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Oct 14, 2011

Binding of Potent Neutralizing Antibody to HIV Hints at New Vaccine Targets

  • Analysis of how a recently identified broadly neutralizing antibody (bnMab) binds to HIV has uncovered a potential target for vaccine development. The antibody, PGT 128, is one of a number isolated from HIV-infected patients by a team led by researchers at the Scripps Research Institute’s IAVI Neutralizing Antibody Center. Of the series of PGT antibodies isolated, PGT 128 is the most broadly neutralizing, and is capable of neutralizing over 70% of globally circulating viruses.  

    However, what appears to make PGT 128 so much more potent than other recently described anti-HIV bnMabs relates to the way the antibody penetrates through a gap in the glycan shield that protects the viral envelope, and binds to two conserve glycans, as well as a beta-strand segment on the V3 loop of the gp120 envelope protein. This triple binding appears to cross-link Env trimer spikes on the viral coat, and prevent it from infecting cells, claim Dennis R. Burton, Ph.D., Ian A. Wilson, Ph.D., and colleagues. Their findings are reported in Science Express in a paper titled “A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield.”

    PGT 128 is one of six bnMAbs (PGTs 125–128, 130–131) isolated by the Scripps-led researchers. The PGT antibodies bind specifically to the Man8/9 glycans on gp120, and potently neutralize across HIV clades, indicating that they may provide protection at relatively low serum concentrations, and the epitopes to which they bind might therefore represent promising vaccine targets. The team’s interest in PGT 128, particularly, arose because the antibody had the broadest neutralizing capability of all the PGT nbMabs, and demonstrates potency that is about an order of magnitude greater than that of other recently described bnMabs, including PG9, PG16, VRC01, and VRCPG04. They therefore carried out crystallization studies to identify the specific binding epitopes for PGT 128.

    The results unexpectedly showed that the antibody engages two different but closely spaced glycans, as well as the C-terminal end of the V3 loop, within the binding site. The primary glycan-binding site is occupied by the high-mannose glycan attached to N332 (Man8/9GlcNAc2), while a secondary glycan-binding site is on the core pentasaccharide attached to N301.

    To see how important the individual glycan binding sites were for epitope recognition, the researchers tested a series of PGT 128 antibody variants that each contained a single amino-acid substitution in each subsite. The results indicated that mutations in the primary glycan binding site (N332) compromised neutralization, gp120 binding, and binding to Man8/9 on a glycan array.

    Mutation of some residues in the secondary site (N301) also resulted in a loss of gp120 binding and virus neutralization, but the affinity of N301 secondary site was too low to detect directly by glycan array. However, mutation of other residues that interact with the mannoses in the secondary binding site had little or no effect on neutralization, suggesting that the interactions with the mannose sugars in the secondary site are not as crucial as the primary GlcNAc interactions.

    Even so, the authors stress, the N301 glycan is still required for the overall high-affinity binding to gp120 and neutralization. Notably, the N301 and N332 glycans are 93% and 73% conserved among HIV isolates, respectively, which accounts for the ability of PGT 128 to neutralize 72% of circulating viruses, they add. Specific interactions of the antibody with V3 were more difficult to evaluate because binding is mediated primarily through backbone hydrogen bonding and van der Waals forces, which can tolerate changes in side chains.

    The envelope protein (Env) of HIV-1 comprises a heterodimer of the gp41 transmembrane glycoprotein and the gp120 surface glycoprotein, and forms trimers on the surface of the viral membrane. In order to gain more insight into the structure of the epitope recognized by PGT 128 in the context of the HIV trimer, the team generated a negative stain reconstruction of a soluble, partially deglycosylated 664G trimer in complex with PGT 128 Fab. This initially showed that three Fabs bind to the trimer, without any close contacts with neighboring gp120 protomers, which indicates that the outer domain epitope is accessible and exposed. Further analysis in addition indicated that the V3 base surface is also exposed.

    The binding affinities of PGT 128 and a second antibody in the family, PGT 127, were then evaluated with respect to their neutralizing potency. Interestingly, the results showed that the neutralization values of PGT 127 and 128 IgGs against the HIV-1JR-FL strain were about 17- and 31-fold lower (i.e., more potent) than their cell-surface trimer-binding values, whereas the neutralization potency of PGT 127 and 128 Fabs correlated strongly with their binding affinity for cell surface HIV-1JR-FL  Env trimers. In addition, while PGT 127 and 128 IgGs bound with similar apparent affinity to cell surface Env trimers as their Fab counterparts, they neutralized approximately 81- and 70-fold more potently, respectively, than their corresponding Fab fragments.

    “These results suggest that PGT 127 and 128 IgGs may cross-link spikes on the surface of the virus, giving an increase in affinity through avidity effects,” the researchers remark. They say the inter-Fab distances observed for PGT 128 Fab-trimer complexes by electron microscopy mean it is unlikely that PGT 128 IgG effects intra-spike cross-linking. The concept of inter-spike, rather than intra-spike, cross-linking is key, they remark, because native Env trimers on the viral surface are scarce: “a possible explanation for this observation is that two or more viral spikes are clustered to form an infectious unit, as proposed previously. In this scenario, neutralization measures binding to infectious Env units, but not to single spikes. This interpretation also requires few infectious units on transfected cells compared to single spikes and that the single spikes are not in close enough proximity for cross-linking to occur.”

    Also of interest was the finding that at antibody concentrations corresponding to 90% neutralization, PGTs 127 and 128 IgGs reduced the half-life of HIV-1JR-FL by approximately 9.7- and 11.2-fold, respectively, whereas the corresponding Fab fragments and a previously discovered bnMab, 2G12 IgG, had little to no effect on viral infectivity decay. This combination of data indicate that inter-spike cross-linking by PGT 127 and 128 IgGs might speed the inactivation of HIV-1JR-FL Env spikes, possibly by inducing conformational changes that disrupt the function of the trimer, which somehow boosts neutralization potency.

    “Here, we provide an example of multivalency achieved through the combination of glycan and protein; the three subsites for N332, N301, and the C-terminal V3 stem are essentially independent, but combine to mediate high-affinity recognition of a glycan-based epitope on HIV Env,” the authors conclude. “Considering the highly exposed nature of this epitope and high conservation of its two glycan and V3 loop backbone components, coupled with recent studies demonstrating that broad and potent serum neutralizing activity is frequently mediated by antibodies that target N332A-sensitive epitopes, it appears that this antigenic region may serve as an attractive vaccine target if appropriate immunogens can be designed.”

     


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