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Journal of General Virology (1991), 72, 369-375. Printed in Great Britain ... Present address: Division of Medical Virology, Institute of. Medical and Veterinary ...
Journal of General Virology (1991), 72, 369-375. Printed in Great Britain

369

Induction of protective immunity with antibody to herpes simplex virus type 1 glycoprotein H (gH) and analysis of the immune response to gH expressed in recombinant vaccinia virus Audrey J. Forrester,* Veronica Sullivan,t Anthony Simmons,:~ Barbara A. Blacklaws,§ Geoffrey L. Smith,II Anthony A. Nash and Anthony C. Minson Div&ion of Virology, Department o f Pathology, University o f Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.

Passive administration of neutralizing monoclonal antibody (MAb) to glycoprotein H (gH) of herpes simplex virus type 1 (HSV- 1) was found to protect mice from an HSV-I strain SC16 challenge infection. To investigate further the protective potential of gH, recombinant vaccinia viruses were constructed which expressed the HSV-1 gH open reading frame under the control of the vaccinia virus 7.5K early/late promoter or the 4b late promoter. Immunization with recombinant viruses, however, did not induce the production of neutralizing antisera and the mice were not protected

from zosteriform spread or the establishment of latent infection following viral challenge. The gH produced by the recombinant vaccinia viruses differed in electrophoretic mobility and antigenicity from authentic HSV-1 gH. Only one of three neutralizing MAbs specific for conformational epitopes on gH was able to immunoprecipitate gH synthesized in recombinant vaccinia virus-infected cells. In addition cell surface expression of gH was not detected in cells infected with the recombinant vaccinia viruses.

Introduction

The antiviral properties of gH-specific MAbs suggest that a good humoral response to gH might be of value in modifying HSV infection in vivo. Most studies of immune responses to HSV have emphasized the importance of cell-mediated immunity (reviewed by Nash et aL, 1985), and in at least one infection model, animals lacking functional B lymphocytes have been shown to recover normally from HSV infection (Simmons & Nash, 1987). Despite the undoubted importance of cell-mediated responses in recovery from HSV infection, there are nevertheless ample data that demonstrate the ability of antibody to protect animals from HSV challenge by several different routes. Thus several studies have shown that passive antibody, administered before or soon after infection, is protective (e.g. Simmons & Nash, 1985; Balachandran et al., 1982). It is uncertain whether antibody acts directly by neutralizing virus and lysing infected cells, or acts indirectly by increasing the efficiency with which inflammatory cells are recruited to the primary infection site, but it appears that protection is best achieved by active immunization with glycoproteins that are major neutralizing targets or by passive administration of antibodies directed against these targets (e.g. Long et al., 1984; Martin et al., 1989; Blacklaws et al., 1990; Balanchandran, 1982). The objective of the work described in this paper was to examine the protective effect of passive immunization

Glycoprotein H (gH) of herpes simplex virus type 1 (HSV-1) is one of three glycoproteins that are essential for virus viability in tissue culture (Cai et al., 1988 ; Ligas & Johnson, 1988; Desai et al., 1988), and homologues of this glycoprotein have been identified in members of all the herpesvirus subfamilies (Davison & Taylor, 1987; Gompels et al., 1988; Cranage et al., 1988; Heineman et al., 1988). gH is required for virus entry (Desai et al., 1988) and is probably involved in cell to cell spread of infectivity since gH-specific monoclonal antibodies (MAbs), in addition to neutralizing free virus, inhibit cell fusion by syncytial strains and prevent intercellular virus transmission (Buckmaster et al., 1984; Gompels & Minson, 1986). Antibodies raised against the varicellazoster virus and Epstein-Barr virus homologues have similar properties implying functional conservation (Keller et al., 1987; Miller & Hutt-Fletcher, 1988). ]"Present address: Harvard MedicalSchool,330 BrooklineAvenue, Boston, Massachusetts 02215, U.S.A. Present address: Division of Medical Virology, Institute of Medical and Veterinary Science, Adelaide SA 5000, Australia. § Present address: Department of Veterinary Pathology,University of Edinburgh, Summerhall, Edinburgh EH9 1QH, U.K. IIPresentaddress: Sir WilliamDunn Schoolof Pathology,University of Oxford, South Parks Road, Oxford OX1 3RE, U.K. 0000-9778 © 1991 SGM

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w i t h a n t i - g H a n t i b o d y or a c t i v e i m m u n i z a t i o n w i t h r e c o m b i n a n t v a c c i n i a viruses e x p r e s s i n g HSV-1 gH. W e found t h a t gH-specific a n t i b o d y p r o t e c t e d efficiently but t h a t v a c c i n a t i o n w i t h r e c o m b i n a n t v a c c i n i a viruses failed to elicit n e u t r a l i z i n g a n t i b o d y a g a i n s t HSV-1 a n d did not protect against challenge with HSV-1..This a p p a r e n t c o n t r a d i c t i o n was e x p l a i n e d b y e x a m i n a t i o n o f g H e x p r e s s e d by r e c o m b i n a n t v a c c i n i a viruses, w h i c h r e v e a l e d t h a t the p r o t e i n was a n t i g e n i c a l l y d i s t i n c t f r o m t h a t f o u n d in HSV-1, a result a n a l o g o u s to t h a t o b t a i n e d w h e n HSV-1 g H was e x p r e s s e d in C o s - I cells ( G o m p e l s & M i n s o n , 1989). C o e x p r e s s i o n o f g H w i t h e i t h e r g D or gB failed to rescue the. a u t h e n t i c a n t i g e n i c f o r m o f gH. Thus, a l t h o u g h a n t i b o d y a g a i n s t HSV-1 is p r o t e c t i v e , an effective m e a n s o f d e l i v e r i n g i m m u n o g e n i c g H is n o t yet available,

Methods Cells and viruses. BHK-2I cells were grown in Glasgow modified Eagle's medium supplemented with 10H tryptose phosphate broth and 10~ newborn bovine serum (ETC). CV-1 cells and 143 thymidine kinase-deficient (TK-) cells were grown in the same medium supplemented with 10% foetal bovine serum. HSV-I strains SC16 and HFEM were propagated and assayed in BHK-21 cells. Vaccinia virus strain WR and recombinant viruses derived from it were propagated in BHK-21 cells and assayed in CV-1 cells. Antibodies. MAbs antibodies LPll, 52S and 53S are specific for HSV-1 gH (Buckmaster et al., 1984; Showalter et al., 1981). MAbs AP7 and LP2 are specific for HSV gD (Minson et al., 1986). Ascites fluids used for passive immunizations contained 3 to 5 mg/ml IgG. AntitrpE-gH is a rabbit serum raised against a tryptophan E-HSV-1 gH fusion protein (Desai et al., 1988). Neutralization assays were performed as described by Minson et al. (1986). Preparation of infected call lysates, immunoprecipitation, detection of enzymes by Western blotting and immunofluorescence were as described by Gompels & Minson (1989). Animal models. All experiments were done with 4- to 6-week-old female BALB/c mice. Two HSV infection models were used. The ear model, in which inoculum is introduced subdermally into the ear pinna is described by Hill et al. (1975). The flank or zosteriform model in which inoculum is scarified into the depilated flank epidermis and forms a zosteriform lesion following spread via the sensory nerves is described by Sydiskis & Schultz (1965) and by Simmons & Nash (1984). Mice were vaccinated by intraperitoneal (i.p.) injection with 10v p.f.u. of recombinant vaccinia virus in 0.1 ml phosphate-buffered saline and were re-vaccinated with the same dose by the same route 10 to 14 days later. HSV challenge infections were performed 7 days later. Passive antibody was introduced as ascites fluid via the tail vein. Virus titres in the infected ear during the acute phase of infection were determined by homogenizing the ear pinnae and storing the homogenate at - 70 °C prior to assaying for infectivity. Establishment of latent infection was determined by removal of the cervical ganglia (CII, CIII, CIV) on the inoculated (left) side of the animal. The pooled ganglia were then incubated for 5 days in ETC, homogenized and assayed for infectivity. Appearance of plaques was taken to indicate establishment of latent infection. Recombinant vaccinia viruses. Recombinant vaccinia viruses VII and VgD52 (Cantin et al., 1987; Cremer et al., 1985) express HSV-1 gB and

gD, respectively and were kindly provided by the authors. Vac YC3 was used as a negative control for vaccination and expresses a schistosome surface antigen (G. Smith, unpublished results). All three recombinants contain the foreign insert within the TK coding sequence and are TK-. The recombinant viruses expressing the HSV-1 gH were constructed using the general strategy described by Mackett et al., (1984) as follows. The gH coding sequence (open reading frame UL22) of HSV-1 strain HFEM (Gompels & Minson, 1988) was isolated on a 3.5 kb NcoI-XhoI fragment from a NcoI/partial XhoI digest (nucleotides 46382 to 42879; McGeoeh et al., 1988), in which the initiating ATG lay within the NcoI site. This fragment was end-repaired and inserted into the Sinai site of pSP64. The coding sequence was then reisolated by digestion with HindlII and XhoI and was inserted into HindlIl/SmaI-digested pGS62 (Cranage et al., 1986) such that the gH coding sequence under the control of the vaccinia virus 7-5K early/late promoter was inserted within the vaccinia virus TK gene. An analogous construct was made in which the gH coding sequence was placed under the control of the vaccinia virus 4b late promoter (Rosel & Moss, 1985) by insertion into pRK19, an insertion vector similar to pGS62 except that the 7.5K promoter sequences are replaced by the 4b promoter (R. K. Kent & G. L. Smith, unpublished results). Before being inserted into pRK19 the HindlII/XhoI gH coding fragment was transferred from pSP64 into M13mpl8 and was modified by sitedirected mutagenesis (Kunkel, 1985) such that the sequence immediately 5' of the initiating ATG was replaced with the sequence TAAATG, the motif found in most vaccinia virus late mRNAs (Rosel et al., 1986). gH coding sequences in pGS62 and pRK19 were introduced into vaccinia virus by transfection and selection for TKrecombinants as described by Mackett et al. (1984). Recombinant vaccinia viruses in which gH was expressed under the control of the 7.5K or the 4b promoter were named Vac7.5-gH and Vac4b-gH respectively.

Results Recombinant

vaecinia viruses

V a c c i n i a virus r e c o m b i n a n t s were c o n s t r u c t e d in w h i c h the HSV-1 g H c o d i n g sequence was p l a c e d u n d e r the control o f either the v a c c i n i a virus 7.5K p r o m o t e r ( V a c 7 . 5 - g H ) or the v a c c i n i a virus 4b p r o m o t e r ( V a c 4 b gH). B H K - 2 1 cells were i n f e c t e d w i t h r e c o m b i n a n t viruses, h a r v e s t e d at various t i m e s o v e r a 16 h p e r i o d a n d lysates were e x a m i n e d for the p r e s e n c e o f g H b y W e s t e r n blotting. T h e results (Fig. 1) show t h a t cells i n f e c t e d w i t h e i t h e r r e c o m b i n a n t a c c u m u l a t e larger a m o u n t s o f g H t h a n cells infected with HSV-1, t h a t g H s y n t h e s i z e d by r e c o m b i n a n t v a c c i n i a viruses a p p e a r s to h a v e an electrophoretic mobility intermediate between that of the i m m a t u r e a n d m a t u r e forms o f g H found in H S V - 1 infected cells a n d t h a t g H e x p r e s s e d by the different r e c o m b i n a n t s is q u a l i t a t i v e l y i n d i s t i n g u i s h a b l e , b u t e x p r e s s i o n f r o m the 4b p r o m o t e r is g r e a t e r t h a n t h a t f r o m the 7.5K p r o m o t e r . T h e 4b p r o m o t e r is active only after viral D N A synthesis a n d drives the e x p r e s s i o n o f a m a j o r virion core c o m p o n e n t (Moss & R o s e n b l u m , 1973; Rosel & Moss, 1985). T h e 7.5K p r o m o t e r is active b o t h before a n d after viral D N A synthesis ( C o c h r a n e t al., 1985; M a c k e t t et al., 1984). T h e a b s e n c e o f g H synthesis at early t i m e s in cells i n f e c t e d w i t h V a c 7 . 5 - g H is

Immunity to HSV-1 gH

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Table 1. Effect of passive immunization of mice with antibodies on zosteriform spread Antibodies

Target

Number of mice

Zosteriform spread

LP 11 LP2 AP7 Immune serum Control ascites

gH gH gD -* -

25 25 25 25 25

0 0 1 0 25

* (-), Not done. Fig. 1. Western blot analysis of gH expressed by recombinant vaccinia viruses. Lysateswere prepared at 2 h (lanes 1 and 2), 4 h (lanes 3 and 4), 6 h (lanes 5 and 6), 8 h (lanes 7 and 8), 16 h (lanes 9, 10 and 11) post-infection (p.i.) from BHK-21 cells infected with Vac7.5-gH (lanes 1, 3, 5, 7 and 9), Vac4b-gH (lanes 2, 4, 6, 8 and 10) or HSV-1 strain HFEM (lane 1l). The samples were then electrophoresed through a 7.5% polyacrylamideget, transferred to nitrocellulose and reacted with trpE-HSV-1 gH fusion antiserum. Cells were infected with recombinant vaccinia viruses at an m.o.i, of 20 and with HSV-I HFEM at an m.o.i, of 10. All lanes were loaded with extracts prepared from approximately 5 x l0 s cells.

probably due to the presence of the sequence T s C T in the gH coding sequence, since the motif T s N T is a transcription termination signal in 'early' vaccinia virus messages (Rohrmann et al., 1986).

Passive immunization Several studies have demonstrated the ability of passively administered M A b or immune sera against HSV to protect against virus challenge but antibodies to HSV-1 gH have not been tested in this way. Before proceeding with active immunization of animals with recombinant vaccinia viruses, we wished to establish that a humoral response to gH would have a protective effect. Groups of mice were inoculated by flank scarification with 5 x 106 p.f.u, of HSV-1 strain SC16 and after 24 h were injected intravenously with 100 ~tl of ascitic fluid containing MAbs specific for gD or gH or with 1 ml of an HSV-1immune mouse serum. Although the monoclonal I g G content of each ascites fluid was not determined, all ascites contained 3 to 5 mg/ml of IgG. In addition ascites fluids containing antibodies LP2 (gD-specific) and LP11 (gH-specific), both of which neutralize HSV-1 in the absence of complement, gave similar dilution endpoints in neutralization tests ( > 1 : 2000, < 1:4000). Control animals received an ascites fluid containing an irrelevant M A b and all such animals developed zosteriform lesions 5 or 6 days after infection. All animals were examined daily until 10 days after infection and the results (Table 1) show that M A b to gH was solidly protective. The efficacy of anti-gD antibodies confirmed previous

Table 2. Effect of immunization with vaccinia recombinants on development of zosteriform lesions and establishment of latency Group

Number of mice

Zosteriform spread

Latent infection

VacT.5-gH Vac4b--gH VgD52 YC3

5 5 5 5

5 5 0 5

5 4 1 5

findings (Simmons & N a s h 1987). Although this is a relatively superficial examination of the effects of passive gH antibody on the infection process, it nevertheless serves to establish that a good humoral response to gH should modulate virus infection and suggests that gH has potential as a protective immunogen.

Immun&ation with recombinant vaccinia virus The efficacy of vaccination with r e c o m b i n a n t s expressing gH was examined by comparison with recombinants expressing gD or with an irrelevant recombinant. Mice were given two vaccinations with 107 p.f.u, of recombinant vaccinia virus by the i.p. route and were challenged with 105 p.f.u, of HSV-1 strain SC16 by flank scarification or with 5 x 104 p.f.u, of SC16 by subdermal inoculation in the ear pinna. The vaccinia virus recombinant expressing gD protected all mice from zosteriform spread of challenge virus and only one of five animals became latently infected (Table 2), a result in agreement with previous findings (Cremer et al., 1985). By comparison, vaccination with Vac7.5-gH or V a c 4 b gH did not modify the challenge infection. Mice challenged by the ear pinna route were killed 5 days after challenge and the virus titre at the challenge site was determined. Only one of five animals vaccinated with the gD recombinant yielded detectable challenge virus from the ear pinna (Fig. 2) while yields of virus from animals vaccinated with Vac7.5-gH or V a c 4 b - g H were not significantly different to those from control mice.

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Fig. 2. Virus clearance from the ear pinnae of mice immunized with recombinant vaccinia viruses. Groups of five mice were injected twice i.p. with a 14 day interval with 107 p.f.u, of VgD52, Vac7.5-gH, Vac4bgH or YC3 then challenged with 5 x 104 p.f.u, of HSV-1 SC16 in the left ear pinna. The ear pinnae were removed 5 days after challenge and remaining virus titred by plaque assay on BHK-21 cells. The results have been expressed as the p.f.u, of virus in each pinnae,

Fig. 4. Western blot analysis of immunoprecipitations of HSV-1 gH. Lysates prepared from BHK-21 cells infected with HSV-I HFEM (lanes 1,4 and 7), Vac7-5 gH (lanes 2, 5 and 8) or Vac4b-gH (lanes 3, 6 and 9) were immunoprecipitated with MAbs 52S (lanes 1 to 3), 53S (lanes 4 to 6) or LPll (lanes 7 to 9). These samples were then electrophoresed through a 7.5% polyacrylamide gel, transferred to nitrocellulose and reacted with polyclonal trpE-HSV-1 gH fusion protein antiserum. Ceils were harvested at 16 h p.i. with an m.o.i, the same as that for the experiment in Fig. 1. Lanes 1, 4 and 7 contain extracts prepared from approximately 1 × 106 cells and lanes 2, 3, 5, 6, 8 and 9 contain approximately 5 × l0 s ceils.

400 ant was effective in inducing neutralizing a n t i b o d y or in modulating the outcome o f challenge infection by the routes used.

30o

Properties of gH synthesized by vaccinia virus recombinants

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16 64 256 1024 4096 Reciprocal serum dilution Fig. 3. Neutralization activity against HSV-I of sera from mice immunized with recombinant vaccinia viruses. Dilutions of sera pooled from mice injected with VgD52 (fq), YC3 (O), Vac4b--gH (11) or Vac7.5-gH (0) were incubated with 5 x 103 p.f.u, of HSV-1 SC16 in the presence of complement for 1 h at room temperature. Remaining viable virus was then titrated in duplicate on BHK-21 cells and the mean plaque number is shown. The endpoints are expressed as the reciprocal of the highest dilution of antiserum that gave a 50~ reduction in plaque number. Pooled sera from parallel groups of vaccinated mice were tested for neutralizing activity against HSV-1. Sera f r o m mice vaccinated with the g D r e c o m b i n a n t contained neutralizing a n t i b o d y with an endpoint o f approximately 1:1024, while sera from animals vaccinated with V a c 7 . 5 - g H or V a c 4 b - g H contained little, if any, neutralizing activity (Fig. 3). Thus neither g H recombin-

The failure o f r e c o m b i n a n t vaccinia virus expressing g H to elicit neutralizing antibody is surprising in view o f the high levels o f expression achieved by c o m p a r i s o n with H S V - l - i n f e c t e d cells and the fact that most M A b s to g H have neutralizing activity (Showalter et al., 1981; Buckmaster et al., 1984). These considerations suggested that the form o f g H expressed by r e c o m b i n a n t vaccinia virus might be different from the authentic molecule expressed by HSV-1, a suggestion reinforced by the slight difference in electrophoretic mobilities shown in Fig. 1. The antigenic characteristics o f g H expressed by HSV-1 and by vaccinia virus recombinants were investigated using three M A b s specific for gH. Antibodies 52S, 53S and L P l l , all of which react with conformationdependent epitopes, were used to precipitate g H f r o m infected cell lysates. After gel electrophoresis and transfer to nitrocellulose the precipitation products were detected using a polyclonal antiserum to a t r p E - g H fusion protein. The results (Fig. 4) show that all three antibodies precipitated g H from lysates of HSV-1infected cells but only antibody 52S precipitated gH from lysates o f cells infected with the r e c o m b i n a n t vaccinia viruses. Immunofluorescence o f BHK-21 cells infected with HSV-1 or with vaccinia virus r e c o m b i n a n t s (Fig. 5)

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Fig. 5. Cellularlocalizationof HSV-1 gH by immunofluorescenceon recombinantvacciniavirus and HSV-1 HFEM-infectedcells. MAb 52S was reacted with either non-permeabilized(a) and permeabilized (b) CV-1 cells infected with HSV-1 HFEM or with permeabilized (c) Vac4b-gH-infectedCV-1 cells.The antibodyreactionwas detectedwith fluoresceinisothiocyanateconjugatedto rabbit anti-mouseimmunoglobulins.

showed that antibody 52S detected gH on the surface and in the cytoplasm of HSV-l-infected cells, but no surface staining was apparent on cells infected with Vac7.5-gH or Vac4b-gH. Staining was limited to the cytoplasm and the nuclear membrane in these cells. It appears that when gH is synthesized by recombinant vaccinia viruses the molecule is antigenically aberrant and is not expressed at the cell surface, but it is unlikely that these abnormalities are due to some adverse effect of vaccinia virus replication on gH synthesis because similar findings were reported by Gompels & Minson (1989) using gH expression plasmids in the temperaturesensitive Cos cell system. In the latter study it was found that the authentic form of gH could be rescued by superinfecting virus, as a result that was interpreted as indicating that the correct synthesis and processing of gH is dependent on other HSV-l-specific functions acting in trans. The finding that aberrant gH is formed when expressed alone in two quite different expression systems supports this interpretation. We have no idea as to the nature of the HSV-specific functions required for authentic gH synthesis, but one obvious possibility is that gH must interact with a second viral glycoprotein. Since, apart from gH, only gB and gD are essential for HSV-1 replication in vitro, any postulated obligatory interaction of a virus-specific glycoprotein with gH must involve either gB or gD. To test this possibility BHK-21 cells were infected with Vac4b--gH either alone or in combination with VII (expressing gB) or VgD52 (expressing gD), each at an m.o.i, of 20. After 16 h the cellular localization of gH was examined by immunofluorescence with antibody 52S and the antigenicity of gH was determined by immunoprecipitation with antibodies 52S, 53S and L-P11. The results were similar to those shown in Fig. 4 and 5; the cellular localization and antigenic characteristics of gH expressed by Vac4b--gH were not modified by coexpression of gB or gD.

Discussion Although gH is a minor component of the HSV-1 envelope, its conservation among all members of the herpesvirus group examined to date implies a central role for this molecule in the infection process. Yet we know little of the function of gH or of the immune response to it, other than that antibodies specific for gH have notable antiviral effects on the virus particle and on the infected cell. Nothing is known of the antibody response to gH in individuals seropositive for HSV, but it seems unlikely that human sera contain high levels of anti-gH antibody because the HSV-1 neutralizing titres of human sera correlate well with anti-gD titres implying that gD antibodies account for most of the neutralizing activity (Cranage et al., 1983). Since it remains impossible to synthesize gH in an authentic antigenic form in the absence of other HSV proteins, direct and reliable measurement of anti-gH antibodies cannot be achieved. The neutralizing and anti-fusion properties of gHspecific antibodies are similar to those of some antibodies against gD (Minson et al., 1986; Gompels & Minson, 1986; Noble et al., 1983), an antigen that has long been recognized as providing protective immunity in experimental animals both by active and passive immunization (Long et al., 1984; Cremer et al., 1985; Berman et al., 1985; Blacklaws et al., 1987; Krishna et al., 1989; Balachandran et al., 1982; Simmons & Nash, 1985). By analogy we might expect gH to be protective and we have demonstrated that LPI 1, a MAb specific for HSV-1 gH, provides efficient protection against zosteriform spread of HSV-1 in the mouse. Active immunization with gH expressed by a vaccinia virus recombinant vector failed either to protect or to elicit neutralizing antibody. This is explained in part by the altered antigenic form of gH expressed in this system. However, at least one neutralizing epitope is present on the

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recombinant gH because the molecule is recognized by 52S, a neutralizing MAb. The failure of gH synthesized by vaccinia virus recombinants to be expressed at the infected cell surface may contribute to its failure as an immunogen. The demonstration that gH when synthesized in two quite different expression systems is antigenically abnormal strengthens the conclusion of Gompels & Minson (1989) that the correct synthesis and processing of gH is dependent on other HSV-specific function(s). Our experiments suggest that neither of the other essential HSV glycoproteins, gB and gD, is involved, although this interpretation of the results should be qualified by the recognition that the vaccinia virus-infected cell might be an unsuitable environment for the relevant interactions to occur. It is generally assumed that the expression of individual virus glycoprotein genes in mammalian vectors results in the synthesis of an authentic protein, but it is clear that this is not always the case. For example~ synthesis of the correct conformational form of influenza virus haemagglutinin requires a functional M2 protein (Hay et al., 1985; Belshe et al., 1988). We have no idea of the requirements for the authentic synthesis of gH, but until this can be achieved, studies of the functions of gH or of the immune response to it will be severely hampered. Finally, we do not know whether the problems we have encountered in expressing HSV-1 gH are associated with an HSV-specific phenomenon, or whether the homologues of gH in other herpesviruses have similar characteristics. However, it has been noted that expression of the gH homologue of human cytomegalovirus (the product of the UL75 open reading frame; Chee et al., 1990) in recombinant vaccinia virus results in failure of cell surface expression (Cranage et al., 1988). Audrey Forrester thanks the Equine Virology Foundation for the award of a training grant. Veronica Sullivan thanks the Medical Research Council, U.K. for a training grant. The work was supported by the Medical Research Council, U.K.

References BALACHANDRAN,N., BACCHETTI,S. 8¢ RAWLS,W. E. (1982). Protection against lethal challenge of BALB/c mice by passive transfer of monoclonal antibodies to five glycoproteins of herpes simplex virus type 2. Infection and Immunity 37, 1132-1137. BELSttE, R. B., HALL-SMITH,M., HALL, C. B., BETIS, R, & HAY, A. J. (1988). Genetic basis of resistance to rimantidine emerging during treatment of influenza virus infection. Journal of Virology 62, 15081512. BERMAN,P. W., GREGORY, T., CRASE,D. & LASKEY,L. A. (1985). Protection from genital herpes simplex virus type 2 infection by vaccination with cloned type 1 glycoprotein D. Science 227, 14901492. BLACKLAWS,B. A., NASH,A. A. & DARBY,G. (1987). Specificity of the immune response of mice to herpes simplex virus glycoproteins B and D constitutively expressed on L cell lines. Journal of General Virology 68, 1103-1114.

BLACKLAWS,B. A., KRISHNA,S., MINSON,A. C. & NASH,A. A. (1990). Immunogenicity of herpes simplex virus type 1 glycoproteins expressed in vaceinia virus recombinants. Virology 177, 727-736. BUCKMASTER, E. A., GOMPELS, U. & MINSON, A. C. (1984). Characterisation and physical mapping of an HSV-1 glycoprotein of approximately 115 x 10~ molecular weight. Virology 139, 408-413. CA1, W., BAOHUA,G. & PERSON, S. (1988). Role of glycoprutein B of herpes simplex virus type 1 in viral entry and cell fusion. Journal of Virology 62, 2596-2604. CANTIN,E. M., EBERLE,R., BALDICK,J. L., MOSS,B., WILLEY,D. E., NOTKINS, A. L. & OPENSHAW, H. (1987). Expression of herpes simplex virus 1 glycoprotein B by a recombinant vaccinia virus and protection of mice against lethal herpes simplex virus 1 infection. Proceedings of the National Academy of Sciences, U.S.A. 84, 59085912. CHEE, M. S., BANKIER,A. T., BECK,S., BOh'NI, R., BROWN,C. M , CESNY, R., HOP-SNELL,T., HUTCHINSON,C. A., KOUZARIDES,T., MARTIGNETTI,J. A., SATCHWELL,S. C., TOMLINSON,P., WESTON, K. M. & BARRELL,B. G. (1990). An analysis of the protein coding content of the sequence of human cytomegalovirus strain AD169. Current Topics in Microbiology and Immunology 154, 125-169. COO-mAN, M. A., PUCI