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Distinct mechanisms of long-term virologic control in two HIV-infected individuals after treatment interruption of anti-retroviral therapy

Nature Medicine volume 27, pages 1893–1898 (2021)Cite this article

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Abstract

Certain infected individuals suppress human immunodeficiency virus (HIV) in the absence of anti-retroviral therapy (ART). Elucidating the underlying mechanism(s) is of high interest. Here we present two contrasting case reports of HIV-infected individuals who controlled plasma viremia for extended periods after undergoing analytical treatment interruption (ATI). In Participant 04, who experienced viral blips and initiated undisclosed self-administration of suboptimal ART detected shortly before day 1,250, phylogenetic analyses of plasma HIV env sequences suggested continuous viral evolution and/or reactivation of pre-existing viral reservoirs over time. Antiviral CD8+ T cell activities were higher in Participant 04 than in Participant 30. In contrast, Participant 30 exhibited potent plasma-IgG-mediated neutralization activity against autologous virus that became ineffective when he experienced sudden plasma viral rebound 1,434 d after ATI due to HIV superinfection. Our data provide insight into distinct mechanisms of post-treatment interruption control and highlight the importance of frequent monitoring of undisclosed use of ART and superinfection during the ATI phase.

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Fig. 1: Levels of plasma viremia after discontinuation of ART and phylogenetic analysis of HIV sequences.
Fig. 2: CD8+ T cell responses to HIV and neutralization capacity of autologous plasma against replication-competent viral isolates.

Data availability

HIV sequences are available in GenBank under accession numbers MT998681–MT998840 (Participant 04) and MW865486–MW865605 (Participant 30). All HIV integration site sequences are available in BioProject under accession number PRJNA720555. External requests for data will be evaluated by the corresponding author, and requests might be subject to National Institutes of Health policies.

Code availability

The pipeline for annotated HIV integration site sequences is available at https://github.com/NCBR-FNLCR/NCBR_hiv_integration.

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Acknowledgements

We are grateful to the study volunteers for their participation in this study. We thank the National Institute of Allergy and Infectious Diseases HIV Outpatient Clinic staff for their assistance in the execution of this study. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Author information

Author notes

  1. Feng Gao

    Present address: Jinan University School of Medicine, Guangzhou, China

  2. Manukumar Honnayakanahalli Marichannegowda

    Present address: Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, USA

  3. These authors contributed equally: Jana Blazkova, Feng Gao.

Authors and Affiliations

  1. Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Jana Blazkova, J. Shawn Justement, Victoria Shi, Emily J. Whitehead, Rachel F. Schneck, Erin D. Huiting, Michael C. Sneller, Susan Moir, Anthony S. Fauci & Tae-Wook Chun

  2. Department of Medicine, Duke University, Durham, NC, USA

    Feng Gao & Manukumar Honnayakanahalli Marichannegowda

  3. Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD, USA

    Kathleen Gittens

  4. Division of Pharmacotherapy and Experimental Therapeutics, University of North Carolina, Chapel Hill, NC, USA

    Mackenzie Cottrell

  5. Maple Leaf Medical Clinic, Toronto ON, Canada

    Erika Benko & Colin Kovacs

  6. NIAID Collaborative Bioinformatics Resource, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Justin Lack

  7. Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research, Frederick, MD, USA

    Justin Lack

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Contributions

J.B., F.G., S.M., A.S.F. and T.-W.C. designed the research. J.B., F.G., M.H.M., J.S.J., V.S., E.J.W., R.F.S., E.D.H., M.C. and T.-W.C. performed experiments. J.B., F.G., M.H.M. and J.L. performed bioinformatic analysis. K.G., E.B., C.K. and M.C.S. contributed to recruitment of study participants. J.B., F.G., M.H.M., J.L., S.M. and T.-W.C. analyzed data. J.B., S.M., A.S.F. and T.-W.C. wrote the manuscript.

Corresponding author

Correspondence to Tae-Wook Chun.

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The authors declare no competing interests.

Additional information

Peer review information Nature Medicine thanks Timothy Henrich, Joseph Wong and Morgane Rolland for their contribution to the peer review of this work. Alison Farrell is the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Longitudinal measurements of CD4+ and CD8+ T cell counts.

Levels of CD4+ and CD8+ T cell counts of study participants are shown following discontinuation of ART.

Extended Data Fig. 2 Genetic analyses of rebounding plasma HIV in Participant 04.

a. Phylogenetic tree analysis of the 3’-half of HIV sequences. The phylogenetic tree without subtype B reference sequences was constructed using the neighbor-joining method and was rooted with the predominant sequences from day 56. The tree reliability was estimated by 1000 bootstrap replicates. The nodes that were supported by more than 80% of bootstrap replicates are indicated by asterisks. The sequences derived at multiple time points are shown in different symbols/colors. b. Genetic divergence of the 3’-half of HIV sequences of rebounding plasma HIV from each time point compared to that from day 56. c. Mutations identified in previously known HLA-restricted CD8+ T cell epitopes. Amino acid substitutions in all seven viral proteins (Vif, Vpr, Vpu, Tat, Rev, Env, and Nef) in the 3’-half of HIV sequences obtained at the time of plasma viral rebound (days 56, 581, 862, and 875) were examined to identify potential escape mutations at previously known HLA (A*03 and B*40)-restricted CD8+ T cell epitopes over time.

Extended Data Fig. 3 Dynamics of HIV reservoirs.

Frequencies of CD4+ T cells carrying total HIV DNA, cell-associated HIV RNA, and replication-competent HIV are shown following discontinuation of ART.

Extended Data Fig. 4 Characterization of HIV-integration landscape/features in the post-treatment controllers.

Integration sites of all HIV proviruses were analyzed in CD4+ T cells from the 2 post-treatment controllers, Participants 04 and 30 (D0) and from 3 chronic-treated HIV-infected individuals on suppressive ART (N01-N03) as controls. a. The relative proportion of HIV integration sites per chromosome in each participant is expressed by the heatmap. Contribution of each chromosome to the total size of human genome (first row of the heatmap) and to the genic regions (expressed by the black bars) are included as references. Distribution of individual HIV-integration sites in each chromosome is depicted by red bars for Participants 04 and 30, and blue bars for Participants N01-N03. b. Proportion of HIV proviruses integrated in the indicated genomic regions. c. Distance in bp between individual HIV-integration sites and the most proximal transcriptional start site (TSS) peak (left) or to the most proximal ATAC-Seq peak indicating accessible chromatin (right). The numbers of unique HIV integration events were 51, 53, 223, 80, and 100 for Participant 04, Participant 30, N01, N02, and N03, respectively.

Extended Data Fig. 5 Phenotypic characterization of T cells.

High-dimensional flow cytometric analyses of longitudinal peripheral blood mononuclear cells (PBMCs) of study participants. a. Frequencies of the activation/exhaustion markers TIGIT, PD-1, CD38 and HLA-DR on CD8+ T cells in Participant 04 (red) and Participant 30 (blue) are shown. P values were determined using an unpaired two-tailed Mann-Whitney test; ns, P > 0.05; **, P = 0.0063; ***, P < 0.001, ns, not significant. P values were adjusted for multiple comparisons. b. Global opt-SNE plots of CD3 + T cells of pooled time points from each study participant (left) and opt-SNE visualization of expression of the indicated markers (right) are shown. c. Opt-SNE map of T cell clusters identified by FlowSOM clustering. Each number indicates a distinct cluster. d. Heatmap showing the level of expression (MFI) within individual clusters. e. Comparison of frequencies of T cells expressing markers associated with indicated clusters are shown. Ten time points per study participant were analyzed. Each measurement was performed once at each time point. P values were determined using an unpaired two-tailed Mann-Whitney test; *, P = 0.011; ***, P < 0.001. P values were adjusted for multiple comparisons.

Extended Data Fig. 6 Neutralization capacity of bNAbs and autologous plasma against replication-competent HIV isolates.

a. Sensitivity of infectious viral isolates to bNAbs. The concentration of all infectious viral isolates was initially determined by HIV p24 ELISA and titration using standard TZM-bl target cells. Each viral isolate was incubated with 10 µg/ml of bNAbs for 90 minutes followed by incubation with TZM-bl cells for 48 hours. Cells were then lysed and substrate was added to measure the luciferase activity. The level of viral suppression by each antibody was determined over media controls. The numbers of independent viral isolates examined on each day were as follows: 12 (day 117) and 11 (day 1,093) for Participant 04 and 10 (day 117), 11 (day 517), 9 (day 705), 12 (day 1,223), 12 (day 1,405), and 24 (day 1,447) for Participant 30. P values were determined using an unpaired two-tailed Mann-Whitney test; ns, P > 0.05; **, P = 0.004; ***, P < 0.001; ns, not significant. P values were adjusted for multiple comparisons. b. Neutralization curves for heat-inactivated plasma against autologous infectious HIV. Heat-inactivated, serially diluted plasma was subjected to the neutralization assay against replication-competent virus at multiple time points. Each measurement was performed in duplicate at each time point.

Extended Data Table 1 Baseline characteristics of study participants
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Supplementary information

Supplementary Information

Supplementary Table 1: Antibodies used for T cell phenotyping and intracellular cytokine staining. Supplementary Fig. 1: Gating strategy for T cell subsets and intracellular cytokine staining

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Blazkova, J., Gao, F., Marichannegowda, M.H. et al. Distinct mechanisms of long-term virologic control in two HIV-infected individuals after treatment interruption of anti-retroviral therapy. Nat Med 27, 1893–1898 (2021). https://doi.org/10.1038/s41591-021-01503-6

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