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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 alpha-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 beta-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.

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 Psi(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 Psi(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.

A series of monoclonal antibodies has been generated against an hexapeptide of sequence IRGERA corresponding to the C-terminal residues 130-135 of histone H3 and three analogues of this model peptide. The analogues correspond to the D-enatiomer, containing only D-residues, and two retro-peptides containing NH-CO bonds instead of natural amide peptide bonds. The chirality of each residue was maintained in the retro-peptide and inverted in the retro-inverso-peptide. Monoclonal antibodies were generated from mice immunized with the analogues coupled to neutral small unilamellar liposomes containing monophosphoryl lipid A as adjuvant. The reactivity of antibodies with the four analogues and with the parent protein H3 was studied in enzyme-linked immunosorbent assay and in a biosensor system. The equilibrium affinity constant (Ka) toward the retro-inverso-peptide of two out of three antibodies of IgG1 isotype induced against the L-hexapeptide was 7-75-fold higher than toward the homologous L-peptide. The range of Ka values of four antibodies of IgG1 and IgG2a isotypes generated against the retro-inverso-peptide was 0.6-1.9 x 10(9) M-1 for both the retro-inverso- and L-peptides. Furthermore, antibodies to the L- and retro-inverso-peptides cross-reacted strongly (in some cases better than with the homologous peptide) with the parent histone H3 and with chromatin subunits containing H3. The results are thus promising in respect to the potential use of retro-inverso-analogues, which are particularly stable, in the design of much more potent synthetic vaccines or to generate antibody probes.

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[Kpsi(CH2N)PR]-TASP, which forms a stable complex with a cell-surface-expressed 95-kDa protein. Here, by using an affinity matrix containing 5[Kpsi(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[Kpsi(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.