An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1

Broadly HIV-1–neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1–infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1–infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patient’s plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells.

Broadly HIV-1–neutralizing antibodies (BnAbs) have been isolated that bind to multiple epitopes on the envelope glycoproteins gp120 and gp41 (reviewed in ref. 1). 2G12 recognizes a posttranslational glycan epitope on gp120 (2, 3). CH103–CH106 (4), b12 (5, 6), VRC01 (7), and numerous other mAbs with VRC01-like characteristics, such as VRC03, VRC-PG04, CH30–CH34, and the HAAD motif antibodies (7–9), recognize the CD4 binding site (CD4bs). HJ16 binds to an epitope near the CD4bs (10). PG9 and PG16, CH01–CH04, and PGT141–PGT145 recognize conformational epitopes in the gp120 V1/V2 region with binding dependence on V2 asparagine residue 160 (11–13). PGT121–PGT123, PGT135–PGT137, PGT125–PGT128, PGT130, and PGT131 recognize a diverse set of carbohydrate or carbohydrate-dependent epitopes in the gp120 V3 region (13). 3BC176 and 3BC315 recognize a conformational HIV-1 spike epitope in the proximity of the V3 loop and the CD4i site that is exposed in part by CD4 binding (14). Finally, 2F5 (2, 15, 16), 4E10 (2, 17), 10E8 (18), and Z13 (19) bind to the membrane proximal external region (MPER) of gp41.

Each of these antibodies displays one or more unusual characteristics, such as polyreactivity with human and/or nonhuman antigens, long heavy chain complementarity-determining region 3 (HCDR3) loops, and high levels of somatic mutations (1, 13, 20–23). These traits suggest that the development of these types of BnAbs may be limited by immune tolerance controls that impede the required tortuous or polyreactive BnAb maturation pathways (20, 21, 24). This notion is supported by a number of observations in 2F5 V_H×V_L knockin mice: targeted expression of the 2F5 V_HDJ_H/V_LJ_L rearrangements triggered a near-complete B cell developmental blockade at the pre-B to immature B cell stage (25); even when clonal deletion mechanisms were circumvented, 2F5 V_HDJ_H/V_LJ_L-expressing broadly neutralizing B cell rescue was limited by κ chain editing and an anergic phenotype (26); and MPER-specific serum neutralizing IgG responses were elicited in 2F5 knockin mice immunized with MPER-lipid complexes by rescuing the anergic self-reactive 2F5-expressing B cells that survived the B cell developmental blockade (26).

We have previously suggested that the paucity of subjects developing BnAbs may be due to the frequent deletion of B cell precursors that acquire autoreactivity during their maturation process (at either early or late stages) and hypothesized that HIV-1–infected subjects with autoimmune diseases might be capable of developing BnAbs in the context of their autoreactive humoral response (24, 26–28). The observations that HIV-1 infection is reported with a disproportionately low frequency among subjects with SLE support this hypothesis (29–34).

To date, there have been no reports of studies of the HIV-1 antibody repertoire of SLE subjects from whom BnAbs have been isolated. Here, we describe CH98, an HIV-1 CD4bs BnAb isolated from an HIV-1–infected individual with SLE (subject CH5329; see Methods). CH98 displayed a number of unusual antibody traits shared by autoimmune disease autoantibodies.

CH5329 plasma neutralization and isolation of CH98. In a multiclade, multitier panel of HIV-1 strains, consisting of 4 tier-1A, 4 tier-1B, and 34 tier-2 isolates, including 9 transmitted/founder viruses, plasma from subject CH5329 neutralized 41 of 42 HIV-1 strains (97.6%), with a mean ID₅₀ titer of 925 (range, 21–9,204; Figure 1A). The only HIV-1 strain that was not neutralized, G.P0402, was a clade G. CH5329 plasma was screened in 2 different but complementary assays for CD4bs activity. CH5329 plasma bound to the HIV-1 Env resurfaced core 3 (RSC3) protein with a mean endpoint titer 5-fold higher than that of the RSC3Δ371I/P363N protein (Figure 1B and Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI73441DS1), indicative of the presence of CD4bs mAbs (7). The RSC3 protein preferentially blocked the neutralization ability of CD4bs mAbs such as VRC01, but not mAbs directed against other sites, such as the gp41-binding 2F5 BnAb. However, the ability of RSC3 protein to block neutralizing activity in HIV-infected sera is rare (35) and indicative of the presence of CD4bs neutralizing antibodies (7, 8). Therefore, the 30%–55% reduction we observed in total serum neutralization against 2 different HIV-1 strains, HxB2 and JRFL (Figure 1C), indicated that CD4bs antibodies markedly contribute to plasma neutralization.

Figure 1

Identification of neutralizing CD4bs antibodies in CH5329 serum. (A) CH5329 plasma and mAb CH98 neutralization profiles, measured as ID₅₀ and IC₅₀,respectively, in TZM-bl assay. Transmitted/founder HIV-1 isolates are italicized. For plasma neutralization: red, ID₅₀ >1,000; orange, ID₅₀ 100 to 1,000; yellow, ID₅₀ >20 and <100; white, ID₅₀ <20 (absence of neutralization). For CH98 BnAb: red, IC₅₀ <1 μg/ml; orange, IC₅₀ 1 to 50 μg/ml; white, IC₅₀ >50 μg/ml (absence of neutralization). (B) Differential binding by CH5329 serum to the RSC3 protein and the CD4bs knockout RSC3Δ371I/P363N, graphed as ELISA endpoint titer (final reciprocal serum dilution with background-corrected OD ≥0.1). (C) CD4bs-directed neutralizing activity in CH5329 serum, shown as percent reduction in ID₈₀ in the presence of RSC3 compared with the knockout RSC3Δ371I/P363N against HIV-1 JRFL and HxB2 strains. Values for the BnAbs VRC01 and 2F5 are included as positive and negative control, respectively. In B and C, error bars represent SEM from 2 independent experiments.

mAb CH98 V_HDJ_H and V_LJ_L gene segments were isolated from a clonal memory B cell culture containing 29 ng/ml IgG at the end of stimulation with culture supernatant that bound to RSC3 but not RSC3Δ371I, and were expressed as a full recombinant IgG1 mAb (11, 36, 37). CH98 neutralized 27 of 43 HIV-1 isolates (62.8%), with a median IC₅₀ neutralization level of 4.2 μg/ml (range, <0.02–49 μg/ml), accounting for 65.9% of the observed plasma neutralization breadth (Figure 1A). No other antibodies were retrieved that bound to RSC3 or neutralized the A.Q23.17 HIV-1 strain, which was selected to screen culture supernatants for heterologous neutralizing activity (see Methods). Thus, the frequency of peripheral blood circulating CD4bs neutralizing antibodies in this subject was estimated to be 0.003%.

Characteristics of CH98. CH98 uses the V_H3–30 gene segment, notably not the V_H1–2 or V_H1–46 segments commonly associated with VRC01-like CD4bs BnAbs (8, 9), paired with the V_λ2–23 light chain gene segment. CH98 is highly mutated, with V_HDJ_H and V_LJ_L mutation frequencies of 25.3% and 15.8%, respectively, and displays a long HCDR3 of 21 aa (Supplemental Table 1). From 454 pyrosequencing, we identified 4 additional V_HDJ_H rearrangements in the CH98 clonal lineage, and all were highly mutated and shared numerous mutations with the CH98 V_HDJ_H (Supplemental Figures 2 and 3).

The CH98 light chain CDR 1 (LCDR1) is 27 nt long and underwent a 15-nt deletion preceded by an activation-induced cytidine deaminase (AID) hotspot (AGT; Supplemental Figure 4). TGT motifs are also present near the start and the end of this deletion (Supplemental Figure 4), raising the hypothesis that slipped-strand synthesis may have occurred at this position. It is also worth noting that the CH98 V_L/J_L gene segment rearrangement had a very long n-region insertion of 24 nt in the LCDR3 that were not encoded by either of the V_L2–23 or J_L3 germline sequences (Supplemental Figure 4).

CH98 epitope mapping. CH98 bound to RSC3 protein (IC₅₀, 0.51 μg/ml), but not to RSC3Δ371I/P363N; in contrast to the CD4bs BnAb VRC01, CH98 did not bind to RSC3 G367R mutant or to the gp120 outer domain stabilized protein OD4.0 (Supplemental Table 2). Binding to WT YU2 gp120 Env (IC₅₀, 0.03 μg/ml) was abrogated by the D368R mutation, which disrupts a key contact residue within the CD4bs, but not by the I420R mutation (IC₅₀, 0.11 μg/ml), which disrupts the CCR5 coreceptor binding site (Supplemental Table 2). Also, CH98 potently neutralized the HIV-1 YU2 strain (IC₅₀, 0.9 μg/ml). Therefore, we epitope mapped CH98 on a panel of YU2 gp120 core protein mutants previously used to map other mAbs binding within and outside the CD4bs (38, 39). We compared the binding profile of CD4bs BnAb CH98 with those of other CD4bs BnAbs — CH103 (4), VRC01 (7), CH31–CH34 (8), and b12 (5, 6) — as well as non-neutralizing CD4bs b6 mAb and CD4-Fc to 31 YU2 gp120 core single-point mutants (Figure 2). Single-point mutations outside the CD4bs did not affect CH98 binding. Within the CD4bs, mutations in position 365 and 368 abrogated binding of all CD4bs BnAbs tested, but not mAb b6, and mutations in positions 282, 461, and 473 reduced CH98 binding to 55%, 64%, and 31%, respectively (Figure 2). Overall, CH98 was the BnAb least sensitive to the mutations represented in the panel, which indicates that these positions are less relevant for CH98 binding to its cognate epitope than for the other CD4bs BnAbs. The only antibodies sensitive to all the mutations that also affected CH98 binding were the BnAbs CH103 and VRC01.

Figure 2

Effect of point mutation of the YU2 core protein on CD4-Fc and on CH98, CH31–CH34, VRC01 and b12 CD4bs BnAbs, and the non-neutralizing CD4bs b6 mAb. The effect of point mutations on the binding to the YU2 core protein of CH98 and other CD4bs mAbs and CD4-Fc is expressed as percentage normalized to binding to the WT YU2 gp120 core protein and color coded as indicated. Positioning within the CD4bs (green), or adjacent or nearby (black), is indicated at far right.

Comparison of CH98 with CD4bs mAbs CH103 and VRC01. CH103 and VRC01 bind to the CD4bs with 2 distinct modes of epitope recognition: CH103 uses a loop dominated mode of interaction including all V_HDJ_H and V_LJ_L CDRs (4), whereas VRC01 contacts the CD4bs by mimicking CD4 with a large footprint that is dominated by HCDR2 and encompasses both heavy and light chains (40). To determine whether CH98 falls into one of these categories, we threaded the CH98 heavy and light chains onto the gp120-complexed structure coordinates of CH103 (4) and VRC01 (40).

CH98 threading on the VRC01 gp120-complexed structure resulted in steric clashes between the LCDR3 of CH98 and the D loop of gp120 (Figure 3A). Relaxation of the model to alleviate the clashes resulted in a complex with a binding energy score of –31.96 Rosetta energy units (REU; see Methods), which was higher (i.e., less favorable) than those of both the solved VRC01-gp120 complex and the CH98 complex modeled in the CH103 mode of recognition (–40.84, and –37.86 REU, respectively). Conversely, CH98 threading onto the gp120-complexed CH103 crystal structure resulted in a model compatible with a CH103-like mode of antigen recognition (Figure 3B). Between the 2 mAbs, aa sequence homology was 36% and 56% for the V_HDJ_H and V_LJ_L, respectively (Supplemental Figure 5 and Supplemental Table 3). This model supported the observed binding data (Figure 2) and predicted that all the heavy and light chain CDRs of CH98 make direct contact with the 5 residues affecting CH98 binding to the YU2 gp120 core (Supplemental Table 4).

Figure 3

Threading of CH98 onto the gp120 complexed structures of CD4bs VRC01 and CH103 BnAbs. (A) CH98 model based on threading onto the gp120 complexed VRC01 crystal structure. The CH98 heavy chain (HC; dark blue) and light chain (LC; light blue) threaded onto the VRC01 structure complexed with gp120 (gray) resulted in an incompatible model, with a clash between the gp120 D loop (red) and the LCDR3 (green). (B) CH98 model based on threading onto the gp120 complexed CH103 crystal structure. The CH98 heavy chain (dark blue) and light chain (light blue) threaded onto the CH103 structure complexed with gp120 (gray) resulted in a model in which HCDR3 (magenta) dominates the binding to the CD4bs region and engages in direct contact with gp120 residues in positions 365, 368, 473, and 282, all of which affect CH98 binding. The residue in position 461 makes direct contact with LCDR1. Each residue that affects CH98 binding in the epitope mapping is illustrated using the corresponding color in Figure 2.

In particular, D461 was predicted to contact LCDR1 (Supplemental Figure 6A), which is 9 aa long and underwent a 5-aa deletion. Therefore, we asked whether a LCDR1 deletion-reverted CH98 would be compatible with the gp120-complexed CH103 mode of recognition. Adoption of the canonical conformation for a LCDR1 of length 14, as defined by North and colleagues (41), allowed the deletion-reverted LCDR1 to avoid steric clashes at the gp120 interface; however, the binding energy score of the complex was less favorable than that of the mature CH98 complex (–35.09 versus –37.86 REU; Supplemental Figure 6B). Our interpretation is that the 15-nt deletion in the CH98 LCDR1 may have played a favorable role during the process of affinity maturation of CH98.

CH98 BnAb reactivity with human proteins. In a panel of 9 autoantigens associated with presence of autoimmune diseases (Sjogren’s syndrome antigens A and B [SSA and SSB, respectively], Smith antigen [Sm], ribonucleoprotein [RNP], centromere B [Cent B], histone, scleroderma 70 [Scl 70] and Jo-1 proteins, and dsDNA), CH98 reacted with dsDNA (Figure 4A). CH98 binding to dsDNA was comparable to that previously observed for the CD4bs BnAb CH103 (Supplemental Figure 7). In the same assay, CH5329 plasma from the same specimen from which CH98 BnAb was isolated was found to be positive for SSA and dsDNA antibodies (mean ± SD, 268 ± 29 and 163 ± 22 response units [RU], respectively; Figure 4B).

Figure 4

Autoreactivity of CH98. (A) CH98 reactivity against common antigens associated with autoimmune diseases, tested with a commercial ANA-II Plus test system. The threshold for positivity was 120 RU (dashed line). (B) CH5329 plasma reactivity against common antigens associated with autoimmune diseases, tested with the commercial ANA-II Plus test system. Results from 3 independent experiments are shown. The threshold for negativity was 100 RU (solid line), and that for positivity was 120 RU (dashed line); values of 100–120 RU were considered equivocal specimens. (C–E) Hep-C IFA staining of BnAb CH98. Antibody concentration was 25 μg/ml, and exposure time was 8 seconds. Original magnification, ×40; inset in D is enlarged ×1.5. (F and G) Reactivity of CH98 on a panel of 9,400 human proteins tested using a ProtoArray 5 microchip, compared with nonautoreactive mAb 151K (F) or palivizumab (G). Values for STUB1 are encircled.

In HEp-2 cell IFA staining, CH98 displayed diffuse fine granular cytoplasmic staining, exhibited stronger binding to dividing cells, and bound to intracellular vesicles in a subset of HEp-2 cells (Figure 4, C–E). CH98 reactivity with 9,400 additional human proteins was screened using microarray technology (28). CH98 showed polyreactivity with numerous human antigens, as measured by 1 log stronger binding to more than 90% of the test proteins than the non-polyreactive control antibodies 151K and palivizumab. CH98 also bound with higher affinity, measured as a >2 log increase in binding compared with antibody controls, to STUB1 (also known as CHIP), an E3 ubiquitin-protein ligase (Figure 4, F and G). Binding of CH98 and CH5329 plasma to STUB1 were confirmed in direct-binding ELISA with an endpoint titer of 3.3 μg/ml for CH98 and plasma titer of 270 (Supplemental Figure 8, A and B). We previously demonstrated that members of the CD4bs BnAb CH103 clonal lineage displayed reactivity with dsDNA and with UBE3A, another E3 ubiquitin-protein ligase (4). Such reactivity was also shared by the CD4bs BnAb VRC01 (4). However, these BnAbs did not bind to STUB1, and CH98 did not bind to UBE3A (data not shown).

Taken together, these findings suggested that the immune system dysregulation that allows for production of autoreactive antibodies during SLE may have played a permissive role in the development and maturation of the BnAb CH98. Moreover, the similarities between CH98 and the SLE autoantibodies of long HCDR3 and dsDNA reactivity are suggestive of similar pools of B cells and mechanisms of their origin.

Here, we describe an individual who, when studied 14 years after HIV-1 transmission and 6 years after onset of SLE, controlled her viral load off antiretroviral therapy (ART) and developed plasma BnAb activity.

An open question regarding the development of BnAbs in HIV-1–positive subjects is whether acquisition of autoreactivity is a common prerequisite for the development of neutralization breadth. We have previously described the CH103 CD4bs BnAb isolated from an SLE-negative, chronically HIV-1–infected individual and demonstrated that antibodies in the CH103 clonal lineage acquired autoreactivity with a number of autoantigens, including dsDNA, concurrently with acquisition of HIV-1 neutralization breadth (4). We have also described an individual in whom the onset of gp41 MPER 2F5-like BnAb plasma activity was temporally associated with the development of plasma dsDNA and Jo-1 autoantigen reactivity, without appearance of any clinical manifestation of autoimmune disease (42). Thus, in some individuals, acquisition of BnAb activity is clearly associated with the acquisition of autoreactivity.

Antibodies to dsDNA are a hallmark of SLE and are present in approximately 70% of subjects with SLE, but only 0.5% of SLE-negative individuals (32). Conversely, dsDNA antibodies are not a typical finding during HIV-1 infection, but have been commonly reported in the plasma of the few SLE subjects with HIV-1 infection (30, 33). The presence of plasma dsDNA antibodies has been documented in subjects diagnosed with SLE both after and before HIV-1 diagnosis (91.7% and 64.3%, respectively) (30). The incidence of plasma BnAbs in these reported cases, however, is unknown.

We report herein a monoclonal HIV-1 BnAb, CH98, isolated from a chronically HIV-1–infected individual who subsequently developed SLE. CH98 neutralized HIV-1 through binding to the CD4bs on HIV-1 gp120 and reacted with dsDNA at levels comparable to those previously observed for the CD4bs BnAb CH103. CH98 accounted for only a portion of the plasma neutralization breadth (65.9%). This observation, in conjunction with the finding that RSC3 plasma absorption only partially abrogated plasma neutralization (30%–55%), implies that other neutralizing antibody specificities contributed to the overall plasma neutralization breadth in this individual. These data are in line with previous observations indicating that plasma neutralization breadth can be mediated by a polyclonal response comprising antibodies with distinct specificities and variable degrees of breadth (43–45). Using a combination of epitope mapping and threading of the CH98 heavy and light chains onto the gp120-complexed structure coordinates of CD4bs BnAbs, we determined that CH98 likely uses a CH103-like Env loop mode of epitope recognition that involves all the heavy and light chain CDRs. Conversely, CH98 threading onto the gp120-complexed structure of VRC01 resulted in steric clashes between the LCDR3 of CH98 and the D loop of gp120. This result was not unexpected: VRC01 and all VRC01-like antibodies had unusually short LCDR3s (5 residues), and it has been suggested that the VRC01-like orientation of gp120 binding does not leave enough space between the antibody and the gp120 D loop to accommodate longer LCDR3s (46). Indeed, CH98 has a 12-residue-long LCDR3, and therefore our model is compatible with this hypothesis.

CH98 was also characterized by a short LCDR1 (9 aa) that underwent a 5-aa deletion during the process of affinity maturation. This 15-nt deletion in CH98 LCDR1 was preceded by an AID hotspot. AID hotspots regulate secondary antibody diversification and are directly involved in the process of somatic hypermutation (47, 48). Since we predicted a less favorable binding energy score of the deletion-reverted CH98 complex compared with the mature CH98 complex, our interpretation is that, similar to our prior observation for CH103 (4), a single macromutation may have improved binding to the cognate epitope. In addition, LCDR1s shorter than 11 aa are rare (46), but commonly found among CD4bs BnAbs (4, 46), and both CH103 and VRC01 also have deletions in their LCDR1s (4, 40). We interpret the disproportionate frequency of short LCDR1s among CD4bs BnAbs as being indicative of a common selection process of CD4bs BnAbs that favors antibodies with shortened LCDR1s.

This is the first chronically infected SLE individual with concomitant HIV-1 infection that we have been able to study, and it was striking that this individual also had robust plasma BnAb activity, given that the incidence of chronically HIV-1–infected subjects with this level of plasma neutralization breadth is only approximately 20% (49–52).

In a literature review from 1981 to 2012 by Carugati and colleagues, of 53 HIV-1/SLE patients, 54.7% were diagnosed with HIV-1 before SLE (33). The diagnosis of HIV-1 in our subject preceded that of SLE, and we cannot conclude that the CH98 maturation was initiated as part of the SLE-induced autoreactive response, nor can we exclude the possibility that silent SLE was present before the time of HIV-1 diagnosis.

When generated, BnAbs appear late (2–4 years) after HIV-1 transmission, and serum neutralization breadth correlates with viral load levels (49). This has been interpreted as an indication that persistent antigen stimulation is required to develop neutralization breadth (53–55). In stark contrast, our study subject developed BnAbs while controlling viremia, which suggests that acquisition of HIV-1 neutralization breadth may not always require chronic stimulation with high levels of HIV-1 viremia. A possible explanation is that the maturation of CH98 was not hindered in this subject by immune tolerance control mechanisms, thus facilitating the acquisition of mutations required to achieve neutralization breadth, possibly driven by exposure to both HIV-1 and autoantigens (21, 24, 27). The low viral load and normal CD4 count of this subject while off ART could not be explained by HIV-1–controlling HLA types (56) or by HIV-1 infection with a nef-defective strain (57, 58); studies are in progress to determine whether the presence of SLE contributed to virus control.

Kobie and colleagues recently identified an autoreactive plasma antibody population (9G4 anti-idiotype antibodies) that is commonly found in SLE subjects and is responsible for serum HIV-1 neutralization breadth in ART-naive, SLE-negative, chronically HIV-1–infected subjects; the authors proposed that this kind of antibody may derive from the same pool of B cells of SLE-induced antibodies (34). The BnAb CH98 differs from 9G4-positive antibodies in that it uses a V_H3–30 gene segment, not V_H4–34, which defines 9G4 antibodies. Therefore, our findings demonstrated that autoreactive antibody populations beyond 9G4-positive antibodies have the potential to broadly neutralize HIV-1 strains in SLE subjects.

In conclusion, we demonstrated that a virus-controller SLE patient with chronic HIV-1 infection developed the highly mutated CD4bs BnAb CH98, and this antibody displayed characteristics typical of SLE-induced autoantibodies. Our findings support the hypothesis that lax mechanisms of tolerance control associated with SLE may have favored the maturation of this BnAb by favoring its evasion from deletion during the process of somatic maturation and the acquisition of reactivity with self-antigens. Moreover, our data support the concept that BnAbs develop from a similar pool of B cells as do autoimmune B cells in SLE.

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We thank Krissey Lloyd, Christina Scholarchuk, Julie Blinn, Andrew Foulger, Fangping Cai, Miroslawa Bilska, Morgan Gilman, and Rekha Bhaskarabhatla for excellent technical support; and Kelly A. Soderberg, Megan McCormick, Jennifer Kirchherr, Celia C. LaBranche, and Janet Mueller for project and/or regulatory management and coordination. We thank the NISC Comparative Sequencing Program of the NIH Intramural Sequencing Center, National Human Genome Research Institute, for 454 pyrosequencing. This study was supported by grants from NIH, NIAID, Center for HIV/AIDS Vaccine Immunology (AI067854), and Center for HIV/AIDS Vaccine Immunology–Immunogen Discovery (AI100645).

Address correspondence to: Mattia Bonsignori, Duke Human Vaccine Institute, Duke University Medical Center, 2 Genome Court, MSRB II, Room 4013, Duke Box 103020, Durham, North Carolina 27710, USA. Phone: 919.681.9739; Fax: 919.684.5230; E-mail: mattia.bonsignori@duke.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Note regarding evaluation of this manuscript: Manuscripts authored by scientists associated with Duke University, The University of North Carolina at Chapel Hill, Duke-NUS, and the Sanford-Burnham Medical Research Institute are handled not by members of the editorial board but rather by the science editors, who consult with selected external editors and reviewers.

Reference information: J Clin Invest. 2014;124(4):1835–1843. doi:10.1172/JCI73441.

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