Research Article

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

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Science  10 Jul 2015:
Vol. 349, Issue 6244, pp. 156-161
DOI: 10.1126/science.aac5894

Steps in the right direction

HIV-1 mutates rapidly, making it difficult to design a vaccine that will protect people against all of the virus' iterations. A potential successful vaccine design might protect by eliciting broadly neutralizing antibodies (bNAbs), which target specific regions on HIV-1's trimeric envelope glycoprotein (Env) (see the Perspective by Mascola). Jardine et al. used mice engineered to express germline-reverted heavy chains of a particular bNAb and immunized them with an Env-based immunogen designed to bind to precursors of that bNAb. Sanders et al. compared rabbits and monkeys immunized with Env trimers that adopt a nativelike conformation. In both cases, immunized animals produced antibodies that shared similarities with bNAbs. Boosting these animals with other immunogens may drive these antibodies to further mutate into the longsought bNAbs. Chen et al. report that retaining the cytoplasmic domain of Env proteins may be important to attract bNAbs. Removing the cytoplasmic domain may distract the immune response and instead generate antibodies that target epitopes on Env that would not lead to protection.

Science, this issue p. 139, 10.1126/science.aac4223, p. 156; see also p. 191


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.

We lack an effective vaccine against HIV, despite its identification more than 30 years ago. An HIV vaccine most likely will need to elicit antibodies capable of neutralizing the majority of the diverse strains circulating in the population. A minority of HIV-infected individuals eventually develop such broadly neutralizing antibodies (bnAbs), but this generally occurs only after years of protracted viral and antibody coevolution (1, 2). Although they fail to control virus in the individuals themselves, passive transfer of recombinant forms of such bnAbs can prevent infection in animal models (38). Hence, there is an expectation that successful elicitation of bnAbs by vaccination before infection will be protective in humans, and developing such a bnAb-based vaccine is a major research goal.

The CD4 binding site (CD4bs) antibody VRC01 (9) and other VRC01-class bnAbs identified in at least seven different donors represent a response with distinguishing features that might be amenable to reproducible vaccine elicitation (1015). In particular, VRC01-class bnAbs share a mode of binding that uses the immunoglobulin heavy (H) chain variable (V) gene segment VH1-2*02 to mimic CD4, in contrast to many antibodies that rely on the CDRH3 (complementarity-determining region 3 of the H chain) loop (10, 14, 16). The VH1-2*02 gene or suitable alternative alleles are present in ~96% of humans (17), and these genes are employed frequently, in ~3% of all human antibodies (18, 19), suggesting that the B cell precursors for a VRC01-class response are generally available for vaccine targeting.

However, several key challenges must be met to induce VRC01-class bnAbs. First, as is true for some but not all classes of HIV bnAbs, the predicted germline precursors of VRC01-class bnAbs lack detectable affinity for native HIV envelope glycoproteins (Env) (10, 12, 17, 2022). To address this problem, we and others have designed germline-targeting immunogens capable of binding and activating VRC01-class precursor B cells in vitro (17, 21). Whether these immunogens can activate precursors in vivo is an open question. Second, VRC01-class bnAbs carry light (L) chains with unusually short CDRL3s (complementarity-determining region 3 of the L chain) composed of five amino acid residues, typically within a CQQYEFF (23) motif (14, 16). The short CDRL3 length is required to avoid clashing with gp120 Env loop D and V5, and amino acids within this motif make specific interactions to stabilize the antibody and to contact gp120 (10, 14, 16). CDRL3s with this length occur in only 0.6 to 1% of human κ antibodies (figs. S1 and S2) (14, 16) and 0.1% of mouse κ antibodies (fig. S2), and the specific amino acid requirements described above will further reduce the frequency of useful L chains. Therefore, a germline-targeting immunogen must be capable of activating relatively rare VRC01-class precursors in the repertoire. Third, VRC01-class bnAbs, like most other HIV bnAbs, are heavily somatically mutated, as a result of chronic stimulation of B cells by successive HIV variants (9, 11, 12, 24). Although engineering approaches can be used to develop less mutated bnAbs (25, 26), it remains clear that vaccine induction of bnAbs will require strategies to induce relatively high mutation levels. This will most likely be achieved by a sequence of different immunogens that successively returns B cells to germinal centers to undergo repeated rounds of affinity maturation (1, 10, 11, 17, 21, 2730). In this view, each immunogen in the sequence, while naturally inducing antibodies of increasing affinity to itself, must induce maturation in memory B cells that enables weak binding to the next immunogen in the sequence. This challenge is particularly acute for the priming step: The germline-targeting prime must not only activate VRC01-class precursors, it must also induce mutations that enable binding to more native-like boost immunogens that have no detectable affinity for the precursors.

To assess the feasibility of meeting the above challenges with a germline-targeting prime, we constructed a knock-in mouse in which the germline-reverted H chain of VRC01 pairs with native mouse L chains, and we conducted immunization experiments in this mouse with an improved version [eOD-GT8 60-subunit self-assembling nanoparticle (60mer)] of a previously described germline-targeting immunogen (17) (see supplementary materials and methods). Responses were interrogated by enzyme-linked immunosorbent assay (ELISA); hybridoma generation; and, most importantly, by antigen-specific B cell sorting to define the pool of memory B cells induced by the immunogens.

VRC01 gH knock-in mice

The true germline precursor is not known for VRC01 or other VRC01-class bnAbs (31). In the knock-in mouse, we approximated the true H chain precursor with a VRC01 germline-reverted H chain (VRC01 gH) composed of the VH1-2*02 and IGHJ1*01 genes assigned by JoinSolver (32) and supported by recent longitudinal analysis of the VRC01 lineage (31), along with the CDRH3 from VRC01 with a single mutation to remove an unpaired cysteine (fig. S3). Though our use of the VRC01 CDRH3 in VRC01 gH (necessary because the germline D gene and V-D and D-J junctions cannot be inferred with confidence) is likely a departure from the (unknown) true germline precursor, the VRC01 CDRH3 plays a relatively minor role in epitope recognition, accounting for only 13.7% of the area buried on the H chain in the VRC01 interaction with gp120 (10) or 10.2% of the area buried on germline-reverted VRC01 in its interaction with eOD-GT6 (17). Furthermore, the CDRH3 in VRC01 is disulfide-bonded to an affinity-matured cysteine in CDRH1, which may serve to stabilize the antibody conformation and increase affinity for gp120; however, this disulfide is not included in VRC01 gH. Thus, the use of this CDRH3 is unlikely to strongly bias the VRC01 gH mouse toward favorable interactions with gp120- or eOD-based immunogens, and we believe the VRC01 gH sequence is a reasonable approximation for the true germ line, for the purpose of evaluating germline-targeting immunogens.

Testing for the ability to stimulate VRC01-class precursor B cells could not be carried out directly in wild-type (WT) mice or other small animals, as none are known to have a VH gene with sufficient similarity to the human VH1-02 germline gene (16, 17). To overcome this limitation, we engineered mice to express a VRC01 gH-chain exon under the control of a mouse VH promoter, introduced by gene targeting into the Igh locus (fig. S4). This targeting to the physiological locus allows normal regulation of H-chain expression, antibody class switching, and somatic mutation. These VRC01 gH mice have similar frequencies of CD19+/B220+ B cells as WT littermates (siblings of knock-in mice that lack the knock-in gene by random chance in breeding male heterozygous knock-in mice with WT females) (Fig. 1A). By next-generation sequencing, the VRC01 gH-chain gene was expressed by ~80% of B cells (Fig. 1B and figs. S5 and S6) and was paired with random mouse L chains generated in the course of normal B cell development (fig. S7). The L chains have similar V gene usage and CDRL3 length distributions as those of WT littermates (fig. S8). Thus, VRC01 gH mice carry germline-reverted bnAb precursor B cells at a frequency appropriate for testing of germline-targeting VRC01-class immunogens, including the eOD-GT8 60mer.

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 with 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 postimmunization. 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 days 5, 10, or 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, or Ribi), along with the number of mice used to test each group. All groups were tested in both VRC01 gH and WT mice, except for BG505 SOSIP, which was tested only in VRC01 gH mice. Five 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).

Analysis of antibody responses to different priming immunogens

VRC01 gH mice were immunized with a single injection of eOD-GT8 60mer, a self-assembling nanoparticle composed of an engineered outer domain from HIV gp120 fused to a lumazine synthase protein (fig. S9). To assess whether VRC01-like germline precursors were primed, we followed antibody responses and sequenced antibody genes of eOD-GT8–reactive B cells that were captured as hybridomas or by cell sorting of eOD-GT8–binding immunoglobulin G (IgG) B cells (Fig. 1C and fig. S10). To investigate the effect of multimeric state, we compared responses to 60-subunit nanoparticles (eOD-GT8 60mers) and trimers (eOD-GT8 3mers). To probe for adjuvant effects, antigens were delivered in three different adjuvants: alum, Iscomatrix [“Isco,” 40-nm–diameter cagelike structures composed of phosopholipids, cholesterol, and saponin that traffic to lymph nodes and can heighten both antibody and T cell responses but contain no known Toll-like receptor (TLR) agonist activity (33)], or Sigma Adjuvant system (“Ribi,” an oil-in-water emulsion containing synthetic trehalose dicorynomycolate and the TLR4 agonist monophosphoryl lipid A). The alum and Ribi immunizations were given by intraperitoneal injection, and the Isco immunizations were delivered subcutaneously per the manufacturer’s recommendations (Fig. 1D). To evaluate whether immunogens bearing an unmodified CD4bs could activate VRC01-like precursors, we tested responses to both the native-like trimer BG505 SOSIP.664 (3437) and eOD17 60mers, nanoparticles presenting a native-like and non–germline-targeting CD4bs on an eOD protein similar to eOD-Base (17), with all glycosylation sites intact.

The eOD-GT8 60mer challenge elicited a CD4bs response in VRC01 gH mice, as their immune serum IgG bound more strongly to eOD-GT8 than to eOD-GT8-KO, a mutant designed to block germline VRC01 binding [D368→R368 (D368R), N279A and mutations to restore the N276 glycosylation site] (Fig. 2A and fig. S11). The IgG response of WT mice, in contrast, was mainly to non-CD4bs epitopes. eOD-GT8 immunogens given in all three adjuvants supported a serum IgG response to CD4bs, though eOD-GT8 60mers were stronger than eOD-GT8 3mers, as assessed by an ELISA area-under-the-curve analysis (i.e., area under the eOD-GT8 reactivity curve minus area under the eOD-GT8-KO curve) (Fig. 2B) and by frequencies of IgG+ memory phenotype B cells that bound eOD-GT8 but not eOD-GT8-KO [eOD-GT8(+)/eOD-GT8-KO(–)] identified by cell sorting (Fig. 2C). eOD-GT8 60mers induced lower frequencies of (non-CD4bs) IgG+ memory phenotype B cells that bound both eOD-GT8 and eOD-GT8-KO [eOD-GT8(+)/eOD-GT8-KO(+)], suggestive of an epitope-specific response (fig. S12). Both BG505 SOSIP.664 trimer and eOD17 60mers elicited weak responses by ELISA and antigen-specific B cell frequencies (Fig. 2, B and C).

Fig. 2 Serum and B cell analysis of antibody responses after 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 ELISA. Sera were titrated for binding to monomers of eOD-GT8 or eOD-GT8-KO. Plotted values represent the mean of OD450 measurements from three different mice for the indicated serum time point (day 28, top; day 42, bottom) at the listed dilutions. Error bars indicate 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 days 28 and 42 sera. Mean and SD (error bars) for three 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 SD (day 42, n = 3 mice), are indicated by bars.

Selection of L-chain partners by the priming immunogen

Priming of the VRC01-class response was revealed in the sequencing data from sorted B cells and hybridomas. B cell sorting recovered 177 IgG H-L paired sequences from days 14 and 42, 167 of which used the VRC01 knock-in H chain (some were unmutated and others had mutations in either or both of the H or L chains, as discussed below). Additionally, 95 [IgG or immunoglobulin M (IgM)] hybridomas were recovered, all of which used the knock-in H chain. Among IgG B cells, this H chain was paired with κ L-chain partners of highly restricted CDRL3 length and Vκ gene usage (Fig. 3). Ninety-two percent (154 of 167) of eOD-GT8(+)/eOD-GT8-KO(–)–sorted IgG B cells using the VRC01 gH had L chains with a CDRL3 length of five amino acids (Fig. 3A and tables S1 and S2), whereas only ~0.1% of naïve (nonimmunized) VRC01 gH B cells or WT mouse B cells had a κ L-chain CDRL3 length of five amino acids (fig. S2). None of the 10 sorted B cells that used an endogenous mouse VH gene contained a five–amino acid CDRL3. Among IgG hybridomas, which were captured as early as day 5 of the response, six of seven hybridomas carried a κ L chain with a five–amino acid CDRL3, each isolated from a different mouse (tables S3 to S6). In contrast, among 88 IgM hybridomas recovered after eOD-GT8 60mer immunization [for which eOD-GT8 affinity was weaker than 100 μM for all but 2 hybridomas, according to surface plasmon resonance (SPR)], only 1 had this CDRL3 signature. This suggests that the initial selection for the unusual CDRL3 length occurred upon class switching. Priming was reproducible, as IgG B cells with five–amino acid CDRL3s were isolated from 20 of 22 mice immunized with eOD-GT8 60mer (14 of 15 mice analyzed by sorting and 6 of 7 mice that produced IgG hybridomas) (table S1). eOD-GT8–binding IgGs preferentially used Vκ genes with a QQY motif at the start of CDRL3 common to mature VRC01-class bnAbs (CQQYEFF) (Fig. 3, B and C, and fig. S13). In contrast, IgM hybridomas used a broad distribution of Vκ and Vλs (table S6), again indicating selection at the class-switch stage. In summary, eOD-GT8 60mer immunization successfully recruited VRC01-like precursors into the T cell–dependent response and promoted the selective IgG class switching of cells carrying desirable L-chain features.

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

(A) Mouse L chains from sorted antigen-specific IgG+ memory phenotype B cells (red) and from hybridomas (blue), as well as mouse L chains from IgM+ antigen-specific hybridomas (orange), were sequenced to identify CDRL3 lengths and mutations from germline mouse κ chains. The distribution of CDRL3 lengths is shown in a histogram compared with known VRC01-class antibodies (black) and the naïve (unimmunized) 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 time points and all adjuvant groups listed in Fig. 1). n, number of L-chain sequences from individual sorted cells, hybridomas, or deep sequencing reads, as indicated. (B) Gene usage is shown for all Vκ genes in antibodies using the VRC01 gH chain and a five–amino acid 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 five–amino acid 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 1653 sequences with five–amino acid CDRL3 found by deep sequencing of four 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 five–amino acid CDRL3, isolated from VRC01 gH mice at day 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) (table S7).

Somatic mutation patterns

A bnAb priming immunogen must not only expand precursor numbers but also promote somatic mutations that allow binding to boosting antigens with closer similarity to HIV Env. We found many somatic mutations among IgG memory phenotype B cells responding to eOD-GT8 60mers and containing a five–amino acid CDRL3, including some L-chain mutations shared with mature VRC01-class antibodies (Fig. 3C). On the L chain, many sequences isolated at day 42 of the response to eOD-GT8 60mer/Ribi achieved a D or E in the VRC01 CQQYEF sequence motif. Sixteen of 47 analyzed IgG memory phenotype B cells had a T-to-G nucleotide mutation in their five–amino acid CDRL3s to introduce a D at position 4 (fig. S14), and several cells had an E at that position.

As the VRC01 gH-chain sequence was known, H-chain mutations were readily identified and could be compared directly to mature VRC01 to identify favorable mutations. To focus exclusively on VRC01-class antibodies, our H chain analysis included only the VH region of Abs that derived from the VRC01 gH chain and contained a five–amino acid CDRL3. Nearly all VRC01-class Abs from day 14 were unmutated. By day 42, however, 53 of 98 VRC01-class Abs contained at least one coding mutation from the starting H-chain sequence (table S7). Among all VRC01-class Abs from days 14 and 42 with at least one coding mutation on the H chain, 55 of 61 contained at least one mutation that is identical to VRC01 (fig. S15), and ≥50% of the mutations in 49 of 61 such Abs were identical to those in one of six VRC01-class bnAbs (12a21, 3BNC60, PGV04, PGV20, VRC-CH31, or VRC01) (Fig. 4A and table S7). In one case, all six coding mutations were identical to mutations found in VRC01-class bnAbs. One particular mutation (H35N) was found in >80% of B cells that had at least one mutation, including cells from 12 different mice and all adjuvant groups (table S2) and including both sorted IgG cells and hybridomas. Examination of the eOD-GT6/GL-VRC01 complex structure [Protein Data Bank identification number (PDB ID): 4jpk] and the gp120/VRC01 complex structure (PDB ID: 3ngb) revealed that the H35N mutation enables a favorable hydrogen bonding interaction with an asparagine on CDRH3 (fig. S16). We also noted differences in mutation levels in different adjuvant groups: Among the day 42 sequences, the percentages of Abs with at least one H-chain coding mutation were 44% (8/18) for alum-immunized mice, 19% (3/16) for Isco-immunized mice, and 67% (42/63) for Ribi-immunized mice (Fig. 4B). Overall, the strong selection of mutations is suggestive of a VRC01-class response, with many mutations identical to those in VRC01-class bnAbs that may help primed cells become cross-reactive to more native-like gp120 molecules. Thus, priming with the eOD-GT8 60mer selected antibody features predicted to improve binding to the CD4bs of Env.

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

(A) A total of 61 mutated H-chain (HC) 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 H-chain amino acid mutations from the germ line. Each circle represents a single H-chain sequence that was isolated by antigen-specific memory phenotype B cell sorting. (B) The total number of amino acid mutations observed in the H chains of antibodies isolated by antigen-specific memory phenotype B cell sorting is listed by adjuvant (Alum, Isco, or Ribi) for spleen and lymph node samples harvested at 14 or 42 days postpriming immunization. Bar graphs are divided by unmutated (white) versus mutated (colored). The mutated bars are divided into Abs with one or two coding mutations (red), three or four coding mutations (blue), or five or six 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.

Antibody affinity for the germline-targeting prime and candidate boost immunogens

T cell–dependent immune responses promote somatic hypermutation and selection for B cells with improved affinity for immunogens, but an additional requirement for an effective bnAb HIV priming immunogen is to promote enhanced affinity for the presumed HIV boosting antigen(s). To assess this aspect of the efficacy of eOD-GT8 60mer priming, we expressed 115 H-L paired sequences that used the VRC01 knock-in H chain and contained a five–amino acid CDRL3 from eOD-GT8(+)/eOD-GT8-KO(–) IgG memory phenotype–sorted B cells (table S7). We then evaluated their binding to eOD-GT8, eOD-GT8-KO, and candidate boosting antigens by SPR. Of the 115 Abs, 72 contained no H- or L-chain mutations from the germ line. These unmutated Abs bound eOD-GT8 with a median dissociation constant (KD) of 32 nM (Fig. 5, A and B, and table S8). Few mutations were required to promote high affinity—most Abs with more than three coding mutations had an affinity too high to measure accurately (KD < 16 pM) (Fig. 5A). Confirming epitope specificity, antibodies for which we could measure a KD for eOD-GT8 showed reduced affinity for eOD-GT8-KO by factors of 36 to 200. We observed intriguing differences among adjuvant groups, with Ribi-immunized mice producing both more Abs (recoverable by sorting) and higher-affinity Abs compared with alum or Isco.

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 the analyte. Data are shown for 42 antibodies from day 14 and 73 antibodies from day 42 after 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 (10 μM) measureable based on the analyte concentration used in the experiment. GL, germ line. (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 the highest affinity for eOD-GT8 (KD < 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 KDs 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 squares; unmutated antibodies are shown as black diamonds.

The eOD-GT8 60mer was designed both to prime germline VRC01-class precursors and to select for mutations that confer cross-reactivity to more native-like gp120 (17). To test whether the latter was effective, we selected the 29 Abs that bound eOD-GT8 with subnanomolar affinity, as well as eight unmutated variants (with average KDs for eOD-GT8), and screened them for binding to more native-like gp120 constructs in both monomer and 60mer form. The Abs with more than three coding mutations not only had improved affinity for eOD-GT8 but in many cases showed affinity for core-e-2CC HxB2 N276D, a conformationally stabilized core gp120 monomer with a near-native CD4bs from strain HxB2 that combines the loop and termini trimming of the “coreE” design (10, 14) with the disulfides and space-fill mutations of the “2CC” design (38) but also lacks the N276 glycan. In total, 23 of 29 Abs that bound with high affinity to eOD-GT8 showed detectable binding to core-e-2CC HxB2 N276D (KD < 100 μM), whereas none of the unmutated Abs did (Fig. 5B and table S8). 60mer nanoparticles of core-e-2CC HxB2 N276D bound to 24 of 29 mutated Abs more strongly than to the monomer by a factor of ~100 due to avidity, but the 60mers also showed no binding to the unmutated Abs. We conclude that priming with eOD-GT8 60mers promotes clonal expansion and facilitates recognition of molecules presenting a near-native CD4bs.

Discussion: The priming problem

A vital goal of rational vaccine design is to understand how to prime naturally subdominant antibody responses in a reproducible manner. Germline-targeting offers one potential strategy to achieve this goal. Here we have used a germline-reverted VRC01 H-chain knock-in mouse model to demonstrate that a germline-targeting immunogen (eOD-GT8 60mer) can activate relatively rare VRC01-class precursors, select productive mutations, and create a pool of memory phenotype B cells that are likely to be susceptible to boosting by more native-like immunogens. In contrast, we found that immunogens bearing a native-like CD4 binding site, including both the eOD17 60mer and the well-ordered BG505 SOSIP.D664 trimer, failed to achieve these goals. These results illustrate the value of an engineered priming immunogen to initiate the development of bnAb lineages by vaccination.

The data in the VRC01 gH mouse model described here have strong potential relevance to human vaccination. Given that (i) VH1-2*02 is expressed in ~80% of B cells in this mouse compared with ~3% of human B cells (18, 19); (ii) the frequency of five–amino acid CDRL3 L chains is 0.1% in VRC01 gH B cells compared with 0.6 to 1% in humans (figs. S1 and S2) (16); and (iii) the CDRH3 requirements are modest for VRC01-class bnAbs (10, 14, 16, 39) and appear to be minimal for VRC01-class precursors, perhaps requiring a length of 11 to 18 amino acids [75% of human Abs (fig. S17)], it is possible that VRC01-class precursors are less frequent in humans compared with the VRC01 gH mouse by a factor of only ~5 (= 80/3 × 0.1/0.6 × 1/0.75). Even if this estimate is off by an order of magnitude or two due to unknown factors, it is also true that humans have orders of magnitude more B cells than mice, hence more potential targets. Therefore, we believe that this study provides strong support for the idea of human clinical testing of the eOD-GT8 60mer, to assess whether this germline-targeting prime can perform similarly in diverse humans. Moreover, the differences observed with different adjuvants in this mouse model—in serum titers, B cell frequencies, selection of favorable mutations, and generation of high-affinity Abs—indicate that testing different adjuvants should be considered in the design of human clinical experiments probing activation of specific classes of precursor B cells.

Having demonstrated that eOD-GT8 60mer immunization initiates a VRC01-class response in this mouse model, several additional developments are probably needed to induce broad neutralizing activity. The eOD-GT8 60mer contains a modified CD4bs to confer germline reactivity and, as such, is probably not capable of selecting all of the H- and L-chain mutations required for bnAb activity against the native CD4bs. Indeed, no neutralizing activity was detected for any of the 8 eOD-GT8 60mer–induced Abs [all with high affinity (KD < 1 nM) for eOD-GT8 and low affinity (1 μM < KD < 100 μM) for core-e-2CC HxB2 N276D] that we tested against a panel of four viruses from clades A and B that included both WT and N276A mutant viruses with increased sensitivity to VRC01-class bnAbs (fig. S18). One design feature of eOD-GT8 is that it lacks the N276 glycan; removal of this glycan is a requirement for germline reactivity (17, 21). However, the N276 glycosylation site is conserved in 94.5% of HIV strains, according to an analysis of 3796 sequences from the Los Alamos HIV database ( Induction of broad neutralization will probably require one or more boosting immunogens bearing a glycan at N276 so as to select mutations to accommodate that glycan (17). On the H chain of VRC01-class bnAbs, mutations in the CDR2, CDR1, FW1, and FW3 are likely required for maximum potency and breadth (24, 40), and native-like Env immunogens will probably be needed to select for these. In sum, boosting with a sequence of increasingly native-like antigens, and potentially including cocktails of different antigens within each boost to mimic the antigenic diversity of the CD4bs, will likely be needed to select the mutations required for VRC01-class bnAb activity. The mouse model presented here, as well as other newly developed VRC01-class knock-in mouse models (41), should aid us to test this notion and can be used to identify the antigens and boosting strategies that work best. Of note, we demonstrated here that a single immunization with the eOD-GT8 60mer induces VRC01-class antibodies with modest affinity for the core-e-2CC HxB2 N276D monomer and 60mer, so these molecules represent promising candidates for the first boost. We are thus mapping the first steps in a sequential strategy for the rational induction of bnAbs against HIV.

Supplementary Materials

Materials and Methods

Figs. S1 to S18

Tables S1 to S8

References (4253)

References and Notes

  1. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  2. Acknowledgments: We thank T. R. Blane, S. Kupriyanov, and G. S. Martin for technical assistance. The data presented in this manuscript are tabulated in the main paper and in the supplementary materials. This work was supported by the IAVI Neutralizing Antibody Consortium and Center (W.R.S. and D.R.B.); the Collaboration for AIDS Vaccine Discovery funding for the IAVI NAC (W.R.S. and D.R.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 R01-AI073148 (D.N.), P01AI081625 (W.R.S.), and CHAVI-ID 1UM1AI100663 (W.R.S. and D.R.B.). IAVI and the Scripps Research Institute are filing a patent relating to the eOD-GT8 immunogens in this manuscript, with inventors J.G.J., D.W.K., S.M., and W.R.S. Materials and information will be provided under a material transfer agreement.
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