immune cells attack

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.


A new HIV Model Could Help Shed Light on Mechanisms of Viral Host Tropism

 

By Asu Erden

 

To better understand the epidemiology of human diseases, we must identify the immunological mechanisms that govern their transmission and enable their jumping from one reservoir to the next. In this regard, animal models have proven useful. Yet the pathogenic mechanisms enabling the interspecies transmission of many diseases remain elusive. This is the case for the Human Immunodeficiency Virus (HIV). Primate and humanized mouse models have helped shed light on the viral mechanisms of HIV. As far as primate models go, pigtailed macaques have been particularly useful since they present the advantage of better mimicking the pathogenesis of AIDS as seen in humans. However, these macaques are not susceptible to HIV-1 since the virus does not normally cause AIDS in this host species. But Hatziioannou et al. recently provided a new stepping-stone to the field when they published their findings of an HIV-1 infection leading to AIDS in pigtailed macaques in the journal Science.

The team of researchers used a modified HIV-1 that encodes Simian Immunodeficiency Virus (SIV) Vif. The latter protein prevents the action of host-specific antiviral enzymes. By passaging this virus and taking advantage of the lack of key antiviral proteins (e.g. TRIM5) in pigtailed macaques, Hatziioannou and her colleagues were able to successfully infect these animals. In one of them, the virus replicated to reach stable high titers. The team decided to deplete this animal of circulating CD8+ T cells to alleviate immune pressure and allow for higher viral titers, since these cells are believed to contribute to the initial control of viremia. By the fourth passage (P4) of this virus in CD8-depleted macaques, AIDS-like pathogenesis became apparent (e.g. sustained high viremia, immune activation in the gut) and eventually animals fully developed the disease (stark loss of CD4+ T cells).

Hatziioannou et al. confirmed that they had obtained a virus capable of causing AIDS in pigtailed macaques by isolating it from P4 animals and inoculating new CD8-depleted animals. The infected macaques developed AIDS confirming that the researchers had developed a virus capable of triggering a pathogenesis similar to what is seen in humans. Moreover, the key time period for CD8+ T cell-depletion was identified to be the acute phase of infection since depletion at the chronic stage did not yield AIDS-like symptoms.

Several signature mutations in the passaged virus’ genome also reflected that Hatziioannou et al. had successfully adapted the virus to acquire characteristics seen in human HIV-1 infection. Single viral genome sequencing revealed that envelope mutations were essential for the aforementioned adaptation in pigtailed macaques. Of specific interest was a deletion in one of the loops of the envelope protein, which is typical of human lentiviral infections but much more rarely observed in non-human primates. Additionally, an insertion mutation in the transmembrane domain of the HIV-1 Vpu immune evasion protein enabled it to immunologically outcompete macaque tetherin, which normally prevents virions from budding from the host cells.

HIV-1 causes AIDS in humans and chimpanzees. The fact that Hatziioannou et al. were able to develop a model of HIV-1-induced AIDS in pigtailed macaques promises to shed light on the key immunological factors at play in the epidemiology of HIV. Their protocol also reinforces the idea that CD8+ T cells play an essential role in the early stages of the pathogenesis, since macaques had to be depleted of this cell subtype during the acute phase of infection to progress to AIDS. Overall, these results highlight the importance of the arms race between the virus and the host. In four passages, Hatziioannou et al.’s modified HIV-1 virus developed the ability to counteract macaque tetherin. Such evolution is required for the virus to spread to new hosts. In the future, studies of HIV-1 in this pigtail macaque model have the potential to provide insight about new prophylactic vaccines and therapeutic drugs against the virus.


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.


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.