The gut mucosal viral reservoir in HIV-infected patients is not the major source of rebound plasma viremia following interruption of highly active antiretroviral therapy

Abstract

Interruption of suppressive highly active antiretroviral therapy (HAART) in HIV-infected patients leads to increased HIV replication and viral rebound in peripheral blood. Effects of therapy interruption on gut-associated lymphoid tissue (GALT) have not been well investigated. We evaluated longitudinal changes in viral replication and emergence of viral variants in the context of T cell homeostasis and gene expression in GALT of three HIV-positive patients who initiated HAART during primary HIV infection but opted to interrupt therapy thereafter. Longitudinal viral sequence analysis revealed that a stable proviral reservoir was established in GALT during primary HIV infection that persisted through early HAART and post-therapy interruption. Proviral variants in GALT and peripheral blood mononuclear cells (PBMCs) displayed low levels of genomic diversity at all times. A rapid increase in viral loads with a modest decline of CD4(+) T cells in peripheral blood was observed, while gut mucosal CD4(+) T cell loss was severe following HAART interruption. This was accompanied by increased mucosal gene expression regulating interferon (IFN)-mediated antiviral responses and immune activation, a profile similar to those found in HAART-naive HIV-infected patients. Sequence analysis of rebound virus suggested that GALT was not the major contributor to the postinterruption plasma viremia nor were GALT HIV reservoirs rapidly replaced by HIV rebound variants. Our data suggest an early establishment and persistence of viral reservoirs in GALT with minimal diversity. Early detection of and therapy for HIV infection may be beneficial in controlling viral evolution and limiting establishment of diverse viral reservoirs in the mucosal compartment.

Fig. 1.

Clinical course and therapy schedule of HIV-infected patients. Patients 116, 134, and 140 initiated therapy during primary HIV infection and discontinued thereafter. Time points for the sample collection of peripheral blood and gut biopsy specimens during HIV infection are shown. Time since putative date of HIV infection is indicated under each time point arrow in weeks (wks), months (mos) (17), and years (yrs). Stars indicate when patients initiated HAART, and bolts indicate when patients interrupted HAART. The time at the top of each time line indicates how much time elapsed from therapy interruption to the next time point when samples were collected.

Fig. 2.

Measurement of HIV loads and CD4⁺ T cell numbers in longitudinal samples of peripheral blood and GALT of HIV-infected patients prior to and post-HAART interruption. (A) CD4⁺ T cell numbers (blue) and HIV RNA copies/ml(red) are plotted across time (in months). Arrows underneath each panel represents the time of GALT sample collection from each patient and represent time points as they are indicated in Fig. 1. The boxed area indicates the time period of therapy interruption. (B) CD4 (blue) and CD8 (red) T-cell percentages from GALT.

Fig. 3.

Measurement of HIV RNA loads in longitudinal gut biopsy specimens from HIV-infected patients. Viral RNA loads in GALT were determined by reverse transcription-PCR (RT-PCR). Time points of sample collection are shown. HI, HAART interruption.

Fig. 4.

HAART interruption leads to alterations in intestinal mucosal gene expression. (A) Hierarchical clustering of genes whose expression was altered in the intestinal mucosa of 3 patients following HAART interruption as compared to baseline gene expression in 3 healthy HIV-negative controls. (B) Pathway analysis of genes up- or downregulated in GALT following HAART interruption indicated a statistical enrichment for genes involved in the cell cycle, ubiquitin ligase conjugation, cytoskeletal activity, host virus interaction, lymphocyte activation, immune response, hemopoiesis, and apoptosis.

Fig. 5.

Mucosal immune response-associated gene expression following HAART interruption. Fold changes in the expression of genes involved in antiviral responses (A) or cytolytic activity (B) in the intestinal mucosa following HAART interruption are shown in heat maps. The transcription profile of genes during HAART interruption was compared to the profiles for HAART-naive patients with acute or chronic HIV infection, patients with long-term HAART administration (>2 years), and long-term HIV-infected nonprogressors.

Fig. 6.

HIV diversity (Pi) and divergence (Dxy) in GALT and peripheral blood prior to and during HAART and at post-HAART interruption. (A to C) HIV diversity plots for patients 116, 134, and 140, respectively. Each color (see key) represents diversity values of HIV population from GALT, PBMC, and plasma compartments during the course of HIV infection. Dashed lines indicate trends from available data. (D) Divergence histogram comparing HIV sequence divergence in plasma and GALT at a pre-HAART time point compared to a post-interruption of HAART time point. Measurements represent the average number of nucleotide substitutions per site between different populations. Bars indicate standard errors.

Fig. 7.

Phylogenetic analysis of V3 region of HIV envelope gene from peripheral blood and GALT from HIV-infected patients. HIV sequences derived from plasma (triangle), PBMC (square), or GALT (circle) from HIV-infected patients (see key for colors representing different time points) were analyzed to construct phylogenetic trees. “PI” indicates post-therapy interruption time points. Trees were constructed using the maximum-likelihood approach by running dnaml from the Phylip 3.68 package. This approach also generates P values for each node in the tree to assess significance, marked below by the use of “*” for P values between 0.01 and 0.05 and “**” for P values below 0.05. Samples from patients 116 (A), 140 (B), and 134 (C) are analyzed.

Fig. 7.

Phylogenetic analysis of V3 region of HIV envelope gene from peripheral blood and GALT from HIV-infected patients. HIV sequences derived from plasma (triangle), PBMC (square), or GALT (circle) from HIV-infected patients (see key for colors representing different time points) were analyzed to construct phylogenetic trees. “PI” indicates post-therapy interruption time points. Trees were constructed using the maximum-likelihood approach by running dnaml from the Phylip 3.68 package. This approach also generates P values for each node in the tree to assess significance, marked below by the use of “*” for P values between 0.01 and 0.05 and “**” for P values below 0.05. Samples from patients 116 (A), 140 (B), and 134 (C) are analyzed.