WO2004042031A2

WO2004042031A2 - Safer attenuated virus vaccines with missing or diminished latency of infection

Info

Publication number: WO2004042031A2
Application number: PCT/US2003/035167
Authority: WO
Inventors: Jeffrey Irvine Cohen; Edward Michael Cox; Lesley M. Pesnicak
Original assignee: The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2002-11-05
Filing date: 2003-11-05
Publication date: 2004-05-21
Also published as: AU2003295398A8; AU2003295398A1; WO2004042031A3

Abstract

Viruses having weakened ability to establish and/or maintain latency and their use as live vaccines are described. The vaccines have one or more alterations in genes that provide continued virus replication but that inhibit latency. The vaccine materials and methods for their construction are exemplified with the varicella zoster virus. Deletion of a significant portion from both copies of the varicella zoster gene ORF63 was shown to inhibit establishment of a latent infection from a live vaccine form of the virus. Other desirable viral antigen encoding sequence(s) and/or cytokine genes advantageously may replace deleted genetic material to enhance a desired immunological response. Aspects of the discovery pertain to live vaccines of other viruses, and can provide a variety of vaccines having greater safety.

Description

38163-0196

Jeffrey I. Cohen

Edward M. Cox

Lesley M. Pesnicak

Safer Attenuated Virus Vaccines with Missing or Diminished Latency of Infection

Field

This disclosure relates to recombinant vaccines, and more particularly relates to live vaccines.

Background

Chickenpox is caused by acute infection with varicella-zoster virus (VZV). The virus spreads throughout the body and enters cells of the nervous system. Latent infection occurs and the virus establishes itself in dorsal root and cranial nerve ganglia. The latent virus subsequently can reactivate and present as zoster (shingles). Researchers and pharmaceutical companies have developed chickenpox vaccines but the side effect of shingles due to the live virus establishing a latent infection is still of concern. The ability of a live virus vaccine to enter and maintain a latent infection phase therefore can compromise the safety of live viral vaccines. Any change to the virus that decreases the probability of establishing or maintaining a latent infection can bring significant public health benefits.

Live vaccines are very popular despite the possibility of latent infection. For example, the live attenuated VZV vaccine based on the "Oka virus" (see, U.S. Patent No. 3,985,615) prevents chickenpox but the virus used in this vaccine can enter a latent infection phase in vaccinated individuals and later cause zoster (Sharrar et al., Wise et al.). The Oka virus is attenuated. However the reason for this attenuation and its significance to the latency problem is unknown. Improved vaccines both for humans and for veterinary care, are needed that comprise altered viruses that present less risk of establishing or maintaining a latent infection and therefore less likely to reactivate. Recombinant DNA technology can be used to alter viruses. For example, as shown in Figure 1 , the VZV genome is 124,884 bp in length (line 1 ) and contains unique 38163-0196

long (UL), unique short (US), internal repeat (IR) and terminal repeat (TR) regions (line 2). Of the VZV genes, ORF63 and ORF70 encode the same ORF63 protein sequence. According to several reports, the ORF63 genes are active during latency of VZV (Mahalingham et al 1996; Lungu et al 1998; Debrus et al 1995; Cohrs et al 2000; Kennedy et al 2000; Kennedy et al 2001 ; Sadzot-Delvaux 1995). However, Sommer et al. reported viral "replication required at least one gene copy" and therefore deletion of the ORF63 gene copies would not be helpful for making a live vaccine (J. Virology 75: 17 p. 8224-8239 Sept. 2001 ). [use "reported" not "have shown" since we showed that replication did NOT require IE63] The ORF63 protein is present in virions (Kinchington et al 1995) and is a phosphorylated protein in VZV infected cells (Ng et al 1994). ORF63 protein can be phosphorylated by the VZV ORF47 protein kinase (Kenyon et al 2001) and by casein kinases I and II (Bontems et al 2002; Stevenson et al 1996). However, the VZV ORF47 protein kinase is not required for VZV replication (Heineman et al 1995). The ORF63 protein contains a nuclear localization signal (Stevenson et al 1996) that may be involved in regular functioning of this protein. ORF63 protein for example localizes to the nucleus in infected cells, and to a lesser extent to the cytoplasm (Debrus et al 1995). In contrast, during latent infection ORF63 protein is located in the cytoplasm (Mahalingham et al 1996); during reactivation the protein moves to the nucleus (Lungu et al 1998). Deletion of the nuclear localization signal or mutations of serine and threonine residues (important for phosphorylation of the protein) in the carboxy half of the protein to alanine residues results in increased localization of the protein to the cytoplasm.

Thus, the search for technical improvements to replication competent vaccines needs new paradigms for selecting genetic weak points and making intelligent changes that can alleviate the latent infection problem.

SUMMARY OF THE DISCLOSURE

Discoveries were made that alleviate the shortcomings reviewed above. One embodiment relates to a virus that has been modified so as to impair its ability to establish latency. One embodiment provides an attenuated live virus vaccine for an 38163-0196

animal such as a mammal, the vaccine having impaired ability to establish latency; comprising a recombinant virus that substantially lacks a phosphoprotein gene that is not required for growth of the virus in cultured cells but is important for establishing a latent infection in the mammal. In embodiments, the gene is homologous to the varicella zoster virus and is found in simian varicella virus, feline herpes 1 , equine herpes 1 , equine herpes 4, pseudorabies virus, canine herpes 1 , bovine herpes 1 , Marek's disease virus (of chickens), Laryngotracheitis virus, Meleagrid herpes virus 1 , or herpes simplex virus. Each such gene can be identified as having sequence similarity to the ORF63 sequence of varicella zoster virus and are particularly contemplated for embodiments. In an embodiment, a gene that encodes a protein sequence portion with sequence homology to the conserved region of the ORF gene product of varicella zoster virus is substantially removed or modified to make a live vaccine of lesser latency. Desirably, the protein sequence encoded by the viral gene is at least 10%, 25%, 27%, 28%, 40%, 45%, 50% or at least 56% identical to the conserved region of the ORF63 gene product. In another embodiment, a live vaccine of a virus that infects nervous systems, but which is not listed here, is constructed by altering a gene of the virus that has a region that is at least 10% identical in amino acid sequence to varicella zoster virus the ORF63 gene in another virus not listed here

Another embodiment provides an attenuated live virus vaccine for a mammal, the vaccine having impaired ability to establish latency; comprising a recombinant virus that substantially lacks a gene for a protein encoded by an mRNA, wherein the mRNA is transcribed during normal latency. Yet another embodiment provides an attenuated live virus vaccine for an animal such as a mammal, the vaccine having impaired ability to establish latency, comprising a recombinant virus that substantially lacks a protein encoding a gene, the gene being encoded by a nucleic acid sequence that is complementary to a nucleic acid and is homologous to the ORF63 gene of varicella- zoster virus.

A further embodiment provides an attenuated live virus vaccine that has impaired ability to establish latency, wherein the virus is selected from the group consisting of herpes simplex virus, varicella-zoster virus, Marek's disease virus and pseudorabies virus. Yet a further embodiment provides an attenuated live virus vaccine against a 38163-0196

target virus that has impaired ability to establish latency, comprising a varicella-zoster virus missing substantial parts of the ORF63 genes and substituted therefore with at least one gene that encodes at least one protein epitope of the target virus.

Another embodiment provides a method for making an attenuated live virus vaccine targeted against a virus and having impaired ability to establish latency, comprising selecting a virus having one or more copies of a dominant latent phase transcribed gene, altering or removing a substantial part of each copy of the latent phase transcribed gene to form an attenuated virus having impaired ability to establish latency, and culturing the attenuated virus to an amount suitable for a vaccine. Desirably, the latent gene has a region that is at least 10%, 25%, 27%, 28%, 40%, 45%, 50% or at least 56% identical to the conserved region of the ORF63 gene product. The gene may be selected on this basis. Yet another embodiment provides a method for making an attenuated live virus vaccine targeted against a virus and having impaired ability to establish latency, comprising selecting a virus having one or more copies of a dominant latent phase transcribed gene, altering or removing a substantial part of each copy of the latent phase transcribed gene to form an attenuated virus having impaired ability to establish latency, and culturing the attenuated virus to an amount suitable for a vaccine. In a desirable embodiment this method selects "one or more copies of an immediate-early gene" by determining which gene of the virus has homology to the ORF63 gene of varicella zoster virus. Other embodiments will be appreciated from a reading of the specification.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. depicts the construction of recombinant VZV with a deletion in ORF63. The four cosmids used to produce infectious virus span the VZV genome (lines 3 and 4). Cosmid Mstll63D (line 5) is deleted for codons 24 to 268 of ORF63 (with a frameshift after codon 268).

Fig. 2. shows the conserved regions of varicella zoster virus and nine other viruses and their comparison with a consensus sequence. 38163-0196

DETAILED DESCRIPTION OF THE DISCLOSURE

It was discovered that modification, particularly by deletion, of a gene encoding the phosphorylated protein found in the cytoplasm during latency of a virus creates an altered virus that can replicate in vitro but has markedly diminished ability to establish a latent infection. Moreover, live vaccines that contain such modified virus should be safer than regular unmodified live virus vaccines. In one embodiment, substantially all (at least 30%, 40%, 50%, 75%, 85%, 90% or more and particularly at least 50%) of the protein coding sequence of all copies of the gene used in the virus or virus vaccine is deleted. In a desirable embodiment, the gene is selected by examination of homology with a conserved region of varicella zoster virus ORF63 gene product. Advantageously, the region is at least 10%, 25%, 27%, 28%, 40%, 45%, 50% or at least 56% identical to the conserved region of the ORF63 gene product. The gene may be selected on this basis.

In another embodiment, a section of the gene that encodes a nuclear localizing region is substantially missing. In another embodiment, a KRRR nuclear localizing sequence is missing. In yet another embodiment, the gene is modified such that phosphorylation of the protein is reduced and the protein has less tendency to enter the nucleus. In yet another embodiment, one or more serines and/or theonines that become phosphorylated are altered to another amino acid that cannot become phosphorylated. In yet another embodiment, two or more of these features are combined. In yet another embodiment, the gene is mutated to decrease the ability of the final protein product from entering the nucleus.

In yet another embodiment, the viral kinase from ORF47, as described by Kenyon et al. is modified or substantially deleted to remove its kinase activity. In yet another embodiment, both the ORF47 kinase gene and the ORF63 genes are modified. In another embodiment where an ORF63 gene homologue from another virus is substantially deleted or modified, a viral kinase in that other virus also is modified or substantially deleted to remove its kinase activity.

In another embodiment, a vaccine is prepared from one or more of the viruses described herein, by combining one or more adjuvants with the virus or viruses in a form suitable for administration. In another embodiment, a vaccine is prepared from one or 38163-0196

more of the viruses described herein, by combining one or more excipients with the virus or viruses in a form suitable for oral delivery. In another embodiment, a vaccine is prepared from one or more of the viruses described herein by forming a sterile suspension of the virus or viruses suitable for administration. In an embodiment, the vaccine is prepared in a form suitable for injection.

It was discovered using the varicella zoster virus genome as a model system that, contrary to knowledge in the field, a gene that encodes a phosphorylated protein, or other homologous protein as described herein, is important for establishing a latent infection (i.e. absence of the gene lowers the frequency of latent infection by at least 50%, and more preferably by at least 75%, 85%, 90%, 95%, 98%, 99% or by even 100%) but is not required for replication in vitro. In a desirable embodiment, each copy of such gene is partly, mostly, or all deleted. In another embodiment, the gene cop(ies) are modified by mutation. In many embodiments one or more copies of a gene are modified (by deletion, mutation or both) the same way and for brevity the ORF63 gene is mentioned by name. That is, the term "ORF63 gene" means both ORF63 and ORF70. A virus having a deletion of the ORF63 gene preferably has the same deletions in both the ORF63 and ORF70 gene copies. A deletion generally leads to ineffective protein, and exact correspondence between both genes ORF63 and ORF70 is desired in many embodiments. Live virus vaccines having impaired propensity to develop a latent infection can be made from a variety of viruses, genes and gene modifications as described herein. Desirable methods for making a virus are described next. Changes to a varicella zoster virus strain are used to exemplify the invention for the chickenpox vaccine.

Gene Selection

A variety of genes are expressed during the latency period of a viral infection. It was discovered during study of the varicella zoster system that a gene that is transcribed with high efficiency and in many studies is the most abundantly transcribed gene (compared to other genes) during viral latency has particularly useful properties in embodiments of the invention. For a given virus or virus strain, such a gene may be 38163-0196

selected using a routine procedure such as the measurement of viral mRNA during latency to determine which viral mRNA is abundant or most abundant.

In yet another embodiment, a gene is selected that has negative transcription regulatory activity. Methods for determining regulatory activity are known. For example, Bontems et al. describes negative transcription regulatory activity found for the ORF63 gene. In another embodiment, a gene that is not required for growth or replication but which is involved in latency is selected for modification. The gene may be determined by preparing mutants such as premature termination or deletion mutants and determining their effects on replication (e.g. synthesis of viral gene(s) or propagation of virus).

In an embodiment of the invention, a gene is chosen having a nucleic acid sequence that shares at least 25% homology with the varicella-zoster ORF63 gene. In another embodiment, the chosen gene shares at least 50%, 66%, 75%, 80%, 85%, 90%, 95% or more homology with the varicella-zoster ORF63 gene. Percent homology is determined by placing the protein coding sequence of the "test" gene to be compared in a best fit alignment next to the protein coding portion of the ORF63 gene. The total number of identical nucleic acids that line up is divided by the total number of nucleic acids of the test gene protein coding region. Homology is evaluated by computer using any of a variety of sequence comparison programs known in the art. Examples of such programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA,

TFASTA, and CLUSTALW as reviewed in U.S. No. 6,472,517. In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST") which is well known in the art.

In another embodiment, a gene homologous to the ORF63 gene is selected from another virus. Homologous genes are known and more are expected to be discovered. For example, the US1 gene of Marek's disease virus as described by Parcells et al., and the analogous gene from the pseudorabies virus as described by Fuchs et al. may be selected for vaccines against those respective viruses. Of course, genes that are less homologous also may be selected. For example, a gene that encodes a protein that becomes phosphorylated may be selected, optionally with a second gene that encodes a viral kinase that acts upon the protein that becomes phosphorylated. Most 38163-0196

advantageous in this respect is the selection of a phosphorylated protein that has negative transcription regulatory activity.

Table 1 shows representative genes having suitable amino acid coding homology to the ORF63 gene of varicella zoster virus that may be selected. The definition of homology is well known to a skilled artisan and can be, for example seen by example on the NCBI conserved domain database. See Fig. 2, which illustrates some homology results that compare a consensus sequence (SEQ ID NO: 1) with the varicella zoster virus ORF63 conserved sequence, (SEQ ID NO: 2), feline herpes sequence (SEQ ID NO: 3), Marek's disease virus sequence (SEQ ID NO: 4), Laryngotracheitis virus sequence (SEQ ID NO: 5), Meleagrid herpes virus 1 sequence (SEQ ID NO: 6), bovine herpes 1 sequence (SEQ ID NO: 7), equine herpes sequence (SEQ ID NO: 8), pseudorabies virus sequence (SEQ ID NO: 9), and simian varicella virus sequence (SEQ ID NO: 10). Other viruses known and to be discovered, particularly that infect ganglia of the nervous system have or are expected to have analogous genes that expressly are intended as embodiments of the invention. Desirably, a viral genome is compared with the consensus sequence shown in Fig. 2 (or a related sequence) and a homology fit of typically at least 10% identity, when found, indicates the presence of a gene that is analogous to the ORF63 gene described herein.

Table 1

VZV ORF63 Protein Homologies with Herpesvirus

Identical Conserved

Virus Animal Amino Acids Amino Acids

Simian varicella virus Rhesus 77/134 (57%) 95/134 (70%)

Feline herpes 1 Cat 77/134 (57%) 95/134 (70%)

Equine herpes 1 Horse 54/120 (45%) 63/120 (52%)

Equine herpes 4 Horse 53/120 (44%) 65/120 (54%)

Pseudorabies virus Pig 46/105 (43%) 56/105 (53%)

Canine herpes 1 Dog 41/96 (42%) 54/96 (56%)

Bovine herpes 1 Cow 37/78 (47%) 44/78 (56%)

Marek's disease virus Chicken 26/92 (28%) 35/92 (38%) 38163-0196

Laryngotracheitis virus Bird 25/86 (29%) 33/86 (38%)

Meleagrid herpesvirus 1 Turkey 22/84 (26%) 32/84 (38%)

Herpes simplex 1 Human 30/85 (35%) 38/85 (44%)

Herpes simplex 2 Human (similar to HSV-1)

A gene also may be selected by virtue of its dual functions during maintenance of latency and during replication. A gene that is needed for replication and has a role in replication but that also has another function during latency is particularly desirable. Also desirable is a gene whose protein product is found mostly in the nucleus during replication but found mostly in the cytoplasm during latency.

A given gene's effect on establishing or maintaining latency may be determined by infecting animals with virus that does not express the gene and determining the amount of viral DNA in the animal at least one month after the infection.

In a related embodiment a gene that is required for growth or replication is mutated to allow growth or replication while interfering with the establishment or continuance of latency. In practice, a large number of mutations may be made to a gene that is suspected to have a strong effect on latency (by virtue of its transcription during latency) followed by a screening assay to select for normal or altered (slower) growth or replication. Altered genes can be further selected for effects on latency. A latency assay may be used to determine the effect on latency as is known to a skilled artisan. In yet another embodiment, a gene that encodes a tegument protein is selected. In yet another embodiment, a gene that generates the most abundant transcripts during latency is selected. In yet another embodiment, a gene that encodes a viral protein that becomes phosphorylated and which generates high levels of transcripts during latency is selected. In each case, the cited gene parameters may be determined by assay of, for example, gene activity by measuring transcribed RNA in the nucleus, measuring mRNA in the cytoplasm, measuring synthesized protein in the cytoplasm, and/or by information from literature reports. The field of molecular virology has advanced to the level where such assays involve only routine experimentation. In the varicella zoster virus example, a desirable gene is ORF63 (both ORF63 and ORF70, the term "ORF63" means both copies of this gene and is used for 38163-0196

convenience). In another embodiment, the viral kinase that phosphorylates this gene and which is not essential for replication, VZV ORF47 (or a gene with at least 10% homology or more to ORF47) is selected. In an advantageous embodiment both ORF63 and ORF47 are selected for modification. Another suitable gene is a gene that has negative transcriptional activity, i.e. that represses gene expression. Such a gene in particular is a preferred candidate to mutate for a vaccine. By way of example, gene ORF63 is expressed in latency, and can turn off other viral genes and may allow latency with limited virus gene expression. It was found experimentally that deletion of this gene reduced the ability of the virus to go latent. Likewise, other genes associated with negative transcriptional activity, either known or to be discovered in the future are good candidates for selection.

A suitable gene also may be selected by determining which viral gene encodes a protein that becomes phosphorylated and/or that is transcribed during latency. In another embodiment, the gene is a viral kinase that phosphorylates a viral protein and that may affect the subcellular distribution of the viral protein. In yet another embodiment, the gene encodes a protein that specifically binds a molecule on or in a particular host cell that the virus inhabits. The term "specifically binds" in this context means that the protein binds to a molecule that is at least ten times more available or present in the host cell compared to a non-host cell, or has at least ten times higher binding affinity to its binding partner in or on the host cell compared with a non host cell. In other embodiments combinations of these conditions are used to select a desirable viral gene.

A variety of other strategies can be used to select a viral gene. A virus that preferentially infects a particular cell type or group in a latent manner often has at least one such gene that has evolved for that particular cell type or group. By modifying the gene(s) as described here, viral replication in vitro may still occur but the virus will have no or diminished ability to establish or maintain latency. Thus, embodiments of the invention, which flow from this understanding include other viruses and genes besides the varicella zoster virus and the ORF63 gene, as will be appreciated by a skilled reader. 38163-0196

Gene Modification

The selected viral gene is modified to remove or inhibit its natural effect in establishing or maintaining latency. For example, one or more base pairs are deleted or otherwise modified and the resulting virus used in formulating a live vaccine having less tendency to establish or maintain a latent infection. In one embodiment the gene lacks a transactivating function for regulating viral infection but is necessary for interacting with the host cell. Accordingly, by removing the activity of this gene, latency can be affected specifically.

Any modification that affects function of the selected gene is useful for decreasing or eliminating the ability of the virus to establish or maintain latency. Most preferably at least 1 , 2, 3, 5, 10, 25, 50, 100, 200, 300, 400, 500 or more base pairs are removed. For the ORF63 gene, advantageously at least a portion of the carboxyl terminal end is removed that contains the RKKK sequence or other sequences that are necessary for proper localization or functioning of the gene product. By removing large blocks of nucleic acid from all copies of the gene in a virus, the modified virus will have less tendency to revert to wild type characteristics. In another desirable embodiment mutation of one or more base pairs is carried out by a regular procedure as is known in the art. For example, PCR-based mutagenesis has been used for mutational analysis of the ORF63 gene, as described by Sommers et al. In a particularly desirable embodiment relevant to phosphorylated proteins such as ORF63, one, or more preferably multiple amino acids are altered to other amino acids and thereby blocks one or more phosphorylations by a kinase. In an embodiment, one or more serines/threonines are altered to prevent or limit phosphorylation by a serine-threonine dependent kinase. By way of example, for the ORF63 protein, at least one, two, three, four, five, six, seven, eight, nine or ten of the serine and threonine amino acids located at Ser-150, Ser-165, Ser-181 , Ser-186, Thr-171 , Ser-173, Ser-185, Thr-201 , Thr-244 and Ser-224 may be converted to other amino acid(s) such as alanine by alternation of the ORF63 and/or ORF70 nucleic acid sequence(s). In another embodiment, other amino acids not mentioned here but that become phosphorylated are modified. 38163-0196

In yet another embodiment, the conformation of the protein (for example, the carboxyl terminus of the ORF63 protein) is modified to inhibit phosphorylation by mutation of the gene. In yet another embodiment, the conformation of the protein that is involved with intermolecular interaction (for example the portion of ORF63 protein that interacts with ORF62 product of varicella zoster virus, as described by Sommers et al.) is modified to inhibit the interaction and thereby interfere with establishment or maintenance of latency. A skilled artisan can determine which portion of a selected protein is involved with intermolecular activities by routine assays. For example, binding assays can be carried out with normal and with mutated forms to determine which mutations decrease binding and thereby inhibit normal protein functioning.

In another embodiment, the nuclear localization signal (if present) of the selected protein is modified or deleted. For the ORF63 genes, the RKKK portion and surrounding amino acids (at least one, two or more on each side) most desirably are deleted. In another embodiment, one or more amino acids are modified. For example, the highly basic RKKK segment may be converted into a less basic or even an acidic segment by changing one or more amino acids to a neutral or acidic form.

In yet another embodiment, multiple small deletions (of between one and 1000) of bases are made in the selected protein encoding gene that includes different portions of the protein. Such deletions may, for example, encompass regions or amino acids that become phosphorylated. In another embodiment, deletions are combined with individual mutations that serve to eliminate phosphorylation sites. Most preferably a virus "substantially lacks" the selected protein gene which has been "substantially deleted." That is, at least 30%, more preferably 50%, and even more preferably at least 60%, 70%, 80%, 90% or 95% of the protein encoding section of the selected gene is deleted. In practice, any portion that affects gene activity may be deleted and have an effect, according to an embodiment of the invention. A "substantial" region of only 8%, 7% or even 5% may be deleted and cause an effect. In practice, however, desirably more than such minimum portion should be deleted.

In a most desirable embodiment one or more deletions are made, which removes protein activity while, at the same time, providing space for adding one or more protein antigen encoding genetic sequences. In a related embodiment a selected viral gene is 38163-0196

at least partly deleted and replaced with sequence(s) that encodes one or more epitopes of another viral protein. A viral protein that is synthesized to a high level and that is packaged into the virus, is particularly desired for this embodiment. For example, enough of a protein that forms a viral capsid (or envelope glycoprotein) may be added in place of the deleted portion in-frame with a promoter and initiation codon to allow expression. A skilled artisan may engineer or select a protein that becomes packaged in the regular capsid (or viral envelope). In a related embodiment a promoter or other regulatory sequence is chosen to allow low enough expression as to avoid formation of unstable virus structures. In yet another embodiment, a cytokine gene is inserted into the site of deletion of a viral genome or even elsewhere in the genome of a viral deletion mutant to improve the immunogenicity of the virus. Such replacement and the effects on immunogenicity are known and readily carried out. For example, insertion of IL-2 into HSV (Ghiasi et al 2002) enhanced the cellular immune response to this virus. Insertion of IL-6 into fowlpox virus (Leong et al 1994) enhanced the systemic and mucosal antibody responses to this virus, and insertion of IL-5 into vaccinia (Ramsay et al 1994) improved the IgA antibody response to this virus. Other cytokine genes may be used as well. Advantageously one or more cytokine genes replaces one or more deletions of a virus used to make a live virus vaccine. The term "ORF" means open reading frame. For example, ORF63 refers to the gene ("ORF63 gene"), its transcript ("ORF63 transcript" or "ORF63 RNA"), or the protein encoded thereby ("ORF63 protein") in different contexts as used in this disclosure and understood by those skilled in the art. Because ORF70 encodes the same ORF63 gene, ORF63 is used to mean both copies of the gene as used throughout this specification unless mentioned otherwise. Other terms are used in their regularly understood meaning. The term "vaccine" as used herein refers to any compound, molecule, agent, or combinations thereof that is capable of establishing immune responsiveness in a user or a patient to an antigenic substance, such as a virus or another pathogen or exogenous stimulus. A vaccine typically contains inactivated antigenic substance such as the inactivated pathogen. The terms Notl A, Notl BD, Mstll A, and Mstll B, as used herein, refer to various cosmids utilized in 38163-0196

transfection experiments to produce desired mutations in various embodiments. These constructs can be made from known methods.

Formation of Live Virus Vaccines A wide variety of vaccines can be prepared that incorporate one or more viruses as described herein. In desirable embodiments a live virus is cultured in cells and harvested to produce a vaccine by combining virus particles with one or more stabilizers in a form that does not extensively denature the virus. A variety of culturing techniques, concentration techniques, stabilizers and vaccine formulations are known and may be adapted to the particular virus type used, as are familiar to the skilled artisan.

Generally, preparation of a stabilized live virus vaccine begins with the culture of the virus in a human diploid cell. The virus culture may be purified, for example, by centrifugation, to obtain a more purified virus fraction. Generally a vaccine stabilizer is then added to the virus fraction, and the mixture diluted. The final desired virus concentration typically will be about 10 to 100,000 PFU (plaque-forming units) and more typically 100 to 10,000 PFU or more of virus content per dose of the stabilized live vaccine. Aliquots of the thus prepared live vaccine may be tested for safety, effectiveness and homogeneity, to confirm eligibility as a vaccine. •

An important factor in vaccine formulation is the stabilizer, as vaccine potency may be adversely affected by concentration and storage conditions. Stabilizers often used for live vaccines of viruses such of measles, rubella and mumps generally include one or more saccharides, amino acids, sugar alcohols, gelatin and gelatin derivatives, to stabilize the virus and, in many cases keep the virus from denaturing during a concentration step. In an advantageous embodiment a recombinant virus described herein may by formulated into a vaccine using a stabilizer or other additive that includes native or recombinant serum albumin for this purpose. U.S. No. 6,210,683 provides representative conditions for this embodiment of the invention. U.S. Nos. 5,728,386, 6,051 ,238, 6,039,958 and 6,258,362 also contain details for stabilizers and methods for more gentle treatment of live virus vaccines. Each of these disclosures, and particularly those portions that describe stabilizer compositions and stabilizing methods are specifically incorporated by reference in their entireties. 38163-0196

After preparation with a stabilizer, the vaccine may be, for example, stored as a lyophilized vaccine, a lyophilized mixed vaccine, a liquid vaccine or a liquid mixed vaccine. Methods for forming these are known. Typically, a lyophilized vaccine is prepared by lyophilizing the vaccine in a vial or an ampule having a volume of about 3 to 30 ml, tightly sealing and storing at a temperature of 5 degrees Centigrade or less. The stored preparation vaccine typically is used according to instructions attached thereto, as a product insert or a notice on the vial or other container. In many cases, a lyophilized vaccine is re-constituted by addition of sterile distilled water before use, and the resultant solution is inoculated by hypodermic injection in an amount, for example, of 0.5 ml per dose. In another embodiment, the vaccine is provided orally.

In an embodiment a modified virus prepared as described herein is less stable than the wild type from which the virus is derived and a more gentle stabilizer is used. For example, a modified virus that contains one or more added genes that encode other antigens may have a larger amount of genetic material than usual and may be more sensitive to denaturation. In one related embodiment the free divalent cation concentration of the stabilizer or final vaccine formulation is reduced, for example, by the addition of EDTA to counteract this instability. U.S. No. 6,039,958 for example provides instructions for lowering the concentration of calcium and magnesium in preparations of live virus vaccines. Other techniques described in the literature that alleviate instability and/or facilitate combinations of multiple viruses in the same formulation may of course be used and are contemplated.

Various embodiments of this disclosure are further described by the following examples, which are illustrative of the embodiments but do not in any manner limit the same. Various molecules, reagents, and instruments referred to in the examples and throughout this disclosure are only representative of the larger intended scope. Equivalent or alternative products as understood by immunologists, virologists, and molecular biologists may be employed to carry out the various embodiments.

Examples 38163-0196

In the examples, Southern blotting and immunoblotting were carried out as follows. Southern blotting assays were performed by isolating VZV DNA from nucleocapsids, cutting the DNA with BamHI, and fractionating the DNA on 0.8% agarose gels. After transferring DNA to a nylon membrane, each blot was probed with a [³²P]dCTP-radiolabeled probe that corresponds to the 4.65 kb BamHI J fragment of the VZV genome that contains the ORF63 gene.

Immunoblotting assays were performed by preparing protein lysates of VZV infected cells, fractionating the proteins on polyacrylamide gels, transferring the proteins to membranes, and incubating the blots with rabbit antibody to ORF63 protein (Ng et al J Virol 1994;68:1350-1359) or mouse monoclonal antibody to glycoprotein E (IgE, Chemicon, Temecula, CA). The membranes were then incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse antibody and imaged using enhanced chemiluminescence (Pierce Chemical Company, Rockfield, IL).

Example 1

Construction of cosmids and transfections. Cosmids VZV Not I A, Not I BD, Mst II A, and Mst II B are derived from the Oka vaccine strain and encompass the entire VZV genome. Upon transfection into cells these cosmids produce infectious virus (Cohen and Seidel, 1993). The short internal repeat of the viral genome contains a first ORF63 gene copy and the short terminal repeat of the genome contains the second copy ORF70, as shown in Fig 1. Both the ORF63 and ORF70 gene copies are located in a Sfi I fragment extending from VZV nucleotides 109,045 to 120,854 (Davison and Scott 1986). To clone the VZV Sfi I fragment, two Sfi I sites were inserted into Bluescript SK+ (pBSSK+) (Strategene, LaJolla, CA). The plasmid pBSSK+ was modified to include the first Sfi I site by cutting it with Spe I and Sma I; and, a double- stranded DNA, derived from CTAGTTGGCCGCGGCGGCCTCCC (SEQ ID NO: 11) and GGGAGGCCGCCGCGGCCAA (SEQ ID NO:12), was inserted into the site. This Sfi I site is compatible with the Sfi I site at VZV nucleotide 109,045. A second Sfi I site, compatible with the Sfi I site at VZV nucleotide 120,854, was created by digesting the modified pBSSK+ plasmid with EcoRI and Hindlll; and, a double-stranded DNA, derived from AATTGTAGGCCGCCGCGGCCA (SEQ ID NO: 13) and 38163-0196

AGCTTGGCCGCGGCGGCCTAC (SEQ ID NO: 14), was inserted into this site. The resulting plasmid was cut with Sfi I; and, the Sfi I fragment from cosmid Mstll A which contains ORF63 and ORF70 (VZV nucleotides 109,045 to 120,854) was inserted to create plasmid pBSSK+Sfil. A modified plasmid pBSSK÷ was constructed in which the NgoM I and Cla I sites were ablated. First, plasmid pBSSK+ was cut with NgoM I and a double-stranded oligonucleotide, derived from CCGGAGAGCCTAGGAGACT (SEQ ID NO: 15) and CCGGAGTCTCCTAGGCTCT (SEQ ID NO: 16), was inserted into the site. The resulting plasmid, pBSSK+Avrll, contains an Avr II site. Plasmid pBSSK+Avrll was cut with Cla I and Kpn I and a single-stranded oligonucleotide-CGGTAC-was inserted into this site to create plasmid pBSSK+AvrlldCla.

The ORF63 gene from plasmid pBSSK+Sfil was inserted into plasmid pBSSK+AvrlldCla. Plasmid pBSSK+Sfil was cut with Spe l-located in pBSSK+ adjacent to the site of the VZV insert-and EcoR l-located at VZV nucleotide 117,034- and the resulting 3.9 kb fragment was inserted into the Spe I and EcoR I sites of pBSSK+AvrlldCla to create plasmid ES. The ORF70 gene from plasmid pBSSK+Sfil was then inserted into plasmid pBSSK+AvrlldCla. Plasmid pBSSK+Sfil was cut with Avr ll-located at VZV nucleotide 112,853 and Hind Ill-located in pBSSK+ adjacent to the site of the VZV insert, and the resulting 3.9 kb fragment was inserted into the Avr II and Hind III sites of pBSSK+AvrlldCla to create plasmid AH.

The ORF63 and ORF70 genes were then deleted from plasmids ES and AH, respectively. Plasmid ES was cut with Hpa I (VZV nucleotide 110,649) and Nae I (VZV nucleotide 111 ,385) and the large blunt ended fragment was ligated to itself. Similarly, plasmid AH was cut with Hpa I (VZV nucleotide 119,247) and Nae I (VZV nucleotide 118,511) and the large fragment was ligated to itself. Mutated plasmid ES was cut with EcoR I and Spe I; and, the ORF63-deleted fragment was inserted in place of the wild- type EcoR l-Spe I fragment into plasmid pBSSK+Sfil. The mutated plasmid AH was cut with Avr II and Hind III, and the ORF70-deleted fragment was inserted in place of the wild-type Avr ll-Hind III fragment of the ORF63 deleted plasmid pBSSK+Sfil. Finally, the ORF63-and-ORF70-deleted plasmid pBSSK+Sfil was cut with Sfi I and inserted in place of the wild-type Sfi I fragment of cosmid MstllA. The resulting cosmid, termed 38163-0196

MstllA-63D, has identical deletions of ORF63 and ORF70 that result in loss of codons 24 to 268; the remaining codons (269-278) are out of frame. Two independently derived clones of plasmids ES and AH were obtained and subsequent reactions were carried out in parallel. Thus, two cosmids, MstllA-63DA and Mstll63-DB, were independently derived.

Recombinant VZV was produced by transfecting human melanoma (MeWo) cells with a plasmid that expresses VZV ORF62 protein and cosmids VZV Notl A, Notl B, Mstll B, and Mstll A or Mstll A-63D. To rescue the deletion in ORF63, a plasmid containing ORF63 and its promoter was constructed. A 5.6 kb Bel I fragment of VZV (nucleotides 106,592 to 112,215) was inserted into plasmid pMAL-p2X (New England Biolabs, Beverly, MA) between nucleotides 445 and 2,645. The resulting plasmid was cut with Bell and the VZV fragment was gel purified.

Example 2 This example describes the deletion of both copies of the ORF63 gene and construction of a rescued virus in which ORF63 is replaced. Cosmids MstllA-63DA and MstllA-63DB were constructed, as described in Example 1 and used to construct viruses with both gene copies ORF63 and ORF70 substantially deleted. Amino acids 24 to 268 of ORF63 and ORF70 were deleted in MstllA-63DA and MstllA-63DB, as shown in Fig. 1. Melanoma cells were transfected with plasmid pCMV62. Cosmids VZV Notl A, Not I BD, Mstll A, and Mstll B showed cytopathic effects. Recombinant Oka VZV (ROka) was produced following transfection. Transfection of cells with plasmid pCMV62 and cosmids VZV Notl A, Not I BD, and MstllA-63DA resulted in cytopathic effects after transfection; the resulting virus was termed ROka63DA. Transfection of cells with plasmid pCMV62 and cosmids VZV Notl A, Not I BD, and MstllA-63DB resulted in cytopathic effects after transfection; the resulting virus was termed ROka63DB. These results indicate that ORF63 is not required for virus replication in vitro.

A rescued virus, in which the two deletions in ROka63DA were repaired, was produced by co-transfecting melanoma cells with 0.5 ug ROka63DA DNA and 1.5 ug of a VZV Bell fragment that contains ORF63 with flanking regions. At the eighth day after 38163-0196

transfection cytopathic effects were observed, and the resulting virus was passaged in melanoma cells. After five rounds of plaque purification, in which virus clones are isolated, a "rescued" virus was isolated and termed ROka63DRA.

To verify that ROka63A, ROka63B, and ROka63DRA had the expected genome structures, Southern blotting was performed using virion DNAs. Digestion of viral DNA from VZV ROka or ROka63DRA with BamHI showed a 4.65 kb fragment, while digestion of ROka63DA or ROka63DB showed a 3.91 kb fragment. Thus, the ORF63 deletion mutants had the expected deletions.

Immunoblotting was performed to confirm that intact ORF63 was not expressed in cells infected with ORF63 deletion mutants. Lysates from cells infected with VZV ROka or ROka63DR contained a 45kDa ORF63 protein, while lysates from cells infected with ROka63DA or ROKa63DB did not contain this protein. As a control, the lysates from all four virus infected cells were shown to contain similar levels of VZV gE. Therefore, cells infected with the ORF63 deletion mutants did not express ORF63 protein.

Example 3

Removal of ORF63 activity affects virus growth in cell culture. VZV growth was determined as follows. Flasks of melanoma cells were infected with 100 to 200 PFU of parental VZV, ORF63 deletion mutants, or rescued virus. At one, two, three, four, and five days after infection, cells were treated with trypsin and serial dilutions made to infect melanoma cells. One week after infection, the cells were fixed and stained with crystal violet. Virus titers were determined by counting infection-associated plaques.

The peak titer of VZV ROka63DA and ROka63DB was about ten-fold less than VZV ROka. In contrast, ROka63DR, in which the ORF63 deletions were repaired, replicated to peak titers that were similar to those of the parental virus, indicating that ORF63 helps sustain normal virus growth.

Example 4 Gene ORF63 of varicella zoster is critical to establish latent viral infection. Four to six week old female cotton rats were inoculated intramuscularly along both sides of 38163-0196

the spine with VZV-infected melanoma cells containing 1.75 x 10⁵ or 3.0 x 10⁵ PFU of parental or ORF63 deletion mutant viruses. The animal protocol used has been described previously (Sato et al. 2002). Six weeks later, the animals were sacrificed and their lumbar and thoracic ganglia removed and pooled from each animal. DNA was isolated from the pooled samples and PCR was performed using 500 ng of pooled ganglia DNA and VZV primers that correspond to the ORF21 gene of VZV (Brunell et al 1999). Serial dilutions of cosmid VZV Notl A, which contains ORF21 , were added to 500 ng of ganglia DNA from uninfected cotton rats and PCR was performed to generate a standard curve for estimating the numbers of latent viral DNA copies. The PCR products were resolved by electrophoresis and Southern blotting with a radiolabeled probe to ORF21 (Brunell et al 1999). Numbers of viral DNA copies were estimated by densitometry using a phosphorimager. The lower limit of detection was 10 copies of viral DNA when mixed with 500 ng of uninfected ganglia DNA.

Initially, animals were inoculated with 1.75 x10⁵ PFU of virus in 50 μl at 6 sites on each side of the spine intramuscularly. VZV DNA was detected in ganglia from 4 of 13 animals inoculated with VZV ROka and 0 of 10 animals infected with ROka63DA. The geometric mean number of VZV copies from PCR-positive ganglia of animals infected with VZV ROka was 48, whereas none of the animals inoculated with VZV ROka63D had detectable VZV in their ganglia. In a comparison study, animals were similarly inoculated with 3.0 x10⁵ PFU of virus. VZV DNA was detected in ganglia from 8 of 12 animals inoculated with VZV ROka and 2 of 14 animals infected with ROka63DA. Accordingly, in total, VZV DNA was detected in 12 of 25 animals infected with VZV ROka and 2 of 24 infected with ROka63DA and thus VZV ORF63 is necessary for the virus to efficiently establish a latent infection. 38163-0196

References

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Brunell, P.A., L. C. Ren, J. I. Cohen, S. E. Straus. 1999. Viral gene expression in rat trigeminal ganglia following neonatal infection with varicella-zoster virus. J. Med. Virol. 58:286-290.

Cohen Jl, Seidel KE. Generation of varicella-zoster virus (VZV) and viral mutants from cosmid DNAs: VZV thymidylate synthetase is not essential for replication in vitro. Proc. Natl. Acad. Sci. USA 1993; 90:7376-7380.

Cohrs R. J., J. Randall, J. Smith, D. H. Gilden, C. Dabrowski, H. van der Keyl, R. Tal- Singer. 2000. Analysis of individual human trigeminal ganglia for latent herpes simplex virus type 1 and varicella-zoster virus nucleic acids using real time PCR. J. Virol 74:11464-11471.

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Debrus S, Sadzot-Delvaux C, Nikkels AF, Piette J, Rentier B. Varicella-zoster virus gene 63 encodes an immediate-early protein that is abundantly expressed during latency. J. Virol. 1995;69:3240-3245.

Fuchs, W., Ehrlich, C, Klupp, B.G., and T.C. Mettenleiter, 2000. Characterization of the replication origin (Ori_s) and adjoining parts of the inverted repeat sequences of the pseudorabies virus genome J. Gen. Virol. 81 : 1539-1543. 38163-0196

Ghiasi. H, Osorio, Y, Perng, G.C., Nesburn, A.B., and S.L. Wechsler, 2002. Overexpression of interleukin-2 by a recombinant herpes simplex virus type 1 attenuates pathogenicity and enhances antiviral immunity. J. Virol. 76: 9069-9078.

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The description, specific examples and data, while indicating exemplary embodiments, are given by way of illustration and are not intended to limit the invention. Various changes and modifications will become apparent to the skilled artisan from this disclosure and are included within the ambit of the invention. The references cited above and throughout this disclosure are herein incorporated by reference in their entireties.

Claims

38163-0196CLAIMS We claim:

1. A live virus vaccine, the vaccine having impaired ability to establish latency; comprising: a recombinant virus that substantially lacks a phosphoprotein gene that is not required for growth of the virus in cultured cells but is important for establishing a latent infection.

2. A live virus vaccine, the vaccine having impaired ability to establish latency; comprising: a recombinant virus that substantially lacks a gene for a protein encoded by an mRNA, wherein the mRNA is transcribed during normal latency.

3. A live virus vaccine, the vaccine having impaired ability to establish latency, comprising: a recombinant virus that substantially lacks a protein encoding gene, the gene being encoded by a nucleic acid sequence that is complementary to a nucleic acid that hybridizes with the ORF63 gene of varicella-zoster virus.

4. The vaccine of any of claims 1 to 3 wherein the substantially lacking gene is selected from the group consisting of the ORF63 gene of varicella-zoster virus, the ORF 70 gene of varicella-zoster virus, the ICP22 gene of the herpes simplex virus, the US1 gene of the Marek's disease virus, and the ICP22 homologue gene of the pseudorabies virus.

5. The vaccine of any of claims 1 to 3 wherein the substantially lacking gene is at least 10% homologous to ORF63 of varicella-zoster virus.

6. The vaccine of any of claims 1 to 4 wherein the substantially lacking gene comprises a nuclear localizing sequence. 38163-0196

7. A live virus vaccine that has impaired ability to establish latency, comprising at least one virus, wherein the at least one virus is selected from the group consisting of herpes simplex virus, varicella-zoster virus, Marek's disease virus and pseudorabies virus.

8. The virus vaccine of claim 7, comprising a varicella-zoster virus that lacks one or more substantial portions of the ORF63 genes.

9. The virus vaccine of claim 8, wherein portions of the ORF63 and ORF70 genes that encode amino acids 24 to 268 have been deleted.

10. A live virus vaccine against a target virus wherein the attenuated live virus has impaired ability to establish latency, and comprises a varicella-zoster virus that lacks substantial parts of the ORF63 genes and has substituted therefore at least one gene that encodes at least one protein epitope of the target virus.

11. The virus vaccine of any of claims 2-6, 8 and 10 wherein at least 75% of the gene is missing.

12. The live virus vaccine of claim 10, wherein the target virus is varicella-zoster virus.

13. The live virus vaccine of claim 10, wherein the target is a herpes virus.

14. The live virus vaccine of claim 10, wherein the target is a virus that infects humans and the at least one protein epitope is a protein from the target virus.

15. A live varicella-zoster virus vaccine that has impaired ability to establish latency, comprising a varicella-zoster virus wherein at least one of the ORF63 and ORF70 genes have been modified to inhibit establishment of latency. 38163-0196

16. The virus vaccine of claim 15 wherein at least one of the ORF63 and ORF70 genes has been substantially deleted.

17. The virus vaccine of claim 15 wherein both of the ORF63 and ORF70 genes have been altered.

18. A vaccine as described in any of claims 1 to 17 further comprising a second modified virus kinase gene having a lower kinase activity against one or more viral proteins.

19. A method for making an attenuated live virus vaccine targeted against a virus and having impaired ability to establish latency, comprising: selecting a virus having one or more copies of a dominant latent phase transcribed gene; altering or removing a substantial part of each copy of the latent phase transcribed gene to form an attenuated virus having impaired ability to establish latency; and culturing the attenuated virus to an amount suitable for a vaccine.

20. The method of claim 19, further comprising adding at least one gene that encodes a protein antigen of the targeted virus or a cytokine.

21. The method of claim 19, wherein the transcribed gene is at least 75% homologous to the ORF63 gene of varicella-zoster virus.

22. The method of claim 19, wherein at least 5% of the latent phase transcribed gene is removed.

23. The method of claim 19, wherein the virus is selected from the group consisting of herpes simplex virus, varicella-zoster virus, Marek's disease virus and pseudorabies virus. 38163-0196

24. The method of claim 19, wherein the virus is varicella-zoster and wherein the nucleic acid that encodes amino acid positions 24 to 268 of the ORF63 and ORF70 genes has been deleted.

25. A method of preventing latent viral infection, comprising administering to a subject in need thereof a vaccine as described in any of claims 1 through 18.

26. A method as described in claim 25, wherein the viral infection is varicella zoster and the vaccine comprises a varicella zoster virus that lacks a complete ORF63 gene.

27. A method as described in claim 25, wherein the vaccine comprises a recombinant virus that comprises a foreign gene that encodes a protein antigen of a virus or a cytokine.

28. A method as described in claim 25, wherein the viral infection is from a virus selected from the group consisting of herpes simplex virus, varicella-zoster virus, Marek's disease virus and pseudorabies virus.

29. A method for preparing a composition useful for preventing infection by a virus, comprising combining a recombinant virus that substantially lacks a phosphoprotein gene that is not required for growth of the virus in cultured cells but is important for establishing a latent infection with a live virus vaccine stabilizer.

30. A method for preparing a composition useful for preventing infection by a virus, comprising combining a recombinant virus that substantially lacks a gene for a protein encoded by an mRNA, wherein the mRNA is transcribed during normal latency with a live vaccine stabilizer.

31. A method for preparing a composition useful for preventing infection by a virus, comprising combining a recombinant virus that substantially lacks a protein encoding gene, the gene being encoded by a nucleic acid sequence that is complementary to a 38163-0196

nucleic acid that hybridizes with the ORF63 gene of varicella-zoster virus, with a live vaccine stabilizer.

32. The method of claim 31 , wherein the nucleic acid that encodes amino acid positions 24 to 268 of the ORF63 and ORF70 genes has been deleted from the recombinant virus.

33. The live vaccine of any of claims 2 and 3, wherein the gene is homologous to the varicella zoster virus ORF63 gene and is found in simian varicella virus, feline herpes 1 , equine herpes 1 , equine herpes 4, pseudorabies virus, canine herpes 1, bovine herpes 1 , Marek's disease virus (of chicken), Laryngotracheitis virus, Meleagrid herpes virus 1 , or herpes simplex virus.

34. The live vaccine of any of claims 2 and 3, wherein the gene product shares at least 10% homology with the ORF63 gene product of the varicella zoster virus.

35. The method of any of claims 19, 30 and 31, wherein the gene is homologous to the varicella zoster virus ORF63 gene and is found in simian varicella virus, feline herpes 1 , equine herpes 1 , equine herpes 4, pseudorabies virus, canine herpes 1 , bovine herpes 1 , Marek's disease virus (of chicken), Laryngotracheitis virus, Meleagrid herpes virus 1 , or herpes simplex virus.

36. The method of any of claims 19, 30 and 31, wherein the gene product shares at least 10% homology with the ORF63 gene product of the varicella zoster virus.