Virus World

NIH-funded study confirms white blood cell subtype as HIV reservoir, suggests new target for cure efforts. A subset of white blood cells, known as myeloid cells, can harbor HIV in people who have been virally suppressed for years on antiretroviral therapy, according to findings from a small study supported by the National Institutes of Health. In the study, researchers used a new quantitative method to show that HIV in specific myeloid cells—short-lived monocytes and longer-lived monocyte-derived macrophages—can be reactivated and infect new cells. The findings, published in Nature Microbiology(link is external), suggest that myeloid cells contribute to a long-lived HIV reservoir, making these cells an important but overlooked target in efforts to eradicate HIV. “Our findings challenge the prevailing narrative that monocytes are too short-lived to be important in cure efforts,” said study author Rebecca Veenhuis, Ph.D.(link is external), an assistant professor of molecular and comparative pathobiology and of neurology at Johns Hopkins University School of Medicine, Baltimore. “Yes, the cells are short-lived, but our follow-up data show that HIV can persist in monocytes over several years in people who are virally suppressed. The fact that we can detect HIV in these cells over such a long period suggests something is keeping the myeloid reservoir going.” The study, led by Veenhuis and colleagues at Johns Hopkins University School of Medicine, was supported by the National Institute of Mental Health, the National Institute of Allergy and Infectious Diseases, and the National Institute on Drug Abuse, all part of NIH. Antiretroviral medications are effective in treating HIV because they prevent the virus from infecting new cells and multiplying. However, HIV may still exist in cells that are in a resting, or latent, state, creating an HIV reservoir. CD4 T cells, a type of white blood cell, are the most well-studied HIV reservoir. Identifying HIV reservoirs is critical to cure efforts, as latent HIV can be reactivated if people stop taking antiretroviral medications. Monocytes are immune cells that circulate in the blood for about 3 days before traveling to tissue in various parts of the body, including the brain, where they can mature into macrophages. To date, it has not been clear whether latent HIV in these cells can become active again and infect other cells. “What’s really important in the long run is understanding how monocytes contribute to the tissue macrophage reservoir,” explained Janice Clements, Ph.D.(link is external), senior author on the study and professor of molecular and comparative pathobiology at Johns Hopkins University School of Medicine. “If monocytes can carry virus to the brain, or lung, or another part of the body and infect resident macrophages that are self-renewing and live almost indefinitely, that’s a real problem.”  

In the study, Veenhuis, Clements, and colleagues first measured HIV DNA in myeloid cells in a sample of 30 participants with HIV, all of whom were virally suppressed and had been on antiretroviral therapy for at least 5 years. They found detectable levels of HIV genetic material in monocytes and macrophages, though the levels were much lower than those observed in CD4 T cells. In some participants, the HIV genetic material found in monocytes was intact, which suggests it may be capable of infecting other cells if reactivated. They then used the new quantitative method they developed to directly measure viral spread from HIV found in myeloid cells. The researchers isolated monocytes from blood samples taken from 10 participants and nurtured the monocytes in cultures that contained antiretroviral drugs, to replicate the participants’ baseline physical state. After the monocytes differentiated into macrophages, the researchers introduced an immune activating agent and then added fresh white bloods cells to allow for the virus to spread to new cells. The researchers collected samples from the cell cultures several times over the next 12 days. They included checkpoints throughout the process to ensure that infected CD4 T cells did not interfere with their measurements. The results showed that cultures from five of the 10 participants had detectable HIV genetic material in monocyte-derived macrophages that could be reactivated to infect other cells and produce more virus. The participants who had these reactivatable reservoirs of HIV in monocyte-derived macrophages had higher overall levels of HIV DNA material.   Follow-up data from three participants showed that this reservoir can be long-lived, harboring latent HIV for months to several years. These reservoirs were stable and could be reactivated over time, indicating that monocyte-derived macrophages could contribute to viral rebound if antiretroviral treatment is disrupted. The researchers note that this study is small and larger studies with more diverse participant pools will be essential to accurately estimate the proportion of people who have latent HIV in myeloid cells. Investigating the mechanisms that replenish the monocyte reservoir over time is a critical next step in this research. “These findings underscore the importance of broadening the scope of HIV cure efforts,” said Joshua A. Gordon, M.D., Ph.D., director of the National Institute of Mental Health. “Shifting away from a sole focus on CD4 T cells to thinking about CD 4 T cells and myeloid cells together, in context, will propel the field toward more promising strategies for eradicating HIV.”

Published in:

 https://doi.org/10.1038/s41564-023-01349-3 

An experiment tests whether the gene-editing technology can stop the virus from replicating, which would ultimately wipe out the infection. 

IN JULY, AN HIV-positive man became the first volunteer in a clinical trial aimed at using Crispr gene editing to snip the AIDS-causing virus out of his cells. For an hour, he was hooked up to an IV bag that pumped the experimental treatment directly into his bloodstream. The one-time infusion is designed to carry the gene-editing tools to the man’s infected cells to clear the virus. Later this month, the volunteer will stop taking the antiretroviral drugs he’s been on to keep the virus at undetectable levels. Then, investigators will wait 12 weeks to see if the virus rebounds. If not, they’ll consider the experiment a success. “What we’re trying to do is return the cell to a near-normal state,” says Daniel Dornbusch, CEO of Excision BioTherapeutics, the San Francisco-based biotech company that’s running the trial. HIV attacks immune cells in the body called CD4 cells and hijacks their machinery to make copies of itself. But some HIV-infected cells can go dormant—sometimes for years—and not actively produce new virus copies. These so-called reservoirs are a major barrier to curing HIV. “HIV is a tough foe to fight because it’s able to insert itself into our own DNA, and it’s also able to become silent and reactivate at different points in a person’s life,” says Jonathan Li, a physician at Brigham and Women’s Hospital and HIV researcher at Harvard University who’s not involved with the Crispr trial. Figuring out how to target these reservoirs—and doing it without harming vital CD4 cells—has proven challenging, Li says. While antiretroviral drugs can halt viral replication and clear the virus from the blood, they can’t reach these reservoirs, so people have to take medication every day for the rest of their lives. But Excision BioTherapeutics is hoping that Crispr will remove HIV for good.

Crispr is being used in several other studies to treat a handful of conditions that arise from genetic mutations. In those cases, scientists are using Crispr to edit peoples’ own cells. But for the HIV trial, Excision researchers are turning the gene-editing tool against the virus. The Crispr infusion contains gene-editing molecules that target two regions in the HIV genome important for viral replication. The virus can only reproduce if it’s fully intact, so Crispr disrupts that process by cutting out chunks of the genome. In 2019, researchers at Temple University and the University of Nebraska found that using Crispr to delete those regions eliminated HIV from the genomes of rats and mice. A year later, the Temple group also showed that the approach safely removed viral DNA from macaques with SIV, the monkey version of HIV. That was an important step toward testing the treatment in people, says Kamel Khalili, a professor of microbiology at Temple University who led the work and is a cofounder of Excision Biotherapeutics. “You don’t want to eliminate the viral genome but at the same time cause any disruption in another part of the human genome and then create another set of problems for the patients,” he says. “We had to make sure that we identified a region within HIV that did not overlap with the human genome.”  Dornbusch thinks this strategy will spare patients from serious side effects and “off-target” edits—unintentional cuts elsewhere in the genome that could cause problems such as cancer. The regions targeted by the company’s Crispr therapy are also in a part of the genome that tends to stay the same even when HIV evolves. That’s important because the virus mutates rapidly, and the researchers don’t want a moving target. This isn’t the first time scientists have tried to use gene editing in the hope of curing people with HIV, but other efforts have focused on a protective mutation in a gene called CCR5. In the 1990s, scientists found that people with this naturally occurring mutation didn’t get HIV when exposed to it. The mutation—known as delta 32—thwarts the virus’s ability to get inside immune cells. In 2009, California-based Sangamo Therapeutics used an older editing technology called zinc finger nucleases to add that protective mutation into patients’ T cells—an important part of the immune system. Those trials have had limited success.

In 2017, Chinese scientists combined Crispr with a bone marrow transplant in an attempt to cure a patient with HIV and leukemia. In a typical transplant, donor stem cells are transferred to a recipient to replace their cancerous blood cells. These cells go on to form new, healthy blood cells. To also address the patient’s HIV, researchers edited the donor stem cells with Crispr to disable CCR5. But after the transplant, only a small percentage of the patient’s bone marrow cells ended up with the desired edit. Then in 2018, Chinese scientist He Jiankui used Crispr to edit the CCR5 mutation into the genomes of twin baby girls to make them resistant to HIV. Fraught with ethical violations, the experiment was widely condemned by scientists. He’s research was suspended by the Chinese government, and he served a three-year prison sentence. While the twins were born healthy, only some of their cells were successfully edited, meaning the girls might in fact not be immune to HIV. As of 2022, two people have now been cured of HIV after receiving bone marrow transplants from donors with the CCR5. Known as the Berlin patient and the London patient, both had cancer and received transplants to treat their disease. But these transplants aren’t a viable option for most people—they’re highly risky, and donors with the delta 32 mutation are scarce. But a third person was declared cured of HIV earlier this year after she received a new type of transplant involving umbilical cord blood.

The Excision trial will eventually enroll nine participants and test three dosage amounts to determine which is most effective. Investigators will measure each person’s viral load and CD4 count before receiving the therapy and after they stop taking antiretroviral drugs. The ultimate goal is to get viral loads down to an undetectable level—that is, less than 200 copies of HIV per milliliter of blood. At this level, HIV can’t be passed on through sex. The challenge for Excision will be getting Crispr to enough cells to bring HIV down to undetectable levels. The company is using an engineered virus to shuttle the gene-editing components to patients’ HIV-infected CD4 cells. But so far, there’s little human data on how well Crispr works when it’s delivered directly to the body. “It’s possible that you get the virus to such low levels that if a person’s immune system were intact, they might be able to keep the virus at bay such that they don’t have to take antiretroviral therapy anymore,” says Rowena Johnston, vice president and director of research for amfAR, the Foundation for AIDS Research.

And even though these drugs are very effective, Johnston says, many people would rather be completely free of the virus. A single Crispr infusion—if it works—would eliminate the need for daily pills. “People with HIV still live with a lot of stigma and internalized shame,” she says. “I think a cure is something that addresses that much better than lifelong therapy, regardless of how easy that therapy becomes.”

An antisense drug developed by GlaxoSmithKline has been shown to suppress the hepatitis B virus (HBV) in a  phase 2 trial, raising hopes of a “functional cure” for the widespread disease. HBV affects an estimated 260 million people worldwide, according to GSK. While drugs to treat it are available – such as Gilead’s polymerase inhibitor Viread (tenofovir) – there is still no curative treatment, and the company hopes that GSK3228836 could fill that void. Most people are able to shake off HBV infection in time, but in a majority of children and a minority of adults, HBV infection can become chronic and lead to cirrhosis as well as liver cancer, whilst also allowing transmission of the virus to others. Chronic HBV kills around 900,000 people every year. In the phase 2a trial, GSK3228836 was able to reduce two biomarkers of chronic HBV infection – hepatitis B surface antigen (HBsAg) and hepatitis B virus DNA – compared to placebo after four weeks treatment. The trial involved four patients previously treated with antivirals and 12 who were new to drug therapy for HBV, and across both groups there were six patients who had a 3-log (99.9%) or greater reduction in HBsAg levels after 29 days. 

In four of the big responders, levels of the biomarker fell below the limit of detection, and in one case that persisted for six months. Meanwhile, five of 12 previously-untreated patients had 2-log (90%) or a greater reduction in HBV DNA. The results are still preliminary but according to GSK show clear signs of a therapeutic effect that will be tested in a phase 2b programme, due to start before the end of the year. The company’s senior vice president for development, Christopher Corsico, said that the results are “a potential step forward toward the goal of assessing sustained functional cure for people with chronic hepatitis B.” The company’s senior vice president for development, Christopher Corsico, said that the results are “a potential step forward toward the goal of assessing sustained functional cure for people with chronic hepatitis B.” Experts talk about a functional cure in HBV – defined as the complete loss of HBsAg – because it is impossible to tell biologically if the virus has been eliminated from the body. That is because viral DNA fragments can lurk in cells, hiding from current drug therapy and evading the immune system. GSK3228836 is designed to target viral DNA directly, which could give it a chance of targeting all traces of the virus in the body, and prevent it from becoming a chronic infection. GSK licensed the drug from longstanding partner Ionis as part of a two-drug deal last year, paying $25 million upfront with milestone payments taking the value of the deal up to $262 million.

If either of the antisense drugs covered by that license make it through to market, that would look like a very good deal, particularly given the sheer scale of HBV infection worldwide. The first drugs that were able to effect a cure for chronic hepatitis C virus (HCV), such as Gilead’s Harvoni (sofosbuvir/ledipasvir), quickly amassed blockbuster revenues, although that tailed off quickly as the pool of available patents declined. Analysts at William Blair have suggested that the market for an HBV cure could be around the same size as HCV, which peaked at around $10 billion a year. There’s competition coming through the industry pipeline though. Last year, Roche paid a whopping $200 million upfront to license an RNA interference drug for HBV from Dicerna called DCR-HBVs (now RG6346), with another $1.47 billion in potential milestones. The drug is in phase 1/2 development. Meanwhile, Johnson & Johnson paid $175 million upfront for Arrowhead Pharma’s RNAi candidate ARO-HBV (now JNJ-3989) which is also in a phase 2 trial.

Researchers from The University of Texas Medical Branch at Galveston have discovered a new potential medication that works with an HIV-infected person's  own body to further suppress the ever present but silent virus that available HIV treatments are unable to combat. The HIV virus gets integrated into the infected person’s genetic coding and establishes a constant dormant infection, creating a big treatment challenge. Because of this, current ART medications fail to cure the virus and when someone stops the drug, the virus almost always begins to multiply and wreak havoc. Drug resistance is also a public health issue with the ART medications. Being able to induce a sustained HIV remission free of ART is an important goal for HIV treatment.

“We are the first to show that human BRD4 protein and its associated machinery can be harnessed to suppress dormant HIV,” said senior author Haitao Hu, UTMB assistant professor in the department of microbiology and immunology. “Our findings are exciting because they not only improve our understanding of the biology of HIV epigenetic regulation, they also present a promising approach for the development of probes and/or therapeutic agents for HIV silencing, hopefully leading to cure of the virus eventually.”

In the laboratory study, the researchers found that the protein BRD4 plays an important role in regulating the production of new copies of the HIV gene. The team successfully designed, synthesized and evaluated a series of small molecules to selectively program BRD4 to suppress HIV and identified a lead compound called ZL0580. They tested the lead molecule in HIV infection models and found that it significantly delayed dormant HIV reactivation after ART cessation in blood cells of ART-treated, HIV infected people. “We will continue to optimize the chemical structure and effectiveness of this class of molecules and conduct safety testing in cellular and animal studies,” said co-senior author Jia Zhou, UTMB professor in the department of pharmacology and toxicology. 

The findings were published on August 1, 2019 in Journal of Clinical Investigation:

 https://doi.org/10.1172/JCI120633

Bepirovirsen, an antisense oligonucleotide therapy aided in preventing chronic hepatitis B in patients in a Phase II trial.  GSK has announced study results from its Phase IIb trial (NCT04449029) of bepirovirsen for chronic hepatitis B (CHB), indicating that the antisense oligonucleotide therapy could help treat CHB independently or in combination with nucleoside/nucleotide analogues (NAs). Bepirovirsen demonstrated clearance of hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA long term, in patients on concurrent NA and those not-on-NA therapy. The current treatment for the condition includes NAs, often taken for life due to their ability to suppress the virus. Chris Corsico, Senior Vice President of Development at GSK: “Today’s results from the study are a promising step forward… We look forward to confirming these findings for bepirovirsen in our Phase III study starting next year, [and] exploring potential sequential therapy options with the aim of helping more people living with chronic hepatitis B achieve functional cure.” The trial investigated the efficacy and safety of 12 or 24 weeks of bepirovirsen treatment on stable NA treatment or not-on-NA treatment at study start. A loading dose of Bepirovirsen was given at day 4 and 11. A dose of 300mg per week was administered for 24 weeks, resulting in 9 percent of patients on NA and 10 percent of patients not-on-NA, achieving HBsAg levels below the Lower Limit of Detection (LLOD) and HBV DNA levels below the Lower Limit of Quantification (LLOQ). Patients with low baseline hepatitis B surface antigen levels responded best to treatment. The findings showed 16 percent of patients on NA and 25 percent not-on-NA achieved appropriate LLOD and LLOQ levels.

Further data on the chronic hepatitis B clinical trial

The research investigating Bepirovirsen was made up of two parallel cohorts. One for patients receiving NA treatment and the other for patients not-on-NA. Volunteers were randomised into 1 of 4 treatment arms within each cohort:

• Arm 1: 300mg with a loading dose for 24 weeks
• Arm 2: 300mg with a loading dose for 12 weeks then 150mg for 12 weeks
• Arm 3: 300mg with a loading dose for 12 weeks then placebo for 12 weeks
• Arm 4: Placebo with a loading dose for 12 weeks then 300mg Bepirovirsen without a loading dose for 12 weeks.

There were no safety signals to prevent further development. Adverse events (AEs) leading to treatment discontinuation occurred in 17 participants, with 0–4 percent frequency in participants on-NA, and 0–7 percent frequency in participants not-on-NA. Serious AEs (SAEs) were reported in six (3 percent) of participants on NA and 11 (5 percent) of participants not-on-NA. One SAE in the on NA and three in the not-on-NA population were considered related to treatment. To obtain data on the drug’s long-term durability, trial participants will be investigated for an additional 33 months. Patients who continue to have no serologic evidence of HBsAg or HBV DNA will have the option of stopping NA therapy, offering the possibility of a functional cure. Results of the Phase IIb trial have been published in the New England Journal of Medicine and will be presented at the American Association for the Study of Liver Diseases’ (AASLD) Liver Meeting 2022.

Results of the Phase 2 trial published in The Lancet (Nov. 8, 2022):

https://doi.org/10.1056/NEJMoa2210027 

Using gene-editing to remove a retrovirus is a step towards a cure for HIV infection in humans. Scientists have used Crispr gene-editing to remove an HIV-like virus from monkey DNA, a major step towards a cure for HIV infection in humans. In a study led by neurovirologist Kamel Khalili of Temple University in Philadelphia, researchers constructed a modified adenovirus containing a Crispr-Cas9 gene-editing system. That 'construct' (called 'AAV9-CRISPR-Cas9') was then injected into rhesus macaque monkeys to deliver the Crispr system into cells. The monkey cells were infected with SIV (Simian Immunodeficiency Virus), a close relative of HIV (Human Immunodeficiency Virus). Both are retroviruses — viral parasites that cut-and-paste their genetic material into a host's DNA. SIV infects macaques and other non-human primates in the same way that HIV infects people, making it a good model for studying retroviral infection — and testing how to remove those viruses from the human genome.

The gene-editing construct was designed to target specific sites where the retrovirus was integrated into the macaque genome. It was able to reach tissues where viruses like SIV and HIV can hide for years without being detected, known as reservoirs, such as bone marrow, lymph nodes, T cells of the immune system and the brain. According to the study, the construct was precise and has a low risk of cutting the wrong places in DNA ('off-target' sites). The research has obvious implications for preventing or treating AIDS (Acquired Immunodeficiency Syndrome) in humans by curing a patient of HIV infection.

Original study published in Nature Comm. (November 27, 2020):

https://doi.org/10.1038/s41467-020-19821-7

A trial of four experimental Ebola treatments carried out in the Democratic Republic of the Congo (DRC) has been stopped early after two of them showed strong signs of being able to save patients’ lives. The preliminary results were reported this morning by Anthony Fauci, director of the U.S. National Institute of Allergy and Infectious Diseases (NIAID) in Bethesda, Maryland, one of the partners in the study. The two treatments will now be made widely available and could help end the yearlong outbreak in the DRC, which has already killed more than 1800 people, scientists say.

The PALM trial (short for the Swahili expression pamoja tulinde maisha, which means “together save lives”) evaluated three Ebola antibody preparations and one antiviral drug in a randomized controlled trial conducted in the midst of the devastating outbreak, which has hit two provinces in the eastern DRC mired in violence. “Today, we have started a new chapter. From now on, we will no longer say that Ebola is not curable,” Jean-Jacques Muyembe-Tamfum, head of the DRC’s National Institute for Biomedical Research in Kinshasa, a partner in the trial, said at the press conference. “This advance will, in the future, help save thousands of lives.” (Muyembe, who was part of the team that discovered Ebola 43 years ago, took over command of the outbreak response in the DRC on 22 July.)

Still, the data on ZMapp were deemed good enough to use the drug as a control in future trials. In the PALM trial, three other trials were compared to ZMapp:

• The monoclonal antibody mAb114, which has its roots in the 1995 Ebola outbreak in Kikwit, DRC. During that episode, Muyembe attempted to treat patients with a mixture of antibodies from Ebola survivors. Years later, researchers at NIAID isolated antibodies from those survivors; mAb114, which is now being developed with Ridgeback Biotherapeutics in Miami, Florida, was the most promising one.
• REGN-EB3, a cocktail of three monoclonal antibodies developed by Regeneron Pharmaceuticals in Eastview, New York. The antibodies were generated by inoculating mice with “humanized” immune systems with the Ebola virus.
• The antiviral drug remdesivir, produced by Gilead Sciences in Foster City, California.

The trial started in November 2018 in four Ebola treatment units in the communities of Beni, Butembo, Katwa, and Mangina, with the aim of enrolling 725 patients. On 9 August, an independent data and safety monitoring board reviewed data for 499 patients and found that REGN-EB3 was much better than ZMapp. Overall, 49% of patients receiving ZMapp—and 53% of those who received remdesivir—died, compared with only 29% of those on REGN-EB3. That difference was big enough to meet the predetermined criterion for stopping the trial early. In the group that received mAb114, mortality was 34%, a rate deemed close enough to that of the Regeneron cocktail that its use should continue.  In the 41% of trial participants who sought treatment early after infection and had lower levels of Ebola virus in their blood, the two new treatments had astonishing success: Mortality plummeted to 6% in the Regeneron antibody group and to 11% with mAb114. (With ZMapp and remdesivir, mortality rates in people with low viral load were 24% and 33%, respectively.) There is far less hope for patients with a high viral load, however: Even with the best treatment, REGN-EB3, their death rate was 60%.