The polyspecific immunoglobulin response to HSV - CiteSeerX

J. Med. Microbiol. - Vol. 25 (1988), 49-57 01988 The Pathological Society of Great Britain and Ireland

The polyspecific immunoglobulin response to HSV- 1 viral proteins: determination of immunogenic proteins and relative antibody titres to individual polypeptides by immunoblotting R. R. McKENDALL, M. PETTIT and W. WOO Departments of Neurology and Microbiology, The University of Texas Medical Branch, Galveston, Texas 77550, USA

Summary.We developed an immunoblotting procedure to characterise the polyspecific immunoglobulin response to the proteins of herpes simplex virus (HSV)-1. We found that 8-20% gradient polyacrylamide gels provided no advantage over fixed 8.5% gels for preparing Western blots for use in immunoblotting. The amount of protein loaded on the gels markedly influenced which proteins were detected by immune serum. The presence of Triton-X-100 0.5% in washes and buffers improved band clarity on immunoblots. In optimal conditions, immune mouse serum reacted with up to 33 HSV- 1 lysate proteins. Six bands or regions appeared to be of major immunogenic reactivity, including the (122-130) x 103-mol.wt region, a 75 x 103-mol.wt protein, the gD region of approximately (56-64) x 103-mol.wt and two non-glycosylated bands at mol. wts( lo3)42 and 44. Minor proteins, more weakly reactive, were detected at 27 other areas. The relative antibody titres in immune mouse serum to the different major regions showed antibody titre to gD > gB/gC > (42/44) x lo3>> P75 >> VP154. Most human sera reacted with all of the major and many of the minor immunogenic proteins but individual sera varied markedly in the proteins recognised. We conclude that immunoblotting is valuable for evaluating immunoglobulin responses to major and minor immunogenic proteins of HSV- 1.

peptides are present at low concentrations in the cell lysate. Isotope incorporation depends on many The humoral immune response to herpes simplex factors which result in differing specific activities virus (HSV) has been studied by numerous techbetween different polypeptides. Some viral proteins niques, including complement fixation, neutralisa- are insoluble and so unavailable in the cell lysate as t ion, ELISA, radioimmunoassay, immuno- usually prepared. precipitation and immunoblotting. The latter two Immunoblotting, on the other hand, with unlatechniques have the advantage of enabling the belled polypeptides electrophoretically separated determination of the individual viral polypeptides and then transferred to nitrocellulose (NC) paper recognised by the polyspecific immunoglobulin. reduces these problems and, furthermore, offers the More than 50 proteins have been described in HSVadvantage of amplification through the use of 1 infected cell lysates. Immunoprecipitation studies secondary or tertiary antibody layers. Despite the of sera from patients with primary or recurrent expected loss of some antigenic sites by denaturaherpes simplex have shown that significantly fewer tion, immunoblotting has been shown to be a very proteins can be demonstrated in precipitates; 7-1 8 sensitive technique for detection of antigens in proteins have been demonstrated in various immany systems. Norrild et al. (1981) in the first munoprecipitation studies (Gilman et al., 1981; HSV-1 immunoblotting studies showed that imZweerink and Stanton, 1981 ; Zweerink and Corey, mune rabbit serum recognised 17 different proteins 1982). Several factors may limit the sensitivity of in an immunoblotting procedure employing 251the immunoprecipitation assay. Some minor polyprotein A. In human sera studies by Eberle and Mou (1983), reactivity with 10-28 HSV-1 proteins was seen. Finally, 4-12 HSV-1 or HSV-2 polypepReceived 20 Oct. 1986; revised version accepted 6 Apr. 1987. Introduction

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R. R. McKENDALL, M. PETTIT AND W. WOO

tides were recognised by human sera in the studies by Lehtinen et al. (1985). Our studies had three purposes: to develop an immunoblotting procedure which could detect as many minor as well as major immunogenic proteins of HSV-1 as possible; to identify the major immunogenic proteins recognised by human and mouse immune serum ;and to determine the relative titres of antibody in immune mouse serum to each major protein. Materials and methods Virus HSV-1 (Heitzman) was originally isolated from a patient with pharyngitis. The isolate was typed as HSV1 by neutralisation kinetics and SDS-polyacrylamide polypeptide pattern with isotopically labelled proteins.

Preparation of immune mouse serum BALB/c male mice, 6 weeks old, were given 1O5 pfu of HSV-1 by injection into the footpad. Two weeks later a second dose of lo6 pfu was given intraperitoneally (ip). After a resting period of one month or more, mice received a third dose of lo7 pfu ip. Serum was then collected and pooled.

Human sera Human serum samples were obtained from a bank of sera from laboratory workers. They were selected from the bank at random without regard to past history of HSV exposure or HSV recurrences.

Incorporation of radiolabelled amino acids into infected cell-lysate proteins

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed according to the standard procedure of Laemmli (1970). An 8.5% w/v acrylamide resolving gel cross linked with bisacrylamide or DATD and a stacking gel of 3-5%w/v bisacrylamide were employed. In some experiments 8-20% linear gradient polyacrylamide was used. HSV- 1 infected cell-lysate proteins were loaded at various concentrations (see Results). Electrophoresis was performed at 15 mA constant current on gels of 0.75 ml thickness by 14 cm length. Gels were dried by heat under vacuum and autoradiography was performed with Kodak XAR film.

Western blotting of HSV-1 infected cell polypeptides Gels prepared as above were assembled against NC paper (Biorad Co., Richmond, CAYUSA). The gel and NC were loaded into the transfer cassette. Proteins were transferred to the NC paper electrophoretically by use of Tris-glycine buffer with methanol 20% v/v as described by Towbin et al. (1979), with a Transphor apparatus (Hoefer Coy San Francisco, CA, USA) at 25 V, 0.2 A overnight. Time and amperage were adjusted in different experiments to determine the most sensitive conditions.

Detection of antibody to H S V-1polypeptides by immunoblotting Western blots containing HSV- 1 polypeptides were cut into strips 2-mm wide and put into plexiglass troughs. Strips were initially wetted with PBS and then blocked for 1 h with bovine serum albumin (BSA) 3% w/v and Triton-X-100 0.5% v/v in PBS. After washing with wash buffer containing BSA 1% w/v and Triton-X-100 0.5% v/v in PBS the strips were overlayed for 1 h with primary antisera diluted 1 in 50 to 1 in 100 000 in immunoblot buffer containing Triton-X-100 03% v/v and BSA 3% w/v in PBS. Affinity purified '251-labelled sheep antimouse or anti-human Ig (Amersham, Arlington Heights, IL, USA) (specific activity 17-20 pCi/pg) was used at 0.045 pCi/ml as a second antibody in immunoblot buffer with an incubation period of 1 h at room temperature. After extensive washing, the strips were dried in air, mounted on filter paper and exposed for 24 h to 17 days to Kodak XAR film for autoradiography.

Human diploid lung fibroblasts, MRC-5, were grown to confluency and infected with HSV-1 at a multiplicity of 10 pfu/cell. One h after infection the residual inoculum was washed away, the cell sheet was incubated for 1 h in PBS to deplete amino acid stores and then 35S-methionine was added at a final concentration of 80 pCi/ml. In some experiments 14C-labelled amino acids were added at a final concentration of 15 pCi/ml or 4C-labelled glucosamine was added at a final concentration of 5 pCi/ml. The labelling was terminated at 24-28 h. The infected cell sheets were washed exhaustively with cold PBS and scraped with a rubber policeman. The infected cells were Results pelleted at 400g4"C and then resuspended in lysate buffer containing SDS 0.1% w/v, Triton-X-100 1% v/v Detection of major immunogenic H S V-1 proteins and deoxycholate 1% w/v, pH 6.8, 0.5 M NaCl. Nuclei immobilised on nitrocellulose ( N C ) paper by probing were pelleted at 10 000 g and the labelled proteins in the with immune mouse serum and 2 5 I-labelled second supernate harvested and frozen at - 70°C until use. The antibody total protein content of lysates was 4.5 mg/ml and the To determine which HSV-1 proteins were recogspecific activity was 0.01-0-1pCi/pg total protein, depending on the radioreagent used in the lysate prepara- nised by antibody in immune mouse serum, 35Smethionine or ''C-glu~~~amine-labelled lysate protion.

IMMUNOGENIC HSV-1 PROTEINS

Panel a

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Panel b

Fig. 1. Autoradiographs of major immunogenic HSV-1 protein bands detected by immunoblotting (a) on Western blots from 8.5% bisacrylamide SDS-PAGE gels and (b) on Western blots of proteins separated by SDS-PAGE with an 8-20% bisacrylamide gradient gel. Strips of NC paper containing unlabelled HSV-1 infected cell proteins were probed with immune mouse serum (lane A) or normal mouse serum (lane B) followed by '251-labelledsheep anti-mouse Ig. 35S-methioninelabelled (a, lane D ; b, lane C) or ''C-glucosamine labelled (a, lane C; b, lane D) HSV-1-infected cell proteins were used as markers to identify the major regions detected on the immunoblot strips. Mol. wts(103)and designations of bands are shown.

teins from HSV- 1-infected cells were used as marker proteins on SDS-polyacrylamide gels which were then electrophoretically blotted on to NC paper. In an adjacent lane, unlabelled HSV- 1 cell-lysate proteins were co-electrophoresed with the labelled proteins and blotted. The NC papers containing the unlabelled proteins were cut into strips and used in an immunoblotting procedure employing immune mouse serum as primary antibody followed by 251-labelledsheep anti-mouse Ig. Fig. l a shows the relationship of the bands detected by immunoblotting to the bands on the Western blot. Mol. wt markers (not shown), run in an adjacent lane, allowed assignment of mol. wts to the major bands. The major regions detected (lane A) include the

(122-1 30) x lo3 region corresponding to the high mol. wt glycoproteins gB and gC, a protein of mol. wt 75 x lo3, a region at (56-64) x lo3 mol. wt which approximates to the location of gD in the ''C-glucosamine labelled lane, and a region containing bands of mol. wt (lo3)42 and 44 which are not glycosylated. In an attempt to select a gel separation system which would maximise detection of both high and low mol. wt proteins, we probed Western blots of proteins separated on 8-20% bisacrylamide linear gradient gel. Fig. 1b shows that no additional major bands of low mol. wt were detected although two bands of mol. wt (lo3) c. 47 and 49 were seen on the gradient gel.

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Determination of optimal protein load on Western blots of HSV-1 cell-lysate proteins To determine the protein concentration needed to detect the major immunogenic proteins on western blots, lanes of 4mm width were loaded with 1-4-22 pg of HSV-1-infected cell-lysate proteins. Fig. 2 shows that 11 pg or more per lane was required to visualise the (42/44) x 103-mol. wt proteins but that as little as 1.4 pg was sufficient to detect gC. Silver staining of the gel after blotting to nitrocellulose indicated a significant level of untransferred proteins (data not shown). We did not observe any evidence for impaired transfer of high mol. wt proteins compared to low mol. wt proteins. For our procedure, 11 or 22 pg total protein/4 mm lane appeared to be optimal to produce high quality Western blots.

Fig. 3. Autoradiograph of major and minor immunogenic HSV1-protein bands detected by immunoblotting on Western blots of proteins separated on 8.5% bisacrylamide SDS-PAGE gels. Strips of NC paper containing unlabelled HSV-1-infected cell proteins were probed with a 1 in 20 dilution of immune (lanes A, B and C) or normal (lane D) mouse serum followed by "'Ilabelled sheep anti-mouse IgG. Lanes A, B and C represent a composite of different photographic exposure times. A radiograph exposure time of 17 days was used to detect the minor immunogenic protein bands. The lower-most band is the dye front (no arrow).

Detection of minor immunogenic H S V-1proteins

Fig. 2. Autoradiograph of an immunoblot detecting HSV-1 proteins loaded in different concentrations. Lanes of an 8.5% SDS-PAGE gel were loaded with 22 pg(A), 11 pg(B), 5.5 pg(c), 2-8 pg(D) or 1.4 pg(E) of HSV-1 infected cell lysate protein and were blotted to NC paper. The Western blot was then probed with immune mouse serum followed bv 251-labelledsheep antimouse Ig.

To detect immunoblot bands due to minor proteins recognised by immune mouse serum we used a high concentration of immune mouse serum (1 in 20 dilution), and exposures of 2 , 5 and 17 days. Fig. 3 shows several bands between the major immunogenic protein regions. Above the major gB and gC region (130 x lo3 mol. wt), five proteins of high mol. wt were detected including the major CaDsid Drotein at 154 x 1O3 mol. wt and 'a Drotein of (175-1 go) x lo3 mol. wt which is most likely ICP4. Between the major regions Of mol' wts (lo3) 130 and 75 there are regions of molwt (lo3> 101-93 and 90-8 1. Below the 75 x 103-mol.wt protein there


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are five minor bands detected with mol. wts (lo’) 72-70, 67, 66, 62, and 59. The long radiograph exposure obscures the clarity of some of the bands in this region but different photographic exposures used in lanes A, B and C compensate for this somewhat. Below the major gD region (mol. wt 56 x lo3)there are three minor bands with apparent mol. wts (lo3) 52, 49 and 48, and below the major region of (42/44) x lo3 mol. wt there are 12 minor bands of mol. wts (lo3) 41, 40, 39.5, 38.5, 36, 35, 34.5, 33, 32, 31, 30 and 29. In all, 27 minor bands were recognised by the immune serum but not by non-immune mouse serum. Three faint bands were noted in the non-immune mouse serum (lane D): the capsid protein VP154, a 73 x 103-mol.wt band and a 45 x 103-mol. wt band. With blots containing uninfected cell-lysate proteins, it was shown that none of the immune serum bands was the result of cross-reactivity with cell proteins (data not shown). Efect of Triton-X-100 on sensitivity of irnrnunoblot detection of antibody to HSV-1 polypeptides Although we had little difficulty detecting antibody to many different HSV-1 polypeptides, some reports had suggested that the denaturing conditions of SDS-PAGE destroyed some antigenic epitopes of blotted proteins and that incubation of N C strips with Triton-X-100 during immunoblotting procedures enhanced antibody binding to HSV-1 proteins by allowing renaturation of some epitopes (Braun et al., 1983). It was, therefore, of interest to examine the effect of Triton-X-100 on band appearance in immunoblotting. Fig. 4 compares strips processed with Triton-X-100 0.5% to strips processed without Triton-X-100. Strips treated with Triton-X-100 for 30 min (lane C ) before being probed with primary antibody showed bands which appeared sharper in most of the major immunogenic regions than strips processed without Triton-X-100 (lane A). Furthermore, most major regions appeared darker, suggesting that more antigen binding sites were available. In particular there was enhanced detection of the 75 x lo3mol.wt protein which appeared much sharper. In the (56-64) x 103-mol. wt region, however, the enhanced darkness obscured two bands (short arrows) which were clearer without Triton-X-100. To determine if the presence of Triton-X-100 in the primary and secondary antibodies would reduce antibody binding, we processed strips with TritonX-100 0.5% present in all solutions including the washes. No reduction in specific antibody binding was detected in strips, including those probed with immune serum (lane E). A significant reduction in

Fig. 4. Effect of Triton-X-100 0.5% on detection of polyspecific antibody binding to individual HSV-1 polypeptides in an immunoblotting procedure with HSV-1 polypeptides separated on an 8.5% DATD-crosslinked SDS-PAGE. Immune (lanes A, C and E) or normal (lanes B, D and F) mouse serum was used to probe NC strips containing HSV-1 polypeptides. All strips were probed with *’I-labelled sheep anti-mouse Ig as second antibody. For strips A and B, PBS with BSA 1% was used for all reagents and washes. Strips C and D were treated similarly except that a 30-min incubation step with Triton-X-100 0.5% with BSA 1% in PBS was inserted before the primary antibody step. For strips E and F, all reagents and washes contained Triton-X-100 0.5% and BSA 1% in PBS. The short arrows indicate two bands which were better visualised in the absence of any Triton-X-100.

nonspecific background was, however, noted in all strips, including those probed with normal mouse serum. Thus, the presence of Triton-X-100 reduced non-specific binding and enhanced specific binding of some bands but other bands may be obscured in these conditions.

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R. R. McKENDALL, M. PETTIT A N D W. WOO

Determination of antibody titres to individual HSV-1 polypeptides by immunoblot ting

To determine the relative antibody titres to individual HSV- 1 polypeptides, immunoblotting was performed in the presence of Triton-X-100 with primary hyperimmune mouse antisera dilutions from 1 in 50 to 1 in 100 000. Fig. 5 is a composite of strip autoradiographs of different exposures and shows that the titre of anti-gD antibody was > 100 000, whereas antibody to gB/ gC and the (42/44) x 103-mol.wt region was barely detectable at the 1 in 100000 dilution. The antiVP154 titre was < 3000 and the anti-p75 was > 20 000 < 100 000. Human serum antibodies to individual HSV-1 polypeptides

Human sera were surveyed and their reactivity compared with the polypeptide reactivity observed with the immune mouse serum; 16 random sera were screened by immunoblotting and 11 had detectable bands. Fig. 6 shows the positive ones (lanes A-K) and one negative serum as a control (lane L). Overall, human and mouse sera recognised the same major immunogenic proteins, except for the 75 x 103-molwt protein. Sera C and D gave results very similar to those obtained with the immune mouse serum. Serum H showed greater reactivity with the gD and (42/44)x 103-mol.wt regions and notably less reactivity with the gB/gC region. Conversely, serum I showed greatest reactivity with the gB/gC region and somewhat less with the gD and (42/44)x 103-mol.wt regions. Serum G had an unusual pattern of faint reactivity with (42/44)x 103-mol.wt and gD regions and greater reactivity with unidentified proteins in the (65-70) x 103-mol.wt range. Thus the individual human specimens show widely different patterns, although most specimens displayed some degree of reactivity with the gB/gC, gD and the (42/44)x 1o3-mo1.wt regions.

Discussion By immunoblotting we have shown that immunological reactivity of 33 major and minor HSV-1 proteins can be detected despite any denaturation resulting from the electrophoretic methods. The sensitivity shown here exceeds that achieved in earlier immunoblot studies (Norrild et al., 198l), probably as a result of greater gel resolution, more complete transfer of protein to paper by electrophoretic methods, and careful determination of optimal protein concentrations on the SDS-polyacrylamide

Fig. 5. Titres of antibody to individual HSV-1 polypeptides detected by binding to Western blots containing HSV- 1-infected cell proteins separated with 8.5% SDS-PAGE crosslinked with DATD. Strips were overlayed with control normal mouse serum at a 1 in 50 dilution (lane A) or with immune mouse serum at dilutions of 1 in 50 (lane B), 1 in 300 (lane C), 1 in 3000 (lane D), 1 in 20 000 (lane E) or 1 in 100 000 (lane F). '251-labelledsheep anti-mouse IgG was used as the second antibody. Triton-X-100 0.5% was used in all reagents and washes. The figure is a composite of autoradiographsof different exposures.

gels. Eberle and Mou (1983) used a sensitive immunoblot system and one of their human sera, HS-3, recognised 28 HSV-1 polypeptides. Sera of other patients in their study reacted with only 10 proteins. We found similar variation between human sera in our studies. Thus, differences in the


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Fig. 6. Human serum antibodies to HSV-1 polypeptides studied by immunoblotting. Individual specimens (lanes A-L) were diluted 1 in 50 for study. '251-labelledsheep anti-human Ig was used as the second antibody.

various immunoblotting studies are also due to the antisera studied. Furthermore, some studies have used peroxidase-conjugated second antibodies (Braun et al., 1983) rather than 2SI-staphylococcal protein A or l2'1-anti-IgG (Norrild et al., 1981; Braun et al., 1983; this study). In our hands, with peroxidase-conjugated second antibody (Vector Labs, Burlingame, CA, USA), the immune serum could be diluted only to 1 in 5000 before all bands became undetectable (McKendall, unpublished observation), whereas some major bands were detectable at a 1 in 100 000 dilution of the same immune mouse serum when * 251-goatanti-mouse Ig was used (Fig. 5). Therefore, immunoblotting

with *'I-labelled reagents, at least those we employed, is more sensitive. We compared our findings of antibody reactivity to individual HSV- 1 polypeptides with published results obtained by immunoprecipitation, the only other technique capable of providing comparable data. One study of sera from seropositive patients with and without facial herpetic recurrences found that 18-22 HSV-1 polypeptides were precipitated (Zweerink and Stanton, 1981) whereas another study of 231 patients found that individual sera recognised, at most, seven polypeptides, although 31 different proteins were recognised by the entire bank of 23 1 sera (Gilman et al., 1981). Serum from

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one patient with herpes encephalitis showed seven bands detectable by immunoprecipitation (Mann and Hilty, 1982). A fourth immunoprecipitation study of HSV-polypeptide reactivity with 50 human sera showed that 12-14 HSV-1 or HSV-2 peptides were precipitated (Norrild, 1985). However, 23 polypeptides were recognised by the same sera when studied in an immunoblotting assay with an 251-proteinA probe (Norrild, 1985). Other immunoprecipitation studies with HSV-2 antigens have shown about 18 precipitated bands with serum from patients with primary genital herpes (Zweerink and Corey, 1982; Ashley and Corey, 1984). While some of the variability in results is due to differences in the immune status of patients and experimental animals, it appears that immunoprecipitation can enable recognition of 7-22 bands whereas immunoblotting allows recognition of up to 33 bands. The major proteins detected in our immunoblots were gC, gB, 75 x lo3, gD (56 x lo3), 44 x lo3 and 42x lo3. The highest titres of antibody were directed at gC, gB, gD and the (44/42) x 103-mol.wt proteins. High titres of antibody to gD and gB are also seen in quantitative immunoelectrop horetic studies on human sera (Vestergaard, 1979). There is generally good agreement between our results and those of Eberle and Mou (1983) except for the 75 x 103-m01.wt protein which was not a major protein in their studies. Correlation with immunoprecipitation studies shows that (1 15-1 30) x lo3mol. wt proteins (probably gB and gC) and gD are the most commonly precipitated HSV- 1 proteins (Gilman et al., 1981 ; Zweerink and Stanton, 1981; Mann and Hilty, 1982).It is of note that the proteins of mol. wts. (lo3) 44 and 42 were not precipitated in these studies, or were only minor proteins in immunoprecipitates. This disparity has previously been recognised by Eberle and Mou (1983). These proteins are probably related to the p40 protein purified from HSV nucleocapsids by Heilman et al. (1981) because monoclonal antisera to p40 (Zweig et al., 1980) bind to a series of proteins ranging in mol. wt (lo3) from 34 to 49 (Eberle et al., 1984, 1985). It is also possible that antibody to viral thymidine kinase (mol. wt 45 x lo3) is responsible for some of the reactivity.

Our finding of a major band of mol. wt 75 x lo3 is of interest in that it has not been recognised as a major band before. The identity of this protein is unknown. It is evidently not a cell protein because it is absent from blots prepared with uninfected cell lysates and because it is present in serum from mice with a primary infection (McKendall, unpublished observations). Therefore the 75 x 103-mol.wt protein is not likely to be a cell protein recognised as a result of the immunisation procedure. Norrild (1985) detected a 77 x 103-mol.wt protein and Gilman et al. (1981) detected proteins of mol. wts (lo3) 73 and 75.5 which reacted with human immune serum but neither report considered the protein to be of major immunogenic importance. The 75 x 103-mol.wt band was not a major band in the panel of human specimens we studied and so may be conspicuous in some species only. The 75 x 103-mol.wt protein in our mouse studies could represent a precursor form of other major immunogenic proteins. A non-glycosylated form of gC obtained by endo-P-N-acetylglucosaminidase H treatment of a high mannose intermediate precursor of gC yields a 75 x 103-mol.wt protein (Wenske et al., 1982). Non-glycosylated gC synthesised in vitro has a mol. wt of 75 x lo3 (Lee et al., 1982). Also, gE purified by affinity chromatography utilising its Fc binding properties shows three forms of gE ranging in mol. wt (lo3) from 65 to 80 (Baucke and Spear, 1979). However it is unlikely that our 75 x lo3mol. wt protein is a form of gE because gE is a minor protein in infected cell lysates and the appearance of the band in our blots is unlike the diffuse bands of gEl or gE2 (Baucke and Spear, 1979). We have shown that immunoblotting, as used by us, is a highly sensitive method of detecting and characterising the HSV-1 polypeptides recognised by immune serum. Great variability in quantitative and qualitative serum antibody responses to HSV1 proteins is apparent in both immunoblotting and immunoprecipitation studies. Further work will be required to identify all the proteins to which antibody is made and to define the conditions which lead to production of antibody to individual polypeptides in one serum and not in another.

REFERENCES

Braun D K, Pereira L, Norrild B, Roizman B 1983 Application of denatured, electrophoretically separated, and immobilized lysates of herpes simplex virus-infected cells for detection of monoclonal antibodies and for studies of the properties of viral proteins. Journal of Virology 46: 103-1 12. Cohen G H, Long D, Eisenberg R J 1980 Synthesis and processing of glycoproteins g D and gC of herpes simplex virus type 1 . Journal of Virofogy36 : 429-439.

Ashley R L, Corey L 1984 Effect of acyclovir treatment of primary genital herpes on the antibody response to herpes simplex virus. Journal of Clinical Investigation 73 : 68 1-688. Baucke R B, Spear P G 1979 Membrane proteins specified by herpes simplex viruses. V. Identification of an Fc-binding glycoprotein. Journal of Virofogy32 : 779-789.

IMMUNOGENIC HSV-1 PROTEINS Eberle R, Mou S-W 1983 Relative titres of antibodies to individual polypeptide antigens of herpes simplex virus type 1 in human sera. Journal of Infectious Diseases 148: 436-444. Eberle R, Mou S-W, Zaia J A 1984 Polypeptide specificity of the early antibody response followingprimary and recurrent genital herpes simplex virus type 2 infections. Journal of General Virology 65: 1839-1843. Eberle R, Mou S-W, Zaia J A 1985 The immune response to herpes simplex virus: Comparison of the specificity and relative titres of serum antibodies directed against viral polypeptides following primary herpes simplex virus type 1 infections. Journal of Medical Virology 16: 147-162. Gershoni J M, Palade G E 1983 Protein blotting: principles and applications. Analytical Biochemistry 131: 1-1 5. Gilman S C, Docherty J J, Rawls W E 1981 Antibody responses in humans to individual proteins of herpes simplex viruses. Infection and Immunity 34: 880-887. Heilman C J, Zweig M, Hampar B 1981 Herpes simplex virus type 1 and 2 intracellular p40: Type-specific and crossreactive antigenic determinants on peptides generated by partial proteolysis. Journal of Virology 40: 508-5 15. Laemmli U K 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 : 680685. Lee G T-Y, Pogue-Geile K L, Pereira L, Spear P G 1982 Expression of herpes simplex virus glycoprotein C from a DNA fragment inserted into the thymidine kinase gene of this virus. Proceedings of the National Academy of Sciences of the USA 79: 6612-6616. Lehtinen M, Koivisto V, Lehtinen T, Paavonen J, Leinikki P 1985 Immunoblotting and enzyme-linked immunosorbent assay analysis of serological responses in patients infected with herpes simplex virus type 1 and type 2. Zntervirology 24: 18-25. Mann D R, Hilty M D 1982 Antibody response to herpes simplex virus type 1 polypeptides and glycoproteins in primary and recurrent infection. Pediatric Research 16 : 176180. Norrild B 1980 Immunochemistry of herpes simplex virus

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glycoproteins. Current Topics in Microbiology and Zmmunology 90: 68-106. Norrild B 1985 Humoral response to herpes simplex virus infections. In: Roizman B, Lopez C (eds) The Herpesviruses, vol4. Plenum Press, New York, NY pp 69-86. Norrild B, Pedersen B, Roizman B 1981 Immunological reactivity of herpes simplex virus 1 and 2 polypeptides electrophoretically separated and transferred to diazobenzyloxymethyl paper. Infection and Immunity 31 : 660-667. Pauli G, Ludwig H 1977 Immunoprecipitation of herpes simplex virus type 1 antigens with different antisera and human cerebrospinal fluids. Archives of Virology 53 : 139-1 55. Preston V G , Coates J A V, Rixon F J 1983 Identification and characterization of a herpes simplex virus gene product required for encapsidation of virus DNA. Journalof Virology 45: 10561064. Ratner J J, Smith K 0 1980 Serum antibodies to herpes simplex virus type 1 during active oral herpes infection. Infection and Immunity 27 : 1 13-1 17. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : Procedure and some applications. Proceedings of the National Academy of Sciences of the USA 76 : 4350-4354. Vestergaard B F 1979 Quantitative immunoelectrophoretic analysis of human antibodies against herpes simplex virus antigens. Infection and Immunity 23: 553-558. Wenske E A, Bratton M W, Courtney R J 1982 Endo-P-Nacetylglucosaminidase H sensitivity of precursors to herpes simplex virus type 1 glycoproteins gB and gC. Journal of Virology44: 241-248. Zweerink H J, Corey L 1982 Virus-specific antibodies in sera from patients with genital herpes simplex virus infection. Infection and Immunity 37: 413-421. Zweerink H J, Stanton L W 1981 Immune response to herpes simplex virus infections : Virus-specific antibodies in sera from patients with recurrent facial infections. Infection and Immunity 31 : 624-630. Zweig M, Heilman C J, Rabin H, Hampar B 1980 Shared antigenic determinants between two distinct classes of proteins in cells infected with herpes simplex virus. Journal of Virology 35 : 644-652.