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Interference with a conformational change in the haemagglutinin molecule of influenza virus by antibodies as a possible neutralization mechanism
Interference with a conformational change in the haemagglutinin molecule of influenza virus by antibodies as a possible neutralization mechanis, m Hiroshi Kida, Sayaka Yoden, Mikinori Kuwabara*, and Ryo Yanagawa A possible mechanism of neutralization of influenza virus by antibodies to the haemagglutinin molecule is proposed in addition to the generally accepted mechanism of blocking attachment to host cell receptors. This proposed mechanism involves interference with a low-pH-induced conformational change in the haemagglutinin molecule by bivalent binding of antibodie~ which results in inhibition of the fusion step in the viral replication process. Keywords:Viruses: influenza virus; haemagglutinin; conformation: neutralization
Introduction Four non-overlapping antigenic areas on the haemagglutinin molecule of A/seal/Massachusetts/I/80 (H7N7) influenza virus were defined by operational antigenic mapping using a panel of antigenic variants selected in the presence of monoclonal antibodiesL Monoclonai antibodies belonging 'to groups I and II neutralized infectivity and inhibited haemagglutination of the virus. On the other hand, monoclonal antibodies belonging to groups III and IV failed to inhibit haemaglutination of the virus, yet effectively neutralized viral infectivity in different host cell systems. How do the antibodies which fail to inhibit haemagglutination neutralize infectivity of the virus? Some possible explanations are that higher affinity interaction is required for haemagglutination-inhibition compared to neutralization, that there are different receptor binding sites for erythrocytes and host tissue cells, or that neutralization by these antibodies takes place at a step later than attachment. Since the binding affinities of the antibodies were indistinguishable ~, such different biological functions of monoclonal antibodies in the different groups could not be attributed to differences in affinity. In neutralization tests, using chick embryos and chick fibroblast cells in addition to M D C K and HRT-18 cells, similar results were obtained, suggesting that the seCond possibility is also unlikely, although it has not been completely Department of Hygiene and Microbiology, and *Department of Radiation Biology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan 0264-410X/85/030219-04 $03.00 © 1985 Butterworth ~ Co (Publishers) Ltd.
excluded. Antibodies of groups Ill and IV were, therefore, presumed not to interfere with attachment of the receptor sites for either erythrocytes or host cells, but to restrict infectivity of the virus by interference with fusion or some other replication step. Since influenza virus has been found to cause haemolysis and cell fusion under acidic conditions 2-4, it has been proposed that in intracellular vesicles such as lysomes` the viral m e m b r a n e fuses with the cellular membrane, resulting in transfer of the viral genome to the cytop!asm5,6. A conformational change in the haemagglutinin molecule has been shown to occur at pH values corresponding to optimal for m e m b r a n e fusion or haemolysis activity of the virus ~,~. Therefore, the effect of the antibodies on the fusion process was examined by determining their ability to inhibit virusinduced haemolysis and a conformational change in the haemagglutinin molecule of the virus.
Results and discussion
Inhibition o f virus-induced haemolysis with monoclonal antibodies to different antigenic areas on the haemagglutinin molecule o f A/seal/Massachusetts/1/80 (H7N7) influenza virus Seal influenza virus causes maximal haemolysis at pH 5.8-5.99. The inhibitory activity of monoclonal antibodies, specific to each of the different non-overlapping antigenic areas on the haemagglutinin molecule of the virus, against virus-induced haemolysis was examined. As shown in Table 1, haemolysis was clearly inhibited with monoclonal antibodies in each of the
Vaccine, Vol. 3, Supplement 1985
219
Neutralization of influenza virus" H. Kida e t al. Table 1
Haemolysis-inhibition of A / s e a V M a s s a c h u s e t t s / 1 / 8 0
influenza virus w i t h monoclonal antibodies Monoclonal antibody
©
~--C
Titres of haemolysis-inhibition
OH
Group
Number
I
55/2 58/2
230 450
II
71/4 46/6
320 450
Figure 1
III
105/2 3/5 8/4
160 160 260
Detection o f a low p H - i n d u c e d conformational change in the haemagglutinin molecule by a spin-labelling m e t h o d
IV
81/6
320
To prove the above hypthesis, an attempt was made to detect a conformational change in the haemagglutinin molecule at the pH optimum of virus-induced haemolysis using a spin-labelling method combined with electron spin resonance (e.s.r.). The haemagglutinin of seal virus was purified from Triton X-100disrupted virus by sucrose density gradient centrifugation and spin-labelled` A nitroxide compound, N-(2,2,5,5-tetramethyl-3-carbonyl- pyrroline- l-oxyl)-imidazole, was synthesized according to the method of Barratt et al. 1°, and used for spin-labelling of the haemagglutinin. Tyrosine is thought to be mainly labelled according to the reaction shown in Figure 1, although the possibility that hydroxide groups at carbohydrate moieties are also spin-labelled cannot be excluded. Figure 2 shows e.s.r, spectra of spin-labelled haemagglutinin at various pH values. The spectrum consists of two components, namely, L and S, corresponding to a strongly immobilized and a weakly immobilized component, respectively. With decreasing pH values, the presence of signal L became clearer. Thus an examination was made of the changes in the height of component L, indicated as HI, which was normalized by the height of the central peak Ho at various pH values. From the e.s.r, spectra obtained in three independent experiments, HJH o ratios were plotted as shown in Figure 3. The curve clearly shows an extensive increase of H/Ho with the pH value
I
i
.o
Titres are expressed as reciprocals of the dilution of the antibody preparation that cause a 5 0 % inhibition of haemolysis induced
by 2 0 0 haemagglutinating doses of the virus
four groups. Even though monoclonal antibodies belonging to groups III and IV did not inhibit haemagglutination of the virus, they effectively inhibited the haemolysis induced by the virus at pH 5.9, to the same extent as did the antibodies of groups I and lI. The haemagglutination-inhibition activity of the monoclonal antibodies was examined at pH 5.9 (Table 2). Haemagglutination of intact virus or haemagglutinin rosettes was not completely inhibited by any of the monoclonal antibodies at this pH, although partial inhibition was sometimes observed. These results suggest that at this pH a conformational change occurred in the haemagglutinin molecule. On the other hand. haemagglutination-inhibition was observed when the antigens were incubated with the antibodies at pH 7.0 and then the pH was later shifted to 5.9. These data suggest that antibody binding prohibits the low-pH-induced conformational change in the haemagglutinin molecule. It was therefore assumed that neutralization with these antibodies, which failed to inhibit haemagglutination of the virus, took place by interference with the low-pH-induced conformational change in the haemagglutinin molecule, resulting in inhibition of the fusion step of the viral replication process.
.o
Spin-labelling of the haemagglutinin molecule
Table 2 Comparative haemaggIutination-inhibition assays of A / s e a l / M a s s a c h u s e t t s / I / 8 0
influenza virus with monoclonal antibodies at
pH 7.0 and pH 5.9 HI titres w i t h Monoclonal antibody Group
Number
Haemagglutinin rosettes at pH
Intact virus at pH 7.0 12 25 51
800 600 200
5.9
7.0/5.9 a
-
25 51 51
600 200 200
5 5
120 120
7.0
I
9/1 55/2 58/2
II
71/4 46/6
Ill
105/2 14/3 3/5 8/4
-
-
-
IV
81/3
-
-
-
5
81/6
-
-
-
2 560
5 120 2 560
(-) (-)
12 25 102
800 600 400
20 480 10 2 4 0 25
600 320 25 600 2 560 120
5.9
7.0/5.9 a
-
25 51 204
600 200 800
(-) (-)
51 51
200 200
-
51
(-) (-)
12 8 0 0
200 400 51 2 0 0 12 8 0 0
5 120
Titres are expressed as reciprocals of the dilution of the antibody preparation that cause complete inhibition of four haemagglutinating doses of the antigens. -, Titre of less than 100; (-), partial inhibition of haemagglutination was observed apH was shifted to 5.9 after antigens were incubated with monoclonal antibodies at pH 7.0 for 30 min at room temperature, and then
erythrocytes were added
220
Vaccine,
V o l . 3. S u p p l e m e n t
1985
Neutralization o f influenza virus" H, IQ'da et al. .
L
A
.pH 7.0 pH 5.8
_
pH5.6 p
pH50 pH4.0 8G
~r
5"
Figure 2 E.s.r, spectra of spin-labelled haemagglutinin at various pH values ,
14-
e
13-
0
7
e
I
I
5
4
pH
12-
Figure 4 Effect of antibody-binding on the change of H1/Ho ratios of e.s.r, spectra of the spin-labelled haemagglutinin when pH was decreased from 7.0. O, H 1 / H 0 ratio of the spectrum of the haemagglutinin; O, H 1 / H 0 ratio of the spectrum of the haemagglutinin bound with monoclonal antibody 8 1 / 6
11
% K 10 o
I
6
9-
=E
8.
Figure 3 H1/Ho ratios of e.s.r, spectra of the spin-labelled haemagglutinin at various pH values. 0 , H1/H o ratio of the spectrum when pH was decreased from pH 7.0; A,, H 1 / H 0 ratio of the spectrum when pH was increased from pH 4.0
sible. The haemagglutinin was first exposed at pH 4.0, and then with increasing pH the change in the e.s.r. spectra of the haemagglutinin was analysed. The figure indicates that the low-pH-induced conformational change in the haemagglutinin molecule is irreversible. Since a conformational change in the haemagglutinin was thus detected by e.s.r., this method could be used to examine whether antibody-binding had any effect on the low-pH-induced change. In preliminary experiments, which were performed in order to examine the effect of the group IV antibody, 81/6, results were obtained showing that the rate of change in the spectra was suppressed by antibody binding
decreasing below 5.8, which is optimal for haemolysis activity of the virus. This figure confirms that a conformational change occurred in the haemagglutinin molecule at low pH. An investigation was then made of whether or not this low-pH-induced change is rever-
These findings strongly support the hypothesis that interference with a low-pH-induced conformational change in the haemagglutinin molecule by antibodybinding results in inhibition of the fusion step in the viral replication process.
7. •
•
60 ,
,
,
;.
pH
(Figure 4).
Table 3
Biological activities of Fab fragments and IgG molecules of the monoclonal antibodies to the haemagglutinin molecule of A / s e a l / M a s s a c h u s e t t s / I / 8 0 influenza virus Monoclonal antibody (Group)
Concentrations (~M)
Neutralization titres
HI titres
Haemolysis- inhibition
58/6
(I)
Fab IgG
6.6 3.3
60 3700
16 1024
+ +
71/4
(11)
Fab IgG
6.2 3.6
120 3700
512 1024
+ +
8/4
(111)
Fab IgG
9.8 5.7
<10 1O00
<8 32
+
(IV)
Fab IgG
13.6 6.1
<10 1000
<8 <8
+
81/6
Neutralization titres are expressed as reciprocals of the antibody dilutions which cause 50% plaque reduction of 200 p.f.u, of the virus. HI titres are expressed as reciprocals of the antibody dilutions which cause complete inhibition of four haemagglutinating doses of the antigens
Vaccine, Vol. 3, S u p p l e m e n t 1 9 8 5
221
Neutralization o f influenza virus." I-/. IO'da et al.
Is bivalent binding of antibodies to the haemagglutinin of influenza virus required for neutralization of viral infectivity? We then studied whether or not bivalent binding of these antibodies is required to exhibit interference with the conformational change in the haemagglutinin under acidic condition". The data obtained are summarized in Table 3. Fab fragments of the IgG antibodies belonging to groups I and II inhibited haemagglutination of the virus and virus-induced haemolysis at pH 5.9 and neutralized viral infectivity. On the other hand, Fab fragments of group III and IV antibodies could no longer neutralize viral infectivity nor inhibit haemolysis, although it was demonstrated by ELISA that they bound to the virus at the same extent as did the intact IgG antibodies (data not shown). However, infectivity of the virus bound with Fab fragments of groups III and rV antibodies was effectively neutralized by addition of anti-Fab fragment antibodies at titres comparable to those of IgG molecules (data not shown). These findings indicate that bivalent binding of the IgG antibodies of groups III and IV is required for neutralization of viral infectivity through a proposed mechanism by which these antibodies interfere with a low-pH-induced conformational change, which results in inhibition of the fusion step of the viral replication process.
References Kida, H., Brown, L.E. and Webster, R.G. Biological activity of monoclonal antibodies to operationally defined antigenic regions on the haemagglutinin molecule of A/seaV
222
Vaccine, Vol. 3. S u p p l e m e n t 1 9 8 5
2 3
4 5 6 7
8
9
10 11
Massachusetts/I/80 (H7N7) influenza virus. Virology 1982, 122, 38 Huang, R.T.C., Rott, R. and Klenk, H.-D. Influenza viruses cause hemolysis and fusion of cells. Virology 1981, 110, 243 Lenard, J. and Miller, D.K. pH-Dependent hemolysis by influenza, Semliki Forest virus, and Sendal virus. Virology 1981, 110, 479 Maeda, T. and Ohnishi, S. Activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes. FEBS Lett, 1980, 122, 283 Matlin, K.S., Reggio, H., Helenius, A. and Simons, K. Infectious entry pathway of influenza virus in a canine kidney cell line. J. Cell. Biol. 1981, 9 1 , 6 0 1 Yoshimura, A., Kuroda, K., Kawasaki, K., Yamashina, S., Maeda, T. and Ohnishi, S. Infectious cell entry mechanism of influenza virus. J. Virol. 1982, 43, 284 Sato, S.B., Kawasaki, K. and Ohnishi, S. Hemolytic activity of influenza virus haemagglutinin glycoproteins activated in mildly acidic environments. Proc. Nat/ Acad. Sci. USA 1983, 80, 3153 Skehel, J.J., Bayley, P.M., Brown, E.B., Martin, S.R., Waterfield, M.D., White, J.M., Wilson, I.A. and Wiley D.C. Changes in the conformation of influenza virus haemagglutinin at the pH optimum of virus-mediated membrane fusion. Proc. Nat/ Acad. Sci. USA 1982 7 9 968 Kida, H., Webster, R.G. and Yanagawa, R. Inhibiiton of virusinduced hemolysis with monoclonal antibodies to different antigenic areas on the haemagglutin molecule of A/seaV Massachusetts/I/80 (H7NT) influenza virus. Arch. Viro/. 1983, 76, 91 Barrat, M.D., Dodd, G.H. and Chapman, D. A spin label for tyrosine residues. Biochirn. Biophys. Acta 1969, 194, 600 Yoden, S., Kida, H. and Yanagawa, R. Is bivalent binding of monoclonal antibodies to different antigenic areas on the haemagglutinin of influenza virus required for neutralization of viral infectivity? Arch. Virol. (in press)
Introduction Four non-overlapping antigenic areas on the haemagglutinin molecule of A/seal/Massachusetts/I/80 (H7N7) influenza virus were defined by operational antigenic mapping using a panel of antigenic variants selected in the presence of monoclonal antibodiesL Monoclonai antibodies belonging 'to groups I and II neutralized infectivity and inhibited haemagglutination of the virus. On the other hand, monoclonal antibodies belonging to groups III and IV failed to inhibit haemaglutination of the virus, yet effectively neutralized viral infectivity in different host cell systems. How do the antibodies which fail to inhibit haemagglutination neutralize infectivity of the virus? Some possible explanations are that higher affinity interaction is required for haemagglutination-inhibition compared to neutralization, that there are different receptor binding sites for erythrocytes and host tissue cells, or that neutralization by these antibodies takes place at a step later than attachment. Since the binding affinities of the antibodies were indistinguishable ~, such different biological functions of monoclonal antibodies in the different groups could not be attributed to differences in affinity. In neutralization tests, using chick embryos and chick fibroblast cells in addition to M D C K and HRT-18 cells, similar results were obtained, suggesting that the seCond possibility is also unlikely, although it has not been completely Department of Hygiene and Microbiology, and *Department of Radiation Biology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan 0264-410X/85/030219-04 $03.00 © 1985 Butterworth ~ Co (Publishers) Ltd.
excluded. Antibodies of groups Ill and IV were, therefore, presumed not to interfere with attachment of the receptor sites for either erythrocytes or host cells, but to restrict infectivity of the virus by interference with fusion or some other replication step. Since influenza virus has been found to cause haemolysis and cell fusion under acidic conditions 2-4, it has been proposed that in intracellular vesicles such as lysomes` the viral m e m b r a n e fuses with the cellular membrane, resulting in transfer of the viral genome to the cytop!asm5,6. A conformational change in the haemagglutinin molecule has been shown to occur at pH values corresponding to optimal for m e m b r a n e fusion or haemolysis activity of the virus ~,~. Therefore, the effect of the antibodies on the fusion process was examined by determining their ability to inhibit virusinduced haemolysis and a conformational change in the haemagglutinin molecule of the virus.
Results and discussion
Inhibition o f virus-induced haemolysis with monoclonal antibodies to different antigenic areas on the haemagglutinin molecule o f A/seal/Massachusetts/1/80 (H7N7) influenza virus Seal influenza virus causes maximal haemolysis at pH 5.8-5.99. The inhibitory activity of monoclonal antibodies, specific to each of the different non-overlapping antigenic areas on the haemagglutinin molecule of the virus, against virus-induced haemolysis was examined. As shown in Table 1, haemolysis was clearly inhibited with monoclonal antibodies in each of the
Vaccine, Vol. 3, Supplement 1985
219
Neutralization of influenza virus" H. Kida e t al. Table 1
Haemolysis-inhibition of A / s e a V M a s s a c h u s e t t s / 1 / 8 0
influenza virus w i t h monoclonal antibodies Monoclonal antibody
©
~--C
Titres of haemolysis-inhibition
OH
Group
Number
I
55/2 58/2
230 450
II
71/4 46/6
320 450
Figure 1
III
105/2 3/5 8/4
160 160 260
Detection o f a low p H - i n d u c e d conformational change in the haemagglutinin molecule by a spin-labelling m e t h o d
IV
81/6
320
To prove the above hypthesis, an attempt was made to detect a conformational change in the haemagglutinin molecule at the pH optimum of virus-induced haemolysis using a spin-labelling method combined with electron spin resonance (e.s.r.). The haemagglutinin of seal virus was purified from Triton X-100disrupted virus by sucrose density gradient centrifugation and spin-labelled` A nitroxide compound, N-(2,2,5,5-tetramethyl-3-carbonyl- pyrroline- l-oxyl)-imidazole, was synthesized according to the method of Barratt et al. 1°, and used for spin-labelling of the haemagglutinin. Tyrosine is thought to be mainly labelled according to the reaction shown in Figure 1, although the possibility that hydroxide groups at carbohydrate moieties are also spin-labelled cannot be excluded. Figure 2 shows e.s.r, spectra of spin-labelled haemagglutinin at various pH values. The spectrum consists of two components, namely, L and S, corresponding to a strongly immobilized and a weakly immobilized component, respectively. With decreasing pH values, the presence of signal L became clearer. Thus an examination was made of the changes in the height of component L, indicated as HI, which was normalized by the height of the central peak Ho at various pH values. From the e.s.r, spectra obtained in three independent experiments, HJH o ratios were plotted as shown in Figure 3. The curve clearly shows an extensive increase of H/Ho with the pH value
I
i
.o
Titres are expressed as reciprocals of the dilution of the antibody preparation that cause a 5 0 % inhibition of haemolysis induced
by 2 0 0 haemagglutinating doses of the virus
four groups. Even though monoclonal antibodies belonging to groups III and IV did not inhibit haemagglutination of the virus, they effectively inhibited the haemolysis induced by the virus at pH 5.9, to the same extent as did the antibodies of groups I and lI. The haemagglutination-inhibition activity of the monoclonal antibodies was examined at pH 5.9 (Table 2). Haemagglutination of intact virus or haemagglutinin rosettes was not completely inhibited by any of the monoclonal antibodies at this pH, although partial inhibition was sometimes observed. These results suggest that at this pH a conformational change occurred in the haemagglutinin molecule. On the other hand. haemagglutination-inhibition was observed when the antigens were incubated with the antibodies at pH 7.0 and then the pH was later shifted to 5.9. These data suggest that antibody binding prohibits the low-pH-induced conformational change in the haemagglutinin molecule. It was therefore assumed that neutralization with these antibodies, which failed to inhibit haemagglutination of the virus, took place by interference with the low-pH-induced conformational change in the haemagglutinin molecule, resulting in inhibition of the fusion step of the viral replication process.
.o
Spin-labelling of the haemagglutinin molecule
Table 2 Comparative haemaggIutination-inhibition assays of A / s e a l / M a s s a c h u s e t t s / I / 8 0
influenza virus with monoclonal antibodies at
pH 7.0 and pH 5.9 HI titres w i t h Monoclonal antibody Group
Number
Haemagglutinin rosettes at pH
Intact virus at pH 7.0 12 25 51
800 600 200
5.9
7.0/5.9 a
-
25 51 51
600 200 200
5 5
120 120
7.0
I
9/1 55/2 58/2
II
71/4 46/6
Ill
105/2 14/3 3/5 8/4
-
-
-
IV
81/3
-
-
-
5
81/6
-
-
-
2 560
5 120 2 560
(-) (-)
12 25 102
800 600 400
20 480 10 2 4 0 25
600 320 25 600 2 560 120
5.9
7.0/5.9 a
-
25 51 204
600 200 800
(-) (-)
51 51
200 200
-
51
(-) (-)
12 8 0 0
200 400 51 2 0 0 12 8 0 0
5 120
Titres are expressed as reciprocals of the dilution of the antibody preparation that cause complete inhibition of four haemagglutinating doses of the antigens. -, Titre of less than 100; (-), partial inhibition of haemagglutination was observed apH was shifted to 5.9 after antigens were incubated with monoclonal antibodies at pH 7.0 for 30 min at room temperature, and then
erythrocytes were added
220
Vaccine,
V o l . 3. S u p p l e m e n t
1985
Neutralization o f influenza virus" H, IQ'da et al. .
L
A
.pH 7.0 pH 5.8
_
pH5.6 p
pH50 pH4.0 8G
~r
5"
Figure 2 E.s.r, spectra of spin-labelled haemagglutinin at various pH values ,
14-
e
13-
0
7
e
I
I
5
4
pH
12-
Figure 4 Effect of antibody-binding on the change of H1/Ho ratios of e.s.r, spectra of the spin-labelled haemagglutinin when pH was decreased from 7.0. O, H 1 / H 0 ratio of the spectrum of the haemagglutinin; O, H 1 / H 0 ratio of the spectrum of the haemagglutinin bound with monoclonal antibody 8 1 / 6
11
% K 10 o
I
6
9-
=E
8.
Figure 3 H1/Ho ratios of e.s.r, spectra of the spin-labelled haemagglutinin at various pH values. 0 , H1/H o ratio of the spectrum when pH was decreased from pH 7.0; A,, H 1 / H 0 ratio of the spectrum when pH was increased from pH 4.0
sible. The haemagglutinin was first exposed at pH 4.0, and then with increasing pH the change in the e.s.r. spectra of the haemagglutinin was analysed. The figure indicates that the low-pH-induced conformational change in the haemagglutinin molecule is irreversible. Since a conformational change in the haemagglutinin was thus detected by e.s.r., this method could be used to examine whether antibody-binding had any effect on the low-pH-induced change. In preliminary experiments, which were performed in order to examine the effect of the group IV antibody, 81/6, results were obtained showing that the rate of change in the spectra was suppressed by antibody binding
decreasing below 5.8, which is optimal for haemolysis activity of the virus. This figure confirms that a conformational change occurred in the haemagglutinin molecule at low pH. An investigation was then made of whether or not this low-pH-induced change is rever-
These findings strongly support the hypothesis that interference with a low-pH-induced conformational change in the haemagglutinin molecule by antibodybinding results in inhibition of the fusion step in the viral replication process.
7. •
•
60 ,
,
,
;.
pH
(Figure 4).
Table 3
Biological activities of Fab fragments and IgG molecules of the monoclonal antibodies to the haemagglutinin molecule of A / s e a l / M a s s a c h u s e t t s / I / 8 0 influenza virus Monoclonal antibody (Group)
Concentrations (~M)
Neutralization titres
HI titres
Haemolysis- inhibition
58/6
(I)
Fab IgG
6.6 3.3
60 3700
16 1024
+ +
71/4
(11)
Fab IgG
6.2 3.6
120 3700
512 1024
+ +
8/4
(111)
Fab IgG
9.8 5.7
<10 1O00
<8 32
+
(IV)
Fab IgG
13.6 6.1
<10 1000
<8 <8
+
81/6
Neutralization titres are expressed as reciprocals of the antibody dilutions which cause 50% plaque reduction of 200 p.f.u, of the virus. HI titres are expressed as reciprocals of the antibody dilutions which cause complete inhibition of four haemagglutinating doses of the antigens
Vaccine, Vol. 3, S u p p l e m e n t 1 9 8 5
221
Neutralization o f influenza virus." I-/. IO'da et al.
Is bivalent binding of antibodies to the haemagglutinin of influenza virus required for neutralization of viral infectivity? We then studied whether or not bivalent binding of these antibodies is required to exhibit interference with the conformational change in the haemagglutinin under acidic condition". The data obtained are summarized in Table 3. Fab fragments of the IgG antibodies belonging to groups I and II inhibited haemagglutination of the virus and virus-induced haemolysis at pH 5.9 and neutralized viral infectivity. On the other hand, Fab fragments of group III and IV antibodies could no longer neutralize viral infectivity nor inhibit haemolysis, although it was demonstrated by ELISA that they bound to the virus at the same extent as did the intact IgG antibodies (data not shown). However, infectivity of the virus bound with Fab fragments of groups III and rV antibodies was effectively neutralized by addition of anti-Fab fragment antibodies at titres comparable to those of IgG molecules (data not shown). These findings indicate that bivalent binding of the IgG antibodies of groups III and IV is required for neutralization of viral infectivity through a proposed mechanism by which these antibodies interfere with a low-pH-induced conformational change, which results in inhibition of the fusion step of the viral replication process.
References Kida, H., Brown, L.E. and Webster, R.G. Biological activity of monoclonal antibodies to operationally defined antigenic regions on the haemagglutinin molecule of A/seaV
222
Vaccine, Vol. 3. S u p p l e m e n t 1 9 8 5
2 3
4 5 6 7
8
9
10 11
Massachusetts/I/80 (H7N7) influenza virus. Virology 1982, 122, 38 Huang, R.T.C., Rott, R. and Klenk, H.-D. Influenza viruses cause hemolysis and fusion of cells. Virology 1981, 110, 243 Lenard, J. and Miller, D.K. pH-Dependent hemolysis by influenza, Semliki Forest virus, and Sendal virus. Virology 1981, 110, 479 Maeda, T. and Ohnishi, S. Activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes. FEBS Lett, 1980, 122, 283 Matlin, K.S., Reggio, H., Helenius, A. and Simons, K. Infectious entry pathway of influenza virus in a canine kidney cell line. J. Cell. Biol. 1981, 9 1 , 6 0 1 Yoshimura, A., Kuroda, K., Kawasaki, K., Yamashina, S., Maeda, T. and Ohnishi, S. Infectious cell entry mechanism of influenza virus. J. Virol. 1982, 43, 284 Sato, S.B., Kawasaki, K. and Ohnishi, S. Hemolytic activity of influenza virus haemagglutinin glycoproteins activated in mildly acidic environments. Proc. Nat/ Acad. Sci. USA 1983, 80, 3153 Skehel, J.J., Bayley, P.M., Brown, E.B., Martin, S.R., Waterfield, M.D., White, J.M., Wilson, I.A. and Wiley D.C. Changes in the conformation of influenza virus haemagglutinin at the pH optimum of virus-mediated membrane fusion. Proc. Nat/ Acad. Sci. USA 1982 7 9 968 Kida, H., Webster, R.G. and Yanagawa, R. Inhibiiton of virusinduced hemolysis with monoclonal antibodies to different antigenic areas on the haemagglutin molecule of A/seaV Massachusetts/I/80 (H7NT) influenza virus. Arch. Viro/. 1983, 76, 91 Barrat, M.D., Dodd, G.H. and Chapman, D. A spin label for tyrosine residues. Biochirn. Biophys. Acta 1969, 194, 600 Yoden, S., Kida, H. and Yanagawa, R. Is bivalent binding of monoclonal antibodies to different antigenic areas on the haemagglutinin of influenza virus required for neutralization of viral infectivity? Arch. Virol. (in press)