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Proc. Natl. Acad. Sci. USA Vol. 84, pp. 5908-5912, August 1987 Medical Sciences Expression of 1 glycoprotein B by a recombinant virus and protection of mice against lethal 1 infection (viral latency//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 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 MATERIALS AND METHODS and expressed under the control of a vaccinia virus promoter. The mature gB glycoprotein produced by the vaccinia virus 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 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 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 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 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. Immulon II microtiter plates (Dynatech, start site, immediately downstream of which is Alexandria, VA) were coated with purified HSV-1 gB diluted an EcoRI site into which heterologous genes can be inserted in carbonate buffer. Immediately prior to use, coated wells (33). This vaccinia transcriptional unit is flanked by vaccinia were blocked with 2% bovine serum albumin in Pi/NaCl for virus thymidine kinase (TK) gene sequences that facilitate 1 hr at 37°C. Mouse sera were diluted in the same buffer, and homologous recombination into the TK locus of the vaccinia 50 ,ul was added to each well. After a 2-hr incubation at 37°C, virus genome. Insertion of the vaccinia HSV-1 gB transcrip- plates were washed with 0.05% Tween 20 in Pi/NaCl, and tional unit into the vaccinia virus genome was achieved by bound murine IgG was detected with a commercial avidin- transfecting vaccinia virus-infected CV-1 cells with the plas- biotin kit (Vector Laboratories, Burlingame, CA). Optical mid PXR-5V, and then selecting TK- recombinant vaccinia density (OD490) values greater than twice the value obtained virus progeny on TK- cells in the presence of BrdUrd. True with serum from nonimmunized mice were considered sig- recombinant TK- vaccinia virus was distinguished from nificant. spontaneous TK- vaccinia mutants by screening for the Immunization of Animals and HSV-1 Challenge. Male presence of HSV-1 gB DNA sequences. A recombinant BALB/c mice (The Jackson Laboratory; 6-8 weeks old) vaccinia virus that expressed the HSV-1 gB gene was isolated were immunized with 3 x 107 pfu of recombinant vaccinia and plaque-purified thrice in TK- cells in the presence of HSV-1 gB virus designated V11 or recombinant vaccinia BrdUrd. A single plaque was then used to prepare under HSV-1 gD virus designated V52 or recombinant vaccinia nonselective conditions a large stock of the recombinant influenza hemagglutinin virus designated V36. Immuniza- vaccinia gB virus, which was designated V11. tions were by the intradermal route at the base of the tail. Sera Expression of HSV-1 gB Glycoprotein. The pgB and gB from immunized mice were collected 4 weeks after immuni- glycoproteins comprise the same polypeptide chain but differ zation and pooled for each immunization group. HSV-1- in the extent of glycosylation (1). To characterize the gB neutralizing antibody titers were determined by a comple- protein expressed by the vaccinia recombinant, Vero cells ment-independent plaque-reduction assay. Mouse serum was were infected with vaccinia virus recombinant V11 or V36, diluted serially 1:1 with 500 ,l of medium, and then 100 pfu which express the HSV-1 gB glycoprotein and the influenza of HSV-1 in 500 ,ul of medium was incubated with each serum hemagglutinin glycoprotein, respectively, or with HSV-1. dilution for 1 hr at 37°C. An aliquot of 400 ,ul was then added Cell extracts prepared from infected and uninfected Vero to monolayers of Vero cells and plaqued by using cells were resolved by NaDodSO4/PAGE, and the separated methylcellulose. End-points were expressed as the reciprocal were transferred to nitrocellulose membranes. Ni- of the highest serum 1:1 dilution reducing the number of trocellulose strips, each containing identical sets of proteins, plaques by 50%. Six weeks after immunization, mice were were treated first with rabbit anti-HSV antiserum, rabbit challenged by the i.p. injection of 1 x 104 pfu of HSV-1 anti-gB antiserum, or normal rabbit serum and then with (McKrae strain) and were observed for an indication of 125I-SPA. In the autoradiogram shown in Fig. 2 Left, a major mortality for 14 days. band corresponding to the pgB glycoprotein and a minor band corresponding to the gB glycoprotein were visible only in RESULTS V11- or HSV-1-infected cells and were detected only with monospecific anti-gB serum or anti-HSV serum. HSV-1- Construction of Recombinant Vaccinia gB Virus. Alteration infected cell extract treated with anti-HSV serum detected of the HSV-1 gB gene and its insertion into the vaccinia numerous additional HSV-1 antigens, whereas V36-infected vector pKB was achieved as outlined in Fig. 1. Our strategy cells and uninfected cells did not react with any of the antisera Downloaded by guest on September 27, 2021 5910 Medical Sciences: Cantin et al. Proc. Natl. Acad. Sci. USA 84 (1987)

7O W

"- ATG C- -m- crXCGJ MP- 8A Pst + Sol (POX-2 - M13mp-8

I...... In vitro mutagenesis 5'- TAGTCCCCCC MT TCC CAC CCC CCC -3' Mutogenic Oligo 3- ATCACCCCCC TAC CCC CTC CCC CCC-5' HSV Template JrMNet Arg Clu Cly Alo

Eco + Sal 0- ) Eco+ Sol (I) wa

T4 DNA Ligase MP-8B

t ct tL PXR-5

o Eco + Bom Klenow DNA polymerose + 4 dNTP s t t Eco Eco linkers T4 DNA Ligase Eco

0 0 tk W t) 5'- ATC AAT TCC CAC CCC CCC -3' pKB-gB Fusion 3'- TAC TTA ACC CTC CCC CCC- 5' Met Ass Ser Clv mly Ala

FIG. 1. Construction of a recombinant vaccinia virus vector for insertion of the HSV gB-coding sequence into vaccinia virus. The HSV-1 gB-coding sequence is contained in the 3.4-kb Xho I/BamHI fragment in the plasmid POX-2. Only those restriction sites relevant to various plasmid constructs are shown (not to scale). The details of the various plasmid constructs leading to the recombinant vaccinia virus vector PXR-5V (Left Bottom) are described in the text. The sequence of the mutagenic 25-mer oligonucleotide (indicated by A in Right Top) and that of the corresponding target region in the gB gene are shown. Mismatches corresponding to the new EcoRI site are indicated by black dots above the nucleotides. In vitro oligonucleotide-directed mutagenesis was carried out by the double-primer method using the M13 universal sequencing primer (B in Right Top). Mutant phage were identified by hybridization with the 32P-labeled mutagenic oligonucleotide (A) and verified by restriction analysis (MP-8B in Right Middle). The in-frame fusion resulting from insertion of the modified gB gene into the vaccinia virus vector pKB is shown (Right Bottom) together with the amino acid sequence of the mutant gB polypeptide.

used. Fig. 2 Left also shows that the pgB and gB immunofluorescence was seen only with V11- and HSV- glycoproteins produced by the vaccinia recombinant V11 infected cells treated with anti-gB serum (Fig. 3). Cells have electrophoretic mobilities that are indistinguishable infected with V36 (influenza HA) and treated with anti-gB from those of authentic HSV-1 pgB and gB. serum or V11-infected cells treated with normal rabbit serum To ascertain whether gB produced by the recombinant were negative (not shown). vaccinia virus V11 was glycosylated, cells infected with V11, Immunological Response and Protection from an HSV-1 V36, and HSV-1 as well as uninfected cells were labeled with Challenge. An ELISA assay was used to measure the level of ['4C]glucosamine. Detergent-soluble antigens from these serum antibodies capable of specific binding to purified cells were immunoprecipitated with rabbit anti-HSV serum, HSV-1 gB. BALB/c mice were immunized by the intrader- rabbit anti-gB serum, or normal rabbit serum. NaDodSO4/ mal route with 3 x 107 pfu of each of the vaccinia virus PAGE analysis of the immunoprecipitated proteins showed recombinants V11 (gB), V52 (gD), or V36 (influenza HA). that the gB glycoprotein produced by the vaccinia recombi- Sera were collected and pooled 4 weeks after viral inocula- nant V11 was both glycosylated and had an electrophoretic tion and were assayed for anti-gB antibodies (Table 1). Only mobility identical to authentic HSV-1 gB (Fig. 2 Right). No mice immunized with the V11 recombinant or HSV-1 devel- proteins from V36-infected or uninfected cells were bound by oped serum antibodies to gB. Neutralizing antibody titers the antisera used, even though V36 does produce a major were lowerfor V11 (gB)-immunized mice-as compared to V52 glycosylated species (Fig. 2 Right). (gD)-immunized mice. Mice were then challenged by i.p. Incorporation ofgB into the . In a productive injection of HSV-1. There was 90% survival in mice immu- HSV-1 infection, the gB glycoprotein is incorporated into the nized with either V11 (gB) or V52 (gD), whereas a high degree various membrane systems of the cell, including the plasma of mortality occurred in nonimmunized mice and in mice membrane, where gB is expressed on the external surface of immunized with V36. the cell. Immunofluorescence studies were undertaken to determine whether gB specified by the vaccinia recombinant DISCUSSION V11 was similarly expressed on the plasma membrane of infected cells. Coverslip cultures of CV-1 cells having ap- The recent development of vaccinia virus as a eukaryotic proximately the same number of cells mock-infected or expression vector (23) capable of faithfully directing expres- infected with V11 (gB), V52 (gD), or HSV-1 were treated with sion of a variety of foreign genes has resulted in the rabbit anti-gB serum followed by fluorescein-conjugated goat construction of live vaccinia virus recombinants that are anti-rabbit IgG. The same pattern of strong membrane capable of protectively immunizing animals against infec- Downloaded by guest on September 27, 2021 Medical Sciences: Cantin et al. Proc. Nati. Acad. Sci. USA 84 (1987) 5911

HSV Vil V36 Anti-HSV Anti-gB NRS

1 2 3 4 1 2 3 4 1 2 3 4 HSV V36 V I HSV U V36 VAl HSV U V36 V11 HSV U s31 1,,... 1

i _ gC _S _ Ws... ill PgB---- E ' ___ pg B

: **; gD- gD- pgD w _n.r......

FIG. 2. Characterization of HSV-1 gB glycoprotein synthesized by the recombinant vaccinia virus V11 (gB). (Left) Immunoblot analysis. Vero cell monolayers were infected with the vaccinia influenza recombinant (lanes V36) or with the vaccinia gB recombinant (lanes V11) or with HSV-1 (F) (lanes HSV). After 16 hr, cell extracts were prepared from infected and uninfected cells and resolved on a 10% polyacrylamide gel. The separated polypeptides were electrophoretically transferred to nitrocellulose strips and then treated sequentially with anti-HSV-1 serum or anti-gB serum or normal rabbit serum (NRS) and then with 1251-SPA. Reactive antigens were visualized by autoradiography. The leftmost lane labeled HSV shows ['4C]glucosamine-labeled HSV-1 glycoproteins resolved in the same gel. Lanes U contained uninfected cell polypeptides. (Right) Immunoprecipitation. Vero cells infected with V11, V36, or HSV-1 were metabolically labeled with ['4C]glucosamine for 20 hr. Cytoplasmic antigens (lane 1) were immunoprecipitated with rabbit anti-HSV-1 antiserum (lane 2) or rabbit anti-gB antiserum (lane 3) or normal rabbit serum (lane 4), and the immune precipitates were resolved on 10% polyacrylamide gels. An autoradiogram is shown, and the mobility of the gB, gC, and gD HSV-1 glycoproteins is indicated. tions with HSV-1 and HSV-2 (21, 22), rabies virus (34, 35), production of virus-neutralizing antibody, and a reduction in hepatitis (36), influenza virus (37), and vesicular antibody response to gB- has been correlated with decreased stomatitis virus (38). protective immunity against intracranial challenge with HSV- Recent studies (39) indicate that in HSV-1-infected cells, 1 (9, 39, 40). A protective effect against both HSV-1 and gB and gC are the most prominent viral cell surface antigens HSV-2 infection in mice has been demonstrated by active and that an immunoprecipitating antibody specific for these antigens predominates in sera from immunized mice. Fur- thermore, gB has been shown to be important for the Table 1. Immunization with recombinant vaccinia viruses: Serum antibody titers and resistance to lethal HSV-1 challenge Serum antibody titert HSV-1 chailenger Neutrali- Mice, Immunization* zation Anti-gB no. % mortality§ Nonimmunized <4 <10 10 100 Vaccinia V52 (gD) 256 <10 20 5 Vaccinia V11 (gB) 32 .160 17 6 Vaccinia V36 (influenza HA) ND ND 10 70 ND, not determined. *BALB/c mice were immunized by the intradermal injection at the base of the tail with 3 x i07 pfu of the vaccinia virus recombinants. tMice were bled 4 weeks after immunization, sera were pooled, and antibody titers were determined as described. tMice were challenged 6 weeks after immunization by i.p. injection of 1 x 104 pfu of HSV-1 (McKrae strain). Animals were monitored for signs of mortality for 14 days. FIG. 3. Cell-surface immunofluorescent staining ofgB. Coverslip §By Fisher's exact test, the difference between nonimmunized and cultures of CV-1 cells infected with the recombinant vaccinia gB V36-immunized mice or between V52- and V11-immunized mice virus V11 (A), HSV-1 (C), or the recombinant vaccinia gD virus V52 was not significant (P = 0.05), whereas, the difference between (D) and uninfected cells (B) were treated first with rabbit anti-gB nonimmunized and V11 (or V52)-immunized mice or between V36- serum and then with goat anti-rabbit fluorescein-conjugated IgG. and V11 (or V52)-immunized mice was significant (P - 0.001). Downloaded by guest on September 27, 2021 5912 Medical Sciences: Cantin et al. Proc. Natl. Acad. Sci. USA 84 (1987)

immunization with purified gB glycoprotein of HSV-1 and 7. McGeoch, D. J., Dolan, A., Donald, S. & Rixon, F. J. (1985) J. passive immunization with gB-specific monoclonal antibody Mol. Biol. 1$1, 1-13. (40-42). Clearly, gB is an important inducer of protective 8. Pederson, B. (1982) J. Virol. 43, 395-402. 9. Courtney, R. J. (1984) in Immunology of Herpes Simplex Virus immunity to HSV infection in addition to playing an essential Infection, eds. Rouse, B. T. & Lopez, C. (CRC, Boca Raton, FL), role in the process of HSV penetration into cells. pp. 34-44. A gene encoding a truncated HSV-1 gB glycoprotein has 10. Spear, P. G. (1985) in Immunochemistry of Viruses: The Basis for recently been expressed in yeast, and although the antigen was Serodiagnosis and , eds. van Regenmortal, M. H. V. & not displayed on the cell surface, yeast cell extracts were Neurath, A. R. (Elsevier, Amsterdam), pp. 435-445. 11. Bishop, G. A., Glorioso, J. & Schwartz, S. A. (1983) J. Exp. Med. capable ofeliciting gB-specific serum-neutralizing antibodies to 157, 1544-1561. HSV-1 when used to immunize guinea pigs (43). We chose, to 12. Carter, V. C., Schaffer, P. A. & Tevethia, S. S. (1981) J. Immunol. construct a recombinant vaccinia virus that would express 126, 1655-1660. authentic HSV-1 gB on the surface of infected cells and would 13. Nash, A. A., Leung, K. N. & Wildy, P. (1985) in The result in presentation of this antigen to the immune system in a Herpesviruses, ed. Roizman, B. (Plenum, New York), Vol. 4, pp. manner analogous to that in a normal HSV-1 infection. 87-100. 14. DeLuca, N., Bzik, D. J., Bond, V. C., Person, S. & Snipes, W. To achieve efficient expression ofthe HSV-1 gB gene using (1982) Virology 122, 411-432. vaccinia virus late transcriptional regulatory sequences, we 15. Little, S. P., Jofre, J. T., Courtney, R. J. & Schaffer, P. A. (1981) fused the gB-encoding sequences in frame to a vaccinia Virology 115, 149-160. virus-derived translational initiation codon (ATG). This was 16. Sarmiento, M., Haffey, M. & Spear, P. G. (1979) J. Virol. 29, achieved by in vitro oligonucleotide-directed mutagenesis 1149-1158. and resulted in the substitution of serine for and 17. Kousoulas, K. G., Pellett, P. E., Pereira, L. & Roizman, B. (1984) arginine the Virology 135, 379-394. insertion of one amino acid, asparagine, so that the gB 18. Pellett, P. E:, Biggin, M. D., Barrle, B. & Roizman, B. (1985) J. polypeptide was now one amino acid longer (Fig. 1). How- Virol. 56, 807-813. ever, the altered amino acids are in the portion of the gB 19. Kitamura, K., Namazue, J., Campo-Vera, H., Ogino, T. & precursor polypeptide that is thought to contain a signal Yamanishi, K. (1986) Virology 149, 74-82. sequence directing the transport of gB to the cell surface (5, 20. Snowden, B. W. & Halliburton, I. W. (1985) J. Gen. Virol. 66, this 2039-2044. 6, 44, 45). Although portion is cleaved during maturation 21. Cremer, K. J., Mackett, M., Wohlenberg, C., Notkins, A. L. & (47), alteration in the putative signal sequence could affect Moss, B. (1985) Science 228, 737-740. the maturation of gB and the localization of gB to the cell 22. Paoletti, E., Lipinskas, B. R., Samsonoff, C., Mercer, S. & membrane. This was not anticipated because the changes Panicalia, D. (1984) Proc. Natl. Acad. Sci. USA 81, 193-197. involved only the first three terminal amino acids. Evidence 23. Mackett, M., Smith, G. L. & Moss, B. (1982) Proc. Nall. Acad. that gB maturation and localization is normal comes from our Sci. USA 79, 7415-7419. 24. Holmes, D. S. & Quigley, M. (1981) Anal. Biochem. 114, 193-197. studies characterizing the V11 recombinant. First, gB pro- 25. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular duced by the recombinant V11 is antigenic and glycosylated, : A Laboratory Manual (Cold Spring Harbor Laboratory, and comigrates in NaDodSO4/PAGE with authentic HSV-1 Cold Spring Harbor, NY), pp. 86-96. (F) gB (Fig. 2). Second, the gB glycoprotein is expressed on 26. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. the cell-surface membrane after infection of cells with the Sci. USA 74, 5463-5467. recombinant vaccinia virus V11 immunization 27. Messing, J. & Vieira, J. (1982) Gene 19, 269-276. (Fig. 3). Third, 28. Williams, S. A., Slatko, B. E., Moran, L. S. & DeSimone, S. M. of mice with V11 results in production of serum antibodies (1986) Biotechniques 4, 138-147. that bind specifically to purified HSV-1 gB in an ELISA assay 29. Eberle, R. & Courtney, R. J. (1980) J. Virol. 35, 902-917. and that are capable of neutralizing HSV-1 infectivity (Table 30. Eberle, R. & Mou, S.-W. (1983) J. Infect. Dis. 148, 436-444. 1). Moreover, immunization with V11 protected mice against 31. Eberle, R. & Courtney, R. J. (1981) Infect. Immun. 31, 1062-1070. a lethal HSV-1 challenge with an efficacy equivalent to that 32. Smith, M. (1985) Annu. Rev. Genet. 19, 423-462. 33. Chakrabarti, S., Brechling, K. & Moss, B. (1985) Mol. Cell. Biol. 5, of the vaccinia gD recombinant virus. Taken together, these 3403-3409. results suggest that cleavage of the leader peptide occurred 34. Wiktor, T. J., MacFarlan, R. I., Reagan, K. J., Dietzschold, B., and that the mature gB glycoprotein expressed by V11 is Curtis, P. J., Wunner, N. H., Kieny, M. P., Lathe, R., LeCocq, identical to that of authentic gB expressed by HSV-1. J. P., Mackett, M., Moss, B. & Koprowski, H. (1984) Proc. Natl. Whether this recombinant vaccinia gB virus is capable of Acad. Sci. USA 81, 7194-7198. preventing the establishment of a latent HSV-1 infection and 35. Kieny, M. P., Lathe, R., Drillien, R., Spehner, D., Skory, S., Scmitt, D., Wiktor, T., Koprowski, H. & LeCocq, J. P. (1984) a comparison of its effectiveness in this regard with the Nature (London) 312, 163-166. vaccinia gD virus (21) needs further study. The recombinant 36. Moss, B., Smith, G. L., Gerin, J. L. & Purcell, R. (1984) Nature virus also should be useful for determining the role of gB in (London) 311, 67-69. cell-mediated immunity. 37. Smith, G. L., Murphy, B. R. & Moss, B. (1983) Proc. Natl. Acad. Sci. USA 80, 7155-7159. We thank Laurie Salazar for technical assistance and Donna Fox 38. Mackett, M., Yilina, T., Rose, J. K. & Moss, B. (1985) Science for typing and editorial assistance. This work was supported in part 227, 433-435. National Institutes of Health Grant Al 22302. 39. Glorioso, J., Schroder, C. H., Kumel, G., Szczesiul, M. & Levine, by M. (1984) J. Virol. 50, 805-812. 40. Chan, W. L. (1983) Immunology 49, 343-352. 1. Spear, P. G. (1984) in The Herpesviruses, ed. 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Claesson-Welsh, L. & Spear, P. C. (1987) J. Virol. 61, 1-7. Downloaded by guest on September 27, 2021