Proc. Natl. Acad. Sci. USA Vol. 84, pp. 5908-5912, August 1987 Medical Sciences Expression of herpes simplex virus 1 glycoprotein B by a recombinant vaccinia virus and protection of mice against lethal herpes simplex virus 1 infection (viral latency/expression vector/neutralizing antibody/protective immunity) EDOUARD M. CANTIN*t, RICHARD EBERLE*, JOSEPH L. BALDICKt, BERNARD Mosst, DRU E. WILLEY*, ABNER L. NOTKINS§, AND HARRY OPENSHAW* *Department of Neurology, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010; tLaboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, and §Laboratory of Oral Medicine, National Institute of Dental Research, Bethesda, MD 20205 Communicated by Rachmiel Levine, April 20, 1987 (received for review February 4, 1987) ABSTRACT The herpes simplex virus 1 (HSV-1) strain F expresses HSV-1 gB and induces a protective immune gene encoding glycoprotein gB was isolated and modified at the response in mice. 5' end by in vitro oligonucleotide-directed mutagenesis. The modified gB gene was inserted into the vaccinia virus genome MATERIALS AND METHODS and expressed under the control of a vaccinia virus promoter. The mature gB glycoprotein produced by the vaccinia virus Viruses and Cells. HSV-1 strain F [HSV-1 (F)] was prop- recombinant was glycosylated, was expressed at the cell sur- agated in Vero cell monolayers, and virus released into the face, and was indistinguishable from authentic HSV-1 gB in medium was concentrated by ultracentrifugation. Vaccinia terms of electrophoretic mobility. Mice immunized intrader- virus (WR strain) was propagated in Hela cells and purified mally with the recombinant vaccinia virus produced gB- from cytoplasmic extracts by sucrose gradient centrifuga- specific neutralizing antibodies and were resistant to a lethal tion. HSV-1 challenge. Isolation by DNA. Vaccinia virus DNA was isolated as described (23). Plasmid DNA was prepared from small-scale The herpes simplex virus 1 (HSV-1) genome codes for at least cultures by the procedure of Holmes and Quigley (24). six antigenically distinct glycoproteins: gB, gC, gD, gE, gH, Large-scale plasmid DNA was isolated by the Triton X-100 and gG, all of which appear to have homologues in herpes lysis technique, and the plasmid DNA was further purified on simplex virus 2 (HSV-2) (1, 2). The DNA sequence of all six CsCl/EtBr density gradients (25). glycoprotein genes has been determined (2-7); although Plasmid Constructions and Oligonucleotide-Directed Muta- much structural information has been obtained, relatively genesis. Construction of recombinant plasmids and transfor- little is known about the function of these glycoproteins (1). mation of Escherichia coli were essentially as described by As integral surface components of both the virion envelope Maniatis et al. (25). The gene encoding gB was isolated from and the infected cell plasma membrane (1, 8), the HSV the BamHI G fragment (0.345-0.399 map units), which was glycoproteins induce both humoral and cell-mediated pro- subcloned from the HSV-1 (F) Bgl 11 (1) fragment cloned in tective immune responses and serve as targets for nonspecific phage Charon 28 (E.M.C., unpublished data). DNA was defense mechanisms (1, 9-13). sequenced by the dideoxy chain-termination technique The only glycoprotein gene in which temperature-sensitive (26-28). In vitro oligonucleotide-directed mutagenesis was by (ts) mutants have been mapped is gB (14-16). Mutant virions the double-primer method (32, 46) using single-stranded M13 produced at the nonpermissive temperature (39°C) are en- phage DNA as template. veloped and capable of adsorbing to cells, but penetration Immunoblotting, Radioimmunoprecipitation, and Protein requires a fusagenic agent such as polyethylene glycol (15, Analysis. Infected and uninfected Vero cell extracts were 16). In addition to this essential role in penetration of virus prepared for immunoblot analysis by using procedures pre- into the cell, gB has been shown to be involved in the fusion viously described in detail (29, 30). Infected cell polypeptides of infected-cell membranes resulting in syncytia formation, electrophoretically separated in NaDodSO4/polyacrylamide and one of four syn loci in HSV-1 has been mapped to the gels were electrophoretically blotted onto nitrocellulose coding sequence specifying the cytoplasmic domain of gB membranes at 500 mA for 1.5 hr. After transfer, the nitro- (14, 17). Clearly, gB is multifunctional, and the fact that it is cellulose sheets were blocked with bovine serum albumin and the only HSV-1 glycoprotein that appears to be conserved to treated with antiserum, and the bound antibody was detected various degrees in many mammalian alphaherpesviruses with 125I-labeled staphylococcal protein A (125I-SPA) as (refs. 18-20; and R.E., unpublished observation) supports described (30). After drying the nitrocellulose membrane, the contention that it plays an important role in the infection bound 125I-SPA was detected by autoradiography with Ko- process (16). dak X-Omat AR-2 film. Recently, a recombinant vaccinia virus expressing gD has Antigens used in radioimmunoprecipitation assays were been shown to immunize mice effectively against lethal and prepared from infected Vero cell monolayers metabolically latent HSV infection (21, 22). It seems likely, however, that labeled with [14C]glucosamine (2.5 /XCi/ml; 1 ttCi = 37 kBq) a clinically effective vaccine might require immunization from 4 to 24 hr after infection as described (31). Antigens against multiple antigens. Here, we report the construction were preadsorbed with SPA (Pansorbin; Calbiochem) for 1 hr and characterization of a recombinant vaccinia virus that Abbreviations: HSV-1 and HSV-2, herpes simplex viruses 1 and 2; HSV-1 (F), HSV-1, strain F; pfu, plaque-forming unit(s); SPA, The publication costs of this article were defrayed in part by page charge staphylococcal protein A; BrdUrd, 5-bromo-2-deoxyuridine; TK, payment. This article must therefore be hereby marked "advertisement" thymidine kinase; ts, temperature sensitive. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5908 Downloaded by guest on September 27, 2021 Medical Sciences: Cantin et al. Proc. Natl. Acad. Sci. USA 84 (1987) 5909 at room temperature. SPA was then removed by centrifuga- was to convert the gB translational initiation codon (ATG) tion at 12,000 x g for 5 min, and the antigen preparation was into an EcoRI site so that, upon ligation to EcoRI-cleaved treated with rabbit antiserum for 1 hr at 370C and for 6 hr at pKB (33), an in-frame fusion to a vaccinia-derived transla- 40C. Immune complexes were precipitated by the addition of tional initiation codon (ATG) would result. A 3.4-kilobase SPA; after an extensive washing, the immune precipitates (kb) BamHI/Xho I fragment (0.345-0.368 map unit) contain- were analyzed by NaDodSO4/PAGE as described (31). ing the entire HSV-1 (F) gB gene was subcloned from the All NaDodSO4/polyacrylamide gels were either 10% BamHI G fragment to give the plasmid POX-2. A 1.2-kb Sal acrylamide cross-linked with DATD (N,N'-diallyltartardia- I/Pst I fragment (0.360-0.368 map unit) comprising se- mide, Aldrich) or 8% acrylamide cross-linked with methyl- quences encoding the amino-terminal region of gB was enebisacrylamide (29). subcloned into M13mp8 to give M13mp8A. The insert was Membrane Immunofluorescence. Coverslip cultures of sequenced from the Pst I site to just past the gB ATG CV-1 (African green monkey kidney) cells in 60-mm plates initiation codon by the dideoxy technique. The sequence were seeded 36 hr before infection so as to approach obtained was completely homologous to the published se- confluency at the time of staining. Monolayers were infected quence for HSV-1 (F) gB between nucleotide residues 562 with an input multiplicity of 0.1 plaque-forming unit (pfu) per and 838 (5). The DNA sequence was used to design a cell, and the virus was allowed to adsorb for 1 hr at 370C, after mutagenic oligonucleotide (25-mer) to convert the gB ATG to which the cells were washed with phosphate-buffered saline an EcoRI site (Fig. 1). In vitro mutagenesis was carried out (PBS; 0.01 M phosphate/0.15 M NaCI, pH 7.2) and mainte- by using single-stranded M13mp8A phage DNA as a tem- nance medium was added. At 18 hr after infection, coverslips plate, and the mutation was verified by restriction enzyme were washed three times with PBS at room temperature, analysis. The eventual result ofthis alteration (Fig. 1) was the excess PBS was removed, and 15 pl of diluted rabbit conversion of the second amino acid residue from arginine to antiserum was added. After 30 min at room temperature in a serine and insertion of a new amino acid, asparagine, such humidified chamber, coverslips were washed three times in that the gB polypeptide was one amino acid longer. PBS, and the procedure was repeated with fluorescein To reconstruct the HSV-1 gB gene (Fig. 1), the 960-base- isothiocyanate-conjugated goat anti-rabbit IgG. After the pair (bp) EcoRI/Sal I fragment from M13mp8B was ligated to final washings, coverslips were mounted on slides with a 4:1 the large EcoRI/Sal I fragment isolated from POX-2; after (vol/vol) ratio of PBS and glycerol. Fluorescence was ob- transformation of E. coli, the plasmid PXR-5 was obtained. served within 2 hr on a Zeiss microscope. Photographs were The 3.2-kb EcoRI/BamHI insert fragment from PXR-5 was taken with Kodak Ektachrome daylight ASA 1000 film. The isolated and cloned into the EcoRI site of the vaccinia preparation and characterization of the antisera used have plasmid vector pKB to give PXR-5V. The vaccinia pKB been described (29). vector comprises the 11-kDa late vaccinia promoter and ELISA Assay.