An engineered mutant of HIV-1 gp120 formulated with adjuvant Quil A promotes elicitation of antibody responses overlapping the CD4-binding site

Abstract

A major priority in HIV vaccine research is the development of an immunogen to elicit broadly neutralizing antibodies (NAbs). Monoclonal antibody (mAb) b12 is one of now several broadly neutralizing mAbs that bind epitopes overlapping the CD4-binding site (CD4bs) on HIV-1 gp120 and that serve as templates to engineer effective immunogens. We are exploring a strategy whereby extra glycans are incorporated onto gp120 to occlude the epitopes of non-neutralizing mAbs while maintaining exposure of the b12 site. Immunizing with these so-called hyperglycosylated gp120s is hypothesized to preferentially elicit b12-like NAbs. Here, the effects of two adjuvants, monophosphoryl lipid A (MPL) and Quil A, on eliciting b12-like responses when formulated with a new hyperglycosylated mutant, ΔN2mCHO(Q105N), is presented. Sera from ΔN2mCHO(Q105N)_MPL immunized animals bound the homologous antigen ΔN2mCHO(Q105N) with greater preference than sera from ΔN2mCHO(Q105N)_QuilA immunized animals, demonstrating the modulation of antibody fine specificity by these two adjuvants. We also found that sera from ΔN2mCHO(Q105N)_QuilA immunized animals bound best to a resurfaced HIV gp120 core protein on which non-CD4bs epitopes are substituted with non-HIV residues, suggesting that these sera contain a relatively larger fraction of CD4bs-specific antibodies. Consistent with these data, inhibition assays revealed epitope overlap with the binding sites of the CD4bs-specific antibodies b12, b13 and VRC03. Unexpectedly, these sera did not exhibit significant neutralizing activity against a set of HIV-1 primary strains. Our results show that although formulating mutant ΔN2mCHO(Q105N) with Quil A promotes the elicitation of CD4bs-directed antibodies relative to wild-type gp120, tweaking of the immunization regimen is needed to yield robust, CD4bs-focused NAbs.

Fig. 1

Locations of modifications on HIV-1 gp120 to focus CD4bs antibody responses: Structure of the JR-FL gp120 core (PDB ID 2B4C) denoting the locations of glycan attachment sites (naturally occurring (yellow) and those inserted for hyperglycosylation (orange)) and engineered Ala substitutions (light blue). The location of residues that upon alanine substitution result in ≥50% reduction in b12 binding to gp120 (ref. [28]) are shown in green. The location of Gly-Asp-Met-Arg (GDMR) residues at the center of the CD4bs that are necessary for binding of several non-NAbs, but not b12 (ref. [28]), is also marked (dark khaki).

Fig. 2

Mutant Q105N limits exposure of antibody epitopes overlapping the CD4bs. The antigenicity of hyperglycosylated mutant Q105N relative to gp120wt (JR-FL) was determined using mAbs F105, b6, VRC01, b13, B4e8, b12, 2G12, CD4-IgG2 and VRC0. CD4-IgG2 was used as a surrogate for CD4. Error bars denote the signal ranges from replicate wells.

Fig. 3

Serum antibody binding to JR-FL gp120wt and Q105N antigens. Antibodies elicited by gp120wt or Q105N formulated in adjuvant MPL bind preferentially to their respective homologous antigen. Sera from gp120wt_QuilA animals also bind preferentially to JR-FL gp120wt. Filled symbols indicate serum binding to the respective homologous antigen whereas open symbols indicate binding to heterologous antigen. Error bars denote the standard error of mean from replicate wells. Statistical analyses show that Quil A induces higher overall antibody titres regardless of antigen. **, p < 0.01; ****, p < 0.0001.

Fig. 4

Subclass profile of immune sera. Binding of sera from mice immunized with gp120wt or Q105N was detected with noted subclass-specific secondary antibodies. The results show that Quil A elicited higher levels of IgG2 when formulated with either gp120wt or Q105N. Error bars denote the standard error of mean from replicate wells. *, p < 0.05; ****, p < 0.0001.

Fig. 5

Sera from animals immunized with hyperglycosylated mutant Q105N mixed with Quil A elicit greater CD4bs-directed responses. (A) All sera bind fairly equally to construct XOD6, indicating that all contain antibodies to the outer domain portion of gp120. (B) The Q105N_QuilA formulation elicited a significantly higher proportion of antibodies to epitopes overlapping the CD4bs relative to gp120wt mixed with the same adjuvant as judged by stronger affinity for the RSC3 mutant. **, p < 0.01; ***, p < 0.001. (C) None of the sera bound significantly less to mutant RSC3Δ371I compared to RSC3, suggesting that the elicited CD4bs-specific antibodies do not require Ile371 for binding. Error bars denote the signal ranges from replicate wells.

Fig. 6

Dissection of the CD4bs specificities of serum antibodies. Sera were serially diluted (starting at 1:50) and incubated on plates for 1 h. The indicated mAbs (diluted to 10 μg/ml) were then added to the plates and these incubated for a further 1 h. MAb binding was quantified using AP-conjugated anti-human F(ab’)₂-specific secondary antibody (Jackson ImmunoResearch). Sera from animals immunized with Q105N inhibited the binding of mAbs b12, b13 and VRC03 to a greater extent than gp120wt animals, suggesting that the epitopes of the serum antibodies overlap most with the epitopes of these CD4bs mAbs. Error bars denote the signal ranges from replicate wells.

Fig. 7

Sera from gp120wt and Q105N animals exhibit poor neutralizing activity. The neutralizing activity of the immune sera was assessed using the tier 1B viruses SS1196 (subtype B) and ZM197M (subtype C) and the tier 2 virus JR-FL (subtype B). SIVmac239 was used as a control for non-specific neutralizing activity. The sera (1:10) were mixed with an equal volume of virus and incubated for 1 h at 37 °C before adding to target cells. The dashed line denotes 50% neutralization. Error bars denote the signal ranges from replicate wells.