Why is there no cure for HIV yet? The virus attacks CD4+ T cells for which antiretroviral therapies have been developed which successfully inhibit viral replication. The standout problem is the latent HIV reservoir: intact, replication competent viruses which infect CD4+ T cells and lie dormant, evading the immune system. This means that when ARV therapy is stopped, the virus reactivates leading to relapse.
Only a small fraction of HIV in the body is ‘replication-competent’ meaning that measuring the total HIV DNA also captures instances of defective virus, containing deletions and hypermutations. This leads to an overestimation of the true copies of the intact virus in the body. When developing therapies to completely eradicate HIV, it is essential to measure the intact fraction of virus as this includes latent HIV reservoirs.
The development of digital PCR (dPCR) integrity assays is one solution. Integrity assays use multiple probes to detect deletions and hypermutations in the HIV DNA. The process involves placing two assays, one in blue and one in green. If there is a deletion or mutation in one of the regions, only one of the probes will bind, resulting in a single signal. However, if both probes bind, it indicates that the HIV DNA is intact, and the signal becomes orange.
To further enhance detection accuracy, a dark probe is used for hypermutations. This probe is highly specific for hypermutations but does not have a fluorophore, allowing it to capture hypermutated fractions within the negatives.
Trypsteen developed this concept further by developing a three-colour system which allows for better visualization of hypermutated viruses. By adding a red colour, researchers can see how many viruses are hypermutated, providing valuable information for treatment intervention studies.
The integrity analysis was improved further still by including more assays. A five-colour system has been developed, which uses five markers inside a single well. This system combines total HIV DNA and intact DNA assays to increase accuracy in clinical trials where patient samples are scarce.
