Binding of inferred germline precursors of broadly neutralizing HIV-1 antibodies to native-like envelope trimers

Virology 486 (2015) 116–120

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Brief Communication

Binding of inferred germline precursors of broadly neutralizing HIV-1 antibodies to native-like envelope trimers Kwinten Sliepen a,1, Max Medina-Ramírez a,1, Anila Yasmeen b, John P. Moore b, Per Johan Klasse b, Rogier W. Sanders a,b,n a b

Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10065, USA

art ic l e i nf o

a b s t r a c t

Article history: Received 3 July 2015 Returned to author for revisions 2 August 2015 Accepted 3 August 2015 Available online 1 October 2015

HIV-1 envelope glycoproteins (Env) and Env-based immunogens usually do not interact efficiently with the inferred germline precursors of known broadly neutralizing antibodies (bNAbs). This deficiency may be one reason why Env and Env-based immunogens are not efficient at inducing bNAbs. We evaluated the binding of 15 inferred germline precursors of bNAbs directed to different epitope clusters to three soluble native-like SOSIP.664 Env trimers. We found that native-like SOSIP.664 trimers bind to some inferred germline precursors of bNAbs, particularly ones involving the V1/V2 loops at the apex of the trimer. The data imply that native-like SOSIP.664 trimers will be an appropriate platform for structure-guided design improvements intended to create immunogens able to target the germline precursors of bNAbs. & 2015 Elsevier Inc. All rights reserved.

Keywords: Broadly neutralizing antibodies Germline HIV-1 Env SOSIP Vaccine

To be effective, a HIV-1 vaccine should elicit bNAbs that target the trimeric Env spike on the virion surface (van Gils and Sanders, 2014). No Env immunogen has been able to elicit bNAbs in animals or humans, but
20% of HIV-1-infected patients do eventually develop these antibodies after
2–3 years, and some exceptional patients develop bNAbs within a year (van den Kerkhof et al., 2014). Longitudinal analyses have shown that bNAbs generally emerge through a co-evolutionary process that is driven by iterative cycles of HIV-1 escape from more narrowly focused NAbs, followed by renewed Ab affinity maturation (Doria-Rose et al., 2014; Liao et al., 2013). To generate bNAbs by vaccination, it may be necessary to mimic such affinity maturation pathways (Haynes et al., 2012). Initiating any particular bNAb lineage requires activating the naïve B cells through their B cell receptor, i.e. the unmutated germline antibody (Haynes et al., 2012). For this to happen in a vaccine setting, the Env-based immunogen should, therefore, be capable of binding germline antibodies that have the potential to evolve into bNAbs. A complication is that most HIV-1 isolates appear incapable of

n Corresponding author at: Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands. E-mail addresses: [email protected] (K. Sliepen), [email protected] (M. Medina-Ramírez), [email protected] (A. Yasmeen), [email protected] (J.P. Moore), [email protected] (P.J. Klasse), [email protected] (R.W. Sanders). 1 These authors contributed equally to the work.

http://dx.doi.org/10.1016/j.virol.2015.08.002 0042-6822/& 2015 Elsevier Inc. All rights reserved.

interacting with the germline versions of bNAbs, which may be the outcome of how HIV-1 immune evasion strategies have evolved over time. In consequence, most recombinant Env proteins also cannot engage the inferred germline precursors of known bNAbs (gl-bNAbs) (Hoot et al., 2013; McGuire et al., 2013a), either because they adopt non-native conformations or because they are derived from viruses that also lack the required reactivity. The problem is not universal, in that some Env proteins based on autologous founder virus sequences isolated from the patient from which a particular bNAb was isolated can sometimes bind the germline precursor of that bNAb (Doria-Rose et al., 2014; Liao et al., 2013; Lynch et al., 2015). Furthermore, Env immunogens can be specifically engineered to have such properties (Dosenovic et al., 2015; Jardine et al., 2013, 2015; McGuire et al., 2013b). Recently, several soluble, recombinant SOSIP.664 Env trimers from clades A (isolate BG505), B (isolate B41) and C (isolates ZM197M and DU422) have been described (Pugach et al., 2015; Sanders et al., 2013; Julien et al., 2015). Electron microscopy imaging, glycan profiling and antigenicity studies show that these SOSIP.664 trimers mimic the virion-associated Env trimer (Pritchard et al., 2015; Pugach et al., 2015; Sanders et al., 2013; Julien et al., 2015). In addition, the BG505 and B41 SOSIP.664 trimers have induced consistent NAb responses against the autologous tier 2 viruses, which has not been achieved by non-native Env immunogens (Sanders et al., 2015). Whether native-like trimers such as the above SOSIP.664 proteins can interact with gl-bNAbs is clearly relevant to strategies

K. Sliepen et al. / Virology 486 (2015) 116–120

intended to induce neutralization breadth. There are reasons to believe that trimers that do so may be desirable. First, only nativelike trimers consistently present several quaternary structuredependent bNAb epitopes at the V1V2-apex or the gp120/gp41 interface (Blattner et al., 2014; Huang et al., 2014; Sanders et al., 2013). Second, native-like trimers force the appropriate restrictions on the selection of Abs with the correct, trimer-compatible angles of approach, and thereby limit the exposure of immunodominant non-neutralizing epitopes that could interfere with the triggering of the desired bNAb germline (McGuire et al., 2014; Sanders et al., 2013; Tran et al., 2014). We have, therefore, assessed whether the BG505, B41 and ZM197M SOSIP.664 trimers can interact with a set of 15 gl-bNAbs. Epitope-tagged SOSIP.664-D7324 or SOSIP.664-His trimers, expressed in 293F cells, were purified by PGT145 bNAb-affinity chromatography (Pugach et al., 2015). We used ELISA and, in some cases, surface plasmon resonance (SPR) methods to assess trimer binding to 15 gl-bNAbs, targeting five distinct Env epitope clusters: the CD4 binding site (CD4bs) (VRC01, 3BNC60, 1NC9, CH103, CH31); the glycan-dependent V3 cluster (PGT121, PGT128); the V1V2-apex (PG9, PG16, PGT145, VRC26.09, CH01) (Doria-Rose et al., 2014; West et al., 2014); the gp120/gp41 interface (PGT151, 35O22) (Blattner et al., 2014; Huang et al., 2014); gp41 (3BC315) (Lee et al., 2015). We did not test binding to gp120 monomers or uncleaved gp140 proteins, since the mature versions of PG9, PG16, PGT145, VRC26.09, PGT151, 35O22 and 3BC315 have been reported to bind these proteins very inefficiently (Blattner et al., 2014; Doria-Rose et al., 2014; Huang et al., 2014; Ringe et al., 2013; Yasmeen et al., 2014; Lee et al., 2015). The sequences of germline versions of PG9, PGT145, PGT151, 35O22, PGT128, 1NC9, 3BC315 were inferred using the IMGT/V QUEST online tools (Giudicelli et al., 2004) (Table 1). We note that PG9 and PG16 are clonal relatives and originate from the same germline precursor (Pancera et al., 2010). However, it is difficult to infer an accurate germline sequence of the long heavy chain complementarity-determining regions 3 (HCDR3) of these antibodies. Therefore, we used two different germline precursors of the PG9/16 lineage: gl-PG16 was based on the previously published sequence (Pancera et al., 2010) and gl-PG9 was inferred as described above. The gl-bNAb sequences were synthesized by Genscript, cloned into the pVRC8400 expression vector, transfected into 293F cells and then purified by a protein A/G agarose

117

column (Thermo Scientific). The mature and germline versions of PG9, PG16 and PGT145 were expressed in the presence of exogenous tyrosylprotein sulfotransferase 1 (TPST1) to ensure they were tyrosine-sulfated (Julien et al., 2013). The germline versions of 3BNC60, VRC01, CH31, CH103, PGT121, CH01 and VRC26 were kindly provided by colleagues (Table 1). The ELISA for measuring Ab-trimer binding was modified from a published method (Sanders et al., 2013), as follows: 3 μg/ml of SOSIP.664D7324 proteins were diluted in Tris-buffered saline pH 7.5 (TBS) containing 10% fetal calf serum (FCS), and captured on D7324 Abcoated plates. Mature and gl-bNAbs were serially diluted in caseinblocking solution (Thermo Scientific). Half-maximal binding Ab concentrations (EC50) were derived using Graphpad Prism (version 5.01). All 15 mature bNAbs bound to all three SOSIP.664-D7324 trimers, which is mostly consistent with the antigenicity profiles reported previously (Pugach et al., 2015; Sanders et al., 2013; Julien et al., 2015) (Table 2). We note that, here, the mature versions of PGT151 and 35O22 were reactive with the B41 SOSIP.664 trimers in ELISA (Table 2), which was not seen in our previous study (Pugach et al., 2015). The difference may arise because we increased the assay sensitivity by using a higher input concentration of the B41 SOSIP.664-D7324 trimer (i.e., 3 mg/ml vs. 0.3 mg/ml, previously) and we used a different blocking solution (i.e., casein blocking solution vs. 2% milk, previously). The BG505 SOSIP.664-D7324 trimer bound to gl-PG9 and its somatic relative gl-PG16 (EC50: 1.0 and 15 μg/ml, respectively), its ZM197M counterpart bound to gl-PG9 (EC50: 6.8 μg/ml) but not glPG16, while the B41 trimer bound neither glPG9 nor glPG16 (Fig. 1 and Table 2). Compared to gl-PG16, gl-PG9 contains more tyrosines that are potentially sulfated, which could increase affinity for the cationic groove at the trimer apex. This might explain the increased reactivity of gl-PG9 with BG505 and ZM197M SOSIP.664 trimers. The BG505 and B41 trimers also bound to gl-CH01 (EC50: 0.64 and 1.0 μg/ml respectively). Gl-VRC26 and gl-PGT145, which bind to a similar epitope as PG9 and PG16, but have entirely different HCDR3 loops and are derived from different germline genes, did not bind to any of the SOSIP.664 trimers. We observed a remarkably high affinity interaction between gl-3BC315 and the BG505 and ZM197M trimers (EC50: 0.27; 0.54 μg/ml respectively) (Fig. 1 and Table 2). We note, however, that the HCDR3 of the germline- and mature versions of 3BC315 were identical, since it was not possible to reliably infer the germline HCDR3 sequence from the mature

Table 1 Putative gene usage and CDR3 sequences of the gl-bNAbs used in this study. Antibody

VH-gene n

PG9 PG16 PGT145 VRC26.09

V3-33 05 V3-33n05 V1-8n01 V3-30n18

CH01 VRC01 3BNC60 CH31 CH103 1NC9 PGT121 PGT128 PGT151 35O22 3BC315

V3-20n01 V1-2n02 V1-2n02 V1-2n02 V4-59n01 V1-46n01 V4-59n01 V4-39n07 V3-30n04 V1-18n02 V1-2n02

a

HCDR3a

JH-gene b

AREAGGPDYRNGYNYYDFWSGYYTYYYMDV AREAGGPIWHDDVKYYDFNDGYYNYHYMDV GSKHRLRDYFLYNEYGPNYEEWGDYLATLDVc CAKDLGESENEEWAYDYYDFSIGYPGQ DPRGVVGAFDIW GTDYTIDDQGIRYQGSGTFWYFDL ARGKNSDYNWDFQH ARQRSDFWDFDL ARAQKRGRSEWAYAH SLPRGQLVNAYFDY QDSDFHDGHGHTLRGMFDYc TLHGRRIYGIVAFNEWFTYFYMDV FGGEVLRFLEWPKPAWFDPb ARMFQESGPPRLDRWSGRNYYYYYGMDVc GLLRDGSSTWLPYLc PMRPVSHGIDYSGLFVFQFc

n

VL-gene n

LCDR3a

JL-gene n

Source

Reference

J6 03 J6n03 J6n02 J3n02

LV2-14 01 LV2-14n01 KV2-28n01 LV1-50n02

SSYTSSSTLV SSYTSSSTLV MQALQTPWT GTWDSSLSAGGVF

LJ3 02 LJ3n02 KJ1n01 LJ1-02

IMGT P. Kwong IMGT J. Mascola

N.A. Pancera et al., 2010 N.A. Doria-Rose et al., 2014

J2n01 J2n01 J2n01 J4n02 J4n02 J4n02 J6n03 J5n02 J6n02 J4n02 J3n01

KV3-20n01 KV3-11n01 KV1-33n01 KV1-33n01 LV3-1n01 LV1-47n01 LV3-21n02 LV2-8n01 KV2D-29n02 LV2-14n02 LV2-23n02

CQQYGSSPYTF CQQYEFF CQQYEFI CQQYETF CQAWDSFSTFV CAAWDDSLSGPVF QVWDSRVITKWV SSYAGNWDVV MQSKDFPLT SSYTSSSGCV CSYANYDKLI

KJ1n01 KJ2n01 KJ3n01 KJ2n01 LJ1n01 LJ2n01 LJ3n02 LJ2n01 KJ4n01 LJ1n01 LJ3n01

B. Haynes&H. Liao W. Schief M. Nussenzweig B. Haynes&H. Liao B. Haynes&H. Liao IMGT M. Nussensweig IMGT IMGT IMGT IMGT

Bonsignori et al., 2011 Jardine et al., 2013 Scheid et al., 2011 Wu et al., 2011 Liao et al., 2013 N.A. N.A. N.A. N.A. N.A. N.A.

The CDR3 was defined based on www.bnaber.org when available or by using www.imgt.org. The N region of the junction between the genes V and D was taken from the mature sequence, since reversion was not possible. The rest of the sequence was inferred by combining D and J genes. c The CDR3 sequence was taken from the mature version whenever the D gene generated by IMGT did not provide an acceptable match to the mature nucleotide sequence. b

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K. Sliepen et al. / Virology 486 (2015) 116–120

Table 2 Summary of EC50 values (in ng/ml) derived from D7324-capture ELISA with different SOSIP.664 trimers. For comparison, midpoint neutralization concentrations (IC50 in ng/ml) of the sequence-matched Env-peudotyped viruses are also indicated.

BG505 Germline 935 13100 >25000 >25000 640 >25000 >25000 >25000 >25000 >25000 >25000 >25000 >25000 >25000 538

PG9 PG16 PGT145 VRC26.09 CH01 VRC01 3BNC60 1NC9 CH103 CH31 PGT121 PGT128 PGT151 35O22 3BC315

Mature 45 * 8* 83 * <10 301 * 70 * 34 * 251 * 2572 * 14 * 15 * 11 * 1 8 1259 *

SOSIP.664 Germline >25000 >25000 >25000 >25000 1003 >25000 >25000 >25000 >25000 >25000 >25000 >25000 >25000 >25000 >25000

ZM197M pseudovirus

Mature 242 65 67 1204 44 34 55 355 2412 32 147 46 126 1052 102

Mature 560 * 80 * 60 >25000 N.D. 1380 * 11 1961 >25000 26 920 * 414 300 * >25000 >25000

SOSIP.664 Germline 6769 >25000 >25000 >25000 >25000 >25000 >25000 >25000 18090 >25000 >25000 >25000 >25000 >25000 266

pseudovirus

Mature 103 77 27 50 2044 7 58 128 143 48 35 6 30 463 124

Mature 393 * 49 * 393 * 4* N.D. 329 * 77 1006* 18222 358 35 * 33 * 2* 2816 * 3549 *

EC50 (ng/ml): <250 250-2500 2500-25000 >25000

Mature

OD450

values obtained from (Sanders et al., 2013) for BG505; (Pugach et al., 2015) for B41; (Julien et al., 2015) for ZM197M N.D.: not determined

Germline

*: IC

Mature 40 31 12 31 58 9 21 209 179 13 61 12 28 379 144

B41 pseudovirus

OD450

gp41 and V3 interface glycan

CD4bs

V1V2 apex

SOSIP.664

Antibody (ng/ml)

Fig. 1. Representative binding curves of a panel of mature and gl-bNAbs tested in the same ELISA. We note that all mature (bottom) and gl-bNAbs (top) generated comparable and low background signals in this ELISA format (“mock”: the wells contained only 10% FCS in TBS), except gl-CH103, for which the background was considerably higher (“gl-CH103 mock”).

version. As the HCDR3 of 3BC315 is known to make important hydrophobic contacts with the gp41/gp120 interface (Lee et al., 2015), this interaction may contribute to the high affinity the gl3BC315 antibody has for the BG505 and ZM197M trimers. However, as gl-3BC315 did not bind to the B41 trimers, there are complexities to the Ab-trimer binding events that remain to be understood, such as the involvement of topologically proximal glycans in either the formation or the occlusion of the epitope (Lee et al., 2015). None of the three trimers bound the gl-bNAbs targeting the glycan-dependent V3 epitopes (PGT121 and PGT128). They also did not interact detectably with the VRC01, 3BNC60, 1NC9 or CH31 gl-bNAbs against the CD4bs, perhaps because of the shielding effects of various trimer glycans. An example is the clash between the N276 glycan and the gl-VRC01 light chain (McGuire et al., 2013b). While gl-CH103 was prone to generating a high level of non-specific background signals in ELISA (Fig. 1, “gl-CH103 mock”), we did detect some specific binding of this antibody to the ZM197M trimers (EC50: 18.1 μg/ml) although not to their BG505 and B41 counterparts (Fig. 1 and Table 2). To corroborate the binding of gl-PG16 to the BG505 SOSIP.664D7324 trimers, we performed SPR analyses using the His-tagged version of the same trimers. The data were fitted with a bivalent

model, and binding parameters for the monovalent component in the bivalent model are given (Yasmeen et al., 2014). We confirmed that gl-PG16 bound to BG505 SOSIP.664-His trimers, although with a lower affinity (dissociation constant, Kd ¼320 nM) than mature PG16 (Kd ¼24 nM) (Fig. 2A). The reduced affinity arises because kon (on-rate constant) and koff (off-rate constant) were
5-fold lower and
3-fold higher, respectively, for gl-PG16 (Fig. 2B). As the binding stoichiometry was also lower for gl-PG16 (Sm ¼0.4) than for mature PG16 (Sm ¼1.3) only a subset of the trimers can bind glPG16, perhaps because of variation in the presence or composition of particular glycan sites (Fig. 2B). The difference in binding to BG505 SOSIP.664 between gl-PG16 and mature PG16 were more substantial in ELISA (EC50 values¼13.1 μg/ml and 0.031 μg/ml, respectively) than in SPR (Kd ¼320 nM and 24 nM, respectively). This can probably be explained by the high off-rate, which results in loss of binding signal in ELISA during washing steps. We note that the BG505 trimers are based on a transmitter/founder virus sequence that is similar in the relevant regions to viruses isolated from the donor of the PG9 and PG16 bNAbs (Hoffenberg et al., 2013; Sanders et al., 2013). This sequence homology may help explain why gl-PG9 and gl-PG16 bind to the BG505 trimers. In contrast, but consistent with the ELISA data, gl-VRC01 did not bind to BG505 SOSIP.664-His trimers in the SPR assay (Fig. 2A).

K. Sliepen et al. / Virology 486 (2015) 116–120

A germline

Kd = 320 nM 200

mature

Kd = 24 nM

150

100

Response difference (RU)

B

PG16 150

119

100

50 50

0

bNAb

0

0

300

600

900

0

300

600

900

VRC01 350

germline

276

no binding

202

500 400 300

128

200 100

54 0

0 0

300

600

900

mature

0

300

Kd = 0.039 nM

600

1000 nM 500 nM 250 nM 125 nM 62.5 nM 31.2 nM 15.6 nM 3.9 nM

PG16 (Germline) PG16 (Mature) VRC01 (Germline) VRC01 (Mature)

Number of replicates (n=2) (n=3)

k (1/Ms) 2.3 10

k (1/s) 7.0 10

k (1/RUs) 4.7 10

k (1/s) 1.3 10

K (nM) 3.2 10

K (RU) 2.7 10

S

± 4.6 . 10

± 3.2 10

± 3.9 10

± 2.8 10

± 3.7

± 0.07

1.1 10

2.6 10

3.4 10

6.5 10

1.7 10

1.3

± 4.4 10

±2.6 10

± 1.8 10

± 4.3 10

± 78 24 ±3

± 3.6 10

± 0.02

0.4

(n=1)

No Binding

(n=3)

5.9 10

2.0 10

3.5 10

1.8 10

3.9 10

4.9 10

2.2

± 5.3 10

± 1.4 10

± 1.2 10

± 7.9 10

± 2.9 10

± 8.0 10

± 0.1

900

Time (s)

Fig. 2. Antibody binding to BG505 SOSIP.664 by SPR analysis. (A) Kinetic binding curves obtained by SPR and derived using the mature and gl- versions of PG16 and VRCO1, and chip-immobilized BG505 SOSIP.664-His trimers. (B) Tabulated summary of the SPR analyses performed on BG505 SOSIP.664.

Gl-bNAb sequences are based on in silico predictions. Whether these inferred gl-bNAbs truly represent the in vivo naïve B cell receptors remains to be determined. Nevertheless, this exploratory study indicates that various SOSIP.664 trimers can bind to several gl-bNAbs, mainly ones that are directed to the V1/V2 loops. Hence these trimers are good starting points for engineering immunogens that more efficiently activate naïve B cell receptors and thereby initiate pathways that lead to the eventual emergence of bNAbs.

Funding MMR is a recipient of a fellowship from the Consejo Nacional de Ciencia y Tecnologıa (CONACyT) of Mexico (CONACyT 186397 and 208235). RWS is a recipient of a Vidi Grant from the Netherlands Organization for Scientific Research (NWO) (Vidi 917.11.314) and a Starting Investigator Grant from the European Research Council (ERC-StG-2011-280829-SHEV). This work was also supported by NIH Grants P01 AI082362, P01 AI110657 and R37 AI36082 (JPM).

Acknowledgments We thank Pia Dosenovic, Michel Nussenzweig, Larry Liao, Barton Haynes, Bill Schief, John Mascola and Peter Kwong for providing antibodies; Jean-Philippe Julien for providing the TPST1 plasmid; Steven de Taeye and Alba Torrents de la Peña for providing BG505, B41 and ZM197M SOSIP.664-D7324 trimers; Ronald Derking and Marit van Gils for providing virus neutralization data; and Judith Burger for technical support. References Blattner, C., Lee, J., Sliepen, K., Derking, R., Falkowska, E., de la Peña, A., Cupo, A., Julien, J.-P., van Gils, M.J., Lee, P.S., Peng, W., Paulson, J.C., Poignard, P., Burton, D.R., Moore, J.P., Sanders, R.W., Wilson, I.A., Ward, A.B., 2014. Structural delineation of a quaternary, cleavage-dependent epitope at the gp41–gp120 interface on intact HIV-1 env trimers. Immunity 40, 669–680. http://dx.doi.org/10.1016/j. immuni.2014.04.008. Bonsignori, M., Hwang, K.-K., Chen, X., Tsao, C.-Y., Morris, L., Gray, E., Marshall, D.J., Crump, J.A., Kapiga, S.H., Sam, N.E., Sinangil, F., Pancera, M., Yongping, Y., Zhang, B., Zhu, J., Kwong, P.D., O’Dell, S., Mascola, J.R., Wu, L., Nabel, G.J., Phogat, S., Seaman, M.S., Whitesides, J.F., Moody, M.A., Kelsoe, G., Yang, X., Sodroski, J., Shaw, G.M., Montefiori, D.C., Kepler, T.B., Tomaras, G.D., Alam, S.M., Liao, H.-X., Haynes, B.F., 2011. Analysis of a clonal lineage of HIV-1 envelope V2/V3 conformational epitope-specific broadly neutralizing antibodies and their inferred unmutated common ancestors. J. Virol. 85, 9998–10009. http://dx. doi.org/10.1128/JVI.05045-11. Doria-Rose, N.A., Schramm, C.A., Gorman, J., Moore, P.L., Bhiman, J.N., DeKosky, B.J., Ernandes, M.J., Georgiev, I.S., Kim, H.J., Pancera, M., Staupe, R.P., Altae-Tran, H.R.,

Bailer, M.K., Crooks, E.T., Cupo, A., Druz, A., Garrett, N.J., Hoi, K.H., Kong, R., Louder, M.K., Longo, N.S., McKee, K., Nonyane, M., O’Dell, S., Roark, R.S., Rudicell, R.S., Schmidt, S.D., Sheward, D.J., Soto, C., Wibmer, C.K., Yang, Y., Zhang, Z., Mullikin, J.C., Binley, J.M., Sanders, R.W., Wilson, I.A., Moore, J.P., Ward, A.B., Georgiou, G., Williamson, C., Abdool Karim, S.S., Morris, L., Kwong, P.D., Shapiro, L., Mascola, J.R., 2014. Developmental pathway for potent V1V2-directed HIVneutralizing antibodies. Nature 509, 55–62. http://dx.doi.org/10.1038/nature13036. Dosenovic, P., von Boehmer, L., Escolano, A., Jardine, J., Freund, N.T., Gitlin, A.D., McGuire, A.T., Kulp, D.W., Oliveira, T., Scharf, L., Pietzsch, J., Gray, M.D., Cupo, A., van Gils, M.J., Yao, K.-H., Liu, C., Gazumyan, A., Seaman, M.S., Björkman, P.J., Sanders, R.W., Moore, J.P., Stamatatos, L., Schief, W.R., Nussenzweig, M.C., 2015. Immunization for HIV-1 broadly neutralizing antibodies in human Ig knockin mice. Cell 161, 1505–1515. http://dx.doi.org/10.1016/j.cell.2015.06.003. Giudicelli, V., Chaume, D., Lefranc, M.P., 2004. IMGT/V-QUEST, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis. Nucleic Acids Res. 32, 435–440. http://dx.doi.org/10.1093/nar/ gkh412. Haynes, B.F., Kelsoe, G., Harrison, S.C., Kepler, T.B., 2012. B-cell-lineage immunogen design in vaccine development with HIV-1 as a case study. Nat. Biotechnol. 30, 423–433. http://dx.doi.org/10.1038/nbt.2197. Hoffenberg, S., Powell, R., Carpov, A., Wagner, D., Wilson, A., Kosakovsky Pond, S., Lindsay, R., Arendt, H., Destefano, J., Phogat, S., Poignard, P., Fling, S.P., Simek, M., Labranche, C., Montefiori, D., Wrin, T., Phung, P., Burton, D., Koff, W., King, C. R., Parks, C.L., Caulfield, M.J., 2013. Identification of an HIV-1 clade A envelope that exhibits broad antigenicity and neutralization sensitivity and elicits antibodies targeting three distinct epitopes. J. Virol. 87, 5372–5383. http://dx. doi.org/10.1128/JVI.02827-12. Hoot, S., McGuire, A.T., Cohen, K.W., Strong, R.K., Hangartner, L., Klein, F., Diskin, R., Scheid, J.F., Sather, D.N., Burton, D.R., Stamatatos, L., 2013. Recombinant HIV envelope proteins fail to engage germline versions of anti-CD4bs bNAbs. PLoS Pathog. 9, e1003106. http://dx.doi.org/10.1371/journal.ppat.1003106. Huang, J., Kang, B.H., Pancera, M., Lee, J.H., Tong, T., Feng, Y., Imamichi, H., Georgiev, I.S., Chuang, G.-Y., Druz, A., Doria-Rose, N.A., Laub, L., Sliepen, K., van Gils, M.J., de la Peña, A.T., Derking, R., Klasse, P.J., Migueles, S.A., Bailer, R.T., Alam, M., Pugach, P., Haynes, B.F., Wyatt, R.T., Sanders, R.W., Binley, J.M., Ward, A.B., Mascola, J.R., Kwong, P.D., Connors, M., 2014. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41–gp120 interface. Nature 515, 138–142. http://dx.doi.org/10.1038/nature13601. Jardine, J., Julien, J.-P., Menis, S., Ota, T., Kalyuzhniy, O., McGuire, A., Sok, D., Huang, P.-S., MacPherson, S., Jones, M., Nieusma, T., Mathison, J., Baker, D., Ward, A.B., Burton, D.R., Stamatatos, L., Nemazee, D., Wilson, I.A., Schief, W.R., 2013. Rational HIV immunogen design to target specific germline B cell receptors. Science 340, 711–716. http://dx.doi.org/10.1126/science.1234150. Jardine, J.G., Ota, T., Sok, D., Pauthner, M., Kulp, D.W., Kalyuzhniy, O., Skog, P.D., Thinnes, T.C., Bhullar, D., Briney, B., Menis, S., Jones, M., Kubitz, M., Spencer, S., Adachi, Y., Burton, D.R., Schief, W.R., Nemazee, D., 2015. Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen. Science 80, 1–12. http://dx.doi.org/10.1126/science.aac5894. Julien, J.-P., Lee, J.H., Ozorowski, G., Hua, Y., de la Peña, A.T., de Taeye, S.W., Nieusma, T., Cupo, A., Yasmeen, A., Golabek, M., Pugach, P., Klasse, P.J., Moore, J.P., Sanders, R.W., Ward, A.B., Wilson, I.A., 2015. Design and structure of two HIV-1 clade C SOSIP.664 trimers that increase the arsenal of native-like Env immunogens. Proc. Natl. Acad. Sci. USA. 112, 11947–11952. http://dx.doi.org/10.1073/ pnas.1507793112. Julien, J.-P., Lee, J.H., Cupo, A., Murin, C.D., Derking, R., Hoffenberg, S., Caulfield, M.J., King, C.R., Marozsan, A.J., Klasse, P.J., Sanders, R.W., Moore, J.P., Wilson, I.A., Ward, A.B., 2013. Asymmetric recognition of the HIV-1 trimer by broadly neutralizing antibody PG9. Proc. Natl. Acad. Sci. USA 110, 4351–4356. http://dx. doi.org/10.1073/pnas.1217537110. Lee, J.H., Leaman, D.P., Kim, A.S., de la Peña, A.T., Sliepen, K., Yasmeen, A., Derking, R., Ramos, A., de Taeye, S.W., Ozorowski, G., Klein, F., Burton, D.R., Nussenzweig,

120

K. Sliepen et al. / Virology 486 (2015) 116–120

M.C., Poignard, P.C., Moore, J.P., Klasse, P.J., Sanders, R.W., Zwick, M.B., Wilson, I. A., Ward, A.B., 2015. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike. Nat. Commun. 6, 8167. http://dx.doi.org/ 10.1038/ncomms9167. Liao, H.-X., Lynch, R., Zhou, T., Gao, F., Alam, S.M., Boyd, S.D., Fire, A.Z., Roskin, K.M., Schramm, C.A., Zhang, Z., Zhu, J., Shapiro, L., Mullikin, J.C., Gnanakaran, S., Hraber, P., Wiehe, K., Kelsoe, G., Yang, G., Xia, S.-M., Montefiori, D.C., Parks, R., Lloyd, K.E., Scearce, R.M., Soderberg, K.A., Cohen, M., Kamanga, G., Louder, M.K., Tran, L.M., Chen, Y., Cai, F., Chen, S., Moquin, S., Du, X., Joyce, M.G., Srivatsan, S., Zhang, B., Zheng, A., Shaw, G.M., Hahn, B.H., Kepler, T.B., Korber, B.T.M., Kwong, P.D., Mascola, J.R., Haynes, B.F., Becker, J., Benjamin, B., Blakesley, R., Bouffard, G., Brooks, S., Coleman, H., Dekhtyar, M., Gregory, M., Guan, X., Gupta, J., Han, J., Hargrove, A., Ho, S., Johnson, T., Legaspi, R., Lovett, S., Maduro, Q., Masiello, C., Maskeri, B., McDowell, J., Montemayor, C., Mullikin, J., Park, M., Riebow, N., Schandler, K., Schmidt, B., Sison, C., Stantripop, M., Thomas, J., Thomas, P., Vemulapalli, M., Young, A., 2013. Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature 496, 469–476. http://dx.doi.org/10.1038/ nature12053. Lynch, R.M., Wong, P., Tran, L., O’Dell, S., Nason, M.C., Li, Y., Wu, X., Mascola, J.R., 2015. HIV-1 fitness cost associated with escape from the VRC01 class of CD4 binding site neutralizing antibodies. J. Virol.89, 4201–4213. http://dx.doi.org/ 10.1128/JVI.03608-14. McGuire, A.T., Dreyer, A.M., Carbonetti, S., Lippy, A., Glenn, J., Scheid, J.F., Mouquet, H., Stamatatos, L., 2014. Antigen modification regulates competition of broad and narrow neutralizing HIV antibodies. Science 346, 1380–1383. http://dx.doi. org/10.1126/science.1259206. McGuire, A.T., Glenn, J.A., Lippy, A., Stamatatos, L., Doms, R.W., 2013a. Diverse recombinant HIV-1 Envs fail to activate B cells expressing the germline B cell receptors of the broadly neutralizing anti-HIV-1 antibodies PG9 and 447-52D. J. Virol. 88, 2645–2657. http://dx.doi.org/10.1128/JVI.03228-13. McGuire, A.T., Hoot, S., Dreyer, A.M., Lippy, A., Stuart, A., Cohen, K.W., Jardine, J., Menis, S., Scheid, J.F., West, A.P., Schief, W.R., Stamatatos, L., 2013b. Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies. J. Exp. Med. 210, 655–663. http://dx.doi. org/10.1084/jem.20122824. Pancera, M., McLellan, J.S., Wu, X., Zhu, J., Changela, A., Schmidt, S.D., Yang, Y., Zhou, T., Phogat, S., Mascola, J.R., Kwong, P.D., 2010. Crystal structure of PG16 and chimeric dissection with somatically related PG9: structure–function analysis of two quaternary-specific antibodies that effectively neutralize HIV-1. J. Virol. 84, 8098–8110. http://dx.doi.org/10.1128/JVI.00966-10. Pritchard, L.K., Vasiljevic, S., Ozorowski, G., Seabright, G.E., Cupo, A., Ringe, R., Kim, H.J., Sanders, R.W., Doores, K.J., Burton, D.R., Wilson, I.A., Ward, A.B., Moore, J.P., Crispin, M., 2015. Structural constraints determine the glycosylation of HIV-1 envelope trimers. Cell Rep. 11, 1604–1613. http://dx.doi.org/10.1016/j. celrep.2015.05.017. Pugach, P., Ozorowski, G., Cupo, A., Ringe, R., Yasmeen, A., de Val, N., Derking, R., Kim, H.J., Korzun, J., Golabek, M., de Los Reyes, K., Ketas, T.J., Julien, J.-P., Burton, D.R., Wilson, I.A., Sanders, R.W., Klasse, P.J., Ward, A.B., Moore, J.P., 2015. A native-like SOSIP.664 trimer based on an HIV-1 subtype B env gene. J. Virol. 89, 3380–3395. http://dx.doi.org/10.1128/JVI.03473-14. Ringe, R.P., Sanders, R.W., Yasmeen, A., Kim, H.J., Lee, J.H., Cupo, A., Korzun, J., Derking, R., van Montfort, T., Julien, J.-P., Wilson, I.a., Klasse, P.J., Ward, A.B.,

Moore, J.P., 2013. Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation. Proc. Natl. Acad. Sci. USA 110, 18256–18261. http://dx.doi.org/10.1073/pnas.1314351110. Sanders, R.W., Derking, R., Cupo, A., Julien, J.P., Yasmeen, A., de Val, N., Kim, H.J., Blattner, C., de la Peña, A.T., Korzun, J., Golabek, M., de los Reyes, K., Ketas, T.J., van Gils, M.J., King, C.R., Wilson, I.A., Ward, A.B., Klasse, P.J., Moore, J.P., 2013. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. PLoS Pathog. 9, e1003618. http://dx.doi.org/10.1371/journal.ppat. 1003618. Sanders, R.W., van Gils, M.J., Derking, R., Sok, D., Ketas, T.J., Burger, J.A., Ozorowski, G., Cupo, A., Simonich, C., Goo, L., Arendt, H., Kim, H.J., Lee, J.H., Pugach, P., Williams, M., Debnath, G., Moldt, B., van Breemen, M.J., Isik, G., MedinaRamirez, M., Back, J.W., Koff, W.C., Julien, J.-P., Rakasz, E.G., Seaman, M.S., Guttman, M., Lee, K.K., Klasse, P.J., LaBranche, C., Schief, W.R., Wilson, I.A., Overbaugh, J., Burton, D.R., Ward, A.B., Montefiori, D.C., Dean, H., Moore, J.P., 2015. HIV-1 neutralizing antibodies induced by native-like envelope trimers. Science 349, aac4223, http://dx.doi.org/10.1126/science.aac4223. Scheid, J.F., Mouquet, H., Ueberheide, B., Diskin, R., Klein, F., Oliveira, T.Y.K., Pietzsch, J., Fenyo, D., Abadir, A., Velinzon, K., Hurley, A., Myung, S., Boulad, F., Poignard, P., Burton, D.R., Pereyra, F., Ho, D.D., Walker, B.D., Seaman, M.S., Bjorkman, P.J., Chait, B.T., Nussenzweig, M.C., 2011. Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science 333, 1633–1637. http://dx.doi.org/10.1126/science.1207227. Tran, K., Poulsen, C., Guenaga, J., de Val Alda, N., Wilson, R., Sundling, C., Li, Y., Stanfield, R.L., Wilson, I.a., Ward, A.B., Karlsson Hedestam, G.B., Wyatt, R.T., 2014. Vaccine-elicited primate antibodies use a distinct approach to the HIV-1 primary receptor binding site informing vaccine redesign. Proc. Natl. Acad. Sci. USA 111, E738–E747. http://dx.doi.org/10.1073/pnas.1319512111. van den Kerkhof, T.L.G.M., Euler, Z., van Gils, M.J., Boeser-Nunnink, B.D., Schuitemaker, H., Sanders, R.W., 2014. Early development of broadly reactive HIV-1 neutralizing activity in elite neutralizers. AIDS 28, 1237–1240. http://dx.doi.org/ 10.1097/QAD.0000000000000228. van Gils, M.J., Sanders, R.W., 2014. In vivo protection by broadly neutralizing HIV antibodies. Trends Microbiol. 22, 550–551. http://dx.doi.org/10.1016/j.tim. 2014.08.006. West, A.P., Scharf, L., Scheid, J.F., Klein, F., Bjorkman, P.J., Nussenzweig, M.C., 2014. Structural insights on the role of antibodies in HIV-1 vaccine and therapy. Cell 156, 633–648. http://dx.doi.org/10.1016/j.cell.2014.01.052. Wu, X., Zhou, T., Zhu, J., Zhang, B., Georgiev, I., Wang, C., Chen, X., Longo, N.S., Louder, M., McKee, K., O’Dell, S., Perfetto, S., Schmidt, S.D., Shi, W., Wu, L., Yang, Y., Yang, Z.-Y.Z., Yang, Z.-Y.Z., Zhang, Z., Bonsignori, M., Crump, J.a., Kapiga, S.H., Sam, N.E., Haynes, B.F., Simek, M., Burton, D.R., Koff, W.C., Doria-Rose, N.a., Connors, M., Mullikin, J.C., Nabel, G.J., Roederer, M., Shapiro, L., Kwong, P.D., Mascola, J.R., 2011. Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing. Science 333, 1593–1602. http://dx.doi.org/ 10.1126/science.1207532. Yasmeen, A., Ringe, R., Derking, R., Cupo, A., Julien, J.-P., Burton, D.R., Ward, A.B., Wilson, I.A., Sanders, R.W., Moore, J.P., Klasse, P.J., 2014. Differential binding of neutralizing and non-neutralizing antibodies to native-like soluble HIV-1 Env trimers, uncleaved Env proteins, and monomeric subunits. Retrovirology 11, 41. http://dx.doi.org/10.1186/1742-4690-11-41.