The budding and the fusion processes of the enveloped animal virus Semliki Forest virus serve the purpose of transporting its nucleocapsid, containing its genome, from the cytoplasm of an infected cell into that of an uninfected one. We show here that, in the infected cell, the viral membrane (spike) proteins p62 and El are organized as heterodimers which are very resistant to dissociation in acidic conditions. In contrast, the mature form of the heterodimer, E2E1, which is found in the virus particle and which is generated by proteolytic processing of p62, is very prone to dissociate upon treatment with mildly acidic buffers. We discuss the possibility that this difference in behavior of the intracellular precursor form and the mature form of the spike protein complex represents an important regulatory mechanism for the processes involving membrane binding around the nucleocapsid during budding and membrane release from the nucleocapsid at the stage of virus fusion.

Fifteen monoclonal antibodies (MAs) directed against either the El or E2 glycoprotein of Semliki Forest virus (SFV) were characterized by immunoglobulin subclass, pl traject, hemagglutination inhibition, neutralization of infectious virus, and protection against virulent infection in mice. All MAs except UM8.4 (immunoglobulin M [IgM]) belonged to various subclasses of IgG and predominantly to IgG2a, but all were unique as indicated by their banding patterns in isoelectric focusing. Competitive binding assays with these MAs revealed the presence of at least six distinct antigenic determinants (epitopes) on the El glycoprotein and five epitopes on the E2 glycoprotein. Two of the epitopes on El, as defined by the properties of the MAs, were associated with hemagglutination inhibition (Elc and Eld), three were associated with neutralization (Ela, Elb, and Elf), and five were associated in various degrees with protection (Ela, Elb, E1c, Ei', and Elf) of mice against virulent SFV infection. With the MAs against E2, the epitopes on E2 were similarly defined. Epitopes E2b and E2e were associated with hemagglutination inhibition, E2c and E2d were associated with neutralization, and three epitopes were associated with in vivo protection (E2', E2C, and E2d). Furthermore, for each MA the relative avidity to purified SFV was determined with an enzyme-linked immunosorbent assay. The binding of some MAs to purified SFV was enhanced by a second MA. The relative avidities of individual MAs did not correlate with their neutralizing capacities. From the results, we suggest that the amino acid sequence which makes up determinant E2d and is recognized by the highly protective MA UM5.1 is an excellent candidate for the production of a synthetic vaccine.

SUMMARYTwo 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. 1 a). 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...

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Both neutralizing and nonneutralizing immunoglobulin G2a monoclonal antibodies (MAs) directed against the E2 glycoprotein of Semliki Forest virus (SFV) protected mice prophylactically and therapeutically against virulent SFV infection. The neutralizing MAs, however, conferred protection to mice at lower doses than did nonneutralizing MAs. The antibody-dependent, complement-mediated cytolysis of SFV-infected L cells was effectuated by both kinds of antibodies, but again neutralizing MAs were more effective. Removal of the Fc part of the neutralizing MA UM 5.1 by pepsin digestion resulted in a 100-fold reduction of the neutralization titer (104 versus 106) and a complete loss of its capacity to mediate antibody-dependent, complement-mediated cytolysis. Passive protection of infected mice occurred only after administration of relatively high doses of F(ab')2 of MA UM 5.1 (30.0 ,ug versus 0.1 ,ug). F(ab')2 fragments prepared from the nonneutralizing MA UM 4.2 had lost their protective capacity completely. Surprisingly, the nonneutralizing MA UM 4.2 retarded virus growth in mouse fibroblasts (L cells), although inhibition was at much higher doses than with the neutralizing MA UM 5.1. Furthermore, both MAs promoted the uptake of virulent SFV in the Fc receptor-bearing WEHI-3 cells. The results suggest that nonneutralizing MAs protect mice not only by antibody-dependent, complement-mediated cytolysis but also by growth inhibition and enhanced uptake of SFV in the nonpermissive macrophages of BALB/c mice. This hypothesis is supported by the absence of viremia in recipients of nonneutralizing MA UM 4.2 at 24 h after infection.