Abstract
In the antibody mediated prevention (AMP) trials, the broadly neutralizing antibody (bNAb) VRC01 demonstrated protective efficacy against susceptible HIV strains. To understand how VRC01 shaped breakthrough infections, deep sequencing was performed on 172 participants (>100,000 gag-Δpol and rev-env-Δnef sequences), at diagnosis and over time, in the placebo and treatment arms of the African (HVTN703/HPTN081; NCT02568215) and Americas/Europe (HVTN704/HPTN085; NCT02716675) cohorts. A high frequency of multilineage infections was detected (38%), including co-infection with both VRC01 sensitive and resistant viruses. This high frequency is largely accounted for by low-abundance lineages. Although VRC01 does not significantly affect the genetic transmission bottleneck compared to placebo, higher VRC01 doses trend towards greater VRC01 neutralization differences among co-infecting lineages. Two-thirds of multilineage infections showed evidence of recombination at the diagnostic timepoint. In the treatment group there is evidence of recombinant viruses preferentially inheriting resistance-associated mutations. This study provides critical insights into viral genetic and antigenic diversity that needs to be targeted to achieve protection, and highlights the role of recombination in facilitating escape.
Data availability
The neutralization and other data underlying the findings of this manuscript are publicly available at the public-facing HVTN data repository at Harvard Dataverse (https://doi.org/10.7910/DVN/3VG3JO), in the files “v703_survival_wk80_tau_sieve.tab” and “v704_survival_wk80_tau_sieve.tab”. The data dictionary for these files is “README_survival.txt”. All individual participant data have been deidentified. All final GP and REN sequences were deposited in GenBank with Accession numbers: PX890939-PX026359; ON890939-ON891092; PX184492-PX150096 ON980967- ON980814; OR508960- OR508936; OQ912897-OQ912888.1. The GenBank accession numbers for the HIV Env clones used in the TZM-bl target cell neutralization assay are: HVTN 703/HPTN 081 sequences, ON890939–ON891092; HVTN 704/HPTN 085 sequences, ON980814–ON980967.
Code availability
Some analyses required custom code written in R. This code is archived for public access at https://github.com/cmagaret/williamson_breakthrough_paper.
References
Corey, L. et al. Two Randomized Trials of Neutralizing Antibodies to Prevent HIV-1 Acquisition. N. Engl. J. Med. 384, 1003–1014 (2021).
Juraska, M. et al. Prevention efficacy of the broadly neutralizing antibody VRC01 depends on HIV-1 envelope sequence features. Proc. Natl. Acad. Sci. 121, e2308942121 (2024).
Mendoza, P. et al. Combination therapy with anti-HIV-1 antibodies maintains viral suppression. Nature 561, 479–484 (2018).
Mkhize, N. N. et al. Neutralization profiles of HIV-1 viruses from the VRC01 Antibody Mediated Prevention (AMP) trials. PLoS Pathog. 19, e1011469 (2023).
Wagh, K. et al. Optimal combinations of broadly neutralizing antibodies for prevention and treatment of HIV-1 Clade C infection. PLoS Pathog. 12, e1005520 (2016).
Mahomed, S. et al. Clinical trials of broadly neutralizing monoclonal antibodies in people living with HIV - a review. AIDS Res Ther. 22, 44 (2025).
Schommers, P. et al. Restriction of HIV-1 escape by a highly broad and potent neutralizing antibody. Cell 180, 471–489.e422 (2020).
Gieselmann, L. et al. Identification of a broad and potent V3 glycan site bNAb targeting an N332(gp120) glycan-independent epitope. Nat. Immunol 27, 572–585 (2026).
Julg, B. et al. Safety and antiviral activity of triple combination broadly neutralizing monoclonal antibody therapy against HIV-1: a phase 1 clinical trial. Nat. Med 28, 1288–1296 (2022).
Bar, K. J. et al. Effect of HIV antibody VRC01 on viral rebound after treatment interruption. N. Engl. J. Med. 375, 2037–2050 (2016).
Crowell, T. A. et al. Safety and efficacy of VRC01 broadly neutralising antibodies in adults with acutely treated HIV (RV397): a phase 2, randomised, double-blind, placebo-controlled trial. Lancet HIV 6, e297–e306 (2019).
Zhang, L. Q. et al. Selection for specific sequences in the external envelope protein of human immunodeficiency virus type 1 upon primary infection. J. Virol. 67, 3345–3356 (1993).
Gottlieb, G. S. et al. HIV-1 variation before seroconversion in men who have sex with men: analysis of acute/early HIV infection in the multicenter AIDS cohort study. J. Infect. Dis. 197, 1011–1015 (2008).
Delwart, E. L., Sheppard, H. W., Walker, B. D., Goudsmit, J. & Mullins, J. I. Human immunodeficiency virus type 1 evolution in vivo tracked by DNA heteroduplex mobility assays. J. Virol. 68, 6672–6683 (1994).
Abrahams, M. R. et al. Quantitating the multiplicity of infection with human immunodeficiency virus type 1 subtype C reveals a non-poisson distribution of transmitted variants. J. Virol. 83, 3556–3567 (2009).
Keele, B. F. et al. Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc. Natl. Acad. Sci. USA 105, 7552–7557 (2008).
Baxter, J. et al. Inferring the multiplicity of founder variants initiating HIV-1 infection: a systematic review and individual patient data meta-analysis. Lancet Microbe 4, e102–e112 (2023).
Carlson, J. M. et al. HIV transmission. Selection bias at the heterosexual HIV-1 transmission bottleneck. Science 345, 1254031 (2014).
Zhu, T. et al. Genotypic and phenotypic characterization of HIV-1 patients with primary infection. Science 261, 1179–1181 (1993).
Fenton-May, A. E. et al. Relative resistance of HIV-1 founder viruses to control by interferon-alpha. Retrovirology 10, 146 (2013).
Parrish, N. F. et al. Phenotypic properties of transmitted founder HIV-1. Proc. Natl. Acad. Sci. USA 110, 6626–6633 (2013).
Haaland, R. E. et al. Inflammatory genital infections mitigate a severe genetic bottleneck in heterosexual transmission of subtype A and C HIV-1. PLoS Pathog. 5, e1000274 (2009).
Selhorst, P. et al. Cervicovaginal inflammation facilitates acquisition of less infectious HIV variants. Clin. Infect. Dis. 64, 79–82 (2017).
Boeras, D. I. et al. Role of donor genital tract HIV-1 diversity in the transmission bottleneck. Proc. Natl. Acad. Sci. USA 108, E1156–E1163 (2011).
Ashokkumar, M. et al. In vitro replicative fitness of early transmitted founder HIV-1 variants and sensitivity to Interferon alpha. Sci. Rep. 10, 2747 (2020).
Chopera, D. R. et al. Transmission of HIV-1 CTL escape variants provides HLA-mismatched recipients with a survival advantage. PLoS Pathog. 4, e1000033 (2008).
Stekler, J. D. et al. Transmission of HIV-1 drug resistance mutations within partner-pairs: a cross-sectional study of a primary HIV infection cohort. PLoS Med. 15, e1002537 (2018).
Mbunkah, H. A. et al. Low-abundance drug-resistant HIV-1 variants in antiretroviral drug-naive individuals: a systematic review of detection methods, prevalence, and clinical impact. J. Infect. Dis. 221, 1584–1597 (2020).
Westfall, D. H. et al. Optimized SMRT-UMI protocol produces highly accurate sequence datasets from diverse populations-Application to HIV-1 quasispecies. Virus Evol. 10, veae019 (2024).
Mullins, J. I. et al. Long-read deep sequencing reveals high rates of multilineage transmission and rapid viral population changes in acute HIV infection. bioRxiv, https://doi.org/10.1101/2025.10.15.682663 (2025).
Westfall, D. H. et al. Optimized SMRT-UMI protocol produces highly accurate sequence datasets from diverse populations—application to HIV-1 quasispecies. Virus Evol. 10, veae019 (2024).
Juraska, M. et al. Prevention efficacy of the broadly neutralizing antibody VRC01 depends on HIV-1 envelope sequence features. Proc. Natl. Acad. Sci. USA 121, e2308942121 (2024).
Rolland, M. et al. Molecular dating and viral load growth rates suggested that the eclipse phase lasted about a week in HIV-1 infected adults in East Africa and Thailand. PLoS Pathog. 16, e1008179 (2020).
Mansky, L. M. & Temin, H. M. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J. Virol. 69, 5087–5094 (1995).
Gilbert, P. B. et al. Neutralization titer biomarker for antibody-mediated prevention of HIV-1 acquisition. Nat. Med. 28, 1924–1932 (2022).
Rossenkhan, R. et al. Bayesian estimation of HIV acquisition dates for prevention trials. mBio 16, e0188125 (2025).
Bricault, C. A. et al. HIV-1 neutralizing antibody signatures and application to epitope-targeted vaccine design. Cell Host Microbe 25, 59–72.e58 (2019).
Dingens, A. S., Arenz, D., Weight, H., Overbaugh, J. & Bloom, J. D. An antigenic atlas of HIV-1 escape from broadly neutralizing antibodies distinguishes functional and structural epitopes. Immunity 50, 520–532.e523 (2019).
LaBranche, C. C. et al. HIV-1 envelope glycan modifications that permit neutralization by germline-reverted VRC01-class broadly neutralizing antibodies. PLoS Pathog. 14, e1007431 (2018).
Barnes, C. O. et al. A naturally arising broad and potent CD4-binding site antibody with low somatic mutation. Sci. Adv. 8, eabp8155 (2022).
Falkowska, E. et al. PGV04, an HIV-1 gp120 CD4 binding site antibody, is broad and potent in neutralization but does not induce conformational changes characteristic of CD4. J. Virol. 86, 4394–4403 (2012).
Schommers, P. et al. Dynamics and durability of HIV-1 neutralization are determined by viral replication. Nat. Med 29, 2763–2774 (2023).
Foulkes, C. et al. Assessing bnAb potency in the context of HIV-1 envelope conformational plasticity. PLoS Pathog 21, e1012825 (2025).
Cohen, P. et al. Resistance mutations that distinguish HIV-1 envelopes with discordant VRC01 phenotypes from multi-lineage infections in the HVTN703/HPTN081 trial: implications for cross-resistance. J. Virol. 99, e0173024 (2025).
Shriner, D., Rodrigo, A. G., Nickle, D. C. & Mullins, J. I. Pervasive genomic recombination of HIV-1 in vivo. Genetics 167, 1573–1583 (2004).
Gottlieb, G. S. et al. Dual HIV-1 infection associated with rapid disease progression. Lancet 363, 619–622 (2004).
Grobler, J. et al. Incidence of HIV-1 dual infection and its association with increased viral load set point in a cohort of HIV-1 subtype C-infected female sex workers. J. Infect. Dis. 190, 1355–1359 (2004).
Janes, H. et al. HIV-1 infections with multiple founders are associated with higher viral loads than infections with single founders. Nat. Med. 21, 1139–1141 (2015).
Miner, M. D., Corey, L. & Montefiori, D. Broadly neutralizing monoclonal antibodies for HIV prevention. J. Int AIDS Soc. 24, e25829 (2021).
Sarzotti-Kelsoe, M. et al. Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1. J. Immunol. Methods 409, 131–146 (2014).
Cohen, Y. Z. et al. Neutralizing activity of broadly neutralizing anti-HIV-1 antibodies against clade B clinical isolates produced in peripheral blood mononuclear cells. J. Virol. 92, e01883-17 (2018).
Landovitz, R. J. et al. Tail-phase safety, tolerability, and pharmacokinetics of long-acting injectable cabotegravir in HIV-uninfected adults: a secondary analysis of the HPTN 077 trial. Lancet HIV 7, e472–e481 (2020).
Eshleman, S. H. et al. Characterization of human immunodeficiency virus (HIV) infections in women who received injectable cabotegravir or tenofovir disoproxil fumarate/emtricitabine for HIV prevention: HPTN 084. J. Infect. Dis. 225, 1741–1749 (2022).
Bekker, L. G. et al. Twice-yearly lenacapavir or daily F/TAF for HIV prevention in cisgender women. N. Engl. J. Med 391, 1179–1192 (2024).
Kelley, C. F. et al. Twice-yearly lenacapavir for HIV prevention in men and gender-diverse persons. N. Engl. J. Med. 392, 1261–1276 (2025).
van Zyl, G. et al. Lenacapavir-associated drug resistance: implications for scaling up long-acting HIV pre-exposure prophylaxis. Lancet HIV 12, e732–e736 (2025).
Elias, A., Pasin, C., Smuk, M., Paterson, A. & Orkin, C. M. Outcomes of real-world initiation of long-acting injectable cabotegravir and rilpivirine (LA-I CAB + RPV) in individuals with viraemia: a systematic review of baseline characteristics, virological failure outcomes and discontinuations. HIV Med. 1, 42–57 (2026).
Gunst, J. D. et al. Impact of a TLR9 agonist and broadly neutralizing antibodies on HIV-1 persistence: the randomized phase 2a TITAN trial. Nat. Med 29, 2547–2558 (2023).
Cale, E. M. et al. Neutralizing antibody VRC01 failed to select for HIV-1 mutations upon viral rebound. J. Clin. Invest 130, 3299–3304 (2020).
Huang, Y. et al. Pharmacokinetics analysis of serum and rectal tissue concentrations of a pair of anti-HIV monoclonal antibodies, VRC01 and VRC01LS, in adults without HIV. J. Clin. Pharmacol. 65, 1506–1516 (2025).
Lemos, M. P. et al. Enhanced and sustained biodistribution of HIV-1 neutralizing antibody VRC01LS in human genital and rectal mucosa. Nat. Commun. 15, 10332 (2024).
Julg, B. et al. Safety and antiviral effect of a triple combination of HIV-1 broadly neutralizing antibodies: a phase 1/2a trial. Nat. Med 30, 3534–3543 (2024).
Lorenzi, J. C. C. et al. Neutralizing activity of broadly neutralizing anti-HIV-1 antibodies against primary African isolates. J. Virol. 95, e01909-20 (2021).
Sneller, M. C. et al. Combination anti-HIV antibodies provide sustained virological suppression. Nature 606, 375–381 (2022).
Edgar, R. C. Muscle5: High-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny. Nat. Commun. 13, 6968 (2022).
Giorgi, E. E. et al. Estimating time since infection in early homogeneous HIV-1 samples using a Poisson model. BMC Bioinform. 11, 532 (2010).
Furlong, J. C., Darley, P. D., Deng, W., Mullins, J. I. & Bumgarner, R. E. Phylobook: a tool for display, clade annotation and extraction of sequences from molecular phylogenies. Biotechniques 76, 263–274 (2024).
Lewitus, E. & Rolland, M. A non-parametric analytic framework for within-host viral phylogenies and a test for HIV-1 founder multiplicity. Virus Evol. 5, vez044 (2019).
Song, H. et al. Tracking HIV-1 recombination to resolve its contribution to HIV-1 evolution in natural infection. Nat. Commun. 9, 1928 (2018).
Dong, K. L. et al. Detection and treatment of Fiebig stage I HIV-1 infection in young at-risk women in South Africa: a prospective cohort study. Lancet HIV 5, e35–e44 (2018).
Robb, M. L. et al. Prospective study of acute HIV-1 infection in adults in East Africa and Thailand. N. Engl. J. Med. 374, 2120–2130 (2016).
Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321 (2010).
Deng, W. et al. DIVEIN: a web server to analyze phylogenies, sequence divergence, diversity, and informative sites. Biotechniques 48, 405–408 (2010).
Montefiori, D. C. Measuring HIV neutralization in a luciferase reporter gene assay. Methods Mol. Biol. 485, 395–405 (2009).
Seaton, K. E. et al. Pharmacokinetic serum concentrations of VRC01 correlate with prevention of HIV-1 acquisition. EBioMedicine 93, 104590 (2023).
Huang, Y. et al. Pharmacokinetics and predicted neutralisation coverage of VRC01 in HIV-uninfected participants of the Antibody Mediated Prevention (AMP) trials. EBioMedicine 64, 103203 (2021).
Jonckheere, A. R. & Bower, G. H. Non-parametric trend tests for learning data. Br. J. Math. Stat. Psychol. 20, 163–186 (1967).
Acknowledgements
We thank the participants of the HVTN703/HPTN081 and HVTN704/HPTN085 clinical trials; the clinical site staff; the protocol development and study implementation teams; NIH/NIAID Vaccine Research Center for the clinical development and manufacturing of the study product. This work was supported by the National Institutes of Health https://www.niaid.nih.gov/: UM1 AI068614 to L.C. at HVTN, FHCC; UM1 AI068635 to P.B.G., Y.H., H.J. at HVTN, SDMC, FHCC; UM1 AI068618 to M.J.M. at HVTN, FHCC; UM1 AI068619 to M.S.C. at HPTN; UM1 AI068613 to M.S.C. at HPTN; UM1 AI068617 to M.S.C. at HPTN; R01 AI152115 to C.W., P.B.G., P.L.M., and L.M. P.L.M. and C.W. and their teams are supported by the South African Medical Research Council Strategic Health Innovations Department. P.L.M. is supported by the South African Research Chairs Initiative of the Department of Science and Innovation and the National Research Foundation (grant no. 98341). D.B.R. is funded by K25 AI155224 and R01 AI186721-01. This study was supported in part by the Bill & Melinda Gates Foundation (CAVD; grant 1032144 to D.M.; and INV-016189 to J.I.M. B.M. was supported, in part, by the Swedish Research Council (2018-02381) and the NIH NIAID (R01 AI157854—subaward). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The sponsor had no role in study design, data collection, analysis or manuscript writing.
Author information
Authors and Affiliations
Contributions
C.W., J.I.M., P.B.G., L.Corey, M.S.C designed the research; J.I.M., J.H.W., C.W., C.M., C.A.M., E.E.G., M.R., R.R., T.B., B.M., J.H., D.H.W. developed or designed the methodology; D.H.W., N.N.M., L.C., H.Z., T.Y., A.G.-N., N.N., R.T., P.C., B.L., H.K., S.B. performed the research assays; S.T.K., J.A.H., L.M., D.M., S.I., N.M., M.J.M., M.S.C, L.Corey, P.B.G., L.B., M.R., R.E.B. provided study materials, reagents, computer resources or other analysis tools; C.W., J.I.M., C.M., C.A.M., E.E.G., T.B., B.M.; D.Y., W.D., R.R., A.P., S.E., N.M., M.J.M., L.M., P.L.M., M.S.C., L.Z., D.B.R., B.M., C.A.M., A.de.C., J.L., A.Y., A.L., B.M., R.E.B., P.T.E., N.B., D.M., W.D. performed data curation or analysis; J.I.M., C.W., C.M., C.A.M., E.E.G., M.D.M. wrote the paper; C.W., C.M., C.A.M., E.E.G., M.R., D.H.W., A.Y., W.D., R.R., N.N.M., L.C., H.Z., T.B., A.P., B.M., T.Y., A.G.N., N.N., B.T., P.C., B.L., H.K., S.B., M.J., H.B., A.de.C., M.D.M., J.L., C.M., N.B., D.M., Y.H., L.Z., D.B.R., B.M., S.T.K., J.A.H., L.M., D.M., R.E.B., P.L.M., P.T.E., S.E., N.M., M.J.M., M.S.C., L.Corey, P.B.G., J.I.M. reviewed and edited the paper.
Corresponding authors
Ethics declarations
Competing interests
MR: This work was supported by a cooperative agreement between The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., and the U.S. Department of the Army [W81XWH-18-2-0040]. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense, or the Department of Health and Human Services. The remaining authors have nothing to declare.
Peer review
Peer review information
Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Williamson, C., Moodley, C., Magaret, C.A. et al. Influence of the broadly neutralizing antibody VRC01 on HIV breakthrough virus populations in antibody-mediated prevention trials. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70888-0
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-026-70888-0
