Immunogenic properties of a trimeric gp41-based immunogen containing an exposed membrane-proximal external region

Abstract

The membrane-proximal external region (MPER) of HIV-1 gp41 is an attractive target for vaccine development. Thus, better understanding of its immunogenic properties in various structural contexts is important. We previously described the crystal structure of a trimeric protein complex named gp41-HR1-54Q, which consists of the heptad repeat regions 1 and 2 and the MPER. The protein was efficiently recognized by broadly neutralizing antibodies. Here, we describe its immunogenic properties in rabbits. The protein was highly immunogenic, especially the C-terminal end of the MPER containing 4E10 and 10E8 epitopes (671NWFDITNWLWYIK683). Although antibodies exhibited strong competition activity against 4E10 and 10E8, neutralizing activity was not detected. Detailed mapping analyses indicated that amino acid residues critical for recognition resided on faces of the alpha helix that are either opposite of or perpendicular to the epitopes recognized by 4E10 and 10E8. These results provide critical information for designing the next generation of MPER-based immunogens.

Evaluation of a novel multi-immunogen vaccine strategy for targeting 4E10/10E8 neutralizing epitopes on HIV-1 gp41 membrane proximal external region

“…To identify immunogenic linear epitopes, ELISAs were conducted with overlapping 10-mer peptide sets biotinylated either at the N- or C-terminal ends as we previously reported (Banerjee et al, 2016; Habte et al, 2015). After the first immunization, very little response was detected (Fig.…”

“…3A and 3B) as previously described (Banerjee et al, 2016; Habte et al, 2015). As noted previously, the D674A mutation affects the helical conformation of the peptide (Banerjee et al, 2016; Habte et al, 2015); thus, results should be interpreted with some caution. For R2, the four residues that were most affected besides D674 were F673, N677, W678 and L679.…”

“…Residues important in R3 were N671, W672, F673 and N677; the first three of which are absolutely critical for 10E8. Of all the immunogens we evaluated to date (Banerjee et al, 2016; Habte et al, 2015), this is the first time we targeted all three of these residues. For R4, the pattern was similar to R3, except that W6762 was not targeted.…”

“…The pHR1-HR2-28×3 construct is based on a pET-21a vector. HR1-HR2-28×3 was expressed in E. coli , solubilized with 8M urea, and purified using Ni-NTA as we previously described (Banerjee et al, 2016; Habte et al, 2015; Qin et al, 2014). The fusion protein was cleaved with thrombin (GE Healthcare; cat#27-0846-01) and the gp41-28×3 fragment was purified using Ni-NTA, dialyzed into PBS (pH 8.0) and stored at −80°C.…”

“…Despite having short, linear, simple alpha-helical epitopes, efforts to develop a vaccine that can induce 4E10/10E8-like bnAbs have been unsuccessful (see (Banerjee et al, 2016; Habte et al, 2015) and references therein). Since all of the immunogens evaluated could bind 4E10/10E8, the failure to induce similar bnAbs is not because antigens could not assume the correct epitope structures.…”