DNA prime/canarypox boost-based immunotherapy of chronic hepatitis B virus infection in a chimpanzee.

There are about 200 million chronic hepatitis B virus (HBV) carriers at high risk of development of cirrhosis and hepatocellular carcinoma. Termination of the carrier state may avert these risks. We have investigated immunotherapy for chronic HBV infection in a chimpanzee HBV carrier using recombinant DNA-based immunization followed by a recombinant canarypox booster. One week after the booster, HBV DNA declined greater than 400-fold and remained undetectable by the quantitative polymerase chain reaction (PCR) assay for 186 weeks. Plasma levels of hepatitis B surface antigen (HBsAg) declined for only a short time. The decline in HBV DNA correlated with a boost in gamma interferon production without a corresponding boost in cytotoxic T lymphocyte levels, and decline in the transcriptional template or covalently closed circular DNA level. Confirmation of these findings requires further studies in chimpanzees and/or in humans.

DNA prime-canarypox boost with polycistronic hepatitis C virus (HCV) genes generates potent immune responses to HCV structural and nonstructural proteins.

DNA vaccination was employed to study immune responses to hepatitis C virus (HCV) proteins. As an immunizing strategy, we studied immune responses of BALB/c (H-2d) and C57BL/6 mice (H-2b) to HCV genes delivered intramuscularly as a polycistronic construct capsid/E1/E2/NS2/NS3 (pRC/C-NS3) encoding 5 structural and nonstructural proteins. We also evaluated canarypox virus containing the same HCV genes as a means for potentiating immune responses to naked DNA. Our results indicate that mice that received a polycistronic pRC/C-NS3 with canarypox booster had enhanced antibody and cellular responses to HCV proteins. Immunodominant CD8(+) T cell responses to several HCV structural and nonstructural proteins, characterized by cytotoxicity and interferon (IFN)-gamma production or IFN-gamma production without significant cytotoxicity, were observed in both strains of mice. The combination of naked DNA with a nonreplicating canarypox booster encoding HCV polycistronic pRC/C-NS3 genes appears to diversify and enhance T cell responses to HCV proteins.

Hepatitis C virus-specific CTL responses in PBMC from chimpanzees with chronic hepatitis C: determination of CTL and CTL precursor frequencies using a recombinant canarypox virus (ALVAC).

The aim of this study was to evaluate HCV-cytotoxic T lymphocyte response from PBMC in bulk CTL assays and in CTL precursor analyses using in vitro stimulation with canarypox virus (ALVAC) expressing HCV-capsid/E1/E2/NS2/NS3 antigens. Canarypox virus is naturally host-range restricted and does not replicate or cause cytopathology on mammalian cells. PBMC were obtained from four chimpanzees with chronic hepatitis C infection and one uninfected chimpanzee. CTL from bulk culture of PBMC and CTL precursor frequencies were found in three of the four chronically infected chimpanzees using ALVAC in vitro stimulation. No CTL response was detected in PBMC from the uninfected chimpanzee. The precursor frequencies of CTL specific for capsid, NS2 and NS3 proteins ranged between 1/2663 and 1/27202. No correlation was observed between percent cytolysis in bulk culture and CTL precursor frequencies. This method may prove useful in assessing the correlation between HCV-CTL response and virological or histological status.

Host-range restriction of vaccinia virus E3L-specific deletion mutants.

The vaccinia virus (VV) E3L gene product functions as a dsRNA binding protein that is involved in conferring an interferon-resistant phenotype upon the virus. Studies with a vaccinia virus (VV) E3L- deletion mutant (vP1080) have also demonstrated that the E3L gene product is critical for productive replication on certain cell substrates. While E3L was found to be nonessential for replication in chick embryo fibroblasts (CEFs), virus specifically deleted of E3L was found to be replication deficient in Vero, HeLa, and murine L929 cells. Further, the temporal block in replication appears to differ in these cell systems, as evidenced by the observed timing of protein synthesis inhibition. In Vero cells infected with the VV E3L- mutant, there was no detectable protein synthesis after 2 hr post-infection, whereas in L929 cells normal protein patterns were observed even at late times post-infection. Expression of a heterologous dsRNA binding protein, the reovirus sigma 3 protein, by the E3L- mutant virus restored near wild-type growth characteristics, suggesting the critical nature for regulating dsRNA levels in VV-infected cells.

Poxvirus-based vaccine candidates for cancer, AIDS, and other infectious diseases.

Over the past 12 years, the poxvirus vector technology has provided scientists with valuable reagents to achieve high-level expression of proteins, to address questions of structure-function relationship of specific polypeptides, to investigate the immunobiology of specific pathogens, and to develop recombinant vaccine candidates. It is this last role that has drawn enthusiasm from the medical community because of the potential this technology has to provide novel approaches for addressing urgent needs in human and veterinary medicine. From one perspective, the safety issues surrounding the use of vaccinia-based vaccine candidates have been addressed with the development of the NYVAC and ALVAC vectors. Evaluation of these novel poxvirus vectors are in progress to determine their potential impact on cancer and infectious disease.

Reversal of the interferon-sensitive phenotype of a vaccinia virus lacking E3L by expression of the reovirus S4 gene.

The vaccinia virus (VV) E3L gene, which encodes a potent inhibitor of the interferon (IFN)-induced, double-stranded RNA (dsRNA)-dependent protein kinase, PKR, is thought to be involved in the IFN-resistant phenotype of VV. The E3L gene products, p25 and p20, act as inhibitors of PKR, presumably by binding and sequestering activator dsRNA from the kinase. In this study we demonstrate that VV with the E3L gene specifically deleted (vP1080) was sensitive to the antiviral effects of IFN and debilitated in its ability to rescue vesicular stomatitis virus from the antiviral effects of IFN. Infection of L929 cells with E3L-minus virus led to rRNA degradation typical of activation of the 2'-5'-oligoadenylate synthetase/RNase L system, and extracts of infected cells lacked the PKR-inhibitory activity characteristic of wild-type VV. The reovirus S4 gene, which encodes a dsRNA-binding protein (sigma 3) that can also inhibit PKR activation by binding and sequestering activator dsRNA, was inserted into vP1080. The resultant virus (vP1112) was partially resistant to the antiviral effects of IFN in comparison with vP1080. Further studies demonstrated that transient expression of the reovirus sigma 3 protein rescued E3L-minus VV replication in HeLa cells. In these studies, rescue by sigma 3 mutants correlated with their ability to bind dsRNA. Finally, vP112 was also able to rescue the replication of the IFN-sensitive virus vesicular stomatitis virus in a manner similar to that of wild-type VV. Together, these results suggest that the reovirus S4 gene can replace the VV E3L gene with respect to interference with the IFN-induced antiviral activity.

Protection of mice and swine from pseudorabies virus conferred by vaccinia virus-based recombinants.

Glycoproteins gp50, gII, and gIII of pseudorabies virus (PRV) were expressed either individually or in combination by vaccinia virus recombinants. In vitro analysis by immunoprecipitation and immunofluorescence demonstrated the expression of a gII protein of approximately 120 kDa that was proteolytically processed to the gIIb (67- to 74-kDa) and gIIc (58-kDa) mature protein species similar to those observed in PRV-infected cells. Additionally, the proper expression of the 90-kDa gIII and 50-kDa gp50 was observed. All three of these PRV-derived glycoproteins were detectable on the surface of vaccinia virus-PRV recombinant-infected cells. In vivo, mice were protected against a virulent PRV challenge after immunization with the PRV glycoprotein-expressing vaccinia virus recombinants. The coexpression of gII and gIII by a single vaccinia virus recombinant resulted in a significantly reduced vaccination dose required to protect mice against PRV challenge. Inoculation of piglets with the various vaccinia virus-PRV glycoprotein recombinants also resulted in protection against virulent PRV challenge as measured by weight gain. The simultaneous expression of gII and gp50 in swine resulted in a significantly enhanced level of protection as evaluated by weight evolution following challenge with live PRV.

NYVAC: a highly attenuated strain of vaccinia virus.

A highly attenuated vaccinia virus strain, NYVAC (vP866), was derived from a plaque-cloned isolate of the Copenhagen vaccine strain by the precise deletion of 18 open reading frames (ORFs) from the viral genome. Among the ORFs deleted from NYVAC (vP866) are two genes involved in nucleotide metabolism, the thymidine kinase (ORF J2R) and the large subunit of the ribonucleotide reductase (ORF I4L); the gene encoding the viral hemagglutinin (ORF A56R); the remnant (ORF A26L) of a highly expressed gene responsible for the formation of A-type inclusion bodies; the disrupted gene (ORFs B13R/B14R) normally encoding a serine protease inhibitor; and a block of 12 ORFs bounded by two known viral host range regulatory functions (ORFs C7L through K1L). Within this block a secretory protein (ORF N1L) implicated in viral virulence and a functional complement 4b binding protein (ORF C3L) are encoded. The ORFs were deleted in a manner which prevents the synthesis of undesirable novel gene products. The attenuation characteristics of the derived NYVAC strain were compared in in vitro and in vivo studies with those of the Western Reserve (WR) laboratory strain, the New York City Board of Health vaccine strain (Wyeth), the parental plaque-cloned isolate (VC-2) of the Copenhagen vaccine strain used to derive NYVAC, and the avipox virus canarypox (ALVAC), which is naturally restricted for replication to avian species. The NYVAC strain was demonstrated to be highly attenuated by the following criteria: (a) no detectable induration or ulceration at the site of inoculation on rabbit skin; (b) rapid clearance of infectious virus from the intradermal site of inoculation on rabbit skin; (c) absence of testicular inflammation in nude mice; (d) greatly reduced virulence as demonstrated by the results of intracranial challenge of both 3-week-old or newborn mice; (e) greatly reduced pathogenicity and failure to disseminate in immunodeficient (nude or cyclophosphamide treated) mice; and (f) dramatically reduced ability to replicate on a variety of human tissue culture cells. Despite these highly attenuated characteristics, the NYVAC strain, as a vector, retains the ability to induce strong immune responses to extrinsic antigens.

Vaccinia virus encodes a protein with similarity to glutaredoxins.

Recently, we have reported the complete nucleotide sequence of vaccinia virus (Goebel, S. J., Johnson, G. P., Perkus, M. E., Davis, S. W., Winslow, J. P., and Paoletti, E. 1990, Virology 179, 247-266). Approximately 2.2 kbp leftward of the large subunit of ribonucleotide reductase resides a 108-amino acid open reading frame, O2L (nt 62,851-62,528) with significant similarity to known glutaredoxins. The deduced amino acid sequence of open reading frame O2L is 28.7% identical to the yeast and Escherichia coli proteins and greater than 40% identical to various mammalian glutaredoxins. Similar patterns of hydrophobicity as well as alpha-helix and beta-sheet potentials suggest that O2L and the glutaredoxins share a similar secondary structure. Furthermore, a common function is inferred by the presence of a highly conserved redox-active site.

Deletion of 55 open reading frames from the termini of vaccinia virus.

Each copy of the inverted terminal repeat of vaccinia virus consists of 8 kb of DNA containing 9 ORFS flanked near the terminus of the genome by 4 kb of repetitive DNA which in turn contains blocks of tandem repeats. Using plasmids containing repetitive DNA as the external arm, we have generated deletions at both the left and the right termini of the vaccinia genome. We report here the engineered deletion within a single vaccinia virus of 32.7 kb of DNA (including 38 ORFS) from the left terminus and 14.9 kb of DNA (including 17 ORFS) from the right terminus.

Vaccinia virus host range genes.

A gene encoding an 18-kDa polypeptide (ORF C7L) located in the vaccinia virus HindIII C fragment was shown to be functionally equivalent to previously described host range gene (ORF K1L) spanning the HindIII K/M fragment junction. Either C7L or K1L host range gene is necessary and sufficient by itself to allow replication of vaccinia virus on human cells. Deletion of both C7L and K1L genes from the wild-type vaccinia genome is required to derive a virus deficient for replication on human cells. Further, an ORF encoding a 77-kDa polypeptide derived from cowpox (CP77kDa) and previously shown to allow vaccinia to overcome the restriction for replication on Chinese hamster ovary cells could substitute for the vaccinia host range genes C7L and K1L in permitting replication of the virus on human cells. Additionally, the three unique host range genes C7L, K1L, and CP77kDa were functionally equivalent for vaccinia replication on pig kidney cells, but not on rabbit kidney cells.

The complete DNA sequence of vaccinia virus.

The complete DNA sequence of the genome of vaccinia virus has been determined. The genome consisted of 191,636 bp with a base composition of 66.6% A + T. We have identified 198 "major" protein-coding regions and 65 overlapping "minor" regions, for a total of 263 potential genes. Genes encoded by the virus were located by examination of DNA sequence characteristics and compared with existing vaccinia virus mapping analyses, sequence data, and transcription data. These genes were found to be compactly organized along the genome with relatively few regions of noncoding sequences. Whereas several similarities to proteins of known function were discerned, the function of the majority of proteins encoded by these open reading frames is as yet undetermined.

Coexpression by vaccinia virus recombinants of equine herpesvirus 1 glycoproteins gp13 and gp14 results in potentiated immunity.

The equine herpesvirus 1 glycoprotein 14 (EHV-1 gp14) gene was cloned, sequenced, and expressed by vaccinia virus recombinants. Recombinant virus vP613 elicited the production of EHV-1-neutralizing antibodies in guinea pigs and was effective in protecting hamsters from subsequent lethal EHV-1 challenge. Coexpression of EHV-1 gp14 in vaccinia virus recombinant vP634 along with EHV-1 gp13 (P. Guo, S. Goebel, S. Davis, M. E. Perkus, B. Languet, P. Desmettre, G. Allen, and E. Paoletti, J. Virol. 63:4189-4198, 1989) greatly enhanced the protective efficacy in the hamster challenge model over that obtained with single recombinants. The inoculum doses (log10) required for protection of 50% of hamsters were 6.1 (EHV-1 gp13), 5.2 (EHV-1 gp14), and less than 3.6 (vaccinia virus recombinant expressing both EHV-1 glycoproteins [gp13 and gp14]).

Expression in recombinant vaccinia virus of the equine herpesvirus 1 gene encoding glycoprotein gp13 and protection of immunized animals.

The equine herpesvirus 1 (EHV-1) gene encoding glycoprotein 13 (gp13) was cloned into the hemagglutinin (HA) locus of vaccinia virus (Copenhagen strain). Expression of the gp13 gene was driven by the early/late vaccinia virus H6 promoter. Metabolically radiolabeled polypeptides of approximately 47 and 44 kilodaltons and 90 kilodaltons (glycosylated form) were precipitated with both polyclonal and gp13-specific monoclonal antibodies. Presentation of gp13 on the cytoplasmic membrane of cells infected with the recombinant gp13 vaccinia virus was demonstrated by immunofluorescence of unfixed cells. Inoculation of the recombinant gp13 vaccinia virus into guinea pigs induced neutralizing antibodies to both EHV-1 and vaccinia virus. Hamsters vaccinated with the recombinant gp13 vaccinia virus survived a lethal challenge with the hamster-adapted Kentucky strain of EHV-1. These results indicate that expression in vaccinia virus vectors of EHV-1 gp13, the glycoprotein homolog of herpes simplex virus gC-1 and gC-2, pseudorabies virus gIII, and the varicella-zoster virus gpV may provide useful vaccine candidates for equine herpesvirus infections.

Cloning and expression of foreign genes in vaccinia virus, using a host range selection system.

A simple selection system has been developed for the cloning and expression of open reading frames in vaccinia virus. The selection system is based on a conditional lethal (host range) mutant of vaccinia virus. A deletion mutant of the vaccinia virus WR strain was generated by insertion of the neomycin resistance gene from transposon Tn5 and selection with the antibiotic G418. This deletion recombinant, vP293, lacked approximately 21.7 kilobases of DNA beginning 3.8 kilobases from the left end of the genome, vP293, was capable of plaquing on primary chicken embryo fibroblasts and two monkey cell lines (BSC-40 and Vero) but was defective in replication in the human cell line MRC-5. Insertion of the host range gene K1L into vP293 restored the ability to grow on MRC-5 cells. A series of plasmids were constructed which in addition to the K1L gene contained a vaccinia virus early-late promoter, H6, followed by a unique polylinker sequence, translational initiation and termination signals, and an early transcription termination signal. These plasmids, pHES1 through 4, allowed for rapid single-step cloning and expression of any open reading frame when recombined in vivo with vP293 and scored for growth on MRC-5 cells.

Antigen-presenting capacity of epidermal cells infected with vaccinia virus recombinants containing the herpes simplex virus glycoprotein D, and protective immunity.

We studied the association of herpes simplex type 1 (HSV-1) glycoprotein D (gD-1) expression in epidermal cells (EC) with virus-specific immunity and protection of mice from fatal HSV-2 challenge. Vaccinia virus recombinants containing gD-1 under the control of an early (VP176) or late (VP254) vaccinia virus promoter were used. Mature gD-1 protein was expressed in VP176-infected EC and they had accessory cell function for HSV-2-induced T cell proliferation of immune lymph node cells (LNC). It was not expressed in VP254-infected EC and they did not act as accessory cells. LNC from VP176- but not VP254-immunized mice proliferated in response to HSV antigen and only VP176-immunized mice had complete long-term protection from HSV-2 challenge.

Antibody-dependent cellular cytotoxicity is directed against both the gp120 and gp41 envelope proteins of HIV.

To define the target antigens for antibody-dependent cellular cytotoxicity (ADCC), assays were performed using affinity-purified human immunoglobulin (Ig) or polyclonal rabbit sera directed against specific proteins of HIV. ADCC was not found using affinity-purified anti-core (p25) human Ig or sera obtained from rabbits hyper-immunized with recombinant p25. However, when affinity-purified human Ig or rabbit antisera specific for the envelope glycoproteins, gp120 or gp41, were used in ADCC assays, killing of HIV-infected cells was observed. These results indicate that antibodies in the infected individual that mediate ADCC are directed against both the gp120 and gp41 HIV envelope proteins and not against the viral core protein.

Regulation of expression of herpes simplex virus (HSV) glycoprotein D in vaccinia recombinants affects their ability to protect from cutaneous HSV-2 disease.

The effect of regulation of herpes simplex virus (HSV) type 1 glycoprotein D (gD-1) gene expression on HSV-specific immune response and protection from cutaneous HSV-2 disease was studied using vaccinia virus recombinants containing gD-1 under the control of early (VP176) or late (VP254) vaccinia virus promoters. Expression of gD-1 in VP176-infected cells was first observed at 2 h after infection. It did not depend on viral DNA replication. In VP254-infected cells, gD-1 was first observed at 24 h after infection and its expression depended on DNA replication. Immunized guinea pigs had similar titers of HSV-specific neutralizing antibody. However, HSV-specific T cell responses were significantly higher in VP176- than in VP254-immunized animals as determined by lymphoproliferation (P less than .005) and delayed type hypersensitivity (P less than .01). The reduced T cell responses of VP254-immunized guinea pigs correlated with poor gD-1 expression in VP254-infected antigen presenting cells (splenic adherent and epidermal cells). Immunization with VP176, but not with VP254, protected guinea pigs from primary (P less than .0005) and recurrent (P less than .0005) cutaneous HSV-2 lesions.