Patent Application
20060008470
The present invention relates to vaccines having an increased level of safety comprising recombinant vaccinia viruses containing an inactivated E3L region. The invention further relates to methods for stimulating a protective immune response in an immunized host using the vaccines of the invention. The invention is based on the discovery that vaccinia virus mutants having deletions in the E3L region exhibit dramatically reduced pathogenesis while remaining highly immunogenic. In addition, the invention relates to modified recombinant vaccinia viruses engineered to express heterologous polypeptides and the use of such viruses in vaccines designed to stimulate a protective immune response against such polypeptides in a host.
Vaccinia virus is a member of the poxvirus family of DNA viruses. Vaccinia virus has been used successfully to immunize against smallpox, resulting in worldwide eradication of smallpox. Many different strains of vaccinia virus exist and the different strains demonstrate varying degrees of immunogenicity and are implicated with a variety of different complications, such as post-vaccinial encephalitis and generalized vaccinia. Thus, the use of vaccinia virus recombinants as expression vectors and particularly as vaccines and anticancer agents raises safety concerns associated with introducing live recombinant viruses into the environment.
Poxviruses including vaccinia virus are used extensively as expression vectors since the recombinant viruses are relatively easy to isolate, have a wide host range, and can accommodate large amounts of DNA. The vaccinia virus genome contains nonessential regions into which exogenous DNA can be incorporated. Exogenous DNA has been inserted into the vaccinia virus genome using well-known methods of homologous recombination. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA and homologous plasmid DNA bearing the gene of interest (see, for example, U.S. Pat. No. 6,372,455). DNA molecules (e.g., plasmids, naked DNA, viral vectors, and poxviruses) have been used for insertion and expression of foreign genes. The resulting recombinant vaccinia viruses are useful as vaccines and anticancer agents.
A critical objective in vector development is to create a so called “attenuated vector” for enhanced safety, so that the vector may be employed in an immunological or vaccine composition. Thus, a balance between the efficiency and the safety of a vaccinia virus-based recombinant vaccine is extremely important. The recombinant virus must present the immunogen(s) in a manner that elicits a protective immune response in the vaccinated host but lacks any significant pathogenic properties. Virulence of vaccinia virus recombinants in a variety of host systems has been attenuated by the deletion or inactivation of certain vaccinia virus genes that are nonessential for virus growth. Replication-competent strains of vaccinia virus currently used against smallpox are interferon-resistant (Thacore and Younger, 1973, Virology 56:505-11).
Type I interferons are induced upon viral infection and constitute an integral part of the host cell's antiviral response (Samuel, 2001, Clin Microbiol Rev 14(4):778-809, table of contents). Double-stranded RNA (dsRNA), which is produced during most viral infections, but otherwise absent from cells, is believed to directly activate human interferon regulatory factor 3 (IRF-3; from an inactive state), thereby triggering transcriptional activation of IFN (Wathelet et al., 1998, Mol Cell 1(4):507-18; Lin et al., 1998, Mol Cell Biol 19:2986-96; Sato et al., 1998, FEBS Lett 452:112-16; Weaver et al., 1998, Mol Cell Biol 18:1359-68; Yoneyama et al., 1998, EMBO J. 17:1087-95; (Nguyen et al., 1997, Cytokine Growth Factor Rev 8(4):293-312). The rate-limiting step in this process is C-terminal phosphorylation of IRF-3 by an uncharacterized virus activated kinase (VAK) activity (Servant et al., 2001, J Biol Chem 276(1):355-63).
Two of the best characterized IFN-induced proteins are the dsRNA dependent enzymes, PKR and 2′-5′ oligo adenylate synthetase (OAS) (Jacobs and Langland, 1996, Virology 219(2):339-49). PKR is a protein kinase consisting of an amino-terminal dsRNA-binding domain and a carboxy-terminal catalytic domain and is activated by autophosphorylation in a process mediated by dsRNA (Bryan, 1999, Oncogene 18:6112-6120; Clemens and Ella, 1997, J Interferon Cytokine Res 17(9):503-24). Following activation, PKR phosphorylates various substrates including the a subunit of protein synthesis initiation factor 2, eIF-2α (Samuel, 1979, Proc Natl Acad Sci USA 76(2):600-4). Phosphorylation of eIF-2α inhibits translation in general by impairing the eIF-2B-catalyzed guanine nucleotide exchange reaction (Clemens and Elia, 1997, J Interferon Cytokine Res 17(9):503-24). Thus, this inhibition blocks viral replication at the level of protein synthesis (Gale, 1998, Mol Cell Biol 18(2):859-71).
Activated OAS polymerizes ATP to produce 2′-5′ linked oligoadenylates (Rebouillat and Hovanessian, 1999, J Interferon Cytokine Res 19(4):295-308). These oligoadenylates subsequently activate a potent antiviral enzyme, RNase L, which cleaves single-stranded RNAs (Baglioni et al., 1979, Biochemistry 18(9), 1765-70; Silverman and Cirino, 1997, Gene Regulation (Morris, D. R., Hartford, J. B., eds), 295-309, John Wiley & Sons). IFN treatment of cells elevates the level of OAS and RNase L but these proteins remain enzymatically inactive until dsRNA is produced upon viral infection (Sen, 2000, Semin Cancer Biol 10(2):93-101). Both PKR and OAS activation result in an inhibition of viral, and at times, host protein synthesis (Jacobs and Langland, 1996, Virology 219(2):339-49).
Both PKR and OAS are targets of viral systems that attempt to defeat host cell resistance. For example, the vaccinia virus (VV) E3L and K3L gene products inhibit PKR (Clemens and Elia, 1997, J Interferon Cytokine Res 17(9):503-24). The viral E3L protein is a dsRNA-binding protein that blocks autoactivation of PKR by sequestering dsRNA activators of PKR (Shors et al., 1997, Virology 239(2):269-76) and possibly interacting directly with the eIF-2α-binding region of PKR (Sharp et al., 1998, Virology 250(2):302-15). E3L is a potent inhibitor not only of the PKR kinase, but also of OAS (Rivas et al., 1998, Virology 243(2):406-14). The E3L gene encodes two related proteins, p20 and p25 (Chang et al., 1992, Proc Natl Acad Sci USA 89(11):4825-9; Yuwen et al., 1993, Virology 195(2):732-44). These gene products are early viral proteins containing a dsRNA-binding motif and are required for providing interferon resistance to the virus (Watson et al., 1991, Virology 185:206-16; Chang et al., 1992, Proc Natl Acad Sci USA 89(11):4825-9; Kibler et al., 1997, J Virol 71(3):1992-2003).
The VV E3L gene products consist of amino-terminal and carboxy-terminal domains, separated by a trypsin-sensitive spacer region (Ho and Shuman, 1996, Virology 217(1):272-84). The C terminal domain contains one copy of a conserved dsRNA-binding motif and is required for dimerization of the protein. Mutational analysis demonstrates that the C-terminal domain is required for dsRNA binding and PKR inhibitory activity seen in VV infected cells (Chang and Jacobs, 1993, Virology 194(2):537-47; Ho and Shuman, 1996, J Virol 70(4):2611-4).
The N-terminal domain of E3L shares significant sequence homology with the eukaryotic RNA-editing enzyme ADAR1, which catalyzes the deamination of adenosine residues that are present in dsRNA, or in secondary structures of predominantly ssRNA (Patterson et al., 1995, Virology 210(2):508-11). The amino-terminal 45% of EM, upstream of the dsRNA-binding domain, is not essential for replication of vaccinia virus in several different cell lines in culture (Kibler et al., 1997, J Virol 71(3):1992-2003; Shors et al., 1997, Virology 239(2):269-76). The amino terminus of E3L proteins has also been reported to directly interact with the catalytic domain of PKR, suggesting that this interaction may be required for the function of E3L protein (Romano et al., 1998, Mol Cell Biol 18(12):7304-16). The E3L gene products are the only VV gene products known to localize to both the nucleus and cytoplasm of infected cells (Yuwen et al., 1993, Virology 195(2):732-44; Chang et al., 1995, J Virol 69(10):6605-8). Sequences at the amino-terminus of E3L are necessary for accumulation of E3L products in the nucleus. These results suggest that cytoplasmic, but not nuclear, accumulation of the E3L gene products is required for efficient viral replication in cells in culture.
The E3L gene also confers a broad host range to VV enabling it to replicate in several cell types including HeLa, Vero and L cells (Chang et al., 1995, J Virol 69(10):6605-8; Shors et al., 1997, Virology 239(2):269-76). Deletion of the E3L gene from vaccinia virus produces a recombinant virus that is interferon-sensitive and highly debilitated for replication in cells in culture (Jacobs and Langland, 1996, Virology 219:339-49). VV deleted of the E3L gene (VVΔE3L) has a severely reduced host-range phenotype in that it does not replicate in human HeLa, and monkey kidney COS, CV-1, or BSC-40 cells, even in the absence of IFN treatment (Beattie et al., 1996, Virus Genes 12(1), 89-94). Interferon sensitivity is exemplified by VVΔE3L's sensitivity to pretreatment of rabbit kidney RK-13 cells with IFN-β and its inability to rescue Vesicular Stomatitis Virus from the effects of IFN (Shors et al., 1998, Virology 239(2):269-76).
VVΔE3L infection induces apoptosis in HeLa cells in an IFN-independent manner (Lee and Esteban, 1994, Virology 199(2):491-6; Kibler et al., 1997, J Virol 71(3):1992-2003). VVΔE3L infection also induces apoptosis in IFN-treated RK-13 cells (Kibler et al., 1997, J Virol 71(3):1992-2003). Infection with VVΔE3L leads to activation of PKR and increased phosphorylation of eIF2α (Beattie et al., 1995, Virology 210(2), 254-63; Beattie et al., 1995, J Virol 69(1), 499-505) in HeLa cells irrespective of IFN treatment, and in IFN-treated RK-13 cells. Infection of several cells with VVΔE3L leads to rRNA degradation typical of activation of the OAS/RNase L pathway (Beattie et al., 1995, J Virol 69(1), 499-505). Thus, both PKR-mediated and OAS-mediated antiviral defense mechanisms are active in cells infected with VVΔE3L.
It has been shown that recombinant vaccinia viruses in which the E3L gene is replaced by a gene encoding an E3L homolog from the orf virus, a poxvirus of the genus parapoxvirus that infects sheep, goats and humans, are immunogenic but have decreased pathogenicity in mice relative to wild-type vaccinia virus (U.S. Pat. No. 6,372,455). When administered intranasally, these recombinant viruses replicated to high titers in nasal tissues, but did not spread to the lung or brain and had reduced neurovirolence.
Development of enhanced vectors having enhanced transcription and/or expression which are attenuated continues to be a desirable goal, especially since attenuation may raise expression levels and/or persistence. Thus, there remains a need in the art for the development of vectors that have reduced pathogenicity while maintaining immunogenicity.
The iridoviruses are large DNA viruses that share many features of replication with the poxviruses, including cytoplasmic transcription and DNA synthesis (Jacobs, 2000, Translational control CH 35, 1-21 (CSHL Press)). They encode an eIF2α homolog (Yu et al., 1999, Virus Res 63(1-2):53-63). Essbauer et al., 2001 have analyzed the eIF2α of several iridoviruses of fish and frogs (Virus Genes 23(3):347-59). eIF2α homologous sequences of European catfish virus (ECV I-III), European sheatfish virus (ESV), and frog virus-3 (FV-3) had a length of 780 nucleotides. At the N-terminus (amino acid 1-100), the iridoviral eIF2α showed a significant homology to the N-termini of cellular initiation factor 2-α of various species and full-length poxyiral K3L protein. The eIF2α iridoviral protein had 37% identity with and 48% similarity to the N-terminus of human eIF2α and 32% identity with and 48% similarity to the K3L protein of VV. Several sites were highly conserved in all eukaryotic, iridoviral and poxyiral proteins. Serine 51 of cellular eIF2α, which is phosphorylated by PKR, could not be found in any viral proteins. At amino acids 15-89, the iridovirus protein reveals a homology to the S1 domain of ribosomal proteins (Bycroft et al., 1997, Cell 88(2):235-42). The C-terminus of the iridoviral proteins (amino acid 100-end) has no homology to any known protein (Essbauer et al., 2001, Virus Genes 23(3):347-59).
The homology of poxyiral and iridoviral proteins does not include 19 residues that flank serine phosphorylation site 51 and that are perfectly conserved from yeast to humans. The pentapeptide KGYID motif, which is important for the interaction of K3L of VV with the PKR is modified to KGYVD in all iridoviral eIF2α amino acid sequences. Since the C-terminus of ranaviral eIF2α reveals no homology to any known protein, it remains unclear whether a truncated form (N-terminal 100 amino acids) of the iridovirus protein could be functional and also why these polypeptides are longer than their poxyiral homologs (Essbauer et al., 2001, Virus Genes 23(3):347-59). Thus, it is unclear whether the iridovirus homolog is acting as an eIF2α kinase inhibitor, or given its large size, as an alternative eIF2α-like translation initiation factor.
Ambystoma tigrinum virus (ATV) is a member of the genus ranavirus in the family Iridoviridae, which was isolated from diseased tiger salamanders (Ambystoma tigrinum stebbinsi). ATV genome sequencing has yielded the sequence of a gene with homology to the eukaryotic translation initiation factor, eIF2α. The role of this gene, if any, in ATV's ability to suppress antiviral host cell responses had not previously been determined.
The present invention provides vaccines comprising a recombinant vaccinia virus from which the region encoding the E3L gene product has been inactivated and a suitable carrier. The recombinant vaccinia virus of the invention may comprise exogenous DNA. This exogenous DNA may encode a gene product. A nonlimiting example of gene products that may be encoded is a polypeptide, e.g., an epitope to which a protective immune response is desired. Another nonlimiting example of a gene product that may be encoded is a ribonucleic acid or polypeptide bearing some desirable property.
In some embodiments of the invention, vectors having reduced pathogenicity while maintaining immunogenicity have been prepared. Recombinant vaccinia viral vectors were prepared wherein the E3L gene was replaced by the eIF2α gene from ATV. The recombinant virus is interferon sensitive, but possesses a broad host range; is able to inhibit the PKR pathway but not the OAS pathway and IRF-3 phosphorylation. Without being limited to any particular mechanism of action, these results indicate that the ATV eIF2α homolog acts as a novel PKR inhibitor by causing proteolytic degradation of PKR and it provides the salamander virus, ATV, with a novel gene to counteract host defenses. The compositions of the invention are useful for eliciting an immune response to smallpox virus and other molecules.
The present invention provides vaccines comprising a recombinant vaccinia virus from which the region encoding the E3L gene product has been inactivated. Such inactivation may result from partial or complete deletion of the E3L region or, alternatively, substitution of nucleotides within the E3L region that result in inactivation of the E3L gene product.
The E3L gene product of the vaccinia virus is a 190 amino acid polypeptide. The E3L gene codes for several functions including a dsRNA-binding protein, a Z-DNA-binding protein, and dimerization. Amino acids 118-190 have been implicated in dsRNA binding, as disclosed by Chang and Jacobs (1993, Virology 194:537-547). Amino acid numbering as used herein is adopted from Goebel et al., 1990, Virology 179:247-66, 577-63.
According to the invention “deletion of the E3L gene” and its grammatical equivalents refer to a vaccinia virus wherein a nucleic acid encoding all 190 amino acids or a subset of the 190 amino acids of E3L are not present. According to the invention, if the vaccinia virus having a deletion in the E3L gene has a residual nucleic acid encoding a subset of the 190 amino acids of E3L, said residual nucleic acid is incapable of producing a functional gene product or the gene product is incapable of binding dsRNA. The ability of the E3L gene product to bind to dsRNA can be determined by binding assays known in the art and disclosed, for example, by Chang et al., 1993, Virology 194:537.
According to the invention, “recombinant vaccinia virus” refers to a vaccinia virus having a deletion or, alternatively, nucleotide substitutions in the E3L gene. The term includes vaccinia virus wherein a heterologous nucleic acid is substituted for the E3L gene.
Replication-competent strains of vaccinia virus currently used for vaccination against smallpox are interferon-resistant (Thacore and Younger, 1973, Virol. 56(2):505-511). Deletion of the E3L gene from vaccinia virus results in a virus that is interferon-sensitive, but also is highly debilitated for replication in many cells in culture (Jacobs and Langland, 1996, Virolology 219(2):339-349). This virus is also dramatically less virulent in both immune competent (
The present invention provides modified poxviruses in which genes that code for certain inhibitors have been substituted for the poxvirus E3L gene or that contain a modified version of the gene. These modified viruses have been found to replicate normally in cells, but display dramatically decreased pathogenesis. These viruses replicate to high titers in nasal tissues, but have a decreased propensity to spread to the lungs and brain and have decreased neurovirulence. These vectors can be used to protect against subsequent infection with vaccinia virus and, therefore, have utility in vaccination against various diseases including smallpox.
The invention further provides a safe replication-competent vector for expression of heterologous proteins. Specifically, the invention provides recombinant vaccinia viral vectors comprising the recombinant vaccinia virus described above and further containing exogenous, i.e., nonvaccinia virus, DNA. Exogenous DNA may encode any desired product, including for example, an antigen, an anticancer agent, or a marker or reporter gene product. The recombinant vaccinia virus may further have deletions or inactivations of nonessential virus-encoded gene functions. Nonessential gene functions are those which are not required for viral replication in a host cell. The exogenous DNA is preferably operably linked to regulatory elements that control expression thereof. The regulatory elements are preferably derived from vaccinia virus.
The present invention further provides a recombinant vaccinia virus, wherein the virus comprises a salamander ATV eIF2α homolog. According to some nonlimiting embodiments of the invention, this virus lacks a portion of the E3L gene. Thus, the invention provides, in some nonlimiting embodiments, a recombinant vaccinia virus in which a portion of the E3L gene is replaced with the eukaryotic initiation factor 2a gene (eIF2α) of Ambystoma tigrinum virus (ATV). These recombinant viruses may be interferon sensitive, but possess a broad host range, thus partially rescuing the phenotype of VV deleted for E3L gene. These viruses may inhibit PKR by proteolytic degradation of PKR. Infection with this virus may lead to activation of IRF-3, which is a transcription factor responsible for the induction of IFN in virus infected cells. Thus, this virus may block the activity of PKR, but cannot block the induction of IFN. Subsequent IFN sensitivity of this virus may occur through alternative IFN-induced, antiviral activity, possibly involving OAS.
According to some nonlimiting examples of the invention, replacing the E3L gene of VV with the eIF2α homolog partially restored the wild type phenotype to the recombinant virus. The E3L gene of VV provides IFN resistance, a wide host range phenotype and inhibits apoptosis (Kibler et al., 1997, J Virol 71(3):1992-2003; Shors et al., 1997, Virology 239(2):269-76). It also functions as an inhibitor of PKR (Chang et al., 1992, Proc Natl Acad Sci USA 89(11):4825-9; Romano et al., 1998, Mol Cell Biol 18(12):7304-16), OAS (Rivas et al., 1998, Virology 243(2):406-14) and IRF-3 phosphorylation (Smith et al., 2001, J Biol Chem 276(12):8951-7). Thus, recombinant viruses of the invention may resemble the wtVV in having a broad host range and in inhibiting PKR activity. At the same time recombinant viruses of the invention may also resemble VVΔE3L in being IFN sensitive and leading to OAS activity and IRF-3 translocation to the nucleus.
The recombinant vaccinia virus of the present invention may be constructed by methods known in the art, and preferably by homologous recombination. Standard homologous recombination techniques utilize transfection with DNA fragments or plasmids containing sequences homologous to viral DNA, and infection with wild-type or recombinant vaccinia virus, to achieve recombination in infected cells. Conventional marker rescue techniques may be used to identify recombinant vaccinia virus. Representative methods for production of recombinant vaccinia virus by homologous recombination are disclosed by Piccini et al., 1987, Methods in Enzymology 153:545.
For example, the recombinant vaccinia virus of a preferred embodiment of the present invention may be constructed by infecting host cells with vaccinia virus from which the E3L gene has been deleted, and transfecting the host cells with a plasmid containing a nucleic acid encoding gene product of interest flanked by sequences homologous to the left and right arms that flank the vaccinia virus E3L gene. The vaccinia virus used for preparing the recombinant vaccinia virus of the invention may be a naturally occurring or engineered strain. Strains useful as human and veterinary vaccines are particularly preferred and are well-known and commercially available. Such strains include Wyeth, Lister, WR, and engineered deletion mutants of Copenhagen such as those disclosed in U.S. Pat. No. 5,762,938. Recombination plasmids may be made by standard methods known in the art. The nucleic acid sequences of the vaccinia virus E3L gene and the left and right flanking arms are well-known in the art, and may be found for example, in Earl et al., 1993, in Genetic Maps: locus maps of complex genomes, O'Brien, ed., Cold Spring Harbor Laboratory Press, 1.157 and Goebel et al., 1990, supra. The amino acid numbering used herein is adopted from Goebel et al., 1990, supra. The vaccinia virus used for recombination may contain other deletions, inactivations, or exogenous DNA as described hereinabove.
Following infection and transfection, recombinants can be identified by selection for the presence or absence of markers on the vaccinia virus and plasmid. Recombinant vaccinia virus may be extracted from the host cells by standard methods, for example by rounds of freezing and thawing.
The resulting recombinant vaccinia virus may be further modified by homologous recombination to provide other deletions, inactivations, or to insert exogenous DNA.
The recombinant vaccinia viruses and compositions of the present invention may be used as expression vectors in vitro for the production of recombinant gene products, or as delivery systems for gene products, as human or veterinary vaccines, or anticancer agents. Such utilities for recombinant vaccinia viruses are known in the art, and disclosed for example by Moss, 1996, “Poxyiridae: The Viruses and Their Replication” in Virology, Fields et al., eds., Lippincott-Raven, Philadelphia, pp. 2637-2671.
The present invention further provides a method of making a recombinant gene product comprising subjecting a recombinant vaccinia viral vector having a deletion of the E3L gene and further comprising exogenous DNA that encodes the recombinant gene product operably linked to the control of regulatory elements that control expression thereof, to conditions whereby said recombinant gene product is expressed, and optionally recovering the recombinant gene product. In a preferred embodiment, the recombinant gene product is an antigen that induces an antigenic and/or immunogenic response when the gene product or a vector that expresses it is administered to a mammal.
The present invention further provides vaccines for providing immunological protection against vaccinia virus, or heterologously expressed polypeptides, wherein said vaccines comprise a recombinant vaccinia viral vector and a carrier. The term carrier as used herein includes any and all solvents, diluents, dispersion media, antibacterial and antifungal agents, microcapsules, liposomes, cationic lipid carriers, isotonic and absorption delaying agents, and the like. Suitable carriers are known to those of skill in the art. The vaccine compositions of the invention can be prepared in liquid forms, lyophilized forms or aerosolized forms. Other optional components, e.g., stabilizers, buffers, preservatives, flavorings, excipients and the like, can be added. In addition, adjuvants may be used to boost or augment immune responses. Optionally, the vaccine may be formulated to contain other active ingredients and/or immunizing antigens.
Also included in the invention is a method of vaccinating a host, including but not limited to mammals such as a humans, against vaccinia virus infection or heterologously expressed proteins with the novel vaccine compositions of the invention. The vaccine compositions, including one or more of the recombinant vaccinia viruses described herein, are administered using routes typically used for immunization, i.e., subcutaneous, oral, or nasal administration, in a suitable dose. The dosage regimen involved in the method for vaccination, including the timing, number and amounts of booster vaccines, will be determined considering various hosts and environmental factors, e.g., the age of the patients, time of administration and the geographical location and environment.
All documents cited throughout this specification are incorporated herein in their entirety by reference.
This invention will be better understood from the following examples. However, one skilled in the art will readily appreciate the specific materials and results described are merely illustrative of, and are not intended to, nor should be intended to, limit the invention as described more fully in the claims which follows thereafter.
The β-galactosidase gene under the control of the vaccinia virus 11 k promoter was cloned into PMPE3delGPT plasmid. This plasmid was used for in vivo recombination with the WR strain of vaccinia virus. Recombinants were isolated by three rounds of plaque purification on a monolayer of BHK-13 cells stained with X-Gal. Deletion of the E3L gene was confirmed by western blotting with E3L specific antibody. PMPE3LdelGPT plasmid, the process of in vivo recombination and isolation of recombinants have been previously described in Kibler et al. (1997, J Virol 71(3):1992-2003).
Groups of 5 C57BL/6 mice were infected with different doses of wild type (wt) vaccinia virus (VV) and VV deleted for the E3L gene (VVΔE3L), by intranasal route. There was 100% survival of mice infected with the highest dose (106) of the mutant virus while wtVV had an LD50 or approximately 103 pfu. The mutant VV construct was over 1000-fold less pathogenic than wtVV (
Groups of 3 C57BL/6 mice were infected with 106 plaque-forming units of wtVV and VVΔE3L by intranasal route. Tissues were harvested, processed and titrated in RK-13 cell line. The figure represents the average titer per gram of tissue of the 3 mice infected with each virus. Wild type VV was detected in the nasal turbinates, lungs, and brain by 5 days post infection. VVΔE3L was detected in the nasal turbinates, but they did not spread to the lung and brain. 4 of the 5 VV mutants replicated to high titers in the nose following infection (
Groups of 5 C57BL/6 mice were infected with different doses of wtVV and VVΔE3L by intracranial injection. The infected mice were observed for 2 weeks following infection and all mortalities were recorded. VVΔE3L was greater than 4 logs less neurovirulent than wtVV (
Groups of 5 SCID mice were infected intranasally with different doses of wtVV or with VVΔE3L. The LD50 for wtVV was less than 100 pfu. VVΔE3L did not kill mice, even at the highest dose administered (
Groups of 5 C57BL/6 mice were immunized with different doses (ranging from 10 to 10,000 pfu) of recombinant vaccinia virus expressing the β-galactosidase gene in place of VV E3L. One month later the immunized mice and the unimmunized controls (mock) were challenged with 106 pfu of wtVV. The results depicted in
Low doses of VVΔE3L (10, 100, and 1000 pfu) were administered by intranasal route to C57BL/6 mice. The noses of 3 mice infected with each dose of each virus were harvested on post-infection days 2, 4, and 6. The harvested tissues were processed and titrated in RK-13 cell line. The average viral load in the nose of mice infected on each of those days is shown in
Groups of 5 C57BL/6 mice were immunized with different doses (ranging from 10 to 10,000 pfu) of UV-inactivated VV. One month later the immunized mice and the unimmunized controls (mock+wtVV) were challenged with 106 pfu of wtVV. Weight loss was used as an indicator of disease due to wtVV. Severe weight loss was recorded in all the mice vaccinated with the UV-inactivated virus (
Groups of 5 C57BL/6 mice were infected intranasally with 104 pfu of wtVV (approximately 1 LD50). One day later animals were immunized with 106 pfu of VVΔE3L. All unimmunized animals died, while animals immunized post-exposure with VVΔE3L survived (
RK-13 cells were set down at 50% confluency in 6 well plates. Four hours later the media overlaying the cells were replaced with media containing different doses of rabbit interferon (480 U/μl, Lee Biomolecular) so that the final concentration of IFN ranged from 0 U/ml to 1000 U/ml of cell culture media. Sixteen hours following IFN treatment these cells were infected with 100 pfu of each virus. Wt VV was used as a positive control since it is resistant to the effects of IFN. VVdelE3L was used as a negative control. The infected cells were incubated at 37° C. and 5% CO2 for 36-48 hours following which they were stained with 0.1% crystal violet in 20% ethanol for 1 minute at room temperature. The plaques were counted in each well and plaque reduction with increasing doses of IFN was computed as a percentage.
VVΔE3L was sensitive to IFN and unable to plaque in RK-13 cells pretreated with 100 and 1000 U/ml of rabbit IFNβ. This virus was also unable to plaque in HeLa cells even in the absence of IFN treatment (Beattie E et al., 1996, Virus Genes 12(1), 89-94).
Rabbit kidney (RK-13) cells were maintained in Eagle's Minimal Essential Medium (Gibco BRL) supplemented with 5% fetal bovine serum (Irvine Scientific), 50 μg/ml gentamycin sulfate and 0.1 mM non-essential amino acids and vitamins (Gibco BRL). Baby Hamster Kidney (BHK-21) cells were maintained in the same media containing the same amount of non-essential amino acids, vitamins and gentamycin but supplemented with 10% fetal bovine serum. HeLa cells (S3 HeLa cells from ATCC and S3 HeLa cells generously provided by Frederick) were grown in Dulbecco's Modified-Minimal Essential Medium (DMEM) (Gibco BRL) containing 5% fetal bovine serum and 50 μg/ml gentamycin sulfate. Unless otherwise notified, HeLa cells used in this study were S3 HeLa cells from Frederick. All cells were incubated at 37° C. in the presence of 5% CO2.
The eIF2α homolog gene from Ambystoma tigrinum virus (ATV) kindly provided by James Jancovich, was recombined into the E3L locus of vaccinia virus (VV) deleted for ESL (VVΔE3L), using the plasmid pMPE3LΔgptMCS (previously described by Kibler et al., 1997, J Virol 71(3):1992-2003). The eIF2α homolog gene was attained by PCR from the plasmid pSTBlue-1 (clone was kindly provided by James Jancovich), with primers containing a BamHI site at the 5′end and a SalI at the 3′ end of the gene (5′ ATT AGG ATC CGC CAT GGC ACA CAA CAG GTT TTA CAG 3′, SEQ ID NO:4; 5′ ATT AGT CGA CAT ATC ACA CAA AGG GGC ACA GTC C3′, SEQ ID NO:5). The PCR products were gel extracted (Qiagen), purified and digested with BamHI and SalI, and ligated into pMPE3LΔgptMCS digested with the same enzymes. Colonies were screened by restriction digests with BamHI and SalI and also by sequencing. Thus this ligation resulted in the formation of pMPE3LΔgptMCS-ATV eIF2α.
In vivo recombination (IVR) was performed as described by Kibler et al. (1997, J Virol 71(3):1992-2003). The recombination vector pMPE3LΔgptMCS has the E. coli gpt gene as a selectable marker and regions homologous to the flanking arms of the E3L gene in VV on either side of the multiple cloning site to facilitate recombination into the E3L locus. Ecogpt is the E. coli guanosine phosphoribosyl transferase gene. This gene allows conversion of xanthine to guanine monophosphate (GMP), without needing the intermediate steps of converting inosine monophosphate (IMP) to xanthine monophosphate (XMP), ultimately resulting in GMP. This is useful in selecting recombinants in the presence of mycophenolic acid (MPA), which is responsible for inhibiting eukaryotic production of GMP, by blocking the conversion of IMP to XMP. Adding xanthine and MPA to the ecogpt selection media, pressure can be applied to select for recombinants containing the pMPE3LΔgptMCS plasmid with the desired insert. In the presence of MPA only the viruses expressing the gpt gene are able to grow thus enabling selection of recombinants.
VVΔE3L, the virus used for recombining this gene into the E3L locus of VV, has a LacZ gene in place of E3L thus allowing for blue white selection of the recombinant virus using X-gal substrate at a final concentration of 200 μg/ml. Subconfluent BHK cells were used for both transfection and in vivo recombination. Cells were set down in 35 mm dishes and were pretreated for 30 minutes with 2 ml of Opti-MEM (Gibco BRL) at 37° C. The cells were transfected with 1 μg of plasmid DNA using LipofectACE (Gibco BRL) as per manufacturer's instructions. The cells were simultaneously infected with VVΔE3L at a multiplicity of infection (moi) of 0.05, allowed to incubate for 1 hour, with rocking at 37° C., 5% CO2 and overlaid with Opti-MEM containing 1% FBS. At 36 hours post infection, the cells were harvested, pelleted by centrifugation at 1000 rpm, 4° C. for 10 minutes and resuspended in 200 μl of 1 mM Tris HCl pH 8.8. Cells were then subjected to three rounds of freezing and thawing in order to release the virus from the cells.
The resultant recombinant virus from above was used to infect 60 mm dishes of confluent BHK cells which had been pretreated for 6 hours prior to infection with ecogpt selection media (complete BHK media containing 10% FBS, 10 μg/ml MPA, 250 μg/ml xanthine and 15 μg/ml hypoxanthine). The infection was carried out for 1 hour and then overlaid with 5 ml of ecogpt selection media. 48-72 hours post infection, the liquid overlay media was replaced with 1.5% agarose supplemented with complete 2×MEM media, 10 μg/ml MPA, 250 μg/ml xanthine, 15 μg/ml hypoxanthine and 200 μg/ml X-gal (Gold Biotechnology, Inc.). 6 hours later blue plaques were picked into 200 μl of 1 mM Tris HCl pH 8.8 and three rounds of freeze thaws were completed. This whole process was repeated three times in order to pick a purified blue plaque. After this selection, the removal of the ecogpt media causes a second recombination event to occur. By removing the MPA selective pressure, VVΔE3L either removes the lacZ gene in place of the E3L gene, keeping the ATV eIF2α homolog gene or, it can remove the ATV eIF2α homolog gene and retain the original lacZ in place. Virus that has retained the lacZ gene will appear as blue plaques in the presence of X-gal, and viral plaques that have retained the ATV eIF2α homolog gene will be clear or colorless. The fourth round of plaquing was done in RK-13 cells in the absence of ecogpt selection media and when plaques were visible the cells were overlaid with 1.5% agarose, complete 2×MEM media and 0.2 mg/ml X-gal. Both clear and blue plaques were visible. 6 hours later the clear plaques were picked in 200 μl of 1 mM Tris HCl pH 8.8 and plaque purified for two more rounds when only clear plaques were visible.
In order to make a working stock of virus, purified plaques must be amplified. BHK cells were set down in five 100 mm dishes with a confluency of 100%. Each dish was infected with a mixture of 40 μl of the viral plaque resuspended in 160 μl of 1×MEM, 2% FBS and 50 μg/ml gentamycin sulfate. After one hour of infection at 37° C., 5% CO2 cells were overlaid with complete BHK media. The infection was allowed to proceed to 100% cytopathic effect (CPE). Cells were then harvested and pelleted by centrifugation at 1000 rpm, 4° C., for 10 minutes. The supernatant was removed and the pellet was resuspended in 1 ml of 1×MEM containing 2% FBS and 50 μg/ml gentamycin sulfate. Three rounds of freeze thaws were done to release the virus from the cells and the cells were centrifuged at 1000 rpm, 4° C., for 10 minutes to spin down the cell debris. Aliquots of 200 μl of the supernatant were transferred into cryogenic tubes and stored at −80° C.
RK-13 cells were used to calculate the titer of the virus in plaque-forming units per ml (pfu/ml). Cells were set down at 100% in 6 well plates and infected with serial dilutions of the amplified virus. The dilutions ranged from 10−3 to 10−8 and each well of the plate was infected with 100 μl of each dilution. The infection was allowed to proceed until the plaques were visible. Cells were then stained with 0.1% crystal violet in 20% ethanol. The number of plaques were counted in a well that has 30-300 plaques and the titer was determined by multiplying the number of plaques with the dilution factor.
100 μl of ATV virus stock of 108 pfu/ml titer was used to extract DNA for viral PCR. To the 100 μl of virus stock, 100 μl of phenol was added and centrifuged at 10000 rpm for 5 minutes at 4° C. The aqueous layer from this step was re-extracted with 50 μl phenol and 50 μl chloroform isoamyl alcohol (24:1) and centrifuged as above. The aqueous layer from this step was again extracted with 100 μl chloroform isoamyl alcohol (24:1) and precipitated with 2.5 volumes of 100% ethanol and 1/10 volume of 3 M sodium acetate, pH 5.2. The DNA was rinsed twice with 70% ethanol, dried and resuspended in 10 μl glass distilled water.
The ATV eIF2α, gene was amplified from this viral DNA by PCR using primers that anneal to the flanking regions of the E3L gene (5′CGAACCACCAGAGGATG 3′ (SEQ ID NO:6) and 5′ TAGTCGCGTTAATAGTACTAC 3′ (SEQ ID NO:7)). The amplified product was run on 0.8% GTG agarose (FMC Bioproducts) gel and the band was cut out. The DNA was extracted from the band of interest by first freezing the band overnight at −80° C. and then thawing it at 37° C. for 30 minutes. The supernatant was separated by centrifugation at 4° C. for 15 minutes at 10,000 g and subjected to ethanol precipitation. The DNA was resuspended in 10 μl of glass distilled water. 1 μl of each of the primers (10 μM stock) used for PCR was added to 5 μl of DNA and sequenced by Dideoxy method.
The eIF2α homolog from ATV has sequence homology to the eukaryotic translation initiation factor, eIF2α and also to the K3L of vaccinia virus (
RK-13 cells were seeded at 50% confluency in 6 well plates. 4 hours later the wells were treated with 0, 1, 10, 100 and 1000 U/ml of rabbit interferon (Lee Biomolecular). 16 hours post interferon treatment, cells were infected with 200 pfu of wtVV, VVΔE3L, and VVΔE3L/ATV eIF2α virus. Wild type vaccinia virus (wtVV) was used as a positive control as it is resistant to the effects of IFN and VVΔE3L was used as a negative control as it is sensitive to IFN. The infected cells were incubated at 37° C., 5% CO2 for 24 hours. Then they were stained with 0.1% crystal violet in 20% ethanol. The plaques were counted in each well and plaque reduction with increasing doses of IFN was computed as a percentage.
Wild type vaccinia virus (wtVV) is resistant to the antiviral effects of IFN and plaques in RK-13 cells pretreated with rabbit interferon up to 1000 units/ml. It has been shown that the N-terminal domain of E3L is necessary and sufficient for IFN resistance (Kibler et al., 1997, J Virol 71(3):1992-2003; Shors et al., 1997, Virology 239(2):269-76). VVΔE3L is IFN sensitive (Shors et al., 1997, Virology 239(2):269-76) and is totally unable to replicate in the presence of 100 units/ml in RK-13 cells. To determine if the eIF2α homolog can provide IFN resistance to VVΔE3L, 6 well plates of confluent RK-13 cells were pretreated with 0, 1, 10, 100 and 1000 units/ml of rabbit IFN. Each well was infected with 200 pfu of wtVV, VVΔE3L, VVΔE3L/ATV eIF2α, and stained with crystal violet once plaques were visible. The number of plaques were counted and recorded in Table 1.
Note. HeLa cells and RK-13 cells (+/−IFN) were infected with 200 Pfu's of wt VV, VVΔE3L, VVΔE3L/ATV eIF2α and 24 hours post infection plates were stained with 0.1% crystal violet in 20% ethanol and the number of plaques were counted and shown in the table above.
wtVV has a broad host range and is able to form plaques in HeLa cells while VVΔE3L is unable to replicate in HeLa cells, and therefore has a limited host range. The ability of the recombinant virus (VVΔE3L/ATV eIF2α) to plaque in HeLa and RK-13 cells was determined by infecting 6 well plates of these cell lines at 90% confluency. Cells were infected with serial dilutions of each of the viruses. In some assays, cells were infected with 200 pfu of the virus. wtVV and VVΔE3L were used as positive and negative controls respectively. Infection was continued until the formation of visible plaques, following which cells were stained with 0.1% crystal violet in 20% ethanol and the plaques were counted.
The recombinant virus was able to form plaques in RK-13 cells but unable to plaque in HeLa cells. The plaquing efficiency in HeLa cells is shown in Table 1. Despite this, it has a broad host range as it replicates like wtVV in HeLa cells and is described in the next section.
The activation of the two IFN-induced enzymes, PKR and OAS, leads to shutoff of protein synthesis in the infected cell. Activated PKR phosphorylates the a subunit of translation initiation factor, eIF2, thus effectively blocking protein synthesis (Levin and London, 1978, Proc Natl Acad Sci US A 75:1121-25; Samuel, 1979, Proc Natl Acad Sci USA 76(2):600-4). To confirm the IFN sensitivity in RK-13 cells and inability to plaque in HeLa cells of the recombinant virus (VVΔE3L/ATV eIF2α), in vivo labeling assays, using [35S] methionine, were performed in RK-13 cells and HeLa cells in the presence and absence of 100 units/ml of IFN.
60 mm dishes containing confluent monolayers of RK-13 cells in the presence or absence of 100 U/ml of rabbit interferon and HeLa cells in the presence or absence of 100 U/ml of human α A/D interferon were mock infected or infected with wtVV, VVΔE3L and VVΔE3L/ATV eIF2α at a moi of 5. The infected cells were allowed to incubate for 5 hours and 30 minutes. Cells were washed twice with 1×PBS and incubated for 30 minutes (at 37° C., 5% CO2) in 2 ml labeling media (methionine-free, serum-free MEM (Sigma)) to starve the cells of methionine. Then the starvation media was removed, cells were washed twice with 1×PBS and incubated in labeling media containing 50 μCi/ml [35S] methionine (Dupont NEN) for another one hour. Labeling media was removed and the cells were scraped into 1×PBS. The cells were pelleted by centrifugation and lysed in 100 μl of NP40 lysis buffer (20 mM HEPES, pH 7.5, 120 mM KCl, 5 mM MgCl2, 1 mM DTT, 10% glycerol and 0.5% NP40). The cells were incubated on ice for 10 minutes and pelleted by centrifugation. The supernatant was stored at −80° C. and run onto a 12% SDS-PAGE gel. The gel was stained in Coomassie blue, destained, dried and exposed to X-ray film for 24-48 hours.
A shutoff of translation in VVΔE3L in RK-13 cells treated with IFN and in HeLa cells, at 6 hours post infection (hpi), but not in mock infected cells or cells infected with wtVV can be seen from
40-50% confluent HeLa cells were seeded in 60 mm dishes and were mock infected or infected with wtVV, VVΔE3L, VVΔE3L/ATV eIF2α at a moi of 5. Six hours post infection (hpi) the cells were scraped into the medium and pelleted by centrifugation at 1000 rpm, 4° C., for 10 minutes. The supernatant was removed and the cell pellet was washed in 1 ml of 1×PBS. The infected cells were then lysed using 100 μl of RIPA buffer (1×PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SIDS and 50 mM sodium fluoride), incubated on ice for 10 minutes and then centrifuged at 10,000 g at 4° C. for 10 minutes. 80 μl of the supernatant was transferred to a fresh microfuge tube containing 80 μl of 2×SDS sample buffer and stored at −80° C. until use in western blot analysis.
Cytoplasmic extracts from the above assay were subjected to SDS PAGE electrophoresis in a 12% polyacrylamide gel at 188 volts for 1 hour. The proteins were transferred to nitrocellulose membrane (Osmonics) at 100 volts for 45 minutes in transfer buffer (10 mM CAPS, pH 11.0, 20% methanol, 14 mM P-ME). The nitrocellulose membrane was blocked for one hour in blotto with milk (20 mM Tris-HCl, pH 7.8, 180 mM NaCl, 3% carnation nonfat dry milk). The blot was incubated overnight at 4° C. with the primary antibody raised in rabbit (1:500 dilution), which recognizes only the phosphorylated form of eIF2 (Research Genetics). The primary antibodies were removed and the blot was washed 3 times with blotto containing milk for 1 hour at room temperature. Then the blot was probed with 1:5000 dilution of goat anti-rabbit IgG-peroxidase conjugate (Sigma). The secondary antibodies were removed and the blot was washed 3 times for 10 minutes each in blotto with milk, and then washed 3 times for 5 minutes each in blotto without milk. The blot was developed by chemiluminiscence.
In response to virus infection, PKR is activated by dsRNA and in turn phosphorylates eIF2α, thus inhibiting protein synthesis. To determine if the recombinant virus can lead to phosphorylation of eIF2α, HeLa cells were mock infected or infected with wtVV, VVΔE3L, VVΔE3L/ATV eIF2α at a moi of 5. Cells were harvested at 6 hpi and the resulting total cell lysates were analyzed by chemiluminiscent western blot as described in Examples 1 and 23. eIF2α was found to be phosphorylated in cells infected with VVΔE3L but not with wtVV or VVΔE3L/ATV eIF2α (
Virus infection leads to the production of dsRNA in the infected cell. dsRNA is required for the activation of PKR which then becomes autophosphorylated and acts on its substrates. PKR activation can be easily detected by looking for its phosphorylation using a chemiluminiscent western blot.
HeLa cells were seeded at 50% confluency in 60 mm dishes and mock infected or infected with wtVV, VVΔE3L and VVΔE3L/ATV eIF2α at a moi of 5. Cells were harvested at 2, 4 and 6 hpi by scraping cells into the medium and pelleting by centrifugation at 1000 rpm, 4° C., for 10 minutes. The supernatant was removed and the cell pellet was washed in 1 ml of 1×PBS. The infected cells were then lysed using 100 μl of RIPA buffer (1×PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM sodium fluoride), incubated on ice for 10 minutes and then centrifuged at 10,000 g at 4° C. for 10 minutes. 80 μl of the supernatant was transferred to a fresh microfuge tube containing 80 μl of 2×SDS sample buffer and stored at −80° C. until use in western blot analysis.
Whole cell extracts were then analyzed for PKR activation by a western blot using anti-PKR antibody which recognizes both phosphorylated and non-phosphorylated PKR. Phosphorylated form is recognized by a shift on the gel as it runs higher compared to the non-phosphorylated form. Whole cell extracts were then run on 10% SDS-PAGE gel, proteins were transferred onto the nitrocellulose membrane and probed with 1:1000 dilution of mouse anti-PKR antibody (Transduction labs). The blot was developed by chemiluminiscence. The same assay was also performed by treating cells with Cytosine β-D arabinofuranoside (AraC, Sigma, 5 mg/ml stock) to a final concentration of 40 μg/ml, one hour prior to the infection. Arac was also added to the inoculum and also to the medium used to overlay HeLa cells after infection. Infected cells were harvested 6 hpi and analyzed in the same way as described above.
On the other hand, infection with VVΔE3L/ATV eIF2α leads to the degradation of PKR by 4 hpi (lane 4), thus indicating that somehow the eIF2α homolog is acting as a PKR inhibitor. To determine if this degradation is mediated by dsRNA or not, HeLa cells were pretreated with AraC for one hour prior to infection. AraC inhibits DNA synthesis, thus blocking the formation of dsRNA at late times post infection. Cells were then mock infected or infected with wtVV, VVΔE3L, VVΔE3L/ATV eIF2α at a moi of 5 and harvested at 6 hpi. The cell extracts were then analyzed by western blot following the same procedure as described above for PKR activation.
It has been demonstrated that activation of endogenous RNase L or increased production of enzymes in the 2-5A pathway causes degradation of rRNA into characteristic discrete fragments that correlated with apoptosis (Diaz-Guerra et al., Virology 227:220-228; Diaz-Guerra et al., 1997, Virology 236:354-363). The following assay was performed to investigate the activity of 2-5A pathway in the recombinant virus infected cells.
100 mm dishes with confluent monolayers of S3 HeLa cells from both ATCC and Frederick were mock infected or infected with wtVV, VVΔE3L, VVΔE3L/ATV eIF2α at a moi of 5. 18 hpi cells were scraped into the media and pelleted by centrifugation. The pellets were washed with 1×PBS and pelleted again. After removing all of the supernatant from the pellet, the cells were resuspended in 500 μl of RNase-free Tris-buffered saline (TBS) and immediately followed by 1:1 phenol: chloroform extraction. The sample was centrifuged for 10 minutes at 4° C. at 10,000 rpm. The top layer was transferred to a sterile eppendorf tube and a 1:1 dilution of chloroform:isoamyl alcohol was added. The samples were again centrifuged and the top layer was transferred into a sterile eppendorf tube. The samples were then treated with 1/10 volume of DEPC treated 3 M sodium acetate, pH 5.0 and then 2.5 volumes of ethanol was added. The RNA was allowed to precipitate for 30 minutes at-80° C. The samples were centrifuged at 10,000 rpm, 4° C. for 30 minutes to pellet the RNA. The RNA pellet was dried and resuspended in 20 μl of 0.5% SDS-DEPC water. For analysis, denatured RNA was fractionated on 1.8% formaldehyde-agarose gels and stained using ethidium bromide buffer.
Equal amounts of RNA were fractionated in 1.8% agarose-formaldehyde gels as shown in
Single step growth curves were performed in HeLa cells. HeLa cells were seeded at 80% confluency in 60 mm dishes and infected with wtVV, VVΔE3L and VVΔE3L/ATV eIF2α at a moi of 5. After one hour, the inoculum was removed by washing cells with 1×PBS and overlaid with complete HeLa media. Cells were harvested at 0 hpi and 30 hpi by scraping cells into the media and pelleting down by centrifugation at 1000 rpm, 4° C. for 10 minutes. The pellet was resuspended in 200 μl of 1 mM Tris HCl, pH 8.8 and subjected to three rounds of freeze thaws. The resultant virus was titered in RK-13 cells.
As shown in
HeLa cells were seeded at 50% confluency in 60 mm dishes and infected with wtVV, VVΔE3L and VVΔE3L/ATV eIF2α at a moi of 0.01. After one hour, the inoculum was removed by washing cells with 1×PBS and overlaid with complete HeLa media. Cells were harvested at 0 hpi and 72 hpi by scraping cells into the media and pelleting down by centrifugation at 1000 rpm, 4° C. for 10 minutes. The supernatant was saved and the pellet was resuspended in 200 μl of 1 mM Tris HCl, pH 8.8 and subjected to three rounds of freeze thaws. The resultant virus was titered in RK-13 cells.
As shown in
One way to test for IFN induction is to treat HeLa cells with the cultured supernatant from the multi-step growth curve (Example 26) along with the presence or absence of anti IFN-β antibody and then infect those cells with Vesicular Stomatitis Virus (VSV). The rationale behind this is, since VSV is IFN sensitive, it should not grow in the supernatant-treated cells if there is IFN present in that supernatant. Conversely, VSV should grow in those cells which have anti IFN-β antibody along with the cultured supernatant.
Sub confluent HeLa cells were mock treated or treated with the cultured supernatant from the above multi-step growth curve in the presence or absence of 3000 neutralization units of human anti IFN-β antibody (Calbiochem). 16 hours post treatment, cells were infected with 100 pfu of Vesicular Stomatitis Virus (VSV). After one hour the cells were overlaid with 1.5% agarose supplemented with complete 2×MEM media. 24 hours later cells were overlaid with 1.5% agarose supplemented with complete 2×MEM media containing neutral red solution (1.5 ml of a 3.33 g/L solution (GIBCO) per 50 ml of medium) and 6 hours later the number of plaques of VSV were counted.
It is evident from
Another way to test for IFN induction is to look for IRF-3 phosphorylation. IRF-3 is a transcription factor that is activated upon virus infection by phosphorylation (Lin et al., 1998, Mol Cell Biol 19:2986-96; Sato et al.; 1998, FEBS Lett 452:112-16; Weaver et al., 1998, Mol Cell Biol 18:1359-68; Yoneyama et al., 1998, EMBO J17:1087-95), translocates to the nucleus (Sato et al., 1998, FEBS Lett 452:112-16; Yoneyama et al., 1998, EMBO J17:1087-95) and leads to the induction of IFN-α and IFN-β genes (Juang et al., 1998, Proc Natl Acad Sci USA 95:9837-42; Sato et al., 1998, FEBS Lett 452:112-16; Schafer et al., 1998, J Biol Chem 273:2714-20; Yoneyama et al., 1998, EMBO J. 17:1087-95).
HeLa cells were seeded at 80% confluency in 100 mm dishes and infected with wtVV, VVΔE3L and VVΔE3L/ATV eIF2α at a moi of 5. Cells were harvested at 6 hpi and nuclear extracts were prepared as described elsewhere (Servant et al., 2001, J Biol Chem 276(1):355-63). The extracts were then run on 10% SDS-PAGE gel, proteins were transferred onto the nitrocellulose membrane and probed with rabbit anti-IRF-3 antibody (kindly provided by Dr. Michael David, University of California at San Diego). The western blot was developed by chemiluminiscence.
IRF-3 was detected in the nucleus only in VVΔE3L and VVΔE3L/ATV eIF2α infected cells but not in mock or wtVV infected cells (
4 male C57BL/6 mice were immunized with different doses (ranging from 2 to 5,000 plaque forming units) of recombinant vaccinia virus with ATV eIF2α homolog in place of its E3L and with 1000 pfu's of VVΔE3L. One month later the immunized mice and the unimmunized controls (mock) were challenged with a 106 pfu of wt VV. Weight loss was used as an indicator of disease due to wt VV. Severe weight loss was observed in the unimmunized control. Weight loss of mice vaccinated with 2 and 20 pfu of VVΔE3L/ATV eIF2α was not significantly different from mock-vaccinated mice challenged with wt VV but both 300 and 5000 pfu were significantly different (
Four C57BL/6 mice were infected with each dilution of VVΔE3L/ATV eIF2α and 104 dilution of wt VV as indicated in the figure. Deaths were recorded. The LD50 of wt VV was observed on day 9 post infection. The LD50 of VVΔE3L/ATV eIF2α could not be calculated as even the highest dose did not kill 50% of the mice (
The present invention relates, in part, to experiments directed to elucidating the role of the eIF2α homolog from ATV, a member of the genus Ranavirus, family Iridoviridae. Several isolates exhibit a gene encoding a protein with homology to eukaryotic eIF-2α and the corresponding VV protein, K3L (Essbauer et al., 2001, Virus Genes 23(3):347-59). eIF2α homolog from ATV has 35% identity with and 55% similarity to the human eIF2α protein and 30% identity with and 51% similarity to the K3L protein of vaccinia virus. Its N-terminus shows significant homology to the amino terminus of human eIF2α protein and to the full length K3L protein. K3L of VV binds to PKR as a pseudosubstrate (Sharp et al., 1997, Eur J Biochem 250(1):85-91) and acts as an inhibitor of PKR. Since a high sequence similarity exists between K3L and eIF2α homolog, the latter could potentially act as a PKR inhibitor.
A vaccinia virus system was chosen to characterize eIF2α homolog from ATV. VV is a useful recombinant vector as it is easy to delete and insert genes from its genome. The E3L gene of VV confers the interferon resistance phenotype to the virus. So VV deleted of the E3L gene has turned out to be an excellent system for in vivo analysis of function of exogenous proteins (Jacobs et al., 1998, Methods: A comparison to methods in enzymology 15:225-232). Insertion of ATV eIF2α homolog into the E3L locus of VV allows its ability to regulate the IFN response to be examined. Even though ATV eIF2α is homologous to K3L of VV, it was the E3L gene of VV that was replaced with ATV eIF2α homolog because the phenotypic characteristics of VVΔE3L are more readily observable and allow examination the entire IFN response. The results of this work established that the eIF2α homolog from iridoviruses acts a novel inhibitor of PKR by leading to its proteolytic degradation.
Nothing in this example should be construed to limit the scope of the invention. Rather, this example is provided merely to illustrate further aspects that are within the contemplation of the invention.
Poliovirus infection leads to the degradation of PKR and degradation requires both an RNA and protein component. Poliovirus infected cell extracts pretreated with trypsin, RNase A, or RNase III were no longer able to degrade PKR, suggesting that a protein and an RNA component with both single-stranded and double-stranded characteristics was required for PKR degradation (Black et al., 1993, J Virol 67(2), 791-800). By in vitro experiments, it was also shown that kinase activation was not a prerequisite for the degradation of PKR by poliovirus and that preincubation with dsRNA could block the poliovirus-mediated degradation of PKR (Black et al., 1993, J Virol 67(2), 791-800). The evidence suggests that a similar phenomenon must be occurring in VVΔE3L/ATV eIF2α, infected cells by which the eIF2α homolog is able to induce the degradation of PKR and this degradation might be mediated by dsRNA. As proposed by Black et al., 1993 (J Virol 67(2), 791-800), it is possible that in Poliovirus infected cells, PKR undergoes some conformational change upon interaction with dsRNA and this alteration in structure then enables a cellular protease to degrade the kinase or alternatively, the dsRNA and protease act in concert, possibly as a ribonucleoprotein complex, to proteolyze the kinase directly and completely. It is interesting to note that the Ranaviruses with an eIF2α homolog might regulate PKR by a similar mechanism.
Cellular protein synthesis is reduced in FV-3 (type species of the genus Ranavirus) infected fish cells and phosphorylation of the a subunit of eIF2 has been observed in these infected cells thus suggesting that translational shut-off may be due to activation of a kinase that selectively phosphorylates eIF2α (Chinchar and Dholakia, 1989, Virus Res 14(3):207-23). While selectively blocking host cell translation, FV-3 can synthesize more than 60 virus-specific polypeptides (Chinchar and Yu, 1990, Virus Res 16(2):163-74). These results suggest that the selective translation of FV-3 messages in virus-infected cells may partly be due to the higher translational efficiency of viral messages.
IRF3/7 activation can be stimulated by dsRNA and a novel cellular kinase distinct from PKR is responsible for its phosphorylation. E3L protein of VV exerts its anti-IFN effect by inhibiting not only PKR but also the distinct IRF3/7 kinase(s) (Smith et al., 2001, J Biol Chem 276(12):8951-7). While wtVV inhibits IRF-3 phosphorylation, infection with VVΔE3L leads to its phosphorylation. Infection with VVΔE3L/ATV eIF2α also leads to the phosphorylation of IRF-3 suggesting that the ATV eIF2α homolog is not able to inhibit the induction of IFN. Translocation of IRF-3 to the nucleus was seen even in the absence of PKR in VVΔE3L/ATV eIF2α infected cells, supporting the data that PKR is not the kinase responsible for phosphorylating IRF-3.
To test for the anti-IFN activity of iridoviral eIF2α, chicken embryo fibroblast (CEC-32) cells were transfected with iridoviral eIF2α and then infected with VSV (Essbauer et al., 2001, Virus Genes 23(3):347-59). Their results found no significant differences between the viability of control cells and eIF2α transfected CEC-32 cells. Also, their attempts to counteract the antiviral effects of chicken IFN by iridoviral eIF2α have failed. The results indicate that the ATV eIF2α can partially counteract the antiviral effects of mammalian IFN by inhibiting PKR. It is possible that the ATV eIF2α homolog is able to recognize mammalian PKR, suggesting the presence of a PKR-like enzyme in salamanders. Subsequently, ATV eIF2α might be involved in evading host defenses in salamanders.
Even though the eIF2α homolog is homologous to K3L of VV, the homolog has a better effect than K3L as the recombinant virus was able to partially rescue VVΔE3L. Since the C-terminus of the homolog has no homology to any known protein, further work is needed to explore the function of the C-terminus. Since K3L binds to PKR and acts as a pseudosubstrate, it would be interesting to see if the homolog can also interact directly with PKR. KGYID (amino acids 73-78 of SEQ ID NO:2), the motif required for interaction of K3L with PKR (Kawagishi et al., 1997, Mol Cell Biol 17(7):4146-58; Kawagishi et al., 2000, Virology 276(2):424-34; Sharp et al., 1997, Eur J Biochem 250(1):85-91) is modified to KGYVD (amino acids 81-85 of SEQ ID NO: 1) in all iridoviral eIF2α proteins. This modification may have an effect on the possible interaction of eIF2α homolog with PKR.
The results suggest that the salamander virus ATV contains a novel gene that may counteract host defenses, emphasizing that there is an evolutionary significance in obtaining this gene in its genome. Since the eIF2α homolog was able to inhibit part of the mammalian IFN system, salamanders may have an antiviral state similar to mammals.
This application claims the benefit of U.S. Provisional Application Nos. 60/397,907 filed Jul. 24, 2002 and 60/399,815 filed Jul. 31, 2002. The contents of these Applications and the documents cited therein are hereby incorporated herein in their entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60397907 | Jul 2002 | US | |
| 60399815 | Jul 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US03/21764 | Jul 2003 | US |
| Child | 11022477 | Dec 2004 | US |
