Fusion intermediates of HIV-1 gp41 as targets for antibody production: design, synthesis, and HR1-HR2 complex purification and characterization of generated antibodies.

The objective of this project was to study the interaction between HR1 and HR2, the stability of the complex formed, and to characterize the antibodies produced against monomeric HR1 and HR2 peptides as well as the HR1-HR2 complex. In this work, HR1 was mimicked by peptide N36, and HR2 was mimicked by peptide C34L and its analogues C34M2, C34M3, and C34D. Whereas C34M2 and C34M3 are partially composed of D-amino acids, C34D has same sequence as C34L, but is assembled entirely of D-amino acids. Using CD analysis, SPR assays, and gel filtration chromatography, we demonstrate the physical interaction between N36 and C34L and its analogues C34M2 and C34M3, but not C34D. We show that the HR1-HR2 complex is formed rapidly (<1 min) and remains stable, as demonstrated by its inability, in contrast to each free peptide, to inhibit the formation of syncytia. To generate antibodies with predetermined specificity against the transiently exposed intermediate that corresponds to the six-helix bundle structure, purified preformed HR1-HR2 complex was used, in parallel with monomeric HR1 and HR2 peptides, as immunogens in mice. Although the produced antibodies recognize total HIV-1 envelope glycoproteins in ELISA, they are unable to neutralize HIV-1-mediated fusion at 37 °C. However, if the incubation with these antibodies is carried out at 27 °C, a temperature that allows stabilization of the transient intermediate complex, anti-peptide antibodies are able to bind their corresponding domains in HeLa cells expressing HIV-1 gp41 in co-culture with HeLa CD4-CCR5/CXCR4 during the dynamic mechanism of membrane fusion. In agreement with the latter results, these antibodies, if previously incubated for 2 h at 27 °C, are able to strongly neutralize HIV-1 entry by membrane fusion, as shown by their ability to block the formation of syncytia.

Development and characterization of peptidic fusion inhibitors derived from HIV-1 gp41 with partial D-amino acid substitutions.

The aim of this study was to design synthetic peptides with D-amino acid substitutions that mimic the human immunodeficiency virus (HIV) gp41 HR2 region. The objective was to develop new and active C34 analogue peptides by introducing D-amino acid point substitutions at nonessential sites for HR1-HR2 interaction without disrupting the structure of the peptide. Herein we report a study with C34L peptide analogues, including the enantiomer peptide C34D, the retro-inverso analogue (RI), and two peptides with D-amino acid point substitutions (C34M2 and C34M3). Our results show that, with the exception of RI, these peptides adopt an alpha-helical structure and are, like C34L, able to interact with HR1, mimicked by the N36 peptide. Furthermore, we show that modifications introduced in C34M2, but not in C34M3, enhance its resistance to trypsin-mediated hydrolysis and increase the stability of C34M2 in physiological medium. Interestingly, our results show that C34 peptide analogues C34M2 and C34M3, but not C34D and its RI analogue, retain their ability to inhibit HIV-1 replication with an efficiency similar to that of the C34L peptide. These data underscore the interest in using D-amino acids at specific sites in the C34 peptide sequence and may lead to a new strategy for the development of more stable and active anti-HIV-1 peptidic drugs.