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Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations

Nature volume 517, pages 381–385 (2015)Cite this article

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Abstract

Despite antiretroviral therapy (ART), human immunodeficiency virus (HIV)-1 persists in a stable latent reservoir1,2, primarily in resting memory CD4+ T cells3,4. This reservoir presents a major barrier to the cure of HIV-1 infection. To purge the reservoir, pharmacological reactivation of latent HIV-1 has been proposed5 and tested both in vitro and in vivo6,7,8. A key remaining question is whether virus-specific immune mechanisms, including cytotoxic T lymphocytes (CTLs), can clear infected cells in ART-treated patients after latency is reversed. Here we show that there is a striking all or none pattern for CTL escape mutations in HIV-1 Gag epitopes. Unless ART is started early, the vast majority (>98%) of latent viruses carry CTL escape mutations that render infected cells insensitive to CTLs directed at common epitopes. To solve this problem, we identified CTLs that could recognize epitopes from latent HIV-1 that were unmutated in every chronically infected patient tested. Upon stimulation, these CTLs eliminated target cells infected with autologous virus derived from the latent reservoir, both in vitro and in patient-derived humanized mice. The predominance of CTL-resistant viruses in the latent reservoir poses a major challenge to viral eradication. Our results demonstrate that chronically infected patients retain a broad-spectrum viral-specific CTL response and that appropriate boosting of this response may be required for the elimination of the latent reservoir.

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Figure 1: CTL escape variants dominate the latent reservoir of CP-treated but not AP-treated patients.
Figure 2: CD8+ T cells pre-stimulated with a mixture of Gag peptides eliminate autologous CD4+ T cells infected with autologous HIV-1 from resting CD4+ T cells.
Figure 3: CD8+ T cells targeting unmutated epitopes, not epitopes with identified escape mutations, eliminate CTL escape variants.
Figure 4: Broad-spectrum CTLs suppress in vivo replication of HIV-1 from the latent reservoir of the same patients in patient-derived humanized mice.

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Acknowledgements

We thank all study participants. We thank J. Blankson for critical advice to the project; L. Alston and R. Hoh for coordinating patient recruitment; J. Alderman, C. Weibel and E. Henchey for technical assistance in the animal study. We thank the National Institutes of Health (NIH) AIDS Reagent Program for providing HIV-1 consensus B peptides. R.F.S. is supported by the Howard Hughes Medical Institute, by the Martin Delaney CARE and DARE Collaboratories (NIH grants AI096113 and 1U19AI096109), by an ARCHE Collaborative Research Grant from the Foundation for AIDS Research (amFAR 108165-50-RGRL), by the Johns Hopkins Center for AIDS Research (P30AI094189), and by NIH grant 43222. L.S. is supported by NIH grant T32 AI07019. R.A.F. is supported by the Bill and Melinda Gates Foundation and the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, 21205, Maryland, USA

    Kai Deng, Mihaela Pertea, Christine M. Durand, Jun Lai, Holly L. McHugh, Janet D. Siliciano & Robert F. Siliciano

  2. Center for Computational Biology, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, 21205, Maryland, USA

    Mihaela Pertea & Steven L. Salzberg

  3. Department of Immunobiology, Yale University School of Medicine, New Haven, 06510, Connecticut, USA

    Anthony Rongvaux, Till Strowig, Richard A. Flavell & Liang Shan

  4. Department of Chronic Disease Epidemiology, Yale School of Public Health, New Haven, 06510, Connecticut, USA

    Leyao Wang

  5. Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, 21205, Maryland, USA

    Gabriel Ghiaur

  6. Deep Sequencing and Microarray Core, Johns Hopkins University School of Medicine, Baltimore, 21205, Maryland, USA

    Haiping Hao

  7. Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, 21205, Maryland, USA

    Hao Zhang & Joseph B. Margolick

  8. Regeneron Pharmaceuticals Inc., Tarrytown, 10591, New York, USA

    Cagan Gurer, Andrew J. Murphy, David M. Valenzuela & George D. Yancopoulos

  9. Department of Medicine, University of California, San Francisco, San Francisco, California 94110, USA,

    Steven G. Deeks

  10. Department of Medicine, Yale University School of Medicine, New Haven, 06510, Connecticut, USA

    Priti Kumar

  11. Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, 21205, Maryland, USA

    Steven L. Salzberg

  12. Howard Hughes Medical Institute, New Haven, 06510, Connecticut, USA

    Richard A. Flavell

  13. Howard Hughes Medical Institute, Baltimore, 21205, Maryland, USA

    Robert F. Siliciano

Authors
  1. Kai Deng
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Contributions

K.D., L.S., R.A.F. and R.F.S. conceived and designed the research studies; K.D., J.L., H.Z., J.B.M. and L.S. performed the in vitro experiments; K.D., A.R., L.W., C.G., A.J.M., D.M.V., G.D.Y., T.S., P. K. and L.S. performed animal experiments; C.M.D., G.G., H.L.M. and S.G.D. provided patient samples; K.D., M.P., L.W., H.H., J.D.S., S.L.S., L.S. and R.F.S. analysed data; K.D., L.S. and R.F.S. wrote the manuscript.

Corresponding authors

Correspondence to Richard A. Flavell, Liang Shan or Robert F. Siliciano.

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Competing interests

C.G., A.J.M., D.M.V. and G.D.Y. are employees and shareholders of Regeneron Pharmaceuticals, Inc.

Extended data figures and tables

Extended Data Figure 1 CTL escape variants dominate the latent reservoir of CP-treated HIV-1-positive individuals, but not AP-treated individuals.

Frequency of variants in Gag CTL epitopes in proviruses from resting CD4+ T cells. Results of all 25 patients tested are shown. Only optimal CTL epitopes relevant to each patient’s HLA type are listed in linear positional order on the x-axis. Results from both PacBio (left bar) and MiSeq (right bar) sequencing platforms are shown for each epitope. The absence of bars above a listed epitope indicates that only wild-type sequences were detected. For each mutation in a CTL epitope, information regarding the effect of the mutation on CTL recognition from the Los Alamos National Laboratory (LANL) HIV Molecular Immunology Database or from ELISpot assays described in Methods was used to assign the mutation to one of the categories indicated at the bottom. See Methods for definitions of these categories.

Extended Data Figure 2 Characterization of CTL responses against HIV-1 Gag epitopes by interferon-γ ELISpot.

Results of seven patients tested are shown. The peptides tested are listed for each patient in each graph. Error bars represent s.e.m., n = 3.

Extended Data Figure 3 Partial Gag sequences from proviral DNA and outgrowth virus from resting CD4+ T cells from eight CP-treated patients.

CTL epitopes with no observed variation are highlighted in blue. Documented escape mutations (red shading), inferred escape mutations (yellow shading), diminished response (pink shading), susceptible form (green shading) or undetermined variations (grey shading) in relevant optimal epitopes are indicated. See Methods for definitions of these types of mutations. This figure supplements Fig. 1e, as a total of nine CP-treated patients were tested.

Extended Data Figure 4 CD8+ T cells pre-stimulated with a mixture of consensus B Gag peptides eliminate autologous CD4+ T cells infected with autologous HIV-1 from resting CD4+ T cells.

a, HIV-1 isolated from ART-treated individuals replicates as well as the laboratory strain virus BaL. p24 values represent mean of three replicates. Error bars represent s.e.m., n = 3. b, CD8+ T cells are not stimulated after co-culture with PHA-activated CD4+ T cells. c, A representative flow cytometric analysis of CTL-mediated killing after co-culture of infected CD4+ T cells with autologous CD8+ T cells. CTL activity is measured by the percentage of Gag-positive, CD8-negative cells after 3 days of co-culture relative to cultures without CD8+ T cells. d, Pre-stimulated CD8+ T cells eliminate autologous infected CD4+ T cells more efficiently than non-stimulated CD8+ T cells. All results were normalized to the CD4 only control group. Error bars represent s.e.m., n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant (P > 0.05), paired t-test.

Extended Data Figure 5 The elimination of infected CD4+ T cells is mediated by direct killing by autologous CD8+ T cells.

a, Killing of infected CD4+ T cells is enhanced by increased effector to target ratios for both pre-stimulated and non-stimulated CD8+ T cells. b, Killing of the infected CD4+ T cells depends on direct cell–cell contact between CD4+ T cells and CTLs. All results were normalized to the CD4+ only control group. Error bars represent s.e.m., n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant (P > 0.05), paired t-test.

Extended Data Figure 6 Viral dynamics and depletion of CD4+ T cells in humanized mice.

a, Viral dynamics in CP18-infected MIS(KI)TRG mice. CP18-derived MIS(KI)TRG mice were infected with autologous HIV-1. Plasma HIV-1 RNA levels were measured from day 0 to day 56. b, Depletion of CD4+ T cells in peripheral blood of HIV-1 BaL-infected mice. MIS(KI)TRG mice engrafted with fetal liver CD34+ cells were infected with HIV-1 BaL. The CD4 to CD8 ratio in peripheral blood was measured by fluorescence-activated cell sorting (FACS) from day 0 to day 29 after infection. Error bars represent s.e.m., n = 5. c, Depletion of CD4+ T cells in spleen of HIV-1 BaL-infected mice. MIS(KI)TRG mice engrafted with fetal liver CD34+ cells were infected with HIV-1 BaL. The CD4 to CD8 ratio in spleen was measured by FACS 20 days after infection. Medians and P values from Mann–Whitney test are shown. d, Detection of cell-associated HIV-1 RNA in T cells and macrophages/monocytes. CD3+ and CD14+ human cells from HIV-1-infected MIS(KI)TRG mice from spleen and lung were purified by FACS. CD3−CD14− cells were also collected as controls. Cell-associated HIV-1 RNA was quantified by gag-specific quantitative polymerase chain reaction (qPCR). Error bars represent s.e.m., n = 3. *P < 0.05, unpaired t-test.

Extended Data Figure 7 HIV-1 infection occurs in peripheral blood and tissues in humanized mice.

a, Engraftment levels of MIS(KI)TRG mice with fetal liver or patient CD34+ cells. b, Memory CD4+ T cells are detected in MIS(KI)TRG mice after infection. MIS(KI)TRG mice were infected with HIV-1 BaL. Peripheral blood and indicated tissues from infected mice were collected at 20 days post-infection. Memory CD4+ T cells were determined by CD45RO staining. c, Total number of cell-associated HIV-1 DNA in blood and tissues. DNA from peripheral blood or indicated tissues was isolated for the measurement of total amount of cell-associated HIV-1 DNA by real-time PCR. b, c, Medians and P values from Mann–Whitney test are shown.

Extended Data Figure 8 Broad-spectrum CTLs suppress in vivo infection of patient-derived humanized mice with autologous latent HIV-1.

The generation of patient CP36-derived humanized mice is described in Fig. 4. Mice were infected with autologous viruses at 6 weeks old. CD8+ T cells from patient CP36 were pre-stimulated with the mixture of Gag peptides or left untreated for 6 days in vitro, and were injected into mice intravenously 9 days after infection. Plasma HIV-1 RNA (a) and HIV-1 DNA (b) in peripheral blood were measured by real-time PCR.

Extended Data Table 1 Characteristics of study subjects
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Deng, K., Pertea, M., Rongvaux, A. et al. Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations. Nature 517, 381–385 (2015). https://doi.org/10.1038/nature14053

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