Citation for published version (APA): Sliepen, K. H. E. W. J. (2016). HIV-1 envelope trimer fusion proteins and their applications

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1 UvA-DARE (Digital Academic Repository) HIV-1 envelope trimer fusion proteins and their applications Sliepen, K.H.E.W.J. Link to publication Citation for published version (APA): Sliepen, K. H. E. W. J. (2016). HIV-1 envelope trimer fusion proteins and their applications General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam ( Download date: 31 Aug 2018

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3 Chapter 5 Binding of inferred germline precursors of broadly neutralizing HIV-1 antibodies to nativelike envelope trimers Kwinten Sliepen 1*,Max Medina-Rami rez 1*, Anila Yasmeen 2, John P. Moore 2, Per Johan Klasse 2, Rogier W. Sanders 1,2 1. Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands 2. Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10065, USA * Co-first author Virology, :

4 Chapter 5 Abstract 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. 88

5 Germline bnab binding to SOSIPs Main text To be effective, a HIV-1 vaccine should elicit bnabs that target the trimeric Env spike on the virion surface [1]. 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 [2]. 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 [3,4]. To generate bnabs by vaccination, it may be necessary to mimic such affinity maturation pathways [5]. Initiating any particular bnab lineage requires activating the naïve B cells through their B cell receptor, i.e. the unmutated germline antibody [5]. 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 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) [6,7], 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 [3,4,8]. Furthermore, Env immunogens can be specifically engineered to have such properties [9 12]. 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 [13 15] Electron microscopy imaging, glycan profiling and antigenicity studies show that these SOSIP.664 trimers mimic the virion-associated Env trimer [13 16]. 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 [17]. Whether native-like trimers such as the above SOSIP.664 proteins can interact with gl-bnabs is clearly relevant to strategies intended to induce neutralization breadth. There are reasons to believe that trimers that do so may be desirable. First, only native-like trimers consistently present several quaternary structure-dependent bnab epitopes at the V1V2-apex or the gp120/gp41 interface [13,18,19]. 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 [13,20,21]. 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 [14]. We used ELISA and, in some cases, surface plasmon resonance (SPR) methods to assess trimer binding to 15 glbnabs, targeting five distinct Env epitope clusters: the CD4 binding site (CD4bs) (VRC01, 3BNC60, 1NC9, CH103, CH31); the glycan-dependent V3 cluster (PGT121, PGT128); 5 89

6 Chapter 5 the V1V2-apex (PG9, PG16, PGT145, VRC26.09, CH01) [4,22]; the gp120/gp41 interface (PGT151, 35O22) [18,19]; gp41 (3BC315) [23]. 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 [4,18,19,23 25]. Table 1. Putative gene usage and CDR3 sequences of the gl-bnabs used in this study. Antibody VH-gene HCDR3 a JH-gene VL-gene LCDR3 a JL-gene Source Reference PG9 V3-33 n 05 AREAGGPDYRNGYNYYDFWSGYYTYYYMDV b J6 n 03 LV2-14 n 01 SSYTSSSTLV LJ3 n 02 IMGT N.A. PG16 V3-33 n 05 AREAGGPIWHDDVKYYDFNDGYYNYHYMDV J6 n 03 LV2-14 n 01 SSYTSSSTLV LJ3 n 02 P. Kwong Pancera et al., 2010 PGT145 V1-8 n 01 GSKHRLRDYFLYNEYGPNYEEWGDYLATLDV c J6 n 02 KV2-28 n 01 MQALQTPWT KJ1 n 01 IMGT N.A. VRC26.09 V3-30 n 18 CAKDLGESENEEWAYDYYDFSIGYPGQ J3 n 02 LV1-50 n 02 GTWDSSLSAGGVF LJ1-02 J. Mascola Doria-Rose et al., 2014 DPRGVVGAFDIW CH01 V3-20 n 01 GTDYTIDDQGIRYQGSGTFWYFDL J2 n 01 KV3-20 n 01 CQQYGSSPYTF KJ1 n 01 B. Haynes&H. Liao Bonsignori et al., 2011 VRC01 V1-2 n 02 ARGKNSDYNWDFQH J2 n 01 KV3-11 n 01 CQQYEFF KJ2 n 01 W. Schief Jardine et al., BNC60 V1-2 n 02 ARQRSDFWDFDL J2 n 01 KV1-33 n 01 CQQYEFI KJ3 n 01 M. Nussenzweig Scheid et al., 2011 CH31 V1-2 n 02 ARAQKRGRSEWAYAH J4 n 02 KV1-33 n 01 CQQYETF KJ2 n 01 B. Haynes&H. Liao Wu et al., 2011 CH103 V4-59 n 01 SLPRGQLVNAYFDY J4 n 02 LV3-1 n 01 CQAWDSFSTFV LJ1 n 01 B. Haynes&H. Liao Liao et al., NC9 V1-46 n 01 QDSDFHDGHGHTLRGMFDY c J4 n 02 LV1-47 n 01 CAAWDDSLSGPVF LJ2 n 01 IMGT N.A. PGT121 V4-59 n 01 TLHGRRIYGIVAFNEWFTYFYMDV J6 n 03 LV3-21 n 02 QVWDSRVITKWV LJ3 n 02 M. Nussensweig N.A. PGT128 V4-39 n 07 FGGEVLRFLEWPKPAWFDP b J5 n 02 LV2-8 n 01 SSYAGNWDVV LJ2 n 01 IMGT N.A. PGT151 V3-30 n 04 ARMFQESGPPRLDRWSGRNYYYYYGMDV c J6 n 02 KV2D-29 n 02 MQSKDFPLT KJ4 n 01 IMGT N.A. 35O22 V1-18 n 02 GLLRDGSSTWLPYL c J4 n 02 LV2-14 n 02 SSYTSSSGCV LJ1 n 01 IMGT N.A. 3BC315 V1-2 n 02 PMRPVSHGIDYSGLFVFQF c J3 n 01 LV2-23 n 02 CSYANYDKLI LJ3 n 01 IMGT N.A. a The CDR3 was defined based on when available or by using b 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. The sequences of germline versions of PG9, PGT145, PGT151, 35O22, PGT128, 1NC9, 3BC315 were inferred using the IMGT/V QUEST online tools [26] (Table 1). We note that PG9 and PG16 are clonal relatives and originate from the same germline precursor [27]. 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 [27] and gl-pg9 was inferred as described above. The glbnab sequences were synthesized by Genscript, cloned into the pvrc8400 expression vector, transfected into 293F cells and then purified by a protein A/G agarose 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 [28]. 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 [13], as follows: 3 μg/ml of SOSIP.664-D7324 proteins were diluted in Tris-buffered saline ph 7.5 (TBS) containing 10% fetal calf serum (FCS), and captured on D7324 Ab-coated plates. Mature and gl-bnabs were serially diluted in casein-blocking solution (Thermo Scientific). Halfmaximal binding Ab concentrations (EC 50 ) 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 [13,14] (Julien et al. in press) (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 [14]. 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 µg/ml vs. 0.3 µg/ml, previously) and we used a different blocking solution (i.e., casein blocking solution vs. 2% milk, previously). 90

7 118 K. Sliepen et al. / Virology 486 (2015) Germline bnab binding to SOSIPs Table 2. Summary of EC 50 values (in ng/ml) derived from D7324-capture ELISA with different SOSIP.664 trimers. For comparison, midpoint neutralization concentrations (IC 50 in ng/ml) of the sequence-matched Envpeudotyped viruses are also indicated. V1V2 apex CD4bs V3 glycan gp41 and interface BG505 B41 ZM197M SOSIP.664 pseudovirus SOSIP.664 pseudovirus SOSIP.664 pseudovirus Germline Mature Mature Germline Mature Mature Germline Mature Mature PG * > * * PG * > * > * PGT145 > * > > * VRC26.09 > <10 > >25000 > * CH * N.D. > N.D. VRC01 > * > * > * 3BNC60 > * > > NC9 > * > > * CH103 > * > > CH31 > * > > PGT121 > * > * > * EC 50 (ng/ml): PGT128 > * > > * <250 PGT151 > > * > * O22 > > >25000 > * BC * > > * >25000 *: IC 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 OD Mature OD 450 Antibody (ng/ml) Figure 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 ). 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 ). The BG505 SOSIP.664-D7324 trimer bound to gl-pg9 and its somatic relative gl-pg16 (EC 50 : 1.0 and 15 μg/ml, respectively), its ZM197M counterpart bound to gl-pg9 (EC 50 : 6.8 μg/ml) but not gl-pg16, 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 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 (EC 50 : 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 (EC 50 : 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 version. As the HCDR3 of 3BC315 is known to make important hydrophobic contacts with the gp41/gp120 interface [23], this interaction may contribute to the high affinity the gl-3bc315 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 91

8 Chapter 5 K. Sliepen et al. / Virology 486 (2015) A PG germline K d = 320 nm 200 K d = 24 nm mature B Response difference (RU) VRC01 germline no binding mature K d = nm Time (s) 1000 nm 500 nm 250 nm 125 nm 62.5 nm 31.2 nm 15.6 nm 3.9 nm bnab Number of k k replicates (1/Ms) (1/s) PG16 (n=2) (Germline) ± ± PG16 (n=3) (Mature) ± ± VRC01 (Germline) (n=1) No Binding VRC01 (Mature) (n=3) ± ± Figure Fig. 2. Antibody 2. Antibody binding to BG505 binding SOSIP.664to by SPR BG505 analysis. SOSIP.664 (A) Kinetic binding by curves SPR obtained analysis. by SPRA. andkinetic derived using binding the maturecurves and gl- versions obtained of PG16 and by VRCO1, SPR and chip-immobilized derived using BG505the SOSIP.664-His mature trimers. and gl- (B) Tabulated versions summary of PG16 of the SPR and analyses VRCO1, performed and onchip-immobilized BG505 SOSIP.664. BG505 SOSIP.664-His trimers. B. Tabulated summary of the SPR analyses performed on BG505 SOSIP.664. R.S., Schmidt, S.D., Sheward, D.J., Soto, C., Wibmer, C.K., Yang, Y., Zhang, Z., glycans in either the formation or the occlusion of Mullikin, the J.C., epitope Binley, J.M., Sanders, [23]. 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., None of the three trimers bound the gl-bnabs Mascola, J.R., targeting Developmentalthe pathway glycan-dependent for potent V1V2-directed HIVneutralizingantibodies. Nature 509, V3 epitopes (PGT121 and PGT128). They also did Dosenovic, not P., interact von Boehmer, L., detectably Escolano, A., Jardine, with J., Freund, the N.T., VRC01, Gitlin, A.D., McGuire, A.T., Kulp, D.W., Oliveira, T., Scharf, L., Pietzsch, J., Gray, M.D., Cupo, A., 3BNC60, 1NC9 or CH31 gl-bnabs against the CD4bs, van Gils, M.J., perhaps Yao, K.-H., Liu, because C., Gazumyan, A., of Seaman, the M.S., shielding Björkman, P.J., Sanders, R.W., Moore, J.P., Stamatatos, L., Schief, W.R., Nussenzweig, M.C., Immunization for HIV-1 broadly neutralizing antibodies in human Ig knockin effects of various trimer glycans. An example is the clash between the N276 glycan and mice. Cell 161, Giudicelli, V., Chaume, D., Lefranc, M.P., IMGT/V-QUEST, an integrated software the gl-vrc01 light chain [9]. While gl-ch103 was prone programto for immunoglobulin generating and T cell a high receptor V-J level and V-D-J of rearrangement analysis. Nucleic Acids Res. 32, nonspecific background signals in ELISA (Fig. 1, gl-ch103 gkh412. mock ), we did detect some specific Haynes, B.F., Kelsoe, G., Harrison, S.C., Kepler, T.B., B-cell-lineage immunogen binding of this antibody to the ZM197M trimers design (ECin vaccine 50 : 18.1 development μg/ml) with HIV-1 although as a case study. not Nat. Biotechnol. to their 30, BG505 and B41 counterparts (Fig. 1 and Table 2). 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. To corroborate the binding of gl-pg16 to the BG505 SOSIP.664-D7324 trimers, we R., Parks, C.L., Caulfield, M.J., Identification of an HIV-1 clade A envelope that exhibits broad antigenicity and neutralization sensitivity and elicits performed SPR analyses using the His-tagged version antibodies targeting of the three same distinct epitopes. trimers. J. Virol. The 87, data were doi.org/ /jvi fitted with a bivalent model, and binding parameters Hoot, S., McGuire, for A.T., the Cohen, monovalent K.W., Strong, R.K., Hangartner, component L., Klein, F., Diskin, in R., Scheid, J.F., Sather, D.N., Burton, D.R., Stamatatos, L., Recombinant HIV the bivalent model are given [24]. We confirmed envelope that gl-pg16 proteins fail to engage bound germline to versions BG505 of anti-cd4bs SOSIP.664- bnabs. PLoS Pathog. 9, e His trimers, although with a lower affinity (dissociation Huang, J., Kang, B.H., constant, Pancera, M., Lee, KJ.H., d = Tong, 320nM) T., Feng, Y., than Imamichi, mature 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., PG16 (K d = 24 nm) (Fig. 2A). The reduced affinity arises because k on (on-rate constant) and 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., Broad and potent HIV-1 neutralizationhigher, by a humanrespectively, antibody that binds thefor gp41 gp120 gl-pg16 interface. (Fig. Nature k off (off-rate constant) were ~5-fold lower and ~3-fold 515, B). As the binding stoichiometry was also lower Jardine, for J., gl-pg16 Julien, J.-P., Menis, (SS., m =0.4) Ota, T., Kalyuzhniy, than O., for McGuire, mature A., Sok, D., PG16 Huang, P.-S., MacPherson, S., Jones, M., Nieusma, T., Mathison, J., Baker, D., Ward, A.B., (S m =1.3) only a subset of the trimers can bind gl-pg16, Burton, D.R., perhaps Stamatatos, L., because Nemazee, D., of Wilson, variation I.A., Schief, W.R., in the Rational HIV immunogen design to target specific germline B cell receptors. Science 340, presence or composition of particular glycan sites (Fig. 2B). The difference in binding to 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., BG505 SOSIP.664 between gl-pg16 and mature PG16 Adachi, Y., were Burton, D.R., more Schief, substantial W.R., Nemazee, D., in ELISA Priming a(ec broadly 50 neutralizing antibody response to HIV-1 using a germline-targeting immunogen. Science than 80, in SPR (K d values = 13.1 mg/ml and mg/ml, respectively) = 320 nm and 24 nm, Julien, J.-P., Lee, J.H., Ozorowski, G., Hua, Y., de la Peña, A.T., de Taeye, S.W., Nieusma, respectively). This can probably be explained by T., the Cupo, A., high Yasmeen, off-rate, A., Golabek, M., which Pugach, P., results Klasse, P.J., Moore, in J.P., loss Sanders, of R.W., Ward, A.B., Wilson, I.A., Design and structure of two HIV-1 clade C binding signal in ELISA during washing steps. We SOSIP.664 note trimers thatthe increase BG505 the arsenaltrimers of native-like are Env immunogens. based Proc. Natl. Acad. Sci. USA. 112, pnas on a transmitter/founder virus sequence that is similar in the relevant regions to viruses 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., isolated from the donor of PG9 and PG16 [13,29]. Ward, A.B., This sequence Asymmetric recognition homology of the HIV-1 trimer may byhelp broadly neutralizing antibody PG9. Proc. Natl. Acad. Sci. USA 110, explain why gl-pg9 and gl-pg16 bind to the BG505 doi.org/ /pnas trimers. In contrast, but consistent Lee, J.H., Leaman, D.P., Kim, A.S., de la Peña, A.T., Sliepen, K., Yasmeen, A., Derking, with the ELISA data, gl-vrc01 did not bind to BG505 R., Ramos, SOSIP.664-His A., de Taeye, S.W., Ozorowski, trimers G., Klein, in F., Burton, the D.R., SPR Nussenzweig, assay (Fig. 2A). 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 k (1/RUs) ± ± ± k (1/s) ± ± ± K (nm) ± ± ± K (RU) ± ± ± S 0.4 ± ± ± 0.1 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, 92

9 Germline bnab binding to SOSIPs B cell receptors and thereby initiate pathways that lead to the eventual emergence of bnabs. 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 neutraliza- tion data; and Judith Burger for technical support. Funding MMR is a recipient of a fellowship from the Consejo Nacional de Ciencia y Tecnologıa (CONACyT) of Mexico (CONACyT and ). RWS is a recipient of a Vidi Grant from the Nether- lands Organization for Scientific Research (NWO) (Vidi ) and a Starting Investigator Grant from the European Research Council (ERC-StG SHEV). This work was also supported by NIH Grants P01 AI082362, P01 AI and R37 AI36082 (JPM). 5 References 1. van Gils MJ, Sanders RW. In vivo protection by broadly neutralizing HIV antibodies. Trends Microbiol. 22(10), (2014). 2. van den Kerkhof TLGM, Euler Z, van Gils MJ, Boeser-Nunnink BD, Schuitemaker H, Sanders RW. Early development of broadly reactive HIV-1 neutralizing activity in elite neutralizers. AIDS. 28(8), (2014). 3. Liao H-X, Lynch R, Zhou T, et al. Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature. 496(7446), (2013). 4. Doria-Rose NA, Schramm CA, Gorman J, et al. Developmental pathway for potent V1V2-directed HIVneutralizing antibodies. Nature. 509(7498), (2014). 5. Haynes BF, Kelsoe G, Harrison SC, Kepler TB. B-cell lineage immunogen design in vaccine development with HIV-1 as a case study. Nat. Biotechnol. 30(5), (2012). 6. Hoot S, McGuire AT, Cohen KW, et al. Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bnabs. PLoS Pathog. 9(1), e (2013). 7. McGuire AT, Glenn JA, Lippy A, Stamatatos L, Doms RW. 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 D. J. Virol. 88(5), (2013). 8. Lynch RM, Wong P, Tran L, et al. HIV-1 Fitness Cost Associated with Escape from the VRC01 Class of CD4 Binding Site Neutralizing Antibodies. J. Virol. 89(8), (2015). 9. McGuire AT, Hoot S, Dreyer AM, et al. Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-cd4 binding site antibodies. J. Exp. Med. 210(4), (2013). 10. Jardine J, Julien J-P, Menis S, et al. Rational HIV immunogen design to target specific germline B cell receptors. Science. 340(6133), (2013). 11. Jardine JG, Ota T, Sok D, et al. Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen. Science. 349(6244), (2015). 12. Dosenovic P, von Boehmer L, Escolano A, et al. Immunization for HIV-1 Broadly Neutralizing Antibodies in Human Ig Knockin Mice. Cell. 161(7), (2015). 13. Sanders RW, Derking R, Cupo A, et al. 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(9), e (2013). 14. Pugach P, Ozorowski G, Cupo A, et al. A native-like SOSIP.664 trimer based on an HIV-1 subtype B env gene. J. Virol. 89(6), (2015). 15. Julien J-P, Lee JH, Ozorowski G, et al. Design and structure of two HIV-1 clade C SOSIP.664 trimers that 93

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