HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen

Abstract

Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.

Fig. 1. Development of eOD-GT8

(A) Model of germline-reverted VRC01 (gray surface) interacting with eOD-GT7 (cartoon) with the 58 positions subjected to deep mutational scanning shown as magenta, green and orange spheres representing the three mutagenized linear segments. Binding enrichments, the ratio of the frequency of a mutation in the top 10% binding population to the frequency of the same mutation in all cells displaying eOD-GT7, were computed for each mutation on eOD-GT7 for germline-reverted VRC01 and are shown as a heatmap on the right, in which blue indicates unfavorable mutations, red indicates favorable mutations and white indicates the amino-acid residue in eOD-GT7. (B) The combined binding enrichments from independent yeast display screens for 18 germline-reverted VRC01-class bnAbs are shown as a multi-dimensional heatmap in which the color scale from yellow to red indicates increasing favorable average enrichment and the symbol sizes reflect the breadth of enrichment, the number of germline-reverted Abs with enriched binding for each point mutation. If enriched, the eOD-GT7 amino-acid residue is indicated by crosses. (C) Sequence logos depicting amino acids at each of 16 positions in the combinatorial library (top), the sequences selected from the combinatorial library for improved binding to germline-reverted VRC01-class bnAbs (middle), and the final sequence of eOD-GT8 (bottom). (D) SPR dissociation constants measured for both germline-reverted and mature VRC01-class bnAbs against eOD-GT6 and eOD-GT8. Solid blue lines show geometric mean measured over all the data, using the value K_D = 100 μM for samples with K_D > 100 uM; dashed blue lines show geometric means computed for the 8 germline-reverted Abs or 12 bnAbs for which K_Ds < 100 μM could be measured for both eOD-GT6 and eOD-GT8. The red dotted line signifies the limit of detection for our SPR instrument (16 pM); K_Ds below this value were measured by KinExa.

Fig. 2. eOD-GT8-binding VRC01-class naïve B cells exist in healthy human donors

(A) eOD-GT8⁺ naïve CD19⁺IgG⁻ B cells. (B) eOD-GT8⁺ B cell frequency (C) and eOD-GT8 KO⁽⁻⁾ cells among eOD-GT8⁺ B cells in individual donors. (D) VH1-2 usage among eOD-GT8⁺/eOD-GT8 KO⁽⁻⁾ sorted B cells (n=173) versus control B cells. VH1-2 (red) allele frequencies are indicated. (E) B cells expressing a 5-aa L-CDR3 among VH1-2⁺ B cells isolated by eOD-GT8 versus control B cells. (F) L-CDR3 sequence logos of VRC01-class bnAbs (top), VRC01-class naïve precursors (middle), and control B cells (bottom). (G) L-CDR1 lengths of 27 VRC01-class naïve B cells. (H) Light chain V gene usage of 27 VRC01-class naïve B cells. Known VRC01-class bnAb Vκ are red. (I) H-CDR3 lengths of VRC01-class naïve B cells versus control B cells. (J) Total B cells screened and VRC01-class naïve B cells found in 15 individuals. (K) Poisson distribution modeling of the number of VRC01-class naïve B cells. Vertical lines show the 2.5% and 97.5% quantiles. (L) SPR dissociation constants for eOD-GT6 or eOD-GT8 binding to VRC01-class or non-VRC01-class Abs derived from eOD-GT8-sorted human naïve B cells. Solid red lines indicate geometric mean.

Fig. 3. Structural analysis of eOD-GT8 and human germline antibody VRC01c-HuGL2 complex

(A) Crystal structures of VRC01c-HuGL2+eOD-GT8 (LC: blue, HC: salmon and eOD-GT8: orange) and of mature VRC01+gp120 (PDB ID: 3NGB in white) shown in the same orientation, showing eOD-GT8 superimposed on gp120, and showing only the antibody Fv regions for clarity. (B) Comparison of the H-CDR2 and L-CDR3 conformations from the structures in (A). (C) Comparison of buried surface areas for the V_H and V_L residues of VRC01c-HuGL2 and mature VRC01+gp120, in their bound forms. (D) Comparison of H-CDR2 and L-CDR3 conformations of unliganded and eOD-GT8-liganded VRC01c-HuGL2 Fab. All atoms of V_H and V_L were aligned. In the left image, H-CDR2 and L-CDR3 are shown as sticks; in the right image the CDRs are shown according to B-factors reporting local structural flexibility using a relative scale in which increasing wire thickness and warmness of color (blue to red) indicates increasing mobility. (E) Crystal structure of unliganded eOD-GT8 shown in cartoon and surface (left) and a superposition of unliganded and VRC01c-HuGL2-bound forms of eOD-GT8 (right; Cα RMSD = 0.4 Å).