses is achieved using thymidine kinase (TK) negative mutants. In such embodiments, a vector containing the HSV TK gene (UL23) disrupted and inactivated by the insertion of a tegument or envelope gene is created. The desired antigenic sequence is cloned into the tegument or envelope gene and the vector is recombined with HSV. Recombinant viral particles are rendered TK negative and may be selected for growth in medium containing bromodeoxycytidine.

Description of the Figures

In order that the invention may be more fully understood, reference is made, by way of example only, to the accompanying figures, in which :

Figure Ia shows a Herpes Simplex Virus infectious virion particle.

Figure Ib shows a HSV L-particle

Figure Ic shows a HSV PREP

Figure 2a shows a HSV L-particle expressing a non HSV antigen in the envelope.

Figure 2b shows a HSV L-particle expressing a non HSV antigen in the tegument.

Figure 3 shows the HHV-I genome showing (a) the positions of genes US6/gD and US7/gI and (b) the structure of a recombinant virus with the insertion of restriction sites to allow for the cloning of new sequences into amino acid positions 244 & 288 of gD and a selectable marker (eGFP/ Zeocin) between US6 and US7.

Figures 4a and 4b show the sequence (4a) and position (4b) of primers for the amplification of the 913 bp HHV-I gD PCR product. Figure 5 shows plasmid maps for the two toolkit vectors pGDTK-

244nm and pGDTK-288nm.

Detailed description of the invention

The invention relies upon the use of HSV particles and HSV VLPs as vaccine delivery vectors. In particular, the invention relates to the use of HSV VLPs to deliver vaccines against HCMV and EBV. More specifically, the invention relates to the use of HSV particles and VLPs to deliver polyepitopes of HCMV and EBV. The polyepitope of HCMV may optionally be further combined with a B cell epitope from HCMV, preferably the gB antigen, either fused to the polyepitope or separate therefrom.

Particularly preferred VLPs are the HSV L-particle and the HSV PREP. From very early in their replication cycle, viruses such as herpes viruses produce two different types of virus particles known as heavy (H) and light (L) particles. H-particles are able to spread and initiate new infections whereas L-particles lack viral DNA and its associated proteins and are, therefore, non-infectious. Herpes Simplex Virus 1 (HSV-I) has been shown to produce L-particles. Such particles contain most, if not all of the envelope and tegument proteins of the H-particle (Szilagyi & Cunningham, 1991).

L-particles have been shown to enhance infectivity of HSV-I and related viruses due to their incorporation of proteins capable of inducing the lytic cycle from otherwise non-replicating molecules within infected cells (Dargan & Subak-Sharpe, 1997).

It has also been demonstrated that adsorption and penetration of HSV-I may be adversely affected by too high a proportion of L-particles per cell to be infected; more than 10,000 L-particles per cell has an adverse effect on penetration of HSV-I, while more than 1,000 L-particles per cell has an inhibitory effect on adsorption (Dargan & Subak-Sharpe, 1997).

Other non-infectious particles derivable from HSV-I include PREPS (pre- viral DNA replication envelope particles), which can be made by blocking DNA replication during HSV-I infection (Dargan, Patel & Subak-Sharpe, 1995).

In a preferred configuration of the invention in its various aspects described above, the non-infectious viral particles, e.g. L-particles and/or PREPS, are derived from alpha herpesviruses, for example HSV-I, HSV-2, EBV, pseudo rabies, VZV. HCMV polyepitopes for expression and delivery by the vectors of the invention may include any number of CTL epitopes and epitopes from any number of HCMV proteins. CTL epiptopes may be derived from proteins showing dominant reactivities, for example UL83, UL44, UL122, UL55 and UL123. However epitopes from other proteins may also be included. In a preferred embodiment the HCMV polyepitope to be expressed and delivered is the 46 epitope polyepitope described in Zhong et al. (supra), comprising the epitopes listed in Table 1. In alternative embodiments, the HCMV polyepitope may include any sub-selection of the epitopes listed in table 1.

In preferred embodiments, the HCMV polyepitope is covalently linked to the HCMV gB protein or a truncated form of the HCMV gB protein, to allow expression of the polyepitope and the HCMV gB protein as a single chain, fused to an envelope or tegument protein of the VLP. In other embodiments the HCMV polyepitope is expressed separately from the gB protein. In yet further embodiments, the gB protein is not included and only the polyepitope is expressed.

In embodiments in which the B cell epitope (such as gb) and the polyepitope are fused together and expressed as a single chain, that chain may be expressed as a fusion with a tegument protein, for example UL46 or VP22. In some instances, more than one tegument protein is fused to the single chain, for example both UL46 and VP22 carry the single chain as fusion proteins.

In embodiments in which a B cell epitope such as gB is included, but is expressed separately from the polyepitope, any pattern of fusion with the tegument or envelope is envisaged. Thus, in some embodiments the polyepitope is expressed as a fusion with a tegument protein, for example vhs-UL41, UL46 or VP22, and the B cell epitope is expressed as a fusion with an envelope protein, for example gD. In some such embodiments, the polyepitope is expressed in several copies, as a fusion with more than one tegument protein, for example vhs-UL41, UL46 and VP22.

In alternative embodiments, the B cell epitope may be expressed as a fusion with a tegument protein, for example UL41, UL46 or VP22, and the polyepitope is expressed as a fusion with an envelope protein, e.g. gD.

In embodiments in which only a polyepitope is included, the polyepitope may be expressed as a fusion with any one or more tegument protein or envelope protein. For example, the polyepitope may be expressed as a fusion with UL46 and/or VP22 and/or gD. In order to maximise the load of polyepitope carried, it is preferred to insert more than one copy of the polyepitiope into the HSV particle or VLP. Preferably, each of gD, UL41, UL46 and VP22 will carry a fusion to the polyepitope.

When the polyepitope contains a large number of epitopes it may be quite a large molecule. In some cases it is desirable to split the polyepitope into two or more pieces and to insert those pieces into different sites for fusion with e.g. different tegument or envelope proteins, so as to replace a non-essential protein such as VP22.

The preferred EBV polyepitope for expression and delivery by the vectors of the invention is the polyepitope described in WO 96/03144.

Polyepitopes and other epitopes (e.g. B cell epitopes) may be inserted into a HSV genome using standard recombinant technology methodologies, for example as described in Sambrook, J., et al. (1989) Cloning : A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. Conveniently, recombinant HSV genomes, into which polyepitopes and other epitopes have been inserted may be selected using the selection methods of the current invention.

Selection methods of the current invention involve the generation of "toolkit" vectors which are used to recombine with an HSV genome. In some instances, the toolkit vectors provide a cloning site for the antigen of interest (i.e. the polyepitope or other epitope) into a tegument or envelope protein. The cloning site may be at either the 3' end, the 5' end or at an internal position within the gene for the tegument or envelope protein. Also provided is a selectable marker gene, such as an enhanced Green Fluorescent Protein gene (eGFP) or an antibiotic, e.g. Zeocin, resistance gene ( in the case of Zeocin resistance - ZeoR). The marker gene is positioned in the toolkit vector in such a way that upon recombination of the vector with an HSV genome the selectable marker gene is inserted, together with the tegument or envelope/antigen gene fusion, into the recombinant viral genome. Recombinant viral particles may then be selected on the basis of the phenotype of the marker gene (e.g. antibiotic resistance or green fluorescence).

In alternative methods, the selection may be based upon the disruption of a phenotype. For example the toolkit vector may be engineered to include a disrupted HSV gene, whose disruption may be selected for. In one embodiment the disrupted gene is the thymidine kinase gene (UL23). The gene may be disrupted by any known means. Conveniently, the gene is disrupted by the insertion of nucleic acid encoding a tegument or envelope protein, into which it is desired to insert the antigen of interest.

Upon recombination of the toolkit vector with an HSV genome, the disrupted gene recombines with its counterpart in the genome, disrupting that counterpart and resulting in a virus that is negative for that gene. Where the disrupted gene is thymidine kinase, recombinant viruses are, therefore, TK negative and may be selected for on medium containing bromodeoxycytidine.

The invention will be further understood with reference to the following non-limiting examples.

Example 1

The following example describes the creation of a "toolkit" vector suitable for the insertion of novel DNA sequences into the envelope glycoprotein D (gD) of Human Herpesvirus 1 (HHV-I) as a functional fusion protein, and the subsequent selection of recombinant viruses. The HHV-I glycoprotein D is a structural component of the virus envelope that is essential for virus entry into host cells (Highlander, S. L. et al. Neutralizing monoclonal antibodies specific for Herpes simplex virus glycoprotein D inhibit virus penetration. J. Virol. 61, 3356-3364 (1987)). Through its binding to the herpes virus entry mediator (HVEM) protein, gD blocks the interaction of this protein with the B- and T-lymphocyte attenuator (BTLA) protein. The fusion of viral antigens to the region of gD which interacts with HVEM has been shown to increase the immune response to the antigen by inducing more potent T and B cell responses (Lasaro, M.O., Tatsis, N., Hensley, S. E., Whitbeck, J. C, Shih-Wen, L., Rux, JJ., Wherry, EJ., Cohen, G. H., Eisenberg, RJ. & Ertl, H. C. Nature Medicine 14, 205-212 (2008)). For this reason the HHV-I gD was chosen as the site into which novel antigens would be introduced in order to make recombinant L-particles and PREPs displaying these antigens as a fusion protein with gD.

The gene coding for gD (US6) is in an open reading frame located between position 138419 and 139603 of the HHV-I genome (GenBank X14112). The aim of this project was to create a vector containing the sequence for US6, into which restriction sites had been engineered to allow the insertion of antigen sequence, together with a selectable marker, such as enhanced green fluorescent protein (eGFP) or Zeocin resistance (ZeoR) between the 3' end of US6 and the start of US7 (Figure 3).

1. Amplification and cloning of HHV-I gD

Oligonucleotide primers HSVgDfI & HSVgDr2b (Figure 4a) were designed to amplify a 913 base pair (bp) region of the HHV-I genome between positions 138887 and 139784 of the genome (GenBank X14112). This sequence includes the terminal 715 bases of gD together with a 198 bp section of the inter-genic region between U_S6 and U_S7 (Figure 4b). The PCR product was amplified using Platinum Pfx DNA polymerase (Invitrogen) and the purified product cloned using a Zero Blunt PCR cloning kit (Invitrogen) to give the plasmid pT0P0gD2.

2. Vector Engineering

The Notl restriction site was removed from pT0P0gD2 by digestion of the vector with Notl. The digested vector was then treated with the large fragment of DNA polymerase 1 to create blunt ends. These were then ligated together to create the vector pTOPOgD2ΔNot which lacked the Notl site present in pT0P0gD2. This digestion, blunt end generation and re- ligation process was repeated with BamHl, then Xbal, to give the vector pTOPOgD2ΔBNX which lacked BamHl, Notl and Xbal restriction sites.

Using site directed mutagenesis BamHl and Xbal restriction sites were introduced into the 913 bp gD sequence in pTOPOgD2ΔBNX. The plasmid DNA was methylated then amplified using the primers gDGFPbxba and gDGFPr (Appendix 1) and cloned into E.coli DH5α -T1^R cells. The resulting plasmid (pTOPOgD2bx) had a BamHl site introduced 8 bp after the stop codon of gD, with an associated Xbal site 3 bp after the BamHl site. This allows the insertion of a selectable marker sequence into the inter-genic region between US6 and US7.

Two different positions within the gD sequence were chosen for the insertion of novel transgenic fusion sequences. These were at amino acid

(αα) position 244 and αα position 288, corresponding to positions 139148

& 139280 of the HHV-I genome. The oligonucleotide primer pairs gD244nm/gD244r and gD288nm/gD288r (Appendix 1) were used to introduce Notl and MIuI restriction sites at aa sites 244 & 288 respectively using a GeneTailor Site-Directed Mutagenesis System (Invitrogen). The resulting plasmids were designated pGD-TK244nm and pGDTK-288nm

(Figure 5). Example 2

Positive selection of recombinant HHV-I viruses expressing novel antigens can be made using thymidine kinase (TK) negative mutants.

To achieve this, a vector is created in which the viral U_L46 gene (vpll/12 tegument protein), together with associated promoter sequences and polyadenylation signals, is inserted into the U_L23 (TK) gene in such a way as to inactivate the TK function. Novel antigenic sequence is then inserted into the U_L46 gene to form a fusion protein as previously described. Recombination of this vector with HHV-I results in TK negative virus, which can be selected for by growth in medium containing bromodeoxycytidine.

This approach can also be used to generate recombinant viruses with the novel antigen fused to the envelope glycoprotein D (gD) by replacing the U_L46 sequence in the U_L23 gene with the U_S6 sequence together with its associated promoter and polyadenylation signals.

Further modification of this system can be made by replacing the HHV-I promoter sequences in the U_L46 or U_S6 genes with highly expressed mammalian promoters such as the CMV promoter. This will ensure high levels of expression of the recombinant protein which can give elevated levels of this in the viral particle.

This approach also allows for the insertion of a novel antigen into a second site within a recombinant HHV-I. For example, where a recombinant HHV-I has already been made by GFP selection of a vpll/12 fusion protein, a second fusion protein could be made with the gD of that virus using TK selection and vice versa for gD/GFP mutants and TK selection of a second insertion into vpll/12.

Other mammalian and non-mammalian herpes or other virus strains (e.g. bacculovirusj may be used to provide VLPs, e.g. animal (e.g. mammal) specific VLPs as will be appreciated by the skilled person.

Claims

1. The use of a Herpes Simplex Virus (HSV) or HSV Virus Like Particle (VLP) to express one or more non-HSV antigens, wherein said non HSV antigens comprise a cancer antigen, a Human Cytomegalovirus (HCMV) polyepitope or an Epstein-Barr Virus (EBV) polyepitope.

2. A use of Claim 1 wherein the said one or more non-HSV antigen comprises a HCMV polyepitope comprising epitopes from more than one HCMV protein antigen.

3. A use of Claim 1 wherein the said one or more non-HSV antigen comprises an EBV polyepitope comprising epitopes from more than one EBV protein antigen.

4. A use of any preceding Claim wherein said HSV or HSV VLP expresses an HCMV polyepitope and an EBV polyepitope.

5. A use of any preceding Claim wherein the HSV VLP is an L-particle or a PREP.

6. A use of any preceding Claim wherein at least one non-HSV antigen is expressed as a fusion protein, fused to a tegument or envelope protein of the HSV or HSV VLP.

7. A use of Claim 6 wherein the tegument protein is selected from UL41, UL46 and VP22.

8. A use of Claim 6 or Claim 7 wherein the envelope protein is HSV gD.

9. A use of Claim 2 wherein said one or more non HSV antigen further comprises a HCMV B cell epitope, preferably gB.

10. A use of Claim 9 wherein the gB epitope is fused to the HCMV polyepitope.

11. A use of Claim 9 wherein the gB epitope is expressed in a form not fused to the HCMV polyepitope.

12. A use of Claim 10 wherein the HCMV polyepitope/gB epitope fusion is expressed as a fusion with a tegument protein or an envelope protein.

13. A use of Claim 12 wherein the HCMV polyepitope/gB epitope fusion is expressed as a fusion with more than one different tegument protein or envelope protein.

14. A use of Claim 11 wherein the HCMV polyepitope is expressed as a fusion with a tegument protein and the gB epitope is expressed as a fusion with an envelope protein.

15. A use of Claim 11 wherein the HCMV polyepitope is expressed as a fusion with an envelope protein and the gB epitope is expressed as a fusion with a tegument protein.

16. A use of Claim 2 or Claim 14 wherein the HCMV polyepitope is expressed as a fusion with more than one different tegument protein.

17. A use of Claim 2 wherein the HCMV polyepitope is expressed in more than one part, as separate fusion proteins with more than one different tegument and/or envelope protein.

18. An HSV or HSV VLP which expresses one or more non-HSV antigens, wherein said non HSV antigens include a cancer antigen, a Human Cytomegalovirus (HCMV) polyepitope or an Epstein-Barr Virus (EBV) polyepitope.

19. An HSV or HSV VLP of Claim 18 further characterised by any of the features of Claims 2 to 17.

20. An HSV of HSV VLP of Claim 18 or Claim 19 for use as a vaccine.

21. The use of an HSV or HSV VLP of Claim 18 or Claim 19 in the manufacture of a vaccine medicament, optionally for the treatment or prevention of HCMV or EBV infection.

22. A vector for use in the manufacture of an HSV or HSV VLP which expresses a non-HSV antigen, said vector comprising a selectable marker gene and nucleic acid portion encoding an HSV tegument or envelope protein, said nucleic acid portion including a cloning site for in frame fusion of a further nucleic acid portion encoding said non-HSV antigen.

23. A vector of Claim 22 wherein said selectable marker gene is an enhanced green fluorescent protein (eGFP) gene, or an antibiotic, e.g. Zeocin, resistance gene.

24. A vector for use in the manufacture of an HSV or HSV VLP which expresses a non-HSV antigen, said vector comprising a nucleic acid portion encoding an HSV thymidine kinase gene, said nucleic acid portion including a cloning site for the introduction further nucleic acid portion encoding said non-HSV antigen, such that introduction of said further nucleic acid portion results in the functional disruption of the thymidine kinase gene.

25. A vector for use in the manufacture of an HSV or HSV VLP which expresses a non-HSV antigen, said vector comprising a nucleic acid portion encoding an HSV thymidine kinase gene, into which either the UL46 gene and/or gD gene has been inserted such that introduction of said further nucleic acid portion results in the functional disruption of the thymidine kinase gene.

26. A vector according to Claim 25, wherein said inserted UL46 and/or gD genes include a cloning site for the introduction further nucleic acid portion encoding said non-HSV antigen, preferably together with HSV or non HSV promoter sequence and/or a polyadenylation signal.

27. A process for the manufacture of an HSV or HSV VLP which expresses a non-HSV antigen, comprising inserting a nucleic acid portion encoding said non-HSV antigen into the cloning site of a vector of any of Claims 22 to 26, and effecting recombination between the vector containing the nucleic acid encoding the non-HSV antigen and an HSV genome.

28. A method of Claim 25 wherein recombinant HSV viruses encoding said non-HSV antigen are selected using the selectable marker gene where present, or by growth in medium containing bromodeoxycitidine.