Neutralizing and Non-neutralizing Monoclonal Antibodies ... - CiteSeerX

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aminopterin-thymidine medium (Littlefield, 1964) and antibody-producing clones by plaque titration and enzyme-linked immunosorbent assay (ELISA). Plaque ...
J. gen. Virol. (1983), 64, 1405-1408. Printed in Great Britain

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Key words: SFV/monoclonal antibodies/protective immunity

Neutralizing and Non-neutralizing Monoclonal Antibodies to the E 2 Glycoprotein of Semliki Forest Virus Can Protect Mice from Lethal Encephalitis By W l L A. M. B O E R E , * B A R R Y J. B E N A I S S A - T R O U W , M. H A R M S E N , C. A. K R A A I J E V E L D AND H. S N I P P E Laboratory of Microbiology, State University of Utrecht, Catharijnesingel 59, 3511 GG Utrecht, The Netherlands

(Accepted 19 January 1983) SUMMARY

Two monoclonal antibodies (UM 4.2 and UM 5.1) directed against the glycoprotein E2 of Semliki Forest virus (SFV) are described; both belong to the IgG2a isotype but are of different idiotype. Analysis employing isoelectric focusing resulted in different focusing patterns for both monoclonals (UM 4.2, pI 8; UM 5.1, pI 7.2). They further differed in their ability to neutralize virus. The UM 4.2 antibodies were inactive in neutralization, while the UM 5.1 antibodies exceeded conventional mouse hyperimmune serum in this respect. Both monoclonal antibodies, however, were able to protect mice passively from a lethal infection with SFV. Based on the amount of protein, the UM 5.1 antibodies were 100-fold more effective than the UM 4.2 antibodies in mouse protection tests. Immunization of mice with an avirulent strain of Semliki Forest virus (SFV) results in production of a heterogeneous population of antibodies which are able to interfere with different viral activities like infectivity and haemagglutination (Dalrymple et al., 1976; Helenius et al., 1976). For an analysis of the role played by the individual membrane glycoproteins El, E2 and E 3 of SFV (Garoffet al., 1974), monospecific antibodies are required. We produced a panel of monoclonal antibodies (MA) directed against the glycoproteins E1 and E2. Two MA specific for the E2 glycoprotein but differing in biological properties are studied in this paper. BALB/c mice were immunized with the avirulent SFV strain MRS MP 192/7 and spleen ceils were subsequently fused with P3-NS 1-1Ag4-1 (NS 1) myeloma cells, using the method described by Fazekas de St. Groth & Scheidegger (1980). Hybrids were selected in hypoxanthineaminopterin-thymidine medium (Littlefield, 1964) and antibody-producing clones by plaque titration and enzyme-linked immunosorbent assay (ELISA). Plaque titration and the plaque reduction test (PRT) have been described previously (Kraaijeveld et al., 1979b). ELISA was performed in Terasaki plates (Falcon Plastics) which had been coated with 10 ~tl amounts per well of 0-3 to 0-5 ~tg of purified inactivated SFV in 0.1 M-carbonate-bicarbonate buffer pH 9.6. Samples of hybridoma culture fluid (5 ~tl) were screened for anti-SFV antibody using goat antimouse IgG or IgM antibodies conjugated with alkaline phosphatase (Tago, Burlingame, Ca., U.S.A.). The substrate disodium p-nitrophenyl phosphate (Sigma) was added and the test was scored visually after 10 min. Positive cultures were subcloned by limiting dilution and tested again for anti-SFV activity. Positive clones were injected into pristane-primed female BALB/c mice (0.5 ml pristane intraperitoneally 1 to 2 weeks before injection of cells) at a dose of 1.0 × 106 to 5-0 x 106 cells/mouse. The antibody specificity in the resulting ascitic fluid was identified by immunoblotting (Fig. 1a). Two clones, UM 4.2 and UM 5.1, showed specificity for the E2 glycoprotein ofSFV. Hyperimmune mouse serum served as a control and reacted with both glycoproteins E1 and E2 as expected. Antibody subclasses were determined by ELISA in Terasaki plates coated with rabbit antisera specific for mouse immunoglobulin subclasses (Miles Laboratories). After incubation 0022-1317/83/0000-5422$02.00 © 1983 SGM

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1406 (a)

1

2

3

4

(b)

pI

1

2

3

E2

E~

Capsid

5.85

Fig. 1. Biochemical characterization of monoclonal antibodies to the E 2 glycoprotein of Semliki Forest virus (SFV). (a) Immunoblots of SFV probed with monoclonal antibodies and mouse hyperimmune serum. SFV was solubilized in sample buffer with 1 ~ SDS without 2-mercaptoethanol and glycoproteins were separated in 10~ polyacrylamide slab gels by SDS~el electrophoresis at 50 mA in Tris-borate buffer (0-09 M-Tris, 0.08 M-boric acid, 2.5 mM-EDTA, 2% SDS, pH 8.4) until the bromophenol blue dye marker had reached the bottom of the gel. After electrophoresis proteins were electrophoretically transferred to nitrocellulose sheets (Bio-Rad) at 10 V for 16 h (Vaessen et al., 1981). These were then soaked in PBS~4~ bovine serum albumin (BSA) for 1 h at 37 °C to prevent nonspecific adsorption of protein. Lanes were cut in strips and each strip was incubated at 37 °C with 0-5 ml hybridoma culture fluid diluted 1 : 5 in PBS-4~ BSA or with a 1 : 100 dilution of immune serum. After extensive washing blots were incubated for 1 h at 37 °C with 2.5 ml PBS-4~ BSA containing goat antimouse IgG-alkaline phosphatase conjugate (Tago) diluted 1:1000. After washing again the bound enzyme was visualized by soaking the blots in a solution of 1 mg naphthol AS-MX phosphate disodium salt (Sigma) and 3 mg 4-aminodiphenylamine diazonium sulphate (Sigma) per ml 0-2 M-Tris-HC1 pH 9.1 containing 10 mM-MgC12. After about 5 rain blue-coloured spots developed and the reaction was terminated by washing with tap water. Lane 1, viral proteins stained with Coomassie Brilliant Blue R250; lane 2, mouse hyperimmune serum; lane 3 and 4, anti-SFV hybridoma supernatant UM 4.2 and UM 5.1 respectively. (b) Isoelectric focusing of Protein A-purified monoclonal antibodies. Focusing was performed with 1~ agarose isoelectric focusing gels in pharmalyte buffer (pH 3 to 10) using the horizontal system (Pharmacia) according to manufacturer's instructions. The anolyte buffer was 0.05 M-H2SO4; the catholyte buffer was 1 M-NaOH. Focusing was performed during 1500 to 2000 volt hours. The gel was stained with Coomassie Brilliant Blue R250. Lane 1, reference kit; lane 2, antibodies UM 4.2; lane 3, antibodies UM 5.1.

T a b l e 1. Properties o f monoclonal immune ascitic fluid and hyperimmune serum Monoclonal antibodies A

• Protein specificity Immunoglobulin subclass PRT titre* FLISA titret Minimal amount of purified antibody required to protect mice fully against 10 LDs0 units of SFV

~ Hyperimmune serum

UM 4.2

UM 5.1

Ez IgG2a < 1

E2 IgG2a 3 × 106

El and E 2 NT~ 10a

10 4

10 5

10 3

10 txg

0.1 I.tg

NT

* The reciprocal of the highest dilution of a monoclonal antibody or serum causing 50~ plaque reduction was called the PRT titre. t The reciprocal of the highest dilution of antiserum resulting in maximum colour development was taken as the ELISA titre. :~NT, Not tested.

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with hybridoma culture fluid mouse antibodies were detected by horseradish peroxidase (HRPO)-conjugated rabbit anti-mouse immunoglobulins (DAKO, Copenhagen, Denmark). Both clones were of the IgG2a isotype. The antibodies eluted at pH 4.6 from a Protein A Sepharose column (Pharmacia) which is the reported value for IgG2a isolation (Ey et al., 1978). In isoelectric focusing gels both Protein A-purified antibodies showed the limited banding patterns characteristic of monoclonal antibodies (Roehrig et al., 1980) (Fig. 1 b); the UM 4.2 antibodies focused between pH 8 and 7.5 while the U M 5.1 antibodies focused between pH 7.2 and 6.7. This difference may be related to idiotypic differences between the two IgG2a clones (Williamson, 1976; Bloor et al., 1982). Ascitic fluids of UM 4.2 and UM 5.1 were tested for their ability to neutralize virus in a plaque reduction test. While UM 4.2 antibody neutralized neither the virulent nor the avirulent strain of SFV, U M 5.1 neutralized SFV to a very high dilution (Table 1), even in comparison to hyperimmune mouse serum. Addition of complement in in vitro P R T had no effect on the neutralization titre although both MA clones are of the complement-binding IgG2a subclass (Neuberger & Rajewsky, 1981). A lower neutralization titre for hyperimmune serum as compared to fluid containing MA has also been reported by Chanas et al. (1982) for antibodies to Sindbis virus. These authors reasoned that due to aggregation of virus particles by nonneutralizing antibodies, the virus might be inaccessible for neutralizing antibodies resulting in a lower titre for mouse hyperimmune serum. Perhaps there are some topographically distinct sites on the E2 glycoprotein which all bind MA but only one of these sites is a target for neutralization (Massey & Schochetman, 1981). Injection of 0.1 ml ascitic fluid protected mice against lethal encephalitis caused by 10 LD50 units of SFV (16 p.f.u.). Subsequently, groups of six mice were injected intravenously with 0.1 ml of 10-fold dilutions of purified MA 2 h before intraperitoneal challenge, starting with 1000 txg/mouse. It was calculated that 10 ktg of UM 4.2 was protective against 10 LD50 units of SFV (Table 1). UM 5.1 antibody, however, was 100-fold more effective on a weight basis, 0-1 btg protein being already protective. The UM 4.2 antibodies which do not neutralize in vitro are nevertheless able to protect mice in vivo. A similar phenomenon was reported by Schmaljohn et al. (1982) for non-neutralizing MA directed against the closely related Sindbis virus and by Balachandran et al. (1982) for herpes simplex virus type 2. The latter authors explain the in vivo protection by suggesting a mechanism of antibody-dependent cell-mediated cytolysis (ADCC). Elimination of SFV is based on neutralization of infectivity by antibodies which bind to E2 determinants on the virion (Dalrymple et al., 1976). Non-neutralizing MA may enhance considerably the uptake of virus by macrophages (Halstead & O'Rourke, 1977; Peiris et al., 1982) leading to a diminished viraemia. Since SFV does not replicate in mouse macrophages (Kraaijeveld et al., 1979a), our non-neutralizing MA UM 4.2 may lead to in vivo protection by a process of opsonization. Protection provided by MA UM 4.2 and UM 5.1 is specific; no interferon activity as measured with a vesicular stomatitis virus PRT could be detected in native ascitic fluids. Moreover, interferon was not detected in mouse serum after injection of ascitic fluid or in vitro after incubation with L-cells (results not shown). Our MA did not show cross-reactivity with another alphavirus (Sindbis virus) in ELISA. Also, MA to Sindbis virus glycoprotein E 2 did not react with SFV (Roehrig et al., 1982). Immunization and screening experiments were carried out with the avirulent strain of SFV, while protection experiments were performed using a virulent challenge strain. Antigenic differences may exist between these two strains. However, both MA are effective in protection against the virulent strain of SFV which means that the recognized epitopes on the E2 glycoprotein must be very similar in both virus strains.

The authors gratefullyacknowledgeMr H. de Jong for the performance of the isoelectric focusingexperiments. This study was supported by the Foundation for Medical Research FUNGO, project no. 13-27-67.

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Short communication REFERENCES

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(Received 7 September 1982)