In some people with HIV, defective viruses that are produced by a subset of infected immune cells can significantly raise the person’s viral load despite excellent adherence and there being no resistance. Although the defective viral particles cannot infect new cells, their mere presence may be partly responsible for heightened inflammation. While the clinical implications are unknown, in cases of persistently detectable viral load in the absence of resistance and other causes, changing treatment may not always be necessary nor effective suggests the paper published in the Journal of Clinical Investigation.
Although everyone adherent to HIV treatment experiences sporadic activation of the virus that produces very few (1 to 3) copies that are detectable only by ultrasensitive assays, about one in 250 people are estimated to experience persistent viraemia (some level of detectable virus constantly or frequently present in the blood, usually below 1000 copies). These cases may be frustrating for both the care provider and the person on treatment and may lead to changes of treatment and use of more complex regimens. However, even after that, the virus may remain detectable despite optimal treatment adherence.
Defective viruses contain genetic errors that make them incapable of effective self-multiplication. Several previous studies have already hinted at the possibility of defective viruses being at the root of persistent viraemia when there is no other risk factor. The present study takes a more in-depth look at the specific causes of persistently detectable virus. It found similar defects in the same place in all viruses from four participants on long-term antiretroviral therapy (ART).
In cases of detectable virus, it is essential to first assess and rule out other possible reasons such as insufficient adherence, existing drug resistance, drug-drug interactions, and drug-gene interactions. Some studies have linked persistently detectable virus to a higher risk of treatment failure and development of resistance; however, the link may not be so straightforward. When defective virus is released from the reservoir (the body sites and cells where the virus stays in hiding) and cannot infect new cells, according to the study senior author Dr Francesco Simonetti, it should not be able to develop resistance regardless of the viral load.
Participants
Four participants who were experiencing persistently detectable viral load agreed to provide past data and samples for analysis. Their age ranged from 58 to 63 years, two were African American and the other two were White. Their time since diagnosis ranged between 15 and 32 years, while that on treatment ranged between 8 and 27 years. They all had stable CD4 counts above 600 cells and their viral load at the last measurement ranged from 20 to 3400 copies. The fourth participant was an extreme case of non-suppressible viral load with the highest copy numbers, while the viral load of the other three fluctuated around 100 copies. All participants had treatment optimisation (replacing or adding more drugs to their regimens) in the past that did not make any difference in supressing the virus. However, the fact that their CD4 counts had remained stable over many years suggests the virus in their blood was not infecting and killing their cells.
The study
The investigators at the Johns Hopkins University had to perform several steps to identify whether the defective virus came from newly infected cells or from reservoir cells. They characterised the size of the reservoir and its diversity in the four participants. Then they investigated the match between the virus that contributed to the detectable viral load in blood and the ‘parent’ provirus (a DNA copy of the virus that merges with our own DNA and remains integrated until the cell dies) that is present in the reservoir cells. Once this was clear, they went on to characterise the virus and the defects across different sequences detected from the same participant and from the other participants. Using that information, they tried to understand the mechanisms by which the defective viruses were produced and whether they could infect new cells. Finally, they tried to understand how the defective viruses were selected for and what their advantages were.
Identifying the source of the detectable virus
Immune cells exist as dozens of different subsets with specific features such as long-lived memory cells, which are the preferred target of HIV in establishing its reservoir. However, when they are activated by a foreign agent such as a virus, they can clone themselves – this is called clonal expansion. In this way, a small number of cells can grow into millions of identical cells. If they contain an HIV provirus in them, they will simultaneously multiply the number of proviruses as they clone. Because the cellular machinery is much more accurate in copying genetic material than the virus’ copying machinery, all the proviruses will be identical, as will be the viruses that come from them. A self-multiplying virus will make mistakes which will lead to a very diverse population of virus particles. This is how scientists can determine that the virus spread by clonal expansion rather than infection of new cells, it is also how they can match the virus present in blood and the provirus present in the reservoir cells.
Characterising the defects of the viruses
The investigators analysed the viral diversity of the participants’ reservoirs to determine whether the detectable virus was a result of infection of new cells. While the reservoirs of the participants contained different quasispecies (different variants of the same virus resulting from mutations that happen after infection), the virus particles responsible for the detectable viral load in the four participants were mostly identical and contained very similar defects. This confirmed the idea that they came only from a subset of the reservoir cells infected with this defective provirus.
To understand the defect, if we imagine the virus as a long strip, one end is called three-prime and the other is five-prime; this shows the direction of reading of the virus’ genetic code. Both ends of this strip contain regulatory regions that are responsible for activating the genes in the middle, protecting the virus, and merging it with our cells’ DNA. The five-prime leader sequence (dubbed 5’-L) is responsible for orchestrating the activation of the dormant virus, its proper packaging into its shell so that it can infect new cells, and many other critical steps in the virus’ life cycle. A virus with a defective 5’-L sequence is expected to be incapable of making copies of itself, as it should not be able to activate or arrange the steps of its life cycle. However, surprisingly, this study found that it is the 5’-L defective virus that caused high levels of detectable virus in the four participants.
Defective viruses lack critical components
Viruses are among the simplest biological agents, which means that in a small amount of genetic code they need to encode many and multifunctional elements to deal with our extremely complex immune systems. If we imagine that the virus’ genetic code is a tiny recipe book, but we need to cook a very diverse menu for an entire royal wedding, we will have to get creative. The way the virus does that is called alternative splicing. That is the ability of the virus to use the same recipe (code), but tweak and rearrange it in order to get a completely different ‘dish’.
The 5’-L defective viruses seem to have lost some of their ability to perform the right kind of alternative splicing that will produce the Env (envelope) protein that coats the virus and is essential to enter new cells. Although they were still able to produce Env, it was in very tiny amounts, insufficient to infect new cells.
Due to a similar loss of alternative splicing, the defective virus particles seemed to be unable to produce the Rev protein either, which helps transport the virus’ ‘recipes’ outside the nucleus of our cells so it can be ‘cooked’ and go on to infect new cells.
Escaping immune surveillance
HIV drugs are excellent in blocking the virus that is actively multiplying, however they cannot act on a non-multiplying virus that is released from the provirus in the reservoir. The immune system is expected to counteract the additional virus that is released from the reservoir, but does not seem to work for these four individuals. Hence, the investigators tried to understand whether these defects gave the virus a selective advantage that helped it go unnoticed by the immune system.
Given the fact that one of the major triggers for the immune system is the Env protein, the low levels of Env present on these defective viruses seemed to give the particles an advantage as they were not very good at triggering immune responses.
The next question was why this only happens in some but not all people on HIV treatment. The 5’-L defects are found in 5 to 10% of all people on ART, but detectable virus is much less common. The authors of the paper hypothesise that in these people, by some chance – perhaps another infection or immune trigger – exactly the subset of immune cells containing the defective provirus became activated and started to produce large numbers of these defective viruses.
Conclusion
The study sheds light onto a previously grey area in our understanding of the causes and mechanisms of persistently detectable viral load. It proposes a more detailed analysis of virus features in routine care for people experiencing this issue. It also suggests that treatment intensification may be ineffective and unnecessary in those people.
However, the expertise and equipment necessary for such characterisation is currently unavailable in most laboratories and healthcare facilities. Also, the lack of understanding and consensus on which defective viruses can be ignored as harmless and which ones can cause productive infections despite the defects makes possible applications more challenging.
Besides, there are other possible reasons for detectable virus, such as insufficient drug adherence, drug interactions and resistance. They should always be assessed first.
Although defective, these viral particles may trigger inflammatory responses. Newer approaches may need to be developed in order to stop or suppress the production of these particles. Currently, there is no therapy that can address these rather rare cases of persistently detectable viral load. Fortunately, the defective viruses seem to be unable to evolve as they do not infect new cells and their effect on health and disease progression should be limited, if any.
The question of whether this type of detectable viral load affects ‘Undetectable equals Untransmittable’ (U=U) is still open to exploration. However, most people experiencing this issue usually have less than 200 copies which means they are untransmissible. For those with higher viral loads like the fourth participant, although the virus seems to be non-infectious, it is too early to know for sure.
White JA et al. Clonally expanded HIV-1 proviruses with 5′-leader defects can give rise to nonsuppressible residual viremia. Journal of Clinical Investigation 133(6):e165245, 2023 (open access).