Priming HIV-1 broadly neutralizing antibody precursors in human Ig loci transgenic mice

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Science  30 Sep 2016:
Vol. 353, Issue 6307, pp. 1557-1560
DOI: 10.1126/science.aah3945


A major obstacle to a broadly neutralizing antibody (bnAb)–based HIV vaccine is the activation of appropriate B cell precursors. Germline-targeting immunogens must be capable of priming rare bnAb precursors in the physiological setting. We tested the ability of the VRC01-class bnAb germline-targeting immunogen eOD-GT8 60mer (60-subunit self-assembling nanoparticle) to activate appropriate precursors in mice transgenic for human immunoglobulin (Ig) loci. Despite an average frequency of, at most, about one VRC01-class precursor per mouse, we found that at least 29% of singly immunized mice produced a VRC01-class memory response, suggesting that priming generally succeeded when at least one precursor was present. The results demonstrate the feasibility of using germline targeting to prime specific and exceedingly rare bnAb-precursor B cells within a humanlike repertoire.

It is widely thought that vaccine elicitation of sustained levels of potent broadly neutralizing antibodies (bnAbs) may protect humans against HIV (1, 2). HIV bnAbs from natural infection show extensive somatic mutation (19), and this maturation is required for cross-reactivity to diverse isolates: bnAb inferred-germline variants typically show detectable affinity for few if any HIV envelope protein (Env) antigens tested (35, 7, 1021). Most wild-type Env proteins are therefore poor immunogens to prime bnAb responses (35, 17, 22, 23). Reliable vaccine initiation of bnAb responses will likely require design or discovery of “germline-targeting” priming immunogens with appreciable affinity for bnAb germline precursors (35, 10, 11, 17, 2229). Furthermore, consistent bnAb priming may only be possible for bnAb precursors present at reasonable frequency in all or most vaccine recipients (27).

Proof of principle that a germline-targeting immunogen can prime relatively rare bnAb-precursor B cells was shown with the eOD-GT8 60-subunit self-assembling nanoparticle (60mer) that targets precursors of CD4–binding- site–directed VRC01-class bnAbs (26). VRC01-class precursors are defined by their use of a heavy-chain VH1-2*02 (or *03 or *04) gene and light chains with unusually short complementarity-determining region 3 (CDR3) loops of five amino acids (5, 3032). In a transgenic mouse model expressing the germline-reverted VRC01 heavy chain paired with wild-type mouse light chains (VRC01 gH mouse), in which the frequency of VRC01-class precursor B cells was estimated to be higher than in humans by a factor of as little as ~5, a single immunization with eOD-GT8 60mer activated VRC01-class precursors and generated VRC01-class memory responses in nearly all immunized mice, whereas control immunogens presenting a native CD4 binding site failed to activate such precursors (26). The eOD-GT8 60mer activated target precursors less robustly in a different VRC01-class inferred-germline heavy-chain transgenic mouse (25). Several properties of both mouse models lowered the bar for germline targeting compared to the challenges confronted in a human: elevated bnAb precursor frequency, reduced competition from other B cell specificities, limited diversity of bnAb precursors owing to their uniform recombined heavy chains, and an affinity advantage conferred on bnAb precursors due to their mature H-CDR3s.

To better model the conditions for initiation of a VRC01-class bnAb response in humans, we investigated immunization with eOD-GT8 60mer in Kymab mice transgenic for the complete unrearranged human antibody germline gene repertoire (33). Kymab mice contain human heavy chains paired with either human kappa light chains (HK mice) or human lambda light chains (HL mice), or both (HKL mice). To determine the level of difficulty for VRC01-class bnAb priming in Kymab mice, we first measured the frequency of VRC01-class precursors in the above mouse strains. Using B cell sorting methods, we previously detected eOD-GT8–specific VRC01-class precursors at a frequency of 1 in 2.4 million among human naïve B cells expressing kappa light chains (27). Using similar methods to probe a total of ~300 million naïve B cells from the spleens and lymph nodes of unimmunized HK and HL mice (n = 3 each), we were unable to isolate any VRC01-class B cells, suggesting that the frequency of such B cells was considerably lower than in humans. The frequency of the VH1-2*02, *03, or *04 alleles among HK B cells was previously measured as 0.9% (33, 34), lower than the frequency in humans (2.9 ± 1.3%) (fig. S1) (35, 36) by a factor of only ~3. Therefore, to explain the reduced frequency of VRC01-class B cells in Kymab mice, we analyzed the light-chain variable gene (VL) usage and CDR3 length distributions from immunoglobulin M–positive (IgM+) or IgG+ B cells for five HK mice and four healthy humans by next-generation sequencing (NGS) (37). The kappa light chain–CDR3 (L-CDR3) length distributions were broadly similar in HK mice and humans (Fig. 1A), but the frequency of five–amino acid L-CDR3s was lower in HK mice (0.018 ± 0.013%) than in humans (0.95 ± 0.57%) by a factor of ~50 (Fig. 1B). The cumulative frequency of Vκ genes used by known VRC01-class bnAbs [IGVK3-20, IGVK1-33, and IGVK3-15 (32, 38)] was 24.0 ± 3.5% in HK mice, similar to the 23.2 ± 4.9% that we measured in humans (Fig. 1C). However, the frequency of five–amino acid L-CDR3s associated with known VRC01-class kappa chains was reduced by a factor of ~300 in HK mice (0.00089 ± 0.00079%) compared to humans (0.27 ± 0.13%) (Fig. 1D). Restricting the analysis to light chains with five or fewer VL nucleotide mutations produced similar conclusions (fig. S2). Whatever the cause, our data indicate that VRC01-class precursors are less frequent in HK mice than in humans by a factor of 150 to 900.

Fig. 1 Frequency analysis of VRC01-class light chains and precursors in Kymab HK mice.

(A) Light-chain L-CDR3 length distributions in humans and HK mice. (B) Frequencies of five–amino acid L-CDR3s in humans and HK mice. (C) Frequencies of known VRC01-class bnAb light-chain Vκ genes in humans and HK mice. (D) Frequencies of known VRC01-class bnAb Vκ genes with five–amino acid L-CDR3s in humans and HK mice. (E) Modeled distributions of the number of VRC01-class precursors per HK mouse for average precursor frequencies of 0.2 or 1.3 per HK mouse, corresponding to frequencies of 1 in 360 million or 1 in 60 million HK B cells, respectively. One in 60 million is likely to be an upper bound on the frequency, and so the distribution is likely to be shaped more like that shown for 1 in 360 million (see text). In (B) to (D), points represent frequencies for individual humans or mice (each sequenced once), and bars represent mean ± SD for n = 4 humans or n = 5 HK mice.

Although we previously detected eOD-GT8–specific VRC01-class precursors at a frequency of 1 in 2.4 million naïve human kappa light-chain B cells, by correcting for cell sorter and polymerase chain reaction (PCR) losses, we estimated the true frequency as 1 in 400,000 (27). Based on this number and the calculations above, we conclude that the frequency of VRC01-class precursors in HK mice is unlikely to be higher than 1 in 60 million (=150 × 400,000) B cells and might be as low as 1 in 360 million (=900 × 400,000) B cells. The spleens of HK and HL mice (7 to 18 weeks of age) contain 50 million B220+ B cells, of which ~60% are mature B cells (33) that are thought prepared to respond to antigen. By also accounting for the lymph nodes and periphery, we estimate that each mouse contains ~75 million B cells, of which 45 million are mature. Thus, we expect a very low average frequency of 0.2 (≈75/360) to 1.3 (≈75/60) eOD-GT8–specific VRC01-class precursors per HK mouse at any given time. Modeling the number of precursors per mouse with Poisson distributions predicts that 27 to 82% of HK mice will have zero precursors and the remainder one to four precursors (Fig. 1E). Thus, priming VRC01-class responses in HK mice appears substantially more difficult than in humans, as the precursor frequency is lower (by a factor of 150 to 900) and the number of precursors per individual is lower [by 3000 to 30,000 precursors (27)]. Frequency analysis in one HL mouse and two HKL mice reached similar conclusions that were only slightly more favorable for VRC01-class priming (table S1).

We conducted immunization experiments to determine if eOD-GT8 60mer could prime rare VRC01-class precursors in Kymab mice. Our first experiment (Fig. 2A), in both HK and HL mice (28 mice each), evaluated differences in antigen dose (20 versus 4 μg), adjuvant formulation, and route (Iscomatrix/subcutaneous versus Sigma Adjuvant system, also known as “Ribi”/intraperitoneal), and time point of analysis (14 days versus 42 days after immunization). Unimmunized mice (three HK and three HL) and mice immunized with 20 μg of non–germline-targeting eOD-17 60mer in Iscomatrix were controls. Serum enzyme-linked immunosorbent assay showed robust responses to eOD-GT8 [endpoint titers of (1 to 5) × 10−5 and half-maximal inhibitory concentrations of (5 to 13) × 10−4 at day 42] and lower responses to eOD-GT8 KO (fig. S3), a mutant with reduced binding to VRC01-class antibodies (26), indicating epitope-specific responses (fig. S4) (37). Spleens and lymph nodes from culled mice were processed, stained, and single-cell sorted for IgM/IgD memory B cells that bound to eOD-GT8 tetramers but not to eOD-GT8 KO tetramers (27, 37). Only eOD-GT8 60mer-immunized mice showed evidence of epitope-specific (eOD-GT8+/eOD-GT8 KO) memory B cells (Fig. 2B). A control sort of eOD-17 60mer-immunized samples with eOD-17/eOD-17-KO probes identified memory B cells reactive with eOD-17 but not eOD-GT8.

Fig. 2

B cell sorting analysis reveals reproducible epitope-specific responses to eOD-GT8 60mer prime. (A) Overview of two immunization experiments in Kymab mice. (B) Frequencies of epitope-specific memory B cells at days 14 or 42 after priming in experiment 1. (C) Same type of data as in (B) but for experiment 2, in which analysis was carried out at day 42 only. In (B) and (C), points represent frequencies measured for a single mouse, each measured once, and bars represent mean ± SD for all data in each column of the graph.

We performed a second experiment (Fig. 2A), in HK and HKL mice, with eOD-GT8 60mer, eOD-17 60mer, the native-like trimer BG505 SOSIP (3944), and eOD-GT8 d41m3 60mer, a variant of eOD-GT8 60mer with disulfide stabilization and modification of the underlying nanoparticle (fig. S5). The results for eOD-GT8 d41m3 60mer are combined with those of eOD-GT8 60mer, as the two were indistinguishable (fig. S6). Although epitope-specific memory B cell frequencies were slightly (factor of ~3) lower in the second experiment, potentially because of sorting with a different eOD-GT8 KO bait [eOD-GT8 KO2 (fig. S3)], comparison of frequencies between groups (Fig. 2C) supported the findings from the first experiment (Fig. 2B).

To determine whether or not the epitope-specific memory B cells generated by eOD-GT8 60mer immunization contained VRC01-class memory B cells, RNA from single eOD-GT8+/eOD-GT8 KO sorted memory B cells was reverse transcribed and IgG variable genes were amplified and sequenced by NGS (37). The two experiments yielded a total of 10,816 wells from which unique heavy- and/or light-chain nucleotide sequences could be determined, and of these, 3122 wells contained a heavy- and light-chain pair (2526 pairs for eOD-GT8 60mer-immunized mice, 537 pairs for control-immunized mice, and 59 pairs for unimmunized mice) (Fig. 3A and tables S2 to S4). From these sequences, we identified 28 nucleotide-unique (26 amino acid–unique) VRC01-class heavy-light paired memory responses among 29% (17 of 58) of eOD-GT8 60mer-immunized mice (aggregating across different doses, adjuvants, time points, types of mice, and the two experiments) (Fig. 3B, fig. S7, and table S5). In contrast, we detected no VRC01-class responses from 32 control mice. eOD-GT8 60mer–induced VRC01-class responses were found at approximately similar frequencies in HK, HL, and HKL mice (Fig. 3C).

Fig. 3 Analysis of antibody sequences from epitope-specific memory B cells.

(A) Summary of all sequence information obtained from experiments 1 and 2. Of the 33 VRC01-class pairs, 28 were identified as unique in nucleotide sequence. (B) Number of eOD-GT8 60mer-immunized or control mice from which at least one VRC01-class pair was isolated by B cell sorting (+) or no VRC01-class pairs were isolated (-). Data are aggregated from all animals and conditions in experiments 1 and 2, with a total of 90 mice. The P value was calculated by using Fisher’s exact test. (C) Number of eOD-GT8 60mer-immunized HK, HL, or HKL mice for which at least one VRC01-class pair was isolated (red, with percentages listed in white) or no VRC01-class pairs were isolated (gray). In (D) and (E), the same analysis was performed as in (B) and (C), respectively, but with five–amino acid L-CDR3 light chains instead of VRC01-class pairs. (F) L-CDR3 sequence logos for VRC01-class bnAbs (top row), eOD-GT8 60mer-induced VRC01-class paired antibodies (second row), eOD-GT8 60mer-induced five–amino acid L-CDR3s (third row), and naïve Kymab mice (bottom row), shown separately for kappa light chains (left column) and lambda light chains (right column). (G) L-CDR1 length distribution for eOD-GT8 60mer-induced VRC01-class antibodies (red) and all LCs in (A) (black). (H) Light chain Vκ and Vλ gene usage for eOD-GT8 60mer-induced VRC01-class antibodies. Red bars denote genes used by, or highly similar to those used by, known VRC01-class antibodies. (I) H-CDR3 length distribution for eOD-GT8 60mer-induced VRC01-class antibodies.

Whereas only 21% (28/136) of paired sequences with VH1-2*04 heavy chains had L-CDR3s of five amino acids, 88% (28/32) of paired sequences with a five–amino acid L-CDR3 included a VH1-2*04 heavy chain (Fig. 3A), suggesting that five–amino acid L-CDR3s in memory B cells may serve as a proxy for VRC01-like responses. Therefore, we examined the frequency of five–amino acid L-CDR3s among all paired and unpaired light-chain sequences. We identified 62 light chains with 5-amino acid L-CDR3s from 48% (28 out of 58) of eOD-GT8 60mer-immunized mice, whereas we found no five–amino acid L-CDR3s among control mice (Fig. 3, A and D). eOD-GT8 60mer induced memory B cells with five–amino acid L-CDR3s at substantial frequencies in all three types of mice (Fig. 3E). All combinations of dose, adjuvant, and time point produced VRC01-class responses and five–amino acid L-CDR3 responses (fig. S8). Given the very low frequency of VRC01-class precursors, these results indicate that VRC01-class priming by eOD-GT8 60mer was highly efficient and may have succeeded in all or most mice in which at least one precursor was present.

The VRC01-class antibodies induced by eOD-GT8 60mer shared other characteristic features of VRC01-class bnAbs in addition to the VH1-2 alleles and five–amino acid L-CDR3. The L-CDR3 is a key site of affinity maturation in VRC01-class bnAbs (32), and the 28 VRC01-class antibodies showed clear signs of selection toward bnAb sequences in both kappa and lambda L-CDR3 (Fig. 3F). In addition, 21 of 28 VRC01-class pairs had L-CDR1 lengths matching those of the germline VL genes of known VRC01-class bnAbs (Fig. 3G) (32). Further, 18 of these 28 antibodies used known VRC01-class VL genes (Fig. 3H), and the H-CDR3 lengths among the VRC01-class pairs (9 to 16 amino acids) were similar to those of known VRC01-class bnAbs (12 to 18 amino acids) (Fig. 3I).

Consistent with a VRC01-class binding mode, all 20 VRC01-class antibodies that we expressed bound to eOD-GT8 but had no detectable affinity for either eOD-GT8 mutant, eOD-GT8 KO, or eOD-GT8 KO2 (fig. S9). These VRC01-class antibodies had geometric mean (GM) affinity for eOD-GT8 of 134 nM [geometric standard deviation (GSD), 9.4], a factor of 25 higher than the geometric mean eOD-GT8 affinities of VRC01-class antibodies isolated from naïve human B cells (3.4 μM; GSD, 5.6) (27), possibly due to maturation of the immunogen-induced antibodies (mean ± SD mutation levels were 0.8 ± 1.1% in VH and 1.5 ± 1.1% in VL). eOD-GT8 60mer-induced VRC01-class antibodies in the VRC01 gH mouse had similar mutation levels (VH: 1.3 ± 3.2%; VL:2.1 ± 1.9%) but higher GM affinity for eOD-GT8 by a factor of 22 (GM, 6.0 nM; GSD, 38.7) (26), probably due at least in part to the engineering of eOD-GT8 for ultrahigh affinity (9 pM) to germline-reverted VRC01 (27). Thus, the Kymab antibody affinities are likely better predictors of the affinities of human responses to a single immunization of eOD-GT8 60mer.

Ultimate elicitation of bnAbs will probably require sequential boosting with more native-like epitope variants to select sufficient bnAb-like mutations (1, 4, 5, 10, 16, 17, 22, 23, 25, 26). Consistent with this expectation and with results of eOD-GT8 60mer priming experiments in knock-in mice (25, 26), the primed VRC01-class antibodies in this study showed no affinity for the native-like trimer BG505 SOSIP (fig. S10) and no neutralizing activity against the VRC01-sensitive HXB2 HIV strain from which eOD-GT8 was derived (fig. S11). That only ~1% (28/2526) of epitope-specific antibodies were VRC01-class (Fig. 3A) suggests that immunofocusing strategies may be needed to suppress competing responses during boosting (1, 45)—though in humans, the higher VRC01-class precursor frequency may mitigate this challenge.

Germline targeting is a promising vaccine strategy, but developing suitable model systems to evaluate targeting of human germline B cells is difficult. Kymab human immunoglobulin loci transgenic mice offer a more stringent and human-like model compared to most knock-in mice. Although Kymab mice underrepresent the frequency of VRC01-class precursor B cells compared to humans and possess an average of at most 1.3 such precursors per mouse, the eOD-GT8 60mer still proved capable of priming. The seemingly high targeting efficiency of eOD-GT8 60mer in this mouse model encourages human testing wherein the VRC01-class precursor frequency among B cells and the number of precursors per individual are more favorable for bnAb priming. The results of this study should also encourage germline targeting for other bnAbs with lower human precursor frequencies.

Supplementary Materials

Materials and Methods

Fig. S1 to S11

Tables S1 to S6

References (4650)

References and Notes

  1. Among human VH1-2 alleles, Kymab mice only contain VH1-2*04. This was misreported as *02 in (33).
  2. See supplementary materials on Science Online.
Acknowledgments: We thank P. Kellam for comments on the manuscript. This work was partially funded by IAVI with the generous support of the U.S. Agency for International Development, Ministry of Foreign Affairs of the Netherlands, and the Bill & Melinda Gates Foundation; a full list of IAVI donors is available at (W.R.S., D.R.B). This work was also supported by the Bill and Melinda Gates Foundation (G.A.F., A.B.); the Ragon Institute of MGH, MIT, and Harvard (D.R.B. and W.R.S.); the Helen Hay Whitney Foundation (J.G.J.); and National Institute of Allergy and Infectious Diseases grants P01 AI094419 (W.R.S.) and CHAVI-ID 1UM1AI100663 (W.R.S., D.R.B.). The data presented in this manuscript are tabulated in the main paper and in the supplementary materials. All mice were maintained, and all procedures carried out, under United Kingdom Home Office License 70/8718 and with the approval of the Sanger Institute Animal Welfare and Ethical Review Body. The VRC01-class paired sequences described in this study were deposited in GenBank under accession numbers KX814864 to KX814919. Materials and information concerning the immunogens are available by material transfer agreement from the Scripps Research Institute. IAVI and the Scripps Research Institute have filed a patent (U.S. PCT Application no. PCT/US2016/038162) relating to the eOD-GT8 immunogens in this manuscript, which included inventors J.G.J., D.W.K., S.M., and W.R.S. W.R.S. is a cofounder and stockholder in Compuvax Inc., which has programs in non-HIV vaccine design that might benefit indirectly from this research. D.R.B. is a paid consultant of IAVI, which may benefit from this research. The Kymab mouse strains described are corporate assets protected by multiple patents; access to these mice is available through licensing. A.W., E-C. L., A.B., and G.F. hold equity in Kymab, Ltd., and Kymab, Ltd., holds patents and patent applications related to the Kymouse technology and the use of that technology in vaccine characterization. A.B. and G.F. are officers of Kymab, Ltd., which may benefit from this research.
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