Patent Application
20080279886
This disclosure relates to recombinant vaccines, and more particularly relates to live vaccines.
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. Pat. 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
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.
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 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 a varicella zoster virus gene, such as the ORF63 gene.
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 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 live virus vaccine, having impaired ability to establish latency, comprising a recombinant virus that has at least one intact ORF62 gene but that substantially lacks a nucleic acid sequence that is complementary to a nucleic acid that hybridizes with the ORF62 gene of varicella-zoster virus. Desirably, the nucleic acid sequence includes at least a portion of the amino terminal protein sequence encoding the end of the ORF62 region. This region may correspond to, for example, the upstream sequence only, or the upstream sequence plus coding region of 0-10 amino acids, 10-25 amino acids, 20-40 amino acids, 30-50 amino acids, 40-60 amino acids, 50-70 amino acids, 75-100 amino acids or more.
According to an embodiment, an amino terminal portion of the ORF62 protein is coded for and expressed. Expression leads to the translation into a partial protein, which then alters regulation of the viral genome, in a manner that allows replication to wild type titers despite inhibition of latency. The inhibition of latency may arise, for example, by an alteration to ORF63 gene sequence(s), to alteration of one or more ORF62 gene sequences, or both. In an embodiment, replication of a virus is enhanced without increasing latency by addition of the partial ORF62 gene sequence to the virus.
Another embodiment provides a vaccine, wherein the recombinant virus further substantially lacks a gene 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. Yet another embodiment provides a vaccine, wherein the recombinant virus is impaired for latency but replicates to wild-type titers in vitro. Another embodiment provides a live virus vaccine, the vaccine having impaired ability to establish latency, and comprising a recombinant virus that has at least one intact ORF62 gene but that substantially lacks a nucleic acid sequence that is complementary to a nucleic acid that hybridizes with the ORF62 gene of varicella-zoster virus. In yet another embodiment, the vaccine comprises a recombinant virus that further substantially lacks a gene 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.
Another embodiment provides a vaccine, wherein the recombinant virus has an additional partial copy of a gene selected from the group consisting of the ORF62 gene (for a mutant involving the varicella-zoster virus), the ICP4 gene which is the homolog of VZV ORF62 (for a mutant involving the herpes simplex virus), the ICP4 gene of the Marek's disease virus (for a mutant involving the Marek's disease virus), and the ICP4 gene of pseudorabies virus (for a mutant involving the pseudorabies virus).
Another embodiment provides a vaccine, wherein the recombinant virus further substantially lacks the ORF63 gene of the varicella-zoster virus and also has an additional partial copy of the ORF62 gene. A recombinant herpes simplex virus would substantially lack the IPC22 gene and also have an additional partial copy of the ICP4 gene. A recombinant Marek's disease virus or pseudorabies virus would substantially lack the ICP22 gene homologs and also have a partial copy of the ICP4 gene homologs.
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.
It was discovered that modification, particularly by deletion, of a gene encoding a protein such as a 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 a varicella zoster virus ORF62 gene product. In another embodiment, the gene is selected by examination of homology with a conserved region of a 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 compared gene product. Moreover, particularly in the case of selection of an ORF62 homology, a desirable gene advantageously contains a deletion on the carboxyl terminal (encoded protein) side of the gene. In this preferred case, the percent homology is calculated without reference to the deleted sequence. The gene may be selected or designed on this basis.
In a desirable embodiment, a VZV or HSV is made having at least one full length and one altered form of ORF62 or ICP4 (a homolog of the ORF62 gene product). The altered form desirably misses at least 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40% or more of the carboxyl terminal end, although other alterations can be carried out that will have at least some beneficial effect on the protein. In an embodiment, the virus additionally has a ICP22 gene deletion. A live vaccine can be made from the altered virus and is particularly contemplated. In another embodiment, a section of a gene homologous to the ORF62 and/or ORF63 gene products and 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 a desirable embodiment an ORF62 gene of 1310 amino acid coding region or a similar sized homologous protein is truncated to remove 0-10%, 10-25%, 25-35%, 35-50%, 50-65% or more of the carboxyl terminal side of the protein. One embodiment that was found to work well was the truncation of a 1310 amino acid coding region to an 840 amino acid coding region. In another embodiment the remaining amino terminal side region is between 700-840 amino acids, 600-700 amino acids, 500-600 amino acids, or even less.
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 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 or altered protein. In the case of the ORF63 and ORF70 genes an exact correspondence between both genes ORF63 and ORF70 is desired in many embodiments. In the case of ORF62 gene modification, it was surprisingly found that addition of a modified form (removal of carboxyl terminal end) can provide normal replication while preserving latency. 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.
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 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 or ORF62 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 or ORF62 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 or ORF62 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. Pat. 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.
One or more copies of a gene may be a) mutated or otherwise altered and/or b) added to an existing viral genome. For example, a gene fragment comprising the coding region and upstream sequence may be added to an existing viral nucleic acid that contains normal ORF62.
In another embodiment, a gene homologous to the ORF63 gene and/or ORF62 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. Various genes homologous to ORF62 may be altered as will be appreciated by a skilled artisan. For example, ICP4 of HSV or the corresponding ICP4 protein in pseudorabies virus or Marek's disease virus may be altered, particularly by deletion in the carboxyl terminal side of the protein and inserted into the corresponding virus to make a vaccine against the virus. Of course, genes that are less homologous also may be selected. For example in the case of ORF63, 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 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. Table 2 shows representative genes having suitable amino acid coding homology to the ORF62 gene of varicella zoster virus. 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
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 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.
Another suitable gene is a gene that has positive transcriptional activity, i.e. that activates gene expression. Such a gene in particular is a preferred candidate to truncate and insert into a vaccine. By way of example, gene ORF62 is expressed in latency. While ORF62 turns on other viral genes during virus replication, a truncated form of the protein may interfere with the activity of the wild-type protein, and may turn off other viral genes. It was found experimentally that insertion of a truncated form of the ORF62 gene reduced the ability of the virus to go latent. Likewise, other genes associated with positive transcriptional activity, either known or to be discovered in the future may be used 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.
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.
In yet another embodiment, the conformation of the protein is modified by altering the carboxyl terminus of the protein. For example, ORF63 can be 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, although alteration by inserting a truncation of the carboxyl terminal protein encoding portion of the ORF62 gene (or another viral gene that like ORF62 turns on gene expression) is particularly desirable. 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 a 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 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 NotI A, NotI BD, MstII A, and MstII B, as used herein, refer to various cosmids utilized in transfection experiments to produce desired mutations in various embodiments. These constructs can be made from known methods.
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. Pat. No. 6,210,683 provides representative conditions for this embodiment of the invention. U.S. Pat. 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.
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. Pat. 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.
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, Calif.). The membranes were then incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse antibody and imaged using enhanced chemiluminescence (Pierce Chemical Company, Rockfield, Ill.).
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
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+AvrII, contains an Avr II site. Plasmid pBSSK+AvrII was cut with Cla I and Kpn I and a single-stranded oligonucleotide—CGGTAC—was inserted into this site to create plasmid pBSSK+AvrIIdCla.
The ORF63 gene from plasmid pBSSK+SfiI was inserted into plasmid pBSSK+AvrIIdCla. Plasmid pBSSK+SfiI was cut with Spe I—located in pBSSK+ adjacent to the site of the VZV insert—and EcoR I—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+AvrIIdCla to create plasmid ES. The ORF70 gene from plasmid pBSSK+SfiI was then inserted into plasmid pBSSK+AvrIIdCla. Plasmid pBSSK+SfiI was cut with Avr II—located at VZV nucleotide 112,853 and Hind III—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+AvrIIdCla 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 I-Spe I fragment into plasmid pBSSK+SfiI. 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 II-Hind III fragment of the ORF63 deleted plasmid pBSSK+SfiI. Finally, the ORF63-and-ORF70-deleted plasmid pBSSK+SfiI was cut with Sfi I and inserted in place of the wild-type Sfi I fragment of cosmid MstIIA. The resulting cosmid, termed MstIIA-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, MstIIA-63DA and MstII63-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 NotI A, NotI B, MstII B, and MstII A or MstII A-63D. To rescue the deletion in ORF63, a plasmid containing ORF63 and its promoter was constructed. A 5.6 kb Bcl I fragment of VZV (nucleotides 106,592 to 112,215) was inserted into plasmid pMAL-p2X (New England Biolabs, Beverly, Mass.) between nucleotides 445 and 2,645. The resulting plasmid was cut with BclI and the VZV fragment was gel purified.
This example describes the deletion of both copies of the ORF63 gene and construction of a rescued virus in which ORF63 is replaced. Cosmids MstIIA-63DA and MstIIA-63 DB 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 MstIIA-63DA and MstIIA-63 DB, as shown in FIG. 1. Melanoma cells were transfected with plasmid pCMV62. Cosmids VZV NotI A, Not I BD, MstII A, and MstII B showed cytopathic effects. Recombinant Oka VZV (ROka) was produced following transfection. Transfection of cells with plasmid pCMV62 and cosmids VZV NotI A, Not I BD, and MstIIA-63DA resulted in cytopathic effects after transfection; the resulting virus was termed ROka63DA. Transfection of cells with plasmid pCMV62 and cosmids VZV NotI A, Not I BD, and MstIIA-63 DB resulted in cytopathic effects after transfection; the resulting virus was termed ROka63 DB. 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 BclI fragment that contains ORF63 with flanking regions. At the eighth day after 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 ROka63 DB 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 45 kDa ORF63 protein, while lysates from cells infected with ROka63DA or ROKa63 DB 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.
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 ROka63 DB 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.
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 the spine with VZV-infected melanoma cells containing 1.75×105 or 3.0×105 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 NotI 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 1.75×105 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 3.0×105 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.
As described above, a VZV virus was made with just a 21 amino acid deletion in ORF63. Separately, A VZV virus “ROka63NLS” was made with the 21 amino acid deletion plus a further rearrangement in ORF62 as shown in
A rearrangement in the genome due to one copy of DNA inserting non-homologously was shown by Southern analysis and by sequencing. The sequenced region from left to right in the first IR region revealed VZV nucleotides 107,336 to 106,614 followed by 112,214-111,379 and then followed by 111,334-109,085 (nucleotide sequence numbers based on Davison and Scott). Here in this case, 107,336 to 106,614 were shown to be in the ORF62 amino portion of the protein. The ORF62 protein is between 105,204 and 109,133 and the amino portion is at 109,133. The schematic in
The region 112,214 to −111,379 encodes the portion of the VZV genome between ORFs 64 and 65, all of ORF64, and the carboxy portion of ORF63 The nucleotides 111,334 to 109,085 encode the amino portion of ORF63, the region between ORF63 and ORF62, and the amino terminus of ORF62. Accordingly, the entire ORF62 region is indicated as being encoded, followed by the region between ORF62 and ORF61, ORF61, and the other regions indicated on the schematic in
The region between 111,379 and 111,334 was purposely deleted in ORF63, and encodes the nuclear localization signal of the ORF63 protein. One IR is intact, and the other IR has the insertion. The rearrangement described above may involve the second IR region, instead of the first IR region
A comparison of the two viruses with wild type showed that the latter recombinant virus with the ORF62 rearrangement is impaired for latency yet grows well. See
In another embodiment, multiple strains of VZV are prepared by modifying ORF62 by rearrangements, while leaving ORF63 intact.
Lungu, O., C. A. Panagiotidis, P. W. Annunziato, A. A. Gershon, S. J. Silverstein. 1998. Aberrant intracellular localization of varicella-zoster virus regulatory proteins during latency. Proc. Natl. Acad. Sci. USA 95:7080-7085.
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.
This application claims priority to provisional application Ser. No. 60/583,399, filed Jun. 29, 2004, the contents of which are incorporated herein in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2005/021788 | 6/22/2005 | WO | 00 | 1/3/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60583399 | Jun 2004 | US |
