* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download The role of carbohydrate in the antigenic and immunogenic structure
Adoptive cell transfer wikipedia , lookup
Immunoprecipitation wikipedia , lookup
Immune system wikipedia , lookup
Gluten immunochemistry wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
Innate immune system wikipedia , lookup
Adaptive immune system wikipedia , lookup
Multiple sclerosis research wikipedia , lookup
Human cytomegalovirus wikipedia , lookup
Henipavirus wikipedia , lookup
Hepatitis B wikipedia , lookup
Autoimmune encephalitis wikipedia , lookup
Immunocontraception wikipedia , lookup
DNA vaccination wikipedia , lookup
Anti-nuclear antibody wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Molecular mimicry wikipedia , lookup
Immunosuppressive drug wikipedia , lookup
Journal of General Virology (1990), 71, 2053-2063. Printedin Great Britain 2053 The role of carbohydrate in the antigenic and immunogenic structure of bovine herpesvirus type 1 glycoproteins gI and glV S. van Drunen Littel-van den Hurk, 1 G. Hughes 1 and L. A. Babiuk ~,2 1Veterinary Infectious Disease Organ&ation and 2Department o f Veterinary Microbiology, University o f Saskatchewan, Saskatoon, Saskatchewan, Canada S 7 N 0 WO The role of carbohydrate in the antigenic and immunogenic structure of bovine herpesvirus type 1 (BHV-1) glycoproteins gI and glV was investigated. Deglycosylated proteins induced a significantly lower antibody response in rabbits than native glycoproteins suggesting that the immunogenicity of several epitopes on gI and glV is carbohydrate-dependent. Loss of carbohydrate from gI also resulted in a significantly decreased ability to induce a serum neutralizing antibody response to BHV-1, due to modifications in three distinct carbohydrate-containing continuous epitopes. Similarly, in vitro lysis of BHV-l-infected cells was significantly reduced when antibodies raised against deglycosylated gI were employed; this was attributed to changes in two of the three carbohydrate-dependent neutralizing epitopes on gI. The oligosaccharides may be directly involved as actual components of these continuous epitopes, rather than in stabilization of the conformation of the protein. In contrast, carbohydrate removal from gIV did not have a significant effect on the capacity to stimulate a neutralizing antibody response. Accordingly, none of the neutralizing epitopes on gIV appeared to be carbohydrate-dependent. Similarly, lysis of virus-infected cells was not significantly reduced when antibodies specific for deglycosylated rather than native gIV were used. In contrast to the humoral response, the delayed-type hypersensitivity response was stronger in rabbits immunized with deglycosylated proteins than in those inoculated with native glycoproteins gI or gIV. Consequently, the carbohydrates on gI and gIV may play a dual role in the host's immune recognition and response by contributing to certain epitopes, but masking others. The implications for the development of a subunit vaccine against BHV-1 are discussed. Introduction the plasma membrane of the host cell and are able to mediate cell fusion (Manservigi et al., 1977; Noble et al., 1983), complement factor C3b (Friedman et al., 1984) and immunoglobulin G (Johnson & Feenstra, 1987) binding and immune destruction of virus-infected cells (Norrild et al., 1980; Carter et al., 1981 ; Bishop et al., 1984; Rouse & Horohov, 1984). Four (sets of) glycoproteins have been identified in BHV-1 : gI, a 130K disulphide-linked 74K/55K heterodimer; glI, a 108K glycoprotein; gill, a 180K/91K dimeric glycoprotein; and glV, a 140K/71K dimeric glycoprotein (van Drunen Littel-van den Hurk et al., 1984; Collins et al., 1984; Marshall et al., 1986; Hughes et al., 1988). Glycoproteins gI, gill and gIV all stimulate the production of neutralizing antibodies in mice, rabbits and cattle, and they all serve as targets for antibodydependent, complement-mediated lysis of virus-infected cells (Collins et al., 1984; van Drunen Littel-van den Hurk et al., t984; van Drunen Littel-van den Hurk & Babiuk, 1985b; Marshall et al., 1986; Okazaki et al., 1986; Trepanier et al., 1986; Israel et al., 1988). Several Bovine herpesvirus type 1 (BHV-1), a member of the alphaherpesvirinae subfamily, is an economically important pathogen in cattle. It is the causal agent of a variety of disease conditions, including respiratory infections, conjunctivitis, vulvovaginitis, abortions, encephalitis and generalized systemic infections (Gibbs & Rweyemamu, 1977). Recovery from most herpesvirus infections, including BHV-1 infection, is mediated by a variety of cellular and humoral immune responses (Rouse & Babiuk, 1978). Viral glycoproteins are the major structural components present in the envelope of herpesviruses. They play an important role in the virus-host relationship as they are involved in the recognition, attachment (Little et al., 1981; Fuller & Spear, 1985) and penetration (Sarmiento et al., 1979; Fuller & Spear, 1987) of the virus into susceptible cells, as well as in viral neutralization (Vestergaard & Norrild, 1979; Glorioso et al., 1984). During infection the viral glycoproteins are inserted into 0000-9399 © 1990SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 19:19:39 2054 S. van Drunen Littel-van den Hurk, G. Hughes and L. A. Babiuk studies have shown that the ability to mediate virus neutralization as well as destruction of virus-infected cells is localized in a number of different epitopes on each of these three glycoproteins (Collins et al., 1984; van Drunen Littel-van de Hurk et al., 1985; Okazaki et al., 1986; Marshall et al., 1988; Hughes et al., 1988). Glycoproteins gI, glII and glV have also been identified as the major immunogens recognized by cattle infected with BHV-1 (Collins et al., 1985; van Drunen Littel-van den Hurk & Babiuk, 1986a). Furthermore, each of glycoproteins gI, gill and glV, individually or in combination, was able to induce protective immunity in cattle against challenge with BHV-1 (Babiuk et al., 1987). Oligosaccharides are integral components of many viral envelope and eukaryotic cell surface proteins. Although the precise function(s) of the carbohydrate moieties of these proteins remains unknown, they appear to play significant roles in specific recognition phenomena, as well as protein folding and conformation (Berger et al., 1982; Sharon & Lis, 1982). Evidence for the glycoprotein nature of BHV-1 glycoproteins gI, glI, gill and glV has been obtained by the incorporation of radiolabelled sugars (van Drunen Littel-van den Hurk et al., 1984) and their reactivity with a variety of lectins (personal observation). Treatment of these glycoproteins with endoglycosidases (van Drunen Littel-van den Hurk & Babiuk, 1986b) and incorporation of glycosylation inhibitors in the tissue culture medium during BHV-1 infection (van Drunen Littel-van den Hurk & Babiuk, 1985 a) have shown that glycoproteins gI and glI contain N-linked sugars, whereas gill and glV have both N-linked and O-linked carbohydrates. Cloning and sequencing data have indicated the presence of six potential asparagine-linked glycosylation sites on gI, four on gill and three on glV (Whitbeck et al., 1988; T. J. Zamb, unpublished results). Glycoproteins gI and glV are primary targets for neutralizing and protective antibodies (Babiuk et al., 1987) and potentially for cellular immune responses as well (unpublished observations). Studies on several viruses like influenza virus, respiratory syncytial virus and Rauscher leukaemia virus, have indicated that the carbohydrate moiety of viral glycoproteins can have a strong influence on functional (Basak & Compans, 1983; Lambert, 1988) as well as antigenic and immunogenic activities (Alexander & Elder, 1984; Elder et al., 1986). In contrast, the carbohydrates did not appear to play a major role in the antigenic structure of the E protein of flaviviruses, a lightly glycosylated protein (Winkler et al., 1987). The objective of this study was to analyse the contribution of the carbohydrate portion of glycoproteins gI and glV to their ability to induce an immune response to BHV-1. Our experiments indicate that loss of carbohydrate results in a reduced neutralizing antibody response to BHV-1. In the case of gI, this was due to a change in three defined carbohydrate-dependent neutralizing epitopes. Similarly, lysis of BHV-l-infected cells appeared to be at least partially mediated by carbohydrate-dependent epitopes. In contrast, the cellular immune response as measured by a delayed-type hypersensitivity (DTH) reaction was stronger following immunization with deglycosylated protein than after inoculation with native glycoprotein, suggesting that carbohydrate may mask at least some epitopes involved in T cell recognition or induction. Methods Cells, virus and glycoproteins. Madin-Darby bovine kidney (MDBK) cells were grown as monolayers in Eagle's MEM (Gibco) supplemented with 10 % foetal bovine serum (FBS; G ibco). Strain P8-2 of BHV-1 was propagated in MDBK cells and quantified by plaque titration in microtitre plates with an antibody overlay as previously described (Rouse & Babiuk, 1974). Glycoproteins gI and gIV were purified from virus-infected cell lysate as previously described (van Drunen Littelvan den Hurk & Babiuk, 1985b). Glyeosidase digestion ofglyeoproteins. Glycoprotein gI was deglycosylated by treatment with peptide:N-glyeosidase F (N-glycanase; Genzyme) or with endo-fl-N-acetylglucosaminidase F (endo F from Flavobaeterium meningosepticurn; New England Nuclear) whereas gIV was treated with N-glycanase or endo F, followed by neuraminidase (from Clostridium perfringens; Sigma) and endo-fl-N-acetylgalactosaminidase (O-glycanase; Genzyme). Incubation with N-glyeanase was carried out in 0.2 M-sodium phosphate buffer pH 8-6, 50 mM-EDTA, whereas digestion ~vith endo F was performed in 0-1 M-sodium phosphate buffer pH 6.1, 50 mM-EDTA. Usually, 10 Ixg of glycoprotein at a concentration of 0.5 mg/ml was digested with 1 unit (U) of enzyme for 20 h at 37 °C. Neuraminidase digestion was performed in 0.02 MTris-maleate, 1 raM-calcium acetate, 10 mM-D-galactoso-gammalactone for 1 to 3 h at 37 °C. Ten mg of purified glycoprotein was incubated with 0.1 U of neuraminidase. Subsequently, O-glycanase treatment was carried out for 6 h at 37 °C under the same buffer conditions, using 10 mU for 10 ~tg of glycoprotein. For analysis by discontinuous SDS-PAGE the proteins were precipitated with ice-cold acetone, resuspended in electrophoresis sample buffer (0-625 M-TrisHC1 pH 6.8, 1.25% SDS, 12-5% glycerol, 0,15 M-2-mercaptoethanol, 0"00125~ bromophenol blue) and boiled for 1 min. For rabbit immunizations the proteins were dialysed against PBS. Polyacrylamide gel electrophoresis and immunoblot analysis. SDSPAGE was carried out in 7.5% discontinuous slab gels (Laemmli, 1970) under reducing conditions as described previously (van Drunen Littelvan den Hurk et al., 1984). The Western blotting technique of Burnette (1981) was used as previously described (van Drunen Littel-van den Hurk et al., 1984). After electrophoresis, afffinity-purified gl was electrophoretically transferred to nitrocellulose sheets, using 25 raMsodium phosphate buffer pH 6-8 as the electrode sotuIion. Subsequently, the instructions for use of the Bio-Rad immunoblot assay kit were followed. Lectin binding assay. Polystyrene microtitre plates (Immulon 2, Dynatech Laboratories) were coated overnight at room temperature with 200 ~tl per well of purified glycoprotein or deglycosylated protein Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 19:19:39 Carbohydrate role in B H V - 1 glycoproteins (0.5 ~tg/ml) in coating buffer (0.05 M-NaHCO 3 pH 9.6). After coating, the plates were washed four times in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST). Two hundred ~tl of biotinylated lectin (1 mg/ml; Vector Laboratories) serially diluted in PBST was added to each well and incubated for 2 h at 37 °C. Alternatively, purified glycoprotein and deglycosylated protein (0.5 Ixg/ml) were serially diluted and incubated with 200 ~tl of biotinylated lectin (10 l-tg/ml). The plates were washed four times and the wells were incubated with avidin DH-biotinylated alkaline phosphatase H complex (Vectastain ABC-AP kit; Vector Laboratories) according to the manufacturer's instructions. The reaction was allowed to continue for 30 min at room temperature, whereafter the absorbance at 450 nm was determined on a Dynatech Microelisa reader (Model MR580). Immunization of rabbits. The glycoproteins and deglycosylated proteins were diluted in PBS and emulsified in an equal volume of Freund's adjuvant (Gibco). Groups of two New Zealand white rabbits were given one immunization with each of the glycoproteins or deglycosylated proteins at different concentrations in Freund's complete adjuvant, followed by a booster immunization in Freund's incomplete adjuvant 4 weeks later. They were bled 3 weeks after each immunization. ELISA. The ELISA was performed essentially as described previously (van Drunen Littel-van den Hurk et al., 1984; van Drunen Littel-van den Hurk & Babiuk, 1985b). To determine the antibody responses of rabbits immunized with glycoproteins or deglycosylated proteins, microtitre plates were coated with 0.05 rtg purified glycoprotein or 1 ~tg purified BHV-I per well. Affinity-purified, horseradish peroxidase (HRPO)-conjugated goat anti-rabbit Ig (Boehringer Mannhelm) was used at a dilution of 1 : 2000 for detection. To analyse the reactivity of monoclonal antibodies with deglycosylated and native gI and gIV, microtitre plates were coated with 0-05 Bg of native or deglycosylated protein per well. Affinity-purified HRPO-conjugated goat anti-mouse IgG (Boehringer Mannheim) was used at a dilution of 1 : 1000 for detection. Results Deglyeosylation o f glycoproteins g I and g l V A s g l y c o p r o t e i n gI c o n t a i n s o n l y N - l i n k e d c a r b o h y d r a t e s , w h e r e a s g I V has b o t h N - a n d O - l i n k e d s u g a r s ( v a n D r u n e n L i t t e l - v a n d e n H u r k & B a b i u k , 1985a, 1986b), e n d o g l y c o s i d a s e s u s e d to g e n e r a t e d e g l y c o sylated proteins dgI and dgIV were chosen accordingly. Incubation with both endo F and N-glycanase, which remove N-linked carbohydrates, decreased the apparent M r o f g I f r o m 1 3 0 K / 7 4 K / 5 5 K to 1 0 3 K / 5 5 K / 5 0 K (Fig. 1). B e c a u s e t h e size o f t h e d e g l y c o s y l a t e d s p e c i e s c o r r e s p o n d e d w e l l w i t h t h e a p p a r e n t M r o f 1 0 5 K r e p o r t e d for t h e p o l y p e p t i d e b a c k b o n e o f gI ( v a n D r u n e n L i t t e l - v a n d e n H u r k & B a b i u k , 1986b), t h i s s u g g e s t s c o m p l e t e r e m o v a l o f c a r b o h y d r a t e f r o m gI. A f t e r s e q u e n t i a l t r e a t m e n t w i t h e n d o F or N - g l y c a n a s e , n e u r a m i n i d a s e and O-glycanase, which remove O-linked oligosacc h a r i d e s , t h e a p p a r e n t M r o f g I V d e c r e a s e d f r o m 7 1 K to 6 0 K a n d a c c o r d i n g l y t h e size o f t h e d i r n e r c h a n g e d f r o m 1 4 0 K to 120K (Fig. 1). T h i s is c o n s i s t e n t w i t h t h e r e m o v a l o f v i r t u a l l y all c a r b o h y d r a t e s as the p o l y p e p t i d e b a c k b o n e o f g I V has a n a p p a r e n t M r o f 5 8 K ( v a n D r u n e n L i t t e l - v a n d e n H u r k & B a b i u k , 1986b). (a) i :i ~ .5 :i: (b) Competitive antibody b&ding assay (CBA). The CBA was based on the ELISA modified as previously described (van Drunen Littel-van den Hurk et al., 1985). The percentage of competition was calculated using the formula 100 x (A - B)/A, where A is absorbance in the absence of competitor antibody and B is absorbance in the presence of competitor monospecific antibody, a modification of the formula described by Kimura-Kuroda & Yasui (1983). Delayed-type hypersensitivity (DTH) assay. Seven weeks after the second immunization the rabbits were inoculated subcutaneously with 1 ~g of homologous antigen (gI or gIV) in one ear and with 1 ~tg of heterologous antigen (gild in the other ear. The DTH response of the rabbits was followed by measuring ear thickness with a dial thickness gauge (Mitutoyo) at the time of inoculation and for 4 days afterwards. The increase in thickness of the test ear, inoculated with homologous antigen, was corrected for naturally occurring variation by subtracting increase or decrease in thickness of the control ear, inoculated with heterologous antigen, and expressed in ~tm. ::':: " ,;t i 00K,. .16KI~ 97K~" Serum neutralization (SN) test and antibody and complement (C') mediated (AbC) cytolysis. The neutralization titres of the rabbit sera and the ability of the rabbit sera to mediate complement-dependent cell lysis were determined as previously described (Babiuk & Rouse, 1975). The SN titres were expressed as the reciprocal of the highest serum dilution causing a 50% reduction of plaques relative to the virus control. Specific release was calculated as 100× (release by Ab + C" - release by control without Ab or C')/(total releasable s~Cr - release by control). 2055 66K~ 43Ki~ _ ...... (dimer) i/ '#': ~ .,g v .... .......... ._5: Fig. 1. Deglycosylation of gI and g]V. Affinity-purified glycoproteins gI (a) and gIV (b) were fractionated by SDS-PAGE (7.5%) before ( - ) and after (+) endoglycosidase treatment and visualized by staining with Coomassie blue. The positions of glycoproteins gIa, gIb, gIc, gIV and gIV dimer are indicated in the right margins. Mr shifts, occurring as a result of deglycosylation, are shown with arrows. Positions of Mr markers are indicated in the left margins. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 19:19:39 2056 S. van Drunen Littel-van den Hurk, G. Hughes and L. A. Babiuk Table 1. Reactivity of a panel of lectins with native and deglycosylated glycoproteins gI and glV ELISA* titre against Lectin Sugar specificity Concanavalin A Lens culinarbr agglutinin Pisum sativum agglutinin Ricinus communis agglutinin I Bandeiraea simplifolica agglutinin I Wheatgerm agglutinin Succinylated wheatgerm agglutinin Peanut agglutinin Soybean agglutinin Dolichus biflorus agglutinin Ulex europaeus agglutinin c~-Mant ~t-Man or-Man Gal, [GalNAc] ct-Gal GlucNAc, N A N A GlucNAc Gal(fll,3)-GalNAc ct,fl-GalNAc(terminal), [Gal] ct-GaINAc • -Fuc gI dgI gIV dgIV 7200 2400 2400 2400 1800 2400 < 50 150 75 <50 < 50 <50 <50 <50 <50 < 50 <50 < 50 <50 < 50 <50 < 50 3200 3200 3200 800 800 800 800 200 200 3200 800 <50 <50 <50 <50 < 50 <50 < 50 <50 < 50 <50 < 50 * ELISA titres were expressed as the reciprocal of the highest dilution that still gave a reading of at least 0.01. The titres against the unglycosylated control antigen, BHV-1 VP8, were ~<75. ~"See text for explanation of abbreviations. The removal of carbohydrate to yield the more rapidly migrating species after endoglycosidase treatment was verified by a lectin-binding assay in which the ability of a panel of lectins to bind to the native and deglycosylated proteins was assessed. Table 1 shows that lectins specific for ~-mannose (a-Man), galactose (Gal) and sialic acid (NANA) bound to gI, but not to dgI. Similarly, lectins specific for a-Man, ~-Gal, N-aeetylglucosamine (GlucNAc), ~-N-acetylgalactosamine (GalNAc) and ~-fucose (~-Fuc) bound to gIV, but not to dgIV. Binding of these lectins to dgI and dgIV was at a level comparable to that of binding to an unglycosylated control antigen, BHV-1 VP8, whereas their reactivity with gI and gIV was at least 10-fold higher. In a similar assay, the limit of antigen detection by this panel of lectins was determined. The lectins that were reactive in the previous assay (Table 1) detected as little as 3.1 to 12.5 ng of gI or gIV whereas they did not react with >~100 ng of dgI, dgIV or VP8. Effect of carbohydrate removal on the antibody response to gI and gIV In order to examine the role of carbohydrate in the humoral antiviral response, we tested the relative efficacy of gI and gIV and their deglycosylated counterparts to elicit an antibody response in rabbits. The sera of these rabbits were analysed for the presence of total antibody, virus-neutralizing antibody and antibody mediating lysis of BHV-l-infected cells. As shown in Fig. 2 (a), one injection of at least 1 gg of native or deglycosylated protein induced a detectable serum antibody titre as measured by ELISA. After the booster immunization 28 days later there was a further increase in the antibody levels. By contrast, a dose of 0.1 ~tg induced a detectable antibody response only after two immunizations, which was at least 100-fold lower than that obtained with two doses of at least 1 gg of immunogen. Fig. 2(a) also indicates that the total antibody response was significantly lower (P < 0.01) in rabbits immunized with deglycosylated proteins than in those vaccinated with native glycoproteins gI and gIV. The mean difference between the antibody responses to gI and dgI was 4.5-fold, ranging from two- to 10-fold depending on the concentration of immunogen. The difference between the gIV and dgIV antibody responses ranged from two- to 8-5-fold, with a mean of 3.5-fold. In the absence of complement, two doses of at least 1 gg of gI, gIV or dgIV were sufficient to induce reasonable serum neutralizing antibody titres, whereas two doses of 50 gg of dgI were required to obtain a detectable neutralizing antibody response (Fig. 2b). In the presence of guinea-pig complement the titres increased two- to fourfold. The neutralizing antibody response of the rabbits immunized with dgIV was fourfold (ranging from two- to 10-fold) lower than the response of the gIV-vaccinated animals, which is in good agreement with the difference in total antibody response. However, animals immunized with dgI had 10-fold (ranging from two- to 16-fold, P < 0.05) lower neutralizing antibody titres than gI-immunized animals, indicating that carbohydrate removal has a relatively strong impact on the neutralizing epitopes of gI. In Fig. 2(c) the percentage specific SlCr release as a result of AbC lysis of BHV-l-infected MDBK cells is shown. It is apparent that the gIV/dgIV-specific antibodies mediate cell lysis more efficiently than the gI/dgIspecific antibodies~ A similar pattern was observed for Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 19:19:39 Carbohydrate role in BHV-1 glycoproteins (a) I gI ~ I dgI I I I gIV I I dgIV 0'1 0-1 I - 5"0 1"0 50"0 (b) gl I 0"1 1"0 5"0 50"0 • I dgI l'0 I 5-0 50"0 glV I ~ I -- 3- 5-0 1"0 50"0 I dgIV I 2 1 (c) F 0-I neutralizing antibody titres between gI/dgI- and gIV/ dglV-specific sera (Fig. 2b). The capacity to lyse virusinfected cells was fivefold (ranging from two- to 12-fold) lower in dgI-specific sera than in gI-specific sera, which appeared to be a significant difference (P < 0.02) and is in accordance with the observed reduction in total antibody titre following deglycosylation. T h e ability to mediate cell lysis was reduced threefold (ranging from 1.5- to fourfold) in dglV-specific sera as c o m p a r e d to glVspecific sera, which corresponds to the decrease in total and neutralizing antibody titres. I 0"1 0 2057 5-0 1'0 50"0 I' 0-I 5-0 l '0 50.0 I I dgI I P, 0.1 1.0 5-0 50-0 I glV I 0.I 1"0 5-0 50"0 I dglV 40 :.1I 30 20 •~ 10 Analysis of epitopes affected by deglycosylation Previously, we developed a panel o f monoclonal antibodies specific for seven different epitopes on gI and eight different epitopes on glV (van D r u n e n Littel-van den H u r k et al., 1985; H u g h e s et al., 1988). The properties of these monoclonal antibodies are s u m m a r ized in Table 2. In order to analyse the influence o f carbohydrate on the i m m u n o g e n i c i t y o f specific epitopes on glycoproteins gI and glV, the reactivity o f the rabbit sera with these epitopes was determined in CBAs. Both gI- and dgI-specific rabbit antibodies recognized epitopes I, II, I I I and V, as they both c o m p e t e d successfully and at the same level with monoclonal a n t i b o d y conjugates 1B10, 3F3, 1 E l l and 1F10, respectively. However, dgI-specific antibodies c o m p e t e d with monoclonal antibodies 1F8, 5G2, 3 G l l and 6 G l l at m u c h lower levels than gI-specific antibodies, indicating that the i m m u n o g e n i c i t y o f epitopes IVa, I V b and IVc, respectively, is dependent u p o n the presence o f carbohydrate (Fig. 3). N o difference between the m a x i m u m level o f competition reached by the glV- and dglV-specific rabbit antibodies was observed with any of the monoclonal antibodies, indicating that none o f the epitopes which have been defined to date, Ia, Ib, II, I I I a , I I I b , I I I c , I I I d and IV, are c a r b o h y d r a t e - d e p e n d e n t (Fig. 4). Effect of deglycosylation on the antigenicity of gI and glV 0.1 1-0 5.0 50-0 0q 1-0 5-0 50"0 . 0-1 1"0 5"0 50.0 0-I 1"0 5-0 50.0 Concentration of immunogen (p.g) Fig. 2. Antibody responses to glycoproteins gI and glV before and after deglycosylation. Rabbits were immunized with increasing concentrations of (glyco)protein and boosted 4 weeks later. They were bled 3 weeks after each immunization. (a) The total antibody response was determined in an ELISA using gl or glV as the antigen. Lower (white) bars show the ELISA titres after the first immunization and the upper (shaded) bars those after the second immunization. The ELISA titres were expressed as the highest dilution that still gave a reading of 0-1 at 450 nm. The difference between gI and dgI-specific antibody titres, as well as between glV- and dglV-specific antibody titres was significant (P < 0-01; analysis of variance analysis). (b) The serum The ability o f specific monoclonal antibodies to bind to deglycosylated gI and gIV was analysed in an E L I S A , in neutralizing antibody titres were determined for the second bleed in the absence (lower, white bars) and presence (shaded bars) of guinea-pig complement. The titres were expressed as a 50~ endpoint using 100 p.f.u, of BHV-1. The difference between gI- and dgI-specific antibody titres was significant (P < 0.05; paired t-test). (c) The AbC lysis was determined at a 1: 10 dilution of the second bleed and a 1 : 40 dilution of guinea-pig complement and expressed as percentage specific s~Cr release. The difference in percentage specific 51Cr release between gIand dgI-specific sera was significant (P < 0.02; paired t-test). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 19:19:39 2058 S. van Drunen Littel-van den Hurk, G. Hughes and L. A. Babiuk l 80 ' ~ ~ ~ i I r 3F3 1B10 ~ " 60 q 40c T a b l e 2. Properties o f monoclonal antibodies to glycoproteins g I and g l V Antibody designation Antigen specificy* Epitope specificityt Isotype IB10 gI I II III IVa IVb IVb IVc IVc V V IgG1 IgG2b IgG2a IgG2a IgG2b IgG2a IgG1 IgM IgG2a IgG2a Ia Ib II IIIa IIIb Illc IIId IV lgG 1 IgG1 IgG 1 IgG 1 IgG1 lgG1 IgG1 IgG1 3F3 20 I 80 I I I IF8 1El 1 1E 11 1F8 5G2 3G 11 5G11 6G 11 1F10 2C5 136 glV 9D6 3E7 60~ 10C2 4C1 2C8 3C1 3D9S q --. 20 O 3GI1 5G2 ~o 80 7 Conformation dependence - + + + + - + + + - * Antigen specificities were determined by imunoprecipitation, immunoblot analysis and ELISA (van Drunen Littel-van den Hurk et al., 1984; Hughes et al., 1988). ~-Epitope specificities were determined by competition binding assays (van Drunen Littel-van den Hurk et al., 1985; Hughes et al., 1988) and the epitopes of gI have been mapped using deletion mutants (Fitzpatrick et al., 1990). Table 3. Reactivity of gI-specific monoclonal antibodies with 60 native and deglycosylated g I 40 Antibody designation ELISA* titre against 20 i 6Gll 80 1FI0 ]- 60 Epitope specificityt 1B10 3F3 1Ell 1F8 5G2 3Gll 5Gll 6Gll 1F10 2C5 I II III IVa IVb IVb IVc IVc V V gI 2.5 x 1-6 x 4x 1.6 × 1.6 × 1× 4x 1.6 × 4x 1.6 x dgI 104 106 105 106 106 105 105 106 105 106 4x 2.5 x 1.6 × 1x 6-4 × 4x 1x 1.6 × 1-6 x 4x 102 104 106 102 103 102 l0 s 103 106 105 * ELISA titres were expressed as the reciprocal of the highest dilution that still gave a reading of at least 0.1. The titre of gI-specific rabbit serum was 3 x 105 both against gI and dgI. t Epitope specificity of the monoclonal antibodies was determined using competition binding assays (van Drunen Littel-van den Hurk et al., 1985). 40 20 pn 0 1 2 3 4 o 1 2 3 Dilution of competitor (log~0) 4 Fig. 3. Competition binding assays of sera from rabbits immunized with 50 ~tg of gl (C)) or with 50 Ixg of dgI ( 0 ) with HRPO-conjugated gI-specific monoclonal antibodies 1B10 (epitope I), 3Iz3 (epitope II) 1Ell (epitope III), 1F8 (epitope IVa), 5G2 (epitope IVb), 3 G l l (epitope IVb), 6 G l l (epitope IVc) and 1F10 (epitope V). an attempt to identify changes in antigenicity induced by carbohydrate removal. Table 3 shows that binding of monoclonal antibodies 1El 1 (epitope III), 1F10 and 2C5 (epitope V) to gI is independent of or perhaps even slightly enhanced after deglycosylation. In contrast, removal of carbohydrates resulted in strongly reduced binding of monoclonal antibodies 1F8 (epitope IVa), Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 19:19:39 Carbohydrate role in BHV-1 glycoproteins 1B10 +0 80 1Ell 2059 1F8 136 9D6 - ~ 200K 60 gla I~ pgla ~" ~'l16K "~97K 20 glb I ~ "~ 66K 80 10C2 3E7 glc 60 '~ 43K 40 20 g + 80 • 60 :• ::•• ". Z:• •!.:%+£¸:• i•::? •: .... Fig. 5. Immunoblot analysis of gI-specific monoclonal antibodies. Affinity-purified gI was electrophoretically separated into gla, pgla, glb and glc, transferred to nitrocellulose and incubated with monoclonal antibodies 1B10, 1Eli and IFS. Monoclonal antibodies 3F3 and 1F10 reacted like 1Ell, whereas 5G2, 3 G l l , 5 G l l and 6GI1 reacted like 1F8. Positions of M~ markers are shown in the right margin. 40 20 80 60 40 20 0 1 2 3 4 0 1 2 3 Dilution of competitor (log~0) 4 Fig. 4. Competition binding assays of sera from rabbits immunized wtih 50 lag of glV (O) or with 50 lag of dglV ( 0 ) with HRPOconjugated glV-specific monoclonal antibodies 136 (epitope Ia), 9D6 (epitope Ib), 3E7 (epitope II), 10C2 (epitope Ilia), 4C1 (epitope IIIb), 2C8 (epitope IIIc), 3C1 (epitope IIId) and 3D9S (epitope IV). 5G2, 3G11 (epitope IVb), 5G11 and 6G11 (epitope IVc), which is in agreement with the reduced ability of epitopes IVa, IVb and IVc to induce an immune response in rabbits. Monoclonal antibodies IB10 (epitope I) and 3F3 (epitope II) bound with slightly reduced efficiency to the deglycosylated protein as compared to the native glycoprotein, which, however, was not reflected in their capacity to induce an antibody response in rabbits. In order to investigate the extent of carbohydrate dependence, the reactivity of gI-specific monoclonal antibodies with gIa and pgla (high-mannose intermediate form of gI) was determined in a Western blot assay. Fig. 5 shows that monoclonat antibodies 1B 10 and 1Ell reacted with pgIa, whereas monoclonal antibody 1F8 did not recognize the high-mannose intermediate Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 19:19:39 2060 S. van Drunen Littel-van den Hurk, G. Hughes and L. A. Babiuk 250 t (a) (b) I ~200 :~ 15o~ 100 50 0 1 2 3 4 0 1 2 3 4 Days post-challenge Fig. 6. DTH responses to glycoproteins gI (a) and glV (b) before and after deglycosylation. Rabbits that had been primed with 5 ~tg gI (11), dgI ([~), gIV (11) or dgIV ([~) were injected with 1 gg of homologous antigen (gI or gIV) in one ear and with 1 gg of heterologous antigen (gIII) in the other ear. The DTH response was determined by measuring ear thickness before and after inoculation. The homologous response was corrected for variation in ear thickness by subtracting the heterologous response and expressed as increase in ear thickness in gin. The difference in increase in ear thickness between gIV- and dgIVimmunized rabbits was significant ( P < 0.05; analysis of variance analysis). precursor. Monoclonal antibodies 3F3, 1F10 and 2C5 showed the same reactivity pattern as 1Ell, whereas monoclonal antibodies 5G2, 3Gll, 5Gll and 6 G l l reacted like 1F8. This not only confirms the data from the ELISA, but also suggests that the carbohydrates in epitopes IVa, IVb and IVc need to be processed for maximum antigenicity and immunogenicity. No changes in antigenicity were observed after carbohydrate removal from gIV (ELISA data not shown), which again suggests that epitopes Ia, Ib, II, IIIa to d and IV are carbohydrate-independent. The role of carbohydrate in the D T H response The effect of carbohydrate removal on the cell-mediated immune response was determined by measuring the DTH response following ear challenge in rabbits that had been immunized with 5 gg of native or deglycosylated glycoprotein. Fig. 6 indicates that the DTH response was 1-5-fold higher in animals immunized with dgI than in animals that received gI. The difference in DTH response between gIV- and dgIV-immunized animals was twofold, which appeared to be significant (P < 0.05). Discussion It has been shown for a number of viruses that the carbohydrate moiety of the viral glycoproteins can have a strong influence on functional as well as antigenic activities (Basak & Compans, 1983; Lambert, 1988; Alexander & Elder, 1984; Elder et al., 1986). Since the major glycoproteins of BHV-1 are heavily glycosylated (van Drunen Littel-van den Hurk & Babiuk, 1985a, 1986b), we were interested in exploring the contribution of the carbohydrate portion of these glycoproteins in the induction of an immune response to BHV-1, both at the humoral and at the cellular level. Following treatment of gI and gIV with the appropriate endoglycosidases, the Mr values of the resulting deglycosylated proteins corresponded well with those previously determined (van Drunen Littel-van den Hurk & Babiuk, 1986b) for the polypeptide backbones of these glycoproteins, indicating that complete or virtually complete deglycosylation had been achieved. This was confirmed by the absence of any reactivity of the deglycosylated proteins with a series of lectins, which did show a strong binding affinity for native gI and gIV. The humoral immune response to the deglycosylated proteins appeared to be significantly lower than that against the native glycoproteins, suggesting that the immunogenicity of at least some epitopes on gI and gIV is carbohydrate-dependent. However, in the case of gIV, the neutralizing antibody response and the ability of the antibodies to mediate cell lysis were not significantly reduced, indicating that most functional epitopes on this glycoprotein were unaffected by deglycosylation. In the case of gI, the capacity to mediate cell lysis and the neutralizing antibody response was significantly reduced by loss of carbohydrate. Consequently, the immunogenicity of the neutralizing epitopes on gI appeared to be preferably affected by deglycosylation, or the response to the most strongly neutralizing epitopes may have been modified. Alternatively, the decreased neutralizing capacity of antibodies to dgI may be due to the induction of a different immunoglobulin subclass by the deglycosylated protein. This possibility is presently under investigation. Seven different epitopes on gI, as well as eight epitopes on glV, have been mapped in our laboratory, using two panels of monoclonal antibodies in competition binding assays (van Drunen Littel-van den Hurk et al., 1985; Hughes et al., 1988). However, the decreased induction of glV-specific antibodies after carbohydrate removal from this glycoprotein could not be attributed to any of these epitopes. The reactivity of monoclonal antibodies to these epitopes also remained unchanged after deglycosylation of gIV, suggesting that both the antigenic and the immunogenic structure of the eight epitopes on gIV, seven of which are neutralizing, is carbohydrateindependent. In contrast, three of the carbohydrate-dependent epitopes on gI were shown to correspond to neutralizing Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 19:19:39 Carbohydrate role in B H V - 1 glycoproteins epitopes IVa, IVb and IVc (van Drunen Littel-van den Hurk et al., 1985). Two of these epitopes, IVa and IVb, are also involved in infected cell lysis. Epitope IVc either does not mediate cell lysis, or the IVc-specific monoclonal antibodies 5Gll and 6GI1 are of the wrong immunoglobulin subclass to mediate lysis activity. Monoclonal antibodies to these epitopes also exhibited reduced binding to gI after carbohydrate removal, indicating that both antigenicity and immunogenicity of epitopes IVa, IVb and IVc are carbohydrate-dependent, in contrast with the carbohydrate-independence of epitopes I, II, III and V. Epitope IV has been mapped to the amino terminus of gI between residues 68 and 119 (Fitzpatrick et al., 1990). This region contains a potential N-linked glycosylation site at position 105 of the gI sequence, further suggesting carbohydrate dependence of this epitope. As these epitopes appear to be continuous, as is evident from their reactivity in a Western blot assay under denaturing conditions, the carbohydrate is probably involved as a component of the epitopes, rather than in stabilization of this glycoprotein. This is supported by the observation that the carbohydrate side-chains of these epitopes have to be processed in order to be fully recognized by the monoclonal antibodies specific for epitopes IVa, IVb and IVc. Alternatively, these epitopes may seem to be continuous because they are protected from complete denaturation by the presence of the carbohydrates. In this case, protection is only completely achieved by the processed oligosaccharides. Removal or partial absence of carbohydrates would then result in loss of protection, as well as of antigenic and immunogenic structure. In contrast to the humoral immune response, the cellmediated immune response, as measured by a DTH reaction, was higher in rabbits immunized with deglycosylated proteins than in those inoculated with native glycoproteins gI and gIV. This indicates that the carbohydrates may mask the induction or recognition of certain T cell epitopes, particularly on glV, as the DTH response between gIV and dglV immunized animals was significantly different. The presence of carbohydrate can influence the antigenicity and immunogenicity of viral glycoproteins in a number of ways. The immunological reactivity of avian myoblastosis virus gp85 appears to depend on an intact carbohydrate side-chain (van Eldik et al., 1978). In addition, the reactivity of polyclonal and monoclonal antibodies to the influenza virus haemagglutinin and the gp70 of Rauscher murine leukaemia virus was dramatically decreased following endo F treatment (Alexander & Elder, 1984). In these cases, carbohydrate may either be directly involved as part of an epitope or it may stabilize a certain conformation of the protein. However, the carbohydrate on gp70 may not be needed to elicit a 2061 neutralizing antibody response and in fact may even protect the virus from immune surveillance during infection (Elder et al., 1986). Similar observations were reported for the gp51 of bovine leukaemia virus (Portetelle et al., 1980, Bruck et al., 1982). By contrast, the reactivity of polyclonal and monoclonal antibodies defining eight epitopes on the E protein of a flavivirus was not affected by endo F treatment of this glycoprotein (Winkler et al., 1987). In the case of BHV-1, the effect of carbohydrate was studied at both the humoral and the cellular level of the immune response. Our data suggest that the carbohydrate moieties of glycoproteins gI and glV do play a role in the host's immune recognition and response. Deglycosylation of these glycoproteins resulted in a decreased ability to elicit antibodies involved in virus neutralization and killing of virus-infected cells and an increased capacity to induce a DTH response in rabbits. These results indicate that the carbohydrates on gI and glV may play a dual role in the host's immune recognition and response to these glycoproteins by contributing to certain epitopes required for the B cell response, but masking other epitopes involved in T cell recognition. In the mouse system, two types of helper T cell clones have been identified (Mossmann et al., 1986; Kurt-Jones et al., 1987). The first type of helper T cell, designated TH1, produces interferon 7 and interleukin-2 (IL-2), which stimulate growth of T cells, and the second type, TH2, synthesizes IL-4 and IL-5, which stimulate B cell growth and antibody responses. The potential existence of such types of helper cells in the rabbit system would explain the different effects of deglycosylation of gI and glV on the antibody and DTH responses. TH1 analogues might recognize epitopes that are masked by the carbohydrates, whereas TH2 analogues might recognize epitopes in conjunction with carbohydrates. Both glycoproteins gI and glV are good candidates for a subunit vaccine to BHV-1 (Babiuk et al., 1987). In order to be produced efficiently they will have to be expressed in a system employing a vector such as vaccinia virus, baculovirus or others. We have shown that vaccinia virus recombinant glycoproteins and authentic BHV-1 glycoproteins are glycosylated in a very similar manner (van Drunen Littel-van den Hurk et al., 1989). However, glycoproteins expressed in a baculovirus system have been reported to lack terminal carbohydrates (Luckow & Summers, 1988). The observation that the carbohydrates may be needed as part of at least three neutralizing epitopes on gI, whereas most neutralizing epitopes of glV appear to be carbohydrateindependent, may have important implications for choosing appropriate expression systems for the production of these glycoproteins. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 19:19:39 2062 S. van Drunen Littel-van den Hurk, G. Hughes and L. A. Babiuk We thank Melanie Elliott and Terry Beskorwayne for their technical assistance, the animal support staff at VIDO for care and handling of the animals, Richard Harland for assistance with the statistical analyses, and Marilee Hagen for typing his manuscript. This work was supported by grants from the Medical Research Council and the National Science and Engineering Council of Canada, Farming for the Future and the Saskatchewan Agricultural Research Foundation. References ALEXANDER, S. & ELDER, J. H. (1984). Carbohydrate dramatically influences immune reactivity of antisera to viral glycoprotein antigens. Science 226, 1328-1330. BABIUK, L. A. & ROUSE, B. T. (1975). Defence mechanisms again_st bovine herpesvirus : relationships of virus-host cell events to susceptibility to antibody-complement lysis. Infection and Immunity 12, 958-963. BABIUK,L. A., L'ITALIEN,J., VANDRUNENLITTEL-VANDEN HURK, S., ZAMB,T., LAWMAN,M. J. P., HUGHES,G. & GIFFORD, G. A. (1987). Protection of cattle from bovine herpesvirus type-1 (BVH-1) infection by immunization with individual viral glycoproteins. Virology 159, 57-66. BASAK,S. & COMPANS,R. W. (1983). Studies on the role of glycosylation in the functions and antigenic properties of influenza virus glycoproteins. Virology 128, 77-91. BERGER, E. G., BUDDECKE, E., KAMERLING, J. P., KOBATA, A., PAULSON, J. C. & VLIEGENTHART, J. F. G. (1982). Structure, biosynthesis and functions of glycoprotein glycans. Experientia 38, 1129-1258. BISHOP, G. A., MARLIN, S. D., SCHWARTZ,S. A. & GLORIOSO,J. C. (1984). Human natural killer cell recognition of herpes simplex virus type-1 glycoproteins: specificity analysis with the use of monoclonal antibodies and antigenic variants. Journal of Immunology 133, 22062214. BRUCK, C., PORTETELLE, D., BURNY, A. & ZAVADA, J. (t982). Topographical analysis by monoclonal antibodies of BLV-gp 51 epitopes involved in viral functions. Virology 122, 353-362. BURNETTE,W. N. (1981). "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analytical Biochemistry 112, 195-203. CARTER, V. C., SCHAFFER, P. A. & TEVETHIA, S. S. (1981). The involvement of herpes simplex type-1 glycoproteins in cell-mediated immunity. Journal of Immunology 126, 1655-1660. COLLINS,J. K., BUTCHER,A. C., RIEGEL,C. A., MCCRANE,V., BLAIR, C. D., TERAMOTO, Y. A. & WINSTON, S. (1984). Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1. Journal of Virology 52, 403-409. COLLINS, J. K., BUTCHER, A. C. & RIEGEL, C. A. (1985). Immune response to bovine herpesvirus type 1 infections: virus-specific antibodies in sera from infected animals. Journal of Clinical Microbiology 21, 546-552. ELDER, J. H., MCGEE, J. S. & ALEXANDER,S. (1986). Carbohydrate side chains of Rauscher leukemia virus envelope glycoproteins are not required to elicit a neutralizing antibody response. Journal of Virology 57, 340-342. FITZPATRICK, D. R., REDMOND, M. J., ATTAH-POKU, S. K., VAN DRUNEN LITTEL-VANDEN HURK, S., ZAMB, T. J. & BABIUK,L. A. (1990). Mapping of ten epitopes on bovine herpesvirus type-1 glycoproteins gI and gIII. Virology 175, 145-157. FRIEDMAN,H. M., COHEN, G. H., EISENBERG,R. J., SEIDEL, C. A. & CINES, D. B. (1984). Glycoprotein C of herpes simplex virus type-I acts as a receptor for the C3b complement component on infected cells. Nature, London 309, 633-635. FULLER, A. O. & SPEAR, P. G. (1985). Specificities of monoclonal and polyclonal antibodies that inhibit adsorption of herpes simplex virus to cells and lack of inhibition by potent neutralizing antibodies. Journal of Virology 55, 475-482. FULLER, A. O. & SPEAR, P. G. (1987). Anti-glycoprotein D antibodies that permit adsorption but block infection by herpes simplex virus 1 prevent virion-cetl fusion at the cell surface. Proceedings of the National Academy of Sciences, U.S.A. 84, 5454-5458. GIBBS, E. P. J. & RWEYEMAMU,M. M. (1977). Bovine herpesviruses I. Bovine herpesvirus-1. Veterinary Bulletin, London 47, 317-343. GLORIOSO,J., SCHRt)DER,C. H., KUMEL,G., SZCZESIUL,M. & LEVINE, M. (1984). Immunogenicity of herpes simplex virus glycoproteins gC and gB and their role in protective immunity. Journal of Virology 50, 805 812. HUGHES, G., BABIUK,L. A. & VAN DRUNENL!TTEL-VANDEN HURK, S. (1988). Functional and topographical analyses of epitopes on bovine herpesvirus type-I glycoprotein glV. Archivesof Virology 103, 47-60. ISRAEL,B., MARSHALL,R. L. & LETCHWORTH,G. J., III (1988). Epitope specificity and protective etficacy of the bovine immune response to bovine herpesvirus-1 glycoprotein vaccines. Vaccine 6, 349-356. JOHNSON,D. C. & FEENSTRA,V. (1987). Identification of a novel herpes simplex type-1 glycoprotein which complexes with gE and binds immunoglobulin Journal of Virology 61, 2208-2216. KIMURA-KURODA,J. & YASUI, K. (1983). Topographical analysis of antigenic determinants on envelope glycoprotein V3(E) of Japanese encephalitis virus, using monoclonal antibodies. Journal of Virology 45, 124-132. KURT-JONES,E. A., HAMBERG,S., OHARA,J., PAUL, W. E. & ABBAS, A. K., (1987). Heterogeneity of helper/inducer T lymphocytes. Journal of Experimental Medicine 166, 1774-1787. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680-685. LAMBERT,D. M. (1988). Role of oligosaccharides in the structure and function of respiratory syncytial virus glycoproteins. Virology 164, 458-466. LITTLE, S. P., JOFRE,J. T., COURTNEY,R. J. & SCHAFFER,P. A. (1981). A virion-associated glycoprotein essential for infectivity of herpes simplex virus type-1. Virology 115, 149-160. LUCKOW,V. A. & SUMMERS,M. D. (1988). Trends in the development of baculovirus expression vectors. Bio/Technology 6, 47-55. MANSERVIGt, R., SPEhR, P. G. & BUCHAN, A. (1977). Cell fusion induced by herpes simplex virus is promoted and suppressed by different viral glycoproteins. Proceedingsof the National Academy of Sciences, U.S.A. 74, 3913-3917. MARSHALL,R. L., RODRIGUEZ,L. L. & LETCHWORTH,G. J., III (1986). Characterization of envelope proteins of infectious bovine rhinotracheitis virus (bovine herpesvirus l) by biochemical and immunological methods. Journal of Virology 57, 745-753. MARSHALL, R. L., ISRAEL, B. A. & LETCHWORTH,G. J., III (1988). Monoclonal antibody analysis of bovine herpesvirus-1 glycoprotein antigenic areas relevant to natural infection. Virology 165, 338 347. MOSSMANN,T. R., CHERWlNSKI,H., BOND, M. W., GEIDLIN, M. A. & COFFMAN,R. A. (1986). Two types of murine helper T cell clones. I. Definition according to profiles of lymphokine activities and secreted proteins. Journal of Immunology 136, 2348. NOBLE, A. G., LEE, G. T.-Y., SPRAGUE,R., PARISH, M. L. & SPEAR, P. G. (1983). Anti-gD monoclonal antibodies inhibit cell fusion induced by herpes simplex virus type-1. Virology 129, 218-224. NORRILD, B., SHORE,S. L., CROMEANS,T. L. & NAHMIAS,A. J. (1980). Participation of three major glycoprotein antigens of herpes simplex virus type-1 early in the infectious cycle as determined by antibodydependent cell-mediated cytotoxicity. Infection and Immunity 28, 38-44. OKASAKI, K., HONDA, E., MINETOMA, T. & KUMAGAI, T. (1986). Mechanisms of neutralization by monoclonal antibodies to different antigenic sites on the bovine herpesvirus type-1 glycoproteins. Virology" 150, 260-264. PORTETELLE,D., BRUCK,C., MAMMERICKX,M. & BURNY,A. (1980). In animals infected by bovine leukemia virus (BLV) antibodies to envelope glycoprotein gp51 are directed against the carbohydrate moiety. Virology 105, 223-233. ROUSE, B. R. & BABIUK, L. A. (1974). Host responses to infections bovine rhinotracheitis. III. Isolation and immunological activities of bovine T lymphocytes. Journal of Immunology 113, 1391-1398. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 19:19:39 Carbohydrate role in BHV-1 glycoproteins RousE, B. R. & BABIUK, L A. (1978). Mechanisms of recovery from herpesvirus infections. Canadian Journal of Comparative Medicine 42, 414-427. ROUSE, B. R. & HOROHOV, D. W. (1984). Cytotoxic T lymphocytes in herpesvirus infections. Veterinary Immunology and Immunopathology 6, 35-66. SARMIENTO, M., HAFFEY, M. & SPEAR, P. G. (1979). Membrane proteins specified by herpes simplex virus III. Role of glycoprotein VP7 (B2) in virion infectivity. Journal of Virology 29, 1149 1158. SHARON, N. & LIS, H. (1982). Glycoproteins: research booming on long-ignored ubiquitous components. Chemicaland Engineering News 59, 21-44. TREPANIER, P., MINOCHA, H. C., BASTIEN,Y., NADON, F., SEGUIN, C., LUSSlER, G. & TRUDEL, M. (1986). Bovine herpesvirus-l: strain comparison of polypeptides and identification of a neutralizing epitope on the 90-kilodalton hemagglutinin. Journal of Virology 60, 302-306. VAN DRUNEN LITTEE-VAN DEN HURK, S. & BABIUK, L. A. (1985a). Effect of tunicamycin and monensin on biosynthesis, transport and maturation of bovine herpesvirus type-1 gtycoproteins. Virology 143, 104-ii8. VAN DRUNEN LITTEL-VAN DEN HURK, S. & BABIUK, L. A. (1985b). Antigenic and immunogenic characteristics of bovine herpesvirus type-1 glycoproteins GVP 3/9 and GVP 6/11a/16, purified by immunoadsorbent chromatography. Virology 144, 204 215. VAN DRUNEN LITTEL-VAN DEN HURK, S. & BABIUK, L. A. (1986a). Polypeptide specificity of the antibody response after primary and recurrent infection with bovine herpesvirus-1. Journal of Clinical Microbiology 23, 274 282. VAN DRUNEN LITTEL-VAN DEN HURK, S. & BABIUK, L. A. (1986b). Synthesis and processing of bovine herpesvirus-1 glycoproteins. Journal of Virology 59, 401-410. 2063 VAN DRUNEN LITTEL-VAN DEN HURK, S., VAN DEN HURK, J. V., GILCHRIST, J. E., MISRA, V. & BABIUK,L. A. (1984). Interactions of monoclonal antibodies and bovine herpesvirus type-1 (BHV-1) glycoproteins: characterization of their biochemical and immunological properties. Virology 135, 466-479. VAN DRUNEN LITTEL-VAN DEN HURK, S., VAN DEN HURK, J. V. & BABIUK, L. A. (1985). Topographical analysis of bovine herpesvirus1 glycoproteins: use of monoclonal antibodies to identify and characterize functional epitopes. Virology 144, 216-227. VAN DRUNEN LITTEL-VAN DEN HURK, S., ZAMB, T. • BABIUK, L. A. (1989). Synthesis, cellular location and immunogenicity of bovine herpesvirus-1 glycoproteins gI and gIV expressed by recombinant vaccina virus. Journal of Virology 63, 2159 2168. VANELDIK, L. J., PAULSON,J. C., GREEN, R. W. & SMITH,R. E. (1978). The influence of carbohydrate on the antigenicity of the envelope glycoprotein of avian myeloblastosis virus and B77 avian sarcoma virus. Virology 86, 193-204. VESTERGAARO, B. F. & NORRILD, B. (1979). Crossed immunoelectrophoretic analysis and viral neutralizing activity of five monospecific antisera against five different herpes simplex virus glycoproteins. International Agency Research Cancer Science Publications 24, 225234. WmTBECK, J. C., BELLO, L. J. & LAWRENCE, W. C. (1988). Comparison of the bovine herpesvirus gI gene and the herpes simplex virus type-1 gB gene. Journal of Virology 62, 3319-3327. WINKLER, G., HEINZ, F. & KUNZ, C. (1987). Studies on the glycosylation of flavivirus E. proteins and the role of carbohydrate in antigenic structure. Virology 159, 237 243. (Received 28 November 1989; Accepted 23 April 1990) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 19:19:39