Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion

Abstract

The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.

Immunological Characterization of Plant-Based HIV-1 Gag/Dgp41 Virus-Like Particles

“…Devoid of the immunodominant gp120 and the N-terminal regions of gp41, we hypothesized that such VLPs would present the MPER in a conformation that approximate the pre-fusion intermediate stage [ 2 ]. Interestingly, recent structural analyses of a similar gp41 construct containing the MPER and the transmembrane domains of gp41 (MPR-TM) reveal that indeed, the neutralizing epitopes of 4E10 and 2F5 are accessible to these monoclonal antibodies when the protein is presented in a non-denatured trimeric state [ 2 , 55 ]. The antibodies bind to the MPR-TM with sub-nanomolar affinities as determined by competitive ELISA and surface plasmon resonance [ 2 , 55 ].…”

A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles

“…Such VLPs display the MPER of gp41 without steric hindrance from gp120, without the immunodominant epitopes on both Env subunits, and with a higher antigen load per VLP than what exists in an HIV virion (Kessans et al, 2016). A similar construct has been shown by us to be a likely trimer with its bnAb epitopes exposed (Gong et al, 2015). When administered to mice, these VLPs elicit both serum IgG and mucosal IgA to Gag and dgp41 antigens (Kessans et al, 2016).…”

Designing a Novel Multiepitope Vaccine from the Human Papilloma Virus E1 and E2 Proteins for Indonesia with Immunoinformatics and Molecular Dynamics Approaches

“…Solubility can be improved by using fusion proteins such as Fh8, glutathione- S -transferase (GST), maltose-binding protein, and other fusion proteins . In another study, the fusion protein was proven to increase the solubility of HIV-1 vaccine proteins …”

“… 37 In another study, the fusion protein was proven to increase the solubility of HIV-1 vaccine proteins. 38 …”