VIROLOGY

188, 93-l

Antigenic

01 (1992)

N to H Conversion

of Poliovirus

by a Monoclonal

Antibody

at Low Ionic Strength

I. DELAET, R. VRIJSEN, AND A. BOEYi’ Department

of Microbiology

and Hygiene,

Vr(ie Universiteit

Received

25,

hly

Brussel,

199 1; accepted

Laarbeeklaan

December

103, B- 1090 Brussels,

Belgium

18, 199 1

Monoclonal antibody 35-lf4 at low ionic strength converted native virions (N antigen) to noninfectious H-antigenic, empty capsids. The reaction was stoichiometric, as the amount of N antigen that could be converted to H was limited to an average of 2 virions per molecule of antibody. The antibody remained associated with virus aggregates after antigenic conversion. Using antibody immobilized onto protein A-bearing staphylococci, it could be shown that the loss of antigen-converting power was concomitant with the loss of antigen-binding ability. Only a small amount of viral protein (equivalent to 0.02 empty capsid per molecule of antibody) remained attached to the antibody. Heating to 56” caused most of this material to be released and restored the antibody’s antigen-binding and antigen-converting abilities. Several possible explanations for the heat-reversible inactivation of the antibody are discussed. o iSS2 Academic Press,

Inc.

INTRODUCTION

ble the empty capsids formed by thermal denaturation in sedimentation coefficient (80 S), antigenicity (H), and p/(6.3) (Brioen ef al., 198513). The H-antigenic, empty particles induced by antibody 35-lf4 do not retain antibody. It was therefore presumed that the antibody responsible for the neutralization of a virion was freed, as expressed in the phrase “hit-and-run neutralization” (Brioen et a/., 198513). If the antibody really escapes without inactivation, it is expected to interact with new virions in a catalytic process. The existence of catalytic antibodies is now well recognized (see Blackburn et a/., 1989 and Lerner et a/., 1991 for reviews). However, this prediction was not borne out by our results: as will be shown in this paper, there is a limit to the amount of N antigen that can be converted to H by an aliquot of antibody 35-lf4. After this limit is reached, the antibody can no longer achieve antigen conversion. As will be shown, the loss of the antigen-converting power is tied to the loss of N antigen-binding capacity.

The neutralization of poliovirus by monoclonal antibodies may involve different mechanisms. Former studies in this laboratory emphasized the importance of aggregation: virions are joined into polymers by antibody bridges, thus reducing the number of infectious units (Brioen eT al., 1983; Thomas et al., 1985, 1986). The aggregated virions suffer no irreversible damage: when they are released from the aggregates, they regain full infectivity (Thomas et al., 1985, 1986). Physical features, such as the virus’s isoelectric point (pl), may be altered after reaction with antibody (Mandel, 1976); but the extent of pl conversion is not correlated with the degree of neutralization, and the two are not necessarily connected (Brioen et al., 1985a). Studies of antibody-virus interactions are invariably conducted under standard physiological conditions, i.e., at the ionic strength and pH of circulating blood. But antibodies are also found on mucosal surfaces and in various secretions where entirely different conditions may prevail; and the mechanisms of neutralization may, in turn, be entirely different under these conditions. Our monoclonal antibody 35-lf4 exemplifies the influence of external conditions on the mechanism of neutralization: at physiological ionic strength, this antibody neutralizes poliovirus exclusively by aggregation (Thomas eta/., 1985); but at low ionic strength, a totally different mechanism prevails: the virions (160 S, N antigen, pl 7.2) are converted to noninfectious empty capsids. These capsids are devoid of RNA and resem’ To whom

reprint

requests

should

MATERIALS

AND METHODS

Virus The labeling of type 1 (Mahoney) poliovirus with [3H]leucine, [35S]methionine, or [3H]uridine and the virus purification were as described by Everaert et a/. (1989). The purified virus was 100% bound by 35-lf4 and other N-specific monoclonal antibodies in immunoprecipitation tests (Vrijsen et al., 1983), whereas no measurable binding occurred with H-specific antibodies; it will therefore be referred to as N antigen. The virus concentration was determined spectropho-

be addressed.

93

0042-6822/92 Copyright All rights

$3.00

0 1992 by Academic Press. Inc. of reproduction I” any form resewed.

94

tometrically, 1961).

Monoclonal

DELAET, VRIJSEN, AND BOEYi

assuming EIFO = 81.6 (Charney et a/.,

antibodies

Three murine IgG monoclonal antibodies were used (Brioen et al., 1982): (1) 35-1 f4, a N-specific, neutralizing antibody directed against site 2 (M. Ferguson, personal communication). The purification of this antibody and its 14Clabeling by reductive methylation were performed according to Thomas et al. (1985). It was verified that the labeling process caused no measurable loss in titer; 45% of the radioactivity was associated with poliovirus-specific antibody (as defined by Thomas et al., 1985). This percentage times the protein concentration was considered to represent the concentration of poliovirus-specific antibody. The specific radioactivity of 14C-labeled 35-lf4 was 200 cpm/ pmol of polio-specific antibody, corresponding to 1.8 14C-methyl groups per IgG molecule. (2) 33-5b5, a Nspecific, neutralizing antibody directed against site 3B (B. Rombaut, personal communication). (3) 39-5b4, a H-specific, nonneutralizing antibody recognizing capsid polypeptide VP1 (Brioen et al., 1982).

Immobilized

antibody

(ImAb)

A 10% (w/v) suspension of protein A-bearing, formaldehyde-fixed Staphylococcus aureus (strain Cowan I) in PBS (137 mlVI NaCI, 2.7 ml\/l KCI, 9.5 mM sodium phosphate, 0.05% NP-40, pH 7.3; p = 0.16) was prepared as described by Kessler (1975). 14C-labeled or unlabeled antibody 35-lf4 (10 pmol) in 0.5 ml PBS was incubated with 0.1 ml of fixed staphylococci. After 30 min at room temperature the mixture was centrifuged for 5 min at 9980 g. The pellet containing the bound antibody was washed twice and finally resuspended in 0.5 ml PBS or Low Ionic Strength Buffer (LISB, i.e., 0.6 mM sodium phosphate, 0.1 mM EDTA, 0.05% NP-40, pH 7.3, p = 0.002). When labeled antibody was used, 45 to 50% of the input radioactivity, including all the N-specific antibody, was bound to the bacteria. The amount of poliovirus-specific antibody bound to the staphylococci corresponded to 400 molecules of antibody per bacterium, or 1% of the IgG binding capacity (Kessler, 1975).

lmmunoprecipitation

titer of antibody

(a) free antibody. The titer was measured essentially as described by Vrijsen et al. (1983). Briefly, serial dilutions of the antibody were incubated with 0.05 pmol of radiolabeled N antigen in 0.45 ml PBS. After 1 hr at room temperature 0.2 ml of a 10% suspension of fixed staphylococci was added. After a further 30 min at room temperature the bacteria-antibody-virus complexes were pelleted by a 5-min centrifugation at

9980 g. The distribution of the radioactivity between the pelleted cocci and the supernatant was determined for each antibody dilution and expressed as percentage of input. The antibody dilution that caused a 50% drop in the radioactivity of the supernatant was taken as the titer of the antibody. (b) ImAb. The procedure was exactly the same as that with free antibody, except that the bacteria were present from the start.

Antigenic

N to H conversion

(a) Antigenic conversion by free antibody. The antigenie conversion was started by incubating 3H-labeled N antigen and 14C-labeled or unlabeled antibody 351f4 in 0.45 ml LISB (F = 0.002) at 37”. At stated times, 0.05 ml 1OX concentrated PBS was added to raise the ionic strength to p = 0.16, thereby stopping the antigenie conversion. Fixed staphylococci (0.2 ml) was added, and after 30 min at room temperature the bacteria were pelleted. The amount of N and H antigen in the supernatant was determined using an anti-N and an anti-H monoclonal antibody (see next section). The antigenic conversion was determined as the radioactivity bound by the anti-H antibody, expressed as percentage of input. It was constantly verified that the radioactivity of the original pellet (after incubation of the 35-lf4-virus mixture), plus the amounts of radioactivity precipitated separately from the supernatant by the anti-N and anti-H antibodies, equaled the input radioactivity, meaning that individual particles were either exclusively N- or exclusively H-antigenic. No evidence for the formation of particles of double (N and H) antigenicity (Rombaut et al., 1985) was found in the present work. (b) Antigenic conversion by IrnAb. The procedure was exactly the same as that with free antibody, except that the bacteria were present from the start.

Determination of the amount the supernatant

of N and H antigen

in

Two monoclonal antibodies directed against N antigen (antibody 33-5b5) and H antigen (antibody 39-5134) were used (see above). The immunoprecipitation procedure was as followed: 0.08-ml samples were mixed with 0.01 ml of antibody 39-5b4 or 33-5b5 and incubated for 1 hr at room temperature. Fixed staphylococci (0.04 ml) was added, and after 30 min at room temperature the bacteria were removed by centrifugation. Fifty microliters of the supernatant was used for radioactivity assay, and the amount of N and H antigen in the original samples was computed (Vrijsen et al., 1983).

N TO

--g -

loo-

”

H CONVERSION

OF

POLIOVIRUS

95

.

-

a

$

b

p =0.002

C

p=O.O06

)1=0.15

B z’ s

50---c

I! 3 F I

0-,

I 0

I 4

I

L 8

F I

L 1

TIME

1

I

I

I

4

8

I 4

1

I

I 8

(HOURS)

FIG. 1. Antigenic N to H conversion as a function of time and ionic strength. 3H-labeled virus (0.5 nl\/l) (N antigen) was incubated at 37” with (solid circles) or without (open circles) 2.5 nM “‘C-labeled antibody 35-lf4. Three different buffers were used which contained 0.6 (a), 2.0 (b), or 60 mlLl (c) sodium phosphate, pH 7.3, supplemented with 0.1 mM EDTA and 0.05% NP-40. At the indicated times the amount of N and H antigen was determined using N- and H-specific monoclonal antibodies (see Material and Methods). The antrgenic conversion is expressed as the percentage of H antigen formed.

RESULTS Antigenic

conversion

by free antibody

Preliminary experiments confirmed the requirements for the conversion of N to H antigen by monoclonal antibody 35.lf4 as originally described by Brioen eta/. (1985b). In particular, the ionic strength had to be 0.002 or lower. Raising the ionic strength from p = 0.002 (Fig. 1a) to 0.006 (Fig. 1b) caused a severe reduction in the rate of antigenic conversion; and at physiological ionic strength (p = 0.16) the antigenic conversion was not significantly above background (Fig. lc). The spontaneous conversion of N to H antigen in the absence of antibody was independent of ionic strength; it proceeded rapidly in the first 2 hr of incubation and more slowly thereafter (Fig. 1). The amount of spontaneous conversion after 4 hr varied from 24 to 31 O/oin successive experiments. Each antigenic conversion experiment included a control without antibody for appropriate correction and computation of the amount of antibody-dependent conversion. The maximum amount of N antigen that could be converted to H by a given amount of 35-lf4 antibody was determined by incubating 1 .O nM virus with different antibody concentrations and measuring the formation of H antigen at different times (Fig. 2). The amount of H antigen formed after 4 hr increased with the antibody concentration. However, up to 0.25 nM antibody, the molar ratio of H antigen formed per molecule of antibody was roughly constant at 2 virions per molecule of antibody. Several additional experiments of this type yielded similar results: conversion of N to H antigen stopped

after each molecule of poliovirus-specific antibody had caused conversion of an average of 1.8 to 2.3 virions.

Conversion of 160 S to 80 S particles by free antibody In an attempt to find out why conversion stopped, 14C-labeled antibody was allowed to react to exhaustion with an excess of 3H-labeled virus (5.2 virions per molecule of poliovirus-specific antibody), and formation of H antigen was measured at different times (Fig. 3, inset). Conversion was completed after 4 hr, and the antibody-dependent antigenic conversion amounted to 1.9 virions per molecule of antibody. Samples col-

0.50

nM

35-lF4

0.25 0.13 0.06 0.00

1

0

2

4 TIME

(hrs

6

8

)

FIG. 2. Antigenic conversion as a function of the antibody/vrrus ratio. %labeled virus (1 .O n/@ was incubated in LISB (p = 0.002) at 37” with the following concentrations of ‘Wabeled antibody 35. lf4: 0.00 (open circles), 0.06 (solid circles), 0.13 (open triangles). 0.25 (solrd triangles), or 0.50 nM(open squares). At the times stated, the antrgenic conversion was determined (see Materials and Methods).

96

DELAET,

VRIJSEN,

AND

BOEYi

a

b

3H CPM -

Am 160 S

80s

I

10

! ‘:I

20

30

10

20

30

FRACTIONS FIG. 3. Reaction of free antibody with an excess of antigen: sedimentation profile and antigenic N to H conversion. 3H-labeled virus (12.5 nM) was incubated in LISB at 37” with 2.4 nM 14C-labeled antibody (a) or without antibody (b). After 4 hr, a sample was taken and layered onto a 15-30% sucrose gradient in LISB. Centrifugation was for 3 hr at 1 10,000 g in a MSE swing-out rotor. (Solid circles) 3H-virus; (open circles) 14C-antibody. Arrows at 80s and 160s show the locations of heated and native virions. Agg, material collected on a l-ml cushion of 70% Nycodenz. (Inset) Kinetics of antigenic conversion (see Materials and Methods). (Solid triangles) With antibody (corresponds to a); (open triangles) no antibody (corresponds to b)

lected at this time were analyzed by sucrose gradient centrifugation without raising the ionic strength. Figure 3 shows the sedimentation profiles of the viral label after incubation with and without antibody. In the presence of antibody (Fig. 3a) 58% of the 160 S material had been converted to 80 S empty capsids, and smaller amounts to virion oligo- and polymers. In the absence of antibody (Fig. 3b), only 240/o of the radioactivity had become 80 S, and no oligomers were formed (a small amount of aggregated material, as seen near the tube’s bottom, is always found in purified preparations; Boeye and De Rees, 1989). The amounts of antibody-dependent conversion to H antigen (37%) or to 80 S (34%) were similar, suggesting that the antigenic conversion and the formation of 80 S may be linked. Forty-five percent of the 14C radioactivity remained associated with the virion oligo- and polymers, corresponding to all the poliovirus-specific antibody in the preparation (see Materials and Methods). As was previously observed at physiological ionic strength, no 35lf4 antibody remained associated with 80 S material or with unpolymerized (160 S) virions (Brioen et al., 1983; Thomas et a/., 1985). The amount of 160 S converted to 80 S after 4 hr in an antibody-dependent process was 1.8 virion per molecule of specific antibody, practically the same figure as found for the N to H conversion in the same experi-

ment; and there was no further change up to 16 hr of incubation (the sedimentation profiles of the 16-hr samples were virtually identical to those shown in Fig. 3). The results presented in Fig. 3 show that in the presence of excess antigen the antibody cosedimented with the viral material in the oligo- and polymer region. This aggregation may be the reason why antigenic conversion stops. An alternative approach is to use antibody immobilized on protein A-bearing staphylococci. Such ImAb can be expected to avoid aggregation. Moreover, they can easily be collected, washed free of unbound antigen, and resuspended in fresh medium with or without antigen. Antigenic

conversion

by ImAb

The immunoprecipitation titer of ImAb (see Materials and Methods) was repeatedly compared with that of the original preparation of free antibody (adjusted to the same volume) and consistently found to be lower by a factor of 2 (results not shown). The converting capacities of free and immobilized antibody were similarly compared; it was found that each molecule of ImAb caused conversion of 0.8 virion, compared to 2 virions per molecule of free antibody (results not shown). Thus, the antibody had lost roughly 6/l 0 of its

N TO

H CONVERSION

OF

97

POLIOVIRUS

antigenic-converting power as a result of immobilization onto bacteria, a loss similar to the twofold reduction in immunoprecipitation titer.

Recycling

of ImAb

Mixtures of 2.2 nn/l ImAb in LISB and variable concentrations of N antigen were incubated for 6 hr at 37”; at the end of this period, the bacteria were collected and resuspended in fresh medium with the initial N antigen concentration for a second, third, or fourth round of incubation. In each round, the conversion of N to H antigen was measured and corrected for spontaneous conversion (32% in these experiments). The results of four independent experiments (not shown) can be summarized as follows: (1) when the initial molar ratio of N antigen to ImAb exceeded 0.8, the antibody-dependent conversion in the first round was between 0.7 and 0.8 virion per molecule of ImAb; and there was no significant antibody-dependent conversion in the following rounds; (2) when the initial molar ratio was between 0.2 and 0.8, antibody-dependent conversion occurred in the first 2 or 3 rounds, but the total antibody-dependent conversion again amounted to only 0.7-0.8 virion per molecule of ImAb; (3) with an initial ratio lower than 0.2, the same amount of antibody-dependent conversion was observed in 4 successive rounds. These results show that the termination of antigenic conversion by ImAb at low ionic strength depended exclusively on the total amount of conversion that the antibody had effected, whether the antigen was provided in one or several portions.

Heat-induced

recovery of the exhausted

antibody

To find out whether the immunoprecipitation titer of the antibody was reduced concomitantly with the loss of its antigen-converting power, ImAb was reacted in LISB with an excess of N antigen (2 virions per molecule of specific antibody), collected by centrifugation, and titrated by immunoprecipitation as described under Materials and Methods. As shown in Fig. 4, the ImAb had a titer of 1:8 before reaction with virus; thereafter, even the undiluted suspension bound less than 50% of the antigen. Thus the loss of converting power was accompanied by a severe lowering of the immunoprecipitation titer. When a sample of ImAb whose immunoprecipitation titer had been lowered by reaction with an excess of N antigen was heated at 56”, the original titer was nearly regained (Fig. 4). As shown in Table 1 (section l), the antibody also fully regained its power to convert N to H antigen upon heating. It may be noted that the conversion during the second round was limited to 0.5 virion

ANTIBODY-

LOADED

STAPH,

DILUTIONS

FIG. 4. Titration of ImAb by immunoprecipitation: effect of previous reactron with virus and of heating to 56”. Two identical mixtures of 5 nM unlabeled virus and 2.5 nM %labeled ImAb in LISB were incubated for 16 hr at 37”. At the end of this period the antigenic conversion was stopped by raising the ionic strength to ~1 = 0.16. In one, the bacteria were collected and the ImAb was immedrately resuspended and serially drluted in PBS (solid squares). In the second, the bacteria were first resuspended in 0.2 ml PBS and heated for 20 min at 56” before collection and serial dilution (solid triangles). As a control, 2.5 nM %labeled ImAb in LISB was incubated without virus for 16 hr at 37”, collected, and simrlarly drluted in PBS (open squares). Each serial dilution was mixed with 0.0 13 nM Wlabeled N antigen to determine the titer of the ImAb (see Materials and Methods).

per immobilized antibody molecule instead of 0.7 to 0.8 as found in previous experiments. This decline was probably due to the multiple washing and collecting operations that the ImAb underwent in the course of recycling experiments. As shown by a control experiment, the heating of ImAb that had not previously been reacted with virus neither reduced nor enhanced its converting capacity (Table 1, section 2).

Relation between the antigen-binding converting capacities of ImAb

and antigen-

The results just reported showed that the immunoprecipitation titer and the antigenic-converting power of ImAb were both reduced upon reaction with an excess of antigen at low ionic strength and restored upon heating at 56”. This suggests that the loss of convert ing power resulted from the loss of the antibody’s ability to bind virions. The quantitative link between the antibody’s binding and converting capacities was examined in the following experiments, exploiting the fact that at physiological ionic strength the N antigen is bound by ImAb but not converted to H, so that the residual binding capacity of the antibody can be measured in the absence of conversion. ImAb was first reacted with virus in PBS and the amount of bound antigen was determined. The ImAb-antigen complexes were then incubated in LISB and the antigenic

98

DELAET,

VRIJSEN,

TABLE 1 RESTORATIONOFCONVERTING

POWERBYHEATTREATMENT Antigenic

Section 1

Heating before 2nd incubation +

2

+

Percentagea

conversion Virions converted per Ab moleculeb

36 85

0.04 0.53

85 83

0.53 0.51

Note. Section 1: Two identical mixtures of 5.0 nlL? unlabeled virus and 2.5 null %labeled ImAb (virus/antibody ratio = 2.0) in LISB were incubated for 16 hr at 37”. At the end of this period the reaction was stopped by raising the ionic strength to Jo = 0.16, and the bacteria were collected by centrifugation. In one mixture, the ImAb were resuspended in the original volume of LISB, which now contained 2.5 n/k! 3H-labeled virus (virus/antibody ratio = 1 .O). In the second mixture, the bacteria were heated for 20 min at 56” in 0.2 ml PBS, again collected, and resuspended in LISB containing 2.5 nn/l 3H-labeled virus (virus/antibody ratio = 1.0). Both mixtures were incubated for 6 hr at 37”, and the antigenic conversion was determined (see Materials and Methods). Section 2: Control without virus during the first incubation. a Includes 32% spontaneous conversion. b Corrected for spontaneous conversion.

conversion was measured. After this first round the ImAb was reused in a second round: it was again reacted with virus and treated exactly as in the first round, to again determine its binding and converting capacities. Table 2 shows the results of two experiments. In the first experiment, an equimolar amount of ImAb and N antigen was used. The ImAb bound an average of 0.39 virion per antibody molecule. Upon further incubation at low ionic strength, 97% of the bound N antigen was converted and released as H antigen. In the second round the antibody bound only 0.17 virion per molecule, but all bound N antigen was again converted to H upon incubation at low ionic strength. In the second experiment, the ImAb was initially reacted with a fivefold molar excess of antigen; under these conditions, the antibody bound 0.48 virion per molecule. Upon incubation at low ionic strength, all the bound N antigen was converted to H. In the second round the binding capacity was almost entirely lost. However, when the ImAb was heated at 56” before the second round, its binding capacity and the converting capacity were both restored. These results showed that the loss of converting power and its recover upon heating correlated with the depletion and recovery of the capacity to bind virions. Retention of viral material by exhausted ImAb The vast majority of the H antigen was released by the antibody, remaining in the supernatant after the

AND

BOEYi

bacteria and the ImAb were collected by centrifugation. Even so, when the antibody was reacted with an excess of 3H-labeled N antigen, approximately 2% of the input radioactivity remained associated with the antibody (Table 3), and the possibility that this material might be blocking further binding of antigen was examined. At least 90% of the radioactivity retained by the antibody was released upon heating at 56” (Table 3). The polypeptide composition of the material retained by the antibody, or released at 56”, was examined by SDS-PAGE (Laemmli, 1970). In both cases, the same VP1 -2-3 pattern was found as that in control virus (results not shown; results regarding VP4 were inconclusive). The RNA/protein composition of the retained material was estimated using a mixture of viruses labeled with [35S]methionine and [3H]uridine. It was reasoned that if the retained material consisted of whole virions, it would have the same 3H/35S ratio as the input virus mixture. However, this prediction was not borne out by the results, as only 0.3% of the input 3H was retained, versus 2.4% of the input 35S(Table 4). It was concluded that the material retained by the antibody, or released at 56”, mainly consisted of viral capsid proteins, with a minor amount of RNA. The amount of viral protein retained by the antibody after reaction corresponded to the protein content of only 2 virions or empty capsids per 100 molecules of specific antibody. For such a small amount of antigen to block the majority of antibody molecules it must have been divided into small packages (for instance, if the protein material consisted of “protomers,” representing l/60 of a virion’s protein shell, the protein complement of a single virion could theoretically block up to 60 antibody molecules). However, when material released at 56” was analyzed by sucrose gradient ultracentrifugation, only 80 S particles and some aggregated material were found (Fig. 5). In high-performance size-exclusion chromatography (Foriers et al., 1990) the same material coeluted with empty capsids and virions, except for a small amount of presumably aggregated material which eluted even faster (results not shown). It is concluded that the traces of viral material retained by exhausted antibody probably consisted mainly of empty capsids and that these were too few to account for the inactivation of the antibody. DISCUSSION The findings with antibody 35-lf4, either free or immobilized on protein A-bearing staphylococci, established that the conversion of N to H antigen at low ionic strength was stoichiometric, rather than catalytic. Each molecule of free antibody converted an average of only 2 virions to H antigen. With ImAb this average was reduced to 0.7-0.8 virions per molecule of antibody. The causes of this reduction may be trivial (e.g.,

N TO

H CONVERSION

OF

TABLE RELATION BFIWEEN THE ANTIGEN-BINDING

Expt

Round

1

2

Initial molar ratio virions/Ab

99

2

AND ANTIGEN-CONVERTING Virions

Heating before 2nd round

POLIOVIRUS

bound

CAPACITIES OF IMAB

in PBS

Virions

Percentage

Vinons bound per Ab molecule

converted

In LISB Virions converted per Ab molecule

Percentage

1 2

1.14 1.14

-

34 15

0.39 0.17

97 94

0.38 0.16

1 2

0.00 1.14

-

35

0.40

95

0.38

1 2 2

5.00 5.00 5.00

9.6 0.4 9.0

0.48 0.02 0.45

96 ND” 96

0.46

+

0.43

1 2

0.00 5.00

-

8.8

0.44

95

0.42

Note. Experiment 1, Round 1: Two mixtures of 2.5 nn/l 3 H-labeled virus and 2.2 nM ‘%-labeled antibody (virus/antibody ratio: 1.14) in PBS were incubated for 1 hr at room temperature. The bacteria were then collected and the amount of bound virus was measured and expressed as percentage of input. The pelleted bacteria, carrying the ImAb-antigen complexes, were resuspended in the original volume of LISB and incubated at 37”. After 6 hr the ionic strength was raised top = 0.16 to stop conversion; in one mixture the antigenic converslon was determined (see Materials and Methods), in the second mixture the bacteria were collected and reused in a second round. Round 2: The ImAb was resuspended in the original volume of PBS containing 2.5 null 3H-labeled virus and further treated exactly as in round 1; the percentage of bound antigen and the antigenic conversion were again determined. Experiment 2: Three mixtures of 2.5 n/v! ‘H-labeled virus and 0.5 nM ‘%-labeled antibody (virus/antibody ratio = 5.00) in PBS were incubated at room temperature for 1 hr. Two mixtures were treated as in Expt. 1. In the third mixture the ImAb was collected after the first round, resuspended in 0.2 ml PBS, and heated 2 min at 56” before the second round. Experiments 1 and 2 were both accompagnied by controls where the virus was omitted in the first round. a Not determined for lack of radioactivity.

incorrect orientation of the antibody molecule, steric hindrance by the bacterial surface). Using ImAb it was possible to conduct experiments in media of either low (p = 0.002) or physiological (p = 0.16) ionic strength. Any reduction in antigen-convert-

TABLE RETENTION AND HEAT-INDUCED

ing power at p = 0.002 was matched by a similar reduction in antigen-binding power (at p = 0.16). The observations suggest that an immobilized antibody molecule that has effected conversion of a virion can no longer bind N antigen.

3

RELEASE OF VIRAL MATERIAL Percentage

BY EXHAUSTED ANTIBODY of Input

radioactivity

Expt

Input wm

Initially bound to lmAba

Released upon heating at 56”“

1 2 3 4 5 6

33,340 64,786 97,065 40,019 99,040 71,202

2.0 N.D. 1.7 1.7 2.3 2.5

2.0 2.3 1.6 1.7 2.2 2.4

Note. Two raised to p = initially bound the supernatant to determine QG Corrected 0.3%).

Still bound after heating” 0.1 0.1 0.2 0.1 0.3 0.1

identical mixtures of 2.2 nM 3H-labled virus and 2.2 nM ImAb in LISB were Incubated at 37”. After 16 hr the Ionic strength was 0.16 and the bacteria were collected. In one sample, the radioactivity of the pellet was measured to determine the percentage of antigen. In the second sample, the bacteria were resuspended in 0.2 ml PBS and heated for 20 min at 56”. After centrifugation, and pellet radioactlvities were expressed as percentage of input. Simultaneous control experiments were done without antibody unspecific binding to the bacteria (see footnotes a-c). for unspecific binding by subtraction of the binding observed in a control experiment without antlbody (a, 0.4%; b, 0.1%; c,

100

DELAET,

VRIJSEN, TABLE

AND

BOEYi

4

RNA CONTENT OF MATERIAL RETAINED BY EXHAUSTED ANTIBODY Percentage Composition

of input

Label [35S]Methionine [3H]Uridine

of input radioactivity

mixture cm

Initially bound to ImAb”

Released upon heating at 56”

34,000 1 17,000

2.4 0.3

2.2 0.3

Still bound after heatingC 0.3 0.0

Note. A mixture of [35S]-methionine-labeled, [3H]uridine-labeled and unlabeled virus was prepared to yield 117,000 cpm 3H and 34,000 cpm 35S/pmol. Samples containing 2.2 nn/t virus mixture and 2.2 nM unlabeled ImAb in LISB were incubated for 16 hr at 37” and further treated as detailed in Table 4. a,b.c Corrected for unspecific binding by subtraction of binding observed in a control experiment without antibody (3H-virus: a, 0.4%; b, 0.1%; c, 0.3% and 35S-virus: a, 0.4%; b, 0.3%; c, 0.1%)

Our findings suggest that the limitation in the converting power of immobilized antibody is not simply due to blocking of the antibody by antigen remnants. An alternative possibility is that an antibody molecule which interacts with a virion to cause its conversion to H antigen undergoes a conformational modification such that it can no longer interact with antigen. The observed reactivation by heat might then correspond to the restoration of the original conformation. The physical conditions required for restoration are currently being studied. There are some interesting similarities between our results and those that were obtained using antibodies with esterolytic activity. Although most of these anti-

CPM

250

1 I\ FRACTIONS

FIG. 5. Sedimentation profile of material released from exhausted ImAb upon heating. A mixture of 2.2 nlM3H-labeled virus and 2.2 nn/r unlabeled ImAb in LISB was incubated for 16 hr at 37”. At the end of this period the antigenic conversion was stopped by raising the ionic strength to ~1 = 0.16. The bacteria were collected, resuspended in 0.2 ml PBS, heated for 20 min at 56”, and again pelleted. The supernatant was layered onto a 15-3096 sucrose gradient. Centrifugation was for 2 hr at 170,000 g in a MSE swing-out rotor, Arrows, see Fig. 3.

bodies act catalytically, some are known to promote the reaction in a stoichiometric way. Some esterolytic antibodies were able to cause hydrolysis of only 2 molecules of substrate (Kohen eta/., 1980; Tramontano et al., 1986). Similar to the report here, these results suggest that the interaction of antibody with the antigen (substrate molecule or virion) may cause the inactivation of one of the IgG’s two paratopes. In analogy with the heat reactivation described in this paper, the esterolytic activity of the antibody could be restored by alkaline or hydroxylamine treatment, or by the use of an ion-exchange column. It is not clear at present why an antibody was capable of virion modification only at low ionic strength. Destabilization of the viral capsid may be important, as the antigenic conversion was inhibited by the capsid stabilizing drug WIN 517 1 1 (unpublished results). Ionic bonds are also known to play an important role in antigen-antibody interactions (Davies and Padlan, 1990); and these interactions are dependent on the ionic strength of the medium. After testing 34 neutralizing monoclonal antibodies, we found a second one besides 35lf4 which also effected the N to H conversion at low ionic strength; thus this property is neither a majority feature of antibodies nor a rarity. It is possible that antibodies that cause antigenic conversion may occur more frequently in nature than in collections of monoclonal antibodies, depending on the screening method. They might be overlooked by tests (e.g., ELISA) which depend on the antibody remaining bound to the antigen.

ACKNOWLEDGMENTS The authors thank B. Rombaut, P. Kronenberger, and A. Foriers for constructive comments and A. De Rees, M. De Pelsmaecker, and S. Peeters for their technical assistance. I.D. is a Research Assistant of the National Fund for Screntific Research (Belgium).

N TO H CONVERSION OF POLIOVIRUS

REFERENCES BLACKBURN.G. M., KANG, A. S., KINGSBURY. G. A., and BURTON,D. R. (1989). Catalytic antibodies. Biochem. J. 262, 381-390. BoEYC~,A., and DE REES, A. (1989). Wall effects in sucrose density gradient centrifugation of viruses. Arch. Viral. 107, 77-84. BRIOEN,P.. SIJENS,R. J., VRIJSEN,R., ROMBAUT, B.. THOMAS, A. A. M., JACKERS,A., and BOEY~, A. (1982). Hybndoma antibodies to poliovirus N and H antigen. Arch. Viral. 74, 325-330. BRIOEN,P., DEKEGEL,D., and BOEY~, A. (1983). Neutralization of poliovirus by antibody-mediated polymerization. Virology 127, 46% 468. BRIOEN,P., THOMAS, A. A. M., and BOEY& A. (1985a). Lack of quantitative correlation between the neutralization of poliovirus and the antibody mediated pl shift of the virions. J. Gen. Viral. 66, 609613. BRIOEN,P., ROMBAUT, B., and BOEY~,A. (1985b). Hit-and-run neutralization of poliovirus. J. Gen. Viral, 66, 2495-2499. CHARNEY,J., MACHLOWITZ,R., TYTELL, A. A., SAGIN.J. F., and SPICER, D. S. (1961). The concentration and purification of poliomyelitis virus by the use of nucleic acid precipitation. Virology 15, 269280. DAVIES, D. R.. and PADLAN, E. A. (1990). Antibody-antigen complexes. Annu. Rev. Biochem. 59, 439-473. EVERAERT,L., VRIJSEN,R.. and BOEY~,A. (1989). Eclipse products of poliovirus after cold-synchronized infectlon of HeLa cells. Virology 171,76-82.

FORIERS,A., ROMBAUT, B., and BOEY~, A. (1990). Use of high-performance size-exclusion chromatography for the separation of poliovirus and subviral particles. J. Chromatogr. 498, 105-l 1 1. KESSLER,S. W. (1975). Rapid isolation of antigens from cells with a

101

staphylococcal protein A-antibody adsorbent: Parameters of the interaction of antibody-antigen complexes with protein A. J. Immunol. 115, 1617-1624. KOHEN, F., KIM, J. B., LINDNER, H. R., ESHHAR, Z., and GREEN, B. (1980). Monoclonal immunoglobulin G augments hydrolysis of an ester of the homologous hapten. FEBS Len. 111, 427-431. hEMMLI. U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Narure 227,680-685. LERNER,A. R., BENKOVIC,S. J., and SCHULTZ. P. G. (1991). At the crossroads of chemistry and immunology: Catalytic antibodies. Science

252,

659-667.

MANDEL, 8. (1976). Neutralization of poliovirus: A hypothesis to explain the mechanism and the one-hit character of the neutralization. Virology 69, 500-510. ROMBAUT, B., VRIJSEN,R., and BOEY~,A. (1985). Stabilization by host cell components and Mg*+ of the neutralization epitopes of poliovirus. J. Gen. Viral. 66, 303-307. THOMAS, A. A. M., BRIOEN,P., and BOEY~, A. (1985). A monoclonal antibody that neutralizes poliovirus by cross-linking virions. J. Viral. 54, 7-l 3. THOMAS, A. A. M., VRIJSEN,R., and BOEY~, A. (1986). Relationship between poliovirus neutralization and aggregation. 1. Viral. 59, 479-485.

TRAMONTANO, A., JANDA, K. D., and LERNER,R. A. (1986). Chemical reactivity at an antibody binding site elicited by mechanistic design of a synthetic antigen. Proc. Nat/. Acad. Sci. USA 83, 67366740. VRIJSEN,R., ROMBAUT, B., and BOEY~,A. (1983). A simple quantitative protein A micro-immunoprecipitation method: Assay of antibodies to the N and H antigens of poliovirus. /. Immunol. Methods 59, 217-220.