T.A.M. Oosterlaken

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.

A neutralization-inhibition enzyme immunoassay for anti-idiotypic antibodies that block monoclonal antibodies neutralizing Semliki Forest virus

In this paper we compare a solid-phase enzyme immunoassay (EIA) with a neutralization-inhibition enzyme immunoassay (NI-EIA) for the determination of anti-idiotypic antibodies against Semliki Forest virus (SFV)-neutralizing monoclonal antibodies (MAs) UM 5.1 (IgG2a) and UM 1.4 (IgG2a). Against these MAs strong immune sera were induced in female BALB/c mice by two subcutaneous injections, 3 weeks apart, with keyhole limpet hemocyanin coupled MA mixed with the saponin Quil A. Rabbit immune sera were prepared by intracutaneous injections of purified MA mixed with either FCA (first immunization) or IFA (second and third immunization). In the NI-EIA serum is preincubated with neutralizing MA, in wells of 96-well plates, before SFV is added. Binding of anti-idiotypic antibodies to MA results in a diminished capacity of that MA to neutralize SFV. After 1 h incubation with SFV L cells are added and residual infectious virus is allowed to multiply for 6 h at 37 degrees C. Then the monolayers are fixed with glutaraldehyde and subsequently SFV is quantified with a horseradish peroxidase-labelled SFV-specific MA. Low absorbance values indicate that the neutralizing capacity of MA is intact and that blocking antibodies were not present in serum. In contrast high absorbance values indicate that blocking (anti-idiotypic) antibodies had abrogated the neutralizing capacity of MA. With the strongly neutralizing MA UM 5.1 as idiotypic antigen the NI-EIA proved to be at least as sensitive as the solid-phase EIA. Furthermore both normal mouse serum-absorbed rabbit immune sera and mouse immune sera were not cross-reactive in both solid-phase EIA and NI-EIA.

Efficient induction of Semliki Forest virus and mumps virus neutralizing anti-anti-idiotypic antibodies using Quil A as adjuvant

Rabbit anti-idiotypic sera were prepared against Semliki Forest virus (SFV) neutralizing monoclonal antibody (MAb) UM 1.13 and mumps virus neutralizing MAb UM 10B. From these sera anti-idiotypic antibodies were purified by ammonium sulphate precipitation and subsequent affinity column chromatography. Anti-iso- and anti-allotypic antibodies were removed by binding to normal mouse serum immunoglobulins coupled to CNBr activated Sepharose. Peak protein fractions eluted from columns loaded with homologous MAb were used for anti-anti-idiotypic immunization of BALB/c mice to raise virus neutralizing anti-anti-idiotypic antibodies. Two intracutaneous immunizations, five weeks apart, with affinity purified rabbit polyclonal anti-idiotypic antibody (40 micrograms protein per animal) coupled to keyhole limpet hemocyanin and mixed with the adjuvant Quil A (50 microliters per animal) were sufficient to evoke neutralizing antibodies against either virus. Moreover the mice who developed SFV neutralizing serum antibodies upon anti-idiotypic immunization all survived an otherwise lethal challenge with virulent SFV.

Blocking by anti-idiotypic antibodies of monoclonal antibody mediated protection in mice against encephalomyocarditis virus induced diabetes and lethal disease

SummaryMonoclonal antibodies (MA) UM 21.1 and UM 21.2 protect mice against encephalomyocarditis virus (D-variant) induced diabetes and lethal disease. MA-mediated protection in vivo as well as neutralization in vitro could be specifically blocked by anti-idiotypic antibodies.

Blocking by anti-idiotypic antibodies of monoclonal antibody-mediated protection against lethal Semliki Forest virus in mice.

Semliki Forest virus‐(SFV) neutralizing monoclonal antibodies (MoAbs), produced after fusion of spleen cells from BALB/c mice and myeloma cell line P3‐X63‐AG8.653 or SP2/0, were used for anti‐idiotypic immunization of female BALB/c mice. Two intracutaneous immunizations (2 × 40 μg per animal), 3 weeks apart, with keyhole limpet haemocyanin‐conjugated MoAbs mixed with the saponin Quil A were sufficient to induce high levels of anti‐idiotypic antibodies in the circulation of these mice with the capacity to block specifically in vitro MoAb‐mediated virus neutralization. Anti‐idiotypic antibodies against SFV‐neutralizing MoAbs, either passively transferred or actively acquired by immunization, are also able to abrogate (specifically) passive immunity, mediated by critical protective doses of MoAb, in mice against infection with a lethal strain of SFV. Furthermore we confirmed by intervention with anti‐idiotypic serum in vivo that an SFV‐ neutralizing MoAb exerts its greatest protective effect during the first 2 days of infection.

Competition binding assay in cell culture for identification of epitopes on enveloped and naked viruses.

Virus infected monolayers, in wells of 96-well plates, could be used as antigen in competition binding assays to identify epitopes on respectively Semliki Forest virus, encephalomyocarditis virus and mumps virus.

Detection by a solid phase independent enzyme immunoassay of monoclonal anti-idiotypic antibodies against monoclonal antibodies neutralizing Semliki Forest virus and encephalomyocarditis virus

Neutralization inhibition enzyme immunoassay (NI-EIA) was evaluated for its usefulness to detect monoclonal anti-idiotypic antibodies (ab2mAbs) against idiotypic monoclonal antibodies (ab1mAbs) neutralizing either Semliki Forest virus (SFV) or encephalomyocarditis virus (EMCV). Purified ab1mAbs were coupled to keyhole limpet hemocyanin (KLH) mixed with the adjuvant Quil A and then injected intracutaneously into homologous BALB/c mice. Successful fusions were performed 5, 6 and 7 days after intracutaneous booster immunizations of these mice. Ab2mAbs in hybridoma supernatant fluids were detected by their capacity to block virus neutralization by ab1mAbs in NI-EIA. Two stable ab2mAb producing hybridomas were obtained against SFV neutralizing mAb UM 1.13, and twelve against EMCV neutralizing mAb UM 21.1.

Detection of a shared idiotope on two encephalomyocarditis virus neutralizing monoclonal antibodies by neutralization inhibition enzyme immunoassay

Idiotypic cross-reactivity between encephalomyocarditis virus (EMCV) neutralizing monoclonal antibodies UM 21.1 (IgG2b) and UM 21.3 (IgG2a) was detected by neutralization inhibition enzyme immunoassay using polyclonal and monoclonal anti-idiotypic antibodies. One strongly cross-reactive anti-idiotypic monoclonal antibody, designated 21.1A5 (IgG2b), might recognize a recurrent idiotope on EMCV neutralizing antibodies but it did not induce EMCV neutralizing anti-anti-idiotypic antibodies in homologous BALB/c mice.

A vaccine against Semliki Forest virus consisting of a monoclonal anti-idiotypic antibody cross-linked to a protein which contains virus-specific T-helper cell epitopes

A recombinantly expressed protein, consisting of cro-beta-galactosidase at the N-terminus and amino acid residues 115 to 151 of the E2 membrane of Semliki Forest virus (SFV) at the C-terminus containing two T-helper cell epitopes of SFV, was cross-linked with glutaraldehyde to a noninternal image monoclonal anti-idiotypic antibody (ab2 alpha MAb) able to induce SFV-neutralizing anti-anti-idiotypic (ab3) antibodies in BALB/c mice. This vaccine, which might potentially induce SFV-specific T-helper cell memory, established in BALB/c mice a state of protective immunity against virulent SFV within 10 days of immunization. A steady rise in serum neutralization titre occurred from day 7 to day 28 after primary anti-idiotypic immunization, levelling off thereafter. In primarily immunized mice significant rises of serum neutralization titres, which could be indicative for an operational T-helper cell memory, were not observed after challenge on day 35 with virulent SFV. The results suggest that SFV is neutralized by ab3 antibodies shortly after challenge, preventing, thereby, virus multiplication to levels sufficient to provoke a measurable booster response.

Requirements for Induction of Semliki Forest Virus Neutralizing Antibodies by a Non-Internal Image Monoclonal Antibody

Recently we identified a noninternal image monoclonal anti-idiotypic antibody (ab2β MAb), designated 1.13A321 (IgG1), that, cross-linked by glutaraldehyde to keyhole limpet haemocyanin (KLH) and combined with the adjuvant Quil A, was able to evoke Semliki Forest Virus (SFV) neutralizing anti-anti-idiotypic (ab3) antibodies in BALB/c mice (Oosterlaken et al, 1991; Oosterlaken et al, 1992). These antibodies protected mice against an otherwise lethal infection with SFV. In this study conditions for induction of SFV-neutralizing ab3 antibodies were investigated. It is shown that cross-linking of 1.13A321 to itself by glutaraldehyde is sufficient to evoke SFV-neutralising ab3 antibodies. Furthermore a recombinantly expressed protein consisting of cro-β-galactosidase at the N-terminus and aminoacid residues 115 to 151 of the E2 membrane protein of SFV at the C-terminus was used as carrier molecule (Snijders et al, 1989). The SFV fragment contains two T-helper cell epitopes which might potentially provide an SFV specific T-cell memory upon immunization (Snijders et al, 1992). Furthermore the potential of ab2 MAb 1.13A321 as carrier for a linear B-cell epitope of SFV was investigated.