production and characterization of neutralizing monoclonal antibodies against poliovirus type 1, 2,...

Production and characterization of neutralizing monoclonal antibodies against poliovirus type 1, 2, and 3

PIERRE PAYMENT,' MICHEL TRUDEL, LISE THIBODEAU, AND JACQUELINE LECOMTE Centre de recherche en virologie, Znstitut Armand-Frappier, C. P. 100, Succursale L.D.R., Ville de Laval (Qukbec),

Canada H7N 423

Received October 7, 1988

Accepted February 1, 1989

PAYMENT, P., TRUDEL, M., THIBODEAU, L., and LECOMTE, J. 1989. Production and characterization of neutralizing mono- clonal antibodies against poliovirus type 1, 2, and 3. Can. J. Microbiol. 35: 550-553.

Neutralizing monoclonal antibodies were produced against a reference vaccine or a reference wild strain of poliovirus type 1, 2, and 3. After 26 fusions, 55 monoclonal antibodies were obtained with serotype I as the immunizing antigen, 180 with serotype 2, and 115 with serotype 3. The neutralizing activity of these monoclonal antibodies was tested first with the two reference strains and then if reactive, against a panel of 10 well-characterized strains of each serotype, 5 vaccinelike (VL) and 5 nonvaccinelike (NVL). All monoclonal antibodies were type specific without reactivity with any of the heterologous strains. There was a wide range of reactivity within the strains of each serotype. Several monoclonal antibodies to serotype 1 reacted with all type 1 strains, while several neutralized strongly all VL strains and weakly one or more of the NVL strains. Most of the 180 monoclonal antibodies to serotype 2 neutralized to various degrees all strains of this serotype and about half reacted very strongly with all homologous strains whether VL or NVL. None could differentiate all VL and NVL homologous strains. Of the 115 monoclonal antibodies to serotype 3, several monoclonal antibodies neutralize to various levels all homolo- gous strains and some can differentiate VL and NVL strains.

Key words: poliovirus, monoclonal antibody, neutralization, serodifferentiation.

PAYMENT, P., TRIJDEL, M., THIBODEAU, L., et LECOMTE, J. 1989. Production and characterization of neutralizing mono- clonal antibodies against poliovirus type 1, 2, and 3. Can. J. Microbiol. 35 : 550-553.

Les auteurs ont produits des anticorps monoclonaux neutralisants contre une souche de rkfkrence vaccinale (VL) ou une souche non vaccinale (NVL) des poliovirus type 1, 2 et 3. Aprb 26 fusions rhssies, 55 anticorps monoclonaux ont kt6 obte- nus contre le strotype 1, 180 contre le strotype 2 et 115 contre le strotype 3. L'activitt neutralisante de ceux-ci a d'abord kt6 vtrifik contre les deux souches de rkfkrenie, puis en prtsence d'une activitt intkressante, contre cinq souches apparentkes aux souches vacinales (VL) et cinq souches apparentkes au souches non-vaccinales (NVL) de chaque skrotype. Tous les anti- corps monoclonaux obtenus ne rkagissent qu'avec les souches homologues: aucune rkaction n'a t t t observke avec les souches hktkrologues. Pour chaque skrotype une grande g a m e de niveau de rtaction a t t t observke. Parmi les anticorps monoclonaux dirigkes contre le strotype 1, plusieurs rkagissent fortement avec toutes les souches homologues, alors que d'autres sont forte- ment spkcifiques pour les souches VL, bien que montrant une activitk plus faible avec I'une ou plusieurs des souches NVL. La plupart des 180 anticorps monoclonaux rkagissant avec le strotype 2 neutralisent h des degrts divers toutes les souches de ce skrotype et, prks de la moitit reconnaissent fortement les souches homologues VL ou NVL. Aucun anticorps mono- clonal ne permet la difftrenciation complkte des souches VL des souches NVL. Parmi les 115 anticorps monoclonaux dirigks contre le skrotype 3, plusieurs neutralisent ii des niveaux difftrents les souches homologues et certains permettent la diffk- renciation des souches VL et NVL.

Mots clks : poliovirus, anticorps monoclonal, neutralisation, strotypage.

Introduction Poliomyelitis has been controlled by vaccination with live

(Sabin) or attenuated (Salk) vaccines (Evans 1984). Polio- myelitis has not, however, been eradicated and could reappear if the vaccination level in the population is lowered owing to improper vaccination or to the appearance of new strains. The origin-of these strains has been postulated to be a reversion of vaccinal strains during passage in man or the introduction of wild strains by infected individuals returning from abroad. In countries where poliomyelitis is under control, it becomes critical to determine rapidly whether strains from the infre- quent cases of paralytic poliomyelitis are vaccinelike or non- vaccinelike. Because polioviruses are represented by three relatively well-defined serotypes, it has been possible to differentiate serotypes by antisera prepared in laboratory animals. However, the differentiation of wild neurovirulent strains from those of vaccinal origin is more difficult because the differences are of molecular origin and subtle. Not one of the tests has been found to correlate perfectly with the neuro-

'Author to whom all correspondence should be addressed. Printed in Canada I Imprime au Canada

virulence of the strains tested, but using a combination of the tests has permitted greater accuracy in determining the related- ness of the strains especially through the use of adsorbed anti- sera and of neutralization kinetics (Nakano et al. 1978; McBride 1959; Van Wezel and Hazendonk 1979). Molecular analysis can provide additional information on the virological and epidemiological characteristics of the virus (Kew et al. 1980; Ferguson et al. 1986), and using a battery of well studies, monoclonal antibodies (MAbs) could be utilized to rapidly characterize poliovirus isolates (Crainic et al. 1983).

Materials and methods Virus strains

Six reference strains of poliovirus were used for hybridorna preparation. They were obtained from the Viral Vaccine Quality Control Laboratory of Institut Armand-Frappier (Laval, Qukbec, Canada). These include the nonvaccine reference strains of the inacti- vated Salk vaccine, Mahoney (type l), MEF-1 (type 2), and Saukett (type 3), and the Sabin oral vaccine strains Sabin 1 (LS-c,2ab), Sabin 2 (P712,Ch,2ab), and Sabin 3 (Leon 12a,b). Virus stocks for mouse inoculation were prepared by infecting Hep-2 cells in the absence of fetal bovine serum (FBS), and after destruction of the

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PAYMENT ET AL. 55 1

TABLE 1. List of strains and their characteristics

McBride testa Van Wezel testa - Temperature

Virus Strain Name or no. Year Origin VL NVL VL NVL markerb

Type 1 Sabin 1 Mahoney

NVL- 10 NVL- 11 NVL-12 NVL- 13 NVL-14

VL-15 VL-16 VL-17 VL- 18 VL-19

Type 2 Sabin 2 MEF-1

NVL-20 NVL-2 1 NVL-22 NVL-23 NVL-24

VL-25 VL-26 VL-27 VL-28 VL-29

Type 3 Sabin 3 Saukett

NVL-30 NVL-3 1 NVL-32 NVL-33 NVL-34

VL-35 VL-36 VL-37 VL-38 VL-39

Ontario Ontario Ontario Ontario Ontario

Quebec Saskatchewan Manitoba Saskatchewan Manitoba

Missouri California England Quebec Quebec

Ontario Quebec Saskatchev Manitoba Ontario

ran

Leon

New Brunswick Quebec Germany Saskatchewan Quebec

Quebec Ontario Ontario Ontario Ontario

'McBride test (McBride 1959) and Van Wezel adsorbed antisenurn test (Van Wezel and Hazendonk 1979). Reported as the neutralization index with the results grouped as vaccinelike (VL) and nonvaccinelike (NVL).

b~eplication (+) or inhibition (-) when grown at 42°C.

monolayers, the supernatants were clarified by centrifugation at 10000 x g and were frozen at 70°C until required. The infectious titer of the supernatants was about lo8 plaque-forming units per millilitre.

Thirty well-characterized strains of poliovirus were obtained from the Bureau of Biologics (Laboratory Center for Disease Control, Health and Welfare Canada, Ottawa). The characteristics of these strains are presented in Table 1. For each poliovirus serotype, there are five non-Sabin-like (NVL) strains and five Sabin-like (VL) strains. Each virus strain was passaged once on Vero cells and infected supernatants were clarified by low-speed centrifugation and kept frozen in aliquots at -70°C.

were harvested from the mice and fused with P3-Ag4 NS1 mouse myeloma cells. Supernatants of growing hybridomas were screened by neutralization using the six reference strains.

Neutralization tests Supernatants of growing hybridomas were first tested for reactions

with the homologous reference type and then retested with the 6 reference strains and the 30 characterized strains. For the neutraliza- tion test, 100 TCID,, of virus were reacted with twofold dilutions of the hybridoma supernatant to be tested. All neutralization tests were performed in microtitration plates using 0.025 mL of virus or 0.025 mL hybridoma supernatant and 0.100 mL of a suspension of Vero cells at lo5 cells1mL in tissue culture medium. Plates were incubated at 37OC in a humidified incubator for 3 to 5 days and the appearance of cytopathic effect was monitored. The neutralization titer was expressed as the reciprocal of the final dilution that pro- tected 50% of the cell cultures.

Production and screening of hybridomas Female BALBIc mice were immunized by inoculation (i.p.1i.m.) of

undiluted clarified virus-infected cell supernatants mixed with an equal amount of complete Freund's adjuvant, followed by reinocula- tion (i.p.1i.m.) without adjuvant 7 days later. Inoculated mice were subsequently kept for 3 to 12 months, then given an i.v. booster inoculation of the same viral antigen. Three days later, spleen cells

Ascitic fluid production Ascitic fluid was produced by inoculating lo6 hybridoma cells to

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552 CAN. J . MICROB IIOL. VOL. 35, 1989

TABLE 2. Neutralizing activitya of ascitic fluids prepared from hybridomas produced against poliovirus type 1

PP12b

Strain

Sabin 1 Mahoney NVL-10 NVL-11 NVL- 12 NVL- 13 NVL- 14

VL-15 196608 327680 20480 229376 VL- 16 163840 327680 8192 20480 VL- 17 163840 327680 10240 262144 VL- 18 163840 327680 8192 196608 VL-19 114688 327680 12288 262 144

'Titers are expressed as the end-pont dilution neutralizing 100 TCID,. of the indicated virus.

b~mmunizing antigen, Sabin 1.

BALBIc mice pretreated with Pristane 10 days before inoculation. Ascitic fluids were collected aseptically and tested for neutralizing activity as described above.

Results Over a period of 4 years, 26 fusions were performed and 16

resulted in the production of 350 viable hybridomas. We selected stable clones secreting poliovirus-specific neutraliz- ing monoclonal antibodies. None of these clones produced detectable neutralizing antibodies against heterologous sero- types. The neutralizing activities of selected monoclonal anti- bodies produced as ascitic fluids are listed in Tables 2, 3, and 4.

Poliovirus type 1 Among the hybridomas selected from fusion PP-12

(immunizing antigen, Sabin I), several clones only reacted with Sabin 1 vaccine strains and with strains VL-15 to VL-19 which were Sabin 1 like. Some clones recognized epitopes from wild strains and reacted with the Mahoney strain as well as with strains NVL-10 and (or) NVL-11. Other monoclonal antibodies (for example, PP-12 1A412A1) recognized all poliovirus type 1 strains whether wild or vaccinal. Other clones produced antibodies which reacted with an epitope present on strain NVL-10 but not on other NVL strains (PP-12 2G211D6).

Poliovirus type 2 The patterns of reactivity observed with poliovirus type 2

strains were more diversified. Using the Sabin 2 strain as the immunizing antigen, we obtained several clones that produce antibodies which react at different intensities with all the homologous strains tested. Several clones from fusion PP-17 were specific for Sabin 2 strain without cross-reactivity with the MEF-1 strain. They did react, however, at a low level with almost all type 2 strains, but at a higher level with strains NVL-23 and NVL-24. Using the MEF-1 strain as the immunizing antigen, reactivity patterns were similar to those obtained using Sabin 2 as the immunizing antigen. Hybri- domas secreting antibodies that reacted with all strains of poliovirus type 2 were obtained. None of the monoclonal anti- body produced permitted a clear differentiation of the Sabin

and MEE-1 reference strains or of the NVL and VL strains.

Poliovirus type 3 With poliovirus type 3 Saukett strain as the immunizing anti-

gen, monoclonal antibodies were obtained which permitted the clear differentiation of VL and NVL strains (PP-16 clone 1A10) or the recognition of all strains (PP-16 clone 2E3). When the Sabin 3 reference strain was the immunizing agent, strains NVL-30, NVL-31, and NVL-32 were not recognized while strains NVL-33 and NVL-34 were strongly recognized by several monoclonal antibodies (FN-S3 clone 7C4).

Discussion In an attempt to correlate structure and pathogenicity, Kew

et al. (1980) made a comparative analysis of tryptic peptides derived from capsid proteins of poliovirus type 1 wild (Mahoney) and vaccine (Sabin) strains. This analysis showed that three of the four capsid proteins of the attenuated virus were different from those of the wild neurovirulent strain. These mutations, altering a given epitope, cannot be identified easily by using polyclonal antisera.

The advent of hybridomas and monoclonal antibodies has opened an era of accuracy down to the level of epitopes responsible for the immunological characteristics of proteins. Because it was already known that vaccine and wild strains could be related on an immunological basis (McBride 1959; Van Wezel and Hazendonk 1979), monoclonal antibodies were a very good choice for the differentiation of poliovirus strains. Several papers have been published on the production and characterization of monoclonal antibodies to individual poliovirus serotype. In 1983, the report of Crainic et al. (1983) indicated the feasibility of such a method for the differ- entiation of homologous strains and the estimation of their relatedness to vaccinal strains. More recently, the reactivities of several strain-specific monoclonal antibodies were com- pared using T-1 oligonucleotide maps of RNA from different strains (Ferguson et al. 1986). Serotypes 2 and 3 were shown to correlate with the T-1 oligonucleotide maps, suggesting the potential value of monospecific antibodies in the characteriza- tion of poliovirus isolates.

During the course of the present project, all clones produced neutralizing antibodies against the immunizing strain as well as against the homologous strains. The neutralization titers observed were very different from clone to clone, but several clones permitted rapid identification of the serotype. Intratypic differentiation was obtained for serotypes 1 and 3; several clones can differentiate vaccine strains (VL) from wild strains (NVL). For serotype 2, such a clear-cut intratypic differentia- tion was not possible, even if some clones had reactivities approaching it. None of the monoclonal antibody produced permitted a clear differentiation of the Sabin and MEF-1 refer- ence strains or of the NVL and VL strains. The fact that most monoclonal antibodies raised against type 2 strains can neutralize almost all isolates, often with comparable titers, suggests that the neutralizing epitopes recognized are quite conserved for type 2 strains. This hypothesis is consistent with the results obtained on the antigenic variation of poliovirus.

The objective of this study was to prepare and characterize monoclonal antibodies that could differentiate vaccinal and nonvaccinal isolates. We have been able to produce such monoclonal antibodies against poliovirus type 1 and type 3. For poliovirus type 2, we were unable to produce a mono- clonal antibody that alone can clearly differentiate the VL

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PAYMENT ET AL.

TABLE 3. Neutralizing activitya of ascitic fluids prepared with hybridomas that produce monoclonal antibodies against poliovirus type 2

PPIOb PP17d CN,"

Virusstrains 2E212E9 1E2lA1 lA211C6 2B41A7D12 lG3IGllB4

Sabin 2 65536 < 2 327680 10240 32768 MEF-1 229376 512 655360 2560 96

NVL-20 131072 64 655360 1792 1280 NVL-21 229376 160 524288 24576 640 NVL-22 196608 380 917504 1280 56 NVL-23 40960 10240 3 120 20 24576 NVL-24 65536 16384 28672 48 32768

VL-25 229376 112 524288 28672 1536 VL-26 229376 224 524288 32768 1024 VL-27 98304 640 524288 28672 1792 VL-28 196608 320 327680 7168 512 VL-29 98304 3072 655360 3072 12

'Titers are expressed as the end-point dilution neutralizing 100 TCID,, of the indicated virus. b~mmunizing antigen, MEF-1. CImmunizing antigen, Sabin 2. dImmunizing antigen, Sabin 2.

TABLE 4. Neutralizing activitya of ascitic fluids ~ r e ~ a r e d with hvbri- Acknowledgments - domas that produce konoclokd antibodies against boliovirus Gpe 3 This work was supported by grant 6605-2032-54 of the

PP16b FN-S3' National Health Research and Development Program of ppl3,d Health and Welfare Canada. The authors wish to thank Nicole

Strains 2E31F9F12 1AlOlA5G3 7C411C4 1A211B2 1E8lG4 Filion and the uersonnel of the Hvbridoma Section for their skilful work in the production and evaluation of the reactivity

Sabin 3 192 Saukett 640 57344 5120 < 2 2560

128 40960 5120 10240 of the hundreds of hybridomas produced.

"Titers are expressed as the end-point dilution neutralizing 100 TCID,, of the indi- cated virus.

bImmunizing antigen, Saukett. 'Immunizing antigen, Saukett. d~mmunizing antigen, Sabin 3,

strains and the NVL strains. However, similar results could be achieved using a combination of two different monoclonal antibodies. These monoclonal antibodies will now be made available to the scientific community for further characteriza- tion and use. We hope that the process of identifying and char- acterizing poliovirus strains will be facilitated by their use.

CRAINIC, R., COUILLIN, P., BLONDEL, B., CABAU, N., BOUE, A., and HORODNICEANU, E 1983. Natural variation of poliovirus neutrali- zation epitopes. Infect. Irnmun. 41: 1217 - 1225.

EVANS, A. S. 1984. Criteria for control of infectious diseases with poliomyelitis as an example. Prog. Med. Virol. 29: 141 - 165.

FERGUSON, M., MAGRATH, D. I., MINOR, P. D., and SCHILD, G. C. 1986. WHO collaborative study on the use of monoclonal anti- bodies for the intratypic differentiation of poliovirus strains. Bull. WHO, 64: 239-246.

KEW, 0 . M., PALLANSCH, M. A., OMILIANOWOSKI, D. R., and RUECKERT, R. R. 1980. Change in three of the four coat proteins of oral poliovaccine strains. J. Virol. 33: 256-263.

MCBRIDE, W. D. 1959. Antigenic analysis of poliovirus by kinetic studies of serum neutralization. Virology, 7: 45-58.

NAKANO, J. H., HATCH, M. H., THIEME, M. L., and NOTTAY, B. 1978. Parameters for differentiating vaccine-derived and wild poliovirus strains. Prog. Med. Virol. 24: 178-206.

VAN WEZEL, A. L., and HAZENDONK, A. G. 1979. Intratypic sero- differentiation of poliomyelitis strains by strain-specific antisera. Intervirology, 11: 2 -8.

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