Timing of very early ART to limit early HIV-related mucosal dysfunction
Simon Collins, HIV i-Base
A paper published recently by Alexandra Schuetz and colleagues as an open access article in PLoS Pathogens is important for providing important data on the timing of early antiretroviral treatment (ART). The results highlight that in the context of the very early window after infection, each day might be time-critical to limit mucosal damage to the gastrointestinal (GI) tract. 
This is an ongoing, prospective, open-label Thai study that enrolled 42 people between May 2009 and March 2012 who were diagnosed either prior to or shortly after HIV seroconversion (Fiebig stages I-V) as part of the RV304/SEARCH 013 study. The study included optional sigmoid biopsy of gut-associated lymphoid tissue (GALT) at baseline and month six and the option to start early treatment.
The participants were gay men (n=35), bisexual men (n=4) and heterosexual women (n=3) with 75% of participants infected with HIV recombinant subtype CRF01_AE. Median age was 29 (range 19 to 48). Median time since infection was an estimated 16 days (+/-SD 6.6) with 13, 4, 21, 1 and 3 participants diagnosed in Fiebig stages I, II, III, IV and V respectively. Due to low numbers, participants with stages I/II and IV/V were combined for the analyses. In people using ART, treatment was started an average of 3 days post-enrolment (range 0 to 5 days). At baseline, median CD4 and viral load were 465 cells/mm3 (range 132 to 1127) and 5.5 log copies/mL (range 2.8 to 7.7), respectively.
In addition, 10 HIV negative people (matched for age, gender and risk group) and 5 treatment naive HIV positive people in chronic infection (CHI) – mean 298 days (SD +/– 154) duration of infection; Fiebig VI, p31 positive), were enrolled as controls.
The Fiebig classification for stages of seroconversion includes median (95%CI) cumulative days since infection of 5.0 (3.1, 8.1), 10.3 (7.1, 13.5), 13.5 (10.0, 17.0), 19.1 (15.3, 22.9) and 88.6 (47.4, 129.8), for stages I, II, III, IV and V respectively. Diagnostic results include being PCR positive from stage I, p-24 positive from stage II, third generation ELISA positive from stage III, Western blot (WB) indeterminate at stage IV and WB determined (2 out of 3: p24, p41, p120; but p31 negative) at stage V.
Within weeks of HIV infection, an early CD4 depletion in GALT occurs and subsequent increased levels of microbial translocation (“leaky gut”) are generally hypothesised as playing a key roll in persistent immune activation during chronic HIV infection.
Gut mucosal function was determined by number and function of Th17 cells which play an essential role in maintaining the gut epithelium by responding to extracellular bacterial and fungi and in producing IL-22 which enhances epithelial regeneration.
Reduced immune and gut function
In a complicated data set looking at absolute and percentage CD4 and CD8 counts and related cellar sub-sets in both plasma and gut tissue and Th17/Th22 cells, results tended to show significant differences by Fiebig stage III.
For example, at baseline the percentage of CD4 cells in gut tissue and plasma were already established depending on time from infection: 49% FI/II, 35% FIII and 17% FIV/V compared to 18% in CHI and 56% in HIV negative controls in sigmoid colon and 35% FI/II, 26% F III, 21% F IV/V compared to 18% in CHI and 53% in HIV negative in blood.
The timing for the decrease in proportion of Th17 cells (in the subset of patients with viable mononuclear cells from sigmoid tissue) was 12.8% in FI/II to 7.9% in FIII, and 2.3% in F IV/V. This compared to 0.9% in the CHI and 13.5% in the HIV negative control groups. Similar results were reported for Th22 cells: 2.9% FI/II, 1.3% in FIII and 0.4% in F IV/V, compared to 3.6% in HIV negative controls.
In gut tissue, the frequencies of IL-17 and IL-17/IL-22 producing CD4 cells also correlated positively with CD4 cells (p < 0.001) and inversely with colonic viral load (p<0.03).
Th17 cell function was assessed by co-expression of IFN-alpha, IL-2 and/or IL-22 and showed a dramatic loss from 6.5% in FI/II to 0.3% in FIII (p=0.02) with these polyfunctional cells entirely depleted in the chronically infected control group.
A reduced proportion of Th17 cells was inversely correlated with plasma markers of immune activation: C-reactive protein (r = –0.42, p=0.03), hyaluronic acid (r = –0.53, p=0.003), TNF-alpha (r = –0.49, p=0.03) and IP-10 (r = –0.71, p<0.001). However, no correlations were seen with biomarkers for intestinal damage (I-FABP), microbial translocation (LPS and sCD14) or coagulation (D-dimer).
Impact of early ART
After six months on ART, 29 participants had a second sigmoid biopsy: 14 from stage FI/II and 15 stage FIII. All patients experienced CD4 increases and had undetectable viral load in plasma (with 28/29 undetectable in sigmoid tissue).
Participants treated at FI/II maintained polyfunctional TH17 cells, with no loss of either total TH17 cells or the proportion of triple cytokine producing Th17 cells post-ART. Participants treated during FIII showed a restoration of TH17 cells (7.9% pre- to 10.2% post-ART, p=0.05) but not a restoration of functional triple-cytokine producing cells, which remained comparable to untreated controls in chronic infection.
However, treatment at FIII significantly decreased plasma levels of CRP (from 1343 to 483 pg/mL, p=0.02) and D-dimer (from 359 to 146 pg/mL, p<0.001). Markers of activated (HLA-DRCD38+) CD8 T cells that were significantly higher in both FI/II and FIII participants in plasma and colon tissue prior to ART, only normalised in people treated during FI/II. Although participants treated at FIII had reductions in CD8 activation, this failed to normalise after 6 months treatment (sigmoid colon: 5.0% post-ART vs 8.9% pre-ART, p<0.001; vs 0.1% for HIV negative controls, p<0.001; peripheral blood: 9.0% post-ART vs 15% pre-ART, p=0.003; vs 3.0% HIV negative, p<0.001).
While the researchers noted that limitations of the study included relatively low numbers for some sample analyses, and for the control group, and that a control group of patients treated during chronic infection would also be important, they emphasised that their cohort is currently unique in regard to early diagnosis, sampling and responses to early ART.
Similar to other research, this study reported that the depletion of CD4 and CD4+CCR5+, including in the lamina propria (main effector GI site) and correlating viral HIV viral load, often occurs within days of HIV infection, and prior to Fiebig stages I/II.
Treatment during FI/II normalised immune activation and some other important markers of mucosal immunity and function were protected when ART was started during FI/II, but not when treatment was delayed to FIII. However, while such early diagnosis may still be rare, aligning these cases to a research setting with this focus could have important clinical as well as research outcomes.
A significant and increasing percentage of people in the UK are diagnosed during acute HIV infection. Of these, a recent patient survey presented at BHIVA 2015 indicated a positive interest in starting earlier treatment that was not matched by being offered early treatment.  Conversely, a similar survey of health providers attitudes by the same researchers, showed than some doctors are reluctant to offer treatment in early infection, and do not always follow BHIVA guidelines to discuss the roll of TasP in this patient group. 
The RIVER study is currently enrolling UK patients diagnosed in early infection and will follow patients irrespective of whether ART is used. This cohort will be an important opportunity to contribute data to the field of HIV cure research. 
In people diagnosed at later stages of acute infection, the study also reports that early ART might have plausible benefits, including reduced CD8 activation compared to chronic infection (if not normalised compared to HIV negative).
As BHIVA guidelines include the recommendation to discuss the benefit of ART in reducing HIV infectiousness to all HIV positive people, including at the initial diagnosis, greater awareness of this early clinical window is an area of patient care that could perhaps be improved. 
- Schuetz A et al. Initiation of ART during early acute HIV infection preserves mucosal Th17 function and reverse HIV-related immune activation. PLoS Pathogens, December 2014: 10(12)e1004543.
- Fox J et al. UK clinicians’ approach to ART in primary HIV infection; comparison with the BHIVA guidelines. 21st Annual BHIVA Conference, 21-24 April 2015, Brighton. Oral abstract O13.
- Fox J et al. UK survey of HIV-positive people’s attitudes towards research. 21st Annual BHIVA Conference, 21-24 April 2015, Brighton. Poster abstract P39
- RIVER study. Research In Viral Eradication of HIV Reservoirs. EudraCT number 2014-001425-32.
- BHIVA guidelines, British HIV Association guidelines for the treatment of HIV-1-positive adults with antiretroviral therapy 2012, (updated November 2013). HIV Medicine (2014), 15 (Suppl. 1), 1–85. DOI: 10.1111/hiv.12119.