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An antigenic map of the haemagglutinin of the influenza hong kong subtype (H3N2), constructed using mouse monoclonal antibodies
Molecular Immunology, Vol. 21, Printed in Great Britain
No. I, pp. 663-671,
1984 0
0161~5890/84 $3.00 + 0.00 1984 Pergamon Press Ltd
AN ANTIGENIC MAP OF THE HAEMAGGLUTININ OF THE INFLUENZA HONG KONG SUBTYPE (H3N2), CONSTRUCTED USING MOUSE MONOCLONAL ANTIBODIES P. A. UNDERWOOD CSIRO Division of Molecular Biology, P.O. Box 184, North Ryde, N.S.W. 2113, Australia (First received 3 February 1984; accepted in revised form 14 March 1984)
Abstract-Panels of monoclonal antibodies were raised to the haemagglutinin of strains of the Hong Kong subtype of influenza (H3N2), namely Hong Kong 1968, England 1972, Port Chalmers 1973, Victoria 1975 and Texas 1977. The probable binding sites of individual antibodies in each panel were determined by correlation of cross-reactivity with 16 heterologous strains from the same subtype, and amino acid differences between their haemagglutinin molecules. This led to an approximation of the “average repertoire” of the BALB/c mouse to influenza type A haemagglutinin. Comparative data of the cross-reactivity of whole mouse sera suggested that the monoclonal panels derived from spleen cells were a fair representation of circulating antibody. Variation in the cross-reactivities of individual sera approached that of individual monoclonal antibodies. The relevance of this finding to selection of new virus variants in the human population was discussed. Heterospecific antibodies were detected in some monoclonal panels. Specific amino acid changes which could be responsible for such activity were identified.
INTRODUCTION The use of monoclonal
antibodies as tools to study antigenic structures is rapidly becoming commonplace. The surface antigens of influenza A viruses have been intensively studied, particularly the haemagglutinin (HA) molecule. This 63,000 mol. wt glycoprotein is the major surface antigen of the virus and comprises two subunits, HA1 and HA2. The former contains most of the antigenic activity (Brand and Skehel, 1972; Eckert, 1973). Each of the five influenza A subtypes which have caused pandemics in man exhibits a process of sequential antigenic drift, giving rise to a series of immunologically distinct but closely related virus strains. Determination of the RNA sequences coding for the HA of a number of strains of the Hong Kong (H3N2) subtype [see Both et al. (1983)] has revealed cumulative changes in a large number of amino acids in HAl. Elucidation of the 3-dimensional structure of the HA molecule (Wilson et al., 198 1) revealed many of these changes to be clustered in close physical proximity on the surface of HA1 and led to the postulate of four-five distinct antigenic areas (Wiley et al., 1981). This was in broad agreement with the definition of three-four discrete operational determinants on the HA1 of representatives of four different subtypes (Webster and Laver, 1980b; Gerhard et al., 1981; Nakajima and Kendal, 1981; Jackson et al., 1982). These conclusions were derived from analysis of reactivity patterns of panels of monoclonal antibodies, with mutant virus strains produced in vitro under pressure of individual monoclonal antibodies. Breschkin et al. (1981) and Jackson et al. (1982) described four topographical determinants on 663
the HAS of two different H3N2 strains by determination of the degree of mutual competition between pairs of monoclonal antibodies. Competitive binding studies also detected considerable overlap between these topographical sites and led to the proposal that the surface of the HA molecule might be described as an antigenic continuum (Lubeck and Gerhard, 1981; Jackson et al. 1982; Underwood, 1982). This idea was also supported by Green et al. (1982) who found that a series of synthetic peptides representing most of the surface of the HA1 molecule were all antigenic, and that antibodies raised to such peptides were capable of binding to the whole HA molecule and to intact virus. In a previous report I described the assignment of target areas (possible binding sites) of a large panel of monoclonal antibodies to the HA molecule of the H3N2 strain NT60/68 (Underwood, 1982). This was achieved by correlating the cross-reactivity patterns of individual monoclonal antibodies (or similar groups) with known amino acid sequence differences in HA1 of natural and laboratory virus variants. The results supported the idea of an antigenic continuum with emphasis (in terms of the proportion of the total panel reactive with different areas) on one or two particular regions. The virus NT60/68 is a representative of the earliest strains of the Hong Kong subtype. In the present study, panels of monoclonal antibodies were produced to five subsequent Hong Kong strains whose dates of isolation ranged from 1968 to 1977. The target areas of these antibodies were mapped to determine whether the regions of particular antigenic emphasis changed with progressive evolution of the virus, or whether the particular distribution observed
P. A.
664
UNDERWOOD
for NT60/68 represented a general antigenic map of the Hong Kong subtype as perceived by the BALB/c mouse. MATERIALS AND METHODS
Viruses
remove non-specific inhibitors of haemagglutination (Jensen, 1961). Cross-reactivity profiles were performed as for the HK68 monoclonal panel (see later). Analysis of cross-reactivity pro$les antibodies
of monoclonal
The cross-reactivity profile of each antibody in a particular panel was obtained by calculating the relative HI titres against 16 heterologous viruses, compared with that against the homologous virus. Mainline strains [international reference strains and The degree of relatedness between any two antibodies very closely related lateral variants-see Both et al. in a particular panel, and the classification of the (1983)]. X31 recombinant of Aichi/2/68 (HK68), panel into groups of similar cross-reactivity, was Northern Territory/60/68 (NT68), England/878/69 performed by methods of numerical taxonomy as England/42172 Port (ENG69), (ENG72), previously described (Underwood, 1982). Amino Chalmers/l~73 (PC73), Hanover/l/74 (HAN74), South Australia/54174 (SA74), V~ctor~a~3175 acids affecting the binding of each antibody group were identified by correlation of the cross-reactivity (VIC75), TEX/BEL recombinant of Texas/l/77 patterns of the antibodies, with clusters (in the (TEX77) and Bangkok/l/79 (BK79). 3-dimensional structure of HAl) of amino acid Terminal strains [antigenically distinct and indifferences between the heterologous and homoloconsistently isolated-see Both et al. (1983)J. gous viruses. Such amino acids defined antibody Queensland/7/70 (Q70), Hong Kong/107/71 (HK71), “target areas”, which obeyed the constraints of antiMemphis/102/72 (MEM72 = MEM/PR8 recombinant), Artenay/l/74 (ART74) and Victoria/l 12/76 body binding sites known to date (Amzel and Poljak, 1979). The target area was comprised of a core region (VIC76). and a peripheral region (East et al., 1980), described Laboratory mutants (Fazekas de St. Groth and in detail previously (Underwood, 1982). Hannoun, 1973). 375/17, 34C, 29C and 30D. The number at the end of each mainline or terminal In brief, the core region was defined by physically strain denotes the year of isolation, e.g. Aichi/2/68 close (within 2nm in the 3-dimensional structure) amino acids which changed in viruses which consistwas isolated in 1968. ently were prevented from binding to any member of Viruses were grown and purified as described prethe antibody group. The periphery included residues viously (Underwood, 1982). adjacent to the core, which changed in viruses exVirus and antibody titrafio~s. Haemagglutination hibiting variable degrees of binding with individual and haemagglutination-inhibition (HI) titrations members of the antibody group. None of these were performed as previously described (Underwood, 1982). particular amino acid differences could be shared by viruses exhibiting full reactivity with all members of Preparation of monoclonal antibodies the antibody group. In most cases this limited the possible position of core + periphery to one, or someBALB/c mice received ip injections of 1000 haemagglutinating units (HAU) of purified virus. At times two, areas of the HA1 molecule for each least 4 weeks Later, booster injections (iv or ip) of antibody group. Such an area is referred to as a “target area” rather than a binding site, since, 10,00~2O,O~HAU were given and the spleens removed for fusion after 34 days. Between 5 and 20 although likely, the coincidence of the two is not proven. mice were used for each virus strain. Hybridomas, using the Sp2/0 myeloma line (Shulman et al., 1978) RESULTS and spleens of mice immunised with HK68, ENG72, PC73, WC75 or TEX77 were prepared, and monoAntibody target areas clonal antibodies produced and purified as previously described (Underwood, 1982). For each fusion, samA summary of the grouping of the various monoples of several spleens were used to maximise the donal panels is shown in Table 1 and details of the target areas of two individual panels are shown in diversity of monoclonal antibodies produced. Therefore, the antibody panels represented the response of Table 2. For the sake of brevity detailed target areas for the remaining three panels are not shown. Simithe strain BALB/c to each virus strain, rather than larly cross-reactivity patterns of individual groups are that of individual mice. Production of immune sera. Groups of 15 BALB/c not shown but can be deduced from the sequence data used to identify target areas, given in Table 3. mice received doses of 1000 HAU of HK68 virus Summarised target areas for all the monoclonal either intraperitoneally or subcutaneously (SC). After panels are given in Table 4. The relative positions of 1 month booster injections of 1000, 10,000 or 20,~O or amino acid residues included in target areas, in the HAU were given either intraperitoneally 3-dimensional structure of the HA1 molecule, are SC+ intravenously. Animals were bled 7 days later and the sera treated with trypsin and periodate to shown in Fig. 1. The egg-adapted epidemic strains of the A/H3N2 subtype were as follows.
Antigenic
map of haemagglutinin Table
1. Summary
Panel raised to virus
of influenza
of analysis
of the five monoclonal
No. of antibodies in panel
HK68 ENG72 PC73 VIC75 TEX77 Total
Hong Kong subtype (H3N2)
No. of target areas defined
75 13 22 25 31
12 12 22 23 9
I66
118
665
panels” No. of antibody groups 7 6 I 8 6
“Viruses in the heterologous panel used to place antibodies in common crossreactivity groups: HK68, 375/17, 34C, 29C, 30D, ENG69, Q70, HK71, ENG72, MEM72, PC73, VIC75, VIC76, TEX77, HAN74, ART74 and SA74. Sequence data (Table 2) available for the first 14 of these were used to identify the target areas of the individual antibody groups. BK79 was used in place of 34C to analyse the TEX77 panel.
A total of three heteroclitic (heterospecific) antibodies were detected amongst the five antibody panels. These antibodies displayed considerably increased (400%) HI titres for particular heterologous virus strains. One antibody from the VIC75 panel bound more avidly to WC76 than to VIC75. The target area of this antibody includes HA 1 residue 155 [Table 2(b), group 81. VIC76 has a change in position 157 from Ser to Leu. An antibody binding closely to the Leu side chain could still be accommodated by the smaller Ser side chain, but with lower binding energy. A similar effect was observed with two antibodies from the PC73 panel. These antibodies also appeared to bind in the 155-160 region [Table 2(a), group 51 and showed increased affinity for ENG72 and MEM72. The residue likely to be responsible is No. 160 which is Thr in the latter two strains and Ala in PC73. Antibodies binding closely to the Thr side chain could be accommodated by the smaller Ala with reduced binding energy. Similar heteroclitic antibodies (to cytochrome c and myoglobin respectively) have been described by Urbanski and Margoliash (1977) and East et al. (1982~). Table 2. Target
Considerable difficulty was experienced in preparing monoclonal antibodies to ENG72 virus. This particular strain was very labile in the sucrose density gradient purification procedure (unpublished observations). Factors associated with this instability may have reduced the immune response of the mice to the native HA 1. The monoclonal antibodies raised to TEX77 were very unreactive with all of the virus strains tested. Twenty of the 3 1 antibodies examined did not crossreact (titres < 1% of homologous) at all with even the closest relative, BK79. The number of reactivity groups identified for each panel of antibodies was very similar (Table I), even when the panel contained less than 20 antibodies. The target areas they addressed showed many features in common (Table 4). All panels contained the highest proportion of antibodies directed to the tip of the HA1 molecule [site B (Fig. l)]. In general, the second most common target area was the interface region between adjacent HA1 molecules [site D + AB (Fig. l)]. Reactivities to the side face BA and the loop A (Fig. 1) were present in low numbers, or absent in
areas of two representative Target
Group
No. of antibodies
Core (a) Antibodies
I 2 3 4
6 4 4 2
5
2
6
2
7
2
I
6 4
229, 230, 220/186 188, 198 155, 157, 159 155, 157, 158 (129, 159) 155, 157, 158, 160’ 189, (159, 129) 157, 158 or 145, 146 188, 189. 201 (b) Antibodies
2 3 4 5 6 7 8
4 2 2 3
I 1
155, (159, 129) 155, 157, 158 (159, 129) 158, 159 155, 157, 158, 193 229, 230, 186/220 144 132 155, 157’
monoclonal
panels
area’ Periphery
Area code”
to PC73 182, 226, 1291159 188, 189, 158, 129 188, 189
D B B B B
189 137
B A B
to VIC75 157. 158
129 226 145, 146
B B B B AB A BA B
“Target areas identified by position of amino acids in primary sequence of HAl. “Target area code after Wiley e! al. (1981), Underwood (1982) and Fig. I. ‘Amino acid change likely to account for heteroclitic activity in this antibody group (see text).
-
2 3 9 10 31 34 50 53 54 62 63 78 81 83 122 126 129 132 133 137 143 141 145 146 1.55 156 157 158 159 160 164 172 174 182 186 188 189 193 197 198 201
Amino acid*
Ser Asn Gin Ser Gln Ala Arg
IS0
Asp Phe
GUY GhI ASII A.Sn Pro G’Y Ser GUY Thr LYS Ser GUY Ser Thr Leu
Asp Val Asn Thr Thr Thr
IS0
LYs Asn Asn
IS0
Asp Leu Ser Thr Asp
HK68
Val
Asp
ASII
375117
GUY
Val
Asn
34c
Val
Asp
Asn
29C
IS0
Vat
Asp
ASfl
30D
Val
Asp
Asp
AStI
AW
ENG69
Val
GlU
GlY
Asn
070
Table 3. Variable
Thr
Asp
Val
A%
Asp
Giu Gill
Tht
LYS
HK71
Vdl
Asp
Val
TY~
TY~
‘W
Tht
Asn
Asp
Val
Ala
Asp
Asn
Asn GlY
AStl
Phe
PC73
Asp
Asn
Asn GUY
Asn
MEM72
LYS
Asp LYS Asn
Ser Val
Gin
TY~
Asp
Ser
ASII
LYS
Gh
Asn
Asn
WC75
1968.-1979”
Asp
AStl
ASfl GUY
Asn
ENG72
amino acids in HA1 of H3N2 subtype,
LYS
Asp LYS Asp
Ser Val
Gin
Leu
TYT
Asp Asn
Ser
Lys Asn AStt
AW GUY
Asp
Asn
WC76
Asp LYS Asn
Val
GUY
GlU
TY~
Asp Asn
TY~
Asn Asn
LYS
Am GUY
Arg Asp Ser LYS
Asp LYS Asn Arg
Val
GUY
LYS
GIU
Ser TY~ Se1 Asp Asn Ser TY~ GlU
LYS Asn Asn
Lys Asn GUY
Arg Asp Ser
Asn
Aslt
Asn
Asn
BK79
l’EX77
D+AB D+AB D+AB, B B B B B B
B
E DfAB D+AB, BA B, BA BA A D+AB, A A A A A B B B B B, BA B, BA D+AB
Area code
Antigen& map of haemagglutinin of influenza Hong Kong subtype (W3N2)
667
some panels. Changes in site E (Fig. 1) appeared to be correlated with changes in the interface region (Underwood, 1982) and could not be separately resolved with panels to viruses isolated later than 1972. Antibodies affected by changes in site C [Fig. l(b)] were not detected in any panel. Experiments with whole sera In order to test whether the average crossreactivities observed with the monoclonal panels reflected those in circulating serum, cross-reactivity patterns of mouse sera derived from several different inoculation regimes were compared with the monoclonal panels. The results for sera and monoclonal antibodies raised to HK68 are shown in Table 5. It is clear that the variation in the response of individual mice is very great. It is also clear that the ranges of cross-reactivity are similar for each immunisation regime. What is surprising is that the range of cross-reactivities observed between individual sera is nearly as great as that displayed by individual monoclonal antibodies. This indicates that the repertoires of individual mice may be largely restricted to one or two target areas. The means of the six immunisation groups were not normally distributed, indicating that five mice were insufficient to give a true representation of the average response. The mean crossreactivities of the mono~lonal antibodies, however, fall largely within, or very close to, the range of the means of the groups of sera. Exceptions were crossreactivities towards HK71 and 29C which were both considerably higher with the monoclonals than with whole sera. Either the spleens used for fusion contained fewer cells reactive towards HA1 residues 132 and 226 than serum antibodies; or such cells possessed some growthjhybridisation disadvantage; or their products were of low affinity. In the latter case, when mixed in serum, their contribution towards average cross-reactivity, would be lower than expected from their proportional representation in the cell population. With these two exceptions, the HK68 monoclonal panel gave a fair representation of the average BALB/c serum response. DISCUSSION
In a previous report I described the assignment of target areas (possible binding sites) of a large panel of monoclonal antibodies to the HA molecule of the H3 strain NT60/68 (Underwood, 1982). This was achieved by correlating the cross-reactivity patterns of individual monoclonal antibodies (or similar groups) with the 3-dimensional location of known amino acid sequence differences in both natural and laboratory variants. This type of antigenic mapping by correlation of cross-reactivity of monoclonal antibodies with sequence differences in variants of the antigen has been described for several other protein antigens [Schroer
668
P. A. UNDERWWD
Table 4. Proposed antibody target areas on HAI of the H3N2 subtype: representation mo”oclo”al DaneIs Interface
Side Top of molecule B
Target arean Contributory’ amino acid residues in HA1 Monoclonal panels to HK68 NT68“ ENG72 PC73 VIC75 TEX77
BA
188-198 201
155-160 188-193 129
155-160 129
0'
44.0
0
12.8 0 27.3 0 8.6
36.8 61.5 36.4 8.0 5.7
0 0 0 60.0 2.9
126132 159-160
0
11.2 15.4 0 4.0 0
of antibodies to each target area in different
D 160, 174. 122. 242,
D+AB 164 207 126 244
0
23.4 1.7 0 0 0
21.9 0 0 21.3 0 5.7
Loop A
Hinge C
137, 220 226 229, 230 182, 186
143-146 133, 137
5fS-54 215, 287
12.0 6.4 0 0 8.0 0
12.0 2.4 0 9.1 12.0 2.9
AB
0
E
62-63 78 81, 83
1.33 8.8 1.7 0 0 0
“Area letter codes from Wiley et al. (1981) and Underwood (1982). ‘Contribution of amino acids deduced from above references and present data. ‘Percentage of panel of monoclonal antibodies affected by changes in the designated area (difference in total from 100% represents unplaced antibodies). Numbers under area E are also included in the interface region since changes in area E appear to affect antibodies binding in this region (see text). dData from Underwood (1982).
(1981), beef insulin; Berzofsky et al. (19826), sperm whale myoglobin; East et al. (1982b), human myoglobin; Stamatoyannopoulos et af. (1981), human haemoglobin; Smith-Gill et al. (1982) chicken lysozyme; Boulain et al. (1982), Nuju nigricolh toxin; Al Moudallal (1982) tobacco mosaic virus protein]. The underlying assumption in this type of approach is that the overall 3-dimensional structure or related variant proteins is highly conserved, and that amino
acid differences are likely to cause only localised perturbations. This has been demonstrated by X-ray crystallography in the case of species variants of lysozyme (Smith-Gill et a/., 1982). Results of such studies have led to the hypothesis of a universal potential for the complete surface of an antigen to be antigenic in an appropriate host (Urbanski and Margoliash, 1972; White et al., 1978; East et al., 19826; Todd et al., 1982; Leach, 1983). The surface of the influenza HA molecule also appears to exhibit this potential (Lubeck and Gerhard, 1981; Jackson et al., 1982, Green et al., 1982; Underwood, 1982). In the present study the mapping of antibody target areas on the H3 influenza HA was extended to
(b)
Fig. I. Drawings of the alpha carbon skeleton of regions of antigenic importance of the HA1 chain of Aichi68 (taken from stereo drawings loaned by Dr D. Wiley). (a) and (b) represent opposite views of the molecule. Area coding after Wiley et al. (1981) and Underwood (1982).
Antigenic
map
of haemagglutinin
of influenza
Hong
Kong subtype (H3N2)
669
Table 5. Comparison of cross-reactivities of monoclonal antibodies and whole mouse sera, raised to HK68 Serum means Ranges of % cross-reactivity of mouse sera for each immunisation group Virus 375117 ENG69 470 HK71 ENG72 MEM72 PC73 WC75 VIC76 TEX71 34C 29C 30D HAN74 ART74 SA74
sc 1” 43-132 29-100 543 >12 47-l 15 14-50 <4-15 <422 <&15 <4-14 76-185 22-50 <4-22 t‘v35 <45 13-38
SC 10
sc 20
ip 1
ip 10
ip 20
4-150 <4-163 <4-25 <4-12 4300 <4107 <47 <4-41 <4-5 147 lW300 ~476 <+41
50-100 12-100 747 t49 50-100 <‘&47 <4-9 t423 t49 <49 10&200 < 450 <&9 <4-9 <4-9 <4-20
31-93 18-50 6-15 <4-12 14-66 l-12 2-l 1
22-100 l&l07 <4-38 tk20 19-100 <&13 <4-11 <4-8 14-E ~4-8 87-130 547 &14 tr150 <4-8 ~423
47-93 41-93 <4-29 <4-12 38-100 12-27 <4-9 <4-9 <&9 <49 71-187 12-50 4623
y0 cross-reactivity” of monoclonals
OVerall mean
Range of group means
Individual range
Overall mean
72.3 64.1 19.8 6.4 70.1 16.0 5.2 6.0 3.9 4.2 107 24.2 9.2 11.1 3.6 11.6
62-86 55-92 1l-34 5-9 32-112 4-30 3-9 l-13 l-8 2-8 91-145 1l-30 &13 5-19 l-7 5-28
< 4200 <4170 <‘&I5 tk80 t4-150 <‘&IO0 <4-95 <4-50 <&18 <4-18 <4-180 <4-150 c&50
93.5 91.3 10.0 21.3’ 49.3 12.8 11.1 5.5 2.7 2.6 80.2 49.5’ 4.8 21.2 3.2 3.5
“Heterologous HI titre as percentage of the homologous titre to HK68. ‘Immunisation scheme. SC:subcutaneous prime, subcutaneous/intravenous boost. ip: intraperitoneal prime and boost. The number represents the dose in thousands of HAU. ‘These means lie well outside the range of the serum means.
monoclonal panels raised against five virus strains isolated between 1968 and 1977. With each panel, a similar distribution pattern of target areas to that previously described for NT68 (Underwood, 1982) was observed. This finding validates the assumption that no major conformational changes in the HA take place during evolution of the virus, which has yet to be demonstrated by X-ray crystallography. This means that although about 10% of the amino acids of HA1 have changed between 1968 (HK) and 1977 (TEX), and most of these changes are in surface residues, the mouse is still stimulated to produce the same topological repertoire of antibody molecules with the same proportional emphasis on the various target areas. Most of the exposed surface of the HA molecule appeared to be antigenic, although some individual epitypes were absent from the smaller panels. In each case the greater proportion of antibodies recognised changes on the tip of the HA molecule [site B of Wiley et al. (1981)). Target areas in the two components of the interface (site D) were also present in high numbers (see Table 4 and Fig. 1). Caton et al. (1982) analyzed the binding sites of monoclonal antibodies to a strain from a different subtype (HONl), employing a large number of laboratory mutants with known sequence changes rather than field strains, and obtained very similar results. Both studies also identified lower numbers of antibodies reacting with the loop (A) and an interaction between area E and the loop containing residue 122 (Fig. 1 and Table 4). Neither study identified any antibodies reactive with residues corresponding to site C. A recent analysis of sequence differences of the HAI of strains of yet another subtype (HlNl), evolved from A/USSR/77 emploqing laboratory-selected mutants, has identified target areas corresponding to sites B and D (Kendal et al., 1983).
The very similar distribution of target areas and similar proportional emphasis described by Caton et al. (1982) for the HO HA, and in the present report for H3, suggests that this pattern approximates the average repertoire of BALB/c mice to all subtypes of influenza type A HA, a correlation not hitherto demonstrated. Both studies have used large monoclonal panels and large numbers of animals to produce them, in order to optimise the representation of the complete BALB/c repertoire. This is an important consideration and Yewdell and Gerhard (198 1) have also suggested that different immunisation regimes and different organ sources of immune lymphocytes should be used. Analysis of cross-reactivity patterns of individual mouse sera reported in the present study indicates that large (> 20) panels of monoclonal antibodies are representative of the in viuo antibody repertoire. This has not been previously demonstrated although it has been suggested. Moreover, the variation in crossreactivity patterns observed between individual whole sera approached that observed between different individual monoclonal antibodies. This suggests that the antibody repertoires of individual mice may be restricted to one or two target areas. These findings are in agreement with Staudt and Gerhard (1983), who demonstrated differences between the repertoires of monoclonal panels derived from different individual mice. Natali et al. (1981) reported individual differences between human sera to a late H3N2 virus strain when titrated against virus variants possessing an amino acid change in only one haemagglutinin epitope. Restriction of epitope recognition has been reported for some rabbit and ferret sera raised to H3N2 and HlNl strains respectively (Webster and Laver, 1980a; Nakajima and Kendal, 1981). In those two reports, however, individual variation in epitope recognition was not demonstrated.
P. A. UNDERWOOD
670
The variation in individual mouse repertoires reported here supports the hypothesis that influenza evolution progresses by an accumulation of mutations in different epitopes as the virus passes sequentially through individuals possessing different antibody repertoires. Acknowledgements-1 am grateful to Dr G. W. helpful discussion, and to Mrs R. MacDonald, N. Miss P. Bean for expert technical assistance, and Drummond and Mrs M. Dowell for preparing script.
Both for Villa and to Mrs J. the type-
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Gerhard W., Yewdell J. and Frankel M. E. (1981) Antigenic structure of influenza virus haemagglutinin defined by hybridoma antibodies. Nature, Lond. 290, 7 13-7 17. Green N., Alexander H., Olson A., Alexander S., Shinnick T. M., Sutcliffe J. G. and Lerner R. A. (1982) Immunogenic structure of the influenza virus hemagglutin. Cell 28, 477487. Jackson D. C., Murray J. M., White D. 0. and Gerhard W. (1982) Enumeration of antigenic sites of influenza virus hemagglutinin. Infect. Immunity 37, 912-918.
Jensen K. E. (1961) Diagnosis of influenza by serological methods. Am. Rev. resp. Dis. 83, 120-124. Kendal A. P., Cox N. J., Nakajima S., Nakajima K., Raymond L., Caton A., Brownlee G. and Webster R. G. (1983) Analysis of the antigenic structure of influenza A/USSR/90/77 hemagglutinin. Presentation at Symposium on Molecular Biology of Viruses, Moscow, June 1983. Laver W. G., Air G. M., Webster R. G., Gerhard W., Ward C. W. and Dooheide T. A. A. (1980) The antiaenic sites on influenza virus haemagglutiiin: studies on their structure and variation. In Structure and Variation in Infruenza Virus (Edited by Laver W. G. and Air G. M.), pp. 190-198. Elsevier, New York. Leach S. J. (1983) How antigenic are antigenic peptides? Biopolymers 22, 425440. Lubeck M. D. and Gerhard W. (1981) Topographic mapping of antigenic sites on the influenza A/PR/8/34 virus haemagglutinin using monoclonal antibodies. Virology 113, 64-72. Moss B. A., Underwood P. A., Bender V. J. and Whittaker R. G. (1980) Antigenic drift in the haemagglutinin from various strains of influenza virus A/Hong Kong/68 (H3N2). In Structure and Variation in Influenza Virus (Edited by Laver W. G. and Air G. M.), pp. 329-338. Elsevier, New York. Nakajima S. and Kendal A. P. (1981) Antigenic drift in influenza A/USSR/90/77 (H 1N I) variants selected in vitro with monoclonal antibodies. Virology 113, 656-662. Natali A., Oxford J. S. and Schild G. C. (1981) Frequency of naturally occurring antibody to influenza virus antigenie variants selected in vitro with monoclonal antibody. J. Hyg., Camb. 87, 185-191. Schroer J. A. (1981) Hybridoma antibody recognition of the insulin molecule. In Monoclonal Antibodies in Endocrine Research (Edited by Fellows R. and Eisenbarth G.), pp. 167-179. Raven Press, New York. Shulman M., Wilde C. D. and Ktjhler G. (1978) A better cell line for making hybridomas secreting specific antibodies. Nature, Lond. 276, 269-270. Sleigh M. J., Both G. W., Underwood P. A. and Bender V. J. (1981) Antigenic drift in the haemagglutinin of the Hong Kong influenza subtype: correlation of amino acid changes with alterations in viral antigenicity. J. Virol. 37, 845-853. Smith-Gill S. J., Wilson A. C., Potter M., Prager E. M., Feldmann R. J. and Mainhart C. R. (1982) Mapping the antigenic epitope for a monoclonal antibody against lysozyme. J. Immun. 128, 314-322. Stamatoyannopoulos G., Lindsley D., Farquhar M., Bruce M., Papayannopoulou T. H., Nute P. E., Serjeant G. R. and Lehmann H. (1981) Mapping of antigenic sites on human haemoglobin by means of monoclonal antibodies and haemoglobin variants. Luncet 1981, 952-954. Staudt L. M. and Gerhard W. (1983) Generation of antibody diversity in the immune response of BALB/c mice to influenza virus haemagglutinin. J. exp. Med. 157, 687-704. Todd P. E. E., East I. J. and Leach S. J. (1982) The immunogenicity and antigenicity of proteins. Trends biothem. Sci. 7, 212-216. Underwood P. A. (1982) Mapping of antigenic changes in the haemagglutinin of Hong Kong influenza (H3N2)
Antigenic map of haema~lutinin
of influenza Hong Kong subtype (H3N2)
strains using a large panel of monoclonal antibodies. J. gen. Viral. 62, 153-169. Urbanski G. J. and Margoliash E. (1977) Topo~aphic determinants on cytochrome c. J. Immun. 118, 1170-l 180. Verhoeyen M., Fang R., Min Jou W., Devos R., Huylebroeck D., Samas E. and Fiers W. (1980) Antigenic drift between the haemagglutinin of the Hong Kong inftuenza strains A/Aichi/2/68 and AiVictoria/3/75. Nature. Land. 286, 771-776. Webster R. G. and Laver W. G. (1980~) Antigenic drift in Hong Kong (H3N2) influenza viruses: selection of variants with potential epidemiological significance using monoclonal antibodies. In Structure and Variation in fn@uenza Yirus (Edited by Laver W. G. and Air G. M.), pp. 283-292. Elsevier, New York. Webster R. G. and Laver W. G. (1980h) Determination of the number of non-overlapping antigenic areas on Hong
671
Kong (H3N2) influenza virus hemagglutinin with monoclonal antibodies and the selection of variants with potential epidemiological significance. Viro~ugy 104, 139-148. White T. J., ibrahimi 1. M. and Wilson A. C. (1978) Evolutionary substitutions and the antigenic structure of globular proteins. Nature, Land. 274, 92-94. 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 mvolvement in antigenie variation. Nature, Land. 289, 373-378. Wilson I. A., Skehel J. J. and Wiley D. C. (1981) Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature, tend. 289, 366-373. Yewdell J. W. and Gerhard W. {198l) Antigenic characterization of viruses by monoclonal antibodies. A. Rev. Microbial. 35, 185-206.
No. I, pp. 663-671,
1984 0
0161~5890/84 $3.00 + 0.00 1984 Pergamon Press Ltd
AN ANTIGENIC MAP OF THE HAEMAGGLUTININ OF THE INFLUENZA HONG KONG SUBTYPE (H3N2), CONSTRUCTED USING MOUSE MONOCLONAL ANTIBODIES P. A. UNDERWOOD CSIRO Division of Molecular Biology, P.O. Box 184, North Ryde, N.S.W. 2113, Australia (First received 3 February 1984; accepted in revised form 14 March 1984)
Abstract-Panels of monoclonal antibodies were raised to the haemagglutinin of strains of the Hong Kong subtype of influenza (H3N2), namely Hong Kong 1968, England 1972, Port Chalmers 1973, Victoria 1975 and Texas 1977. The probable binding sites of individual antibodies in each panel were determined by correlation of cross-reactivity with 16 heterologous strains from the same subtype, and amino acid differences between their haemagglutinin molecules. This led to an approximation of the “average repertoire” of the BALB/c mouse to influenza type A haemagglutinin. Comparative data of the cross-reactivity of whole mouse sera suggested that the monoclonal panels derived from spleen cells were a fair representation of circulating antibody. Variation in the cross-reactivities of individual sera approached that of individual monoclonal antibodies. The relevance of this finding to selection of new virus variants in the human population was discussed. Heterospecific antibodies were detected in some monoclonal panels. Specific amino acid changes which could be responsible for such activity were identified.
INTRODUCTION The use of monoclonal
antibodies as tools to study antigenic structures is rapidly becoming commonplace. The surface antigens of influenza A viruses have been intensively studied, particularly the haemagglutinin (HA) molecule. This 63,000 mol. wt glycoprotein is the major surface antigen of the virus and comprises two subunits, HA1 and HA2. The former contains most of the antigenic activity (Brand and Skehel, 1972; Eckert, 1973). Each of the five influenza A subtypes which have caused pandemics in man exhibits a process of sequential antigenic drift, giving rise to a series of immunologically distinct but closely related virus strains. Determination of the RNA sequences coding for the HA of a number of strains of the Hong Kong (H3N2) subtype [see Both et al. (1983)] has revealed cumulative changes in a large number of amino acids in HAl. Elucidation of the 3-dimensional structure of the HA molecule (Wilson et al., 198 1) revealed many of these changes to be clustered in close physical proximity on the surface of HA1 and led to the postulate of four-five distinct antigenic areas (Wiley et al., 1981). This was in broad agreement with the definition of three-four discrete operational determinants on the HA1 of representatives of four different subtypes (Webster and Laver, 1980b; Gerhard et al., 1981; Nakajima and Kendal, 1981; Jackson et al., 1982). These conclusions were derived from analysis of reactivity patterns of panels of monoclonal antibodies, with mutant virus strains produced in vitro under pressure of individual monoclonal antibodies. Breschkin et al. (1981) and Jackson et al. (1982) described four topographical determinants on 663
the HAS of two different H3N2 strains by determination of the degree of mutual competition between pairs of monoclonal antibodies. Competitive binding studies also detected considerable overlap between these topographical sites and led to the proposal that the surface of the HA molecule might be described as an antigenic continuum (Lubeck and Gerhard, 1981; Jackson et al. 1982; Underwood, 1982). This idea was also supported by Green et al. (1982) who found that a series of synthetic peptides representing most of the surface of the HA1 molecule were all antigenic, and that antibodies raised to such peptides were capable of binding to the whole HA molecule and to intact virus. In a previous report I described the assignment of target areas (possible binding sites) of a large panel of monoclonal antibodies to the HA molecule of the H3N2 strain NT60/68 (Underwood, 1982). This was achieved by correlating the cross-reactivity patterns of individual monoclonal antibodies (or similar groups) with known amino acid sequence differences in HA1 of natural and laboratory virus variants. The results supported the idea of an antigenic continuum with emphasis (in terms of the proportion of the total panel reactive with different areas) on one or two particular regions. The virus NT60/68 is a representative of the earliest strains of the Hong Kong subtype. In the present study, panels of monoclonal antibodies were produced to five subsequent Hong Kong strains whose dates of isolation ranged from 1968 to 1977. The target areas of these antibodies were mapped to determine whether the regions of particular antigenic emphasis changed with progressive evolution of the virus, or whether the particular distribution observed
P. A.
664
UNDERWOOD
for NT60/68 represented a general antigenic map of the Hong Kong subtype as perceived by the BALB/c mouse. MATERIALS AND METHODS
Viruses
remove non-specific inhibitors of haemagglutination (Jensen, 1961). Cross-reactivity profiles were performed as for the HK68 monoclonal panel (see later). Analysis of cross-reactivity pro$les antibodies
of monoclonal
The cross-reactivity profile of each antibody in a particular panel was obtained by calculating the relative HI titres against 16 heterologous viruses, compared with that against the homologous virus. Mainline strains [international reference strains and The degree of relatedness between any two antibodies very closely related lateral variants-see Both et al. in a particular panel, and the classification of the (1983)]. X31 recombinant of Aichi/2/68 (HK68), panel into groups of similar cross-reactivity, was Northern Territory/60/68 (NT68), England/878/69 performed by methods of numerical taxonomy as England/42172 Port (ENG69), (ENG72), previously described (Underwood, 1982). Amino Chalmers/l~73 (PC73), Hanover/l/74 (HAN74), South Australia/54174 (SA74), V~ctor~a~3175 acids affecting the binding of each antibody group were identified by correlation of the cross-reactivity (VIC75), TEX/BEL recombinant of Texas/l/77 patterns of the antibodies, with clusters (in the (TEX77) and Bangkok/l/79 (BK79). 3-dimensional structure of HAl) of amino acid Terminal strains [antigenically distinct and indifferences between the heterologous and homoloconsistently isolated-see Both et al. (1983)J. gous viruses. Such amino acids defined antibody Queensland/7/70 (Q70), Hong Kong/107/71 (HK71), “target areas”, which obeyed the constraints of antiMemphis/102/72 (MEM72 = MEM/PR8 recombinant), Artenay/l/74 (ART74) and Victoria/l 12/76 body binding sites known to date (Amzel and Poljak, 1979). The target area was comprised of a core region (VIC76). and a peripheral region (East et al., 1980), described Laboratory mutants (Fazekas de St. Groth and in detail previously (Underwood, 1982). Hannoun, 1973). 375/17, 34C, 29C and 30D. The number at the end of each mainline or terminal In brief, the core region was defined by physically strain denotes the year of isolation, e.g. Aichi/2/68 close (within 2nm in the 3-dimensional structure) amino acids which changed in viruses which consistwas isolated in 1968. ently were prevented from binding to any member of Viruses were grown and purified as described prethe antibody group. The periphery included residues viously (Underwood, 1982). adjacent to the core, which changed in viruses exVirus and antibody titrafio~s. Haemagglutination hibiting variable degrees of binding with individual and haemagglutination-inhibition (HI) titrations members of the antibody group. None of these were performed as previously described (Underwood, 1982). particular amino acid differences could be shared by viruses exhibiting full reactivity with all members of Preparation of monoclonal antibodies the antibody group. In most cases this limited the possible position of core + periphery to one, or someBALB/c mice received ip injections of 1000 haemagglutinating units (HAU) of purified virus. At times two, areas of the HA1 molecule for each least 4 weeks Later, booster injections (iv or ip) of antibody group. Such an area is referred to as a “target area” rather than a binding site, since, 10,00~2O,O~HAU were given and the spleens removed for fusion after 34 days. Between 5 and 20 although likely, the coincidence of the two is not proven. mice were used for each virus strain. Hybridomas, using the Sp2/0 myeloma line (Shulman et al., 1978) RESULTS and spleens of mice immunised with HK68, ENG72, PC73, WC75 or TEX77 were prepared, and monoAntibody target areas clonal antibodies produced and purified as previously described (Underwood, 1982). For each fusion, samA summary of the grouping of the various monoples of several spleens were used to maximise the donal panels is shown in Table 1 and details of the target areas of two individual panels are shown in diversity of monoclonal antibodies produced. Therefore, the antibody panels represented the response of Table 2. For the sake of brevity detailed target areas for the remaining three panels are not shown. Simithe strain BALB/c to each virus strain, rather than larly cross-reactivity patterns of individual groups are that of individual mice. Production of immune sera. Groups of 15 BALB/c not shown but can be deduced from the sequence data used to identify target areas, given in Table 3. mice received doses of 1000 HAU of HK68 virus Summarised target areas for all the monoclonal either intraperitoneally or subcutaneously (SC). After panels are given in Table 4. The relative positions of 1 month booster injections of 1000, 10,000 or 20,~O or amino acid residues included in target areas, in the HAU were given either intraperitoneally 3-dimensional structure of the HA1 molecule, are SC+ intravenously. Animals were bled 7 days later and the sera treated with trypsin and periodate to shown in Fig. 1. The egg-adapted epidemic strains of the A/H3N2 subtype were as follows.
Antigenic
map of haemagglutinin Table
1. Summary
Panel raised to virus
of influenza
of analysis
of the five monoclonal
No. of antibodies in panel
HK68 ENG72 PC73 VIC75 TEX77 Total
Hong Kong subtype (H3N2)
No. of target areas defined
75 13 22 25 31
12 12 22 23 9
I66
118
665
panels” No. of antibody groups 7 6 I 8 6
“Viruses in the heterologous panel used to place antibodies in common crossreactivity groups: HK68, 375/17, 34C, 29C, 30D, ENG69, Q70, HK71, ENG72, MEM72, PC73, VIC75, VIC76, TEX77, HAN74, ART74 and SA74. Sequence data (Table 2) available for the first 14 of these were used to identify the target areas of the individual antibody groups. BK79 was used in place of 34C to analyse the TEX77 panel.
A total of three heteroclitic (heterospecific) antibodies were detected amongst the five antibody panels. These antibodies displayed considerably increased (400%) HI titres for particular heterologous virus strains. One antibody from the VIC75 panel bound more avidly to WC76 than to VIC75. The target area of this antibody includes HA 1 residue 155 [Table 2(b), group 81. VIC76 has a change in position 157 from Ser to Leu. An antibody binding closely to the Leu side chain could still be accommodated by the smaller Ser side chain, but with lower binding energy. A similar effect was observed with two antibodies from the PC73 panel. These antibodies also appeared to bind in the 155-160 region [Table 2(a), group 51 and showed increased affinity for ENG72 and MEM72. The residue likely to be responsible is No. 160 which is Thr in the latter two strains and Ala in PC73. Antibodies binding closely to the Thr side chain could be accommodated by the smaller Ala with reduced binding energy. Similar heteroclitic antibodies (to cytochrome c and myoglobin respectively) have been described by Urbanski and Margoliash (1977) and East et al. (1982~). Table 2. Target
Considerable difficulty was experienced in preparing monoclonal antibodies to ENG72 virus. This particular strain was very labile in the sucrose density gradient purification procedure (unpublished observations). Factors associated with this instability may have reduced the immune response of the mice to the native HA 1. The monoclonal antibodies raised to TEX77 were very unreactive with all of the virus strains tested. Twenty of the 3 1 antibodies examined did not crossreact (titres < 1% of homologous) at all with even the closest relative, BK79. The number of reactivity groups identified for each panel of antibodies was very similar (Table I), even when the panel contained less than 20 antibodies. The target areas they addressed showed many features in common (Table 4). All panels contained the highest proportion of antibodies directed to the tip of the HA1 molecule [site B (Fig. l)]. In general, the second most common target area was the interface region between adjacent HA1 molecules [site D + AB (Fig. l)]. Reactivities to the side face BA and the loop A (Fig. 1) were present in low numbers, or absent in
areas of two representative Target
Group
No. of antibodies
Core (a) Antibodies
I 2 3 4
6 4 4 2
5
2
6
2
7
2
I
6 4
229, 230, 220/186 188, 198 155, 157, 159 155, 157, 158 (129, 159) 155, 157, 158, 160’ 189, (159, 129) 157, 158 or 145, 146 188, 189. 201 (b) Antibodies
2 3 4 5 6 7 8
4 2 2 3
I 1
155, (159, 129) 155, 157, 158 (159, 129) 158, 159 155, 157, 158, 193 229, 230, 186/220 144 132 155, 157’
monoclonal
panels
area’ Periphery
Area code”
to PC73 182, 226, 1291159 188, 189, 158, 129 188, 189
D B B B B
189 137
B A B
to VIC75 157. 158
129 226 145, 146
B B B B AB A BA B
“Target areas identified by position of amino acids in primary sequence of HAl. “Target area code after Wiley e! al. (1981), Underwood (1982) and Fig. I. ‘Amino acid change likely to account for heteroclitic activity in this antibody group (see text).
-
2 3 9 10 31 34 50 53 54 62 63 78 81 83 122 126 129 132 133 137 143 141 145 146 1.55 156 157 158 159 160 164 172 174 182 186 188 189 193 197 198 201
Amino acid*
Ser Asn Gin Ser Gln Ala Arg
IS0
Asp Phe
GUY GhI ASII A.Sn Pro G’Y Ser GUY Thr LYS Ser GUY Ser Thr Leu
Asp Val Asn Thr Thr Thr
IS0
LYs Asn Asn
IS0
Asp Leu Ser Thr Asp
HK68
Val
Asp
ASII
375117
GUY
Val
Asn
34c
Val
Asp
Asn
29C
IS0
Vat
Asp
ASfl
30D
Val
Asp
Asp
AStI
AW
ENG69
Val
GlU
GlY
Asn
070
Table 3. Variable
Thr
Asp
Val
A%
Asp
Giu Gill
Tht
LYS
HK71
Vdl
Asp
Val
TY~
TY~
‘W
Tht
Asn
Asp
Val
Ala
Asp
Asn
Asn GlY
AStl
Phe
PC73
Asp
Asn
Asn GUY
Asn
MEM72
LYS
Asp LYS Asn
Ser Val
Gin
TY~
Asp
Ser
ASII
LYS
Gh
Asn
Asn
WC75
1968.-1979”
Asp
AStl
ASfl GUY
Asn
ENG72
amino acids in HA1 of H3N2 subtype,
LYS
Asp LYS Asp
Ser Val
Gin
Leu
TYT
Asp Asn
Ser
Lys Asn AStt
AW GUY
Asp
Asn
WC76
Asp LYS Asn
Val
GUY
GlU
TY~
Asp Asn
TY~
Asn Asn
LYS
Am GUY
Arg Asp Ser LYS
Asp LYS Asn Arg
Val
GUY
LYS
GIU
Ser TY~ Se1 Asp Asn Ser TY~ GlU
LYS Asn Asn
Lys Asn GUY
Arg Asp Ser
Asn
Aslt
Asn
Asn
BK79
l’EX77
D+AB D+AB D+AB, B B B B B B
B
E DfAB D+AB, BA B, BA BA A D+AB, A A A A A B B B B B, BA B, BA D+AB
Area code
Antigen& map of haemagglutinin of influenza Hong Kong subtype (W3N2)
667
some panels. Changes in site E (Fig. 1) appeared to be correlated with changes in the interface region (Underwood, 1982) and could not be separately resolved with panels to viruses isolated later than 1972. Antibodies affected by changes in site C [Fig. l(b)] were not detected in any panel. Experiments with whole sera In order to test whether the average crossreactivities observed with the monoclonal panels reflected those in circulating serum, cross-reactivity patterns of mouse sera derived from several different inoculation regimes were compared with the monoclonal panels. The results for sera and monoclonal antibodies raised to HK68 are shown in Table 5. It is clear that the variation in the response of individual mice is very great. It is also clear that the ranges of cross-reactivity are similar for each immunisation regime. What is surprising is that the range of cross-reactivities observed between individual sera is nearly as great as that displayed by individual monoclonal antibodies. This indicates that the repertoires of individual mice may be largely restricted to one or two target areas. The means of the six immunisation groups were not normally distributed, indicating that five mice were insufficient to give a true representation of the average response. The mean crossreactivities of the mono~lonal antibodies, however, fall largely within, or very close to, the range of the means of the groups of sera. Exceptions were crossreactivities towards HK71 and 29C which were both considerably higher with the monoclonals than with whole sera. Either the spleens used for fusion contained fewer cells reactive towards HA1 residues 132 and 226 than serum antibodies; or such cells possessed some growthjhybridisation disadvantage; or their products were of low affinity. In the latter case, when mixed in serum, their contribution towards average cross-reactivity, would be lower than expected from their proportional representation in the cell population. With these two exceptions, the HK68 monoclonal panel gave a fair representation of the average BALB/c serum response. DISCUSSION
In a previous report I described the assignment of target areas (possible binding sites) of a large panel of monoclonal antibodies to the HA molecule of the H3 strain NT60/68 (Underwood, 1982). This was achieved by correlating the cross-reactivity patterns of individual monoclonal antibodies (or similar groups) with the 3-dimensional location of known amino acid sequence differences in both natural and laboratory variants. This type of antigenic mapping by correlation of cross-reactivity of monoclonal antibodies with sequence differences in variants of the antigen has been described for several other protein antigens [Schroer
668
P. A. UNDERWWD
Table 4. Proposed antibody target areas on HAI of the H3N2 subtype: representation mo”oclo”al DaneIs Interface
Side Top of molecule B
Target arean Contributory’ amino acid residues in HA1 Monoclonal panels to HK68 NT68“ ENG72 PC73 VIC75 TEX77
BA
188-198 201
155-160 188-193 129
155-160 129
0'
44.0
0
12.8 0 27.3 0 8.6
36.8 61.5 36.4 8.0 5.7
0 0 0 60.0 2.9
126132 159-160
0
11.2 15.4 0 4.0 0
of antibodies to each target area in different
D 160, 174. 122. 242,
D+AB 164 207 126 244
0
23.4 1.7 0 0 0
21.9 0 0 21.3 0 5.7
Loop A
Hinge C
137, 220 226 229, 230 182, 186
143-146 133, 137
5fS-54 215, 287
12.0 6.4 0 0 8.0 0
12.0 2.4 0 9.1 12.0 2.9
AB
0
E
62-63 78 81, 83
1.33 8.8 1.7 0 0 0
“Area letter codes from Wiley et al. (1981) and Underwood (1982). ‘Contribution of amino acids deduced from above references and present data. ‘Percentage of panel of monoclonal antibodies affected by changes in the designated area (difference in total from 100% represents unplaced antibodies). Numbers under area E are also included in the interface region since changes in area E appear to affect antibodies binding in this region (see text). dData from Underwood (1982).
(1981), beef insulin; Berzofsky et al. (19826), sperm whale myoglobin; East et al. (1982b), human myoglobin; Stamatoyannopoulos et af. (1981), human haemoglobin; Smith-Gill et al. (1982) chicken lysozyme; Boulain et al. (1982), Nuju nigricolh toxin; Al Moudallal (1982) tobacco mosaic virus protein]. The underlying assumption in this type of approach is that the overall 3-dimensional structure or related variant proteins is highly conserved, and that amino
acid differences are likely to cause only localised perturbations. This has been demonstrated by X-ray crystallography in the case of species variants of lysozyme (Smith-Gill et a/., 1982). Results of such studies have led to the hypothesis of a universal potential for the complete surface of an antigen to be antigenic in an appropriate host (Urbanski and Margoliash, 1972; White et al., 1978; East et al., 19826; Todd et al., 1982; Leach, 1983). The surface of the influenza HA molecule also appears to exhibit this potential (Lubeck and Gerhard, 1981; Jackson et al., 1982, Green et al., 1982; Underwood, 1982). In the present study the mapping of antibody target areas on the H3 influenza HA was extended to
(b)
Fig. I. Drawings of the alpha carbon skeleton of regions of antigenic importance of the HA1 chain of Aichi68 (taken from stereo drawings loaned by Dr D. Wiley). (a) and (b) represent opposite views of the molecule. Area coding after Wiley et al. (1981) and Underwood (1982).
Antigenic
map
of haemagglutinin
of influenza
Hong
Kong subtype (H3N2)
669
Table 5. Comparison of cross-reactivities of monoclonal antibodies and whole mouse sera, raised to HK68 Serum means Ranges of % cross-reactivity of mouse sera for each immunisation group Virus 375117 ENG69 470 HK71 ENG72 MEM72 PC73 WC75 VIC76 TEX71 34C 29C 30D HAN74 ART74 SA74
sc 1” 43-132 29-100 543 >12 47-l 15 14-50 <4-15 <422 <&15 <4-14 76-185 22-50 <4-22 t‘v35 <45 13-38
SC 10
sc 20
ip 1
ip 10
ip 20
4-150 <4-163 <4-25 <4-12 4300 <4107 <47 <4-41 <4-5 147 lW300 ~476 <+41
50-100 12-100 747 t49 50-100 <‘&47 <4-9 t423 t49 <49 10&200 < 450 <&9 <4-9 <4-9 <4-20
31-93 18-50 6-15 <4-12 14-66 l-12 2-l 1
22-100 l&l07 <4-38 tk20 19-100 <&13 <4-11 <4-8 14-E ~4-8 87-130 547 &14 tr150 <4-8 ~423
47-93 41-93 <4-29 <4-12 38-100 12-27 <4-9 <4-9 <&9 <49 71-187 12-50 4623
y0 cross-reactivity” of monoclonals
OVerall mean
Range of group means
Individual range
Overall mean
72.3 64.1 19.8 6.4 70.1 16.0 5.2 6.0 3.9 4.2 107 24.2 9.2 11.1 3.6 11.6
62-86 55-92 1l-34 5-9 32-112 4-30 3-9 l-13 l-8 2-8 91-145 1l-30 &13 5-19 l-7 5-28
< 4200 <4170 <‘&I5 tk80 t4-150 <‘&IO0 <4-95 <4-50 <&18 <4-18 <4-180 <4-150 c&50
93.5 91.3 10.0 21.3’ 49.3 12.8 11.1 5.5 2.7 2.6 80.2 49.5’ 4.8 21.2 3.2 3.5
“Heterologous HI titre as percentage of the homologous titre to HK68. ‘Immunisation scheme. SC:subcutaneous prime, subcutaneous/intravenous boost. ip: intraperitoneal prime and boost. The number represents the dose in thousands of HAU. ‘These means lie well outside the range of the serum means.
monoclonal panels raised against five virus strains isolated between 1968 and 1977. With each panel, a similar distribution pattern of target areas to that previously described for NT68 (Underwood, 1982) was observed. This finding validates the assumption that no major conformational changes in the HA take place during evolution of the virus, which has yet to be demonstrated by X-ray crystallography. This means that although about 10% of the amino acids of HA1 have changed between 1968 (HK) and 1977 (TEX), and most of these changes are in surface residues, the mouse is still stimulated to produce the same topological repertoire of antibody molecules with the same proportional emphasis on the various target areas. Most of the exposed surface of the HA molecule appeared to be antigenic, although some individual epitypes were absent from the smaller panels. In each case the greater proportion of antibodies recognised changes on the tip of the HA molecule [site B of Wiley et al. (1981)). Target areas in the two components of the interface (site D) were also present in high numbers (see Table 4 and Fig. 1). Caton et al. (1982) analyzed the binding sites of monoclonal antibodies to a strain from a different subtype (HONl), employing a large number of laboratory mutants with known sequence changes rather than field strains, and obtained very similar results. Both studies also identified lower numbers of antibodies reacting with the loop (A) and an interaction between area E and the loop containing residue 122 (Fig. 1 and Table 4). Neither study identified any antibodies reactive with residues corresponding to site C. A recent analysis of sequence differences of the HAI of strains of yet another subtype (HlNl), evolved from A/USSR/77 emploqing laboratory-selected mutants, has identified target areas corresponding to sites B and D (Kendal et al., 1983).
The very similar distribution of target areas and similar proportional emphasis described by Caton et al. (1982) for the HO HA, and in the present report for H3, suggests that this pattern approximates the average repertoire of BALB/c mice to all subtypes of influenza type A HA, a correlation not hitherto demonstrated. Both studies have used large monoclonal panels and large numbers of animals to produce them, in order to optimise the representation of the complete BALB/c repertoire. This is an important consideration and Yewdell and Gerhard (198 1) have also suggested that different immunisation regimes and different organ sources of immune lymphocytes should be used. Analysis of cross-reactivity patterns of individual mouse sera reported in the present study indicates that large (> 20) panels of monoclonal antibodies are representative of the in viuo antibody repertoire. This has not been previously demonstrated although it has been suggested. Moreover, the variation in crossreactivity patterns observed between individual whole sera approached that observed between different individual monoclonal antibodies. This suggests that the antibody repertoires of individual mice may be restricted to one or two target areas. These findings are in agreement with Staudt and Gerhard (1983), who demonstrated differences between the repertoires of monoclonal panels derived from different individual mice. Natali et al. (1981) reported individual differences between human sera to a late H3N2 virus strain when titrated against virus variants possessing an amino acid change in only one haemagglutinin epitope. Restriction of epitope recognition has been reported for some rabbit and ferret sera raised to H3N2 and HlNl strains respectively (Webster and Laver, 1980a; Nakajima and Kendal, 1981). In those two reports, however, individual variation in epitope recognition was not demonstrated.
P. A. UNDERWOOD
670
The variation in individual mouse repertoires reported here supports the hypothesis that influenza evolution progresses by an accumulation of mutations in different epitopes as the virus passes sequentially through individuals possessing different antibody repertoires. Acknowledgements-1 am grateful to Dr G. W. helpful discussion, and to Mrs R. MacDonald, N. Miss P. Bean for expert technical assistance, and Drummond and Mrs M. Dowell for preparing script.
Both for Villa and to Mrs J. the type-
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