Abstract
In the RV144 gp120 HIV vaccine trial, decreased transmission risk was correlated with Abs that reacted with a linear epitope at a lysine residue at position 169 (K169) in the HIV-1 envelope (Env) V2 region. The K169 V2 response was restricted to Abs bearing Vλ rearrangements that expressed aspartic acid/glutamic acid in CDR L2. The AE.A244 gp120 in AIDSVAX B/E also bound to the unmutated ancestor of a V2-glycan broadly neutralizing Ab, but this Ab type was not induced in the RV144 trial. In this study, we sought to determine whether immunodominance of the V2 linear epitope could be overcome in the absence of human Vλ rearrangements. We immunized IgH- and Igκ-humanized mice with the AE.A244 gp120 Env. In these mice, the V2 Ab response was focused on a linear epitope that did not include K169. V2 Abs were isolated that used the same human VH gene segment as an RV144 V2 Ab but paired with a mouse λ L chain. Structural characterization of one of these V2 Abs revealed how the linear V2 epitope could be engaged, despite the lack of aspartic acid/glutamic acid encoded in the mouse repertoire. Thus, despite the absence of the human Vλ locus in these humanized mice, the dominance of Vλ pairing with human VH for HIV-1 Env V2 recognition resulted in human VH pairing with mouse λ L chains instead of allowing otherwise subdominant V2-glycan broadly neutralizing Abs to develop.
Introduction
The RV144 ALVAC/AIDSVAX B/E gp120 vaccine trial demonstrated an estimated 31% efficacy (1), and epidemiologic data indicated that efficacy was highest when the envelope (Env) of infecting virus matched the vaccine Env at lysine residue at position 169 (K169) in the V2 region of gp120 (2). Additionally Env V2-reactive Abs were shown to be a correlate of reduced transmission risk in RV144 vaccinees (3). The importance of Abs that recognize the V2 epitope at K169 was further underscored when four Abs isolated from RV144 subjects that recognized the K169 V2 determinant were shown to mediate killing of CD4+ T cells infected by primary isolate HIV-1 strains by Ab-dependent cell-mediated cytotoxicity (4). Despite the use of two Vλ gene segments (Vλ3–10 and Vλ6–57), all four RV144-derived Abs contained a germline glutamic acid–aspartic acid (ED) motif in their respective L chain second CDRs (CDR L2). The crystal structure of two of these Abs, CH58 and CH59, in complex with V2 peptides revealed that the ED motif formed stabilizing salt bridges with two lysine residues in the V2 loop, including with K169 (4).
Recognition at K169 by Abs with the CDR L2 ED motif was also a hallmark of the HIV-1 Env V2 response elicited by three independent rhesus macaque HIV-1–immunization regimens, including two regimens that used RV144 immunogens (5). Rhesus macaque Abs targeted to the V2 K169 determinant predominantly used (66%) L chains containing the macaque Vλ gene segment orthologous to human Vλ3–10; this ortholog is the only VL gene in rhesus that contains a CDR L2 ED motif (5). We concluded that the phylogenetic conservation of V gene segments that contain the Vλ3–10-like CDR L2 ED motif implies a fitness advantage in pathogen recognition by the primate adaptive immune system (5). That the CDR L2 ED motif was independently used by V2 K169 Abs in multiple subjects, different species, and following distinct immunization protocols strongly implies that V2 K169 recognition is limited by a restricted set of paratopic structural solutions (4, 5).
Another group of Abs that bind to V2 at K169 are the V2-glycan broadly neutralizing Abs (bnAbs) (6, 7); these bnAbs bind an epitope on the Env that includes glycans at the N156 and N160 positions, as well as the V2 polypeptide chain (7, 8). V2-glycan Abs arise during infection but, to date, have not been induced by vaccination (9, 10). Induction of a V2-glycan bnAb is a preferred vaccine response, because bnAbs were shown to be protective in nonhuman primate infection models (11). A component in the RV144 vaccine, AE.A244 gp120, also expressed an epitope bound by mature V2-glycan bnAbs and the V2-glycan bnAb CH01 germline unmutated ancestor (4). Thus, in the RV144 vaccine trial, despite the vaccine immunogen expressing two types of V2 epitopes that involve K169, one for a linear Ab-dependent cell-mediated cytotoxicity epitope and one for a bnAb V2-glycan epitope, only the linear V2 peptide Ab response was dominant.
In this study, we investigated whether the immunodominance of the nonneutralizing linear V2 epitope would diminish in the absence of Vλ gene segments carrying the CDR L2 ED motif. Is the HIV-1 Env V2 K169 determinant intrinsically immunodominant or is the high frequency of Ab responses to this determinant controlled by the Ab repertoire? To address this question, we immunized humanized VelocImmune mice with the RV144 immunogens (1). VelocImmune mice carry the complete human IgH and Igκ variable loci in place of the endogenous IgH and Igκ variable loci, respectively (12, 13). These humanized animals retain the endogenous Igλ loci; however, in contrast to humans and macaques, none of the mouse Vλ gene segments have a CDR L2 with the ED motif (14). Consequently, although these mice primarily (≥90%) use human Igκ VJ rearrangements to produce BCRs and Ab responses, they cannot produce the germline ED motif BCRs that dominate human and macaque Ab responses to the V2 K169 epitope (4, 5). Remarkably, we found that, although VelocImmune mice lacked Vλ gene segments with the CDR L2 ED motif, they mounted robust humoral responses against the HIV-1 Env V2 that were dominated by chimeric Abs composed of human VDJ H chain rearrangements paired with mouse λ L chain rearrangements. However, K169 was not included in the epitope of these V2 Abs, which was consistent with our previous findings that the ED motif is necessary for precise recognition of K169. We conclude that the immunodominance of linear V2 epitopes is largely due to intrinsic factors and that the promotion of rare Ab responses to the V2-glycan bnAb epitope cannot be achieved simply by modulation of the available Ig L chain repertoire.
Materials and Methods
Immunization of humanized mice
Three Regeneron VelocImmune mice were immunized i.p. with 25 μg of AE.A244 gp120Δ11 (15) and 80 μl of the adjuvant STR8S-C nine times at weeks 0, 2, 5, 7, 9, 11, 14, 16, and 18. Mouse RE503 was fusion boosted at week 30, mouse RE504 was fusion boosted at week 40, and mouse RE505 was immunized a tenth time at week 40 and then fusion boosted at week 57. The STR8S-C adjuvant was formulated as previously described (16), with concentrations of TLR agonists modified for mouse (0.5 mg/ml of R848, 0.2 mg/ml of CpG oligodeoxynucleotides).
Mice were housed in the Duke University vivarium in a pathogen-free environment with 12-h light/dark cycles at 20–25°C. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use committee at Duke University Medical Center under the National Institutes of Health/Public Health Service Policy on Humane Care and Use of Laboratory Animals.
Ab isolation
Mouse serum was screened for binding to AE.A244 gp120Δ11 (15) and A244 V1V2 tags (4). Then B cell hybridomas were generated according to methods described previously (17). Two mice, RE504 and RE505, yielded successful hybridoma fusions, whereas a third mouse (RE503) did not because of technical issues. Hybridoma supernatants were then assayed for AE.A244 gp120Δ11, AE.A244 V1V2 tags binding, and neutralization to virus isolates AE.92TH023 or AE.CM244. A total of 8.57 × 107 splenocytes was harvested from mouse RE504. Sixteen 96-well plates (with 80 wells containing cells) with ∼66,000 splenocytes per well were screened. One hundred and thirty-two wells were positive for A244 gp120Δ11 and/or AE.A244 V1V2 tags binding and/or neutralization. After secondary screening to confirm reactivity, 57 wells were positive. A representative set of wells was selected based on highest reactivity to put into limiting dilutions to confirm activity from a single clone. The resulting five clones were purified according to methods described previously (17) and yielded Abs RE504-46, RE504-60, RE504-97, RE504-117, and RE504-125. A total of 6.27 × 107 splenocytes was harvested from mouse RE505. Eleven 96-well plates (with 80 wells containing cells) with ∼71,000 splenocytes per well were screened. Seventy-five wells were positive for A244 gp120Δ11 and/or AE.A244 V1V2 tags binding and/or neutralization. A representative set of wells was selected based on reactivity, and secondary screening for binding was performed on this selected set to confirm reactivity. Those clones that repeated were put into limiting dilutions. The resulting seven clones were purified and yielded Abs RE505-11, RE505-22, RE505-58, RE505-70, RE505-23, RE505-27, and RE505-33.
ELISA binding and epitope mapping
ELISA binding assays were performed as previously described (18). V2 Ab epitopes were mapped by alanine scanning mutagenesis of the A244 V2 peptide (LRDKKQKVHALFYKLDIVPIED). Amino acid positions were included in the epitope if the log of the area under the binding curve (LogAUC) of the alanine-mutated V2 peptide was reduced to <25% of the LogAUC value of the binding to the wild-type V2 peptide.
Biolayer interferometry
Biolayer interferometry measurements were made using the ForteBio OctetRed 96 instrument and streptavidin sensors. Data analysis was performed using ForteBio evaluation software. The peptide sensors were prepared by incubating streptavidin sensor tips in biotinylated V2 peptide, AE.A244 171 (LRDKKQKVHALFYKLDIVPIED), at 5 μg/ml for 5 min. The sensors tips were washed in PBS for 60 s prior to obtaining a baseline. Affinity measurements were made by dipping the peptide sensors in RE505Fab and CH59Fab, ranging in concentration from 0.5 to 10 μg/ml, for 600 s with a subsequent dissociation length of 600 s. A non-HIV Env-specific Fab 7968 was used as a negative control to subtract out nonspecific binding to peptide sensors. Using ForteBio evaluation software, subtracted binding curves for each Fab were fit using a 1:1 Langmuir model to obtain the association and dissociation rate constants, ka and kd, and the equilibrium dissociation constant, KD.
Neutralization
Neutralization activity of Abs was measured in the TZM-bl cell-based neutralization assay, as described previously (19).
Peptide microarray
Peptide microarray analysis was performed with modifications from a previously reported protocol using a Tecan HS4000 Hybridization Workstation (20, 21). Peptide libraries consisting of 15-mers overlapped by 12 aa were printed onto glass slides, covering the full length of consensus gp160 Env from clades A, B, C, D, CRF01_AE, and CRF02_AG. Sequences for all peptides in the library were published previously (21, 22). mAbs were tested at 40 μg/ml, with the exception of Abs 505-11 and 505-22, which were tested at 60 μg/ml. The signal intensity of each spot was defined as the median 645-nm foreground measurement after spot background subtraction. Spot background is defined as the belt located 3× the diameter around each spot. The positivity criterion for epitope binding is more than two peptides in the epitope bind with intensities higher than 99th percentile binding intensity of all 2058 peptides in the array library. mAbs that only bound peptide regions known to have higher nonspecific binding with intensities at the top 99th percentile were defined as negative for linear epitope binding.
Protein production
RE505-22 Fab was recombinantly produced as previously described for other anti-HIV Abs (4, 5, 23, 24). Briefly, chains were generated by PCR, using L and H chain genes as templates with appropriate primer pairs, and cloned into pcDNA3.1/hygro (25). Recombinant Fabs were produced in 293T cells by transient cotransfection with H chain and L chain gene plasmids. Recombinantly expressed Fab was captured with CaptureSelect Fab λ affinity matrix (BAC) using 10 mM sodium phosphate (pH 7.2), 150 mM NaCl, 0.05% azide as the loading buffer and 100 mM glycine (pH 2.4), 150 mM NaCl, 0.05% azide as the eluting buffer. RE505-22 Fab was further purified via size exclusion chromatography on a Superdex 200 pg 26/60 column (GE Life Sciences) in a buffer of 10 mM HEPES (pH 7.2), 50 mM NaCl, 0.02% azide. Peak Fab-containing fractions were pooled, concentrated, and buffer exchanged as necessary.
Crystallography
Purified RE505-22 Fab was brought to a concentration of 12.9 mg/ml in double distilled H2O. A gp120164–182 peptide bearing an acetylated N terminus and amidated C terminus was dissolved in double distilled H2O to 100 mg/ml. Fab and peptide were mixed at a 1:3 molar ratio and diluted to a total protein concentration of 14.2 mg/ml. Crystals were observed over a reservoir of 0.2 M NaF, 20% PEG 3350 at a temperature of 20°C. Crystals were cryoprotected by soaking briefly in mother liquor supplemented with 10% ethylene glycol and then cryocooled in liquid nitrogen. Diffraction data were collected at the Southeast Regional Collaborative Access Team beamline at the Advanced Photon Source (Argonne, IL), with an incident beam of 1 Å in wavelength. The data were reduced in HKL-2000 (26). Matthews analysis suggested two Fab molecules in the asymmetric unit (27). The structure was phased by molecular replacement in PHENIX (28) using source models chosen by high sequence homology: the H chain of anti-HIV Ab CH59 (PDB:4HPY) (4) and the L chain of an Ab against parathyroid hormone-related protein (PDB:3FFD) (29). Rebuilding and real-space refinements were done in Coot (30) with reciprocal space refinements in PHENIX (31) and validations in MolProbity (32). During the rebuilding and refinement process, significant positive peaks were observed in the difference electron density maps in the vicinity of the paratope, which were interpreted to represent bound peptide; however, a single conformation or even a single tracing of the peptide backbone could not be confidently established in the structural model. Unaveraged omit maps and phase improvement using noncrystallographic symmetry averaging did not improve the density of the peptides. Additional datasets on isomorphous crystals also failed to resolve any bound peptides. Difference fo-fc maps calculated at lower resolution (3.0 Å) still showed contiguous tracings with strong positive peaks, ruling out the possibility that the features could be explained as a network of water molecules or other ions present in the crystallization experiment. Coordinates and structure factors were deposited in the Protein Data Bank under accession code 5KG9. Structural image rendering and root-mean-square deviation calculations were performed using PyMOL (version 1.8; Schrödinger).
Sequence analysis
A multistep approach was used to search in mammalian species for candidate orthologs of VL3-10 that included the CDR L2 ED motif. First, we used BLAST (33) with the rhesus IGLV3-10 ortholog, IGLV3-17 (5), as the query against the RefSeq genomic database (34). The top 5000 hits from the BLAST search were retained, and the highest similarity to IGLV3-10 for each species was included in the dataset of orthologs. To account for potential allelic differences at the ED positions, if a suboptimal BLAST hit within a species included the ED motif, that hit was retained in the dataset of candidate orthologs for that species. If no ED motif was found within VL3-10 candidate orthologs, we applied a second step using the BLAT Web server (35, 36) to find additional potentially orthologous alleles within fully sequenced mammalian genomes. Two BLAT queries were constructed: one using as the query sequence the non-ED–containing hit from the BLAST run described above and one using as the query sequence the rhesus IGLV3-17 sequence. If any hits from the BLAT runs resulted in sequences that included the CDR L2 ED motif, they were included in the dataset. If no hits for a species included the CDR L2 ED motif, the hit with the highest similarity to VL3-17 was included for comparison. A multiple sequence alignment was created from the dataset using the MAFFT program (37).
Results
To characterize L chain κ and λ usage in the naive B cell repertoire in VelocImmune mice, we isolated CD38+B220+ naive splenic B cells and determined that 91.2% of this population bore κ L chains (Fig. 1A). This observation is virtually identical to the ≈95% expected κ usage for laboratory mice (38). We subsequently immunized three VelocImmune mice by i.p. injection nine times over 18 wk with an HIV-1 Env gp120 identical to AE.A244 gp120, a gp120 component of the RV144 HIV vaccine (15). Mice were rested for 30 wk and given one or two boost immunizations by i.p. injection. Seventeen days after boosting, we quantified serum IgG that bound the A244 gp120 immunogen and a recombinant Env fragment that contained the V1 and V2 loops, termed A244 V1V2 tags (4). We observed serum IgG binding to A244 gp120 and A244 V1V2 tags in all three mice (Fig. 1B). Next, in two mice (RE504 and RE505) we generated B cell hybridomas from splenocytes to identify and profile mAbs representative of serum IgG activity. Hybridoma supernatants were assayed for binding to A244 gp120 and A244 V1V2 tags and HIV-1 neutralization. We selected mAbs that bound A244 gp120, A244 V1V2 tags, and/or neutralized HIV-1 AE.92TH023.
Characterization of L chain usage in naive mice and sera response in immunized mice. (A) Naive B cells (CD38+B220+) derived from the spleen of a VelocImmune mouse (left panel) expressed 91.2% κ L chains (right panel). A representative plot of two independent experiments is shown. (B) Sera in all three immunized mice (RE503 to RE505) were observed to be reactive to A244 gp120 (left panels) and A244 V1V2 tags (right panels), an Env fragment construct that included the V2 epitope. Sera reactivity increased over time in each mouse for both Ags. Line marker colors correspond to immunization time points from blue (early) to red (late).
Twelve Abs from this set were successfully cloned and purified for further characterization. Five of the twelve gp120-reactive mAbs bound A244 V1V2 tags (Fig. 2A, 2B), and binding was not dependent on the presence of V2 glycans (Supplemental Fig. 1A). Of these, four bound V2 peptides (Fig. 2C, Supplemental Table I). These V2-binding Abs were isolated from mouse RE505 and shared the same VH gene segment (VH3-9) used by the human mAbs CH59 and HG10 recovered from RV-144 vaccinees that recognize the K169 V2 epitope. Remarkably, the four VelocImmune V2 Abs did not bear human κ L chains but rather mouse λ L chains encoded by rearrangements of the mouse Vλ3-1 gene segment (Table I).
Chimeric humanized mouse Abs bind a gp120 V2 linear epitope. Binding of the isolated Abs to A244 gp120 (A) and an A244 V1V2 construct (B). (C) Only the four chimeric humanized mouse Abs (red lines) bound V2 peptide. Binding was measured by ELISA, and values are reported as OD at 450 nm or the natural LogAUC.
The usage of endogenous mouse λ L chains was unexpected given the low frequency of λ L chain usage in the VelocImmune mouse (Fig. 1A). Mouse Vλ3-1 does not have a CDR L2 ED motif but rather has a Lys-Asp pair in the CDR L2 and is the mouse Vλ gene with the highest sequence identity to human Vλ3-10 (Supplemental Table II). In addition, the CDR L2 in the murine Vλ3-1 gene segment was four residues longer than in human Vλ3-10. The four V2 Abs induced in the VelocImmune mouse were clonally related (Table I). Thus, this clonal lineage represents expansion of an improbable human heavy mouse λ chimeric Ab response that shared similarities with the human RV144 vaccine–induced V2 mAb CH59 (4), despite the VelocImmune mouse lacking an L chain repertoire with a germline-encoded ED motif. Given that these Abs lacked an ED motif, we next asked how the V2 linear epitope could be engaged without it. To address this question, we turned next to characterizing the V2 epitopes of these Abs, as well as structural analysis.
V2 recognition by ED motif–containing Abs is mediated by electrostatic interaction via salt bridge formation between the ED motif in the CDR L2 and two positively charged lysines, including K169, in the V2 loop (4, 5). The substitution of a positively charged lysine for a negatively charged glutamate in the composition of the CDR L2 of the chimeric V2 Abs described in this study has the potential to alter the electrostatic interaction component of V2 recognition. To characterize changes in how the four chimeric Abs may recognize the V2 loop with a CDR L2 that lacks the ED motif, we next performed epitope mapping by ELISA with a set of alanine-scanning mutants of an AE.A244 V2 peptide (LRDKKQKVHALFYKLDIVPIED). Compared with the human ED motif containing V2 Abs induced in the RV144 trial, the chimeric mice V2 Abs elicited in VelocImmune mice were markedly less sensitive to K169A mutation and recognized an epitope shifted away from K169 toward the C terminus of the V2 loop (Table II, Supplemental Fig. 1B–E). The structure of CH59 in complex with a V2 peptide showed that the C-terminal half of the V2 was primarily contacted by the H chain, whereas the N-terminal half, marked by multiple positively charged lysines, was contacted by the L chain, specifically by salt bridges formed with the CDR L2 ED motif (4). The epitope mapping of RE505-22 is consistent with a similar mode of recognition as CH59, whereby usage of a VH3–9 rearranged H chain mediates interaction with the C-terminal half of the V2. However, because of a lack of an ED motif in the RE505-22 L chain, N-terminal V2 recognition is altered, and K169 is not fully engaged by complementary electrostatic interactions in RE505-22. Such an alteration in V2 recognition could reduce overall binding of RE505-22; indeed, we observed that RE505-22 binds with nearly an order of magnitude lower affinity to the V2 peptide than CH59 (Supplemental Fig. 1F–H).
RE505-22 weakly neutralized the easy-to-neutralize (tier 1) Clade AE virus 92TH023.6 in the TZM-bl neutralization assay but failed to neutralize a difficult-to-neutralize tier 2 Clade AE virus CM244 or other Clade C viruses (Supplemental Table III). This neutralization breadth for RE505-22 was similar to CH59 (4); however, RE505-22 was observed to be an order of magnitude less potent neutralizer of 92TH023.6.
Structural comparison between human/mouse V2 and human V2 Abs
To define the structural differences in the CDR L2 orientation of the human Ig knock-in mouse V2 Abs, we determined the structure of Fab RE505-22. We cocrystallized RE505-22 with V2 peptide (ELRDKKQKVHALFYKLDIV), and the Ab structure was solved to 2.3 Å resolution (Fig. 3A, Table III). Although the presence of the V2 peptide in the asymmetric unit was confirmed in the difference electron density map (Fig. 3B), the peptide was disordered, and its structural coordinates could not be resolved. Therefore, the RE505-22 Ab Fab structure represents the bound Ab conformation. The conformation of RE505-22 used for recognition of V2 could be compared with the bound conformation of the highly similar human V2 Ab CH59 (Fig. 3C, left panel). Because of their high sequence similarity due to shared VH3-9 usage, the structure of the RE505-22 H chain was nearly identical to that of the CH59 H chain (0.32 Å Cα root-mean-square deviation). However, pairing with different L chains resulted in key structural differences between RE505-22 and CH59, with the largest differences observed in the L chain CDRs (Fig. 3C). Of particular interest to V2 recognition is the CDR L2, which, in CH59, contains the ED motif that forms salt bridges with lysines, including K169, in the N-terminal half of the V2 peptide. In RE505-22, the CDR L2 is four residues longer than the CDR L2 of CH59 due to the usage of mouse Vλ3-1, which encodes for a CDR L2 with a length of 7 aa. Interestingly, Vλ3-1 is the only gene segment in the mouse L chain repertoire of this length. All other CDR L2s in the mouse L chain repertoire are 3 aa in length. The pairing of the only VL gene segment with a long CDR L2 could be the result of selection, such that lysine 52 (K52) of the Lys-Asp pair in the RE505-22 CDR L2 is oriented away from the critical like-charged lysines in the V2 epitope. Superposition of the RE505-22 and CH59 L chains demonstrated that K52 and aspartic acid at position 53 (D53) do not share the structural positions of the ED motif of CH59 (Fig. 3C). Therefore, if the Abs recognize V2 in the same orientation, K52 may be positioned far enough away from K169 and K171 that repulsive forces would be limited. This model of RE505-22 interaction is consistent with the evidence that the presence of K52 does not completely abrogate V2 binding. In addition, it is consistent with data that a K52E mutation introduced in RE505-22 to substitute in the ED motif did not improve binding or neutralization and did not alter the epitope from wild-type RE505-22 (Supplemental Fig. 1I, Supplemental Table III).
Structure of the bound conformation of RE505-22. (A) Structure of the bound conformation of the RE505-22 Fab. H chain is colored blue, and L chain is colored gray. (B) The difference electron density map in the paratope of the RE505-22 Fab shows strong peaks and contiguous features; nonetheless, bound peptide could not be built into the structure model with confidence. L and H chain carbons are colored as in (A) behind a semitransparent surface (white). The electron density map (green mesh) is contoured at 3sigma level. (C) Superposition of the CH59 L chain (cyan) onto the RE505-22 Fv domain L chain (gray) showed structural differences in the CDRs. A four-residue insertion alters the conformation of the RE505-22 CDR L2 (left panel), such that the positively charged K52 is oriented away from key positively charged V2 lysines (K169 and K171). E49 in RE505-22 adopts a similar position and orientation as E50 in CH59, which interacts with K171 in the V2 in the CH59-V2 complex. A sequence alignment of the CDR L2 region in RE505-22 and CH59 reflective of this structural alignment is shown at the bottom (left panel).
The superposition of the CDR L2s of CH59 and RE505-22 also revealed that E50 of the ED motif in CH59 is close in position to E49 of RE505-22, which was not apparent from our sequence alignments first performed in the absence of the RE505-22 structural information. E50 of CH59 forms a salt bridge with K171 of V2; thus, similar positioning of E49 would suggest an explanation for the inclusion of K171 in the RE505-22 epitope (Table II). It is notable that Vλ3-1 is also the only mouse Vλ gene segment with a negatively charged residue encoded at position 49. The CDR L2 of RE505-22 lacks a negatively charged residue in the position corresponding to D51 of CH59, the residue that forms a salt bridge with K169; this provides a rationale for why K169 is not included in the RE505-22 epitope (Table II).
Presence of the CDR L2 ED motif in mammals other than the mouse
Previously, we showed that the CDR L2 ED motif is highly conserved in primate phylogeny (5). However, the endogenous mouse λ repertoire does not include a Vλ gene segment with high sequence similarity and, thus, is not orthologous to Vλ3-10; also, no mouse Vλ gene segments encode for an ED in the CDR L2 (14). Therefore, we were interested in determining whether early common ancestors of mouse and humans had an ortholog of Vλ3-10 in their repertoire, thus implying that these λ repertoire traits were lost during the line of descent to mouse, or whether Vλ3-10 orthologs and the CDR L2 ED motif simply arose later in Mammalia evolution. We searched for potential Vλ3-10 orthologs in the RefSeq database of genomic sequences (34) and observed >60% identity to the Vλ3-10 aa sequence in a broadly divergent group of organisms, including species of mammals, reptiles, and birds (Fig. 4). The observation of Vλ3-10 orthlogs containing the CDR L2 ED motif throughout the mammalian lineage, including in the Tasmanian devil, a marsupial, suggests that an ED motif was present in the mammalian lineage from at least the time of speciation of placentals and marsupials, which was estimated to have occurred 162 million years ago (39). We also observed a Vλ3-10 ortholog with the CDR L2 ED motif in the brown rat, which has a recent common ancestor with mouse. The Vλ3-10 ortholog in rat has 78% identity to human, whereas the highest-identity mouse λ V gene segment, VL4-1, has only 46% identity. This stands in striking contrast to the 95% identity between Vλ3-10 orthologs of human and rhesus macaques, which diverged in the same order of time as mice and rats (5). Taken together, these data suggest that there was a loss of the orthologous Vλ3-10 gene segment during the line of descent in mouse and that it occurred after a relatively recent divergence from the common ancestor with rat.
CDR L2 ED motif is conserved throughout Mammalia lineage. Amino acid sequence alignment of human Vλ3-10 to the highest-similarity sequence from a representative set of animals with sequenced genomes. Dots represent amino acid residue matches. ED motif positions are shown in red. Mammals with sequences that include the CDR L2 ED motif are shown in blue. Amino acid sequence identity to human Vλ3-10 is shown in the far right column. Mouse Vλ3-1 is included for reference.
Discussion
A V2-directed response to RV144 immuogens is conserved between humans and rhesus macaques due to the common utilization of a recognition component, the CDR L2 ED motif. It is this component that interacts with a critical residue, K169, that is correlated with reduced transmission risk in the RV144 trial (5). In this study, we showed that immunization with the same RV144 vaccine immunogens in a humanized mouse model that lacks the human Vλ repertoire and, consequently, the CDR L2 ED motif, nevertheless generates a robust V2-directed response. The V2-targeting Abs from this mouse model that were elicited had a remarkable similarity to those induced in the RV144 vaccine trial, utilizing the same human VH gene segment and a close alternative endogenous mouse Vλ gene segment as human RV144 Ab CH59. We showed that the V2 epitope response in the humanized mice was shifted away from the K169 residue. Structural determination revealed a mechanism by which the mouse Ab could recognize the V2 without a principal interaction between the ED motif and K169. Thus, the absence of the ED motif does not prohibit a similar V2 response as that observed in humans, but it altered the precise footprint of that Ab response away from a critical residue identified to be important for transmission risk.
The V2 response to RV144 immunogens in the VHDJH, VκJκ VelocImmune humanized mouse is essentially the closest alternative to CH59 in humans that is available in the VH/Vκ humanized mouse repertoire. This remarkably conserved response not only demonstrates the viability of humanized mouse models to recapitulate human Ab responses in a vaccination setting, it also demonstrates the extraordinary selective pressure for the immune system to respond to the same immunogen in a strikingly similar fashion.
These observations have implications for HIV vaccine design. First, if a strain-specific effector function-mediating response can be protective, it stands that broadening the diversity of such a response within the V2 epitope could increase vaccine efficacy. Immunizing with a diverse panel of V2 sequences could leverage the immunodominance of the V2 response to elicit several V2-targeting Abs that, individually, may be narrowly specific but, in combination, are broad enough to be protective against multiple strains of infecting virus. In this regard, we showed recently that a pentavalent B/E/E/E/E gp120 vaccine boost of the ALVAC used in the RV144 trial protected against a mucosal R5 SHIV challenge (T. Bradley, J. Pollara, S. Santra, N. Vandergrift, S. Pittala, C. Bailey-Kellogg, X. Shen, R. Parks, D. Goodman, A. Eaton, H. Balachandran, L.V. Mach, K.O. Saunders, J. Weiner, R. Scearce, L.L. Sutherland, S. Phogat, J. Tartaglia, S.G. Reed, S.-L. Hu, J.F. Theis, A. Pinter, D.C. Montefiori, T.B. Kepler, N.L. Michael, T.J. Suscovich, G. Alter, M.E. Ackerman, M.A. Moody, H.X. Liao, G. Tomaras, G. Ferrari, B.T. Korber, and B.F. Haynes, unpublished observations). Second, if an easy-to-induce strain-specific dominant V2 polypeptide response outcompetes early ancestors of V1V2 glycan bnAb lineage targeting bnAb lineages, such as CH01, PG9, and VRC-26, one strategy that was proposed is termed B cell lineage design, in which sequential Envs targeted at lineage members are administered to selectively drive bnAb development (40). Alternatively, strategies to select against a CH59-like response might be necessary for bnAb induction. Strategies, such as Ag-induced germinal center B cell apoptosis (41, 42), could be used to negatively select CH59-like Abs, theoretically providing a survival advantage for V1V2-glycan bnAb lineage members.
Disclosures
C.A.K., L.E.M, and A.J.M are employed by Regeneron Pharmaceuticals, Inc. B.F.H. holds a patent with Regeneron Pharmaceuticals, Inc. related to vaccine development in VelocImmune mice. The other authors have no financial conflicts of interest.
Acknowledgments
We thank Melissa Cooper for performing neutralization assays. Crystallography was performed in the Duke University X-ray Crystallography Core Lab. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract Number W-31-109-Eng-38. Southeast Regional Collaborative Access Team supporting institutions may be found at http://www.ser-cat.org/members.html.
Footnotes
This work was supported by a Center for HIV/AIDS Vaccine Immunology-Immunogen Discovery grant (UMI-AI100645) from the National Institutes of Health/National Institute of Allergy and Infectious Diseases/Division of AIDS.
The coordinates and structure factors presented in this article have been submitted to the Protein Data Bank under accession number 5KG9.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- bnAb
- broadly neutralizing Ab
- CDR L2
- L chain second CDR
- ED
- glutamic acid–aspartic acid
- Env
- envelope
- K52
- lysine 52
- K169
- lysine residue at position 169
- LogAUC
- log of the area under the binding curve.
- Received October 4, 2016.
- Accepted November 18, 2016.
- Copyright © 2017 by The American Association of Immunologists, Inc.
References
In this issue
