Tools to Assess HIV Eradication
As clinical research is moving toward HIV eradication trials, we need acurate biological tools to measure the reservoir of persistent replication competent viruses.
In a recent paper, Susanne Eriksson and collaborators have compared eleven different approaches for quantitating persistent HIV-1 in patients on cART, involving seven analytical approaches and four different kinds of tissue samples (1).
The study involved 30 patients; 10 started cART during acute or early infection while the remaining 20 started cART during chronic infection.
The average duration of viral load suppression on cART was 5.8+/-2.5 years in the first group and 8.0+/-4.2 years in the second group.
The viral outgrowth assay was defined as the reference assay for measuring the HIV reservoir, giving results on the order of 0.1-10 IUPM in most patients on long term cART.
Viral outgrowth assay
Replication-competent HIV was recovered from purified resting CD4+ T cells from peripheral blood in 20/30 patients, at a geometric mean titer of 0.64 IUPM.
The mean frequency of latently infected cells was significantly lower in patients starting cART at acute or early infection compared to patients starting cART at chronic infection (0.28 vs 0.97, p=0.048).
It is important to note that the limit of detection of this assay is determined by the input number of resting CD4+ T cells.
HIV-1 DNA in PBMC
Unfractionated PBMCs were analyzed by using a droplet PCR approach. HIV-1 DNA was detected in 28/30 PBMC samples. Values varied over a 2 log range with a mean geometric mean value of 74 copies/106 PBMC.
Values were generally lower in patients starting cART at acute/early infection compared to chronic infection but the difference did not reach statistical significance.
There was essentially no correlation between this assay and the viral outgrowth assay
HIV-1 DNA in purified resting CD4+ T cells
HIV-1 DNA was detected in 14/16 samples with a geometric mean titer of 186 copies/106 resting CD4+ T cells.
No significant correlation was found between the frequency of resting CD4+ T cells harboring HIV DNA and the time between initial infection and suppression of viral replication with cART.
There was only a modest correlation with results of the viral outgrowth assay in the whole population, and no correlation in patients treated at chronic infection.
Integrated HIV-1 DNA in PBMC and purified resting CD4+ T cells
There was a highly significant positive correlation between the level of integrated HIV-1 DNA in PBMC and the frequency of latently infected resting CD4+ T cells determined in the viral outgrowth assay.
HIV-1 DNA and RNA in rectal biopsy samples
19 patients were evaluated.
HIV-1 DNA was detectable in all samples, with a geometric mean titer of 3015 copies/106 CD4+ T cells.
The level of HIV-1 DNA in rectal CD4+ T cells was not significantly correlated with the frequency of latently infected resting CD4+ T cells in blood as measured by the viral outgrowth assay.
There was a significant correlation between the level of HIV-1 DNA in rectal biopsy samples and the frequency of infected cells in peripheral blood as measured by digital droplet PCR.
HIV-1 RNA was also detected by qRT-PCR in all samples, with a geometric mean level of 1985 copies/106 rectal CD4+ T cells.
The geometric mean RNA/DNA ratio was 0.68. RNA/DNA ratios in rectal biopsies were significantly correlated with the frequency of resting CD4+ T cells in peripheral blood that scored in the viral outgrowth assay.
Circles were detected in 9/30 PBMC samples, with a geometric mean level in these 9 samples of 6.8 copies/106 PBMCs.
Levels of 2-LTR circles in PBMC or resting CD4+ T cells were not significantly correlated with infected cell frequencies measured by the viral outgrowth assay.
Single copy assay for residual viremia
Residual viremia was detected in 20/30 subjects, with a geometric mean level of 0.78 copies/ml. No obvious correlation was found with the viral outgrowth assay.
Residual viremia was not correlated with the level of infection in PBMCs by droplet digital PCR, the level of infection of CD4+ T cells in the rectal biopsies, or the level of 2LTR circles.
Consequently, PCR based assays gave infected cell frequencies at least 2 log higher than the viral outgrowth assay. This difference may reflect the presence of defective genomes. The ratio of infecetd cells determined by viral outgrowth and PCR-based assays varied dramatically between patients. This is an indication that the proportion of defective genomes is not constant. The study also raises the issue of the dynamic range needed for an assay to assess HIV eradication.
Eriksson S, Graf EH, Dahl V, Strain MC, Yukl SA, et al. (1013) Comparative analysis of measures of Viral reservoirs in HIV-1 eradication Studies. PLoS Pathog 9(2): e:1003174. doi:10.1371/journal.ppat.1003174
Key words: HIV biological tools, HIV curative strategies, HIV cure, HIV eradication, HIV persistence, HIV reservoirs, HIV workshop