Two Distinct Broadly Neutralizing Antibody Specificities of Different Clonal Lineages in a Single HIV-1-Infected Donor: Implications for Vaccine Design Mattia Bonsignori,a David C. Montefiori,a,c Xueling Wu,i Xi Chen,a Kwan-Ki Hwang,a Chun-Yen Tsao,a Daniel M. Kozink,a Robert J. Parks,a Georgia D. Tomaras,a,c John A. Crump,b,d,f,g Saidi H. Kapiga,h Noel E. Sam,f Peter D. Kwong,i Thomas B. Kepler,a,e Hua-Xin Liao,a John R. Mascola,i and Barton F. Haynesa Duke Human Vaccine Institute,a Division of Infectious Diseases and International Health, Department of Medicine,b Department of Surgery,c Duke Global Health Institute,d and Center for Computational Immunology Duke University Medical Center,e Durham, North Carolina, USA; Kilimanjaro Christian Medical Centre, Moshi, Tanzaniaf; Kilimanjaro Christian Medical College, Tumaini University, Moshi, Tanzaniag; London School of Hygiene and Tropical Medicine, London, United Kingdomh; and Vaccine Research Center, National Institute of Allergies and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USAi
Plasma from a small subset of subjects chronically infected with HIV-1 shows remarkable magnitude and breadth of neutralizing activity. From one of these individuals (CH0219), we isolated two broadly neutralizing antibodies (bnAbs), CH01 and VRCCH31, from two clonal lineages of memory B cells with distinct specificities (variable loop 1 and 2 [V1V2] conformational specificity and CD4-binding site specificity, respectively) that recapitulate 95% of CH0219 serum neutralization breadth. These data provide proof of concept for an HIV-1 vaccine that aims to elicit bnAbs of multiple specificities. ecent studies have shown that ⬃20% of subjects chronically infected with HIV-1 mount broadly neutralizing antibody (bnAb) responses and that 1 to 3% of subjects have exceptional magnitude and breadth of neutralizing activity (level 3 neutralizers) (4–6, 19, 20). It is unclear if such plasma neutralization breadth is mediated by many (17) or only one or a few (23, 24) antibody specificities. A number of bnAbs have been isolated from HIV-1-infected individuals, and these include those directed to a variable loop 1 and 2 (V1V2) conformational (quaternary) epitope, to the CD4-binding site (CD4bs), and to outer domain glycans, all on the gp120 surface unit of the HIV-1 envelope glycoprotein (2, 3, 21, 22, 24). Additional bnAbs target the membrane-proximal external region (MPER) of gp41 (13, 26). While evidence suggests that bnAbs to the gp41 MPER are limited by tolerance mechanisms, the V1V2 conformational and CD4bs antibodies are generally less polyreactive (2, 8, 10, 12, 22). However, they both present unusual characteristics: the V1V2 conformational bnAbs have long heavy-chain complementarity-determining region 3 (HCDR3) (2, 10, 14, 15), whereas the CD4bs bnAbs display a high degree of somatic mutation (10, 12, 17, 18, 24) and appear to derive from restricted VH gene families (18, 24). Strategies that allow for highly specific serologic and/or neutralization assays to determine the epitopes of plasma bnAbs are now well established (1, 6, 9, 23), and recent studies suggest that a single HIV-1-infected subject can make bnAbs of multiple specificities (20). If both V1V2 conformational and CD4bs antibodies could be isolated from the same individual and demonstrated to recapitulate the serum neutralizing activity, this would provide direct evidence in support of a polyvalent bnAb HIV-1 vaccine strategy. For this study, we selected a chronically infected individual (CH0219) whose plasma displays extraordinary broad and potent neutralization and includes antibody specificities directed against MPER, CD4bs, CD4-induced (CD4i), gp120 core, and V1V2 conformational epitopes (20). While responses against the CD4bs and a PG9-like V1V2 conformational epitope appeared to be responsible for most of the breadth, only a limited number of mapping reagents were available to confirm this observation (20). Because
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polyvalent neutralizing antibody responses may be a key consideration for HIV-1 vaccine development, and considering the exceptional breath of serum neutralization in this subject, we interrogated the IgG⫹ memory B-cell repertoire of donor CH0219 to isolate and characterize the antibodies that recapitulated serum neutralization. By using a clonal memory B-cell culture system (2), we previously identified four bnAbs (CH01, CH02, CH03, and CH04), members of the same clonal lineage, binding to a V1V2 conformational epitope (2). The CH01 through CH04 bnAbs neutralized 36% to 46% of 91 cross-clade HIV-1 isolates, which represented a subset of strains also neutralized by PG9 (2). Another clone of five CD4bs-specific bnAbs (VRC-CH30, VRC-CH31, VRC-CH32, VRC-CH33, and VRC-CH34) was isolated from the same donor by antigen-specific B-cell sorting of individual IgG⫹ memory B cells reactive with RSC3 but not RSC3⌬371 (25). VRCCH30 through VRC-CH34 neutralized 75% to 95% of a multisubtype panel of viruses with breadth comparable to that of VRC01 (25). Figure 1 shows the phylogenetic tree of the VRC-CH30 to -CH34 bnAb clonal lineage. Analysis of the V(D)J rearrangements of the CH01 to CH04 and VRC-CH30 to -CH34 sequences demonstrated that the two clones use distinct VH genes (VH3 and VH1, respectively) (Table 1), that the frequency of somatic mutations of the VRC-CH30 to -CH34 VH chains (23 to 24%) is approximately twice that of CH01 to CH04 (12 to 14%) (Table 1), and that, conversely, CH01 to CH04 have substantially longer HCDR3s (24 amino acids [aa] versus 13 aa, according to the Kabat numbering system [7]) (Table 1). These data demonstrate that the two clones
Received 19 December 2011 Accepted 23 January 2012 Published ahead of print 1 February 2012 Address correspondence to Mattia Bonsignori,
[email protected], or Barton F. Haynes,
[email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.07163-11
0022-538X/12/$12.00
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FIG 1 Phylogenetic tree of the VRC-CH30 to VRC-CH34 monoclonal antibodies. The tree shows the evolutionary distances of the V(D)J nucleotide sequences of the VRC-CH30 to VRC-CH34 monoclonal antibodies and is rooted on the nucleotide sequence of the unmutated common ancestor (UCA) sequence. It is important to note the great distance of each of the mature antibodies from the common UCA sequence, as we previously observed for the CH01 through CH04 MAbs (2).
did not share a genetic background, relative to VH genes, and suggest that they likely evolved independently. We have previously shown that the neutralization profiles of the individual bnAbs isolated within each of these two clones are comparable (2, 25). Therefore, we used CH01 and VRC-CH31 as representative of the respective clonal lineages to ask if these two bnAbs, either alone or in combination, could recapitulate the breadth of serum neutralization seen in donor CH0219. Neutralization assays using TZM-bl target cells were performed as previously described (2, 11). Figure 2 shows the 50% inhibitory concentrations (IC50; g/ml) of bnAbs CH01 and VRC-CH31 and their combination at a 1:1 ratio and the 50% inhibitory dose (ID50; 1/dilution) titer of the donor CH0219 serum against a multiclade panel of 97 Env-pseudotyped viruses (pseudoviruses), including tier 1, tier 2, and transmitted/founder HIV-1 strains from clades A, B, C, AG, AE, G, and D. The serum neutralized 91/97 (94%) pseudoviruses. CH01 neutralized 43/96 (45%) of the tested pseudoviruses. VRC-CH31 had broader neutralization than CH01,
with 81/97 (84%) of HIV-1 strains neutralized. Thirty-three pseudoviruses were neutralized by both monoclonal antibodies (MAbs), indicating that both epitopes were coexpressed on the virion surface in a conformation recognized by the cognate antibody. Conversely, 41 pseudoviruses were neutralized by VRCCH31 but not by CH01, and 5 were neutralized by CH01 only. As suggested by previous data on plasma (20), the spectra of neutralization of each of the two MAbs alone did not recapitulate the breadth of serum neutralization (Fig. 2). However, when CH01 and VRC-CH31 were combined at a 1:1 ratio, they neutralized 86/91 (95%) of HIV-1 strains neutralized by donor CH0219 serum (indicated with the symbol ✓ in Fig. 2). The five HIV-1 strains that were not neutralized by the combination of CH01 and VRC-CH31 bnAbs, AE.NP03, D.3016, G.X2088, C.Du422, and B.62357 (indicated with the symbol ⫻ in Fig. 2), showed relatively weak serum neutralization (ID50 values of 294, 282, 61, 27, and 23, respectively; Fig. 2). Indeed, the ability of donor CH0219 to generate additional antibody specificities with some degree of HIV-1 neutralizing activity was formally proven by the isolation from IgG⫹ memory B-cell cultures of anti-V3 loop (CH19) and antigp41 (CH11) MAbs with narrow spectra of neutralization: CH19 neutralized exclusively clade B tier 1 viruses, and CH11 neutralized only two tier 2 viruses out of 97 tested (data not shown). While the CH11 and CH19 antibodies neutralized pseudoviruses that were also neutralized by the combination of CH01 and VRCCH31, it is plausible that other antibody specificities with restricted breadth of neutralization were made that account for the 5% of serum neutralization missed by the combination of CH01 and VRC-CH31. The combination of CH01 and VRC-CH31 weakly neutralized three pseudoviruses, B.6240, AE.703357, and C.ZM53 (IC50s of 10, 18, and 7.7 g/ml, respectively), that were not neutralized by the serum (ID50, ⬍20; indicated with a ⌬ in Fig. 2), suggesting either that the CH01 and VRC-CH31 serum antibody concentrations were not sufficient to generate detectable levels of neutralization for these strains or that there was some type of interference in epitope recognition by other nonneutralizing antibodies. Overall, the numbers of HIV-1 strains neutralized by the serum and the combination of CH01 and VRC-CH31 were strongly associated (P ⫽ 0.007; Fisher’s exact test) (Fig. 3A). Also, the neutralization titers of the serum and the combination of CH01 and VRC-CH31 strongly correlated (R ⫽ 0.7, P ⬍ 0.0001; linear correlation) (Fig. 3B). We previously demonstrated the substantial
TABLE 1 Characteristics of the V-heavy and V-light chains of the monoclonal antibodies CH01 to CH04 and VRC-CH30 to VRC-CH34 isolated from memory B cells of donor CH0219 V-heavy chain
V-light chain
MAb
V
D
J
Mutation HCDR3 GenBank frequency (%) length (aa) Isotype accession no. V
CH01 CH02 CH03 CH04 VRC-CH30 VRC-CH31 VRC-CH32 VRC-CH33 VRC-CH34
3-20 3-20 3-20 3-20 1-2 1-2 1-2 1-2 1-2
3-10 3-10 3-10 3-10 3-16 3-16 3-16 3-16 3-16
2 2 2 2 4 4 4 4 4
13 14 12 12 23 24 23 23 22
a
24 24 24 24 13 13 13 13 13
IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1
JQ267523 JQ267524 JQ267525 JQ267526 JN159434 JN159435 JN159436 JN159472 JN159473
3-20 3-20 3-20 3-20 1-33 1-33 1-33 1-33 1-33
J
Mutation LCDR3 GenBank frequency (%) length (aa)a / accession no. Reference
1 1 1 1 2 2 2 2 2
10 14 11 9 16 15 17 17 17
9 9 9 9 5 5 5 5 5
JQ267519 JQ267520 JQ267521 JQ267522 JN159437 JN159438 JN159439 JN159470 JN159471
2 2 2 2 25 25 25 25 25
LCDR3, light-chain complementarity-determining region 3.
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FIG 2 Comparison of the neutralization profiles of the serum of donor CH0219 and MAbs CH01 and VRC-CH31 either alone or in combination. Neutralizing activity was tested on a panel of 97 Env-pseudotyped lentiviruses, which comprised tier 1 and tier 2 isolates as well as transmitted founder viruses from multiple clades. CH01 and VRC-CH31 MAbs were tested both alone and in combination at a 1:1 ratio. Monoclonal antibody activity is shown as IC50 (g/ml) and color coded based on the following IC50 ranges: ⬍0.1 (red), 0.1 to 1 (orange), 1.1 to 10 (bright yellow), 11 to 50 (straw yellow), and ⬎50 (white). Serum activity is shown as ID50 titer (1/dilution). Serum neutralization resistance was defined as an ID50 titer less than 20. Concordance of neutralization between the serum and the combination of the CH01 and the VRC-CH31 MAbs is noted as follows: ✓, sensitivity to both serum and the MAb combination; ⫻, sensitivity to serum only; ⌬, sensitivity to the MAb combination only; ⫽, neutralization resistance to both serum and MAb combination. Part of the data for CH01 MAb alone has been previously published (2).
contribution of V1V2 conformational epitope and CD4bs antibodies to CH0219 serum neutralizing activity (20). To evaluate the levels of serum antibodies with CH01-like and VRC-CH31like specificities, we measured the ability of CH0219 serum to
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compete binding of biotin-labeled CH01 and VRC-CH31 MAbs to the E.A244 gp120 envelope glycoprotein (16) and calculated their concentrations based on standard curves measuring the blocking obtained with cold CH01 and VRC-CH31 MAbs. At a
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FIG 3 Correlation analysis of neutralization breadths and titers of donor CH0219 serum and the combination of CH01 and VRC-CH31 MAbs. (A) Contingency table showing the numbers of isolates neutralized by serum of donor CH0219 and/or the combination of CH01 and VRC-CH31 MAbs. (B) Linear regression analysis comparing IC50s of the CH01 and VRC-CH31 combination (y axis) and serum ID50 values (x axis).
1:12.5 dilution, CH0219 serum blocked 74.3% and 50.1% of CH01 and VRC-CH31 MAb binding, respectively (data not shown). The serum concentrations of CH01-like and VRC-CH31like antibodies were 70.4 ⫾ 8.1 (mean ⫾ standard deviation) CH01 g/ml equivalents and 42 ⫾ 10.5 VRC-CH31 g/ml equivalents, respectively. In both cases, the serum levels of CH01-like and VRC-CH31-like antibodies estimated from standard curves of CH01 and VRC-CH31 MAbs are higher than the concentration needed to mediate neutralization in the TZM-bl assay (Fig. 2). These findings suggest that, within the context of the polyclonal anti-envelope response, the combination of CH01 and VRCCH31 is responsible for the majority of the neutralizing activity observed in the serum. Remarkably, the combination of CH01 and VRC-CH31 bnAbs achieved a near panneutralization (89/97; 92%) of the panel of Env-pseudotyped viruses, which is broader than the neutralization achieved by the two bnAbs alone (Fig. 3A and Table 1). The combination of CH01 and VRC-CH31 bnAbs, tested at a 1:1 ratio, did not display any appreciable additive or synergistic effect on the magnitude of neutralization: in fact, 87 of the 89 viruses (97.8%) were neutralized by the combination of CH01 and VRC-CH31 bnAbs with a titer comparable to the most potent of the two bnAbs (Fig. 2). However, for 22 viruses, the neutralization titer of a single bnAb was already at or below the lower limit of detection (IC50 ⫽ 0.02 g/ml), and in these cases, a synergistic or additive effect could not be ruled out. In only one case was the IC50 of the bnAb combination ⬎1 order of magnitude higher than that of the most potent bnAb (for A.191955, the CH01 IC50 was ⬍0.02 g/ml and
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the CH01 plus VRC-CH31 IC50 was 0.1 g/ml; Fig. 2). Therefore, we conclude that the combination of CH01 and VRC-CH31 bnAbs does not have detrimental effects on the magnitude of neutralization of each of the individual antibodies and that the combination broadens the overall spectrum of in vitro neutralization. The findings presented here are relevant for vaccine development design in three aspects: first, the clones of the two bnAbs produced by subject CH0219 came from different VH genes and did not present similarities in terms of accumulation of somatic mutations or HCDR3 length; second, the combination of two bnAbs from each of the two clones is sufficient to confer near-panHIV-1 neutralization; and, third, our data suggest that vaccine formulations capable of inducing bnAbs of both specificities will have an advantage over monovalent formulations in inducing broader neutralization. In conclusion, we present the first isolation of two clonal lineages of bnAbs with distinct specificities from memory B cells of a single individual. Two bnAbs from these clones largely recapitulate the breadth of the donor’s serum neutralization and achieve near pan-HIV-1 neutralization, implying that a vaccine capable of inducing bnAbs against both the CD4bs and the V1V2 conformational epitope could achieve broader HIV-1 neutralization than a vaccine inducing only one of the two specificities. These observations demonstrate that the concurrent development of bnAbs targeting diverse regions of the gp120 envelope glycoprotein in the memory B-cell compartment of a single subject is possible. Such in vivo cooperative regulation of two different bnAbs provides proof of concept supporting the design of polyvalent vaccines. ACKNOWLEDGMENTS This work was supported by NIH, NIAID, the Division of AIDS with the Center for HIV/AIDS Vaccine Immunology (CHAVI; grant U19 AI067854), and by the Intramural Research Program of the Vaccine Research Center, NIAID, NIH.
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