Using data from treatment interruption studies, Dr Behzad Etemed and colleagues have compared the immunological response between post-treatment controllers and others living with HIV following analytical treatment interruptions. Post-treatment controllers are able to maintain viral suppression off ART. By comparing an exhaustive list of immunological variables, the researchers found post-treatment controllers mount different responses to HIV both on and then off antiretroviral therapy (ART) to other people.
This research is novel in that it analyses different facets of the immune response and develops our understanding of what makes post-treatment controllers able to suppress viral replication in the absence of ART. The findings pave the way for research that could help fine-tune future trials whilst also providing further insight to how and why some people can live with HIV despite stopping treatment.
The team, mostly based out of Harvard University and Massachusetts Institute of Technology, used data from people initially identified as post-treatment controllers in the Control of HIV After Medication Pause (CHAMP) study. CHAMP was designed to review viral dynamics after stopping treatment by evaluating data from 14 separate AIDS Clinical Trials Group studies that included an analytical treatment interruption (ATI). An ATI is required to evaluate the effect of an intervention. It involves some participants being given a vaccine or antibody infusion for example, and then all participants stopping their ART under close supervision. The viral rebound that occurs afterward is compared between groups to measure how successful, if at all, the intervention was at inhibiting viral replication. A small number of people, independent of the intervention given, do not experience a viral rebound and these people are termed post-treatment controllers (PTC) - a phenomenon first documented by French researchers in 2013.
The study
A PTC (post-treatment controller) was classed as anybody with a viral load of less than 400 copies/ml on at least two thirds of follow-up blood tests taken more than 24 weeks from the treatment interruption.
Non controllers (NCs) were identified as those who had available stored samples and did not meet the criteria for PTC – participants who, like most people, experience viral rebound once they stop taking medication. The response of the virus and the immune system of individuals was evaluated during the “early” and “late” periods following ATI. Defining “early” and “late” differed between the two groups – it was felt that due to the more rapid viral rebound and consequent re-initiation of ART in NCs, the samples analysed in the NC group at the “early” stage should be taken at 4 weeks post-ATI, in contrast to 12 weeks for PTCs. For similar reasons, but also to examine the viro-immunological landscape following the transient viral rebound seen in some PTCs, late ATI profiling was performed at 16 and 96 weeks for NCs and PTCs respectively.
"Findings from this work could provide insights for identifying those people who could control the virus after stopping treatment."
Fifty-nine participants overall were included – 22 PTCs and 37 NCs. Baseline characteristics of gender, sex, race, duration of ART and percentage who initiated ART during early infection were comparable between the groups. Around three out of ten patients in each group had received an intervention prior to the ATI (i.e. a vaccine or antibody infusion), though this study did not account for these interventions in its analysis. Not all participants were included in each part of the sub-analysis due to incomplete data.
Etemed and colleagues used data from stored samples and compared different immune factors between the groups. They analysed viral load trajectory, reservoir dynamics, types of immune cells and the inflammatory markers they produce. Combining their findings in these sub-group analysis allowed them to identify the factors that make an individual more or less likely to become a post-treatment controller.
Their findings have numerous implications. First, they could be used in trials of interventions to differentiate between people who do not rebound during ATI because of the intervention versus those who would not have rebounded anyway. Second, they could be used in those reluctant to continue ART to see if they would maintain viral control without medications, although the cost of measuring all the variables might be prohibitive. Third, by identifying the immune make-up that permits post-treatment control, scientists could try and replicate this environment, for example by attempting to increase CD4 gag specific activity or inducing NK cell specific responses.
Viral load
PTCs and NCs both experienced viral rebounds after stopping treatment. NCs had a higher peak viral load (4.3 vs 2.5 log10 copies). This difference in viral load persisted at early and late ATI time points. When accounting for the duration of HIV infection before ART initiation, those who started treatment earlier had similar trajectories of viral rebound as those starting later.
As expected, ART levels remained undetectable during ATI in PTCs. In previous studies, certain genetic variants of part of the immune system called the Human Leukocyte Antigen system (proteins that help identify “foreign” material such as viruses and bacteria in order so that they may be destroyed) were found to be more common in elite controllers. However, the distribution of different HLA variants was even between PTCs and NCs in this study.
HIV reservoir
For those with available samples – 11 PTCs and 13 NCs, HIV DNA was used as a marker of the size of the viral reservoir. Whilst on ART (pre-ATI), the viral reservoirs in the two groups were not significantly different. During treatment interruption though, HIV DNA was significantly higher in NCs. The team clarified that the proportion of intact (capable of making infective virions) and defective (no potential to make working virions) HIV DNA did not differ between groups. This is important as it shows defective virus is not the reason that PTCs maintain better viral control off treatment.
Whilst HIV-DNA is a good measure of the size of the viral reservoir, un-spliced cell associated RNA (CA-RNA) reflects active transcription of the reservoir itself. PTCs maintained stable levels of CA-RNA at both early and late time points, whilst NCs demonstrated rapid increases in CA-RNA during all time points of the ATI. By comparing the CA-RNA/HIV DNA ratios, the much higher ratio in the NCs reflects much greater increases in the plasma viral load after treatment interruption.
To summarise, during ATI the non-controllers experience rapid transcription of their viral reservoir, which in turn increases the size of the viral reservoir itself.
T-cell composition, immune activation, and exhaustion
CD4+ cells act as messengers, instructing other immune cells to attack or not attack – they have no direct ability to “kill” other cells or pathogens. They may recruit CD8+ cells amongst others to undertake the killing required, for example to help neutralise cells that have become infected with virus or another pathogen.
The team reviewed whether the types of T-cells differed between PTCs and NCs both before and during an ATI. Ten PTCs and 25 NCs had T-cell subsets available for comparison.
CD4+ counts were comparable between the two groups pre-ATI though PTCs had a significantly higher CD4% (the proportion of CD4+ from total T-cell population ) and CD4/CD8 ratio.
During early ATI both PTCs and NCs demonstrated increases in the levels of CD8+ T-cell activation, although only those from the NC group reached statistically significant levels. NCs also had significant increases in activation levels of total CD4+, CD4+ central memory and CD4+ effector memory T-cell activation. In contrast, PTCs maintained stable levels of these same cell subtypes.
Specific T-cell responses
T-cells that have developed specifically to counter HIV were compared between the two groups. HIV has evolved so that it preferentially targets these HIV specific T-cells. Gag is a protein found on the surface of HIV virions and is used as a target by T-cells. Pre-ATI there were comparable levels of gag-specific CD4+ and CD8+ T cells, except for gag-specific TNF-alpha CD8+ cells which were significantly higher in NCs.
During early ATI the gag-specific CD4+ IFN-gamma T cell levels became significantly higher in PTCs, followed by modestly higher levels of CD4+ IL2 T-cells. Pre-ATI, individuals with higher levels of CD4+ IFN-gamma were more likely to have lower levels of CA-RNA. This relationship persisted during ATI with lower rebound viral load and CA-RNA in those with higher levels of both CD4+ IFN-gamma IL-2 T cells. No gag-specific CD8+ cells were correlated with CA-RNA levels. It appears that CD4+ gag specific cells may play a role in maintaining restriction of the viral reservoir in PTCs, whilst the CD8+ dominant response seen in NCs may be unhelpful.
Natural killer cells
Natural killer (NK) cells are increasingly implicated in in HIV control. They are cells that have the capacity to directly kill infected cells or tumour cells without the need for these target cells to specifically present themselves as harmful. NK cells again are differentiated by the kind of proteins they express on their surface, each of these proteins contributing to the cell’s different role. PTCs had significantly higher levels of NK activation (measured by %CD38+ and %CD69+ surface protein markers) pre-ATI.
Across both PTCs and NCs, higher levels of NK activation, measured by %CD38+, were associated with less CA-RNA pre-ATI, i.e. less activation of the HIV viral reservoir. During early ATI, as the number of CD69+ NK cells as a percentage of total NK cells increased, there was less viral rebound. These results suggest a role for NK cells in the restriction of the viral reservoir pre-ATI and the suppression of viral rebound during early ATI.
Inflammatory markers
The authors describe how certain proinflammatory markers have been associated with illness and death in people with HIV. Proinflammatory markers are chemical signals or messengers produced by immune cells to help them identify invading organisms or cancer cells.
Pre-ATI there was no difference in the inflammatory markers studied between PTCs and NCs. During treatment interruption interleukin 10 (IP10) and interferon (IFN)-gamma-induced protein 10 rose significantly in NCs compared to PTCs. NCs also experienced a significant increase in the levels of sCD163, IFN-gamma and interleukin 10, particularly during the early ATI period. The inflammatory response in the NCs was more strongly correlated with viral reservoir size and rebound than in PTCs. The researchers feel these results indicate that VL rebound and HIV reservoir expansion are more strongly associated with a “hyperinflammatory” environment in NCs but not PTCs.
Predicting who will become a post-treatment controller
By integrating the above information, the team used a statistical pairing technique to assess whether they could predict before an ATI which individuals would become PTCs during ATI. They used data from eight PTCs and 20 NCs who had complete data for all factors available.
Fourteen features were identified. The most important (determined as contributing >5% to the outcome) were:
- Higher pre-ATI CD4%
- Higher pre-ATI CD4/CD8 ratio
- Higher pre-ATI CD56- NK activation
- Lower CD4+ senescence (%CD4+TDEM)
- Lower CD4+ T Cell exhaustion
- Lower CA-RNA.
Measuring these immune markers in anyone living with HIV should therefore be able to tell us who might control the virus after stopping ART, whilst also giving scientists more avenues to explore in terms of replicating the same immune environment in others.
Conclusion
The study has a few limitations. The influence of an intervention such as a vaccine given before ATI, even though distributed evenly between the NCs and PTCs, is difficult to account for. Second, the numbers involved in each sub-analysis were exceedingly small and so causative mechanisms behind the correlations found are difficult to prove based on the data included. Third, participants were probably taking different types of ART and it is unclear how this may have affected the results.
Findings from this work could provide insights for identifying those people who could control the virus after stopping treatment. More useful, in identifying the ideal immune landscape of a PTC, the study could support developments that aim to replicate this landscape in more people.
Etemad B et al. HIV post-treatment controllers have distinct immunological and virological features. PNAS 120 (11) e2218960120, 2023.