WO2005092374A2

WO2005092374A2 - Recombinant herpes simplex virus and uses therefor

Info

Publication number: WO2005092374A2
Application number: PCT/EP2005/003639
Authority: WO
Inventors: Barbara Ensoli; Antonella Caputo; Peggy Marconi
Original assignee: Istituto Superiore di Sanita ISS
Current assignee: Istituto Superiore di Sanita ISS
Priority date: 2004-03-22
Filing date: 2005-03-22
Publication date: 2005-10-06
Anticipated expiration: 2006-09-22
Also published as: WO2005092374A3; GB0406389D0

Abstract

Provided is a replication-deficient HSV encoding a heterologous antigen, and a vaccine comprising the HSV. The vaccine may be directed to viruses, intracellular bacteria or tumours. Also provided is an anticancer vaccine comprising replication-deficient HSV encoding an angiogenesis inhibitor, a cytokine and a suicide gene. A challenge model for screening candidate antigens is also provided, as is an UL13 mutant HSV vector. Further provided are HSV vectors for expression of Hex A and NTFs, and methods comprising said HSV.

Description

RECOMBINANT HERPES SIMPLEX VIRUS AND USES THEREFOR

The present invention relates to the use of the herpes simplex virus (HSN) in a vaccine capable of inducing a CD8⁺ immune response in an animal and in a method of screening heterologous candidate antigens for use in vaccines.

The use of vaccination to combat disease is well known and has, in some cases, such as smallpox, been so successful that it has lead to the eradication of the disease to which it is targeted. Niruses are often used in vaccines, either in an attenuated form or in a fully functional form.

Viruses are often referred to as vectors and are frequently used to deliver antigens. A large number of viral vectors are known for use in vaccines. These may include poxvirus- based vectors, alphavirus self- replicating vectors, adenovirus, and lentivirus, for instance. Many of these viral vectors are known for their capacity to induce in vivo strong Thl and CTL responses, as well as antibody titres against various antigens, such as HIV-1 gene products.

HSV vectors for use in prophylaxis against, for instance, viral infections, show several advantages. HSV vectors have been shown to elicit strong and durable immune responses by various routes of inoculation; the viral DΝA persists inside the host's cell nucleus as an episomal element, thus eliminating safety concerns arising from possible random integration of the viral genome into the host's DΝA.

However, despite recent advances in vaccine technology, bacteria, viruses and cancers still result in a high proportion of fatalities. In particular, viruses such as HIV have resulted in the deaths of millions of people worldwide whilst tumours leading to cancers are one of the principal causes of death, particularly in the western world.

Accordingly, there remains a need in the art for new vaccines, preferably that may be used with a variety of antigens.

We have, surprisingly, found that HSV is capable of inducing a strong immune response when encoding a heterologous antigen. Thus, in a first aspect, the present invention provides a vaccine capable of inducing a

CD8 immune response in an animal, the vaccine comprising replication-deficient HSV, the

HSV comprising polynucleotides encoding a heterologous antigen.

Preferably, the polynucleotides are DNA, although RNA is also envisaged. A heterologous antigen is an antigen that is not derived from HSV.

Cytotoxic T cell activation is part of the so-called cellular immune response, and leads to mature cytotoxic T lymphocytes (CTLs) specific for a particular antigen. This is part of the MHC1/CD8⁺ antigen processing, recognition and clearance pathway. Mature CTLs, aided by CD4⁺ Helper T cells, recognise antigens presented on an infected or cancerous cell, leading to the lysis of said cell.

The HSV, having been introduced or administered to an animal as part of a vaccine, enters suitable cells, such as endothelial, mucosal or nerve cells, and proteins encoded within its genome are expressed. In the present invention, the HSV genome encodes a heterologous antigen. The expression of the antigen in the infected cell induces a CD8 immune response as the antigen is processed in the cell, such that a fragment or peptide thereof is presented on the cell surface in combination with a MHC Class 1 molecule. As is well known, immature CTLs with Class 1 -restricted antigen-specific T cell receptors and CD 8 molecules on their cell surfaces, bind to the peptide-MHCl complex, leading to cytokine release, and CTL proliferation and differentiation. This in turn leads to mature and memory CTLs specific for the presented peptide and, therefore, the antigen. Such an immune response can be easily recognised by the increased levels in the blood of cytokines such as IL-1 and IL-2, together with the large increase in the number of CTLs.

The animal to which the vaccine is administered may be any animal capable of inducing such an immune response, but preferably a mammal, more preferably a mouse or a monkey, and most preferably a human.

The antigen, when expressed in the host or patient induces an immune response, preferably a CD8⁺ response. The antigen may be functional or non-functional protein or a fragment thereof, provided that it comprises at least one epitope and can, therefore, be bound by antibodies or raised to the antigen or lymphocytes specific for the antigen. The vaccine may be administered by any means known in the art. Preferably, the vaccine is adapted for the means of administration. The vaccine may be suitably formulated together with a pharmaceutically acceptable carrier or diluent, such that it may be administered orally, transdermally, or through a mucous membrane such as the nose or rectum. . It is, however, preferred that the vaccine is administered intravenously, intraperitoneally, subcutaneously, intramuscularly or intradermally. It will be understood that the formulation of the vaccine is dependent upon the preferred means of administration.

The vaccine may be administered as a so-called "single-shot" vaccine, wherein the vaccine is preferably administered once to the same animal. However, the skilled person will appreciate that the vaccine can also be administered repeatedly as part of a repeated vaccination regimen. Alternatively, it is also preferred that the vaccine forms part of a prime- boost regimen. Such a regimen may be a homologous or a heterologous prime-boost regimen.

In a homologous prime-boost regimen, the vaccine may be administered at separate times, the first occasion being the "prime" and subsequent occasions being the "boosts".

In the case of a heterologous regimen, the vaccine may be administered together with, but preferably separate from, a further means of inducing an immune response. This further means of inducing an immune response preferably comprises the same antigen as the present vaccine, which may be in the form of a protein or peptide. Alternatively, this further means of inducing an immune response may comprise a polynucleotide encoding the antigen, for instance a plasmid or a non-HSV viral vector, such as an adenovirus, poxvirus, lentivirus or retro virus.

The HSV, due to the size of its genome, can accommodate more than one inserted gene, in general. Accordingly, the HSV of the present invention may encode more than one antigen, although this depends on the size of the genes. The further antigens may be derived from the same source as the first antigen, for instance multiple epitopes from the same protein or, alternatively, different proteins derived from the same source. For instance, should the vaccine be directed towards HIV, then the antigen may be the entire ENV HIV protein or any of its peptides, and the further antigens may be either separate peptides from the same ENV (clades A, B or C) protein or may be derived from other HIV proteins such as the GAG, REV, POL or NEF proteins. Alternatively, the antigens encoded by the HSV may also be derived from separate sources. For instance, one of the antigens may be derived from HTV, whilst the other may be derived from another (non-HSV) virus, an intracellular bacterium or may be a tumour-associated antigen (TAA).

Furthermore, the HSV may encode one or more antigens as well as any number of further proteins, such as Cytokines, that serves to enhance or regulate the immune response to the antigen or antigens. Alternatively, the HSV may encode a co-factor or inhibitor that may have a regulatory effect on the patient's immune response or other biological processes, such as cell cycle. The HSV may also encode an enzyme that has an activity in the infected cell once transcribed, such as a suicide gene.

The HSV according to this aspect of the present invention is replication-deficient or defective. Accordingly, the HSV is not capable of copying or replicating itself in its entirety once it has entered the host cell. Therefore, once the virus has entered the host cell, its lifecycle is effectively over and further host cells cannot be infected as no "daughter" virions are produced. Accordingly, it is preferred that the HSV is substantially or essentially avirulent in the patient. In particular, it is preferred that the HSV is unable to trans-induce immediate-early gene expression.

However, it is essential that the lifecycle of the HSV vector is not so affected that the DNA encoding the antigen is not expressed. The HSV may be rendered replication-incapable by a number of means known in the art. The impairment of replication is preferably by mutation or alteration of the genes or promoters responsible for HSV replication. Such a mutation may be by insertion, deletion or rearrangement, for instance by U. V. knock out.

Preferably, the vector is rendered replication-deficient by replacement of the genes or promoters responsible for HSV replication by another gene.

Whilst it is preferred that the virulence, and preferably the toxicity, of the HSV virus is substantially reduced, and preferably completely eradicated, by the step of rendering the HSV replication-deficient, it will be appreciated that targeted mutation or replacement of the viral genome, or parts thereof, is preferred so that the remainder of the HSV genome is retained, thus helping to induce a stronger immune response against the HSV, which preferably aids an immune response to the HS V-encoded antigen via a synergistic mechanism.

In particular, it is preferred that one or all of the following immediate-early genes, ICP4, ICP22 and ICP27 are mutated in such a way that their expression is prevented or that the protein expressed is non-functional. The VHS (Wirion Host Shutoff) structural protein may also be mutated with the same effect. Accordingly, a preferred embodiment is the triple mutant ICP4^", ICP22^" and ICP27^", which may also, optionally, include a mutated VHS gene (VHS^").

It is particularly preferred that the heterologous polynucleotides, such as those encoding the antigen or further genes (which may include a further antigen or a cytokine or suicide gene), for instance, are provided as independent expression cassettes, inserted at different loci in the HSV genome, and preferably at HSV genes that are responsible for immediate early protein expression, preferably also rendering the HSV replication-deficient. For instance, the HIN-1 env gene may be inserted in the US1 locus (encoding ICP22 protein) and the HIN-1 tat gene may be inserted in the UL41 locus, and so on. Thus, a unique vector backbone is achieved. The backbone is represented by the ICP4-ICP22-ICP27- mutant HSV-1.

It is particularly preferred that, where the HSN comprises polynucleotides encoding more than one heterologous gene (for instance an antigen and a cytokine) these heterologous genes are under the control of separate promoters in separate independent expression cassettes. Preferably, each independent expression cassette is used to mutate, disrupt and preferably replace an immediate early gene, and preferably UL13, but most preferably one of ICP4, ICP22, ICP27 and UL41 (encoding the NHS protein of HSN).

Viral mutants deleted simultaneously for the IE genes encoding ICP4, ICP22 and ICP27 showed substantially reduced cytotoxicity compared with viruses deleted for ICP4 alone or ICP4 in combination with either ICP22, ICP27 or ICP47 (see Krisky DM et al., Gene Ther. 1998 Dec;5(12):1593-603.).

The UL13 locus of HSV-1 is in the position 28502-26946 (complementary strand, see the ΝC_001806 NCBI reference and SEQ ID NO. 1). The UL 13 is given separately as SEQ TD NOS. 2 and 3. The UL41 locus (encoding VHS protein) is found at 92637-91168 of the above sequence, and also as SEQ ID NO. 4. The UL54 locus (encoding ICP27 protein) is found at 113734-115272 of the above sequence, and also as SEQ ID NO. 5.

The first RSI locus (encoding ICP4 protein) is found at 131128-127232 of the above sequence, and also as SEQ TD NO. 6. The second RSI locus (there are two of them, localised in the two short repeated regions of the HSV-1 genome, both encoding the ICP4 protein) is found at

147105-151001 of the above sequence, and also as SEQ ID NO. 7. The US1 locus (encoding

ICP22 protein) is found at 132644-133906 of the above sequence, and also as SEQ ID NO. 8.

The US4 locus (encoding gG protein) is found at 136744-137460 of the above sequence, and also as SEQ ID NO. 9. The US5 locus (encoding gJ protein) is found at 137731-138009 of the above sequence, and also as SEQ TD NO. 10.

Thus, it is preferred that the HSV is a mutant, wherein at least one of the following loci are mutated: UL13, UL41 (encoding VHS protein), UL54 locus (encoding ICP27 protein), US1 (encoding ICP22 protein), US4 (encoding gG protein), US5 (encoding gJ protein) and either or both of the two RSI loci (encoding ICP4 protein).

Preferably, these loci are mutated, disrupted or replaced, preferably by insertion of heterologous nucleotides encoding proteins that it is desired to express, as discussed elsewhere.

Therefore, the vaccine is preferably capable of inducing a CD8 immune response in an animal, the vaccine comprising replication-deficient HSV, the HSV comprising polynucleotides encoding a heterologous antigen, where-in the HSV is mutated at at least one of the following loci: UL13, UL41 (encoding VHS protein), UL54 locus (encoding ICP27 protein), US1 (encoding ICP22 protein), US4 (encoding gG protein), US5 (encoding gJ protein) and either or both of the two RSI loci (encoding ICP4 protein). Thus, the HSV is preferably UL13-, UL41-, UL54-,US1-,US4-,US5-RS1-, and preferably a combination of at least two of these. In other words, it is preferred that the HSV comprises mutations in any of SEQ ID NOS. 3, 4, 5, 6, 7, 8, 9 or 10.

Most preferred is the mutant HSV which is UL54-, US1- and RS1-, where the HSV comprises mutations in SEQ ID NOS 5, 6, 7 and 8, i.e. 5, 8 and 6 or 7.

The HSV is preferably rendered replication-defective by incorporating non-reverting mutations into mandatory viral genes, such that the HSV maintains the immunogenicity of wild-type HSV, but is much safer.

Non-reverting mutations are preferred in comparison with mutations that are capable of reversion. Mutations that are capable of reversion usually involve only minimal changes in the original DNA sequence, such as transitions, transversions, or frame shifts. Thus, whilst any mutation or disruption of the viral genome that is sufficient to render the HSV substantially avirulent is preferred, it is more preferable that the mutation is large enough to render the HSV substantially avirulent permanently.

An advantage of the deletion of a series of viral immediate early genes such as ICP4, ICP22 and ICP27, is that this not only substantially reduces cytotoxicity, it also enhances long-term transgene expression.

These HSV mutants showed a reduced immunogenicity, due to their inability to replicate and to spread in the host, but still retain the capability to infect a wide range of tissues and host species.

In addition, these recombinant replication-deficient viral vectors can sustain high expression of any foreign or heterologous genes, such as an antigen to which the vaccine is directed, under homologous or heterologous promoters, such as HSV-1 or HCMV, respectively (references 47 and 48 in Example 5). Furthermore, their large genome can accommodate a relatively large antigen, or even multiple antigens (reference 49 in Example 5), which can be simultaneously expressed. Recent studies also indicate that pre-existing immunity against HSV infection does not compromise its efficacy as a vaccine vector (references 50 and 51 in Example 5).

The DNA or polynucleotides encoding the antigen is preferably under the control of a promoter and even more preferably under the additional control of an enhancer. Such genetic elements are well known in the art. Thus, the DNA encoding the antigen may form together with such elements an expression cassette.

Most preferably, the DNA or polynucleotides are under the control of an HSV immediate early promoter. However, it is also preferred that the antigen is under the control of a promoter such as the human Cytomegalovirus (CMV) promoter.

Preferably, the promoter is inducible, preferably by an extrinsic factor. This allows the administrator of the HSV, such as a health official, to induce expression of the antigen or heterologous DNA encoded by the HSV when required, which may be some time after infection of the patient by HSV.

Thus, it is also envisaged that two or more promoters can be used, both controlling separate or different heterologous genes. The first promoter, for instance, may lead to high levels of expression of the first gene on infection of the patient by HSV, i.e. on administration of a vaccine comprising the HSV of the present invention, whilst a second promoter controlling expression of the other gene, is induced at a later stage, for instance as part of a later "boost" as further discussed below. The inducer could be taken orally, for instance, which may be more acceptable than the preferred means of administration of the HSV itself, which may be intravenously.

Thus, the heterologous DNA encoded by the HSV is under the control of at least one, and preferably at least two, inducible promoters. Preferably, the inducer of the promoter is an extrinsic factor. A suitable example of such a promoter and inducer system is the Tet-on/Tet- off promoter system, which is induced by the absence of tetracycline in the diet.

However, whilst it is envisaged that expression of the antigen or heterologous DNA may be delayed under the control of a suitable promoter such as the Tet-on/Tet-off promoter, this is not generally preferred.

What is preferred is that the promoter used results in an immediate and high level of antigen protein or peptide expression, such that the antigen is preferably co-temporaneously expressed with an immune response raised against the HSV vector itself.

Indeed, it is a preferred embodiment of the present invention that the antigen or heterologous DNA is expressed as soon as possible after the vaccine is administered or introduced into the animal. Without being bound by theory, it is thought that there is a synergistic effect between the use of HSV, which itself raises an HSV-directed immune response, and the antigen-specific immune response. It is thought that the presence of HSV viral vectors may result in a stronger antigen-specific immune response, as both are likely to raise MHC1/CD8 cellular immune responses. Therefore, it is preferred that both the anti- HSV and the antigen-specific response are raised at the same time.

As mentioned above, it is particularly preferred that, where the HSV comprises polynucleotides encoding more than one heterologous gene (for instance an antigen and a cytokine) these heterologous genes are under the control of separate promoters in separate independent expression cassettes. Preferably, each independent expression cassette is used to mutate, disrupt and preferably replace an immediate early gene, and preferably UL13, but most preferably one of ICP4, ICP22, ICP27 and UL41 (encoding the VHS protein of HSV). Whilst reference is made herein to antigens, it will be understood that the present invention relates to any heterologous DNA or genetic material that it is wished to be expressed. Thus, it will be understood that the terms are interchangeable, unless otherwise apparent.

The antigen may be derived from a number of different sources, as mentioned above. Preferably, the antigen may be derived from a disease-causing agent. This disease-causing agent may be a fungus or a parasite, such as Plasmodium ssp.

Preferably, the antigen may be derived from a virus, an intracellular bacterium, or a tumour. Suitable viruses include HIV, in which case the antigen is preferably derived from the POL, ENV (clades A, B or C), REV or NEF HIV proteins or peptide fragments thereof. It is particularly preferred that the antigen is derived from the HIV GAG protein.

Example 6 shows that whilst recombinant HSV-1 derived vaccines may only be weak inducers of CD4 T helper dependent antibody responses, they are capable of activating or inducing efficient long-term CD8 T cell responses. We show that the T0H:gag and TO-tat replication-defective HSV-1 recombinant vectors were able to elicit a Gag- and Tatspecific immune response respectively in immunized mice, although the breadth of the response was different depending on the site of inoculation.

The antigen is preferably not derived from SIV, unless the HSN further encodes a cytokine or suicide gene, or both, as discussed elsewhere.

It is also preferred that the antigen is derived from an intracellular bacterium. These may be, for instance, Listeria, Salmonella or M. tuberculosis bacteria.

Furthermore, the antigen may be derived from a tumour or pre-cancerous cell which may be in the process of becoming cancerous. Preferably, the antigen is a tumour-associated antigen (TAA), which are well known in the art.

The tumour may be a neoplasia, glioma or glioblastoma and is preferably derived from cancerous endothelial or CΝS cells. However, it is also preferred that the antigen is derived from any cancerous cell, including those associated with AIDS-related dysfunctions, such as Kaposi's sarcoma, as discussed below. Preferably, the HSN is Heφes Simplex Virus - type 2 and, even more preferably, Herpes Simplex Virus - type 1, also known as HHV (Human Herpes Virus).

It is also preferred that the HSV further encodes a cytokine. Suitable cytokines will be known to the skilled person. However, these may, preferably, include Interleukins or Colony- Stimulating Factors, in particular those that promote a Thl Type response in Macrophages, ΝK Cells, and/or induction of IFΝ-Gamma production.

Particularly preferred are the cytokines IL-12 and GM-CSF. Granulocyte macrophage-colony stimulating factor (GM-CSF) is a potent stimulator of macrophages and Dendritic Cells which are important antigen-presenting cells involved in the induction of immune response. _. Literleukin 12 (IL-12), is a heterodimeric Thl cytokine with strongly immunomodulatory properties that promotes the proliferation of T cells, ΝK cells and tumour-infiltrating lymphocytes (TIL cells). In addition, IL-12 can induce a cascade of other cytokines and chemokines which possesses significant antiangiogenic properties.

It is, therefore, particularly preferred that the HSV comprises DΝA encoding a cytokine as well as an antigen. It is particularly preferred that when the antigen is derived from SIN, the HSN also comprises DΝA that encodes a cytokine.

An advantage of the HSV expressing a cytokine is that cytokines are, in general, toxic and it is therefore preferable that they are not administered systemically. According to the present invention, the cytokine is expressed locally in the relevant cell, therefore reducing the need for systemic administration.

It is also particularly preferred that the HSV comprises DΝA encoding a suicide gene.

This may be in combination with the antigen alone (or other heterologous DΝA), or, even more preferably, the HSV comprises DΝA encoding a suicide gene, an antigen and a_. cytokine.

The suicide gene may be a gene encoding viral or bacterial enzymes that converts an inactive form of a drug ("prodrug") into toxic anti-metabolites capable of inhibiting nucleic acid synthesis, for example. Suicide genes code for enzymes that convert nontoxic compounds (prodrugs) into toxic products. Gene therapy with suicide genes is considered one of the most powerful approaches for cancer treatment: tumor cells transduced with suicide genes can be eliminated upon treatment with the prodrug. Many suicide gene therapy approaches have been successfully used in animal models of cancer, and are currently being tested in clinical trials. Among more than 30 suicide genes described, the most preferred are the HSVltk and the cytosine deaminase (CD) genes, as these are thought to be the most potent and widely used.

Preferably, the suicide gene is a humanized suicide gene, as these are thought to have an improved cytotoxic activity.

One of the suicide genes most used to convert a non-toxic pro-drug into a toxic molecule is the Herpes simplex TK. Virus-originated HSV-TK gene is different from that of mammals. Its product, thymidine kinase, is able to metabolize the nontoxic prodrug, GCV, into a monophosphate derivative, then phosphorylate it further into GCV triphosphate. This metabolite is incorporated into replicating DNA strands and acts as both a DNA synthesis inhibitor and a cell cycle blocker, finally leading to cell apoptosis and cell death. The therapeutic effect of this system is also based on a "bystander effect" whereby HSV-TK gene modified cells are toxic to nearby unmodified cells when exposed to the antiviral drug GCV. The mechanism underlying this "bystander effect" is complex, but the primary mechanism is thought to be "metabolic cooperation" involving formation of gap junctions between cells that permit the passage of small molecules from one cell to another (Reference 39 from expt 2).

Thus, it is particularly preferred that the suicide gene is the HSV-1 tk gene, as not only is "native" to the HSV, thus obviating any potential complications with the HSV genetic machinery, but the suicide gene is already present in HSV-1, so it does not need to be inserted by a user, thus simplifying the recombination process. Of course, the skilled person will appreciate that some care must be taken to ensure that the HSV-1 tk gene is not mutated or disrupted, so that the functional enzyme is produced.

In this instance, the animal is administered Ganciclovir at a suitable point, preferably co- temporaneously with the administration of the vaccine. As is known, Ganciclovir is relatively non-toxic. It will be appreciated that suitable amounts of Ganciclovir will need to be administered, such that sufficient Ganciclovir is available for conversion into the toxic form in all HSV infected cells.

A further example of a suitable suicide gene is the bacterial cytosine deaminase (CD) system, for instance the Escherichia coli cytosine deaminase/5-fluorocytosine (CD/5-FC) system. The combination of HSV-TK/GCV and CD/5-FC has proven to be very potent for experimental brain tumour treatment.

The TK system confers sensitivity to the respective pro-drug and the cytotoxic effect acts synergistically with combined expression of cytokines (references 16 and 40 in Experiment 2). Accordingly, it is preferred that the suicide gene and the cytokine gene are expressed co-temporaneously and, furthermore, that they are preferably expressed at the same time as the antigen. Thus, it is also preferred that at least two and, preferably three, of these genes are within different independent expression cassettes.

Where the antigen is derived from a tumour, it is also preferred that the vaccine further comprises DNA encoding an angiogenic inhibitor.

Furthermore, in a further aspect of the present invention, there is provided a vaccine capable of inducing an anti-cancer effect in an animal, comprising replication-deficient HSV whose DNA encodes an angiogenic inhibitor, a cytokine and a suicide gene. Preferably, the effect comprises an anticancer CD8⁺ immune response. Preferably, the HSV may also encode an antigen, preferably a tumour-associated antigen.

In both aspects, the vaccine preferably also comprises DNA encoding connexin 43 (CX43) which is involved in the formation of gap junctions, as this can increase the efficacy of the suicide gene, in particular the TK suicide gene, by enhancing bystander killing (references 2 and 29 from Experiment 2). Preferably, the antitumour response is selective and attacks the primary tumour, inhibits metastasis, prevents recurrence, and does not promote drug resistance.

Preferred anti-angiogenesis factors include angiostatin, kringle 5 (K5), or endostatin. Preferably, these areendostatin:: angiostatin or endostatin-kringle5 fusion proteins cloned in UL41 locus combined with IL-12 or GM-CSF,in US1 locus of HSV. . iό Angiogenesis is a complex process that includes endothelial cell proliferation, migration and differentiation, degradation of extra-cellular matrix, tube formation, and sprouting of new capillary branches. This process is tightly regulated by angiogenic factors and an unbalance between angiogenic stimulators and inhibitors leads to progression of many diseases, including tumour growth (Reference 14 from expt 2)

Angiostatin is one of the first identified endogenous specific inhibitors of tumour- related endothelial cell proliferation; it contains the first four disulfide-linked structures of plasminogen (Pgn), known as kringle structures (Reference 8 from expt 2). Smaller fragments of angiostatin display differential effects on suppression of endothelial cell growth but integrity of the kringle structures of angiostatin is required to maintain its inhibitor potency (References 25, 31 from expt 2).

Accordingly, it is preferred that the anti-anigiogenesis factor is Angiostatin or a functional variant thereof, wherein the integrity of the kringle structures of Angiostatin is retained.

Kringle 5 (K5) is another fragment of Pgn that, although related to the other four kringles in Pgn, is not present in angiostatin. K5 is a specific inhibitor of endothelial-cell proliferation and appears to be more potent than angiostatin (Reference 7 from expt 2). The combination of angiostatin and K5 exerts a synergistic inhibitory effect on endothelial cell proliferation. In parallel to the discovery of angiostatin, a proteolytic fragment of type XVIII was identified: endostatin (Reference 32 from expt 2). The identification of agents that inhibit angiogenesis represents a potential therapeutic approach for the treatment of solid tumours. An angiogenic switch has been hypothesized to be responsible for transition from a slow, dormant phenotype to a faster, more aggressive one, although recent studies have demonstrated the ability of even a few tumour cells to recruit neovessels. Endothelial cell proliferation, migration and tube formation are critical early events during angiogenesis, and their inhibition would be expected to affect the angiogenic process. Here, we present data showing that angiostatic proteins expressed by HSV vectors inhibit endothelial cell proliferation, migration and tube formation in vitro and reduce tumour angiogenesis in vivo. Endostatin is another protein with a potent angiostatic activity (Reference 12 from expt 2). Anti-tumour immunotherapy approaches are also rapidly evolving as we increase our understanding of the molecular events involved in the host's antitumor response (Reference

27 from expt 2).

One of the causes of the tumour establishment may also be regarded as the outcome of the absence or ineffectiveness of an anti-tumour immune response. There are various reasons for failure of this kind, and a sort of "blindness" for neoplastic cells. However it has been shown that in some cases "sight" can be restored by helping the immune system to recognize a tumour and to mount an effective reaction against it (Reference 33 from expt 2). One way of doing this is offered by the use of cytokines released by the transduced tumour cells (Reference 9, 20 from expt 2). hiterleukin 12 (IL12), is preferred.

The suicide genes approach increases the possibility of rendering cancer cells more sensitive to chemotherapeutics or toxin agents (Reference 21, 36 from expt 2). Most suicide genes currently under investigation mediate sensitivity by encoding viral or bacterial enzymes that convert inactive form of a drug ("prodrug") into toxic anti-metabolites capable of inhibiting nucleic acid synthesis, as discussed above.

Preferred examples of suicide genes are the viral thymidine kinase (TK) and the bacterial cytosine deaminase (CD) discussed above.

The cytokine Granulocyte macrophage-colony stimulating factor (GM-CSF), also discussed above, is a potent stimulator of macrophages and DCs which are important antigen- presenting cells involved in the induction of immune response (Reference 26 from expt 2). However, it has also been found that the combined effect of GM-CSF with TK suicide gene increases the anti-tumour effects leading to prolonged survival and partial protection against a subsequent tumour challenge; this effect may be due to the combination of HSVtk/GCV- induced tumour cell death and tumour GM-CSF production attracting a greater number of host APCs that take up antigens derived from the dying tumour cells and cross-present them to the host's immune system (Reference 5 from expt 2). Thus, a vaccine comprising HSV comprising DNA encoding both HSV tk and GM-CSF, is particularly preferred.

Preferably, the activities of the various genetic elements encoded by the HSV, whether heterologous or homologous to the HSV, are synergistic. An example of this is the HSV tk enzyme in combination with GM-CSF, the effects of which are greater than the sum of their separate parts.

A further preferred example is that, in the case that the vaccine is directed to a tumour and the HSV encodes an angiogenic inhibitor, a cytokine and a suicide gene, it is preferred that the anti-tumour effect of the vaccine is greater than three separate vaccines comprising only one of these elements. It is preferred that the synergy applies in all aspects and embodiments of the invention according to the present application, where appropriate. In particular, it is preferred that the combination of expression of an antigen with a cytokine and/or with a suicide gene, is synergistic.

It will, of course, be understood that certain genetic elements, such as the suicide gene or the angiogenesis inliibitor may not themselves be responsible for raising a CD8 response, but that they nevertheless contribute to the effect of the vaccine which is to clear the infected or tumour cell.

By genetic element, it is meant a gene, encoding a protein having a functional effect in the patient. The genetic element may be heterologous to the HSV, such as an antigen, but it may also be homologous to HSV, i.e. derived therefrom, such as the tk gene.

In a further aspect, the present invention also provides a method for screening putative vaccines comprising a candidate antigen in a non-human animal, the method comprising administering said vaccine to said animal and determining whether an immune response is successfully elicited to the antigen by subsequently administering a pathogenic amount of HSV comprising a polynucleotide encoding the antigen.

Preferably, the antigen is first administered by means other than by replication- defective HSV, preferably by means of a plasmid comprising DNA encoding the antigen. In other words, it is preferred that the putative vaccine is not replication-defective HSV, but may, preferably, be a plasmid encoding the antigen.

In this aspect, it is particularly preferred that the HSV used is replication competent, so as to provide a suitably strong challenge to the host animal. It will be appreciated that if the challenge to the host is not strong enough to kill a non-immunised host, then the model will not be as efficient at screening for antigens, as further tests on the host will be required to determine whether any initial protective immunity was raised against the antigen.

Preferably, the animal is a mouse. The HSV may, preferably, be any HSV, including HSV-1 or HSV-2. Preferably, the antigens are as described above, although HTV and SIV viral antigens are particularly preferred. Where HSV-2 is used, it is particularly preferred that the VHS locus is mutated, preferably by deletion, although this should, preferably, not lead to a significant reduction in the replicative ability of the HSV.

A host is first vaccinated with a candidate antigen, where the antigen is a potential candidate for use in a vaccine regimen. To test whether the host has raised an immune response, that preferably leads to long-lasting protective immunity, the host is then further challenged with a replication competent HSV virus, which has been manipulated to express the antigen. The antigen is a heterologous, non-HSV, antigen.

The host is preferably highly susceptible to HSV infection and dies when injected with a lethal dose of the replication-competent virus. However, if the host has previously raised an immune response to the antigen, then HSV infected cells, which express the antigen, will be recognised by the immune system and destroyed, by CTLs, for instance. However, if no immune response was previously raised, then the host will die, indicating that the candidate is not particularly suitable for use in vaccines, at least in that host. Suitable antigens can then be used in vaccine trials in other hosts, such as humans.

For instance, a host is first vaccinated with a candidate antigen, where the antigen is a potential candidate for use in a vaccine regimen. To test whether the host has raised an immune response, that preferably leads to long-lasting protective immunity, the host is then further challenged with a replication competent HSV virus, which has been manipulated to express the antigen. The antigen is a heterologous, non-HSV, antigen.

A host, for instance a mouse, is first vaccinated against the candidate antigen. This may be by any means known in the art, except that replication-deficient HSV are, preferably, not to be used. For instance, the antigen may be delivered in the form of a plasmid comprising DNA, encoding the antigen, or by a viral vector, such as an adenovirus or pox virus. Alternatively, the antigen may be delivered in its native state. If the antigen is a protein, it may be injected, for instance, directly into the mouse as a protein, rather than as DNA encoding the antigen. In this example and Example 4, the HIV Tat protein is used. Having been vaccinated with the antigen, the mouse is then further challenged with a lethal dose of replication competent HSV encoding Tat, for instance by means of an HTV expression cassette inserted into the genome of the HSV virus under the control of a suitable promoter, as discussed elsewhere in the application. If the earlier Tat vaccination has raised a specific anti-Tat protective immunity, the mouse's immune system should respond to the HSV- Tat infection. This response will be detectable from the sera of the mouse, for instance by the increased presence of lymphocytes. If particularly successful, the mouse will rapidly clear the introduced HSV and antigen.

However, if the mouse did not initially raise protective or memory immunity against the Tat antigen, then the mouse should die in 10-15 days.

In this way, suitable candidate antigens can be quickly and rapidly identified.

HSV-1 or HSV-2 expressing the antigen may be used. The antigen is preferably an HTV antigen, as discussed above, but particularly preferred are tat, gag (clade B), and env (clades A, B, and C).

In the HSV-based challenge system, the antigen is introduced according to a previously described method (Marconi et al., Proc. Natl. Acad. Sci. USA, 1996) in a HSV locus which is not essential for HSV-1 and HSV-2 replication (e.g. UL41, US4, US5). These recombinant viruses are able to replicate and to induce disease and death in mice after inoculation of a lethal dose by systemic and mucosal routes. Survival of immunized mice 10-15 days after challenge with a lethal dose of recombinant HSV-1 expressing the vaccine antigens is the efficacy endpoint.

Another HSV-based challenge system is based on HSV-2, or genital herpes, which is more pathogenic in the genital tract as compared to HSV-1.

In a still further aspect, the present invention also provides HSV comprising a mutation in the UL13 protein kinase encoded by HSV, the mutation reducing levels of the HSV ICPO protein. Preferably, the HSV comprises a deletion in the UL13 locus of HSV-1 that encodes for protein kinase. The UL13 sequence is in the viral genome in the locus that goes from position 26946 to 28502 of PubMed nucleotide sequence NC_001806 rwww.ncbi.nlm.nih. eov). SEQ ID NO. 3, and the deletion is in the coding sequence where are deleted 164 bp from Sphl- 27508 to EcoRV-27672.

The nucleotide sequence of UL13 is given in SEQ ID NO. 2 and protein sequence is given in SEQ ID NO. 3. SEQ ID NO. 3 is the nucleotide sequence for the HSV-1, taken from PubMed nucleotide sequence NC_001806.

The deletion is accomplished by insertion of non-native expression cassette encoding the Escherichia coli lacZ gene. In the new deletion, the expression cassette containing LacZ can be substituted by another non-native expression cassette encoding other gene of interest. The new deletion in the mutated vectors increases the capacity of the vector to contain another non-native expression cassette and reduces the residual toxicity of the multiple mutated HSV vector. In cells infected with a mutant lacking UL13, restricted infected cells accumulate reduced levels of regulatory protein ICPO and several late viral proteins. UL13 is involved in the posttranslational modification of the immediate early gene ICPO. ICPO is phosphorylated by the UL13 protein kinase.

A preferred plasmid comprising the UL13 mutant is SK131acZ, as described in Example 10.

Therefore, the present invention provides a vector comprising a deletion in the UL13 sequence of HSV-1 encoding a protein kinase, between position 27508 and 27672 of SEQ ID NO. 1, to provide a mutant lacking UL13 or a function variant thereof, thereby reducing levels of regulatory protein ICPO and several late viral proteins. Therefore, the mutant preferably cannot encode the protein according to SEQ ID NO.3.

A functional variant is a protein having substantially the same sequence as the reference sequence, except that it may comprise a number of mutations or amino acid changes, whilst still retaining substantially the same functional activity, preferably 50% or more activity.

The mutation may affect the coding sequence of UL13, so that the protein encoded lack activity due to imprecise folding, for instance, or the mutation may effect the promotion of expression of the UL13 protein, whether fully functional or otherwise. Thus, it is preferred that the mutant has either none or reduced UL13 activity, whether this is due to reduced expression, reduced functionality of the expressed protein, or both. An advantage of an HSV vaccine according the present invention is that it may be used, as described above and in the accompanying Examples, with a wide variety of substantially different antigens, but still lead to the induction of a strong CD8+ immune response to the antigen

A particular advantage of an HSV vaccine according to the present application is that it may be used a "single-shot" vaccine, making it more acceptable to the vaccinee, cheaper to produce and results in a vaccination regimen that is easier to administer.

, The present invention further provides HSV transformed according to the present invention or for use in a vaccine or screening method of the present invention.

The present invention also provides a host non-human animal or transformant to which the present anti-pathogen or anti-cancer vaccine invention has been applied. This may be an animal to which a vaccine according to the present invention has been administered, or animal which has been used in a screening method according to the present invention. Preferably, the animal is a mouse or rat.

HSV is a complex human neurotrophic virus with several characteristics which make it attractive as a vector for gene therapy (reference 6 from Example 1). Firstly, HSV replication-deficient mutants have been generated by the inactivation of genes essential for the virus life cycle. Furthermore, HSV replication deficient viruses efficiently infect almost all cell types at a relatively low multiplicity of infection.

HSV has a large genome (152 kb) which can accommodate up to 50 kb of exogenous DNA, thus allowing incorporation of multiple transgenes. High titer stocks of replication deficient vectors can also be generated using cell lines which complement the essential HSV functions in trans. Indeed, highly deficient vectors due to the deletion of multiple essential genes have been created. These vectors show minimal cytotoxicity when infecting cells in vitro and in vivo (reference 24, 28 from Example 1).

People with AIDS are particularly prone to develop various cancers, especially those caused by viruses such as Kaposi's sarcoma and cervical cancer, or cancers of the immune system known as lymphomas. These cancers are usually more aggressive and difficult to treat in people with AIDS. Signs of Kaposi's sarcoma in light-slrinned people are round brown, reddish, or purple spots that develop in the skin or in the mouth. In dark-skinned people, the spots are more pigmented.

FGF is one of the prominent cytokines expressed by AIDS-KS cells. The ability of FGF to induce these lesions is augmented (in a synergistic fashion) by the HIV protein Tat, which is secreted by HIV-infected cells ( Reference 27 from expt 3). During acute HIV infections, Tat is released from infected cells. In its extracellular form, Tat stimulates HIV gene expression, the growth of cells derived from Kaposi's sarcomas, angiogenesis and the promotion of tumour metastasis, the development of lymphoid hyperplasia, the secretion of TGF, TNF alpha and beta, IL-6, the malignant transformation of keratinocytes, the inhibition of IL-2 and IL-2 receptor gene expression, and the inhibition of the anti-viral alpha/beta interferon system.

The cell growth-promoting activity and the, virus-transactivating effects of extracellular Tat are mediated by different pathways. Tat is known to activate endothelial cells and to be a powerful angiogenic growth factor. Expression of Tat in transgenic mice induces Kaposi sarcoma-like lesions, squamous cell papillomas and carcinomas, adenocarcinomas of skin adnexa glands, and B-cell lymphomas. Very small amounts of extracellular Tat (0.1 ng/ml) may mimic VEGF by activating its receptor. This would explain a number of AIDS-related dysfunctions associated with endothelial cells, such as Kaposi's sarcoma, arteriopathy, and intravascular coagulopathy or hypercoaguloability of the blood.

The present invention also provides, in a further aspect, a therapy for inherited disorders with neurological implication, such as Tay-Sachs (TS) disease, which requires an active enzyme to be produced in the central nervous system to restore the cellular dysfunction caused by the defected gene.

We used a replication-deficient Herpes simplex vector encoding for the hexosaminidase (Hex) A alfa-subunit (HSV-T0 αHex) and delivered it into the internal capsule of the TS brain animal model. With this gene transfer strategy we re-established the Hex A activity and removed the GM2 ganglioside storage in both injected and controlateral hemisphere and in the cerebellum only one month after treatment. The high efficiency of transduction of HSV-T0 αHex, the correct expression of the transgene, together with the site of injection within the brain, make this gene transfer strategy suitable for the treatment of lysosomal storage disorders with neurological involvement.

Accordingly, there is provided replication-deficient HSV whose DNA encodes hexosaminidase (Hex) A, preferably the alfa-subunit (HSV-TO αHex), and a method of expressing Hex A in a subject, preferably in the subject's brain, preferably by administration of said HSV, preferably so that the HSV is delivered to the internal capsule. Preferably, the levels of GM2 storage are reduced.

Therapy for neurodegenerative lysosomal Tay-Sachs (TS) disease requires an active Hexosaminidase (Hex) A production in the central nervous system and a therapeutic approach that can be efficacious and can act faster than human disease progression. We combined the efficacy of a non-replicating Herpes simplex vector encoding for the Hex A alpha-subunit (HSV- TOalphaHex) and the anatomic structure of the brain internal capsule to optimally distribute the missing enzyme. For the first time, with this gene transfer strategy, we re-established the Hex A activity and the GM2 ganglioside storage in both injected and controlateral hemispheres, in the cerebellum and spinal cord of TS animal model in one month of treatment. In our studies we do not observed adverse effects due to the viral vector, injection site or gene expression and, based on the results, we fill confident that the same approach can be applied to similar diseases involving an enzyme defect.

We focused our attention on the rapid restoration of Hexosaminidase A activity and the reduction of GM2 ganglioside storage in the CNS since they represent the first cause of this metabolic alteration.

Neurotrophic factors (NTFs) are known to govern the processes involved in CNS cell proliferation and differentiation. Thus, they represent very attractive candidates for use in the study and therapy of neurological disorders. Administration of multiple NTFs has been reported to stimulate progenitors located in the periventricular region and in the parenchyma to replace CA1 neurons damaged by ischemia. We have employed replication-deficient herpes simplex virus- 1 (HSV-l)-based vectors engineered to express any kind of NTFs and synergized their effect by expressing the NTFs in one vector. We constructed recombinant Herpes virus based- vectors capable of expressing fibroblast growth factor (FGF-2), ciliary neurotrophic factor (CNTF), brain derived neurotrophic factor (BDNF) and glial derived neurotrophic factor M&C FOLIO: WPP896 β58 P(ΛEP 2005/003639 22 (GDNF) alone or in combinations. Therefore, according to a further aspect of the present invention, there is provided replication-deficient HSV whose DNA encodes at least one neurotrophic factor (NTF), and a method of expressing the NTF in a subject comprising administering said HSV.

There is also provided the use of replication-deficient HSV, as described herein, comprising DNA encoding at least one Neurotrophic Factor (NTF) in medicine. Also provided is the use of such HSV the treatment of neurodegenerative disease, preferably Tay-Sachs Disease (TSD), by gene therapy.

Surprisingly, we have shown the feasibility of use of HSV-1 vectors for obtaining long- term biological effects in an in vivo system.

Preferred NTF's are selected from the group consisting of : nerve-growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3,-4,-5, and 6 and glial-derived neurotrophic factor (GDNF).

Most preferably, the NTF is FGF-2 or BDNF. However, we have also shown that synerg occurs between different NTFs, particularly between FGF-2 and BDNF. Thus, it is also preferred th both of these are encoded by the HSV.

We have also shown that this occurs at the single cell level. In fact, the insertion of expressio cassettes for both NTFs in the same viral backbone ensures transfection of both genes of interest i every infected cell.

Also provided is a method of controlling or manipulating CNS cell proliferation, differentiatio and migration by using appropriate combinations of NTFs encoded by the replication-deficient HSV This allows for the development of new strategies for the gene therapy of neurological disorder characterized by or associated with loss of specific CNS cell types.

The present invention will now be illustrated by the following examples. References regarding the UL13 mutant vector

1. Bruni, R., B. Fineschi, W. O. Ogle, and B. Roizman. 1999. A novel cellular protein, p60, interacting with both herpes simplex virus 1 regulatory proteins ICP22 and ICPO is modified in a cell-type-specific manner and Is recruited to the nucleus after infection. J Virol 73:3810-7.

2. Long, M. C, V. Leong, P. A. Schaffer, C. A. Spencer, and S. A. Rice. 1999. ICP22 and the UL13 protein kinase are both required for herpes simplex virus-induced modification of the large subunit of RNA polymerase II. J Virol 73:5593-604.

3. Morrison, E. E., Y. F. Wang, and D. M. Meredith. 1998. Phosphorylation of structural components promotes dissociation of the herpes simplex virus type 1 tegument. J Virol 72:7108-14.

4. Moss man, K. L., H. A. Saffran, and J. R. Smiley. 2000. Heφes simplex virus ICPO mutants are hypersensitive to interferon. J Virol 74:2052-6.

5. Ng, T. I., W. O. Ogle, and B. Roizman. 1998. UL13 protein kinase of heφes simplex virus 1 complexes with glycoprotein E and mediates the phosphorylation of the viral Fc receptor: glycoproteins E and I. Virology 241:37-48.

6. Ogle, W. O., T. I. Ng, K. L. Carter, and B. Roizman. 1997. The UL13 protein kinase and the infected cell type are determinants of posttranslational modification of ICPO. Virology 235:406-13.

7. Ogle, W. O., and B. Roizman. 1999. Functional anatomy of heφes simplex virus 1 overlapping genes encoding infected-cell protein 22 and US 1.5 protein. J Virol 73 ^OS- IS.

8. Paludan, S. R., and S. C. Mogensen. 2001. Virus-cell interactions regulating induction of tumor necrosis factor alpha production in macrophages infected with heφes simplex virus. J Virol 75:10170-8.

9. Purves, F. C, W. O. Ogle, and B. Roizman. 1993. Processing of the heφes simplex virus regulatory protein alpha 22 mediated by the UL13 protein kinase determines the accumulation of a subset of alpha and gamma mRNAs and proteins in infected cells. Proc Natl Acad Sci U S A 90:6701-5.

10. Roizman, B. 1999. HSV gene functions: what have we learned that could be generally applicable to its near and distant cousins? Acta Virol 43:75-80.

11. Shibaki, T., T. Suzutani, I. Yoshida, M. Ogasa ara, and M. Azuma. 2001. Participation of type I interferon in the decreased virulence of the UL13 gene-deleted mutant of heφes simplex virus type 1. J Interferon Cytokine Res 21:279-85.

Example 1 : replication-deficient HSV vectors in treatment of intracelleular bacterial infection

Major histocompatibility complex (MHC) class I-restricted CD8⁺ T cells recognizing antigenic peptides derived from pathogens play major roles in protection against intracellular bacteria like Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes (for a review, see references 15 and 16). Vaccines utilizing inactivated or recombinant bacteria have been demonstrated to elicit both CD4- and CD8-T-cell activation (16), but they seem to be inefficient stimulators of effector (25) and memory (34) T cells if compared side by side with live bacteria (25) or recombinant viral vaccines (34). In this respect, inactivated intracellular bacterial vaccines are similar to natural M. tuberculosis (44) and L. monocytogenes (33) infections, which fail to induce sufficient immunological memory to prevent recurrent infections. DNA vaccines have been demonstrated to induce protection against infections with M tuberculosis (43) and L. monocytogenes (9).

However, it was demonstrated that combining DNA vaccines with attenuated bacteria (12), protein antigen (42), or modified vaccinia virus (28) in heterologous prime-boost vaccination protocols could further optimise protection. In other words, DNA vaccines alone are not sufficient to induce maximal protective immunity and, in a clinical setting, might require complex vaccination arrangements. Similarly, recombinant vaccinia virus could elicit only limited protection against intracellular bacteria and delayed, rather than prevented death after infection (2). Thus, efficacious "single-shot" vaccines to intracellular infection have yet to be developed.

While most intracellular bacteria replicate in maturation-arrested phagosomes (17), L. monocytogenes bacteria egress from the phagosome and gain access to the cytoplasm of infected cells (37). This led initially to the assumption that, because their antigens have access to the cytosolic MHC class I presentation pathway, only the latter would induce CD8-Tcell responses. In contrast, bacteria localized in phagosomes were thought to preferentially trigger CD4-T-cell responses via the phagosomal MHC class II presentation pathway. However, it has been demonstrated in mice (39) as well as in humans (41) that cytotoxic T lymphocytes (CTL) are important players in the control of both M. tuberculosis and L. monocytogenes infections (24, 29), and human MHC class I peptides derived from M. tuberculosis were recently identified (6). In addition to lysis of infected cells, CTL expressing granulysin have been shown to directly kill extracellular bacteria (11, 40). Thus, CD8 T cells are the effector cells of choice for intracellular.

Replication-deficient (10, 30) or disabled infection with single-cycle heφes simplex virus (HSV) (3, 27) has been shown to induce strong irnrnune responses against HSV-derived antigens.

hi order to examine the potential of HSV vaccines for vaccination against intracellular bacteria, we used recombinant replication-defective HSV-1 (rHSV-1), which was modified to encode OVA (ovalbumin) as a model antigen. We have demonstrated that in contrast to gene gun DNA vaccination, rHSV-1 induce neither strong humoral responses nor CD4-T-cell responses specific for the model antigen OVA. However, when ex vivo CD8-T-cell responses were compared, rHSV- 1 was much more potent than DNA vaccination and resulted in immediate as well as long-term memory protection against infection with recombinant L. monocytogenes expressing OVA. Thus, rHSV-1 represents a "single-shot" vaccine inducing long-term protective CTL responses.

FIG. 1. Visualization of antigen-specific CTL expansion after vaccination. PBL were stained with anti-CD8-APC and H^"2K /OVA257-264 tetramers-phycoerythrin and analysed by flow cytometry before (a) and 7 days after vaccination with pcDNA3-OVA (b), T0H-OVA (c), or T0-GFP (4 x 10⁶ virus particles i.v.) (d). The percentage of CD8⁺ Tet⁺ CTL among PBL is indicated on each plot, (e) The frequency of antigen-specific cells among peripheral blood CD8⁺ T cells was determined by H- 2Kb/OVA257-264 teframer staining for pcDNA3-OVA-, T0H-OVA-, and TO-GFP-immunized mice as shown (a to d). Data represent average values +/- standard deviations (error bars) obtained from five mice per group at each time point, p.i., postinfection

FIG. 2. Protection against L.m.-OVA infection, (a) C57BL/6 mice were left ummmunised or vaccinated with pcDNA3-OVA, T0H-OVA, or T0-GFP (4 x 10⁶ virus particles i.v.) (day 0) and infected i.v. with 5 x 10⁴ (two times the LD₅₀) L.m.-OVA cells 8 days later. H^"2K /OVA257-264 tetramer staining for OVA-specific CD8⁺ T cells in blood was performed at day 7 postvaccination (b), and survival of the mice was monitored for 10 days after infection (c). At day 10 postinfection (day 18 postimmunisation), spleens of surviving mice were analysed for presence of Tet⁺ CTL (d). The bar graphs (b and d) depict the percentage (mean +/- standard deviation [error bars]) of Tet- positive cells among total CD8⁺ T cells (five mice/group), (e) As a control for the antigen-specificity of protection, vaccinated mice were infected with 5 x 10⁴ wild-type L. monocytogenes cells (not expressing OVA), and survival was monitored. Abbreviations: no imm, no immunization; p.i., postinfection

FIG. 3. Protection against infection with high dose of L.m.-OVA. C57BL/6 mice were vaccinated with T0H-OVA, TO-GFP (4 x 10⁶ virus particles i.v.), or ρcDNA3-OVA and infected with 10⁵ (four times the LD50) L.m.-OVA cells 7 days postvaccination. Mice were sacrificed 3 days after infection, and bacterial counts in spleen (a) and livers (b) were determined from organ homogenates (n = 3 to 4 mice per group), (c) Differentially vaccinated mice (three to four per group) were infected with 5 x 10⁵ (20 times the LD₅₀) L.m.-OVA cells, and survival was monitored for 13 days.

FIG. 4. Long-term protection against L.m.-OVA infection, (a) Experimental protocol: C57BL/6 mice were vaccinated with pcDNA3OVA, T0H-OVA, or TO-GFP (4 x 10⁶ virus particles i.v.) and infected i.v. with 5 x 10⁴ (two times the LD₅₀) L.m.-OVA cells 6 weeks later, (b) The percentage of H-2Kb/OVA257-264 tetramer-specific cells among CD8⁺ T lymphocytes in the blood was determined by flow cytometry at the days indicated, (c) Survival of the mice was monitored daily; no death occurred after day 7 postinfection. (d) On day 7 after infection (day 49 postvaccination), spleen, mesenteric (mes), inguinal, and brachial lymph nodes were isolated and analysed by flow cytometry; inguinal and brachial lymph nodes (ing. and bra. LN) were pooled for each mouse. The bar graphs depict the percentage (mean +/- standard deviation [error bars]) of H-2Kb/OVA257-264 tetramer-positive cells among CD8⁺ T cells (n = 4 to 5 per group).

FIG. 5. Monitoring of OVA-specific CD4⁺-T-cell responses in vivo following rHSV-1 vaccination. BALB/c mice received 2.5 x 10⁶ naϊve DOl l.lO cells at day -1 and were immunized with gene gun (pcDNA3-OVA), T0H-OVA, or TO-GFP (4 x 10⁶ virus particles) i.v. at day 0. The frequency of DOl l.lO T cells present in the inguinal lymph nodes and spleen was measured by flow cytometry. a-c) Cell suspensions were stained with anti-CD4, and KJl-26-fluorescein isothiocyanate (DOll.lO TCR-specific). The percentages of CD4⁺/KJ126⁺ cells in lymph nodes of control (TO-GFP)-, pcDNA3-OVA-, and TOH-OVA-immunized mice at day 5 postimmunization are indicated in the dot plots. Kinetics of DOll.lO T-cell expansion is shown for lymph nodes (d) and spleen (e). In d and e, the total cell numbers +/- standard deviations (error bars) of CD4⁺ KJ1- 26⁺ cells are indicated (three to four animals per group). FIG. 6. OVA-specific antibody responses in immunized BALB/c mice. Sera were obtained from mice immunized with pcDNA3-OVA, T0H-OVA, or TO-GFP (4 x 10⁶ virus particles i.v.) at the indicated time points postvaccination, and OVA-specific antibody serum levels were determined by enzyme-linked immunosorbent assay. Preimmune serum (day 0) from each group was determined with a pool of sera. Results are expressed as the mean of optical density at 450 nm (O.D. 450 nm) +/- standard deviation (error bars) from at least three individual mice per group.

Protective vaccination against bacteria with a facultative intracellular life style is dependent on the induction of cytotoxic T-cell responses (17, 39). Since natural Etsterzα-derived CTL epitopes have not been identified in H-2^b mice, we studied and visualized the vaccine efficacy of rHSV-1 vectors by using the model antigen OVA; mice were vaccinated with rHSV-1 encoding, OVA and then challenged with recombinant OVA-expressing L. monocytogenes. As a control we used gene gun DNA vaccination, which has been shown to induce reliable protective CTL-mediated immunity to intracellular L. monocytogenes infection (9). Analysis of vaccinated mice with H-2K -OVA peptide tetramers revealed that rHSV-1 vaccines induce strong activation and expansion of OVA- specific CD8⁺ T cells, resulting in peak frequencies much greater than those observed following gene gun vaccination (Fig. 1). These data are in concordance with recent reports showing that DNA vaccines induce very low numbers of epitope-specific CTL which are poorly detectable ex vivo by flow cytometry after a single DNA vaccination via either gene gun (1) or intramuscular application (45).

At the peak of the response following T0H-OVA vaccination, 2 to 5% of all CD8⁺ T cells in the blood and spleen were specific for OVA257-264. This frequency is two to threefold lower than that shown in a recently published report for HSV-1 glycoprotein epitope gB498-505 specific CTL in H-2^b mice infected with replication competent HSV-1 (7). he lower frequencies of specific CTL in our system might be explained by our use of a replication-deficient (ICP4-, ICP22-, and ICP27- triple mutant) rHSV-1 vaccine, which reduces the number of infected cells compared to replication competent HSV-1.

The efficiency of a vaccine is directly proportional to its capacity to activate T cells and to generate a memory T-cell pool. Development of memory T cells has been shown to be directly proportional to the intensity of the primary response (31), and in several viral infection models, the viral dose correlated positively with T-cell memory development (23, 34, 35). Recombinant replication- deficient mutant HSV-1 not only induced strong expansion of CD8⁺ CTL (Fig. 1), it also proved to be an extremely efficient vaccine against infection with an intracellular bacterium.

Gene gun DNA vaccination protected equally well when mice were challenged with low but lethal doses of bacteria, but following infection with higher doses of Listeria, only TOH-OVA-vaccinated animals showed low bacterial counts in spleen and liver (Fig. 3 a) and were completely protected (Fig. 3c). Thus, as a consequence of the low frequencies of memory precursor CTL generated in gene gun-vaccinated mice, the response to a large infectious challenge was insufficient to offer protection (Fig. 4).

These data are in line with findings that gene gun vaccination can be further enhanced by combining it in heterologous prime-boost protocols with other vaccines (12, 28, 42); rHSV-1 vaccine, on the other hand, is by itself a very efficient vaccine to even high doses of infection with intracellular bacteria.

Interestingly, TOH-OVA, unlike DNA vaccination, did not induce significant CD4 T helper cell or antibody responses specific for the recombinant antigen. This may be irrelevant, as protection against intracellular bacterial pathogens such as L. monocytogenes is largely CTL mediated (17, 39). Although antibody responses could be important to neutralize L. monocytogenes immediately after bacterial entry into the host and might therefore be considered a prerequisite for the establishment of an efficient long-term vaccination effect against intracellular bacteria (8), a lack of pathogen-specific antibodies did not negatively affect the ability of HSV-1 -derived vaccines to provide long-term memory protection in our system.

Many different recombinant viral vector systems, including alpha-, adeno-, pox- and poliovirus systems, have been developed for vaccination. One concern common to all these vaccines is that their efficiency might be negatively affected by pre-existing immunity to the viruses, as has proven true in the case of adenovirus (38) and poxvirus vectors (14). An advantage of the present invention is that HSV-1 -derived vectors are not affected by pre-existing immunity to HSV-1 (4). In the light of 75% of the adult human population have been previously exposed to HSV-1, this is an important prerequisite for the efficient usage of these vaccines (13). In addition, the strength of CTL response we observed upon vaccination with TOH-OVA indicates that, in contrast to other vaccines tested in the infectious Listeria model (16), further homologous or heterologous prime-boost protocols maybe superfluous in the case of rHSV-1.

We have evaluated the efficacy of rHSV-1 vaccines in mice and demonstrate that a single vaccination is sufficient to elicit a protective response specific for a vaccine-encoded, nonviral protein. We have directly compared the cellular and humoral responses elicited by rHSV-1 vaccines and gene gun DNA vaccination and demonstrate significant differences in the strength and quality of the elicited responses.

We demonstrate for the first time that HSV-1 -derived vectors induce strong CTL responses, making them a promising candidate for vaccines against intracellular^" bacterial infection.

MATERIALS AND METHODS

Mice. All mice were bred and maintained under standard conditions in the animal facilities of the Institute for Immunology, Ludwig-Maximilians-University Munich; the Institute for Microbiology, Immunology and Hygiene, Technical University Munich; or the Department of Pharmacy, University of Ferrara. DOll.lO mice (expressing transgenic T-cell receptors [TCR] specific for OVA323-339/MHC class II I-A^d) were obtained from Jackson Laboratory, Bar Harbor, Maine.

Plasmid construction and preparation of recombinant, replication-deficient HSV-1 vaccines.

A BamHiyXhoI fragment of rabbit βglobin was cloned into a BamHI/XhoI-opened pcDNA3 vector (Invitrogen) to create pcDNA3-βglobin. The pcDNA3-OVA vector encoding the secreted form of chicken ovalbumin (OVA) was constructed by cloning a 1.9-kb EcoRI fragment from the plasmid pAc-Neo-OVA (provided by F. Carbone, Melbourne, WEHI, Australia), which contained the entire coding sequence of OVA, into the EcoRI site of pcDNA3-βglobin. Plasmids were prepared from Escherichia coli with Qiagen (Hilden, Germany) Mega Kits. A recombination plasmid (pB410H:OVA) was constructed by introduction of HCMV-βglobin OVA cDNA expression cassette into the U141 locus of HSV-1. The cDNA under the transcriptional confrol of the human Cytomegalovirus promoter was inserted in a Smal/Xbal-opened pBBSK plasmid between the two UL41 fragments (map positions 93,858 to 92,230 and 91,631 to 90,145) 100 bp downstream of the HSV immediate-early ICPO promoter. This plasmid (pB410H:OVA) was recombined with the genome of TOZGFP using the previously described Pac-facilitated lacZ substitution method (19). TOZGFP is a nonreplication HSV viral vector background that has low toxicity due to the deletion in three immediate early genes (ICP4 and ICP27, which are essential for viral replication, and ICP22) with cDNA encoding GFP inserted into the ICP22 locus and an insertion of LacZ in the UL41 locus. The recombination was carried out using standard calcium phosphate fransfection of 5 μg of viral DNA and 1 μg of linear recombination plasmid pB410H:OVA. Transfection and isolation of the recombinant virus was performed in 7b Vero cells (African green monkey kidney cells CCL81; American Type Culture Collection, Manassas, Va.) capable of providing the essential ICP4 and ICP27 HSV gene products. The recombinant virus containing the OVA cDNA was identified by isolation of a clear plaque phenotype after X-Gal (5-bromo-4-chloro-3-indolyl-β- D-galactopyranoside) staining. TOH-OVA virus was purified by three rounds of limiting dilution and the presence of the fransgene was verified by Southern blot analysis. Viral stocks of the TOH- OVA virus and the control vector TO-GFP (derived from TOZGFP without lacZ reporter gene in UL41 locus) were prepared and titrated using 7b cells.

Adoptive transfer.

DOll.lO cells were prepared from lymph nodes and spleens of transgenic mice. Briefly, spleen and lymph nodes were taken out, and single-cell suspensions were prepared. Erythrocytes were removed by osmotic lysis, and after determining the percentage of DOll.lO TCR-trahsgenic T cells by flow cytometry, 2.5 x 10⁶ transgenic T cells were injected intravenously into the recipient mice.

Vaccination.

Naked DNA immunization was performed by gene gun administration (Bio-Rad Laboratories, Hercules, Calif.). Cartridges of DNA-coated gold particles were prepared according to the manufacturer's instructions. For each preparation, gold particles (25 mg; diameter, 1 μm) were coated with 200 μg of DNA. Mice were anaesthetized prior to vaccination with a mixture of Ketavet/Rompun in phosphate-buffered saline (PBS). A total of 8 μg of plasmid DNA was delivered to the shaved abdominal skin of adult mice with a discharge pressure of 400 lb/in². For TOH-OVA vaccination, frozen virus stocks were thawed on ice, diluted in PBS to 4 x 10⁶ T0H- OV A/200 μl, and injected intravenously (i.v.).

Enzyme-linked immunosorbent assay. For the detection of OVA-specific antibodies, 96-well microtiter plates (Nunc Maxisoφ, Nunc, Wiesbaden, Germany) were coated with OVA (15 μg ml; Sigma Chemical Co., St. Louis, Mo.) at room temperature overnight. Plates were blocked (PBS, 0.5% milk powder, 0.05% NaN3), and immune sera (diluted 1:100 in blocking buffer) were incubated for 2 h at room temperature. After washing five times with PBS, horseradish peroxidase-labelled second-step goat sera specific for mouse immunoglobulin M (IgM) or IgG (Serotec Ltd., Oxford, England) or IgGl or IgG2a (Southern Biotechnology Assoc. Inc., Birmingham, Ala.) in PBS (0.5% milk powder, 0.05% Tween20) was added and incubated for 2h. After five washing steps, the amount of bound antibody was determined by addition of substrate solution (1 mM 3,3',5,5' tetramethylbenzidine, 30% H₂O₂ [0.3 μl/ml] in 0.2 M potassium acetate). The reaction was stopped by addition of 2 N H₂SO_4> and the absorbance at 450 nm was determined with a V_max microplate reader (Molecular Devices Coφoration, Sunnyvale, Calif).

MAbs, tetramers, and flow cytometry.

Lymphocytes were analysed using the following monoclonal antibodies (MAbs): anti-CD4- PerCP (L3T4), anti-CD8a-PerCP (Ly2), and anti-CD62L (Mel-14) from PharMingen (San Diego, Calif.) and KJl-26-fluorescein isothiocyanate specific for DOll.lO TCR, anti-CD8a- APC (Ly2), and anti-CD44-PE from Caltag (Burlingame, Calif). Biotinylated MAbs were detected with streptavidin-APC (Caltag). Analytic flow cytometry was performed on a FACScalibur (Becton Dickinson, Mountain View, Calif.), and the data were analysed using CellQuest software (Becton Dickinson). Tetrameric H2-K /Ova ₅ .₂₆₄ complexes were generated as previously described (5). In brief, refolded and biotinylated MHC-peptide complexes were multimerised with the addition of phycoerythrin-conjugated sfreptavidin (Molecular Probes, Eugene, Oreg.). Tetrameric complexes were stored at 2 mg/ml at 4°C in PBS (pH 8.0) containing 0.03% sodium azide, pepstatin (1 μg/ml), leupeptin (1 μg/ml), and 1 mM EDTA. The reagents were frequently tested on antigen-specific T-cell lines to document staining quality.

Listeria.

Mice were infected i.v. with L. monocytogenes expressing the secreted form of OVA (36) (L.m.- OVA, kindly provided by Hao Chen, Philadelphia, Pa.). Viable bacterial counts within spleen and liver were determined by homogenizing the respective tissue in PBS containing 0.05% Triton X- 100 and plating on brain heart infusion agar plates (Life Technologies, Gaithersburg, Md.). L. monocytogenes colonies were identified by their characteristic moφhology and by Gram staining. RESULTS

Induction of CTL responses by rHSV-1 vaccines. The goal of the present study was to investigate the capacities of rHSV-1 vectors to induce protective primary and long-term immune responses against intracellular bacterial infection in vivo. Earlier attempts to use HSV-1 -derived vectors in gene therapy approaches were complicated by viral cytotoxicity and transient expression of transgenes. Replication-defective HSV-1 strains, in which nonreverting mutations have been incoφorated into mandatory viral genes, retain the immunogenicity of wild-type HSV but are much safer. Deletion of a series of viral immediate early genes such as ICP4, ICP22, and ICP27 substantially reduces cytotoxicity and enhances long-term transgene expression (21).

Further studies have shown that these or similar mutant vectors do not interfere with MHC class I expression in the infected neurons (20) or fibroblasts (46), an important consideration with respect to antigen presentation. For our studies, a replication-incompetent, low-cytotoxicity, ICP4^", ICP22^" , and ICP27^" triple mutant HSV-1 virus was modified to express the model antigen chicken OVA under control of the human Cytomegalovirus (CMN) promoter. This virus (T0H-ONA) was used to vaccinate mice. As positive vaccination control, we employed gene gun DΝA vaccination, a method previously proven to confer CD8-T cell-mediated protective immunity against bacterial challenge (9). The plasmid used for this approach was pcDΝA3-ONA, containing the same CMN- OVA-expression cassette as TOH-OVA.

Using OVA as a model antigen, we first investigated the capacity of the different vaccines to induce OVA-specific CD8 T-cell activation and expansion in vivo. With tetrameric H2-K /Ova₂₅₇. ₂₆ complexes (Tet), we determined that the frequency of OVA-specific Tet⁺ CD8⁺ T cells in normal nonimmune C57BL/6 mice was, on average, 0.05% of all peripheral blood lymphocytes (Fig. la). This percentage did not increase upon gene gun (0.03%) (Fig. lb) or control rHSV-1 (TO-GFP) vaccination (0.02%) (Fig. Id). In contrast, TOH-OVA induced a 10-fold expansion of OVA-specific CTL compared to the background (0.59%) (Fig. lc). When the kinetics of CD8⁺ Tet⁺ T-cell expansion in peripheral blood was analysed as a percentage of total CD8⁺ T cells (Fig. le), a weak, but statistically significant (Student's t test, P = 0.0026) expansion was detectable at day 7 after gene gun vaccination (0.43% +/- 0.15%) compared to negative-control mice vaccinated with TO-GFP (0.145% +/- 0.03%). However, a significant expansion after gene gun vaccination could not be observed in all of our experiments (see Fig. 4). In contrast, TOH-OVA vaccination induced >21-fold expansion of Tet⁺ CD8⁺ CTL over background (Fig. le) (3.03% +/- 0.57%) (except as noted, values are presented as means +/- standard deviations). At the peak of the response (day 7), the frequency of Tet⁺ CD8⁺ T cells induced by TOH-OVA was sevenfold larger than following gene gun vaccination. While levels of Tet⁺ CD8⁺ CTL remained elevated in TOH-OVA-vaccinated mice for more than 50 days, the expansion in gene gun-vaccinated mice was transient and short-lived, declining rapidly to background levels (Fig. le).

As CD8-T-cell expansion in animals vaccinated with TOH-OVA was variable between different experiments, we could not observe elevated levels of Tet CD8⁺ CTL for such a long period in each experiment performed (see Fig. 4b). When the peripheral blood of the same animals was analysed for nonspecific (Tet-) augmentation of the CD8⁺ T-cell compartment, TOH-OVA and TO- GFP induced an approximately twofold increase of Tet+ CD8+ T cells (data not shown). This effect was probably due to a proinflammatory response in rHSV-1 -vaccinated mice and was not observed in the DNA-vaccinated group. These results show that TOH-OVA induce a severalfold stronger expansion of antigen-specific CD8⁺ T cells compared to gene gun vaccination.

Recombinant HSV-1 vaccination protects against L. monocytogenes infection.

While infection of mice with sublethal doses of J. monocytogenes leads to rapid clearance of the bacterium and long-lived protective immunity, larger doses result in death of the animals within a few days. Despite a relatively weak induction of antigen-specific CTL expansion, as demonstrated in Fig. 1, gene gun DNA vaccination has been reported to provide protective immunity against L. monocytogenes infection in mice (9). We set out to discover whether vaccination with TOH-OVA would have similar protective properties.

To directly compare both vaccination strategies, we challenged rHSV-1 or gene gun-vaccinated mice with a lethal dose (5 x 10⁴, two times the 50% lethal dose [LD₅₀]) of J. monocytogenes genetically modified to express the model antigen OVA (L.m. OVA [36]). In order to determine the expansion of OVA-specific CTL after vaccination and L.m.-OVA challenge, the experimental protocol was designed as shown in Fig. 2a, including Tet analysis at two stages during the experiment. Tet analysis at day 7 post vaccination confirmed the findings shown in Fig. 1; a strong increase of Tet⁺ CD8⁺ CTL could be detected in TOH-OVA immunized mice, while DNA vaccination induced a weak, but significant (P < 0.05) increase compared to nonimmunised mice (Fig. 2b). Despite the relatively weak expansion of Tet CTL following gene gun vaccination, all mice from this group were protected from the subsequent L.m.-OVA challenge, whereas all control animals (unimmunised or control-vaccinated with TO-GFP) died between day 3 and day 6 postinfection (Fig. 2c). TOH-OVA-vaccinated mice were also fully protected (Fig. 2c).

These results demonstrate that under these conditions of bacterial challenge (low dose) the strength of the CD8⁺-T-cell response did not correlate with protection. Elevated levels of Tet⁺ CD8⁺ CTL were detectable in both gene gun- and TOH-OVA-vaccinated mice 18 days postvaccination (10 days postMection, Fig. 2d). While the average of Tet⁺ CD8⁺ CTL was higher in DNA-vaccinated mice than in the TOH-OVA-vaccinated group, the difference was not statistically significant (Fig. 2d). To control the specificity of protection, we infected gene gun and TOH-OVA-vaccinated mice with wild-type L. monocytogenes not expressing the model antigen OVA (Fig. 2e).

Neither group was protected, and all animals died from infection. Together with the results from confrol vaccination using TO-GFP (Fig. 2c), where mice were not protected from lethal infection with L.m.-OVA, these data exclude the possibility that protection induced by TOH- OVA is due to unspecific HSV-1 -mediated effects. These findings indicate that TOH-OVA induce strong CTL responses specific for recombinant antigen and are sufficient to protect mice from subsequent bacterial infection.

Protection from infection with a high dose of X. monocytogenes is induced by HSV-1 vaccines, but not by DNA vaccination.

Although DNA vaccination induced much lower numbers of circulating antigen-specific Tet ⁺ CTL compared to TOH-OVA, both groups were protected from a lethal (two times the LD50) L.m.-OVA challenge (Fig. 2). We set out to determine whether DNA vaccination would also provide protection from higher doses of L. monocytogenes. We therefore repeated the experiment shown in Fig. 2a, mfecting the mice with a twofold higher dose of L.m.-OVA (10⁵ cells, four times the LD₅₀).

When spleens and livers were analysed 3 days postinfection for presence of viable Listeria, striking differences between gene gun- and TOH-OVA-vaccinated mice became evident: spleens from two out of three TOH-OVA-vaccinated animals were completely free of bacteria, while bacterial counts in the third were approximately 6,000-fold lower than those from gene gun-vaccinated mice (Fig. 3a). The latter had bacterial numbers similar to the control-vaccinated (TO-GFP) group. Livers of the same mice showed a similar picture, with an average of >430-fold lower bacterial counts in livers of TOH-OVA-vaccinated compared to gene gun- or control virus (TO-GFP)-vaccinated mice (Fig. 3b).

In order to test if these differences in bacterial numbers correlate with survival, we infected vaccinated mice with 10-fold more L.m.-OVA (5 x 10⁵ cells, 20 times the LD₅₀) than the amounts used for the experiment depicted in Fig. 2, and then we monitored survival (Fig. 3 c). With this high dose, a direct relationship between the frequency of antigen-specific Tet⁺ CTL induced by the vaccine and protection from infection became clear. The TOH-OVA-vaccinated mice survived without signs of disease (data not shown), while all gene gun- and TO-GFP -vaccinated mice died (Fig. 3c).

These data show that while the DNA vaccine induces protection against infection with a low, but lethal dose of L.m.-OVA, TOH-OVA induces much stronger responses capable of protecting against a much more potent infectious dose.

Long-lived CTL immunity to L. monocytogenes induced by recombinant replication-deficient HSV-1.

The induction of long-term memory responses is a prerequisite for optimal vaccine efficacy. In order to test if TOH-OVA is able to induce antigen-specific long-term protection against intracellular bacterial infection, we vaccinated mice with TOH-OVA or control vaccines and challenged the mice 6 weeks later with a lethal dose of L.m.-OVA (5 x 10⁴ cells, two times the LD ₀). Expansion of Tet⁺ CD8⁺ CTL was monitored in the blood following vaccination and challenge. As observed previously (Fig. 1 and 2), only TOH-OVA induced strong expansion of Tet⁺ CD8⁺ CTL 7 days postvaccination (Fig. 4b). This increase was transient and after the contraction of Tet⁺ CTL, lower levels of Tet⁺ CD8⁺ CTL could be detected in the vaccinated mice 5 weeks after vaccination (day 39) (Fig. 4b).

Following challenge with L.m.-OVA, changes were first detectable at day 5 (day 47) (Fig. 4b), with significantly elevated levels of Tet+ CD8+ CTL in TOH-OVA-vaccinated animals compared to the gene gun group or controls (P< 0.001); more than 20% of all CD8 T cells in the blood of TOH-OVA-vaccinated mice were Tet ⁺. Elevated levels of antigen-specific Tet+ CD8+ CTL in gene gun-vaccinated mice could be detected at day 49, 7 days postinfection, where they represented approximately 10% of all CD8⁺ CTL, an expansion sixfold lower than that of the TOH- OVA group (Fig. 4b).

Despite these differences in antigen-specific CTL frequency, both TOH-OVA- and gene gun DNA- vaccinated mice were well protected from low-dose (5 x 10⁴ cells) L.m.-OVA challenge, showing no signs of serious illness. In contrast, all control TO-GFP-vaccinated mice died within 6 days postinfection (Fig. 4c).

At day 7 postinfection, we sacrificed the surviving mice and analyzed spleens and livers for presence of L. monocytogenes. The spleens of all animals from both surviving groups were bacteria free (data not shown). However, while the livers of TOH-OVA-vaccinated mice were also free of L. monocytogenes, 50% of the gene gun-vaccinated mice had not yet cleared the bacteria (3 x 10⁴ to 9 x 10⁴ bacteria per liver; data not shown). These data indicate that despite survival of all mice from both groups, the TOH-OVA vaccine results in more complete or, alternatively, more rapid bacterial clearance.

The different frequencies of antigen-specific CTL observed in TOH-OVA- versus gene gun- vaccinated mice (Fig. 4b) is a systemic phenomenon, observed not only in the peripheral blood, but also in spleens of surviving mice (day 49) (Fig. 4d). The mesenteric lymph nodes of TOH- OVA immunized mice also contained significantly more Tet⁺ CD8⁺ CTL than detected in gene gun- vaccinated animals (P< 0.005), although the difference was not as great as observed in spleen or blood (Fig. 4d). This analysis excludes the possibility that the differences in frequencies of Tet⁺ CD8⁺ CTL detected in peripheral blood lymphocytes (PBL) following TOH- OVA or gene gun vaccination were a result of differential homing properties of the stimulated T cells. 5 1

Taken together this analysis shows that TOH-OVA-derived vaccines induce potent long-term protection against infection with the facultative intracellular bacterium L. monocytogenes and generate a large memory CTL pool capable of clearing even large bacterial loads very efficiently. In contrast, gene gun vaccination induces lower frequencies of specific CTL sufficient for the protection of mice from challenge with lower but lethal L isteria-dosss but are less efficient at achieving complete bacterial clearance.

Induction of antigen-specific CD4⁺ T helper responses and antibodies by recombinant replication-deficient HSN-1.

In order to directly monitor the capacity of TOH-OVA to induce CD4⁺-T-cell responses against their encoded recombinant antigen in vivo, we vaccinated mice that had previously received adoptively transferred TCR-transgenic OVA-specific DOl l.lO CD4⁺ T cells (18). DOl l.lO transgenic T cells recognize the OVA323-339 peptide in the context of MHC class II I-A^d and can be detected with the clonotypic TCR-specific MAb KJ1-26 facilitating the monitoring of their activation and expansion following vaccination with the specific antigen. While control vaccinated mice contained very few CD4 ⁺ KJ1-26 ⁺ T cells (TO-GFP) (Fig. 5a and d), the percentages increased 5- to 10-fold and total numbers increased 10- to 30-fold in the draining lymph nodes in gene gun- vaccinated animals (Fig. 5b and d) 5 days after vaccination.

In contrast, vaccination with TOH-OVA did neither augment the frequencies (Fig. 5c) nor cell numbers (Fig. 5d) of antigen-specific DOl l.lO T cells. Simultaneously, we monitored the activation status of DOll.lO T cells with a MAb specific for CD44, a cell surface marker modulated from moderate levels on naive T cells to high expression on activated T cells (data not shown). While CD4 ⁺ KJl-26⁺ T cells demonstrated a highly activated phenotype following gene gun vaccination, in lymph nodes of control (T0-GFP-), and T0H-OVA- immunized mice they did not gain an activated phenotype (data not shown). A CD4-T-cell response, following i.v. TOH-OVA vaccination would be expected primarily in the spleen.

Suφrisingly, however, the kinetics of DOll.lO expansion in the spleen following TOH-OVA vaccination was also not significantly different from the control immunized mice during the first 5 days postvaccination (Fig. 5e). A twofold expansion of CD4⁺ KJ1-26 ⁺ T cells (Fig. 5e) with high levels of CD44 (data not shown) was first detected in the spleens of TOH-OVA-vaccinated mice at day 7 post- vaccination compared to gene gun- vaccinated animals. Other vaccination routes for TOH-OVA such as intraperitoneal, subcutaneous, and intradermal routes did not induce greater expansion of antigen-specific CD4 T cells (data not shown). These findings indicate that rHSV-1 vaccines elicit poorer CD4 T helper responses than gene gun vaccination.

Poor induction of CD4 helper T-cell responses by HSV-1 -derived vaccines could result in similarly weak induction of T dependent antibody responses. To address this possibility, we analysed the sera of vaccinated mice for OVA-specific antibodies. While none of the vaccinations resulted in significant levels of specific IgM (Fig. 6), specific IgG was induced at later time points after vaccination (Fig. 6). The recombinant virus (TOH-OVA) and gene gun immunization induced similar IgG levels 17 days postvaccination, but only gene gun irrmiunization resulted in further increase at later time points (Fig. 6). No specific IgG was detected following control (TO- GFP) immunization (Fig. 6). Further analysis of IgG subclasses revealed that gene gun vaccination induces a Th2-dominated response with OVA-specific IgGl antibodies (Fig. 6), while a weak Thl- like IgG2a-dominated response is induced by TOH-OVA (Fig. 6). These data show that vaccination with rHSV-1 -derived vaccines leads to the induction of a significantly weaker humoral response than gene gun immunization with additional skewing of the response towards Thl .

Thus, recombinant HSV-1 -derived vaccines are weak inducers of CD4 T helper and antibody responses but activate a large and efficient CD8 ⁺ T-cell response.

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43. Tascon, R. E., M. J. Colston, S. Ragno, E. Stavropoulos, D. Gregory, and D. B. Lowrie. 1996. Vaccination against tuberculosis by DNA injection. Nat. Med. 2:888-892.

44. van Rie, A., R. Warren, M. Richardson, T. C. Victor, R. P. Gie, D. A. Enarson, N. Beyers, and P. D. van Helden. 1999. Exogenous reinfection as a cause of recurrent tuberculosis after curative freatment. N. Engl. J. Med. 341:1174-1179.

45. Wolkers, M. C, M. Toebes, M. Okabe, J. B. Haanen, and T. N. Schumacher. 2002. Optimizing the efficacy of epitope-directed DNA vaccination. J. Im munol. 168:4998-5004.

46. York, I. A., C. Roop, D. W. Andrews, S. R. Riddell, F. L. Graham, and D. C. Johnson. 1994. A cytosolic heφes simplex virus protein inhibits antigen presentation to CD8~ T lymphocytes. Cell 77:525-535.

Example 2 : Replication-deficient HSV comprising angiogenic inhibitors. cytokines and suicide genes for use in Anticancer treatments

Methodology for Example 2

Construction of HSV vectors expressing cytokine molecules.

HSV vectors are created containing mIL12 or mGM-CSF provided by Invivogene. Production and secretion of cytokines molecules following viral infection of various cell lines will be confirmed by western blot and the quantification will be performed by specific ELISA kits.

The vectors are generated by inserting the transgene expression cassettes, in which the cytokine genes are under the transcriptional control of the strong human cytomegalovirus immediate early promoter (HCMV), into the Usl locus of HSV genome by homologous recombination with the method previously described (Krisky D, Marconi et al. 1997). TL12 (frivivogen) or GM-CSF sequences will be introduced in the same viral locus to avoid differences in gene expression due to promoters and viral location.

The vectors are constructed by insertion of the IL12 or GM-CSF genes expression cassettes into the background of the THZ-1 vector (THIL12 and THGM-CSF). THZ-1 is a recombinant virus deleted in the ICP4, ICP27 and ICP22 immediate early genes with the lacZ reporter gene under HCMV promoter, in the ICP22 locus. To this end THZ-1 viral DNA and the recombinant plasmids containing the cytokine genes are co-transfected into a complementing cell line (7b), which provides, in trans, the essential viral genes ICP4 and ICP27.

Screening progeny viruses by Southern blot analysis will identify the new recombinants for the presence of the transgenes. Positive isolates will be purified following three rounds of limiting dilution and the recombinant viruses tested for their ability to express the transgenes following infection. The cytokine expression will be assayed by ELISA kit. In parallel, another vector is constructed in which GM-CSF is inserted either under HCMV promoter or under the transcriptional confrol of the Matrix Metalloproteinase 9 (MMP-9) tumor specific promoter in order to express the cytokine only in tumor cells.

The gene expression cassettes are introduced into the background of the vector THZ.5, which is a mutant deleted in the ICP4, ICP27 and ICP22 immediate early genes and in the non-essential gene UL13 of HSV genome that contains LacZ reporter gene under HCMV promoter (THZ.5). This vector (T-MMP9GM-CSF) as well as all the others in this proposal is based on a recombination methodology developed in J.C. Glorioso's laboratory, which results in high frequency recombinants (22, 28). The reason for the construction of these two last vectors is to have their expression cassettes in a different viral location from IL12 insertion locus.

By genetic crossing, a furhter vector is obtained, which carries both cytokine genes GMCSF and/or IL12 (THGM-CSF/IL12 or T-MMP9GM-CSF/IL12). GM-CSF is expressed under tumor-specific promoters in order to increase vector expression only in targeted tumor cells and avoid the toxicity induced by an over-expression of this cytokine. The cytokine expression of all the above described vectors is detected and measured by ELISA.

Construction of HSV vectors co-expressing cytokine and antiangiogenic molecules.

The vectors expressing mIL12 or mGM-CSF are combined with vectors, constructed in the previous project and expressing the angiostatic fusion protein, Endostatin- Angiostatin or Endostatin-Kringle5, in different ways. In vitro, production and secretion of the antiangiogenic and cytokines molecules following viral infection of various cell lines is confirmed by western blot and the cytotoxic activity of TK will be evaluated in presence of GCV with standard MTT assay.

In order to generate multiple transgene vectors, known angiostatic vectors are genetically crossed with the vectors constructed above:

(a) TO endostatinangiostatin in UL41 (TOendo-angio) with THIL12 or THGM-CSF; (b) TO endostatin-kringle5 in UL41 (TOendo-kringle) with THIL12 or THGM-CSF.

All these recombinant viruses have also their own HSV-TK gene where the TK endogenous promoter has been substituted with the HSV immediate early promoter ICP4 in order to ensure the expression of this suicide gene in a replication-defective HSV mutant. We will identify the recombinants by screening progeny viruses by Southern blot analysis for the transgenes sequences. Positive isolates are purified following three rounds of limiting dilution and the protein expression will be determined by Western blot analysis and the cytokine expression will be detected and measured by ELISA. Construction of HSV vectors co-expressing cytokine, suicide genes and Cx43.

The vectors expressing IL12 or GM-CSF are combined with vectors expressing TK and Cx 43 gene. The tumor cell lines are infected with the cytokine vectors and the secreted proteins quantified by ELISA kits. The cytotoxic activity of TK is evaluated in presence of GCV with standard MTT assay.

The THIL12 or THGM-CSF vectors are genetically crossed with the mutant virus that carries Cx43 in the UL41 locus (29) to create T0CX-IL12 or TOCX-GMCSF. These mutants have their natural TK under the ICP4 promoter. Progeny viruses are then screened for the presence of the transgenes by Southern blot analysis and the protein expression identified by Western blot analysis. Cytokine expression is identified by ELISA.

These two last vectors are used to compare the effect of the suicide genes in combination with cytokine genes with or without coexpression of Cx 43. The cytotoxic activity of the suicide genes and any synergistic effect with Cx43 is determined by MTT assay.

Tumor cells are infected with the control vector (THZ4) and with the vectors expressing TK or TK and Cx43. The cells are infected at different MOIs and plated in 96well plates in presence or absence of GCV. At 24h intervals the cell viability is determined by MTT assay. The results are plotted as the percentage of survival relative to mockinfected cells.

Biological activity of the vector-encoded anti-angiogenic molecules

ECV304 and primary endothelial cell lines HUVEC are treated with the recombinant basic fibroblast growth factor (rbFGF) and vascular endothelial growth factor (rVEGF) in order to induce angiogenesis. Subsequently, different tumor cell lines are infected with vectors expressing anti-angiogenic factors in different combinations and tested their antiproliferative and antimigratory activity on ECV304 and on HUNEC cells, using MTT-based assays, conventional migration chambers and specific tube formation assays. Νon-replicative HSV-1 based vectors are able to express biologically active angiogenesis inhibitors and suicide genes. Anti-proliferative activity in vitro is used to evaluate the biological activity of the recombinant proteins. Inhibition of endothelial cell proliferation is determined by using ECV304 and HUVEC cells. We have established experiments to estimate the capacity of these angiostatic molecules to inhibit migration and formation of capillary-like tubules by endothelial cells. Co-culture assays are set up with tumor cells infected with the recombinant viruses and the endothelial cells to investigate the:

(1) Proliferation of endothelial cells by MTT assay, which determines the metabolic activity of mitochondria and correlates well with the number of viable cells. Media from LLC, B16 and GL- 216 (2*10β) cells infected with the recombinant vectors at MOI of 2 is collected and overlaid on ECV304 or HUVEC. bFGF 3ngr/ml is added as angiogenic stimulus. Negative controls are represented by ECV304 or HUVEC cells freated with medium obtained from non-infected tumor cells or infected with a control vector (THZ1 or THZ5). After an incubation of 5 days, ECV304 growth or HUVEC is determined by a colorimetric, tetrazolium-based (MTT) assay.

(2) Migration through transwell systems. LLC, B16 and Gl-216 (2*10s cells/ well) is seeded in the lower compartments of 24 well transwell systems and infected (MOI 2) with the HSV1- based vectors; 48h post infection, bFGF 25ngr/ml is added as a chemotactic stimulus. ECV304 (6000 cells /well) and HUVEC (3000cells/well) cells are placed in the upper chambers; after 5 hours incubation, cells migrated from the upper to the lower filter surface are fixed and counted. Results are expressed as percentage of cell migration, compared with migration induced by conditioned media from uninfected tumor cells; 10 Qgr/ml of recombinant human antiangiogenic proteins (angiostatin and endostatin) is used as a positive control.

(3) Differentiation of endothelial cells into tubules in presence and absence of T0-Endo::Angio and/or T0-Endo::Kringle. The endothelial tube formation is evaluated and visualized through a commercial kit. The cells (a mixture of fibroblasts and endothelial cells) are infected with the vectors (MOI 1) at days 1, 4, 7 and 10 post cells seeding. Positive kit confrol are treated with VEGF 2 ngr/ml; negative control are treated with suramin 20 mM or the viral vector control. At the 11^th day post seeding the cells are incubated with anti-CD31 antibody, in order to evidence and to count the formed tubes.

In vivo studies with HSV vectors expressing antiangiogenic proteins, cytokines and HSV TK. Brief Summary: following tumor implantation in the right flank of C57B1/6, the palpable tumor mass is treated with PBS solution as negative control, vectors without therapeutic genes as vector confrol, vectors expressing anti-angiogenic, cytokine and suicide genes, developed in above. The tumors that examined in these studies are: (i) BL16 melanoma tumor cells (3, 4, 10); (ii) Lewis lung carcinoma (LLC) cells (19, 34, 38) and (iii) murine GL-261 gliomas model (1, 35); all tumor models are syngenic on C57BL/6 mice. Three different types of tumor are considered to compare the different antitumor immune responses induced by exposing various neoplasias to the same therapeutic freatment. Microvessel moφhology, neovascularization and infiltrating cell phenotypes are analyzed on tumor tissue sections using immunohistochemical techniques. The tumors implanted in the right flank will allow us to test the efficacy of the different recombinant vectors by monitoring the tumor mass growth with calipers over the course of the experiments. To determine the number of cytokine secreting cells, spleens and tumors are dissected and the cytokine in situ production is carried out by ELISPOT method. If these vectors are efficient, the tumor volume decreases and the immune response against the tumor is increased.

Murine Lewis lung carcinoma (LLC), B16 melanoma and GL-261 glioma cells are implanted in the right flank of 7 weeks old C57BL/6 mice to directly test the efficacy of angiostatic genes, cytokines and TK suicide gene following prodrug adminisfration and to analyze the anti-tumor treatment response related to the tumor model. After implantation tumors are treated with PBS solution and vectors without therapeutic genes as negative control and with recombinant vectors expressing cytokines molecules alone and/or in combination with anti-angiogenic and suicide genes. The group of mice treated with combined therapy is compared with animals inoculated with vectors expressing only angiostatic molecules or only cytokines or TK/GCV. Physiologic solution and pro-drugs are administered intraperitoneally and treatment will start when tumors are palpable. Tumor volumes are measured with a digital calliper every two days; each time point will represent the average of 6 mice in each group.

Some mice are sacrificed at different times post tumor cells injection. Histologic analysis is done on tumor sections to define, microvessel moφhology and neovascularization and tumor infilfrates using immunohistochemical techniques. Furthermore to determine also the number of cytokine secreting cells, spleens and tumors are dissected and the cytokine released will be carried out by ELISPOT method. The end point to use three different tumor cell lines is to test this multifactorial therapy and to verify the susceptibility of the different tumors to respond to these angiostatic, cytokines and TK molecules and to analyze if their synergistic effect is equally efficient in eradicating different neoplasies.

REFERENCES for Example 2

1. Andreansky, S. S., B. He, G. Y. Gillespie, L. Soroceanu, J. Markert, J. Chou, B. Roizman, and R. J. Whitley. 1996. The application of genetically engineered heφes simplex viruses to the freatment of experimental brain tumors. Proc Natl Acad Sci U S A 93: 11313-8. 2. Asklund, T., I. B. Appelskog, O. Ammeφohl, I. A. Langmoen, M. S. Dilber, A. Aints, T. J. Ekstrom, and P. M. Almqvist. 2003. Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene. Exp Cell Res 284:185-95. 3. Asselin-Paturel, C, N. Lassau, J. M. Guinebretiere, J. Zhang, F. Gay, F. Bex, S. Hallez, J. Leclere, P. Peronneau, F. Mami-Chouaib, and S. Chouaib. 1999. Transfer of the murine interleukin-12 gene in vivo by a Semliki Forest virus vector induces B16 tumor regression through inhibition of tumor blood vessel formation monitored by Doppler ulfrasonography. Gene Ther 6:606-15. 4. Berenstein, M., S. Adris, F. Ledda, C. Wolfmann, J. Medina, A. Bravo, J. Mordoh, Y. Chernajovsky, and O. L. Podhajcer. 1999. Different efficacy of in vivo heφes simplex virus thymidine kinase gene transduction and ganciclovir treatment on the inhibition of tumor growth of murine and human melanoma cells and rat glioblastoma cells. Cancer Gene Ther 6:358-66. 5. Brockstedt, D. G., M. Diagana, Y. Zhang, K. Tran, N. Belmar, M. Meier, A. Yang, F. Boissiere, A. Lin, and Y. Chiang. 2002. Development of anti-tumor immunity against a non- immunogenic mammary carcinoma through in vivo somatic GM-CSF, IL-2, and HSVtk combination gene therapy. Mol Ther 6:627-36. 6. Burton, E. A., D. J. Fink, and J. C. Glorioso. 2002. Gene delivery using heφes simplex virus vectors. DNA Cell Biol 21:915-36. 7. Cao, Y., A. Chen, S. S. An, R. W. Ji, D. Davidson, and M. Llinas. 1997. Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J Biol Chem 272:22924-8. 8. Cao, Y., R. W. Ji, D. Davidson, J. Schaller, D. Marti, S. Sohndel, S. G. McCance, M. S. O'Reilly, M. Llinas, and J. Folkman. 1996. Kringle domains of human angiostatin. Characterization of the anti-proliferative activity on endothelial cells. J Biol Chem 271:29461-7. 9. Castleden, S. A., H. Chong, I. Garcia-Ribas, A. A. Melcher, G. Hutchinson, B. Roberts, I. R. Hart, and R. G. Vile. 1997. A family of bicisfronic vectors to enhance both local and systemic antitumor effects of HSVtk or cytokine expression in a murine melanoma model. Hum Gene Ther 8:2087-102. 10. Chatterjee, S. K., H. Qin, S. Manna, and P. K. Tripathi. 1999. Recombinant vaccinia virus expressing cytokine GM-CSF as tumor vaccine. Anticancer Res 19:2869-73. 11. Duda, D. G., M. Sunamura, L. Lozonschi, T. Kodama, S. Egawa, G. Matsumoto, H. Shimamura, K. Shibuya, K. Takeda, and S. Matsuno. 2000. Direct in vitro evidence and in vivo analysis of the antiangiogenesis effects of interleukin 12. Cancer Res 60:1111-6. 12. Ergun, S., N. Kilic, J. H. Wurmbach, A. Ebrahimnejad, M. Fernando, S. Sevinc, E. Kilic, F. Chalajour, W. Fiedler, H. Lauke, K. Lamszus, P. Hammerer, J. Weil, H. Herbst, and J. Folkman. 2001. Endostatin inhibits angiogenesis by stabilization of newly formed endothelial tubes. Angiogenesis 4:193-206. 13. Folkman, J. 2003. Angiogenesis inhibitors: a new class of drugs. Cancer Biol Ther 2:S127- 33. 14. Folkman, J. 2002. Role of angiogenesis in tumor growth and metastasis. Semin Oncol 29:15-8. 15. Griscelli, F., H. Li, A. Bennaceur-Griscelli, J. Soria, P. Opolon, C. Soria, M. Perricaudet, P. Yeh, and H. Lu. 1998. Angiostatin gene transfer: inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest. Proc Natl Acad Sci U S A 95:6367-72. 16. Guo, S. Y., Q. L. Gu, Z. G. Zhu, H. Q. Hong, and Y. Z. Lin. 2003. TK gene combined with mIL-2 and mGM-CSF genes in treatment of gastric cancer. World J Gasfroenterol 9:233-7. 17. Herbst, R. S., M. Hidalgo, A. S. Pierson, S. N. Holden, M. Bergen, and S. G. Eckhardt. 2002. Angiogenesis inhibitors in clinical development for lung cancer. Semin Oncol 29:66- 77. 18. Hess, S. D., N. K: Egilmez, N. Bailey, T. M. Anderson, E. Mathiowitz, S. H. Bernstein, and R. B. Banker! 2003. Human CD4+ T cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of IL-12 to kill tumors in situ by indirect effects of IFN-gamma. J Immunol 170:400-12. 19. Jia, S., F. Zhu, H. Li, F. He, and R. Xiu. 2000. Anticancer freatment of endostatin gene therapy by targeting tumor neovasculature in C57/BL mice. Clin Hemorheol Microcirc 23 :251-7. 20. Kaφoff, H. M., D. Kooby, M. D'Angelica, J. Mack, D. H. Presky, M. D. Brownlee, H. Federoff, and Y. Fong. 2000. Efficient cotransduction of tumors by multiple heφes simplex vectors: implications for tumor vaccine production. Cancer Gene Ther 7:581-8. 21. Kirn, D., I. Niculescu-Duvaz, G. Hallden, and C. J. Springer. 2002. The emerging fields of suicide gene therapy and virotherapy. Trends Mol Med 8:S68-73. 22. Krisky, D. M., P. C. Marconi, T. Oligino, R. J. Rouse, D. J. Fink, and J. C. Glorioso.

1997. Rapid method for construction of recombinant HSV gene transfer vectors. Gene Ther 4:1120-5. 23. Krisky, D. M., P. C. Marconi, T. J. Oligino, R. J. Rouse, D. J. Fink, J. B. Cohen, S. C. Watkins, and J. C. Glorioso. 1998. Development of heφes simplex virus replicationdefective multigene vectors for combination gene therapy applications. Gene Ther 5:1517- 30. 24. Krisky, D. M., D. Wolfe, W. F. Goins, P. C. Marconi, R. Ramakrishnan, M. Mata, R. J. Rouse, D. J. Fink, and J. C. Glorioso. 1998. Deletion of multiple immediate-early genes from heφes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons. Gene Ther 5:1593-603. 25. Li, M., X. Huang, Z. Zhu, Q. Zhao, M. Wong, and E. Gorelik. 2002. The therapeutic efficacy of angiostatin against weakly- and highly-immunogenic 3LL tumors. In Vivo 16:577- 82. 26. Lissoni, P., E. Fumagalli. F. Malugani, A. Ardizzoia, R. Bucovec, G. Tancini, and G. S. Gardani. 2001. Stimulation of IL-12 secretion by GM-CSF in advanced cancer patients. J Biol Regul Homeost Agents 15 : 163 -5. 27. Mackensen, A., A. Lindemann, and R. Mertelsmann. 1997. h munostimulatory cytokines in somatic cells and gene therapy of cancer. Cytokine Growth Factor Rev 8:119-28. 28. Marconi, P., D. Krisky, T. Oligino, P. L. Poliani, R. Ramakrishnan, W. F. Goins, D. J. Fink, and J. C. Glorioso. 1996. Replication-defective heφes simplex virus vectors for gene transfer in vivo. Proc Natl Acad Sci U S A 93 : 11319-20. . 29. Marconi, P., M. Tamura, S. Moriuchi, D. M. Krisky, A. Niranjan, W. F. Goins, J. B. Cohen, and J. C. Glorioso. 2000. Connexin 43-enhanced suicide gene therapy using heφesviral vectors. Mol Ther 1:71-81. 30. Moriuchi, S., T. Oligino, D. Krisky, P. Marconi, D. Fink, J. Cohen, and J. C. Glorioso.

1998. Enhanced tumor cell killing in the presence of ganciclovir by heφes simplex virus type 1 vector-directed coexpression of human tumor necrosis factor- alpha and heφes simplex virus thymidine kinase. Cancer Res 58:5731-7. 31. Moser, T. L., M. S. Stack, M. L. Wahl, and S. V. Pizzo. 2002. The mechanism of action of angiostatin: can you teach an old dog new tricks? Thromb Haemost 87:394-401. 32. O'Reilly, M. S., T. Boehm, Y. Shing, N. Fukai, G. Vasios, W. S. Lane, E. Flynn, J. R. Birkhead, B. R. Olsen, and J. Folkman. 1997. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88:277-85. 33. Oppenheim, J., and H. Fujiwara. 1996. The role of cytokines in cancer. Cytokine Growth Factor Rev 7:279-88. 34. Oshikawa, K., A. L. Rakhmilevich, F. Shi, P. M. Sondel, N. Yang, and D. M. Mahvi. 2001. Interleukin 12 gene transfer into skin distant from the tumor site elicits antimetastatic effects equivalent to local gene transfer. Hum Gene Ther 12:149-60. 35. Parker, J. N., G. Y. Gillespie. C. E. Love, S. Randall, R. J. Whitley, and J. M. Markert. 2000. Engineered heφes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors. Proc Natl Acad Sci U S A 97:2208-13. 36. Springer, C. J. 2003. Introduction to Vectors for Suicide Gene Therapy. Methods Mol Med 90:29-46. 37. Sumimoto, H., K. Tani, Y. Nakazaki, T. Tanabe, H. Hibino, M. S. Wu, K. Izawa, H. Hamada, and S. Asano. 1998. Superiority of interleukin- 12-transduced murine lung cancer cells to GM-CSF or B7-1 (CD80) transfectants for therapeutic antitumor immunity in syngeneic immunocompetent mice. Cancer Gene Ther 5:29-37. 38. Tanaka, M., Y. Saijo, G. Sato, T. Suzuki, R. Tazawa, K. Satoh, and T. Nukiwa. 2000. Induction of antitumor immunity by combined immunogene therapy using IL-2 and IL-12 in low antigenic Lewis lung carcinoma. Cancer Gene Ther 7:1481-90. 39. van Dillen, I. J., N. H. Mulder, W. Vaalburg, E. F. de Vries, and G. A. Hospers. 2002. Influence of the bystander effect on HSV-tk/GCV gene therapy. A review. Curr Gene Ther 2:307-22. 40. Vile, R. G., S. Castleden, J. Marshall, R. Camplejohn, C. Upton, and H. Chong. 1997. Generation of an anti-tumour immune response in a non-immunogenic tumour: HSVtk killing in vivo stimulates a mononuclear cell infiltrate and a Thl -like profile of intratumoural cytokine expression. Int J Cancer 71:267-74. _{M&C FOLIO : WPP89}658 PC EP 2005/003639 53

Example 3 : HSV-based vectors for the treatment of HIV-associated tumours, such as neoplasias

As described in Example 2, different elements are combined to enhance tumor destruction: angiostatic factors promoting tumor regression along with enzyme-directed prodrug activation (tumor suicide) and/or IL-12 cytokine.

In the present approach, two type of vectors are created and tested for their ability to eliminate the ATDS-associated tumor:

(i) a replication-defective HSV vector, which expresses a fusion anti-angiogenic molecule, such as angiostatin and endostatin and

(ii) a replication-defective HSV vector, which expresses endostatin and kringle 5.

Both vectors have TK suicide gene that has been shown to improve the efficiency of anti-tumor gene therapy. Furthermore, new therapeutic combinations are accomplished by coupling, in the same vectors, the genes with angiostatic modulatory effect with the cytokine IL12 gene, which has a strongly immunomodulatory properties (40, 66). 11-12 promotes the proliferation of T cells, NEC cells and tumor-infiltrating lymphocytes (TIL cells), in addition, it can induce a cascade of other cytokines and chemokines which possesses significant antiangiogenic properties (17, 59).

In this particular example, are developed:

(1) HSV vectors containing multiple anti-angiogenic factors, and

(2) anti-angiogenic molecules to decreasing Tat-related angiogenesis in ATDS-associated tumors.

The anti-angiogenic and the suicide genes are expressed under the control of human cytomegalovirus immediate early promoter (HCMV) or HSV ICP4 immediate early promoter. These promoters have been chosen for their relative short time expression, which will eliminate concerns over-dosing and long term expression of these proteins. These vectors are tested in vitro, for their cytotoxic activity, in presence of ganciclovir (GCV) and for the biological activity of anti-angiogenic factors.

Their biological function is assessed by evaluation of the antiproliferative and antimigratory activity on endothelial-like ECV304 and HUVEC cells incubated with supematants derived from T53 BKV/Tat cells (that express Tat) (20) infected with the recombinant vectors with or without angiostatic genes.

Subsequently, the vectors are tested, in vivo, in appropriate animal models. The BDF and nude Balb/c mice are inoculated with T53 BKV/Tat derived tumor cell line and subsequently the tumor mass are treated with the vectors expressing: endostatin/angiostatin, endostatin/kringle genes, or both recombinant vectors, with or without addition of GCV. The mice are treated in order to inhibit tumor growth and reduce its mass.

References for Example 3

1. Albini, A., G. Barillari, R. Benelli, R. C. Gallo, and B. Ensoli. 1995. Angiogenic properties of human immunodeficiency virus type 1 Tat protein. Proc Natl Acad Sci U S A 92:4838-42.

2. Albini, A., R. Soldi, D. Giunciuglio, E. Giraudo, R. Benelli, L. Primo, D. Noonan, M. Salio, G. Camussi, W. Rockl, and F. Bussolino. 1996. The angiogenesis induced by HIV-1 tat protein is mediated by the Flk-1/KDR receptor on vascular endothelial cells. Nat Med 2:1371-5.

3. Altavilla, G., C. Trabanelli, M. Merlin, A. Caputo, M. Lanfredi, G. Barbanti- Brodano, and A. Corallini. 1999. Morphological, histochemical, immunohistochemical, and ultrastructural characterization of tumors and dysplastic and non-neoplastic lesions arising in BK virus/tat transgenic mice. Am J Pathol 154: 1231-44.

4. Barbanti-Brodano, G., R. Sampaolesi, D. Ca pioni, L. Lazzarin, G. Altavilla, L. Possati, L. Masiello, R. Benelli, A. Albini, and A. Corallini. 1994. HIV-1 tat acts as a growth factor and induces angiogenic activity in BK virus/tat transgenic mice. Antibiot Chemother 46:88-101.

5. Barillari, G., C. Sgadari, C. Palladino, R. Gendelman, A. Caputo, C. B. Morris, B. C. Nair, P. Markham, A. Nel, M. Sturzl, and B. Ensoli. 1999. mflammatory cytokines synergize with the HIV-1 Tat protein to promote angiogenesis and Kaposi's sarcoma via induction of basic fibroblast growth factor and the alpha v beta 3 integrin. J Immunol 163:1929-35. 6. Benelli, R., M. Morini, C. Brigati, D. M. Noonan, and A. Albini. 2003. Angiostatin inhibits extracellular HIV-Tat-induced inflammatory angiogenesis. hit J Oncol 22:87-91.

7. Bergers, G., and L. E. Benjamin. 2003. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 3:401-10.

8. Bergers, G., and D. Hanahan. 2002. Combining antiangiogenic agents with metronomic chemotherapy enhances efficacy against late-stage pancreatic islet carcinomas in mice. Cold Spring Harb Symp Quant Biol 67:293-300.

9. Boehm, T., J. Folkman, T. Browder, and M. S. O'Reilly. 1997. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390:404-7.

10. Brem, S. 1999. Angiogenesis and Cancer Confrol: From Concept to Therapeutic Trial. Cancer Confrol 6:436-458.

11. Browder, T., C. E. Butterfield, B. M. Kraling, B. Shi, B. Marshall, M. S. O'Reilly, and J. Folkman. 2000. Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer. Cancer Res 60:1878-86.

12. Buonaguro, L., G. Barillari, H. K. Chang, C. A. Bohan, V. Kao, R. Morgan, R. C. Gallo, and B. Ensoli. 1992. Effects of the human immunodeficiency virus type 1 Tat protein on the expression of inflammatory cytokines. J Virol 66:7159-67.

13. Bussolino, F., A. Albini, G. Camussi, M. Presta, G. Viglietto, M. Ziche, and G. Persico. 1996. Role of soluble mediators in angiogenesis. Eur J Cancer 32A:2401-12.

14. Campioni, D., A. Corallini, G. Zauli, L. Possati, G. Altavilla, and G. Barbanti- Brodano. 1995. HIV type 1 extracellular Tat protein stimulates growth and protects cells of BK virus/tat transgenic mice from apoptosis. AIDS Res Hum Retroviruses 11:1039- 48.

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Example 4: Challenge Model

A critical issue in the field of vaccine development is to have suitable animal models to test the safety, irnmunogenicity and efficacy of a vaccine formulation.

We provide a method, e.g. for pathogens such HIV-1 - that doesn't have a murine challenge model - to test the efficacy of a protein-, DNA- , viral or bacterial vector-based vaccine in the mouse to select, in a time- and cost-efficent manner (as compared to the non-human primate model), the most promising vaccine formulation and vaccination protocol that deserve further development, e.g. clinical development.

The present challenge model is based on recombinant replication-competent HSV-1 or HSV-2 expressing the vaccine antigen, e.g. HIV antigens.

The method is generally useful in pre-clinical vaccine research, for example, in investigations aimed at studying the efficacy of any vaccine candidate. Safety, immunogenicity and protection experiments will be performed in animal models (e.g murine) that may be of outmost relevance to speed up vaccine candidate testing and transition to clinical trials.

In the HSV-based challenge system, the antigen is introduced in a HSV locus which is not essential for HSV-1 and HSV-2 replication (e.g. UL41, Us4, Us5) (Marconi et al., Proc. Natl. Acad. Sci. USA, 1996). These recombinant viruses are able to replicate and to induce disease and death in mice after inoculation of a lethal dose by systemic and mucosal routes. Survival of immunized mice 10-15 days after challenge with a lethal dose of recombinant HSV-1 expressing the vaccine antigens is the efficacy endpoint.

In the HSV-1- based system, the vaccine antigen (e.g. HIV-1 genes [tαt, gag (clade B), and env (clades A, B, and C)] is introduced in the UL41 locus of the HSV-1. Alternatively, the vaccine antigen may be introduced in the Us5 locus of HSV-1.

Another HSV-based challenge system is based on HSV-2, or genital heφes, which is more pathogenic in the genital tract as compared to HSV-1. Balb-c mice inoculated in the vagina or in the footpad are highly susceptible to HSV-2 infection. Although both wild-type HSV-1 and HSV-2 infect and induce disease in mice after infra- vaginal inoculation, the lethal dose of HSV-1 is 1-2 logs higher (depending on the strains) than that of HSV-2. Thus, replication competent HSV-2 viruses expressing the vaccine antigens may he more efficacious as a vaginal challenge model, as compared to recombinant HSV-1.

The HSV virus carrying a deletion in a non-essential gene, such as UL41, Us4 or Us5 locus, is be generated by homologous recombination of the HSN-1 or HSV-2 genome with a plasmid carrying the LacZ gene flanked by HSV-1 or HSV-2 sequences. The deleted virus is then used to create the recombinant HSV-1 or HSV-2 viruses carrying the e.g. HlV-vaccine antigens in the deleted locus. The new recombinant HSN-1 or HSV-2 viruses are analyzed by Southern blot and PCR, and tested for expression of the transgenes e.g. by immunofluorescence and western blot. Large scale stocks of each recombinant virus are then produced, titrated in vitro, in suitable cell cultures, and in Balb/c mice to determine the lethal dose (LDl 00) following systemic and mucosal (i.e intra- vaginal, intra- rectal) inoculation.

MATERIAL AND METHODS

Cells. Vero (African green monkey kidney) and Balb-c (mouse) cells were maintained in Dulbecco's modified minimal essential medium (BioWhittaker) supplemented with 5% fetal bovine serum, 1% Glutamine and 1% penicillin/streptomycin.

Bacteria. Escherichia coli (Stratagene) strain DH5α was used in plasmid cloning procedures. Bacteria were grown in Luria-Bertani medium (for liquid culture) or in Luria-Bertani agar plates, both supplemented with antibiotics as appropriate (Ampicillin 100 μg/ml or Kanamycin 50 μg/ml).

Plasmids. pET28a-modi-Nef plasmid was kindly provided by V. Erfle (GSF, Munich, Germany), pKCMV-p37 (Gag p24 + pl7) and pKCMV-gpl60 (Env clade A, B, C separately) by B. Wahren (Karolinska Institutet, Stockolm, Sweden). p41plasmid (a pBlueScript comprising the HSV-UL41 flanking regions), alone and with the expression cassette containing LacZ gene has been previously described (Krisky DM et al, 1997). pcDNA 3.1^" plasmid was purchased from Invitrogen, pTZ18U plasmid from Sigma.

Animals. Balb-c female mice (5-6 weeks old) were purchased from Charles River (Milan, Italy) Replication competent viruses expressing LacZ. The virus HSV-1 Δ41 or HSV-2 Δ41-1 were created by insertion of a deletion in the UL41 locus of the HSV-1 and HSV-2 genome according to the method previously described (Krisky DM et al, 1997). Briefly, a plasmid containing the HSV ICPO-immediated early promoter driving the expression of the lacZ reporter gene (transgene cassette), cloned into the UL41 locus of HSV (Pad restriction endonuclease site), was constructed. In this plasmid, therefore, the transgene cassete is flanked by HSV-1 or HSV-2 UL41 viral sequenses. Next, the recombinant virus was created by homologous recombinatio between the palsmid and the HSV genome. To this puφose Vero cells were co-transfected with the plasmid and either the HSV-1 wild-type or HSV-2 wild-type genome DNA. The recombinant virus carrying the deletion in the UL41 locus were selected and isolated based on lacZ gene expression, according to standard procedures (Krisky DM et al, 1997 ). The correct insertion was confirmed by Southern blot analysis.

The same procedure was used to generate the viruses HSV-l/Us5Δ-l or HSV-2/Us5Δ-l and HSV-2/Us4Δ-2 with a deletion in the Us5 locus of HSV-1 and HSV-2, corresponding to glycoprotein JI (gJl), or in the Us4 locus of HSV-2, corresponding to glycoprotein G (gG). Briefly, an expression cassette containing the human cytomegalovirus (HCMV)-immediate early promoter-lacZ gene flanked by Pmel restriction endonuclease recognition sites was cloned in a plasmid containing viral sequences Us4 or Us5.

This plasmid was co-transfected in Vero cells either with LV-HSV-1 wild-type strain or G- HSV-2 wild-type strain.

Plasmid constructs. In order to have the genes of interest under the control of the HCMV promoter and between the UL41 flanking regions, two cloning steps were necessary. For construction of plasmids p41-Tat, p41-Nef, p41-Gag, p41-EnvA, p41-EnvB and p41-EnvC, the following procedure was carried out. The tat gene was excided from original plasmid pCV-tat (Arya et al., Science 1985) with Pstl and then ligated into the Pstl site of plasmid pTZ18U (Sigma) to generate the intermediate expression plasmids pTZ18U-Tat. Plasmid pB410-tat was then constructed by introduction of the HIV-1 tat cDNA (350 pb) from pTZ18U-Tat into the UL41 HSV1 sequences (HSV genomic positions 90.145-91.631 and 92.230-93.858) of plasmid pB41, which has been described elsewhere (Krisky DM et al, 1997), under the transcriptional control of the HSV1 immediate-early ICPO promoter. The nef gene was excided from plasmid pET28a-modi-Nef with Xbal/EcoRI, while the other genes (gag and env) were excided with Sall/EcoRI from pKCMV-p37 and pKCMV-gpl60, respectively. The fragments were then ligated into the Xbal/EcoRI (nef) or XhoI/EcoRI (gag and env) site of pcDNA 3.1^" (InVitrogen) to generate intermediate expression plasmids (pcDNA3.1- Nef, pcDNA3.1-Gag, pcDNA3.1-EnvA, pcDNA3.1-EnvB and pcDNA3.1-EnvC) where the nef, gag and env genes are under the transcriptional control of the HCMV-immediate early promoter. Fragments with HIN genes under HCMV promoter were then cleaved from the pcDΝA3.1- derived plasmids with NruI/EcoRI and ligated into the Sma ΕcoRI sites of the pB41 plasmid.

HSV-1 recombinant vectors expressing HIV-1 proteins. LV-LacZ viral genome was isolated from infected Vero cell lysates by the proteinase K / phenol-chloroform extraction procedure (Sambrook J. et al, 1989). Cotransfections of LV-LacZ viral DNA, cleaved with Pad (in order to excide Lac Z), and the above described plasmids containing the HIV genes, linearized with Notl, were performed in Vero cells using calcium phosphate precipitation technique (Knipe DN et al, 1979), then in vitro titer was assessed with the methyl-cellulose method as previously described (Fendrick JL et al, 1983). After each co-fransfection, recombination percentage was evaluated by using LacZ as a reporter gene.

Plaque purification was carried out by 3-4 Limiting Dilution (LD) rounds (Krisky et al. 1997), each followed by a Southern-Blot (SB) or Dot Blot (DB) analysis to confirm the presence of the transgenes and their correct insertion into the viral genome.

Analysis of the expression of the transgenes from the HSV-1 recombinant virus. Vero and Balb-c cells were infected with 1 multiplicity of infection (m.o.i.) of recombinant virus, harvested at different time points and diluted in loading buffer containing protease inhibitors and dithiothreitol (DTT). Cell extracts, corresponding to 10 μg of total proteins, were loaded onto 12% SDS-polyacrylamide gel and analyzed by western blot using specific monoclonal antibodies (ARP 3061 1:1000 for Gag and EVA 307 1:250 for EnvC) and a goat-anti-mouse IgG antibody HRP-conjugate (Sigma) (1:2500). Antibody immunocomplexes were detected by ECL Western Blot detection kit (Amersham, Pharmacia Biotech). Controls were represented by uninfected cells and cells infected at m.o.i. of 1 with HSV-1 wild-type virus.

Recombinant proteins and MAbs were obtained from the Centralized Facility for AIDS Reagents (EVA Program), UK. Large-scale virus production. In order to have a stock of serum-free purified virus, cells (24 to 32 150 mm² tissue culture flasks) were infected with 0.02 multiplicities of each virus and harvested once the infection was well spread (approximately 24 hours post-infection). After collection of the viral particles present in the culture medium and in the cells (disrupted by repeated freezing and thawing), the recombinant HSV viruses were purified with Optiprep® gradient (Axis-Shield) according to the manufacturer's instructions, diluted in PBS, divided in 30-100 μl aliquots and stored at - 80°C. The in vitro titer was assessed with the methyl-cellulose method as previously described (Fendrick JL et al, 1983).

Vaginal challenge. One week before challenge, female Balb-c mice are treated with 2 mg/100 μl of Depo-Provera^® (Depo-medroxy-progesterone acetate; Pharmacia & Upjohn) given subcutaneously in the neck. This drug brings the animals at the same estrous stage, minimizing infection variability (Kaushic C et al, 2003). The day of the challenge, the virus is thawed in ice, sonicated for 5 seconds, and briefly stored on ice. Mice are anaesthetized with 5% isoflurane to allow scraping of the vagina with a pipe scraper (in order to remove the mucus that could trap the virus) and then inoculated with the purified virus using a pipette-tip. Onset and follow-up of the infection is then observed for at least 15 days.

RESULTS

HSV-1 ΔUL41

Recombinant HSV-1 viruses carrying HIV-1 genes [tαt, gag, env (clade B) and env of clade A and C] in the UL41 locus, namely LV-Tat, LV-Gag, LV-EnvA, LV-EnvB, LV-EnvC (Figure 7) were generated and characterized by Southern blot analysis, as described in the materials and methods section. Expression of HIV-1 proteins was assessed following infection of Vero cells or Balb/c fibroblasts with the HSV-1 recombinant viruses by western blot, 24 and 48 hours post- infection. Controls were represented by cells infected with the LV-lacZ vector or non-infected cells.

To determine the lethal dose (LD) of recombinant HSV-1 viruses expressing HIN-1 antigens and the best route of adminsitration, the following preliminary experiments were carried out. Female Balb-c mice are more susceptible to HSN infection as compared to C57BL6_. mice (Lundberg P et al, 2003), thus all experiments were carried out in Balb-c mice.

A first experiment was carried out to determine the range of the infectious dose capable of killing the mice and the best route of administration. Mice were inoculated with 1.4 x 10⁶, 1.4 x 10⁷, 1.4 x 10⁸ pfu of LV-Tat infraperitoneally (in 100 μl of physiologic solution/mouse) or intravaginally (10 μl mouse). Mice, that received the virus infra- aginally, were inoculated subcutaneously with Depo-Provera^® 2mg/100μl, five days before the treatment. This drug transforms a proliferative endometrium into a secretive endometrium, by inhibiting hypophysis in gonadofropin secretion and then hampering follicular maturation and ovulation. Inoculation of Depo-Provera^® brings the mice at the same estrous stage and render them more susceptible to HSV infection (Kaushic C et al, 2003).

After infection (day 0), animals were observed daily for the onset of symptoms and death. Figure 8 summarizes the results of this preliminary experiment. None of the animals infected infraperitoneally died, whereas mice inoculated infra-vaginally were highly susceptible to LV- Tat infection (5/7 died). Similar results were obtained in mice inoculated infraperitoneally with LV-Gag (Figure 11). Thus, the intra-vaginal route was selected for further experiments aimed at determining the LD of the HSV recombinant viruses.

To this goal, 10 mice were infected infra- vaginally with 1.5 x 10⁸ pfu/10 μl, 3 x 10^s pfu/20 μl, 6 x 10⁸ pfu/40 μl, from a viral stock that had a titer of 1.5 x 10¹⁰ pfu/rnl. Figure 9 A shows that LV-Tat killed 9/10 of the mice at the dose of 3 x 10⁸ pfu. The reason why a lower mortality was observed with the dose of 6 x 10⁸ pfu may depend on a technical problem, namely that to infect with the dose of 6 x 10 it was necessary inoculate 40 μl of viral solution. This volume is too large and most of treated mice leaked some of the virus. Thus, these results indicate that the maximum volume that can administered should not exide 10 μl. However, since the virus stock titers is usually not higher thatn 10¹⁰ pfu/ml, we tested a different approach to determine the lethal dose 100. Five days after the treatment with Depoprovera, mice were divided into three groups and inoculated mfra- vaginally. One group was inoculated with 3 x 10 pfu/10 μl of LV- Tat at day 1, the second group was inoculated with same dose both at day 1 and at day 2, and the third group received the same virus dose at days 1, 2 and 3. Mice were then observed daily for disease onset and death. As shown in Figure 9B, at day 12 after infection 7/7 treated mice died when they were inoculated once. In contrast, at day 12 after infection 5/7 and 6/7 died in the groups inoculated twice or three times, respectively. This different outcome is probably dependent on a lower capability of the cells to be infected 24 and 48 hours after the first infection. This result was also corifirmed in independent experiments where mice were inoculate infra-vaginally with the same dose (1.35 x 10¹⁰ pfu/10 μl) of LV-Gag once at day l or twice (day 1 and day 2). Indeed as shown in Figure 11, the single inoculation killed 9/10 mice, whereas only 4/10 died after receiving a double dose of virus. These results indicate that the multiple-infection approach is not useful. At present, procedures to obtain virus stocks with titers higher that 10¹⁰pfu/ml are being developed in order to be able to inoculate a dose higher that 3 x 10⁸ pfu/10 μl, which will likely lead to 100% death of the treated mice.

By using the same protocol, it was demonstrated that LV-Env C killed 8/10 of the mice at both 4 x 10⁷ and 8 x 10⁷ doses (Fig.10).

To determine the feasibility of this murine challenge model, a preliminary pilot experiment was carried out. First, mice (n=10) were immunized with 1 μg of pCV-tat plasmid DNA alone or delivered by the K2 cationic block copolymer (Caputo et al., Vaccine 2001) at week 0 and 4, and boosted with 1 μg of HIV-1 Tat protein in alum at weeks 8 and 10. Control mice were inoculated with the backbone pCV-0 plasmid DNA alone or combined with K2 and boosted with alum alon. Two weeks after last immunization were challenged with LV-Tat and observed for disease onset and death. The results of this experiment are promising since 3/10 died in the group immunized with pCV-Tat ± K2 at day 22 after challenge, as compared to 4/10 which died in the group inoculated with pCV-0 ± K2.

HSV-1 ΔUs5

In order to create HSV-1 recombinant viruses which are more pathogenic that HSV-1 ΔUL41, recombinant HSV-1 deleted in a different non-essential viral locus, eg the Us5 locus corresponding to glycoprotein JI (gJl), have been generated. These virus expresses the lacZ reporter gene under the control of HCMV-imediate early gene promoter (Figure 12) and is currently beign tested for pathogenicity in mice in comparison to HSN-lΔUL41-lacZ and wild- type HSN-1. DISCUSSION

In order to test the efficacy of HIV/ AIDS vaccines in mouse is necessary to create a challenge model ad hoc, since this animal cannot be infected by HIN (Hunter E, 1997).

To date, various challenge systems have been developed, especially based on pseudoviruses like HINl/Murine Leukemia Virus - HIVl/MuLV (Hinkula J et al, 2004) or HlVl/Vaccinia (Shinoda K et al, 2004). The first one is carried out by administering to the mice syngeneic (C57) HIN-1 MuLVinfected spleen cells: they produce HIV-1 pro viral DΝA, able to infect mice spleen cells. The successfully vaccinated mice cleared HIV-1 infected spleen cells 10-14 days after the challenge. The second one is based on infravenous inoculation of vaccinated Balb-c mice with replication competent vaccinia virus encoding HIV-1 genes. The end-point is the inhibition of viral growth.

With this work has been developed a new challenge model based on HSV1 recombinant viruses expressing HIV antigens: in this case, successfully vaccinated animals would survive to the intravaginal challenge with recombinant HSVs, while the others would die 10-15 days after virus administration.

The selection of this virus is based on many evidences: particularly, HSV can infect and establish a latency in mice, but also kill them if given at the appropriate dose (Kuklin et al, 1998) and its genome is well known, so it is quite easy to handle with it.

Importantly, the ability of HSV to kill mice would be useful to easily assay the efficacy of the vaccination.

The results obtained with the recombinant HIV/HSV-1 with deletion in UL41 indicate that such viruses are able to infect and kill mice, if administrated at the appropriate dose and through the right route of inoculation. The data obtained from the chllenge experiment also suggest that not all the recombinant viruses have the same behavior: if they encode for a structural protein (like Env or Gag) the titer needed for mice killing seem to be lower than the one necessary with the virus expressing a regulatory protein as Tat, that is known to have a plethora of effect on the immune system and on the cell cycle and to be implied in the confrol of viral gene expression, replication and pathogenicity. oy However these LV-HIN viruses have shown to kill the animals the dose to be use is very high to respect to LV wild-type virus suggesting that the mutation in vhs gene (UL41) of HSV-1 or HSV-2 viruses attenuates the pathogenicity of the wild-type viruses. New viruses have been constructed with deletion in sequences that should do not reduces replication and susceptibility in mice. These LV-HSV-1 or G-HSV2 new mutants are deleted in a small portion of glycoprotein gGl that is a non-essential for viral replication. Mutations in Us4 (gG) or Us5 (gJ) should do not interfere with HSV replication in vivo allowing the use of low viral doses with higher efficiency.

Figure Legends for Example 4

FIG.7

HSV-1 recombinant vectors expressing HIV-1 genes in the UL41 locus.

FIG.8

Mice inoculated (A) by intravaginal or (B) intraperitoneal route with three different doses of LV- Tat.

LV-Tat virus, given infravaginally at the dose of 1.4 x 10⁸, is able to kill 5/7 mice. None of the tested doses is able to kill the mice if given infraperitoneally.

FIG.9

(A). Mice were inoculated by the intravaginal route with three different doses of LV-Tat. (B) Mice were inoculated by the intravaginal route with the same dose of LV-Tat given once, twice or three times.

FIG.10

Mice inoculated by the intravaginal route with two different doses of LV-Env C.

FIG.11

Mice inoculated by the intravaginal route with the same dose of LV-Gag given once or twice. In addition, mice were inoculated with the same dose by the intraperitoneal rout. FIG.12

Graphic map of (A) HSV-1 LV strain-derived pgJl plasmid with lacZ expression cassette that was inserted as follows: the HSV-1 fragment Sail 136308-Hindm 138345 was first cloned into ρTZ18 plasmid = pgJl vector; pgJl was cut with Sphl (HSV-1 137626)-NruI (HSV-1 137729) and ligated with lacZ expression cassette excised with Nrul-Sphl from pcDNA3.1/Hygro/lacZ plasmid (Invitrogen); (B) graphic map of either LV HSVl or G HSV2 strain structures containing the lacZ gene in Us5 or Us4 loci, respectively.

Example 5 : Expression of Human Immunodeficiency Virus type 1 tat from a replication-deficient Herpes simplex type 1 vector induces antigen-specific T cell responses

Introduction

Despite the strong efforts to halt the HIV pandemic and to reduce the deaths for AIDS, it is clear that it is of extreme importance, as an alternative or additional strategy to HAART [1], to develop an anti-HIV vaccine capable of inducing an efficient anti-viral immunity and/or keeping under control the ongoing disease in the infected individuals [2-4]. There is growing evidence about the importance of both cellular and humoral immune responses against HIV proteins in AIDS patients in controlling the disease progression [5,6], giving rise to a more favorable, clinical outcome [7]. It has been previously shown that a broad CTL response against various viral proteins [8], both structural and regulatory, plays a major role in the clearance of initial viremia [9,10] and in the containment of viral replication during the following stages of infection [11]. The wide genetic variability across the HIN-1 clades and also the high mutation rate of structural genes, resulting in the presence of multiple protein forms even in the same individual [12], has turned the HIN vaccine research direction towards to the more conserved regulatory proteins, Tat, Rev and Νef, as possible components of a new combined vaccine [13-17]. In particular, it has been shown that the CTL response against the HIV-1 Tat protein inversely correlates with progression of disease in the infected individuals, i.e. the patients exhibiting stronger cytotoxic activity against Tat are frequently either non-progressors or have a slower disease progression in comparison with the patients showing little or no anti-Tat CTL response [2,11,18,19]. In addition, recent studies have shown that immunization with HIV-1 Tat (protein or DΝA) confers protection againstthe challenge with the highly pathogenic virus SHIN89-6A in monkey and that protection correlates with the presence of anti-Tat specific CTLs [13,15,20-23]

In order to develop new vaccination strategies able to enhance the cellular immunity towards Tat as well as other HIN-1 proteins [24,25], a large number of viral vectors expressing HIV or SIV antigens are under investigation [26,27], and among them poxvirus-based vectors which have been the most studied as HIV vaccine viral vectors [28], the alphavirus self-replicating vectors [29,30], the adenovirus [31-33], the lentivirus [34-36] or the heφes simplex virus vectors [37- 39]. Despite the general concerns regarding safety issues using live viral recombinants, the overall results with these different viral vectors indicate that they might be good candidates for the development of an anti-HIN-1 vaccine. Many of these viral vectors have been reported for their capacity to induce strong in vivo Thl and CTL responses, as well as high antibody titers, against various HIN-1 gene products [40-43].

In particular, the heφes simplex virus (HSV) vectors show several advantages for prophylaxis against viral infections. They have been shown: i) to elicit strong and durable immune responses by various routes of inoculation [39,44]; ii) the viral DΝA persists inside the host's cell nucleus as an episomal element, thus eliminating the safety concerns deriving from the random integration of the viral genome into the host's DΝA; iii) they carry the tk gene, encoding the viral thymidine kynase, that, in case of undesired effects, can be used, in combination with specific antiviral drugs, to kill the virus-harbouring cells. The use of HSV vectors requires the development of mutated viruses that are genetically stable, uncapable of replicating in the CΝS and of spreading in immuno-compromised individuals, not transmissible from immunized individual by contacts and, at the same time, capable of inducing protective immunity against the disease. Thus, replication-defective heφes simplex viruses characterized by the simultaneous deletion of multiple viral functions, including the immediate-early proteins ICP4, ICP27, ICP22 and the structural protein VHS (viral host shutoff) have been developed [45,46]. These HSV mutants show a reduced cytotoxicity , due to their inability to replicate and to spread in the host, but maintain the capability to infect a wide range of tissues and host species, h addition, these recombinant replication-defective vectors sustain high expression of the exogenous genes under homologous or heterologous promoters (HSV-1 or HCMV, respectively) [47,48], and because of their large genome can be arranged to simultaneously express multiple antigens [49]. Moreover, recent studies indicate that the pre-existing immunity against HSV infection does not compromise its efficacy as a vaccine vector [50,51].

In the present experiment, we investigated the ability of a recombinant replication-defective HSV-1 vector encoding the HIN-1 Tat protein, either alone (homologous prime-boost regimen) or in combination with Tat protein (heterologous prime-boost regimen), to induce long-term Tat- specific immune responses in a murine model. The results demonstrated that both homologous and heterologous prime-boost regimens elicited broad anti-Tat specific immune responses characterized by the presence of the Tat-specific cytotoxic T cells and also anti-Tat antibodies. 2. Materials and methods

2.1 Generation of plasmids and recombinant replication-defective HSV-1 1 vectors

Plasmid pCN-tat, expressing the HIN-1 tαt cDΝA (HTLN-IIIB isolate, subtype B) has been previously described [52,53]. Plasmid DΝA was purified from Escherichia coli by using Qiagen endotoxin free Maxi Kit (Qiagen, Hilden, Germany).

Plasmid pB410-tat was constructed by introduction of the HIN-1 tat cDΝA (350 pb) from pCV- tat into the UL41 locus of plasmid HSN-1 pB41 that has been described elsewhere [47]. The tat cDΝA under the transcriptional control of the HSN immediate-early ICPO promoter was inserted into EcoRI/Xbal sites of pB41 plasmid between the two UL41 HSN fragments (HSN genomic positions 90.145-91.631 and 92.230-93.858) [54]. The TOZGFP is a replication-defective HSN-1 viral vector having a low cytotoxicity due to the deletion of three immediate early genes (ICP4, ICP27, which are essential for viral replication and ICP22 which is not) and contains the gfp gene in the ICP22 locus and also the LacZ gene in the UL41 locus as marker genes. Plasmid pB410-tat was constructed to genetically recombine with the genome of the TOZGFP viral vector using the previously described Pac-facilitated lacZ substitution method [54]. The generation of recombinant viruses was carried out using the standard calcium phosphate transfection procedure with 5 μg of TOZGFP viral DΝA and 1 μg of linear plasmid pB410-tat. Transfection and isolation of the recombinant viral progeny was performed in 7b cells as previously described [45,55].

The recombinant virus TO-tat containing the tat cDΝA was first identified by isolation of a clear plaque phenotype after X-gal staining. The TO-tat virus was purified by three rounds of limiting dilution technique and the presence of the transgene was confirmed by Southern blot analysis. Viral stocks of the TO-tat and of the control vector TO-GFP (derived from TOZGFP without lacZ reporter gene in UL41 locus) were prepared and titrated using Vero-ICP4 and ICP27 stabely- transfected 7b cells [55].

2.2 Cell lines

The Vero-derived cell line, termed 7b, expresses the HSV-1 immediate early genes ICP4 and ICP27 required for virus replication [45,55]. . The monkey kidney fibroblast or (Vero), the 7b cells, the P815 murine (H-2^d) mastocytoma cell line, the baby hamster kidney cells (BHK) and the fibroblasts balb/c cells were all cultured in DMEM (Euroclone, Grand Island, NY) supplemented with 10% FBS (Euroclone), 2mM L-glutamine, 100 Dg/ml penicillin and 100 TJ/ml streptomycin. 7b cells were subjected montly to two-weeks long selection with 1 mg/ml G418 (Sigma). Human HeLa 3T1 cells were grown in DMEM and 10% FBS; these cells contain an integrated copy of plasmid HIV-LTR-CAT where expression of the choramphenicol acetyl fransferase (CAT) reporter gene is achievable only in the presence of Tat, indispensable for fransactivating the HIN-LTR promoter driving CAT [56]. Splenocytes from immunized and control mice were cultivated in RPMI 1640 (Euroclone) supplemented with 10% Hyclone (Euroclone), 50 μM β-mercaptoethanol, and 10 mM HEPES.

2.3 Western blot analysis

Tat protein expression from the TO-tat vector was analyzed in murine fibroblast cell line BALB/c cells (lxlO⁶ cells) infected with TO-tat virus at multiplicity of infection 1 (m.o.i.l). Cell extracts, corresponding to 10 μg of total proteins, were loaded on 12% SDS-polyacrylamide gel and analyzed by Western blot using a rabbit anti-Tat polyclonal serum (Intracel) at 1:1000 dilution and a mouse anti-rabbit HRP-conjugated secondary antibody (Sigma) at 1 :4000 dilution. Tat antibody immunocomplexes were detected by ECL Western Blot detection kit (Amersham, Pharmacia Biotech). Controls were represented by uninfected cells and cells infected at m.o.i. of 1 with TO-GFP recombinant vector.

2.4 CAT assay

Expression of functional Tat protein was determined by Cat assay as follows: HeLa 3T1 cells (lxlO⁶) were infected in suspension with TO-tat and control vector TO-GFP, at different m.o.i. (from 0.01 to 1) for 1 hour at 37°C under mild shaking. After infection, cells were washed twice with complete medium to eliminate the virus particles that did not infect the cells, plated onto 6 well-plates, and cultured at 37°C for 24 and 48 hours. After incubation, cells were disrupted by sonication, supematants were collected and cellular debris removed by cenfrifugation. CAT expression was measured after normalization to protein content (100 μg aliquots) as previously described [57].

2.5 HIV Tat protein expression, purification, and manipulation.

Recombinant HIN Tat from the HTLV-IIIB isolate (subtype B) was expressed in Escherichia coli, purified to homogeneity by heparin-affinity chromatography and high-performance liquid chromatography and stored lyophilized at -80°C as described [58]. Purified Tat protein had full biological activity in several assays [14,59,60]. As Tat is sticky, easily oxidable and photo- and thermo-sensitive, it was resuspended at 2 μg/ml in degassed PBS containing 0,1% BSA immediately before use and handled on ice and in the dark, with degassed buffer pre-flushed plasticware.

2.6 Virus stock purification

HSV-1 stocks were prepared by infecting 4x10^s 7b complementing cell lines with 0,05 MOI of TO-tat and TO-GFP viruses in suspension in 15 ml of medium for 1 hour at 37°C under mild agitation. When a 100% cytopathic effect was evident, cells were collected and centrifuged at 2000 φm for 15 minutes. The supematants were spun at 20.000 rpm in JA20 rotor (Beckman) for 30 minutes to collect the vims. The cellular pellets were resuspended in 2 ml of medium, subjected to three cycles of freeze-thawing (-80°C/37°C) and a single burst of sonication, to release the viral particles. The virus was further purified by density gradient centrifugation (Opti Prep; Life Technologies, Inc.) and resuspended in PBS-A IX. Viral stocks were titered as previously described (55) and stored at -80°C. Titles averaged between 2xl0⁸ to 2xl0⁹ plaque forming unit pfu/ml

2.7 Animals and immunization protocols

Animals were handled according use was according to national guidelines and institutional policy. Six weeks old BALB/c (H-2^d) female mice were purchased from Harlan Italy and immunized after one week according to the protocols described below.

First immunization protocol: mice were immunized with 4xl0⁴ pfu or 4xl0⁶ pfu of TO-tat purified vims by subcute (s.c.)-injection on the left flank. Control animals were injected with PBS. The animals were boosted s.c. at weeks 2, 4, and 9 after priming immunization.

Second immunization protocol: mice were immunized with 4xl0⁶ PFU of TO-tat vims or PBS, s.c. on the left flank or intranasally (i.n.) The animals were boosted s.c. and i.n. respectively at weeks 2, 4, and 9 after priming.

Third immunization protocol: mice were immunized s.c. with 4xl0⁶ PFU of TO-tat vims or PBS. Animals were boosted s.c. at weeks 2, 4, and 9, or at weeks 4 and 8 after priming. Fourth immunization protocol: mice were primed with 4xl0⁶ PFU of TO-tat vims by s.c. route, or with recombinant Tat protein (2 μg/mouse) by the intradermal route (i.d.). Confrol animals were injected with PBS. All mice were boosted s.c. at weeks 2and 9 after priming with 4xl0⁶ PFU of TO-tat vims.

The above-described doses of recombinant vims or protein were administered in 100 μl for the s.c. (one site) and i.d. (50 μl / site) routes, and in 10 μl (5 μl/ nostril) for the i.n. route. In each immunization protocol, six mice per group were sacrificed at day 14 after each boost immunization to collect spleens, vaginal fluids and blood samples for analysis of the immune responses of individual mice.

2.8 Serology

Anti-Tat IgG antibodies were measured by enzyme-linked immunosorbent assay (ELISA). The concentration of the recombinant protein used for coating was 1 μg/ml diluted in 0.05 M carbonate buffer (pH 9.6-9.8), and 100 μl/well were added to 96-well immunoplates (Nunc- Immunoplate F96 Polysoφ, Nunc, Naperville, IL). The plates were sealed and incubated in the dark for 18 to 20 hours at 4°C. Prior to use, the plates were extensively washed with 0.05% Tween 20 in PBS-A IX and blocked for 90' at 37°C with 3% bovine serum albumin (BSA) in PBS-A IX. Sera were two-fold diluted in PBS-A IX containing 3% BSA and each sample was ran in triplicate wells (100 μl/well). After incubation at 37°C for 90', the plates were washed and immunocomplexes were detected with 100 μl/well of HRP-sheep anti-mouse IgG (Amersham Life Science) diluted 1:1000. After incubation at room temperature for 90 minutes, the wells were washed, and 100 μl/well of ABTS (Sigma) were added as HRP substrate. The reaction was blocked with 100 μl of 0.1 M citric acid per well. The absorbance was measured at 405 nm in an automated plate reader (ELX-800, Bio-Tek Instruments, Winooski, UT). Absorbance higher than the control group (PBS- injected) mean + 3SD values were considered positive.

2.9 Splenocyte purification

Mice spleens were disrupted with 2ml syringe plungers using 70 μm pores cell strainers (Falcon), resuspended in PBS IX with 2 mM EDTA and, after 15' cenfrifugation at 1500 φm, treated with red blood cell lysis buffer (100 mM NH4C1, 10 mM KHCO3, 10 μM EDTA) for 4 minutes at room temperature and finally washed with RPMI 1640 medium (Euroclone) containing 3% of heat-inactivated FBS. Cells were resuspended in RPMI 1640 complete medium with 10% Hyclone, counted using tripan blue exclusion method and incubated in a humidified 5% CO₂ atmosphere at 37°C at the final concentration of 5xl0⁶/ml. 2.10 Lymphoproliferation assays

Splenocytes (2xl0⁵ cells/well) were cultured in 96 well plates in the presence of 1 μg/ml and 5 μg/ml of recombinant Tat protein, 10 μg/ml of Con A (ICN) or culture medium alone as positive and negative controls respectively. After 48 hours, bromodeoxyurindine (BrdU) was added (10 μM/final concenfration) to the plates. BrdU incoφoration was determined after ON incubation with BrdU by using a cell proliferation ELISA system (Amersham Pharmacia Biotech) according to the manufactures instructions.

2.11 CTL assays

Mice splenocytes were co-cultivated at 1.5:1 ratio with naive syngeneic stimulator splenocytes, previously irradiated at 30 Gy, in the presence of 1 μg/ml of purified recombinant Tat protein. Recombinant IL-2 (10 U/ml) was added to the cells after 3 days of culture. ^5I Chromium release assays were performed at day 6 of culture using P815 target cells, preincubated overnight with 2 μg/ml of Tat, as previously described [61], with additional 10μg of protein during the ⁵¹Cr (100 μCi/target) labeling step. After 4 hours incubation of effector and target cell at 37°C, supematants were harvested and the ⁵¹Cr released by the lysed target cells was quantified using a γ-counter. Specific percent cell lyses was calculated according to the following formula:

specific % cell lysis= 100 x fcpm [sample release]- (cpm spont. rel. (min) [62]) (cpm [max release]- cpm [min release])

where the minimum is represented by the spontaneous release of the ⁵¹Cr isotope from the target cells and the maximum release is obtained by addition of 50% solution of Triton X-100 to the target cells.

2.12 Cytokine ELISA

The cytokine profile was determined in culture supematants of mice splenocytes (2,5x10⁶/l ml in 48 well plate) cultured with 1 μg/ml of recombinant Tat protein, 10U hIL-2 from day 3. At day 3 and 6 of culture standard sandwich ELISA tests were performed, using antibodies and recombinant standard proteins purchased from ENDOGEN. The concenfration of the anti-IFNγ antibody used for coating was 1 μg/ml diluted in 0.05 M carbonate buffer (pH 9.6-9.8), and for anti-IL-4 2 μg/ml diluted in PBS IX. 100 μl/well of capture antibody solution were added to 96- well plates (Nunc-Immunoplate). The plates were sealed, incubated ON at 4°C and blocked for 60 minutes at room temperature with 4% BSA in PBS IX. Cell culture supematants were tested in triplicates undiluted or diluted 1:10. The concentration of the anti-IFNγ and anti-IL-4 biotinilated antibodies was 0.4 μg/ml and HRP-streptavidin was used at 1:6000 (IFN-γ) or 1:20.000 (IL-4) dilution. TMB (100 μl/well) was added as chromogen substrate. Reaction was blocked with IN HCl. The absorbance was measured at 450 nm in an automated plate reader (ELX-800, Bio-Tek Instruments, Winooski, UT).

3. Results

3.1 Construction of recombinant TO-tat replication-defective HSV-1 vector and analysis of Tat expression A replication defective HSV-1 virus was modified in order to express the HIV-1 Tat protein under the confrol of the HSV-1 immediate early (ICPO) promoter (TO-tat) (Fig.13). This recombinant vims named TO-tat was obtained by homologous recombination of the pB410-tat plasmid with the HSV-1 TOZ-GFP triple mutant vims into UL41 HSV-1 locus. The presence of the tat gene in the HSV-1 genome was determined by Southern blot analysis. Expression of Tat protein was assessed by infecting Balb/c fibroblasts with TO-tat replication-defective HSV-1 recombinant vector and Tat expression analysed by Western blot after 24, 48 and 72 hours post- infection. Controls were represented by cells infected with the TO-GFP vector or non-infected cells. As shown in the Figure 14A the TO-tat vector expressed Tat at high levels. Tat expression was detected already 24 hours after infection and increased up to 72 hours post infection. Similar patterns of Tat expression were detected in Vero, BHK, HeLa and P815 cells (data not shown).

To determine whether TO-tat infected cells release Tat in the extracellular medium and whether released Tat is taken up by uninfected cells, Balb/c fibroblast were grown for 4, 6, 8, 12 and 18 hours with cell-free culture supematants (collected at 48 hours post infection) of TO-tat infected cells. Internalization of Tat by Balb/c cells was then analysed at each time-point on cell lysates by Western blot. Negative control was represented by Balb/c cells cultured for 8 hours with supernatan derived from TO-GFP infected cells. As shown in figure 14B TO-tat infected cells release Tat in the extracellular medium, and this extracellular released protein is efficiently taken up by uninfected cells. Tat is promptly detected intracellularly in uninfected Balb/c cells after 4 hours incubation with Tat-containing supematants and its uptake increases up to 8 hours incubation. After 12 and 18 hours Tat become undetectable likely because cells, which have internalized the protein, had already processed by that time. To determine whether Tat produced by the TO-tat vector is biologically active, HeLa3Tl cells, containing an integrated copy of the CAT reporter gene under the transcriptional control of the HIV-LTR promoter, and in which CAT expression occurs only in the presence of bioactive Tat, were infected with different m.o.i. (0.01-1) of TO-tat or TO-GFP confrol vector. CAT expression was measured 24 and 48 hours post-infection. As shown in Table 1, high levels of CAT expression were detected already at 24 hours after infection, even at the lowest m.o.i. of 0.01. These results confirm that Tat is expressed at high levels promptly after infection of cell with TO-tat vector (Fig. 14A) and indicate that it is biologically active (Tablel).

Table 1 : Analysis of the biological activity of Tat expressed by TO-tat ^a

^a HeLa 3T1 cells containing an integrated CAT gene under the control of the Tat-inducible HIN- 1 LTR were infected at various multiplicities of infection (m.o.i.). ^b Results are expressed as percentage (%) of acetylation at 24 and 48 hours post infection with TO- tat or TO-GFP vectors.

In conclusion these results indicate that the replication-defective HSV-1 TO-tat vector expresses high amount of bioactive HIV-1 Tat upon infection of various cell lines. The Tat protein has been shown to be released in the extracellular medium and efficiently taken up by neighboring cells where it is further processed, presumably by entering both MHC class I and class II patways, suggesting that this vector may represent an useful delivery system for Tat- vaccination.

3.2 Analysis of the immune response induced in mice by TO-tat immunization Different vaccination protocols were tested in order to determine the optimal route as well as the dose of recombinant TO-tat vector required for induction of efficient anti-Tat cell-mediated and humoral immune responses. In the first experiment mice were immunized with 4x10 or with 4x10 pfu by the s.c. route and boosted at weeks 2, 4 and 9 after priming. The analysis of the immune responses elicited in animals , have demonstrated that only the group of mice immunized with the higher dose of TO- tat recombinant vims was able to mount a significant response against Tat (data not shown). From these preliminary studies the dose of 4xl0⁶ pfu recombinant vims was chosen for the subsequent experiments.

In a second set of experiments we analyzed the immune responses elicited by TO-tat administered parenterally or mucosally. Mice were vaccinated with 4xl0⁶ pfu of TO-tat by s.c. or i.n. inoculation. Mice were boosted with the same dose of TO-tat s.c. or i.n. at weeks 2, 4 and 9 after priming (Fig. 15A). The results of this experiment, shown in figures 15B and 15C, indicate that only mice vaccinated with TO-tat by the s.c. route developed a specific immune response to Tat. Antigen-specific CTL responses and TNFγ production were detected in mice vaccinated s.c. with TO-tat, on the contrary, mice vaccinated i.n. with TO-tat did not develop any specific anti-tat response in a fashion similar to control mice injected with PBS. IL-4 production was barely detectable or undetectable in all groups (Fig. 15C).

A possible explanation for the different responses after s.c. and i.n. immunization, may be that immunization by i.n. route might require a higher dose of recombinant virus in order to induce a specific response against Tat or that the immunization by i.n. route should be implemented with an adequate mucosal adjuvant to- achieve the desired effect.

In a third set of experiments we tested the immune responses elicited by s.c. vaccination with 4xl0⁶ pfu of TO-tat using two different schedules of immunization. Mice were boosted at weeks 2, 4 and 9 after the priming time (schedule 1) or at weeks 4 and 8 after the first immunization time (schedule 2) (Fig. 16A). The results, shown in figure 16B, indicate that an effective antigen-specific CTL response was developed only in mice receiving the first boost 2 weeks after the priming immunization as compared to mice receiving the first boost 4 weeks after the priming. Similarly, INFγ production was significantly higher in mice vaccinated according to time schedule 1 (Fig. 16C).

Based on the results of the second and third set of experiments, we next compared immune responses elicited by homologous and heterologous prime/boosts immunization schedules. Animals were inoculated with purified Tat protein by the intradermal route and boosted twice with 4xl0⁶ TO-tat viral particles by s.c. route (heterologous regimen) (Fig. 17A). Alternatively mice were immunized three times with 4x10⁶ pfu of TO-tat vims by s.c. route (homologous regimen) (Fig. 17A). The results indicate that one effective CTL response was elicited only by the homologous (vaccination regimen) and not by the heterologous immunization regimen (Fig. 17B). Similarly, INFγ production was higher in mice vaccinated with the homologous regimen (Fig.l7C). The production of IL-4 was either barely detectable or undetectable in all groups of mice (Fig. 17C). The differences in the immunological response between homologous and heterologous regimen may depend on the fact that (no adjuvant was used with the protein inoculation), as expected, the viral TO-tat vector has proven to be much more efficient in the priming of a Thl -like immune response, characterized by anti-Tat CTL activity and INFγ induction.

Tat-specific T cell proliferation was evaluated by BrdU incoφoration in mice splenocytes cultured with recombinant Tat. No significant T cell proliferation was detected in TO-Tat immunized animals at any viral dose, administration route or vaccination protocol (data not shown), suggesting that HSV-1 derived vaccines are weak inducers of T-dependent antibody responses. To directly examine this possibility, we tested in sera of vaccinated mice for anti-Tat IgG presence. As shown in figure 18, only mice vaccinated with heterologous regimen developed low to intermediate levels of anti-Tat IgG antibodies. These results are in agreement with the analyses of IL-4 production indicative of the Th2-like immune response induction shown in figures 15, 16 and 17.

These data indicate that recombinant HSV-1 derived vaccines are only weak inducers of CD4 T helper dependent antibody responses, whereas they activate efficient long-term CD8 T cell responses.

4. Discussion

Currently, many vector systems are being developed for use in vaccine design, and among them those based on adenovims, poxvims, alphavims and heφes simplex vims [28,37,63,64]. The efficacy of all of these vectors might potentially be affected by the preexisting immunity to viral antigens in host. Nevertheless, at least the last two [63] vector systems, namely alpha-^" and heφes simplex viral recombinants, have been reported to elicit antigen-specific immune response despite preexisting immunity against viral antigens in host [50,51,65]. Moreover, two different recombinant vectors based on heφes simplex vims were shown to efficiently transduce tumors in mice previously exposed to HSV infection after direct intratumoral inoculation [66,67]. However, intravenous delivery of an HSV-1 recombinant vector has been reported to reduce its capability to transduce a murine tumor [68], which is in accordance with the recent data [69] indicating complement C3 as an important factor in serum antiviral activity. The above data suggest that the recombinant HSV vector administration route is extremely important in determining the efficacy of fransgene delivery to the host cells, whether it is a vaccination antigen, a cytokine or a suicide gene. Another concern regarding the use of HSV recombinants as vaccination vectors is based on the fact that the viral Vhs protein (viral host shutoff) encoded by UL41 gene has been shown to block dendritic cell (DC) maturation and thus inhibit the immune response against the vector-delivered transgene [70]. The elimination of the UL41 locus from the viral genome was reported in the same paper to allow DC activation and also to stimulate the antigen specific T-cell response in vitro.

In the present experiement, we describe the constmction and the in vitro and in vivo characterization of the replication defective HSV-1 -based TO-tat vector devoid of multiple immediate early regulatory genes and also containing a deletion in the UL41 locus obtained by the fransgene (E. coli lacZ reporter gene first and then HIN-1 tat) insertion. The TO-tat replication incompetent HSN-1 background conserves the capacity to infect cells of different origins, both dividing and non dividing, as efficiently as a wild-type heφes vims, but is much safer due to the incapacity to replicate and to induce the expression of viral proteins after infection.

Western blot analysis has shown that the TO-tat vector was able to express high quantity of HIN- 1 Tat protein upon infection of various cell lines. The protein maintained its biological activity, as we demonstrated by CAT assay. Furthermore, the Tat protein was released in the extracellular medium by the TO-tat vector-transduced cells and was readily taken up by uninfected cells cultivated in presence of supematants obtained from the former. We injected the TO-tat vector at peripheral, i.e. subcutaneous sites in order to avoid the antiviral serum activity and following vector inoculation in Balb/c mice we investigated its capacity to induce primary as well as long- term immune response against HIV-1 Tat protein, alone or in a combined immunization regimen with the purified recombinant Tat protein. We show that the TO-tat replication-defective HSV-1 recombinant vector was able to elicit a Tat- specific immune response in immunized mice, although the breadth of the response was different depending on the viral dose, site of inoculation, and timing of the boosts.

After the first in vivo experiments performed in order to establish the optimal dose and route, as well as the frequency of adminisfration of the recombinant TO-tat viras required to obtain an anti-Tat specific immunity, we examined the alternative vaccination priming mechanisms, based on the use of purified recombinant protein, followed by one or more recombinant vims boosts. The homologous TO-tat virus prime/boost regimen, when injected by s. c. route, was characterized by significant Tat-specific cytolytic activity, high quantity of IFN-γ released by the in vitro Tat-restimulated lymphocytes and no T cell proliferation nor anti-Tat antibody production above background levels, all compatible with the hypothesis of the predominance of Thl -type on Th2-type immunity. A previous study performed by T. Brocker and our group has demonstrated the efficacy of recombinant HSV-1 vaccines in mice by using another viral vector that we constructed, with the same genetical background as TO-Tat, expressing chicken ovalbumin [71]. A single vaccination, with a strong antigen as ovalbumine, was sufficient to elicit a protective immune response characterized by high frequency of both primary and memory antigene-specific CTL response. Poor induction of CD4 helper T cell responses and the similarly weak induction of T-dependent antibodies were observed. Apparently, the lack of antibodies did not negatively affect the ability of HSV-1 derived vaccines to give long-term protection against L. monocytogenes expressing Ova. The results obtained with Ova viral vector seem to support the data we collected following the homologous prime/boost TO-tat immunizations in murine model.

On the other hand, the purified Tat protein prime/TO-tat viras boost heterologous vaccination regimen was not so efficient in inducing the Tat-specific cellular immunity, resulting in generally low, yet still detectable CTL activity and INF-γ production in the restimulated lymphocyte cultures after the complete time course of the immunizations. Furthermore, low to intermediate levels of anti-Tat antibodies were detected in the sera of immunized mice. It is likely that, in order to improve the overall, i.e. both cellular and humoral, efficacy of the heterologous vaccination model, we will need to associate an adjuvant agent to the recombinant protein during the priming inoculations. Also, another protein or recombinant viras boost might be required to obtain an efficient and complete immune response against a weakly immunogenic HIN-1 Tat protein. The appealing properties of replication incompetent HSN-1 -based vectors inducing strong CTL response, both in murine and in simian model, against foreign genes delivered by viral particles have made them very promising candidates for potential anti-HIV-l and also other viral or intracellular bacterial pathogens vaccine development. Further studies will have to be performed in order to investigate the feasibility of HSN-1 vectors application for human use, too.

Example 5 Figure Legends

Fig. 13 Schematic representation of pBlueScript plasmid containing UL41-ICP0-tat cassette (A) and of the TO-GFP and TO-tat HSN-1 replication defective vector (B). A: schematic representation of pBlueScript plasmid, containing tat cDΝA under the confrol of HSN-1 ICPO promoter flanked by HSN UL41 sequences (pB410-tat). The homologous recombination event between viral DΝA of TOZ-GFP and pB410-tat gave raise to the TO-tat recombinant vims. Expression of GFP gene is driven by HCMV IE promoter. The black squares symbolize the IE genes (ICP4, 27, 22) and other genes that are deleted in the HSV backbone. The white squares symbolize the terminal and internal repeats of the HSV genome delimiting the unique regions (U_L: unique long; Us: unique short)

Fig. 14 Western blot analysis of Tat protein expressed by the TO-tat HSV-1 vector. A: Balb/c cells infected with 1 m.o.i of TO-tat and analyzed at 24 (lane 1), 48 (lane 2) and 72 (lane 3) hours post-infection. Control cells were infected with 1 m.o.i. of the TO-GFP vector (lane 4). Recombinant Tat protein was loaded as the positive confrol at 20 ng (lane 5), 50 ng (lane 6). B: analysis of Tat protein uptake in Balb/c cells cultured with media obtained from TO-tat infected cells. Cell lysates of Balb/c cells after 4 (lane 1), 6 (lane 2), 8 (lane 3), 12 (lane 4) and 18 (lane 5) hours incubation with cell-free medium from TO-tat infected cells are shown. Positive controls were the recombinant Tat protein (50 ng) (laneό) and Balb/c cell lysates after infection with 1 m.o.i. of TO-tat (lane7). In lane 8, Balb/c cultured with supernatant derived from Balb/c TO-GFP infected cells, and lane 9, Balb/c cell lysates infected with 1 m.o.i. of TO-GFP, were loaded as negative controls. The arrow works the 14 KDalton Tat protein.

Fig. 15 Analysis of anti-Tat immune responses elicited by vaccination with 4.x 10⁶ pfu of TO-Tat HSV-1 replication defective vims subcutaneously (s.c.) or infranasally (i.n.). A: schematic representation of the homologous prime/boost immunization schedule. Mice were boosted at weeks 2, 4 and 9 after the first inoculation.

B: Tat-specific CTL response analyzed by ⁵¹Chromium release assays; C: INFγ and IL-4 production in cell-free culture supematants of mice splenocytes after 6 days in vitro culture. In B and C the data shown in charts mean results conespond to the mean (± SD) of individual mice.

Fig. 16 Analysis of anti-Tat immune response elicited by s.c. vaccination with 4.xl0⁶ pfu of T0-

Tat HSV-1 replication defective viras in different immunization schedules.

A: schematic representation of the homologous prime/boost immunization schedule. Mice were immunized with TO-tat and boosted at weeks 2,4 and 9 (schedule 1) or at weeks 4 and 8 after the first inoculation (schedule 2).

B: Tat-specific CTL responses mesured by ⁵¹Chromium release assays; C: INFγ and IL-4 production in cell-free culture supematants obtained from mice splenocytes after 6 days in vitro culture with recombinant Tat at 1 μg/ml. In B and C results are represented as the mean (± SD) of individual mice.

Fig. 17 Analysis of anti-Tat immune responses after homologous or heterologous prime/boost vaccination regimens with 4.x 10⁶ pfu of TO-Tat HSV-1 replication defective vector.

A: schematic representation of the prime/boost regimens. Mice were either immunized s.c. with

TO-tat and boosted with the same vector at weeks 2 and 8 after the priming (homologous regimen) or primed with 2 μg of recombinant Tat protein i.d. and boosted with TO-tat viras s.c. at weeks 2 and 8 (heterologous regimen).

B: Tat-specific CTL responses analyzed by ⁵¹Chromium release assays.

C: INFγ and IL-4 production in cell-free culture supematants of murine splenocytes collected after 6 days of in vitro culture with 1 μg/ml recombinant Tat. Mean (± SD) of individual mice are shown in charts B and C.

Fig. 18 Tat-specific IgG antibodies in the sera of Balb/c mice immunized with TO-tat replication- defective HSV-1 vector. Sera were obtained from mice primed with TO-tat and boosted with the same viras (4xl0⁶ viras particles) s.c, or from those that have been Tat protein-primed and boosted with TO-tat. Confrol mice were injected with PBSX1. ELISA assays were performed as described in the material and methos section andthe absorbabce values of four representative mice/group are shown References for Example 5

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Micellar-type complexes of tailor-made synthetic block copolymers containing the HIV-1 tat DNA for vaccine application. Vaccine 2002, 20(17-18), 2303-2317. Shiver, J. A non-replicating adenoviral vector as a potential HIN vaccine. Res Initiat Treat Action 2003, 8(2), 14-16. Marovich, M.A., Mascola, J.R., Eller, M.A. et al. Preparation of clinical-grade recombinant canarypox-human immunodeficiency viras vaccine-loaded human dendritic cells. J Infect Dis 2002, 186(9), 1242-1252. Pushko, P., Parker, M., Ludwig, G.N., Davis, Ν.L., Johnston, R.E. & Smith, J.F. Replicon-helper systems from attenuated Venezuelan equine encephalitis viras: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo. Virology 1997, 239(2), 389-401. Ali, S.A., Lynam, J., McLean, C.S. et al. 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Samady, L., Costigliola, E., MacCormac, L. et al. Deletion of the virion host shutoff protein (vhs) from heφes simplex vims (HSV) relieves the viral block to dendritic cell activation: potential of vhs- HSV vectors for dendritic cell-mediated immunotherapy. J Virol 2003, 77(6), 3768-3776. Lauterbach, H., Kerksiek, K.M., Busch, D.H. et al. Protection from bacterial infection by a single vaccination with replication-deficient mutant heφes simplex vims type 1. J Virol 2004, 78(8), 4020-4028. Example 6: Non-replicative HSV vectors expressing SIV Gag

Expression of SIV-1 Gag from a replication-deficient Herpes simplex type 1 vector

In order to develop new vaccination strategies able to enhance the cellular irnmunity towards Tat as well as other HIV-1 proteins, a large number of viral vectors expressing HIV-1 or SIV-1 antigens are under investigation, and among them poxviras-based vectors which have been the most studied as HIN vaccine viral vectors, the alphavims self-replicating vectors, the adenovims, the lentivims or the heφes simplex viras vectors. Despite the general concerns regarding safety issues using live viral recombinants, the overall results with these different viral vectors indicate that they might be good candidates for the development of an anti-HIN-1 vaccine. Many of these viral vectors have been reported for their capacity to induce strong in vivo Thl and CTL responses, as well as high antibody titers, against various HIN-1 gene products.

In particular, the heφes simplex type-1 viras (HSN-1) vectors show several advantages for prophylaxis against viral infections. They have been shown: i) to elicit strong and durable immune responses by various routes of inoculation; ii) the viral DΝA persists inside the host's cell nucleus as an episomal element, thus eliminating the safety concerns deriving from the random integration of the viral genome into the host's DΝA; iii) they carry the tk gene, encoding the viral thymidine kinase, that, in case of undesired effects, can be used, in combination with specific antiviral drags, to kill the virus-harbouring cells.

MATERIAL AND METHODS

Many procedures, such as Southern blot, vector construction (including DNA extraction, isolation, restriction digestion, ligation, etc.), cell culture, transfection and infection of cells, protein assays (such as ELISA, Western blotting, β-galactosidase and CAT assays) are techniques performed by those of skill in the art. However, viruses employed in the following invention deserve specific description.

Viruses

We are using an ICP4^", ICP27^", and ICP22^" vims having an ICP4-immediate early promoter-tk gene expression cassette at the UL24 locus known in art (Marconi, P., Krisky, D., Oligino, T., Poliani, P.L., Ramakrishnan, R., Goins, W.F., Fink, D.J., 8c Glorioso, J.C. (1996). Replication- defective heφes simplex viras vectors for gene transfer in vivo. Proc Natl Acad Sci U S A, 93(21), 11319-11320.).

The HSN-1 vectors have been modified by insertion of heterologous expression cassettes encoding for HIN-1 proteins at the UL41 or ICP22 locus of HSN-1 replication defective vector. A DΝA fragment comprising an HSN ICPO-immediate early promoter-lacZ expression cassette flanked by Pad restriction endonuclease recognition sites and sequences homologous to UL41 for HSN-1 was co-trasfected in 7b cells with HSN-1 mutant. Recombinants were screened for lacZ expression, and the correct insertion was confirmed with Southern blot analysis.

Generation of plasmids and recombinant replication-defective HSV-1-SIV-gag vector

Plasmid p239SpSp5', expressing the SIV-1 gag cDΝA (obtained from NTH AIDS Reagent Program, Catalog number 829) has been previously described (Kestler HW III, Kodama T, Ringler D, Marthas M, Pedersen Ν, Ratner A, Regier D, Sehgal T, Daniel M, King Ν, Desrosiers RC, (1990). hiduction of AIDS by molecularly cloned vims. Science, 248: 1109-1112.). Plasmid DΝA was purified from Escherichia coli by using Qiagen endotoxin free Maxi Kit (Qiagen, Hilden, Germany).

Plasmid pB410-gag was constructed by introduction of the 1.9 kbp SIV-1 gag cDΝA (SIV_mac239 proviras genome) after subcloning the sequence respectively in pSP72 and pcDΝA3-Gag expression cassette into the UL41 locus of HSV-1. The cDNA under the transcriptional confrol of the human cytomegalovirus promoter (NruI-EcoRI) was inserted in a S αI/EcoR/-opened pBBSK-plasmid between the two UL41 fragments (map positions 93,858-92,230 and 91,631- 90,145) 100 bp downstream of the HSN-1 immediate-early ICPO promoter. The CTΕ sequence was inserted downstream of the expression cassette in EcoRI site. This plasmid (pB410Hgag) was recombined with the genome of TO-GFP using the previously described Pac-I facilitated LacZ substitution method (Krisky et al., 1997). TO-GFP is a non-replicative HSV-1 viral vector that has low toxicity due to the deletion in three immediate early genes (ICP4, ICP27, which are essential for viral replication, and ICP22) with cDΝA encoding GFP inserted into the ICP22 locus and an insertion of LacZ in the UL41 locus. The recombination was carried out using standard calcium phosphate fransfection of 5 μg of viral DΝA and 1 μg of linear recombination plasmid pB410Hgag. Transfection and isolation of the recombinant vims was performed in 7b Vero cells (African green monkey kidney cells CCL81: ATCC, Rockville, MD) capable of providing the essential ICP4 and ICP27 HSN-1 gene products. The recombinant vims containing the gag cDΝA was identified by isolation of a clear plaque phenotype after X-gal staining. The viras was purified by three rounds of limiting dilution and the presence of the transgene was verified by Southern blot analysis.

Construction of HSV-1 recombinant vectors expressing murine GM-CSF

Mouse-GM-CSF sequence (916 bp) derived from CMV-pal/BLSK kindly given by Malter S. (Blood 86,7,1995:2551-58) was subcloned as Smal-Xbal fragment into a pBluescript (pBSSK) plasmide. The cytokine gene was then cut Clal-Xbal and inserted in a plasmid named PB5 under the trascriptional confrol of HCMV promoter. PB5 is a pBSSK plasmid containing the Usl HSV- 1 sequences encoding the ICP22 immediate early protein, as previously described (ref: Krisky DM, et al. Development of heφes simplex vims replication-defective multigene vectors for combination gene therapy applications. Gene Ther 1998;5:1517-1530). The fransgene expression cassette was inserted into the Usl locus of HSV-1 genome into the background of the THZ1 vector by homologous recombination generating the cytokine recombinant vector. THZ-1 is a recombinant vims deleted in the ICP4, ICP27 and ICP22 immediate early genes with the LacZ reporter gene, under HCMV promoter, in the ICP22 locus. THZ-1 viral DΝA and the recombinant plasmid containing the gmcsf gene have been co-transfected into 7b complementing cell line. The TH-gmcsf viras was purified by three rounds of limiting dilution and the presence of the transgene was verified by Southern blot analysis. Western blot analysis and ELISA of the recombinant vims-infected cell lines was performed in order to assess the transgene expression.

Construction of HSV-1 recombinant vectors expressing SIV-1 Gag and murine GM-CSF

The vector TOHgag Hgmcsf containing SIV-1 gag in UL41 HSV-1 locus and gmcsf in Usl HSV-1 locus was created by genetically crossing the TOHgag and THgmcsf vectors. 7b cells plated on 60 mm petri dishes were infected with 3 m.o.i. of TOHgag and THgmcsf viruses and harvested 18 hours post infection. The mixture of viruses derived from the co-infection was titrated, and the viral vector containing both SIV-1 gag and murine gmcsf genes was isolated by Southern blot screening.

Expression of the transgene product

SIV-1 Gag protein expression was analysed in Vero cells (1x10° cells/well in 6 well plates) infected with 1 m.o.i. TOHgag vims. At 12, 24, 48 and 72 hours after infection cell exfracts corresponding to 10 μg of total protein, were run onto 12% SDS-polyacrilamide gel and transferred by elecfroblotting to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The blots were stained with a mouse monoclonal anti-SIN-p27specific antibody (ENA 643, Centralised facility for AIDS Reagents), diluted 1:2000 and a goat anti mouse IgG HRP- conjugate secondary antibody (ΝA931, Amersham) diluted 1:2500. Immunocomplexes were detected by ECL western blot detection kit (Amersham, Pharmacia Biotech). Controls was represented by Vero cells infected with 1 m.o.i. of TO-GFP recombinant vector and collected at 48 hours after infection.

Virus stock purification

HSV-1 stocks were prepared by infecting 4xl0⁸ 7b complementing cells with 0,05 M.O.I. of TOHgag and TO-GFP viruses in suspension in 15 ml of medium for 1 hour at 37°C under mild agitation. When a 100% cytopathic effect was evident, cells were collected and centrifuged at 2000 rprα for 15 minutes. The supematants were spun at 20.000 φm in JA20 rotor (Beckman) for 30 minutes to collect the viras. The cellular pellets were resuspended in 2 ml of medium, subjected to three cycles of freeze-thawing (-80°C/37°C) and a single burst of sonication, to release the viral particles. The vims was further purified by density gradient centrifugation (Opti Prep; Life Technologies, Inc.) and resuspended in PBS-A IX. Viral stocks were titered and stored at -80°C Titles averaged between 2x10 to 2x10 plaque forming unit (pfu)/ml.

Animals and immunization protocols

Animals were handled according to national guidelines and institutional policy. Six weeks old BALB/c (H-2^d) female mice were purchased from Harlan Italy and immunized when they were 7 weeks old, according to the protocols described below.

Mice infection with the replication defective HSV vectors.

Female 7-weeks old BALB/c mice (Harlan Italy) were primed by subcutaneous (s.c.) or intradermal (i.d.) route with TOHgag or TO-GFP (4xl0⁶ viras particles/100 μl of PBS-A lx).

Groups of mice were boosted s.c or i.d. after 15 days with the same dose of immunogen.

The recombinant viruses were administered in 100 μl for the s.c. (one site) and i.d. (50 μl / site) routes. Seven mice per group were sacrificed on day 14 and after the boost on day 28 to collect spleens, vaginal fluids and blood samples for analysis of the immune responses of individual mice. Serology

Anti-Gag IgG antibodies were measured by enzyme-linked immunosorbent assay (ELISA) as previously described (O'Hagan D. et al. J. of Virology 2001, vol.75 (19): 9037-9043) . The concenfration of the recombinant protein used for coating was 5 μg/ml diluted in PBS- A IX, and 50 μl/well were added to 96-well immunoplates (Nunc-Immunoplate Maxisoφ, Nunc, Naperville, IL) and incubated over night, in the dark at 4°C Prior to use, the plates were extensively washed with 0.1% Tween 20 in PBS-A IX and blocked for 60' at 37°C with 1% normal serum goat in 0.1% Tween 20 and PBS-A IX. Sera were 1:100 diluted in blocking buffer and each sample was run in triplicate wells (50 μl/well). After incubation at 37°C for 120', the plates were washed and immunocomplexes were detected with 50 μl/well of anti-mouse IgG HRP conjugate (Amersham NA931) diluted 1:20.000 in blocking buffer. After incubation at 37°C for 60 minutes, the wells were washed, and incubated at room temperature for 30 minutes with 50 μl/well of TMB (Sigma) as HRP substrate. The reaction was blocked with 50 μl of HCL IN per well. The absorbance was measured at 450 nm with an automated plate reader (ELX-800, Bio-Tek Instruments, Winooski, UT). The positive control was represented by a recombinant Gag (SIN p27-GST fusion protein - NIBSC EVA 643) and the negative control was represented by the sera from mice injected with T0GFP vector and PBS-A lx. Absorbance values higher than the control group (PBS-A lx injected) mean + 3SD values were considered positive.

Splenocyte purification

Mice spleens were disrupted with 2ml syringe plungers using 70 μm pores cell strainers (Falcon), resuspended in PBS-A IX with 2 mM EDTA and, after 15' cenfrifugation at 1500 rpm, treated with red blood cell lysis buffer (100 mM NH4C1, 10 mM KHCO3, 10 μM EDTA) for 4 minutes at room temperature and finally washed with RPMI 1640 medium (Euroclone) containing 3% of heat-inactivated fetal bovine serum. Cells were resuspended in RPMI 1640 complete medium with 10% Hyclone, counted using tripan blue exclusion method and incubated in vertical T25 flasks in a humidified 5% CO₂ atmosphere at 37°C at the final concenfration of 2,5x10⁶ cells/ml.

Lymphoproliferation assays

Splenocytes (2x10⁵ cells/well) were cultured in 96 well plates in the presence of 1 and 5μg/ml of each gag peptide, divided in two pools, or with, 10 μg/ml of Con A (ICN) or culture medium alone as positive and negative controls respectively. After 48 hours, bromodeoxyuridine (BrdU) was added (10 μM/final concenfration) to the plates. BrdU incoφoration was determined after an over night incubation by using a cell proliferation ELISA system (Amersham Pharmacia Biotech) according to the manufacturer's instructions.

CTL assays

Mice splenocytes were co-cultivated at 1.5:1 ratio with naive syngeneic stimulator splenocytes, previously irradiated at 30 Gy and pre-incubated for one hour with in the presence of 10 μg/ml of the pool 1 or the pool 2 of Gag peptides. Recombinant IL-2 (10 U/ml) was added to the cells after 3 days of culture. ⁵¹Chromium release assays were performed at day 6 of culture using P815 target cells, preincubated with 10 μg/ml of each Gag peptide and 100 μCi of Na₂CrO₃ (NEN). After 4 hours incubation of effector and target cells at 37°C, supematants were harvested and the ⁵¹Cr that was released by the lysed target cells was quantified using a γ-counter. Specific percent cell lyses was calculated according to the following formula:

specific % cell lysis= 100 x (cpm [sample release]- cpm [min. release]) (cpm [max release]- cpm [min release])

where the minimum is represented by the spontaneous release of the ⁵¹Cr isotope from the target cells and the maximum release is obtained by addition of 5% solution of Triton X-100 in PBS-A lx to the target cells.

Cytokine ELISA

The cytokine profile was determined in culture supematants of mice splenocytes (2,5x10⁶/l ml in 48 well plate) cultured with 10 μg/ml of each gag peptide, 10U hIL-2 from day 3. At day 3 and 6 of culture standard sandwich ELISA tests were performed, using antibodies and recombinant standard proteins purchased from ENDOGEN. The concentration of the anti-IFNγ antibody used for coating was 1 μg/ml diluted in 0.05 M carbonate buffer (pH 9.6-9.8). The anti- IL-4 antibody was diluted in PBS-A IX at 2 μg/ml. 100 μl/well of capture antibody solutions were added to 96-well plates (Nunc-Immunoplate PolySoφ). The plates were sealed, incubated ON at 4°C and blocked for 60 minutes at room temperature with 4% BSA in PBS IX. Cell culture supematants were tested in triplicates undiluted or diluted 1:10. The concenfration of the anti-IFNγ and anti-IL-4 biotinilated antibodies was 0.4 μg/ml and HRP-streptavidin was used at 1:6000 (IFN-γ) or 1:20.000 (IL-4) dilution. TMB (100 μl/well) was added as chromogen substrate. Reaction was blocked with IN HCl. The absorbance was measured at 450 run in an automated plate reader (ELX-800, Bio-Tek Instruments, Winooski, UT). Results

Construction of recombinant replication-defective HSV-1 vectors

A replication defective HSV-1 viras was modified in order to express the SIV-1 Gag protein under the control of HCMV immediate early promoter, 100 bp downsfream of the HSV immediate-early ICPO promoter (Fig.l9A). This recombinant viras named TOHgag was obtained by homologous recombination of the pB410-gag plasmid with the HSV-1 TOZ-GFP triple mutant viras into UL41 HSV-1 locus. The presence of the gag gene in the HSV-1 genome was confirmed by Southern blot analysis.

The recombinant viral vector expressing murine GM-CSF was constracted by co-transfecting a plasmid (PB5-gmcsf), which contains gmcsf gene under HCMV promoter flanked by ICP22 HSV-1 sequences, with THZ1 recombinant backbone viras into 7b complementing cell line. THZ-1 is a recombinant viras deleted in the ICP4, ICP27 and ICP22 immediate early genes with the lacZ reporter gene, under the control of HCMV promoter, in the ICP22 locus (Fig.l9B).

The vector TOHgag/Hgmcsf containing the gag gene in UL41 HSV locus and gmcsf in Usl HSV locus was created by genetically crossing the above vectors (Fig.l9C).

Analysis of Gag expression

Expression of Gag protein was assessed by infecting Vero fibroblasts with T0H:gag replication- defective HSV-1 recombinant vector and Gag expression analysed by Western blot after 12, 24, 48 and 72 hours post-infection. Controls were represented by cells infected with the TO-GFP vector or non-infected cells. As shown in the Figure 20 the T0H:gag vector expressed Gag at high levels. The protein expression was revealed by a mouse monoclonal anti-SIV-p27specific antibody (EVA 643, Centralised facility for AIDS Reagents), diluted 1:2000 and a goat anti mouse IgG HRP-conjugate secondary antibody (NA931, Amersham) diluted 1:2500. Immunocomplexes were detected by ECL

Analysis of the immune response induced in mice by TOHgag immunization

Two different routs of immunization were tested (Fig. 21 A) in order to determine the optimal route of recombinant TOHgag vector required for induction of efficient anti-Gag cell-mediated and humoral immune responses. Two pools of peptides were used to perform all the immune assays (Fig. 2 IB)

Mice were immunized with 4x10⁶ pfu by the route and boosted also s.c. or i.d. two weeks after priming. The analysis of the immune responses elicited in animals, has demonstrated that only the group of mice immunized i.d. after the priming was able to mount a significant response against Gag (Fig. 22). On the contrary from the first sacrifice, the group of mice immunized by s.c. route after the s.c. boost has demonstrated a significant and much stronger immune response in comparison with the i.d. prime/boost vaccination regimen (Fig. 23A and 5B).

High levels of INF-γ were detected in splenocytes of mice vaccinated s.c. or i.d. with TOHgag, while mice vaccinated s.c. or i.d. with TO-GFP did not develop any specific anti-Gag response in a fashion similar to control mice injected with PBS-A lx (Fig.24A and 25A). IL-4 production was low but detectable in the mice immunized with both TOHgag groups (Fig. 24B and 25B).

Gag-specific T cell proliferation was evaluated by BrdU incoφoration in mice splenocytes cultured with Gag peptides. No significant T cell proliferation was detected in TOHgag immunized animals at any viral administration route suggesting that HSV-1 derived vaccines are weak inducers of T helper-mediated responses, and among them the antigen-specific antibody production. To directly test this hypothesis, we tested sera of vaccinated mice for anti-Gag IgG presence. The absence of anti-Gag specific antibodies and the low levels of IL-4 secreted by splenocytes in vitro are indicative of the Thi-like immune response induction, characterized by high IFN-γ production and CTL activity, as shown in figures 24 and 25.

We show that the T0H:gag replication-defective HSV-1 recombinant vector was able to elicit a Gag-specific immune response in immunized mice, although the breadth of the response was different depending on the site of inoculation.

Figure Legends for Example 6 Fig. 19 Schematic representation of recombinant TOHgag viras. A: schematic representation of HSN-1 genome, containing SIN-1 gag cDΝA under the control of HCMV fused to 100 bp of HSV-1 ICPO promoter into HSV-1 UL41 locus. Expression of GFP gene is driven by HCMV IE promoter. The red squares symbolize the IE genes (ICP4, 27, 22) and the green squares other genes that are deleted in the HSV-1 backbone. The white squares symbolize the terminal and internal repeats of the HSV-1 genome delimiting the unique regions (U_L: unique long; Us." unique short)

Fig. 20 Western blot analysis of SIV-1 Gag protein expressed by the TOHgag HSV-1 vector. Vero cells were infected with 1 m.o.i of TOHgag and TO-GFP (as negative confrol). Primary antibody: anti-SIV-1 p27 lmicrogr/ml (EVA 643, Centralised facility for AIDS Reagents); secondary antibody: antimouse IgG HRP-conjugate (ΝA931V, Amersham) 1:2500. Lanes 1-4: T0GAG infected Vero cells, 72, 48, 24 and 12h post infection, respectively; lane 5: MW marker; lane 6: T0GFP infected Vero cells; lane 7: uninfected Vero cells

Fig. 21 (A) Schematic representation of the immunization schedule. Mice were boosted two weeks after the first inoculation.

(B) Peptides used for the immune response assays the green dots are the peptides present in the pooll, the red dots are the peptides in pool 2.

Fig. 22 Gag-specific CTL response analyzed by ⁵¹ Chromium release assay after the 1^st sacrifice, 14 days after priming. Target cells were pulsed with the pool 1 of Gag peptides.

Fig. 23 Gag-specific CTL response analyzed by ⁵¹ Chromium release assay after the 2^nd sacrifice, 14 days after boosting. (A) The target cells were pulsed with the pool 1 of Gag peptides; (B) The target cells were pulsed with the pool 2 of Gag peptides. Gag peptide pools 1 and 2 are represented in fig. 2 IB

Fig. 24 A and B represent respectively INF-γ and IL-4 production post priming in cell-free culture supematants of mice splenocytes after 3 and 6 days of in vitro culture in presence of Gag pool 1 peptides. Fig. 25 A and B represent respectively INF-γ and LL-4 production post boosting in cell-free culture supematants of mice splenocytes after 3 and 6 days of in vitro culture in presence of Gag pool 1 peptides. The data shown in charts correspond to the mean values of different groups of mice.

Example 7 Herpetic vector inhibiting angiogenesis and inducing cell suicide in gliomas

The present example relates- to anticancer therapy, particularly to a multimodal tumor therapy treatment conseming anti-angiogenic, suicide genes and cytokine genes.

BRIEF SUMMARY

The example relates to the use of non-replicate HSV vectors to combine angiogenic inhibitors genes (e.g., angiostatin, endostatin and kringle 5 fusion proteins) with cytokines such as GMCSF or IL12 together with a HSV-tk suicide gene in the same vector to increase their synergistic effects. This strategy will combine three different modalities: (i) the use of angiostatic factors promoting tumor regression along, with (ii) the enzyme-directed prodrug activation (tumor suicide) and (iii) the local production of cytokines, to overcome the inadequate release of tumor antigens and the defect in antigen presentation, and to increase the immune response against the tumor. The last two approaches are devised to enhance the tumor destruction, the inhibition of metastasis and to prevent recurrences.

The method is useful in synergize the effect of cytokines with HSV-TK suicide gene and with antiangiogenesis molecules carried in one Heφes simplex vims based (HSV) vector. These new vectors will improve the ability of the immune system to reject the tumors and should be capable of eradicating malignancies, without toxicity towards healthy tissues.

MATERIAL AND METHODS

Construction of HSV recombinant vectors expressing angiostatic fusion proteins

Two angiostatic recombinant vectors have been constracted: one containing the human endostatinXVIII:: angiostatin fusion gene (T0H-endo::angio) and the other containing the human endostatinXVIII::Kringle5 fusion gene (T0H-endo::kringle5), both purchased from InvivoGen (pGT60 and pGT64 plasmids). The genes have been subcloned in EcoRV under the HCMV promoter in a pCDNA shuttle plasmid. The expression cassettes from the derived plasmids pcDNA3ΔNotI/endo-angio e pcDNA3ΔNotI/endo-lringle5 were cut Nrul-Xbal and inserted in Smal-Xbal of a plasmid, named p41, contajLtώig the UL41 HSV flanking sequences with HCMN promoter downstream of 100 pb ICPO promoter. The fransgene expression cassettes were inserted into the UL41 locus of HSV genome by replacing the LacZ gene present in the TOZGFP background vector by homologous recombination generating the angiostatic recombinant vectors. The recombinant vimses were identified by isolation of a clear plaque phenotype after X-gal staining. To purify positive isolates we have performed three rounds of limiting dilution. Progeny vimses have been screened by Southern blot analysis to identify recombinants containing the angiostatic genes.

Construction of HSV recombinant vectors expressing GMCSF

Mouse-GMCSF sequence derived from CMV-pal/BLSK kindly given by Malter S. (Blood 86,7,1995:2551-58) was subcloned as Smal-Xbal fragment into a pBluescript (pBSSK) plasmid. The cytokine gene was then cut Cla-Xba and inserted in a plasmid named PB5 under the transcriptional control of HCMV promoter. PB5 is a pBSSK containing the Usl HSV-1 sequences corresponding to the ICP22 immediate early gene previously described (ref: Krisky DM, et al. Development of heφes simplex viras replication-defective multigene vectors for combination gene therapy applications. Gene Ther 1998;5:1517-1530). The transgene expression cassette was inserted into the Usl locus of HSV genome into the backbone of the THZ1 vector by homologous recombination, generating the cytokine recombinant vector. THZ-1 is a recombinant viras deleted in the ICP4, ICP27 and ICP22 immediate early genes with the LacZ reporter gene, under HCMV promoter, in the ICP22 locus. THZ-1 viral DNA and the recombinant plasmid containing the gmcsf gene have been co-transfected into 7b complementing cell line. The recombinant virus TH-gmcsf containing the gmcsf cDNA was identified by isolation of a clear plaque phenotype after X-gal staining. The TH-gmcsf virus was purified by three rounds of limiting dilution and screened by Southern blot analysis to identify the. new recombinant. Western blot analysis and ELISA of the recombinant virus-infected cell lines were performed in order to assess the fransgene expression.

Construction of HSV recombinant vectors expressing angiostatic fusion proteins and GMCSF

The vector T0Hendo::angio/Hgmcsf and T0Hendo::kringle5/Hgmcsf containing the angiostatic fusion proteins in UL41 HSV locus and gmcsf ^'in Usl HSV locus was created by genetically crossing the above vectors (T0H:endo::angio or T0H:endo::kringle5 and TH:gmcsf). 7b cells plated on 60 mm petri dishes were infected with 3MOI of T0H:endo::angio or T0H:endo::kringle5 and TH:gmcsf virases and harvested 18 hours post infection. The mixture of viruses derived from the co-infection was titrated, and by Southern blot screening the viral vectors containing both genes were isolated. The T0Hendo::angio Hgmcsf and TOHendo::kringle/Hgmcsf vimses were purified by three rounds of limiting dilution and the genes expression was confirmed by Western blot. analysis.

In vitro expression of antiangiogenic proteins

Antiangiogenic fusion proteins were detectable by Western blot in cell culture and supematants of Vero and LLC tumor cells following the infection by T0Hendo::angio and T0Hendo::kringle5 vectors.

The media were harvested adding 1 μg/ml aprotinin. Individual cell monolayers were scraped in Tris-buffered saline (TBS), the cell pellet treated with lysis buffer (0.1 M NaCl, 0.01 M Tris.- HC1 pH 7.0, 0.001 EDTA pH 8.0, 1% Triton X-100, 1 μg/ml aprotinin, 100 μg/ml PMSF), sonicated and cleared by cenfrifugation.

Media from Vero (green African monkey renal fibroblast) cells or from LLC (Lewis lung carcinoma) murine tumor cells infected at multiplicity of 2 with T0Hendo::angio vector and with T0Hendo::kringle5 vector were harvested at 24, 48 and 72 hours post infection, loaded on 12% SDS-polyacrylamide gel and analyzed by Western blot.

The proteins were detected with a rabbit polyoclonal antibody specific for the human endostatin (Oncogene Research Products, Boston, USA) 1:1000 diluted and a mouse anti-rabbit IgG PA- conjugated secondary antibody (Promega Coφoration, USA) at 1:2500 dilution. Antibody immunocomplexes were detected by ECL Western Blot detection kit (Amersham, Pharmacia Biotech). Controls were represented by uninfected cells and cells infected at m.o.i. of 2 with TO- GFP recombinant vector.

In vitro biological activity of antiangiogenic proteins: effect on endothelial cell proliferation

The recombinant HSV-1 vectors encoding endostatin:: angiostatin (T0Hendo::angio) and endostatin: :kringle5 (T0Hendo::kringle5), as well as TO-GFP control vector were used to infect LLC cells at multiplicity of one. 48 hours later, LLC cell culture supematants were collected from the control and vector-infected cells and tested on human umbilical vein endothelial cells (HUVEC) at different LLC-conditioned vs. normal non conditioned HUVEC medium ratios. HUVEC viability was determined after 5 days by a colorimetric, tetrazolium-based (MTT) assay. HUVEC cells were cultured in a 96 well plate (3000 cells/well) and 50μl/well of conditioned medium was added. Plates were incubated at 37°C for 5 days and then 25μl of MTT (1- (4, 5 dimetiltiazol-2 -il)- 3, 5- dimetilformazane, Sigma M5655) 2,5 mg/m) were added. The precipitate was solubilized with SDS20%-DMF50% (pH 4.7). Absorbance values were measured at 570 nm using a microtifre plate specfrophotometer (Titertek Multiskan, Flow Laboratories, Irvine, UK).

In vitro biological activity of antiangiogenic proteins: effect on endothelial cell migration

To evaluate the migration was used a transwell system (Corning) with 8 μm polycarbonate membrane pretreated.

LLC tumor cells (0,8x10 cells / well) were seeded in the lower compartments of the 6 well transwell system and infected at multiplicity of 2 with various recombinant vectors; 48h post infection, recombinant bFGF 25ngr/ml (Oncogene Research Products, Boston, USA) was added as a chemotactic stimulus. Primary HUVEC cells (4000 cells /well) in Ml 99 + FCS 0.5% pretreated with DilisC (l, -dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate, Fluka) fluorescent tracer (5μgr/ml) were placed in the upper chambers; after 5 hours of incubation at 37°C, 5% CO₂, cells that migrated from the upper to the lower filter surface were fixed with 4% of parafolmadeyde and counted. Results were expressed as percentage of cell migration, relative to the migration induced by conditioned media from uninfected LLC, which was assumed as 100% of HUVEC migration. 10 μgr/ml of recombinant human antiangiogenic proteins (angiostatin and endostatin) were used as positive controls.

In vitro biological activity of antiangiogenic proteins: effect on tube formation in primary human endothelial + fibroblast co-cultures.

The antiangiogenic biological effect on tube formation was tested with an ANGIOKIT (ZHA- 1000, TCS) system. Human fibroblasts and endothelial cells co-cultured in a 24 well plate constitute the ANGIOKIT system. The co-cultured cells have been infected with the vectors THZ4, TOendo-angio, T0endo-kringle5 e TOendo-angio+TO endo-kringle5 at multiplicity of 1 on days 1, 4, 7 and 10 after cell seeding. Positive and negative controls were freated with recombinant VEGF 2ngr/ml and suramin 20mM, respectively. At the 11^th day post seeding, samples have been fixed with ethanol 70% and then incubated with the primary antibody anti- CD31 and a secondary antibody AP-conjugated. Antibody immunocomplexes were detected by BCIP and NBT substrates in order to show primitive blood vessels (tubes) that had been formed by the endothelial cells. All reagents including the cells were provided by TCS Cell Works 1U4

Tubule Staining Kit.

Cytotoxic activity of HSV-1 TK suicide gene following prodrug Ganciclovir (GCV) addition to the cell culture media

LLC tumor cells (2500 cells/well) and primary endothelial HUNEC cells (3000 cells/well) were infected at multiplicity of 1 with T0Hendo::angio, T0Hendo::kringle5 or TK-negative viral confrol (THZ.4) in 96-well plates. Prodrug ganciclovir (G2536, Sigma) was then added at 5 μgr/ml. Proliferation was evaluated at 24, 48, 72 and 96 hours post infection with a standard tetrazolium-based (MTT) assay. Uninfected cells were also used as negative confrol. Absorbance values were measured at 570 nm using a microtifre plate specfrophotometer (Titertek Multiskan, Flow Laboratories, Irvine, UK).

In vivo experiment :

Local treatment of pre-established LLC tumors with recombinant vectors

6 weeks old C57B1/6 female mice were injected subcutaneously with 0,5xl0⁶/100μl LLC cells and treated with antiangiogenic and suicide genes expressing vectors when the tumors became palpable. The vectors (10⁶ plaque forming units/1 OOμl) were administered every other day, directly into the tumors. The prodrug GCN was injected daily, at lOOmg/kg (i.e. 2,5 mg/mouse in lOOμl) by intraperitoneal route. Tumor volumes were measured with a digital caliper 3 times a week; each time point represents the average of 6 mice per group.

Tumor growth inhibition by implantation of vector-infected LLC cells

LLC (1*10⁶ cells/1 OOμl) were infected at multiplicity of 1 with the recombinant viral vectors, and then injected into the left flanks of C57B1/6 mice. lOOμl of Ganciclovir (lOOmg/kg) was administered i.p. for a week afterwards. Tumor volumes were measured with a digital calliper every two days; each time point represents the average of 12 mice per group.

Tumor challenge

Mice injected with LLC infected with T0Hendo::angio + T0Hendo::kringle5 that have shown complete tumor regression were divided in two groups: the follow-up group and LLC-challenge group; the last one has been injected with 1*10⁵ LLC in the right flanks. Secondary tumors were 11D treated locally with T0Hendo::angio + T0Hendo::kringle5, in presence or in absence of GCN.

Results

The human endostatinXNIII:: angiostatin fusion gene and the human endostatinXVIII::Kringle5 fusion gene, both purchased from InvivoGen as pGT60 and pGT64 plasmids, have been cloned in a plasmid containing UL41 HSN flank sequences under the HCMV promoter downstream of lOOpb of ICPO promoter. Both transgenes at the present have been introduced in the same viral locus to avoid differences in gene expression due to promoters and viral location (Fig.26A).

The fransgene expression cassettes were inserted into the UL41 locus of TOZ-GFP HSN vector genome backbone by homologous recombination, generating the angiostatic recombinant vectors. TOZ-GFP is a recombinant viras deleted in the ICP4, ICP27 and ICP22 immediate early genes with the GFP reporter gene, under HCMN promoter, in the ICP22 locus and the lacZ reporter gene, under ICPO promoter, in the UL41 locus. To this end TOZ-GFP viral DΝA and the recombinant plasmids containing the angiostatic genes have been co-fransfected into a complementing cell line (7b), which provides, in trans, the essential viral genes ICP4 and ICP27.

The recombinant viral vector expressing GMCSF was constracted by co-transfecting a plasmid (PB5-gmcsf), which contains gmcsf gene under HCMN promoter flanked by ICP22 HSN1 sequences, with THZ1 recombinant backbone viras into 7b complementing cell line. THZ-1 is a recombinant virus deleted in the ICP4, ICP27 and ICP22 immediate early genes with the LacZ reporter gene, under HCMN promoter, in the ICP22 locus (Fig.26B).

The vectors TOHendo::angio/Hgmcsf and T0Hendo::lαingle/Hgmcsf containing the angiostatic fusion proteins in UL41 HSN locus and gmcsf gene in US1 HSN locus were created by genetically crossing the above vectors (T0H:endo::angio or T0H:endo "kringle and TH:gmcsf) (Fig.26C).

Progeny virases have been screened by Southern blot analysis to identify recombinants that contain the angiostatic or cytokine or both genes. Finally, western blot analysis of the recombinant vimses-infected cell lines was performed in order to assess the transgenes expression. 1U6

Antiangiogenic fusion proteins were detectable by Western blot in cell culture supematants of Nero and LLC tumor cells following the infection by T0Hendo::angio and T0Hendo::kringle5 vectors. In the media, it was evident the release of angiostatic proteins expressed both in the cleaved (20 kDa endostatin) and in the uncleaved form (58 kDa endostatin:: angiostatin and 35 kDa endostatin: :kringle 5) (Fig.27).

The effect of conditioned media obtained from infected Lewis lung carcinoma (LLC) cells on primary human endothelial cell line (HUVEC) growth was evaluated. Media from LLC (2*10⁶) cells infected with the recombinant vectors at MOI 2 were collected and overlaid on HUVEC. bFGF 3ngr/ml was added as angiogenic stimulus. Negative confrols were represented by HUVEC cells treated with medium obtained from non-infected LLC or LLC infected with the confrol vector (THZ4). After 5 days incubation HUVEC viability was determined by a colorimetric, tetrazolium-based (MTT) assay. All samples were run in quadruplicate (Fig.28).

We have performed appropriate experiments to study the effect of angiostatic molecules on HUVEC migration toward a chemotactic stimulus. LLC (800.000 cells / well) were seeded in the lower compartments of 6 well transwell systems and infected (MOI 2) with the HSVl- based vectors; 48h post infection, bFGF 25ngr/ml was added as a chemotactic stimulus. HUVEC (4000 cells/well) were placed in the upper chambers; after 5 hours incubation, cells migrated from the upper to the lower filter surface were fixed and counted. Results were expressed as percentage of cell migration, relative to the migration induced by conditioned media from uninfected LLC, which was assumed as 100% of HUVEC migration. 10 μgr/ml of recombinant human antiangiogenic proteins (angiostatin and endostatin) were used as a positive control. Numerical values of HUVEC were: more than 100% for THZ4; 18% for T0Hendo::angio; 13% for T0Hendo::kringle5; 15% for T0Hendo::angio and T0Hendo::kringle5, indicating a significant inhibition of migration (Fig.29).

We also performed experiments to study the inhibitory effects both on tubules formation as well as anastomosis of newly formed vessels by infecting endothelial cells with vectors expressing the angiostatic factors. The cells (a mixture of fibroblasts and endothelial cells) supplied by (AngioKit- TCS CellWork) were infected with the vectors (MOI 1) at days 1, 4, 7 and 10 post cells seeding. Positive kit control: VEGF 2 ngr/ml; negative kit confrol: suramin 20 mM; viral vector control: THZ4. At the 11^th day post seeding the cells have been incubated with anti-CD31 AP-labeled antibody, in order to put in evidence and count the formed tubes (Fig.30).

We have tested the cytotoxic activity of HSV-1 TK suicide gene co-express in the angiostatic vectors following prodrag Ganciclovir (GCV) addition to the cell culture media. LLC tumor cells (2500 cells/well) and primary endothelial HUVEC cells (3000 cells/well) were infected at multiplicity of 1 with T0Hendo::angio, TOHendo "kringle 5 or TK-negative viral control (THZ.4) in 96-well plates. Prodrag ganciclovir was then added at 5 μgr/ml. Proliferation was evaluated with an MTT assay (Fig.31).

In vivo to further characterize the angiostatic effect of the fusion proteins produced by the viral vectors T0Hendo::angio and T0Hendo::kringle5 in combination with the suicide genes we used the LLC tumor model (ATCC) which is one of the best known models to study tumor angiogenesis. We performed two different experiments repeated several times. In the first one, in an attempt to reduce or block the tumor mass growth, we induced tumor formation by injecting LLC cells subcutaneously in the right flank of C57bl/6 syngenic mice. Next we treated the tumor masses with the suicide genes and the angiogenesis inhibitors expressing-vectors, followed by the adminisfration of GCV at different doses and times. In the second experiment we infected in vitro LLC cells with the endostatin-angiostatin, endostatin-kringle5 and confrol HSV vectors and then inoculated them into the mice flanks, and monitored the tumor mass development; The use of these systems in vivo on pre-established tumors led to a reduction (but not suppression) in the rate of tumor mass growth and the regression effect is more efficient when the GCV is given 24 hours instead of 48 hours post viral injection (Fig.32A and B). The in vitro infection of the tumor cells prior to their implantation, instead, halt the growth of primary tumor, with subsequent complete regression of the formed mass in vivo (Fig.33).

Mice injected with LLC infected with T0Hendo::angio + T0Hendo::kringle5 which have shown complete tumor regression were divided in the follow-up (A) and LLC-challenge (B) groups. Secondary tumors were treated locally with T0Hendo::angio + T0Hendo::kringle 5, in presence or in absence of GCV. Mice with rapidly growing tumors present significant spleen enlargement, also observed in the pre-established tumor treatment-model (Fig.34).

Further example of the construction of a replication-deficient recombinant HSV for use in an anticancer therapy A DNA fragment comprising an HSV ICPO-immediated early promoter-lacZ expression cassette flanked by Pad restriction endonuclease recognition sites and sequences homologous to UL41 for HSV-1 was co-transfected in 7b cells with HSV-1 mutant. Recombinants were screened for LacZ expression, and the correct insertion was confirmed with Southern blot analysis.

The resulting recombinant viras was used, as backbone, to construct the vimses to use in an anticancer therapy. The recombination was designed to remove the LacZ expression cassette from the vimses to be substituted with expression cassettes containing the HIV gene either under ICPO-IEp promoter or HCMV-IEp promoter.

Thus, isolates were screened with X-gal staining, and white plaques were selected and screened by Southern blot hybridization using a probe hybridizing the specific gene (e.g., endostatin, angiostatin, kringleS, gm-csfi etc.). To confinn the protein expression from the virases, cells were infected at MOI of 1 and the cellular extract and the supernatant were assayed, by standard Western blot, for the presence of the non-native proteins.

Vectors containing more then one non-native expression cassette were obtained by genetic crosses between the recombinant vimses. Such manipulations are known in art.

ICP4^", ICP27^", and ICP22^" HSV vectors having an ICP4-immediate early promoter-TK expression cassette at the UL24 locus, are known (Marconi, P., Krisky, D., Oligino, T., Poliani, P.L., Ramakrishnan, R., Goins, W.F., Fink, D.J., & Glorioso, J.C. (1996). Replication-defective heφes simplex vims vectors for gene transfer in vivo. Proc Natl Acad Sci USA, 93(21), 11319- 11320.) The replication defective HSV-1 vectors are modified by having non-native expression cassettes encoding for HIV proteins at the UL41 or ICP22 locus.

Conclusions for Example 7 T0Hendo::angio and TOHendo "kringle 5 viral vectors express the suicide HSV-TK gene and are able to kill the infected cells in presence of GCV in vitro; TOHendo ::angio and TOHendo "kringle 5 are able to exert significant inhibitory activity on proliferation and migration of human endothelial cells in vitro, as well as tubes formation in mixed cultures of endothelial cells and fibroblasts; treatment of pre-established LLC tumors in vivo with TOHendo ::angio and TOHendo: :kringle 5 vectors is capable of reducing the tumor growth rate, but is not able to completely arrest it; antitumoral effect is further increased by systemic adminisfration of GCV; infection of LLC tumor cells with TOHendo ::angio and TOHendo:. -kringle 5 before their in vivo implantation induces the complete regression of primary tumors, both in presence and in absence of GCV; complete tumor regression was also observed when the implantation of LLC cells infected with the TK-alone expressing vector was followed by GCV treatment; experiments in murine model suggest that in vivo LLC rumor destruction occurs either directly by the TK/GCV activity, or indirectly by local delivery of antiangiogenic molecules (antioangiogenic vectors in absence of GCV), or by both mechanisms together (TK and antiangiogenic vectors in presence of GCV); however, the efficiency of treatment is limited by the tumor mass dimensions and is more successful early in the tumor establishing phase.

Figure legends for Example 7

FIG.26 Schematic representation of recombinant vectors

A) Human endostatin: .-angiostatin (TOHendo: :angio) and endostatin: :kringle 5 (TOHendo "kringle 5) genes were cloned in the UL41 locus of the TO-GFP backbone vector, both under the HCMV promoter; HSV-1 thymidine kinase (TK) gene is located in its natural UL23 locus, under ICP4 IE (instead of the native early) promoter control.

B) Murine gmcsf gene, under the HCMV promoter, has been cloned in ICP22 locus of THZ.1 backbone vector.

C)_. Recombinant virases expressing angiostatic and cytokine genes in one vector derived from the genetic crossing between the vectors A and B.

FIG. 27 In vitro expression of antiangiogenic proteins

A. media from Vero (green African monkey renal fibroblast) cells infected at multiplicity of 2 with TOHendo "angio vector at 24, 48 and 72 hours post infection (lanes 1, 2, 3), with TO-GFP negative control vector (lane 4) and with TOHendo: :kringle 5 vector at 24, 48 and 72 hours post infection (lanes 5, 6, 7). Lanes 8, 9 and 10 show MW marker, recombinant human endostatin (positive control) and uninfected Vero cell culture medium, respectively.

B. media from LLC (Lewis lung carcinoma) murine tumor cells infected with TOHendo:: angio vector at 24, 48 and 72 hours post infection (lanes 1, 2, 3), with TO-GFP negative confrol vector (lane 4) and with TOHendo: :kringle 5 vector at 24, 48 and 72 hours post infection (lanes 5, 6, 7). Lanes 8, 9 and 10 show MW marker, recombinant human endostatin (positive confrol) and uninfected LLC cell culture medium, respectively.

FIG.28 In vitro biological activity of antiangiogenic proteins: effect on endothelial cell proliferation

The recombinant HSV-1 vectors encoding endostatin:: angiostatin (TOHendo:: angio) and endostatin: :kringle 5 (TOHendo: :kringle 5), as well as THZ.4 confrol vector were used to infect LLC cells at multiplicity of one. 48 hours later, LLC cell culture supematants were collected from the confrol and vector-infected cells and tested on human umbilical vein endothelial cells (HUVEC) at different LLC-conditioned vs. normal non conditioned HUNEC medium ratios. HUNEC cell proliferation was evaluated after 5 days by MTT assay.

FIG. 29 In vitro biological activity of antiangiogenic proteins: effect on endothelial cell migration.

LLC tumor cells (0,8xl0⁶cells / well) were seeded in the lower compartments of 6 well transwell systems and infected at multiplicity of 2 with various recombinant vectors; 48h post infection, bFGF 25ngr/ml was added as a chemotactic stimulus. Primary HUNEC (4000 cells /well) were placed in the upper chambers; after 5 hours of incubation at 37°C, 5% CO₂, cells that migrated from the upper to the lower filter surface were fixed and counted. Results are expressed as a ratio of HUNEC cells migrated towards vector-infected LLC cells and n° of HUVEC cells migrated towards uninfected LLC cells.

FIG. 30 In vitro biological activity of antiangiogenic proteins: effect on tube formation in primary human endothelial + fibroblast co-cultures.

Human fibroblasts and endothelial cells co-cultured in a 24 well plate have been infected with the angiostatic vectors. Positive and negative controls were represented by VEGF 2 ngr/ml and suramin 20 mM, respectively. The panels show the stained primitive blood vessels (tubes) that had been formed by the endothelial cells after 11 days of co-culture (40x magnification).

FIG. 31 Cytotoxic activity of HSV-1 TK suicide gene following prodrag Ganciclovir (GCV) addition to the cell culture media.

LLC tumor cells (2500 cells/well) and primary endothelial HUNEC cells (3000 cells/well) were infected at multiplicity of 1 with TOHendo:: angio, TOHendo "kringle 5 or TK-negative viral control (THZ.4) in 96-well plates. Charts A, B: cytotoxicity of TK + antiangiogenic factors (TOHendo:: angio and TOHendo "kringle 5) -expressing vectors in presence (A) and in absence (B) of Ganciclovir on LLC tumor cells; charts C, D: cytotoxicity of TK + antiangiogenic factors (T0Hendo::angio and TOHendo: :kringle 5) -expressing vectors in presence (C) and in absence (D) of Ganciclovir on primary endothelial HUNEC cells.

FIG. 32 In vivo local treatment of pre-established LLC tumors with the recombinant TK and antiangiogenic proteins- expressing vectors.

6 weeks old C57B1/6 female mice were injected subcutaneously with 0,5xl0⁶ LLC cells and treated with antiangiogenic and suicide genes expressing vectors when the tumors became palpable. The vectors (10⁶ plaque forming units/lOOμl) were administered every other day, directly into the tumors. The prodrag GCN solution was injected 24 hours (chart A) or 48 hours (chart B) post viral injection and administered daily, at lOOmg/kg (i.e. 2,5 mg/mouse in lOOμl) by intraperitoneal route. Tumor volumes were measured with a digital caliper 3 times a week; each time point represents the average of 6 mice per group.

FIG. 33 In vivo tumor growth inhibition by implantation of vector-infected LLC cells LLC (1*10⁶ cells) were infected at multiplicity of 1 with the recombinant viral vectors, and then injected into the left flanks of C57B1/6 mice. Ganciclovir (lOOmg/kg) was administered i.p. for a week afterwards. Tumor volumes were^' measured with a digital caliper every two days; each time point represents the average of 12 mice per group. Mice injected with LLC infected with TOHendo:: angio + TOHendo ::kringle5 have shown complete tumor regression. Images show representative primary tumor-injection sites (left flanks) of mice belonging to different groups at the time points indicated by the black arrows.

FIG. 34 In vivo follow up and tumor challenge growth inhibition by implantation of vector- infected LLC cells.

Mice injected with LLC infected with TOHendo:: angio + TOHendo: :kringle5 that have shown complete tumor regression were divided in the follow-up (A) and LLC-challenge (B) groups. For the challenge group, secondary tumors were treated locally with TOHendo:: angio + TOHendo: :kringle5, in presence or in absence of GCN. Representative images of secondary tumors (challenge; right flanks) are shown in (B) chart, together with the spleens. Mice with rapidly growing tumors present significant spleen enlargement, also observed in the pre- established tumor treatment-model. PAGE INTENTIONALLY LEFT BLANK

Example 8: Gene Therapy For Nervous System : Defective Hsv Vector Expressing Fgf2 And Bdnf And Synergic Effect On In Vitro Proliferation And Differentiation

This Example realtes to the use of non-rep licate HSV vectors: (i) to treat acquired Neurological Diseases such as Parkinson's disease, Alzheimer's disease, Amyotrophic lateral sclerosis in the CNS and in the peripheral nervous system (PNS) by combining neurotrophic factors genes together in the same vector to increase their synergistic effects or (ii) to treat Neurodegenerative diseases with genetic involvement such as Tay-Sachs Disease (TSD) by expressing a missing enzyme.

Background

Understanding the molecular basis and the physiopathology of neurological diseases has become obtainable through the discovery of new gene or gene products involved in neuronal pathways and following the development of powerful tools and techniques applicable to cellular and molecular aspects in neurobiology. One of the major breakthroughs, towards the evaluation of novel strategies for anatomical and functional regeneration in the central nervous system (CNS), is the concept that neurotrophic and inhibitory proteins govern the processes involved in brain and spinal cord repair.

The CNS, with its unique complex structures consisting of a variety of cell types and tracts kept in a privileged environment separated from the blood system by the blood-brain barrier (BBB) represents a barrier to physiopatholocical studies and to gene therapy.

Recent isolation and identification of an increasing number of trophic factors has led to a series of studies of these factors in the CNS. Neurotrophins represent a large family consisting of nerve-growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotroρhin-3,-4,-5, and 6 and glial-derived ^' neurotrophic factor (GDNF). Neurotrophins affect a large number of neuronal cells in developing and adult neuronal populations of the peripheral nervous system (PNS) and CNS and play important roles in proliferation and differentiation of cortical progenitorscells to a particular lineage. Furthermore, neurotrophins can provide neuroprotection to neurons axotomized by traumatic injury or by neurotoxins. Two other growth factors involved in progenitor cell proliferation and in graded stages of neuronal differentiation belong to the cytokines family and are the basic fibroblast growth factor (bFGF or FGF-2) and the ciliary neurotrophic factor (CNTF).

The development of viral-based vectors as gene-transfer vehicles has made possible to deliver genes into defined cell population in the CNS. Because the heφes simplex viras (HSN) is a neurofropic virus, the HSN system seems to be the most suitable tool to deliver foreign genes to the nervous system.

Replication deficient HSN-1 based vectors have several characteristics which make them particularly attractive for gene transfer to neurons: (a) HSN-1 has an intrinsic neurotropism; b) vectors can be grown to high titer without the generation of replication competent viras (c) second generation vectors have low cytotoxicity for neurons; (d) replication-deficient viruses have been demonstrated to establish latency in the cell body of motor neurons following inoculation of muscles in the extremities of animals; (e) HSV contains an endogenous promoter system which has been shown to remain active long-term albeit low levels in neurons; (f) vectors can be engineered to contain multiple transgene expression cassettes.

Materials and Methods

Animals Newborn Sprague-Dawley rat pups (P1-P3), were used for in vitro experiments. Animals were housed under standard conditions: constant temperature (22-24°C) and humidity (55-65%), 12 h dark-light cycle, free access to food and water. Procedures involving animals and their care were carried out in accordance with European Community and national laws and policies.

Preparation of viral vectors

Cell culture and virus stock production. The Vero cells (African green monkey kidney cells from ATCC, Rockville, MD, USA; CCL81) and Vero-derived cell line, termed 7b, that expresses the HSV-1 immediate early genes ICP4 and ICP27 required for viras replication (ref: Krisky, D.M., Marconi, P.C, Oligino, T., Rouse, R.J., Fink, D.J., and Glorioso, J.C. (1997). Rapid method for construction of recombinant HSV gene transfer vectors. Gene Therapy 4:1120- 1125. / ref: Marconi, P., Krisky, D., Oligino, T. et al. Replication-defective heφes simplex viras vectors for gene transfer in vivo. Proc Natl Acad Sci U S A 1996, 93(21), 11319-11320), were grown in Dulbecco's modified essential medium (D-MEM, EuroClone Ltd., U.K.) supplemented with glutamine, antibiotics (PSN) and 10% fetal calf serum (FCS); 7B cells were maintained under G418 (1 mg/ml; Sigma) selection. PC12, rat pheochromocytoma cells (ATTC, Rockville, MD, USA; CRL-1721), were maintained in RPMI medium supplemented with 10% FCS. Oligodendrocyte-type 2 astrocyte precursor cells (O-2A) were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with NI supplement (1 ml/100 ml, Sigma), penicillin/streptomycin 2%, L-glutamine (1 ml 100 ml) and biotin (1 g/100 ml, Sigma). Viras stocks were prepared as previously described (Krisky, D., Marconi P, Goins, W.F., and Glorioso, J.C. Development of Replication-Defective Heφes Simplex Viras Vectors. Chapter in Methods in Molecular Medicine. Gene Therapy Protocols. P.Robbins (ed.) Humana Press Inc. Totowa, N.J. 1996).

Plasmid and vector constructions.

Different vectors were used, as described below.

TH.bFGF and THZ HSV-1 -derived vectors

The first one (TH:bFGF vector) contained the sequences for the rat ovarian basic fibroblast growth factor (FGF-2) cDNA (A. Baird, The Whittier Institute, La Jolla, CA, USA) were placed downstream of the HCMV IE promoter and 5' to the polyadenylation sequences from the bovine growth hormone. The second construct (thereafter referred as control vector) contained the Escherichia Coli LacZ gene driven by the HCMV immediate early promoter (THZ vector). All expression cassettes were inserted into the thymidine kinase (TK) locus on a triple deleted recombinant HSV-1 vector lacking three different immediate early genes (ICP4, ICP27, ICP22) using the previously described Pad recombination method (ref: Krisky, D.M., Marconi, P.C, Oligino, T., Rouse, R.J., Fink, D.J., and Glorioso, J.C (1997). Rapid method for constraction of recombinant HSV gene transfer vectors. Gene Therapy 4:1120-1125.), and showing low cytotoxicity and high level of fransgene expression (ref: Krisky DM, et al. Development of heφes simplex viras replication-defective multigene vectors for combination gene therapy applications. Gene Ther 1998;5:1517-1530 / ref: Marconi P, et al. Replication-defective heφes simplex viras vectors for neurotrophic factor gene transfer in vitro and in vivo. Gene Ther 1999;6:904-912)

TO.-BDNF/GFP, TOZGFP and TH:FGF/0:BDNF/GFP HSV-1-derived vectors l it)

Plasmid pB410-BDNF. was constructed by introduction of the rat brain-derived neurotrophic factor (bdnf) cDNA (pb) from pBluscript-BDNF (kindly given by Marco Riva, Milan) into the HSV flank sequences .of pB41 plasmid that has been described elsewhere (ref: Krisky, D.M., Wolfe, D., Goins, W.F. et al. Deletion of multiple immediate-early genes from heφes simplex viras reduces cytotoxicity and permits long-term gene expression in neurons. Gene Ther 1998, 5 (12), 1593-1603.). The bdnf cDNA under the transcriptional control of the HSV immediate-early ICPO promoter was inserted into Xbal sites of pB41 plasmid between the two UL41 HSV fragments (HSV genomic positions 90.145-91.631 and 92.230-93.858) (ref: Krisky, D.M., Marconi, P.C, Oligino, T., Rouse, R.J., Fink, D.J., and Glorioso, J.C (1997). Rapid method for constraction of recombinant HSV gene fransfer vectors. Gene Therapy 4:1120-1125.). Plasmid pB410-BDNF was recombined within the genome of the TOZGFP viral vector using the previously described Pac-facilitated lacZ substitution method (ref: Krisky, D.M., Marconi, P.C, Oligino, T., Rouse, R.J., Fink, D.J., and Glorioso, J.C. (1997). Rapid method for constraction of recombinant HSV gene fransfer vectors. Gene Therapy 4:1120-1125.). The TOZGFP is a replication-defective HSV-1 viral vector this vector has low toxicity due to the deletion of three immediate early genes (ICP4, ICP27, which are essential for viral replication and ICP22) and contains the gfp (green fluorescence protein) gene into the ICP22 locus and the LacZ gene in the UL41 locus as marker genes. The production of recombinant virases was carried out using the standard calcium phosphate transfection procedure with 5 μg of viral DNA and 1 μg of linear plasmid pB410-BDNF. Transfection and isolation of the recombinant virus was performed in 7b cells as previous described.

The recombinant viras T0:BDNF/GFP containing the bdnfcDNA was identified by isolation of a clear plaque phenotype after X-gal staining. The T0:BDNF/GFP viras was purified by three rounds of limiting dilution and the presence of the fransgene was verified by Southern blot analysis. Viral stocks of the TO: BDNF/GFP and of the control vector TO-GFP (derived from TOZGFP without lacZ reporter gene in UL41 locus) were prepared and titrated using Vero- 7b cells (ref: Marconi, P., Krisky, D., Oligino, T. et al. Replication-defective heφes simplex vims vectors for gene transfer in vivo. Proc Natl Acad Sci USA 1996, 93(21), 11319-11320).

The vector TH:FGF/0:BDNF/GFP containing FGF-2 in Tk HSV locus and BDNF in UL41 HSV locus was created by genetically crossing the above vectors (TH-FGF2 and T0:BDNF/GFP). 7b cells plated 60 mm petri dishes were infected with 3MOI of TH:FGF and TO: BDNF virases and harvested 18 hours post infection. The mixture of virases derived from the co-infection was titrated, and the viral vector containing both genes was isolated by Southern blot screening. The TH:FGF/0:BDNF/GFP viras was purified by three rounds of limiting dilution and the genes expression were confirmed by Western blot analysis.

Transgene expression in vitro

Vero cells were plated in 6-well plates (2 10⁵ cells/well). One day after plating, some wells were infected at a MOI of 1.0 with TH:FGF-2, T0:BDNF/GFP, TH:FGF/0:BDNF/GFP, THZ, TOZ. One to seven days after infection, individual cell monolayers were scraped in Tris-buffered saline (TBS), the cell pellet treated with lysis buffer (0.1 M NaCl, 0.01 M Tris.-HCl pH 7.0, 0.001 EDTA pH 8.0, 1% Triton X-100, 1 g/ml aprotinin, 100 g/ml PMSF), sonicated and cleared by centrifugation. The presence of FGF-2, BDNF and FGF/ BDNF was determined by Western blot and ELISA assays.

Western blot analysis

FGF and BDNF protein expression from the TH:FGF-2, T0:BDNF/GFP, TH:FGF/0:BDNF/GFP vectors was analyzed in Vero cells (lxlO⁶ cells) infected with 1 MOI of the respectively virases. Cell exfracts, corresponding to 10 g of total proteins, were ran onto 12% SDS-polyacrylamide gel and analyzed by western blot. The FGF protein was reveled using a mouse anti-basic human FGF monoclonal antibody (Transduction Laboratories, Lexington, Kentucky) at 1:250 dilution and a mouse anti-mouse HRP-conjugated secondary antibody (NA931V, Amersham) at 1:2500 dilution. Immunocomplexes were detected by ECL Western Blot detection kit (Amersham, Pharmacia Biotech). The BDNF protein was reveled using a polyclonal rabbit anti human BDNF (S.Cmz, SC546) 1:500, which reacts also with rat BDNF and a detected using an anti-rabbit IgG HRP- conjugate (NA934V, Amersham) l:250.0.and TMB substrate. Controls were represented by uninfected cells and cells infected with 1 MOI of the THZ or TOZGFP recombinant vectors.

ELISA.

Direct quantification of FGF-2 (R&D System, Minneapolis, MN, USA) and BDNF was determined using an ELISA kit in which 96-wells microplates were coated respectively with a monoclonal anti-human FGF-2, which cross-reacts with the rat FGF-2 and with polyclonal rabbit anti human BDNF (S.Cruz, SC546). Briefly, 200 μl of the FGF-2 or BDNF standard and the supematants or the clarified lysates from TH:FGF-2, T0:BDNF/GFP, TH:FGF/0:BDNF/GFP, THZ, TOZ infected cells and Vero uninfected cells were added to each well, and the plates were incubated for 2 hours at room temperature. After washing, 200 μl of perqxidase-conjugate monoclonal anti-human FGF-2 or polyclonal anti-human BDNF were added to each well. Incubation was continued for two hours at room temperature. After washing, enzymatic activity was detected for FGF by adding the substrate solution for 20 min at room temperature. To detect BDNF was added an anti-rabbit IgG, HRP conjugate ((NA934V, Amersham) at 1:1000 dilution and TMB substrate. Absorbance values were measured at 450 nm using a microtitre plate specfrophotometer (Titertek Multiskan, Flow Laboratories, Irvine, UK).

Biological activity in vitro

Purification of progenitor cells. Immediately after euthanizing the newborn pups (n=20) by CO₂ inhalation, meninges-free cerebral hemispheres were removed, fransfened in Hank's balanced salt solution (HBSS, Gibco) and washed twice. Dissociate cell culture were obtained using a modification of previously described procedures (Avellana-Adalid V. et al. Journal of Neuroscience Research 1996).

Brains were minced and digested in 10 ml HBSS with 0.0025% frypsin/EDTA (Gibco). After 20 min at 37°C, 5 ml fetal bovine serum (FBS, to stop trypsin action) and 30 ml HBSS were added and the whole was centrifuged for 10 min at 1,500 r.p.m. The supernatant was discarded and the digested tissue was mechanically dissociated by two passages through cell strainers (mesh size 100 micrometer and 70 micrometer). Fresh HBSS was added to help cells through, reaching a final volume of 32 ml. Dissociated cells were layered on 4 preformed Percoll (Sigma) gradients. Percoll is a density gradient material consisting of colloidal silica coated with polyvinylpyrrolidone. Percoll gradients are suitable to sediment the cells in isopycnic banding. Cells are thus separated according to buoyant density. The density of cells and subcellular particles is a function of their water content and is very sensitive to the colloid osmotic pressure, pH and ionic strength. Progenitor cells are separated rapidly from postmitotic cells using density gradients because they have densities higher than differentiated cells.

To each Percoll self-generating density gradient, the solution was made by mixing 9 ml Percoll, lml HBSS/10X, 10ml HBSS/1X and was centrifuged at 14,000 r.p.m. for 30 min at 4°C using swing-out rotors. The cell suspension was then centrifuged at 14,000 t.p.m. for 15 min at 4°C After centrifugation, the material was fractionated in four gross fractions. From the top to the bottom of the tube, the fractions were: a) a clear supernatant, b) a white, thin, myelin-like layer, c) a large, slightly opalescent fraction, and d) a layer of red blood cells on top of a relatively dense-appearing pellet. Fractions a and b were vacuum aspirated and eliminated. Fraction c i iy consisted of the enriched progenitor fraction. This was carefully pipetted out, harvested and washed by two or more rinses in mixture of Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with NI supplement (1 ml/100 ml, Sigma), penicillin/streptomycin 2%, L- glutamine (1 ml 100 ml) and biotin (1 g/100 ml, Sigma).

The purified cell population obtained consisted of.oligodendrocyte-type 2 astrocyte precursor cells and neurons precursors in the form of steady, proliferating homotypic aggregates called "neurospheres".

Neurosphere induction, maintenance and characterization.

The purified cell population was seeded at high cell density (1.5xl0⁵ cell/cm²) in uncoated plastic tissue culture flasks (T25) and fed with DMEM-N1 (supplemented as described above) and 2% fetal bovine serum (FBS). Flasks were kept at 37°C in a humidified 9% CO₂/91% air atmosphere for 72 hr. After elimination of adhering cells, the neurospheres were disaggregated by gentle trituration with a micropipet and the cells were counted in a hemacytometer. The progenitor cells were resuspended in aliquots of 5x10⁵ cells and these aliquots were infected with 5xl0⁵ pfu TH-lac Z, TH-FGF2, TO- BDNF/GFP or TH-FGF2/0- BDNF/GFP recombinant vimses (i.e., at a MOI of 1.0). Infected cells were incubated at 37°C for 30 min, resuspended in serum-free DMEM, layered in 100 1 droplets onto glass coverslips precoated with 100 g/ml polyomithine (posed into 12-well tissue culture plates) and allowed to attach for 1 hr before adjusting the medium to the final volume of 2 ml per well. Finally, cells were cultured in serum- free DMEM without other growth factors for 4 weeks, to dissect out the effects of the neurotrophic factors produced by the vectors.

Immunocytochemistry.

Following the 4 week culture on polyomithine-coated glass, Ops were washed with PBS IX, fixed with 4% paraformaldehyde in PBS for 20 min and rinsed with PBS IX. The cells were preincubated with PBS containing 0.15% Triton X-100 for 5 min, rinsed twice and then blocked in 10 mg/ ml bovine serum albumin (BSA) in PBS for 30 min. Sister coverslips were then incubated for 45 min at room temperature in PBS containing a primary antibody: anti-GFAP (rabbit polyclonal, 1:500. dilution, Sigma), anti-MAP2 (mouse monoclonal, 1:1000 dilution; Sigma) or anti-CNPase (mouse monoclonal 1 mg/ml, Chemicon). Finally, coverslips were washed twice with PBS for 10 min, incubated with TR-conjugated secondary goat anti-mouse antibody (1:250) for 45 min at room temperature, and washed three times with PBS. The last wash contained 10 ng/ ml 4', 6-diamidino-2-phenylindole (DAPI, Sigma) in PBS. This was used as a fluorescent counterstain for cell nuclei. Coverslips were mounted using Gel/ Mount™ (Biomeda).

Results

Vectors

A schematic representation of the vectors employed in this study is shown in Fig. 35. All these vectors are triple mutants, defective for the ICP4, ICP27 and ICP22 immediate-early genes, that ensure efficient expression of the fransgene. Although this mutant background contains some degree of residual toxicity for some cell types, this phenomenon does not appear to be long- lasting nor to heavily affect neurons. For FGF-2, we used a previously characterized vector, TH- FGF2, in which the fransgene was introduced into the tk (UL23) locus of the control (TH-lacZ) vector by digestion of the viral DNA and by homologous recombination with an expression cassette containing sequences coding for FGF-2 under the confrol of the human cytomegalovirus immediate-early (HCMV IE) promoter. For BDNF, we used a vector, TO- BDNF/GFP, in which the transgene was introduced into the UL41 locus of another confrol vector (TO-lacZ/GFP) by digestion of the viral DNA and by homologous recombination with an expression cassette containing sequences coding for BDNF under the control of the ICPO promoter. The rationale for choosing these two distinct promoters was to mimic a sequential activation of FGF-2 and BDNF: in fact, the HCMV IE promoter ensures robust, but very transient, transgene expression, while the ICPO promoter provides a longer-lasting expression. The double mutant (TH-FGF2/0- BDNF/GFP) was obtained by crossing over TH-FGF2 and TO- BDNF/GFP (Fig.35). TH-lacZ and TO-lacZ/GFP were used as controls.

Transgene expression in vitro

To evaluate whether cells infected with the recombinant viruses could express the conesponding NTFs and to quantitate the transgene levels, ELISA assays were performed on total proteins extracted from Vero cell monolayers infected with the different vectors at a multiplicity of infection (MOI) of 1.0. Vero cells were infected with TH-FGF2, TO-BDNF/GFP, TH- FGF2/0:BDNF/GFP or their respective confrols (TH-lacZ and TO-lacZ/GFP), and FGF-2 or BDNF expression was determined at different times post-infection (1 to 4 days). In cells infected with either NTF vector, we observed a robust expression of the transgene up to 4 days, whereas in cells infected with the control (lacZ) vectors the expression of NTFs was virtually absent (Fig. 36). These data have been confirmed in a Western blot analysis (Fig.37).

Biological activity in vitro

To determine the biological activity of the NTF proteins expressed by the transduced cells and, further, to determine if the combination of FGF-2 and BDNF exerted different effects compared with either NTF alone, we use a dissociated cell culture system to isolate progenitor cells (neurospheres) and study their biological effect. We infected progenitor cells with TH-FGF2, T0:BDNF/GFP, TH-FGF2/0:BDNF/GFP or with the control vector, TH-lacZ. Neurospheres were maintained in culture for 3 days before being infected with the different vectors (MOI of 1.0) and transferred on a polyornithin substrate in a minimum essential, serum-free DMEM medium, not containing any trophic factor. Following infection, cells were maintained in this minimum medium for 4 weeks before analysis. This approach allowed testing the residual trophic, proliferative and differentiative capacity of the cells, ensured solely by the NTFs produced by the viral vectors.

To determine the differentiation pattern of the cells surviving 4 weeks in the minimum medium, we performed immunocytochemical analysis employing cell-line specific antibodies (Fig.38): type 2 asfrocytes were identified on the basis of expression of GFAP (glial fibrillary acid protein); neurons on MAP2 (microtubule-associated protein-2); oligodendrocytes on CNPase (2',3'-cyclic nucleotide-3' phosphodiesterase). The cells were also checked for the infection of HSV-1 based vectors with the polyclonal antibody anti HSV As described above, confrol cells and cells infected with the control vector TH-lacZ or the FGF-2 expressing vector TH-FGF2 or the BDNF expressing vector TO- BDNF/GFP differentiated in different proportion in a mixed culture containing GFAP-positive cells (presumably asfrocytes), CNPase- and MAP2-positive cells (presumably oligodendrocytes and neurons) (Fig.39) The vector expressing FGF-2 and BDNF together (TH-FGF2/0- BDNF/GFP) proliferate and differentiated in a significant proportion in neurons (Fig. 40 A and B).

Discussion

The present data demonstrate the feasibility of use of HSV-1 vectors for obtaining long-term biological effects in an in vivo system; extend previous observations that synergies occur between different NTFs, by showing a synergistic effect between FGF-2 and BDNF, and indicating that this occurs at the single cell level: in fact, the insertion of expression cassettes for both NTFs in the same viral backbone ensures transfection of both genes of interest in every infected cell; suggest that it will be possible to manipulate CNS cell proliferation, differentiation and migration by using appropriate combinations of NTFs: if achieved, such results would form the basis for the development of new strategies for the gene therapy of neurological disorders characterized by or associated with loss of specific CNS cell types.

Example 8 Figure Legends

FIG. 35. Graphic map of the viras TH-FGF2/0-BDNF/GFP, a triple mutant containing the rat FGF-2 gene in the tk locus, the rat BDNF gene in the UL41 locus, and the GFP cDNA in the ICP22 locus. The FGF-2 and GFP genes are driven by the HCMV IE promoter; the BDNF gene is driven by ICPO IE promoter.

FIG. 36. Time course of BDNF expression in vitro as determined by ELISA. The NTF expression was assayed on clarified lysates from TOZ-GFP, and TO-BDNF/GFP infected Neuro2A cells (1 pfu/cell) using an anti-human BDNF antibody. Cells were harvested every 24 h up to 3 days

FIG.37 A Western blot analisys for BDNF: by infecting at moi 1 Vero cells with TH- FGF2/0:BDNF/GFP, TOBDNF (as positive control) and TOGFP (as negative control). Primary antibody: Polyclonal rabbit anti human BDNF (S.Cruz, SC546) 1:500, which reacts also with rat BDNF; secondary antibody: antirabbit IgG HRP- conjugate (NA934V, Amersham) 1 :2500. Lane 1: . TOGFP infected Vero cells, 24h post infection; lane 2: MW marker; lanes 3-5: TH- FGF2/0:BDNF/GFP Vero infected cells (three vectors); lane7: THFGF Vero infected cells; lane 8: non-infected Vero cells; lane9: recombinant human BDNF protein (Promega, G1491), 50ngr. B Western blot analisys for FGF; by infecting at moi 1 Vero cells with TH-FGF2/0:BDNF/GFP, THFGF (as positive confrol) and THZ4 (as negative confrol). Primary antibody: Polyclonal mouse anti human FGF2 (Transduction laboratories F14220) 1:500; secondary antibody: antimouse IgG HRP- conjugate (NA931V, Amersham) 1:2500. Lane 1: THZ4 infected cells; lane 2: MW marker; lanes 3-5: TH-FGF2/0:BDNF/GFP Vero infected cells (three vectors); lane 7: non-infected Vero cells; lane 8: THFGF infected Vero cells, 24h post infection; lane 9: TOBDNF infected cells; lanelO: recombinant human FGF2 protein (Sigma, F0291), lOOngr

FIG.38 Schematic representation of the progenitore maturation ant the cellular markers that are acquiring during the differentiation in the diffemt lineages

FIG.39 Graphic representation of proliferation and differentiation of progenitor cells infected with the different viral vectors.

FIG. 40. In vitro differentiation of progenitors infected with TH-FGF2/0:BDNF/GFP vector expressing FGF-2 and BDNF together. Cellular immunofluorescence experiments performed progenitors isolated from the newborn rat CNS infected with TH-FGF2/0:BDNF/GFP and maintained in minimum essentail, serum-free DMEM medium for 4 weeks. Neurons were identified on the basis of expression of MAP2.

Example 9: A direct gene transfer strategy via brain internal capsule delays the progression of Tay-Sachs disease and revert the altered phenotype

The abbreviations used are: TS, Tay-Sachs; HexA, β-hexosaminidase A; MUG, 4-methylumbelliferyl β-N-acetylglucosaminide; MUGS, 4-methyumbelliferyl β-N-acetylglucosamine-6-sulfate;

Therapy for neurodegenerative lysosomal Tay-Sachs (TS) disease requires an active Hexosaminidase (Hex) A production in the central nervous system and a therapeutic approach that can be efficacious and can act faster than human disease progression. We combined the efficacy of a non-replicating Heφes simplex vector encoding for the Hex A alpha-subunit (HSN- TOalphaHex) and the anatomic structure of the brain internal capsule to optimally distribute the missing enzyme. For the first time, with this gene fransfer strategy, we re-established the Hex A activity and the GM2 ganglioside storage in both injected and controlateral hemispheres, in the cerebellum and spinal cord of TS animal model in one month of treatment. In our studies we do not observed adverse effects due to the viral vector, injection site or gene expression and, based on the results, we fill confident that the same approach can be applied to similar diseases involving an enzyme defect.

INTRODUCTION Tay-Sachs disease (TS) is a GM2 gangliosidosis due to the deficiency of the α- subunit of β-hexosaminidase A ^l'² (HEXA gene). In the most severe and typical clinical form, rapid mental and motor deterioration, starting within the first year of life, leads to death within 2-3 years. Pathological features include wide neurodegeneration and neuronal lipid storages. Currently, TS treatment is restricted to supportive care and appropriate management of intervening problems³.

In mammalian tissues, β-hexosaminidase (Hex, EC 3.2.1.52) exists in two major forms: Hex A (αβ structure) and Hex B (ββ structure) ^λ'². The homodimer αα, Hex S, represents the residual Hex activity in Sandhoff disease patients, a type 0 GM2 gangliosidosis due to inherited defects in the HEXB gene^4"7. Only Hex A, in the presence of the GM2-activator protein, hydrolyses the β-GalNAc-(l-4)-β-Gal glycosidic linkage of the GM2 ganglioside¹'^7"10.

Therapy for lysosomal storage disorders with neurological involvement such as TS disease requires production and distribution of the missing enzyme into the Central Nervous System (CNS). Several therapeutic approaches allow restoring the enzymatic activity in many key tissues (kidney, liver, spleen, etc.) but the reduction of the GM2 ganglioside deposits in the CNS is difficult to achieve^11"16. In fact, CNS is kept in a privileged environment, separated from the blood system by the blood-brain barrier (BBB), that represents an obstacle to therapy. A further obstacle stems from the observation that the fibroblasts from TS patients are not cross- corrected in vitro by simply adminisfration of the missing enzyme ¹⁷ and hider recent data supporting the concept that therapeutic approaches that heighten expression of the lacking enzyme in macrophages/microglia serves to enhance the corrective therapeutic potential ^18,19. Moreover, since TS pre-clinical diagnosis is an as fortunate as sporadic event, a therapeutic approach could be effective if it is able to delay the acute phase of the disease. Therefore restoration of Hex A activity and reduction of GM2 ganglioside storage represent one of the first major goal.

Here we proposed an in vivo gene transfer strategy for the production and the distribution of the HEXA gene into the CNS of TS animal model . We used a non-replicating Heφes simplex viral vector¹⁹ since the HSV-1 has the ability to infect a wide variety of cell types in non-replicating phase, e.g. neurons, and the intrinsic capacity to be transported in a retrograde manner to motor and/or sensory neuronal cell body following peripheral inoculation^20"21. We identified the internal capsule as a suitable site of injection. This is a connection structure mainly involved in motor pathways which are described highly impaired in human TS, that is also considered a very rare cause of Amyotrophic Lateral Sclerosis²²'²³.

Our data clearly showed that the combination of the internal capsule anatomic features, together with the described viral properties, allow a wide distributions of the Hex A activity and the reversion of the GM2 ganglioside storage in both injected and controlateral brain hemispheres. Interestingly these results were also obtained for the first time in the cerebellum and in the spinal cord suggesting that this approach is effective in the retardation of the progression of the disease. In our studies we do not observed adverse effects due to the viral vector, injection site or gene expression and based on the results that we have obtained we fill confident that the same approach can be applied to similar diseases involving an enzyme defect.

RESULTS Construction of herpes simplex viral vector encoding for Hex -subunit. We produced the non- replicating heφes simplex viral vector containing the human Hex α-subunit cDNA under the control of the ICPO promoter (HSV-TOαHex) (Fig.41a) ^24"25. ICPO promoter, that is an immediate early viral promoter, is up-regulated to extremely high levels in a background of a triple mutant replication-deficient vector. The expression cassette, containing the cDNA surrounded by UL41 flanking sequences of HSV, has been recombined into UL41 locus of the

TOZ viral vector (ICP4", 27", 22", UL417LacZ) (Fig.41a). We evaluated the insertion of the Hex α-subunit transgene into the viral DNA by southern blot (data not showed).

We used the HSV-TOαHex (therapeutic viral vector) and the HSV-TOZ (control viral vector) to infect the CNS of TS mice.

Validation of HSV-TO Hex viral vector by transduction ofTS organotypic brain slices. To test the efficacy of HSV-TOαHex viral vector, we transduced organotypic brain slices from TS mice either with the therapeutic or with the control vector, as described in method section. We evaluated the Hex specific activity in slice exfracts by using the fluorogenic substrate MUGS, which is hydrolyzed only by Hex isoenzymes containing the α-subunit. 3Days TS organotypic brain slices transduced with the therapeutic viral vector displayed MUGS activity comparable to that found in wild-type slices (Fig.41b). We detected no MUGS activity either in the untransduced or in the transduced with control vector organotypic brain slices from TS mice (Fig.41b).

In vivo gene transfer strategy for TS disease. We injected the vector in the internal capsule of the left-brain hemisphere (coordinates: - 0.34 mm to bregma, 1.4 mm mediolateral, and 3.8 mm of depth). We designed an experimental plan composed of five groups of five months old TS mice. Group 1. TS + HSV-TOαHex, 2,5x10°^" total PFU; group 2. TS + HSV-TOαHex, 5xl0⁶ total PFU; group 3. TS + HSV-TOZ, 5xl0⁶ total PFU; group 4, untreated TS mice; group 5, WT mice.

Therapeutic effect of the HSV-TOaHex viral vector in treated TS mice. 4 weeks after the injection, we sacrificed the mice, separated the brain hemispheres, the cerebellum and the spinal cord. We dissected each brain hemisphere in 4 rosfro-caudal 2.5 mm thick sections and the spinal cord in 3 antero-posterior segments. We analyzed each brain section, as well as cerebellum and spinal cord, for Hex A activity and for GM2 ganglioside content.

Brain hemispheres analysis. We evaluated the Hex specific activity in brain section by using the fluorogenic substrate MUGS. In Fig.42a we reported the Hex specific activity toward the MUGS substrate in each brain section of treated and untreated animals. All TS mice injected with the therapeutic viral vector display MUGS activity values according to the activity measured in WT. The injection of TS mice with the highest PFU resulted in the highest increase of the MUGS specific activity. We detected no MUGS activity neither in TS mice brain infected with the control viral vector nor in untreated TS mice. Interestingly, MUGS activity was restored both in the inoculated hemisphere and in controlateral one.

We verified the conect fonnation of Hex A isoenzyme into the brain of freated mice combining the chromatographic analysis on DEAE-cellulose with a specific enzymatic assay employing two different substrates, MUG, hydrolyzed by both α- and β-subunit, and MUGS, hydrolyzed only by α-subunit yields information regarding the subunit composition of the Hex isoenzymes. Under our experimental conditions, Hex B, unretained by the column, is simply eluted with void volume, while Hex A and Hex S are eluted by a linear gradient of NaCl ^6,17]27.

We reported a representative Hex isoenzyme pattern of the brain sections of all mice (Fig.42c). Murine TS brain sections, fransduced with both HSV-TOαHex viral vector doses (2,5x10⁶ PFU and 5xl0⁶ PFU) have Hex A and an Hex isoenzyme pattern comparable to that in WT mice (Fig.42c). All transduced brain sections displayed similar Hex isoenzyme pattern demonstrating the restoration of the enzymatic defect throughout the hemisphere. Chromatography also showed the restoration of Hex A isoenzyme in the controlateral TS hemisphere (data not shown).

We analyzed each hemisphere sections for the GM2 ganglioside content by Thin Layer Chromatography (TLC). A representative gangliosides chromatographic pattern of treated and untreated brain sections is reported (Fig.43a). Densitometric analysis of the TLC by ID Image Analysis Software of all considered mice, demonstrated the disappearance of the accumulated GM2 ganglioside in TS mice treated with the highest dose of the HSV-TOαHex viral vector and a decrease of the stored ganglioside in animals treated with the lower dose of the therapeutic viral vector (Fig.43c and 43d). These data were observed in all brain sections (both the injected and the controlateral hemispheres) and suggest a dose-dependent gene transfer reversion of the altered phenotype. Moreover these data are consistent with the observed distribution of the Hex A activity.

Cerebellum- analysis. We found the presence of the MUGS activity (Fig.42b) and Hex A isoenzymes (data not showed) also in the cerebellum of TS mice treated with the therapeutic viral vector. As for the brain hemispheres, the GM2 ganglioside storage disappeared in the cerebellum of TS mice treated with the highest dose of the HSV-TOαHex viral vector and it was partially removed in TS mice treated with the lower dose of the same vector (Fig.43b, 43c).

Spinal cord analysis.

Distribution of the HSV-TOZ viral vector in treated TS mice. We analyzed the diffusion of the vector in the brain after treating TS mice with a number of PFU of the HSV-TOZ viral vector conesponding to the highest dose of the therapeutic viral vector used. Animals were sacrificed after 72h and 1 month. We follow the viral vector distribution by monitoring the X-gal staining²⁸ in coronal (Fig.44a) transversal (Fig.44b) and sagittal (Fig.44c) brain serial sections. Results demonstrated a wide viral vector spreading in both injected and uninjected hemisphere.

We observed blue cells in all the brain area. Signal starts from second section on glass A2 and extends since all sections collected on glass A14 (total glasses 15); this means that signals extent for 6 mm in coronal sectioning (see methods). Interestingly the sagittal brain sections showed blue cells in the midbrain moφhologically of undoubted neuronal identity, cerebellum and controlateral hemisphere (Fig.44c and Fig.45a). The presence of X-gal staining in the Lateral ventricle, IIP ventricle, Silvius Aqueductus, Coφus Callosufn, indicated that a little part of the vector also spread from the site of injection to further brain areas (Fig. 45b).

We observed blue cells also in the spinal cord. In the Fig.45c we report representative X-Gal staining of spinal cord saggittal sections of freated TS mice. We found the blue signal in all the encephalic (C) and thoracic trunk (T) of the spinal cord and very few cells also in the dorsal trunk.

DISCUSSION In this study we delineate a new therapeutic approach for the genetic lysosomal storage disorder Tay-Sachs (TS) disease. Bearing in mind the rapid neurodegeneration and the high lethality of this disease we propose a plan able to delay the acute phase of the disease and to enhance the emergency service gap. We focused our attention on the rapid restoration of Hexosaminidase A activity and the reduction of GM2 ganglioside storage in the CNS since they represent the first cause of this metabolic alteration. We hypothesized that reduction/prevention of the neural deposits obtained by direct delivery of the missing enzyme into the CNS, could slow down the TS clinical features.

We design a strategy combining the potentiality of the HSV-1 non replicating viral vector and the anatomic structure of an injection site to allow a wide distribution of Hex A into the

CNS.

We delivered a non-replicating heφes-derived viral vector encoding for the Hex A α- subunit cDNA into the brain internal capsule of TS mice² to transduce the CNS cells in vivo. This injection site seemed particularly suitable been constituted by numerous fiber bundles which make it possible to reach several CNS areas with a single vector adminisfration. Pyramidal tract connects the frontal motor cortex with the midbrain, spinal cord and cerebellum, while the "higher thalamic peduncle" connects the cortex with the thalamus. Other "minor" components 97 connect capsular fibers with the parietal, occipital, and temporal cortex . We combined anatomical features of the internal capsule with properties of the heφetic vectors: i) intrinsic capacity to be transported in a retrograde manner to neuronal cell bodies; ii) ability to infect a wide variety of cell types in non replicating phase, especially neurons; iii) ability, being the transgene under the control of ICPO promoter, to allow a sustained transgene expression¹⁹'^20,28"30 which is a target for activation by neuronal transcription factors in neurons that have undergone a damaging insult³⁰. These features may give to this HSV promoter the interesting ability to enhance fransgene expression under pathological conditions.

All these HSV features with the chosen site of injection, have allowed a long and strong transgene expression and a wide spreading of the missing enzyme into the brain after a single injection (Fig.42 -45).

We evaluated the efficacy of our strategy by injecting the animal model of TS disease¹⁸ with the HSV-TOαHex. We used five months old TS mice because they clearly present GM2 13U ganglioside storages in the brain. In contrast with human patients, as the hydrolysis of GM2 ganglioside to GA2 by a murine sialidase, TS mice express a mild form of the disease ¹⁹'²⁹. They do not show neurologic symptoms, however they are lacking in Hex A activity (Fig.42) and present storage of GM2 ganglioside in the brain (Fig.43). Therefore these mice represent a suitable experimental model for testing the effectiveness of our approach on slowing the progression of the disease.

We found a decrease in GM2 ganglioside storage in brain of all TS mice treated with the therapeutic vector consistent with viral vector administration dose. One month after injection, we observed the absence of the GM2 ganglioside storage only in TS mice injected with the highest dose of HSV-TOαHex (Fig.43). We assume that results obtained with the two different viral doses are due to the different amount of enzyme production. In fact, TS mice inoculated with the lower dose of HSV-TOαHex resulted in a 40% of MUGS activity levels related to normal WT mice while the MUGS activity produced in the animals treated with the highest dose of HSV- TOαHex was 60-70% respect to WT value. The lower level of the Hex A activity was sufficient to hydrolyze the GM2 ganglioside but it was not sufficient to completely remove the lipid storage. We hypothesized that either it is need a grater number of cells that express the enzyme or the lower viral dose could need a longer time to completely remove the storage material.

All freated TS mice showed the restoration of GM2 ganglioside in both brain hemispheres - injected and non-injected one - and in the cerebellum indicating a wide diffusion of the therapeutic enzyme. Interestingly, our gene transfer strategy restores the enzymatic activity in TS mice in a range of value comparable to that observed in the WT mice.

Results obtained by distribution studies with HSV-TOZ control vector into the CNS of TS mice are consistent with the Hex A activity spreading. We found the presence of X-Gal positive brain cells in both injected and non-injected hemispheres (Fig.44, 45). In particular the presence of positive blue cells within the thalumus, coφus callosum, optic chiasma, indicated that the viral vector distribution mimic the internal capsule architecture²⁷. Cellular phenotype analysis (data not showed) suggested that viral vector fransduced most neurones, asfrocytes and olygodendrocytes in the injection site, but positive cells far from the injection site showed a neuronal phenotype (Fig.45a). Interestingly we found for the first time X-gal positive cells also in the cerebro-dorsal trunlc of the spinal cord (Fig. 45c) confirming the potentiality of this site of injection on the distribution of the transgene in all the CNS. The wide enzyme distribution observed is of particular relevance in that we have previously 17 demonstrated, in vitro, the absence of cross-correction m TS fibroblasts Secreted recombinant Hex A was able to hydrolyze the GM2 ganglioside to GM3 in an in vifro assay, but it did not correct the metabolic defect of TS fibroblasts. It seemed that an additional intracellular alteration, perhaps related to the endocytic sorting of the enzyme does occur in this disease, as reported for other lysosomal storage disorders^33"34. Via brain internal capsule the α-subunit gene was directly delivered within the neural cells overcoming in part the cross-correction mechanism. However, we have to hypothesize that secreted enzyme from transduced cells could be up-taken and correctly reached the lysosome of neighboring cells by specific neural endocytic path-way in vivo ³⁵. X-Gal positive cells in the ventricular regions (lateral ventricle, 111° ventricle and Silvius Aqueduct; Fig. 45b) indicate that the diffusion of the therapeutic viral vector in the contolateral hemisphere is in part via cerebro-spinal fluid flow³⁶. Moreover a cross-correction active mechanism via cerebro-spinal fluid flow may also contribute to the therapeutic effect. A previous work by Consiglio and coworkers , have demonstrated the presence and the distribution of the recombinant arylsulphatase A in all tested brain areas and suggested that the enzyme is transported from the site of injection to the other hemisphere through cerebral commessures. According to this data, Passini and collaborators demonstrated the presence of the β-glucuronidase in the hippocampus area of controlateral hemisphere in MPS VII animals treated with an adeno-associated viral vector encoding for the missing enzyme³⁸.

In this challenge our data represent a novelty since they are the first evidence of the distribution of a therapeutic viral vector in all the CNS and suggest that the anatomic structure of the brain may be a useful tool for therapy for genetic neurodegenerative disorders. In our studies we do not observed adverse effects due to the viral vector, injection side or gene expression and based on the results that we have obtained we fill confident to express the opinion that the same approach can be applied to similar diseases involving an enzyme defect.

MATERIALS AND METHODS

Materials.

The animal model of TS disease was generated by Yamanaka et al. (18) and bred in our laboratory. C57/bl6 mice were from Charles-River, Italy. The fluorogenic substrates 4- methylumbelliferyl-derivative of β-N-acetylglucosaminide (MUG), of β-N-acetylglucosamine-6- sulphate (MUGS) of 4-methyl-umbelliferone, 5-bromo-4-cloro-2-indlyl-βD-galactopiranoside (X-Gal), Nonidet NP40, agarose gel for electrophoresis, the 2,2,2-tribromoethanol, the 2-methyl- 2-butanol, GM2, GM3, and gangliosides mix type III from bovine brain, resorcinol, were from Sigma Chemical Co. Bovine seric albumin and Bio-Rad protein assay reagent were from Bio- Rad Laboratories, DE-52 DEAE-Cellulose was from Whatman Biochemicals. Thin-layer 20x20 CM plates TLC, were form E.Merck A.G. (Darmstadt, Germany). The medium for tissue culture was from Euro-Clone, Celbio Lab., fetal calf serum was from Mascia Brunelli, penicillin/streptomycin was from Gibco BRL. All other reagents were of analytical grade.

Construction of a suitable herpes vector for Hex a-subunit cDNA transfer. The non-replicating HSV-1 viral vector, which expresses the α-subunit cDNA of Hex A, has been created using the Pad recombination system according to the methods of Krisky ²⁶. The α-subunit cDNA of Hex A was cloned under the transcriptional control of ICPO promoter. The expression cassette, containing the cDNA surrounded by UL41 flanking sequences of HSV, has been recombined into UL41 locus of TOZ viral vector (ICP4", 27", 22", UL417LacZ). TOZ vector derived from the replication-deficient T.l, defective for ICP4, ICP27, ICP22 immediate early genes and for UL41 gene where the β-Galactosidase cDNA, under the control of the ICPO IE promoter and flanked by Pad restriction sites (which are not present into the viral genome), was inserted into the vhs (UL41) locus. Potential recombinant viras (TOαHex) was identified by a "clear plaque" phenotype after the X-Gal staining since the insertion of the transgene constract into the viral genome have eliminated the LacZ gene. The TOαHex recombinant vims was purified by three rounds of limiting dilution and verified by Southern Blot analysis for the presence of the α- subunit cDNA of Hex A and for the correct insertion of the named gene in the viral genome.

TS organotypic brain slices preparation and transduction with the HSV-TOaHex. The organotypic brain slice were produced and cultured under standard condition defined in Malgaroli's laboratories. For each experiment, at least three animals and 18 slices were used. After decapitation, 5 months-old TS and wild-type mice (C57/bl6) brains were dissected out into cold Gey's balanced salt solution containing 5 mg/ml glucose. Brain coronal slices (500 μm thick) were cut on a Mcllwain tissue chopper and transferred into membranes of 30 mm Millipore culture inserts with 0.45 μm pore size (Millicell; Millipore, Bedford, MA). Slices were maintained in culture in six- well plates containing 1 ml of medium at 37°C in an atmosphere of humidified 5% CO2. The medium was composed of 50% basal medium with Earle's salts (Invitrogen, Gaithersburg, MD), 25% HBSS (Invitrogen), 25% horse serum (Invitrogen), L- glutamine (1 mM), and 5 mg/ml glucose.

After 24h the medium was changed with new fresh medium and 1 μl either of HSV-TO Hex (PFU= 1 x 10⁶) or of HSV-TOZ (PFU= 1 x 10⁶) was delivered as a drop on the top of oraganotypic slice. Slices were then maintained in culture and collected at different time points (3^th, 7 ^th, 14^th days). Some slices were homogenized in 10 mM Na/phospate buffer pH 6.0 for the determination of the Hex activity some were used to monitor the viral vector distribution. In this case slices were stained with the X-Gal substrate according to the manufacture protocol.

TOalϊex direct injection into the brain internal capsule of TS mice. Five groups of 5 months old animals were injected with two different doses of TOαHex into the internal capsule of the left-brain hemisphere of the TS mice. Mice were anesthetized with 0.02 ml/g body weight of 2,2,2-tribromoethanol and 2-methyl-2-butanol and placed on the Styrofoam platform of a stereotaxic injection apparatus (David Kopf Instruments, Tujunga, California, USA). The skull was exposed by a 10-mm incision in the midline. The injection coordinates for the internal capsule were -0.34 mm to bregma, 1.4 mm mediolateral, and.3.8 mm of depth. These coordinates were chosen in order to minimize vector leakage to the ventricular space. Each injection was 5 μl total, and the injection speed was 0,lμl/min. The injections were carried out using a needle capillary (1.2 mm x 0.6 mm) attached to a Hamilton syringe. The injections were delivered at a rate of O.lμl/min, and the needle was slowly withdrawn after an additional 5 minutes. The scalp was closed by suture.

HSV-TOZ viral vector distribution. One month after injection some mice were sacrificed by cardiac perfusion. The left ventricle was cannulated, an incision was made in the right atrium, and the animals were perfused with 2% paraformaldehyde in PBS until the outflow run clear then the brain included in ornithyne carbamoyl fransferase (O.C.T. TM Compound TISSUE- TEK, Sakamura, The Netherlands) after exposure at 5% - 30% glucose gradient and finally sectioned on a cryostat into 15 μm thick serial sections). β-Gal positive cells were assayed through X-Gal staining (39) . Animal experimentation protocols were approved by the HSR Institutional Animal Care. We collected brain serial sections into four series of glasses (A-B-C- D) so that, for example, section nr.1 on glass B3 was collected immediately after section nr.1 on glass A3 and immediately before of section nr.l on glass C3. In this way, staining only V_Λ of the sections (A) we checked the beta-gal staining distribution (1 section every 60 um) along the whole brain extension. We collected also spinal cord section.

Brain extracts. At sacrifice some mice were decapitated and the brain hemispheres dissected in 4 rosfro-caudal 2,5 mm sections and cerebellum. Organotypic brain slices and brain sections, were homogenized in a Potter Elveheim type homogenizer in lOmM-sodium phosphate buffer, pH 6.0, containing 0.1 % (v/v) Nonidet NP40 detergent and sonicated. The lysates were centrifuged at 12,000 φm Eppendorf microfuge for 20 min and supematants used as tissue extracts for enzyme analysis. All procedures were carried out at 4°C

^-Hexosaminidase activity assay. Enzyme activity was determined by using two fluorogenic substrates: 3 mM MUG or MUGS in 0.1 M-cifrate/0.2 M-disodium phosphate buffer at pH 4.5 ' ' . Fluorescence of the liberated 4-methylumbelliferone was measured on a Perkin Elmer LS3 fluorimeter (excitation 360 nm, emission 446 nm). β-Hexosaminidase isoenzymes analysis. Tissue extracts were analyzed by the ionic-exchange chromatography on DEAE-cellulose

. The chromatography was performed by using 1 ml column equilibrated with 10 mM-Na phosphate buffer, pH 6.0 (buffer A). The flow rate was 0.5 ml/min. Enzyme activity retained by the column was eluted by a linear gradient of NaCl (0.0-0.5 M in 40 ml of buffer A). Finally, the column was eluted with 1.0 M-NaCl in the same buffer. Fractions (1ml) were collected and assayed for the Hex activity.

Gangliosides extraction and quantitative determination. Gangliosides were extracted from mouse brain and cerebellum (10-70 mg of tissue) by using the method of Folch⁴⁰ as modified by Hess and Rolde⁴¹. Briefly, the weighted frozen tissue was thawed, manually homogenized in a 1 ml potter-Elvehjem homogenizer with Teflon pestle and to the mash was added 2:1 chloroform- methanol mixture (v/v). in a volume 20 fold the tissue weight. The obtained homogenate was centrifuged 10 minutes at 5000 φm in an Eppendorf Centrifuge 5415D and the supernatant was recovered. This cmde extract was partitioned by adding 20% of its volume of re-distilled water and mixing. The two phase were separated by centrifugation for 15 minutes at 3000 φm in a microfuge: the upper phase was accurately recovered and the interface rinsed with a few tenths μl of theoretical upper phase (3:48:47 chloro form-methanol- water); the lower phase was re- extracted with a volume of theoretical upper phase containing 0.015 M of KC1 in water. After centrifugation, the recovered upper phase was combined with the first one and dried. The total ganglioside concentration was determined by resorcinol-HCl method according to Svennerholm with 85:15 butyl acetate-butyl alcohol as extiactant⁴² and added to an eppendorf tube containing an equal volume of appropriately diluted upper phase re-suspended in water, mixed well, and heated at 100 °C in a thermo-bloc for 15 minutes. After addition of 1 ml of extiactant and mixing, the samples were cooled on ice water and centrifuged for 3 minutes at 5000 φm. The solvent layer was recovered and measured at 580 nm wavelengths using a Shimadzu UV- Visible Recording Specfrophotometer (UV-160A). The sialic acid concentration was determined by comparison with a standard curve.

TLC analysis of gangliosides. Aliquots of each sample corresponding to 3 μg of sialic acid were re-suspended in chloroform-methanol (1:1 v/v) and spotted on a TLC Silica gel plate previously washed in acetone. The chromatography analysis was carried out, according to Dreyfus⁴³ method, by using three consecutive runs, using different migration solvent, with plates being systematically dried between each run. The ganglioside pattern was quantified by densitometry scanning of the TLC plates stained with the resorcinol-HCl reagent (Svennerholm), using the ID Image Analysis Software (Amersham Pharmacia Biotech)

Other analytical methods - Proteins were measured by the method of Bradford⁴⁴ using the serum bovine albumin as standard. X-Gal staining was carried out according to the method described in the manufacture procedure (Boheringer Lab).

LEGEND TO THE FIGURES

Fig.41 Recombinant HSV-1 non-replicating viral vector containing Hex α-subunit cDNA. a) Recombinant HSV-1 non-replicating viral vector was produced as described in Material and Method; α-subunit Hex cDNA or β-Galactosidase cDNA (LacZ) are expressed under the ICPO promoter. b) Hex activity was assayed towards the synthetic substrate MUGS. One unit (U) is the amount of enzyme that hydrolyses 1 μmol/min of substrate at 37°C ^TS, Tay-Sachs mice; EϋDTS- TOαHex, Tay-Sachs mice transduced with the HSV-TOαHex; I WT, C57 B16 mice; 0 TS-T0Z, Tay-Sachs mice transduced with the HSV-TOZ.

Fig.42 Hex A activity in TS mice brain transduced with HSV- TOαHex viral vector.

Hex activity was assayed towards the synthetic substrate MUGS in brain section exfracts a) and cerebellum b); LI, L2, L3, L4 and RI, R2, R3, R4 are the left and right brain hemisphere sections respectively. D Tay-Sachs mice; S Tay-Sachs mice injected with the lower HSV- TOαHex PFU; ED Tay-Sachs mice injected with the highest HSV-TOαHex PFU; ■ WT, C57/B16 mice; Tay-Sachs mice infected with the HSV-TOZ. c) DEAE-cellulose chromatography analysis of Hex A in a representative mouse brain section exfract: TS, Tay-Sachs mice; TS -TOαHex*, Tay-Sachs mice injected with the lower HSV- TOαHex PFU; TS-TOαHex**, Tay-Sachs mice injected with the highest HSV-TOαHex PFU; WT, C57/B16 mice. Fractions (1ml) were collected and assayed for Hex activity towards the two substrates MUG (•) and MUGS (O).

Fig.43 Decrease of GM2 ganglioside in TS brain sections transduced with HSV-TOαHex viral vector.

Gangliosides TLC pattern of a representative brain section (a) and cerebellum (b) of treated and untreated mice. Lines: std, Ganglioside Standards from bovine brain supplemented with GM2 and GM3; GM2, pure GM2; TS-TOαHex*, Tay-Sachs mice injected with the lower HSV- TOαHex PFU; TS-TOαHex**, Tay-Sachs mice injected with the highest HSV-TOαHex PFU; WT, C57/B16 mice; TS, Tay-Sachs mice.

Densitometric ID Image Analysis Software of the GM2 ganglioside content in brain sections c) and d) and cerebellum c). _-2 Tay-Sachs mice injected with the lower HSV-TOαHex PFU; D Tay- Sachs mice injected with the highest HSV-TOαHex PFU. LI, L2, L3, L4 and RI, R2, R3, R4 are the left and right brain hemisphere sections respectively, C is the cerebellum. Results are expressed as % of removed GM2 ganglioside with respect to the GM2 content of the corresponding TS brain section. Data are representative of three independent experiments.

Fig. 44 HSV-TOZ distribution into the mouse central nervous system. Serial brain sections were produced dissecting animals in coronal, transversal and sagittal orientation. Sections were stained with the X-Gal substrate, a) Coronal brain sections; al-a9: representative coronal serial sections; a4: site of injection (IC, internal capsule) b) Transversal brain sections; bl-b8, representative transversal serial sections c) Sagital brain sections; cl-c8, representative sagittal serial sections DF:dark field; BF: brigth field Fig. 45 HSV- TOZ distribution into the mouse central nervous system: relevant brain area - High magnification of significative brain area: a) AA: tela choroidea of third ventricule, thalamus (pulvinar); Bl', antirior hom of the lateral ventricule; CC, coφus callosum,tela chorioidea of third ventricule, thalamus; DD', Fourth ventricle and cerebellar vermis; EE', genu of the internal capsule; GG', cerebellar peduncle, ependimal channel, pyramis. b) AA', thalamus, internal capsule, posterior thalamic radiation.; BB', white anterior commissure, coφus callosum; DD', putamen, nucleo caudato, anterior arm internal capsule; EE', third ventricle, globus pallidum, internal capsule; FF', mid brain.;GG', hippocampus, controlateral hemisphere; HH', fimbria, controlateral hemisphere. c) A, spinal cord sagittal section; BB', CC, DD', EE', representative spinal cord of the encephalic- thoracic spinal cord section: BB', C3 section, CC, CX section, DD' T3 section, EE', T8 section

References for Example 9

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7. Hepbildikler,S.T., SandhoffR., Kolzer, M.,Proia,R.L. & Sandhoff,K.P. hysiological substrates for human lysosomal beta -hexosaminidase S. J.Biol.Chem. 277, 2562-72 (2001),

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10. Kolter, T., Doering, T., Wilkening, G., Werth, N. & Sandhoff, K. Recent advances in the biochemistry of glycosphingolipid metabolism. Biochem.Soc.Trans., 27, Review, 1087-92 (1999)

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14. Liu Y, et al., Alleviation of neuronal ganglioside storage does not improve the clinical course of the Niemann-Pick C disease mouse. Hum. Mol. Genet., 9, 1087-92 (2000)

15. Eto,Y.& Ohashi,T. Gene therapy/cell therapy for lysosomal storage disease. J.Inherit.Metab.Dis., 23, 293-8 (2000)

16. Desnick RJ. & Schuchman E.H. Enzyme replacement and enhancement therapies: lessons from lysosomal disorders. Nat. Rev. Genet.,3. 954-66 (2002)

17. Martino S., et al. Absence of metabolic cross-correction in Tay-Sachs cells: implications for gene therapy; J Biol Chem.,277, 20177-84 (2002)

18. Yamanaka S., et al. Targeted dismption of the Hexa gene results in mice with biochemical and pathologic features of Tay-Sachs disease. Proc Natl Acad Sci U S A, 91, 9975-9 (1994)

19. Marconi P., et al. Replication-defective HSV vectors for gene transfer in vivo. Proc. Natl. Acad. Sci., USA, 93, 11319-11320 (1996)

20. Burton E.A., Huang S., Goins W.F. &Glorioso J.C. Use of the heφes simplex viral genome to constract gene therapy vectors. Methods Mol. Med.,76, 1-31 (2003)

21. Wolfe D., et al. Engineering heφes simplex viras vectors for CNS applications. Exp Neural. 159(l):34-46. Review (1999)

22. Drory VE, Birnbaum M, Peleg L, Goldman B, 8c Korczyn AD. Hexosaminidase A deficiency is an uncommon cause of a syndrome mimicking amyofrophic lateral sclerosis. Muscle Nerve. 28, 109-12 (2003)

23. Richard W. Orrell, MD; and Denise A. Figlewicz, PhD.Clinical implications of the genetics of ALS and other motor neuron diseases. Neurology,57, 11 (2001)

24. Krisky D.M., et al. Development of Heφes simplex viras replication-defective multigene vectors for combination gene therapy applications. Gene Therapy, 5: 1517-1530. (1998)

25. Krisky D.M., et al. Deletion of multiple immediate-early genes from heφes simplex viras reduces cytotoxicity and permits long-term gene expression in neurons. Gene Therapy, 5, 1593- 1603 (1998)

26. Martino S., Emiliani C,Orlacchio A., Hosseini R. & Stirling, J .L. BetaN- acetylhexosaminidases A and S have similar sub-cellular distributions in HL-60 cells. Biochim Biophys Acta; 1243, 489-95 (1995)

27. Grey H. & Lewis, W.H. Gray's Anatomy of the Human Body. Twentieth Edition,. Philadelphia: Lea & Febiger, 1918; New York: Bartleby (2000)

28. Burton E.A., Hong C.S.& Glorioso J.C. The stable 2.0-kilobase intron of the heφes simplex vims type 1 latency-associated transcript does not function as an antisense repressor of ICPO in non neuronal cells. J Virol. 77, 3516-30 (2003).

29. Loiacono, CM., Taus, N.S., & Mitchell, W.J. The heφes simplex vims type 1 ICPO promoter is activated by viral reactivation stimuli in trigeminal ganglia neurons of transgenic mice. J. Nuro Virology, 9, 336-345 (2003)

30. La Vail J.H., Tauscher A.N., Aghaian E., Harrabi O. & Sidhu S.S. Axonal transport and sorting of heφes simplex viras components in a mature mouse visual system. J. Virol.77, 6117- 26 (2003)

31. Taniike M., et al. Neuropathology of mice with targeted disruption of Hexa gene, a model of Tay-Sachs disease; Acta Neuropathol (Berl), 89, 296-304 (1995)

32. Sango K, et al, Mice lacking both subunits of lysosomal bet -hexosaminidase display gangliosidosis and mucopolysaccharidosis. Nat. Genet.14, 348-52 (1996)

33. Le Borgne R. & Hoflack B. Protein transport from the secretory to the endocytic pathway in mammalian cells. Biochim.Biophys.Acta., 1404, 195-209.(1998)

34. Marks D.L. 8c Pagano R.E. Endocytosis and sorting of glycosphingolipids in sphingolipid storage disease; Trends Cell. Biol.,12, 605-13 (2002)

35. Gosh, P., DahmsN.M. & Komefeld, S.Mannose-6-phosphate receptors: new twists in the tale. Nat.Rev.Mol. Cell.BioL, 4, 202-12. Review (2003)

36. Brinker,T., Ludemann,W., Berens von Rautenfeld,D. & Samii.M. Dynamic properties of lynphatic pathways for the absoφtion of cerebrospinal fluid. Acta Nerapat, 94, 493-498 (1997)

37. Consiglio A., et.al. In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice. Nat. Med., 7, 310-6 (2001)

38. Passini M.A., Lee E.B., Heuer G.G. & Wolfe J.H. Distribution of a lysosomal enzyme in the adult brain by axonal transport and by cells of the rostral migratory stream. J. Neurosci., 22, 6437-46 (2002)

39. Sanes, J.R., Rubenstein, J.L., & Nicolas, J.F. Use of a recombinant retroviras to study post-implantation cell lineage in mouse embryos. EMBO, 5., 3133-42 (1986).

40. Folch, J., Lees, M. & Sloan Stanley,G.H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226,497-509 (1957)

41. Hess, H.H., & Rolde, E. Fluorometric assay of sialic acid in brain gangliosides. J. Biol. Chem., 239, 3215-3220 (1964)

42. Svennerholm, L. Quantitative estimation of sialic acids II. A colorimetric resorcinol- hydrochloric acid method. Biochim. Biophys. Acta., 24, 604-611 (1957)

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44. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-54 (1976).

Example 10 : SK131acZ plasmid construction The following is a scheme for producing the SK131acZ constract. The mutation in the viral UL13 locus creates a new site for the exogenous DNA insertion.

1. , HSV-1 fragment Kpnl 17793-KρnI 28630 was cloned into Kpnl site of ρSP72 plasmid

2. HSV-1 fragment Bglll (then blunt) 25380-KpnI 28630 was cut from pSP72

3. pBSSK- plasmid was cut BamHI (then blunf)-KpnI

4. HSV-1 fragment Bglll (blunt) 25380-KpnI 28630 was inserted into pBSSK- plasmid BamHI (blunt)-KpnI = SKFIIUL13 plasmid

5. SKFIIUL13 plasmid was cut EcoRV (HSV-1 27672)-SphI (HSV-1 27508) cut Sphl - Nral lacZ expression cassette from pcDNA3.1/Hygro/lacZ (available from Invitrogen) and clone into SKFIIUL13 EcoRV-Sphl = SK131acZ vector.

The SK131acZ constract is shown in Figure 46.

Claims

Claims:

1. A vaccine capable of inducing a CD8⁺ immune response in an animal, the vaccine comprising replication-deficient HSV, the HSV comprising polynucleotides encoding a heterologous antigen.

2. A vaccine according to claim 1, wherein the polynucleotides are DNA.

3. A vaccine according to claim 1 or 2, wherein the vaccine comprises at least one further heterologous antigen.

4. A vaccine according to any preceding claim, wherein the HSV is substantially avirulent in the patient.

5. A vaccine according to any preceding claim, wherein the HSV is mutated at at least one of the following loci: UL13, UL41, UL54 locus, USl, US4 , US5 and either or both of the two RSI loci.

6. A vaccine according to any preceding claim, wherein the HSV comprises mutations in HSV genes or loci as defined in any of SEQ ID NOS. 3, 4, 5, 6, 7, 8, 9 or 10.

7. A vaccine according to any preceding claim, wherein the HSV is UL54- US 1 - RS 1 -.

8. A vaccine according to any preceding claim, wherein one or all of the following immediate-early genes, ICP4, ICP22 and ICP27 are mutated in such a way that their expression is prevented or that the protein expressed is non-functional.

9. A vaccine according to any preceding claim, wherein the HSV is the triple mutant ICP4^" ICP22^" ICP27^".

10. A vaccine according to any preceding claim, wherein the HSV is the VHS^" mutant.

11. A vaccine according to any preceding claim, wherein the HSV is replication-deficient due to incoφoration of non-reverting mutations into mandatory viral genes, such that the HSV substantially maintains the immunogenicity of wild-type HSV.

12. A vaccine according to any preceding claim, wherein the polynucleotides encoding the antigen under the control of a promoter and/or an enhancer.

13. A vaccine according to claim 12, wherein the polynucleotides are under the control of an HSV immediate early promoter.

14. A vaccine according to any preceding claim, wherein the antigen is derived from a disease-causing agent.

15. A vaccine according to claim 14, wherein the disease-causing agent is a fungus or a parasite.

16. A vaccine according to claim 14, wherein the antigen is derived from a viras

17. A vaccine according to claim 16, wherein the virus is HIV, and the antigen is derived from the GAG, POL, ENV, REV or NEF HIV proteins or peptide fragments thereof

18. A vaccine according to any preceding claim, wherein the antigen is not derived from SIV, unless the HSV further encodes a cytokine or suicide gene, or both.

19. A vaccine according to claim 14, wherein the antigen is derived from an intracellular bacterium.

20. A vaccine according to claim 14, wherein the antigen is a tumour-associated antigen (TAA).

21. A vaccine according to claim 14, wherein the tumour is a neoplasia, glioma, glioblastoma or Kaposi's sarcoma.

22. A vaccine according to any preceding claim, wherein the HSV is Heφes Simplex Virus, type 1.

23. A vaccine according to any preceding claim, wherein the HSV further encodes a cytokine.

24. A vaccine according to claim 23, wherein the cytokine is an Interlueukin or Colony- Stimulating Factor

25. A vaccine according to claim 24, wherein the cytokine is IL-12 or GM-CSF.

26. A vaccine according to any preceding claim, wherein the HSV further encodes a suicide gene.

27. A vaccine according to any preceding claim, wherein the HSV encodes a suicide gene, an antigen and a cytokine.

28. A vaccine according to claim 26 or 27, wherein the suicide gene is HSVltk gene.

29. A vaccine according to claim 28, which is adapted for administration with the nontoxic prodrug, Ganciclovir.

30. A vaccine according to claim 26 or 27, wherein the suicide gene is the cytosine deaminase gene,

31. A vaccine according to any preceding claim, wherein the vaccine further encodes an angiogenic inhibitor.

32. A vaccine capable of inducing an anti-cancer effect in an animal, comprising replication- deficient HSV whose DNA encodes an angiogenic inhibitor, a cytokine and a suicide gene.

33. A vaccine according to claim 32, wherein the vaccine is capapble of inducing an anticancer CD8⁺ immune response.

34. A vaccine according to claim 33, wherein the HSV also encodes a tumour-associated antigen.

35. A vaccine according to any preceding claim, wherein the HSV further encodes connexin 43.

36. A vaccine according to claim 31 or 32, wherein the angiogenic inhibitor is selected from the group consisting of: angiostatin, kringle 5, and endostatin.

37. A vaccine according to claim 37, wherein the angiogenic inhibitor is a fusion protein.

38. A vaccine according to any preceding claim, wherein the animal to which the vaccine is administered is selected from the group consisting of a mouse, a monkey, or a human.

39. A vaccine according to any preceding claim, wherein the vaccine is formulated together with a pharmaceutically acceptable carrier or diluent.

40. A vaccine according to any preceding claim, wherein the vaccine is adapted for administration by any of the following means: orally, fransdermally, through a mucous membrane, intravenously, infraperitoneally, subcutaneously, intramuscularly or intradermally.

41. A vaccine according to any preceding claim, which is adapted for administration as part of a prime-boost regimen.

42. A vaccine according to claim 41, wherein the regimen is a heterologous prime-boost regimen.

43. A vaccine according to claim 42, wherein the vaccine is administered separately from a further means of inducing an immune response, wherein the further means of inducing an immune response comprises the antigen or a polynucleotide encoding the antigen.

44. A method for screening putative vaccines comprising a candidate antigen in a non-human animal, the method comprising administering said vaccine to said animal and determining whether an immune response is successfully elicited to the antigen by subsequently administering a pathogenic amount of HSV comprising a polynucleotide encoding the antigen.

45. A method according to claim 44, wherein the antigen is first administered by means other than by replication-defective HSV

46. A method according to claim 45, wherein the antigen is first administered by means of a plasmid comprising DNA encoding the antigen.

47. A method according to any of claims 44- 48, wherein the putative vaccine is a plasmid encoding the antigen.

48. A method according to any of claims 44-47, wherein the animal is a mouse.

49. A method according to any of claims 44-48, wherein the antigens are derived from HIV or SIV viral antigens.

50. A method according to claim 49, wherein the antigen is derived from HIV tat, gag, and env.

51. A non-human animal as defined in any of claims 44-50.

52. A vector comprising a mutation position between nucleotides 27508 to 27672 of the UL13 protein kinase encoded by SEQ ID NO. 3, the mutation reducing levels of the HSV ICPO protein.

53. A vector comprising a mutation at position 27508 to 27672 in the UL13 protein kinase encoded by HSV, the mutation reducing phosphorylation of the HSV ICPO protein.

54. A replication-deficient HSV whose DNA encodes hexosaminidase A, or a subunit thereof.

55. A method of expressing Hex A in a subject, comprising administering the HSV of claim 54.

56. A replication-deficient HSV whose DNA encodes at least one neurotrophic factor.

57. A method of expressing an NTF in a subject, comprising administering the HSV of claim 56.

58. HSV as defined in any preceding claim.

59. An HSV genome mutated so that the HSV is a replication-deficient HSV, as defined in any of claims 1-43 and 52-58.