Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (Piccini et al., 1987).
Specifically, the recombinant poxviruses are constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of poxviruses such as the vaccinia virus and avipox virus described in U.S. Pat. Nos. 4,769,330, 4,722,848, 4,603,112, 5,110,587, and 5,174,993, the disclosures of which are incorporated herein by reference.
First, the DNA gene sequence to be inserted into the virus, particularly an open reading frame from a non-pox source, is placed into an E. coli plasmid construct into which DNA homologous to a section of DNA of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA containing a nonessential locus. The resulting plasmid construct is then amplified by growth within E. coli bacteria (Clewell, 1972) and isolated (Clewell et al., 1969; Maniatis et al., 1982).
Second, the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential region of its genome, of foreign DNA sequences. The term "foreign" DNA designates exogenous DNA, particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the exogenous DNA is placed.
Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA. In certain viruses RNA may replace DNA. Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.
Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.
However, recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome. Additional strategies have recently been reported for generating recombinant vaccinia virus.
Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable. The second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.
Vaccinia virus has been used successfully to immunize against smallpox, culminating in the worldwide eradication of smallpox in 1980. In the course of its history, many strains of vaccinia have arisen. These different strains demonstrate varying immunogenicity and are implicated to varying degrees with potential complications, the most serious of which are post-vaccinial encephalitis and generalized vaccinia (Behbehani, 1983).
With the eradication of smallpox, a new role for vaccinia became important, that of a genetically engineered vector for the expression of foreign genes. Genes encoding a vast number of heterologous antigens have been expressed in vaccinia, often resulting in protective immunity against challenge by the corresponding pathogen (reviewed in Tartaglia et al., 1990a).
The genetic background of the vaccinia vector has been shown to affect the protective efficacy of the expressed foreign immunogen. For example, expression of Epstein Barr Virus (EBV) gp340 in the Wyeth vaccine strain of vaccinia virus did not protect cottontop tamarins against EBV virus induced lymphoma, while expression of the same gene in the WR laboratory strain of vaccinia virus was protective (Morgan et al., 1988).
A fine balance between the efficacy and the safety of a vaccinia virus-based recombinant vaccine candidate is extremely important. The recombinant virus must present the immunogen(s) in a manner that elicits a protective immune response in the vaccinated animal but lacks any significant pathogenic properties. Therefore attenuation of the vector strain would be a highly desirable advance over the current state of technology.
A number of vaccinia genes have been identified which are non-essential for growth of the virus in tissue culture and whose deletion or inactivation reduces virulence in a variety of animal systems.
The gene encoding the vaccinia virus thymidine kinase (TK) has been mapped (Hruby et al., 1982) and sequenced (Hruby et al., 1983; Weir et al., 1983). Inactivation or complete deletion of the thymidine kinase gene does not prevent growth of vaccinia virus in a wide variety of cells in tissue culture. TK.sup.- vaccinia virus is also capable of replication in vivo at the site of inoculation in a variety of hosts by a variety of routes.
It has been shown for herpes simplex virus type 2 that intravaginal inoculation of guinea pigs with TK.sup.- virus resulted in significantly lower virus titers in the spinal cord than did inoculation with TK.sup.+ virus (Stanberry et al., 1985). It has been demonstrated that herpesvirus encoded TK activity in vitro was not important for virus growth in actively metabolizing cells, but was required for virus growth in quiescent cells (Jamieson et al., 1974).
Attenuation of TK.sup.- vaccinia has been shown in mice inoculated by the intracerebral and intraperitoneal routes (Buller et al., 1985). Attenuation was observed both for the WR neurovirulent laboratory strain and for the Wyeth vaccine strain. In mice inoculated by the intradermal route, TK.sup.- recombinant vaccinia generated equivalent anti-vaccinia neutralizing antibodies as compared with the parental TK.sup.+ vaccinia virus, indicating that in this test system the loss of TK function does not significantly decrease immunogenicity of the vaccinia virus vector. Following intranasal inoculation of mice with TK.sup.- and TK.sup.+ recombinant vaccinia virus (WR strain), significantly less dissemination of virus to other locations, including the brain, has been found (Taylor et al., 1991a).
Another enzyme involved with nucleotide metabolism is ribonucleotide reductase. Loss of virally encoded ribonucleotide reductase activity in herpes simplex virus (HSV) by deletion of the gene encoding the large subunit was shown to have no effect on viral growth and DNA synthesis in dividing cells in vitro, but severely compromised the ability of the virus to grow on serum starved cells (Goldstein et al., 1988). Using a mouse model for acute HSV infection of the eye and reactivatable latent infection in the trigeminal ganglia, reduced virulence was demonstrated for HSV deleted of the large subunit of ribonucleotide reductase, compared to the virulence exhibited by wild type HSV (Jacobson et al., 1989).
Both the small (Slabaugh et al., 1988) and large (Schmidtt et al., 1988) subunits of ribonucleotide reductase have been identified in vaccinia virus. Insertional inactivation of the large subunit of ribonucleotide reductase in the WR strain of vaccinia virus leads to attenuation of the virus as measured by intracranial inoculation of mice (Child et al., 1990).
The vaccinia virus hemagglutinin gene (HA) has been mapped and sequenced (Shida, 1986). The HA gene of vaccinia virus is nonessential for growth in tissue culture (Ichihashi et al., 1971). Inactivation of the HA gene of vaccinia virus results in reduced neurovirulence in rabbits inoculated by the intracranial route and smaller lesions in rabbits at the site of intradermal inoculation (Shida et al., 1988). The HA locus was used for the insertion of foreign genes in the WR strain (Shida et al., 1987), derivatives of the Lister strain (Shida et al., 1988) and the Copenhagen strain (Guo et al., 1989) of vaccinia virus. Recombinant HA.sup.- vaccinia virus expressing foreign genes have been shown to be immunogenic (Guo et al., 1989; Itamura et al., 1990; Shida et al., 1988; Shida et al., 1987) and protective against challenge by the relevant pathogen (Guo et al., 1989; Shida et al., 1987).
Cowpox virus (Brighton red strain) produces red (hemorrhagic) pocks on the chorioallantoic membrane of chicken eggs. Spontaneous deletions within the cowpox genome generate mutants which produce white pocks (Pickup et al., 1984). The hemorrhagic function (u) maps to a 38 kDa protein encoded by an early gene (Pickup et al., 1986). This gene, which has homology to serine protease inhibitors, has been shown to inhibit the host inflammatory response to cowpox virus (Palumbo et al., 1989) and is an inhibitor of blood coagulation. The u gene is present in WR strain of vaccinia virus (Kotwal et al., 1989b). Mice inoculated with a WR vaccinia virus recombinant in which the u region has been inactivated by insertion of a foreign gene produce higher antibody levels to the foreign gene product compared to mice inoculated with a similar recombinant vaccinia virus in which the u gene is intact (Zhou et al., 1990). The u region is present in a defective nonfunctional form in Copenhagen strain of vaccinia virus (open reading frames B13 and B14 by the terminology reported in Goebel et al., 1990a,b).
Cowpox virus is localized in infected cells in cytoplasmic A type inclusion bodies (ATI) (Kato et al., 1959). The function of ATI is thought to be the protection of cowpox virus virions during dissemination from animal to animal (Bergoin et al., 1971). The ATI region of the cowpox genome encodes a 160 kDa protein which forms the matrix of the ATI bodies (Funahashi et al., 1988; Patel et al., 1987). Vaccinia virus, though containing a homologous region in its genome, generally does not produce ATI. In WR strain of vaccinia, the ATI region of the genome is translated as a 94 kDa protein (Patel et al., 1988). In Copenhagen strain of vaccinia virus, most of the DNA sequences corresponding to the ATI region are deleted, with the remaining 3' end of the region fused with sequences upstream from the ATI region to form open reading frame (ORF) A26L (Goebel et al., 1990a,b).
A variety of spontaneous (Altenburger et al., 1989; Drillien et al., 1981; Lai et al., 1989; Moss et al., 1981; Paez et al., 1985; Panicali et al., 1981) and engineered (Perkus et al., 1991; Perkus et al., 1989; Perkus et al., 1986) deletions have been reported near the left end of the vaccinia virus genome. A WR strain of vaccinia virus with a 10 kb spontaneous deletion (Moss et al., 1981; Panicali et al., 1981) was shown to be attenuated by intracranial inoculation in mice (Buller et al., 1985). This deletion was later shown to include 17 potential ORFs (Kotwal et al., 1988b). Specific genes within the deleted region include the virokine N1L and a 35 kDa protein (C3L, by the terminology reported in Goebel et al., 1990a,b). Insertional inactivation of N1L reduces virulence by intracranial inoculation for both normal and nude mice (Kotwal et al., 1989a). The 35 kDa protein is secreted like N1L into the medium of vaccinia virus infected cells. The protein contains homology to the family of complement control proteins, particularly the complement 4B binding protein (C4bp) (Kotwal et al., 1988a). Like the cellular C4bp, the vaccinia 35 kDa protein binds the fourth component of complement and inhibits the classical complement cascade (Kotwal et al., 1990). Thus the vaccinia 35 kDa protein appears to be involved in aiding the virus in evading host defense mechanisms.
The left end of the vaccinia genome includes two genes which have been identified as host range genes, K1L (Gillard et al., 1986) and C7L (Perkus et al., 1990). Deletion of both of these genes reduces the ability of vaccinia virus to grow on a variety of human cell lines (Perkus et al., 1990).
Two additional vaccine vector systems involve the use of naturally host-restricted poxviruses, avipoxviruses. Both fowlpoxvirus (FPV) and canarypoxvirus (CPV) have been engineered to express foreign gene products. Fowlpox virus (FPV) is the prototypic virus of the Avipox genus of the Poxvirus family. The virus causes an economically important disease of poultry which has been well controlled since the 1920's by the use of live attenuated vaccines. Replication of the avipox viruses is limited to avian species (Matthews, 1982) and there are no reports in the literature of avipoxvirus causing a productive infection in any non-avian species including man. This host restriction provides an inherent safety barrier to transmission of the virus to other species and makes use of avipoxvirus based vaccine vectors in veterinary and human applications an attractive proposition.
FPV has been used advantageously as a vector expressing antigens from poultry pathogens. The hemagglutinin protein of a virulent avian influenza virus was expressed in an FPV recombinant (Taylor et al., 1988a). After inoculation of the recombinant into chickens and turkeys, an immune response was induced which was protective against either a homologous or a heterologous virulent influenza virus challenge (Taylor et al., 1988a). FPV recombinants expressing the surface glycoproteins of Newcastle Disease Virus have also been developed (Taylor et al., 1990; Edbauer et al., 1990).
Despite the host-restriction for replication of FPV and CPV to avian systems, recombinants derived from these viruses were found to express extrinsic proteins in cells of nonavian origin. Further, such recombinant viruses were shown to elicit immunological responses directed towards the foreign gene product and where appropriate were shown to afford protection from challenge against the corresponding pathogen (Tartaglia et al., 1993a,b; Taylor et al., 1992; 1991b; 1988b).
In 1983, human immunodeficiency virus type 1 (HIV1) was identified as the causative agent of AIDS. Twelve years later, despite a massive, worldwide effort, an effective HIV1 vaccine is still not available. Recently, however, several reports have suggested that an efficacious HIV1 vaccine may be attainable. For example, macaques have been protected against a simian immunodeficiency virus (SIV) challenge by a vaccination protocol involving a primary immunization with a vaccinia virus recombinant expressing the SIV gp160 glycoprotein and a booster immunization with purified SIV gp160 glycoprotein (Hu et al., 1992). In addition, chimpanzees have been protected against an HIV1 challenge with an HIV1 gp120 subunit vaccine (Berman et al, 1990). Chimps have also been protected against an HIV1 challenge by a vaccination protocol involving multiple injections of either inactivated HIV1, gp160 and/or V3 peptide or gp160, p17 (a Gag protein) and/or V3 peptide (Girard et al., 1991). A similar protocol involving multiple injections of gp160, p17, p24 (a Gag protein), Vif, Nef and/or V3 peptide has also protected chimps against a challenge of HIV1-infected cells (Fultz et al., 1992). Furthermore, chimps have been passively protected by the infusion of HIV1 V3-specific antibodies (Emini et al., 1992).
Most of these vaccination protocols have focused on eliciting an immune response against the HIV1 or SIV envelope glycoprotein, or more specifically, against the V3 epitope of the envelope glycoprotein. Unfortunately, different strains of HIV1 exhibit extensive genetic and antigenic variability, especially in the envelope glycoprotein. Therefore, an effective HIV1 vaccine may need to elicit an immune response against more than one HIV1 antigen, or one epitope of one HIV1 antigen.
Contrary to the extensive sequence variability observed in B-cell epitopes, T-cell epitopes are relatively conserved. For example, cytotoxic T-lymphocytes (CTL) clones, isolated from an HIV1-seronegative individual vaccinated with a vaccinia virus recombinant expressing HIV1 gp160 (LAI strain) and boosted with purified HIV1 gp160 (LAI), lyse target cells expressing the HIV1 MN or RF envelope glycoprotein as efficiently as cells expressing the HIV1 LAI envelope glycoprotein (Hammond et al., 1992). Therefore, a vaccine that elicits an immune response against relatively conserved T-cell epitopes may not only be more efficacious against a homologous challenge, but also more efficacious against a heterologous challenge.
HIV1-seronegative individuals have been vaccinated with an ALVAC recombinant (vCP125) expressing HIV1 gp160, in a prime-boost protocol similar to the regimen used to vaccinate macaques against SIV. These ALVAC-based protocols demonstrated the ability of vCP125 to elicit HIV1 envelope-specific CD8.sup.+ CTLs and to enhance envelope-specific humoral responses observed following a subunit booster (Pialoux et al., 1995). These results justify the rationale for a recombinant ALVAC-based HIV1 vaccine.
Individuals infected with human immunodeficiency virus type 1 (HIV1) initially generate a relatively dynamic and extensive antiviral immune response, including HIV1-specific neutralizing antibodies and HIV1-specific CTLs. Despite these responses, however, the vast majority of HIV1-infected people eventually succumb to HIV1-associated diseases. Since the immune response generated by most HIV1-infected people is not protective, generation of an effective immune response may necessitate that the immune response be modulated or redirected against HIV1 epitopes that are not normally or efficiently seen by HIV1-infected individuals.
Approximately 40% of the HIV1-specific antibody in HIV1-seropositive individuals capable of binding HIV1-infected cells is specific to the third variable region (V3) of the HIV1 envelope glycoprotein (Spear et al, 1994). These results indicate that the V3 loop is 1) highly immunogenic and 2) exposed on the surface of infected cells. The amino acid sequence of the V3 loop varies considerably between different HIV1 isolates. Therefore, a moderate level of sequence variation does not appear to alter the structure or immunogenicity of this region of the envelope glycoprotein. Since the V3 loop is highly immunogenic and its structure and immunogenicity is not severely affected by sequence variation, this region of the envelope glycoprotein may be useful as an immunogenic platform for presenting normally non-immunogenic linear HIV1 epitopes or heterologous epitopes to the immune system.
Sera from HIV1-seropositive individuals can neutralize lab-adapted strains of HIV1. These sera can also neutralize primary HIV1 isolates (although 100.times. higher titers are required). Conversely, sera from individuals vaccinated with HIV1 gp120 can neutralize lab-adapted strains of HIV1 (although 10.times. higher titers relative to sera from seropositive individuals are required), but can not neutralize (at assayable levels) primary isolates (Hanson, 1994). A significant portion of the neutralizing activity found in sera from seropositive and gp120-vaccinated individuals appears to be specific to the V3 loop (Spear et al., 1994; Berman et al., 1994). Since the V3 loop is hypervariable and since antibodies against this region may not neutralize primary isolates or heterologous strains of HIV1, it may be necessary to develop vaccines that elicit an immune response against epitopes other than the V3 loop, epitopes that can neutralize a broad spectrum of HIV1 strains, including primary isolates.
A monoclonal antibody capable of neutralizing primary HIV1 isolates, as well as a broad spectrum of lab-adapted HIV1 strains, has been isolated (Conley et al., 1994; Katinger et al., 1992). The epitope recognized by this monoclonal antibody has been mapped between amino acids 662 and 667 of HIV1 gp41 and has the amino acid sequence, ELDKWA (Buchacher et al, 1994). Approximately 80% of the HIV1 strains from which sequence information has been derived, including strains from the various HIV1 clades, express the core binding sequence of this epitope, LDKW (Conley et al., 1994). Therefore, unlike the V3 loop, this epitope appears to be relatively well conserved. Unfortunately, this epitope does not appear to be very immunogenic in its normal configuration. Only approximately 50% of HIV1-seropositive individuals have a detectable antibody response to the region of gp41 containing this epitope (Broliden et al., 1992).
It can thus be appreciated that provision of an immunodeficiency virus recombinant poxvirus, and of an immunogenic composition which induces an immunological response against immunodeficiency virus infections when administered to host, particularly a composition having enhanced safety such as NYVAC or ALVAC based recombinants containing coding for any or all of HIV1gag(+pro)(IIIB), gp120(MN)(+transmembrane), nef(BRU)CTL epitopes, pol(IIIB)CTL epitopes; for instance, HIV1gag(+pro)(IIIB), gp120(MN)(+transmembrane), nefCTL1, nefCTL2, pol1(PolCTL1), pol2(PolCTL2), pol3(PolCTL3), ELDKWA or LDKW epitopes, (SEQ ID NOS:.147 and 148) especially in an immunogenic configuration, or any combination thereof, for example all of them in combination, would be a highly desirable advance over the current state of technology. ALVAC, TROVAC, NYVAC, and vCP205 (ALVAC-MN120TMG) were deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., 20852, USA: NYVAC under ATCC accession number VR-2559 on Mar. 6, 1997; vCP205 (ALVAC-MN120TMG) under ATCC accession number VR-2557 on Mar. 6, 1997; TROVAC under ATCC accession number VR-2553 on Feb. 6, 1997 and, ALVAC under ATCC accession number VR-2547 on Nov. 14, 1996.