Two recent studies have helped to explain how patients taking highly active antiretroviral therapy (HAART) but who have low, detectable viral loads can maintain stable CD4 cell counts and viral loads despite the presence of replicating HIV. The studies, presented in the January 3rd edition of AIDS and in the February 1st edition of The Journal of Infectious Diseases show that this group of patients has high levels of activated CD4 and CD8 T-cells with anti-HIV activity.
Low-level viraemia
Although the aim of HAART is to reduce viral loads to below the limits of detection, usually 50 copies/ml, this cannot be achieved in some patients. However, patients with detectable but low viral loads (under 10,000 copies/ml) often maintain stable CD4 cell counts and have a low risk of progression to AIDS despite detectable levels of virus in the blood, and the presence of resistance to anti-HIV drugs.
The virus found in these patients is likely to be resistant to some or all of the HIV drugs being taken. While these mutant strains of HIV often have an impaired capacity for replication, it remains uncertain whether this is sufficient to explain the stability of viral loads and CD4 cell counts. These two studies were undertaken to investigate the roles of CD4 and CD8 T-cells in controlling HIV replication.
HIV-specific T-cell responses
Investigators from Paris set out to assess the role of cells with specific anti-HIV activity, by examining the responses of T-cells extracted from HIV-positive patients to purified HIV proteins (Alatrakchi 2005). They found that the levels of HIV-specific CD4 T-cells, which release chemical messengers to co-ordinate the immune response against HIV, as well as CD8 T-cells, which kill cells infected with the virus, are higher in patients with low but detectable viral loads.
The researchers selected three groups of adult HIV-positive patients who had been receiving HAART for at least 18 months. Twenty-nine patients had persistently undetectable viral loads (below 200 copies/ml), 28 had low viral loads (below 10,000 copies/ml) and 14 had high viral loads (above 10,000 copies/ml).
“We found that patients with stable low viral loads had significantly stronger HIV-specific CD4 and CD8 T-cell responses than patients with full viral suppression and patients with uncontrolled viral replication under HAART,” state the investigators. “Continuous moderate immunologic stimulation by low-level viral replication helps to maintain potent HIV-specific responses that may play a key role in virus control.”
The investigators measured the number of T-cells that produced the activity marker interferon gamma in response to the HIV proteins p24 and gp160, using an enzyme linked immunosorbent spot assay (ELISpot). Patients with low viral loads had significantly more CD4 T-cells that were actived in response to p24 (median of 131 cells per million peripheral blood mononuclear cells) than those with undetectable (47 cells, p = 0.012) or high viral loads (23 cells, p = 0.047).
In contrast, the responses of the cells to cytomegalovirus or tuberculosis proteins were similar in the three groups. “These differences…suggested that changes in p24 responses were more consistent and did not simply reflect general losses of immune responses to pathogens,” conclude the authors.
However, the cells’ responses to the HIV protein gp160 were similar across all three groups, although the study’s authors do not offer an explanation for the difference between the response to gp160 and p24.
The levels of CD8 T-cells activated in response to stimulation with fragments of the HIV proteins Gag and reverse transcriptase were also higher in patients with low viral loads than those with undetectable viral loads (1340 vs. 410 cells; p = 0.001). However, they did not differ significantly from those with high viral loads (518 cells; p = 0.138).
The researchers also examined the proliferation of T-cells in response to HIV proteins as a marker for their anti-HIV activity. CD4 T-cell proliferation was significantly greater in patients with low viral loads than those with undetectable viral loads both for p24 (p = 0.004) and for gp120 (p = 0.007). However, they were similar to those from patients with high viral loads. As observed using the ELISpot technique, responses to cytomegalovirus and tuberculosis were similar across the three groups.
Finally, the investigators observed that there was an inverse relationship between viral load and the number of anti-HIV CD8 T-cells. “Higher frequencies of CD8 T-cells directed against pools of HIV-gag and reverse transcriptase peptides were associated with lower plasma viral load in both the low viral load and high viral load groups,” they state. This suggests that the presence of low levels of HIV in the blood stimulates CD8 T-cells to fight back against the virus.
“Low level ongoing virus production appropriately stimulates both CD4 and CD8 T-cell responses to HIV and contributes to viral control,” they conclude.
Links to CD4 cell count
Findings from the second study reflected those of the French investigation, confirming that patients with low but detectable viral loads had increased levels of activated T-cells. The investigators, from Copenhagen, argue that this keeps viral loads under control, but also prevents CD4 cell count gains by accelerating the activity-induced death of CD4 T-cells.
“Although low-level viraemia did not result in obvious treatment failure, it was associated with reductions in CD4 gain and with T-cell activation,” they conclude. “Periods of even low-level viraemia in patients receiving HAART are negatively associated with immune reconstitution, either directly or through induction of immune activation.”
The investigators wished to observe the CD4 and CD8 cell counts and viral loads of 101 HIV-positive patients on HAART every three months for two years. All of the patients had viral loads below 200 copies/ml at the start of the study, but only 33 had ‘undetectable’ viral loads (below 20 copies/ml) at each measurement.
Every six months, the researchers also measured the levels of T-cells in the patients’ blood. They found that the patients who had at least one detectable viral load measurement had higher levels of CD8 T-cells (p = 0.007) than the patients who were always undetectable. They also found that these patients had higher levels of HIV’s genetic material (‘proviral DNA’) in cells.
When they analysed the types of T-cells found in the blood from the patients, the investigators saw that patients with at least one detectable viral load measurement had more ‘activated’ CD8 T-cells than patients who never had a detectable viral load (p = 0.001). These T-cells, which were identified by the presence of the CD38 receptor on the cell surface, have become active against a particular foreign molecule or ‘antigen’ that they have met in the body.
The investigators also found more ‘memory’ CD8 T-cells (p = 0.020) in these patients. These are long-lived cells that have previously been active against an antigen and are awaiting the possible reappearance of the same antigen, in order to mount a rapid attack if a subsequent infection occurs.
Similar effects were seen when they examined CD4 T-cells, with more activated (p = 0.016) and memory cells (p = 0.009) in the patients with detectable viral loads.
The research group went on to compare the viral loads of patients with changes in CD4 cell count, finding a significant association between higher viral load and smaller increases in CD4 cell count (p
“The present study is the first, to our knowledge, to demonstrate a negative association between even low-level viraemia and immune reconstitution in patients receiving HAART,” comment the authors. “The concentration of total CD8 cells, activated CD8 cells, memory CD4 cell and memory CD8 cells and in proviral DNA level…were higher in the detectable viral load patients, indicating that low-level viraemia may be associated with T-cell activation and a higher proviral DNA level.”
The researchers also investigated whether any of the T-cell measurements could predict the risk of a detectable viral load in the future. After carrying out a univariate model analysis, they found that for every twofold increase in the numbers of activated CD4 T-cells, the risk of a patient becoming detectable increased by 39% (p = 0.018).
The proportion of ‘early differentiated’ CD4 T-cells was also associated with an increased risk of detectability (p = 0.037), but the opposite trend was seen for ‘late differentiated’ CD4 T-cells (p = 0.036). There were no significant effects of CD8 T-cell levels.
“Early differentiated CD4 cells have a high proliferative capacity but no cytotoxic [cell-killing] potential, whereas late differentiated CD4 cells have a reduced proliferative capacity but a cytotoxic potential,” explain the researchers. “Thus, cytotoxic late-differentiated CD4 cells possibly exhibit cytotoxicity against HIV-infected cells.”
The investigators stress that their findings do not indicate whether detectable viral load is responsible for, or a consequence of the differences in T-cell numbers. “Since the investigated virological and immunological variables were measured concurrently, cause-and-effect inferences simply cannot be made,” they warn.
Alatrakchi N et al. Persistent viral load on antiretroviral therapy is associated with T cell-mediated control of HIV replication. AIDS 19: 25-33, 2005.
Ostrowski SR et al. Low-level viremia and proviral DNA impede immune reconstitution in HIV-1-infected patients receiving highly active antiretroviral therapy. J Infect Dis 191: 348-357, 2005.