Can HIV be Cured? — Jones Laboratory

By Brad Jones

The human immunodeficiency virus has inspired decades of intensive research into virology, immunology, and vaccines. © iStockphoto

**Figure 2. Prevalence, new cases, and deaths from HIV/AIDS** Deaths reached a peak in 2005 and 2006 and continue to fall, as do new infections, while the number of people living with HIV continues to rise. (Note: To fit all three metrics on the same graph, the number of people living with HIV has been divided by 10). © IHME, Global Burden of Disease

In early 2020, the COVID-19 pandemic upended the lives of people worldwide as the novel coronavirus SARS-CoV-2 spread around the globe. For many of us, this was not our first time witnessing a pandemic caused by a novel virus; the human immunodeficiency virus (HIV) (Fig. 1) pandemic, and the associated disease Acquired Immunodeficiency Syndrome (AIDS), have affected hundreds of millions of lives around the globe (Fig. 2). These viruses differ in their modes of transmission, which have shaped the two pandemics very differently. SARS-CoV-2 is readily transmitted through aerosols or droplets in the air or on surfaces, necessitating measures such as lockdowns, social distancing, and mask wearing to slow transmission. HIV, in contrast, is spread predominantly through sexual intercourse or the sharing of contaminated needles, or—in the early days of the pandemic—through transfusions with contaminated blood products. This made for a much slower initial spread of HIV, with approximately 60 years between the estimated time of first transmission to humans from chimpanzees in the 1920s (in the Congo basin) and the first awareness of the disease that came to be known as AIDS in 1981. Consistent with its mode of transmission, the early impacts of HIV were concentrated in certain communities and regions, with gay men bearing the intial brunt. In fact, the cluster of illnesses that we currently know as AIDS—which includes certain rare cancers, pneumonia, and infection by opportunistic pathogens that do not otherwise cause illness in healthy adults—was briefly and inaccurately termed gay-related immunodeficieny, or GRID. Although certain communities, often marginalized, continue to be disproportionately affected, the cumulative global impact of HIV/AIDS has been staggering, with an estimated 36.3 million total deaths and 1.7 million new cases in 2019. Of the estimated 49.5 million people infected with HIV today, twothirds reside in sub-Saharan Africa, where the impact continues to be devastating. By way of grim contrast, at the time of this writing, more than 6 million people have lost their lives to COVID-19.

The Search for a Vaccine

In the early 1990s, hopes were high that a vaccine for HIV was on the horizon. In contrast to the remarkable success of SARS-CoV-2 vaccines, however, these hopes for HIV have been dashed time and time again when clinical trials showed vaccines failing to provide appreciable protection from infection. Several reasons probably account for this difference in vaccine success, foremost among them that HIV and SARS-CoV-2 are fundamentally different viruses. Within the virus particles themselves, both HIV and SARS-CoV-2 have genomes made of RNA. However, whereas the SARSCoV- 2 genome only ever exists in this RNA form, HIV is a retrovirus—meaning that it reverse transcribes its RNA genome into DNA and then inserts that DNA into our own DNA genomes. Once this insertion has occurred, the viral genetic material is an inseparable part of the host cell that it has infected, and it is eliminated only when the host cell dies (Fig. 3).

**Figure 3. HIV replication cycle** This illustration, which we also saw in the Week 2 essay “Viral Life Cycles,” shows the steps by which a HIV viral particle uses a host cell’s machinery to replicate itself. © NIAID

These host cells are primarily human CD4+ T-cells, which have a central role in regulating our immune systems. Without them, our immune systems can’t do their jobs properly. The infection and subsequent death of CD4+ T-cells underlies the immunodeficiency that is the hallmark of AIDS (Fig. 4). Perhaps the greatest challenge to developing an HIV vaccine is the fact that the reverse transcription process is highly prone to errors. Simply put, HIV is sloppy and makes mistakes (mutations) every time it copies its own genome into genome (RNA into DNA). Most of these mistakes are harmful to the virus. However, some mutations change the virus in a way that prevents it from being recognized by an immune response, including one elicited by vaccination. Similarly, when the delta variant of SARS-CoV-2 emerged, we saw that its mutations reduced the vaccines’ ability to protect against the variant. However, SARS-CoV-2 has a very slow mutation rate, which allows such variants of concern to emerge on a global scale. HIV, in contrast, mutates so fast that many, many variants emerge even within one infected individual! Some of these HIV variants almost invariably escape from most immune responses.

**Figure 4. HIV Progression.** During the early, acute phase of HIV infection, the virus multiplies rapidly. Multiplication slows as the infection progresses to chronic infection, but as chronic infection progresses, CD4 cells decrease, and the immune system is increasingly damaged, HIV rises again until the disease progresses to AIDS. © NIH

Hope does exist for an HIV vaccine. One approach is to try to coax the immune system to produce broadly neutralizing antibodies, special antibodies from which HIV has a very hard time escaping. Scientists have identified these antibodies, but causing the immune system to produce them in response to a vaccine has proven challenging. Still, many researchers consider this a solvable problem, and scientists around the world continue to work on it. Although HIV vaccine efficacy trials have had disappointing results, the study of HIV has nevertheless been a source of scientific triumph. It has resulted in the development of drugs called antiretrovirals, which have saved millions of lives (Fig. 5). These drugs act to inhibit multiple stages of the HIV replication cycle, for example by blocking the reverse transcription of HIV RNA into DNA.

**Figure 5. Antiretroviral agents for HIV** (A) **Entry inhibitors** work outside the cell. They prevent HIV from entering the CD4 cell by blocking binding or fusion of HIV with the CD4 cell membrane. If HIV cannot enter the CD4 cell, it is unable to replicate. (B) **Non-nucleoside reverse transcriptase inhibitors** bind to reverse transcriptase and inhibit the enzyme, stopping HIV replication by preventing formation of HIV DNA. These drugs act in a completely different way from nucleoside/nucleotide analogues. (C) **Nucleoside/nucleotide analogues** act as false substrate for reverse transcriptase, causing chain termination. The resulting DNA is incomplete and prevents HIV replication. (D) **Integrase inhibitors** block the integration of HIV and cell DNA. This process prevents HIV replication. (E) **Protease inhibitors** work at the last stage of the HIV replication cycle. They prevent HIV from being successfully assembled and released from the infected CD4 cell. © NIAID / QPP (adapted by AMNH)

The Search for a Cure

The ability of HIV to mutate to escape from antibodies and other immune responses parallels its ability to mutate to become resistant to any single drug. However, researchers found in the late nineties that a cocktail of three different drugs that target different parts of the virus, administered simultaneously, is enough to block any attempts by the virus to escape by mutation. Such combination antiretroviral therapy is now the standard of care for people with HIV. Those who have access to these drugs can reduce their viral burden to levels that cannot be detected by clinical tests, and they can generally expect to live long and healthy lives. Strikingly, intensive research in recent years has demonstrated that 6 these individuals with undetectable viral loads cannot pass the virus on to their sexual partners, a scenario captured by the message “Undetectable = Untransmittable” or “U = U.” These same drugs can also be taken by people who are not infected by HIV. They serve to protect these individuals from infection if they are exposed to HIV, an approach termed pre-exposure prophylaxis, or PrEP.

Despite the tremendous success of antiretroviral drugs, it has been disappointing to learn that even decades of effective combination therapy are insufficient to cure HIV infection. Any interruption in the use of these drugs results in the rapid rebound of viral replication and the reinitiation of progression toward AIDS. Antiretroviral therapy is therefore a lifelong commitment. Sadly, it comes with considerable cost as well as drug side effects (though considerably less than the early drugs of the late nineties). To further improve the lives of people with HIV, scientists are now aiming to move beyond the need for lifelong antiretroviral therapy by seeking to cure infection. The nature of the virus makes this a truly daunting task, as we will explore below. Yet we know that it is possible, as at least two individuals have been cured—albeit under extraordinary and dangerous circumstances. The questions now are, can cures be achieved that are safe and less onerous than daily medication? And can these cures be scaled up to reach all people in need?

Timothy Ray Brown: The First Person Cured of HIV

The seed of the first cure for HIV was sown by a different pandemic from the distant past. We do not know which pathogen (likely a virus or bacteria) was responsible. However, the prevalence of a “delta32” mutation in a gene called CCR5 in European populations shows us that individuals who possessed this mutation were preferentially spared. This mutation comprises a 32-nucleotide deletion that gives rise to a shorter, defective version of the CCR5 protein. As it turns out, CCR5 is a critical co-receptor for HIV— essentially a door that the virus uses to enter a cell (Fig. 6). It has been known for many years that the rare individuals who are homozygous for this CCR5-delta32 mutation (that is, who have two copies of the mutation) are extremely resistant to infection with most HIV strains. (Some HIV strains use a different co-receptor, CXCR4, and can still infect these individuals.) Timothy Ray Brown did not start out having a CCR5-delta32 mutation, but through poor fortune and a physician’s initiative, this would change.

**Figure 6. HIV infection and resistance** When CCR5-delta32 is present instead of CCR5, the HIV particle is unable to use it to enter the cell, thus protecting the cell against infection. © NIAID (adapted by AMNH)

In 2007, Brown—who had been living with HIV since 1995—was diagnosed with acute myeloid leukemia. He had to face the difficult and risky treatment of a bone marrow transplantation: Doctors would use chemicals and radiation to destroy his immune system, then replace it with immune cells generated from the bone marrow of a donor. His physician, Dr. Gero Hütter, saw an opportunity. Knowing the scientific literature around the CCR5 mutation, he was able to find a matched donor who was a CCR5-delta32 homozygote. After the complex and onerous transplant procedures, Brown had an immune system that was devoid of CCR5, and for the subsequent 13 years, his HIV never came back. Sadly, in 2020 Brown’s leukemia relapsed, and he died. At least one more individual, also with leukemia, has also been cured of HIV in this way, with others now being monitored.

These cases have shown us that HIV can be cured by destroying and replacing the immune system, but apart from the dire circumstances of leukemia, this difficult and risky solution is not warranted. A safe cure will instead require the targeted removal of HIV from the immune system, specifically the CD4+ Tcells in which HIV integrates its DNA permanently into the genome. Gene therapies that seek to artificially generate delta32-like mutations in the CCR5 gene are being pursued, through gene editing platforms such as CRISPR-Cas9. However, delivering these mutagens to a substantial proportion of the infected cells in a person’s body will require considerable advancement beyond current technologies. At present, our best bet appears to be to enable the human system to remove HIV from itself—specifically, by making use of immune cells called cytotoxic T-cells. Encouragingly, recent findings suggest that in very rare individuals, this may have occurred naturally.

The Shock-and-Kill Approach

Cytotoxic T-cells (CTL) are a subset of white blood cells that are specialized in killing virally infected cells with exquisite precision. Every CTL is specific for a different peptide (a fragment of a protein) and recognizes these on the surface of a cell, where they are presented by (bound to) a host protein called major histocompatibility complex I. Cells that are producing HIV proteins are thus subject to being recognized by CTL. Once a CTL recognizes an HIV peptide, it releases the cytotoxic proteins perforin and granzyme, which punch holes in the target cell and induce it to die (Fig. 7). HIV elicits a very robust CTL response naturally, but this fails to eliminate infection. Understanding why is key to improving upon its action with new therapies.

**Figure 7. Cytotoxic T-cells (CTL)** A group of killer T-cells (outer three cells) surround a cancer cell (center, blue). All the cells in this image are tagged with fluorescent dyes. Signaling molecules (pink) indicate that the CTLs have found a target. To kill it, they will use cytotoxic chemicals stored in vesicles (red), which they will deliver through the microtubule cytoskeleton (green). © NIH

The ability of HIV to reverse transcribe its RNA genome into DNA, and then insert it into the human genome of an infected cell, creates a particular barrier that prevents CTL from curing infection. Since CTL rely on detecting viral peptides, they are only able to recognize a cell that is actively producing viral proteins. Not all infected cells produce viral proteins at all times, however. A cell that possesses HIV DNA but is not expressing proteins is termed a latently infected cell. In people on antiretroviral therapy, many infected cells persist in such latent states and thus are poorly visible to CTL. If antiretroviral therapy is stopped, however, latently infected cells can reactivate to reseed viral replication. To make these cells visible to CTL, and thus subject to elimination, researchers can “shock” them out of latency with drugs known as latency reversing agents—the shock-and-kill approach to cure.

While these approaches can be effective in vitro (in the test tube) against latently infected cells, substantial initial enthusiasm for shock-and-kill strategies has receded following a series of clinical trials that failed to show reductions in reservoirs of HIV-infected cells in study participants. While efforts are under way to strengthen both the latency reversal and CTL response aspects of these approaches, findings from our lab have led us to propose an alternative hypothesis.

Infected Cells that Resist CTL

The scenarios in which shock-and-kill approaches have been effective generally use models of latency, where CD4 cells are infected at high levels and then allowed to return to a resting/latent stage in vitro. In an attempt to bridge the chasm between this success and the failure of shock-and-kill in clinical trials, we have taken a new, in-between approach. We are studying whether we can actually take real CD4+ Tcells from blood samples of people with HIV and specifically eliminate infected cells to “cure” an infection simulated in the test tube, using shock-and-kill approaches (Fig. 8).

**Figure 8. At work in the Jones lab** At left, Brad Jones performs an assay called digital droplet PCR to measure the levels of residual HIV infection in people treated with antiretroviral therapy. At right, Evan McNeil (top), Eva Stevenson (center), and Uche Chukwukere (bottom) isolate and grow cytotoxic T-cells (CTL), which they will use to study mechanisms of CTL resistance. © Brad Jones

The scarcity of infected cells in the blood of such individuals, on the order of 5 cells per mL of blood, posed a technical challenge to such experiments. We overcame this through leukapheresis, a procedure in which 5 L of a study participant’s blood is run through an instrument over the course of two hours, retaining the white blood cells in a sample container while the other blood components flow directly back into the individual. We were surprised to find that even when we applied very strong shock and- kill combinations—potent latency reversing agents and CTL—the reservoirs of HIV-infected cells in these samples were not reduced.

Interestingly, when we grew cultures of virus from these samples and used these to infect additional CD4+ T-cells from these same donors, the same CTL that had failed to reduce the reservoirs were able to efficiently kill these newly infected cells. Was there something special about the HIV-infected cells that persist in people? These results led us to think so. The persistent cells may be intrinsically resistant to CTL; even when they are exposed to CTL by potent latency-reversing agents, they are not killed. Simply put, hiding from the immune system through latency may not be the full story of how HIV persists. Rather, some infected cells may be visible on an ongoing basis to the immune system but are nonetheless hard to kill, for reasons that we are only beginning to understand.

Although resistance to CTL is a relatively new concept in the context of HIV, it is familiar in the setting of cancer. Certain cellular properties are known to enable cancer cells to resist CTL, and this resistance can arise through multiple mechanisms. Indeed, our HIV studies have already revealed some commonalities with cancer, highlighting the potential for cross-fertilization of these fields of study. Specifically, we have discovered that a gene known to favor the survival of cells, BCL-2, is expressed at higher levels in cells that are infected with HIV than in their uninfected counterparts. BCL-2 is a regulator of cell death, which is one pathway through which CTL kill infected cells. We have gone on to show that by adding Venetoclax, a cancer drug that inhibits BCL-2, to our shock-and-kill experiments, we have been able to eliminate some infected cells. Current research by us and others is building upon these results. Some studies are testing whether Venetoclax can help reduce reservoirs of HIV-infected cells in 10 animal models. Other studies are identifying additional mechanisms that may help HIV-infected cells survive, and thus identifying other therapeutic targets for drug development.

The Road Ahead

Achieving a cure for HIV through the targeted elimination of all or most infected CD4+ T-cells, while sparing uninfected cells, is a daunting task and a long-term goal. But progress is being made as a result of the dedication of study participants, scientists, clinicians, and funding agencies. Initial signs of success are emerging in animal models, where more than one type of therapeutic intervention has succeeded in enabling the immune systems of animals to control HIV replication without antiretroviral drugs. It is reasonable to expect that similar approaches will soon yield at least some partial success in human clinical trials. Perhaps an intervention will allow trial subjects’ immune systems to moderately suppress the virus without the aid of drugs. Perhaps an intervention will partially eliminate reservoirs of HIV-infected cells. We can hope that these milestones, when they come, will point the way toward improved iterations of these therapeutics and will yield novel insights into what it will take to cure HIV infection. The path to the ultimate goal of a cure for HIV has been and will continue to be a rich source of scientific discovery, furthering our fundamental understanding of how the immune system works. Indeed, many articles have been written on how the knowledge, expertise, techniques, resources, and infrastructure of the HIV biomedical research enterprise have contributed to understanding SARS-CoV-2 infection and to the rapid development of vaccines to address this most recent global viral pandemic. Some 2,500 years passed between the time when a pandemic selected for the CCR5 mutation and when scientists took advantage of this mutation to cure a patient of HIV. In contrast, less than a year elapsed between knowledge of SARS-CoV-2 and emergency authorization of safe and effective vaccines, achieved in part through advances catalyzed by HIV. While novel viral pandemics will continue to periodically affect humanity for the foreseeable future, scientific progress stands to continue to reduce the impacts of these outbreaks as long as this progress is paired with engagement between scientists and the broader public at each stage of the journey.

--- This essay was authored for the AMNH online course Virology and Epidemiology in the Time of COVID- 19, a part of Seminars on Science (learn.amnh.org), a program of online graduate-level professional learning courses for K-12 educators. Created with the support of the City of New York Department of Health and Mental Hygiene. © 2022 City of New York