AC3 La Sota/octylglucoside IgGl. +. +. +. +. HD5 La Sota/octylglucoside IgG2. +. +. +. +. JB6 La Sota/octylglucoside IgGl. +. ±. ±. +. 4A4. La Sota. IgG2A +. +. +. +.
JOURNAL OF VIROLOGY, Mar. 1986, p. 1198-1202 0022-538X/86/031198-05$02.00/0 Copyright © 1986, American Society for Microbiology
Vol. 57, No. 3
Monoclonal Antibodies to Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Newcastle Disease Virus: Relationship between Glycosylation and Reactivity GHYSDAEL,lt MARTINE GONZE,' ARSENE BURNY,"2 AND GUY MEULEMANS3*
LE LONG,' DANIEL PORTETELLE,12 JACQUES
Department of Molecular Biology, University of Brussels, 1640, Rhode-St-Genese,' Faculty of Agronomy, 5800 Gembloux,2 and National Institute for Veterinary Research, 1180 Uccle,3 Belgium Received 20 May 1985/Accepted 6 September 1985
Eighteen hybridoma lines obtained by immunization of mice with Newcastle disease virus (NDV) lentogenic strain La Sota or velogenic strain Italien produced hemagglutinating monoclonal antibodies. The 18 monoclones were divided into four groups according to their reactivity toward native hemagglutiniin neuraminidase protein (HN), nonglycosylated HN precursor, and heat-denatured HN blotted on nitrocellulose membranes. Only group II reagents were reactive toward their targets in all conditions tested. They were considered sequence-specific antibodies. Group I antibodies did not require glycosylation but lacked reactivity towards the denatured glycosylated antigen. Monoclonal antibodies from group III recognized only the native HN. Group IV was made up of a single monoclone that lacked reactivity with NDV Italien but recognized the La Sota strain in hemagglutination inhibition and enzym,e-linked immunosorbent assays. Five hybridoma lines produced monoclonal antibodies which neutralized viral infectivity but failed to inhibit hemagglutination. One monoclonal antibody obtained after immunization of mice with NDV La Sota showed a low neutralization index versus NDV Italien. Four monoclonal antibodies derived from mice immunized with NDV Italien showed higher neutralization indices towards this strain. Neither the denatured F protein nor its nonglycosylated precursor was reacted against by the five monoclonal antibodies.
ically conserved and other regions subject to changes (15). These observations support the generally held opinion that all NDV strains and isolates form an antigenically homologous group (19) and, at the same time, account for variability among strains and support the idea that differences in pathogenicity could correspond to differences in at least some antigenic determinants. The biological significance of neuraminidase activity remains uncertain, as a second-step revertant of a temperature-sensitive mutant of NDV displayed a slight advantage over the wild-type virus during a single reproductive cycle, despite a small disadvantage in attachment and a major defect in elution (21). Besides the number of glycoproteins inserted into the hydrophobic matrix M, virus infectivity is conditioned by the glycosylation of these glycoproteins. Even if unglycosylated forms of the glycoproteins were found to be relatively stable in infected cells (13), a 30-fold decrease of infectivity was observed in the systems in which virions could be assembled with nonglycosylated glycoproteins (1). In this study, we produced 18 anti-HN monoclonal antibodies and classified them into four differently reactive groups. Five monoclonal antibodies with virus neutralization activity were also obtained. They reacted only with native F glycoprotein. All anti-F and 7 of 18 anti-HN antibodies (group III) were unable to precipitate the unglycosylated, precursor of the target glycoprotein. Among the anti-HN antibodies that did not require glycosylation of the antigen, eight (group II) reacted with denatured HN blotted on nitrocellulose membranes; two others (group I) were devoid of reactivity in these conditions. Viruses. The virus strain Italien used in this study was obtained from W. H. Allan, International Reference Laboratory, Weybridge, United Kingdom. The La Sota strain was
Newcastle disease virus (NDV) belongs to the paramyxovirus group of enveloped, negative-stranded RNA viruses. NDV virions are made of three envelope and three core proteins (2-4). Envelope proteins include a large glycoprotein with both hemagglutinin and neuraminidase activities (HN), a smaller glycoprotein with cell-fusing activity (F), and a nonglycosylated protein (M) localized at the inner surface of the envelope. F is synthesized as a precursor, Fo that after cleavage yields F, and F2 components, which are held together by disulfide bonds. Generation of F, and F2 is required for F function in cell-to-cell fusion, hemolysis, and virus penetration (7, 12, 14, 16) and for fusion of the virus with liposomes (8). The active site of protein F for membrane-fusing activity is the amino acid sequence at the N-terminal region of F1. Synthetic oligopeptides with the same amino acid sequence react with about 3.0 x 106 receptor sites
on
the cell membrane and inhibit membrane-
fusing activity by competing with the F, polypeptide for such sites (18). Use of monospecific sera to both HN and F has shown that cell fusion was secondary to virus-to-cell adsorption and that adsorption and hemolysis were separable processes (16). HN is a multifunctional hemagglutinin-neuraminidase glycoprotein. That both functions reside on the same protein was demonstrated by use of monospecific polyclonal antibodies to purified virion proteins (16) and by monoclonal antibodies to HN9(9, 15, 19, 20). From the latter studies, at least four determiniants were delineated on HN, two of them overlapping. When a number of NpV strains were screened, it was observed that there might be regions of HN antigen* Corresponding author. t Present address: Institut Pasteur de Lille, Unitd INSERM 186, Oncologie Moldculaire, Lille, France.
1198
NOTES
VOL. 57, 1986
TABLE 1. Classification of anti-HN monoclonal antibodies by their reactivity with strain Italien in RIP and Western blot tests Monoclonal anti-
body group
Isotype Origin of antigen'
of
mono-
cloneb
and clone
IgGl IgG2A
+ +
± +
AC3 La Sota/octylglucoside IgGl HD5 La Sota/octylglucoside IgG2 JB6 La Sota/octylglucoside IgGl 4A4 IgG2A La Sota 4C3 La Sota IgG2A 4D2 La Sota IgG2A 4D6 La Sota IgG2A La Sota IgGl 5C1
+ + + + + + + +
+ + ± +
7B2 8C11
Italien Italien
Antigen precipitation in RIP' Reaction with Italien HN HN HNj P52 in WBd
+ +
II
III IA3 2B6 SA1 5H1 6C2 6C6 8E8
La Sota/octylglucoside La Sota La Sota Italien La Sota Italien Italien
IV 7D4
La Sota
IgGl IgGl IgGA2 IgG2A IgG2A IgG2A IgGl
+ +
±
+ + + + + + +
+
+ + + + +
+ +
-
-
-
-
-
-
+ + + + +
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+ + +
+
IgGl
a Italien, Intact NDV Italien; La Sota/octylglucoside, octylglucoside extract of NDV La Sota; La Sota, intact NDV La Sota. b Isotype was determined by double-agar gel immunodiffusion against reference commercial antisera specific for each subclass of mouse immunoglobulin (Meloy Laboratories). IgG, immunoglobulin G. HN was obtained in experiments in the absence of tunicamycin; HNb and P52 were obtained from extracts of tunicamycin-treated cells. Symbols: +, Precipitation or recognition; ±, weak precipitation; -, no precipitation or recognition. d For method, see the text. WB, Western blot.
a commercial vaccine. The viruses were grown in the allantoic fluid of 9- to 11-day-old embryonated eggs from
specific-pathogen-free chicks. Harvest of the allantoic fluid was performed 48 and 96 h postinoculation for NDV Italien and La Sota, respectively. The virus was purified by sucrose density gradient centrifugation (20 to 40% sucrose in 0.01 M Tris hydrochloride-0.1 M NaCl-0.001 M EDTA, [pH = 7.4] [TNE]) at 50,000 x g for 3 h. The virus band was recovered and extensively dialyzed against TNE or phosphate buffer (0.02 M, pH 7.2), and the protein concentration was determined by the method of Lowry et al. (11). Monoclonal antibodies. Hybrid cell lines producing monoclonal antibody to the envelope glycoproteins of NDV La Sota and Italien were selected after fusion of myeloma cells SP 2/0-Ag 14 with immune BALB/c spleen cells. For immunization, intact virions or virions treated at pH 7.2 with octylglucoside (13) were used. The viral suspension or the octylglucoside extract (0.7 mg of protein in 1 ml of phosphate-buffered saline [PBS; pH 7.2]) was mixed with 1 ml of complete Freund adjuvant and injected intraperitoneally (0.4 ml) and intradermally (1.6 ml) at multiple sites along the spinal cord. This injection was followed by a booster 8 weeks later and 4 days before the fusion experiment. Fusion
1199
was carried out with a 50% polyethylene glycol 4000 solution in phosphate buffer (pH 7.2), and fused cells were grown in RPMI medium containing sodium pyruvate (0.001 M), glutamine (0.004 M), kanamycin (200 ,ug/ml), amphotericin B (Fungizone; 2.5 ,ug/ml), nonessential amino acids (0.1 mM), hypoxanthine (0.1 mM), aminopterin (0.4 ,uM), thymidine (16 ,uM), heat-inactivated horse serum (10%), and fetal calf serum (5%). Immunoglobulin-producing hybridomas were identified by enzyme-linked immunosorbent assay, hemagglutination inhibition (HI), and virus neutralization (data not shown). They were cloned twice with a semisolid medium containing 0.18% agarose. Immunoglobulin-producing clones were identified by precipitation of immunoglobulins with a 1:1 mixture of rabbit and goat anti-mouse sera made in 0.18% agarose. Ascites fluid was obtained by intraperitoneal injection of 107 hybridoma cells into mice that had been pristane (2,6,10,14-tetramethylpentadecane) primed at least 10 days
before injection. Immunoprecipitation assays. Baby hamster kidney cells (BHK-21) were trypsinized and infected in suspension by NDV Italien at a multiplicity of 10 PFU per cell in 2 ml of minimal essential medium (MEM)-HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; 20 mM; pH 7.4) for 30 min at room temperature and then incubated at 37°C with 8 ml of MEM-HEPES per flask. Actinomycin D (2 ,ug/ml) was added at 2.5 h postinfection. 35S-methionine labeling was performed at 5 h postinfection with 40 ,uCi/ml in 5 ml of MEM-HEPES without methionine supplemented with actinomycin D and dialyzed fetal calf serum. After 1 h of labeling, the infected cells were washed twice with TNE (0.01 M Tris hydrochloride, pH 7.4, 0.15 M NaCl, 0.001 M EDTA) and lysed with 1 ml of lysis buffer (TNE with 1% Triton X-100) for 10 min at 4°C per flask. The lysate was then spun in the Eppendorf centrifuge for 15 min at 4°C, brought to 5 ml with radioimmunoprecipitation (RIP) buffer (TNE buffer with 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]), and clarified by centrifugation in the Spinco SW50.1 rotor at 36,000 rpm for 60 min at 4°C. In experiments involving tunicamycin (13), the drug was added to infected cells at 4 h postinfection and kept in the culture at 2 ,ug/ml during labeling. For immunoprecipitation assays, the monoclonal antibody to be tested (4 jil) was mixed with 100 ,ul of clarified lysate; the solution was brought to 400 RI with RIP buffer and incubated for 16 h at 4°C. Rabbit anti-mouse immunoglobulin serum was added and allowed to react for 1 h at 4°C. Immune complexes were adsorbed onto 50 ,ul of a 10% suspension of inactivated Staphylococcus aureus and allowed to react for 30 min at 4°C. The bacterial suspension was washed twice in RIP buffer, once in a 1:1 RIP-TNE buffer, and once in TNE. The complexes were dissolved in 50 ,ul of sample buffer (0.05M Tris hydrochloride [pH 6.8], 1% SDS, 1% 2mercaptoethanol, 10% [vol/vol] glycerol), heated at 100°C for 3 min, and centrifuged to eliminate S. aureus. The supernatants were electrophoresed in 10% SDS-polyacrylamide gels. After electrophoresis, the gels were fixed, stained for fluorography, dried, and exposed with Kodak X-AR5 films. Western blots. NDV Italien viral proteins (200 ,ug) were dissolved in sample buffer and heated at 100°C for 3 min. The proteins were separated by electrophoresis in a 10% polyacrylamide-SDS gel and transferred to a nitrocellulose filter by the method of Towbin et al. (22). Transfer was for 2 h at 480 mA in 0.025 M Tris-0.2 M glycine buffer (pH 8.3).
1200
NOTES
J. VIROL.
After transfer, the nitrocellulose filter was soaked at room temperature in 100 ml of PBS (pH 7.4) with 1% bovine serum albumin for 30 min. The filter was then cut into strips that were incubated for 16 h, with gentle shaking, with appropriate sera (1:1,250 for monoclonal antibodies or 1:100 for a polyclonal serum) in 5 ml of reaction buffer (PBS buffer, pH 7.4, with 1% bovine serum albumin and 0.2% Tween 80). The strips were then washed three times by soaking in PBS buffer (pH 7.4)-0.2% Tween 80 and incubated for 3 h with rabbit anti-mouse serum (except for strips already incubated with rabbit anti-Italien serum or normal rabbit serum) diluted 1:1,000 in 5 ml of reaction buffer. After 3 washings with PBS-Tween buffer, the strips were incubated for 4 h with 3 ,uCi of 125I-protein A in 3 ml of reaction buffer. The strips were finally washed six times with PBS-Tween buffer, dried, and autoradiographed with Kodak X-AR5 film. Protein A was iodinated at a specific radioactivity of 35 ,uCi/,ug of protein by the method of Greenwood et al. (6). Results and discussion. The specificity of the antibodies was determined by immunoprecipitation of NDV Italieninfected [35S]methionine-labeled BHK-21 cell lysates (Tables 1 and 2; Fig. 1 and 3) by HI and virus neutralization (data not shown). When these criteria were combined, the 23 monoclonal antibodies were divided into the following two categories: (i) 18 antibodies directed against HN (Table 1) and (ii) 5 antibodies reacting with F (Table 2). (i) Monoclonal antibodies directed against HN. The 18 monoclonal antibodies identified as anti-HN by RIP and HI tests were divided into four groups (Table 1; Fig. 1). That individual clones within each group reacted with the same epitope was initially inferred from similar HI titers measured on 13 velogenic, 2 mesogenic, and 38 lentogenic strains (data not shown). Group I contained monoclones 7B2 and 8C11. They precipitated native HN, the nonglycosylated HN precursor HN4,, and P52. They were unreactive, however, towards HN in Western blots (Fig. 2), suggesting that group I antibodies were raised against a conformational epitope, made up of a sugar-free amino acid sequence, which was largely independent of protein glycosylation but sensitive to heat denatur-
R
Origin of
antigena
Isotype of mono-
2C1 1C3 8B1 n°3 10F2 12C4
La Sota Italien Italien Italien Italien
IgGl IgGl IgGl IgG2A IgG2B
clone"b
tralizain-
precipitation in
RIP"
F antigen recognized in WB'
-
-
dex'in2 F P52
tion
1.47 3.90 3.20 2.17 3.20
+ + + + +
-
-
-
-
-
-
-
-
a See Table 1, footnote a. b See Table 1, footnote b. c Expressed as the last dilution (in causing 50% plaque reductior NDV Italien (102.7 PFU/ml in Eagle MEM) was mixed with an equal volume of serial dilutions of ascites fluid previously inactivated at 56°C for 30 min. After 2 h of incubation at 37°C, 0.2 ml of the different virus-ascites dilution mixtures were plated on five 60-mm plates of chicken embryo fibroblasts. After adsorption for 60 min, the cells were washed with MEM and overlaid with 0.6% agarose in MEM. Plaques were scored on day 3 after being stained with neutral red. d F was obtained in experiments in the absence of tunicamycin; P52 was obtained from extracts of tunicamycin-treated cells. Symbols: +, Precipitation or recognition; -, no precipitation or recognition. e WB, Western blot.
log,0)
GR.II
GRi l
GR. IV
ciCti
4D6)
(5A11
(7D4)
MW
MO
xI100
-200
L
HN - 925
HNq0
- 69 p
F,
- 46
Pi-,
- 30
TM
+
-
+
FIG. 1. Autoradiogram of an SDS-polyacrylamide gel of 35Smethionine-labeled proteins of NDV Italien labeled in the presence (+) or absence (-) of tunicamycin (TM). Immunoprecipitation was carried out with rabbit polyclonal serum raised against NDV Italien octylglucoside extract (lanes R) and monoclonal antibodies 8C11, 4D6, SA1, and 7D4, representatives of groups I, II, III, and IV, respectively (see also Table 1). The background of the precipitation reaction was obtained with normal mouse serum (lanes Mo). Molecular weight estimates of NDV proteins were essentially those published previously (5, 13) (L, 200,000; HN, 74,000; HN+, 67,000; Fo, 66,000; P, 59,000; FI-NP, 56,000; P52, 52,000; M, 40,000; F2, 10,000).
ation in the presence of mercaptoethanol (1%) and SDS (1%) followed by adsorption onto nitrocellulose membranes. Monoclonal antibodies of group II showed reactivity towards HN antigen whether native, unglycosylated, or even blotted after hot SDS-mercaptoethanol denaturation. It was 1 2 3 4 5 6 7 8 9 101112 131415161718 A BC 0 E NWR r
TABLE 2. Reactivity of anti-F monoclonal antibodies with NDV Italien in neutralization, RIP, and Western blot tests Monoclonal antibody
GR.I
HN-
I
F1
I
-
FIG. 2. Behavior of monoclonal antibodies with strain Italien polypeptides in Western blot experiments. Immune reactions involved (i) HN monoclones from group I (7B2 and 8C11, lanes 1 and 2, respectively), group ll (AC3, HD5, JB6, 4A4, 4C3, 4D2, 4D6, and SC1, lanes 3 through 10, respectively), group III (IA3, 2B6, 5A1, SH1, 6C2, 6C6, and 8E8, lanes 11 through 17, respectively), and group IV (7D4, lane 18); (ii) anti-F monoclones (2C1, 1C3, 8B1 no. 3, 10F2, and 12C4, lanes A through E, respectively); (iii) normal mouse serum (lane Mo); (iv) rabbit polyclonal anti-NDV proteins (lane R); and (v) normal rabbit serum (lane r).
NOTES
VOL. 57, 1986 R
A
B
C
D
Mo i
E
low
-2o HN HN0 \
F.-. \ FF. -
P52
-923
_m .
M -0
-Aat--
TM
-
-
_
__ -+
+ - + -
-
_ 46 -48 30
+ - +
FIG. 3. Autoradiogr am of an SDS-polyacrylamide gel of 35smethionine-labeled prolteins of NDV Italien labeled in the presence (+) or absence (-) of tiunicamycin (TM). Immunoprecipitation was serum (lanes R); monoclones 2C1 carried out with rabbit I (lanes A), 1C3 (lanes E ), 8B1 (lanes C), 10F' (lanes D), and 12C4 (lanes E ); and normal mouse serum (lanes Mo). For molecular weights of NDV proteil ns, see the legend to Fig. 1.
polyclonal
thus inferred that m onoclonal antibodies of this group behaved as probes for a sequential rather than a conformational determinant. Precipitation of P52, cleaved and
nonglycosylated Fo ( 13), occurred with monoclonal antibodgroups I ai nd II and solely from extracts of tunicamycin-treated cells. This result was interpreted as a coprecipitation due to interactions of P52 with regions of HN4, not available orn HN, the native glycosylated protein. Group III antibodices reacted only with the native glycosylated HN protein. Their lack of reactivity versus the nonglycosylated HN suggested that the recognized epitope was configurational a nd glycosylation dependent. The structure of the polysacclharide moiety being mostly cell determined (17), one wou ld expect antibodies against HN and F glycoproteins to sho w only limited specificity if the carbohydrate contributed (directly to the reactive epitope. Such a behavior was not olbserved. It was thus tentatively concluded that polysacc haride chains influenced the folding of polypeptides and in(directly contributed to the building of epitopes (12). ies of
uniquebehavior among our a a Monoclonal 7D4 s showed as it did not precipitate any form of monoclonal antibodi(es, asit strain Italien HN (Tzable 1). It reacted, however, in HI and enzyme-linked immuinosorbent assays in which strain La Sota was the source of antigen (data not shown). The M protein, a highly hydrophobic molecule, was precipitated by all m onoclonal antibodies used except when the lysate was prepa red from tunicamycin-treated cells. M and L were precipita ted even by normal mouse serum. (ii) Monoclonal anitibodies directed against F. The search
;es,
for anti-F
did
monoclonail antibodies was based on
virus neutralization a ssays
ssays
positivityInin
nonacstivity
accompanied by accompanied by no activity In HHII
tests.
Immunization of miice with NDV La Sota yielded only one monoclonal antibodly (2C1) with neutralization activity against strain Italien but devoid of HI activity. A second experiment was set iup in which the immunogen was NDV Italien. Four hybridc )mas (1C3, 8B1 no. 3, 10F2, and 12C4) were isolated that di d not affect the HI test but neutralized the virus. Consequeritly, these five monoclones were taken as good anti-F candi4 dates. The name, isotype neutralization index, and immunopre-
datesu.
cipitation capacities (of each of the five monoclonal antibodies are listed in Tabl4 e 2. All five antibodie5s precipitated protein F1 (Fig. 3) but
1201
lacked reactivity towards P52, the presumed nonglycosylated precursor of protein F (Fig. 3, lanes A, B, C, D, and E). None of the five monoclonal antibodies reacted with F protein in Western blot experiments (Fig. 2, lanes A, B, C, D, and E). Anti-La Sota monoclonal 2C1, even if slightly active in seroneutralization tests, was able to precipitate NDV Italien F, protein (Fig. 3). The other four monoclonal 'antibodies raised against NDV Italien exhibited much stronger neutralization capacity in in vitro experiments. We thus tentatively concluded that (i) the five monoclonal antibodies with virus neutralization activity were indeed raised against determinants of protein F1, and (ii) the five antibodies were recognizing conformational determinants, whose spatial structure depended upon adequate glycosylation of the peptide backbone of F1 protein. None of the five monoclonal antibodies was able to bind to nonglycosylated F precursor; similarly, none of these antibodies could react with denatured F1 protein transferred to nitrocellulose (Fig. 2, lanes A, B, C, D, and E). It will be instructive to compare activities of the five monoclonal antibodies both in vivo and in vitro. To that end, the five antibodies are being checked for their capacity to protect chicks from NDV Italien infection. When better characterized, the available monoclonals will also be instrumental in studies aimed at identification of epitopes engaged in the fusion reaction of cleaved F protein with liposomes, as was described by Hsu et al. (8) for Sendai virus. Immediate and detailed comparison with the reaction involving cell membranes will also be feasible. Production of anti-F monoclonal antibodies has met so far with little success (19, 20). The availability of more of these reagents will facilitate thedetailed characterization of biologically important epitopes on F1 and provide efficient ways to identify subtle variations between virus strains, especially when these techniques are combined with gene cloning and sequencing techniques. This work was helped financially by a contract from Institut pour l'Encouragement de la Recherche Scientifique dains l'Industrie et I'Agriculture-Smith Kline Recherche Industrie Therapeutique; L.L. held a fellowship from Relations Culturelles Internationales de la Communaute Franqaise de Belgique and received financial support
from the
Foundation David and Alice van
Buuren.
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NOTES
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