Impact of multi-targeted antiretroviral treatment on gut T cell depletion and HIV reservoir seeding during acute HIV infection.

BackgroundLimited knowledge exists on early HIV events that may inform preventive and therapeutic strategies. This study aims to characterize the earliest immunologic and virologic HIV events following infection and investigates the usage of a novel therapeutic strategy.Methods and findingsWe prospectively screened 24,430 subjects in Bangkok and identified 40 AHI individuals. Thirty Thais were enrolled (8 Fiebig I, 5 Fiebig II, 15 Fiebig III, 2 Fiebig IV) of whom 15 completed 24 weeks of megaHAART (tenofovir/emtricitabine/efavirenz/raltegravir/maraviroc). Sigmoid biopsies were completed in 24/30 at baseline and 13/15 at week 24. At baseline, the median age was 29 years and 83% were MSM. Most were symptomatic (87%), and were infected with R5-tropic (77%) CRF01_AE (70%). Median CD4 was 406 cells/mm(3). HIV RNA was 5.5 log(10) copies/ml. Median total blood HIV DNA was higher in Fiebig III (550 copy/10(6) PBMC) vs. Fiebig I (8 copy/10(6) PBMC) (p = 0.01) while the median %CD4+CCR5+ gut T cells was lower in Fiebig III (19%) vs. Fiebig I (59%) (p = 0.0008). After 24 weeks of megaHAART, HIV RNA levels of <50 copies were achieved in 14/15 in blood and 13/13 in gut. Total blood HIV DNA at week 0 predicted reservoir size at week 24 (p<0.001). Total HIV DNA declined significantly and was undetectable in 3 of 15 in blood and 3 of 7 in gut. Frequency of CD4+CCR5+ gut T cells increased from 41% at baseline to 64% at week 24 (p>0.050); subjects with less than 40% at baseline had a significant increase in CD4+CCR5+ T cells from baseline to week 24 (14% vs. 71%, p = 0.02).ConclusionsGut T cell depletion and HIV reservoir seeding increases with progression of AHI. MegaHAART was associated with immune restoration and reduced reservoir size. Our findings could inform research on strategies to achieve HIV drug-free remission

HIV-1 protease inhibitors do not interfere with provirus transcription and host cell apoptosis induced by combined treatment TNF-alpha plus TSA

HIV-1 latency represents a major hurdle to the complete eradication of the virus from patients under highly active anti-retroviral therapy (HAART) regimens. One solution to this problem would be to eliminate the latently infected cellular reservoirs by forcing gene expression in presence of HAART to prevent spreading of the infection by the newly synthesized viruses. Many studies have reported that a combination of a histone deacetylase inhibitor (HDACi) (i.e. TSA, NaBut, Valproic acid,...) with a pro-inflammatory cytokine (i.e. TNF alpha, IL-1,...) reactivates in a synergistic manner HIV-1 transcription in latently infected cells. The aim of the present study was to determine whether HIV-1 protease inhibitors (PIs) used in HAART (such as Saquinavir, Indinavir, Nelfinavir, Lopinavir, Ritonavir and Amprenavir) could interfere with the potential purge of the cellular reservoirs induced by a combined treatment involving TSA and TNF alpha. We showed, in two HIV-1 latently infected cell lines (ACH-2 and U1) that all PIs efficiently inhibited release of mature viral particles but did neither affect cell apoptosis nor NF-kappa B induction and HIV-1 transcription activation following combined treatment with TNF alpha + TSA. This study is encouraging in the fight against HIV-1 and shows that PIs should be compatible with an inductive adjuvent therapy for latent reservoir reduction/elimination in association with efficient HAART regimens. (c) 2007 Elsevier Inc. All rights reserved

HIV DNA Set Point is Rapidly Established in Acute HIV Infection and Dramatically Reduced by Early ART

HIV DNA is a marker of HIV persistence that predicts HIV progression and remission, but its kinetics in early acute HIV infection (AHI) is poorly understood. We longitudinally measured the frequency of peripheral blood mononuclear cells harboring total and integrated HIV DNA in 19 untreated and 71 treated AHI participants, for whom 50 were in the earliest Fiebig I/II (HIV IgM−) stage, that is ≤2 weeks from infection. Without antiretroviral therapy (ART), HIV DNA peaked at 2 weeks after enrollment, reaching a set-point 2 weeks later with little change thereafter. There was a marked divergence of HIV DNA values between the untreated and treated groups that occurred within the first 2 weeks of ART and increased with time. ART reduced total HIV DNA levels by 20-fold after 2 weeks and 316-fold after 3 years. Therefore, very early ART offers the opportunity to significantly reduce the frequency of cells harboring HIV DNA

Primers used for <i>gag</i> and <i>pol</i> gene DNA identification in the peripheral blood of HIV-1 JRCSF infected humanized NSG mice.

<p>Primers used for <i>gag</i> and <i>pol</i> gene DNA identification in the peripheral blood of HIV-1 JRCSF infected humanized NSG mice.</p

Engraftment potential of CD34⁺ hematopoietic cells.

<p>(A) Flow cytometric analysis of murine and human CD45⁺ cells in the peripheral blood of NSG mice of two different groups, representative profiles of the mice engraftment levels after 12 and 22 weeks after CD34⁺ cells transplantation. (B) Engraftment levels of human CD45⁺ cells in peripheral blood up to 306 days after transplantation in group 1 and group 7. (C) White blood cell counts at 12 and 22 weeks post-engraftment in group 7 mice along with human control.</p

HIV induced immune activation and expression levels of multiple activation markers on CD4⁺ and CD8⁺.

<p>Blood samples were obtained from mice before and 6 week after HIV-1 JRCSF infection. (A) Beta-2-microglobline levels were measured using ELISA before (Pre) and after HIV-infection (Post) (n = 4) and at same time intervals in engrafted but noninfected mice. (B) CD69 (C) HLA-DR (D) PD-1 and (E) CD27 (F) CCR5 levels were evaluated on CD4⁺ and CD8⁺ cells. (G) LPS levels in humanized mice 2 and 8 weeks post-HIV infection. Each circle and square represent one mouse at the indicated time point. Means are shown in solid lines. <i>P</i> values were determined by unpaired student’s t-tests. ** indicates a <i>P</i> value<0.001 and *** indicates a <i>P</i> value<0.0001.</p

An improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rgammanull mice allows HIV replication and development of anti-HIV immune responses.

Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rgamma(null) (NSG) and NOD/SCID/IL2Rgamma(null) (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials

Transplantation conditions and engraftment levels of human CD45⁺ cells after 22 weeks in various groups of NSG mice.

<p>Transplantation conditions and engraftment levels of human CD45⁺ cells after 22 weeks in various groups of NSG mice.</p

Engraftment levels of human cells in peripheral blood (PB), spleen, lymph nodes (LNs) and bone marrow (BM) of humanized NSG mice.

<p>Peripheral blood and organs were collected at 12 and 22 weeks after CD34⁺ cell transplantation in group 1 and group 7 mice. Percentage of human and murine CD45 cells was calculated on lymphoid gate. Percentage of human CD19⁺, CD3⁺, CD4⁺ and CD8⁺ cells were calculated out of human CD45⁺ cells.</p

Human cell differentiation in humanized NSG mice.

<p>(A) Absolute number of human cells in lymph nodes, bone marrow and spleens of group 1 and group 7 mice. (B) Mesenteric and brachial lymph nodes from male mice obtained after 24 weeks of CD34⁺ cell transplantation in group 7 mice. (C) Flow cytometric analysis of engraftment percentage of CD3⁺ T cells and CD19⁺ B cells at 12 and 22 weeks after transplantation of CD34⁺ cells in representative mice from group 1 and group 7. (D) Flow cytometric analysis of CD4⁺CD8⁺, CD4⁺ and CD8⁺ population in thymus of 22 weeks engrafted mice. (E) Flow cytometric analysis of CD45RA⁺ and CD45RO⁺ positive CD3 cells 22 weeks after engraftment in mice from group 7. (F) Representative profile CD11c⁺ cells in spleen and CD14⁺ population in bone marrow and blood of group 7 mice 22 weeks after CD34⁺ cell transplantation.</p