Amazing Science

A 53-year-old man in Germany has become the third person with HIV to be declared cleared of the virus after a procedure that replaced his bone marrow cells with HIV-resistant stem cells from a donor. For years, antiretroviral therapy (ART) has been given to people with HIV with the aim of lowering the virus to almost undetectable levels and preventing it from being transmitted to other people. But the immune system keeps the virus locked up in reservoirs in the body, and if an individual stops taking ART the virus can begin replicating and spreading. A true cure would eliminate this reservoir, and this is what seems to have happened for the latest patient, whose name has not been released. The man, who is being referred to as the ‘Düsseldorf patient’, stopped taking ART in 2018 and has remained HIV-free since. But the risks associated with the procedure mean it is unlikely to be widely used in its current form.

The stem-cell technique involved was first used to treat Timothy Ray Brown, often referred to as the Berlin patient. In 2007, he had a bone marrow transplant, in which those cells were destroyed and replaced with stem cells from a healthy donor, to treat acute myeloid leukemia. The team treating Brown selected a donor with a genetic mutation called CCR5Δ32/Δ32, which prevents the CCR5 cell-surface protein from being expressed on the cell surface. HIV uses that protein to enter immune cells, so the mutation makes the cells effectively resistant to the virus. After the procedure, Brown was able to stop taking ART and remained HIV-free until his death in 2020. In 2019, researchers revealed that the same procedure seemed to have cured the London patient, Adam Castillejo. And, in 2022, scientists announced that they thought a New York patient who had remained HIV-free for 14 months might also be cured, although researchers cautioned that it was too early to be certain. Ravindra Gupta, a microbiologist at the University of Cambridge, UK, who led the team that treated Castillejo, says the latest study “cements the fact that CCR5 is the most tractable target for achieving a cure right now”.

An mRNA vaccine has been found to induce antibody responses against all 20 known subtypes of influenza A and B in mice and ferrets. An experimental vaccine has generated antibody responses against all 20 known strains of influenza A and B in animal tests, raising hopes for developing a universal flu vaccine. Influenza viruses are constantly evolving, making them a moving target for vaccine developers. The annual flu vaccines available now are tailored to give immunity against specific strains predicted to circulate each year. However, researchers sometimes get the prediction wrong, meaning the vaccine is less effective than it could be in those years. Some researchers think annual flu jabs could be replaced by a universal flu vaccine that is effective against all flu strains. Researchers have tried to achieve this by making vaccines containing protein fragments that are common to several influenza strains, but no universal vaccine has yet gained approval for wider use. Now, Scott Hensley at the University of Pennsylvania and his colleagues have created a vaccine based on mRNA molecules – the same approach that was pioneered by the Pfizer/BioNTech and Moderna covid-19 vaccines. mRNA contains genetic codes for making proteins, just like DNA. The vaccine contains mRNA molecules encoding fragments of proteins found in all 20 known strains of influenza A and B – the viruses that cause seasonal outbreaks each year. The strains have different versions of two proteins on their surface, haemagglutinin (H) and neuraminidase (N), which are targeted by immune responses. But even within one strain, such as H1N1, there can be slight variations in these proteins, so the version in the universal vaccine will not exactly match every possible variant.

In tests in mice, the team found that the animals generated antibodies specific to all 20 strains of the flu virus, and these antibodies remained at a stable level for up to four months. In another test, the team gave mice the universal flu vaccine or a dummy vaccine containing code for a non-flu protein. A month later, they infected them with either one of two variants of the H1N1 flu virus, one with an H1 protein that was very similar to the version of the protein in the vaccine, and one with a more distinct version. All the mice given the flu vaccine survived exposure to the virus with the more similar protein and 80 per cent survived being infected with the more distinct variant. All of the mice given the dummy vaccine died around a week after infection with either variant. Another group of mice were given an mRNA vaccine targeted only to the precise flu strain they were exposed to, and all of this group survived over the same time period. This suggests the universal flu vaccine would offer less protection against new variants of the 20 flu strains than an annual vaccine matched to new forms of the virus, says Albert Osterhaus at the University of Veterinary Medicine Hannover in Germany, who wasn’t involved in the study. The researchers also tested the universal vaccine in ferrets with similar results.

“The mouse and ferret models for influenza are as good as animal models get. The animal data are promising and thus a good indication of what will happen in humans,” says Peter Palese at the Icahn School of Medicine at Mount Sinai in New York. A key benefit of mRNA vaccines is that they can easily be scaled up compared with other approaches which rely on growing influenza viruses in chicken eggs or in the lab, says Palese. “For generating a basic immunity against epidemic or pandemic influenza virus strains in the future, this strategy could offer an option if longevity [of immunity] in humans is confirmed,” says Osterhaus. “Definitely these animal data are promising and merit further exploration in clinical studies. Given previous studies with candidate universal flu vaccines in human trials, it is hard to predict what the clinical data will bring,” says Osterhaus. “This 20-HA mRNA vaccine was tested in ferret animals, which is highly significant and may hold promise for protecting against future emerging flu strains against severe disease in humans,” says Sang-Moo Kang at Georgia State University.

Study cited published in Science (Nov. 24):

https://doi.org/10.1126/science.abm0271 

New Research Finds That Viruses May Have “Eyes and Ears” on Us

The newly-found, widespread ability of some viruses to monitor their environment could have implications for antiviral drug development. New research indicates that viruses are using information from their environment to “decide” when to sit tight inside their hosts and when to multiply and burst out, killing the host cell. The work has important implications for antiviral drug development. Led by the University of Maryland Baltimore County (UMBC), the study was recently published in Frontiers in Microbiology. A virus’s ability to sense its environment, including elements produced by its host, adds “another layer of complexity to the viral-host interaction,” says Ivan Erill. He is senior author on the new paper and professor of biological sciences at UMBC. Right now, viruses are taking advantage of that ability to their benefit. But he says that in the future, “we could exploit it to their detriment.”

Monkeypox has infected more than 77,000 people in more than 100 countries worldwide, and—similar to COVID-19—mutations have enabled the virus to grow stronger and smarter, evading antiviral drugs and vaccines in its mission to infect more people. Now, a team of researchers at the University of Missouri have identified the specific mutations in the monkeypox virus that contribute to its continued infectiousness. The findings could lead to several outcomes: modified versions of existing drugs used to treat people suffering from monkeypox or the development of new drugs that account for the current mutations to increase their effectiveness at reducing symptoms and the spread of the virus. Kamlendra Singh, a professor in the MU College of Veterinary Medicine and Christopher S. Bond Life Sciences Center principal investigator, collaborated with Shrikesh Sachdev, Shree Lekha Kandasamy and Hickman High School student Saathvik Kannan, to analyze the DNA sequences of more 200 strains of monkeypox virus spanning multiple decades, from 1965, when the virus first started spreading, to outbreaks in the early 2000s and again in 2022. "By doing a temporal analysis, we were able to see how the virus has evolved over time, and a key finding was the virus is now accumulating mutations specifically where drugs and antibodies from vaccines are supposed to bind," Sachdev said. "So, the virus is getting smarter, it is able to avoid being targeted by drugs or antibodies from our body's immune response and continue to spread to more people."

Needles in a haystack

Singh has been studying virology and DNA genome replication for nearly 30 years. He said the homology, or structure, of the monkeypox virus is very similar to the vaccinia virus, which has been used as a vaccine to treat smallpox. This enabled Singh and his collaborators to create an accurate, 3D computer model of the monkeypox virus proteins and identify both where the specific mutations are located and what their functions are in contributing to the virus becoming so infectious recently. "Our focus is on looking at the specific genes involved in copying the virus genome, and monkeypox is a huge virus with approximately 200,000 DNA bases in the genome," Singh said. "The DNA genome for monkeypox is converted into nearly 200 proteins, so it comes with all the 'armor' it needs to replicate, divide and continue to infect others. Viruses will make billions of copies of itself and only the fittest will survive, as the mutations help them adapt and continue to spread."  Kannan and Kandasamy examined five specific proteins while analyzing the monkeypox virus strains: DNA polymerase, DNA helicase, bridging protein A22R, DNA glycosylase and G9R. "When they sent me the data, I saw that the mutations were occurring at critical points impacting DNA genome binding, as well as where drugs and vaccine-induced antibodies are supposed to bind," Singh said. "These factors are surely contributing to the virus' increased infectivity. This work is important because the first step toward solving a problem is identifying where the problem is specifically occurring in the first place, and it is a team effort."

The evolution of viruses

Researchers continue to question how the monkeypox virus has evolved over time. The efficacy of current CDC-approved drugs to treat monkeypox have been suboptimal, likely because they were originally developed to treat HIV and herpes but have since received emergency use authorization in an attempt to control the recent monkeypox outbreak. "One hypothesis is when patients were being treated for HIV and herpes with these drugs, they may have also been infected with monkeypox without knowing, and the monkeypox virus got smarter and mutated to evade the drugs," Singh said. "Another hypothesis is the monkeypox virus may be hijacking proteins we have in our bodies and using them to become more infectious and pathogenic." Singh and Kannan have been collaborating since the COVID-19 pandemic began in 2020, identifying the specific mutations causing COVID-19 variants, including Delta and Omicron. Kannan was recently recognized by the United Nations for supporting their 'Sustainable Development Goals,' which help tackle the world's greatest challenges. "I could not have done this research without my team members, and our efforts have helped scientists and drug developers assist with these virus outbreaks, so it is rewarding to be a part of it," Singh said. "Mutations in the monkeypox virus replication complex: Potential contributing factors to the 2022 outbreak" was recently published in Journal of Autoimmunity. Co-authors on the study include Shrikesh Sachdev, Athreya Reddy, Shree Lekha Kandasamy, Siddappa Byrareddy, Saathvik Kannan and Christian Lorson.

Research cited published in Journal of Autoimmunity (Dec. 22, 2022):

https://doi.org/10.1016/j.jaut.2022.102928 

A recent experiment tests whether the gene-editing technology CRISPR can stop the virus from replicating, which would ultimately wipe out the HIV infection. In July, an HIV-positive man became the first volunteer in a clinical trial aimed at using the CRISPR gene editing technology to snip the HIV 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 anti-retroviral drugs he’s been on to keep the virus at undetectable levels. Then, investigators will wait 12 weeks to see if the virus rebounds and becomes detectable again. 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 CD4-positive immune cells in the body 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 copies of the virus. 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 hide-out reservoirs, so people have to take medication every day for the rest of their lives. But Excision BioTherapeutics is hoping that this CRISPR-based approach will remove HIV for good.

CRISPR is being used in several other studies to treat a handful of conditions that arise from various 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 even 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 involving zinc finger nucleases to add that protective mutation into patients’ T cells—an important part of the immune system. Those trials have had only 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 during their lives. 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 anti-retroviral 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 anymore. 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.”

Ancient genomes from the herpes virus that commonly causes lip sores – and currently infects some 3.7 billion people globally – have been uncovered and sequenced for the first time by an international team of scientists led by the University of Cambridge.   Latest research suggests that the HSV-1 virus strain behind facial herpes as we know it today arose around five thousand years ago, in the wake of vast Bronze Age migrations into Europe from the Steppe grasslands of Eurasia, and associated population booms that drove rates of transmission. Herpes has a history stretching back millions of years, and forms of the virus infect species from bats to coral.

Despite its contemporary prevalence among humans, however, scientists say that ancient examples of HSV-1 were surprisingly hard to find. The authors of the study, published in the journal Science Advances, say the Neolithic flourishing of facial herpes detected in the ancient DNA may have coincided with the advent of a new cultural practice imported from the east: romantic and sexual kissing. “The world has watched COVID-19 mutate at a rapid rate over weeks and months. A virus like herpes evolves on a far grander timescale,” said co-senior author Dr Charlotte Houldcroft, from Cambridge’s Department of Genetics. “Facial herpes hides in its host for life and only transmits through oral contact, so mutations occur slowly over centuries and millennia. We need to do deep time investigations to understand how DNA viruses like this evolve,” she said. “Previously, genetic data for herpes only went back to 1925.” The team managed to hunt down herpes in the remains of four individuals stretching over a thousand-year period, and extract viral DNA from the roots of teeth. Herpes often flares up with mouth infections: at least two of the ancient cadavers had gum disease and a third smoked tobacco.

The oldest sample came from an adult male excavated in Russia’s Ural Mountain region, dating from the late Iron Age around 1,500 years ago. Two further samples were local to Cambridge, UK. One a female from an early Anglo-Saxon cemetery a few miles south of the city, dating from 6-7th centuries AD. The other a young adult male from the late 14th century, buried in the grounds of medieval Cambridge’s charitable hospital (later to become St. John’s College), who had suffered appalling dental abscesses. The final sample came from a young adult male excavated in Holland: a fervent clay pipe smoker, most likely massacred by a French attack on his village by the banks of the Rhine in 1672. “We screened ancient DNA samples from around 3,000 archaeological finds and got just four herpes hits,” said co-lead author Dr Meriam Guellil, from Tartu University’s Institute of Genomics. “By comparing ancient DNA with herpes samples from the 20th century, we were able to analyse the differences and estimate a mutation rate, and consequently a timeline for virus evolution,” said co-lead author Dr Lucy van Dorp, from the UCL Genetics Institute. Co-senior author Dr Christiana Scheib, Research Fellow at St. John’s College, University of Cambridge, and Head of the Ancient DNA lab at Tartu University, said: “Every primate species has a form of herpes, so we assume it has been with us since our own species left Africa.”

“However, something happened around five thousand years ago that allowed one strain of herpes to overtake all others, possibly an increase in transmissions, which could have been linked to kissing.” The researchers point out that the earliest known record of kissing is a Bronze Age manuscript from South Asia, and suggest the custom – far from universal in human cultures – may have travelled westward with migrations into Europe from Eurasia. In fact, centuries later, the Roman Emperor Tiberius tried to ban kissing at official functions to prevent disease spread, a decree that may have been herpes-related. However, for most of human prehistory, HSV-1 transmission would have been “vertical”: the same strain passing from infected mother to newborn child. Two-thirds of the global population under the age of 50 now carry HSV-1, according to the World Health Organization.

For most of us, the occasional lip sores that result are embarrassing and uncomfortable, but in combination with other ailments – sepsis or even COVID-19, for example – the virus can be fatal. In 2018, two women died of HSV-1 infection in the UK following Caesarean births. “Only genetic samples that are hundreds or even thousands of years old will allow us to understand how DNA viruses such as herpes and monkeypox, as well as our own immune systems, are adapting in response to each other,” said Houldcroft. The team would like to trace this hardy primordial disease even deeper through time, to investigate its infection of early hominins. “Neanderthal herpes is my next mountain to climb,” added Scheib.

Publisjhed in Science Advances (July 27, 2022):

https://doi.org/10.1126/sciadv.abo4435 

A study employing CRISPR/Cas9 to explore the evolutionary beginnings of some giant viruses finds evidence that their large genomes arose from gene duplications.  Most viruses are small and carry minimal genomes. Even one of the largest small viruses, Vaccinia, measures merely one-fiftieth the size of a pollen grain and contains only 270 genes.

Giant viruses flout these rules. With sizes that rival small bacteria and genomes that contain thousands of genes, their complexity emulates that of cellular life. How these viruses came to be so large has been the subject of much debate. Now, scientists are finally poised to unravel the mystery of their evolutionary origins, thanks to a suite of CRISPR/Cas9-based tools described in a Nature Communications paper from January.

“It was by chance that we encountered the first giant virus,” says Chantal Abergel, a virologist at Aix-Marseille University in France. “It was Mimivirus, and it was actually mistaken for a bacterium.” In the 20 years since that discovery, virologists have prioritized exploring the diversity of giant viruses. Now that they’ve found a fair few, the focus has shifted towards studying their evolution in more detail with molecular biology techniques.

Evolutionary biologists have grappled over two possible origins of giant viruses. One possibility is that they were once cellular organisms that shrunk physically and genetically over time. But most virologists now suspect giant viruses grew out of much smaller ones—though the evidence supporting either hypothesis is scant.

To begin addressing this origin question, Abergel decided to examine how the essential genes in the Pandoravirus genome are distributed. In cellular organisms, essential genes are scattered throughout the genome—so if giant viruses are essentially reduced cells, one would expect a similar pattern. Alternatively, if the genes are clumped, that could indicate the viruses’ large genomes started out in a more compact form. One way to locate a virus’s essential genes is to knock out genes one at a time to find the ones that are needed for virus production. But to do that with a giant virus, Abergel needed a gene-editing system that worked in members of the group. With the help of Hugo Bisio, a postdoctoral researcher in Abergel’s lab, and colleagues at Aix–Marseille University, Abergel used a CRISPR/Cas9-based gene-editing system to modify the genome of the amoeba Acanthamoeba castellanii and the giant virus Pandoravirus neocaledonia, which infects it.

The CRISPR/Cas9 system was designed to delete specific genes and consists of two guide RNAs and a Cas9 scission enzyme. Similar to other CRISPR/Cas9 systems, each guide RNA contains 17 to 20 bases designed to bind to one specific location on the genome of the giant virus or the amoeba, allowing the Cas9 scission enzyme to cut the genome at that site. The amoeba A. castellanii contains 25 copies of each chromosome, making it difficult to design an efficient CRISPR/Cas9 system that could delete each gene copy. To overcome this issue, the researchers modified their CRISPR/Cas9 system to generate a chain reaction. Each time DNA was cut to remove a gene, a DNA segment encoding the Cas9 enzyme and the guide RNAs responsible for the cut would take the place of the missing gene in the genome. This allowed gene deletions to repeat and propagate until all copies were removed.

Once they optimized their CRISPR/Cas9 system, the team deleted each gene separately from the Pandoravirus genome and measured the resulting change in virus production, in order to determine how important each gene is to the virus’s lifecycle. They found that essential genes clustered together at one end of the genome and were segregated from nonessential genes at the other end. This level of gene orderliness has not been seen in viruses, according to Bisio. Even bacterial genomes aren’t quite so tidy: While they do group genes with linked functions together into gene clusters known as operons, these tend to be dispersed throughout the genome rather than grouped all together in one spot. Bisio says the cluster of essential genes may echo a smaller “core genome” of an ancient virus. This genome could have become elongated through multiple rounds of gene duplication that were biased in one direction to produce an additional set of spare nonessential genes. This could explain how modern-day giant viruses came to possess thousands of genes. “Our data indicate that complex viruses arose from smaller and simpler ones,” Bisio tells The Scientist in an email—noting that it will take further research to determine whether that’s true of all giant viruses or just Pandoravirus. Other studies found that some genes in giant viruses were usurped from their amoeba hosts, suggesting gene exchange is another way giant viruses increased in size. The team then set their sights on one of the many evolutionary mysteries of Pandoravirus: its lack of a capsid. Small viruses package their genomes into capsids made of viral proteins. While some giant viruses, such as Mimivirus, continue this tradition, others, including Pandoravirus, do not. If giant viruses did indeed evolve from smaller ones, there could be traces of capsid proteins hiding in their genomes. So, the researchers set out to study the function of potential capsid protein remnants in a close cousin of Pandoravirus, the smaller Mollivirus, which can also infect A. castellanii.

Researchers have suspected that a Mollivirus protein called ml_347 evolved from a capsid gene based on its gene’s sequence and predicted 3D shape. So, the team investigated its function by deleting the gene using their CRISPR/Cas9 system. They found that the gene is important for Mollivirus assembly, which the authors say is intriguing given its possible capsid ancestry. It’s possible that, as capsids were lost in giant virus evolution, obsolete capsid genes were adapted for new assembly functions. Frederik Schulz, an evolutionary biologist with the DOE Joint Genome Institute in California who wasn’t involved with the study but who has worked with Chantal Abergel in the past, tells The Scientist that the findings align with recent discoveries. “There was a debate for a long time [about] how giant virus[es] evolved,” he says. “The working hypothesis in the previous years was that they evolved from smaller viruses, and that’s exactly what Chantal and her team could show and confirm using their CRISPR/Cas9 gene-editing approach.” Schulz notes that it will be exciting to see the CRISPR/Cas9 technology introduced into other host species, such as algae, which would allow researchers to expand research into a greater variety of giant viruses. He also points out that the system only works for viruses that replicate in a host cell’s nucleus, while most giant viruses replicate in cytoplasmic structures called viral factories, which the Cas9 enzyme and guide RNAs can’t penetrate. Still, Bisio says there’s much left to discover in Pandoravirus. “[This CRISPR/Cas9 technology is] a goldmine to find new functions,” he says—one that he and his colleagues are eager to employ to tease apart what all the virus’s genes do. 

Cited research published in Nat. Comm. (Jan. 26, 2023):

https://doi.org/10.1038/s41467-023-36145-4 

Precision-controlled CAR-T-cell immunotherapies could be used to tackle a range of tumor types.

Elaborately engineered immune cells can not only recognize cancer cells, but also evade defenses that tumors use to fend off attacks, researchers have found. Two studies recently published in Science1,2 build on the success of chimeric antigen receptor (CAR)-T cancer therapies, which use genetically altered T cells to seek out tumors and mark them for destruction. These treatments have the potential to lead to long-lasting remission, but are not successful for everyone, and have so far been effective against only a small number of cancers.

To bolster the power of CAR-T therapies, researchers have further engineered the cells to contain switches that allow control over when and where the cells are active. The hacked cells produce a protein that stimulates T cells, to counteract immunosuppressive signals that are often released by tumors. Both studies are a tour de force in T-cell engineering and highlight the direction that researchers want to push CAR-T-cell therapy, says systems immunologist Grégoire Altan-Bonnet at the US National Cancer Institute. “We know a lot of the parts, now it’s being able to put them together and explore,” he says. “If we engineer the system well, we can really put the tumors into checkmate.”

Engineered immune cells

T cells typically patrol the body, looking for foreign proteins displayed on the surface of cells. Such cells could be infected with a virus, for example, or they could be tumor cells that are producing abnormal, cancer-associated proteins. A class of T cells called killer T cells can then destroy the abnormal cells. CAR-T therapies involve genetically engineering T cells from a person with cancer to produce CARs, which are proteins that recognize the proteins displayed by tumor cells. The approach has been approved to treat some leukemias, lymphomas and myelomas. But researchers have been pursuing ways to make the treatments safer and more effective, and to expand their use to other diseases.

Highly mutated cancers respond better to immune therapy

In one of the new studies, Ahmad Khalil, a synthetic biologist at Boston University in Massachusetts, and his colleagues wired a complex system of 11 DNA sequences into CAR T cells. The resulting genetic circuits can be switched on and off using already-approved drugs, which allows researchers to control when and where the hacked T cells are active, as well as their production of a protein called IL-2, which stimulates immune responses. The other group of researchers, led by synthetic biologist Wendell Lim at the University of California, San Francisco, programmed CAR T cells to produce IL-2 only when the engineered T cell encounters a cancer cell. The team found that this IL-2 production was most efficient at fighting tumors in mice with pancreatic cancer when it was activated through a pathway that was separate from the one used to recognize the cancer cell — a detail that could help in shaping future therapies, says Andrea Schietinger, a tumor immunologist at Memorial Sloan Kettering Cancer Center in New York City.

Phages probably picked up DNA-cutting systems from their microbial hosts, and may use them to fight other viruses. A systematic sweep of viral genomes has revealed a trove of potential CRISPR-based genome-editing tools.

CRISPR–Cas systems are common in the microbial world of bacteria and archaea, where they often help cells to fend off viruses. But an analysis1 published on 23 November 2022 in Cell finds CRISPR–Cas systems in 0.4% of publicly available genome sequences from viruses that can infect these microbes.

Researchers think that the viruses use CRISPR–Cas to compete with one another — and potentially also to manipulate gene activity in their host to their advantage. Some of these viral systems were capable of editing plant and mammalian genomes, and possess features — such as a compact structure and efficient editing — that could make them useful in the laboratory.

“This is a significant step forward in the discovery of the enormous diversity of CRISPR–Cas systems,” says computational biologist Kira Makarova at the US National Center for Biotechnology Information in Bethesda, Maryland. “There is a lot of novelty discovered here.”

DNA-cutting defenses

Although best known as a tool used to alter genomes in the laboratory, CRISPR–Cas can function in nature as a rudimentary immune system. About 40% of sampled bacteria and 85% of sampled archaea have CRISPR–Cas systems. Often, these microbes can capture pieces of an invading virus’s genome, and store the sequences in a region of their own genome, called a CRISPR array. CRISPR arrays then serve as templates to generate RNAs that direct CRISPR-associated (Cas) enzymes to cut the corresponding DNA. This can allow microbes carrying the array to slice up the viral genome and potentially stop viral infections. Viruses sometimes pick up snippets of their hosts’ genomes, and researchers had previously found isolated examples of CRISPR–Cas in viral genomes. If those stolen bits of DNA give the virus a competitive advantage, they could be retained and gradually modified to better serve the viral lifestyle. For example, a virus that infects the bacterium Vibrio cholera uses CRISPR–Cas to slice up and disable DNA in the bacterium that encodes antiviral defences2.

Molecular biologist Jennifer Doudna and microbiologist Jillian Banfield at the University of California, Berkeley, and their colleagues decided to do a more comprehensive search for CRISPR–Cas systems in viruses that infect bacteria and archaea, known as phages. To their surprise, they found about 6,000 of them, including representatives of every known type of CRISPR–Cas system. “Evidence would suggest that these are systems that are useful to phages,” says Doudna. The team found a wide range of variations on the usual CRISPR–Cas structure, with some systems missing components and others unusually compact. “Even if phage-encoded CRISPR–Cas systems are rare, they are highly diverse and widely distributed,” says Anne Chevallereau, who studies phage ecology and evolution at the French National Centre for Scientific Research in Paris. “Nature is full of surprises.”

Small, but efficient

Viral genomes tend to be compact, and some of the viral Cas enzymes were remarkably small. This could offer a particular advantage for genome-editing applications, because smaller enzymes are easier to shuttle into cells. Doudna and her colleagues focused on a particular cluster of small Cas enzymes called Casλ, and found that some of them could be used to edit the genomes of lab-grown cells from thale cress (Arabidopsis thaliana), wheat, as well as human kidney cells. The results suggest that viral Cas enzymes could join a growing collection of gene-editing tools discovered in microbes.

Although researchers have uncovered other small Cas enzymes in nature, many of those have so far been relatively inefficient for genome-editing applications, says Doudna. By contrast, some of the viral Casλ enzymes combine both small size and high efficiency. In the meantime, researchers will continue to search microbes for potential improvements to known CRISPR–Cas systems.

Makarova anticipates that scientists will also be looking for CRISPR–Cas systems that have been picked up by plasmids — bits of DNA that can be transferred from microbe to microbe. “Each year we have thousands of new genomes becoming available, and some of them are from very distinct environments,” she says. “So it’s really going to be interesting.”

Published in Nature (Nov. 23, 2022): https://doi.org/10.1038/d41586-022-03837-8

Reports of myocarditis, penile necrosis, and atypical/persistent rash requiring amputation.  Be alert for potentially severe manifestations of monkeypox in patients who are immunocompromised or co-infected with HIV, the CDC told healthcare workers on Thursday.

"People who are immunocompromised due to HIV or other conditions are at higher risk for severe manifestations of monkeypox than people who are immunocompetent," the agency said in a health advisory to its Health Alert Network, adding that "the HIV status of all sexually active adults and adolescents with suspected or confirmed monkeypox should be determined."

Of the patients with severe manifestations "for whom CDC has been consulted," the majority had HIV with CD4 counts below 200 cells/mL, indicating "substantial immunosuppression," the agency noted. These severe manifestations have included cases involving atypical or persistent rash requiring amputation of an extremity; secondary bacterial or fungal infections; lesions in sensitive areas resulting in severe pain or urethral or bowel strictures; and co-morbidities involving organ systems, including myocarditis, transverse myelitis, bowel lesions, penile necrosis, and others. The news arrives as the U.S. records its third known monkeypox-associated death in Ohio. To date, over 25,000 monkeypox cases have been reported in the U.S., 38% of which involved patients co-infected with HIV. Researchers have highlighted monkeypox severity and outcomes in people with HIV before.

A recent study published in The Lancet about a 2017-2018 monkeypox outbreak in Nigeria reported seven deaths in a population of 122 patients, with four of the seven co-infected with HIV.  More recently, papers on the current outbreak have reported conflicting results about the association between monkeypox severity and HIV co-infection. CDC data involving more than 1,300 monkeypox patients showed a higher rate of hospitalizations for those with HIV (8% vs 3% for those without HIV), with higher rates for those whose HIV was not virally suppressed. Conversely, German researchers reported that the clinical characteristics in HIV-infected versus non-infected monkeypox patients were largely similar. The new CDC advisory recommends that monkeypox treatment include optimization of immune function for people with immunocompromising conditions, such as limiting the use of immunosuppressive medications when "not otherwise clinically indicated," and offering antiretroviral therapy for those living with HIV. 

Severe immunocompromising conditions can including those with autoimmune disease where immunodeficiency is a clinical component; leukemia or lymphoma, transplant recipients; and those treated with high-dose steroids, alkylating agents, antimetabolites, radiation, or tumor necrosis factor (TNF) inhibitors.  On a case-by-case basis, medications such as oral and intravenous tecovirimat (Tpoxx), cidofovir or brincidofovir, and vaccinia immune globulin intravenous should be considered, "although there are no data on effectiveness in treating human monkeypox with these medical countermeasures," according to the CDC. Clinicians should gather repeat lesion swabs in patients with persistent monkeypox DNA, and continue tecovirimat beyond 14 days (but not more than 90 days), "until there is clinical improvement," the advisory stated. Modifications to the dose, frequency, and duration of tecovirimat may be necessary, depending on the patient's clinical condition, disease progression, or therapeutic response. The advisory encouraged clinicians to consult with the CDC Monkeypox Response Clinical Escalations team when appropriate.  The following severe manifestations seen in monkeypox patients were reported to the CDC, although the advisory did not include information about HIV status:

• Atypical or persistent rash with coalescing or necrotic lesions, or both, some of which have required extensive surgical debridement or amputation of an affected extremity

• Lesions on a significant proportion of the total body surface area, which may be associated with edema and secondary bacterial or fungal infections among other complications

• Lesions in sensitive areas (including mucosal surfaces such as the oropharynx, urethra, rectum, and vagina) resulting in severe pain that interferes with activities of daily living

• Bowel lesions that are exudative or cause significant tissue edema, leading to obstruction

• Severe lymphadenopathy that can be necrotizing or obstructing (such as in airways)

• Lesions leading to stricture and scar formation resulting in significant morbidity such as urethral and bowel strictures, phimosis, and facial scarring

CDC Health Advisory published September 29, 2022

Low cortisol levels and herpes-virus reactivation are associated with prolonged COVID-19 symptoms, preliminary research suggests.  Researchers looking for biological drivers and markers of long COVID have linked the syndrome to herpes viruses, as well as to reduced levels of a stress hormone1. The exploratory study drew both praise and criticism from scientists contacted by Nature. “A major weakness of this paper is the very small sample size,” says infectious-disease specialist Michael Sneller at the US National Institute of Allergy and Infectious Diseases (NIAID) in Bethesda, Maryland. “I would take with a huge grain of salt all these findings unless they are confirmed in larger studies.” But Danny Altmann, an immunologist at Imperial College London who was not involved in the study, says such reports are exactly what the long-COVID research community needs. “We’re really desperate to work out which drugs to test” for long COVID, he says. “The more studies we have like this, the more it enables us to race off and plan appropriate randomized, controlled trials.” The study, posted on the preprint server medRxiv, has not yet been peer reviewed.

The disease that lingers

Long COVID consists of a constellation of sometimes debilitating symptoms that last for months or years after a SARS-CoV-2 infection. It is common — affecting anywhere from 5% to 50% of people who contract COVID-19 — but researchers have few clues to its causes. The latest study, led by immunobiologist Akiko Iwasaki at Yale School of Medicine in New Haven, Connecticut, involved performing tests that generated thousands of data points for each of 215 participants in an attempt to reveal key immunological features that distinguish people with long COVID from healthy individuals. The study included 99 people with long COVID — those who reported persistent symptoms after SARS-CoV-2 infection — and 116 healthy participants who had either never had COVID-19 or had recovered after SARS-CoV-2 infection. Most strikingly, the study found that in the long-COVID group, levels of cortisol, a stress hormone that has a role in regulating inflammation, blood sugar levels and sleep cycles, were about 50% lower than in healthy participants. The authors also found hints that in people with long COVID, Epstein–Barr virus, which can cause mononucleosis, and varicella-zoster virus, which causes chickenpox and shingles, might recently have been ‘reactivated’. Both of these viruses are in the herpes family, persist indefinitely in the body after infection and can start to multiply again after a period of quiescence. People in the long-COVID group also had unusual levels of certain types of blood and immune cell, as well as dysfunctional immune cells, suggesting that their immune systems are working overtime.  Iwasaki cautions that the results do not suggest that low cortisol levels or herpes viruses cause long COVID, but that their association with the syndrome warrants further study. The findings build on previous reports associating low cortisol levels and Epstein–Barr virus with long COVID, including a Cell paper2 published in March.

With only 215 participants, the Iwasaki’s study is relatively small, and only a subset of the participants were included in some parts of the work. For example, in the analysis of cortisol levels, the researchers included all 99 people with long COVID, but only 40 of the 116 healthy people. “The small number of controls compared to the long-COVID group is problematic” because it makes the study less statistically robust, says Sneller, who is leading a separate study on the drivers of long COVID. Iwasaki says that samples were collected from the remaining healthy volunteers later and are being sent for analysis this week. Another limitation of the study is that Iwasaki’s team did not analyse participants’ samples for the presence of the Epstein–Barr and varicella-zoster viruses, instead measuring levels of antibodies against the viruses. Members of the long-COVID group had higher levels of these antibodies than did the healthy people, which suggests a more recent reactivation of the virus, Iwasaki says. However, the only way to determine definitively whether there is current reactivation is to test for viral DNA in the blood.  Iwasaki’s team is now running such tests. Her group also plans to test participants’ cortisol levels throughout the day, because the stress hormone’s levels tend to fluctuate, and to examine how immune features vary at different time points after infection. “This is an exploratory, hypothesis-generating study, and clearly not the conclusion to what long COVID is, but it does begin to open doors,” says Iwasaki.

Published in Nature (August 25, 2022):

https://doi.org/10.1038/d41586-022-02296-5