Daniel Chargelegue

Peptide mimics of a conformationally constrained protective epitopes of respiratory syncytial virus fusion protein

AIMS To identify peptides that mimic (mimotopesi conformational and protective epitopes of RSV fusion protein and to assess their efficacy as immunogens and potential vaccines. MATERIAL AND METHODS An 8-mer solid-phase (TG resin) library was screened with a neutralising and protective RSV fusion protein specific monoclonal antibodies (Mab-19). After selection of positive beads, reactive sequences were identified by microsequencing and 8-mer peptides were synthesised. Improvement of binding was analysed by amino acid replacement using the SPOTs method. RESULTS Mabs were not able to bind to the free and soluble peptides, nor did these peptides induce anti-RSV specific antibodies. However, several peptides re-synthesised on a TG resin (to produce de-protected 8-mer peptides linked to the resin) or as SPOTs reacted specifically. Therefore it was critical to be able to reproduce this conformation in order to use these mimotopes as immunogens and potential vaccines. Using C-terminal constrained versions of the mimotopes, strong binding of one of the Mabs to the peptides was demonstrated by surface-plasmon resonance. Immunisation of Balb/c mice with these peptide-mimics produced anti-sera that: (1) reacted specifically with RSV; (2) inhibited the binding of the Mab to the virus; (3) neutralised RSV in vitro with high titres (range: 80-640); and (4) reduce significantly the viral load in the lungs of mice challenged with RSV (P < 0.01). CONCLUSIONS This report demonstrates for the first time that: (1) a protective epitope of the conserved RSV fusion protein can be mimicked by synthetic peptides; and (2) immunisations with these mimotopes induced specific anti-RSV neutralising antibodies and reduced viral load in vivo. These results represent a novel concept for the development of a vaccine against RSV.

Development of a sensitive ELISA for HIV-1 p24 antigen using a fluorogenic substrate for monitoring HIV-1 replication in vitro

A two-site sandwich fluorescent-ELISA was optimized for the detection of HIV-1 p24 antigen produced by lymphoid cells infected with HIV-1 in vitro. To improve the sensitivity of the ELISA, a combination of streptavidin-beta-galactosidase and a fluorogenic substrate (4-methylumbelliferyl-beta-D-galactopyranoside) was employed for the enzymatic detection stage. Using recombinant p24 as standard antigen, a two-step assay detected as little as 0.7 pg/ml (3.10(-14) M) with an upper limit of 10,000 pg/ml. This detection range (approx. 50-70-times greater than ELISAs using a chromogenic detection) permitted an accurate and straightforward quantitation of p24 in culture supernatants. Overall, the fluorescent-ELISA had increased detectability, sensitivity and efficiency over existing ELISAs for HIV-1 p24.