Mimicking the Structure of the V3 Epitope Bound to HIV-1 Neutralizing Antibodies

Abstract

The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 is a target for virus neutralizing antibodies. The V3 sequence determines whether the virus will manifest R5 or ×4 phenotypes and use the CCR5 or C×CR4 chemokine co-receptor, respectively. Previous NMR studies revealed that both R5- and ×4-V3 peptides bound to antibodies 0.5β and 447-52D form β-hairpin conformations with the GPGR segment at the turn. In contrast, in their free form, linear V3 peptides and a cyclic peptide consisting of the entire 35-residue V3 loop were highly unstructured in aqueous solution. Herein we evaluated a series of synthetic disulfide constrained V3-peptides in which the position of the disulfide bonds, and therefore the ring size, was systematically varied. NMR structures determined for singly and doubly disulfide constrained V3-peptides in aqueous solution were compared with those found for unconstrained V3JRFL and V3IIIB peptides bound to 447-52D and to 0.5β, respectively. Our study indicated that cyclic V3 peptides manifested significantly reduced conformational space compared to their linear homologues and that in all cases cyclic peptides exhibited cross strand interactions suggestive of β-hairpin like structures. Nevertheless, the singly constrained V3-peptides retained significant flexibility and did not form an idealized p-hairpin. Incorporation of a second disulfide bond results in significant overall rigidity and in one case, a structure close to that of V3MN peptide bound to 447-52D Fab was assumed and in another case a structure close to that formed by the linear V3IIIB peptide bound to antibody 0.5β was assumed.

An optimally constrained V3 peptide is a better immunogen than its linear homolog or HIV-1 gp120

“…R304 and K305 have been found to be critical for the interactions of several HIV-1 neutralizing antibodies with V3(Pantophlet et al, 2008) and therefore it was important to include them in our immunogen design. To constrain a peptide to the 447-52D bound conformations of V3, a disulfide bond could be inserted at positions 301, 303, 305 or 307 of the N-terminal strand of the V3 loop (Mester et al, 2009; Mor et al, 2009). Residue I307 is part of the V3 epitope recognized by 447-52D, and interacts extensively with this antibody (Sharon et al, 2003).…”

“…305 and 306), and C-terminal residues found downstream of 318/319 (i.e. 319 and 320) to a β-hairpin conformation (Mor et al, 2009). We therefore decided to test peptides constrained using disulfide bonds involving residues 301, 303 or 305.…”

“…However, such peptides, and even peptides representing the entire 35-residues V3 region with its native disulfide bond at its base, are mostly flexible in solution and except for a transient β-turn formed by the GPGR segment do not have a well-defined secondary structure (Chandrasekhar, Profy, and Dyson, 1991; Mor et al, 2009; Zvi, Hiller, and Anglister, 1992). As a result of this flexibility, unconstrained V3 peptides used as immunogens likely induce a broad distribution of antibodies, many of which will not recognize the native conformation of the corresponding region in gp120.…”

“…In previous studies we used disulfide bonds to constrain the V3 to the conformation recognized by the 447-52D antibody (Mor et al, 2009). We showed that some of the constrained peptides exhibited higher affinity to 447-52D Fab in comparison with linear V3 peptides (Mester et al, 2009).…”

Surface plasmon resonance for vaccine design and efficacy studies: recent applications and future trends

“…This uncharacteristically conserved V region structural constraint is needed to mediate interaction with chemokine receptors during the infection process [16] and by virtue of its conserved nature is acknowledged as a target for neutralizing antibodies. Immunization with the linear V3 peptide epitope was shown to elicit a degree of bnAb response [17] but recent efforts have been made to focus the immune response by partially constraining the V3 peptide using two engineered disulfide bonds in a b-hairpin mimicking bnAb-bound V3 context [18]. The number and location of the disulfide bonds in the constrained V3 peptide were systematically varied and the impact of these changes on the intrinsic kinetic parameters defining the binding affinity of 447-52D was assessed in a highly parallel analysis of multiple constrained V3 peptide variants [19].…”