Adi Moseri

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

Synthetic peptides offer an attractive option for development of a V3-directed vaccine. However, immunization with flexible linear peptides may result in an immune response to multiple conformations, many of which differ from the native conformation of the corresponding region in the protein. Here we show that optimization of the location of a disulfide bond in peptides constrained to mimic the beta-hairpin conformation of the V3, yields an immunogen that elicits a 30-fold stronger HIV-1 neutralizing response in rabbits compared with the homologous linear V3 peptide. The HIV-1 neutralizing response elicited by the optimally constrained peptide is also significantly stronger than that elicited by a gp120 construct in which the V3 is exposed. Neutralization of an HIV-1 strain that shares only 72% identity with the immunizing peptide was demonstrated. The most effective immunogen was also able to neutralize primary isolates that are more resistant to neutralization such as SS1196 and 6535.

NMR observation of HIV-1 gp120 conformational flexibility resulting from V3 truncation.

The envelope spike of HIV‐1, which consists of three external gp120 and three transmembrane gp41 glycoproteins, recognizes its target cells by successively binding to its primary CD4 receptor and a coreceptor molecule. Until recently, atomic‐resolution structures were available primarily for monomeric HIV‐1 gp120, in which the V1, V2 and V3 variable loops were omitted (gp120core), in complex with soluble CD4 (sCD4). Differences between the structure of HIV gp120core in complex with sCD4 and the structure of unliganded simian immunodeficiency virus gp120core led to the hypothesis that gp120 undergoes a major conformational change upon sCD4 binding. To investigate the conformational flexibility of gp120, we generated two forms of mutated gp120 amenable for NMR studies: one with V1, V2 and V3 omitted (mutgp120core) and the other containing the V3 region [mutgp120core(+V3)]. The TROSY‐1H‐15N‐HSQC spectra of [2H,13C,15N]Arg‐labeled and [2H,13C,15N]Ile‐labeled unliganded mutgp120core showed many fewer crosspeaks than the expected number, and also many fewer crosspeaks in comparison with the labeled mutgp120core bound to the CD4‐mimic peptide, CD4M33. This finding suggests that in the unliganded form, mutgp120core shows considerable flexibility and motions on the millisecond time scale. In contrast, most of the expected crosspeaks were observed for the unliganded mutgp120core(+V3), and only a few changes in chemical shift were observed upon CD4M33 binding. These results indicate that mutgp120core(+V3) does not show any significant conformational flexibility in its unliganded form and does not undergo any significant conformational change upon CD4M33 binding, underlining the importance of V3 in stabilizing the gp120core conformation.

Detection of intermolecular transferred‐NOEs in large protein complexes using asymmetric deuteration: HIV‐1 gp120 in complex with a CCR5 peptide

Weak protein–protein and protein–ligand interactions play important roles in biological recognition. In many cases, simplification of structural studies of large protein complexes is achieved by investigation of the interaction between the protein and a weakly binding segment of its protein ligand. Detection of pairwise interactions in such complexes is a major challenge for both X‐ray crystallography and nuclear magnetic resonance. We demonstrate that transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to ~ 100 kDa. Using this approach, we revealed interactions between tyrosine residues of a 27‐residue peptide (deuterated at Ile and Val residues) corresponding to the N‐terminal segment of the human C‐C chemokine receptor 5 (CCR5) chemokine receptor, and a 43 kDa construct of gp120 envelope protein of human immunodeficiency virus type 1 (deuterated on all aromatics) complexed with a cluster of differentiation 4‐mimic miniprotein. The complex was present mostly as a dimer as determined by T2 relaxation measurements. The TRNOE crosspeaks in the ternary complex were assigned to the specific Tyr protons in the CCR5 peptide and to methyl protons, predominantly of isoleucine residues, and also of leucine and/or valine residues of gp120. The TRNOE/asymmetric deuteration method benefits from the sensitivity of the homonuclear NOESY experiment and does not suffer the sensitivity losses associated with isotope‐edited/isotope‐filtered approaches that rely on magnetization transfer between protons and heteronuclei that are bonded to them. The technique can be widely applied for studying large protein complexes that exhibit fast off‐rates.

The C4 region as a target for HIV entry inhibitors – NMR mapping of the interacting segments of T20 and gp120

The peptide T20, which corresponds to a sequence in the C‐terminal segment of the HIV‐1 transmembrane glycoprotein gp41, is a strong entry inhibitor of HIV‐1. It has been assumed that T20 inhibits HIV‐1 infection by binding to the trimer formed by the N‐terminal helical region (HR1) of gp41, preventing the formation of a six helix bundle by the N‐ and C‐terminal helical regions of gp41. In addition to binding to gp41, T20 was found to bind to gp120 of X4 viruses and this binding was suggested to be responsible for an alternative mechanism of HIV‐1 inhibition by this peptide. In the present study, T20 also was found to bind R5 gp120. Using NMR spectroscopy, the segments of T20 that interact with both gp120 and a gp120/CD4M33 complex were mapped. A peptide corresponding to the fourth constant region of gp120, sC4, was found to partially recapitulate gp120 binding to T20 and the segment of this peptide interacting with T20 was mapped. The present study concludes that an amphiphilic helix on the T20 C‐terminus binds through mostly hydrophobic interactions to a nonpolar gp120 surface formed primarily by the C4 region. The ten‐ to thousand‐fold difference between the EC50 of T20 against viral fusion and the affinity of T20 to gp120 implies that binding to gp120 is not a major factor in T20 inhibition of HIV‐1 fusion. Nevertheless, this hydrophobic gp120 surface could be a target for anti‐HIV therapeutics.

An extended CCR5 ECL2 peptide forms a helix that binds HIV‐1 gp120 through non‐specific hydrophobic interactions

C‐C chemokine receptor 5 (CCR5) serves as a co‐receptor for HIV‐1. The CCR5 N‐terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing extracellular loops 1 and 3 (ECL1 and ECL3) were found to inhibit HIV‐1 infection. The aromatic residues in the C‐terminal half of an ECL2 peptide were shown to interact with gp120. In the present study, we found that, in aqueous buffer, the segment Q188–Q194 in an elongated ECL2 peptide (R168–K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two‐dimensional saturation transfer difference NMR spectroscopy and dynamic filtering studies revealed involvement of Y187, F189, W190 and F193 of the helical segment in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule, and therefore could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions that are not representative of the interactions of the intact CCR5 receptor. The HIV‐1 inhibition by ECL2 peptides, as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV‐1 co‐receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV‐1 entry inhibitors.

Immunofocusing using conformationally constrained V3 peptide immunogens improves HIV-1 neutralization

V3-directed antibodies are present in practically all HIV-1 infected patients and in individuals vaccinated with gp120. The levels of maternal V3-directed antibodies were recently shown to correlate with reduced mother to child transmission, and V3 IgGs were found to be a negative correlate of risk in the RV-144 human trial. mAb directed to the tip of the V3 are capable of broad neutralization of Tier-1 and some Tier-2 viruses. Here we report an immunofocusing approach using conformationally constrained V3 peptides of different lengths. Immunofocusing with short constrained V3 peptides following immunizations with long constrained V3 peptides resulted in sera with improved neutralization of Tier-1B viruses in comparison with immunizations with the long constrained peptide alone. Immunizations only with the short constrained peptide were ineffective. Our results demonstrate that immunofocusing with constrained V3 peptides of different lengths could improve the induction of HIV-1 neutralizing antibodies.

Defining specific residue-to-residue interactions between the gp120 bridging sheet and the N-terminal segment of CCR5: applications of transferred NOE NMR

Infection by HIV‐1 requires protein–protein interactions involving gp120, CD4 and CCR5. We have previously demonstrated that the transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to ~ 100 kDa. Here, using this approach, we reveal interactions between tyrosine residues of a 27‐residue peptide corresponding to the N‐terminal segment of the CCR5 chemokine receptor, and a dimeric extended core YU2 gp120 envelope protein of HIV‐1 complexed with a CD4‐mimic miniprotein. The TRNOE crosspeaks in the ternary complex were assigned to the specific Tyr protons in the CCR5 peptide and to methyl protons of isoleucine, leucine and/or valine residues of gp120. Site directed mutagenesis combined with selective deuteration and TRNOE resulted in the first discernment by a biophysical method of specific pairwise interactions between gp120 residues in the bridging sheet of gp120 and the N‐terminus of CCR5.

Synergism between a CD4-mimic peptide and antibodies elicited by a constrained V3 peptide.

Due to the different mechanisms HIV-1 has evolved to escape from a neutralizing antibody response it has been extremely challenging to develop an effective anti-HIV-1 vaccine. The V3 region of the gp120 HIV-1 envelope glycoprotein has been considered as one of the possible targets for an anti-HIV vaccine. It is well known that the V3 region of gp120 is at least partially masked in circulating strains and becomes exposed only after CD4 binding. However, when the virus is bound to surface CD4, steric hindrance prevents effective neutralization by V3-directed antibodies. Here we have used a 27-residue CD4-mimetic peptide in combination with immune sera elicited by an optimally constrained V3 peptide to enhance neutralization of a panel of clade B viruses. We observed strong synergism between the immune sera and the CD4-mimetic in the neutralization of tier 1 and a representative tier 2 clade B virus suggesting that the constrained V3 peptide immunogen correctly mimics the V3 conformation even in tier 2 clade B viruses. This synergy should improve the potential of CD4-mimetic compounds for preexposure prophylaxis and in the treatment of HIV-1-infected patients who usually manifest high titers of V3-directed antibodies. Moreover, constrained V3 immunogens elicit immune sera that may neutralize HIV in synergy with CD4 binding site antibodies that expose V3 and the coreceptor binding site.

P19-28. The V3 region of HIV-1: from NMR to vaccine design

Background The V3 loop is one of the few epitopes to which broadly neutralizing antibody response can be directed. The most potent and most broadly neutralizing anti-V3 antibody to date is the human monoclonal antibody 447-52D. Using NMR spectroscopy, we studied the conformation of several V3 peptides in complex with 447-52D. The flexible V3 peptides were found to adopt a β-hairpin conformation when bound to this antibody. Using disulfide bonds we constrained V3 peptides to adopt a conformation similar to those of V3 peptides bound to 447-52D.