Challenges in detecting HIV persistence during potentially curative interventions: a study of the Berlin patient

Abstract

There is intense interest in developing curative interventions for HIV. How such a cure will be quantified and defined is not known. We applied a series of measurements of HIV persistence to the study of an HIV-infected adult who has exhibited evidence of cure after allogeneic hematopoietic stem cell transplant from a homozygous CCR5Δ32 donor. Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV DNA or RNA was detected in peripheral blood mononuclear cells (PBMC), spinal fluid, lymph node, or terminal ileum, and no replication-competent virus could be cultured from PBMCs. However, HIV RNA was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in ART-suppressed patients. It was not possible to obtain sequence data from plasma or gut, while an X4 sequence from PBMC did not match the pre-transplant sequence. HIV antibody levels were readily detectable but declined over time; T cell responses were largely absent. The occasional, low-level PCR signals raise the possibility that some HIV nucleic acid might persist, although they could also be false positives. Since HIV levels in well-treated individuals are near the limits of detection of current assays, more sensitive assays need to be developed and validated. The absence of recrudescent HIV replication and waning HIV-specific immune responses five years after withdrawal of treatment provide proof of a clinical cure.

Conflict of interest statement

Michael Busch has consulted for Gen-Probe (now owned by Hologic), which provided support for some of the virologic measurements performed in this study. Christopher Bentsen is currently employed by Bio-Rad Laboratories, Inc. and received salary, benefits and stock. He oversaw the testing of samples in this paper with FDA approved tests from Bio-Rad. These relationships do not alter our adherence to all PLoS Pathogens policies on sharing data and materials.

Figures

Figure 1. Timeline for clinical treatments and study samples.

Figure 2. Fluorescence-activated cell sorting (FACS) strategies for PBMC samples (A-G) and GI tract samples (H-M; ileum shown as example).

For PBMC, all cells were included (panel A), doublets excluded (panel B), and residual non-viable cells excluded by LIVE/DEAD violet cell staining ("Viab dump," panel C). Low-frequency CCR5+ events were then collected in one sorting tube (inside box gate, panel D). Of the remaining, CCR5- events (outside box gate, panel D), CD3+ events negative for CD14 and CD11c were included for further gating (inside polygon gate, panel E), with remaining events collected in a second sorting tube (outside polygon gate, panel E). CCR5-CD3+ events negative for CD14 and CD11c that were also CD8- (panel F) and T cell receptor-γδ-, CD20-, and CD56- ("Lin dump," panel G) were collected in a third sorting tube as presumptive CD4+ T cells. Remaining CD3+ events that were either CD8+ or Lin dump+ were combined in a fourth sorting tube. For ileum and rectum, all cells were included (panel H) and then doublets excluded (panel I). Viable CD45+ events were included for further gating (inside polygon gate, panel J), with all events outside this gate collected in one sorting tube as non-hematopoietic cells. CD3+ events negative for CD14 and CD11c were included for further gating (inside polygon gate, panel K), with remaining events collected in a second sorting tube (outside polygon gate, panel K). CD3+ events negative for CD14 and CD11c that were also CD8- (panel L) and T cell receptor-γδ-, CD20-, and CD56- ("Lin dump," panel M) were collected in a third sorting tube as presumptive CD4+ T cells. Remaining CD3+ events that were either CD8+ or Lin dump+ were combined in a fourth sorting tube. Numbers in upper-right corners of flow plots indicate the percentages of events on plots falling inside gates shown.

Figure 3. HIV-specific antibodies.

Blood from four time points was tested for HIV specific antibody levels using the HIV-1/2 VITROS assay (3A), a detuned version of the HIV-1 VITROS assay (3B), and the Limiting Antigen avidity assay (3C). The y-axis shows the relative level of total HIV-specific antibody, as expressed as the signal to cutoff ratio (3A–B) or normalized optical density, ODn (3C). The x axis represents months since transplant. In Figure 3A, the dotted line represents the diagnostic HIV antibody assay cut-off level used to classify individuals as HIV-positive or HIV-negative. For purposes of comparison, HIV antibody responses were also measured in HIV-uninfected adults, untreated HIV-infected adults, and ART-treated chronically-infected adults using the detuned HIV-1 VITROS assay (Figure 3D). The bars in the scatterplots represent the median and interquartile ranges of distributions of seroreactivity for each group. Finally, samples from the Berlin Patient were tested for antibodies to other infectious diseases (3E). Tests included antibodies to CMV (strong positive, above the limit of detection), EBV, measles, and hepatitis B (all within the range of detection) as well as VZV, mumps, rubella, and toxoplasmosis (all negative, below the limit of detection). Only the results within detectable range of the assay are shown. S/CO = signal/cutoff ratio; ODn = normalized optical density; AI = antibody index.

Figure 4. HIV Gag-specific cell mediated immune responses.

PBMC were obtained from the Berlin Patient (solid red circles), HIV-uninfected adults (open black circles in 4A–B), chronically HIV-infected adults on long term ART with undetectable plasma viral loads (open black circles, 4C–D), and elite controllers (open black circles, 4E–F). PBMC were stimulated with CMV pp65 or HIV Gag peptide pools, and flow cytometry was used to measure the percentage of CD4+T cells (4A, 4C, 4E) or CD8+T cells (4B, 4D, 4F) with intracellular staining for interferon-γ, tumor necrosis factor-α, IL-2, or CD107. The y axis shows the percent of T cells that express each cytokine in response to HIV Gag. The solid red circle indicates the Berlin Patient, while open black circles indicate individuals from comparator groups.