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Science. Author manuscript; available in PMC 2016 December 12. Published in final edited form as: Science. 2016 May 20; 352(6288): 997–1001. doi:10.1126/science.aaf0972.
HIV-1 Therapy with Monoclonal Antibody 3BNC117 Elicits Host Immune Responses against HIV-1
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Till Schoofs1,*, Florian Klein1,2,3,*, Malte Braunschweig1,4,*, Edward F. Kreider5, Anna Feldmann6, Lilian Nogueira1, Thiago Oliveira1, Julio C. C. Lorenzi1, Erica H. Parrish5, Gerald H. Learn5, Anthony P. West Jr7, Pamela J. Bjorkman7, Sarah J. Schlesinger1, Michael S. Seaman8, Julie Czartoski9, M. Juliana McElrath9, Nico Pfeifer6, Beatrice H. Hahn5, Marina Caskey1, and Michel C. Nussenzweig1,10 1Laboratory
of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA
2Laboratory
of Experimental Immunology, Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany
3Department
I of Internal Medicine, Center of Integrated Oncology Cologne-Bonn, University Hospital Cologne, 50937 Cologne, Germany 4Albert
Ludwigs University of Freiburg, Freiburg, Germany
5Departments
of Medicine and Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
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6Department
of Computational Biology and Applied Algorithmics, Max Planck Institute for Informatics, Campus E14, 66123 Saarbrücken, Germany 7Division
of Biology, California Institute of Technology, Pasadena, CA 91125, USA
Correspondence should be addressed to: [email protected]. *These authors contributed equally
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Supplementary Materials Materials and Methods Figure S1 Dynamics of neutralization potency and breadth in each individual Figure S2 Dynamics of neutralization breadth by group of study Figure S3 Comparison of clinical parameters between groups of study subjects Figure S4 Correlation between starting neutralization (AUC) and neutralization increase (AUC) Figure S5 Neutralization profile comparison of AUC change in 3BNC117-treated individuals with neutralization profile of 3BNC117 Figure S6 Pairwise diversity over time (amino acid level) Figure S7 Viral evolution in subjects 2A3, 2C5, 2C4 and 2E2–2E5 Figure S8 Viral sequence evolution in subject 2A1 Figure S9 Viral sequence evolution in subjects 2C5 and 2E1–2E5 FigureS10 Full 3BNC117 contact site logo plots for subjects 2A1 and 2E1 Supplementary Tables Table S1 Clinical characteristics of 3BNC117-treated subjects and untreated viremic subjects Table S2 Autologous neutralization data of patient IgG against PBMC co-culture virus Table S3 Panel of pseudoviruses used to determine neutralizing activity Table S4 Heterologous TZM.bl neutralization data of purified patient IgG (IC50, AUC) Table S5 Summary measures table breadth and potency Day 0 and Wk24 Table S6 TZM.bl neutralization data over time in subject 2A3 (IC50) Table S7 Analysis of potential confounding variables Table S8 Rank correlation and p-values for Spearman rank correlation of patient and 3BNC117 neutralization Table S9 Number of gp160 nucleotide sequences included in phylogenetic analyses Table S10 Envelope amino acid residues under significant selection in each individual Table S11 TZM.bl neutralization data of CMV-env pseudotyped viruses (IC50)
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8Center
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for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
9Vaccine
and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
10Howard
Hughes Medical Institute, The Rockefeller University, New York, NY 10065, USA
Abstract
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3BNC117 is a broad and potent anti-HIV-1 neutralizing antibody that targets the CD4 binding site on the viral envelope spike. When administered passively, this antibody can prevent infection in animal models and suppress viremia in HIV-1-infected individuals. Here we report that HIV-1 immunotherapy with a single injection of 3BNC117 impacts host antibody responses in viremic subjects. In comparison to untreated controls that showed little change in their neutralizing activity over a six-month period, 3BNC117 infusion significantly improved neutralizing responses to heterologous tier 2 viruses in nearly all study participants. We conclude that 3BNC117-mediated immunotherapy enhances host humoral immunity to HIV-1. Development of serum neutralization breadth during HIV-1 infection typically occurs several years after infection and is seen as a continuum with ~50% of HIV-1-infected individuals developing some level of broad neutralization and a small fraction of individuals acquiring serum neutralizing activity of extraordinary breadth and potency (1–4). Antibody cloning experiments revealed that this activity is due to one or more potent broadly neutralizing antibodies (bNAbs) that target one or more epitopes on the viral spike protein, gp160 (1, 5–10).
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bNAbs show exceptional breadth and potency in vitroand can protect against or suppress active infection in humanized mice (11–13) and macaques (14, 15). Moreover, in a phase I clinical trial, a single injection of 3BNC117, a CD4-binding-site specific bNAb (6) was safe and effective in suppressing HIV-1 viremia by an average of 1.48 logs (16). In addition to direct effects on target cells and pathogens, antibody-mediated immunotherapies have the potential to engage the host immune system and induce both innate and adaptive immune responses (17). In particular the Fc domains of antibodies interact with receptors on innate cells such as natural killer (NK) cells and phagocytes to enhance the clearance of viral particles and the killing of infected cells (18). To test the hypothesis that bNAb-mediated immunotherapy can enhance immunity to HIV-1 in humans, we examined the serologic responses to the virus in individuals who received 3BNC117.
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A single 3BNC117 infusion was administered to HIV-1-infected individuals at doses of 1, 3, 10, or 30 mg/kg (Fig. 1A, Table S1A) (16, 19). To determine whether 3BNC117 therapy is associated with changes in viral sensitivity and serologic responses to autologous viruses, we cultured HIV-1 from peripheral blood mononuclear cells (PBMCs) of 9 viremic individuals before (d0) and 4 weeks (wks) after 3BNC117 infusion (16). On d0, all but one of the cultured viruses were sensitive to 3BNC117 with IC50 values ranging from 0.09 - 8.8
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µg/ml (Fig. 1B and (16)). At wk 4, we found increased resistance to 3BNC117 in most individuals indicating selection for viral escape variants (Fig. 1B and (16)). When the same viral isolates were tested for sensitivity to the matched individual’s immunoglobulins (IgG) obtained before (d0) and 24 wks after 3BNC117 infusion (Fig. 1A), we found increased neutralizing activity in the wk 24 IgG against both d0 and wk 4 autologous viruses (p=0.0078, Fig. 1C, Table S2). Thus, while 3BNC117 infusion selected for 3BNC117-resistant HIV-1 variants, neutralizing antibody responses continued to develop against autologous viruses (20).
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To test for changes in heterologous neutralizing activity following 3BNC117 treatment, we assayed patients’ d0 and wk 24 IgG against a panel of tier 1 (n=1) and tier 2 (n=12) HIV-1 pseudoviruses that included globally circulating HIV-1 strains (21) (Fig. 2, Table S1, S3, S4). Neutralizing activity was compared between the two time points by measuring the area under the neutralization curve for subjects’ isolated IgG against each virus (AUC) (Table S4B). 15 subjects that received 3BNC117 were not on anti-retroviral therapy (ART) and had starting viral loads from 640 - 53,470 copies/ml (Table S1A). Control IgGs were obtained from 36 viremic individuals who did not receive 3BNC117 and had starting viral loads ranging from 150 – 303,200 copies/ml (Fig. 2, Table S1B).
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During a 6-month observation period, control individuals’ neutralizing activity showed no consistent improvement in either breadth or potency (Fig. 2A and B, S1A, S2, Table S4, S5A) (4, 22). In contrast, all but one of the 15 viremic individuals infused with 3BNC117 showed increased breadth and/or potency against the pseudovirus panel at wk 24 (p=7.1 × 10−7, Fig. 2A, S1B, S2, Table S4, S5, S6). The absolute change in neutralizing activity varied between viruses and individuals, ranging from small effects to dramatic increases as observed in patient 2A3 for viral strain Q769.d22 (Fig. 2C, Table S4, S5, S6). Significant differences were also evident between 3BNC117-treated and control groups regardless whether sera from all individuals were considered in aggregate, or examined against individual viruses (p=1.9 × 10−9, Fig. 2B, D). In addition to viremic subjects, we examined 12 individuals that received 3BNC117 while on ART, with no detectable or low-level viremia (20 µg/ml), yet they were ~6.5-fold more sensitive to wk 24 IgG versus d0 IgG (Fig. S7). In
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conclusion, viremic individuals receiving 3BNC117 produced antibodies to autologous viruses that were both sensitive and resistant to 3BNC117. While exceptional broadly neutralizing antibodies to HIV-1 develop only sporadically in a fraction of infected individuals, most HIV-1 infected individuals develop some level of neutralization breadth (1–4). Here we show that 3BNC117 immunotherapy accelerates this process. This boost in heterologous breadth occurs irrespective of demographic, virologic, or dosage factors and was associated with both transient and lasting changes to the viral quasispecies. Of note, neutralization improvements observed were modest in most individuals, potentially owing to the transient nature of therapy with a single antibody as well as the short timeframe of observation.
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Although the effect of 3BNC117 on neutralizing responses to heterologous HIV-1 viruses may seem surprising, anti-HIV-1 antibodies have been associated with enhanced immunity in infants born to HIV-1-infected mothers that have circulating anti-HIV-1 antibodies and macaques treated with monoclonal antibodies or neutralizing serum (29–31). How passively administered antibodies to HIV-1 accelerate the emergence of bNAbs is not completely understood. One possibility is that 3BNC117 infusion selected for viral variants with altered antigenic properties, which in turn stimulated new B cell lineages (23–25, 32– 34). A second possibility is that immune complexes formed by 3BNC117 and circulating viruses act as potent immunogens, a phenomenon that is believed to be responsible for the enhanced CD8+ T cell immunity to tumor antigens in individuals receiving monoclonal antibody based immunotherapy (35–37).
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Irrespective of the mechanism(s), the enhanced antibody response found in individuals receiving 3BNC117 therapy indicates that immunotherapy boosts host immunity to HIV-1. Moreover, the finding that antibody responses to heterologous tier 2 viruses develop in nearly all 3BNC117-treated individuals suggests that host genetics or a specific viral envelope sequence do not limit the development of neutralizing antibodies to HIV-1.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
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We thank all study participants who devoted time to our research. We thank the Rockefeller University Hospital Clinical Research Support Office, the nursing staff for patient care and recruitment, the clinical study group of the Infectious Disease Division at the University Hospital Cologne, and all members of the Nussenzweig lab for helpful discussions. We thank M. Schechter and C. Baro for technical assistance, P. Fast and H. Park for Clinical monitoring, E. Gotschlich and B. Coller for input on study design and P. Hraber for helping with LASSIE analyses. The data reported in this study are tabulated in the main paper and in the supplementary material. Envelope single genome sequencing data can be downloaded from GenBank (Accession numbers KX027737 – KX028736). This work was supported in part by the Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery (CAVD) Grants OPP1032144 (M.S.S.), OPP1092074 and OPP1124068 (M.C.N), the Robertson Foundation to M.C.N., NIH Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID) 1UM1 AI100663-01 (M.C.N) and 1UM1 AI00645 (B.H.H.), the University of Pennsylvania Center for AIDS Research (CFAR) Single Genome Amplification Service Center P30 AI045008 (B.H.H.), NIH grants UM1AI068618 (MJM), UM1AI069481 (MJM), F30 AI112426 (E.F.K), and HIVRAD P01 AI100148 (P.J.B.). T.S. is supported by a Deutsche Forschungsgemeinschaft postdoctoral fellowship (SCHO 1612/1-1). F.K. is supported by the HeisenbergProgram of the DFG (KL 2389/2-1), the European Research Council (ERC-StG639961), and the German Center for
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Infection Research (DZIF), partner site Bonn-Cologne, Cologne, Germany. M.B. is supported by the German National Academic Foundation. J.C.L. is supported by an award from CNPq "Ciencia sem Fronteiras" Brazil (248676/2013-0). M.C.N. is a Howard Hughes Medical Investigator and an inventor on U.S Patent Application No. 14/118,496 filed by Rockefeller University related to 3BNC117.
References and Notes
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1. Klein F, et al. Antibodies in HIV-1 vaccine development and therapy. Science. 2013; 341:1199– 1204. [PubMed: 24031012] 2. West AP Jr, et al. Structural insights on the role of antibodies in HIV-1 vaccine and therapy. Cell. 2014; 156:633–648. [PubMed: 24529371] 3. Mikell I, et al. Characteristics of the earliest cross-neutralizing antibody response to HIV-1. PLoS pathogens. 2011; 7:e1001251. [PubMed: 21249232] 4. Hraber P, et al. Prevalence of broadly neutralizing antibody responses during chronic HIV-1 infection. AIDS. 2014; 28:163–169. [PubMed: 24361678] 5. Wu X, et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science. 2010; 329:856–861. [PubMed: 20616233] 6. Scheid JF, et al. Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science. 2011; 333:1633–1637. [PubMed: 21764753] 7. Walker LM, et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature. 2011; 477:466–470. [PubMed: 21849977] 8. Klein F, et al. Broad neutralization by a combination of antibodies recognizing the CD4 binding site and a new conformational epitope on the HIV-1 envelope protein. J Exp Med. 2012; 209:1469– 1479. [PubMed: 22826297] 9. Bonsignori M, et al. Two Distinct Broadly Neutralizing Antibody Specificities of Different Clonal Lineages in a Single HIV-1-infected Donor: Implications for Vaccine Design. Journal of virology. 2012 10. Scheid JF, et al. Broad diversity of neutralizing antibodies isolated from memory B cells in HIVinfected individuals. Nature. 2009; 458:636–640. [PubMed: 19287373] 11. Klein F, et al. HIV therapy by a combination of broadly neutralizing antibodies in humanized mice. Nature. 2012; 492:118–122. [PubMed: 23103874] 12. Horwitz JA, et al. HIV-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice. Proceedings of the National Academy of Sciences of the United States of America. 2013; 110:16538–16543. [PubMed: 24043801] 13. Pietzsch J, et al. A mouse model for HIV-1 entry. Proceedings of the National Academy of Sciences of the United States of America. 2012; 109:15859–15864. [PubMed: 23019371] 14. Barouch DH, et al. Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys. Nature. 2013; 503:224–228. [PubMed: 24172905] 15. Shingai M, et al. Antibody-mediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia. Nature. 2013; 503:277–280. [PubMed: 24172896] 16. Caskey M, et al. Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117. Nature. 2015; 522:487–491. [PubMed: 25855300] 17. Bournazos S, Ravetch JV. Fcgamma receptor pathways during active and passive immunization. Immunol Rev. 2015; 268:88–103. [PubMed: 26497515] 18. Lu C-L. Enhanced clearance of HIV-1-infected cells by anti-HIV-1 broadly neutralizing antibodies in vivo. Science. 2016 19. Materials and methods are available as supplementary materials on Science Online 20. Wei X, et al. Antibody neutralization and escape by HIV-1. Nature. 2003; 422:307–312. [PubMed: 12646921] 21. deCamp A, et al. Global panel of HIV-1 Env reference strains for standardized assessments of vaccine-elicited neutralizing antibodies. Journal of virology. 2014; 88:2489–2507. [PubMed: 24352443] 22. Deeks SG, et al. Neutralizing antibody responses against autologous and heterologous viruses in acute versus chronic human immunodeficiency virus (HIV) infection: evidence for a constraint on
Science. Author manuscript; available in PMC 2016 December 12.
Schoofs et al.
Page 7
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
the ability of HIV to completely evade neutralizing antibody responses. Journal of virology. 2006; 80:6155–6164. [PubMed: 16731954] 23. Liao HX, et al. Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature. 2013; 496:469–476. [PubMed: 23552890] 24. Doria-Rose NA, et al. Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies. Nature. 2014; 509:55–62. [PubMed: 24590074] 25. Moore PL, Williamson C, Morris L. Virological features associated with the development of broadly neutralizing antibodies to HIV-1. Trends Microbiol. 2015; 23:204–211. [PubMed: 25572881] 26. Zhou T, et al. Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01. Science. 2010; 329:811–817. [PubMed: 20616231] 27. Klein F, et al. Somatic mutations of the immunoglobulin framework are generally required for broad and potent HIV-1 neutralization. Cell. 2013; 153:126–138. [PubMed: 23540694] 28. Hraber P, et al. Longitudinal Antigenic Sequences and Sites from Intra-Host Evolution (LASSIE) Identifies Immune-Selected HIV Variants. Viruses. 2015; 7:5443–5475. [PubMed: 26506369] 29. Goo L, Chohan V, Nduati R, Overbaugh J. Early development of broadly neutralizing antibodies in HIV-1-infected infants. Nat Med. 2014; 20:655–658. [PubMed: 24859529] 30. Haigwood NL, et al. Passive immunotherapy in simian immunodeficiency virus-infected macaques accelerates the development of neutralizing antibodies. Journal of virology. 2004; 78:5983–5995. [PubMed: 15140996] 31. Ng CT, et al. Passive neutralizing antibody controls SHIV viremia and enhances B cell responses in infant macaques. Nat Med. 2010; 16:1117–1119. [PubMed: 20890292] 32. Moore PL, et al. Evolution of an HIV glycan-dependent broadly neutralizing antibody epitope through immune escape. Nat Med. 2012; 18:1688–1692. [PubMed: 23086475] 33. Gao F, et al. Cooperation of B cell lineages in induction of HIV-1-broadly neutralizing antibodies. Cell. 2014; 158:481–491. [PubMed: 25065977] 34. Bhiman JN, et al. Viral variants that initiate and drive maturation of V1V2-directed HIV-1 broadly neutralizing antibodies. Nat Med. 2015; 21:1332–1336. [PubMed: 26457756] 35. Wang TT, et al. Anti-HA Glycoforms Drive B Cell Affinity Selection and Determine Influenza Vaccine Efficacy. Cell. 2015; 162:160–169. [PubMed: 26140596] 36. Bournazos S, DiLillo DJ, Ravetch JV. The role of Fc-FcgammaR interactions in IgG-mediated microbial neutralization. J Exp Med. 2015; 212:1361–1369. [PubMed: 26282878] 37. DiLillo DJ, Ravetch JV. Differential Fc-Receptor Engagement Drives an Anti-tumor Vaccinal Effect. Cell. 2015; 161:1035–1045. [PubMed: 25976835] 38. Ratcliffe SJ, Shults J. GEEQBOX: A MATLAB toolbox for generalized estimating equations and quasi-least squares. J Stat Softw. 2008; 25:1–14. 39. Gilbert PB, Rossini AJ, Shankarappa R. Two-sample tests for comparing intra-individual genetic sequence diversity between populations. Biometrics. 2005; 61:106–117. [PubMed: 15737083] 40. Giorgi EE, Bhattacharya T. A note on two-sample tests for comparing intra-individual genetic sequence diversity between populations. Biometrics. 2012; 68:1323–1326. author reply 1326. [PubMed: 23004569] 41. Deng W, et al. DIVEIN: a web server to analyze phylogenies, sequence divergence, diversity, and informative sites. Biotechniques. 2010; 48:405–408. [PubMed: 20569214] 42. van 't Wout AB, Schuitemaker H, Kootstra NA. Isolation and propagation of HIV-1 on peripheral blood mononuclear cells. Nature protocols. 2008; 3:363–370. [PubMed: 18323807] 43. Seaman MS, et al. Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. Journal of virology. 2009; 84:1439–1452. [PubMed: 19939925] 44. Li M, et al. Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. Journal of virology. 2005; 79:10108–10125. [PubMed: 16051804]
Science. Author manuscript; available in PMC 2016 December 12.
Schoofs et al.
Page 8
Author Manuscript Author Manuscript
45. West AP Jr, et al. Computational analysis of anti-HIV-1 antibody neutralization panel data to identify potential functional epitope residues. Proceedings of the National Academy of Sciences of the United States of America. 2013; 110:10598–10603. [PubMed: 23754383] 46. Kryazhimskiy S, Rice DP, Jerison ER, Desai MM. Microbial evolution. Global epistasis makes adaptation predictable despite sequence-level stochasticity. Science. 2014; 344:1519–1522. [PubMed: 24970088] 47. Larkin MA, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007; 23:2947–2948. [PubMed: 17846036] 48. Kearse M, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012; 28:1647–1649. [PubMed: 22543367] 49. Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9:772. 50. Guindon S, et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic biology. 2010; 59:307–321. [PubMed: 20525638] 51. Nickle DC, et al. HIV-specific probabilistic models of protein evolution. PloS one. 2007; 2:e503. [PubMed: 17551583] 52. Kirchherr JL, et al. High throughput functional analysis of HIV-1 env genes without cloning. J Virol Methods. 2007; 143:104–111. [PubMed: 17416428]
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Author Manuscript Author Manuscript Figure 1. Virus sensitivity to 3BNC117 and autologous antibody responses
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A. Graph displays kinetics of 3BNC117 antibody decay in HIV-1-infected individuals as determined by a validated anti-idiotype ELISA (16). Shown are mean values of patients infused in each respective dose group. Each patient sample was measured in duplicates. Gray shaded area indicates lower level of accuracy of the assay (2 µg/ml). Red arrows indicate the timepoints of IgG purification. B. Autologous virus sensitivity to 3BNC117 before (day 0, grey) and 4 wks (black) after 3BNC117 infusion. Y-axis shows IC50s for 3BNC117 on viral culture supernatants from PBMCs determined by TZM.bl assay. Neutralization assays performed in duplicates. C. Graph shows the AUC of the neutralization curves of purified IgGs obtained from sera on day 0 (orange) or wk 24 (green) against day 0 (left panel) or wk 4 (right panel) autologous viruses. Neutralization assays performed in duplicates. p-values determined by Wilcoxon signed-rank test.
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Figure 2. Heterologous antibody responses
A. Graph shows the difference in overall AUC (mean AUC change) per individual in TZM.bl assays against 13 heterologous viruses (see 2D) for day 0 vs. wk 24 IgG obtained from 36 untreated viremic controls (mean sampling interval: 26.8 wks), 15 viremic individuals infused with 3BNC117 (mean sampling interval: 24.1 wks), and 12 ART-treated individuals receiving 3BNC117 infusion (mean sampling interval: 24.0 wks) (16). Neutralization assays performed in duplicates. p-values determined by unpaired Wilcoxon test (rank-sum test). B. Graph shows the aggregated differences in AUC between d0 and wk
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24 IgG assayed by TZM.bl for all viruses and all individuals. Each dot represents a single AUC difference for a single virus from one individual displayed in A. Colored bars represent mean of all AUCs. Whiskers show standard deviation. p-values determined using generalized estimating equations (38). C. Graph shows 3BNC117 antibody levels (ELISA, white) and TZM.bl neutralization titer (green) against tier 2 strain Q769.d22 in subject 2A3 D. Mean AUCs of IgGs of all individuals at d0 (grey) and wk 24 (color of respective group) for each HIV-1 pseudovirus tested. Changes in neutralization of viremic control individuals without 3BNC117 infusion are shown in yellow (left). Change in neutralization of 3BNC117-treated individuals shown in dark (off ART, middle) and light blue (on ART, right). p-values determined using unpaired Wilcoxon test (rank-sum test). Red stars indicate significant p-values after Bonferroni-correction (threshold p < 0.0038).
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Author Manuscript Author Manuscript Author Manuscript Figure 3. HIV-1 quasispecies diversity before and after 3BNC117 infusion
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A. Maximum likelihood phylogenetic tree of single genome-derived env gene sequences from d0 plasma, before therapy with 3BNC117 (Table S9). Asterisks indicate bootstrap values of 100%. Individual viral sequences are color coded as indicated. B. Scatter plots depicting pairwise nucleotide sequence diversity of plasma env sequences on d0, and wk 4 (2E5, wk 6), 12 and 24 after infusion. Each dot represents the pairwise genetic difference between two sequences at a given timepoint. Colored bars indicate median diversity, while black bars indicates the interquartile range. p-values were determined using a two-sample Ustatistic based Z-test (39–41). C. Graph shows the relationship between d0 mean heterologous neutralizing AUC against a panel of tier 1 (n=1) and tier 2 (n=12) viruses (xaxis) and the median pairwise nucleotide diversity for each patient (y-axis).
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Author Manuscript Author Manuscript Author Manuscript Figure 4. Antibody responses to the evolving viral quasispecies
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A. Maximum-likelihood phylogenetic trees of single genome-derived env gene sequences from subjects 2A1 and 2E1 sampled on d0 and wk 4, 12, and 24 after 3BNC117 infusion (left). Clades with bootstrap support ≥ 70% are indicated by a black star and are arbitrarily named groups A-D in case of subject 2A1. Bar graphs (middle) indicate the timepoints from which sequences in the tree are derived. Heat maps (right) show the 3BNC117 IC50, d0 IgG IC50 and wk 24 IgG IC50 values against autologous pseudoviruses using env sequences as indicated by colored stars. Neutralization assays performed in duplicates. B. Sequence logo plots illustrating longitudinal amino acid changes in and around known 3BNC117-contact residues (26, 27) in subject 2A1 and 2E1. Letters indicate deviations from the d0 consensus shown at the top, whereas white spaces indicate agreement with the d0 consensus. Colors indicate basic (dark blue) and acidic (red) residues and a turquoise “O” is used instead of
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“N” to indicate a potential N-glycosylation site. Logo plots were generated using LASSIE (28). + symbols indicate 3BNC117 contact residues confirmed by two crystal structures (26, 27).
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Science. Author manuscript; available in PMC 2016 December 12. Published in final edited form as: Science. 2016 May 20; 352(6288): 997–1001. doi:10.1126/science.aaf0972.
HIV-1 Therapy with Monoclonal Antibody 3BNC117 Elicits Host Immune Responses against HIV-1
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Till Schoofs1,*, Florian Klein1,2,3,*, Malte Braunschweig1,4,*, Edward F. Kreider5, Anna Feldmann6, Lilian Nogueira1, Thiago Oliveira1, Julio C. C. Lorenzi1, Erica H. Parrish5, Gerald H. Learn5, Anthony P. West Jr7, Pamela J. Bjorkman7, Sarah J. Schlesinger1, Michael S. Seaman8, Julie Czartoski9, M. Juliana McElrath9, Nico Pfeifer6, Beatrice H. Hahn5, Marina Caskey1, and Michel C. Nussenzweig1,10 1Laboratory
of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA
2Laboratory
of Experimental Immunology, Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany
3Department
I of Internal Medicine, Center of Integrated Oncology Cologne-Bonn, University Hospital Cologne, 50937 Cologne, Germany 4Albert
Ludwigs University of Freiburg, Freiburg, Germany
5Departments
of Medicine and Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
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6Department
of Computational Biology and Applied Algorithmics, Max Planck Institute for Informatics, Campus E14, 66123 Saarbrücken, Germany 7Division
of Biology, California Institute of Technology, Pasadena, CA 91125, USA
Correspondence should be addressed to: [email protected]. *These authors contributed equally
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Supplementary Materials Materials and Methods Figure S1 Dynamics of neutralization potency and breadth in each individual Figure S2 Dynamics of neutralization breadth by group of study Figure S3 Comparison of clinical parameters between groups of study subjects Figure S4 Correlation between starting neutralization (AUC) and neutralization increase (AUC) Figure S5 Neutralization profile comparison of AUC change in 3BNC117-treated individuals with neutralization profile of 3BNC117 Figure S6 Pairwise diversity over time (amino acid level) Figure S7 Viral evolution in subjects 2A3, 2C5, 2C4 and 2E2–2E5 Figure S8 Viral sequence evolution in subject 2A1 Figure S9 Viral sequence evolution in subjects 2C5 and 2E1–2E5 FigureS10 Full 3BNC117 contact site logo plots for subjects 2A1 and 2E1 Supplementary Tables Table S1 Clinical characteristics of 3BNC117-treated subjects and untreated viremic subjects Table S2 Autologous neutralization data of patient IgG against PBMC co-culture virus Table S3 Panel of pseudoviruses used to determine neutralizing activity Table S4 Heterologous TZM.bl neutralization data of purified patient IgG (IC50, AUC) Table S5 Summary measures table breadth and potency Day 0 and Wk24 Table S6 TZM.bl neutralization data over time in subject 2A3 (IC50) Table S7 Analysis of potential confounding variables Table S8 Rank correlation and p-values for Spearman rank correlation of patient and 3BNC117 neutralization Table S9 Number of gp160 nucleotide sequences included in phylogenetic analyses Table S10 Envelope amino acid residues under significant selection in each individual Table S11 TZM.bl neutralization data of CMV-env pseudotyped viruses (IC50)
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for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
9Vaccine
and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
10Howard
Hughes Medical Institute, The Rockefeller University, New York, NY 10065, USA
Abstract
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3BNC117 is a broad and potent anti-HIV-1 neutralizing antibody that targets the CD4 binding site on the viral envelope spike. When administered passively, this antibody can prevent infection in animal models and suppress viremia in HIV-1-infected individuals. Here we report that HIV-1 immunotherapy with a single injection of 3BNC117 impacts host antibody responses in viremic subjects. In comparison to untreated controls that showed little change in their neutralizing activity over a six-month period, 3BNC117 infusion significantly improved neutralizing responses to heterologous tier 2 viruses in nearly all study participants. We conclude that 3BNC117-mediated immunotherapy enhances host humoral immunity to HIV-1. Development of serum neutralization breadth during HIV-1 infection typically occurs several years after infection and is seen as a continuum with ~50% of HIV-1-infected individuals developing some level of broad neutralization and a small fraction of individuals acquiring serum neutralizing activity of extraordinary breadth and potency (1–4). Antibody cloning experiments revealed that this activity is due to one or more potent broadly neutralizing antibodies (bNAbs) that target one or more epitopes on the viral spike protein, gp160 (1, 5–10).
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bNAbs show exceptional breadth and potency in vitroand can protect against or suppress active infection in humanized mice (11–13) and macaques (14, 15). Moreover, in a phase I clinical trial, a single injection of 3BNC117, a CD4-binding-site specific bNAb (6) was safe and effective in suppressing HIV-1 viremia by an average of 1.48 logs (16). In addition to direct effects on target cells and pathogens, antibody-mediated immunotherapies have the potential to engage the host immune system and induce both innate and adaptive immune responses (17). In particular the Fc domains of antibodies interact with receptors on innate cells such as natural killer (NK) cells and phagocytes to enhance the clearance of viral particles and the killing of infected cells (18). To test the hypothesis that bNAb-mediated immunotherapy can enhance immunity to HIV-1 in humans, we examined the serologic responses to the virus in individuals who received 3BNC117.
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A single 3BNC117 infusion was administered to HIV-1-infected individuals at doses of 1, 3, 10, or 30 mg/kg (Fig. 1A, Table S1A) (16, 19). To determine whether 3BNC117 therapy is associated with changes in viral sensitivity and serologic responses to autologous viruses, we cultured HIV-1 from peripheral blood mononuclear cells (PBMCs) of 9 viremic individuals before (d0) and 4 weeks (wks) after 3BNC117 infusion (16). On d0, all but one of the cultured viruses were sensitive to 3BNC117 with IC50 values ranging from 0.09 - 8.8
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µg/ml (Fig. 1B and (16)). At wk 4, we found increased resistance to 3BNC117 in most individuals indicating selection for viral escape variants (Fig. 1B and (16)). When the same viral isolates were tested for sensitivity to the matched individual’s immunoglobulins (IgG) obtained before (d0) and 24 wks after 3BNC117 infusion (Fig. 1A), we found increased neutralizing activity in the wk 24 IgG against both d0 and wk 4 autologous viruses (p=0.0078, Fig. 1C, Table S2). Thus, while 3BNC117 infusion selected for 3BNC117-resistant HIV-1 variants, neutralizing antibody responses continued to develop against autologous viruses (20).
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To test for changes in heterologous neutralizing activity following 3BNC117 treatment, we assayed patients’ d0 and wk 24 IgG against a panel of tier 1 (n=1) and tier 2 (n=12) HIV-1 pseudoviruses that included globally circulating HIV-1 strains (21) (Fig. 2, Table S1, S3, S4). Neutralizing activity was compared between the two time points by measuring the area under the neutralization curve for subjects’ isolated IgG against each virus (AUC) (Table S4B). 15 subjects that received 3BNC117 were not on anti-retroviral therapy (ART) and had starting viral loads from 640 - 53,470 copies/ml (Table S1A). Control IgGs were obtained from 36 viremic individuals who did not receive 3BNC117 and had starting viral loads ranging from 150 – 303,200 copies/ml (Fig. 2, Table S1B).
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During a 6-month observation period, control individuals’ neutralizing activity showed no consistent improvement in either breadth or potency (Fig. 2A and B, S1A, S2, Table S4, S5A) (4, 22). In contrast, all but one of the 15 viremic individuals infused with 3BNC117 showed increased breadth and/or potency against the pseudovirus panel at wk 24 (p=7.1 × 10−7, Fig. 2A, S1B, S2, Table S4, S5, S6). The absolute change in neutralizing activity varied between viruses and individuals, ranging from small effects to dramatic increases as observed in patient 2A3 for viral strain Q769.d22 (Fig. 2C, Table S4, S5, S6). Significant differences were also evident between 3BNC117-treated and control groups regardless whether sera from all individuals were considered in aggregate, or examined against individual viruses (p=1.9 × 10−9, Fig. 2B, D). In addition to viremic subjects, we examined 12 individuals that received 3BNC117 while on ART, with no detectable or low-level viremia (20 µg/ml), yet they were ~6.5-fold more sensitive to wk 24 IgG versus d0 IgG (Fig. S7). In
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conclusion, viremic individuals receiving 3BNC117 produced antibodies to autologous viruses that were both sensitive and resistant to 3BNC117. While exceptional broadly neutralizing antibodies to HIV-1 develop only sporadically in a fraction of infected individuals, most HIV-1 infected individuals develop some level of neutralization breadth (1–4). Here we show that 3BNC117 immunotherapy accelerates this process. This boost in heterologous breadth occurs irrespective of demographic, virologic, or dosage factors and was associated with both transient and lasting changes to the viral quasispecies. Of note, neutralization improvements observed were modest in most individuals, potentially owing to the transient nature of therapy with a single antibody as well as the short timeframe of observation.
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Although the effect of 3BNC117 on neutralizing responses to heterologous HIV-1 viruses may seem surprising, anti-HIV-1 antibodies have been associated with enhanced immunity in infants born to HIV-1-infected mothers that have circulating anti-HIV-1 antibodies and macaques treated with monoclonal antibodies or neutralizing serum (29–31). How passively administered antibodies to HIV-1 accelerate the emergence of bNAbs is not completely understood. One possibility is that 3BNC117 infusion selected for viral variants with altered antigenic properties, which in turn stimulated new B cell lineages (23–25, 32– 34). A second possibility is that immune complexes formed by 3BNC117 and circulating viruses act as potent immunogens, a phenomenon that is believed to be responsible for the enhanced CD8+ T cell immunity to tumor antigens in individuals receiving monoclonal antibody based immunotherapy (35–37).
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Irrespective of the mechanism(s), the enhanced antibody response found in individuals receiving 3BNC117 therapy indicates that immunotherapy boosts host immunity to HIV-1. Moreover, the finding that antibody responses to heterologous tier 2 viruses develop in nearly all 3BNC117-treated individuals suggests that host genetics or a specific viral envelope sequence do not limit the development of neutralizing antibodies to HIV-1.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
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We thank all study participants who devoted time to our research. We thank the Rockefeller University Hospital Clinical Research Support Office, the nursing staff for patient care and recruitment, the clinical study group of the Infectious Disease Division at the University Hospital Cologne, and all members of the Nussenzweig lab for helpful discussions. We thank M. Schechter and C. Baro for technical assistance, P. Fast and H. Park for Clinical monitoring, E. Gotschlich and B. Coller for input on study design and P. Hraber for helping with LASSIE analyses. The data reported in this study are tabulated in the main paper and in the supplementary material. Envelope single genome sequencing data can be downloaded from GenBank (Accession numbers KX027737 – KX028736). This work was supported in part by the Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery (CAVD) Grants OPP1032144 (M.S.S.), OPP1092074 and OPP1124068 (M.C.N), the Robertson Foundation to M.C.N., NIH Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID) 1UM1 AI100663-01 (M.C.N) and 1UM1 AI00645 (B.H.H.), the University of Pennsylvania Center for AIDS Research (CFAR) Single Genome Amplification Service Center P30 AI045008 (B.H.H.), NIH grants UM1AI068618 (MJM), UM1AI069481 (MJM), F30 AI112426 (E.F.K), and HIVRAD P01 AI100148 (P.J.B.). T.S. is supported by a Deutsche Forschungsgemeinschaft postdoctoral fellowship (SCHO 1612/1-1). F.K. is supported by the HeisenbergProgram of the DFG (KL 2389/2-1), the European Research Council (ERC-StG639961), and the German Center for
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Infection Research (DZIF), partner site Bonn-Cologne, Cologne, Germany. M.B. is supported by the German National Academic Foundation. J.C.L. is supported by an award from CNPq "Ciencia sem Fronteiras" Brazil (248676/2013-0). M.C.N. is a Howard Hughes Medical Investigator and an inventor on U.S Patent Application No. 14/118,496 filed by Rockefeller University related to 3BNC117.
References and Notes
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1. Klein F, et al. Antibodies in HIV-1 vaccine development and therapy. Science. 2013; 341:1199– 1204. [PubMed: 24031012] 2. West AP Jr, et al. Structural insights on the role of antibodies in HIV-1 vaccine and therapy. Cell. 2014; 156:633–648. [PubMed: 24529371] 3. Mikell I, et al. Characteristics of the earliest cross-neutralizing antibody response to HIV-1. PLoS pathogens. 2011; 7:e1001251. [PubMed: 21249232] 4. Hraber P, et al. Prevalence of broadly neutralizing antibody responses during chronic HIV-1 infection. AIDS. 2014; 28:163–169. [PubMed: 24361678] 5. Wu X, et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science. 2010; 329:856–861. [PubMed: 20616233] 6. Scheid JF, et al. Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science. 2011; 333:1633–1637. [PubMed: 21764753] 7. Walker LM, et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature. 2011; 477:466–470. [PubMed: 21849977] 8. Klein F, et al. Broad neutralization by a combination of antibodies recognizing the CD4 binding site and a new conformational epitope on the HIV-1 envelope protein. J Exp Med. 2012; 209:1469– 1479. [PubMed: 22826297] 9. Bonsignori M, et al. Two Distinct Broadly Neutralizing Antibody Specificities of Different Clonal Lineages in a Single HIV-1-infected Donor: Implications for Vaccine Design. Journal of virology. 2012 10. Scheid JF, et al. Broad diversity of neutralizing antibodies isolated from memory B cells in HIVinfected individuals. Nature. 2009; 458:636–640. [PubMed: 19287373] 11. Klein F, et al. HIV therapy by a combination of broadly neutralizing antibodies in humanized mice. Nature. 2012; 492:118–122. [PubMed: 23103874] 12. Horwitz JA, et al. HIV-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice. Proceedings of the National Academy of Sciences of the United States of America. 2013; 110:16538–16543. [PubMed: 24043801] 13. Pietzsch J, et al. A mouse model for HIV-1 entry. Proceedings of the National Academy of Sciences of the United States of America. 2012; 109:15859–15864. [PubMed: 23019371] 14. Barouch DH, et al. Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys. Nature. 2013; 503:224–228. [PubMed: 24172905] 15. Shingai M, et al. Antibody-mediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia. Nature. 2013; 503:277–280. [PubMed: 24172896] 16. Caskey M, et al. Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117. Nature. 2015; 522:487–491. [PubMed: 25855300] 17. Bournazos S, Ravetch JV. Fcgamma receptor pathways during active and passive immunization. Immunol Rev. 2015; 268:88–103. [PubMed: 26497515] 18. Lu C-L. Enhanced clearance of HIV-1-infected cells by anti-HIV-1 broadly neutralizing antibodies in vivo. Science. 2016 19. Materials and methods are available as supplementary materials on Science Online 20. Wei X, et al. Antibody neutralization and escape by HIV-1. Nature. 2003; 422:307–312. [PubMed: 12646921] 21. deCamp A, et al. Global panel of HIV-1 Env reference strains for standardized assessments of vaccine-elicited neutralizing antibodies. Journal of virology. 2014; 88:2489–2507. [PubMed: 24352443] 22. Deeks SG, et al. Neutralizing antibody responses against autologous and heterologous viruses in acute versus chronic human immunodeficiency virus (HIV) infection: evidence for a constraint on
Science. Author manuscript; available in PMC 2016 December 12.
Schoofs et al.
Page 7
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
the ability of HIV to completely evade neutralizing antibody responses. Journal of virology. 2006; 80:6155–6164. [PubMed: 16731954] 23. Liao HX, et al. Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature. 2013; 496:469–476. [PubMed: 23552890] 24. Doria-Rose NA, et al. Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies. Nature. 2014; 509:55–62. [PubMed: 24590074] 25. Moore PL, Williamson C, Morris L. Virological features associated with the development of broadly neutralizing antibodies to HIV-1. Trends Microbiol. 2015; 23:204–211. [PubMed: 25572881] 26. Zhou T, et al. Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01. Science. 2010; 329:811–817. [PubMed: 20616231] 27. Klein F, et al. Somatic mutations of the immunoglobulin framework are generally required for broad and potent HIV-1 neutralization. Cell. 2013; 153:126–138. [PubMed: 23540694] 28. Hraber P, et al. Longitudinal Antigenic Sequences and Sites from Intra-Host Evolution (LASSIE) Identifies Immune-Selected HIV Variants. Viruses. 2015; 7:5443–5475. [PubMed: 26506369] 29. Goo L, Chohan V, Nduati R, Overbaugh J. Early development of broadly neutralizing antibodies in HIV-1-infected infants. Nat Med. 2014; 20:655–658. [PubMed: 24859529] 30. Haigwood NL, et al. Passive immunotherapy in simian immunodeficiency virus-infected macaques accelerates the development of neutralizing antibodies. Journal of virology. 2004; 78:5983–5995. [PubMed: 15140996] 31. Ng CT, et al. Passive neutralizing antibody controls SHIV viremia and enhances B cell responses in infant macaques. Nat Med. 2010; 16:1117–1119. [PubMed: 20890292] 32. Moore PL, et al. Evolution of an HIV glycan-dependent broadly neutralizing antibody epitope through immune escape. Nat Med. 2012; 18:1688–1692. [PubMed: 23086475] 33. Gao F, et al. Cooperation of B cell lineages in induction of HIV-1-broadly neutralizing antibodies. Cell. 2014; 158:481–491. [PubMed: 25065977] 34. Bhiman JN, et al. Viral variants that initiate and drive maturation of V1V2-directed HIV-1 broadly neutralizing antibodies. Nat Med. 2015; 21:1332–1336. [PubMed: 26457756] 35. Wang TT, et al. Anti-HA Glycoforms Drive B Cell Affinity Selection and Determine Influenza Vaccine Efficacy. Cell. 2015; 162:160–169. [PubMed: 26140596] 36. Bournazos S, DiLillo DJ, Ravetch JV. The role of Fc-FcgammaR interactions in IgG-mediated microbial neutralization. J Exp Med. 2015; 212:1361–1369. [PubMed: 26282878] 37. DiLillo DJ, Ravetch JV. Differential Fc-Receptor Engagement Drives an Anti-tumor Vaccinal Effect. Cell. 2015; 161:1035–1045. [PubMed: 25976835] 38. Ratcliffe SJ, Shults J. GEEQBOX: A MATLAB toolbox for generalized estimating equations and quasi-least squares. J Stat Softw. 2008; 25:1–14. 39. Gilbert PB, Rossini AJ, Shankarappa R. Two-sample tests for comparing intra-individual genetic sequence diversity between populations. Biometrics. 2005; 61:106–117. [PubMed: 15737083] 40. Giorgi EE, Bhattacharya T. A note on two-sample tests for comparing intra-individual genetic sequence diversity between populations. Biometrics. 2012; 68:1323–1326. author reply 1326. [PubMed: 23004569] 41. Deng W, et al. DIVEIN: a web server to analyze phylogenies, sequence divergence, diversity, and informative sites. Biotechniques. 2010; 48:405–408. [PubMed: 20569214] 42. van 't Wout AB, Schuitemaker H, Kootstra NA. Isolation and propagation of HIV-1 on peripheral blood mononuclear cells. Nature protocols. 2008; 3:363–370. [PubMed: 18323807] 43. Seaman MS, et al. Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. Journal of virology. 2009; 84:1439–1452. [PubMed: 19939925] 44. Li M, et al. Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. Journal of virology. 2005; 79:10108–10125. [PubMed: 16051804]
Science. Author manuscript; available in PMC 2016 December 12.
Schoofs et al.
Page 8
Author Manuscript Author Manuscript
45. West AP Jr, et al. Computational analysis of anti-HIV-1 antibody neutralization panel data to identify potential functional epitope residues. Proceedings of the National Academy of Sciences of the United States of America. 2013; 110:10598–10603. [PubMed: 23754383] 46. Kryazhimskiy S, Rice DP, Jerison ER, Desai MM. Microbial evolution. Global epistasis makes adaptation predictable despite sequence-level stochasticity. Science. 2014; 344:1519–1522. [PubMed: 24970088] 47. Larkin MA, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007; 23:2947–2948. [PubMed: 17846036] 48. Kearse M, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012; 28:1647–1649. [PubMed: 22543367] 49. Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9:772. 50. Guindon S, et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic biology. 2010; 59:307–321. [PubMed: 20525638] 51. Nickle DC, et al. HIV-specific probabilistic models of protein evolution. PloS one. 2007; 2:e503. [PubMed: 17551583] 52. Kirchherr JL, et al. High throughput functional analysis of HIV-1 env genes without cloning. J Virol Methods. 2007; 143:104–111. [PubMed: 17416428]
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Author Manuscript Author Manuscript Figure 1. Virus sensitivity to 3BNC117 and autologous antibody responses
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A. Graph displays kinetics of 3BNC117 antibody decay in HIV-1-infected individuals as determined by a validated anti-idiotype ELISA (16). Shown are mean values of patients infused in each respective dose group. Each patient sample was measured in duplicates. Gray shaded area indicates lower level of accuracy of the assay (2 µg/ml). Red arrows indicate the timepoints of IgG purification. B. Autologous virus sensitivity to 3BNC117 before (day 0, grey) and 4 wks (black) after 3BNC117 infusion. Y-axis shows IC50s for 3BNC117 on viral culture supernatants from PBMCs determined by TZM.bl assay. Neutralization assays performed in duplicates. C. Graph shows the AUC of the neutralization curves of purified IgGs obtained from sera on day 0 (orange) or wk 24 (green) against day 0 (left panel) or wk 4 (right panel) autologous viruses. Neutralization assays performed in duplicates. p-values determined by Wilcoxon signed-rank test.
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Figure 2. Heterologous antibody responses
A. Graph shows the difference in overall AUC (mean AUC change) per individual in TZM.bl assays against 13 heterologous viruses (see 2D) for day 0 vs. wk 24 IgG obtained from 36 untreated viremic controls (mean sampling interval: 26.8 wks), 15 viremic individuals infused with 3BNC117 (mean sampling interval: 24.1 wks), and 12 ART-treated individuals receiving 3BNC117 infusion (mean sampling interval: 24.0 wks) (16). Neutralization assays performed in duplicates. p-values determined by unpaired Wilcoxon test (rank-sum test). B. Graph shows the aggregated differences in AUC between d0 and wk
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24 IgG assayed by TZM.bl for all viruses and all individuals. Each dot represents a single AUC difference for a single virus from one individual displayed in A. Colored bars represent mean of all AUCs. Whiskers show standard deviation. p-values determined using generalized estimating equations (38). C. Graph shows 3BNC117 antibody levels (ELISA, white) and TZM.bl neutralization titer (green) against tier 2 strain Q769.d22 in subject 2A3 D. Mean AUCs of IgGs of all individuals at d0 (grey) and wk 24 (color of respective group) for each HIV-1 pseudovirus tested. Changes in neutralization of viremic control individuals without 3BNC117 infusion are shown in yellow (left). Change in neutralization of 3BNC117-treated individuals shown in dark (off ART, middle) and light blue (on ART, right). p-values determined using unpaired Wilcoxon test (rank-sum test). Red stars indicate significant p-values after Bonferroni-correction (threshold p < 0.0038).
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A. Maximum likelihood phylogenetic tree of single genome-derived env gene sequences from d0 plasma, before therapy with 3BNC117 (Table S9). Asterisks indicate bootstrap values of 100%. Individual viral sequences are color coded as indicated. B. Scatter plots depicting pairwise nucleotide sequence diversity of plasma env sequences on d0, and wk 4 (2E5, wk 6), 12 and 24 after infusion. Each dot represents the pairwise genetic difference between two sequences at a given timepoint. Colored bars indicate median diversity, while black bars indicates the interquartile range. p-values were determined using a two-sample Ustatistic based Z-test (39–41). C. Graph shows the relationship between d0 mean heterologous neutralizing AUC against a panel of tier 1 (n=1) and tier 2 (n=12) viruses (xaxis) and the median pairwise nucleotide diversity for each patient (y-axis).
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Author Manuscript Author Manuscript Author Manuscript Figure 4. Antibody responses to the evolving viral quasispecies
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A. Maximum-likelihood phylogenetic trees of single genome-derived env gene sequences from subjects 2A1 and 2E1 sampled on d0 and wk 4, 12, and 24 after 3BNC117 infusion (left). Clades with bootstrap support ≥ 70% are indicated by a black star and are arbitrarily named groups A-D in case of subject 2A1. Bar graphs (middle) indicate the timepoints from which sequences in the tree are derived. Heat maps (right) show the 3BNC117 IC50, d0 IgG IC50 and wk 24 IgG IC50 values against autologous pseudoviruses using env sequences as indicated by colored stars. Neutralization assays performed in duplicates. B. Sequence logo plots illustrating longitudinal amino acid changes in and around known 3BNC117-contact residues (26, 27) in subject 2A1 and 2E1. Letters indicate deviations from the d0 consensus shown at the top, whereas white spaces indicate agreement with the d0 consensus. Colors indicate basic (dark blue) and acidic (red) residues and a turquoise “O” is used instead of
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“N” to indicate a potential N-glycosylation site. Logo plots were generated using LASSIE (28). + symbols indicate 3BNC117 contact residues confirmed by two crystal structures (26, 27).
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