Evolution of influenza virus variants recapitulated by serial human-to-human transmission through human Lung Airway Chip culture devices
By Benjamin Boettner
(BOSTON) – Influenza virus was the cause of the flu pandemic of 1918 that killed over 20 million people world-wide, and different variants continue to cause new epidemic flu outbreaks every year that threaten the health and livelihoods of many. The Centers for Disease Control and Intervention (CDC) estimate that influenza has resulted in between 9 million and 45 million illnesses, 140,000 and 810,000 hospitalizations, and 12,000 and 61,000 deaths annually since 2010. Especially, people with chronic illnesses or compromised immune systems, pregnant women, people 65 years and older, and children younger than 5 years old are at risk.
What makes influenza so difficult to control is the fact that this virus mutates rapidly in humans and some animals, and thus constantly evolves. This in turn blunts or even obliterates the efficacy of vaccines and antiviral therapeutics. To outrace the virus, vaccine and drug developers have to continually create new and more effective therapies against mutant variants that have become resistant to earlier treatments. To predict the emergence of new variants, researchers rely on ferrets as a model because they can be infected by the virus and develop disease symptoms that are more similar to those in humans than other animal models. However, because the ferret’s immune system differs from that of humans, the current success rate of predicting resistance to anti-viral drugs and vaccines remains fairly low.
Now, a team in the Bioinspired Therapeutics & Diagnostics Platform led by Founding Director Donald Ingber, M.D., Ph.D. at Harvard’s Wyss Institute for Biologically Inspired Engineering have used their microfluidic human Lung Airway-on-a-Chip (Airway Chip) culture device to mimic viral evolution during human-to-human transmissions, and demonstrate the appearance of influenza virus variants that evolve to escape attack with antiviral drugs. Some of the spontaneously emerging mutations were already found in patients, while other ones have not been described before. The findings are reported in Microbiology Spectrum.
“Our demonstration that human Lung Chips can be used to reconstitute influenza virus evolution in vitro should enable other researchers to rapidly and comprehensively identify variants of any potential pandemic respiratory virus, including SARS-CoV-2, that are likely to emerge in patients. This could give medicine a significant advantage in the relentless race with these viruses and help accelerate the identification of next-generation therapeutics and prophylactics,” said Ingber, who also is the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS).
In 2017, the Wyss Institute, with support from the National Institutes of Health (NIH) and the Defense Advanced Research Projects Agency (DARPA), started to use its Organ Chip platform to mimic influenza infection and pathogenesis in vitro, and to identify new drug leads that target the host response. The microfluidic Airway Chip is an optically clear silicone rubber device that contains two parallel microchannels each no wider than a pencil lead which are separated by a porous membrane. In the “airway channel,” the membrane is lined by primary human lung airway epithelial cells cultured under an air-liquid interface that resembles the milieu of the lung’s airways. The opposite “blood channel” is lined with human pulmonary microvascular endothelial cells in the presence of a continuous flow of medium, with or without human immune cells, mimicking the natural perfusion of pulmonary blood vessels with circulating blood.
In a previously published study, the team successfully modeled lung infection with respiratory viruses, including influenza A and a pseudotyped SARS-CoV-2 virus, using their Lung Chip technology, and, in a larger collaborative effort, studied virus-specific responses in lung tissue (host responses), and the effectiveness of new repurposed candidate antiviral drugs.
Our demonstration that human Lung Chips can be used to reconstitute influenza virus evolution in vitro should enable other researchers to rapidly and comprehensively identify variants of any potential pandemic respiratory virus, including SARS-CoV-2, that are likely to emerge in patients.
“To study the evolution of the virus, we started by introducing influenza virus into the airway channel of the device, and adding known antiviral drugs to the blood channel. After a short incubation time, we then passaged infected mucus droplets to another human Airway Chip also treated with drug, and repeated this multiple times to mimic human-to-human transmission, analyzing the virus genome after every passage,” said co-first author and Wyss Postdoctoral Fellow Haiqing Bai, Ph.D. “This enabled us to recover mutations in the viral genome that also have been described to frequently cause resistance to the anti-viral drugs amantadine and oseltamivir in patients, and other ones that have not been described yet but could help expand our understanding of how drug resistance occurs in the virus.” Amantadine targets the influenza virus’ so-called M2 ion channel surface protein, and oseltamivir, which is known under the trade name Tamiflu®, the virus’ neuraminidase surface protein. While resistance of influenza viruses to amantadine is wide-spread, only a small number of viruses have been found to be resistant to oseltamivir, which is why the CDC maintains its recommendation for the drug.
Interestingly, the Wyss study recapitulated this difference in vitro: “Influenza A virus developed significant resistance against amantadine already after eight chip-to-chip passages, while it took more than 25 passages to become resistant to oseltamivir. A third therapeutic called nafamostat, which we had identified as a potential treatment for flu earlier, and which targets the human host response rather than a virus protein, did not induce viral resistance even after 30 passages,” said co-first author Longlong Si, Ph.D. “This shows that our system can be used to evaluate the tendency of the virus to become resistant to a therapeutic or vaccine, and also that targeting host responses rather than the virus may be less likely to result in development of resistance.”
In their previous study, the team actually showed that combining oseltamivir with nafamostat extended oseltamivir’s therapeutic window – the time in which patients can still be effectively treated after they have been infected with influenza – from a two-day to a four-day-period. Si worked as a Postdoctoral Fellow on Ingber’s team at the time of the study, and now is Professor at the Institute of Synthetic Biology of the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences.
In addition, the team also demonstrated that genetic “reassortment,” the process in which the eight distinct segments of the influenza genome can be reshuffled in lung cells that are co-infected by two different viruses, is mimicked by their Airway Chip and leads to drug resistance. Reassortment leads to dramatic changes to viral genomes with entire protein-encoding sequences being swapped between viruses, which has great pandemic potential.
“In this study, we captured changes to the viral genome that occur under the pressure of antiviral drugs. Our approach can be extended to also identify future vaccine-resistant variants, investigate if variants emerge more rapidly in hosts that are predisposed by certain diseases, and find measurable parameters that would allow better matching of specific therapeutics and vaccines with predisposed and previously healthy people, or geographically restricted populations,” said Ingber.
Other authors on the study are Crystal Yuri Oh, and Lei Jin at the Wyss Institute, and former Wyss Senior Staff Scientist Rachelle Prantil-Baun, Ph.D. The study was funded by the NIH under grant #NCATS 1-UH3-HL-141797-01, Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement #HR00111920008, and the Wyss Institute for Biologically Inspired Engineering.