Cure Research Takes Center Stage: Proof of Concept for Activating the Latent Reservoir

Treatment During Early Infection

Theoretically, the easiest targets for cure research might be those patients diagnosed earliest in their infection, who promptly start treatment and who maintain suppressed viraemia for many years.

Although the latent cell reservoir is established within weeks of infection and is likely to be slowly reduced after years on effective ART, in nearly all patients, viraemia rapidly returns within weeks if treatment is interrupted. Even when HIV is reduced to being present in less than 1 in 1.7 billion cells, this is sufficient for systemic infection to quickly be reestablished (within two months) if treatment is stopped. [14]

While levels this low might question the importance of a treatment to target the viral reservoir, they can so far only be achieved with very early treatment and/or many years of viral suppression. The need to reduce the viral reservoir more quickly will be a concern for everyone else who started ART during chronic infection.

Rapid viral rebound without treatment has been widely reported in numerous treatment interruption studies. However, several small cohorts have also reported viral control in a minority of patients, usually in those who initiated treatment in acute infection and maintained undetectable viral load for several years.

Last year at CROI, the ANRS Visconti study reported small numbers of patients who started treatment in early infection (after serconversion, median viral load >100,000 copies/mL), maintained viral suppression for >3 years on treatment and who have subsequently controlled viraemia off treatment for >6 years. [15] This year at CROI similar cases were reported in posters by two other groups.

Maria Salgado and colleagues reported a single case of a patient who initiated treatment during seroconversion (viral load >750,000 c/mL, western blot indeterminate) for three years and after stopping ART has since maintained viral load suppressed to <50 copies/mL off-treatment for more than nine years. Initial and current viral isolates are dual CCR5/CXCR4 tropic and fully replication-competent in vitro. Minimal viral evolution has been detected over the 11 years.

He is reported to currently have low titers of neutralising antibodies to heterologous and autologous HIV-1 isolates, and his CD8+ T cells do not have potent HIV suppressive activity suggesting a mechanism other than CTL-mediated suppression reported in elite controllers. [16]

Alain Lafeuillade from General Hospital, Toulon (who is also one of the key organisers of the International HIV Persistence Workshop that has been meeting every two years since 2003) reported that 17% (8/45) of a cohort of patients treated at seroconversion for a median of 2.2 years (range 1.8 to 4.0) have remained off treatment for more than 10 years, two of whom remain suppressed to <20 copies/mL (median 2,500 copies/mL for the other six). The 37 people who restarted treatment (due to confirmed CD4 decline to <350 cells/mm3) did this after a median of 5.0 years (range 3.0-8.0) off-treatment. The study suggested the protective mechanism could be relate to early ART reducing the HIV reservoir but also emphasised that such responses seem to be rare. [17]

A poster from Joseph Margolick and colleagues reported small differences in viraemia between people diagnosed in early infection (within a year of infection) and randomised to immediate treatment (n=57) year) and those who did not start early treatment (n=24). However, study numbers were very low at the evaluation point (24 months after stopping treatment of 24 months after diagnosis) due to ~20% loss to follow-up and exclusion of people who restarted treatment for other reasons. [18]

Generally small differences were also reported from early treatment in the larger SPARTAC study that randomised almost 400 people (diagnosed within 6 months of infection) to deferred ART or immediate treatment for either 3 months or 12 months, and who then stopped treatment. [19]

However, in the context of eradication research, two oral presentations suggested that early treatment, while too late to prevent the establishment of the viral reservoir, might reduce the pool of latently infected cells.

Maria Buzon and colleagues estimated the size of the viral reservoir in patients treated for more than ten years who initiated ART within 3 months of infection (n=9) and compared levels integrated and total HIV DNA levels to people who started treatment during chronic infection (n=26) and to elite controllers (n=37). [20]

Integrated and total DNA levels were significantly lower in both primary treated (p=0.06 and p=0.001, respectively) and elite controllers (p=0.003 and p<0.0001, respectively) compared to those treated in chronic infection. In addition, the ratio between total and integrated HIV-DNA was significantly lower in early treated and elite controllers (both p=0.04 vs chronic) with no differences between acute and EC groups.

Although patient numbers were small, differences were also reported when comparing how soon treatment had been started with patients treated during Fiebig stage III or IV vs stage V having significantly lower levels of both total and integrated HIV DNA after two years.

An oral presentation by Alan Perelson from the Los Alamos National Laboratory used mathematical modelling to look at the impact of early treatment of 27 people treated during acute infection on the size of the latent reservoir, and the relationship of both to initial viral load and target cell ability. [21]

This study also reported that earlier ART, including earlier during primary infection, had a measurable impact related to the initial size of the reservoir, with patients who already started with very low levels of resting cell infection (who also had low levels of peak viral load) experiencing less change in the reduction of resting cell decay. The model also suggested that CD4 T cell increases in response to successful ART was not increasing the viral reservoir.

Research Into a Functional Cure

Other groups are focusing on immunological interventions that would support a functional rather than eradicating cure.

Pablo Tebas from the University of Pennsylvania, presented additional safety and efficacy results from the use of zinc finger nuclease (ZFN) modification of CD4 cells (using SB-738) to a CCR5-deleted phenotype (in development by Sangamo BioSciences). [22]

This process involves harvesting cells by apheresis, treating them with SB-738 to produce 13-35% of cells with CCR5-detetions in vitro. The cells are then expanded, cryopreserved and 5-30 billion cells are reinfused into the donor patient.

Results were combined from three studies: one in ART responders (baseline CD4 >450 cells/mm3) who subsequently interrupted treatment (group 1, n=6) and two in immune non-responders (baseline CD4 <500 cells/mm3) who have not interrupted treatment (group 2, combined n=15). Initial results from these studies were presented at CROI and ICAAC conferences last year.

Most patients were male, white, mean age 48, with a long history of HIV infection (median 12 and 18 years in group 1 and 2 respectively). Mean CD4 count and CD4:CD8 ratio were 921 (+222) cells/mm3 and 1.4 (+0.6) in group 1 and 335 (+89) cells/mm3 and 0.7 (+0.3) in group 2.

Duration of follow-up is now a mean 325 days (range 90 – 738 days).

After infusion, CD4 cells increased by about 1500 cells/mm3 in group 1 (n=6), these then decreased during the treatment interruption but which remained significantly above baseline during follow-up. CD4 responses in group 2 involved an increase of about 500 cells/mm3 which then dropped by about 200-300 which then remained stable out to over a year in the patients who did not interrupt treatment. The expansion of CD4 cells was associated with increases in IL-2, IL-7 and IL-15.

The CD4:CD8 ratio increased significantly in both groups, normalising and remaining at approximately 1.0 throughout follow-up in the group 1 and increasing to approximately 2.5 for the six patients in group 2 decreasing during the treatment interruptions but then remaining stable.

The modified cells continued to be detected through follow-up at 2% of circulating CD4 cells at 48 weeks for most patients. Levels were higher during the treatment interruption for group 2 and then dropping to 2%. Circulation of cells to other tissue sites was confirmed by multiple rectal biopsies where levels of the CCR5-modified cells were comparable to those in blood or higher throughout follow-up.

During the treatment interruption viral load rebounded over the first 8 weeks to around 100,000 copies/mL in a similar way to other interruption studies dropping by one log during the last 4 weeks off-treatment to levels that were generally higher than pre-ART. After three months, when treatment was restarted, viral load become undetectable again in all six patients. One person, later found to be heterozygous for the delta-32 mutation, had a lower rebound (to 10,000 c/mL) and then resuppressed viral load to undetectable by week 8 and remained undetectable off treatment until restarting as per protocol at week 12.

This group used a new method to measure changes in the viral reservoir based on levels of HIV DNA sensitive to low copy numbers (although unable to distinguish between integrated DNA and 2-LTR circles. They reported no detectable change in 4/6 patients with one person have a transient 4-fold increases at week 12 and 20 during the interruption but returning to baseline levels and one person experiencing a 9-fold increase that returned to baseline 16 weeks after restarting treatment.

Side effects were mild and transient, mainly within 24 hours of the infusion (mild chills, fever, headache, fatigue) but included one report of arthritis lasting a few days and abnormal garlic-like body odour.

Next steps include using immunomodulatory drugs such as cylcophosphamide to promote engraftment and increase the percentage of modified cells and studying other patients who are heterozygous for the delta-32 deletion.

Several studies presented studies where pegylated interferon (peg-IFN) was added to ART prior to stopping HIV treatment and continuing peg-IFN. The results suggested that viral rebound was delayed by the peg-IFN via an immune-mediated rather than antiviral mechanism, but these were small studies with short-term follow up (12 and 24 weeks). [23, 24, 25]