Protective effect of a monoclonal antibody specific for an echovirus cellular receptor in human fibroblast and simian kidney cell lines

© INSTITUTPASTEUR/ELSEVIER Paris 1992

Res. Virol. 1992, 143, 397-400

Protective effect of a monoclonal antibody specific for an echovirus cellular receptor in human fibroblast and simian kidney cell lines A.D. Mbida, B. Pozzetto (*), F. Grattard and O.G. Gaudin Laboratoire de Virologie, Facult6 de M6decine Jacques Lisfranc, Universit~ de Saint-Etienne, 15, rue Ambroise-Pard, 42023 Saint-Etienne Cedex 2 (France)

SUMMARY

We previously reported that infection of KB cells by echoviruses (EV) was inhibited by a KB-derived EV receptor murine monoclonal antibody (mAb 143). This antibody enabled the identification of a cellular receptor common to all echoviruaes (with the exception of EV-22 and -23) and coxsackievirus (CV) A9, but different from the receptor of other picornaviruses. We now present results of cell protection assays conducted with human and simian cell lines different from the KB cell line used for production of mAb 143. When human embryonic lung fibroblasts were pretreated with 150 i~g/ml of mAb 143, EV-11 and CV-A9 were completely inhibited (more than a 2-log difference compared to untreated cells). When the cell protection experiments were performed with Vero cells, the same results were observed with EV-33, but not with EV-22. The protection afforded human fibroblast cells by mAb 143 persisted for at least 5 days after 2-h exposure to 100 TClDso of EV-11. These results suggest that EV receptors can be effectively blocked for prolonged periods in susceptible cells.

Key-words: Echovirus, Enterovirus, Cell receptor; Monoclonal antibody, Human fibroblasts.

Human echoviruses (EV), which currently number 31 distinct antigenic serotypes (Melnick, 1985), are responsible for many symptoms including acute and chronic infections. The preparation from KB cells of a monoclonal antibody designated mAb 143 was previously described (Mbida et al., 1992). This antibody protected KB and other cell lines from infection by 29 serotypes out of the 31 tested (EV-22 and -23 excepted), and protection was extended to coxsackievirus (CV) A9. MAb 143 had no efSubmitted July 21, 1992, accepted November23, 1992. (*) Correspondingauthor.

fect on replication of the three poliovirus serotypes, the six serotypes of CV-B, CV-A16 and CV-A21 and human rhinovirus 14 (Mbida et al., 1992). Differences have been observed in virus and cell specificity in the cell protection assay for most antireceptor mAb (Minor et al., 1984; Crowell et al., 1986; Hsu et al., 1988; Colonno et al., 1986). In the present study, we performed in vitro experiments to evaluate the protective effect of mAb 143 in human embryonic lung

398

A.D. MBIDA E T AL.

fibroblasts and green monkey kidney cells (Vero), two cell lines which differ from those in which mAb 143 was initially produced. In addition, we defined the concentration-related inhibition of virus replication. MAb 143 used in this work was produced from murine ascites fluids as previously described (Hoogenraad et aL, 1987). The concentration-related activity of mAb 143 was determined against (i) EV-I 1 and CV-A9 in human lung embryonic fibroblasts (P2002 line, Flow Laboratories) and (ii) EV-33 in Vero cells. EV-22, also cultured on Vero cells, was used as a negative control. The cells were cultured in 96-well plastic microplates in MEM (P2002 cells) or E199 medium (Vero) supplemented with 2 mM L glutamine, 10 % foetal calf serum, 100 I U / m l penicillin and 100 ~tg/ml streptomycin. EV-11 (Gregory strain) and EV-22 (Harris strain) were prototype strains; EV-33 and CV-A9 were clinical isolates. To evaluate the timing effect of mAb 143 addition, cell monolayers were treated with various concentrations of mAb (0, 50, 100 and 150 ~g/ml) before, at the time of and after virus adsorption. In pre-treatment experiments, quadruplicate monolayers of cells were exposed for 2 h at 37°C to 0.1 ml of diluted mAb 143 per well; the antibody was then removed, and 0.1 ml of virus dilution ranging from 10-l to 10 -6 was added in each well; after a l-h incubation at 37°C, the virus solution was removed, and 0.1 ml of culture medium was added to each well. In simultaneous treatment experiment~, 0.1 ml of virus solution and 0.1 ml of diluted mAb 143 were added at the same time to each well; after a 2-h incubation at 37°C, the contents of each well were removed and replaced by 0.1 ml of culture medium. In post-treatment experiments, 0.1 ml of virus solution was added to each well for 1 h at 37°C; after virus removal, 0.1 ml of diluted mAb 143 was added to each well for 2 h at 370C; the mAb 143 solution was then removed and replaced by 0.1 ml of fresh medium per well. Then, in all experiments, the plates were incubatCPE CV EV

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cytopathic effect. coxsackievirus.

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echovirus.

ed for 48 h at 37"C in a 3 % CO 2 atmosphere, and the cytopathic effect (CPE) was recorded by microscopic examination. Results were expressed as logl0 reduction in titre with reference to untreated virus. Pretreatment with mAb 143 resulted in a concentration-dependent reduction in virus titre for EV-11, EV-33 and CV-A9; as expected, no effect was observed with EV-22 (table I). Simultaneous treatment with mAb 143 and EV-11, EV-33 or CV-A9 resulted in a partial reduction in viral titres with the higher concentrations of antibody (table I). MAb 143 post-treatment did not reduce titres of any viruses (data not shown), indicating that this antibody was unable to displace previously bound virions from the receptor, as did mAb directed against the ICAM-1 receptor of rhinoviruses (Colonno et al., 1986). The degree of inhibition by mAb 143 of a cytopathic effect at different concentrations of EV-11 was determined in quadruplicate for P2002 cells pre-treated with 0.1 ml of culture medium containing 0, 12.5, 25, 50, 100, 150, 200, or 250 ~g/ml of mAb 143. After a 2-h incubation at 37°C, the solution was replaced by 0.1 ml of culture medium containing approximately 100, 250, 500, or 1,000 TCID50 of EV-11. The monolayers were incubated at 37°C in 3 % CO 2 atmosphere for three days. After crystal violet staining, the plates were read spectrophotometrically at 540 nm (Multiscan, Flow Laboratories) and the percentage of viral inhibition was calculated as previously described by Teyssedou et al. (1987). Figure 1 illustrates the concentrationdependent effect of mAb 143 on EV-11 replication: a total inhibition of virus attachment was achieved with 150 ~g/ml of mAb 143 only in the presence of the lowest concentration of EV-11

(100 TCIDs0). To determine the duration of the mAb 143 protective effect on P2002 ceils after EV-11 infection, quadruplicate monolayers of cells were pretreated with 0.1 ml of culture medium containing 0, 12.5, 50, and 150 ~tg/ml o f m A b 143. After 2-h incubation at 37°C, the contents of mAb TCID

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m o n o c l o n a l antibody. tissue c u l t u r e infectious dose.

ECHOVIRUS RECEPTOR BLOCKING B Y mAb 143

399

Table 1. Reduction in viral control titres (logn0TCIDs0/0.1 ml) by 2-h exposure of P2002 (EV-I l and CV-A9) or Vero (EV-33 and EV-22) cell monolayers to 0.1 ml of various concentrations of mAb 143 (l~g/ml)before or at the time of virus addition.

150

Reduction in titre compared to untreated control Pre-treatment Simultaneous treatment 100 50 150 100 50

EV-11 CV-A9 EV-33

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0.5 0.5 0.5

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each well were replaced by 0.1 ml of medium containing approximately 100 TCIDs0 of EV-11. Four identical microplates were incubated at 37°C. A different microplate was stained daily from day 2 to day 5 with crystal violet and read as above. All infected untreated control monolayers showed a complete CPE on day 2 (table II). In contrast, monolayers treated with 150 ~tg/ml of mAb 143 were totally protected through day 5. Monolayers treated with 12.5 and 50 ~g/mI of mAb 143 showed intermediate results (table II). In further experiments, the protective effect of 150 ~g/ml of mAb 143 was shown to be prolonged up to ten days (data not shown).

Pre-treatment of cells with mAb 143 resulted in dose-dependent protection against infection by EV-11, CV-A9, and EV-33, three enterovirus serotypes known to share the same receptor, in contrast with EV-22 (Mbida et al., 1992). These results were obtained with cell fines different from those from which the antibody was derived. A similar finding was reported by Sperber and Hayden (1989) on human fibroblasts with the HeLa-derived rhinovirus receptor murine mAb (Colonno et al., 1986). However, the protective effect of mAb 143 is weak, as illustrated by the high concentrations required to obtain total inhibition of EV replication; furthermore, mAb 143 has to be added

400

A.D. MBIDA ET AL.

Table II. Percentage of CPE of 100 TCIDs0 of EV-11 on P2002 cells pre-treated for 2 h with various concentrations of mAb 143. mAb 143 (;zg/ml) 0 12.5 50 150

Day 2 100 90 50 0

prior to the addition o f virus in order to have a meaningful effect. Despite these limitations, high doses o f m A b 143 were shown to protect cells for prolonged periods (5 days or more) and even to reduce the mortality o f suckling mice infected with EV-9 (unpublished data). These results are encouraging for the further development o f synthetic receptor blocking agents as an approach to the possible early treatment o f echoviral infections.

Effet protecteur d'un anticorps monoclonal sp~cifique du r~cepteur cellulaire des echovirus sur des lign~es de fibroblastes humains et de ceilules de rein de singe Nous avons pr&:ddemment montrd que l'infection de cellules KB par des echovirus (EV) dtait inhibde par un anticorps monoclonal murin dirigd contre un d&erminant membranaire de ces cellules (AcM 143). Cet anticorps a permis l'identification d'un r~cepteur cellulaire commun/l tousles EV - - / t l'exception des sdrotypes 22 et 23 - - et au coxsackfevirus (CV) A9, mais qui diff6re du rdcepteur des autres picornavirus. Nous pr6sentons ici les r6sultats d'expdriences de protection cellulaire r6alisdes sur des ligndes cellulaires humaines et simiennes diffdrentes des cellules KB utilis6es pour la production de l'AcM 143. Quand des fibroblastes pulmonaires embryonnaires humains sont pr6trait6s par 150 izg/ml d'AcM 143, la r6plication de l'EV-11 et celle du CV-A9 sont totalement inhib~es (plus de deux log10 d'6cart par rapport aux t6moins non trait6s). De mSme, les cellules Vero sont prot6g6es de l'infection par I'EV-33 mais ne le sont pas pour celle par l'EV-22. La protection conf6rde aux fibroblastes humains par rAcM 143 persiste au moins 5 jours apr6s une exposition de deux heures ~t 100 doses infectieuses d'EV-I I. Ces r6sultats sugg6rent que les r6cepteurs des EV au niveau de lign6es cellulaires sensibles peuvent ~tre

Percent viral CPE Day 3 Day 4 I00 90 50 0

100 100 79 0

Day 5 100 100 79 0

efficacement bloquds pendant des pdriodes prolongdes. Mots-cids: Echovirus, Ent6rovirus, Rdcepteur celiulaire; Anticorps monoclonal, Fibroblastes humains.

References Colonno, R.J., Callahan, P.L. & Long, W.J. (1986), Isolation of a monoclonai antibody that blocks attachment of the major group of human rhinoviruses. J. ViroL, 57, 7-12. Crowell, R.L., Field, A.K., Scheilf, W.A., Long, W.L., Colonno, R.J., Mapoles, J.E. & Emini, E.A. (1986), Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma ceils by selected enteroviruses through receptor blockade. J. Virol., 57, 438-445. Hoogenraad, N., Helman, T. & Hoogenraad, J. (1987), Production of ascites fluids in mice. J. lmmunol. Methods, 61, 317-320. Hsu, K.L., Longberg-Holm, K., Alstein, B. & Crowell, R.L. (1988), A monoclonal antibody specific for the cellular receptor for the group B coxsackieviruses. J. Virol., 62, 1647-1652. Mbida, A.D., Gaudin, O.G., Sabido, O., Pozzetto, B. & Le Bihan, J.C. (1992), A monoclonal antibody specific for the cellular receptor of echoviruses. Intervirology, 33, 17-22. Melnick, J.L. (1985), Enteroviruses: polioviruses, coxsackieviruses, echoviruses, and new enteroviruses, in "Virology" (B.N. Fields) (pp. 739-794). Raven Press, New York. Minor, P.D., Pipkin, P.A., Hockley, D., Schild, G.C. & Almond, J.W. (1984), Monoclonal antibodies which block cellular receptors of poliovirus. Virus Res., 1, 203-212. Sperber, S.J. & Hayden, F.G. (1989), Protective effect of rhinovirus receptor blocking antibody in human fibroblast cells. Antiviral Res., 12, 231-238. Teyssedou, E., Magar, R., Justewicz, D.M. & Lecomte, J. (1987), Cell-protectivemonoclonal antibody to bovine enterovirus-3 and partial or no activity against other serotypes. J. ViroL, 61, 2050-2053.