Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 842 46 Bratislava, Slovak Republic1
The National Institute for Medical Research, Virology Division, The Ridgeway, Mill Hill, London NW7 1AA, UK2
Author for correspondence: Frantiek Kostolanský. Fax +421 7 547 742 84. e-mail virufkos{at}nic.savba.sk
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Abstract |
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Introduction |
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We described monoclonal antibody (MAb) IIB4 (Russ et al., 1987 ), which displays a rare combination of VN activity and broad cross-reactivity of different intensity with influenza virus A strains of the H3 subtype isolated in a period from 1973 to 1988. Recently we showed that the HA epitope recognized by MAb IIB4 is localized in antigenic site B, situated near the receptor binding site of HA (Wiley et al., 1981
), and includes amino acids 198, 199 and 201 (Betáková et al., 1998
). Here we show that amino acids 155, 159, 188, 189 and 193 also contribute to the MAb IIB4 epitope.
Because highly cross-reactive antibodies bind to HA of various influenza virus strains with different affinity they represent an ideal reagent to readdress the question of how important antibody affinity is for neutralization of virus infectivity. Using MAb IIB4 and a single epitope in antigenic site B in the HA of different influenza viruses we found a strong positive correlation between effective affinity and VN activity of MAb IIB4. Our results are compared with those of other authors. Finally we hypothesize how the affinity influences the proportion of spikes which must be occupied by antibody in neutralized virus preparation.
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Methods |
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Selection of MAb escape variants of viruses.
Serial dilutions of infectious allantoic fluid (10-310-8) were incubated with 10-fold dilutions of MAb IIB4 ascitic fluid (10-1, 10-2) for 2 h at room temperature and inoculated (200 µl) into embryonated hen eggs. After 48 h, allantoic fluid samples with haemagglutination activity were tested for MAb IIB4 binding. Those which lost antibody binding were propagated, after limiting dilutions, in embryonated hen eggs.
Influenza virus HA gene sequencing.
HA genes of virus strain isolates from 1968 to 1985 were sequenced by the dideoxynucleotide chain termination method as described previously (Daniels et al., 1985 ; Grambas et al., 1992
) using primers described by Betáková et al. (1998)
. Partial HA sequences (amino acids 151250) of A/Victoria/3/75, A/Caen/1/84 and A/Mississippi/1/85 strains revealed complete agreement with information available in the GenBank database.
Haemagglutination-inhibition (HI) test.
The HI activity of MAb IIB4 with particular strains was tested by standard procedure, using chick erythrocytes at 0·5% concentration.
Virus neutralization (VN) test.
Amounts of individual influenza virus strains (dilutions of infectious allantoic fluids) used in the VN test were standardized according the quantity of viral nucleoprotein (NP) detected after 18 h of replication in MDCK cells by measuring the binding of iodinated anti-NP MAb 107-L (Vareková et al., 1995
). These concentrations represented approximately 2 haemagglutination units (HU) of virus. The virus was incubated with fourfold dilutions of purified MAb IIB4 (from 10000 ng to 2·5 ng) for 90 min at room temperature. After washing with PBS pH 7·2, MDCK cells on 96-well plates were infected with virus/antibody mixtures for 1 h at room temperature. Cell monolayers were then washed with PBS and cultivation medium containing 0·2% FCS was added. Infected cells were incubated for 17 h at 37 °C, washed with PBS, fixed with 0·05% glutaraldehyde in PBS at 4 °C and permeabilized with 0·1% Triton X-100 for 10 min. Virus replication was evaluated using 125I-labelled MAb 107-L (for iodination procedure see Russ et al., 1978
) and VN titre (VN50) was established as the concentration of MAb IIB4 causing a 50% decrease in viral NP.
MAb IIB4 binding to viruses.
MAb IIB4 binding to viruses of the H3 subtype was determined by ELISA. Purified viruses were adsorbed onto wells of microtitre plates overnight at twofold dilutions (from 300 ng to 10 ng of virus per 100 µl of PBS pH 7·2). After washing (PBS with 0·05% Tween-20) and saturation of wells (with PBS pH 7·2 containing 1% non-fat dry milk), 100 ng of MAb IIB4 was added for 90 min at room temperature. The reaction was detected using swine anti-mouse immunoglobulin conjugated to horseradish peroxidase by measuring the enzyme activity in the presence of activated substrate 1,2-phenylenediamine dihydrochloride (1 mg/ml) with 0·03% H2O2 at 492 nm.
Homologous competitive radioimmunoassay (RIA).
The principle of the RIA was homologous competition of 125I-labelled MAb IIB4 with increasing amounts of unlabelled antibody for binding to virus adsorbed to the solid phase. In these experiments, approx. 3 ng total input of 125I-labelled IIB4 was added in each sample. The amount of purified virus was such that it had less than 50% of maximal binding of 125I-labelled IIB4. Unlabelled MAb IIB4 competitor (2 to 2000 ng) was present in the reaction mixture (100 µl). Incubation was carried out at room temperature for 3 h. After washing, bound radioactivity was measured in a gamma counter.
Effective affinity estimation of MAb IIB4 and epitope occupancy determination.
The effective affinity of MAb IIB4 binding was measured for various H3 strains. However, only those strains whose binding activity with MAb IIB4 was sufficiently high to allow precise measurement could be included in affinity measuring. For effective affinity estimation the data obtained from homologous competitive RIA were used (Rodbard & Feldman, 1975 ; Mucha, 1993
; Vare
ková et al., 1993
). Linear regression was applied to the experimental points [log10(VN50) vs log10(effective affinity of binding)] representing individual virus strains, and the regression coefficient was estimated.
Epitope occupancy rate at equilibrium in virus/MAb preincubation (1 h at room temperature) in the VN test was determined on the basis of the mass action law from the formula:
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where K is effective affinity determined as stated above, q and p are concentrations of antibody combining site and epitope, respectively, and X is epitope occupancy to be calculated. The epitope concentration was estimated on the basis of the virus concentration used in the VN experiment, taking into account a 20% proportion of HA in the virion. Protein concentration in purified virus preparations was estimated by the method of Lowry et al. (1951) using BSA as a standard.
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Results |
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Amino acid sequences of HA.
To determine other amino acids possibly involved in the binding of MAb IIB4 we compared the reactivity pattern of antibody to various H3 strains (Fig. 1). The amino acid sequences of H3 strains are shown in Fig. 3
. Two non-reactive strains, Hong Kong/68 and England/72, differed from all reactive ones in positions 188 and 193, both spatially close to already determined contact amino acids 198, 199 and 201. Therefore, we suppose that positions 188 and 193 might influence MAb IIB4 binding. However, the significance of position 193 remains questionable since the substitution of neutral asparagine (N) to basic lysine (K) present in the highly reactive strain Praha/83 HI+ (Fig. 3
) is remarkable.
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Besides oligosaccharide 246, a second case of indirect influence on the IIB4 epitope is probably represented by tyrosine (Y) 219 in strain Praha/83 HI-. In the HA trimer this position is close to position 201 of the adjacent monomer and might influence the binding of MAb IIB4 to the adjacent monomer (Fig. 2a).
Comparison of the sequences of the closely related strains poorly reactive England/88 and highly reactive Mississippi/85 (both lacking a sugar at 246) revealed three additional amino acid positions possibly involved in MAb IIB4 binding: 155, 159 and 189 (Fig. 3). As shown in Fig. 2
, these residues are also spatially close to positions 198201. The spatial distance between amino acid positions potentially involved in the IIB4 epitope does not exceed 18
. We do not exclude the fact that other even more distant amino acids may be included in the interface area that influences antibody binding. It is obvious that the area delineated by these amino acids is smaller than the buried area (1250
2) in the HAFab interface found by Bizebard et al. (1995)
.
In summary, the crucial amino acid positions forming the IIB4 epitope are 198, 199 and 201. We assume that amino acids 155, 159, 188, 189 and possibly 193 are also involved in the binding of MAb IIB4.
Virus neutralization depends on the effective affinity of MAb binding
The VN activity of MAb IIB4 is related to its ability to inhibit haemagglutination with particular viruses and roughly correlates with its binding reactivity in ELISA (Table 2 and Fig. 1
). To find out how important antibody affinity is for VN we also estimated the effective affinity of MAb IIB4 binding to HA of influenza virus strains included into this study (Table 2
). As shown in Fig. 4
, VN activity was proportional to the effective affinity of binding. The application of linear regression to the experimental points resulted in a regression coefficient greater than 3. A parabolic regression line (solid line on Fig. 4
) provided, however, a better fit to the experimental points. These results thus show a strong positive correlation between effective affinity and VN activity of MAb IIB4. This raises the question of whether occupancy of HA spikes required for VN is independent of the effective affinity of MAb IIB4 binding.
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To evaluate the epitope concentration we determined the amount of virus present in 1 HU of purified virus preparation. The amount of virus in allantoic fluid used in the VN test was determined from its haemagglutination activity. Presuming that there is a 20% proportion of HA in a virion, 2 HU of virus used in the VN test represented the following amounts of HA in particular virus strains: A/Dunedin/4/73, 16 ng; A/Victoria/3/75, 7 ng; A/Bangkok/1/79, 14 ng; A/Praha/2/83 HI+, 7 ng; A/Mississippi/1/85, 9 ng. For these virus strains the epitope occupancy rate required for VN50 was calculated (Fig. 5).
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Provided that the epitope occupancy required for VN is constant, a relationship between binding affinity and VN titre can be described by an equi-occupancy line determined by the law of mass action. A plot of equi-occupancy lines for a given range of effective affinities and MAb concentrations used in the VN experiments described here is shown for three occupancy levels (5%, 50% and 95%) in Fig. 4 (dashed lines). There is a great difference in slopes between theoretical equi-occupancy lines and the experimental curve showing the relationship between VN activity and effective affinity. We conclude from this that the effective affinity of MAb IIB4 strongly influences epitope occupancy required for VN.
Fig. 5 shows the influence of MAb IIB4 affinity on the occupancy rate of HA spikes needed for VN50. For the virus strains to which MAb IIB4 binds with high effective affinity (ranging from 5·6x108 to 6·9x108 l/mol), an occupancy level from 13·5% to 27% was required for VN50. In contrast, for the virus strain with low effective affinity to MAb IIB4 (6·1x107 l/mol) as many as 98% of the spikes must be occupied to neutralize the virus. The virus strain with intermediate effective affinity of binding (1·7x108 l/mol) required 64% occupation of spikes. On the basis of the observed results we suggest that for the IIB4 epitope, the limiting minimal value of antibody binding effective affinity required for VN50 would be about 6x107 l/mol.
Analysing the plots on Figs 4 and 5
, it follows that for MAb IIB4 and its epitope an increase of antibody effective affinity value above ~109 l/mol would not increase VN efficiency, and at an effective affinity value under ~6x107 l/mol (e.g. Praha/83 HI-; Table 2
) no reasonable antibody concentration would result in VN.
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Discussion |
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We used a single MAb binding to an epitope on the HA of several related virus strains of the H3 subtype. Therefore, experiments were influenced neither by antibody variability nor by a different localization of the binding site critical for VN. Several, but not all, amino acid positions on the HA involved in the binding of MAb IIB4 were identified. We suppose that MAb IIB4 binds to the topologically identical epitope on each reactive virus strain, which does not necessarily mean that the interface in the HAMAb IIB4 complex and the number of intermolecular hydrogen bonds are conserved throughout these strains. Possible amino acid changes within the IIB4 epitope could induce changes in its surface structure, therefore it may have variable affinity of binding to the paratope on the MAb IIB4. Moreover, mutations spatially close to the epitope could cause small differences in the surface structure of the IIB4 epitope, hence the affinity of MAb IIB4 binding to the HA may differ for different virus strains even if their IIB4 epitope consists of the same amino acids. Thus, observed variable binding affinity of MAb IIB4 to the topologically identical epitope may be accepted. Our experiments showed a strong positive correlation between the effective affinity of MAb IIB4 and its VN activity; a 10-fold increase in effective affinity corresponded to about a 2000-fold increase in VN titre. Application of linear regression to the experimental points resulted in a regression coefficient greater than 3.
Similar results were described by Nakamura et al. (1993) using the human immunodeficiency virus (HIV) model and a panel of seven MAbs directed to the C4 domain of gp120. These authors concluded that small changes in antibody affinity result in large differences in the inhibitory concentration (IC50)'. Their affinityVN relationship, replotted into (log10-log10) co-ordinates, which are used in our work to present results, gave not only a regression coefficient close to 3, but also a deviation from linearity similar to our results. Langedijk et al. (1991)
used MAbs against an 18 amino acid long gp120-derived peptide which contains a principal neutralization domain of HIV. They found a linear relationship between log10 VN and log10 affinity with a regression coefficient approx. 5/3 (i.e. the slope was less steep than in our influenza model, but still greater than 1). Bachmann et al. (1997)
, using the vesicular stomatitis virus model and a set of MAbs, described a close correlation between neutralizing capacity and affinity in vitro. The regression coefficient of the relationship seems to be just above 1. On the other hand, West et al. (1994)
have not found a clear correlation between neutralizing activity and binding affinities of MAbs specific to respiratory syncytial virus. Particularly, the two MAbs with the best VN activity (binding to the same site) possessed low binding affinity to F protein.
As follows from data mentioned above, the VN vs affinity relationship expressed by regression coefficient differs considerably. However, experiments leading to these conclusions were done on different viral models including different sets of epitopes and corresponding antibodies.
According to the law of mass action, the affinity value determines epitope occupancy by antibody at equilibrium (see Methods, equation 1). This enabled us to determine the occupancy level of HA spikes required for VN50 of examined virus strains. The regression line on Fig. 5 determines the effective affinity vs occupancy relationship: occupancy of HA spikes required for VN50 for particular strains was not constant but varied considerably from 13·5% to 98%, inversely proportional to the effective binding affinity. The occupancy of spikes approaching 10% probably represents the minimal occupancy warranting VN. This is feasible when one assumes a random distribution of MAb IIB4 binding to HA spikes and the localization of the IIB4 epitope on the membrane distal end of the HA molecule. Under such conditions, assuming that one antibody molecule bound to the epitope blocks the whole trimeric HA spike because of spatial proximity of the three epitope sites on the trimer, the occupation of every tenth HA spike may be enough to complete inhibition of binding of virus to cells, most probably due to steric interference, though virus aggregation cannot be excluded particularly in a certain MAb concentration range. This observation is in accordance with Taylor et al. (1987)
who found that for 63 % neutralization of influenza virus infectivity about 70 molecules of monoclonal immunoglobulin G per virus particle are needed (i.e. approx. 10% of HA spikes).
The very broad range of HA occupation needed for a given VN effect may be explained as follows. Apparently the minimum number of HA spikes which must be occupied by antibody (e.g. 10%) is the same for all influenza virus strains neutralized with MAb IIB4. We estimated the initial occupancy of HA in equilibrium before the virusantibody preparations were allowed to interact with MDCK cells. However, following the exposure of the virusMAb mixture to MDCK cells the equilibrium conditions are changed and the initial occupancy of HA spikes, due to dissociation indirectly proportional to the affinity, decreases. Provided that the effective affinity of MAb IIB4 to the particular virus strain is sufficiently high, such a loss of bound antibody can be discounted and the initial occupation (e.g. 13·5%) of HA spikes is enough for VN. In contrast, for an interaction characterized by low effective affinity, the occupancy during incubation with MDCK cells may drop quickly from over 90% to about 10 %. Therefore the initial occupation by an antibody with low effective binding affinity must be much higher than the minimal occupancy required for VN.
Additional in vivo protective experiments using mouse-adapted influenza virus strains interacting with MAb IIB4 with various affinities could support our in vitro observations concerning the strong effect of antibody affinity on its neutralizing activity.
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Acknowledgments |
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Footnotes |
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References |
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Received 24 November 1999;
accepted 27 March 2000.