AIDS Attack: Priming an Immune Response to Conquer HIV

By Esther Cooke, PhD

Infection with HIV remains a prominent pandemic. Last year, an estimated 36.7 million people worldwide were living with HIV, two million of which were newly infected. The HIV pandemic most stringently affects low- and middle-income countries, yet doctors in Saskatchewan, Canada are calling, in September 2016, for a state of emergency over rising HIV rates.

Since the mid-20th century, we have seen vaccination regimes harness the spread of gnarly diseases such as measles, polio, tetanus, and small pox, to name but a few. But why is there still no HIV vaccine?

When a pathogen invades a host, the immune system responds by producing antibodies that recognise and bind to a unique set of proteins on the pathogen’s surface, or “envelope”. In this way, the pathogen loses its function and is engulfed by defence cells known as macrophages. Memory B cells, a type of white blood cell, play a pivotal role in mounting a rapid attack upon re-exposure to the infectious agent. The entire process is known as adaptive immunity – a phenomenon which is exploited for vaccine development.

The cornerstone of adaptive immunity is specificity, which can also become its downfall in the face of individualistic intruders, such as HIV. HIV is an evasive target owing to its mutability and highly variable envelope patterns. Memory B cells fail to remember the distinctive, yet equally smug, faces of the HIV particles. This lack of recognition hampers a targeted attack, allowing HIV to nonchalantly dodge bullet after bullet, and maliciously nestle into its host.

For HIV and other diverse viruses, such as influenza, a successful vaccination strategy must elicit a broad immune response. This is no mean feat, but researchers at The Scripps Research Institute (TSRI), La Jolla and their collaborators are getting close.

The team have dubbed their approach to HIV vaccine design a “reductionist” strategy. Central to this strategy are broadly neutralizing antibodies (bnAb), which feature extensive mutations and can combat a wide range of virus strains and subtypes. These antibodies slowly emerge in a small proportion of HIV-infected individuals. The goal is to steer the immune system in a logical fashion, using sequential “booster” vaccinations to build a repertoire of effective bnAbs.

Having already mapped the best antibody mutations for binding to HIV, Professor Dennis Burton and colleagues at TSRI, as well as collaborators at the International AIDS Vaccine Initiative, set out to prime precursor B cells to produce the desired bnAbs. They did this using an immunogen – a foreign entity capable of inducing an immune response – that targets human germline B cells. The results were published September 8, 2016 in the journal Science.

“To evaluate complex immunogens and immunization strategies, we need iteration – that is, a good deal of trial and error. This is not possible in humans, it would take too long,” says Burton. “One answer is to use mice with human antibody systems.”

The immunogen, donated by Professor William Schief of TSRI, was previously tested in transgenic mice with an elevated frequency of bnAb precursor cells. Germline-targeting was easier than would be the case in humans. In their most recent study, the Burton lab experimented in mice with a genetically humanised immune system, developed by Kymab of Cambridge, UK. This proved hugely advantageous, enabling them to study the activation of human B cells in a more robust mouse model. Burton speaks of their success:

“It worked! We could show that the so-called germline-activating immunogen triggered the right sort of antibody response, even though the cells making that kind of response were rare in the mice.”

The precursor B cells represented less than one in 60 million of total B cells in the Kymab mice, yet almost one third of mice exposed to the immunogen produced the desired activation response. This indicates a remarkably high targeting efficiency, and provides incentive to evaluate the technique in humans. Importantly, even better immunisation outcomes are anticipated in humans due to a higher precursor cell frequency. Burton adds that clinical trials of precursor activation will most likely begin late next year. If successful, development of the so-called reductionist vaccination strategy could one day spell serious trouble for HIV, and other tricky targets alike.

The Discovery of HIV: A Tale of Two Scientists

 

By Elizabeth Ohneck, PhD

In the early 1980s, scientists were struggling to find the cause of a new, rapidly spreading disease called Acquired Immune Deficiency Syndrome, or AIDS. At the time, few thought that a virus could cause this devastating disease. But the work of two labs, that of Robert Gallo at the U.S. National Cancer Institute, and that of Luc Montagnier at the Pasteur Institute in France, would lead to the discovery of the novel human retrovirus HIV in May of 1983 and later establish this virus as the cause of AIDS. They could not, however, predict the drama that would unfold from their discoveries.

 

Both labs originally sought to identify a retrovirus associated with cancer in humans. At the time, they were considered “old fashioned” in this pursuit. Starting in the late 1970s, the general attitude was that microbes no longer posed a major health threat in industrialized countries. In addition, research had yet to uncover a retrovirus that infected humans, much less one that caused cancer.

 

Gallo’s research led to the discovery of interleukin-2, a factor that stimulates T cell growth, allowing T cells to be grown in culture. This method laid the groundwork for Montagnier’s team to recover reverse transcriptase, a retroviral enzyme, from T cells of human cancer patients, providing the long-sought evidence that retroviruses do infect humans and might be associated with cancer. Perseverance paid off when Gallo’s group isolated the first human retrovirus from a patient with T cell leukemia, which they named human T cell leukemia virus, or HTLV.

 

Nevertheless, agents such as fungi, chemicals, and autoimmunity were considered more likely causes of AIDS. But Gallo and Montagnier saw several clues that a virus was to blame. The hallmark of AIDS was a decrease in the levels of T cells carrying the surface antigen CD4, suggesting the causative agent might specifically target CD4+ T cells. In addition, epidemiology studies indicated AIDS was transmitted through blood, sexual activity, and from mother to infant. HTLV exhibited these same characteristics, so they believed HTLV was a likely candidate.

 

Montagnier’s lab began searching for retroviruses from patients with AIDS. While they were able to recover retroviruses from the cells of these patients, the viruses did not react with antibodies against HTLV. Upon comparing electron micrograph pictures of the new virus to HTLV, it was clear to Montagnier that the virus from AIDS patients was different. Montagnier’s group termed this virus LAV. Meanwhile, Gallo’s lab was conducting its own search, and isolated two forms of HTLV from patients with AIDS. One exhibited unusual characteristics, which they called an “aberrant” form. They would later realize that these patients were actually doubly infected with HTLV and HIV.

 

The two research teams had collaborated extensively, so Gallo and Montagnier agreed to publish their findings together in the May 20, 1983 volume of Science. But whether HTLV or LAV actually caused AIDS was still unclear. In 1984, Gallo’s group announced the isolation of a virus related to but distinct from HTLV, termed HTLV-IIIB, from a pooled collection of samples from AIDS patients. This virus could be continuously cultured, allowing thorough study, a problem Montagnier had not been able to overcome with LAV. Gallo also provided convincing evidence HTLV-III viruses in fact caused AIDS, and the HTLV-IIIB isolate was used to develop a blood test to ensure purity of blood bank supplies were uncontaminated and test patients for the presence of the virus.

 

HTLV-IIIB, however, was strikingly similar to an isolate Montagnier’s group was studying, LAI, that also grew robustly in culture, identified before HTLV-III. Such similarity between different isolates would be unusual, as these viruses were found to be highly variable. When it turned out these viruses were essentially the same, an argument ensued. The two labs had exchanged multiple isolates. Had Gallo inappropriately used the Pasteur Institute isolate for the development of the blood test? Who should get credit for the discovery of this new virus? Should the French group have rights to the blood test patent?

 

Finally, President François Metterrand of France and U.S. President Ronald Reagan met to resolve the issues between their scientists and governments. In 1987, it was agreed that the scientists would share the prestige of the discovery and the patent profits of the blood test equally. The names LAI and HTLV-III were exchanged for human immunodeficiency virus, or HIV. Motagnier was credited with its discovery, as he was the first to isolate a pure culture of the virus. Gallo was credited for demonstrating conclusively that HIV causes AIDS. In 1993, scientists at Roche analyzed archived samples of viruses from both labs and discovered that a sample of LAV given to the Gallo’s group by Montagnier, had been contaminated with LAI, identifying the cause of the confusion and officially clearing Gallo of any misconduct. Tempers cooled, and in 2002 Montagnier and Gallo published three papers, one co-authored by both scientists, reviewing the history of HIV/AIDS research and acknowledging the contributions each had made.

 

Unfortunately, the 2008 Nobel Prize in Physiology or Medicine did not reflect this compromise. The prize was awarded to Luc Montagnier and co-researcher François Barré-Sinoussi for the discovery of HIV, and was shared with Harald zur Hausen, who established the link between HPV and cervical cancer. As the Nobel Prize can be split among maximally three people, this meant Robert Gallo was left out. Had the selection committee chosen to focus only on HIV, Gallo almost certainly would have been included. This decision was met with some surprise; Montagnier himself stated the third recipient should have been Gallo.

 

Both scientists are quick to point out the astounding rapidity of HIV research. It took only 2 ½ years from its first identification to establish it as the causative agent of AIDS, and another 2 years to develop a commercially available blood test. In 1987,the first anti-HIV drug, AZT was introduced, soon followed by protease inhibitors and eventually the triple drug therapy used today that has saved countless lives. Despite some tumultuous times, the story of Gallo, Montagnier, and HIV serves as important demonstration of the power and necessity of scientific cooperation.

Microscopy, Mice, and HIV

 

By Elaine To

Monkeys infected with simian immunodeficiency virus (SIV) have been the traditional animal model for the study of HIV pathology. However, SIV does not result in the same immunodeficiency that HIV does, and monkeys are expensive to care for. Mice without immune cells can be engrafted with human immune cells and used instead. The specific model used by Ladinsky et al transfers human fetal thymic and liver tissues along with hematopoietic stem cells. These mice, known as BLT mice, reconstitute human immune cells in significant levels in many tissues, and HIV infection results in T cell depletion.

Ladinsky et al. use a powerful microscopy technique known as cryoelectron tomography in addition to immunofluorescence to understand the characteristics of HIV infection in the small and large intestines in BLT mice. The interior of the small intestine has an upper layer that includes the villi, known as the lamina propria. Between the villi in the lower layer are intestinal crypts, and this is where the majority of HIV viruses were located. Any villi that had evidence of HIV were also adjacent to an infected crypt.

Looking closer, the researchers were able to see individual viruses in the process of budding out of infected cells. It was possible to distinguish between mature and immature viruses based on the differences in internal structures. An examination of the viral pools located outside cells showed that 90% of the pools were mostly mature, but 10% were mostly immature. This is in contrast with previous studies in cell culture showing all viruses found outside cells are mature, indicating a difference in virus maturation or diffusion between cells organized in tissues and cells cultured in vitro.

After some searching, an isolated infected cell was located that was responsible for the production of all viruses in the nearby region. The single cell produced 63 viruses, but the microscopic methods only saw viruses in the same plane as this cell. Regions above and below the cell could not be examined, so the real number of viruses that can result from a single infected cell is likely much more than just 63.

Antibodies targeting CD4 showed that uninfected cells have CD4 on their outer membranes, whereas infected cells have CD4 on their inner endoplasmic reticulum. This supports the previous finding that the HIV protein Vpu causes the internalization of CD4 to prevent newly released HIV viruses from reattaching to the host cell.

There was also evidence of the virological synapse, the phenomenon that happens when a virus budding out of an infected cell immediately contacts a neighboring cell and infects it. Two of the proteins that help bring two cells close together, LFA-1 and ICAM, were found at the cell-cell junction near the actively budding virus.

Lastly, the researchers looked for evidence of the ESCRT proteins, which are known to help release viruses from infected cells. The ESCRT components hCHMP1B, hCHMP2A, and hALIX were found on the thin membranous necks of actively budding viruses. Some budding viruses with thick necks appeared to be in an early stage of budding, and displayed spoke-like projections originating from the virus. These were proposed to be the early components of ESCRT.

Overall, the combination of advanced microscopy with the BLT mouse model revealed new aspects of the process of HIV infection, and showed that conclusions drawn from in vitro cell culture cannot always be assumed to be true in whole animals. Further evidence was also gained for the virological synapse and use of ESCRT proteins to facilitate the spread of HIV within whole organisms.

Cutting out HIV: One Step Closer to the Cure

 

 

Elaine To

Currently, individuals who test positive for HIV are put on highly active antiretroviral therapy (HAART), a cocktail of multiple drugs that inhibit different aspects of the viral life cycle. While there are drugs that prevent the integration of the viral genome into the host cell genome, there is no known mechanism to remove the viral genome post-integration. This is also the reason we cannot completely eradicate HIV from infected individuals—even after HAART treatment, the viral genome persists in inactive memory T cells. In order to address this, Hauber et al. re-engineered the commonly known Cre recombinase enzyme, directing the novel Tre recombinase to target sequences in the HIV long terminal repeat regions. These regions flank the viral genome, allowing Tre recombinase to cut the targeted sequences within these regions and excise the viral genome from the host cell’s genome.

Lentiviral transduction was used to deliver the Tre recombinase vector into cells. The vector was designed to place Tre under the control of a Tat dependent promoter, ensuring only the infected cells that express the HIV protein Tat will express Tre. Flow cytometry was used to analyze HeLa cells infected with HIV that contained blue fluorescent protein. Cells transduced with the Tre vector had fewer blue fluorescing cells while the blue fluorescing population remained stable in cells transduced with a control vector. Immunoblots confirmed the protein expression of Tre in the Tre transduced cells. Additionally, the time course of Tre expression matched the time course of the decreasing blue fluorescence seen in the flow cytometry experiment. PCR and DNA sequencing checked that the exact DNA sequence intended to be cut out was removed in the Tre transduced cells.

Viral gene delivery comes with a fear of deleterious effects on the host cells. The researchers first examined this possibility in Jurkat cells using a re-designed vector that constitutively expresses Tre. Between the Tre and control transduced cells, there were no differences in tubulin expression, growth rate, apoptosis, or cell cycle progression. When this constitutive Tre vector was transduced into CD4+ T cells isolated from a human donor, the cells displayed similar activation and cytokine secretion profiles as compared to the control vector. The Tat dependent and constitutive Tre vectors were both transduced into hematopoietic stem cells (HSCs) without any change on the abilities of the HSCs to differentiate into the expected cell lineages. Karyotyping and comparative genomic hybridization revealed that CD4+ T cells have no Tre dependent genomic aberrations. Lastly, Tre was shown to be incapable of cutting DNA sequences within the host genome that are similar to the targeted HIV LTR sequences.

The core experiments behind this paper are the in vivo studies done in Rag knockout mice, which can be transplanted with human immune cells and used as a humanized animal model. CD4+ T cells were isolated from human donors, transduced with the Tat dependent vector, and transplanted into the mice, which were then exposed to HIV. The mice displayed lower viral counts and higher frequencies of human T cells versus the control transduction vector. Similar results were obtained when mice were given Tre transduced HSCs. Thus, the researchers elegantly show that their engineered Tre recombinase can alleviate the symptoms of HIV infection. However, reliable methods of gene delivery are yet in development, and the inactive memory T cells harboring the latent HIV reservoir do not express Tat, precluding Tre expression. If combined with methods that activate viral protein expression in the presence of HAART, Tre recombinase therapy may yet play an important role in the cure of HIV.

Two for the Price of One: Anti-viral and a Recreational Drug

 

Elaine To

Efavirenz is a frequently prescribed drug for treating HIV with side effects including depression, paranoia, hallucinations, and delusions. While efavirenz is prescribed orally, recent reports show that people have been crushing efavirenz pills and smoking the powder as a recreational drug. The researchers in this article set out to characterize the psychoactive effects of efavirenz, both on a molecular and a behavioral scale. They study efavirenz both in vitro and in vivo to show that the drug’s properties most closely resemble those of lysergic acid diethylamide (LSD) and that this is due to their shared targeting of the serotonin (5HT) pathway.

Hallucinogenic drugs are known to act through binding to specific receptors within the brain, which activates or deactivates certain neural pathways. Knowing what receptors a drug of interest can bind to is therefore a clue for its activity—and the first question that the researchers answer. Using a ligand displacement assay, they show that efavirenz can bind to 5HT2A and 5HT2C. Efavirenz also increases phospholipase C activity and this increase is abrogated by pretreatment with a 5HT antagonist, showing that efavirenz is a 5HT agonist. Other known agonists of the 5HT system are the natural ligand serotonin, antidepressants, and psychoactive drugs including mescaline and LSD.

The researchers followed up on the receptor pharmacology studies with in vivo studies in mice and rats. This allows a comparison of the whole body effects between efavirenz and other psychoactive drugs. Mice that are given efavirenz are less mobile when exposed to a new environment, similar to LSD. However, LSD has this effect at a dose as low as 3 mg/kg whereas the efavirenz dose needed to be 30 mg/kg before an effect was seen. Efavirenz also induces head twitching in wild type mice, but not in 5HT2A knockouts. Head twitching is a characteristic trait of rodents whose 5HT2A pathway has been affected.

The next set of experiments uses rats that are trained to distinguish between an injection of a drug and a saline negative control. Each rat is given an injection of the drug or saline before being placed in an experimental chamber containing two levers. If drug was injected, depressing one lever would release food pellets while depressing the other did nothing, and vice versa for saline. Rats that were trained on 0.1 mg/kg LSD responded similarly on the LSD lever when injected with 30 mg/kg efavirenz, and rats trained on 18 mg/kg efavirenz responded on the efavirenz lever when given 0.1 mg/kg LSD. There were no such associations with rats trained on ecstasy, cocaine, or carisoprodol.

Since rats known to self-administer cocaine did not do so with efavirenz, it was concluded that efavirenz is not an addictive drug. Additional evidence comes from place conditioning studies, where rats who preferentially spend time on a floor associated with cocaine infusions do not behave similarly with a floor associated with efavirenz infusions.

Overall, these studies show that efavirenz acts through the 5HT2A pathway to exert psychoactive effects similar to those of LSD. It is less potent than LSD, however, requiring larger doses to achieve the same effects on mice and rats. Both drugs are also non-addictive. LSD is not associated with long term adverse effects, so hopefully smoking efavirenz would be a similar case. However, efavirenz is often prescribed for HIV treatment in a single pill that contains additional HIV drugs whose activities on the brain are not as well characterized. Consequences aside, the fact that a drug meant to inhibit reverse transcriptase can also act as a psychoactive agent is fascinating, and demonstrates the complexity of pharmacology.

Domesticated HIV Leads to Gene Therapy Success

Alisa Moskaleva

For as long as biomedical scientists have known that DNA mutations cause disease, they wanted to be able to correct them. Dozens of rare but devastating human diseases are caused by a mutation in a single gene. If a mutant form of the gene causes the disease, then giving the patient the correct form of the gene should cure it. This idea is called gene therapy. Now, two papers in the 23 August 2013 issue of Science by Dr. Luigi Naldini and co-workers report prolongation and improvement of quality of life from gene therapy for six children with rare genetic diseases. These papers are not the first to report health benefit from gene therapy and not the first to use the technique of lentivirus-mediated hematopoietic stem cell gene therapy, which involves domesticated HIV and which I’ll explain shortly. However, the papers address two previously incurable diseases, metachromatic leukodystrophy in one paper and Wiskott-Aldrich syndrome in the other, and add to the evidence that the gene-therapy technique that involves something as scary as HIV is safe.

So, what is lentivirus-mediated hematopoietic stem cell gene therapy and what does it have to do with HIV? Let’s start with “lentivirus-mediated.” Lentivirus is a virus with an RNA genome that infects eukaryotic cells. To persist in cells and reproduce, lentivirus integrates its RNA genome by converting it into DNA and adding this DNA into the genome of the host. HIV is a lentivirus that infects T-cells. By replacing the gene in the HIV genome that targets it to T-cells with a gene that recognizes all mammalian cells and further adding a gene of interest, it’s possible to use HIV to integrate the gene of interest into the genome of any mammalian cell. So, if only HIV could be made to stop replicating uncontrollably, gene therapy could use it to deliver correct forms of disease-causing genes to patients’ cells. Over the past twenty years, scientists have domesticated HIV by removing most of its genes, and constructing versions that can infect but can’t replicate. HIV-based lentivirus is now routinely used in research to introduce DNA into mammalian cells and it can be a method of gene therapy. Hematopoietic stem cell gene therapy means that hematopoietic stem cells, the cells that can give rise to red and white blood cells, are the targets of gene therapy.

Why did Dr. Naldini and colleagues target hematopoietic stem cells? Wiskott-Aldrich syndrome is a disease of white blood cells, so it makes sense to deliver the correct form of the disease-causing gene to their source. Metachromatic leukodystrophy is a disease of the brain and the peripheral nervous system, so at first glance brain cells and nerve cells should have been the targets of gene therapy. However, getting the gene to integrate in enough cells requires literally bathing the cells in a high concentration of lentivirus. It’s impossible to achieve a high enough concentration of lentivirus for cells inside the body. Delivering genes to cells inside the body remains one of the biggest challenges in gene therapy. Hematopoietic stem cells are a much easier target because they can be isolated from the body by drawing blood and sorting it. The isolated cells can then be concentrated and bathed in lentivirus before being re-injected into the patient’s bloodstream. In metachromatic leukodystrophy, the disease-causing form of the gene causes the accumulation of a toxin and the correct form destroys it. The authors hoped that hematopoietic stem cells with the correct form of the gene would give rise to cells of the immune system that would migrate to the brain and the peripheral nervous system and scavenge the toxin. Therefore, hematopoietic stem cells are a good target for gene therapy not only because they are easy to isolate but also because they can migrate and help out other cell types that cannot be targeted directly.

However, hematopoietic stem cells have a dark side. When they are injected into the patient’s bloodstream to exert their beneficial effect, they can transform into cancerous cells and cause leukemia. Leukemia was an unfortunate side effect of gene therapy against the Wiskott-Aldrich syndrome and two other rare diseases using a different method of gene delivery called gamma retrovirus that preceded lentivirus. Gamma retrovirus is also a virus that infects eukaryotic cells and integrates its genome into the host genome. Analysis of the genome of cancerous cells revealed that when the gamma retrovirus integrated, a part of its DNA ended up near a cancer-causing gene and, acting like a switch, turned it on. Mindful of this, Dr. Naldini and colleagues analyzed the genome of hematopoietic stem cells from their patients several times after treatment. Reassuringly, they did not find any cells with inappropriately activated cancer-causing genes 1.5 to 2 years after treatment in patients battling metachromatic leukodystrophy and 1.5 to 2.5 years after treatment in patients with Wiskott-Aldrich syndrome. Dr. Naldini and colleagues hypothesize that HIV-based lentivirus may be safer than gamma retrovirus because it has fewer DNA switches that can turn a gene on, and because lentivirus integrates more randomly in the genome than gamma retrovirus. They will be following their patients for years to come.

Is gene therapy with domesticated HIV safe? Only time and more research will tell. But six children who were predicted to die months ago are walking and running, playing and talking. They got a respite from dreadful symptoms, like eczema, internal bleeding, and inability to walk or even to hold up their own head. One fervently hopes for more gene-therapy good news.