Hyperglycosylated Stable Core Immunogens Designed To Present the CD4 Binding Site Are Preferentially Recognized by Broadly Neutralizing Antibodies

Abstract

The HIV-1 surface envelope glycoprotein (Env) trimer mediates entry into CD4 ؉ CCR5 ؉ host cells. Env possesses conserved antigenic determinants, such as the gp120 primary receptor CD4 binding site (CD4bs), a known neutralization target. Env also contains variable regions and protein surfaces occluded within the trimer that elicit nonneutralizing antibodies. Here we engineered additional N-linked glycans onto a cysteine-stabilized gp120 core (0G) deleted of its major variable regions to preferentially expose the conformationally fixed CD4bs. Three, 6, 7, and 10 new NXT/S glycan (G) motifs were engineered into 0G to encode 3G, 6G, 7G, and 10G cores. Following purification, most glycoproteins, except for 10G, were recognized by broadly neutralizing CD4bs-directed antibodies. Gel and glycan mass spectrometry confirmed that additional N-glycans were posttranslationally added to the redesigned cores. Binding kinetics revealed high-affinity recognition by seven broadly neutralizing CD4bs-directed antibodies and low to no binding by non-broadly neutralizing CD4bs-directed antibodies. Rabbits inoculated with the hyperglycosylated cores elicited IgM and IgG responses to each given protein that were similar in their neutralization characteristics to those elicited by parental 0G. Site-specific glycan masking effects were detected in the elicited sera, and the antisera competed with b12 for CD4bs-directed binding specificity. However, the core-elicited sera showed limited neutralization activity. Trimer priming or boosting of the core immunogens elicited tier 1-level neutralization that mapped to both the CD4bs and V3 and appeared to be trimer dependent. Fine mapping at the CD4bs indicated that conformational stabilization of the cores and addition of N-glycans altered the molecular surface of Env sites of vulnerability to neutralizing antibody, suggesting an explanation for why the elicited neutralization was not improved by this rational design strategy. IMPORTANCEMajor obstacles to developing an effective HIV-1 vaccine include the variability of the envelope surface glycoproteins and its high-density glycan shield, generated by incorporation of host (human) glycosylation. HIV-1 does harbor highly conserved sites on the exposed envelope protein surface of gp120, one of which is the virus receptor (CD4) binding site. Several broadly neutralizing antibodies elicited from HIV patients do target this gp120 CD4 binding site (CD4bs); however, gp120 immunogens do not elicit broadly neutralizing antibodies. In this study, we targeted the CD4bs by conformational stabilization and additional glycan masking. We used the atomic-level structure to reengineer gp120 cores to preferentially present the cysteine-stabilized CD4bs and to mask (by glycan) nonneutralizing determinants. Importantly, glycan masking did successfully focus antibody responses to the CD4bs; however, the elicited CD4bs-directed antibodies did not neutralize HIV or bind to unmodified gp120, presumably due to the structure-guided modifications of the modified...

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“…These results demonstrate that enrichment of distinct specificities through MAb isolation can reveal additional neutralizing activities, providing considerably more information from a given pre-clinical or clinical vaccine trial. Furthermore, the higher resolution provided by MAb isolation confirms that Env-based vaccines can elicit CD4bs- and V3-directed neutralizing Abs and is consistent with studies using adsorption and competition assays on unfractionated plasma or serum samples (21, 48, 57–59). Among the cloned MAbs, additional Env sub-specificities identified were composed of gp41 (n=23) and the V1V2 region (n=1).…”

One-step design of a stable variant of the malaria invasion protein RH5 for use as a vaccine immunogen

“…To address this problem, approaches for immunogen stabilization or grafting of immunogenic epitopes onto stable scaffolds have been implemented (15)(16)(17)(18)(19)(20)(21). However, key vaccine immunogens frequently have complex folds with significant flexibility and low stability.…”

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