Antigenic determinants of influenza virus hemagglutinin VIII. Topography of the antigenic regions of influenza virus hemagglutinin determined by competitive radioimmunoassay with monoclonal antibodies

VIROLOGY

113,130-140

(1981)

Antigenic

Determinants

of Influenza Virus Hemagglutinin

VIII. Topography of the Antigenic Regions of Influenza Virus Hemagglutinin Determined by Competitive Radioimmunoassay with Monoclonal Antibodies

A. M. BRESCHKIN,’ J. AHERN, 2kh00l

of Microbiology, University

AND

D. 0. WHITE’

of Melbourne, Parkville,

Victoria $052, Australia

Received December 15, EWO; accepted April I, 1981 Several independent hybrid cell lines (“hybridomas”) that produce monoclonal antibodies against the hemagglutinin (HA) of influenza virus A/Memphis/102/72 were derived in this laboratory. The reactivity of each monoclonal antibody against the virus was characterized by radioimmunoassay (RIA), hemagglutination inhibition (HI), neutralization of infectivity, and avidity of binding. Most hybridoma antibodies reacted with the apoprotein of the HA, and several of these displayed HI and neutralizing activity; one monoclone was directed against the “host antigen” (carbohydrate) and had no HI or neutralizing capacity. This panel of monoclonal antibodies was used to study the topography of epitopes on the HA by means of a competitive RIA; the capacity of unlabeled hybridoma antibodies to block the binding of radiolabeled heterologous monoclones was tested. Some pairs of monoclones competed completely and were therefore judged to be binding in the same antigenic region; other pairs bound noncompetitively to physically distinct regions. The results demonstrate the existence of four distinct antigenie regions. One major region contains epitopes recognized by several monoclones, all of which have substantial HI and neutralizing activity. Two other regions are apparently distinct from but not completely remote from this major region. A fourth region is physically unrelated to the others; monoclones binding to this region had no activity in HI or neutralization assays.

peptide (Laver et aZ., 1979). Based on the reactivity patterns of a panel of monoA number of hybridomas producing an- clonal antibodies with a series of antigenic tibodies against the hemagglutinin (HA) variants, Gerhard et al. (1980) concluded of influenza A virus have been isolated and that there were at least five nonoverlapcharacterized (Koprowski et al., 19’77;Ger- ping antigenic “sites” on A/PR8/8/34 hard et al., 1978) and used to study the (HONl); similarly Webster and Laver antigenic determinants on this important (1980) established that there were at least protein. Monoclonal antibodies have been three independent antigenic areas on A/ employed to select virus variants which no Memphis/l/‘ll. These results were conlonger react with the monoclone used for firmed by measurements of the frequency selection (Gerhard and Webster, 1978). of antigenic variants selected in the presAntigenic variants of influenza A/Memence of various combinations of monophis/l/71 (H3N2) generated in this way clonal antibodies (Yewdell et al., 1979; were shown to have single amino acid sub- Webster and Laver, 1980). stitutions in the sequence of the HA polyWhile these experimental approaches may define the number of antigenic determinants in operational terms, they proi Present address: National Biological Standards vide no information on the spatial distriLaboratory, Parkville, Victoria 3052, Australia. bution of the several antigenic sites on the ‘To whom reprint requests should be addressed. INTRODUCTION

6042-6822/81/110130-11$02.00/O Copyright All rights

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

130

TOPOGRAPHY

OF INFLUENZA

HA DETERMINANTS

131

HA molecule. Only limited data on the ferred to as Aichin-BelN; and the avian strain A/Shearwater/ recombinant topography of the antigenic determinants E.Aust./l/‘lZ(Hav 6)-A/Be1/42(Nl), rehave been obtained. Electron micrographs indicate that “cross-reactive” and “spe- ferred to as Shearwateru-Beln. Viruses cific” antibodies from hyperimmune serum were grown in the allantoic cavity of emall appear to bind in the same general vi- bryonated eggs and purified as described cinity near the tip of the HA spike (Wrig- by Jackson et al. (1979). Cell cultures. The murine myeloma cell ley et al., 1977); whereas Gerhard et al. (1980) have interpreted their finding that lines, P3-NSl/l-Ag4-1 (kindly provided by some monoclones bind more effectively to Dr. M. Howard, Walter and Eliza Hall In“dried” virions than to “wet” ones to mean stitute) and Sp2/0-Ag14 (kindly provided that some determinants are situated fur- by Dr. A. W. Harris, Walter and Eliza Hall ther down the spike. An earlier study from Institute) were cultivated in RPM1 1640 our own laboratory (Russell et al., 1979) medium supplemented with 10% heat-indemonstrated by competitive inhibition of activated fetal calf serum (Flow Laborabinding between strain-specific and cross- tories Australasia Pty. Ltd.) and gentareactive antibodies that different epitopes mycin (30 pg/ml). CV-1 cells, a continuous are situated in the same antigenic region line of African green monkey (kindly proof the HA molecule. vided by Dr. D. McPhee, Commonwealth The aim of the present study was to Scientific and Industrial Research Orgainvestigate the topography of antigenic nization), were cultivated in 199 medium determinants on the HA of influenza supplemented with 10% heat-inactivated A/Memphis/102/72 using a competitive fetal calf serum (FCS), 1.4 mg/ml NaHC03, binding radioimmunoassay and a panel of gentamycin (30 pg/ml), and fungizone (2 hybridoma antibodies derived in this lab- &ml). oratory. In order to relate our monoclones Preparation of hybrid cell lines. Hybrid to those recently defined by a completely cell lines producing antibodies to influenza different type of assay (Webster and Laver, virus were obtained following fusion of 1980), two additional hybridoma antibod- NSl/l or Sp2/0 myeloma cells with imies (kindly provided by Dr. W. Gerhard, mune spleen cells (Kiihler and Milstein, Wistar Institute) were included for com- 1976). Balb/c mice were immunized as deparison. Our experimental approach was scribed by Gerhard et al. (1978). Polyethbased on the rationale that if the epitopes ylene glycol 4000 (Ajax Chemicals, Sydrecognized by two monoclonal antibodies ney) was used as the fusing agent. The are physically close, the binding of one cells were maintained in HAT medium (RPM1 1640 supplemented with 10% FCS, monoclone will sterically block the binding of the second; conversely no blocking will 10m4M hypoxanthine, 4 X 10m5M thymioccur if the epitopes are sufficiently dis- dine, 10e6M deoxycytidine, and 10e5M tant. Using this method, four distinct an- amethopterin) for lo-14 days, after which tigenic regions were detected on the influ- hybrid cells were tested for influenza enza HA molecule. virus antibodies using a solid-phase radioimmunoassay (RIA) described previously (Anders et aZ., 1979). Bound mouse MATERIALS AND METHODS immunoglobulin was detected using ‘%IViruses. The strains of influenza vi- labeled rabbit (IgG) anti-mouse immurus used in these studies were A/Bel/ noglobulin (RAMIG) or ‘251-labeled staph42(HlNl), referred to hereafter as Bel; ylococcal protein A diluted in 0.1 M phosA/Port Chalmers/1/‘73(H3N2), referred phate buffer, pH 8.2. Hybrids that to as P.C.; the “recombinant” virus (ob- produced influenza virus antibody were tained by genetic reassortment), A/Memcloned twice by limit dilution using norphis/lO2/72(H3)-A/Be1/42(Nl), referred mal Balb/c spleen cells as a feeder layer. to as Memn-Belx; the recombinant vi- Hybrid cell lines were considered to prorus A/Aichi/2/68(H3)-A/Be1/42(Nl), re- duce monoclonal antibody only when the

132

BRESCHKIN

pattern of cross-reactivity to several virus strains remained unchanged upon further cloning. High concentrations of monoclonal antibodies were obtained from the sera of Balb/c mice bearing subcutaneous hybridomas. Determination of immunoglobulin class. The class of immunoglobulin to which influenza virus antibody belonged was determined using the two types of RIA assays described above. ‘%I-labeled RAMIG detected all classes of mouse immunoglobulin, whereas ‘l-labeled protein A specifically detected the IgG class. The immunoglobulin class of antibodies which did not bind to protein A was confirmed by immunodiffusion using anti-mouse IgG (Bionetics Laboratories) and anti-mouse IgM-F, (Nordic Immunology). Although hybrid cell lines producing IgM antibody were frequently derived, only IgG hybridoma antibodies were used in this study. Estimation of concentration of monoclonal antibody against iqjhenxa virus. Monoclonal antibody 46A3 (Table 1) was used to establish a standard for the quantitation of monoclonal IgG antibodies. Initially, immunoglobulin from the culture fluid of hybridoma 46A3 was concentrated by ammonium sulfate precipitation (40% saturation) and titrated by radial immunodiffusion (Davis and Ho, 1976) against RAMIG using IgG from myeloma MOPC 21 (kindly provided by Dr. R. Anders, Walter and Eliza Hall Institute) as the standard. Using this as a “standard” influenza virus antibody, a quantitative RIA was developed according to Frankel and Gerhard (1979). A standard binding curve for 46A3 antibody was constructed which could be used to estimate the concentration of IgG antibody against influenza virus present in hybridoma culture fluids or tumor sera. For each monoclonal antibody it was established that lo4 HAU/ml of virus provided an antigen concentration sufficient to bind >99% of the antibody within the range of the standard binding curve. For each assay, a parallel standard binding curve was constructed. Unknown samples were diluted so that the counts per minute bound were within the range for the standard. Assays were routinely

ET AL.

done in duplicate and were generally accurate to + 20%. This assay is valid providing only that the efficiency of detection of different monoclonal IgG antibodies by ‘%I-RAMIG did not vary greatly. Antibody avidity assays. Antibody avidities were determined using a solid-phase RIA described by Frankel and Gerhard (1979), except that virus was adsorbed onto the wells without drying and fixing. ‘%I-RAMIG, present in excess, was used to detect bound antibody. Each assay was done in triplicate wells. Radioiodination. RAMIG, protein A, and monoclonal antibody preparations were radioiodinated using a modification of the chloramine-T method described previously (Russell and Jackson, 19’78). The monoclonal antibodies were prepared from the sera of hybridoma-bearing mice by passing them through a &ml protein ASepharose (Pharmacia South Seas Pty. Ltd.) column in 0.01 M phosphate buffer, pH 8.2 (Ey et al., 1978). The bound IgG was eluted with 0.1 M acetate buffer, pH 4 or 6. The eluate was adjusted to pH 7.4, concentrated with ammonium sulfate (40% saturation), and dialyzed against PBSA. The concentration of monoclonal antibody was then estimated using the quantitative RIA and 10-100 fig was taken for iodination. The proportion of the total ‘%I-IgG which bound to virus in the RIA fell within the range 13-65%, depending on the concentration of monoclonal antibody present in the different preparations. The specific activities of the monoclonal antibodies were 0.5-2.0 &i/pg. Competitive binding assay. Competitive binding assays were performed using a modification of the RIA described above. For each radiolabeled monoclonal antibody, a limiting concentration of antigen was used for competition experiments and was typically 10’ HAU/ml. Nonspecific binding of the labeled antibodies was assayed on wells coated with 5 mg/ml bovine serum albumin (BSA) and was insignificant in all cases. The tumor sera or ascitic fluids containing unlabeled monoclonal antibodies were periodate treated (see below) prior to use.

Memn-Belu

0.9 1.0 1.0 1.0 1.0 1.0 1.0 1.0 < 1.0

(mg/ml)

5 20 3 7 2 40 10 20 7 11 0.7 0.9 0.1 0.9 < 1.2 1.7 0.9 < 0.5

P. Chalmers 1.0 0.9 0.5 < 1.7 < < 0.9 1.0 1.0

Aichin-Beln

Reactivity in RIA*

1.0 < <

C

< < < < < <

2,700 11,000 14,000 16,000 < < < < < 16,500

Be1 Memn-BelN

NY& N.D. N.D. 44,800 23,200

2,000

2,700 11,000 440 <

N>. N.D.

450 2,800 43,000 < < <

2,~

N’D. N.D. N.D.

22,000 < <

600 450 <

P. Chalmers

Neutralization/mg/mld Aich&-Be1 N Memu-BelN

700 2,750 < 16,000 < < < N.D. <

P. Chalmers

HI/mg/mlC

9.0 9.1 8.9 9.4 8.9 9.1 9.1 N.D. N.D. N.D.

Aviditye [M-l (log)]

a Monoclonal antibody concentrations were estimated by a quantitative RIA described in Materials and Methods. b Binding of monoclonal antibodies was assayed on wells costed with lo* HAU/ml of each strain of virus. Values given are the ratios of cpm bound to the strain indicated compared to the priming strain. < indicates that the cpm bound was no greater than to BSA-coated wells. ’ Hemagglutination inhibition (HI) was determined by standard procedures using 4 HAU virus, < indicates that the 50% endpoint was
46A3 5OH7 65ClO 6535 65C3 7HlO 7G9 50All H14-A20 H14-A21

Hybridoma

or ascitic fluid”

Concentration of monoclonal antibody in tumor

REACTIVITY PATTERNSOF MONOCLONAL ANTIBODIES

TABLE 1

c

134

BRESCHKIN

Serial twofold dilutions were mixed with an equal volume of homologous or heterologous ‘l-labeled antibody in PBSA containing BSA (5 mg/ml), and 0.1 ml was applied to virus-coated wells. The amount of unlabeled competing antibody added to each well was estimated by the quantitative RIA. The results are expressed as percentage competition determined by the formula 100 X [l - (cpmbund in the presence of unlabeled antibody + cpmbou,,din the absence of unlabeled antibody)]. In all assays, a homologous competition was included as a positive control. Serological tests. Tumor sera or ascitic fluids were heat-inactivated at 56” and periodate treated (Dowdle, 1976) prior to hemagglutination inhibition (HI) and neutraliation assays. Hemagglutination (HA) and HI were assayed according to Fazekas de St. Groth and Webster (1966). Virus neutralization was assayed by plaque reduction on CV-1 cells. Serial dilutions of monoclonal antibodies in tumor sera were mixed with an equal volume of virus containing loo-150 plaque-forming units (PFU) and incubated for 45 min at 20”. The samples were then assayed for infectivity on a confluent CV-1 cell monolayer overlaid with modified Eagle’s medium containing 0.5% agarose and 10 pg/ml trypsin (Difco, 1:250). After 2-3 days’ incubation at 3’7”, monolayers were fixed with 5% formaldehyde in saline and plaques were counted following staining with 0.5% crystal violet to determine the 50% endpoint. Since nonspecific inhibition of virus infectivity by normal Balb/c serum often occurred at dilutions to 1:500, only those tumor sera with neutralizing titers in excess of 1:lOOOwere regarded as showing specific neutralizing activity. HI and neutralization titers were expressed as the reciprocal of the 50% endpoint divided by the concentration of monoclonal antibody in milligrams per milliliter. RESULTS

Characterization of the Monoclonal Antibodies A set of twice-cloned stable murine hybrid cell lines (“hybridomas”) producing

ET AL.

monoclonal antibodies to the HA of the H3 subtype of influenza virus was derived in this laboratory. By testing the monoclones against a panel of virus strains in RIA (Table l), it was deduced that seven were directed against the polypeptide portion of the HA (the “apoprotein”), whereas one (50All) was probably directed against the carbohydrate “host antigen” which is common to all egg-grown viruses (Laver and Webster, 1966; Jackson et al., 1981). The HA specificity of the antibodies was confirmed by their reaction in RIA against purified isolated HA from Memu-BelN or P. Chalmers (kindly provided by Dr. D. C. Jackson of this laboratory). The anticarbohydrate specificity of 50All was confirmed by its reactivity against the egggrown avian recombinant strain Shearwater-u-BelN. Monoclones H14-A20 and H14-A21, included for comparison, were derived by and kindly provided in ascitic fluid by Dr. W. Gerhard (Wistar Institute) and have been described in detail elsewhere (Webster and Laver, 1980).

Reactivity Patterns of the Monoclonal Antibodies The antigenic specificity of each monoclonal antibody was analyzed by RIA, HI,and neutralization against a panel of naturally occurring H3 variants (Table 1). A variety of reactivity patterns was exhibited: 46A3, 50H7, and H14A21 were cross-reactive for the H3 strains tested; the others were more specific-H14A20 for Aichiu-BeIN, 6535 for Memu-BeiN and the closely related P. Chalmers; 65ClO was relatively specific for Memu-BelN but displayed some reactivity with Aichin-BelN. In agreement with Gerhard et al. (1978), hybridomas derived from spleen cells of mice which had been cross-boosted produced highly cross-reactive antibodies. Surprisingly, 7H10,7G9, and 65C3 showed a higher reactivity to a heterologous strain than to the immunizing strain. The reactivity patterns determined by RIA and HI were not necessarily equivalent because of the large excess of virus antigen (lo4 HAU/ml) used in the RIA, compared to the much lower amount in the

TOPOGRAPHY

OF INFLUENZA

HI assays. Consequently, a cross-reaction of low avidity was more apparent in RIA than in HI. For example, 65ClO showed significantly greater cross-reactivity to Aichiu-BelN in RIA than in HI; the avidity of this monoclone was approximately lOOfold greater to Memn-BelN (9 x 10sM-l) than to Aichin-BelN (1 X 10’ M-l). HI and neutralization tests separated the monoclones into two distinct categories: those with substantial HI and neutralizing activity (4683,50H7,65ClO, 6535, H14A20, H14A21) and those with none (65C3,7HlO, 7G9,50All). One of the latter (50All) was presumably directed against the oligosaccharide sidechains of the HA molecule since it reacted with Bel(H1) virus (Table 1) and Shearwater (HavG)Bel(N1) virus (data not shown). The others (65C3, 7H10, 7G9) are, however, directed against the apoprotein of the HA. Topographical Analysis of Antigenic Detern&ants Six monoclonal antibody preparations were purified from tumor serum or ascitic fluid by protein A-Sepharose chromatography and then radioiodinated. In order to select conditions of maximum sensitivity for a competitive RIA, each lZI-labeled monoclone was assayed at a fixed concentration (approximately 200 ng per well) against varying amounts of virus to select a concentration of virus giving two- to threefold less than maximum antibody binding. This limiting virus concentration (generally 102 HAU/ml) was used in the following experiments in which the spatial relationships between different epitopes on the HA molecule were investigated. Initially, dilutions of various unlabeled hybridoma antibodies were tested for their capacity to block the binding of two ‘%Ilabeled monoclones displaying HI and neutralizing activity; 50H7 was chosen from the “cross-reactive” group and 65ClO was chosen as a relatively “specific” one. Figure 1A reveals that all the HI and neutralizing monoclones tested were capable of inhibiting completely the binding of ‘251-50H7, whereas none of the non-HI, nonneutralizing monoclones could do so. The fact that relatively specific (65C10,

135

HA DETERMINANTS

10

20

30

40 ANTIBODY

10 CLOG,,

20 NG)

30

40

FIG. 1. Competitive binding assays with ‘l-labeled monoclonal antibodies 50H7 (A) and 65ClO (B). The radiolabeled monoclonal antibodies were mixed with the indicated amounts of various unlabeled competing hybridoma antibodies diluted from tumor serum or ascitic fluid, and 0.1 ml was applied to virus-coated wells of microtiter plates. Competitive binding was assayed on Memn-BelN (4683, 50H7, 50Al1, 65C3, 65C10, 6535), Aichin-BelN (H14A20, H14A21), or P. Chalmers (7G9,7HlO). In the absence of competitors, approximately 4 X lo4 cpm (‘%I-50H7) and 3 X lo4 cpm (‘%I-65ClO) were bound.

6535, H14A20) as well as cross-reactive (46A3, H14A21) monoclonal antibodies can totally block the binding of the crossreactive 50H7 suggests that the epitopes recognized by all these monoclones are in the same general region of the HA molecule. In contrast, the epitopes recognized by the non-HI monoclones 65C3,7G9, and 7HlO are topographically distinct from this region. Figure 1B reveals a similar picture; the more specific monoclone 65ClO is completely blocked by all the HI monoclones tested, whether specific or cross-reactive. Neither the non-HI monoclone 65C3 nor the anticarbohydrate monoclone 50All showed any blocking capacity. The results are not markedly affected by the virus strain used in the RIA (Figs. 1A and B), provided of course it is one to which the radiolabeled monoclone binds. The slopes of the curves are comparable; lateral displacement may reflect differences in avidity of the monoclonal antibodies which were not always revealed by the Frankel and Gerhard assay (Table 1). Indeed, this type of competitive RIA would

136

BRESCHKIN

be expected to provide a highly sensitive index of relative avidity. For example, the data in Fig. 1 suggest that the avidities of the monoclones for Memn-BelN can be ordered approximately as follows: 6535 > 65ClO > 46A3 and 50H7, while the avidities for Aichin-BelN may be ranked H14A21> H14A20 and 50H7 > 65ClO. The apparent differences in the avidities of the monoclonal antibodies in the competitive RIA did not significantly interfere with the topographical analysis of the HA using this method. However, it was essential that blocking capacity be tested using a range of concentrations of unlabeled antibody and, in some cases, that reciprocal assays be performed. Figure 2 gives the results of competitive RIAs using radiolabeled monoclones from the non-HI group. It can be seen that only the non-HI monoclone 7G9 showed significant blocking of ‘%I-7HlO. The rather variable but low level of competition observed with the HI monoclones 65C10, 6535, and 46A3 was considered to be nonsignificant. The findings with the other non-HI monoclone were particularly interesting; unlabeled 65C3 failed to block

ANTIBODY

10 ILOG,oNG)

2.0

30

40

FIG. 2. Competitive binding assays with ‘261-labeled monoclonal antibodies 7HlO (A) and 65C3 (B). The radiolabeled monoclonal antibodies were mixed with the indicated amounts of various unlabeled competing hybridoma antibodies diluted from tumor serum, and 0.1 ml was applied to virus-coated wells of microtiter plates. Competitive binding was assayed on Memu-Beln virus in all cases. In the absence of competitors, approximately 5 X 10s cpm (‘%I-7H10) and 8 X 109 cpm (izI-65C3) were bound.

ET AL.

ANTIBODY

CLOG,,

NGI

FIG. 3. Competitive binding assays with ‘%I-labeled monoclonal antibodies H14A21 (A) and H14A20 (B). The radiolabeled monoclonal antibodies were mixed with the indicated amounts of various unlabeled competing hybridoma antibodies diluted from tumor serum or ascitic fluid, and 0.1 ml was applied to viruscoated wells of microtiter plates. Competitive binding was assayed on Memn-Belx (65C3, 65C10, 6535) or Aichin-Belx (46A3,5OH7,50All, H14A20, H14A21). In the absence of competitors, approximately 3 X 10’ cpm (1251-H14A21) and 1 X 10’ cpm (‘2SI-H14A20) were bound.

7H10, but labeled 65C3 was completely blocked by 6535. This suggested that 65C3 reacts with an epitope in an antigenic region which is topographically separate from the region defined by the other nonHI monoclones 7HlO and 7G9. In fact, the 6563 epitope is apparently close to that recognized by 6535 but still distinct from the region to which the other HI monoclones bind. Webster and Laver (1980) have recently distinguished three groups of monoclonal antibodies to the H3 subtype, based on a completely different experimental approach, namely, selection of antigenic variants using hybridoma antibodies. It seemed relevant, therefore, to compare a small number of the monoclones which they used with the collection derived in our own laboratory, by the competitive RIA method. Accordingly, one monoclone from their group II (H14A20) and one from their group III (H14A21) were radioiodinated and tested in a competitive RIA (Fig. 3). HI monoclones 6535, 65C10, and 46A3

TOPOGRAPHY

OF INFLUENZA

HA DETERMINANTS

137

fined by hybridomas 46A3, 50H7, 65C10, 6535, and H14A21; all of these monoclones competed completely and reciprocally against each other in the combinations tested, and all had substantial HI and neutralizing activity. A second region included epitopes defined by 7HlO and 7G9 and was topographically completely separate from the other regions; monoclones 7HlO and 7G9 showed complete competition against each other, but did not compete with any other monoclones, and displayed no HI or neutralizing activity. A third region was defined by monoclone 65C3, which had no detectable HI or neutralizing activity, but was blocked by 6535; thus 65C3 must recognize an epitope that is close to 6535 but distinct from other epitopes in this region. The fourth region was defined by the H14A20 hybridoma, which, uniquely among the monoclonal DISCUSSION antibodies tested, gave nonreciprocal and The -aims of the present study were to only partial competition; hence the epitope recognized by H14A20, though close to develop a panel of hybridoma antibodies agains the HA of the H3 subtype of influthose defined by the other HI monoclones, enza virus and to utilize these monoclones is distinct. Webster and Laver (1980) have recently to study the spatial arrangement of epiobtained evidence for at least three nontopes on this protein. Eight independent overlapping antigenic areas on the HA of hybrid cell lines synthesizing monoclonal A/Memphis/l/7l(H3N2) by the quite difIgG antibodies were derived. As determined by reactivity in RIA against a panel ferent approach of using hybridoma anof virus strains, seven of these monoclones tibodies to select variants. It was therefore were directed against the apoprotein of of particular interest to include in our the HA, and one had anti-host carbohystudy two of the monoclones used by drate specificity. The monoclones were Webster and Laver (1980): H14A20 (from their group II) and H14A21 (their group further characterized by HI and neutralIII). Monoclone H14A21 showed extensive ization assays, which separated them into two groups: Four had substantial HI and competition against the HI monoclones neutralizing activity (4683, 50H7, 65C10, derived in our laboratory, indicating that the epitopes defined by all these hybrid6535), while four had no such activity whatever (65C3, 7H10, 7G9, 50All). Two oma antibodies are in close physical proxadditional hybridoma antibodies (H14A20, imity. It seems quite likely that all of our H14A21) which were derived and kindly HI monoclones belong to group III of provided by Dr. W. Gerhard (Wistar In- Webster and Laver (1980). The monoclones stitute) had HI and neutralization activity within this group display a variety of “spe(Webster and Laver, 1980) and were in- cific” and “cross-reactive” patterns of cluded for comparison. A competitive reactivity to related virus strains within binding RIA was utilized to analyse the the H3 subtype, suggesting that the group topography of the epitopes to which the represents a region (presumably near the monoclones reacted. tip of the spike) which contains overlapOur results demonstrated the presence ping or adjacent epitopes. Alternatively, of four distinct regions on the HA poly- this region may represent a “topographic peptide. One region embraced epitopes de- determinant” (Urbanski and Margoliash,

all completely blocked the binding of ‘%Ilabeled H14A21 (Fig. 3A), but H14A20 achieved only 45% inhibition even at high concentrations. In the reciprocal experiment (Fig. 3B), unlabeled H14A21 did not block iodinated H14A20 at all. Monoclones 50H7 and 46A3 achieved only 25-35s competition against H14A20, although previous results (Fig. 1A) had shown that H14A20 completely blocked ‘?-labeled 50H7. Thus, H14A21 and all of our own HI monoclones apparently react with epitopes which are in close physical proximity. The complex pattern of competitive interactions’ shown by H14A20 suggests that its epitope, while in the same general region as the epitopes to which the other HI monoclones bind, is nevertheless topographically quite distinct.

138

BRESCHKIN

197’7)composed of a core epitope which is affected by “peripheral” residues (East et al., 1980). In the hope of demonstrating whether this antigenic region (and others) can be subdivided further, we are now using Fab fragments from a wider variety of monoclonal antibodies in our competitive binding RIA. The results of competitive binding studies on monoclone H14A20 correlated with the antigenic variant analysis of Webster and Laver (1980) in demonstrating that this group II monoclone recognizes an epitope situated in an antigenic region distinguishable from group III. Nevertheless, the partial and nonreciprocal competitive inhibition of binding of group III hybridomas suggests that this epitope may also be located at or near the tip of the spike. Stone and Nowinski (1980) have proposed that most IgG antibodies bind monovalently to viral antigens, thereby retaining considerable rotational freedom, which allows for noncompetitive binding to physically separate epitopes within the confines of a single antigenic protein. It is conceivable that the orientation of the epitope recognized by H14A20 is such that the monoclone binds bivalently to separate copies of the same epitope on different polypeptide monomers (or different trimeric spikes); this would significantly magnify the blocking effects of H14A20 and possibly account for its ability to hinder accessof the group III monoclones, but not vice versa. If this were the case, it would be predicted that a Fab fragment of the H14A20 antibody would show no competition against the group III monoclones. The two additional antigenic regions defined by our non-HI, nonneutralizing monoclones 7H10, 7G9, and 65C3, would be undetected by the Webster and Laver (1980) variant selection approach, as all their hybridoma antibodies had HI and neutralizing activity. Our results provide the first indication that such antigenic regions exist on the HA of the H3 subtype, although Gerhard et al. (1980) have described similar sites on the HA of A/PR/ 8/34(HlNl). The non-HI monoclones of

ET AL.

Gerhard et al. (1980) reacted preferentially with “dried” rather than “wet” virions, suggesting that these sites are relatively inaccessible in the native virus. The competitive binding RIA used in our studies appeared to be a highly sensitive method for comparing antibody avidities. The observed lateral shifts in the competition curves can be best explained by differences in avidity of the various monoclones; similar effects have been described previously (Stone and Nowinski, 1980). Some of these differences were not apparent from direct avidity measurements using the Frankel and Gerhard (1979) method, the sensitivity of which may have been decreased in our hands by the use of wet virions instead of dried; this modification was introduced because wet conditions were used in the competitive binding assay. It is also possible that under- or overestimates of the concentration of monoclonal antibodies as determined by the quantitative RIA described in this paper, which assumes that the efficiency of detection of different monoclonal IgG antibodies by ‘%I-RAMIG does not vary greatly, could account for some of the lateral shifts in the competitive binding curves. Whatever the explanation, however, it does not affect our conclusions regarding the spatial distribution of epitopes within distinct antigenic regions on the HA. The results presented demonstrate that the competitive binding assay utilizing hybridoma antibodies is a powerful technique for the topographical analysis of complex antigens such as the HA of influenza virus. To date, four distinct antigenic regions have been demonstrated, and it is likely that additional regions will be detected as more monoclonal antibodies are used. A more precise analysis of the physical relationships between distinct antigenie regions and of the arrangement of epitopes within regions may be possible by using Fab fragments of monoclonal antibodies in this type of competitive binding assay. It should be stressed that the antigenic sites delineated by our competitive bind-

TOPOGRAPHY OF INFLUENZA

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ing RIA do not necessarily correspond EY, P. L., PROWSE,S. J., and JENKIN, C. R. (1978). Isolation of pure IgGi, IgG%, and IgGzb immunowith those operationally defined by HI globulins from mouse serum using protein A-sereactivity patterns (Gerhard et al., 1980), pharose. Immunochemistry 15,429-436. selection of variants (Webster and Laver, FAZEKAS DE ST. GROTH, S., and WEBSTER, R. G. 1980), or location of amino acid substitu(1966). Disquisitions on original antigenic sin. I. tions on a three-dimensional model of HA Evidence in man. J. Eacp. Med. 124.331-345. (Wiley et al., 1981). The latter approaches FRANKEL, M. E., and GERHARD,W. (1979). The rapid map the position of amino acid changes, determination of binding constants for antiviral whereas competition RIA maps the sites antibodies by a radioimmunoassay. An analysis of to which antibodies actually bind; the two the interaction between hybridoma proteins and influenza virus. Mol. Immunol. 16,101-106. may not correspond in the case of substitutions which, by altering the conforma- GERHARD, W., CROCE,C. M., LOPES, D., and KoPROWSKI,H. (1978). Repertoire of antiviral antition of the HA molecule, change an antibodies expressed by somatic cell hybrids. Proc. genie site perhaps some considerable Nat. Acad. Sci. USA 75,1510-1514. distance away. A complete understanding W., and WEBSTER,R. G. (1978). Antigenic of the nature, number, and location of the GERHARD, drift in influenza A viruses. I. Selection and charantigenic sites on the influenza virus HA acterization of antigenic variants of A/PR/8/34 will require the interpretation of data ob(HONl) influenza viruses with monoclonal antitained using a variety of experimental apbodies. J. Exp. Med. 148,333-392. proaches. GERHARD,W., YEWDELL,J., and FRANKEL, M. (1980). ACKNOWLEDGMENTS We are particularly indebted to Dr. Walter Gerhard, who provided hybridomas H14A20 and H14A21 for comparison with our own. Miss Joanne Dean and Miss Lorraine Vogels provided technical assistance, while Miss Jacinta Hoare assisted with some of our early attempts to produce hybridomas. We would also like to thank Drs. Margot Anders, David Jackson, Lorena Brown, and Robin Anders for helpful discussions. One of us (A.M.B.) was a recipient of a University of Melbourne Research Fellowship. The work was supported by a grant from the National Health and Medical Research Council of Australia. REFERENCES ANDERS, E. M., PEPPARD,P. M., BURNS,W. H., and WHITE, D. 0. (1979). In vitro antibody response to influenza virus. I. T cell dependence of the secondary response to hemagglutinin. J. Immwwl. 123, 1356-1361. DAVIS, N. C., and HO, M. (1976). Quantitation of immunoglobulins. In “Manual of Clinical Immunology” (N. R. Rose and H. Friedman, eds.), pp. 4-16. American Society for Microbiology, Washington, D. C. DOWDLE,W. R. (1976). Influenza virus. In “Manual of Clinical Immunology” (N. R. Rose and H. Friedman, eds.), pp. 433-437. American Society for Microbiology, Washington, D. C. EAST, I. J., TODD, P. E., and LEACH, S. J. (1980). On topographic antigenic determinants in myoglobins. Mol. Immunol. 17.519-525.

An experimental approach to define the antigenic structures of the hemagglutinin molecule of A/ PR/8/34. In “Proceedings of the International Workshop on Structure and Variation in Influenza Virus, Thredbo, Australia” (W. G. Laver and G. Air, eds.), pp. 273-281. Elsevier/North-Holland, New York. JACKSON,D. C., BROWN,L. E., and WHITE, D. 0. (1981). Antigenic determinants of influenza virus haemagglutinin. VI. Antigenic characterization of the oligosaccharide sidechains from HAi of influenza virus haemagglutinin. J. Cen. Viral. 52, 163168. JACKSON,D. C., BROWN,L. E., WHITE, D. O., DoPHEIDE, T. A. A., and WARD, C. W. (1979). Antigenie determinants of influenza virus hemagglutinin. IV. Immunogenicity of fragments isolated from the hemagglutinin of A/Memphis/72. J. Zmmunoi. 123,2610-2617. K~HLER, G., and MILSTEIN, D. (1976) Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol. 6, 511519. KOPROWSKI,H., GERHARD, W., and CROCE,C. M. (1977). Production of antibodies against influenza virus by somatic cell hybrids between mouse myeloma and primed spleen cells. Proc. Nat. Acad sci. USA 74, 2985-2988.

LAVER, W. G., and WEBSTER,R. G. (1966). The structure of influenza viruses. IV. Chemical studies of the host antigen. Virology 36,104-115. LAVER, W. G., AIR, G. M., WEBSTER,R. G., GERHARD, W., WARD, C. W., and DOPHEIDE,T. A. A. (1979). Antigenic drift in type A influenza virus: Sequence differences in the hemagglutinin of Hong Kong

140

BRESCHKIN

(H3N2) variants selected with monoclonal hybridoma antibodies. Viroh 98,226-237. RUSSELL,R. J., BURNS,W. H., WHITE, D. 0.. ANDERS, E. M., WARD, C. W., and JACKSON,D. C. (1979). Antigenic determinants of influenza virus hemagglutinin. III. Competitive binding of antibodies directed against common and strain-specific antigenie determinants of A/Memphis/72 hemagglutinin. J. ImmunoL 123.825-832. RUSSELL,R. J., and JACKSON,D. C. (1978). Direct solid phase radioimmunoassay for measuring antigenic differences between the hemagglutinins of influenza viruses. J. ImmunoL Methods 22.201-209. STONE,M. R., and NOWINSKI,R. C. (1980). Topological mapping of murine leukemia virus proteins by competition-binding assays with monoclonal antibodies. Virology 100.370-381. URBANSKI, G. J., and MARGOLIASH,E. (1977). Topographic determinants on cytochrome C. I. The com-

ET AL.

plete antigenic structures of rabbit, mouse, and guanaco cytochromes C in rabbits and mice. J. Immunol. 118.1170-1180. WEBSTER,R. G., and LAVER, W. G. (1980). Determination of the number of non-overlapping antigenie areas on Hong Kong (H3N2) influenza virus hemagglutinin with monoclonal antibodies and the selection of variants with potential epidemiological significance. Virology 104,139-148. WILEY, D. C., WILSON,I. A., and SKEHEL, J. J. (1981). Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature (Lcmdo?z) 289,373-378.

WRIGLEY, N. G.,LAvER, W. G., and DOWNIE, J. C. (1977). Binding of antibodies to isolated hemagglutinin and neuraminidase molecules of influenza virus observed in the electron microscope. J. Mol. Biol. 109.405-421.