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    • TILDA does not rely on

    2018-10-23

    TILDA does not rely on the amplification of viral replication and offers similar advantages of the DNA PCR assays with regard to simplicity and small blood volume requirement. It does not require RNA extraction and 96-well plates with serially diluted potassium phosphate monobasic can be stored at −80°C for future quantification in batch analyses, making TILDA well-suited for longitudinal clinical studies. Therefore, TILDA has a number of characteristics that make it attractive for clinical trials, particularly in paediatric studies in which the number of cells that can be obtained are often limited. In addition, TILDA could be used to measure the frequency of infection in rare population of cells, which may facilitate the measurement of the HIV reservoir in tissues. Using TILDA, we found the median frequency of latently infected CD4+ T cells during ART to be 24cells/million, which is 48 times more than the frequency measured by the quantitative viral outgrowth assay. Ho et al. recently reported that the median frequency of intact noninduced proviruses is around 60-fold higher than the frequency of induced proviruses detected in Q-VOA (Ho et al., 2013). Therefore, TILDA may provide an accurate estimate of the size of the latent HIV reservoir, although the precise fraction of the replication competent latent and inducible HIV reservoir captured by TILDA will require further studies. Similarly, evaluating the ability of TILDA to predict viral rebound upon ART interruption will necessitate the development of well-powered clinical trials and cannot be inferred from our current observations. TILDA correlated with several PCR-based assays that measure the size of the latent reservoir, including the quantification of total and integrated HIV DNA in PBMCs. TILDA tended to correlate with the size of the reservoir measured by Q-VOA, but this was not statistically significant. Our data are consistent with a lack of strong correlation between these two measures although the lack of statistical significance may reflect, in part, the sample size. The lack of correlation between the two assays may also result from the fact that Q-VOA is performed on purified resting CD4+ T cells, whereas total CD4+ T cells are used for TILDA determinations. Total CD4+ T cells include cells undergoing homeostatic proliferation which express some activation markers and are not captured in the Q-VOA assay. Ho et al. reported that Q-VOA does not correlate strongly with the frequency of cells harbouring intact non-induced proviruses (Ho et al., 2013), suggesting that it may not provide an accurate estimate of the frequency of latently infected CD4+ T cells. Whether TILDA could provide a better estimate of the size of the replication competent reservoir remains to be determined by using full-length sequencing approaches, similar to the ones used by Ho et al. and others (Ho et al., 2013; Cohn et al., 2015). Using TILDA, we observed that early initiation of ART leads to a reduced size of the viral reservoir after years of viral suppression. This is in line with several reports that have clearly demonstrated the benefit of early therapy on the size of the latent HIV reservoir (Strain et al., 2005; Archin et al., 2012; Buzon et al., 2014; Ananworanich et al., 2012, 2015; Chomont et al., 2009). In addition, we demonstrated that before the initiation of ART, the latent HIV reservoir already represents a substantial fraction of all infected CD4+ T cells and that the size of the reservoir increases with time during untreated HIV infection. This observation further argues for early ART intervention to prevent the establishment and continuous seeding of an increasingly large pool of latently infected CD4+ T cells. Although TILDA constitutes an attractive alternative to other methods to measure the size of the latent HIV reservoir, there are potential limitations associated with the use of our assay. First, although all cells that are releasing viral particles are producing tat/rev msRNA, the opposite may not be necessarily true. We cannot exclude the possibility that some cells that generate a positive signal in TILDA do not produce virions. Indeed, very low levels of nuclear msRNA can be detected in resting CD4+ T cells using ultrasensitive approaches (Lassen et al., 2004, 2006). Therefore, TILDA is likely to overestimate the size of the latent and replication competent reservoir. Notwithstanding this limitation, TILDA is more likely to capture the functional reservoir than other existing PCR-based assays: TILDA positive events result from the transcription of integrated HIV genomes that have an intact LTR (to ensure transcription) and that are not deleted in the two most commonly deleted regions identified in defective genomes namely tat and rev (Ho et al., 1995). Of note, TILDA measures frequencies of latently infected CD4+ T cells close to those predicted to harbour replication competent virus in the study by Ho et al. (2013). Another potential caveat of TILDA is the fact that it relies on the amplification of a highly variable region of the HIV genome, suggesting that the primers and probes used in our assay may not recognize all viral quasispecies. We anticipate that the use of TILDA on HIV clades other than B will require further validation and optimization.