NEW YORK (GenomeWeb) – HIV patients who take antiretroviral (ART) drugs still have a reservoir of latent virus lurking in their T cells. Even when ARTs suppress active viral replication to undetectable levels, this latent reservoir prevents patients from being fully cured of the disease.
According to a team led by Johns Hopkins University School of Medicine researchers, however, current methods for measuring the size of these latent reservoirs may be giving too much weight to the presence of defective proviruses — virus genomes integrated into the DNA of a host cell — which is throwing off researchers' ability to gauge the effectiveness of certain therapies that may be aimed at the virus reservoir.
"To cure HIV, you want to get rid of the proviruses without defects," senior author Robert Siliciano, an infectious disease physician and molecular biologist at Johns Hopkins, said in a statement. "But our work shows that the standard assays used to do that are measuring forms of the virus that are not really relevant to these cure strategies."
As they reported in Nature Medicine, the investigators set out to develop an unbiased single-genome-amplification method to amplify near-full-length proviral genomes from 19 HIV-1-infected adults treated at different stages of infection, in order to clarify how defective proviruses are accumulated and how they persist in patients.
Ten of the patients had initiated ART during the chronic phase of infection. The team found that 98 percent of the proviruses detected in these patients were defective, with the most common defects being internal deletions.
The other subjects examined were treated during the acute or early phase of infection, and here the team found that only 7 percent of the proviruses present were intact, with some 93 percent being defective in some way.
"Because many of the proviruses identified have defects that would preclude high-level expression of viral genes, cells carrying these proviruses would presumably be less susceptible to elimination through viral cytopathic effects or through lysis by cytotoxic T lymphocytes," the authors wrote. "Thus, even individuals treated very early after infection have large numbers of defective proviruses. Taken together, these results demonstrate that defective proviruses arise commonly, accumulate rapidly within two to three weeks after infection and persist in vivo."
A big part of the problem in accurately detecting these proviruses, the team said, is that researchers investigating the effectiveness of experimental HIV treatments often use PCR assays to measure the size of the viral reservoir. Some also use a quantitative viral outgrowth assay (QVOA). However, the team found that PCR overestimates the true size of the latent reservoir by a median of 188-fold in patients who began treatment more than six months after their infection with HIV, and 13-fold in patients who began treatment earlier. And QVOA underestimates the reservoir by a median of 27-fold in patients who began treatment late and 25-fold in those who began treatment early.
However, this new unbiased method of analysis allowed the team to accurately count the proviruses present and distinguish the defective ones from those that are intact.
"Our analyses show that the fraction of intact proviruses is much lower than originally thought, even in individuals who were treated early," the authors added. "Taken together these data suggest that some, if not all, of these intact proviruses are competent for viral replication and that the number of intact proviruses is probably the closest estimate to the true size of the latent reservoir."
Despite the results, first author Katherine Bruner said in a statement, the full sequencing method used in the study "is very helpful from a research standpoint but time-consuming, expensive, and impractical for wide clinical use." So it's not a replacement for PCR or QVOA.
What the results do show, though, is that researchers must learn to reinterpret PCR and QVOA results in order to get a more accurate count of intact proviruses in a given patient, which could also help give a more accurate picture of how effective some experimental therapies really are.