Peilin Li

Differences in HIV Burden and Immune Activation within the Gut of HIV-Positive Patients Receiving Suppressive Antiretroviral Therapy

BACKGROUND The gut is a major reservoir for human immunodeficiency virus (HIV) in patients receiving antiretroviral therapy (ART). We hypothesized that distinct immune environments within the gut may support varying levels of HIV. METHODS In 8 HIV-1-positive adults who were receiving ART and had CD4(+) T cell counts of >200 cells/μL and plasma viral loads of <40 copies/mL, levels of HIV and T cell activation were measured in blood samples and endoscopic biopsy specimens from the duodenum, ileum, ascending colon, and rectum. RESULTS HIV DNA and RNA levels per CD4(+) T cell were higher in all 4 gut sites compared with those in the blood. HIV DNA levels increased from the duodenum to the rectum, whereas the median HIV RNA level peaked in the ileum. HIV DNA levels correlated positively with T cell activation markers in peripheral blood mononuclear cells (PBMCs) but negatively with T cell activation markers in the gut. Multiply spliced RNA was infrequently detected in gut, and ratios of unspliced RNA to DNA were lower in the colon and rectum than in PBMCs, which reflects paradoxically low HIV transcription, given the higher level of T cell activation in the gut. CONCLUSIONS HIV DNA and RNA are both concentrated in the gut, but the inverse relationship between HIV DNA levels and T cell activation in the gut and the paradoxically low levels of HIV expression in the large bowel suggest that different processes drive HIV persistence in the blood and gut. TRIAL REGISTRATION ClinicalTrials.gov identifier: NCT00884793 (PLUS1).

Challenges in Detecting HIV Persistence during Potentially Curative Interventions: A Study of the Berlin Patient

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.

The Distribution of HIV DNA and RNA in Cell Subsets Differs in Gut and Blood of HIV-Positive Patients on ART: Implications for Viral Persistence

Even with optimal antiretroviral therapy, human immunodeficiency virus (HIV) persists in plasma, blood cells, and tissues. To develop new therapies, it is essential to know what cell types harbor residual HIV. We measured levels of HIV DNA, RNA, and RNA/DNA ratios in sorted subsets of CD4+ T cells (CCR7+, transitional memory, and effector memory) and non-CD4+ T leukocytes from blood, ileum, and rectum of 8 ART-suppressed HIV-positive subjects. Levels of HIV DNA/million cells in CCR7+ and effector memory cells were higher in the ileum than blood. When normalized by cell frequencies, most HIV DNA and RNA in the blood were found in CCR7+ cells, whereas in both gut sites, most HIV DNA and RNA were found in effector memory cells. HIV DNA and RNA were observed in non-CD4+ T leukocytes at low levels, particularly in gut tissues. Compared to the blood, the ileum had higher levels of HIV DNA and RNA in both CD4+ T cells and non-CD4+ T leukocytes, whereas the rectum had higher HIV DNA levels in both cell types but lower RNA levels in CD4+ T cells. Future studies should determine whether different mechanisms allow HIV to persist in these distinct reservoirs, and the degree to which different therapies can affect each reservoir.

HIV-1 Drug Resistance in the iPrEx Preexposure Prophylaxis Trial

Background. The iPrEx study demonstrated that combination oral emtricitabine and tenofovir disoproxil fumarate (FTC/TDF) as preexposure prophylaxis (PrEP) protects against HIV acquisition in men who have sex with men and transgender women. Selection for drug resistance could offset PrEP benefits. Methods. Phenotypic and genotypic clinical resistance assays characterized major drug resistant mutations. Minor variants with FTC/TDF mutations K65R, K70E, M184V/I were measured using 454 deep sequencing and a novel allele-specific polymerase chain reaction (AS-PCR) diagnostic tolerant to sequence heterogeneity. Results. Control of primer-binding site heterogeneity resulted in improved accuracy of minor variant measurements by AS-PCR. Of the 48 on-study infections randomized to FTC/TDF, none showed FTC/TDF mutations by clinical assays despite detectable drug levels in 8 participants. Two randomized to FTC/TDF had minor variant M184I detected at 0.53% by AS-PCR or 0.75% by deep sequencing, only 1 of which had low but detectable drug levels. Among those with acute infection at randomization to FTC/TDF, M184V or I mutations that were predominant at seroconversion waned to background levels within 24 weeks after discontinuing drug. Conclusions. Drug resistance was rare in iPrEx on-study FTC/TDF-randomized seroconverters, and only as low-frequency minor variants. FTC resistance among those initiating PrEP with acute infection waned rapidly after drug discontinuation. Clinical Trials Registration. NCT00458393.

Latently-infected CD4+ T cells are enriched for HIV-1 Tat variants with impaired transactivation activity.

The ability of HIV to establish latent infection in CD4+ lymphocytes represents a major barrier to the eradication of HIV. It is not clear what mechanisms favor latent over productive infection, but prior studies have suggested a role for the viral transcription factor Tat or its RNA target, TAR. Using samples from five individuals who were started on ART within 6 months of infection and achieved a viral load <50 (suppressed), we isolated one- and two-exon tat RNA from HIV propagated ex vivo from baseline plasma and from co-cultures of CD4+ T cells obtained at baseline and suppressed time points. Compared to virus from the baseline plasma (mostly from productively-infected CD4+ T cells), virus from the baseline and suppressed co-cultures (mostly from latently-infected cells) had more Tat variants with impaired transactivation activity. These findings suggest that impaired activity in the Tat-TAR axis may contribute to the establishment of latent infection in CD4+ T cells.

Stimulating the RIG-I pathway to kill cells in the latent HIV reservoir following viral reactivation

The persistence of latent HIV proviruses in long-lived CD4+ T cells despite antiretroviral therapy (ART) is a major obstacle to viral eradication. Because current candidate latency-reversing agents (LRAs) induce HIV transcription, but fail to clear these cellular reservoirs, new approaches for killing these reactivated latent HIV reservoir cells are urgently needed. HIV latency depends upon the transcriptional quiescence of the integrated provirus and the circumvention of immune defense mechanisms. These defenses include cell-intrinsic innate responses that use pattern-recognition receptors (PRRs) to detect viral pathogens, and that subsequently induce apoptosis of the infected cell. Retinoic acid (RA)-inducible gene I (RIG-I, encoded by DDX58) forms one class of PRRs that mediates apoptosis and the elimination of infected cells after recognition of viral RNA. Here we show that acitretin, an RA derivative approved by the US Food and Drug Administration (FDA), enhances RIG-I signaling ex vivo, increases HIV transcription, and induces preferential apoptosis of HIV-infected cells. These effects are abrogated by DDX58 knockdown. Acitretin also decreases proviral DNA levels in CD4+ T cells from HIV-positive subjects on suppressive ART, an effect that is amplified when combined with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor. Pharmacological enhancement of an innate cellular-defense network could provide a means by which to eliminate reactivated cells in the latent HIV reservoir.

A comparison of methods for measuring rectal HIV levels suggests that HIV DNA resides in cells other than CD4+ T cells, including myeloid cells.

We compared different techniques for measuring gut HIV reservoirs and assessed for HIV in non-CD4+ T cells. HIV DNA levels were similar when measured from rectal biopsies and isolated rectal cells, while HIV RNA tended to be higher in rectal cells. HIV DNA levels in total rectal cells were greater than those predicted from levels in sorted CD4+ T cells, suggesting a reservoir in non-CD4+ T cells, and HIV DNA was detected in sorted myeloid cells (7/7 subjects).

Site-Specific Differences in T Cell Frequencies and Phenotypes in the Blood and Gut of HIV-Uninfected and ART-Treated HIV+ Adults

Gastrointestinal T lymphocytes are critical for mucosal immunity and HIV pathogenesis, yet little is known about normal T cell numbers and phenotypes in different regions of the gut, or the degree to which ART can restore levels to those of HIV-uninfected individuals. To investigate these questions, we measured T cell frequencies and markers of memory, activation, anergy, and homing in the blood, ileum, and rectum of HIV- and ART-suppressed HIV+ adults. In HIV- individuals, T cell frequencies and phenotypes differed significantly between sites. Compared to HIV- adults, HIV+ adults had lower absolute CD4+T cell counts in the ileal lamina propria and lower relative CD4+T cell counts in the blood and ileum. In the gut, HIV+ adults had a higher proportion of CD38+ CD4+T cells, a lower proportion of terminally-differentiated effector cells, and, in the rectum, a higher proportion of CTLA-4+ CD4+T cells. In HIV+ individuals, relative CD4+T cell numbers in the ileum correlated with the proportion of CTLA-4+ CD4+T cells, whereas in the rectum, they tended to correlate with the proportion of circulating CD4+T cells expressing α4β7 or CCR6. Mechanisms of T cell reconstitution may differ throughout the gut, with homing contributing more in the rectum while ileal reconstitution is associated with mucosal CD4+T cell anergy.

Novel application of Locked Nucleic Acid chemistry for a Taqman assay for measuring diverse human immunodeficiency virus type 1 subtypes.

There remains a need for sensitive and cost-effective assays to monitor therapy in human immunodeficiency virus type-1 (HIV-1) infection. However, the genetic diversity of HIV poses difficulties for traditional real-time PCR assays that require long oligonucleotides probes. LNA™ probes may be useful in overcoming these limits to traditional probe design. A new application of LNA™ chemistry in a Taqman assay applicable to a wide range of HIV-1 subtypes is described. This assay, based on a 13-mer LNA™ probe that matches the majority of HIV-1 sequences in the Los Alamos database, exhibited a wide dynamic range (10(1)-10(7) copies of HIV DNA), high sensitivity (limit of detection of 1 copy of HIV DNA in 10(5) cells), and broad applicability to a range of HIV-1 subtypes (including A, B, C, D, F, H, B/C, and A/E CRFs). Using the LNA™ probe assay, HIV-1 DNA was detected in all dried blood spots (DBS) from treatment naïve HIV-1 positive Ugandan children, and HIV DNA levels significantly correlated with viral RNA levels in plasma (r=0.765, p<0.0001). This approach to Taqman probe design should be explored further for use in diagnosis and monitoring of HIV in resource-limited settings, especially where several subtypes co-circulate.

Advantages of using the QIAshredder instead of restriction digestion to prepare DNA for droplet digital PCR.

The viscosity of genomic DNA can interfere with digital PCR systems that partition samples into oil droplets or microfluidic wells. Restriction digestion may reduce the viscosity, but the process is labor-intensive, and the buffer can alter the conditions for PCR. DNA fragmentation using the QIAshredder (a biopolymer spin column) is faster, may result in more predictable and uniformly-sized fragments, and avoids the need for restriction buffers that can inhibit downstream PCR. In 10 separate head-to-head experiments comparing aliquots of DNA processed using the QIAshredder to those digested with RsaI or BsaJI prior to droplet digital PCR, we found that the copy numbers measured from the QIAshredded DNA tended to be greater than those measured from the digested DNA (average of 1.35-fold compared with BsaJI; P < 0.0001), even for inputs as high as 1.8 μg or dilution down to the single copy level.