New paths, but still a long way to go

After four decades, the search for an HIV “cure” remains elusive. And while antiretroviral therapy (ART) suppresses the virus, it does not eliminate HIV, partly because of the presence of viral reservoirs in CD4 T cells and myeloid cells. That’s why even in a session about research into potential cures at the Conference on Retroviruses and Opportunistic Infections (CROI) in March, presenters used the C-word only when describing research that could be one of many more steps toward a cure.

Four presenters shared promising research that boosts the understanding of the biology of viral reservoirs and possible mechanisms that may, eventually, help clear the virus from people living with HIV (PLWH).

CARD8 and the secondary effect of NNRTIs

The first presenter, Liang Shan, PhD, a professor of immunology at Washington University in St. Louis, summarized research on how a now-common class of anti-HIV drugs—non-nucleoside reverse transcriptase inhibitors (NNRTIs), which inhibit reverse transcriptase (RT) and target early stages of infection—could be used to kill virus particles hiding in reservoirs, even those with intermittent viral expression.

The process uses a mechanism called CARD8 inflammasome, which he said can be a promising approach to clear viral reservoirs for PLWH on ART. “We started with a question: Can our immune system sense immutable components of HIV-1 and trigger cell death?” Shan said. The goal, he explained, was to identify immune sensors that detect the function of HIV key enzymes, because the virus cannot change the functionality of these enzymes to help it replicate.

CARD8 inflammasome activation by HIV protease can act as a “tripwire sensor” that may make sense for reservoir targeting because it is expressed by all lymphocytes, including lymphoid and myeloid.

Shan said the entry of HIV can immediately trigger CARD8 to sense it and “snip” the virus through protease cleavage. The team has demonstrated, both in vivo (in mice) and in vitro (using T cells from PLWH), how the CARD8 inflammasome works by driving CD4 T cell loss as HIV enters the body, he said. Further, in vivo experiments in non-human primates will be needed, Shan concluded.

How NNRTIs could lead HIV to sabotage itself

Presenting the research of a large group of scientists, Tracy Diamond, principal scientist and infectious disease researcher at drug manufacturer Merck, summarized how compounds already in use, NNRTIs, could induce selective cell death in HIV-infected cells through molecules termed TACK, or Targeted Activator of Cell Kill.

“NNRTIs inhibit reverse transcriptase, but some NNRTIs also have the ability to act through this CARD8 mechanism described by Professor Shan,” she said. But this pre-clinical work on TACK molecules did not use currently approved NNRTIs like efavirenz (brand name Sustiva), which is orders of magnitude less potent than what’s needed for cell death. But at larger doses, certain NNRTIs could potentially kill enough HIV-infected cells to deplete the HIV reservoir. Focusing on a previously secondary effect of certain NNRTIs, researchers invented extremely potent reverse transcriptase-targeting TACK molecules. “These differ from standard NNRTIs through monomeric RT-p66, which can start a process of premature intracellular HIV protease activation,” she said. 

NNRTIs have the ability to trigger an HIV booby-trap early inside the cell, where a cellular protein detects HIV protease and recognizes it as a threat, then encourages the virus to sabotage itself. Diamond reiterated that it’s not clinical research, but that a next step could be to demonstrate activity in primary cells and in vivo in viremic mice.

Regarding Merck’s next steps and possible involvement in clinical solutions, she said the company is interested in the mechanism and understanding how that translates to the clinic.

“We’re developing different ways to test how this mechanism could provide benefit in vivo and also considering how to test those hypotheses in clinical studies.”

Using transcriptomics to study rare cells that harbor HIV

Transcriptomics is the study of all RNA molecules in a cell or organism at a given timepoint or condition, unlike genomics, which looks only at DNA. At the same CROI session, Rasmi Thomas, a researcher at the Walter Reed Army Institute of Research, explained how single-cell transcriptomics could be used to identify the host and viral transcripts in individual cells from PLWH.

In single-cell transcriptomics, a cell remains isolated and each cell type has a distinct expression profile. This is important, Thomas explained, because it reveals single-cell heterogeneity and subpopulation expression variability of thousands of individual cells.

“We wanted to use single-cell transcriptomics because… knowledge of viral factors that restrict HIV in cells that become infected is sparse,” she said. “These cells contribute to reservoir seeding and represent a source of rebounding virus after treatment interruption.”

Newer technologies such as single-cell RNA sequencing provide for wider and deeper investigation of rare cells harboring HIV, she said, and such an approach can be used in an unbiased manner to find host factors that restrict HIV in vivo. The ultimate goal, she said, is identifying novel targets for therapeutic intervention.

CD4 mimetics and exposing hidden HIV cells

Introducing his presentation, Andrés Finzi, a professor of microbiology and immunology at the University of Montréal, reminded the audience that in order for virus particles to be killed, they have to be seen. That’s a hurdle because antibodies don’t see infected cells.

“The cells look like they’re sealed in a can,” Finzi said. “We’re looking for a ‘can-opener,’ that once it opens the can will allow HIV-1 cells to be killed efficiently.” A so-called can opener studied by Finzi and his team is CD4 mimetics, which opens the HIV “can,” through the virus envelope glycoprotein gp120/gp41. CD4 mimetics are small organic molecules that bind with the glycoprotein, forcing it to expose more of the virus in an infected cell. Adding antibodies to CD4 mimetics further exposes the cell.

“Once you have this cocktail of antibodies with CD4 mimetic, the effector cells are able to engage and kill,” he said. 

Indoline CD4 mimetics with improved antiviral potency and breadth have been developed. They’re called CJF-III-288, and they’ve displayed favorable pharmacokinetics and toxicology in humanized mice, Finzi said. In these HIV-1 infected mice, a cocktail of CJF-III-288 and CD4i antibodies significantly decreased the size of the viral reservoir, with very little viral rebound. “Something is keeping the virus in check,” he said.

Finzi added that new antibody cocktails have been developed to eliminate infected cells. “It is preliminary research but we’re very excited by this.”

One thing all presenters emphasized is that more investigation in all of these avenues is needed.