Author:

Abstract:

As HIV integrates a provirus into the DNA of long lived memory immune cells, HIV infection can persist for decades in spite of effective combination antiretroviral therapy (cART) with viremia rebounding from these latent reservoirs soon after treatment is interrupted. Therefore, if we want to develop an HIV cure rather than lifelong treatment, a thorough understanding of HIV latency and latent reservoirs will be necessary. While the integration of a provirus is catalysed by the integrase of HIV, the virus uses several host factors on its road to active chromatin in the nucleus. Chief among these host factors is the transcriptional co-activator lens-epithelium-derived-growth factor p75 (LEDGF/p75). Our lab and others have developed small molecule inhibitors of the LEDGF/p75-IN interaction, called LEDGINs, that bind integrase and disrupt various stages in the HIV replication cycle. Though much of their potency is derived from stabilization of IN-IN dimers, which disrupts HIV-1 maturation and leads to the production of defective HIV virions (late effect of LEDGINs), they also inhibit HIV-1 integration (early effect). Starting in 2016, our lab has used various techniques to show that any residual integrants are retargeted away from genes and active chromatin and towards a more repressive chromatin environment. In addition, the resulting reservoir is more prone to latency and more refractory to reactivation. These latency promoting effects of LEDGINs not only make them interesting tools to study HIV latency, but also support the use of LEDGINs as part of a functional cure strategy called "block-and-lock", where HIV is not eliminated from the body, but the virus can be permanently controlled by inducing a state of deep latency in residual HIV reservoirs. In a first project I compared the novel pre-clinical LEDGIN compound GS-9822 with the research compound CX14442 with regards to potency and effect on HIV-1 integration site selection and latency. I showed that GS-9822 potently inhibitis HIV-1 infection and integration, in addition to retargeting residual integrants away from genes and active chromatin and towards a more repressive epigenetic landscape. Using the HIV-1 OGH double reporter virus, I confirmed that GS-9822 increases immediate latency and decreases the reactivation potential of residual proviruses at nanomolar concentrations. That a functional cure strategy may worth pursuing, is suggested by natural models of less pathogenic infections with lentiviruses related to HIV-1, such as SIV and HIV-2. Unfortunately, little is known about HIV-2 latency, though proviral DNA levels in people living with HIV (PLWHIV) appear similar for HIV-1 and HIV-2, but PLWHIV-2 more often spontaneously control viremia below detectable levels. This has led researchers to hypothesize that HIV-2 may have a more latent phenotype. To test this hypothesis, in a second project, I developed a double reporter virus for HIV-2, similar to HIV-1 OGH, called HIV-2 OGH. As this double reporter allows us to differentiate between quiescent and productively infected cells, I next used these viruses to compare HIV-1 and HIV-2 integration sites, proviral expression, latency and response to reactivation. Surprisingly, I demonstrated higher levels of reporter gene expression and a lower latent fraction for HIV-2 than for HIV-1. I also showed that HIV-2 may have an even stronger preference for integration in genes and active chromatin than HIV-1. Lastly, my results also hinted at a higher cytotoxicity for HIV-1 and revealed that HIV-2 is more refractory to reactivation in cell lines. Even though all lentiviral integrases can bind to LEDGF/p75, early generations of LEDGINs were not active on HIV-2 and SIV, as these compounds were specifically designed to disrupt the HIV-1 IN-LEDGF/p75 binding pocket. As GS-9822 was optimized to remain active against HIV-1 LEDGIN resistance mutations that mimic naturally occurring amino acids in the integrases of HIV-2 and SIV that may prohibit effective LEDGIN binding, we hypothesized that GS-9822 may be active against HIV-2 and SIV as well. In a third project I studied the effects of GS-9822 on the HIV-2 and SIV IN-LEDGF/p75 interaction and wild type infection with these viruses in vitro and demonstrated for the first time that LEDGINs inhibit SIV and HIV-2 replication. In addition, for HIV-2, using the HIV-2 OGH construct, I studied whether GS-9822 could inhibit HIV-2 integration and replicate the latency promoting effects of LEDGINs in HIV-2. I demonstrated that GS-9822 inhibits HIV-2 integration and retargets HIV-2 integration away from genes and towards a more repressive chromatin environment and showed that at high LEDGIN concentrations, residual proviruses show reduced reporter gene expression levels, an increased tendency for latency and reduced levels of reactivation. Finally, LEDGF/p75, and more specifically its integrase binding domain, is also the target of a type of anti-nuclear auto-antibody, called anti-DFS70 for its Dense Fine Speckled pattern in indirect immunofluorescence using HEp-2 cells. Though these antibodies have been investigated in quite a few disease states, they can occur in healthy individuals as well, with prevalences varying with age, ethnicity and gender. Researchers have wondered if these antibodies occur in PLWHIV and, if so, if they affect the course of HIV-1 infection. In 2018 Mahroum et. al. reported that they could not detect anti-DFS70 antibodies in a cohort of PLWHIV, while they were detected in healthy controls. We already obtained data from a pilot experiment in South-African PLWHIV, demonstrating the presence of these antibodies in PLWHIV. Therefore, as a final project, I set up a single centre prospective interventional clinical trial, called anti-DFS70 antibodies in HIV infection (ADAHI) at the University Hospital in Leuven to determine the prevalence of these antibodies in PLWHIV and compared them with healthy controls taking HIV pre-exposure prophylaxis (PrEP) and demonstrated that these antibodies do occur in PLWHIV. Still, more research is needed to determine if they can affect HIV infection and disease progression.