Nadia Benkirane

Identification of V3 loop-binding proteins as potential receptors implicated in the binding of HIV particles to CD4(+) cells.

The binding of human immunodeficiency virus (HIV) type 1 particles to CD4+ cells could be blocked either by antibodies against the V3 loop domain of the viral external envelope glycoprotein gp120, or by the V3 loop mimicking pseudopeptide 5[Kψ(CH2N)PR]-TASP, which forms a stable complex with a cell-surface-expressed 95-kDa protein. Here, by using an affinity matrix containing 5[Kψ(CH2N)PR]-TASP and cytoplasmic extracts from human CEM cells, we purified three V3 loop-binding proteins of 95, 40, and 30 kDa, which after microsequencing were revealed to be as nucleolin, putative HLA class II-associated protein (PHAP) II, and PHAP I, respectively. The 95-kDa cell-surface protein was also isolated and found to be nucleolin. We show that recombinant preparations of gp120 bind the purified preparations containing the V3 loop-binding proteins with a high affinity, comparable to the binding of gp120 to soluble CD4. Such binding is inhibited either by 5[Kψ(CH2N)PR]-TASP or antibodies against the V3 loop. Moreover, these purified preparations inhibit HIV entry into CD4+ cells as efficiently as soluble CD4. Taken together, our results suggest that nucleolin, PHAP II, and PHAP I appear to be functional as potential receptors in the HIV binding process by virtue of their capacity to interact with the V3 loop of gp120.

Solution Structure of a Retro-inverso Peptide Analogue Mimicking the Foot-and-Mouth Disease Virus Major Antigenic Site STRUCTURAL BASIS FOR ITS ANTIGENIC CROSS-REACTIVITY WITH THE PARENT PEPTIDE

The antigenic activity of a 19-mer peptide corresponding to the major antigenic region of foot-and-mouth disease virus and its retro-enantiomeric analogue was found to be completely abolished when they were tested in a biosensor system in trifluoroethanol. This suggests that the folding pattern, which is α-helix in trifluoroethanol (confirmed by CD measurement), does not correspond to the biologically relevant conformation(s) recognized by antibodies. The NMR structures of both peptides were thus determined in aqueous solution. These studies showed that the two peptides exhibit similar folding features, particularly in their C termini. This may explain in part the cross-reactive properties of the two peptides in aqueous solution. However, the retro-inverso analogue appears to be more rigid than the parent peptide and contains five atypical β-turns. This feature may explain why retro-inverso foot-and-mouth disease virus peptides are often better recognized than the parent peptide by anti-virion antibodies.

Delineation of a neutralizing subregion within the immunodominant epitope (GH loop) of foot-and-mouth disease virus VP1 which does not contain the RGD motif

The major immunogenic site of foot-and-mouth disease virus (FMDV) is contained in a disordered loop comprising residues 134-158 of capsid protein VP1, located on the surface of the viral particle. Peptides corresponding to this sequence generally elicit protective levels of neutralizing antibodies in guinea pigs. In some instances, however, the level of neutralizing antibodies is low although the level of antibodies against the peptide, determined by ELISA, is as high as that in the sera with high neutralizing antibody titres. In an attempt to ascertain the reason for this difference, we have synthesized on a cellulose membrane 10 overlapping decapeptides, offset by one residue, covering the segment 141-159 of VP1 of two viruses belonging to serotypes A12 and O1, and tested them with guinea pig antisera raised against peptide 141-159, VP1 and FMDV particles (SPOTscan method). With type A, some peptides which were strongly positive with highly neutralizing antisera did not include the RGD triplet located at residues 145-147. In contrast, antisera with low neutralization titres reacted only with decapeptides which included the RGD motif. Moreover, peptide 147-156 coupled to keyhole limpet haemocyanin, but not peptide 141-149 coupled to the same carrier, elicited high levels of neutralizing antibodies in guinea pigs. In the case of serotype O, highly neutralizing antisera to virus reacted in ELISA with peptides 141-150 (containing the RGD motif) and 135-144 (located upstream from the RGD motif). The results suggest that the RGD triplet is not an indispensable constituent of peptides able to elicit a neutralizing antibody response against the virus.

The potential of retro-inverso peptides as synthetic vaccines

Retro-inverso (RI) peptides, also called all-D-retro peptides, have been shown to mimic the antigenic and immunogenic properties of L-peptides successfully. RI peptides corresponding to the loop 141-159 of the VP1 protein of foot-and-mouth disease virus (FMDV) have been synthesized and used to immunize rabbits and guinea pigs. These peptides induced longer-lasting and higher antibody titres in immunized animals than did the corresponding L-peptides and the antibodies cross-reacted strongly with virus particles and with L-peptides. Antisera raised to RI peptides had in vitro virus neutralization titres equal to or better than those obtained after immunization with classical FMDV antigens and L-peptides. In view of their increased stability, RI peptides may overcome some of the shortcomings of synthetic viral vaccines based on L-peptides.

Exploration of Requirements for Peptidomimetic Immune Recognition ANTIGENIC AND IMMUNOGENIC PROPERTIES OF REDUCED PEPTIDE BOND PSEUDOPEPTIDE ANALOGUES OF A HISTONE HEXAPEPTIDE

We present a detailed analysis of the antigenic and immunogenic properties of a series of very stable peptidomimetics of a model hexapeptide corresponding to the C-terminal residues 130-135 of histone H3. Five pseudopeptide analogues of the natural sequence IRGERA were synthesized by systematically replacing, in each analogue, one peptide bond at a time by a reduced peptide bond Ψ(CH2-NH). Three important features of the resulting analogues were examined. First, the analogues were tested in a biosensor system for their ability to bind monoclonal antibodies generated against the parent natural peptide, and their kinetic rate constants were measured. The results show that reduced peptide bond analogues can very efficiently mimic the parent peptide. The position of reduced bonds which were deleterious for the binding was found to depend on the antibody tested, and one monoclonal antibody recognized all five analogues. The equilibrium affinity constant toward reduced peptide bond analogues of four antibodies of IgG1 isotype induced against the parent hexapeptide was higher (up to 670 times) with certain analogues than toward the homologous peptide. Second, immunogenic properties of the five analogues were studied, and it was found that polyclonal antibodies induced against analogues in which Ψ(CH2-NH) bonds were introduced between residues 130-131, 131-132, and 132-133 (R1-R2, R2-R3, and R3-R4) cross-reacted strongly with the cognate protein H3. Third, we tested the protease resistance of analogues. Altogether, the results provide a strong support for the potent applicability of reduced peptide bond pseudopeptides as components of synthetic vaccines and open a new field for the development of immunomodulatory agents.