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. 2020 Sep 15;117(37):22920-22931.
doi: 10.1073/pnas.2004489117. Epub 2020 Sep 1.

B cells expressing authentic naive human VRC01-class BCRs can be recruited to germinal centers and affinity mature in multiple independent mouse models

Deli Huang  1 , Robert K Abbott  2   3 , Colin Havenar-Daughton  2   3 , Patrick D Skog  1 , Rita Al-Kolla  2 , Bettina Groschel  1   3   4 , Tanya R Blane  1 , Sergey Menis  1   3   4 , Jenny Tuyet Tran  1 , Theresa C Thinnes  1 , Sabrina A Volpi  1 , Alessia Liguori  1   3   4 , Torben Schiffner  1   3   4 , Sophia M Villegas  1   3   4 , Oleksandr Kalyuzhniy  1   3   4 , Mark Pintea  1 , James E Voss  1 , Nicole Phelps  1   3   4 , Ryan Tingle  1   3   4 , Alberto R Rodriguez  1 , Greg Martin  1 , Sergey Kupryianov  1 , Allan deCamp  5 , William R Schief  1   3   4   6 , David Nemazee  7 , Shane Crotty  8   3   9
Affiliations

B cells expressing authentic naive human VRC01-class BCRs can be recruited to germinal centers and affinity mature in multiple independent mouse models

Deli Huang et al. Proc Natl Acad Sci U S A. .

Abstract

Animal models of human antigen-specific B cell receptors (BCRs) generally depend on "inferred germline" sequences, and thus their relationship to authentic naive human B cell BCR sequences and affinities is unclear. Here, BCR sequences from authentic naive human VRC01-class B cells from healthy human donors were selected for the generation of three BCR knockin mice. The BCRs span the physiological range of affinities found in humans, and use three different light chains (VK3-20, VK1-5, and VK1-33) found among subclasses of naive human VRC01-class B cells and HIV broadly neutralizing antibodies (bnAbs). The germline-targeting HIV immunogen eOD-GT8 60mer is currently in clinical trial as a candidate bnAb vaccine priming immunogen. To attempt to model human immune responses to the eOD-GT8 60mer, we tested each authentic naive human VRC01-class BCR mouse model under rare human physiological B cell precursor frequency conditions. B cells with high (HuGL18HL) or medium (HuGL17HL) affinity BCRs were primed, recruited to germinal centers, and they affinity matured, and formed memory B cells. Precursor frequency and affinity interdependently influenced responses. Taken together, these experiments utilizing authentic naive human VRC01-class BCRs validate a central tenet of germline-targeting vaccine design and extend the overall concept of the reverse vaccinology approach to vaccine development.

Keywords: HIV; germinal center; germline targeting; immunodominance; vaccine.

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Conflict of interest statement

Competing interest statement: W.R.S. is an inventor on a patent application submitted by IAVI and The Scripps Research Institute that covers the eOD-GT8 immunogen. W.R.S. is involved as a principal investigator of a human clinical trial involving eOD-GT8 60mer (G001). As part of that involvement, W.R.S. is privy to certain clinical trial data, and he was forbidden from sharing that data with the authors of this study during the course of this HuGL study, as is standard for blinded clinical trials. Any conclusions in this manuscript regarding relationships between HuGL mouse models and humans were made by the other authors, without input from W.R.S. related to any clinical trial activities.

Figures

Fig. 1.
Fig. 1.
Naive human B cell BCR VRC01-class H/L knockin mouse models HuGL16, HuGL17, and HuGL18. Figure shows the design and features of HuGL knockin mice. (A) Affinities of VRC01-class BCRs cloned from HIV– healthy human donors (43) highlighting the KD values and LC usage of HuGL16, HuGL17, and HuGL18. (B and C) HC knockins were generated using a conventional targeting strategy as described (13, 47, 48). LC knockin strategies are shown in the schematics, detailing the CRISPR-facilitated targeting strategy used for HuGL18 (B) or HuGL16 and HuGL17 (C), which involved one or two cuts in the Jκ locus, respectively. HR, arms of homology. Flow plots below each schematic show transient transfection analysis of gene targeting efficiencies in a Rag1−/− pro-B cell line using CRISPR ribonucleoproteins carrying the indicated guide RNAs and LC targeting construct (Materials and Methods). (D–H) Characterization of lymphocytes in HuGL mice. (D) Alleles were marked by breeding BCR knockins of interest (C57BL/6 background: Ighb/bCκm/m) to Igha/aCκhu/hu mice, followed by flow cytometry analysis with antibodies that distinguish C regions. (E–H) Evaluation of antigen-binding by B cells of the indicated strains using eOD-GT8 streptavidin tetramers (E) or eOD-GT8 60mer nanoparticles (F), with quantification shown in G and H. Data are representative of multiple litters per HuGL. (E–H) n = individual experiments, n = mice per group. n = 3. n = 1 to 3 mice per experiment. See also SI Appendix, Fig. S1.
Fig. 2.
Fig. 2.
VRC01-class B cells from a BCR knockin mouse with an authentic naive human VRC01-class BCR specificity (HuGL18) can be primed and recruited to GCs. (A) Day 8 ELISA binding curves from CD45.1+ mice that received HuGL18 B cells (1 in 103 precursor frequency) and immunized with eOD-GT8 60mer (Top) or eOD-GT8-KO 60mer (Bottom). eOD-GT8 IgG titers are shown in black, KO IgG titers are shown in red. Responses are shown from individual mice that are representative of two mice per group. Bars indicate SEM. (B) Flow cytometric analysis of total BGC (gated as SSL/B220+/CD4−/CD38−/GL7+) and HuGL18 B cell occupancy of GCs (SSL/B220+/CD4−/CD38−/GL7+/CD45.1−/CD45.2+ cells) of mice immunized with eOD-GT8 60mer (Left), or eOD-GT8-KO 60mer (Right). Day 8 splenic B cells were analyzed. (C) Quantitation of VRC01-class B cells among BGC cells. (D) Flow cytometric analysis of antigen binding (eOD-GT8) by endogenous or HuGL18 BGC cells. Representative of five mice tested. Mice were immunized as in B. (E) Representative histological analysis of GCs. Frozen splenic sections were stained with B220 (blue), TCRβ (green), GL7 (red), and CD45.2 (white) for identification of HuGL18 B cells within GCs. SSL, singlet scatter live. n = 3 (B and C), n = 1 (A, D, and E). n = 3 to 4 mice per experiment.
Fig. 3.
Fig. 3.
Authentic human VRC01-class naive B cell BCRs can be primed by eOD-GT8 60mer and recruited to GCs at rare physiological precursor frequencies. (A) Frequency of splenic BGC cells in mice immunized with eOD-GT8 60mer, or the eOD-GT8-KO variant, when starting from the indicated HuGL18 B cell precursor frequency (PF). Day 8 splenocytes were analyzed and BGC cells were gated as SSL/B220+/CD4−/CD38−/GL7+ and plotted as percent of total B cells (B220+ cells). (B) Representative flow cytometric plots of HuGL18 B cells in GCs utilizing allotype mark. Cells are gated on GCs as indicated in A. (C) Quantitation of HuGL18 B cells in GCs as indicated in B. Experiments are pooled between two experiments (circles and triangles are separate experiments). A total of 25 mice were examined. (D) Representative histological analysis of individual GCs for HuGL18 B cells in individual GCs as indicated in B. Dotted blue line represents starting affinity. (E) Kinetic experiment showing HuGL18 B cell competitiveness within GCs over time. All data are shown pooled from multiple independent experiments. *P < 0.05. n = 3. n = 3 to 4 mice per experiment.
Fig. 4.
Fig. 4.
HuGL18 B cells develop memory after eOD-GT8 60mer immunization. (A) Mice were assessed by flow cytometry for HuGL18 memory B cell formation in the spleen on day 36 postimmunization with eOD-GT8 60mer. eOD-GT8-KO 60mer immunization was done as a negative control. Precursor frequency of HuGL18 naive B cells was 1 in 106 before immunization. Memory B cells were gated as SSL/B220+/CD4−/IgD−/CD38+/GL7−/CD45.2+/CD45.1−. (B) Quantitation of memory B cells in A, as percent of B220+ B cells. (C) Flow cytometric analysis of class switched memory B cells gated in A. (D–E) Quantitation of (D) HuGL18 class switched memory B cells gated as IgM− B cells and (E) HuGL18 IgG1+ memory B cells on day 36 postimmunization. (F and G) Memory B cells phenotypes. (F) Representative flow cytometry and (G) quantitation of indicated memory B cell markers on memory HuGL18 B cells on day 36. Red histograms are total non-BGC cells (SSL/B220+/CD4−/CD38+/GL7−). n = 3. n = 4 to 7 mice per experiment. Mem, memory; Con, control.
Fig. 5.
Fig. 5.
Vk3-20+ VRC01-class B cells develop SHM and affinity maturation after a single priming immunization. Mice were generated with a HuGL18 B cell precursor frequency of 1 in 106 B cells, then immunized with eOD-GT8 60mer. Splenic IgG1+ d16 HuGL18 BGC cells were sorted from eOD-GT8 60mer immunized mice on days 8 to 40 postimmunization and HCs (A, D, and G) and LCs (E, F, and H) were assessed for amino acid SHMs. Cells were gated as SSL/B220+/CD4−/CD38−/GL7+/CD45.2+/CD45.1−/IgG1+/IgD−/CD138−. (A) Distribution of HC mutations over time. S, individual sequences. s = 134 for d8, s = 455 for d16, s = 295 for d36/40. (B) HuGL18 HC amino acid SHM distribution. For comparison, d36 VRC01gHL SHM data from animals with 1 in 106 precursor frequencies immunized with eOD-GT5 60mer (from ref. 27) are overlaid. (C) Specific HuGL18 HC amino acid mutations at d36. Asterisks (*) mark VRC01-class mutations. (D) VRC01-type mutations detected in HuGL18 HCs. A VRC01-class mutation (y axis) was any mutation observed in a representative set of VRC01-class bnAbs (12a12, 3BNC60, PGV04, PGV20, VRC-CH31, and VRC01) (13, 46). The black staircase depicted is a computational estimate of antigen-agnostic mutation accumulation in VH1-2 B cells (46). Each number within each square on the graph represents the number of antibody sequences that contained those specific numbers of mutations. Sequences were recovered from GC B cells that were single-cell sorted (one sequence per cell). The density of coloring is proportional to the stated number of sequences at each point relative to the total sequences analyzed. (E) Total LC amino acid mutations. s = 72 for d8, s = 343 for d16, s = 272 for d36/40. (F) Per residue HuGL18 LC amino acid SHM distribution. D36 VRC01gHL data are shown for comparison, as in B. (G) Alignment of two representative HuGL18 clones recovered on d36 with deletions in H-CDR3, out of seven total clones recovered with deletions in H-CDR3. (H) Alignment of two representative HuGL18 clones recovered on d16 with deletions in l-CDR1, out of 13 total clones recovered with deletions in l-CDR1. (I) Surface plasmon resonance (SPR) measured KD affinities for mAbs recovered from paired d36 HuGL18 GC B cell sequences. Dotted blue line represents affinity of HuGL18 precursor. Red line placed at geometric mean of clones with detectable affinity. Total sequences are from all experiments pooled. n = 2 for d16/d36. n = 1 for d8. n = 3 to 7 mice per experiment. See also SI Appendix, Fig. S4.
Fig. 6.
Fig. 6.
Characterization of HuGL17, a medium-affinity VK1-5+ VRC01-class BCR model. Analysis of total and HuGL17 splenic BGC cells. (A and B) Analysis of HuGL17 responsiveness to eOD-GT8 in vivo at a HuGL17 B cell precursor frequency of 10−3 (“1K” = 1,000). (A) Total (Left) and donor-derived fraction (Right, boxes) of BGC cells after immunization with eOD-GT8 or eOD-GT8-KO. (B) Representative flow plots of antigen-specific and IgG1 class-switched HuGL17 and endogenous (CD45.1+) BGC cells (TCRβ–CD19+CD38–GL7+). (C–L) Longitudinal analysis of mice immunized with eOD-GT8 60mer when HuGL17 precursor frequencies were 1 in 1 million B cells. (C) Longitudinal analysis of total frequency of BGC cells. (D) Representative flow plots enumerating HuGL17 BGC cells. (E) Longitudinal analysis of GC occupation by HuGL17 B cells. Each data point indicates the value measured in one recipient spleen. (F) HuGL17 BGC cell percent among all B cells. (G) Analysis of the number of amino acid replacements per sequence in the HC and LC, respectively, at d16 (n = 197, 172) and d36 (n = 178, 226). (H and I) Analysis of HC VRC01-class mutations on d16 and d36. Analysis was conducted as in Fig. 5D. (J and K) Distribution of replacement mutations as a function of amino acid position. (L) Quantitation of HC replacement mutations d36 (n = 178). (M) SPR measured KD affinities for mAbs recovered from paired d36 HuGL17 GC B cell sequences, with dotted blue line indicating affinity of the HuGL17 precursor. Asterisks (*) mark VRC01-class mutations. n = 3. n = 2 to 7 mice per experiment.
Fig. 7.
Fig. 7.
Precursor frequency and affinity are interdependent in determining competitive success for B cells possessing authentic human naive VCR01-class BCRs. (A) Frequency of HuGL B cells in GCs d8 postimmunization following immunization with eOD-GT8 60mer or eOD-GT8KO 60mer. BGC cells gated as SSL/B220+/CD4−/CD38−/GL7+/CD45.1−/CD45.2+. For comparison purposes between mouse strains, HuGL18 plots (Fig. 3) are reshown here. (B) Quantitation of HuGL B cells in GCs as shown in A. All available data are shown. Data were pooled from multiple independent experiments. Dashed line at 1% is provided as visual aide only for the reader to denote responses greater or less than 1%. (C) Statistical comparisons of HuGL GC B cells over time. Kinetics data shown in C are the average of all data shown in Figs. 3E and 6E. Mice were immunized with eOD-GT8 containing HuGL17 or HuGL18 B cells at a precursor frequency of 1 in 106 B cells.*P < 0.05 ***P < 0.001, ****P < 0.0001. n.s., not significant; n = 3, n = 3 to 4 mice per experiment for A and B. n = 4 to 5 for HuGL17 and HuGL18 each, n = 3 to 7 mice per time point for C.
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