GB2421025A

GB2421025A - HSV vaccination vectors

Info

Publication number: GB2421025A
Application number: GB0427001A
Authority: GB
Inventors: Richard John Anderson; Michael Keith Howard; Andre Van Maurik; Joerg Schneider
Original assignee: Oxxon Therapeutics Ltd
Current assignee: Oxxon Therapeutics Ltd
Priority date: 2004-12-09
Filing date: 2004-12-09
Publication date: 2006-06-14
Also published as: GB0427001D0; WO2006061643A1

Abstract

The invention provides a method for vaccinating a subject against a target pathogen or disease, comprising administering at least one dose of: (a) a first, priming, composition comprising at least one immunogenic epitope associated with the target pathogen or disease, and (b) a second, boosting, composition comprising at least one immunogenic epitope associated with the target pathogen or disease, including at least one immunogenic epitope which is the same immunogenic epitope as the first composition, ```wherein at least one of the first and second composition comprises a nucleic acid encoding said immunogenic epitope in a replication impaired HSV vector, provided that the vector used in the first composition is different to the vector used in the second composition.

Description

VECTOR

The present invention relates to herpesvirus vectors. In particular, the present invention relates to attenuated herpesvirus vectors such as DISC herpesvirus vectors for use in prime-boost immunisation.

The present invention also relates to prime-boost vaccination methods using herpesviral vectors, in particular heterologous prime-boost vaccination regimes employing two different non-replicating viral vector compositions, one of which is a herpesvirus vector.

BACKGROUND

Prime-boost vaccination strategies have been proposed in the art. In general, prime-boost vaccination has been attempted using naked DNA and poxvirus-based vaccines (see EP 0979284).

The most used poxvirus for vaccination is vaccinia, which was used for vaccination against smallpox. In common with the other poxviruses, vaccinia virus resides within the cell cytoplasm where it expresses the proteins needed for viral replication. Recombinant vaccinia can, therefore, deliver foreign antigens to the cytoplasm of mammalian cells, thereby allowing them direct access to antigen processing pathways which leads to presentation of antigen derived peptides in association with MHC Class I and Class II molecules on the cell surface (Moss, 1991, Proc Nati Acad Sci USA 93, 11341-8). This property makes vaccinia useful as recombinant vaccines, particularly for stimulating CD8+ and CD4+ T-cell immune responses.

Concern about the capacity of vaccinia virus to replicate in mammalian cells has limited its clinical use and led to the search for safer alternatives. These include attenuated vaccinia viruses, such as modified vaccinia Ankara (MVA) (Meyer el al, 1991, J Gen Virol 72, 1031-8; Sutter and Moss, 1992, Proc Nati Acad Sci USA 89, 10847-51; Sutter et a!, 1994, Vaccine 12, 1032-40), which undergoes limited replication in human cells * * *.* *.s.

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(Blanchard et a!, 1998, J Gen Virol 79, 1159-67), and the avipox viruses, such as fowlpox, which do not proliferate in mammalian cells (Somogyi et a!, 1993, Virology 197, 439-44).

Herpesviruses have been proposed for use in prime-boost applications as priming agents (see W00224224) and more generally (see W00044410). However, herpesviruses have never been tested in prime-boost vaccination and their effectiveness has never been demonstrated. Herpes Simplex virus is an enveloped, icosahedral and double stranded DNA virus large enough to encode roughly 70 transcripts of which only half are required for viral replication. This suggests ample space for recombinant gene insertions and the delivery of multiple genes in one vector. Like other large DNA viruses, HSV has also evolved a variety of mechanisms to evade the host immune responses to infection. The Fc and complement receptors on the surface of HSV weaken humoral responses while its cell to cell spread and latency in neurons facilitate the evasion of antibody neutralisation and clearance. HSV is also capable of inhibiting antigen presentation to CD8 cells by blocking the viral peptide presentation by the MHC-I of the infected cells. HSV possesses a number of valuable characteristics such as a high transduction efficiency, the ability to infect post-mitotic cells and a large transgenic capacity.

Attenuated HSV vectors are known, which do not replicate in host cells. However, gene expression in such vectors is unreliable. Defective/helper systems can be constructed with blocks at various stages of the replication cycle, which reliably express foreign genes but which are unable to sustain an infection. Defective infectious single cycles (DISC) viruses have been derived from both HSV types 1 and 2 with a block at the late phase, which kill any permissive cells they enter, but are unable to sustain infection by entering further cells (single cycle' viruses). The most tested DISC viruses lack glycoprotein H, a surface glycoprotein essential for virion infectivity and spread of virus from cell to cell by the contact route.

Such HSV vectors have been proposed for vaccination, especially against HW, but no reports of their usefulness in prime-boost protocols have been published. In the art, there is a strong perception that priming with naked DNA and boosting with a poxvirus such as * * *** *.s * * * : * * * S.. * S * * . * S * : : : . * ** ** : * S S S MVA is a very effective route to prime-boost vaccination (e.g. Woodberry et a!., The Journal of Immunology, 2003, 170: 2599-2604). Experiments have failed to show that other prime-boost strategies are consistently significantly better than DNA-MVA approaches.

SUMMARY OF THE INVENTION

We have now determined that HSV vectors can be used to provide highly efficient compositions for prime-boost applications in vaccination. In particular, we have found that HSV vectors are highly efficient boosting agents when used in prime-boost applications, and are as efficient as MVA vectors. HSV vectors therefore provide a viable alternative to MVA. Moreover, HSV DISC mutants are particularly effective as both priming and boosting compositions.

In a first aspect, therefore, there is provided a method for vaccinating a subject against a target pathogen or disease, comprising administering at least one dose of: (a) a first, priming, composition comprising at least one immunogenic epitope associated with the target pathogen or disease, and (b) a second, boosting, composition comprising at least one immunogenic epitope associated with the target pathogen or disease, including at least one immunogenic epitope which is the same immunogenic epitope as the first composition, wherein at least one of the first and second composition comprises a nucleic acid encoding said immunogenic epitope in a replication impaired HSV vector, provided that the vector used in the first composition is different to the vector used in the second composition.

Advantageously, the second composition comprises an HSV vector. The present inventors have observed that HSV vectors provide highly efficient boosting compositions, as efficient as the MVA boosting compositions of the prior art.

Preferably, the first composition comprises an HSV vector. However, where the first and second compositions both comprise HSV vectors, the vectors are different.

* * *s* *** . * * * * * S * *** * * * * * * : : : : * *. * * : * S S S S Advantageously, either the first composition or the second composition, but not both, comprise HSV vectors.

Preferably, the HSV vector undergoes a single round of replication in a target host cell.

HSV vectors which are capable of a single round of replication in a host cell but which cannot infect other cells, for example DISC mutants, have particular advantages in prime- boost applications and have been shown to be as good as or better than the DNA-MVA

"gold standard" of the prior art.

Preferably, the HSV vector has a deletion in the gH gene locus. Deletion of the gU gene in DISC mutants permits single-cycle replication in host cells, without the risk of infection of other cells. Advantageously, only the gH gene is deleted.

* **. * * * * I * * * I * * I S S S.. S S S S * * : : : : * *. *** : S S * In a further aspect, there is provided a method for vaccinating a subject against a target pathogen or disease, comprising administering at least one dose of: (a) a first, priming, composition comprising at least one immunogenic epitope associated with the target pathogen or disease in an HSV vector, and (b) a second composition comprising at least one immunogenic epitope associated with the target pathogen or disease, including at least one immunogenic epitope which is the same immunogenic epitope as the first composition, wherein the vector used in the first composition is different to the vector used in the second composition.

It has been found that HSV vectors and particularly HSV vectors which are capable of undergoing a single round of replication without further infection, such as DISC mutants and particularly those mutants comprising a deletion in the gH gene locus only, have particularly advantageous properties as priming compositions. Such mutants prime more effectively than other vectors, including other HPV vectors.

Advantageously, the vector is a dHIA or dH2A DISC mutant, in which the gH gene alone has been disrupted.

The invention moreover provides for the administration of an immunomodulator in any method as set forth above.

In a further aspect, the invention relates to the use of a replicationimpaired HSV vector to boost a primed immune response in an animal. Advantageously, the replication- impaired HSV vector has a deletion in the gH gene.

For example, the HSV vector is a dH1A or dH2A DISC mutant.

The invention is applicable to the vaccination of animals, especially mammals. More particularly, the invention is applicable to the vaccination of primates, including humans. S..

* * : : * * * S * S * * S.. * * * S * * * : : : : * ** *. : S * In a further aspect, the invention provides a method of boosting a pre-existing immune response in a subject comprising administering a composition comprising a replication- impaired HSV vector to said subject.

In a still further aspect, the invention provides a vaccination kit which comprises: (i) a priming composition which comprises an HSV vector which is dH1A or dH2A; and (ii) a boosting composition for simultaneous, separate or sequential administration.

Moreover, the invention provides a vaccination kit which comprises: (i) a priming composition; and (ii) a boosting composition which comprises an HSV vector.

Preferably, the first and second compositions are capable of expressing the same antigen.

Antigens are inserted into HSV vectors using inactivated gene loci. Preferably, the gH locus is used for insertion. Other gene loci may be used, including ICP4, 1CP22, 1CP47, 1CP27, the Lat region, and combinations thereof.

Moreover, the invention provides the use of a boosting composition or a kit according to previous aspects of the invention in the manufacture of a medicament for treating and/or preventing a disease in a subject.

The methods, kits, compositions etc. according to the invention advantageously elicit cellular immune responses. Preferably, a T-cell immune response is elicited in the subject.

BRIEF DESCRIITION OF THE FIGURES

Figure 1: IFN-y ELISPOT responses elicited by dH1A.LacZ in a single immunization regimen.

Groups of female BALB/c mice (H2d; n=4 mice/group) were immunised intravenously (i.v.) with 1x106 plaque forming units (pfu) of dHIA.LacZ or MVA.LacZ, or * * * *: 1:. * * * . * * S * * S.. * * * S * * * S S S * * * *. * S * S S * * a * * . S intramuscularly (i.m.) with 50tg pCMV.beta DNA vaccine, as indicated. Fourteen days later, the number of IFN-y spot forming cells (SFC) per million splenocytes was determined by IFN-'y ELISPOT using a 3-galactosidase specific CD8 T cell epitope TPH. Columns represent the number of SFC/million SD for four mice per group.

Mice immunized with pCM V-beta elicited stronger TPH-specific IFN-y responses than mice immunized with dH1A.LacZ (P = 1.4x104). However, similar frequencies TPH- specific IFN-y secreting cells were detected in mice immunized with dH1A. LacZ and mice immunized with MVA.LacZ (P= 0.10).

Figure 2: IFN-'y ELISPOT responses elicited by priming with HSV-vectors in a prime-boost immunization regimen.

Groups of female BALB/c mice (n=4 mice/group) were immunised i.v. with lx 106 pfu of HSV virus, or i.m. with 50tg of pCM V-beta DNA vaccine as indicated. Fourteen days later, mice were boosted by i.v. administration of 1x106 pfu of MVA.LacZ, as indicated.

Fourteen days after boosting, the number of IFN-y SFC/million splenocytes was determined (n=4 mice/group) by IFN-y ELISPOT using a -galactosidase specific CD8 T cell epitope TPH. Columns represent the number of SFC/million SD for four mice per group.

Mice primed with dH1A.LacZ, dH1D.LacZ or dH1F.LacZ and boosted with MVA. LacZ elicited stronger TPH-specific IFN-y responses than mice immunized solely with MVA.LacZ (P = <0.05). Similar frequencies of TPH-specific IFNy secreting cells were detected in mice immunized with dHLA.LacZ + MVA. LacZ and mice immunized with pCM V-beta and MVA.LacZ (P= 0.07).

Figure 3: IFN-y ELISPOT responses elicited by dH1A.LacZ in homologous and heterologous prime-boost immunization regimens.

Groups of female BALB/c mice (n=4 mice/group) were immunised i.v. with lx 106 pfu of dH1A.LacZ or with 1x106 pfu of MVA.LacZ, or i.m. with 50tg of pCMV-beta DNA vaccine, as indicated. Fourteen days later, mice were either left alone or boosted by i.v.

administration of lx 106 pfu of dl-I1A.LacZ or MVA.LacZ, as indicated. Fourteen days after boosting, the number of IFN-y SFC/million splenocytes was determined (n=4 mice/group) by 1FN-y ELISPOT using a -galactosidase specific CD8 T cell epitope TPH. Columns represent the number of SFC/million SD for four mice per group.

Mice primed with dH1A.LacZ, elicited stronger TPH-specific IFN-y responses than mice primed with pCMV-beta (P=0.02) when MVA.LacZ was given as a boost. Similar frequencies TPH-specific IFN-y secreting cells were detected in mice immunized with dH1A.LacZ and MVA.LacZ irrespective to the order of administration (P= 0.22).

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DETAILED DESCRIPTION OF INVENTION

"Priming" and "boosting" compositions are defined herein as per the common useage of these terms in the art. A priming composition provides an immune response to an antigen in an organism which has not previously been vaccinated with the antigen, or which has not been previously vaccinated in the vaccination program in question. A boosting composition boosts an already present immune response, which is present due to priming with a priming composition.

Antigens as used herein comprise at least one immunogenic epitope which is characteristic of a disease or pathogen and which is capable of raising an immune response. Preferably, it is a cellular immune response, such as a T cell response.

An HSV vector is a herpes simplex virus which has been incapacitated such that its replication is impaired, as defined herein; the vector also comprises at least one heterologous nucleic acid encoding an immunogenic epitope or antigen.

Viruses and viral vectors The present invention relates to vaccination regimes using non-replicating viral vectors.

Many viral vectors are known in the art which are capable of delivering an nucleotide of interest (NOD via infection of a target cell. Suitable recombinant viral vectors include but are not limited to adenovirus vectors, adeno-associated viral (AAV) vectors, herpes-virus vectors, retroviral vectors, lentiviral vectors, baculoviral vectors, poxviral vectors or parvovirus vectors (see Kestler et all 999 Human Gene Ther 10(10): 1619-32).

Examples of retroviruses include but are not limited to: murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus S *SP: ** *:0 $ S S - $ S S I.. * . * - S S $ * S S S $ * $ S S

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(FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).

A detailed list of retroviruses may be found in Coffin et al("Retroviruses" 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763).

Poxviruses In a preferred embodiment the present invention provides a vaccine, priming or boosting composition which comprises a nonreplicating pox virus vector.

The family of poxviruses can be split into two subfamilies, the Chordopoxvirinae and the Entomopoxviriniae. The Chordopoxvirinae (poxviruses of vertebrates) include orthopoxviruses, parapoxviruses, avipoxviruses, caripoxviruses, leporipoxviruses, suipoxviruses, molluscipoxviruses and yatapoxviruses. A review of poxviruses, their structure and organisation, biological and antigenic properties is given in Murphy et al (1995) Virus Taxonomy Springer Verlag, Vienna pp79-87. The following table (Table 2) gives some examples of species within each genus of the pox virus family.

Table 2

Genus Species Orthopoxvirus buffalopox virus, camelpox virus, cowpox virus, ectromelia virus, monkeypox virus, rabbitpox virus, raccoonpox virus, teterapox virus, vaccinia virus, variola virus, voleopox virus, skunkpox virus, Uasin Gishu disease virus Parapoxvirus bovine papular stomatis virus, orf virus, parapoxvirus of red deer in New Zealand, pseudocowpox virus, Auzduk disease virus, chamois contagious ecthyma, sealpox virus

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S 4e1 ** * : . * : * :. :: . Avipoxvirus canarypox virus, fowipox virus, juncopox virus, mynahpox virus, pigeonpox virus, psittacinepox virus, quailpox virus, sparrowpox virus, starlingpox virus, turkeypox virus, peacockpox virus and penguinpox virus Capripoxvirus goatpox virus, lumpy skin disease virus, sheeppox virus Leporipoxvirus hare fibroma virus, myxoma virus, rabbit fibroma virus, squirrel fibroma virus Suipoxvirus swinepox virus Molluscipoxvirus Molluscum contagiosum virus Yatapoxvirus Yaba monkey tumor virus The present invention provides a vaccination kit which comprises an HSV vector as a priming andlor boosting composition. Where an HSV vector is not used, however, another non-replicating viral vector such as a poxvirus vector may be used.

One of the compositions may act as a "priming" composition, to be administered first, and the other composition may act as a "boosting" composition, to be administered after an appropriate time interval (such as three weeks).

The first and second non-replicating viral vectors should be sufficiently different that no significant cross-reaction occurs.

The two viral vectors may be derived from viruses belonging to different families, for example, a poxviral vector and an HSV vector.

Non-replicating The virus vectors used in the present invention should be non-replicating in the cells of the subject (for example in human cells). The term "non-replicating" or "replication- impaired" as used herein means not capable of replication to any significant extent in the majority of normal subject cells. Viruses which are non-replicating or replicationimpaired may have become so naturally (i.e. they may be isolated as such from nature) or S..

*.. *S* * S * * * * : : * S S * S ** * S S S * * ** ** S ! . . artificially e.g. by breeding in vitro or by genetic manipulation, for example deletion of a gene which is critical for replication. There will generally be one or a few cell types in which the viruses can be grown. A replication-impaired HSV vector, as referred to herein, is a herpes simplex virus in which at least one gene essential for virus replication has been deleted or inactivated.

Some HSV genes essential for viral replication are shown in the table below: Glycoprotein L (ULI) Required for production of infectious virions Major capsid protein VP5 (IlL 19) Required for capsid production Major capsid protein VP23 (IJIL1 8) Required for capsid production Glycoprotein H (UL22) Required for production of infectious virions Major DNA binding protein (1JL29) Required for DNA replication Glycoprotein B (UL27) Required for infectious virus production Major Tegument Protein (UL48) Required for virion production (aTIF, Vmw65) ___________________________________ IE- 175 (RSI) Transcriptional regulator required for early and __________________________________ late gene expression glycoprotein D (US6) Required for infectious virion production 1CP27 (UL54). Post-transcriptional regulator The recombinant non- replicating HSV may be derived from either HSV-1 or HSV-2.

Advantageously, the HSV vector is DISC-HSV (as described by W092/05263 and W0942 1807). Preferably, one of the specific mutants described in the table below is used. dHIA and dH2A both undergo a disabled infectious single cycle of replication whereas the others listed are completely nonreplicating.

Gene deletedlinactivated HSV- HSV- ___________________ 1 2 gH dEllA dH2A gI-I+ICP4 dH1D dH2D gH +ICP4 + 1CP22 + 1CP47 dH1E dH2E gEl +ICP4 + 1CP22 + 1CP47 + 1CP27 dH1F dH2F gEl + ICP4 + 1CP22 + 1CP47 + 1CP27 + LAT dH1J dH2J region _______ _______ * *** *.* S * S * * * S S * * * * : * S * S * *S * S * * * S 55 * * S S * 5 Vaccines The HSV vector of the present invention may be used in a method to treat andlor prevent a disease in a subject.

For example, the HSV vector may be employed in a vaccine which is administered to a subject for prophylactic or therapeutic purposes. The vaccine may also comprise an adjuvant (see below).

It has been found that multiple dose vaccination (for therapy or disease prevention) is often more effective than single doses. A multiple dose vaccination program involves doses of two or more different compositions.

In heterologous vaccination programs, there is a "priming" composition which is administered to the patient first and a "boosting" composition which is administered some time later. The HSV vector of the present invention may be used in a priming composition andlor a boosting composition.

A number of other compositions may be employed in heterologous vaccination programs.

Other compositions include "naked DNA", non-viral vector systems and other viral vector systems.

Naked DNA (or RNA) may be linear or circular (far example, a plasmid). It may be provided in a carrier such as a liposome or in a free form.

Suitable non-viral vectors for use in the priming composition include lipid-tailed peptides known as lipopeptides, peptides fused to carrier proteins such as KLH either as fusion proteins or by chemical linkage, whole antigens with adjuvant, and other similar systems.

If a viral vector system is used, it may be an advantage if it is derived from a different virus (i.e. not HSV) to minimise cross-reaction. The vector may be derived from an avipox virus, such as canary pox, or from a different genus of pox viruses. Particularly * *S.

S.. *S* . S S s * * * * * * * : :. . preferred is an attenuated vaccinia vector system such as MVA or NVYAC. Other suitable viral vectors are vectors based on non-pox viruses, such as adeno virus, herpes virus and Venezuelan equine encephalitis virus (VEE). Suitable bacterial vectors include recombinant BCG and recombinant Salmonella and Salmonella transformed with plasmid DNA (Darji A et a! 1997 Cell 91: 765-775).

Heterologous vaccination regimes The present invention also relates generally to heterologous vaccination regimes using two different nonreplicating viral vectors, one of which is HSV.

The present inventors have shown for the first time that heterologous prime-boost regimes using an HSV boosting vector is efficient in generating an immune response in a murine subject.

Triple and Multiple Regimes The present invention also relates generally to multiply heterologous vaccination regimes, such as triply heterologous regimes, using different non-replicating viral vectors.

The invention thus provides a triple regime comprising administering to a subject three heterologous compositions. Preferably said three compositions each differ from their neighbouring composition. For example, if the first composition comprises X then the second composition will preferably differ from X. Clearly, in this embodiment, it is possible that the third composition may be similar or identical to the first composition.

Preferably all three compositions are different from one another.

In one embodiment, one of the compositions may be a DNA based composition such as a DNA vaccine. Preferably at least the second and third compositions comprise non- replicating viral vectors. At least one of the compositions is an HSV vector. *

I * S * * I :.. : : : * T cell responses The vaccination method or program according to the invention preferably elicits a cellular immune response, advantageously a T-cell immune response, in the subject.

The nature of a T cell immune response can be characterised by virtue of the expression of cell surface markers on the cells. T cells in general can be detected by the present of TCR, CD3, CD2, CD28, CD5 or CD7 (human only). CD4+ T cells and CD8+ T cells can be distinguished by their co-receptor expression (for example, by using anti-CD4 or anti- CD8 monoclonal antibodies).

Since CD4+ T cells recognise antigens when presented by MHC class II molecules, and CD8+ recognise antigens when presented by MHC class I molecules, CD4+ and CD8+ T cells can also be distinguished on the basis of the antigen presenting cells with which they will react.

Within a particular target antigen, there may be one or more CD4+ T cell epitopes and one or more CD8+ I cell epitopes. If the particular epitope has already been characterised, this can be used to distinguish between the two subtypes of T cell, for example on the basis of specific stimulation of the T cell subset which recognises the particular epitope.

CD4+ T cells can also be subdivided on the basis of their cytokine secretion profile. The T11 1 subset (sometimes known as "inflammatory CD4 T cells") characteristically secretes IL-2 and IFN'y and mediates several functions associated with cytotoxicity and local inflammatory reactions. TFI 1 cells are capable of activating macrophages leading to cell mediated immunity. The TH2 subset (sometimes known as "helper CD4 T cells") characteristically secretes 11-4, IL-5, IL-6 and IL-b, and is thought to have a role in stimulating B cells to proliferate and produce antibodies (humoral immunity). a' * 11

* :. * * * a a TH 1 and TH2 cells also have characteristic expression of effector molecules. THI cells expressing membrane-bound TNF and TH2 cells expressing CD4O ligand which binds to CD4O on the B cell.

CD4 and CD8 epitopes from desired antigens may be selected for use in the present invention. CD4+ and CD8+ may be selected by comparison with the literature. For example epitope maps for both HIV and HCV may be found via the the Los Alamos National Laboratories website (http:I/www. lanl. gov/worldview/).

Alternatively, likely epitopes may be predicted using the "ProPred" program (epitope prediction program, employing a matrix based prediction algorithm as disclosed in Sturniolo et al. Nat. Biotechnol. 17. 555561(1999) and Singh and Raghava (2001) Bioinfonnatics, 17(12), 1236-37).

If whole antigens are modified for use in the PrimeBoost regime it is preferred that care is taken so that any modifications of the amino acid sequence do not disrupt the amino acid sequence of known epitopes. We describe this approach in our copending international patent application PCT/GB2004/004038.

CD8+ T cell responses are preferred.

The type of T cell immune response may thus be readily determined, for example using fluorescence activated cell scanning (FACScan).

Target antigens The target antigen may be characteristic of the target disease. If the disease is an infectious disease, caused by an infectious pathogen, then the target antigen may be derivable from the infectious pathogen.

In general, target diseases treated by means of the present invention include infectious diseases and cancer. aa a S *

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Infectious diseases include: hepatitis (strains A, B and C), H1V, AIDS, malaria, influenza, Epstein-Bar virus, measles, tuberculosis, toxoplasmosis, herpes, melanomas, adenovirus infection, meningitis, bilharzia, Candida infection, Chicken pox, Chiamydia infection, Creutzfeldt-Jakob Disease, Cytomegalovirus, dengue fever, dengue haemorrhagic fever, Diphtheria (Corynebacterium diphtheriae Infection), Ebola virus infection, E. coli infection, Epstein-Barr virus, Gonorrhea (Neisseria gonorrhoeae Infection), Hansen's Disease (Leprosy), herpes simplex virus, Helicobacter pylon, Hepatitis A-E, Human Immunodeficiency Virus, Human papilloma virus, Human Parainfluenza Viruses, H.pyloniinfection, Legionellosis: Legionnaire's Disease (LD) and Pontiac Fever, Mumps virus infection, Pneumonia, Poliovirus infection, Rabies virus infection, Respiratory Syncytial virus, Rhinitis, Rubella, Salmonella infection, Smallpox, Streptococcus infections, Syphilis, Tetanus, Typhoid Fever (Salmonella typhi Infection) and West Nile Virus infection Hepatitis and HIV are most preferred.

Cancer includes: Melanoma, Breast Cancer, Prostate Cancer, Bladder Cancer, Colon and Rectal Cancer, Endometnial Cancer, Kidney Cancer (Renal Cell), Leukemia, Lung Cancer, Non-Hodgkin's Lymphoma, Ovarian Cancer, Pancreatic Cancer, Skin Cancer (Non-melanoma) Melanoma is most preferred.

Target Antigens are preferably Antigens of infectious diseases and cancer, particularly those listed above, and particularly the melanoma antigens Tyrosinase, gp 100, Melan-A, MAGE-3, TRP-1 and TRP-2) The target antigen may be an antigen which is recognised by the immune system after infection with the disease. Alternatively the antigen may be normally "invisible" to the immune system such that the method induces a non- physiological T cell response. This may be helpful in diseases where the immune response triggered by the disease is not effective (for example does not succeed in clearing the infection) since it may open up another line of attack.

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* S S S I S S **. S S * I S S S * S S S S I * S S I * * * S 55 I 4 IS * S S S S S Preferred Breast Cancer antigens are MUC- 1, HER2, CEA; Preferred Colon cancer antigens: CEA, MUC-l, MAGE-12, mutant P53; Preferred Cervical cancer antigens: human paplioma virus proteins E6 and E7; Preferred EBV-induced B and T cell lymphomas antigens: EBNA1 and 2, LMP 1; Preferred renal cancer antigens: HER-2 neu, RAGE, MUC-l.

Preferred HPV antigens are viral proteins El-8, Li and L2 Preferred HSV antigens are viral proteins gM, gH, gK, GG, gD Preferred HBV antigens are viral proteins small, middle and large surface antigen, core antigen, polymerase Preferred HCV proteins are viral proteins core protein, envelope protein, NS2, NS3, NS4 and NS5 region The antigen may be a tumour antigen, for example HER2/neu, MUC-1, MAGE-1, MAGE-3 or NY-ESO.

The antigen may be an autoantigen, for example tyrosinase.

In a preferred embodiment of the invention, the antigen is derivable from M tuberculosis.

For example, the antigen may be ESAT6 or MPT63.

In another preferred embodiment of the invention, the antigen is derivable from the malaria-associated pathogen P. Falciparum.

The compositions of the present invention may comprise T cell epitopes from more than one antigen. For example, the composition may comprise one or more T cell epitopes from two or more antigens associated with the same disease. The two or more antigens may be derivable from the same pathogenic organism.

Alternatively, the composition may comprise epitopes from a variety of sources. For example, the ME-TRAP insert described in the examples comprises T cell epitopes from P. falciparum, tetanus toxoid, M. tuberculosis and M bovis.

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* . S S * I I SSS S S I * * S S * S S S S S * S S * S I S S IS S * IS * S S S S S Target Diseases The method of the present invention will be useful for treating and/or preventing a number of diseases, especially those which are susceptible to a T-cell mediated immune response.

In particular, the method of the present invention will be useful in the treatment and/or prevention of diseases which are or are caused by chronic infections, particularly persistent, latent infections.

The compositions described herein may be employed as therapeutic or prophylactic vaccines. Whether prophylactic or therapeutic immunisation is the more appropriate will usually depend upon the nature of the disease. For example, it is anticipated that cancer will be immunised against therapeutically rather than before it has been diagnosed, while anti-malaria vaccines will preferably, though not necessarily be used as a prophylactic.

Pharmaceutical compositions/Vaccines The present invention also relates to a pharmaceutical composition such as a vaccine, priming or boosting agent.

The pharmaceutical composition may also comprise, for example, a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.

In particular, a composition comprising a DNA plasmid vector may comprise granulocyte macrophage-colony stimulating factor (GM-CSF), or a plasmid encoding it, to act as an adjuvant; beneficial effects are seen using GMCSF in polypeptide form. Adjuvants such as QS21 or SBAS2 (Stoute J A et al. 1997 N Engl J Medicine 226: 86-91) may be used with proteins, peptides or nucleic acids to enhance the induction of T cell responses.

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* . I S * * S S.. I I S * * * . * S * * I S S * * * . S * S ** S S 55

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In the pharmaceutical compositions of the present invention, the composition may also be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), or solubilising agent(s).

The pharmaceutical composition could be for veterinary (i.e. animal) usage or for human usage. For veterinary usage, the composition may be used to treat for example mammals (especially cattle) or birds.

Preferably the subject is a mammalian subject, in particular a primate (e. g. human) or ungulate (e.g. cow) subject.

Administration In general, a therapeutically effective daily intradermal or intramuscular dose of the compositions of the present invention is likely to range from i05-101 plaque-forming units (pfu).

Typically, the physician or veterinary surgeon will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case.

There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

Transmission of members of the Chordopoxvirinae may occur by aerosol (Murphy et al (1995) as above). The compositions of the present invention may also be administered by aerosol for inhalation by the subject. The compositions of the present invention may also be conveniently administered by injection, such as intradermal andlor intramuscular injection. In addition, the compositions may be administered using a suitable device into the skin or other tissues (for example using a "gene gun" or similar) .

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Where appropriate, the pharmaceutical compositions can be also be administered in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavernosally, intravenously, or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

It is also possible to administer the compositions of the present invention in sustained release formulations.

Immunomodulators The prime and/or boost may also encode immunomodulators such as cytokines and costimulatory molecules.

Cytokines include GM-CSF (preferred), Interleukin (IL)-1 IL-1f3, [L-2, IL3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-lO, IIL-12, interferon (IFN)- a, IFNj3, IFN-'y, Macrophage Inflammatory Protein (MIP)-1a, MIP1-f3, Tumour Growth Factor (TGF)-f, Tumour Necrosis Factor (TNF)-a and TNF-3.

Costimulatory Molecules include ICAM-l, LFA-3, 4-4BBL, CD59, CD4O, CD7O, VCAM-l and OX-40L.

Examples

INTRODUCTION

* S **I *I* * *** * * I S * * S *5S S I S * S S * * S I S S S S I S * . S S S IS S S IS * S S S S S Herpes simplex virus (HSV) replication defective mutants have been shown to be safe for use in humans and have the potential to be used as vectors for immunotherapy against cancer or against infectious diseases.

The following HSV-1-derived replication defective mutants are evaluated as antigen delivery vectors: dH1A, dH1D and dH1F. The dH1A vector has been attenuated by deletion of the gH gene. In addition to the gH gene, the gene encoding for the infected cell protein-4 (ICP4) has been deleted in the dH1D vector. The gH, ICP4, 1CP22, 1CP47 and 1CP27 genes have been deleted in vector dH1F, rendering this virus the most attenuated of those evaluated.

These attenuated HSV vectors all express the Escherichia coli LacZ gene (encoding - galactosidase) under the control of the hCMV immediate-early (IE1) promoter. The LacZ expression cassette was inserted into the essential glycoprotein H gene (gH) of HSV-1.

OBJECTIVES

The primary objective of this study was to evaluate the immunogenic potential of the attenuated HSV-l viruses to act as priming or boosting agents in heterologous prime- boost immunisation regimens. Accordingly, this study aimed to determine the immunogenicity of dH1A, dH1D and dH1F by measuring the 13- galactosidase IFN-y responses in splenocytes of immunized mice using an IFN-y ELISPOT assay in the following experiments: 1) The immunogenicity of the HSV vectors in a single immunization regimen.

2) The ability of the HSV vectors to function as priming agents in combination with modified vaccinia virus Ankara expressing -galactosidase (MVA.lacZ) in a heterologous prime-boost immunization regimen.

3) The ability of the HSV vectors to function as boosting agents in homologous and heterologous prime-boost immunization regimen with MVA. lacZ.

* * S.. *** S *** S S S 0 S * S 0*0 S S. * . S S * . S S S S S S S * S * S S 05 5 5 *S * S P S S MATERIALS and METHODS Constructs The recombinant poxvirus MVA expressing beta-galactosidase (MVA.LacZ), was kept at -80 C until required. After thawing of the MVA.LacZ, virus was sonicated for exactly 1 minute prior to dilution in sterile PBS to 1x107 plaque forming units (pfu)/mL. The disabled HSV constructs dH1A, dH1D and dH1F were kept at -80 C until required.

pCMV-beta plasmid DNA (lmg/ml) was made by the VDG and kept at -20 C until required.

Mice and Immunisations Female BALB/c mice (H2d; 6 - 8 weeks) were used in all experiments and kept in individually ventilated cages at the Biomedical Services Unit, John Radcliffe Hospital, Oxford, in accordance with the Animals (Scientific Procedure) Act 1986 of the UK. Fifty micrograms of the DNA plasmid, pCMV-beta, was administered by intramuscular (i.m.) injection into both anterior tibialis muscles (25tl per muscle). Mice were anaesthetised prior to i.m. immunisation using Hypnorm (Jensen Pharmaceutical Ltd.) and Midazolam (Hypnoval, Roche Products Ltd.).

Immunological Assays IFN-7ELISPOT Immune responses against the MHC class I-restricted peptide TPH recognised by CD8 T cells were determined in spleen cells by ex vivo IFN-y ELISPOT assay. ELISPOT assays were performed following the standard protocol described in W003/047617. Statistical differences between groups were determined by a single-factor ANOVA using Microsoft Excel 2002.

* p *.d *** * *** S.. * S * * * * . * p * S S S P S I * . . . . p. S * ** * S * * S S Peptide Sequence Antigen MHC-restriction I cell recognition TPH TPHPARIGL -galactosidase H2Ld CD8

RESULTS

Experiment 1: IFN-y ELISPOT responses elicited by dH1A.LacZ in a single immunisation regimen.

Mice immunized with a single i.v. injection of 1x106 pfu of dHIA.LacZ elicited detectable frequencies of TPH-specific IFN-y secreting cells (Figure 1). In contrast, a single immunization with Ad5.CMV.LacZ failed to elicit TPH-specific IFN-7 responses (Figure 1). Thus, dH1A.LacZ proved to be immunogenic in mice after a single i.v. administration.

Experiment 2: IFN-y ELISPOT responses elicited by priming with HSVvectors in a prime-boost immunization regimen.

Priming with any of the attenuated HSV constructs and boosting with MVA. LacZ induced significantly (P<O.05) higher frequencies of TPH-specific IFN-y responses than priming with placebo (PBS) and boosting with MVA. LacZ (Figure 2). Moreover, priming with dH1A.LacZ was found to be more potent than priming with dH1D.LacZ or dHIF.LacZ. The magnitude of the TPH-specific IFN-y response following priming with dH1A.LacZ and boosting with MVA. LacZ was comparable to that of the TPH-specific IFN-y response elicited in mice immunized with a DNA vaccine plasmid (pCMV-beta) and boosted MVA. LacZ. Thus, replication defective HSV vectors proved to be potent priming agents, with dHIA.LacZ (in which only the gH gene has been knocked-out) proving to be the most potent priming agent.

* S **d *** * .** S * * * S S S * . * S * I S I I I S S I I II I * ** Experiment 3: IFN-y ELISPOT responses elicited by dH1A.LacZ in heterologous and homologous prime- boost immunization regimens.

Heterologous prime-boost regimens using dH1A.LacZ were significantly more immunogenic than homologous prime-boost regimens using dH1A.LacZ (Figure 3; p<O.OS). Furthermore, no significant difference (P>0.05) in the frequency of TPH-specific IFN-y secreting cells was detected in mice when dHIA.LacZ was used as a priming or boosting agent in a heterologous regimen with MVA.LacZ.

In contrast to previous observations (Section 4.2), MVA.LacZ-boosted mice primed with dH1A.LacZ elicited stronger IFN-y responses than mice primed with pCMV.beta (p<O.O5).

Thus, dHIA.LAcZ proved to be immunogenic as a priming agent, as well as a boosting agent in heterologous prime-boost regimens.

CONCLUSIONS

The HSV-1-derived replication defective mutant dH1A.lacZ, in which a single gene (gH) has been knocked-out, proved to be as immunogenic as MVA. lacZ after a single administration. This vector was also the most immunogenic priming agent when compared with dH1D.lacZ and dHIF.lacZ, which are more highly attenuated than the dH1A.lacZ vector. Heterologous prime-boost using dH1A.LacZ was more immunogenic than homologous primeboost and this vector elicited equivalent immune responses when used as a priming agent or boosting agent in combination with MVA.lacZ. Taken together, these results indicate that the dH1A construct is an immunogenic delivery system that can be used in heterologous prime-boost immunisation regimes.

The inventors have constructed recombinant vectors which encode CD8+ T cell epitopes which are known to elicit an immune response in BALB/c mice. The epitope used has an * S *SS 555 * *.* * * S S S S I S.. I I I S * S I * I I S S S S I * * I S * S SI * 5 SI MHC-restriction H2Ld and mice are H2'. Therefore the immune response elicited can be attributed to a CD8+ T cell immune response.

Figure 1 shows that di-LIA is a good priming composition.

* dH1A is at least as good as MVA for eliciting a TPH-specific immune response.

Figure 2 shows that DISC-HSV is a good priming composition in a PrimeBoost regime.

* There is no significant difference between the TPH-specific response elicited by dH1A/MVA and the response elicited by DNA/MVA.

This is very surprising as DNA/MVA is considered to be an excellent immunisation regime (as shown by Schneider J, Langermans JA, Gilbert SC, Blanchard TJ, Twigg S, Naitza 5, Hannan CM, Aidoo M, Crisanti A, Robson KJ, Smith GL, Hill AV, Thomas AW. A prime-boost immunisation regimen using DNA followed by recombinant modified vaccinia virus Ankara induces strong cellular immune responses against the Plasmodium falciparum TRAP antigen in chimpanzees. Vaccine. 2001 Sep 14;19(32):4595-602.) DNA is highly immunogenic in mice; higher doses of plasmid DNA are needed to induce an immune response in humans. Immune responses are usually elicited in mice using 0.1- lug of plasmid DNA administered by a gene gun and 10-lOOug administered by injection [Fynan, E. F., Webster, R. G., Fuller, D. H., Haynes, J. R., Santoro, J. C., Robinson, J. L. Proc. Natl. Acad. Sci. U. S. A., 1993, 90, 11478.]. In a clinical trial of a malaria plasmid DNA vaccine, 500-2500ug of plasmid DNA was needed to elicit a CTL response [Wang, R., Doolan, D. L., Le, T. P., Hedstrom, R. C., Coonan, K. M., Charoenvit, Y., Jones, T. R., Hobart, P., Margalith, M., Ng, J., Weiss, W. R., Sedagah, M., De Taisne, C., Norman, J. A., Hoffman, S. L. Science, 1998, 282, 476.]. The 6 pair motif (AACGTT) in plasmid DNA that induces optimal stimulation in mice may not work equally well in humans.

Different CpG motifs might operate in humans [Liang, H., Nishoika, Y., Reich, C. F., Pisetsky, D. S., Lipsky, P. E. J. Clin. Invest., 1996, 98, 1119].

* S *** 55. . *** * S 5 5 * * ** S S S S S S S * S S * S I * S S I I S * * II I S *5 Figure 1 shows that DNA is significantly better than HSV at eliciting an immune response when administered in a single shot. The skilled man would therefore assume that DNA would be significantly better than HSV at priming an immune response in a PrimeBoost regime. It is therefore highly surprising that dl-I1AIMVA is at least as good as DNAIMVA.

dH1A is significantly better than dH1D and dH1F for priming a TPHspecific immune response This is a highly surprising result because we would have expected the viruses with more mutations to be more immunogenic. Some of the inactivated genes are responsible for immune evasion in humans therefore their inactivation would likely make the virus more immunogenic in man.

For example 1CP47 (inactivated in dR1F) is known to affect the expression of MHC class I epitopes (Neumann L, Kraas W, Uebel S, Jung G, Tampe R. The active domain of the herpes simplex virus protein 1CP47: a potent inhibitor of the transporter associated with antigen processing. J Mol Biol. 1997 Oct 3;272(4):484-92.) Its presence hinders the body's ability to recognise HSV and therefore its inactivation would be expected to make the virus more immunogenic. The immune evasion mechanism is thought to proceed via transporter protein TAP (transporter associated with antigen processing).The surprising result could be explained by the fact that the interaction of 1CP47 and TAP has been shown to be highly species-specific since ICP 47 has a 100 fold higher affinity for human TAP than fore murine TAP. The observed results may therefore be due to the fact that dHIA undergo a single cycle of replication whereas dHID and dH1F cannot replicate at all.

Figure 3 shows HSV heterologous PrimeBoost is significantly better than HSV homologous PrimeBoost dH1A/MVA elicits an immune response many times greater than that elicited by dH1A/dHIA - and better than DNA/MVA.

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S* S S * Both DNAIdH1A and MVA/dH1A elicit similar responses to those achieved with DNAIMVA.

This is very surprising as DNA/MVA is considered to be an exceptionally good immunisation regime (see Schneider et al above). DNA is shown by Figure 1 to be significantly better than MVA when used in a single shot. However HSV manages to boost the DNA and MVA responses to a level only usually seen with the highly successful DNA/MVA regime.

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Claims

I. A method for vaccinating a subject against a target pathogen or

disease, comprising administering at least one dose of: (a) a first, priming, composition comprising at least one immunogenic epitope associated with the target pathogen or disease, and (b) a second, boosting, composition comprising at least one immunogenic epitope associated with the target pathogen or disease, including at least one immunogenic epitope which is the same immunogenic epitope as the first composition, wherein at least one of the first and second composition comprises a nucleic acid encoding said immunogenic epitope in a replication impaired HSV vector, provided that the vector used in the first composition is different to the vector used in the second composition.
2. A method according to claim 1, wherein second composition comprises an HSV vector.
3. A method according to claim I or claim 2, wherein the first composition comprises an HSV vector.
4. A method according to any preceding claim, wherein the HSV vector undergoes a single round of replication in a target host cell.
5. A method according to claim 5, wherein the HSV vector has a deletion in the gH gene locus.

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6. A method for vaccinating a subject against a target pathogen or disease, comprising administering at least one dose of: (a) a first, priming, composition comprising at least one immunogenic epitope associated with the target pathogen or disease in an HSV vector, and (b) a second composition comprising at least one immunogenic epitope associated with the target pathogen or disease, including at least one immunogenic epitope which is the same immunogenic epitope as the first composition, wherein the vector used in the first composition is different to the vector used in the second composition.
7. A method according to claim 6, wherein the HSV vector is a DISC mutant.
8. A method according to claim 7, wherein the HSV vector undergoes a single round of replication in a target host cell.
9. A method according to claim 8, wherein the DISC mutant comprises a deletion in the gH gene locus.
10. A method according to any preceding claim, further comprising the administration of an immunomodulator.
11. Use of a replication-impaired HSV vector to boost a primed immune response in an animal.
12. Use of a replication-impaired HSV vector having a deletion in the gH gene to prime an immune response in an animal.
13. Use according to claim 12, wherein the HSV vector is a dHIA or dH2A DISC mutant.
14. Use according to any one of claims 11 to 13 wherein said animal is a mammal.

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15. Use according to claim 14 wherein said mammal is a primate.
16. Use according to claim 15 wherein said primate is a human.
17. A method of boosting a pre-existing immune response in a subject comprising administering a composition comprising a replication-impaired HSV vector to said subject.
18. A vaccination kit which comprises: (i) a priming composition which comprises an HSV vector which is dH1A or dH2A; and (ii) a boosting composition for simultaneous, separate or sequential administration.
19. A vaccination kit which comprises: (i) a priming composition; and (ii) a boosting composition which comprises an HSV vector.
20. A kit according to claim 18 or claim 19, wherein the first and second compositions are capable of expressing the same antigen.
21. The use of a kit according to any of claims 18 to 20 in the manufacture of a medicament for treating andlor preventing a disease in a subject.
22. A method, composition, kit or use according to any preceding claim, which elicits a T-cell immune response in the subject.

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