Identification of linear epitopes on Semliki Forest virus E2 membrane protein and their effectiveness as a synthetic peptide vaccine.

Semliki Forest virus (SFV) infection of mice was used as a model to study the applicability of synthetic peptides containing only linear epitopes as viral vaccines. The identification of linear epitopes with vaccine potential on the E2 membrane protein of SFV was based on the binding of SFV-specific antibodies to a set of overlapping synthetic hexapeptides (Pepscan) representing the whole E2 amino acid sequence. The 14 available E2-specific monoclonal antibodies which were protective in vivo proved to be unsuitable for the identification of linear epitopes because they recognized only conformational epitopes, as indicated by their lack of reactivity with unfolded, reduced E2 protein on immunoblots. Three epitopes were detected with polyclonal anti-SFV serum at amino acid positions 135 to 141, 177 to 185 and 240 to 246 of the E2 protein. Synthetic peptides containing these epitopes were coupled to a carrier protein and tested as a vaccine. Mice immunized with the peptide containing amino acids 240 to 255 of protein E2 were protected against a challenge with virulent SFV but protection of mice immunized with the peptides containing amino acids 126 to 141 or 178 to 186 was only marginally better than that of controls. The prechallenge sera of most peptide-immunized mice reacted with SFV-infected cells but none of these sera neutralized the virus in vitro. However, protection of mice correlated well with SFV-specific antibody titre, suggesting antibody-mediated protection.

The heterodimeric association between the membrane proteins of Semliki Forest virus changes its sensitivity to low pH during virus maturation.

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 E1 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.

Rapid determination of neutralizing antibodies to Semliki Forest virus in serum by enzyme immunoassay in cell culture with virus-specific monoclonal antibodies.

We describe in this study a rapid enzyme immunoassay for the titration of neutralizing antibodies in serum against Semliki Forest virus. For this assay L cells were added to preincubated virus-antiserum mixtures to form monolayers. Six hours after infection by residual, nonneutralized virus, the monolayers were fixed, and the E2 glycoprotein of Semliki Forest virus on the surface of infected cells was quantified with an E2-specific, peroxidase-labeled monoclonal antibody (UM 5.1). The serum antibody titer was defined arbitrarily as the inverse value of that dilution of serum associated with a 25% inhibition of control absorbance values. These titers of both early and later mouse immune sera were similar to those determined in simultaneously performed 50% plaque reduction tests. The results indicate that the enzyme immunoassay (duration, 9 h) is reliable and compares favorably with the conventional plaque reduction test (duration, 25 h) in rapidity, ease of performance, and objectivity.

The role of complement in monoclonal antibody-mediated protection against virulent Semliki Forest virus.

Monoclonal antibodies (MAs), specific for either the E1 or E2 glycoproteins of Semliki Forest virus (SFV), and belonging to various immunoglobulin subclasses (IgM, IgG2a, IgG2b and IgG3), effected lysis of SFV-infected L cells in co-operation with guinea-pig complement. In this antibody-dependent complement-mediated cytolysis (ADCMC) test, IgG1 MAs were not effective although these antibodies recognize the viral antigens on the surface of SFV-infected L cells. The latter was shown with horseradish peroxidase (HRPO)-labelled MAs in a direct enzyme immunoassay. The binding reactivities of HRPO-labelled MAs to infected L cells at selected time-intervals after infection correlated well with the amount of cytolysis in a parallel ADCMC test. Cytolysis was dependent on the duration of incubation with antibodies: more cytolysis was measured after a 4-hr incubation period with MA, starting at 4 hr after infection, compared to a 1-hr incubation period starting after 7 hr of infection. However, in the latter case (1-hr period) the amount of cytolysis measured correlated better to neutralization and/or protection by MAs than after the extended period (4 hr) of incubation. Complement (C3) depletion by cobra venom factor treatment led to a higher mortality and viraemia of mice prophylactically injected with critically protective doses of either the neutralizing MA UM 8.4 (IgM) or the non-neutralizing MA UM 4.2 (IgG2a). The results suggest a co-operative role of MA with complement in mediating protection against SFV. Passive immunization by administration of low amounts (0.1 micrograms/mouse) of neutralizing MA UM 5.1 resulted in protection of normal mice against a lethal infection with SFV. Mice immunosuppressed by cyclophosphamide were not protected by these doses. If the doses were increased however, these mice were protected both prophylactically and therapeutically. These results indicate that, using critical doses of MAs, an intact immune system ensures survival in normal mice after infection with virulent SFV.

Antigenic differences between virulent and avirulent strains of Semliki Forest viruses detected with monoclonal antibodies. Brief report.

Eleven monoclonal antibodies (MAs) reacted strongly in an enzyme immunoassay with virulent Semliki Forest virus (SFV) replicating in L cell monolayers. Three MAs showed a considerably diminished reaction with an avirulent strain of SFV both in enzyme immunoassays and plaque reduction tests.

Enzyme immunoassay of interferon with peroxidase-labelled virus-specific monoclonal antibodies.

To quantify the antiviral effect of interferon (IFN) we applied a mixture of two horseradish peroxidase-labelled monoclonal antibodies, specific for the E1 glycoprotein of Semliki Forest virus, in a direct enzyme immunoassay. This assay is suitable for detection of virus replication in L-cells, seeded as monolayers in 96-well plates. Inhibition of absorbance values caused by IFN was determined in a Flow Titertek Multiskan. Three IFN samples from different sources were titrated simultaneously in the enzyme immunoassay and in the vesicular stomatitis virus plaque reduction test in five consecutive experiments. Titres were calculated as the inverse value of the dilution of IFN causing 25% inhibition of absorbance values and 50% reduction of plaque counts respectively. The results show equality of precision and reproducibility between and within the two assays. However, the enzyme immunoassay is more convenient and objective than the plaque reduction assay.

Determination of inhibitory concentrations of antiviral agents in cell culture by use of an enzyme immunoassay with virus-specific, peroxidase-labeled monoclonal antibodies.

An enzyme immunoassay (EIA) to determine 50% inhibitory concentrations of drugs which suppress Semliki Forest virus replication is described. Inhibition of virus replication was measured in L-cells, seeded as monolayers in 96-well plates by use of horseradish peroxidase-labeled monoclonal antibodies directed against the E1 glycoprotein of Semliki Forest virus. The antiviral agents tested were cycloheximide, tunicamycin, NH4Cl, and disodium cromoglycate. The 50% inhibitory concentration of these antiviral agents was arbitrarily defined as the concentration of drug, in culture medium, associated with 50% reduction of the control absorbance value measured on Semliki Forest virus-infected cells without drug in the culture fluid. Twenty-two hours after infection the 50% inhibitory concentrations of the drugs were 0.2 microgram/ml for cycloheximide, 0.8 microgram/ml for tunicamycin, 0.3 mg/ml for NH4Cl, and 4.9 mg/ml for disodium cromoglycate. These values are similar to those determined by others with conventional methods of virus quantification. This test is sensitive and easy to perform and therefore is suited for large-scale experiments.

Mechanisms of monoclonal antibody-mediated protection against virulent Semliki Forest virus.

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 (10(4) versus 10(6)) 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 micrograms versus 0.1 microgram). 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.

Identification of distinct antigenic determinants on Semliki Forest virus by using monoclonal antibodies with different antiviral activities.

Fifteen monoclonal antibodies (MAs) directed against either the E1 or E2 glycoprotein of Semliki Forest virus (SFV) were characterized by immunoglobulin subclass, pI 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 E1 glycoprotein and five epitopes on the E2 glycoprotein. Two of the epitopes on E1, as defined by the properties of the MAs, were associated with hemagglutination inhibition (E1c and E1d), three were associated with neutralization (E1a, E1b, and E1f), and five were associated in various degrees with protection (E1a, E1b, E1c, E1e, and E1f) 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 (E2a, 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.

Detection of Semliki Forest virus in cell culture by use of an enzyme immunoassay with peroxidase-labeled monoclonal antibodies specific for glycoproteins E1 and E2.

Four noncompeting monoclonal antibodies (MA) directed against either the E1 (UM 8.64 and 8.139) or E2 (UM 8.55 and 8.73) glycoprotein of Semliki Forest virus were purified and labeled with horseradish peroxidase. Each enzyme-labeled MA was tested alone and in combination with others for its sensitivity to detect virus-infected cells. Semliki Forest virus-infected L cells seeded as monolayers in 96-well plates were screened for the virus after incubation with enzyme-labeled MA and a substrate. In this system single enzyme-labeled MA even at high dilution (10(3.0) to 10(4.5] were able to detect virus-infected cells. The sensitivity of the test could be enhanced by combining two noncompeting MA (10(4.5) to 10(5.0]. Combinations of three and four MA were less effective, due to high absorbance values for noninfected cells. The threshold of virus defection was between 10(5) and 10(6) PFU/ml. This test is sensitive and specific and therefore may be useful for diagnostic purposes.

Neutralizing and non-neutralizing monoclonal antibodies to the E2 glycoprotein of Semliki Forest virus can protect mice from lethal encephalitis.

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.

Characterization of the Philadelphia chromosome by gene mapping.

Chinese hamster X human and mouse X human somatic cell hybrid lines were obtained using circulating leucocytes from six chronic myeloid leukemia patients. All six patients carried the Ph1 translocation, t(9q+;22q-), characteristic of chronic myeloid leukemia, in their dividing immature granulocytes. Analysis of independent hybrid clones yielded the following results: 1. The chromosome 9 markers, soluble aconitase and adenylate kinase-1, segregated with the 9q+ derivative. The latter marker has previously been localized to 9q34. 2. The chromosome 22 markers, mitochondrial aconitase, N-acetyl-alpha-D-galactosaminidase, and arylsulfatase-A, also segregated with the 9q+ derivative. Mitochondrial aconitase has recently been assigned to 22q11 leads to 22q13. No evidence was obtained either for reciprocity of the translocation or for variations in breakpoints in different patients. The results reported in this paper provisionally assign the gene for mitochondrial aconitase to a region distal to the breakpoint in 22q11.

Changes in the cholesterol and phospholipid content of mouse spleen after Raucscher leukemia virus infection.

The effect of Rauscher Leukemia Virus (MuLV-R) infection on the lipid composition of mouse spleen from BALB/c mice was investigated. Drastic changes in the lipid composition of the spleen as a result of tumor growth induced by the virus could be demonstrated at 21 days after infection. The molar ratio of cholesterol to phospholipids was found to be low, while a shift within the choline containing phospholipid classes resulted into a lower sphingomyelin and a higher phosphatidyl choline content of the MuLV-R infected spleen. The cholesterol ester content increased more than two-fold during tumor growth, and shifts in the fatty acid patterns of the lipids were demonstrated.