_Amhives
Arch Virol (1991) 116:45-56
Virology © Springer-Verlag 1991 Printed in Austria
Three antibody molecules can bind simultaneously to each monomer of the tetramer of influenza virus neuraminidase and the trimer of influenza virus hemagglutinin D. C. Jackson l, B. S. Crabb 1, P. Poumbourios 1, W. R. Tulip;, and W. G. Laver 3 1Department of Microbiology, University of Melbourne, Parkville 2 CSIRO Division of Biomolecular Engineering, Parkville, Victoria 3John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia
Accepted August 14, 1990
Summary. Trimeric hemagglutinin and tetrameric neuraminidase molecules isolated from influenza virus bind an average of 9 and 13 molecules respectively of monovalent antibody fragments prepared from IgG isolated from polyclonal sera. In each case this represents an average of approximately three molecules of antibody binding to each protomer. Although there is compelling evidence for the presence of multiple adjacent and overlapping epitopes covering the surface of these two viral antigens, steric hindrance ensures that even under saturating conditions only three molecules of monovalent antibody fragments can be simultaneously accommodated on each monomer.
Introduction The hemagglutinin (HA) and neuraminidase (NA) glycoproteins of influenza virus are two of the best characterized proteins in terms of their structure and antigenic properties. Amino acid sequences of the HA and NA from a large number of naturally occurring and laboratory-derived viruses have been determined and the effects that the amino acid substitutions in such variants have on the antigenic and biological properties of each protein have been well documented [-for review, see 1, 10, 38]. Determination of the three-dimensional structure of both HA [40] and NA [-4,33] by X-ray crystallography has also led to an appreciation of the relationship between structure and function of the proteins. Furthermore, solution of the three-dimensional structure of an immune complex between the NA and a monoclonal antibody (MAb) [11, 12, 30] has provided information on the structure of two epitopes of NA. The selection of antigenic variants of NA with MAbs and a comparison of their amino acid sequences, together with analysis of reactivity patterns [35, 37]
46
D.C. Jackson et al.
and results of competitive binding studies [23, 36], has allowed an operational definition o f three or four antigenic sites within the N A molecule. Using similar techniques, four or five antigenic sites have been described on the H A [8, 14, 16, 21, 22, 39] but U n d e r w o o d , using numerical taxonomic methods, has p r o p o s e d the existence of between 6 and 10 overlapping antigenic sites [31, 32]. A l t h o u g h the antigenic structure of both glycoproteins has been the subject of extensive study by a n u m b e r of groups, it is still n o t k n o w n how m a n y antibody molecules are able to bind simultaneously to either of these antigen molecules, although some attempts to answer this question have been m a d e [15,29]. In the present study, the molecular weight of i m m u n e complexes, f o r m e d between these antigens a n d m o n o v a t e n t F a b or Fab' antibody fragments derived from rabbit antisera, were measured in order that the stoichiometry of binding could be determined.
Materials and methods
Hemagglutinin and neuraminidase Hemagglutinin was released from A/Memphis/I/71 (Mere 71) virus by digestion with bromelain [-7] and heads of NA were released by exposure of the avian virus G70c to pronase [25]. Each of these treatments provides a soluble form of viral antigen.
Preparation of lgG Antiserum to NA was raised in rabbits by footpad inoculation of 40 ~tg pure NA in complete Freund's adjuvant (cFA) followed four weeks later by a similar dose of NA. Antiserum to HA was raised in rabbits by two intramuscular inoculations of 50 ~tg antigen emulsified in cFA fourteen days apart followed one month later by 100 jig of purified Mere 71 virus in cFA. In each case, animals were bled l0 days after the final injection and IgG was prepared by affinity chromatography using protein A-Sepharose CL-4B (Pharmacia South Seas Pty. Ltd.). Serum was also obtained from animals prior to immunisation as a source of normal rabbit IgG.
Preparation of monovalent antibodyfragments Monovalent Fab' fragments were prepared from anti-NA IgG by dialysis of IgG against 0.7 M sodium acetate buffer pH 4.0, containing 0.05 M NaC1 followed by exposure to 3% pepsin (Worthington Diagnostic Systems Inc.) for a period of 18 h at 37 °C in the presence of 10raM cysteine hydrochloride. The reaction was stopped by adjusting to pH 7.4 and iodoacetamide was added to a final concentration of t 1 mM. The solution was held in the dark for 1 h and then dialysed exhaustively against phosphate buffered saline containing 0.1% sodium azide (PBSA). Monovalent Fab fragments were obtained from anti-HA IgG by hydrolysis with 1% w/w papain (Worthington Biological Systems Inc.) for 7 h at 37 °C in 0.1 M sodium acetate pH 5.5 containing 3 mM EDTA and 1.5 mM cysteine. Digestion was terminated by addition of iodoacetamide to a final concentration of 2 mM followed by exhaustive dialysis against PBSA. Undigested IgG present in both preparations was separated by passage through protein A-Sepharose and the effluent, containing the monovalent antibody fraction, was finally purified by gel filtration on a coIumn (1 x 30 cm) of Superose 6 using an automated FPLC system (Pharmacia South Seas Pry. Ltd.). The elution profiles of the purified antibody
Immune complexes of influenza virus hemagglutinin and neuraminidase
47
fragments were single symmetrical peaks, indicative of homogenous protein. Analysis by polyacrylamide gel electrophoresis confirmed the homogeneity of the antibody fragment preparations.
Radioiodinations Purified IgG, Fab', Fab and NA were radioiodinated using a modification [19] of the chloramine T method described by Greenwood et al. [17].
Radioimmunoassay A solution-phase radioimmunoassay was used to estimate the concentration of anti-NA Fab' required to saturate NA. A constant amount of radiolabelled NA was treated with serial two fold dilutions of Fab' in PBS containing bovine serum albumin (BSA) at a concentration of 5 mg/ml and 0.05% Tween 20 (BSAsPBST). After overnight incubation at room temperature, antigen-Fab' complexes were isolated by addition of 40 ~tl of a 50% suspension of protein G-Sepharose 4 Fast Flow (Pharmacia South Seas Pty. Ltd.) previously saturated with sheep IgG directed against rabbit F(ab')2. After holding at room temperature for 30 rain with occasional resuspension of the immunoadsorbent, the gel was washed five times with BSAsPBST and the amount of radioactivity associated with the gel determined by solid phase V-scintillation spectrometry.
Sucrose gradient centrifi4gation The sedimentation coefficients (S) of proteins and immune complexes were determined by velocity sedimentation analysis. Linear (10-30%) sucrose gradients (5 ml in PBSA) were assembled in cellulose nitrate tubes (13 x 51 ram). After equilibration at 4 °C for 1 h, 200 gl of sample was layered over the gradient and centrifugation carried out in a Beckman SW55 rotor at 35,000 rpm for 17-18 h at 4 °C. Gradients were fractionated from the bottom and the distribution of radiolabelled material or protein determined by solid phase scintillation spectrometry or by determination of absorbance at 280 nm. Gradients were calibrated using standard proteins with known S values: thyroglobulin, 19.4 S; catalase, 11.3 S; aldolase, 7.3 S; and BSA, 4.4 S.
Gel permeation chromatography The Stokes' radius (a) of various proteins was determined by gel filtration on a column (1 x 30 cm) of Superose 6 using an automated FPLC chromatography system. The column was usually equilibrated in PBSA but in one experiment, where complexes of 125I-HA and Fab were examined under saturating conditions of antibody, the eluant contained Fab at a concentration of 2mg/ml. The flow rate was set at 0.5ml/min and fractions were cut every 0.5rain. Elution profiles were constructed either by on line spectrophotometry at 280 nm or by scintillation spectrometry of individual fractions. The column was calibrated using standard proteins of known Stokes' radii: thyroglobulin, a = 85]~; ferritin, a = 61 A; aldolase, a = 48.1 ,~; and ovalbumin, a = 30.5 ~. Purified virus was used to determine the void volume of the column.
Calculation of molecular weights Values of Stokes' radii, obtained by gel permeation chromatography and values for sedimentation coefficients, obtained by velocity sedimentation were used to derive the relative molecular mass, Mr, for the various species using the expression according to Siegel and Monty [28]: 6 rcN~Isa Mr -1 -up
48
D.C. Jackson et al.
where N is Avogadro's number, u partial specificvolume of the protein (in this study an average value of u = 0.73ml/g was used), s sedimentation constant (cm/sec/dynex 1013), a Stokes' radius (~), and 11 and 9 are the viscosity and density, respectively, of water at 20 °C. Results
Stoichiometry of binding between monovalent antibody J?agments and NA In order to ensure that maximum numbers of antibody molecules were bound to radiolabelled antigen, the concentration of antibody fragments required to saturate radioiodinated antigen was determined. At a concentration of 130 ~tg/ ml Fab' and above, maximum levels of antigen were precipitated indicating saturation of antigen with antibody. This concentration of Fab' was subsequently used for the formation of immune complexes with NA. Normal rabbit IgG was unable to precipitate 125I-NA in this assay. To determine the molecular weight of immune complexes, 125I-NA and antiNA Fab' were mixed and held overnight at room temperature. The mixture was then analysed by (i) sedimentation in continuous sucrose gradients, and (i/) elution from a column (1 x 30 cm) of Superose 6. The behaviour of 12SI-NA and Fab' were also determined individually under identical experimental conditions. The results of these experiments (Fig. 1) show that the relative mobilities of the (125I-NA)-Fab' immune complexes measured by either technique are markedly greater than those of the antigen alone, reflecting an increase in molecular size. Furthermore, the antigen-antibody complex behaved in the same way whether or not antibody was present throughout the gradient or in the eluting buffer (data not shown) suggesting that the interaction between this antibody preparation and the NA is of sufficiently high affinity to prevent dissociation. The values for the Stokes' radius and sedimentation coefficient of 125I-NA, Fab' and the immune complex were calculated by reference to the standard curve. These values are summarised in Table 1. The values of 188kDA obtained for pronase released NA and 48 kDA obtained for Fab' are in good agreement with the values of 196 kDa and 48.2 kDa respectively obtained by a sedimentation equilibrium method [20]. The molecular mass derived for the immune complex formed between 125I-NA and Fab' was 810 kDa indicating that approximately 13 Fab' fragments bound to the tetramer of NA.
Stoichiometry of binding between monovalent antibody fragments and HA Similar experiments to those described above were carried out to examine the stoichiometry of immune complexes formed between HA and monovalent Fab antibody fragments. The concentration of Fab required to saturate HA was determined by exposing a constant amount of 125I-HAto various concentrations of Fab and applying 200 p.1 of each mixture to linear 10-30% sucrose gradients.
Immune complexes of influenza virus hemagglutinin and neuraminidase
49
100
50
0
20
40 60 % Gradient length
100-
80
100
D
8060402oo 0
i 20
10
" ~ ..........I'"............. 30 40 50
Elution time (rain)
Fig. 1. Determination of the sedimentation constant and Stokes' radius for the immune complex formed between radioiodinated NA and rabbit anti-NA Fab'. A Linear 10-30% sucrose gradients in PBSA were overlaid with 300btl of PBSA containing t25I-NA (400,000 cpm) and 40 gg Fab' (F1), or 125I-NA alone (11). Centrifugation was carried out in a Beckmann SW 55 rotor at 35,000 rpm for 17 h at 4 °C. The gradients were fractionated from the bottom and individual fractions assayed for radioactivity. The direction of sedimentation is indicated by the arrow. B 100 gl of sample containing 12SI-NA alone (D), or antigen-antibody complex formed by mixing 125I-NA with 40 gg Fab' (
Arch Virol (1991) 116:45-56
Virology © Springer-Verlag 1991 Printed in Austria
Three antibody molecules can bind simultaneously to each monomer of the tetramer of influenza virus neuraminidase and the trimer of influenza virus hemagglutinin D. C. Jackson l, B. S. Crabb 1, P. Poumbourios 1, W. R. Tulip;, and W. G. Laver 3 1Department of Microbiology, University of Melbourne, Parkville 2 CSIRO Division of Biomolecular Engineering, Parkville, Victoria 3John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia
Accepted August 14, 1990
Summary. Trimeric hemagglutinin and tetrameric neuraminidase molecules isolated from influenza virus bind an average of 9 and 13 molecules respectively of monovalent antibody fragments prepared from IgG isolated from polyclonal sera. In each case this represents an average of approximately three molecules of antibody binding to each protomer. Although there is compelling evidence for the presence of multiple adjacent and overlapping epitopes covering the surface of these two viral antigens, steric hindrance ensures that even under saturating conditions only three molecules of monovalent antibody fragments can be simultaneously accommodated on each monomer.
Introduction The hemagglutinin (HA) and neuraminidase (NA) glycoproteins of influenza virus are two of the best characterized proteins in terms of their structure and antigenic properties. Amino acid sequences of the HA and NA from a large number of naturally occurring and laboratory-derived viruses have been determined and the effects that the amino acid substitutions in such variants have on the antigenic and biological properties of each protein have been well documented [-for review, see 1, 10, 38]. Determination of the three-dimensional structure of both HA [40] and NA [-4,33] by X-ray crystallography has also led to an appreciation of the relationship between structure and function of the proteins. Furthermore, solution of the three-dimensional structure of an immune complex between the NA and a monoclonal antibody (MAb) [11, 12, 30] has provided information on the structure of two epitopes of NA. The selection of antigenic variants of NA with MAbs and a comparison of their amino acid sequences, together with analysis of reactivity patterns [35, 37]
46
D.C. Jackson et al.
and results of competitive binding studies [23, 36], has allowed an operational definition o f three or four antigenic sites within the N A molecule. Using similar techniques, four or five antigenic sites have been described on the H A [8, 14, 16, 21, 22, 39] but U n d e r w o o d , using numerical taxonomic methods, has p r o p o s e d the existence of between 6 and 10 overlapping antigenic sites [31, 32]. A l t h o u g h the antigenic structure of both glycoproteins has been the subject of extensive study by a n u m b e r of groups, it is still n o t k n o w n how m a n y antibody molecules are able to bind simultaneously to either of these antigen molecules, although some attempts to answer this question have been m a d e [15,29]. In the present study, the molecular weight of i m m u n e complexes, f o r m e d between these antigens a n d m o n o v a t e n t F a b or Fab' antibody fragments derived from rabbit antisera, were measured in order that the stoichiometry of binding could be determined.
Materials and methods
Hemagglutinin and neuraminidase Hemagglutinin was released from A/Memphis/I/71 (Mere 71) virus by digestion with bromelain [-7] and heads of NA were released by exposure of the avian virus G70c to pronase [25]. Each of these treatments provides a soluble form of viral antigen.
Preparation of lgG Antiserum to NA was raised in rabbits by footpad inoculation of 40 ~tg pure NA in complete Freund's adjuvant (cFA) followed four weeks later by a similar dose of NA. Antiserum to HA was raised in rabbits by two intramuscular inoculations of 50 ~tg antigen emulsified in cFA fourteen days apart followed one month later by 100 jig of purified Mere 71 virus in cFA. In each case, animals were bled l0 days after the final injection and IgG was prepared by affinity chromatography using protein A-Sepharose CL-4B (Pharmacia South Seas Pty. Ltd.). Serum was also obtained from animals prior to immunisation as a source of normal rabbit IgG.
Preparation of monovalent antibodyfragments Monovalent Fab' fragments were prepared from anti-NA IgG by dialysis of IgG against 0.7 M sodium acetate buffer pH 4.0, containing 0.05 M NaC1 followed by exposure to 3% pepsin (Worthington Diagnostic Systems Inc.) for a period of 18 h at 37 °C in the presence of 10raM cysteine hydrochloride. The reaction was stopped by adjusting to pH 7.4 and iodoacetamide was added to a final concentration of t 1 mM. The solution was held in the dark for 1 h and then dialysed exhaustively against phosphate buffered saline containing 0.1% sodium azide (PBSA). Monovalent Fab fragments were obtained from anti-HA IgG by hydrolysis with 1% w/w papain (Worthington Biological Systems Inc.) for 7 h at 37 °C in 0.1 M sodium acetate pH 5.5 containing 3 mM EDTA and 1.5 mM cysteine. Digestion was terminated by addition of iodoacetamide to a final concentration of 2 mM followed by exhaustive dialysis against PBSA. Undigested IgG present in both preparations was separated by passage through protein A-Sepharose and the effluent, containing the monovalent antibody fraction, was finally purified by gel filtration on a coIumn (1 x 30 cm) of Superose 6 using an automated FPLC system (Pharmacia South Seas Pry. Ltd.). The elution profiles of the purified antibody
Immune complexes of influenza virus hemagglutinin and neuraminidase
47
fragments were single symmetrical peaks, indicative of homogenous protein. Analysis by polyacrylamide gel electrophoresis confirmed the homogeneity of the antibody fragment preparations.
Radioiodinations Purified IgG, Fab', Fab and NA were radioiodinated using a modification [19] of the chloramine T method described by Greenwood et al. [17].
Radioimmunoassay A solution-phase radioimmunoassay was used to estimate the concentration of anti-NA Fab' required to saturate NA. A constant amount of radiolabelled NA was treated with serial two fold dilutions of Fab' in PBS containing bovine serum albumin (BSA) at a concentration of 5 mg/ml and 0.05% Tween 20 (BSAsPBST). After overnight incubation at room temperature, antigen-Fab' complexes were isolated by addition of 40 ~tl of a 50% suspension of protein G-Sepharose 4 Fast Flow (Pharmacia South Seas Pty. Ltd.) previously saturated with sheep IgG directed against rabbit F(ab')2. After holding at room temperature for 30 rain with occasional resuspension of the immunoadsorbent, the gel was washed five times with BSAsPBST and the amount of radioactivity associated with the gel determined by solid phase V-scintillation spectrometry.
Sucrose gradient centrifi4gation The sedimentation coefficients (S) of proteins and immune complexes were determined by velocity sedimentation analysis. Linear (10-30%) sucrose gradients (5 ml in PBSA) were assembled in cellulose nitrate tubes (13 x 51 ram). After equilibration at 4 °C for 1 h, 200 gl of sample was layered over the gradient and centrifugation carried out in a Beckman SW55 rotor at 35,000 rpm for 17-18 h at 4 °C. Gradients were fractionated from the bottom and the distribution of radiolabelled material or protein determined by solid phase scintillation spectrometry or by determination of absorbance at 280 nm. Gradients were calibrated using standard proteins with known S values: thyroglobulin, 19.4 S; catalase, 11.3 S; aldolase, 7.3 S; and BSA, 4.4 S.
Gel permeation chromatography The Stokes' radius (a) of various proteins was determined by gel filtration on a column (1 x 30 cm) of Superose 6 using an automated FPLC chromatography system. The column was usually equilibrated in PBSA but in one experiment, where complexes of 125I-HA and Fab were examined under saturating conditions of antibody, the eluant contained Fab at a concentration of 2mg/ml. The flow rate was set at 0.5ml/min and fractions were cut every 0.5rain. Elution profiles were constructed either by on line spectrophotometry at 280 nm or by scintillation spectrometry of individual fractions. The column was calibrated using standard proteins of known Stokes' radii: thyroglobulin, a = 85]~; ferritin, a = 61 A; aldolase, a = 48.1 ,~; and ovalbumin, a = 30.5 ~. Purified virus was used to determine the void volume of the column.
Calculation of molecular weights Values of Stokes' radii, obtained by gel permeation chromatography and values for sedimentation coefficients, obtained by velocity sedimentation were used to derive the relative molecular mass, Mr, for the various species using the expression according to Siegel and Monty [28]: 6 rcN~Isa Mr -1 -up
48
D.C. Jackson et al.
where N is Avogadro's number, u partial specificvolume of the protein (in this study an average value of u = 0.73ml/g was used), s sedimentation constant (cm/sec/dynex 1013), a Stokes' radius (~), and 11 and 9 are the viscosity and density, respectively, of water at 20 °C. Results
Stoichiometry of binding between monovalent antibody J?agments and NA In order to ensure that maximum numbers of antibody molecules were bound to radiolabelled antigen, the concentration of antibody fragments required to saturate radioiodinated antigen was determined. At a concentration of 130 ~tg/ ml Fab' and above, maximum levels of antigen were precipitated indicating saturation of antigen with antibody. This concentration of Fab' was subsequently used for the formation of immune complexes with NA. Normal rabbit IgG was unable to precipitate 125I-NA in this assay. To determine the molecular weight of immune complexes, 125I-NA and antiNA Fab' were mixed and held overnight at room temperature. The mixture was then analysed by (i) sedimentation in continuous sucrose gradients, and (i/) elution from a column (1 x 30 cm) of Superose 6. The behaviour of 12SI-NA and Fab' were also determined individually under identical experimental conditions. The results of these experiments (Fig. 1) show that the relative mobilities of the (125I-NA)-Fab' immune complexes measured by either technique are markedly greater than those of the antigen alone, reflecting an increase in molecular size. Furthermore, the antigen-antibody complex behaved in the same way whether or not antibody was present throughout the gradient or in the eluting buffer (data not shown) suggesting that the interaction between this antibody preparation and the NA is of sufficiently high affinity to prevent dissociation. The values for the Stokes' radius and sedimentation coefficient of 125I-NA, Fab' and the immune complex were calculated by reference to the standard curve. These values are summarised in Table 1. The values of 188kDA obtained for pronase released NA and 48 kDA obtained for Fab' are in good agreement with the values of 196 kDa and 48.2 kDa respectively obtained by a sedimentation equilibrium method [20]. The molecular mass derived for the immune complex formed between 125I-NA and Fab' was 810 kDa indicating that approximately 13 Fab' fragments bound to the tetramer of NA.
Stoichiometry of binding between monovalent antibody fragments and HA Similar experiments to those described above were carried out to examine the stoichiometry of immune complexes formed between HA and monovalent Fab antibody fragments. The concentration of Fab required to saturate HA was determined by exposing a constant amount of 125I-HAto various concentrations of Fab and applying 200 p.1 of each mixture to linear 10-30% sucrose gradients.
Immune complexes of influenza virus hemagglutinin and neuraminidase
49
100
50
0
20
40 60 % Gradient length
100-
80
100
D
8060402oo 0
i 20
10
" ~ ..........I'"............. 30 40 50
Elution time (rain)
Fig. 1. Determination of the sedimentation constant and Stokes' radius for the immune complex formed between radioiodinated NA and rabbit anti-NA Fab'. A Linear 10-30% sucrose gradients in PBSA were overlaid with 300btl of PBSA containing t25I-NA (400,000 cpm) and 40 gg Fab' (F1), or 125I-NA alone (11). Centrifugation was carried out in a Beckmann SW 55 rotor at 35,000 rpm for 17 h at 4 °C. The gradients were fractionated from the bottom and individual fractions assayed for radioactivity. The direction of sedimentation is indicated by the arrow. B 100 gl of sample containing 12SI-NA alone (D), or antigen-antibody complex formed by mixing 125I-NA with 40 gg Fab' (
