Background:
Combination antiretroviral therapies (cART) have transformed HIV into a chronic
manageable health condition for many PLWH.
Despite potent cART, the vast majority of PLWH have residual of viremia that remains
below the limit of detection of current clinical assays of 20 copies/ml plasma but can
often be detected by highly sensitive assays. A number of studies report that PLWH
compliant with cART, without drug resistance mutations, have ongoing detectable low level
viremia (also referred to as nonsuppressible viremia, NSV), usually from 20-400
copies/ml, ranging in frequency from 1 in 250 to 9% and 10%. In Ontario, over the past
two years, 11.9% of PLWH had two or more consecutive values of low level viremia (LLV)
(personal communication Vanessa Tran, Ontario Public Health labs). Although low level
viremia on compliant cART is felt not to reflect treatment failure, there is evidence
that it may not be clinically insignificant. Recent studies of large cohorts predict a
2-3 fold chance of virologic failure and increased all cause mortality and non-AIDS
events with LLV. White et. al. showed that detectable LLV is due to expansion of clones
containing both defective and replication competent virus, that are proliferating likely
in response to antigens. Thus, individuals with LLV, likely have very large populations
of proliferated clones in their reservoirs. Whether this higher level of daily virus
production sustains inflammation and immune activation is poorly understood. Moreover,
the clinical short-term significance of these findings is unclear, however, these
individuals represent a unique group to further study cure strategies that decrease the
reservoir size.
Recent observations, suggest that some NNRTIs (especially efavirenz) could be repurposed
to kill these virus-producing cells. The HIV protease enzyme is part of a larger gag-pol
protein precursor that is produced as an inactive monomer in the cytoplasm of infected
cells after HIV transcription. In order for the HIV protease to be active, it must
dimerize, and this dimerization only happens within the virus particle that has budded
off the cell, which results in mature, infectious viral particles. It has been shown that
some NNRTIs such as efavirenz can induce intracellular Gag-Pol dimerization and premature
protease activation in the cytosol prior to budding, which can trigger the CARD8 (Caspase
recruitment domain protein 8) inflammasome, resulting in death of HIV-1-producing cells
via pyroptosis. This process occurs when the compound binds to the RT domain of the
Gag-Pol polyprotein, leading to a conformational change that induces its dimerization
within the cell cytoplasm rather than in the virus particle, and the consequent
autocatalytic activity of the protease enzyme. Thus EFV can induce Gag-pol dimerization
in the cytoplasm, which triggers CARD8 sensing and inflammasome activation
Intra-cytoplasmic protease-CARD8 activation would be blocked by the presence of protease
inhibitors (eg. indinavir). Preliminary data from the Simonetti lab (one of the
co-investigators) using CD4+ T cells from people on cART experiencing non-suppressive,
but low level viremia, have shown that micromolar concentrations of efavirenz (EFV) can
significantly reduce virus production upon strong T cell activation by CD3/CD28
stimulation, due to CARD8 sensing of HIV protease activity . In other words, these virus
infected cells were killed in the presence of EFV during immune activation. In control
experiments, with the addition of the protease inhibitor lopinavir, the effect of EFV is
completely abrogated. Experiments were conducted with EFV at 5uM, which can be reached in
plasma in a large fraction of individuals with the recommended full dose (600mg q.d.).
Previous pharmacokinetics studies showed that EFV reaching plasma concentration above the
EC50 required for its killing effect (1uM) and reaches even higher concentrations in
tissues. Recent work reported NNRTI-like molecules with comparable antiviral activity to
EFV but significantly higher potency in inducing dimerization and CARD8-mediated killing.
However, such compounds are still in the early stages of pre-clinical investigation and
their pharmacokinetic profile and activity in vivo has not been explored. This effect, of
killing HIV infected cells by pyroptosis after CARD8 sensing has been termed TACK
(targeted activator of cell killing). EFV represents the best TACK molecule to test
whether the CARD8-inflammasome can be harnessed as a therapeutic option in people living
with HIV on ART EFV is recognized as the most promising and potent FDA approved NNRTI
compound for this purpose. In the proposed study, cART intensification with EFV is used
to induced CARD8 sensing and not to block viral replication.
Hypothesis:
For PLWH, who are adherent to cART, with no documented resistance to their current cART,
or EFV, and who have documented persistent nonsuppressible viremia (NSV), the short-term
addition of Efavirenz, as a TACK molecule [targeted activator of cell killing], will
reduce plasma HIV-1 RNA viral load.
Primary Endpoint Plasma HIV viremia will be compared at baseline (visit 2) after 8 weeks
of EFV. (visit 7) Secondary Endpoints
HIV Viral Reservoirs:
QVOA (replication competent virus outgrowth assay) at baseline and at 2 months of EFV
Intact proviral DNA assay at baseline, during and after 2 months of EFV Cell-associated
HIV RNA at baseline, during and after 2 months of EFV
Immune Activation:
Markers of immune activation and cell death in plasma (baseline and 2 months):
CRP, LDH, IL-1 beta, TNF, IL-6, gasdermin D, Th1 and Th2 cytokines and chemokines Markers
of T cell (CD4 and CD8) activation measured at baseline and 2 months via flow cytometry.
T cell resposnes to HIV proteins at baseline and 2 months.
Study Design:
This is a prospective, non-randomized study wherein each enrolled subject will act as
their own control before and after the introduction of two months of Efavirenz as a TACK
molecule. The study will be at a single site, at Maple Leaf Clinic at Toronto, Unity
Health St. Michael's Hospital, and the University of Toronto. After enrollment, the study
duration is 20 weeks. All subjects will be recruited to Maple Leaf Clinic, Toronto. Blood
draws will occur at Maple Leaf Clinic. Leukapheresis procedures to more intensively study
virus reservoirs and immunity will be optional, and will occur at baseline and 8 weeks.
Baseline is defined as time to have baseline blood draws, and/or leukapheresis, and
individuals will be started on EFV 600 mg PO once daily for 8 weeks, to start the next
day. Participants will be identified prior to the baseline visit at about - 4 weeks prior
to baseline to ensure eligibility. We intend to enroll 14 participants. At the -4 weeks
visit (visit 1), the study pharmacist will review all concomitant medications,
compliance, review current ART regimen and the proposed ART regimen with the addition of
full dose EFV. Potential side effect/toxicities -espcially EFV's known but reversible
neurological effects - and management of them will be discussed. At this time point, a
viral load will be obtained to confirm persistent low level viremia. At baseline, (time
0), if prior VL showed LLV, the following assays will be done: HIV viral load, qVOA, IPDA
for HIV reservoir measurements, cell-associated HIV RNA, plasma and T cell markers of
immune activation, HIV specific T cell immunity. At baseline instead of a 70cc blood
draw, subjects will be offered to participate in leukapheresis (optional) which will
provide larger cells numbers to better interrogate the HIV reservoir. Subsequent visits
at 1 week (V3), 3 weeks (V4), 5 weeks (V5) and 7 weeks (V6) will follow plasma viral load
and reservoirs by IPDA. At 8 weeks (V7), participants will discontinue EFV, and viral
load, HIV reservoirs, plasma and cell markers of immune activation and T cell immunity
will be assayed. Subjects will be offered leukapheresis (optional) to better characterize
HIV reservoir and T cell immune studies. Follow up visits will measure plasma viral loads
and reservoirs at weeks 9 (V8), 12 (V9) and 16 (V10). At all visits, participants will be
reviewed for potential side-effects/toxicities including neuropsychiatric symptoms, and
adherence, and CRF reviewed. Potential side effects will be investigated with blood draws
and treated accordingly (for e.g., rash, provide antihistamines, HC cream, +/-
discontinue EFV if moderate to severe AE by study physician (C. Kovacs). All visits will
take place +/- 3 days of the indicated time point.
Sampling. Blood will be collected using SST/EDTA/ACD tubes for the various assays. At
baseline and 8 weeks, if subjects undergo leukapheresis, only one 5 ml tube of blood for
viral load will be done instead at those time points. Blood volumes are as follows:
screening visit (25 ml), 75 ml at all other visits (if leukapheresis not done). At each
visit, assessment is made of EFV side effects, new or continuing medications, including
cART regimen, and adherence will be assessed. To ensure immune safety, CD4 counts are
done at screening, wk 8 and 16.
Statistical Analysis:
Statistical analyses will be conducted with the support of the Johns Hopkins CFAR
Biostatistics and Epidemiology core (BEM) and the CTN; differences between time points
across the entire cohort will be compared using the Wilcoxon rank-sum or Kruskal-Wallis
test. Within-participant (log-transformed) HIV-1 RNA, DNA, changes between time points
will be estimated with participant-specific linear regression models and compared against
the null hypothesis of no change using the Wilcoxon signed-rank test. The same approach
will be applied to soluble markers of immune activation and inflammation.
EXPECTED RESULTS AND IMPLICATIONS The HIV reservoir fails to decay naturally or using
cART. The proposed research will address translational aspects of HIV persistence with
immediate implications for clinical care. Harnessing CARD8 inflammasome activation with
EFV has the potential to eliminate gag/pol transcriptionally active proviruses as a cause
of LLV, which at present has limited therapeutic options, given that current
antiretroviral agents do not affect virus expression. Reducing LLV could free clinicians
and patients from misguided and potentially harmful attempts at unwarranted regimen
switching, anxiety of drug failure or personal risks of transmissions to sexual partners.
The potential to lower a significant and treatable cause of newly identified adverse
inflammation and chronic immune activation has profound implications for improving
incidence and outcomes of preventable concomitant diseases. If these efforts advance with
discipline, inflammation marker reduction could be used in the HIV treatment clinical
care cascade for the first time. Given that much of the reservoir reflects expansion of
infected clones, such a strategy may be able to eliminate these. Recently cure studies
have experienced participation hesitancy from communities reluctant to agree to cART
interruptions in research. This design could provide safety assurance to volunteers while
satisfying investigator requirements for reliance on verifiable signals, such as IPDA and
vQOA or reduction of LLV. The goal of TACK therapeutics is two fold: 1) to substantially
reduce the reservoir of replication competent viruses; and 2) to reduce ongoing antigenic
stimulation from clones of latently infected cells containing transcriptionally active
but defective viruses interventions that could convert these individuals to undetectable
would be beneficial.
RECRUITMENT A total of 26 participants will be recruited for the study.