The present invention relates to vaccines having an increased level of safety comprising recombinant vaccinia viruses containing an inactivated E3L region. The invention also 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. The invention further relates to an interferon-sensitive recombinant vaccinia virus with broad host range wherein a salamander eIF2.alpha. is inserted into the viral genome in place of at least a portion of the E3L gene.

Description of the Invention

INTRODUCTION

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.

BACKGROUND

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 .alpha. subunit of protein synthesis initiation factor 2, eIF-2.alpha. (Samuel, 1979, Proc Natl Acad Sci USA 76(2):600-4). Phosphorylation of eIF-2.alpha. 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-2.alpha.-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 (VV.DELTA.E3L) 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 VV.DELTA.E3L's sensitivity to pretreatment of rabbit kidney RK-13 cells with IFN-.beta. and its inability to rescue Vesicular Stomatitis Virus from the effects of IFN (Shors et al., 1998, Virology 239(2):269-76).

VV.DELTA.E3L 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). VV.DELTA.E3L infection also induces apoptosis in IFN-treated RK-13 cells (Kibler et al., 1997, J Virol 71(3):1992-2003). Infection with VV.DELTA.E3L leads to activation of PKR and increased phosphorylation of eIF2.alpha. (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 VV.DELTA.E3L 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 VV.DELTA.E3L.

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 eIF2.alpha. homolog (Yu et al., 1999, Virus Res 63(1-2):53-63). Essbauer et al., 2001 have analyzed the eIF2.alpha. of several iridoviruses of fish and frogs (Virus Genes 23(3):347-59). eIF2.alpha. 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 eIF2.alpha. showed a significant homology to the N-termini of cellular initiation factor 2-.alpha. of various species and full-length poxyiral K3L protein. The eIF2.alpha. iridoviral protein had 37% identity with and 48% similarity to the N-terminus of human eIF2.alpha. 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 eIF2.alpha., 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 eIF2.alpha. amino acid sequences. Since the C-terminus of ranaviral eIF2.alpha. 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 poxviral homologs (Essbauer et al., 2001, Virus Genes 23(3):347-59). Thus, it is unclear whether the iridovirus homolog is acting as an eIF2.alpha. kinase inhibitor, or given its large size, as an alternative eIF2.alpha.-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, eIF2.alpha.. The role of this gene, if any, in ATV's ability to suppress antiviral host cell responses had not previously been determined.

SUMMARY OF THE INVENTION

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 eIF2.alpha. 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 eIF2.alpha. 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.

DETAILED DESCRIPTION OF THE INVENTION

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 (FIGS. 1 and 2 and Brandt and Jacobs, 2001, J Virol 75(2): 850-6, see Original Patent) and immune suppressed (FIG. 3, see Original Patent) mice than wild type vaccinia virus. Despite the lack of virulence and poor replication in cells in culture this virus is highly immunogenic (FIG. 4, see Original Patent). Induction of a protective response correlated with replication of this virus in nasal mucosa (FIGS. 5 and 6, see Original Patent). This virus could also protect against challenge with a pathogenic poxvirus when administered one day after challenge (FIG. 7, see Original Patent). Thus, the present invention provides a viral vector that is a safe, effective vaccine for smallpox.

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 eIF2.alpha. 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 2.alpha. gene (eIF2.alpha.) 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 eIF2.alpha. 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 VV.DELTA.E3L 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, "Poxviridae: 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.
 

Claim 1 of 6 Claims

1. A recombinant vaccinia virus comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1.

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