Relationship between Poliovirus Neutralization and Aggregation

Transcription

1 JOURNAL OF VIROLOGY, Aug. 1986, p X/86/8479-7$2./ Copyright 1986, American Society for Microbiology Vol. 59, No. 2 Relationship between Poliovirus Neutraliation and Aggregation ADRI A. M. THOMAS, RAF VRIJSEN, AND ALBERT BOEYE* Dep(aiittUient o Miclrobiology antid Hygiene, Vr-ije Universiitit Brul ssel, B-19 Bruiissels, Belgiuimon Received 9 October 1985/Accepted 5 May 1986 The interaction of mono- and polyclonal neutraliing antibodies with poliovirus was studied. In all cases, neutraliation was due to antibody-mediated virus aggregation, and the unpolymeried virions accounted for the residual infectivity. The effect of papain on previously neutralied virus was to deaggregate the virus to fully infective single virions. With some antibodies, the amount of aggregated virus regressed in the region of greatest antibody excess, even though the virus remained fully neutralied. Under these conditions, noninfective, unaggregated immune complexes were formed. A mutant resistant to one of the monoclonal antibodies was selected. The mutant virions were still bound but no longer aggregated or neutralied by the selecting antibodies. In previous papers, we studied the neutraliation of poliovirus by a single hybridoma antibody (MoAb). This MoAb, 35-1f4, reduced the number of infective units through antibody-mediated virion aggregation (4). After partial neutraliation with MoAb 35-1f4, the residual infectivity was proportional to the remaining amount of unpolymeried virions. The aggregated virions were not intrinsically damaged, for they regained their original sedimentation value of 16S and full infectivity upon dispersion of the aggregates by papain treatment (21). The reduction of infectivity (i.e., by definition, neutraliation) brought about by MoAb 35-1f4 clearly resulted from nondestructive, antibody-mediated virion aggregation. The question which will be addressed in the present paper is whether this conclusion can be generalied. There exists a vast literature dealing with poliovirus neutraliation (e.g., see references 9 and 14). The mechanism proposed by Mandel (13), which is based on an antibody-induced shift in the isoelectric ph of the virus, has been widely accepted but was also recently challenged (7). Emini et al. (11) showed that bivalent binding of the antibody caused neutraliation and pl shift. It was claimed without supporting evidence that nonsaturating, neutraliing amounts of antibody failed to cause virus aggregation (1). Neutraliation associated with stable, monogamous binding of the antibody was demonstrated only for MoAb F7.12 (12). and the residual infectivity of the virions was shown to decrease exponentially as the average number of bound antibody molecules increased. However, MoAb F7.12 also caused aggregation, and it can be seen (12: see Fig. 4 and 6) that this mechanism was the main cause of neutraliation at the medium range of antibody/virion ratios. Baxt et al. (1) showed that at least some monoclonal antibodies directed against foot-and-mouth disease virus neutralied by an aggregation mechanism. The analysis originally performed with MoAb 35-1f4 was extended to all the other neutraliing MoAbs in our own panel (5) and to the MoAbs lc (1) and F7.12 (12). The antibodies of two conventional antisera, previously shown to differ in their ability to shift the isoelectric ph of poliovirus (7), were also included in this study. All of the antibodies tested caused polio virions to aggregate, and the relation of proportionality originally derived for 35-1f4 between the * Corresponding author. 479 residual infectivity and the remaining amount of unpolymeried (16S) virions was generally valid. In the region of extreme antibody excess, however, noninfective, unaggregated immune complexes were formed. MATERIALS AND METHODS Virus neutraliation assay. Type 1 poliovirus strains la/s3 and Mahoney were used (21). The culture of 3H- or "Slabeled virus was as previously described (19, 21). Mutants resistant to MoAb 35-1c3 were isolated as previously described (17). Neutraliation was determined by plaque assay (3) Ȧntibodies. All of the antibodies used are listed in Table 1. The sera and ascitic fluids were assayed for neutraliing activity as previously described (3). The dilutions causing 5% plaque reduction fell between to No plaque reduction with preimmune sera or control ascitic fluids (induced by nonsecreting SP2/ cells) was observed. MoAbs were prepared from ascitic fluids by (NH4)2SO4 precipitation, labeled by reductive methylation with 14C]formaldehyde, and tested for possible inactivation (21); only preparations retaining full activity after '4C labeling were used. Sodium dodecyl sulfate-gel electrophoresis, followed by fluorography, showed that all of the antibody preparations contained at least 8% immunoglobulin. The percentage of 14C binding to excess poliovirus was considered to represent specific antibody content (Table 1). The input ratios mentioned in this paper were corrected for the presence of nonspecific proteins in ascitic fluids and sera. Polyclonal rabbit and guinea pig antibodies (7) were purified and labeled as described above. RESULTS Analysis of neutraliation mixtures. In previous work, we showed the sedimentation profiles of neutraliation mixtures made of 3H-poliovirus and 14C-labeled MoAb 35-1f4. The formation of virion oligomers (antibody-bridged dimers, trimers, etc.) and large aggregates was documented. The analysis was extended to the 12 other neutraliing MoAbs listed in Table 1 and elsewhere (5). Representative sedimentation profiles were selected to document the formation of virion oligomers by using an adequate antibody/virion ratio for each antibody (Fig. 1). In all cases, higher anti- Downloaded from on October 1, 218 by guest

2 48 THOMAS ET AL. TABLE 1. Properties of the antibodies Specific Seii Antibody Isotype" Reference p1 shift" antiendy radioactivity" 35-1h2 IgG lc' IgG 8 NT' 5 24,9 36-5d1O IgM-K 5 + 9( 1, h2 IgG3-K ,35 F7.12v IgG 12 NT 5 5, f4 IgG2a-K 4, , c3" IgG 5 NT NT NT Rabbit NT 7-5 6, Guinea pig NT ,74 Determined by the Ouchterlony double diffusion test. + Means that the antibody at high concentraltion caused the isoelectric pl of poliovirus to shift to approximately ph 4.5. These results were hitherto unpublished or taken from reference 7. See Materials and Methods. d 14 C counts per minute per microgram of p-otein (for the labeling procedure, see Materials and Methods). This antibody, also known as 1BM 55.6, wais kindly provided by R. Crainic and B. Blondel. NT, Not tested. Kindly provided by R. Rueckert. MoAb 35-1c3 bound to N aind H aintigens. aind this double specificity subsisted after recloning by single-cell isolation (21)). This aintibodv was not 14 C labeled. body/virion ratios brought all virus radioactivity down in the pellet. The composition of virus-antibody complexes is given in Table 2. The behavior encountered with all immunoglobulin G (IgG) antibodies except lc, 36-5h2, and F7.12 (see below) is exemplified by MoAb 35-1h2 (Fig. 1A). Oligomers and aggregates were formed, and the residual 16S particles remained free of antibody. MoAb 36-Sd1, the only IgM in our panel, formed so few oligomers that their presence had to be inferred from the antibody profile (Fig. 1C). No dimers were formed by this antibody, whereas they were predominant among the oligomers formed by the IgG antibodies (e.g., Fig. 1A and B). Only one antibody (36-5h2, Fig. 1D) failed to form any oligomers, although it formed large aggregates. Stable association of the antibody with monomeric virions was observed only with MoAbs Ic (Fig. ib), 36-5h2 (Fig. ID), and F7.12 (Table 2) and polyclonal guinea pig antibodies (Fig. 1F). With MoAb lc (Fig. 1B), the peaks in the antibody and virion radioactivity profiles near the 16S position did not coincide exactly, as the antibodies sedimented more slowly than the virions. As a result, the relative antibody content varied from 2. molecules of MoAb lc per virion in the antibody peak fraction to only.15 at the virus peak (Fig. 1B; Table 2). The slower sedimentation of virions carrying a few antibody molecules was probably due to increased friction. Table 2 shows the composition of the virus-antibody complexes sedimenting as single virions, oligomers, or aggregates. The relative antibody content of the aggregates (as defined in the legend to Fig. 1) always exceeded that of the dimers, as well as the input antibody/virion ratio. The oligomers contained at least twice the minimum number of antibody molecules required to link the virions to each other (e.g., one divalent antibody molecule for a dimer or two for a trimer), as was also found with MoAb 35-If4 (21). It was previously reported (12) that MoAb F7.12 formed virion oligomers and that it also bound stably to single virions (two properties not seen together with the MoAbs generated in this laboratory). We confirmed these observations. The results with F7.12 were similar to those obtained with MoAb Ic (Fig. 1B). The fact that 36-5h2 antibody molecules remained bound to 16S particles did not mean that this MoAb neutralied poliovirus by inactivating single virions. At an input ratio of 2.5 antibodies per virion, the infectivity of the mixture was reduced to 1% of that of untreated virus. After ultracentrifugation, it was found that only circa 1% of the virus input sedimented as 16S material. These single virions carried an average of one antibody molecule and retained 8% of the specific infectivity of untreated virus. The remainder of the virus input was recovered as aggregates of low (9%) specific infectivity. Thus, even though MoAb 36-5h2 formed stable complexes with single virions, aggregation was still the main factor in neutraliation by this antibody. For MoAb F7.12, the slope of residual infectivity versus the antibody/virion ratio in monomeric complexes indicated that infectivity was not significantly reduced when only one antibody per virion was bound (12). Our findings with MoAb 36-5h2 therefore do not contradict those of Icenogle et al. (12) Figure 1 and Table 2 also show data on neutraliation (I VOLUME ( ml ) J. VIROL. FIG. 1. Sedimentation profiles of mixtures of 3H-labeled poliovirus and 14C-labeled antibodies. 3H-labeled poliovirus strain la/s3 was used, except for MoAb lc (B), where it was replaced by Mahoney. The final virus concentration was 24 nm. '4C-antibody was added to the virus in the molar ratio shown below. The buffer was PBS-A (14 mm NaCI. 6.5 mm KCI, 5.5 mm phosphate..5 mm MgCIl..1 mm CaCI2 containing.1 mg of bovine serum albumin per ml, final ph 7.2). The mixtures, in a final volume of 1.1l, were incubated for 1 h at 37TC and overnight at 4 C. After taking a 1-p.I sample for plaque assay. we layered the remainder onto 14.5-ml 15 to 3% sucrose gradients in phosphate-buffered saline and centrifuged it for 2.5 h at 85, x g.a at 4 C in an SW3 rotor (Measuring and Scientific Equipment, Ltd.). The pellet was suspended in 3,ul of PBS-A. and all fractions were analyed for radioactivity as previously described (21). Under the conditions of centrifugation used, complexes larger than hexamers reach the bottom of the gradient. These complexes are referred to as aggregates. The amounts of virus and antibody in aggregates are represented by closed and open bars, respectively. The input antibody/virion molar ratios (Ab/V) and residual infectivities (RI) of the mixtures before centrifugation were as follows: A, 2. Ab/V, RI 9%: B. 4.7 Ab/V. RI 85%: C,.5 Ab/V, RI 45%: D, 1.3 Ab/V, RI 55%: E. ).7 Ab/V, RI 65%: F,.4 Ab/V, RI 95%. i a 7 ;5 l3 Downloaded from on October 1, 218 by guest

3 VOL. 59, 1986 POLIOVIRUS NEUTRALIZATION AND AGGREGATION 481 mixtures made with antibodies purified from conventional rabbit and guinea pig antisera. The rabbit antibodies (Fig. 1E) behaved like the majority of the MoAbs; oligomers and large aggregates were formed, and little antibody remained bound to single virions. In contrast, the guinea pig antibodies (Fig. 1F) yielded few oligomers, and more antibody remained bound to 16S virions. Relation of residual infectivity to the amount of 16S virions remaining after neutraliation. Although the amount of virus in oligomers was variable, depending on the antibody used, it never exceeded 5 to 1% of the total virus input. The specific infectivity of the oligomers was reduced but not nil, as was also reported for 35-1f4 (21) and F7.12 (12). Therefore, incorporation of virions into oligomers contributed only slightly to neutraliation. With all of the antibodies listed in Table 1, the specific infectivity of aggregated virus was between 1 and 1% of that of untreated controls (data not shown). The residual infectivity of virus-moab 35-1f4 mixtures correlated well with the percentage of 16S virus remaining after neutraliation, as shown by sucrose gradient centrifugation (21). All available data on antibodies other than 35-1f4 are shown in Fig. 2. In all of the cases studied, the same linear relation was found as for 35-1f4. Thus, at least for all of the antibodies presently studied, the loss of infectivity was mainly due to virion aggregation. Effect of papain on previously neutralied poliovirus. As TABLE 2. Composition of virus-antibody complexes isolated after sucrose centrifugation of neutraliation mixtures Neutraliation mixture Composition of immune complexes comprising monomeric and polymeried poliovirions (antibody/virion molar ratio) Antibody/ virion Antibody inputa Monomers Dimers Aggregates (molar ratio) 35-1h2.9 <.b 2.5 C 1.2 <.2b d <.3b <.3b lc.4.6;.33e NDf ;.71e ND d. 15; 2.e ; 3.e ND dlO.8 <.3 OC (Trimers: 1.2) h2.5.4;.6e o d.4;.8e o ; 1.2e OC 6.6 F Rabbit Guinea pig.37d.4 1.5b ; 4.e atype 1 poliovirus of strain Mahoney was used with MoAbs F7.12 and lc; strain la/s3 was used in all other cases. bowing to trailing of 14C-antibody, these ratios are rough estimates. ' No sufficient dimers (or aggregates) were present. d Data from the profile shown in Fig. 1. ethe first figure refers to the virus peak, and the second refers to the antibody peak (see the text for further details). f ND, Not determined. x 'U U1) to U) n : 5 LL uj C.) RESIDUAL INFECTIVITY (PERCENT) FIG. 2. Relationship between residual infectivity and amount of residual 16S virions after neutraliation. Residual infectivity of neutraliation mixtures was measured as described previously (3). The mixtures were analyed as described in the legend to Fig. 1, and the amount of unpolymeried virions was calculated. O, 36-5d1O; *, 36-5h2; *, 35-1c3;, rabbit antibodies. -, Theoretical expectation assuming infectivity to be exclusively associated with unpolymeried virions , Theoretical expectation assuming that neutraliation was unaccompanied by polymeriation. reported elsewhere (21), papain acting on poliovirus previously neutralied and aggregated by MoAb 35-1f4 caused deaggregation, restoration of infectivity, and release of fully infective, monomeric virions. We repeated the experiment with MoAbs lc, 36-5d1O, and 36-5h2 and polyclonal rabbit antibodies. Enough antibody (four to seven molecules of antibody per virion) was used to aggregate the virus completely (Fig. 3) and to lower the infectivity of the mixtures to 8% of the original or less (Table 1 DA B C D d 36-5h2 Rabbit,,5-< VOLUME (ml) FiG. 3. Deaggregation of neutralied poliovirus by papain. To 2.4 pmol (5, cpm) of 35S-Mahoney virus (A to C and E to G) or.8 pmol (5, cpm) of 3H-laIS3 virus (D and H) the following amounts of antibody were added in a final volume of 1 p.1 of phosphate-buffered saline minus divalent cations, supplemented with 2 mm L-cysteine and 1 mm EDTA. A and E, 18 pmol of 1c; B and F. 1 pmol of 36-Sd1; C and G, 16 pmol of 36-5h2; D and H, 6 pmol of polyclonal rabbit antibodies. After 1 h at 37 C, 12.5 u1l of buffer (A to D) or buffer containing 2.5,ug papain (E to H) was added. After 4 h at 37 C and overnight at 2 C, 1 pu1 was taken to determine the residual infectivity, and the remainder was layered onto 15 to 3% sucrose gradients made in phosphate-buffered saline. Centrifugation was for 3 h (A to C and E to G) or for 2.5 h (D and H) at 85, x at 4C pmin an MSE SW3O rotor. For further details, see the legend to Fig. 1. The arrows indicate positions to which untreated virus sedimented.. Downloaded from on October 1, 218 by guest

4 482 THOMAS ET AL. TABLE 3. Reactivation of poliovirus by papain Residual infectivity Specific infectivity Addition" of mixture %) of 16S particlesb None 1 1 Monoclonal antibodies 1c' 2 lc + papain' dlOc dlO + papain' h2' h2 + papain' NDe 8 Rabbit antibodies' 8 Rabbit antibodies papainc asee the legend to Fig. 3 for details. Ratio of PFU to radioactivity, assayed after sucrose gradient isolation. Sedimentation profile shown in Fig. 3. d No 16S material available. ND, Not done. 3). After papain treatment, infectivity was restored to at least 33%, and the bulk of the virus again sedimented as single virions (Fig. 3) of full specific infectivity (Table 3). Effect of antibody concentration on the formation of large aggregates. The experiments reported above were carried out at high virus concentration (7 x 19 PFU/ml). In this concentration region, the degree of neutraliation depends on the antibody/virion ratio. At lower virus concentrations (i.e., less than 1.5 x 18 PFU/ml), the degree of neutraliation is determined solely by the antibody concentration (percentage law; reference 3). We examined whether aggregation also occurred when the percentage law applied. Therefore, we used the minimum virus concentration allowing radioactivity measurements, i.e., 5 x 17 PFU/ml or.16 nm virus. 1- -i LU CL. -J LU LU U) A B C D 35-1h2 lc 36-5d1 36-5h2- E F G H Rabbit Ginea pig F lf pmol Ab (loglo ) 1 5 m c--4 m r- N 1 > I 5 - FIG. 4. Formation of large poliovirus aggregates by mono- and polyclonal antibodies. To 1, cpm of 3H-poliovirus, i.e.,.3 pmol of Mahoney () or.5 pmol of la/s3 (), the indicated amount of specific antibody was added in a final volume of 1 p.l of PBS-A (see the legend to Fig. 1). After 16 h at 23 C, 1 p.1 was taken for the plaque assay, and the remainder was centrifuged as described in the text. The radioactivity of the supernatant and pellet (suspended by heating at 56 C for 5 min in 1% sodium dodecyl sulfate) was determined and expressed as a percentage of the input. Preimmune sera never aggregated more than 1% of the input virus (data not shown). Neutraliation experiments were done with Mahoney virus. To avoid laborious sucrose gradient analyses, the amount of large aggregates was estimated by a simpler method. When neutralied mixtures were centrifuged in conical 1.5-ml tubes for 1 min at 1, x g, aggregated virus was pelleted, whereas monomeric virions and oligomers remained in the supernatant. Using this procedure, we measured the percentage of virus in the pellet as a function of antibody concentration. In some cases, the residual infectivity of the mixtures was also determined before centrifugation. Figure 4 shows the results. Most MoAbs and sera yielded a three-legged curve. At first, the amount of aggregated virus increased with increasing antibody concentration until total aggregation was reached with most antibodies. At a large antibody excess, however, the amount of pelleted virus tended to regress as the antibody input was further increased. The findings confirmed that all neutraliing antibodies caused virus aggregation. However, whereas some MoAbs caused total aggregation over a wide antibody concentration range (Fig. 4A, D, E, and H), this range was narrower with other MoAbs (Fig. 4B and C), and in two cases the virus was incompletely pelleted at any antibody/virus ratio (Fig. 4F and G). Our findings of incomplete aggregation with antibody F7.12 may seem at variance with those of Icenogle et al. (12), who reported complete aggregation. However, when we applied the same centrifugal conditions as used by them (and by ourselves [Fig. 1]), the virus was indeed fully aggregated. The low-speed centrifugation method evidently yielded a lower estimate of the extent of virus aggregation. Neutraliation curves were determined for four antibodies (Fig. 4B, D, G, and H). They paralleled the rising leg of the aggregation curves; in three cases (Fig. 4 D, G, and H), the neutraliation curve preceded the aggregation curve by about.5 log1. This does not contradict the results in Fig. 2, wo I V 1 8 3' 2 1 A IB =I I.~ N ; 5. -,1- J. VIROL. VOLUME (ml) FIG. 5. Formation of aggregates and single-virion complexes as a,function of antibody concentration. To.3 pmol of Mahoney virus (3,3 cpm) the following amounts of MoAb lc were added in a final volume of 1 p.1 of PBS-A (see the legend to Fig. 1): A, none; B,.5 pmol; C, S pmol; D, 5 pmol. After 16 h at 23 C, 1 pi. was taken for plaque assay, and the remainder was centrifuged for 45 min in an MSE SW6 rotor at 25, X gav in a 15 to 3% sucrose gradient made in phosphate-buffered saline. Fractions of 3 p.l were collected and counted for radioactivity. The bar represents the radioactivity in the solubilied pellet. The residual infectivity of the mixtures before centrifugation was: A, 1%; B, 56%; C,.6%; D,.5%. Downloaded from on October 1, 218 by guest

5 VOL. 59, 1986 POLIOVIRUS NEUTRALIZATION AND AGGREGATION 483 as the supernatants of low-speed centrifugation contained small aggregates in addition to single virions. The virus remained fully neutralied, even when aggregation regressed. In immunoprecipitation assays mediated by Staphylococcus aureus Cowan I (23), MoAb 35-1h2 bound to lais3 but not to Mahoney virus. The reverse was true of F7.12. All of the other antibodies bound to both strains of poliovirus. To assess the possible influence of the virus strain on the results, a comparison was made between the la/s3 and Mahoney viruses with some antibodies that recognied both (Fig. 4B, E, and F). Similar aggregation curves were obtained with both virus strains. Formation of single-virion immune complexes in the region of high antibody excess. The aggregation curve of MoAb Ic (Fig. 4B) illustrated the negative effect of great antibody excesses on aggregation particularly well. This antibody was therefore chosen to study the physical condition of the virions after reacting with a great antibody excess. Labeled poliovirus (.3 nm) was incubated with antibody lc in concentrations ranging from 5 to 5 nm, and the mixtures were centrifuged in 15- to 3% sucrose gradients (Fig. 5). Antibody at a concentration of 5 nm caused all of the input virus to aggregate and neutralied the mixture by more than 99%. With 5 nm antibodies, only 1% of the virus remained in the pellet, and most of the virus radioactivity sedimented slightly slower than native virions (compare Fig. 5, panels A and D). These particles were by no means native virions, however, as the residual infectivity of the mixture was only.5% of the original. To find out whether these particles carried antibody,.3 nm 3H-labeled virus was incubated with 25 nm "4C-labeled antibody lc, and the mixture was centrifuged in a 15 to 3% sucrose gradient. The result is shown in Fig. 6B. The pellet contained only a negligible amount of virus or antibody. As 3- A 3-c _ ~~B 3-D -2 > ~ jo.8 2 ".4 co VOLUME (ml) FIG. 6. Sedimentation of native poliovirus and single-virion complexes in two different sucrose gradients. To.3 pmol of 3H-Mahoney virus either no antibody (A and C) or 25 pmol of '4C-labeled antibody lc (B and D) was added in a final volume of 1,ul of PBS-A (see the legend to Fig. 1). After 18 h at 23 C, 1,u was taken for plaque assay, and the remainder was centrifuged in 15 to 3% (A and B) or 46 to 52% (C and D) sucrose gradients made in phosphate-buffered saline. Gradients A and B were centrifuged for 2.5 h at 4 C at 85, X gav and fractionated into 4-pd fractions. Gradients C and D were run for 17 h at 4 C at 25, X ga, and fractionated into 2-pul fractions. Bars represent the radioactivity in solubilied pellets. Owing to the large admixture of 14C in the top fractions, the 3H radioactivity could not be determined accurately in these fractions (B and D). I- LU LUI. ā J -J C,) 1 5 Ie I X 173 I 1lo1 1 DILUTION OF ASCITIC FLUID FIG. 7. Neutraliation and aggregation of poliovirus by MoAb 35-1c3: comparison of the wild type and a neutraliation-resistant mutant. Ascitic fluid of 35-1c3 was serially diluted 1-fold in phosphate-buffered saline. The dilutions were mixed with 1, cpm of 35S-labeled la/s3 or mutant virus (resistant to neutraliation by 35-1c3). Incubation was for 1 h at 37 C. The amount of virus pelleted was determined as described in the legend to Fig. 4. Virus in pellet: wild type, A; mutant,. Neutraliation: wild type, A; mutant,. in the preceding experiment, the main peak of virus radioactivity sedimented slightly slower than untreated virions (compare Fig. 6A and B). As might be expected from the 7,5-fold molar excess of antibody, the bulk of the antibody remained free. However, some antibody was associated with the virion peak as well as with minor, faster-sedimenting virus material (presumably virion di- and trimers). From the known specific radioactivities of the virus and antibody, it was calculated that each monomeric virion carried about 1 molecules of IgG antibody. It seemed incredible that a mass increase of 15 million (15, x 1) daltons would leave the sedimentation velocity of the virions almost unchanged. However, Icenogle et al. (12) also found no significant change in the sedimentation velocity of virions when 3 molecules of F7.12 antibody were bound to each virion. It was possible that the effect of the increased mass was offset by the lower density and higher frictional coefficient of the immune complexes; if that were the case, increasing the density and viscosity of the medium might be expected to slow the immune complexes more than the native virions. To test this, a mixture of virus and antibody lc was prepared as described above, but centrifugation was carried out in a 46 to 52% sucrose gradient instead of in the usual 15 to 3% gradient. In this highly viscous medium, the immune complexes sedimented about 2% slower than native virions (compare Fig. 6C and D), thus confirming the theoretical expectation. The composition of the immune complexes was unchanged, i.e., about 1 antibody molecules per virion. Relationship between binding, aggregation, and neutraliation of a mutant virus. Mutants resistant to neutraliation by MoAb 35-1c3 were selected within the la/s3 virus strain. Constant amounts of the wild type or the resistant mutant were incubated with various concentrations of MoAb 35-1c3, and aggregation and residual infectivity were measured (Fig. 7). The neutraliation curve of the wild-type virus preceded the upward leg of the aggregation curve by.5 log1o in antibody concentration, as also observed with other MoAbs (see above). The sedimentation profile (data not shown) and aggregation of the wild type were compara- 5 m c ~-q --4 Downloaded from on October 1, 218 by guest

6 484 THOMAS ET AL. ble to those shown in Fig. 1A for MoAb 35-1h2. On the other hand, the mutant was neither measurably neutralied nor aggregated by the selecting MoAb 35-1c3 at any concentration (Fig. 7). Remarkably, protein A-aided immunoprecipitation experiments showed that antibody 35-1c3 still bound to the mutant, although with less affinity than to the wild type. The immunoprecipitation titer of antibody 35-1c3, as measured against the mutant virus, was 1.3 log1 lower than the titer measured against the wild-type antigen. Thus, at the higher antibody concentrations (e.g., at the 1-1 dilution [Fig. 7]), the virions were still recognied by the selecting antibody, but they were neither aggregated nor neutralied. In conclusion, the ability to bind to virions is a necessary but not a sufficient condition of neutraliation; virions are neutralied only if the antibody also causes them to aggregate. DISCUSSION Two situations were distinguished by the antibody concentration. Either the concentration was just sufficient to neutralie the virus by about 2 log1, or it was much greater than that. The region of moderate antibody excess. The ability to cause poliovirus aggregation, formerly documented for MoAb 35-1f4, was found to be a general property of all of the neutraliing MoAbs we examined, which were generated in three different laboratories. All antibodies studied converted 16S virions to aggregates of high antibody content and low residual infectivity. The infectivity loss of partly neutralied mixtures was well correlated with the loss of 16S particles to aggregation. It was concluded that aggregation was the basis of neutraliation by all of the antibodies studied. This conclusion was strengthened when a mutant resistant to neutraliation by one of these antibodies was studied. Even though the antibody still recognied the mutant virions in immunoprecipitation tests, it no longer aggregated or neutralied them. Resistant mutants that were still bound by the selecting antibody were also reported by Blondel et al. (2). It might be argued that aggregation is secondary to neutraliation, i.e., that aggregation occurs only after antibodies bind to single virions and inactivate them. If this were the case, one would expect a substantial contribution of unaggregated, noninfective immune complexes. At least at moderate degrees of neutraliation, at which the amount of 16S material could still be measured, this was not so (Fig. 2). When single virions were released from aggregates by papain digestion, they were fully infective. To reconcile this fact with the hypothesis of preaggregation inactivation requires the additional hypothesis that the damage was reversible by papain. The least contrived explanation is that the virions suffered no intrinsic inactivation, being simply linked into aggregates by the antibody and released by papain. Aggregation was not restricted to experimental conditions involving huge virus concentrations such as those used when neutraliation mixtures are to be analyed by sucrose gradient centrifugation. To show this, the relatively low concentration of 5 x 17 PFU/ml (.16 nm) was used, and a simplified technique was used, in which the aggregates were collected by low-speed centrifugation. Without exception, all antibodies again caused virus aggregation. With most antibodies, the amount of pelleted virus regressed in the region of extreme antibody excess (see below). It was claimed that some MoAbs (including lc) failed to cause poliovirus aggregation (1). However, no mention was made of either the virus or antibody concentration. When a single antibody concentration is used, and this happens to be outside the range at which virus aggregation occurs, one may erroneously conclude that the antibody does not cause aggregation. This possibility is particularly evident for MoAb lc (Fig. 4B). In conclusion, (i) for all antibodies studied, aggregation was the most important and, in most cases, the only known mechanism of neutraliation, and (ii) direct inactivation of unpolymeried 16S particles (12) contributed little to neutraliation. The region of great antibody excess. Neutraliation may occur without aggregation, but only in the presence of an enormous antibody excess (Fig. 5). Whereas antibody Ic at a concentration of 5 nm ensured full aggregation and neutraliation (Fig. SC), a 1-times-higher concentration was required for full conversion to single-virion complexes. Recently, McCullough (16) reported aggregation of footand-mouth disease virus by a monoclonal antibody and the formation of monomeric complexes when 1 times more antibody was used. Our findings are in agreement with the immune lattice theory (15, 18). The theory predicts formation of virion oligomers in the region of antigen excess, of large aggregates in balanced virus-antibody mixtures, and of single virions, surrounded by as many antibody molecules as their valence allows, when the antibody is in great excess. Poliovirus is expected to carry a maximum of 6 molecules of antibody, provided each is attached by a single paratope, as was observed with tobacco mosaic virus in the region of extreme antibody excess (22). That the actual number of antibody molecules per virion was even higher suggests that unspecific binding occurred. The antibody-saturated virions remained noninfective. In conclusion, the occurrence of virus aggregation and the contribution of aggregation to loss of infectivity of the virus population (i.e., neutraliation) depends on at least three kinds of factors: (i) the physicochemical environment: lowering the ionic strength may replace an aggregative by a disruptive mechanism of neutraliation [6]; (ii) the antibody concentration, as discussed above; and (iii) the preference of each monoclonal antibody for mono- or bigamous binding. Monogamous, bivalent binding of MoAbs 35-1f4 [21], 35-1h2, and 36-5d1O was not observed; on the other hand, MoAbs lc, 36-5h2, and F7.12 were capable of both monoand bigamous binding; antibodies incapable of bigamous binding may well exist but have not yet been encountered. ACKNOWLEDGMENTS J. VIROL. We thank R. Crainic, B. Blondel, and R. Rueckert for their kind gifts of ascitic fluids, B. Rombaut for critical comments, and M. De Peismacker, A. De Rees, and S. Peeters for technical assistance. 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