. 2024 Jun;25(6):1073-1082.

doi: 10.1038/s41590-024-01833-w. Epub 2024 May 30.

Vaccination induces broadly neutralizing antibody precursors to HIV gp41

Torben Schiffner^#^{1

2

3

4}, Ivy Phung^#^{3

5}, Rashmi Ray^#⁶, Adriana Irimia^#^{2

3

7}, Ming Tian^#^{8

9}, Olivia Swanson^{1

2

3}, Jeong Hyun Lee^{1

2

3}, Chang-Chun D Lee^{2

3

7}, Ester Marina-Zárate^{3

5}, So Yeon Cho^{2

3

7}, Jiachen Huang^{2

3

7}, Gabriel Ozorowski^{2

3

7}, Patrick D Skog^{1

2

3}, Andreia M Serra^{1

2

3}, Kimmo Rantalainen^{1

2

3}, Joel D Allen¹⁰, Sabyasachi Baboo¹¹, Oscar L Rodriguez¹², Sunny Himansu¹³, Jianfu Zhou¹⁴, Jonathan Hurtado^{1

2

3}, Claudia T Flynn^{1

2

3}, Katherine McKenney^{1

2

3}, Colin Havenar-Daughton^{3

5}, Swati Saha¹², Kaitlyn Shields¹², Steven Schultze¹², Melissa L Smith¹², Chi-Hui Liang^{1

2

3}, Laura Toy^{3

5}, Simone Pecetta⁶, Ying-Cing Lin⁶, Jordan R Willis^{1

2

3}, Fabian Sesterhenn^{1

2

3}, Daniel W Kulp^{1

2

3}, Xiaozhen Hu^{1

2

3}, Christopher A Cottrell^{1

2

3}, Xiaoya Zhou^{1

2

3}, Jennifer Ruiz^{1

2

3}, Xuesong Wang⁶, Usha Nair⁶, Kathrin H Kirsch⁶, Hwei-Ling Cheng^{8

9}, Jillian Davis^{8

9}, Oleksandr Kalyuzhniy^{1

2

3}, Alessia Liguori^{1

2

3}, Jolene K Diedrich¹¹, Julia T Ngo¹⁵, Vanessa Lewis¹⁵, Nicole Phelps^{1

2

3}, Ryan D Tingle^{1

2

3}, Skye Spencer^{1

2

3}, Erik Georgeson^{1

2

3}, Yumiko Adachi^{1

2

3}, Michael Kubitz^{1

2

3}, Saman Eskandarzadeh^{1

2

3}, Marc A Elsliger^{2

3

7}, Rama R Amara^{15

16}, Elise Landais^{1

2

3}, Bryan Briney^{1

2

3

17

18}, Dennis R Burton^{1

2

3

6}, Diane G Carnathan¹⁵, Guido Silvestri^{15

19}, Corey T Watson¹², John R Yates 3rd¹¹, James C Paulson^{1

11}, Max Crispin¹⁰, Gevorg Grigoryan^{14

20

21}, Andrew B Ward^{2

3

7}, Devin Sok^{1

2

3}, Frederick W Alt^{8

9}, Ian A Wilson^{22

23

24}, Facundo D Batista^{25

26}, Shane Crotty^{27

28

29}, William R Schief^{30

31

32

33

34}

Affiliations

¹ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA.
² IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA.
³ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVD), The Scripps Research Institute, La Jolla, CA, USA.
⁴ Institute for Drug Discovery, Leipzig University Medical Faculty, Leipzig, Germany.
⁵ Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA.
⁶ The Ragon Institute of Mass General, MIT and Harvard, Cambridge, MA, USA.
⁷ Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.
⁸ Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.
⁹ Department of Genetics, Harvard Medical School, Boston, MA, USA.
¹⁰ School of Biological Sciences, University of Southampton, Southampton, UK.
¹¹ Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA.
¹² Department of Biochemistry and Molecular Genetics, University of Louisville School of Medicine, Louisville, KY, USA.
¹³ Moderna, Inc., Cambridge, MA, USA.
¹⁴ Department of Computer Science, Dartmouth College, Hanover, NH, USA.
¹⁵ Division of Microbiology and Immunology, Emory National Primate Research Center, Emory University, Atlanta, GA, USA.
¹⁶ Department of Microbiology and Immunology, Emory School of Medicine, Atlanta, GA, USA.
¹⁷ Multi-omics Vaccine Evaluation Consortium, The Scripps Research Institute, La Jolla, CA, USA.
¹⁸ San Diego Center for AIDS Research, The Scripps Research Institute, La Jolla, CA, USA.
¹⁹ Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA.
²⁰ Department of Biological Sciences, Dartmouth College, Hanover, NH, USA.
²¹ Generate Biomedicines, Inc., Somerville, MA, USA.
²² IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA. wilson@scripps.edu.
²³ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVD), The Scripps Research Institute, La Jolla, CA, USA. wilson@scripps.edu.
²⁴ Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. wilson@scripps.edu.
²⁵ The Ragon Institute of Mass General, MIT and Harvard, Cambridge, MA, USA. fbatista1@mgh.harvard.edu.
²⁶ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. fbatista1@mgh.harvard.edu.
²⁷ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVD), The Scripps Research Institute, La Jolla, CA, USA. shane@lji.org.
²⁸ Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA. shane@lji.org.
²⁹ Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, CA, USA. shane@lji.org.
³⁰ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA. schief@scripps.edu.
³¹ IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA. schief@scripps.edu.
³² Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVD), The Scripps Research Institute, La Jolla, CA, USA. schief@scripps.edu.
³³ The Ragon Institute of Mass General, MIT and Harvard, Cambridge, MA, USA. schief@scripps.edu.
³⁴ Moderna, Inc., Cambridge, MA, USA. schief@scripps.edu.

^# Contributed equally.

PMID: 38816615
PMCID: PMC11147780
DOI: 10.1038/s41590-024-01833-w

Vaccination induces broadly neutralizing antibody precursors to HIV gp41

Torben Schiffner et al. Nat Immunol. 2024 Jun.

. 2024 Jun;25(6):1073-1082.

doi: 10.1038/s41590-024-01833-w. Epub 2024 May 30.

Authors

Affiliations

¹ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA.
² IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA.
³ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVD), The Scripps Research Institute, La Jolla, CA, USA.
⁴ Institute for Drug Discovery, Leipzig University Medical Faculty, Leipzig, Germany.
⁵ Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA.
⁶ The Ragon Institute of Mass General, MIT and Harvard, Cambridge, MA, USA.
⁷ Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.
⁸ Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.
⁹ Department of Genetics, Harvard Medical School, Boston, MA, USA.
¹⁰ School of Biological Sciences, University of Southampton, Southampton, UK.
¹¹ Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA.
¹² Department of Biochemistry and Molecular Genetics, University of Louisville School of Medicine, Louisville, KY, USA.
¹³ Moderna, Inc., Cambridge, MA, USA.
¹⁴ Department of Computer Science, Dartmouth College, Hanover, NH, USA.
¹⁵ Division of Microbiology and Immunology, Emory National Primate Research Center, Emory University, Atlanta, GA, USA.
¹⁶ Department of Microbiology and Immunology, Emory School of Medicine, Atlanta, GA, USA.
¹⁷ Multi-omics Vaccine Evaluation Consortium, The Scripps Research Institute, La Jolla, CA, USA.
¹⁸ San Diego Center for AIDS Research, The Scripps Research Institute, La Jolla, CA, USA.
¹⁹ Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA.
²⁰ Department of Biological Sciences, Dartmouth College, Hanover, NH, USA.
²¹ Generate Biomedicines, Inc., Somerville, MA, USA.
²² IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA. wilson@scripps.edu.
²³ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVD), The Scripps Research Institute, La Jolla, CA, USA. wilson@scripps.edu.
²⁴ Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. wilson@scripps.edu.
²⁵ The Ragon Institute of Mass General, MIT and Harvard, Cambridge, MA, USA. fbatista1@mgh.harvard.edu.
²⁶ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. fbatista1@mgh.harvard.edu.
²⁷ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVD), The Scripps Research Institute, La Jolla, CA, USA. shane@lji.org.
²⁸ Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA. shane@lji.org.
²⁹ Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, CA, USA. shane@lji.org.
³⁰ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA. schief@scripps.edu.
³¹ IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA. schief@scripps.edu.
³² Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVD), The Scripps Research Institute, La Jolla, CA, USA. schief@scripps.edu.
³³ The Ragon Institute of Mass General, MIT and Harvard, Cambridge, MA, USA. schief@scripps.edu.
³⁴ Moderna, Inc., Cambridge, MA, USA. schief@scripps.edu.

^# Contributed equally.

PMID: 38816615
PMCID: PMC11147780
DOI: 10.1038/s41590-024-01833-w

Erratum in

Author Correction: Vaccination induces broadly neutralizing antibody precursors to HIV gp41.
Schiffner T, Phung I, Ray R, Irimia A, Tian M, Swanson O, Lee JH, Lee CD, Marina-Zárate E, Cho SY, Huang J, Ozorowski G, Skog PD, Serra AM, Rantalainen K, Allen JD, Baboo S, Rodriguez OL, Himansu S, Zhou J, Hurtado J, Flynn CT, McKenney K, Havenar-Daughton C, Saha S, Shields K, Schultze S, Smith ML, Liang CH, Toy L, Pecetta S, Lin YC, Willis JR, Sesterhenn F, Kulp DW, Hu X, Cottrell CA, Zhou X, Ruiz J, Wang X, Nair U, Kirsch KH, Cheng HL, Davis J, Kalyuzhniy O, Liguori A, Diedrich JK, Ngo JT, Lewis V, Phelps N, Tingle RD, Spencer S, Georgeson E, Adachi Y, Kubitz M, Eskandarzadeh S, Elsliger MA, Amara RR, Landais E, Briney B, Burton DR, Carnathan DG, Silvestri G, Watson CT, Yates JR 3rd, Paulson JC, Crispin M, Grigoryan G, Ward AB, Sok D, Alt FW, Wilson IA, Batista FD, Crotty S, Schief WR. Schiffner T, et al. Nat Immunol. 2024 Jul;25(7):1307. doi: 10.1038/s41590-024-01891-0. Nat Immunol. 2024. PMID: 38877179 Free PMC article. No abstract available.

Abstract

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

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Conflict of interest statement

T.S., O.S., A.M.S., J.Z., F.S., D.W.K., G.G. and W.R.S. are named inventors on patent applications filed by Scripps and IAVI regarding 10E8-GT immunogens. S.H., S.P. and W.R.S. are employees of Moderna. G.G. is an employee of Generate Biomedicines. The other authors declare no competing interests.

Figures

**Fig. 1. 10E8-class bnAb precursors are present in most humans.**
a, Schematic of the epitope scaffold design showing antibody 10E8 (gray) and Env (blue), including the MPER (purple) that was grafted onto an unrelated epitope scaffold (cyan). b, Frequency of 10E8-class IgH precursors in 14 NGS datasets^, of heavy chains from HIV-seronegative humans defined as sequences with genes encoding V_H closely related to 10E8 and HCDR3 lengths of 21–24 aa with a YxFW motif at the correct position. Lines indicate the median and 25 and 75% quantiles; HCs, heavy chains.

**Fig. 2. 10E8-GT immunogens bind diverse 10E8-class precursors.**
a, SPR-measured monovalent K_d values for the scaffold without germline-targeting mutations (MPER) and various 10E8-GT scaffolds (10E8-GT9.2 to 10E8-GT12) binding to mature 10E8, germline-reverted 10E8 (10E8-iGL3), the proposed 10E8 UCA and multiple NGS-derived 10E8-class human precursor heavy chains paired with the germline-reverted 10E8 light chain (NGS). Each symbol represents a different antibody; LOD, limit of detection; NB, no binding. b, SPR-measured monovalent K_d values for 10E8-GT10.2, 10E8-GT11 and 10E8-GT12 binding to different antibodies containing the indicated number of 10E8-class mutations, including fully germline-reverted 10E8 (10E8-iGL3), partially mature 10E8-class antibodies (intermediates) and mature (Mat) 10E8.

**Fig. 3. 10E8-GT immunogens mimic the interaction between 10E8 and the MPER.**
Structures (from left to right) of 10E8 bnAb bound to MPER peptide, 10E8 bnAb bound to T117v2 scaffold, 10E8-iGL1 bound to 10E8-GT4 scaffold, 10E8 bnAb bound to 10E8-GT10.2 scaffold, NGS precursor 10E8-NGS-03 bound to 10E8-GT10.2 scaffold and 10E8-iGL1 bound to 10E8-GT11 scaffold, in which the previously published MPER peptide and T117v2 complexes with 10E8 are shown for comparison. Top, structures shown as cartoon diagrams, aligned on the MPER (purple), with antibody heavy chain in white or yellow, light chain in gray and scaffold in blue. Antibody constant regions are omitted for clarity. Bottom, interaction between the HCDR3 YxFW motif (sticks) and the engineered D_H binding pocket (green) on the scaffold. All structures were determined by crystallography, except for the complex of 10E8 bnAb with 10E8-GT10.2, which was determined by cryo-EM and included a scaffold-specific off-target Fab (not shown) to facilitate image reconstruction.

**Fig. 4. 10E8-GT scaffolds engage 10E8-class HCDR3s in human blood.**
a, Representative flow cytometry staining of 10E8-GT12 double-positive (10E8-GT12⁺⁺; signifying binding to two probes with different fluorochromes; left) and epitope-specific 10E8-GT12⁺⁺10E8-GT12-KO^– (right) CD20⁺CD27^–IgD⁺IgG^– naive B cells from HIV-seronegative donors; SA, streptavidin. b, Frequency of 10E8-GT9⁺⁺ (n = 3 donors), 10E8-GT10.1⁺⁺ (n = 3) and 10E8-GT12⁺⁺ (n = 6) cells among CD20⁺IgG^– naive B cells for 10E8-GT9 and 10E8-GT10 or CD20⁺CD27^–IgD⁺IgG^– naive B cells for 10E8-GT12 that were sorted from HIV-seronegative donors and BCR sequenced using either a Sanger sequencing method (squares) or a 10x Genomics sequencing method (crosses). c, Percentage of 10E8-GT9⁺⁺, 10E8-GT10.1⁺⁺ and 10E8-GT12⁺⁺ naive B cells (CD20⁺IgG⁻ B cells or CD20⁺CD27⁻IgD⁺IgG⁻ B cells, as in b) that are epitope specific (10E8-GT9⁺⁺10E8-GT9-KO⁻, 10E8-GT10.1⁺⁺10E8-GT10.1-KO⁻ or 10E8-GT12⁺⁺10E8-GT12-KO⁻). d, HCDR3 length distribution for human naive BCRs sorted by 10E8-GT9, 10E8-GT10.1 and 10E8-GT12 (average of all donors in each case). NGS datasets (n = 14) of heavy chains from HIV-seronegative humans served as unsorted controls^, where indicated (black). The targeted HCDR3 length range (21–24 aa) is highlighted in gray. The exact HCDR3 length for the 10E8 bnAb is indicated by a tick mark at 22 aa. e, Percentage of 10E8-class HCDR3s (with lengths of 21–24 aa and YxFW at the correct position within HCDR3) among epitope-specific (10E8-GT9⁺⁺10E8-GT9-KO⁻, n = 3 donors; 10E8-GT10.1⁺⁺10E8-GT10.1-KO⁻, n = 4 donors; 10E8-GT12⁺⁺10E8-GT12-KO⁻ n = 6 donors) IgM⁺ BCRs compared to unsorted controls defined in d; *P = 0.03 and ***P = 0.0004. Data were analyzed by Kruskal–Wallis test with a Dunn’s multiple comparison correction. f, Percentage of 10E8-class HCDR3s among all naive IgM⁺ B cells compared to unsorted controls as in e. g, Percentage of 10E8-class and LN01-class IgH precursors among naive IgM⁺ B cells sorted with 10E8-GT12 or unsorted controls defined in d. Lines indicate median values.

**Fig. 5. 10E8-class B cells function in vivo.**
a, Flow cytometry analysis and quantification of B220⁺TCRβ^– B cells, B220^–TCRβ⁺ total T cells, CD4⁺CD8⁻ T cells, CD4⁻CD8⁺ T cells, CD2⁻CD24^hi T0/T1 B cells, CD21^loCD24^lo follicular B cells, CD21^hiCD24^hiCD23⁻ T2 B cells and CD21^hiCD24^hiCD23⁺ marginal zone B (MZB) cells in the spleens of MPER-HuGL18^H mice (n = 7) compared to wild-type (WT) C57BL/6 mice (n = 4). Symbols represent individual animals, and error bars indicate standard deviation. b, Frequency of CD38^loCD95⁺ GC B cells among total B220⁺ B cells (left) and CD45.2⁺ B cells among CD38^loCD95⁺ GC B cells (right) at day 21 after immunization with 10E8-GT10.2 12mer (n = 14) or control 10E8-GT9-KO 12mer (n = 8) in CD45.1 wild-type recipient mice adoptively transferred with 200,000 CD45.2 MPER-HuGL18^H B cells. Symbols represent individual animals; bars indicate mean ± s.d.; ****P < 0.0001. Data were analyzed by two-sided Mann–Whitney test. c, Frequencies of 10E8-GT10⁺⁺ cells among CD38^loCD95⁺CD45.1⁻CD45.2⁺ MPER-HuGL18^H GC cells as in b.

**Fig. 6. mRNA-LNP delivery of 10E8-GT12 nanoparticles primes diverse 10E8-class B cells.**
a, Percentage of 10E8-GT10.1⁺⁺10E8-GT10.1-KO^– (epitope-specific) CD19⁺IgD⁺ naive B cells with 10E8-class HCDR3s for humans (as in Fig. 4f) and hD3-3/J_H6 mice. b, Percentage of 10E8-GT9^–KO⁺⁺10E8-GT9^–, 10E8-GT10.1⁺⁺10E8-GT10.1-KO^– or 10E8-GT12⁺⁺10E8-GT12-KO^– epitope-specific IgG⁺ BCRs with 10E8-class HCDR3s from day 42 after immunization of hD3-3/J_H6 mice with 10E8-GT9-KO 12mer (n = 3), 10E8-GT10.2 12mer (n = 12), 10E8-GT12 12mer (n = 5) or 10E8-GT12 24mer (n = 12) delivered as protein in SMNP, respectively, or 10E8-GT12 24mer delivered by mRNA (n = 11). Symbols represent individual animals, and bars indicate median values. c, Percentage of epitope-specific IgG⁺ BCRs as in b with 10E8-class HCDR3s and at least one proline in position +7 or +8 relative to the YxFW motif from hD3-3/J_H6 mice with >100 sequences. Sequences of genes encoding D_H in mature 10E8 and iGL are shown with the YxFW motif in green, and the targeted prolines are colored red; **P = 0.006. Data were analyzed by two-sided Mann–Whitney test.

**Fig. 7. 10E8-GT immunogens induce 10E8-class responses in NHPs.**
a, Alignment of known rhesus macaque homologs of the human gene encoding D_H3-3. Macaque D-gene residues that differ from the critical YxFW binding motif (orange) that directly contacts the 10E8 epitope are highlighted in red. b, HCDR3 length distribution for 10E8-GT10.2⁺⁺10E8^–GT10.2-KO^– epitope-specific CD20⁺IgG^– naive B cells sorted from unimmunized macaques (n = 9) compared to 10E8-GT10.2⁻ nonbinding BCRs from the same macaques and the human naive BCRs, as in Fig. 4. c, Percentage of 10E8-class HCDR3s among CD20⁺IgG⁻ naive B cells from unimmunized macaques (n = 9) compared to human naive BCRs from Fig. 4 (n = 3) and to rhesus macaque sequences from the Observed Antibody Space (OAS) repository (n = 8). d, Macaque immunization schedule for an escalating dose of 10E8-GT10.2 12mer (n = 8) or 10E8-GT12 12mer (n = 6), indicating analysis of lymph node fine needle aspirates (LN FNA) at weeks –2, 3 and 10 and analysis of blood at weeks 6 and 11 or week 10 for macaques immunized with 10E8-GT10.2 12mer or 10E8-GT12 12mer, respectively. e, Percentage of 10E8-class HCDR3s among 10E8-GT10.2⁺⁺10E8-GT10.2-KO⁻ or 10E8-GT12⁺⁺10E8-GT12-KO⁻ epitope-specific IgG⁺ BCRs from macaques after immunization as in d in the GCs and PBMCs at all time points indicated in d combined for each macaque compared to macaques immunized with stabilized soluble HIV Env (control) at weeks 3, 4, 7 and 10 after immunization. Open symbols represent macaques lacking a permissive D_H3-41 allele; **P = 0.004. Data were analyzed by two-sided Mann–Whitney test. f, Percentage of 10E8-class V_H among IgG⁺CD20⁺IgD⁻ memory BCRs with 10E8-class HCDR3s or among IgG⁺ CD20⁺IgD⁻ memory BCRs lacking the YxFW motif (non-10E8) from macaques after immunization as in d; *P < 0.05. Data were analyzed by Kruskal–Wallis test with a Dunn’s multiple comparison correction; control versus 10E8-class induced by 10E8-GT10.2 12mer, P = 0.03; control versus 10E8-class induced by 10E8-GT12 12mer, P = 0.02. g, Percentage of LN01-class HCDR3s among 10E8-GT10.2⁺⁺10E8-GT10.2-KO⁻ or 10E8-GT12⁺⁺10E8-GT12-KO⁻ epitope-specific IgG⁺ BCRs from macaques after immunization as in d; *P = 0.04. Data were analyzed by two-sided Mann–Whitney test. h, Crystal structure of a 10E8-GT10.2-induced macaque antibody with a YxIW motif from week 3 in complex with 10E8-GT10.2 (right) with the structure of 10E8 bound to peptide (left) shown for reference. Colors are as in Fig. 3.

**Fig. 8. 10E8-class BCRs induced by 10E8-GT nanoparticles bind epitope scaffold 10E8-B1 containing a near-native 10E8 peptide epitope.**
a, SPR-measured monovalent K_d values for 10E8-B1 binding to different antibodies containing the indicated number of 10E8-class mutations, including the 10E8 UCA, 10E8-class human naive precursors isolated by human B cell sorting (human naive), artificial partially mature 10E8-class antibodies (intermediates) and mature 10E8. Each symbol represents an antibody; overlapping data are staggered along the x axis. b, Crystal structure of 10E8-B1 in complex with 10E8 bnAb (right), with the structure of 10E8 bound to peptide (left) shown for reference. Colors are as in Fig. 3. c, SPR-measured monovalent K_d values for 10E8-B1 binding to iGL antibodies or antibodies recovered after immunization of hD3-3/J_H6 mice or macaques with 10E8-GT nanoparticles (after priming). Each symbol represents a different antibody; **P = 0.004. Data were analyzed by a Kruskal–Wallis test with a Dunn’s multiple comparison correction.

**Extended Data Fig. 1. Design and properties of immunogens.**
a, Overview of 10E8-class and LN01-class antibody categories. HCDR3 motifs are shown as regular expressions that were used to query the database. If multiple amino acids were allowed at the same position, they are shown in square brackets; positions in which all amino acids were allowed are indicated as ‘.’. b, Schematic of the development of MPER-GT scaffolds. c, Schematic overview of nanoparticle formation by genetic fusion of the immunogen (T2983⁻GT) to each terminus of the 3-dehydroquinase nanoparticle from *Thermus thermophilus* (NP) via flexible linkers containing exogeneous T-help peptides derived from *Aquifex aeolicus* lumazine synthase (link). The epitope scaffold is shown in light blue, the MPER graft in purple, the linker in green, the nanoparticle in red and glycans in dark blue. d, SEC-MALS traces of 10E8-GT NPs. Normalized UV₂₈₀ absorptions are shown as dotted lines and protein molecular weights of main peaks are shown as solid lines. e, DSC measurements of the indicated monomers and nanoparticles with results from a fit indicated in light grey. f, Amino acid sequences of 10E8-GT epitope scaffolds through generation 7, none of which had the circular permutation present in later generations. Germline-targeting mutations are highlighted in red; N-linked glycosylation sites are blue; the D-gene binding pocket is green; resurfacing and/or solubility enhancing mutations are orange. All sequences are succeeded by a 6x His-tag, unless the protein ends in stop codons (denoted by symbol *). 10E8-GT8.2 through 10E8-GT12 are preceded by a mammalian secretion signal. g, Resurfaced T298v2 sequences compared to previously published T298 32. Colors as in f. h, Amino acid sequences of monomeric immunogens based on resurfaced circularly permutated T298v2, with colors as in f. i, Amino acid sequences of multivalent nanoparticles. Sequences are wrapped over multiple lines. The 3-dehydroquinase is shown in purple; additional T help epitopes are brown; epitope KO mutations are cyan; and all other colors are shown as in f.

**Extended Data Fig. 2. Glycosylation sites on 10E8-GT nanoparticles vary in occupancy.**
Site-specific glycan analysis was measured using the single site glycan profiling (SSGP) and DeGlyPHER methods. Positions of N-linked glycosylation sites are indicated as relative positions within the epitope-scaffold (ES) or the nanoparticle (NP, indicated with a black box). The 24mer contains two independent copies of the scaffold, which cannot be distinguished by either method and therefore averaged values are shown.

**Extended Data Fig. 3. Data collection, refinement and validation.**
a, Data collection and refinement statistics of x-ray crystallography. ^a Numbers in parentheses refer to the highest resolution shell; ^b CC_1/2 = Pearson correlation coefficient between two random half datasets; ^c From MolProbity. b, Summary statistics of data collection, refinement and validation of the cryo-EM reconstruction of 10E8-GT10.2 in complex with 10E8 and W6-10 Fabs. c, Fourier Shell Correlation, d, angular sampling and e, map colored according to local resolution (units Ångstrom) of the cryo-EM reconstruction of 10E8-GT10.2 in complex with 10E8 and W6-10 Fabs.

**Extended Data Fig. 4. Ex vivo evaluation of 10E8-GT scaffolds, related to Fig. 4.**
a, Representative gating scheme. b, Enrichment of HCDR3 lengths among epitope-specific B cells over unsorted controls as in Fig. 4d. c, Frequency of long (>=20aa) HCDR3s among epitope-specific B cells (sorted) as in b, or among total IgM+ naïve B cells (unsorted). Symbols represent n = 3 (GT9), n = 4 (GT10.1), n = 6 (GT12) or n = 14 (unsorted controls) independent donors. * p = 0.046, **** p < 0.0001, Kruskal-Wallis test with Dunn’s multiple comparison correction. d, Percentage of HCDR3s containing the YxFW motif among epitope-specific B cells as in c. * p = 0.03, *** p = 0.0001, Kruskal-Wallis test with Dunn’s multiple comparison correction. e, Percentage of 10E8-class V_H among epitope-specific BCRs with 10E8-like HCDR3s (with length 21-24 aa and YxFW at correct position within HCDR3) within datasets obtained using the 10x Genomics sequencing method as in c. **p = 0.002, two-sided Mann-Whitney test. f, Percentage of IGVL3 family light chains among 10E8-GT12-sorted BCRs that are either 10E8-class (10E8-class H3) or lack the YxFW motif (non-YxFW). n = 6 independent donors, ns not significant, two-sided Wilcoxon test. g, Percentage of 10E8-GT12-specific naive IgM⁺ BCRs with 10E8-class or LN01-class HCDR3s. Symbols represent n = 6 (GT12) or n = 14 (unsorted controls) independent donors. h, Frequency of 10E8-class or LN01-class B cells among IgM+ naive B cells, detected through 10E8-GT12 sorting as in g. i, SPR-measured monovalent K_d values for 10E8-GT9, 10E8-GT10.2, and 10E8-GT12 monomer binding to 10E8-class and non-10E8-class (competitor) antibodies isolated by the respective scaffolds. Symbols represent different antibodies; lines represent median values.

**Extended Data Fig. 5. Poly- and Auto-reactivity of 10E8-class precursors.**
a, Polyspecificity reagent binding as measured by ELISA. PGT121, VRC01 and PGT128 served as negative controls, MPER bnAb 4E10 as a positive control. NGS-1 through −22 correspond to human NGS precursors described in the main text. These are 10E8-class heavy chain (HC) precursors identified from searching next-generation sequencing (NGS) datasets of primarily naive IgM HCs from 14 HIV-seronegative human donors^, paired with the inferred-germline 10E8 light chain (LC); 10E8-HuGL: bona fide HC/LC pairs isolated by epitope-specific sorting of naïve human B cells (n = 25). b, Mean fluorescence intensity (MFI) of antibodies as in a in a HEp-2 cell autoreactivity assay. c, raw images of data shown in b.

**Extended Data Fig. 6. Immunization of MPER-HuGL18^H B cell adoptive transfer recipient mice with 10E8-GT10.2 12mers.**
a, Flow cytometry analysis of bone marrow cells from WT (n = 4) and MPER-HuGL18^H (M18, n = 7) mice; gating strategy shown on the left. B-cell progenitors (B220⁺) were divided into immature (CD43⁺) and mature (CD43⁻) cells. Early (CD43⁺) B-cell progenitors were subdivided into Hardy populations A (CD24⁻BP-1⁻), B (CD24⁺BP-1⁻), and C (CD24⁺BP1⁺). Late (CD43⁻) B-cell progenitors were subdivided into Hardy populations D (IgM⁻IgD⁻), E (IgM⁺IgD^int), and F (IgM⁺IgD+). Right bars represent quantifications of these populations, error bars indicate SD. b, Frequency of CD45.2⁺ B cells among splenic B cells, one day after adoptive transfer of 200,000 CD45.2⁺ MPER-HuGL18^H B cells into CD45.1⁺ WT mice. Symbols represent individual animals, error bars indicate SD. c, Germinal center (GC) response to immunization in CD45.1⁺ WT mice adoptively transferred with 2 × 10⁵ CD45.2⁺ MPER-HuGL18^H B cells on Day 21 after immunization with 10E8-GT10.2 12mer or negative control 10E8-GT9-KO 12mer (KO). Left column shows the frequency of total GC (CD38^loCD95⁺) among B cells gated from SSL; right column shows the frequency of CD45.2 B cells among total GC.

**Extended Data Fig. 7. Generation and characterization of hD3-3/J_H6 mice.**
a, Illustration of genetic modifications in hD3-3/J_H6 mice (not drawn to scale) with hD3-3 and hJ_H6 segments replacing mouse DQ52 and J_H1-4 segments. Sequences of hD3-3 (in red), J_H6 (in blue) and flanking regions are shown below the diagram. b, Characterization of B220⁺ B cell and CD3⁺ T cell populations among lymphocyte/live cell/single cells from homozygous D3-3/J6 mouse spleens by flow cytometry compared to a wild-type 129SVE mouse (WT). c, Characterization of IgM⁺IgD^hi naïve B cells among B cells as in b. d, Characterization of CD23^hiCD21^low follicular (FO) B cells and CD23^lowCD21^hi marginal zone (MZ) B cells among lymphocyte/live cell/single cell/B220⁺CD93^low mature B cells, as in b. e, Characterization of Igk⁺ and Igl⁺ B cells among B cells as in b. f, Fraction of productive IgH rearrangements that contain hD3-3 or mouse D (mD), based on repertoire analysis of three homozygous hD3-3/J_H6 mice. The hD3-3 usage value represents average ± standard deviation. Since hJ_H6 is the sole J_H segment in homozygous hD3-3/J_H6 mice, all V(D)J recombination events contain hJ_H6. Therefore, 16.3% productive rearrangements in homozygous hD3-3/J_H6 mice contain both hD3-3 and hJ_H6. g, HCDR3 length distribution of productive IgH rearrangements that contain hD3-3 and J_H6 as in f. Error bars indicate SD of three independent mice.

**Extended Data Fig. 8. Immunogenicity of 10E8-GT nanoparticles in hD3-3/J_H6 mice.**
a, Representative gating strategy of splenic B cells sorted for sequencing of BCRs from hD3-3/J_H6 mice six weeks after immunization with 10E8-GT12 24mer. b, Percentage of 10E8-GT-binding cells (10E8-GT9-KO⁺⁺, 10E8-GT10.1⁺⁺ or 10E8-GT12⁺⁺⁻) among IgM⁻IgD⁻ B cells, 42 days after immunization of hD3-3/J_H6 mice with 10E8-GT9-KO 12mer (control, n = 3), 10E8-GT12 12mer (n = 12), 10E8-GT12 12mer (n = 5) or 10E8-GT12 24mer (n = 12) delivered as protein in SMNP, respectively, or 10E8-GT12 24mer delivered by mRNA (n = 11). Each symbol indicates an animal, lines indicate median values. c, Percentage of 10E8-class HCDR3s among all IgM⁻IgD⁻ B cells after immunization as in b. d, Enrichment ratio for HCDR3 amino acid (aa) length distribution for epitope-specific (10E8-GT10.1⁺⁺10E8-GT10.1-KO⁻ or 10E8-GT12⁺⁺10E8-GT12-KO⁻) IgG⁺ B cells from animals immunized with the indicated 10E8-GT immunogens, relative to HCDR3 amino acid length distribution for epitope-specific (10E8-GT9⁻KO⁺⁺10E8-GT9⁻) IgG⁺ B cells from animals immunized with 10E8-GT9-KO 12mer as in b. HCDR3 lengths longer than 22 were only found in the 10E8-GT-immunized groups, precluding calculation of enrichment scores for longer HCDR3s. e, Frequency of long (>=20 aa) HCDR3s among epitope-specific IgG⁺ B cells as in d. f, Percentage of HCDR3s containing the YxFW motif among epitope-specific IgG⁺ B cells as in c. *p < 0.05, Kruskal-Wallis test with Dunn’s multiple comparison correction. P value (control vs. GT10 12mer): 0.02, p value (control vs. GT12 24mer): 0.01. g, Fold-change in SPR-measured K_D for 10E8-GT immunogens binding to 10E8-iGL3 upon addition of the indicated HCDR3 mutations. h, Percentage of epitope-specific (10E8-GT9-KO⁺⁺10E8-GT9⁻,10E8-GT10.1⁺⁺10E8-GT10.1-KO⁻, or 10E8-GT12⁺⁺10E8-GT12-KO⁻) with 10E8⁻class or LN01-class HCDR3s among IgG⁺ BCRs from day 42 after immunization of hD3⁻3/J_H6 as in b. Symbols represent individual animals; bars indicate median values.

**Extended Data Fig. 9. Gating strategy used to assess immune responses to 10E8-GT immunogens in NHPs.**
a, Gating scheme for 10E8-GT10.2-specific (10E8-GT10.2⁺⁺) and 10E8-GT10.2 epitope-specific (10E8-GT10.2⁺⁺10E8-GT10.2-KO^–) naïve B cells. b, Gating scheme for 10E8-GT10.2-specific and 10E8-GT10.2 epitope-specific GC B cells. c, Gating scheme for 10E8-GT10.2-specific and 10E8-GT10.2 epitope-specific PBMC-memory B cells.

**Extended Data Fig. 10. Immunogenicity of 10E8-GT NPs in rhesus macaques.**
a, Percentage of GC B cells (CD38⁻CD71⁺) among all B cells (CD3⁻CD20⁺) in fine needle aspirate samples of rhesus macaques 3 or 10 weeks post immunization with 10E8-GT10.2 12mer, as described in Fig. 7. Each symbol represents an independent lymph node (n = 16 from n = 8 independent animals). b, Frequency of epitope-specific (10E8-GT10.2⁺⁺10E8-GT10.2-KO⁻) GC B cells (CD38⁻CD71⁺) observed as a percentage of total B cells (CD3⁻CD20⁺) 2 weeks prior to or 3 or 10 weeks post immunization with 10E8-GT10.2 12mer, as described in Fig. 7. Each symbol represents an independent lymph node (n = 8 for week −2, n = 16 for week 3 and 10). c, Median percent amino acid (aa) mutations in the V_H of epitope-specific (10E8-GT10.2⁺⁺10E8-GT10.2-KO⁻or 10E8-GT12⁺⁺10E8-GT12-KO⁻) BCRs sorted from macaques immunized with 10E8⁻GT10.2 12mer or 10E8-GT12 12mer at the indicated time-points from GC B cells (CD38⁻CD71⁺) or PBMC-memory B cells (CD20⁺IgD⁻) among BCRs with 10E8-class HCDR3s (10E8-class) compared to BCRs lacking the YxFW motif (Competitor). Symbols represent individual animals, bars indicate median values. d, SPR-measured monovalent K_D values for 10E8-GT10.2 binding to selected antibodies with 10E8-class HCDR3s (10E8-class) or lacking the YxFW motif (Competitor) induced by 10E8-GT10.2 12mer isolated from the GCs at week 3 (W3) or 10 (W10) post immunization, compared to available matching inferred-germline (iGL) antibodies. Each symbol represents one antibody. e, SPR-measured monovalent K_D values for 10E8-GT10.2 binding to selected mutated or unmutated antibodies with 10E8-class HCDR3s (10E8-class) or lacking the YxFW motif (Competitor) induced by 10E8-GT10.2 12mer isolated from PBMC-memory (CD20⁺IgD⁻). f, Frequency of 10E8-class IgH among PBMC-memory B cells (CD20⁺IgD⁻) at week 11 (GT10.2, n = 6) or week 10 (GT12, n = 5) post immunization compared to macaques immunized with stabilized soluble HIV envelope protein (control, week 10, n = 4). For comparison, dotted lines indicate median frequencies of VRC01-class precursors 8 weeks after high dose (HD) or low dose (LD) immunization with eOD-GT8 60mer in the IAVI G001 human phase 1 clinical trial, with the caveat that frequencies in G001 were measured among IgG⁺ B cells rather than IgD⁻B cells. Open symbols indicate macaques lacking a permissive D_H3-41 allele. Lines indicate median values.

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References

1. Sok D, Burton DR. Recent progress in broadly neutralizing antibodies to HIV. Nat. Immunol. 2018;19:1179–1188. doi: 10.1038/s41590-018-0235-7. - DOI - PMC - PubMed
1. Yechezkel I, Law M, Tzarum N. From structural studies to HCV vaccine design. Viruses. 2021;13:833. doi: 10.3390/v13050833. - DOI - PMC - PubMed
1. Guthmiller JJ, et al. Broadly neutralizing antibodies target a haemagglutinin anchor epitope. Nature. 2022;602:314–320. doi: 10.1038/s41586-021-04356-8. - DOI - PMC - PubMed
1. Dacon C, et al. Rare, convergent antibodies targeting the stem helix broadly neutralize diverse betacoronaviruses. Cell Host Microbe. 2023;31:97–111.e12. doi: 10.1016/j.chom.2022.10.010. - DOI - PMC - PubMed
1. Jardine J, et al. Rational HIV immunogen design to target specific germline B cell receptors. Science. 2013;340:711–716. doi: 10.1126/science.1234150. - DOI - PMC - PubMed

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Vaccination induces broadly neutralizing antibody precursors to HIV gp41

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