NYVAC: A highly attenuated strain of vaccinia virus

VIROLOGY

188, 217-232

(1992)

NYVAC: A Highly Attenuated

Strain of Vaccinia

Virus

JAMES TARTAGLIA,* MARION E. PERKUS,* JILL TAYLOR,* ELIZABETH K. NORTON,* JEAN-CHRISTOPHE AUDONNET,* WILLIAM I. COX,* STEPHEN W. DAVIS,* JOHANNA VAN DER HOEVEN,* BERNARD MEIGNIER,t MICHEL RIVIERE,+ BERNARD LANGUET,# AND ENZO PAOLETTl*,’ *Virogenetics Corporation, 465 Jordan Road, Rensselaer Technology Park, Troy# New York 12180; tPasteur Merieux, Serums & Vaccins, 154 1, av. Marcel Merieux. 69280 Marcy L’ftoile, France; and +Rhone-Merieux, 254 rue Marcel Merieux, Lyon Cedex 07, France Received December

19, 199 1; accepted

February

5, 1992

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 l4L); 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 B13RIB14R) normally encoding a serine protease inhibitor; and a block of 12 ORFs bounded by two known viral host range regulatory functions (ORFs C7L through Kl L). Within this block a secretory protein (ORF Nl L) 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 0 1992 Academic Press, Inc. responses to extrinsic antigens.

INTRODUCTION

of major concern and vaccination was contraindicated. Correlations existed among the vaccinia virus vaccine strain used, the age of the vaccinee, the immunological status of the vaccinee, and the occurrence of postvaccinial complications (Fenner et al., 1989; Behbehani, 1983). It is difficult to assess the contribution of adventitious agents in the vaccine preparations or other factors to these adverse reactivities. Ironically, concurrent with the eradication of smallpox and the cessation of vaccination, a new use for vaccinia virus was proposed (Panicali and Paoletti, 1982). Utilizing molecular cloning techniques, it became possible to express genes from foreign pathogens in vaccinia virus providing new approaches tovaccination. Nevertheless, the initial enthusiasm regarding the use of genetically engineered vaccinia virus recombinant vaccines for both veterinary and human applications has been tempered by the issue of safety. In order to address the issue of safety, a strategy was developed to genetically engineer a highly attenuated vaccinia virus that would still retain the ability to induce

The introduction of Edward Jenner’s strategy to prevent smallpox was followed by an evolutionary process that culminated, in 1980, in a World Health Organization declaration that smallpox, as a human infectious disease, was eradicated from the globe. This unique success was made possible in great part by the availability of an effective vaccine that was easy and inexpensive to produce, readily administered, and thermally stable. In general, vaccination with vaccinia via dermal scarification caused a relatively localized, selflimiting infection which resulted in a strong and longlasting immunological response (Fenner et a/., 1989). On rare occasions, however, adverse reactions resulted from vaccination. The most severe consequences were exanthematous disease or encephalopathies. Vaccination of immune-compromised individuals or those with skin conditions such as eczema was ’ To whom reprint requests should be addressed 217

0042.6822/92

$3.00

Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved

218

TARTAGLIA

vigorous immunological responses against extrinsic antigens. A number of vaccinia vaccine strains including New York City Board of Health (Wyeth), Copenhagen, Lister, MVA, and CV-1 were evaluated with respect to their relative immunogenic potential as vectors and to their attenuation characteristics. Based on this analysis, the Copenhagen strain demonstrated the desirable balanced characteristics and was chosen for further development. A working hypothesis was developed on the premise that genetic functions associated with virulence were distinct from essential genetic functions involved in viral replication. If this were the case, then the “virulence” genes could be deleted from the vector. Studies from this and other laboratories have defined a number of orthopoxvirus functions associated with virulence. Among these functions are certain enzymatic activities involved with nucleotide metabolism such as thymidine kinase (Buller el al., 1985) and ribonucleotide reductase (Child er al., 1990) a complement binding protein (Kotwal et a/., 1990; Kotwal and Moss, 1988), the viral hemagglutinin (Shida et a/., 1988), serine protease inhibitors (Zhou et al., 1990), as well as other virally encoded proteins (Kotwal et a/., 1989; Patel and Pickup, 1987; Buller et a/., 1988; Beattie et al., 199 1). It has been postulated that these functions enhance the ability of the virus to replicate in quiescent cells, interfere with complement-mediated lysis of infected cells, and prevent the chemotactic signaling of heterophils to foci of viral infections or otherwise subvert host defense mechanisms against viral infections. The basis for selection of the genes deleted in the generation of NYVAC is described below. It has been shown for herpes simplex virus (HSV) type 2 that intravaginal inoculation of guinea pigs with thymidine kinase (rk-) virus resulted in significantly lower virus titers in the spinal cord than did inoculation with tk+ virus (Stanberry et a/., 1985). Jamieson et al. (1974) demonstrated that herpesvirus-encoded tk activity was not important for virus growth in actively metabolizing cells in vitro, but was required for virus growth in quiescent cells. By analogy, deletion of the tk gene from vaccinia virus vaccine strains is particularly desirable to prevent replication of the virus in quiescent cells of the central nervous system, since one of the most serious postvaccination complications observed with vaccinia is encephalitis. More specifically, the attenuation of tk- vaccinia inoculated into mice by the intracranial and intraperitoneal routes was shown by Buller et al. (1985). Attenuation was observed both for the WR neurovirulent laboratory strain and for the Wyeth vaccine strain. Taylor et al. (1991a) have also shown that significantly less tk- vaccinia virus dissemi-

ET AL

nates to other tissues, including the brain, than does tk+ virus following intranasal inoculations of mice. Like the thymidine kinase, ribonucleotide reductase is an enzyme involved with nucleotide metabolism. Loss of the viral-encoded ribonucleotide reductase activity from HSV was shown to severely compromise the ability of the virus to replicate on serum-staNed cells (Goldstein and Weller, 1988) and to reduce virulence relative to wild-type HSV in a mouse model system (Jacobson et al., 1989). Insertional inactivation of the large subunit of ribonucleotide reductase from the WR strain of vaccinia has been shown to attenuate the virus as measured by intracranial inoculation of mice (Child et al., 1990). A number of other orthopoxvirus genes have been implicated in pathogenicity. In particular, a 38-kDa protein, associated with the formation of hemorrhagic pocks on the chorioallantoic membrane of the fertilized chicken egg, was identified in the Brighton Red strain of cowpox virus (Pickup et al., 1984, 1986). This gene shares significant homology with placental plasminogen activator inhibitor and other serine protease inhibitors (Serpins). Expression of the gene has been shown to inhibit the host inflammatory response to cowpox virus, possibly by preventing the generation of chemotactic factors (Palumbo et al., 1989). The 38-kDa Serpin gene exists in a defective form in the Copenhagen strain of vaccinia virus (open reading frames) (ORFs B13R and B14R) (Goebel et al., 1990a,b). The lack of a functional Serpin gene product may, in addition to reducing the virulence of the virus, enhance its immunogenicity as a vector since inactivation of viral-encoded serine protease inhibitors has been shown to enhance the humoral response toward a heterologous gene product expressed byvaccinia virus (Zhou eta/., 1990). Cowpox virus is embedded in cytoplasmic A-type inclusion bodies (ATI) (Kato et al., 1959). The proteinaceous ATI matrix is thought to stabilize the virus in the environment and thus enhance dissemination of the virus from animal to animal (Bergoin and Dales, 197 1; Joklik et a/., 1988). The cowpox genome encodes a 160-kDa protein which forms the matrix of the A-type inclusion bodies (Pate1 and Pickup, 1987; Funahashi et al., 1988). Vaccinia virus, although containing a homologous region in its genome, generally does not produce ATI. In the WR strain of vaccinia, the ATI region of the genome is expressed as a C-terminal truncated protein of 94 kDa (Pate1 et al., 1988). In the Copenhagen strain of vaccinia virus, most of the DNA sequences corresponding to the ATI gene are absent. Only 3’ sequences of the gene exist, and these are fused with sequences upstream from the ATI gene to form ORF A26L (Goebel et al., 1990a,b). The immunodominant vaccinia virus hemagglutinin

A-ENUATED

VACCINIA

(HA), found in highly infectious membrane-associated extracellular virus, has been shown to promote viral binding to host cells and to inhibit fusion of infected cells (Oie et al., 1990). Inactivation of the HA gene resuits in reduced neurovirulence in rabbits inoculated by the intracranial route and smaller lesions in rabbits at the site of intradermal inoculation (Shida et a/., 1988). Two genes, Nl L and C3L, which encode secretory proteins have been implicated in vaccinia virus virulence. Insertional inactivation of N 1L reduces neurovirulence for both normal and nude mice (Kotwal et a/., 1989). ORF C3L encodes a 35-kDa protein with homology to the family of complement control proteins, particularly the complement 4b binding protein (Kotwal and Moss, 1988; Kotwal et al., 1990). Previous work in this and other laboratories (Gillard et a/., 1986; Spehner et a/., 1988; Perkus et a/., 1990) has defined host range regulatory functions responsible for enabling viral replication in specific hosts. Deletion of two vaccinia host range genes, Kl L and C7L (Gillard et al., 1986; Perkus et al., 1990), abrogates or greatly reduces the ability of vaccinia virus to grow on a variety of human cell lines, as well as rabbit kidney and pig kidney cells (Perkus et a/., 1990). In this communication, we report the construction of a highly attenuated vaccinia vector (NYVAC) generated by the precise deletion of 18 ORFs, including a number of viral gene functions associated with virulence. The NYVAC strain was demonstrated to be highly attenuated in comparison with currently existing vaccine strains. Nonetheless, NYVAC retains good potential as a vaccine vector. The availability of the NYVAC vector provides significant progress in addressing the safety concerns of vaccinia virus-based recombinant vaccines.

MATERIALS AND METHODS Cells and viruses The precise origin of the vaccine strain of Copenhagen virus is not known (Glosser, 1989), although records of the strain can be traced to 19 13 at the Serum lnstitut of Copenhagen, Denmark. Studies in 19131914 comparing virulence characteristics of a number of strains indicated that the Copenhagen strain was less virulent than the Munchen, Hamburg, Dresden, Tours, and Christiania strains (Glosser, 1989). The Copenhagen strain was approved for human vaccine use in Denmark and the Netherlands (Glosser, 1989). The particular strain of Copenhagen virus from which NYVAC was derived was obtained from lnstitut Merieux, Lyon, France. Plaques on MRC-5 cells showed a typical population profile of size heterogeneity. A number

VIRUS NYVAC

of typical

well-isolated

219

plaques

were selected

and on MRC-5 monolayers. Genomic DNA was extracted from the resulting stocks and DNA restriction profiles were analyzed. Virus stocks prepared from the clonal isolates were also tested for relative attenuation by intracranial inoculation of mice. These analyses did not demonstrate significant differences between the plaque clones, and one virus stock designated VC-2 was selected for further work. All further plaque purifications and virus amplifications were performed on Vero cell (ATCC No. CCL81) monolayers. Vaccinia strain WR (ATCC No. VRl 19) is a mouse neurovirulent strain derived from the New York City Board of Health strain (Parker et a/., 1941). The virus was plaque purified on CV-1 cells (ATCC No. CCL-/O) and a plaque isolate designated L variant (ATCC No. VR2035) selected as described in Panicali et al. (1981). Virus used in this study was amplified on Vero cell monolayers. Vaccinia strain Wyeth (ATCC No. VR325) is a calfadapted strain whose origin also is the New York City Board of Health strain. The vaccine was marketed by Wyeth Laboratories (Marietta, PA) as DRYVAX and for simplicity is designated here as Wyeth. The virus was not plaque purified before amplification in order to analyze the properties of the entire population. The virus was amplified on either Vero cells or primary chick embryo fibroblast (CEF) cells. The derivations of the NYVAC strain (vP866) and NYVAC-RG (vP879) containing the gene encoding the rabies glycoprotein are described below. Plaquing and amplification of both viruses were performed on Vero cell monolayers. The origin of VV-RG is described in Kieny et al. (1984). The canarypox strain from which ALVAC was derived was isolated from a pox lesion on an infected canary. The virus was first isolated at the Rentschler Bakteriologishes Institut, Lauphein, Wurtemberg, Germany, where it was attenuated by 200 serial passages in CEFs. This attenuated strain (Kanapox) obtained from Rhone Merieux is licensed as a vaccine for canaries in France. At Virogenetics, the virus was subjected to four successive rounds of plaque purification under agarose. One plaque isolate, designated ALVAC, was selected for amplification and used in these studies. All amplifications and plaque titrations were performed on primary CEF cells. The origins of cells used in this study are as follows: (1) Vero cells (ATCC No. CCL8 1) are a line derived from African green monkey kidney; (2) MRC-5 (ATCC No. CCL171) are of human embryonic lung origin; (3) primary CEFs were obtained from lo- to 1 1-day-old embryonated eggs of SPF origin (SPAFAS, Inc., Storrs, CT); (4) HNK are human neonatal kidney cells which have been subcultured for less than five passages

carried through three further plaque purifications

220

TARTAGLIA

(Whittaker Bioproducts, Inc., Walkersville, MD (Cat No. 70-l 51)); (5) HEL 299 are human embryonic lung cells (ATCC No. CCL137); (6) WISH are of human amnion origin (ATCC No. CCL25); (7) Detroit 532 are of human foreskin (Downs Syndrome) origin (ATCC No. CCL54); and (8) JT-1 is a human lymphoblastoid cell line transformed with Epstein-Barr virus as described in Rickinson et al. (1984). Virus stocks used in this study were prepared as follows. Infected cells were collected in 1 mM Tris pH 9.0. Virus was released from the cells by three cycles of freezing and thawing followed by indirect sonication. Large debris was removed by low-speed centrifugation for 10 min. The supernatant was then layered over a 36% sucrose cushion and centrifuged at 70,000 g for 60 min. Virus in the pellet was collected in sterile 1 mn/l Tris, pH 9.0, and deaggregated by indirect sonication before yield was assessed by plaque titration on Vero or CEF monolayers. Virus used for intracranial inoculations was further purified by banding on a 25-40% sucrose gradient for 45 min at 40,000 g. The virus band was collected, diluted in 1 m/v/ Tris, pH 9.0, and concentrated by centrifugation at 70,000 g for 60 min. Virus in the pellet was collected in 1 mM Tris, pH 9.0, deaggregated by indirect sonication, and titrated. In the experiment designed to assess viral yield on cells of human origin, the following procedure was performed. HNK, HEL, MRC-5, WISH, Detroit 532, and JT-1 cell cultures containing 2 X 1O6 cells were mockinfected or infected with the wild-type Copenhagen strain of vaccinia, VC-2, the derivative NYVAC, or ALVAC at a multiplicity of infection of 0.1 PFU per cell. Identical inoculations were also performed on CEF cells which served as a permissive host for all viruses. Inoculations were performed in Eagle’s minimal essential medium (EMEM) containing 2% newborn calf serum (NCS). One set of cultures of each cell line was inoculated with the virus in the presence of 40 pg/ml of cytosine arabinoside (AraC; Sigma Cat No. C6645). Inoculum virus was allowed to adsorb for 60 min at 37”. Following this period, the inoculum was removed and the monolayer washed twice to remove unadsorbed virus. Five milliliters of maintenance medium (EMEM + 2% NCS) with or without AraC was added. For the human-derived cell line infections, three samples, in duplicate, taken at 0 hr postinfection (p.i.) and 72 hr p.i. in the presence and absence of 40 pglml of AraC were obtained. Additional infected cultures of CEF cells were also harvested at 24 and 48 hr p.i. Virus was liberated by three successive cycles of freezing and thawing. Virus yields were then estimated by plaque titration on CEF monolayers under agarose. Duplicates were titrated as individual samples and the results expressed as an average.

ET AL.

DNA cloning

and synthesis

Plasmids were constructed, screened, and grown by standard procedures (Maniatis et a/., 1982; Perkus et al., 1985; Piccini et al., 1987). Restriction endonucleases were obtained from Bethesda Research Laboratories (Gaithersburg, MD), New England Biolabs, Inc. (Beverly, MA), and Boehringer-Mannheim Corp. (Indianapolis, IN). The Klenow fragment of fscherichia co/i polymerase was obtained from Boehringer-Mannheim Corp. Bal-31 exonuclease and phage T4 DNA ligase were obtained from New England Biolabs, Inc. The reagents were used as specified by the various suppliers. Synthetic oligodeoxyribonucleotides were prepared on a Biosearch 8750 or Applied Biosystems 380B DNA synthesizer as previously described (Perkus et a/., 1989). DNA sequencing was performed by the dideoxy-chain termination method (Sanger et a/., 1977) using Sequenase (Tabor and Richardson, 1987) as previously described (Goebel et a/., 1990a,b). DNA amplification by polymerase chain reaction (PCR) for sequence verification (Engelke et al., 1988) was performed using custom synthesized oligonucleotide primers and the GeneAmp DNA amplification Reagent Kit (Perkin-Elmer Corp., Norwalk, CT) in an automated Perkin-Elmer Cetus DNA thermal cycler. Excess DNA sequences were deleted from plasmids by restriction endonuclease digestion followed by limited digestion by Bal-31 exonuclease and mutagenesis (Mandecki, 1986) using synthetic oligonucleotides. Generation

of NYVAC

Previously, we reported the complete nucleotide sequence for the Copenhagen strain of vaccinia virus (Goebel et a/., 1990a,b). This enabled the precise deletion of the 18 open reading frames from the Copenhagen strain to generate NYVAC. All ORFs, restriction fragments and nucleotide designations are presented according to Goebel et al. (1990a,b). Deletion of the vaccinia gene, ORF J2R

virus thymidine

kinase

To delete the vaccinia virus thymidine kinase gene, plasmid pSD460 was constructed. Flanking arms for the deletion of the thymidine kinase gene (J2R) were derived from plasmid pSD406, which contains vaccinia HindIll J in pUC8. Deletion plasmid pSD460 contains a 0.8-kb left vaccinia arm and a 0.5-kb right vaccinia arm flanking a J2R deletion (nt 83,858-84,386). In pSD460 thymidine kinase coding sequences are replaced by a polylinker region. pSD460 was used as donor plasmid for-in viva recombination with VC-2. Recombinant virus

ATTENUATED

VACCINIA

vP410, deleted for the fk gene, was identified by plaque hybridization to 32P-labeled polylinker DNA.

Deletion of the hemorrhagic region, ORFs B13R and B14R To delete the B 13R and B 14R ORFs from vP410, the following constructions were derived. Left and right vaccinia flanking arms for the deletion of the B13R and B14R ORFs region were derived from plasmids pSD419 and pSD422, respectively. pSD419 contains vaccinia S&l G (nt 160,744-173,351) cloned into pUC8. pSD422 contains the contiguous vaccinia WI fragment to the right, SalI J (nt 173,351-l 82,746) cloned into pUC8. To take advantage of the P-galactosidase screening system (Panicali eta/., 1986; Chakrabarti et al., 1985) plasmid pSD479BG was constructed. pSD479BG contains a 0.3-kb left vaccinia arm and a 0.3-kb right vaccinia arm flanking a 3.2-kb BamHl fragment containing the bulk of the /3-galactosidase coding sequences (Shapira et al., 1983) placed under the control of the endogenous vaccinia B13R promoter present in the left vaccinia flanking arm. pSD479BG was used as donor plasmid for in viva recombination with vaccinia virus vP410. Recombinant vaccinia virus vP533 was isolated as a blue plaque in the presence of the chromogenic substrate X-gal. In vP533, the B13R-B14R region is deleted and is replaced by ,%galactosidase. To remove /I-galactosidase sequences from vP533, deletion plasmid pSD486, in whichtheB13RandB14RORFs(nt172,549-173,552) are replaced by a polylinker, was used as donor plasmid for in viva recombination with recombinant vaccinia virus vP533. The resulting vaccinia deletion mutant, vP553, was isolated as a clear plaque in the presence of X-gal.

Deletion of the A-type inclusion region, ORF A26L To delete the ATI region from vP553, the following constructions were derived. pSD4 14 contains vaccinia Sal1 B cloned into pUC8. pSD414 was used as the source of both left (0.9 kb) and right (1.3 kb) vaccinia arms flanking the A26L deletion region. Plasmid pSD493KBG contains a 3.3-kb Bglll cassette containing the E. co/i /+galactosidase gene (Shapira et a/., 1983) under the control of the vaccinia 1 1-kDa promoter (Perkus et a/., 1990; Bertholet et a/., 1985) in the A26L deletion locus. Plasmid pSD493KBG was used in in viva recombination with rescuing virus vP553. Recombinant vaccinia virus, vP581, containing the P-galactosidase gene in the A26L deletion region, was isolated as a blue plaque in the presence of X-gal. Deletion plasmid pMP494A contains a precise deletion of the vaccinia A26L ORF (nt 137,889-l 38,937) flanked

VIRUS NYVAC

221

by left and right vaccinia arms. ln viva recombination between pMP494A and vP581 deleted the P-galactosidase insert and resulted in vaccinia deletion mutant VP61 8.

Deletion of the hemagglutinin gene, ORF A56R To delete the HA gene from vP618, the following plasmids were used. The construction of plasmids pSD466 (pSD466VC), which contains 0.4-kb left and 0.4-kb right vaccinia arms flanking the deletion of the HA gene, and pSD466KBG (pSD466VCBGA) which contains the 1 1K/P-galactosidase expression cassette in the HA deletion, has been described (Guo et a/., 1989). The vaccinia deletion in pSD466 encompasses nucleotides 161 ,185-l 62,053. Plasmid pSD466KBG was used to insert the ,!?-galactosidase gene into deletion mutant vP6 18, resulting in recombinant vaccinia virus, vP708. P-galactosidase sequences were deleted from vP708 using donor plasmid pSD467. pSD467 is identical to pSD466, except that EcoRI, Smal, and BamHl sites were removed from the pUC/vaccinia junction. ln viva recombination between vP708 and pSD467 resulted in vaccinia deletion mutant vP723.

Deletion of the host range gene region, ORFs C7L, C6L, C5L, C4L, C3L, C2L, ClL, Nl L, N2L, MlL, M2L, Kl L To delete the host range region (C7L-Kl L) from vP723, the following plasmids were used. pSD420 (Sal1 H cloned into pUC8) was used as the source of the vaccinia arm to the left of C7L. pSD435 (Kpnl F cloned into pUCl8) was used as the source of the vaccinia arm to the right of Kl L. To provide a substrate for the deletion of the (C7L-Kl L) gene cluster from vaccinia, the E. co/i @-galactosidase gene was first inserted into the vaccinia M2L deletion locus. Plasmid pMP409DBG, containing the P-galactosidase gene under the control of the vaccinia 11 -kDa promoter (Guo et al., 1990), was used as donor plasmid for in viva recombination with rescuing virus vP723. Recombinant vaccinia virus, vP784, containing the P-galactosidase gene inserted into the M2L deletion locus, was isolated as a blue plaque in the presence of X-gal. Deletion plasmid pMPCSK1 A, containing a 0.2-kb left vaccinia arm derived from pSD420 and a 0.6-kb right vaccinia arm derived from pSD435, is deleted for vaccinia sequences (nt 18,805-29,108) encompassing 12 vaccinia ORFs (C7L-KlL) (Goebel et a/., 1990a,b). In viva recombination between pMPCSK1 A and the P-galactosidase gene containing vaccinia recombinant, vP784, resulted in vaccinia deletion mutant vP804, which is deleted for ORFs (C-/L-K1 L) as well as the P-galactosidase gene.

222

TARTAGLIA

Deletion of the gene encoding the large subunit ribonucleotide reductase, ORF 14L

of

In plasmid pSD542KBG, a 3.2.kbSmal cassettecomposed of the E. co/i ,&galactosidase gene (Shapira et al., 1983) under the control of the vaccinia 1 1-kDa promoter (Perkus et al., 1990; Bertholet et al., 1985) is inserted into a partial deletion of l4L coding sequences. pSD524KBG was used to insert the ,&galactosidase gene into deletion mutant vP804, resulting in recombinant vaccinia virus, vP855. Plasmid pSD548 contains a deletion of the entire l4L gene (nt 65,04767,386) replaced by a polylinker region, which is flanked by 0.6-kb vaccinia DNA to the left and 0.6-kb vaccinia DNA to the right. In viva recombination between pSD548 and vP855 resulted in vaccinia deletion mutant vP866 which was isolated as a clear plaque in the presence of X-gal. DNA from recombinant vaccinia virus vP866 was analyzed by restriction digests followed by electrophoresis on an agarose gel. The restriction patterns were as expected. PCR (Engelke et al., 1988) using vP866 as template and primers flanking the six deletion loci detailed above produced DNA fragments of the expected sizes. Sequence analysis of the PCR-generated fragments around the deletion junctions confirmed that the junctions were as expected. Recombinant vaccinia virus vP866, containing the engineered deletions as described above, was designated vaccinia vaccine strain NYVAC (vP866). Insertion of the rabies virus glycoprotein NYVAC and ALVAC

gene into

The gene encoding rabies virus glycoprotein under the control of the vaccinia H6 promoter (Taylor et a/., 1988) was inserted into tk deletion plasmid pSD513. (pSD513 is identical to plasmid pSD460 described above except for an altered polylinker region.) The resulting plasmid, pRW842, was used as donor plasmid for in viva recombination with NYVAC rescuing virus. Recombinant vaccinia virus, VP879 (NYVAC-RG), was identified by plaque hybridization using 32P-labeled DNA probe to rabies glycoprotein coding sequences. ALVAC-RG was generated in a manner similar to that described in Taylor et a/. (199 1b) using ALVAC as the rescue virus. lmmunoprecipitations Preformed monolayers of avian or nonavian cells were inoculated with 10 PFU per cell of parental (NYVAC) or rabies recombinant virus (NYVAC-RG). The inoculation was performed in EMEM free of methionine and supplemented with 2% dialyzed fetal bovine

ET AL.

serum. After a 1-hr incubation, the inoculum was removed and the medium replaced with EMEM (methionine free) containing 20 &i/ml of r5S]methionine. After an overnight incubation of approximately 16 hr, cells were lysed by the addition of bufferA (1% Nonidet P-40, 10 mMTris, pH 7.4, 150 mlL1 NaCI, 1 rnn/l EDTA, 0.019/o sodium azide, 500 units/ml of aprotinin, and 0.02% phenylmethylsulfonyl fluoride). Immunoprecipitation was performed using a rabies glycoprotein-specific monoclonal antibody designated 24-3FlO supplied by Dr. C. Trimarchi, Griffin Laboratories, New York State Department of Health (Albany, NY), and a rat anti-mouse conjugate obtained from BoehringerMannheim Corp. (Cat No. 605-500). Protein A-Sepharose CL-4B obtained from Pharmacia LKB Biotechnology Inc. (Piscataway, NJ) was used as a support matrix. lmmunoprecipitates were fractionated on 10% polyacrylamide gels according to the method of Dreyfuss et a/. (1984). Gels were fixed, treated for fluorography with 1 M Na-salicylate for 1 hr, and exposed to Kodak XAR-2 film to visualize the immunoprecipitated protein species. Sources of animals New Zealand White rabbits were obtained from Hare-Marland (Hewitt, NJ). Three-week-old male Swiss Webster outbred mice, timed pregnant female Swiss Webster outbred mice, and 4-week-old Swiss Webster nude (nu+/nu+) mice were obtained from Taconic Farms, Inc. (Germantown, NY). All animals were maintained according to NIH guidelines. All animal protocols were approved by the institutional IACUC. When deemed necessary, mice which were obviously terminally ill were euthanized. Evaluation

of lesions in rabbits

Each of two rabbits was inoculated intradermally at multiple sites with 0.1 ml of PBS containing 104, 105, 106, 107, or 10’ PFU of each test virus or with PBS alone. The rabbits were observed daily from Day 4 until lesion resolution. Indurations and ulcerations were measured and recorded. Virus recovery from inoculation

sites

A single rabbit was inoculated intradermally at multiple sites with 0.1 ml of PBS containing 106, 1O’, or 1O* PFU of each test virus or with PBS alone. After 1 1 days, the rabbit was euthanized and skin biopsy specimens taken from each of the inoculation sites were aseptically prepared by mechanical disruption and indirect sonication for virus recovery. Infectious virus was assayed by plaque titration on CEF monolayers.

ATTENUATED

Virulence

VACCINIA

in mice

Groups of 10 mice, or 5 in the nude mice experiment, were inoculated intraperitoneally (ip) with one of several dilutions of virus in 0.5 ml of sterile PBS. Groups of 10 young or newborn mice were challenged intracranially (ic) with virus in 0.05 or 0.03 ml, respectively. Mice inoculated with PBS served as controls. All mice were observed daily for mortality or for general signs of disease. Mice found dead the morning following inoculation were excluded from the study due to potential death by trauma. Cyclophosphamide

(CY) treatment

Mice were injected by the ip route with 4 mg (0.2 ml) of CY (SIGMA) on Day -2, followed by virus injection on Day 0. On the following days postinfection, mice were injected ip with CY: 4 mg on Day 1; 2 mg on Days 4,7, and 11; and 3 mg on Days 14, 18,2 1,25, and 28. lmmunosuppression was indirectly monitored by enumerating white blood cells with a Coulter counter on Day 11. The average white blood cell count was 13,500 cells/PI for untreated mice (n = 4) and 4220 cells/~1 for CY-treated control mice (n = 5). Calculation

of LD,,

The lethal dose required to produce 50% mortality (LDsO) was determined by the proportional method of Reed and Muench (Reed and Muench, 1938). Potency testing

of NYVAC-RG

in mice

Four to six-week-old mice were inoculated in the footpad with 50 to 100 ~1 of a range of dilutions (2.08.0 log,, tissue culture infective dose 50% (TCID,,)) of VV-RG (Kieny et a/., 1984) ALVAC-RG, or the NYVACRG. Each group consisted of eight mice. At 14 days postvaccination, the mice were challenged by intracranial inoculation with 15 LD,, of the rabies virus CVS strain (0.03 ml). On Day 28, surviving mice were counted and the protective dose 50% (PDsO)was calculated. RESULTS Derivation

of NYVAC

The NYVAC strain of vaccinia virus was generated from VC-2, a plaque-cloned isolate of the Copenhagen vaccine strain. We have recently reported the complete DNA sequence of VC-2 (Goebel et a/., 1990a,b). With this information, it was possible to engineer precise deletions of individual ORFs or clusters of ORFs encoding genetic functions associated with virulence. To generate NYVAC from VC-2, 18 vaccinia ORFs were

VIRUS NWAC

223

precisely deleted in a series of sequential manipulations as described under Materials and Methods. These deletions were constructed in a manner designed to prevent the appearance of novel unwanted open reading frames. Figure 1 schematically depicts the ORFs deleted to generate NYVAC. Replication studies of NYVAC and ALVAC on human tissue cell lines Previous results defining vaccinia virus host range deletion mutants illustrated that deletion of the KlL and C7L coding regions from the Copenhagen strain of vaccinia virus resulted in >99% reduction in the level of replication on human-derived MRC-5 cells compared to that on monkey kidney-derived Vero cells (Perkus et al., 1990). In order to determine the level of replication of the NYVAC strain of vaccinia virus in cells of human origin, six cell lines were inoculated at an input multiplicity of 0.1 PFU per cell under liquid culture and incubated for 72 hr. The Copenhagen parental clone (VC-2) was inoculated in parallel. Primary CEF cells were included to represent a permissive cell substrate for all viruses. Cultures were analyzed on the basis of two criteria: the occurrence of productive viral replication and expression of an extrinsic antigen. The replication potential of NYVAC in a number of human-derived cells is shown in Table 1. Both VC-2 and NYVAC are capable of productive replication in CEF cells, although NYVAC shows slightly reduced yields. VC-2 is also capable of productive replication in the six human-derived cell lines tested with comparable yields except in the EBV-transformed lymphoblastoid cell line JT-1. In contrast, NYVAC is highly attenuated in its ability to productively replicate in any of the human-derived cell lines tested. Small increases of infectious virus above residual virus levels were obtained from NYVAC-infected MRC-5, Detroit 532, HEL, and HNK cells. Replication on these cell lines was significantly reduced when compared to virus yields obtained from NYVAC-infected CEF cells or with parental VC-2 (Table 1). It should be noted that the yield at 24 hr in CEF cells for both NYVAC and VC-2 is equivalent to the 72-hr yield. Allowing the human cell line cultures to incubate an additional 48 hr (another two viral growth cycles) may, therefore, have amplified the relative virus yield obtained. Consistent with the low levels of virus yields obtained in the human-derived cell lines, MRC-5 and Detroit 532, detectable but reduced levels of NYVAC-specific DNA accumulation were noted. The level of DNA accumulation in the MRC-5 and Detroit 532 NYVAC-infected cell lines relative to that observed in NYVAC-infected CEF cells paralleled the relative virus yields

224

TARTAGLIA

C

NMKF III I ,I

t’ ,’

4’

I

EPOIGLJH II

I

\\ _e-- -a-- JI** \*, 14L Ftibonucleolide

#’ C7L C6L C5L C4L C3L C2L ClL Nl L

s,reductase 1

Host range (16kDa) 17kDa 25kDa 37kDa Complement binding 59kDa 26kDa Secretory (14kDa) N2L 2IkDa MIL 54kDa M2L 25kDa Ki L Host range (33kDa)

(87kDa)

D

III

1

#’

ET AL.

A

I

II ,,, * ,

,

a’ 8’

I #’ a’

\, ‘\

J2R Thymidine kinase (2OkDa)

I

II ,t’ 8:

*’ ,)’ a’

(29kDa)

I3

I

a’ #’ A26L ATI (37kDa) _--A56R

_s--

, : :

,

II ,’ : I I

:

: I : I I : : I I I

l

: : :

,’

:

,’ ’ ;’

_ s -’

Hemagglutinln

: : : :

: , I(

,’

:

II : ‘6\

(35kDa)

a , ’3 I ‘,

: : : : : :

I 1 3 * , \ , \ 1 * , \ 8 , ,

,:

, .’ B13A Serpin B14R Serpln

t (13kDa) (25kDa)

FIG. 1. Derivation of NYVAC. At the top the HindIll restriction map of the vaccinia virus genome (VC-2 plaque isolate, Copenhagen strain) is depicted. Expanded are the six regions of VC-2 that were sequentially deleted in the generation of NM/AC. The deletions were engineered as described under Materials and Methods. Below each deletion locus are listed the ORFs which are deleted from that locus, along with the functions or homologies and molecular weight of their gene products as described in the text.

(data not presented). NYVAC-specific viral DNA accumulation was not observed in any of the other humanderived cells. An equivalent experiment was also performed using the avipox virus, ALVAC. The results of virus replication are also shown in Table 1. No progeny virus was detectable in any of the human cell lines consistent with the host range restriction of canarypox virus to avian species. Also consistent with a lack of productive replication of ALVAC in these human-derived cells is the observation that no ALVAC-specific DNA accumulation was detectable in any of the human-derived cell lines (data not presented).

Expression of rabies glycoprotein human cells

by NYVAC-RG in

In order to determine whether efficient expression of a foreign gene could be obtained in the absence of significant levels of productive viral replication, the same cell lines were inoculated with a NYVAC recombinant expressing the rabies virus glycoprotein in the presence of [35S]methionine as described under Materials and Methods. lmmunoprecipitation of the rabies glycoprotein was performed from the radiolabeled culture lysate using a monoclonal antibody specific for the rabies glycoprotein. Figure 2 illustrates immunoprecipitation of a 67-kDa protein consistent with a fully glycosylated form of the rabies glycoprotein. This species can be seen in lanes c, f, and i, representing, respectively, CEF, HNK, and HEL cells inoculated with NYVAC-RG. No serologically cross-reactive product

was detected in uninfected (lanes a, d, and g) or parental NYVAC-infected (lanes b, e, and h) cell lysates. Although the results of only two human cell lines are shown, equivalent results were obtained with all other human cells analyzed.

Inoculations on the rabbit skin The induction and nature of skin lesions on rabbits following intradermal (id) inoculations have been previously used as a measure of pathogenicity of vaccinia virus strains (Buller et a/., 1988; Child et a/., 1990; Fenner, 1958; Flexner et al., 1987; Ghendon and Chernos, 1964). Therefore, the nature of lesions associated with id inoculation of the vaccinia strains WR, Wyeth, Copenhagen (VC-2), and NYVAC was evaluated by inoculation of two rabbits (A069 and Al 28). The two rabbits displayed different overall sensitivities to the viruses, with rabbit Al 28 displaying less severe reactions than rabbit A069. In rabbit Al 28, lesions were relatively small and resolved by 27 days postinoculation. On rabbit A069, lesions were intense, especially for the WR inoculation sites, and resolved only after 49 days. Intensity of the lesions was also dependent on the location of the inoculation sites relative to the lymph drainage network. In particular, all sites located above the backspine displayed more intense lesions and required longer times to resolve than lesions located on the flanks. All lesions were measured daily from Day 4 to the disappearance of the last lesion, and the means of maximum lesion size and days to resolution were calculated (Table 2). No local reactions were observed

ATTENUATED

VACCINIA

TABLE1 REPLICATIONOF COPENHAGEN(VC-2) NWAC, AND ALVAC IN AVIAN OR HUMAN DERIVEDCELL LINES Yield Cells

Hours postrnfection

CEF

MRC-5

WISH

Detroit

HEL

JT-1

HNK

0 24 48 72 72A" 0 72 72A 0 72 72A 0 72 72A 0 72 72A 0 72 72A 0 72 72A

vc-2

NYVAC

ALVAC

3.8' 8.3 8.6 8.3 <1.4 3.8 7.2 2.2 3.4 7.6 -d

3.7 7.8 7.9 7.7 1.8 3.8 4.6 2.2 3.4 2.2 1.9 3.7 5.4 1.7 3.5 4.6 2.1 3.1 3.1 2.1 3.7 4.5 2.7

4.5 6.6 7.7 7.5 3.1 4.7 3.8 3.7 4.3 3.1 2.9 4.4 3.4 2.9 4.3 3.3 3.6 4.1 4.2 4.4 4.7 3.6 3.7

3.8 7.2 1.7 3.8 7.5 2.5 3.1 6.5 2.4 3.8 7.6 3.1

O/O

Yield”

25

225

VIRUS NYVAC

induration or ulceration. At Day 11 after inoculation, skin samples from the sites of inoculation were excised and mechanically disrupted, and virus was titrated on CEF cells. The results are shown in Table 3. In no case was more virus recovered at this time point than was administered. Recovery of vaccinia strain WR, was approximately 1O6 PFU of virus at each site irrespective of the amount of virus administered. Recovery of vaccinia strains Wyeth and VC-2 was 1O3to 1O4 PFU regardless of the amount administered. No infectious virus was recovered from sites inoculated with NYVAC.

0.25

Comparative virulence in normal 3-week-old newborn mice by the intracranial inoculation

0.0004

Groups of male 3-week-old Swiss Webster outbred mice (normal young mice) and l- to 2-day-old mice (normal newborn mice) were inoculated intracranially with one of several 1O-fold dilutions of NYVAC, ALVAC, Wyeth, VC-2, or WR. Mice were observed daily for 14 days (newborn mice) or 21 days (young mice) after inoculation. In both young and newborn mice, NYVAC and ALVAC are considerably less virulent than the other vaccinia virus strains tested (Table 4). Both NYVAC and ALVAC caused mortality in mice by this route of admin-

1.6

0.125

0.039

and

0.079

a Yield of NYVAC at 72 hr postinfection expressed as a percentage of yield of VC-2 at 72 hr on the same cell line. b Titer expressed as loglo PFU/ml. c Sample was incubated in the presence of 40 pglml of cytosine arabinoside. d Not determined.

a

bcdefgh

i

from sites injected with the control PBS. Ulcerative lesions were observed at sites injected with WR, VC-2, and Wyeth vaccinia virus strains. Significantly, no induration or ulcerative lesions were observed at sites of inoculation with NYVAC. Persistence inoculation

of infectious

virus at the site of

To assess the relative persistence of these viruses at the site of inoculation, a rabbit was inoculated intradermally at multiple sites with 0.1 ml PBS containing 106, 107, or lo8 PFU of VC-2, WR, Wyeth, or NYVAC. For each virus, the lo7 PFU dose was located above the backspine, flanked by the 1O6 and 1O* doses. Sites of inoculation were observed daily for 11 days. WR elicited the most intense response, followed by VC-2 and then Wyeth (Table 3). Ulceration was first observed at Day 9 for WR and Wyeth and at Day 10 for VC-2. Sites inoculated with NYVAC or control PBS displayed no

FIG. 2. lmmunoprecipitation of rabies glycoprotern expressed by NWAC-RG in CEF or human cells. CEF or human cell monolayers were infected with 10 PFUlcell of parental NYVAC or the NYVAC-RG recombinant. lmmunoprecipitations were performed as described under Materials and Methods. An autoradiogram IS shown. Lanes a-c, CEF; lanes d-f, HNK (human neonatal krdney, Whittaker M. A. Broproducts Cat No. 70-151); lanes g-i, HEL (human embryonic lung, ATCC Cat No. CCL1 37); unrnfected: lanes a, d. and g; NYVAC Infected: lanes b, e, and h; NYVAC-RG infected: lanes c, f, and I. Molecular werght markers are indicated in the margrn (200, 97, 68, 45, 29, 18, and 14 kDa).

226

TARTAGLIA

ET AL

TABLE 2

TABLE 4

INDURATIONAND ULCERATIONATTHE SITE OF INTFIADERMALINOCULATION OF THE RABBIT SKIN

INTRACRANIALlN0cuLATl0~ OF 3-WEEK-OLD 0s NEWBORN OUTBRED MICE

Induration Virus strain WR

Wyeth

vc-2

NYVAC

Ulceration

LD,oa

Dosea

Size6

DaysC

Size

Days

Poxvirus strain

1 o4 105 1 O6 107 1O8 104

386 622 1057 877 581 32

30 35 34 35 25 5

88 149 271 204 88 -d

30 32 34 35 26

WR vc-2 Wyeth NYVAC ALVAC

lo5 1O6 10’ lo8 lo4 105 106 10’ 108 lo4

116 267 202 240 64 86 136 167 155 -

15 17 17 29 7 8 17 21 32

3 3 12

15 24 31

105 lo6 lo7 lo8

6 6

10 8

-

-

a PFU of indicated vaccinia virus in 0.1 ml PBS inoculated intradermally into one site. b Mean maximum size of lesions (mm2). c Mean time after inoculation for complete healing of lesion. d No lesion discernable.

istration at the highest doses (3 X 1O* PFU NYVAC; 3.85 X lo* PFU ALVAC) by an undetermined mechanism. Nevertheless, both NYVAC and ALVAC demon-

TABLE 3 PERSISTENCEOF POXVIRUSESAT THE SITE OF INTRADERMALINOCULATION

Virus

lnoculum dose

Total virus recovered

8.0a 7.0 6.0 8.0 7.0 6.0 8.0 7.0 6.0 8.0 7.0 6.0

6.14 6.26 6.21 3.66 4.10 3.59 4.47 4.74 3.97 0 0 0

WR

Wyeth

vc-2

NYVAC

a Expressed

as log,, PFU.

3-week mice 2.5 1.26 X 5.00 x 1.58 X 1.58 X

lo4 lo4 10’ 10’

Newborn

mice

0.4 0.1 1.6 1.58 x 1O6 1 .oo x 1o7

’ Calculated 50% lethal dose (PFU) for newborn and 3-week-old mice by the indicated vaccinia viruses and for ALVAC by the intracranial route.

strate an approximate 4 log,0 improvement in attenuation over Copenhagen or Wyeth. Using an ic challenge of newborn mice as a more sensitive model, both NYVAC and ALVAC are much less virulent than vaccinia strains WR, Wyeth, and VC-2 (Table 4). The two highest doses of NYVAC, 6 X lOa and 6 X 10’ PFU, caused 100% mortality. However, the mean survival time of mice challenged with the highest dose, corresponding to 380 LD,,, was only 2.2 rt 0.5 days (nine deaths on Day 2 and one on Day 4). In contrast, all mice challenged with the highest dose of WR, equivalent to 500 LD,,, survived to Day 4. The rapid onset of mortality in mice challenged with NYVAC, clearly less virulent than WR, raises suspicion about the cause of death in these mice. At the highest dose of ALVAC tested (6.3 X 1O7 PFU), 100% mortality resulted. Mortality rates of 33.3% were observed with a dose of 6.3 X 10” PFU. Histological examination of neural tissues would be required to ascertain whether the deaths resulted from complications not directly attributable to viral toxicity or associated pathology. Significantly, however, both NYVAC and ALVAC demonstrate an approximate 6-7 log,,, improvement in attenuation characteristics compared to WR, Copenhagen, and Wyeth vaccinia strains in this sensitive test. Inoculation of genetically deficient mice

or chemically

immune

lntraperitoneal inoculation of high doses of NYVAC (5 x 1O8 PFU) or ALVAC (1 OS PFU) into nude mice caused no deaths, no lesions, and no apparent disease throughout the loo-day observation period. In contrast, mice inoculated with WR (1 O3 or 1O4 PFU), Wyeth (5 X 10’ or 5 X 10’ PFU), or VC-2 (1 O4 to 1OS PFU) displayed disseminated lesions typical of poxviruses first on the toes and then on the tail followed by severe orchitis in some animals. In mice infected with

ATTENUATED

VACCINIA

TABLE 5 VIRULENCESTUDIESIN IMMUNOCOMPROMISEDMICE LD,oa Poxvirus strain

Nude mice

WR vc-2 Wyeth NYVAC ALVAC

422 >109 1.58 x lo7 >5.50 x lo8 >109

Cyclophosphamldetreated mice 42 <1.65x lo5 1.83 x lo6 7.23 X 10’ 35.00 x lo* *

a Calculated 50% lethal dose (PFU) for nude and cyclophosphamide-treated mice by the indicated vaccinia viruses and for ALVAC by the intraperitoneal route. b Five of 10 mice died at the highest dose of 5 X 10’ PFU.

WR and Wyeth, the appearance of disseminated lesions generally led to eventual death, whereas most mice infected with VC-2 eventually recovered. Calculated LD,, values are given in Table 5. Mice inoculated with VC-2 began to display lesions on their toes (red papules) and 1 to 2 days later on the tail. These lesions occurred between 1 1 and 13 days pi in mice given the highest doses (1 Og, 108, 107, and 1O6 PFU), on Day 16 pi in mice given 1O5 PFU, and on Day 21 pi in mice given 1O4 PFU. No lesions were observed in mice inoculated with lo3 and 10’ PFU during the loo-day observation period. Orchitis was noticed on Day 23 pi in mice given 10’ and 10’ PFU and approximately 7 days later in the other groups (1 O7to 1O4PFU). Orchitis was especially intense in the 10’ and 1O8 PFU groups and, although receding, was observed until the end of the loo-day observation period. Some pox-like lesions were noticed on the skin of a few mice, occurring around 30-35 days pi. Most pox lesions healed normally between 60 and 90 days pi. Only one mouse died in the group inoculated with 10’ PFU (Day 34 pi) and one mouse died in the group inoculated with 10’ PFU (Day 94 pi). No other deaths were observed in the VC+inoculated mice. Mice inoculated with lo4 PFU of the WR strain of vaccinia started to display pox lesions on Day 17 pi. These lesions appeared identical to the lesions displayed by the VC-2-injected mice (swollen toes, tail). Mice inoculated with 1O3 PFU of WR strain did not develop lesions until 34 days pi. Orchitis was noticed only in the mice inoculated with the highest dose of WR (1 O4 PFU). During the latter stages of the observation period, lesions appeared around the mouth and the mice stopped eating. All mice inoculated with 1O4PFU of WR died or were euthanized when deemed necessary between 21 and 31 days pi. Four of the 5 mice injected

227

VIRUS NYVAC

with lo3 PFU of WR died or were euthanized when deemed necessary between 35 and 57 days pi. No deaths were observed in mice inoculated with lower doses of WR (1 to 100 PFU). Mice inoculated with the Wyeth strain of vaccinia virus at the higher doses (5 X 1O7 and 5 X 1O* PFU) showed lesions on toes and tails, developed orchitis, and died. Mice injected with 5 X lo6 PFU or less of Wyeth showed no signs of disease or lesions. Significantly, nude mice inoculated with NYVAC or ALVAC displayed no signs of disease throughout the loo-day observation period. As shown in Table 5, CY-treated mice provided a more sensitive model for assaying poxvirus virulence than did nude mice. LD,, values for the WR, Wyeth, and VC-2 vaccinia virus strains were significantly lower in this model system than in the nude mouse model. Additionally, lesions developed in mice injected with Wyeth, WR, and VC-2 vaccinia viruses, as noted below, with higher doses of each virus resulting in more rapid formation of lesions. As was seen with nude mice, CY-treated mice injected with NYVAC or ALVAC did not develop lesions. However, unlike nude mice, some deaths were observed in CY-treated mice challenged with NYVAC and ALVAC, regardless of dose. These random incidences are being further investigated. Mice injected with all doses of Wyeth (9.5 X 1O4 to 9.5 x 1O* PFU) displayed pox lesions on their tail and/ or on their toes between 7 and 15 days pi. In addition, the tails and the toes were swollen. Evolution of lesions on the tail was typical of pox lesions with formation of a papule, ulceration, and finally formation of a scab. Mice inoculated with all doses of VC-2 (1.65 X 1O5 to 1.65 X 10’ PFU) also displayed pox lesions on their tails and/or their toes analogous to those of Wyeth-injected mice. These lesions were observed between 7 and 12 days postinoculation. Mice injected with 2 15 to 2.15 X 1O4 PFU of the WR vaccinia virus strain displayed pox lesions on their tail and/or their toes between 12 and 16 days postinoculation. No lesions were observed on mice injected with the lower doses of WR virus, although deaths occurred in these groups. Potency testing

of NYVAC-RG

In order to determine that attenuation of the Copenhagen strain of vaccinia virus had been effected without significantly altering the ability of the resulting NYVAC strain to be a useful vector, comparative potency tests were per-formed. In order to monitor the immunogenic potential of the vector during the sequential genetic manipulations performed to attenuate the virus, the rabiesvirus glycoprotein was used as a reporter

228

TARTAGLIA TABLE 6 COMPARATIVEEFFICACYOF NYVAC-RG, VV-RG, AND ALVAC-RG IN MICE Recombinant

PDma

VV-RG ALVAC-RG NYVAC-RG

3.74 3.86 3.70

a Four- to srx-week-old mice were inoculated in the footpad with 50-100 PI of a range of dilutions (2.0-8.0 loglo tissue culture infection dose 50%; TCID,,) of VV-RG (Kieny eta/., 1984). ALVAC-RG, or NYVAC-RG. At Day 14, mice of each group were challenged by intracranial inoculation of 30 PI of a live CVS strain of rabies virus corresponding to 15 lethal doses 50% (LD,,) per mouse. At Day 28, surviving mice were counted and a protective dose 50% (PD,,) was calculated.

extrinsic antigen. The protective efficacy of the vectors expressing the rabies glycoprotein gene was evaluated in the standard NIH mouse potency test for rabies (Seligmann, 1973). Table 6 demonstrates that the PD,, values obtained with the highly attenuated NYVAC vector are identical to those obtained using a Copenhagen-based recombinant containing the rabies glycoprotein gene in the tk locus (Kieny et al., 1984) and similar to PD,, values obtained with ALVAC-RG, a canarypox-based vector restricted for replication to avian species.

DISCUSSION The exact origins of vaccinia virus are unknown (Baxby, 1981). However, many strains of vaccinia virus exist and were utilized during the WHO campaign to eliminate smallpox. The use of some strains (e.g., Berne) was short lived since adverse reactions to vaccination were unacceptably high (Fenner et al., 1989). Others, for example MVA, while producing no significant side effects, induced little or no neutralizing antibody. A balance must exist between safety and efficacy. This was true for the standard smallpox vaccinia vaccines and is no less true for candidate vaccinia recombinant vaccines today. This paper describes the use of genetic engineering to produce a highly attenuated strain of vaccinia virus, NYVAC, which retains its full capacity to express foreign genes and produce a protective immune response in an immunized host. The derivation of NYVAC was accomplished by the precise deletion of 18 endogenous ORFs from the vaccinia genome (Fig. 1). Multiple genes were deleted, recognizing that virulence is most likely a multifactorial phenomenon. Included in these deletions are the ORFs encoding the vaccinia virus thymidine kinase, the large subunit of the ribonucleotide reductase, the

ET AL.

vaccinia hemagglutinin, the truncated homolog of the 94-kDa A-type inclusion gene, two ORFs homologous to the N-terminal and C-terminal regions, respectively, of the 38-kDa serine protease inhibitor (Serpin), a secretory protein, and a complement 4b binding protein. These vaccinia-encoded gene products and/or their homologs encoded by other poxviruses have functions implicated in the pathogenicity of poxviruses (Buller and Palumbo, 199 1; Turner and Moyer, 1990; Dales, 1990). NYVAC was further attenuated by the deletion of the host range regulatory genes, C7L and Kl L. The gene products of C7L and Kl L modify the replication competency of vaccinia virus on cells derived from certain species including human-derived cells, but not others (Gillard eta/., 1986; Perkus eta/., 1990). Results described in Table 1 demonstrate the highly debilitated growth characteristics of NYVAC relative to those of parental Copenhagen (VC-2) in human-derived MRC-5, Detroit 532, HEL, HNK, WISH, and EBV-transformed B cells. The study was also performed in comparison to ALVAC, the designation given to a canarypox virus vector which is naturally host-restricted for replication to avian species. The inability to detect progeny virus from NYVAC-infected WISH and EBV-transformed B cells (Table 1) coupled with the lack of detectable NYVAC-specific DNA accumulation in these infected cells (data not presented) suggest that NYVAC is unable to productively replicate in these cells and that replication is blocked early in the virus replicative cycle (prior to the onset of DNA replication). The demonstration of detectable, albeit low, levels of progeny virus from NYVAC-infected MRC-5 and Detroit 532 cells (Table 1) and the concomitant low level accumulation of NYVAC-specific DNA in these infected cells demonstrate that a complete block in NYVAC replication does not occur in all human-derived cells. Since deletion of these genetic functions downregulates the ability of the virus to replicate on human cells, this provides yet another molecular approach to attenuation. On the other hand, these “essential” regulatory functions are conditional and the virus does not require these functions for amplification on permissive and accepted cell substrates for vaccine production such as primary CEFs. NYVAC, deleted of known virulence genes and having restricted in vitro growth characteristics, was analyzed in animal model systems to assess its attenuation characteristics. These studies were performed in comparison with the neurovirulent vaccinia virus laboratory strain, WR, two vaccinia virus vaccine strains, Wyeth (New York City Board of Health) and Copenhagen (VC-2) as well as with a canarypox virus strain, ALVAC. Together these viruses provided a spectrum of relative pathogenic potentials in the mouse challenge model and the rabbit skin model, with WR being

AnENUATED

VACCINIA

the most virulent strain, Wyeth and Copenhagen (VC-2) providing previously utilized attenuated vaccine strains with documented characteristics, and ALVAC providing an example of a poxvirus whose replication is restricted to avian species. Results from these in viva analyses clearly demonstrate the highly attenuated properties of NYVAC relative to the vaccinia virus strains, WR, Wyeth, and Copenhagen (VC-2) (Tables 2-5). Significantly, the LD,, values for NYVAC were comparable to those observed with the avian host-restricted avipoxvirus, ALVAC. Deaths due to NYVAC, as well as ALVAC, were observed only when extremely high doses of virus were administered via the intracranial route (Table 4). It has not yet been established whether these deaths were due to nonspecific consequences of inoculation of a high protein mass. Results from analyses in immunocompromised mouse models (nude and CY-treated) also demonstrate the relatively high attenuation characteristics of NYVAC, compared to those of the WR, Wyeth, and Copenhagen strains (Table 5). Interestingly, although the LD,, values for Wyeth, Copenhagen, and NYVAC were similar in the nude mouse model, Wyeth- and Copenhagen-challenged mice showed signs of disseminated vaccinia infection. Similar disseminated infections were also observed in CY-treated mice inoculated with the Wyeth and Copenhagen strain. Significantly, no evidence of disseminated vaccinia infection or vaccinial disease was observed in NYVAC-inoculated animals or ALVAC-inoculated animals over the observation period. LD,, values from a mouse intracranial challenge system have previously been reported for vaccinia virus mutants which have a deleted or inactivated tk gene (Buller et al., 1985) HA gene (Shida et al., 1988; Flexner et a/., 1987), vaccinia epidermal growth factor (VEGF) gene (Buller et al., 1988) vaccinia complement 4b binding protein gene (Buller and Palumbo, 1991), 13.8-kDa secretory protein gene (Kotwal et a/., 1989), or large subunit of the ribonucleotide reductase gene (Child et a/., 1990). The most reduced level of pathogenicity was observed for mutants lacking the tk gene, the VEGF gene, or the 13.8-kDa secretory protein (Nl L). LD,, values for these mutant viruses were approximately lO,OOO-fold higher than that of wild-type virus. Minimal increases in the LD,, values (1 O-fold) were observed with the mutants devoid of a functional ribonucleotide reductase or a vaccinia complement binding protein. These results coupled to the LD,, values obtained for NYVAC in the mouse intracranial challenge system (Table 4) suggest that deletion of multiple virulence-associated genes provides a synergistic effect with respect to the reduction in pathogenicity. A similar observation was noted with a mutant virus lack-

VIRUS NYVAC

229

ing both the tk gene and the large subunit of the ribonucleotide reductase (Child et al., 1990). Another measure of the innocuity of NYVAC relative to that of the WR, Wyeth, and Copenhagen vaccinia virus strains was provided by the intradermal administration of these viruses on rabbit skin. Significantly, no local reactions were detected at the sites inoculated with any dose of NYVAC (Table 2). This is in contrast to the induration and ulceration observed following administration of the WR, Wyeth, and Copenhagen vaccinia virus strains. Lesions caused by WR inoculation were, as expected, the most severe. If one considers the rabbit skin reactions caused by the WR, Wyeth, Copenhagen, and NYVAC vaccinia virus strains, a correlation exists between the persistence of the viruses at the sites of inoculation (Table 3) and the severity of the lesions (Table 2). NYVAC, which causes no local reactions, does not persist at the inoculation site and shows no evidence of replication at Day 11 pi. The WR, Wyeth, and Copenhagen vaccinia virus strains all persist until at least 11 days postinoculation and induce ulcerated skin lesions. However, if the results observed with ALVAC, a virus unable to replicate in nonavian species, are considered, it becomes obvious that the ability to replicate at the site of inoculation is not the sole correlate with reactivity since intradermal inoculation of ALVAC caused areas of induration in a dose dependent manner with no ulceration (unpublished observations). Therefore, it is likely that factors other than the replicative capacity of the virus contribute to the formation of the lesions. It is intriguing to speculate that poxvirus-encoded virulence factors induce the formation of lesions. Deletion of one or a combination of the genes as in NYVAC prevents lesion occurrence. Previous reports have shown that deletion of the VEGF gene, the vaccinia HA gene, and the vaccinia tk gene decreased the ability of vaccinia to induce skin lesions on rabbits following intradermal inoculation (Buller et a/., 1988; Flexner et al., 1987). Deletion of the large subunit of the ribonucleotide reductase did not, by itself, reduce the potential of the virus to cause skin lesions compared to wild-type virus. Child et al. (1990) have reported, however, that a vaccinia mutant virus inactivated for both the gene encoding the large subunit of the ribonucleotide reductase and the tk gene was markedly reduced in its ability to induce skin lesions on rabbits. More recently, intradermal inoculation of cows in the neck region with Wyeth-based recombinants containing either the rinderpest virus (RPV) HA or F gene in the tk locus resulted in the formation of typical pox-like lesions at the inoculation site. Inoculation of a Wyeth-based recombinant containing the RPV HA and F genes in the tk and the HA gene loci, respectively, however, did not induce

230

TARTAGLIA

such lesions (Giavedoni e2 al., 1991), although the occurrence of other genomic rearrangements were not assessed. Such a finding again illustrates the multifactorial nature of virulence, in that varying effects can be observed using different virus genetic backgrounds and different animal model systems. Therefore, it is reasonable to conclude that deletion of the large subunit of the ribonucleotide reductase, the tk and HA genes, as well as the host range genes in NYVAC may have contributed to the inability of NYVAC to induce skin lesions on rabbits following intradermal inoculation. Results shown in Tables 4 and 5 further exemplify the multifactorial nature of poxvirus pathogenesis by illustrating that attenuation is not an absolute phenomenon, but a relative one, reflecting the complex interactions between the virus and the host. For instance, the LD,, values for the attenuated vaccine strains, Wyeth and Copenhagen, were found to approach the low LD,, values of the neurovirulent WR strain in immunocompetent mice inoculated via the intracranial route. In the nude and CY-treated test systems, both Wyeth and Copenhagen strains displayed an attenuated phenotype relative to that of the WR strain (Table 5). However, their attenuation profiles relative to each other differed between the nude and the CY-treated animals. Another interesting feature from these data was the fact that lethality could be distinguished from virus dissemination in the host. Although, like NYVAC and ALVAC, intraperitoneal inoculation of the maximum dosage of the Copenhagen strain (10’ PFU) in nude mice did not produce 50% lethality, Copenhagen-inoculated mice developed orchitis as well as skin lesions on their tails and toes. These symptoms are indicative of disseminated vaccinia infection. Significantly, none of the NYVAC- or ALVAC-inoculated mice displayed any of these symptoms. Together the results presented in this communication demonstrate the highly attenuated nature of NYVAC relative to that of WR and the previously utilized vaccinia virus vaccine strains, Wyeth and Copenhagen. In fact, the pathogenic profile of NYVAC, in the animal model systems tested, was similar to that of ALVAC, a poxvirus known to productively replicate only in avian species. The apparently restricted capacity of NYVAC to productively replicate on cells derived from humans (Table 1) and other species, including the mouse, swine, dog, and horse (data not shown), provides a considerable barrier that limits or prevents potential transmission to unvaccinated contacts or to the general environment in addition to providing a vector with reduced probability of dissemination within the vaccinated individual.

ET AL

Significantly, NYVAC-based vaccine candidates have been shown to be efficacious. NWAC recombinants expressing foreign gene products from a number of pathogens have elicited immunological responses toward the foreign gene products in several animal species, including primates (unpublished studies). In particular, a NYVAC-based recombinant expressing the rabies glycoprotein was able to protect mice against a lethal rabies challenge. The potency of the NYVAC-based rabies glycoprotein recombinant was comparable to the PD,, value for a Copenhagen-based recombinant containing the rabies glycoprotein in the tk locus (Table 6). NYVAC-based recombinants have also been shown to elicit measles virus neutralizing antibodies in rabbits and protection against pseudorabies virus and Japanese encephalitis virus challenge in swine (unpublished results). The highly attenuated NYVAC strain confers safety advantages for the development of live poxvirus-based vaccine candidates with human and veterinary applications (Tartaglia et al., 1990). Furthermore, the use of NYVAC as a general laboratory expression vector system may greatly reduce the biological hazards associated with using vaccinia virus.

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