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

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.

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.

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.

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.

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.

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.

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.