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. 2016 Feb 16;113(7):1883-8.
doi: 10.1073/pnas.1522675113. Epub 2016 Feb 8.

Clonally expanded CD4+ T cells can produce infectious HIV-1 in vivo

Francesco R Simonetti  1 , Michele D Sobolewski  2 , Elizabeth Fyne  2 , Wei Shao  3 , Jonathan Spindler  4 , Junko Hattori  4 , Elizabeth M Anderson  4 , Sarah A Watters  4 , Shawn Hill  4 , Xiaolin Wu  3 , David Wells  3 , Li Su  3 , Brian T Luke  3 , Elias K Halvas  2 , Guillaume Besson  2 , Kerri J Penrose  2 , Zhiming Yang  5 , Richard W Kwan  6 , Carter Van Waes  7 , Thomas Uldrick  8 , Deborah E Citrin  9 , Joseph Kovacs  10 , Michael A Polis  6 , Catherine A Rehm  6 , Robert Gorelick  11 , Michael Piatak  11 , Brandon F Keele  11 , Mary F Kearney  4 , John M Coffin  12 , Stephen H Hughes  4 , John W Mellors  2 , Frank Maldarelli  13
Affiliations

Clonally expanded CD4+ T cells can produce infectious HIV-1 in vivo

Francesco R Simonetti et al. Proc Natl Acad Sci U S A. .

Abstract

Reservoirs of infectious HIV-1 persist despite years of combination antiretroviral therapy and make curing HIV-1 infections a major challenge. Most of the proviral DNA resides in CD4(+)T cells. Some of these CD4(+)T cells are clonally expanded; most of the proviruses are defective. It is not known if any of the clonally expanded cells carry replication-competent proviruses. We report that a highly expanded CD4(+) T-cell clone contains an intact provirus. The highly expanded clone produced infectious virus that was detected as persistent plasma viremia during cART in an HIV-1-infected patient who had squamous cell cancer. Cells containing the intact provirus were widely distributed and significantly enriched in cancer metastases. These results show that clonally expanded CD4(+)T cells can be a reservoir of infectious HIV-1.

Keywords: HIV persistence; clonal expansion of infected cells; replication-competent HIV.

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Conflict of interest statement

Conflict of interest statement: J.W.M. is a consultant for Gilead Sciences and owns shares in Co-Crystal, Inc.

Figures

Fig. S1.
Fig. S1.
Clonally expanded cells responsible for low-level viremia emerged from a diverse population of HIV-1–infected cells. (A) Time course of HIV-1 viremia (circles) and CD4+ T-cell numbers (gray diamonds) before and following the initiation of combination antiretroviral therapy. (B) Plasma samples obtained at the time points indicated by colored circles were subjected to SGS (p6-RT) as described, and neighbor joining phylogenetic analysis was performed. (C) Composite neighbor joining tree of all sequences in Fig. S2B. *Bootstrap support ≥80% (16).
Fig. 1.
Fig. 1.
Persistent low-level viremia during cART is due, in part, to a clonal population of HIV-1 that has no drug resistance mutations. (A) Profile of plasma HIV-1 RNA levels starting 9.5 y after initiation of therapy, with annotations for clinical events (arrows), and sampling for SGS of plasma viral RNA (∼1,100 nt p6-RT) indicated by colored circles (14) and triangles for cell-associated HIV-1 DNA. The cART regimen is indicated by the horizontal bars (see Fig. S1 for treatment details). (B) Phylogenetic analysis of plasma viral RNA and cell DNA. (C) Abundance of the AMBI-1 variant in plasma. The percentage of proviruses that are AMBI-1 at the times indicated is shown below the graph.
Fig. S2.
Fig. S2.
Cells from the AMBI-1 clone represent a significant fraction of the infected peripheral lymphocytes. PBMCs from the 9 December 2011 (12.1 y after therapy) time point were subjected to SGS (p6-RT) and neighbor-joining phylogenetic analyses were performed. AMBI-1 represented ∼13% of the total p6-RT sequences recovered from PBMCs at this time point (there were 83 HIV-1 sequences of which 11 were AMBI-1). Real-time PCR amplification for total HIV-1 DNA (17) revealed that there were 209 HIV-1 DNA copies per 106 PBMCs, of which ∼13% were AMBI-1, which would correspond to 27 × 106 PBMCs. The peripheral T-cell count was 1,279 cells/µL, and the total number of PBMCs in this patient was estimated to be 3.3 × 1011, based on total blood volume (Nadler formula) = 5.12 L, assuming that 2% of the total T cells are in the blood. From these estimates, the total number of expanded cells containing AMBI-1 proviruses is calculated to be ∼9 × 106. DNA sequences that correspond to a second clonal virus (OG-1) detected in the ex vivo infectious virus recovery assay were also present. †Hypermutants (5).
Fig. 2.
Fig. 2.
Recovery of infectious HIV-1 from a provirus present in a clonally expanded CD4+ T cells. (A) The AMBI-1 provirus was amplified from CD8-depleted CD4+ T cells in two fragments that overlapped in the IN-vif region, using primers in the flanking host sequence and in HIV (primers named with HXB2 coordinates are listed in Table S3). Sequence analysis revealed ORFs for all HIV-1 genes with no obvious debilitating mutations. Amplified fragments were mixed 1:1 and used to transfect 293T cells with lipofectamine 2000, and the supernatant was used to infect CD8-depleted blasts from a healthy, HIV-negative donor; p24 was measured in culture supernatants by ELISA (Alliance HIV-1 p24 ELISA Kit; Perkin-Elmer). Viral sequences from the culture supernatants were identical to AMBI-1. (B) CD4+ T cells were purified from patient 1 PBMCs, serially diluted, PHA treated, and added to cocultures with irradiated feeder cells and CD8-depleted allogeneic blasts from healthy HIV-negative donors, which were maintained for 28 d as described (SI Materials and Methods); p24 antigen in the culture supernatants was determined weekly. p24-positive wells are shown. p24-negative wells were also negative by an assay for HIV-1 RNA (Roche Taqman v2.0). Infectious units per million (IUPM) were calculated using maximum likelihood estimate (5, 9). (C) HIV-1 sequences obtained from the p24-positive wells on day 21, as indicated by the symbols, were obtained by bulk sequencing of the p6-RT region and subjected to neighbor-joining phylogenetic analysis. Virus recovered from the supernatant (inverted triangle) also underwent full-length sequencing and was identical to AMBI-1. (D) HIV-1 recovered by in vitro cultivation is infectious in subsequent infections. Cell supernatant from one of the p24-containing wells with AMBI-1() was used to infect fresh CD8-depleted blasts from a healthy, HIV-negative donor. p24 levels were measured in culture supernatants at days 1, 7, 10, and 14. Viral RNA sequence (p6-RT) obtained from culture supernatants obtained at day 7 was identical to AMBI-1.
Fig. 3.
Fig. 3.
Cells carrying the AMBI-1 proviruses are widely distributed anatomically and enriched in tumor metastases. (A) H&E-stained section of a metastasis of the invasive squamous cell carcinoma shows lymphocytic infiltration. (Magnification, 200×.) (B) Sections were stained for CD4 expression to demonstrate that the infiltrate includes CD4+ lymphocytes. CD8 staining was negative. (Magnification, 200×.) (C) High magnification (12) of CD4 expressing lymphocytes (arrows) (D) Samples from autopsy specimens were subjected to env-U3 SGS. HIV-1 sequences were amplified from individual metastatic lesions and lymphoid tissues and used for phylogenetic analyses (Fig. S3). The table shows the number of AMBI-1 and other HIV-1 DNA sequences in metastatic lesions and lymphoid tissues; P values were derived from the Fisher exact test.
Fig. S3.
Fig. S3.
Phylogenetic analysis of HIV from autopsy tissues. Frozen samples obtained from the indicated autopsy material were subjected to SGS (env-U3) and neighbor-joining phylogenetic analysis. For ease of identification, large groups of expanded clones are grouped (boxes), and their position in the tree is noted by arrows.
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