HIV-1 VACCINES. Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen

Abstract

A major goal of HIV-1 vaccine research is the design of immunogens capable of inducing broadly neutralizing antibodies (bnAbs) that bind to the viral envelope glycoprotein (Env). Poor binding of Env to unmutated precursors of bnAbs, including those of the VRC01 class, appears to be a major problem for bnAb induction. We engineered an immunogen that binds to VRC01-class bnAb precursors and immunized knock-in mice expressing germline-reverted VRC01 heavy chains. Induced antibodies showed characteristics of VRC01-class bnAbs, including a short CDRL3 (light-chain complementarity-determining region 3) and mutations that favored binding to near-native HIV-1 gp120 constructs. In contrast, native-like immunogens failed to activate VRC01-class precursors. The results suggest that rational epitope design can prime rare B cell precursors for affinity maturation to desired targets.

Fig. 1. Generation of VRC01 gH mice and outline of priming experiments

(A) Flow cytometry analysis of spleen cells showing B cell frequencies in VRC01 gH mice and WT littermates. (B) Next-generation sequencing of splenic cDNA from VRC01 gH mice revealed VH1-2*02 usage compared to mouse VH gene usage. (C) Summary of the time course for experiments and analysis. Mice were given a single prime of immunogen in adjuvant and then serum immune responses were evaluated at days 14, 28, and 42 post-immunization. Two of five mice per group were sacrificed for B cell sorting analysis at day 14, and the remaining three of five mice were sacrificed at day 42. In other animals, splenic B cells were collected at day 5, day 10, or day 31 for hybridoma generation. (D) Overview of the immunization groups listed by immunogen (eOD-GT8 60mer, eOD-GT8 3mer, eOD17 60mer, and BG505 SOSIP), multimeric state (nanoparticle or trimer), and adjuvant (Alum, Iscomatrix, and Ribi), along with the number of mice used to test each group. All groups were tested in both VRC01 gH and wild-type (WT) mice, except for BG505 SOSIP which was tested only in VRC01 gH mice. 5 mice per group were used for B cell sorting and/or ELISA, and an additional 13 VRC01 gH mice were employed for hybridoma generation after immunization with eOD-GT8 60mer (6 for Alum, 7 for Ribi).

Fig. 2. Serum and B cell analysis of antibody responses following priming immunization showed robust responses for the eOD-GT8 60mer

(A) Serum binding titers of VRC01 gH or WT littermate mice immunized with eOD-GT8-60mer nanoparticles in Ribi were measured by enzyme-linked immunosorbent assay (ELISA). Sera were titrated for binding to monomers of eOD-GT8 or eOD-GT8-KO. Plotted values represent the mean of OD450 measurements from 3 different mice for the indicated serum time point (day 28, top; day 42, bottom) at the listed dilutions. Error bars are standard error of the mean (SEM). (B) To determine differences in the level of specificity of antibody responses, the differences between the areas under the eOD-GT8 and eOD-GT8-KO ELISA binding curves were calculated for day 28 and 42 sera. Mean and standard deviation for 3 animals are shown. (C) Frequencies of epitope- or antigen- specific memory phenotype B cells sorted by flow cytometry for each immunization group. The frequency of eOD-GT8⁽⁺⁾/eOD-GT8-KO⁽⁻⁾ cells among all memory phenotype B cells is shown for all groups except for BG505 SOSIP, for which the frequency of BG505 SOSIP+ cells among all memory phenotype B cells is shown. Each point represents a mouse sacrificed at day 14 (left) or day 42 (right). Mean (day 14), or mean and standard deviation (day 42, N=3), are indicated by bars.

Fig. 3. Priming with eOD-GT8 60mer selects for mouse light chains with VRC01-class features

(A) Mouse light chains from sorted antigen-specific IgG+ memory phenotype B cells (red) and from hybridomas (blue) as well as mouse light chains from IgM+ antigen-specific hybridomas (orange) were sequenced to identify CDRL3 lengths and mutations from germline mouse kappa chains. The distribution of CDRL3 lengths is shown in a histogram compared to known VRC01-class antibodies (black) and to the naïve (un-immunized) VRC01 gH mouse antibody repertoire (white). This analysis is based on all sequences using the VRC01 gH-chain from all mice immunized with eOD-GT8 60mers (from all hybridoma or sorting timepoints and all adjuvant groups listed in Fig. 1). (B) Gene usage is shown for all Vκ genes in antibodies using the VRC01 gH-chain and a 5aa CDRL3 recovered by sorting IgG⁺ eOD-GT8⁺/eOD-GT8-KO⁽⁻⁾ memory phenotype B cells at day 14 or 42 from all mice immunized with eOD-GT8 60mers in all adjuvants (table S7). (C) Comparison of the VRC01 CDRL3 sequence with sequences of 5 aa CDRL3s recovered from VRC01 gH mice. Sequences are depicted as sequence logos at the indicated positions, with the size of each letter corresponding to the prevalence of that residue at that position. The “VRC01” sequence logo shows the sequence of the VRC01 CDRL3; the “Naïve Repertoire” sequence logo represents all 1,653 sequences with 5 aa CDRL3 found by deep sequencing of 4 unimmunized VRC01 gH mice (these sequences amount to 0.14% of all 1,169,886 sequences from those mice); the “Unmutated Abs” and “Mutated Abs” sequence logos represent the sets of unmutated (N=84) or mutated (N=70) antibodies, respectively, using the human VH1-2*02 gene and a 5aa CDRL3, isolated from VRC01 gH mice at days 14 or 42 after immunization with eOD-GT8 60mer and Alum, Isco, or Ribi (the red bar at CDRL3 length = 5 in Fig 3A corresponds to these 154 sequences, and see table S7).

Fig. 4. Priming with eOD-GT8 60mer selects for productive heavy chains mutations found in VRC01-class bnAbs

(A) A total of 61 mutated heavy chain sequences from day 14 and 42 eOD-GT8 60mer-immunized VRC01 gH-chain mice (table S7) were evaluated for the number of amino acids that match the mutations found in VRC01-class bnAbs (12a12, 3BNC60, PGV04, PGV20, VRC-CH31 and VRC01) compared to total heavy chain amino acid mutations from germline. Each circle represents a single heavy chain sequence that was isolated by antigen-specific memory phenotype B cell sorting. (B) The total number of amino acid mutations observed in the heavy chains of antibodies isolated by antigen-specific memory phenotype B cell sorting are listed by adjuvant (Alum, Isco, or Ribi) for spleen and lymph node samples harvested at 14 or 42 days post-priming immunization. Bar graphs are divided by unmutated (white) vs mutated (colored). The mutated bars are divided into Abs with 1–2 coding mutations (red), 3–4 coding mutations (blue) or 5–6 coding mutations (orange). The number of mice and the number of antibodies used to compute the frequencies in each bar are listed at the top of the graph.

Fig. 5. Binding affinities of eOD-GT8 60mer elicited antibodies for eOD-GT8 and candidate boost immunogens

(A) eOD-GT8 dissociation constants measured by SPR for 115 eOD-GT8 60mer-elicited antibodies isolated by antigen-specific B cell sorting (table S7). Antibodies were captured on the sensor chip and eOD-GT8 monomer was analyte. Data are shown for 42 antibodies from day 14 and 73 antibodies from day 42 following immunization of VRC01 gH mice with eOD-GT8 60mer. Each point is colored to indicate the type of adjuvant used (Alum, Iscomatrix, and Ribi) in the immunizations. The scale on the y-axis spans from the smallest dissociation constant (16 pM) measureable by our SPR instrument (as stated by the manufacturer) to the highest dissociation constant (10 μM) measureable based on the analyte concentration used in the experiment. (B) Dissociation constants measured by SPR between selected eOD-GT8 60mer-elicited antibodies and candidate boost immunogens. Among the 115 Abs in (A), the 29 antibodies with highest affinity for eOD-GT8 (K_D < 1 nM), along with 8 unmutated antibodies with lower affinity for eOD-GT8, were selected for binding to candidate boosting immunogens (HxB2 core-e 2CC N276D and core BG505 N276D) by SPR. High analyte concentration was used to determine K_Ds up to 100 μM. HxB2 core-e 2CC N276D 60mer nanoparticles were also assayed, with values presented as apparent affinity, due to the avidity between particles and IgG. Mutated antibodies are shown as green open squares while germline antibodies are shown as black open diamonds.