Antigenic Sites of Poliovirus Type 3 Eliciting IgA Monoclonal Antibodies in Orally Immunized Mice

Virology 281, 265–271 (2001) doi:10.1006/viro.2000.0786, available online at http://www.idealibrary.com on

Antigenic Sites of Poliovirus Type 3 Eliciting IgA Monoclonal Antibodies in Orally Immunized Mice Gabriele Buttinelli,* Franco M. Ruggeri,† Jill Marturano,* Francesco Novello,* Valentina Donati,* and Lucia Fiore* ,1 *Laboratory of Virology and †Laboratory of Ultrastructure, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy Received August 7, 2000; returned to author for revision October 12, 2000; accepted December 5, 2000 A panel of neutralizing IgA monoclonal antibodies was produced from mice orally inoculated with poliovirus type 3 Sabin and cholera toxin as adjuvant. Low levels of neutralizing antibodies were elicited in mice after several boosts, but only in the presence of cholera toxin. Characterization of IgA MAbs by neutralization-escape virus mutants showed that all but one neutralizing MAbs against type 3 poliovirus were directed to antigenic site N-AgIII, which was previously found by us to be the major target of mucosal immune response to Sabin 1 in the mouse. Our data indicate that residue 236 of VP3, not previously reported, is also involved in forming site N-AgIII in addition to formerly described VP3 (aa 58–59) and VP1 (aa 286–290) residues. Unlike poliovirus type 1 IgA MAbs, all IgA MAbs herein described neutralized the wild-type parental poliovirus. © 2001 Academic Press Key Words: poliovirus; monoclonal antibody; IgA; mucosal immunity; neutralization; epitope.

INTRODUCTION

the three poliovirus serotypes (Minor et al., 1990; Wiegers et al., 1990; Uhlig et al., 1990). Whereas the antigenic determinants involved in poliovirus neutralization in vitro have been widely characterized with humoral monoclonal antibodies generated from parenterally immunized mice, only limited information is available on whether the same viral epitopes are also accounted for by sIgAs obtainable by mucosal immunization. In a recent study, we generated a panel of hybridoma cell clones secreting IgA-class MAbs against poliovirus type 1 Sabin by oral immunization of normal mice with live virus in combination with cholera toxin (CT) as a mucosal adjuvant (Fiore et al., 1997). Most neutralizing IgA-MAbs investigated were directed to the VP3 epitope at amino acid 59 (N-AgIII), indicating that this site of poliovirus type 1 is particularly efficient in eliciting an immune response also on the intestinal mucosa. None of the MAbs recognized the N-AgI site (aa 89–100) of VP1, confirming that this site is immunorecessive for poliovirus type 1 also in the gut-associated lymphoid tissue. One of the IgA-MAbs selected a virus variant carrying a novel mutation at amino acid 138 in VP2, which, however, was shown to be partially related to the N-AgII loop previously described around VP2 residue 164. Most interestingly, only 2 of 13 MAbs characterized neutralized the wild-type Mahoney strain. To extend knowledge on mucosal immunity to poliovirus, we have undertaken a similar investigation on type 2 and 3 Sabin strains. In this paper, we report the isolation and characterization of seven neutralizing IgA MAbs for Sabin type 3 and one for Sabin type 2 poliovirus, respectively. The MAbs were analyzed in detail by using various

Protection from poliomyelitis greatly relies on humoral neutralizing antibodies elicited by either infection with wild or vaccine poliovirus (OPV) or injection of inactivated vaccine (IPV). Secretory dimeric IgA (sIgA) antibodies produced in the intestine are also likely to play a critical role in protection by preventing extensive virus replication and/or crossing of the gut mucosa (Ogra et al., 1971; Savilahti et al., 1988; Solari et al., 1985). Detailed knowledge of antigenic determinants, especially neutralizing determinants of the viruses, is therefore essential for understanding the mechanisms of antibody-mediated virus neutralization and protection from disease. The poliovirus virion shows an icosahedral capsid composed of 60 copies of proteins VP1, VP2, VP3, and VP4. The precise structure of the capsid has been established for wild poliovirus type 1 Mahoney (Hogle et al., 1985) and for poliovirus type 3 Sabin (Filman et al., 1989) strains by X-ray crystallography, and the antigenic structure of poliovirus has been extensively studied by characterization of neutralizing IgG monoclonal antibodies (MAbs) from parenterally immunized mice. Genome sequencing of neutralization-resistant variants selected by MAbs has identified three major (N-AgI through III) and two minor (N-AgIV and V) neutralization sites due to the contribution of residues from the three capsid polypeptides VP1, VP2, and VP3 with some differences between

1

To whom reprint requests should be addressed at Laboratory of Virology, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy. Fax: (⫹39 06) 49902082. E-mail: [email protected]. 265

0042-6822/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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BUTTINELLI ET AL. TABLE 1 Comparison of Neutralization Titers of IgA Monoclonal Antibodies against Viral Antigenic Variants, Parental Sabin 3, and Leon Type 3 Poliovirus Strains Neutralization titer

MAb Group I 1A4 Group II 1F5 1F9 1G1 2B5 3B9 Group III 1B8

v1A4

v1F5

v1F9

v1G1

v1B8

Sabin 3

Sabin 3 ⫹ tryp

Leon

⬍40

5120

5120

5120

2560

5120

5120

5120

1280 1280 5120 640 1280

⬍40 ⬍40 ⬍40 ⬍40 ⬍40

⬍40 ⬍40 ⬍40 ⬍40 ⬍40

⬍40 ⬍40 ⬍40 ⬍40 ⬍40

2560 5120 2560 640 640

2560 5120 2560 640 1280

2560 5120 2560 640 640

1280 5120 5120 1280 160

2560

5120

5120

5120

40

2560

2560

2560

immunological methods and cross-neutralization with neutralization-escape viral mutants. Nucleotide sequencing of neutralization-escape variants was performed to locate mutations involved in resistance to the antibodies. RESULTS Development of immunity to poliovirus in mice Four groups of three mice each were orally inoculated with Sabin 2 or 3 poliovirus with or without CT as a mucosal adjuvant. Anti-viral neutralizing antibodies were measured in sera after each antigen boost. No antibody response developed in mice receiving poliovirus alone whereas increasing serum antibody titers (up to 1430 for type 3, and 570 for type 2 poliovirus, respectively) were detected in all but one (type 2) mice given poliovirus in combination with CT. Hybridoma screening Fusions were screened by both neutralization and enzyme-linked immunosorbent assays (ELISA), as indicated under Materials and Methods. For type 3 poliovirus, two separate fusions yielded altogether 11 neutralizing (7 IgA and 4 IgG) and 13 nonneutralizing (9 IgA and 4 IgG) MAbs. A neutralizing IgA and 14 nonneutralizing (8 IgA and 6 IgG) MAbs were isolated from a fusion specific for type 2 poliovirus. No IgM antibody-producing clone was obtained. Hybridomas secreting neutralizing IgA MAbs were cloned three times, and antibodies were examined by SDS-PAGE under nonreducing conditions and IgA-specific Western blotting. The presence of both IgA dimers and monomers in cell supernatants (data not shown) was assessed before further investigation. Isolation of neutralization-escape poliovirus mutants Poliovirus neutralization-resistant variants were selected by plaque isolation in the presence of antibody.

Mutants were obtained with five of the seven neutralizing IgA MAbs produced against type 3 and with the only type 2-specific neutralizing IgA MAb. All isolated variants could be grown to similar titers as parental poliovirus on following replication in cell cultures and retained their resistance characteristics upon passage in cell cultures. Neutralization analysis of neutralization-escape poliovirus mutants with IgA MAbs Sabin 3 poliovirus variants were examined for neutralization by the whole panel of IgA MAbs (Table 1). Variants v1A4.1, 2, 3 proved resistant only to the MAb 1A4 antibody used for their selection and were otherwise neutralized by all other anti-PV3 MAbs isolated. MAb 1A4 was arbitrarily assigned to group I of reactivity. Five MAbs (1F5, 1F9, 1G1, 2B5, and 3B9) were assigned to group II of reactivity, because variants v1F5, v1F9, and v1G1 were shown to escape neutralization by all of these MAbs. Conversely, these three variants were neutralized by both MAbs 1A4 and MAb 1B8. Finally, MAb 1B8 was assigned to group III, as it could neutralize all the variants isolated. All IgA MAbs studied neutralized intact as well as trypsin-cleaved Sabin type 3 poliovirus to a similar extent, suggesting that they were directed at antigenic sites of the virion other than N-AgI. All MAbs also neutralized the Leon type 3 wild poliovirus. No MAb showed cross-neutralization with either wild or Sabin type 1 or 2 polioviruses. The only MAb (1G6) selected against poliovirus Sabin 2 neutralized both intact and cleaved type 2 Sabin poliovirus and the wild-type 2 MEF-1 strain. Sequence analysis of neutralization-escape mutants To investigate the IgA MAb epitope specificity, virus variants were subjected to nucleotide sequence analysis of the genomic region encoding the poliovirus capsid proteins (Table 2), and compared with parental Sabin 3

IgA MONOCLONAL ANTIBODIES TO SABIN TYPE 3 POLIOVIRUS

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TABLE 2 Nucleotide and Amino Acid Substitutions in Poliovirus Type 3 Sabin Neutralization-Resistant Variants

MAb group

Polio 3 Sabin variant

Protein

v1A4 v1F5.1 v1F5.2 v1F5.3 v1F9 v1G1 v1B8

VP3 VP1 VP1 VP3 VP3 VP3 VP3

I II

III

Nucleotide change (bp) 1939 3334 3340 2468 2468 2468 1993

or 2 poliovirus strains (Toyoda et al., 1984). To verify whether different mutations could be responsible for resistance to neutralization by any given MAbs, multiple mutant poliovirus plaques were picked and eventually subjected to sequence analysis (three against MAb 1A4, six for 1F5 and 1F9, and seven for 1G1 MAbs). As shown in Table 2, in most cases different plaques yielded neutralization-escape virus variants showing the same base mutation. However, in the case of MAb 1F5, three different mutations were observed among the virus variants isolated, namely G3334C, A3340G, and C2468T. Whereas the first two mutations corresponded to the already established change of amino acids 286–288 of VP1 within N-AgIII, the latter mutation produced a substitution of amino acid 236 in VP3 which had not been previously described. Other MAbs (1F9 and 1G1) belonging to reactivity group II were shown to be only associated with residue 236 in VP3 which, based on results of crossneutralization analysis, is most likely part of the N-AgIII site itself. In the case of MAb 1A4, all variants isolated showed an identical mutation corresponding to N-AgIII at amino acid residue 59 of VP3. Sequencing of v1B8 escape mutants showed mutation of nucleotide 1993 corresponding to amino acid 77 of VP3, which is part of antigenic site N-AgIV. None of the antibodies studied was shown to select for mutations in N-AgI or N-AgII of poliovirus type 3. Sequence analysis of the escape mutant to type 2 poliovirus MAb 1G6 showed mutation of nucleotide U3146C leading to a leucine to serine change at residue

G3A G3C A3G C3T C3T C3T G3C

Amino acid change (aa)

Neutralization site

Ser 3 Asn (59) Arg 3 Ser (286) Asn 3 Asp (288) Leu 3 Phe (236) Leu 3 Phe (236) Leu 3 Phe (236) Asp 3 Asn (77)

N-AgIII N-AgIII N-AgIII N-AgIII N-AgIII N-AgIII N-AgIV

222 of VP1. Mutations in this position have not been described previously; this amino acid is probably part of N-AgII, as it is close to the mutated residues defining this site described by Patel and co-workers (1993). Characterization of poliovirus type 3 N-AgIII site To better define the amino acid residues forming the N-AgIII site of polio type 3, selected virus variants were assayed for neutralization by formerly characterized IgG MAbs (kindly supplied by Dr. Morag Ferguson, NIBSC, South Mimms, UK) 1023 and 840, affected by mutations within VP3 at residues 58–59 of VP3 and within VP1 at residues 286–290 (Minor et al., 1986) and 138, mapping at residues 286–290 of VP1 (Ferguson et al., 1982). The results of these assays are reported in Table 3, where IgA MAbs 1A4 and 1G1 described in this study are also included for comparison. Three distinct patterns of cross-neutralization were shown for the four MAbs toward virus neutralization-escape mutants changed at amino acids 3059, 1286 or 1288, or 3236. Neutralization with MAbs 138 and 1G1 was inhibited by mutations in either 1286–1288 or 3236, but not in 3059. Conversely, MAb 1023, linking site 3059 with site 1286, fully neutralized poliovirus variant v1G1 showing a change of 3236. Finally, neutralization by MAb 1A4 was only affected by a change at position 3059. Overall, these data suggest that site N-AgIII is a complex domain involving both residues 59 and 236 of VP3 and VP1 residues 286–290. A three-dimensional representation of the poliovirus type 3 protomer is reported in Fig. 1, where the position

TABLE 3 Comparison of Neutralization Titers of IgG and IgA MAbs against Viral Antigenic Variants Parental Sabin, and Leon Type 3 Poliovirus Strains MAb IgG

IgA

138 1023 840 1A4 1G1

v1A4

v1F5.1

v1F5.2

v1G1

Sabin

Leon

1280 ⬍40 ⬍40 ⬍40 5120

⬍40 ⬍40 1280 5120 ⬍40

⬍40 ⬍40 40 5120 ⬍40

⬍40 5120 1280 5120 ⬍40

5120 2560 1280 5120 5120

⬍40 ⬍40 1280 5120 2560

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FIG. 1. Poliovirus type 3 Sabin. Ribbon representation of the protomer (a, front view; b, side-view). VP1, VP2, VP3, and VP4 are colored cyan, magenta, green, and white, respectively. Residues 286 of VP1 and residues 235 and 59 of VP3 are shown as spheres and colored yellow, orange, and red, respectively. The distance between residue 286 of VP1 and 235 of VP3 is approx 17 Å; residue 286 of VP1 and 59 of VP3, 21 Å; residue 235 of VP3 and 59 of VP3, 30 Å. The pictures have been generated from the coordinates deposited in the Brookhaven Protein Data Bank (Bernstein et al., 1977) (PDB entry: 1pcv) using the software Insight II. Residue 236 of VP3 is not present in the coordinate file (Filman et al., 1989); as reference, residue 235 has been used.

of amino acids 3059, 3236, and 1286–1288 composing the N-AgIII site is indicated by spheres. The image, presented for description purposes, is based on the crystal structure defined for Sabin 3 poliovirus (Filman et al., 1989) and does not take into account possible perturbations induced by the change of amino acids in the area of N-AgIII. The frontal view of the protomer shows that residues 3059, 3236, and 1286–1288 define a triangle with approximate distances between vertices ranging from a minimum of 16 Å to a maximum of 23 Å, in all cases well within the spanning area of an IgA antibody. DISCUSSION In view of the global eradication of poliomyelitis, immunization with the oral live poliovirus vaccine established by Sabin more than 40 years ago will soon come to an end (de Quadros et al., 1992; Hull et al., 1994; WHO, 1994). As a consequence of the continuous decrease of wild poliomyelitis cases worldwide following the massive efforts put into vaccination during the last decades, the risk of vaccine-associated paralysis, although very low, is rapidly becoming a toll no longer acceptable particularly in developed countries. Also, the shedding of live vaccine polioviruses into the environment and their possible recirculation in humans infected by the oralfecal route may allow novel virus strains to spread in the community, to evolve accumulating genomic mutations, and eventually to change back into neurovirulent polio-

viruses. For these reasons, it can be anticipated that immunization of children will not be stopped for at least several years after achievement of eradication to maintain a broad herd immunity in the global population, although live vaccine will be totally dismissed and replaced by killed vaccines. In fact, the latter have already been introduced in substitution of or in a combined schedule with OPV in many countries worldwide, and such a trend will increase further with time. Despite their unquestionable safety, a major drawback of inactivated poliovirus vaccines is, however, the fact that parenteral immunization may not ensure a mucosal immunity effective enough to halt replication in the gut epithelium and to stop spreading of polioviruses (taken up by vaccinees from the environment or from healthy carriers) with feces (Carlsson et al., 1985; Manor et al., 1999). Therefore, besides the implemented epidemiological surveillance of acute flaccid paralysis cases and environmental monitoring suggested by the WHO, efforts should be also undertaken to implement mucosally deliverable immunogens which should prove able to evoke a local intestinal immune barrier as effective as the one mounted in response to live poliovirus vaccines. To this aim, further knowledge appears needed on if and to what extent the poliovirus antigenic determinants involved in mucosal immunity and elicited by the OPV are also involved in immunity achievable with the current killed vaccines (IPV).

IgA MONOCLONAL ANTIBODIES TO SABIN TYPE 3 POLIOVIRUS

In line with what we previously reported for poliovirus type 1 (Fiore et al., 1997), our present study further confirms that the oral route is not effective in eliciting an immune response to poliovirus in mice unless cholera toxin is included in the antigen preparation. That is not surprising given the absence of poliovirus receptors in the cells of normal mice (Buisman et al., 2000). Conversely, the presence of CT can favor the intimate contact between the virus antigens and the host immune system. The basis for this is still unclear; however, CT is unlikely to act in mediating a possible entry of the virus into the blood stream (Chen et al., 1990; Lycke et al., 1986). In fact, a large part of the hybridoma clones derived in this as well as our previous study on type 1 polio were shown to produce dimeric IgA class antibodies which are typically secreted by B-cells belonging to the mucosa-associated lymphoid tissue (Fiore et al., 1997). Also, the lack of isolation of MAbs directed at site N-AgI, which is destroyed by intestinal trypsin, is in line with the specific involvement of the local gut immune system (Fricks et al., 1985; Roivainen and Hovi, 1987, 1988). Almost all IgA MAbs obtained in this study were directed at site N-AgIII, which was also a primary mucosal target in our previous paper on polio 1 (Fiore et al., 1997). A recent publication on OPV and IPV vaccines in The Netherlands showed that as many as 88.7% of subjects receiving the oral vaccine, versus significantly fewer IPV vaccines, were seropositive with respect to the N-AgIII epitope of PV3 (Herremans et al., 2000), indicating that this site represents an immunodominant mucosal antigenic site in humans. Conversely, antibodies to N-AgI were found to be similarly developed in both groups of subjects. The possibly critical role of site 3-specific mucosal immunity is suggested by the type 3 polio epidemic that occurred in Finland between 1984 and 1985, where most people were shown to have low antibody titers to this site. The presence of an altered neutralization site 1 in the wild-type 3 poliovirus involved may also have contributed to the outbreak (Hovi et al., 1986), since antibodies to site 1 may provide a second line of defense against poliovirus in the blood stream. Looking ahead toward vaccination with killed or recombinant polioviruses, it may thus be necessary to adopt two distinct vaccines suited to elicit optimal antibody responses on the gut mucosa and in the blood stream, respectively. If the established Salk IPV may already be a suitable vaccine for parenteral vaccination, since it elicits a good serum antibody response in particular to N-AgI (Herremans et al., 2000), more efforts may still be needed to implement vaccines with the desired potency and epitope specificity for mucosal use. Also, due to the differences in both the immune cell repertoire and the susceptibility to infection between mice and men, further studies should hopefully be undertaken to address the epitope-specific mucosal immune response to poliovirus in humans, also taking

269

advantage from the recent advancement in recombinant immunoglobulin technology. Differently from poliovirus type 1 IgA MAbs described in our previous study (Fiore et al., 1997), all IgA MAbs to Sabin 3 described here neutralized the corresponding wild-type parental poliovirus. That is probably due to the very closely related amino acid sequence corresponding to N-AgIII and N-AgII between Sabin and wild-type 3 poliovirus. In this respect, given the importance in particular of site 3 for protection, Sabin 3 strain may be more suitable than Sabin 1 as a mucosal vaccine. Unfortunately, this strain is acknowledged as being the most unstable of the three Sabin seeds with respect to neurovirulence, resulting sometimes in OPV vaccine-associated paralysis. Implementation of a mucosally deliverable vaccine suitable for eliciting anti-N-AgIII secretory antibodies might thus also prove successful for preventing PV3 vaccine accidents. Matching genomic analysis of virus variants with their neutralization resistance phenotype, we could presently demonstrate that resistance of poliovirus type 3 to neutralization by the novel IgA MAb 1F5 can originate from change of either amino acids 286–288 of VP1 or amino acid 236 in VP3. Although residues in VP1 had already been reported to be part of site N-AgIII together with residue 59 of VP3 (Minor et al., 1986), amino acid 236 of VP3 represents a new finding. Study of the 3-dimensional structure of the area around this residue reported for Sabin poliovirus type 3 shows that residues 3059, 3236, and 1286-8 are very close to one another, as can be expected for amino acids within a same antigenic site. Our observation that in no case was the binding of any single antibody in this area affected by change of more than two of these residues may indicate that these antibodies bind to their specific epitope according to a precise orientation defining a specific footprint at the surface of the virion. Antibody orientation was previously hypothesized by Page and co-workers (1988) to be critical for neutralization of poliovirus particularly for those antibodies capable of bridging pentamers. MATERIALS AND METHODS Cells and virus HEp-2 cells were grown in Eagle’s minimal essential medium (MEM) supplemented with 5% fetal bovine serum (FBS) at 36°C. The viruses used for production of monoclonal antibodies (MAbs) were the Sabin type 2 and 3 poliovirus strains. Sabin type 1 poliovirus and wild-type 2 (MEF-1) and 3 (Leon) strains were also used to define the neutralization specificity of MAbs. Virus purification and enzyme treatment Polioviruses were purified on a sucrose cushion as previously described (Fiore et al., 1997), resuspended in

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1 to 2 ml of phosphate-buffered saline (PBS), and used for either immunization of mice or coating of ELISA plates. Samples of purified virus preparations were treated with 80 ␮g/ml of purified trypsin (Difco) at 4°C overnight. Western blotting Purified preparations of native or trypsin-treated poliovirus were subjected to 15% SDS-polyacrylamide gel electrophoresis under denaturing conditions, and tested in Western blotting using a 1:500 dilution of pooled rabbit antisera against the three poliovirus serotypes (RIVM Bilthoven). Secondary incubation was carried out with an anti-rabbit IgG (H ⫹ L) antibody conjugated with peroxidase. After washing, detection of bands was performed with an enhanced chemiluminescence assay (ECL, Boehringher Mannheim).

pH 11, and optical densities were measured at a wavelength of 405 nm. Neutralization assays A standard microneutralization assay was used. Approximately 100 TCID 50 of uncleaved or trypsin-treated type 2 or 3 polioviruses was mixed either with a 1:10 dilution of hybridoma cell supernatants for screening of fusions or with serial dilutions of selected antibodies, and the mixtures were incubated for 60 min at 37°C prior to inoculation onto HEp-2 cells in 96-well plates. With a similar protocol, antigenic virus variants (see below) were tested for cross-neutralization by MAbs. Monolayers were scored for CPE daily for 4 days. Neutralizing titers were expressed as the inverse dilution of cell supernatant or ascitis fluid resulting in the absence of CPE in 50% of replica cultures. Isolation of poliovirus antigenic variants

Immunization of mice Six-week-old female Balb/c mice (supplied by Charles River) were orally administered with 100 ␮l of purified Sabin type 2 or 3 polioviruses (containing 10 9 PFU) in the presence of 10 ␮g of purified cholera toxin, and using 0.2 ml sodium bicarbonate to neutralize the acidic pH of the stomach. Mice were boosted 3 to 4 times at 3-week intervals; approximately 10 days after boosting, sera from immunized animals were tested for the presence of neutralizing (Nt) antibody to poliovirus. Fusion Mice received a final oral boost 4 days before fusion. Animals were sacrificed, and the small bowel, the mesenteric lymph nodes, and the spleen were resected and prepared for fusion as described previously (Greenberg et al., 1983). Hybridoma supernatants were screened using a microneutralization test and an enzyme-linked immunosorbent assay (see below). Selected cultures were cloned by limiting dilution using a mouse thymocyte feeder layer, and MAbs were amplified in mouse ascitis. Confirmation of MAb isotype was performed by ELISA using a mouse antibody isotyping kit (Pierce, Rockford, IL). Enzyme-linked immunosorbent assay Microtiter ELISA plates were coated with a mixture of purified, intact, and trypsin-cleaved poliovirus (Sabin type 2 or 3) in PBS, and blocked with 5% FBS/PBS. Wells were incubated with a 1:10 dilution of hybridoma cell supernatants in 1% BSA-PBS for 2 h at 37°C, and secondary incubation was effected with anti-mouse IgA (␣chain) or IgG (H ⫹ L) antibodies conjugated with alkaline phosphatase. The reaction was developed with p-nitrophenol phosphate (Sigma 104) in 10 mM diethanolamine,

Neutralization-resistant poliovirus variants were selected in HEp-2 cell monolayers by plaquing serial dilutions of Sabin type 2 or 3 poliovirus preparations (10 7 to 10 8 PFU) previously neutralized with each single IgA MAb (1:100 dilution of ascitis fluid), and including the antibody (1:1000) in the overlay medium during growth. Single viral plaques were subjected to at least two further cycles of plaque purification before cell culture expansion in antibody-free medium. Virus mutants were designated with the name of the corresponding MAb used for selection preceded by the type “v”. Nucleotide sequencing Sequencing of the capsid region of poliovirus type 2 or 3 variants was carried out as previously described (Fiore et al., 1997). After heat denaturation, genomic RNA was subjected to reverse transcription and cDNA amplification with a panel of synthetic oligonucleotides as primers. Amplified products were purified and sequenced by the DyeDeoxy Terminator cycle sequencing kit (Applied Bio Systems, Perkin-Elmer, Foster City, CA) with the same primers used for amplification. RNAs from Sabin type 2 and 3 parental strains were included as controls. The sequences obtained (GenBank accession numbers AF334949–53) were aligned with the reference strain sequences (Toyoda et al., 1984, last update for PV2 1996). Structural analysis of poliovirus type 3 antigenic sites Structural analysis was performed by using the INSIGHT II molecular modeling package (MSL, San Diego, CA) (Bernstein et al., 1977) and coordinates of the type 3 Sabin strain crystal structure (Filman et al., 1989). ACKNOWLEDGMENTS We thank Dr. Morag Ferguson (NIBSC, South Mimms, UK) for kindly supplying IgG MAbs 138, 1023, 840, against poliovirus type 3 Sabin, Dr.

IgA MONOCLONAL ANTIBODIES TO SABIN TYPE 3 POLIOVIRUS Veronica Morea (IRBM, Pomezia, Italy) for structural analysis of poliovirus antigenic sites, and Mrs. Sabrina Tocchio for editorial assistance. This study was partially supported by grants from the ISS (“Mucosal immunity during infection with polio and other enteroviruses and prevention from the disease,” 2000–2001 art. 12 D.L. 502/92) and from the WHO (“Characterization of polio and other enteroviruses associated with paralytic disease in Italy, Albania and Malta,” 1999-2000 I8/181/ 211(3)A, HQ/99/632C89).

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