Download The role of carbohydrate in the antigenic and immunogenic structure

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
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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
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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.
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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
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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).
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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),
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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
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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
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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.
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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.
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(Received 28 November 1989; Accepted 23 April 1990)
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