Experimental treatment able to control HIV for six months after stopping antiretrovirals
Electron microscopy of a CD4 T lymphocyte, responsible for activating other immune cells infected by HIV (green spheres)
Niaid
In São Paulo, two men with HIV—ages 24 and 49—managed to spontaneously control the HIV virus while suspending their antiretroviral drugs. They were part of a group of five HIV-positive patients who received an experimental treatment designed by the research team of virologist Ricardo Sobhie Diaz, from the Federal University of São Paulo (UNIFESP). The ingenious and innovative therapy had previously been evaluated in an initial clinical trial, completed in mid-2020, conducted with funding from FAPESP and the National Council for Scientific and Technological Development (CNPq). The preliminary results were published in the journal Aids Research and Therapy in January of this year. “When the first signs appeared that the virus might be able to reproduce itself again in these two patients—possibly because some viral reservoirs remained intact—we resumed the preventive use of the antiretroviral cocktail,” Diaz says. “Some researchers thought we could have waited longer to see if the virus would actually reproduce again. We decided not to risk it,” the virologist explains.
Though still far from becoming available for clinical use, this therapeutic strategy has two objectives: to reduce the amount of HIV in the body to the lowest possible level and, with the help of a vaccine, to teach the cells in the body’s defense system to find the remaining viruses—usually lurking in hidden places—and destroy them. Thus, they hope to arrive at what some experts have been calling a “functional cure” for the infection, which would mean the ability to control HIV replication without the need for antiretroviral drugs. This was unimaginable during the first decades of the AIDS pandemic, which has killed 36.3 million people worldwide since the early 1980s. To date there are only three recognized cases of complete cures. These were obtained through much more aggressive treatments in patients with hematological cancer that was treated with chemotherapy followed by bone marrow transplants. If the effects the UNIFESP group obtained are confirmed in future trials, their approach could be more advantageous than the previous method because, in principle, it could be used to treat anyone with HIV, not just those with cancer.
The therapy proposed by the São Paulo team basically involves two stages. The first step uses a cocktail reinforced with two antiretroviral drugs—totaling five or six drugs in this category, instead of the usual three or four—and aims to control virus replication more rigorously. It’s the simplest part of the process, and generally one already mastered by doctors caring for those infected with HIV.
Since the first cases of AIDS were identified in the United States, in 1981, just over 50 compounds have been approved to fight HIV. They interfere with different stages of virus replication, from entering into cells to assembling new copies, and are often used in combinations of two, three, and even four drugs (see box). Correct, continuous use of these combinations, known as antiretroviral cocktails, currently allows about 90% of people under treatment to reduce the blood concentration of HIV in the body to such low levels—usually less than 50 copies per milliliter (mL)—that they become undetectable in some laboratory tests. Individuals who achieve this level can remain healthy for decades without transmitting HIV to others or suffering the immune system damage characteristic of acquired immunodeficiency syndrome (AIDS). That is, of course, as long as the therapy is not interrupted.
NiaidImage of lymph nodes from a person with HIV…Niaid
The largest hurdle comes during the second phase of treatment. Many experts suspect that the reason an efficient method for eradicating the virus that causes AIDS hasn’t been found yet is that a small proportion of the virus load (something like one copy in every 10,000 or 100,000) remains hidden, in a kind of dormant state, inside the immune cells themselves. The preferred targets are usually CD4-type T lymphocytes, considered the “conductors” of the body’s defense system because they coordinate the action of other immune cells. These viral reservoirs arise after the body has become infected, when HIV manages to insert its genetic material into stretches of lymphocyte DNA that aren’t normally read by the cellular machinery. Dubbed the “genomic desert,” these regions of DNA remain silent most of the time. Since the virus doesn’t multiply in this state, cells don’t show signs of being infected and hence go unnoticed by the immune system. Specific conditions, however, can reactivate these regions of the genome and trigger the production of HIV, which begins to spread once again.
The second stage of combating the virus consists precisely in trying to eradicate these reservoirs. “This strategy is quite significant,” said Biochemist Carl Dieffenbach, director of the AIDS Division at the US National Institutes of Health (NIH), in an interview with Pesquisa FAPESP. “The concept has been tried before, with limited success. The methods are better now, and this group is conducting a very important test.”
Working in partnership with researchers from the University of São Paulo (USP), in Brazil, and the Italian National Institute of Health (ISS), in Italy, Diaz’s team planned three other actions against infected cells. “We knew that single interventions didn’t produce good results, so we decided to add those that showed the greatest potential to reduce viral sanctuaries,” says Diaz.
Niaid…and without the virus infection disrupts the distribution of cells in the immune system’s structureNiaid
The first was to include doses of nicotinamide, vitamin B3, in the treatment. The volunteers, men aged 18 to 60 treated at the UNIFESP outpatient clinic, were invited to join the study after maintaining a stable viral load of less than 50 virus copies per mL for six months. They then began receiving doses of the vitamin for 48 weeks, in parallel with the reinforced antiretroviral cocktail. This is because in order to eliminate the latent viruses, they first need to be “woken up” such that they become apparent to the immune system.
In previous tests, Diaz’s team had found that this compound is a potent activator of hibernating HIV. Inside the cell nuclei, vitamin B3 blocks the action of the enzyme histone deacetylase. These enzymes help sections of the DNA molecule to coil around proteins, so they become silent, and this “packaging” prevents the cellular machinery from reading these sections. With vitamin B3 taking the deacetylase out of action, the genetic material remains extended, ready to be transcribed and generate proteins (including viral proteins). As a result, the lymphocytes that previously functioned as hiding places begin to produce copies of the virus and attract the attention of other immune cells. Immunologists have dubbed this strategy “shock and kill.” “This clinical trial was one of the first attempts to use the shock and kill approach to treat people,” Italian immunologist Andrea Savarino of the ISS told Pesquisa FAPESP.
To eliminate the virus’s reservoirs, the researchers added doses of auranofin, a gold salt that has been used for years to combat the swelling and joint pain caused by rheumatoid arthritis. In Rome, Savarino and her team had observed that auranofin effectively eliminated certain virus reservoirs, in particular memory CD4 T lymphocytes. Administered over 24 weeks, the gold salt induced the death of these cells, which could otherwise live for years, preserving sanctuaries of dormant HIV in the body. “Auranofin eliminates memory lymphocytes, but spares naïve lymphocytes, which have never had contact with the virus,” reports Italian-Albanian virologist Iart Luca Shytaj, a visiting researcher at UNIFESP (with FAPESP support) and a participant in the study.
In the attempt to eradicate HIV reservoirs, the final blow was also the most sophisticated. It entailed developing an individualized therapeutic vaccine, made from the patient’s own healthy immune cells (see infographic). This type of immunizing agent doesn’t prevent infection by the virus, but it does help fight it. The researchers selected immature components (monocytes) in the immune system and, in the laboratory, induced them to transform into dendritic cells. These cells perform a special function in the body: they are the immune system’s “scout cells.” Found in greater quantity in the skin, lungs and intestines, tissues that have more direct contact with the external environment, dendritic cells scour their environment and, when they find structures that don’t belong to the organism, for example, a virus or a bacterium, they engulf them, digest them, and then present their antigen fragments to other immune cells. In this manner they teach lymphocytes to recognize invader organisms and fight back more quickly.
Tiago Cardoso
In the clinical trial carried out by the UNIFESP team, the training of dendritic cells was also customized. The researchers exposed each participant’s cells to fragments (peptides) of an HIV protein extracted from their own blood. In this case, they chose the gag protein, the only one that appears on the surface of CD4 T lymphocytes harboring dormant HIV. The objective was to prepare the vaccine cells to signal the other components of the immune system what to search out and destroy.
Using a peptide analysis program available on the internet, Savarino and immunologist Edecio Cunha-Neto, from USP, selected those peptides most likely to be recognized by each participant’s defense system. Even when two individuals carry the same virus, the gag fragments that are exposed on their immune cells may be different. The peptides were then synthesized—the total number ranged from two to six in each case—at the laboratory of biochemist Maria Aparecida Juliano, of UNIFESP, and placed in contact with the dendritic cells before they were reinfused into the participants. Each volunteer received three doses of the cell vaccine, administered 15 days apart, after suspending antiretrovirals, vitamin B3, and auranofin. “Since the cells had been taken from the patients themselves, the vaccine had practically no side effects,” says Shytaj, who also collaborated in the development of Custommune, an enhanced version of the peptide analysis program.
Treatment performance was monitored through blood tests and rectal biopsies. Two of the test subjects were able to temporarily control the virus: one identified by the acronym P27, a 24-year-old man who had been infected three years earlier; and another coded as P29, a 49-year-old individual who had been living with the virus for eight years. DNA analyses of rectal lymphocytes, an important reservoir of HIV, failed to detect viral genetic material for at least six months.
The other three members of the group also showed a reduction in the number of virus reservoirs, but the drop wasn’t enough to eliminate these sanctuaries. “Perhaps the duration of treatment or the dosage wasn’t adequate, so we intend to make changes in an upcoming clinical trial,” explains Diaz.
“The UNIFESP team’s study is one of the most successful yet using this strategy. Theoretically, the group achieved a temporary, six-month functional cure. It’s a very good result,” says virologist Amilcar Tanuri from the Federal University of Rio de Janeiro (UFRJ), who studies the genetic diversity of HIV and the virus’ resistance to drugs, who did not participate in the clinical trial. “The strategy works. Now they need to adjust it to try and increase the remission time. It will be a battle to obtain the maximum effectiveness with the least toxicity possible,” adds the researcher, who in 2018 led a study showing that compounds extracted from the janaúba bush (Euphorbia umbellata) were able to reactivate latent HIV.
The therapeutic vaccine activated some level of immune response in all the participants: the five who received the full treatment (even in individuals who did not completely eliminate the virus reservoirs) and the other five who were given only the boosted cocktail and the vaccine (without the use of vitamin B3 and auranofin). The T lymphocytes in these individuals began to produce chemical compounds released to fight the virus, a sign that they had been activated by the dendritic cells, the researchers report in the Aids Research and Therapy article. The clinical trial also had four other arms, each with five test subjects, who were submitted to different combinations of antiretroviral drugs plus vitamin or gold salt. “The best results were obtained in the group in which all therapies were added together,” says Diaz.
“It’s a fascinating idea to train dendritic cells to target the HIV reservoir,” German hematologist Gero Hütter observes. “It’s a well-known strategy now, especially in the treatment of cancer. Dendritic cells are ‘miraculous,’ they do things we don’t quite understand. Training these cells described in the paper appears to be a promising approach that’s less risky than others.”
A therapeutic vaccine trained immune cells to destroy HIV reservoirs, complementing the action of other drugs
Currently the medical director of an immune and cellular therapy company, Cellex Cell, Hütter became internationally known in the late 2000s for coordinating the procedure that led to the first known case of a cure for HIV. In February 2007, he and his team at the Charité Hospital in Berlin performed a bone marrow transplant on Timothy Ray Brown (1966–2020), an American living in Germany who, in addition to being HIV positive, had leukemia, a cancer of the bone marrow. After having his immune system eradicated by chemotherapy, Brown received bone marrow from a donor with one special feature: their cells lacked the functional version of a protein, CCR5, used by HIV to invade lymphocytes. Known initially only as “the Berlin patient,” Brown remained virus-free until his death in 2020 from leukemia. One year earlier, a second case of a cure for HIV was reported: that of Adam Castillejo, from Venezuela, a patient treated in London who had also received chemotherapy followed by a bone marrow transplant with the altered version of CCR5 to treat his lymphoma. This year, a third case was reported, of an American woman with leukemia who received an umbilical cord stem cell transplant.
Brown’s case (later reinforced by the other similar cases), together with findings of rare individuals who naturally control the virus, and the recognition that many HIV-positive people don’t have access to or cannot adhere to long-term use of antiretrovirals, have over the last decade led the international scientific community to prioritize the search for interventions capable of achieving lasting remission of the virus or even curing the infection. In a review article published in December 2021 in Nature Medicine, a group led by American immunologist Steven Deeks at the University of California at San Francisco enumerated a number of techniques in development. They range from cellular vaccines to the use of synthetic antibodies, from therapies with cells manipulated in the laboratory to gene editing. Several look promising, but none have yet shown big results. “All the new strategies have challenges,” the NIH’s Dieffenbach told Pesquisa FAPESP, adding, “I think a functional cure will be possible at some point.”
The clinical trial carried out at UNIFESP was classified as a phase I/II study and serves as a proof of concept, an indication that the strategy can work. Although encouraging, the results should be viewed with caution. Only studies with a much larger number of people will confirm the approach’s safety and verify its effectiveness. Diaz and his collaborators are already preparing the next step. They plan to start testing an improved version of the treatment on 60 subjects within a few months.
From death sentence to chronic illness In June 1981, medical reports emerged in the United States of an unusual form of pneumonia and a rare and aggressive skin cancer in gay men in San Francisco and New York. These were the first signs of a disease that would later be known as acquired immunodeficiency syndrome, AIDS, caused by a virus that was only identified in 1983 by French virologist Françoise Barré-Sinoussi and his then mentor, French virologist Luc Montagnier (1932–2022), both from the Pasteur Institute in Paris. Their findings were independently confirmed the following year by the team of Robert Gallo, then a researcher at the National Institute of Cancer, in the United States. For their discovery, Barré-Sinoussi and Montagnier shared the 2008 Nobel Prize in Medicine with researchers working on the HPV virus.
During the early years of this pandemic, a diagnosis of HIV infection was practically a death sentence: half of infected adults and children died within 15 months after the first signs of disease. Today, a person with HIV can live as long as individuals who have never had contact with the virus, living into their 70’s. The main reason for this change was the development of antiretrovirals, which transformed a highly lethal infection into a chronic and controllable disease.
The first medication approved by the US Food and Drug Administration to treat the infection was azidothymidine (AZT). Also known as zidovudine, this compound inhibits the functioning of the HIV reverse transcriptase enzyme, which is necessary for viral replication.
Tiago Cardoso
HIV is a retrovirus. Its genes are encoded in an RNA molecule and are only read by the cellular machinery after being converted into DNA by reverse transcriptase. However, the drug by itself proved to be insufficient to defeat the virus.
From the 1990s onwards, new compounds were incorporated almost annually into the anti-HIV arsenal. Today there are more than 50 drugs used to combat the virus. They belong to nine families of antiretrovirals and interfere with four stages of HIV replication: entry into the cell, converting RNA into DNA, inserting the viral DNA into cellular DNA, and assembling new copies of the virus. The treatment usually consists in combining two or three of these compounds (cocktails).
During the four decades of pandemic, almost 80 million people have been infected by HIV around the world, and at least 36.3 million have died as a result. Joint Program Data on HIV and AIDS from the United Nations (UNAIDS) indicate that in mid-2021, 37.7 million people were living with the virus, and 28.2 million of these were receiving antiretroviral therapy. In 2016, AIDS caused one million deaths (see graph). That year, antiretrovirals prevented another 1.2 million from dying.
Projects 1. Use of a dendritic cell vaccine in association with viral reservoir elimination strategies to provide a sterilizing cure for chronic HIV-1 infection in patients receiving antiretroviral treatment (nº 13/11323-5); Grant Mechanism Regular Research Grant; Principal Investigator Ricardo Sobhie Diaz (UNIFESP); Investment R$349,369.21. 2. Production and characterization aDC1 polarizing dendritic cells for an anti-HIV immunotherapy clinical protocol (nº 18/12460-0); Grant Mechanism Postdoctoral Fellowship; Supervisor Alberto José da Silva Duarte (USP); Grant Beneficiary Laís Teodoro da Silva; Investment R$413,127.14. 3. Use of a dendritic cell vaccine in association with viral reservoir elimination strategies to provide a sterilizing cure for chronic HIV-1 infection in patients receiving antiretroviral treatment (nº 19/17461-7); Grant Mechanism Visiting researcher; Principal Investigator Ricardo Sobhie Diaz (UNIFESP); Grant Beneficiary Iart Luca Shytaj; Investment R$146,596.00.
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