Human CD4-binding site antibody elicited by polyvalent DNA prime-protein boost vaccine neutralizes cross-clade tier-2-HIV strains

Abstract

The vaccine elicitation of HIV tier-2-neutralization antibodies has been a challenge. Here, we report the isolation and characterization of a CD4-binding site (CD4bs) specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent DNA prime-protein boost HIV vaccine. HmAb64 is derived from heavy chain variable germline gene IGHV1-18 and light chain germline gene IGKV1-39. It has a third heavy chain complementarity-determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 20 (10%), including tier-2 strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 in complex with a CNE40 SOSIP trimer revealed details of its recognition; HmAb64 uses both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4bs. This study demonstrates that a gp120-based vaccine can elicit antibodies capable of tier 2-HIV neutralization.

Fig. 1. DNA prime and protein boost elicited serum HIV-1 Env specific neutralizing antibody responses targeting the CD4-binding site.

a Immunization scheme. Serum samples were collected 2 weeks after each immunization, and PBMCs were collected 2 weeks after the last immunization. b Development of gp120-specific IgG titers following key immunization steps. c Neutralization with the volunteer serum at peak level against a panel of pseudotyped viruses expressing primary Env antigens from different subtypes of HIV-1 as indicated. d Virus capture competition assay using mAb b12 and volunteer sera. Serum 50% inhibition titer is reported as the reciprocal of serum dilution necessary to inhibit 50% of the virus captured by mAb b12. Source data are provided as a Source Data file.

Fig. 2. Vaccine-elicited antibody HmAb64 targets the CD4-binding site and neutralizes tier-2 HIV-1 strains.

a HmAb64 binding to gp120 Env proteins. HmAb64 showed strong binding to wild-type gp120 antigens from multiple HIV-1 isolates but not to their D368R mutants, indicating CD4-binding site specificity. Data are presented as mean values ± SEM (n = 2 duplicated wells). b Neutralization breadth. HmAb64 neutralized 20 viruses (10%) from diverse clades of the 208-strain NIAID panel. c Neutralization titers (IC₅₀) and tier designation of VRC panel viruses neutralized by HmAb64. d Number and percentage in each tier designation of VRC panel viruses neutralized by HmAb64. Source data are provided as a Source Data file.

Fig. 3. Binding of HmAb64 to HIV-1 gp120 antigens.

a Binding to either autologous gp120 antigens included in the PDPHV vaccine (left) or heterologous gp120 antigens (right). b Biolayer interferometry binding kinetics of HmAb64 (Octet assay). Source data are provided as a Source Data file.

Fig. 4. Binding of HmAb64 to HIV-1 on cell surface.

a Examples of FACS analyses and gating showing the specific binding of HmAb64 to HIV-1 SF162 infected human CD4+ T cells but not to uninfected cells. CD4bs mAb VRC01 was used as the positive control, and normal human IgG was used as the negative control. b Percentage of HmAb64 binding to SF162 infected CD4+ T cells from three individual human donors. Source data are provided as a Source Data file.

Fig. 5. Surface plasmon resonance assay of HmAb64 binding to HIV-1 Env antigens.

a Titration of sCD4 (D1D2 domain) binding to various HIV-1 Env antigens (Bal gp120, A244 gp120, and BG505-SOSIP). b Binding kinetics of HmAb64 to HIV-1 Env antigens. c Binding kinetics of HmAb64 to HIV-1 Env antigens in the presence of increasing amount of sCD4(D1D2). d Binding kinetics of VRC01 to HIV-1 Env antigens in the presence of increasing amount of sCD4(D1D2).

Fig. 6. Cryo-EM structure of HmAb64 in complex with a tier-2 HIV-1 CNE40 Env trimer.

a Cryo-EM density of HmAb64 in complex with CNE40 Env in two perpendicular views. The regions corresponding to gp120, gp41, and N-linked glycans, as well as heavy and light chains of bound HmAb64, were colored light gray, dark gray, green, blue, and light blue, respectively. b Refined cryo-EM structure of HmAb64 in complex with CNE40 Env with the same color usage as in (a) and the CD4-binding loop of gp120 highlighted in orange. c Surface representation of the HmAb64-bound Env trimer with HmAb64 epitope colored blue and the footprint of CD4 outlined in purple. d Comparison of the HmAb64-bound open form Env trimer (blue) with the closed form (gray) and the open occluded form trimer recognized by b12 (teal). Each gp120 and gp41 subunit was represented by two conserved helices, α1/α2, and HR1/HR2, respectively, in cylinders (also see Supplementary Fig. 8). e Superimposition of sCD4 (purple) onto an HmAb64-bound gp120 in the same orientation as boxed in (a). The signature residue of CD4, Phe43, was highlighted in the sticks to indicate the approximate position of the Phe43 cavity. f Key residues in the CD4-binding loop and HmAb64 at the interface between gp120 and HmAb64. g Mapping of HmAb64 epitope on CNE40 gp120. Epitope residues were highlighted in gray shade, with those forming hydrogen bonds colored red and those forming salt bridges underlined. Amino acids that were disordered in the cryo-EM structure were shown in light gray font. Secondary structures of the antibody-binding regions were shown for reference. h Mapping of HmAb64 paratope. Paratope residues were highlighted in the gray shade, with those forming hydrogen bonds colored red and those forming salt bridges underlined. Only two SHM residues were involved in direct contact with gp120. The sequences were numbered according to the Kabat nomenclature.