Virus World

Over four years have passed since the beginning of the COVID-19 pandemic. The scientific response has been rapid and effective, with many therapeutic monoclonal antibodies and small molecules developed for clinical use. However, given the ability for viruses to become resistant to antivirals, it is perhaps no surprise that the field has identified resistance to nearly all of these compounds. Here, we provide a comprehensive review of the resistance profile for each of these therapeutics. We hope that this resource provides an atlas for mutations to be aware of for each agent, particularly as a springboard for considerations for the next generation of antivirals. Finally, we discuss the outlook and thoughts for moving forward in how we continue to manage this, and the next, pandemic.

Published (April 18, 2024):

https://doi.org/10.1016/j.chembiol.2024.03.008

Vaccines and drugs have helped reduce disease severity and blunt the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, ongoing virus transmission, continuous evolution, and increasing selective pressures have the potential to yield viral variants capable of resisting these interventions. Here, we investigate the susceptibility of natural variants of the main protease [Mpro; 3C-like protease (3CLpro)] of SARS-CoV-2 to protease inhibitors. Multiple single amino acid changes in Mpro confer resistance to nirmatrelvir (the active component of Paxlovid). An additional clinical-stage inhibitor, ensitrelvir (Xocova), shows a different resistance mutation profile.

Importantly, phylogenetic analyses indicate that several of these resistant variants have pre-existed the introduction of these drugs into the human population and are capable of spreading. These results encourage the monitoring of resistance variants and the development of additional protease inhibitors and other antiviral drugs with different mechanisms of action and resistance profiles for combinatorial therapy.

Published in Science Advances (March 29, 2023)

The antiviral Molnupiravir (Lageviro) is widely used across the world to treat SARS-CoV-2 infection. Molnupiravir reduces viral replication by inducing mutations throughout the genome, yet in patients that do not clear the infection, the longer-term impact of the drug on virus evolution is unclear. Here, we used a case-control approach to monitor SARS-CoV-2 genomes through time in nine immunocompromised -patients with five treated with Molnupiravir. Within days of treatment, we detected a large number of low-frequency mutations in patients and that these new mutations could persist and, in some cases, were fixed in the virus population. All patients treated with the drug accrued new mutations in the spike protein of the virus, including non-synonymous mutations that altered the amino acid sequence. Our study demonstrates that this commonly used antiviral can supercharge viral evolution in immunocompromised patients, potentially generating new variants and prolonging the pandemic.

Preprint available in  medRxiv (Dec. 22, 2022):

 https://doi.org/10.1101/2022.12.21.22283811 

Lab studies identify resistance mutations in SARS-CoV-2’s protease, and some circulating variants have them.  Prescriptions for Pfizer’s blockbuster drug Paxlovid have skyrocketed in recent weeks. That’s good news for many COVID-19 patients, as the pill has been proven to reduce severe disease from SARS-CoV-2 infections. But a bevy of new lab studies shows the coronavirus can mutate in ways that make it less susceptible to the drug, by far the most widely used of the two oral antiviral drugs authorized to treat COVID-19 in the United States. Researchers have found some of those mutations in variants already circulating in infected people, raising fresh concerns that physicians could soon lose one of their best therapies for fighting COVID-19. Taken together, the studies show that “when you put pressure on the virus it escapes,” says David Ho, a virologist at Columbia University who was among the first to document drug resistance mutations in HIV some 3 decades ago. Ho was not involved with the new studies but is conducting similar work on SARS-CoV-2. Although the newly identified mutations have yet to become widespread, Ho and many other scientists think it’s only a matter of time. “Given the amount of infections out there, it’s going to come,” Ho says. The resistance studies come on the heels of other recent concerns about Paxlovid, which in the United States remains restricted to use in people with risk factors making them more likely to develop severe COVID-19. Confirming anecdotal reports widely reported by media, several studies have found a small percentage of infected people who receive the normal 5-day course initially feel better, only to have their symptoms rebound. And questions have grown about whether Paxlovid helps those who aren’t at high risk of serious disease—Pfizer earlier this month halted a large trial of the drug in standard risk COVID-19 patients because it was failing to show statistically significant protection against death or hospitalization. The U.S. Food and Drug Administration (FDA) granted emergency use authorization for Paxlovid in December 2021. The drug consists of nirmatrelvir, the active antiviral, and ritonavir, a compound that slows the breakdown of nirmatrelvir in the body. Because of bottlenecks in manufacturing nirmatrelvir, Paxlovid’s rollout was slow—doctors in the United States issued only 40,000 or fewer prescriptions per week through mid-April. Since then, prescriptions have surged to more than 160,000 per week, according to the latest numbers from the Centers for Disease Control and Prevention.

That rise creates selective pressure on the virus, favoring mutations that help it survive in the presence of the drug. And because each infected person makes trillions of copies of SARS-CoV-2, the virus has plenty of opportunities to test out different mutations as it replicates.   So far, those mutations don’t seem to have interfered with Paxlovid’s effectiveness. Nirmatrelvir prevents SARS-CoV-2’s main protease (MPRO) from cutting a long precursor molecule made by the virus into shorter active proteins, an essential step in SARS-CoV-2’s reproduction. In February, Pfizer researchers reported in JBC Accelerated Communications that nirmatrelvir remained effective in halting the activity of MPRO in multiple SARS-CoV-2 variants, including Alpha, Beta, Delta, Gamma, Lambda, and Omicron, as well as the original strain.  However, the recent studies suggest the virus is poised to develop resistance—a fate that befalls many antiviral drugs. Two preprints posted on bioRxiv on 7 June, for example, show that SARS-coV-2 grown in the lab quickly gains the ability to avoid nirmatrelvir’s attack. Two research groups independently cultured the coronavirus with low levels of nirmatrelvir, killing some but not all of the virus. Such tests are meant to simulate what might happen in an infected person who doesn’t take the whole regimen of the drug or an immunocompromised patient who has trouble clearing the virus. One of those studies, led by Dirk Jochmans, a virologist at KU Leuven in Belgium, found that after 12 rounds of nirmatrelvir treatment, SARS-CoV-2 accumulated three mutations—at positions 50, 166, and 167 in the string of amino acids that make up MPRO–that reduced the virus’ susceptibility to nirmatrelvir 20-fold, as determined by the dose of drug required to kill half the virus in a sample. The other study, led by Judith Margarete Gottwein, an immunologist at the University of Copenhagen, also spotted potential resistance-conferring mutations at positions 50 and 166 in MPRO . When those mutations occurred together, the virus was 80 times less susceptible to nirmatrelvir. “This tells us what mutations we should be looking for [in patients],” Gottwein says. Indeed, some of these mutations are already in coronavirus-infected people, according to work by Adam Godzik, a bioinformatics expert at the University of California, Riverside. Godzik and his colleagues scoured the GISAID database, a catalog of more than 10 million SARS-CoV-2 genomes sequenced from viruses isolated from infected individuals, searching for amino acid changes at positions in MPRO near where nirmatrelvir binds. In a bioRxiv preprint posted on 30 May, they reported that mutations to amino acids 166 and 167—two of the resistance mutations flagged by the Belgian group—were already in viruses circulating in people. Because these mutations occurred before widespread use of Paxlovid, they likely occurred randomly, Godzik says. However, he adds, they reveal the enzyme has some flexibility at these positions that could help the virus work around the drug.

And the list of potential resistance mutations keeps growing. In a paper posted yesterday on bioRxiv, Jun Wang, a medicinal chemist at Rutgers University and colleagues report 66 common mutations to MPRO near the nirmatrelvir binding site. Like Godzik’s team, they scanned the GISAID database to find altered versions of the protease, but then went a step further. Adding the gene for each of these variants of MPRO to Escherichia coli bacteria, they created supplies of the enzymes for additional tests: first to determine whether each variant still carried out the essential duties of cutting viral proteins, and second to determine whether the mutations allowed MPRO to resist nirmatrelvir. Eleven of the 66 variants retained the protease’s function (the others impaired it), and five of the 11 were resistant to nirmatrelvir, requiring at least a 10-fold increase in the drug to kill half the virus in the sample. One of those variants had a previously seen resistance mutation, at position 166, but the other four had novel workarounds at positions 144, 165, 172, and 192. The bottom line from all this work, Wang says: “It’s just a matter of time before we see resistance emerge.” So, why hasn’t it happened already? One possibility is that not enough people have taken Paxlovid yet to force the virus to mutate. Another explanation, Wang says, is that it may take multiple mutations in MPRO for the virus to get around Paxlovid while remaining both fully functional and easily transmissible. Thus far, adds Aditya Shah, an infectious disease specialist at the Mayo Clinic, studies show that patients who have rebound of symptoms, which happens in just 2% or fewer of those who take the drug, the rebound does not seem to be due to resistance mutations. “It’s reassuring,” Shah says, but not proof the virus won’t eventually find its way around the drug. Pfizer says its Paxlovid regimen may forestall resistance. Patients only take the drug for a short period and typically get a dose “manyfold higher” than that required to prevent the virus from replicating in cells, thereby minimizing the opportunities for the virus to mutate, says Kit Longley, a company spokesperson.

Giving patients multiple antivirals could help prevent resistance by making it harder for the virus to evolve its way around different compounds at the same time, a strategy that has proved highly effective in treating other viruses, including HIV and hepatitis C, Ho says. Two other SARS-CoV-2 antivirals are authorized in the United States, but they have drawbacks. The other oral drug, molnupiravir, has proven considerably less effective than Paxlovid, and has raised safety concerns because it induces random genetic mutations in the virus—that typically stops it from replicating but could also spawn dangerous new variants, some scientists caution. And remdesivir, which interferes with the ability of the virus to copy its genome, is only authorized for hospitalized patients and must be delivered intravenously. A preprint posted on bioRxiv yesterday suggests combining molnupiravir and nirmatrelvir is more effective in combating SARS-CoV-2 infections than either antiviral given alone, at least in mice. But the strategy has yet to be widely embraced by doctors. Meanwhile, pharmaceutical companies are racing to complete clinical trials on additional SARS-CoV-2 antivirals, some targeting MPRO at different sites. But those aren’t available yet. And numerous researchers, including representatives of the nonprofit Drugs for Neglected Diseases Initiative, have complained that Pfizer has not made Paxlovid easily available for trials of combination therapies. The company has said it was planning on doing those studies itself, although some are skeptical. Until more antiviral drugs become available, Paxlovid will remain essentially alone, raising fears that sooner or later it will lose its punch. When pressed by a single antiviral, viruses usually find a way around the drug, Gottwein says. “If it can happen it will happen.” And at least according to the latest lab results, it can happen.

The mutated version of the virus exhibited resistance to the antiviral drug remdesivir.  In a preprint study published on Medrxiv, researchers found that a woman in her 70s who had been treated with the drug had failed to completely clear her Covid-19 infection for several months. A genetic examination of the virus showed that it had mutated during treatment in a manner that reduced the effectiveness of the antiviral drug, manufactured by Gilead. According to Professor Akiko Iwasaki, one of the study’s authors, remdesivir initially brought the patient’s viral load down but it rose again during the course of the remdesivir treatment.  The persistent infection was eventually successfully treated using monoclonal antibodies, according to the study. The woman who exhibited the mutation was immunocompromised as she had previously been treated for non-Hodgkin’s lymphoma. 

Dengue virus (DENV) infects about 400 million people annually around the world, with a high prevalence in tropical and sub-tropical regions.The virus causes diseases ranging from mild dengue fever to severe dengue haemorrhagic fever and dengue shock syndrome.

DENV2 exists as smooth spherical surface particles while growing at the mosquito’s physiological temperature (29 degrees Celsius). It then changes to bumpy surfaced particles at human physiological temperature (37 degrees Celsius). This ability to morph helps the virus to evade the immune system of the human host. Hence, understanding the mechanism behind this is important for therapeutics and vaccine development. “Together with Professor Pei-Yong Shi from UTMB, we found that in laboratory developed DENV2 strains, mutations in the virus’ E protein causes its transformation into bumpy particles. These structural changes can cause vaccines and therapeutics to be ineffective against the virus,” said Ms Xin-Ni Lim, the study’s lead author who is from Duke-NUS’ Emerging Infectious Diseases (EID) Programme.

The team also tested four DENV2 strains obtained from patients. They observed that in contrast to the laboratory adapted viruses, the majority of these clinical strains maintained smooth surface structure at 37 degrees Celsius. However, at 40 degrees Celsius, the temperature of a fever, all virus strains took on a bumpy surface. “Our study gives a new direction to vaccine development and treatment for dengue disease. For prevention of disease through vaccines that are administered to the patient before dengue infection, we should use those that are effective against the smooth surface virus. When it comes to patients displaying fever symptoms, treatment strategies effective against the bumpy surface particles should be implemented,” said Dr Sheemei Lok, Professor, Duke-NUS’ EID and corresponding author of this study.

“This study is a first step towards gaining more insight into how DENV2 reacts and adapts to the host’s immunological defenses. We were also able to use computational modelling approaches to predict why particles from different DENV2 strains are more or less adept at morphing from the smooth to bumpy structures. By better understanding the interactions between the virus and the host, we will be able to develop better therapies and vaccines to treat or prevent infections, and contribute to public health outcomes,” said Dr Peter Bond, Principal Investigator from A*STAR’s BII.  The study’s findings also show that the lab adapted DENV2 may not be a good model for research, as its structure is different from the clinical strains isolated from patients. The team is planning to study the other DENV serotypes to find out if there are any other possible structural changes.

Study published on September 19, 2019 in PLOS Pathogens (Open Access):

https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1007996

Antiviral drugs are used globally as treatment and prophylaxis for long-term and acute viral infections. Even though antivirals also have been shown to have off-target effects on bacterial growth, the potential contributions of antivirals to antimicrobial resistance remains unknown. Herein we explored the ability of different classes of antiviral drugs to induce antimicrobial resistance. Our results establish the previously unrecognized capacity of antivirals to broadly alter the phenotypic antimicrobial resistance profiles of both gram-negative and gram-positive bacteria Escherichia coli and Bacillus cereus. Bacteria exposed to antivirals including zidovudine, dolutegravir and raltegravir developed cross-resistance to commonly used antibiotics including trimethoprim, tetracycline, clarithromycin, erythromycin, and amoxicillin.

Whole genome sequencing of antiviral-resistant E. coli isolates revealed numerous unique single base pair mutations, as well as multi-base pair insertions and deletions, in genes with known and suspected roles in antimicrobial resistance including those coding for multidrug efflux pumps, carbohydrate transport, and cellular metabolism. The observed phenotypic changes coupled with genotypic results indicate that bacteria exposed to antiviral drugs with antibacterial properties in vitro can develop multiple resistance mutations that confer cross-resistance to antibiotics. Our findings underscore the potential contribution of wide scale usage of antiviral drugs to the development and spread of antimicrobial resistance in humans and the environment. Bacteria exposed to antiviral drugs in vitro can develop multiple resistance mutations that confer cross-resistance to antibiotics.

Published (Aug.12, 2023) in Communications Biology:

https://doi.org/10.1038/s42003-023-05177-3

Protease inhibitors are among the most powerful antiviral drugs. Nirmatrelvir is the first protease inhibitor specifically developed against the SARS-CoV-2 protease 3CLpro that has been licensed for clinical use. To identify mutations that confer resistance to this protease inhibitor, we engineered a chimeric vesicular stomatitis virus (VSV) that expressed a polyprotein composed of the VSV glycoprotein (G), the SARS-CoV-2 3CLpro, and the VSV polymerase (L). Viral replication was thus dependent on the autocatalytic processing of this precursor protein by 3CLpro and release of the functional viral proteins G and L, and replication of this chimeric VSV was effectively inhibited by nirmatrelvir.

Using this system, we applied nirmatrelvir to select for resistance mutations. Resistance was confirmed by retesting nirmatrelvir against the selected mutations in additional VSV-based systems, in an independently developed cellular system, in a biochemical assay, and in a recombinant SARS-CoV-2 system. We demonstrate that some mutants are cross-resistant to ensitrelvir and GC376, whereas others are less resistant to these compounds. Furthermore, we found that most of these resistance mutations already existed in SARS-CoV-2 sequences that have been deposited in the NCBI and GISAID databases, indicating that these mutations were present in circulating SARS-CoV-2 strains.

Published in Science Translational Medicine:

https://doi.org/10.1126/scitranslmed.abq7360 

Monoclonal antibodies (mAbs) offer a treatment option for individuals with severe COVID-19 and are especially important in high-risk individuals where vaccination is not an option. Given the importance of understanding the evolution of resistance to mAbs by SARS-CoV-2, we reviewed the available in vitro neutralization data for mAbs against live variants and viral constructs containing spike mutations of interest. Unfortunately, evasion of mAb-induced protection is being reported with new SARS-CoV-2 variants. The magnitude of neutralization reduction varied greatly among mAb–variant pairs. For example, sotrovimab retained its neutralization capacity against Omicron BA.1 but showed reduced efficacy against BA.2, BA.4 and BA.5, and BA.2.12.1. At present, only bebtelovimab has been reported to retain its efficacy against all SARS-CoV-2 variants considered here.

Resistance to mAb neutralization was dominated by the action of epitope single amino acid substitutions in the spike protein. Although not all observed epitope mutations result in increased mAb evasion, amino acid substitutions at non-epitope positions and combinations of mutations also contribute to evasion of neutralization. This Review highlights the implications for the rational design of viral genomic surveillance and factors to consider for the development of novel mAb therapies. In this Review, Carabelli, Robertson and colleagues explore data on the neutralization of globally circulating variants of concern by monoclonal antibodies (mAbs) and discuss how knowledge of the dynamics of viral evasion of mAbs can contribute to viral surveillance and the development of novel mAb treatments, as well as inform predictions of resistance that may arise in the future.

Published in Nature (October 28, 2022):

https://doi.org/10.1038/s41579-022-00809-7 

For the first time, bacteriophages — viruses that kill bacteria — eliminated an antibiotic-resistant mycobacterial lung infection in a man with advanced cystic fibrosis (CF) lung disease, a study reported. As a result, the patient was able to undergo a successful lung transplant after on.  “I am so grateful for the effort, persistence and creativity of all the people who were involved in my treatment,” Jarrod Johnson, the recipient of the lung transplant, said in a press release. “I thought I was going to die. They have literally saved my life.” Added Jerry Nick, MD, study lead and director of the adult CF program at National Jewish Health in Colorado: “This research can serve as a road map for future use of phages to treat patients with severe Mycobacterium abscessus lung infection and to save lives.” The study, “Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection,” was published in Cell. The buildup of thick mucus in the lungs is a hallmark of CF, which can lead to recurrent and damaging bacterial infections and eventually lung failure, requiring transplant. Mycobacteria are a common type of bacteria that can cause tuberculosis, leprosy, and non-tuberculous mycobacterial (NTM) infections. They can be aggressive and difficult to treat, even with combinations of multiple antibiotics over long periods. Johnson is a 26-year-old man with CF who experienced repeated lung infections throughout childhood and, as a result, was admitted to hospitals several times a year. As an adult, his lung function rapidly declined due to persistent infection by Mycobacterium abscessus, which did not respond to antibiotic treatment. It recently dropped below 30% and he needed a transplant. However, due to the ongoing mycobacteria infection, which had spread to his skin and other tissues, and the lack of effective treatments, he was unable to undergo the procedure.

Bacteriophages, or phages, are viruses that selectively target and kill bacteria. There has been a growing interest in using phages to treat antibiotic-resistant bacterial infections in recent years. To date, phages have not been used to treat a mycobacterial infection of the lungs. In this report, Nick and colleagues describe how Johnson’s mycobacteria infection was eradicated by phage therapy, which allowed him to undergo a lung transplant. “We had tried unsuccessfully for years to clear the mycobacterial infection with a variety of antibiotics,” Nick said. “When we used the bacteria’s own natural enemies, we were able to clear the infection which resulted in a successful lung transplant.” Sputum samples were collected from Johnson, M. abscessus was grown in culture, then screened against a panel of phages, which were genetically engineered to improve their bacteria-killing potential. Two specific phages were identified that efficiently killed the bacteria, either alone or in combination, over a wide range of phage and bacterial levels. While he was in the hospital for an acute worsening of his symptoms, the therapeutic phage cocktail was infused into Johnson’s bloodstream twice daily. It was well tolerated, with no reported side effects. He was discharged five days later and continued on two phage treatments a day. Johnson was readmitted to the hospital four times in the year after starting phage therapy because of pulmonary exacerbations. Sputum samples were collected for up to a year, which were largely positive for M. abscessus to day 96 (about three months). However, most of the following cultures were negative for up to one year. At around three months, he began CF treatment with Trikafta.

Although his lung function ranged from 23% to 31% over the year following phage therapy, similar to before treatment, his time in the hospital decreased. Based on the absence of M. abscessus infection, he was listed for a lung transplant one year after phage therapy and underwent a successful transplant soon after, in October 2021. Phage therapy continued throughout his recovery for up to 500 days (about 16 months). CT scans of his lung during phage therapy showed some improvements over time, along with markers for M. abscessus death. There was no evidence of resistance to phage therapy. All M. abscessus samples isolated over the course of his infection were tested against 20 antibiotics, most of which were consistently resistant or demonstrated little variability, “suggesting the combination of phage and antibiotics did not result in greater antibiotic resistance,” the researchers wrote. Lastly, because phage treatment can fail due to the emergence of anti-phage antibodies, his blood was collected and tested. Surprisingly, Johnson had a substantial antibody response to both phages even before treatment, suggesting that “this individual had naturally encountered phages,” the researchers noted. Antibody levels increased modestly in post-treatment blood samples, but he did not mount a substantial additional antibody response to one of the phages until late in the treatment regimen. At that point, sputum cultures were primarily negative for M. abscessus. “The use of phages in very advanced lung disease with the objective to sufficiently control M. abscessus as a bridge to lung transplant may be considered based on this report,” the researchers wrote. The Cystic Fibrosis Foundation and the National Institutes of Health provided funding for the treatment and study.

Cited research published in Cell (May 13, 2022):

https://doi.org/10.1016/j.cell.2022.04.024 

Similar to bacteria evolving resistance to antibiotics, viruses can evolve resistance to vaccines, and the evolution of SARS-CoV-2 could undermine the effectiveness of vaccines that are currently under development, according to a paper published November 9 in the open-access journal PLOS Biology by David Kennedy and Andrew Read from Pennsylvania State University, U.S. The authors also offer recommendations to vaccine developers for minimizing the likelihood of this outcome. "A COVID-19 vaccine is urgently needed to save lives and help society return to its pre-pandemic normal," said David Kennedy, assistant professor of biology. "As we have seen with other diseases, such as pneumonia, the evolution of resistance can quickly render vaccines ineffective. By learning from these previous challenges and by implementing this knowledge during vaccine design, we may be able to maximize the long-term impact of COVID-19 vaccines." The researchers specifically suggest that the standard blood and nasal-swab samples taken during clinical trials to quantify individuals' responses to vaccination may also be used to assess the likelihood that the vaccines being tested will drive resistance evolution. For example, the team proposes that blood samples can be used to assess the redundancy of immune protection generated by candidate vaccines by measuring the types and amounts of antibodies and T-cells that are present.

"Much like how combination antibiotic therapy delays the evolution of antibiotic resistance, vaccines that are designed to induce a redundant immune response—or one in which the immune system is encouraged to target multiple sites, called epitopes—on the virus's surface, can delay the evolution of vaccine resistance," said Andrew Read, Evan Pugh Professor of Biology and Entomology and director of the Huck Institutes of the Life Sciences. "That's because the virus would have to acquire several mutations, as opposed to just one, in order to survive the host immune system's attack." The researchers also recommend that nasal swabs typically collected during clinical trials may be used to determine the viral titer, or amount of virus present, which can be considered a proxy for transmission potential. They noted that strongly suppressing virus transmission through vaccinated hosts is key to slowing the evolution of resistance, since it minimizes opportunities for mutations to arise and reduces opportunities for natural selection to act on those mutations that do arise. 

In addition, the team suggests that the genetic data acquired through nasal swabs can be used to examine whether vaccine-driven selection has occurred. For example, differences in alleles, or forms of genes that arise from mutations, between the viral genomes collected from vaccinated versus unvaccinated individuals would indicate that selection has taken place. "According to the World Health Organization, at least 198 COVID-19 vaccines are in the development pipeline, with 44 currently undergoing clinical evaluation," said Kennedy. "We suggest that the risk of resistance be used to prioritize investment among otherwise similarly promising vaccine candidates."

Publication cited available in PLOS Biology (Nov. 9 , 2020):

https://doi.org/10.1371/journal.pbio.3001000