Fusion Intermediates of HIV‐1 gp41 as Targets for Antibody Production: Design, Synthesis, and HR1–HR2 Complex Purification and Characterization of Generated Antibodies

Abstract

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.

Detection of Receptor-Induced Glycoprotein Conformational Changes on Enveloped Virions by Using Confocal Micro-Raman Spectroscopy

“…Conformational changes of soluble fusion proteins were previously detected at temperatures higher than 37°C (e.g., see reference 12). However, it is well known that class I fusion proteins (such as HIV-1 gp41) can undergo a conformational change from the prefusion to the prehairpin intermediate at 23°C to 27°C (52,53). Thus, to confirm the biological relevance of the Raman spectroscopic conformational change that we observed for NiV-F, we performed a NiV-F-triggering assay to confirm that a receptor-induced conformational change in NiV-F can occur at 25°C.…”

HIV-1 Envelope Glycoproteins Induce the Production of TNF-α and IL-10 in Human Monocytes by Activating Calcium Pathway

“…2C ); (ii) the anti-fusion peptide C34L (10 −6 M) (Fig. 2D ); that efficiently inhibits viral entry 56 ; these results suggest that virus entry is not essential for the induction TNF-α and IL-10 production.

Figure 2 Activation of human monocytes by gp120 is CD4, CXCR4 and MR independent.

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Protein kinase C-delta regulates HIV-1 replication at an early post-entry step in macrophages

“…Indeed, similar levels of p24 were found in cells treated or not with rottlerin (Figure 2G). As a control, to ensure that levels of p24 correspond to intracellular antigen and not to adsorbed viruses after trypsin digestion, we used a known inhibitor of fusion, the C34 peptide [48]. In its presence, the virus continues to bind to its receptors, but it becomes unable to induce membrane fusion.…”