. 2017 Jun 20;46(6):1073-1088.e6.

doi: 10.1016/j.immuni.2017.05.007.

Elicitation of Robust Tier 2 Neutralizing Antibody Responses in Nonhuman Primates by HIV Envelope Trimer Immunization Using Optimized Approaches

Matthias Pauthner¹, Colin Havenar-Daughton², Devin Sok³, Joseph P Nkolola⁴, Raiza Bastidas¹, Archana V Boopathy⁵, Diane G Carnathan⁶, Abishek Chandrashekar⁷, Kimberly M Cirelli², Christopher A Cottrell⁸, Alexey M Eroshkin⁹, Javier Guenaga¹, Kirti Kaushik², Daniel W Kulp¹, Jinyan Liu⁴, Laura E McCoy¹⁰, Aaron L Oom¹¹, Gabriel Ozorowski⁸, Kai W Post⁹, Shailendra K Sharma¹, Jon M Steichen¹, Steven W de Taeye¹², Talar Tokatlian¹³, Alba Torrents de la Peña¹², Salvatore T Butera¹⁴, Celia C LaBranche¹⁵, David C Montefiori¹⁵, Guido Silvestri⁶, Ian A Wilson¹⁶, Darrell J Irvine¹⁷, Rogier W Sanders¹⁸, William R Schief¹⁹, Andrew B Ward⁸, Richard T Wyatt¹, Dan H Barouch²⁰, Shane Crotty²¹, Dennis R Burton²²

Affiliations

¹ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA.
² Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA.
³ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; International AIDS Vaccine Initiative, New York, NY 10004, USA.
⁴ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
⁵ Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁶ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Yerkes National Primate Research Center, Emory University, Atlanta, GA 30322, USA; Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322, USA.
⁷ Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
⁸ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
⁹ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Bioinformatics Core, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
¹⁰ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Division of Infection & Immunity, University College London, London WC1E 6BT, UK.
¹¹ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA; University of California, San Diego, La Jolla, CA 92093, USA.
¹² Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands.
¹³ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
¹⁴ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA.
¹⁵ Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
¹⁶ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA; Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
¹⁷ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA; Departments of Biological Engineering and Materials Science & Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
¹⁸ Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10065, USA; Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands.
¹⁹ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Cambridge, MA 02139, USA.
²⁰ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Cambridge, MA 02139, USA.
²¹ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA; Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, CA 92037, USA. Electronic address: shane@lji.org.
²² Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Cambridge, MA 02139, USA. Electronic address: burton@scripps.edu.

PMID: 28636956
PMCID: PMC5483234
DOI: 10.1016/j.immuni.2017.05.007

Elicitation of Robust Tier 2 Neutralizing Antibody Responses in Nonhuman Primates by HIV Envelope Trimer Immunization Using Optimized Approaches

Matthias Pauthner et al. Immunity. 2017.

. 2017 Jun 20;46(6):1073-1088.e6.

doi: 10.1016/j.immuni.2017.05.007.

Authors

Affiliations

¹ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA.
² Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA.
³ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; International AIDS Vaccine Initiative, New York, NY 10004, USA.
⁴ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
⁵ Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁶ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Yerkes National Primate Research Center, Emory University, Atlanta, GA 30322, USA; Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322, USA.
⁷ Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
⁸ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
⁹ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Bioinformatics Core, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
¹⁰ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Division of Infection & Immunity, University College London, London WC1E 6BT, UK.
¹¹ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA; University of California, San Diego, La Jolla, CA 92093, USA.
¹² Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands.
¹³ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
¹⁴ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA.
¹⁵ Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
¹⁶ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA; Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
¹⁷ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA; Departments of Biological Engineering and Materials Science & Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
¹⁸ Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10065, USA; Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands.
¹⁹ Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Cambridge, MA 02139, USA.
²⁰ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Cambridge, MA 02139, USA.
²¹ Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA; Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, CA 92037, USA. Electronic address: shane@lji.org.
²² Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; IAVI Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Cambridge, MA 02139, USA. Electronic address: burton@scripps.edu.

PMID: 28636956
PMCID: PMC5483234
DOI: 10.1016/j.immuni.2017.05.007

Abstract

The development of stabilized recombinant HIV envelope trimers that mimic the virion surface molecule has increased enthusiasm for a neutralizing antibody (nAb)-based HIV vaccine. However, there is limited experience with recombinant trimers as immunogens in nonhuman primates, which are typically used as a model for humans. Here, we tested multiple immunogens and immunization strategies head-to-head to determine their impact on the quantity, quality, and kinetics of autologous tier 2 nAb development. A bilateral, adjuvanted, subcutaneous immunization protocol induced reproducible tier 2 nAb responses after only two immunizations 8 weeks apart, and these were further enhanced by a third immunization with BG505 SOSIP trimer. We identified immunogens that minimized non-neutralizing V3 responses and demonstrated that continuous immunogen delivery could enhance nAb responses. nAb responses were strongly associated with germinal center reactions, as assessed by lymph node fine needle aspiration. This study provides a framework for preclinical and clinical vaccine studies targeting nAb elicitation.

Keywords: BG505; GC B cells; HIV vaccine; NFL; SOSIP; Tfh cells; germinal centers; nonhuman primates; protein design; rhesus macaques.

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Figures

**Figure 1**
Induction of Potent HIV nAb Titers after Two Immunizations with Env Trimers and Correlations with GC B Cell frequency (A) 0-8-24 week immunization schedule and sampling. (B) BG505 SOSIP EC₅₀ binding IgG titers of BG505-SOSIP.664-immunized RMs (n = 12). Colors indicate individual animals. Vertical dotted lines indicate immunization time points. LOD, limit of detection. (C) BG505 neutralization IC₅₀ titers of BG505-SOSIP.664-immunized RMs over the course of the immunizations (n = 12). Colors are as in (B). (D) Flow-cytometry gating of GC B cells, gated on CD20⁺ B cells. (E) GC B cell frequencies at baseline (BL) and after the first immunization. Points represent individual LNs (n = 24). (F) Flow-cytometry gating of GC Tfh cells, gated on CD3⁺ CD4⁺ T cells. (G) GC Tfh cell frequencies at baseline (BL) and after the first immunization (n = 24). (H) GC B cell frequencies after the first and second immunizations. LNs sampled at both time points are connected by a black line. Red symbols indicate means. (I) GC B cell frequencies (mean of two draining LNs per animal) after the first immunization (week 3) predicts nAb titers after the second immunization (week 10). Red line shows linear regression (n = 12). (J and K) GC B cell frequencies correlate with nAb titers after the second (J) and third (K) immunizations (n = 12). All cell-frequency data represent the mean and SD. See also Figure S1.

**Figure 2**
Subcutaneous Immunizations Induce Stronger Autologous nAb Responses Than Intramuscular Immunizations SOSIP.664 and NFL Env trimers have similar immunogenic properties. (A–F) Comparison of subcutaneous (s.c. or SubQ) and intramuscular (i.m. or IM) immunization routes with BG505 SOSIP.664 as immunogen. (A) BG505 nAb titers after BG505 SOSIP.664 s.c. or i.m. immunization (week 26; n = 12 animals per group). (B and C) GC B cell frequencies (B) and GC Tfh cell frequencies (C) 3 weeks after the first, second, and third s.c. or i.m. immunizations (n = 24 LNs per group). (D and E) BG505 SOSIP (D) and BG505 gp120 (E) IgG binding titers (week 26; n = 12). (F) Evans Blue dye drainage to LNs at 48 hr was scored by visual inspection (left). Dye accumulation in LNs at 72 hr was quantified by dye extraction (right). (G–K) Ab responses in RMs immunized with BG505 SOSIP.664 or BG505 NFL via s.c. injection. (G and H) BG505 nAb titers in BG505-SOSIP.664- and BG505-NFL-immunized RMs (n = 12 and 6, respectively) 2 weeks after the third (G) or second (H) immunization. (I–K) BG505 SOSIP (I), BG505 NFL (J), and BG505 gp120 (K) IgG binding titers 2 weeks after the third immunization (week 26; n = 6 or 12). (L) Ratio of BG505 to SF162 nAb titers 2 weeks after the third immunization (week 26; n = 6 or 12). All nAb titer and ELISA binding Ab data represent geometric mean titers with geometric SD. All cell-frequency data represent the mean and SD. The Evans blue quantification shows mean with SEM. See also Figure S2.

**Figure 3**
Trimer Stabilization Strategies Can Reduce V3-Loop Immunogenicity Modified BG505 SOSIP and BG505 Olio6 constructs were compared with the BG505 SOSIP.664 immunogen. Immunogens are abbreviated as follows: BG505 SOSIP.664, .664 or BG505 WT; BG505 SOSIP.v4.1, v4.1; BG505 SOSIP.v5.2, v5.2; BG505 SOSIP Olio6, Olio6; and BG505 SOSIP Olio6 CD4-KO, Olio6 CD4-KO. Dotted lines indicate LOD. (A) V3-loop protein sequence of BG505 WT, v4.1, v5.2, Olio6, and Olio6 CD4-KO. Changes are in red. (B) BG505 V3-loop peptide binding IgG titers 2 weeks after the third immunization (week 26; n = 6 or 12). (C) BG505 SOSIP binding IgG titers 2 weeks after the third immunization (week 26; n = 6 or 12). (D) Cross-binding analysis of Olio6 and SOSIP.664 (WT) V3-loop peptides (week 26; n = 6). See Figure 3A. (E–G) Tier 1 SF162 nAb titers (E), BG505 nAb titers (F), and their ratio (G) 2 weeks after the third immunization (week 26; n = 6 or 12). (H and I) GC B cell frequencies after the first (H) and second (I) immunizations (n = 24 or 12). (J and K) GC Tfh cell frequencies after the first (J) and second (K) immunizations (n = 24 or 12). (L) Correlation between GC B cell frequency and BG505 nAb titers. Animals are color coded by immunogen (n = 24). (M) Correlation between GC B cell frequency and BG505 SOSIP binding IgG titers (n = 24). All nAb titer and ELISA binding Ab data represent geometric mean titers with geometric SD. All cell-frequency data represent the mean and SD. See also Figure S3.

**Figure 4**
Extended Immunogen Release Induces Higher nAb Titers Than Conventional Immunization (A–E) Immunogen doses of 100 or 20 μg s.c. immunizations of BG505 SOSIP.664. (A) BG505 nAb titers at week 26 (n = 6 or 12). (B) BG505 SOSIP binding titers at week 26 (n = 6 or 12). (C) Kinetics of BG505 nAb titers. (D and E) GC B cell (D) and GC Tfh cell (E) frequencies after the first, second, and third immunizations. (F–L) Bolus (conventional) versus continuous immunogen delivery of BG505 SOSIP.v5.2 immunogen. (F) Immunization schedule and sampling for continuous antigen delivery using osmotic pumps. (G) BG505 nAb titers in animals immunized by osmotic pump (red) or conventional bolus (Conv, black) (^∗p < 0.05; ^∗∗p < 0.01; n = 6). (H) Peak BG505 nAb titers after the third immunization (n = 6). (I and J) GC B cell (I) and GC Tfh cell (J) frequencies after the first, second, and third immunizations. (K) Proliferation of GC Tfh cells at week 11. Flow cytometry was gated on CXCR5^hi PD-1^hi GC Tfh cells. (L) Frequency of Ki67⁺ GC Tfh cells at week 11 (n = 12). All nAb titer and ELISA binding Ab data represent geometric mean titers with geometric SD. All cell-frequency data represent the mean and SD. See also Figure S4.

**Figure 5**
Liposomal Presentation of Env Trimer Immunogen Induces nAb Titers Comparable to Those of Soluble Immunogens RMs were immunized with soluble (Sol) BG505 Olio6 CD4-KO or BG505 Olio6 CD4-KO covalently conjugated to liposomes (Lipo). (A) BG505 nAb titers in RMs 4 weeks after the second immunization (n = 6). (B and C) BG505 SOSIP (B) or V3-loop peptide (C) binding IgG titers 2 weeks after the second immunization (week 10; n = 6). (D and E) GC B cell (D) and GC Tfh cell (E) frequencies after the first or second immunization (n = 12). All nAb titer and ELISA binding Ab data represent geometric mean titers with geometric SD. All cell-frequency data represent the mean and SD. See also Figure S5.

**Figure 6**
GC Dynamics across All Immunized Animals (A–C) GC B cell frequency (A), GC Tfh cell frequency (B), and ratio of GC B cells to GC Tfh cells (C) for all study animals at baseline (BL) and after the first, second, and third immunizations. Points represent individual LNs (n = 144; 72 animals × 2 LNs). (D–F) Subpopulations of GC Tfh cells. (D) Flow cytometry of GC Tfh cell sub-populations. Gates were set on the basis of total CD4⁺ T cells (Figure S6B). (E) Frequency of FoxP3⁺ GC Tfr cells. (F) Frequency of CXCR3⁺ GC Tfh cells. (G) GC B cell frequencies separated by gender. (H) BG505 nAb titers separated by gender. All nAb titer data represent geometric mean titers with geometric SD. All cell-frequency data represent the mean and SD. See also Figure S6.

**Figure 7**
NHPs Immunized with Env Trimer Recognize Diverse Neutralization Epitopes and Generate Some Neutralization Breadth (A and B) BG505 nAb epitope mapping of sera from high-BG505-neutralizer RMs. (A) Fold difference of nAb titer in the presence and absence of competitor proteins (linear BG505 V3-peptide or BG505 D368R gp120) in sera at week 26. (B) Fold difference in nAb titers between mutant and WT BG505 pseudoviruses at week 26. (C) Neutralization breadth on a 12-virus global panel. (D) Neutralization of BG505 and MG505 A2 nAb titers at week 26 (n = 78). (E) Correlation of BG505 and MG505 A2 nAb titers at week 26 (n = 12). See also Figure S7.

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References

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Elicitation of Robust Tier 2 Neutralizing Antibody Responses in Nonhuman Primates by HIV Envelope Trimer Immunization Using Optimized Approaches

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Elicitation of Robust Tier 2 Neutralizing Antibody Responses in Nonhuman Primates by HIV Envelope Trimer Immunization Using Optimized Approaches

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