Abstract
On October 5, 2020, Dr. Harvey J. Alter, Dr. Michael Houghton, and Dr. Charles M. Rice received the Nobel Prize in Physiology or Medicine for the discovery of hepatitis C virus (HCV). It is not at all surprising that these outstanding scientists were recognized for this monumental achievement when one considers the impact this virus has had on global health. Anyone who has worked in the field of HCV can appreciate the magnitude of effort that was required to find this virus and cure it, but for those who may not be familiar with the history of HCV research, it is a story of undying perseverance that is motivational and inspirational. Given the unfortunate reality that this recognition comes in the middle of a global viral pandemic, the importance of research into virology and viral immunology is appropriately emphasized by the awarding of science's highest honor to the discovery of this globally relevant virus.
My own introduction to HCV happened in 1999 when my MSc supervisor at the time, Dr. Michael Grant, advised me to look for a laboratory for my PhD in which I could possibly do some HCV research in addition to my HIV focus. I remember him saying an interesting article had just been published in Science about “HCV replicons,” and he thought they were going to change the landscape of HCV research. At that time, I was doing my MSc in HIV research and had absolutely no idea what a replicon was, but I remember wondering why so little was known about HCV, and why it was, in my naive opinion, so far behind the HIV field with respect to basic virology and antiviral development. Little did I know. In 2003, when I told my PhD supervisor, the late Dr. Mark Wainberg, that I was switching to HCV for my postdoctoral research, he said, “Why would you want to work on HCV? It doesn't even integrate! That virus will be over in five years!” Mark would ultimately be right, that HCV was indeed a curable virus, but it took longer than five years, and HCV research still is not over.
The strongest theme I see in the field of HCV research is perseverance. Finding HCV was not easy, working with HCV was not easy, curing HCV was not easy, and even now, making an effective vaccine against HCV is proving not to be easy. What impresses me most about the thousands of scientists around the world who contributed to the study of this virus, even before it was discovered, was, and still is, their absolute unwillingness to quit. These scientists leaped along developing system after system to study HCV and never gave up until they ultimately cured it.
Finding the Virus
The story of HCV started similar to that of most viruses, some people get sick, and nobody knows why, just as we saw with AIDS, and now again with COVID-19. Eventually someone finds the new virus that is causing the new disease, but what has changed most over the years, at least for some viral diseases, is the amount of time between these two events. AIDS was first observed in 1981, and the virus was discovered soon after in 1984. COVID-19 was originally described in 2019, and the causative agent was identified less than one month later, which shows how fast science can move now compared with decades ago. Non-A, non-B (NANB) post-transfusion hepatitis (PTH) was first reported in 1975, but it was not until 1989 that an association with a virus was found. This 14-year search was the first chapter of many in the decades of perseverance that ultimately led to the discovery of curative antiviral agents for the treatment of HCV infection.
The story began in 1975 when Harvey Alter, Paul Holland, and Bob Purcell demonstrated that 90% of the PTH patients they studied were serologically negative for hepatitis A and B viruses. Then in 1978, this same group, then including Dr. Hans Popper, showed that plasma or serum from patients with NANB PTH could transmit hepatitis to chimpanzees. Even that early in the story the authors concluded that NANB hepatitis seemed to be due to a transmissible agent that could persist and remain infectious for long periods. NANB hepatitis was later found to be transmissible to humans when it was shown in 1981 that 6 out of 15 individuals who received malaria-rich blood in a 1969 prison volunteer study of malaria transmission subsequently developed hepatitis. Finally, in 1989, using an eloquent blind immunoscreening cDNA cloning strategy and a tour de force of effort, Dr. Michael Houghton and his team published the isolation of a small ∼100 bp cDNA clone (5-1-1) that hybridized to an ∼10 kb RNA molecule that was present only in NANB hepatitis samples. They also found that clone 5-1-1 encoded a protein that was reactive with antibodies found only in the context of NANB hepatitis infection. The isolation of this clone allowed them to walk the genome to identify the novel hepatitis virus, HCV. When I asked Dr. Houghton to comment on receiving the Nobel Prize for this finding, he said, “My success was based on working with very good colleagues at Chiron, Dr. Qui-Lim Choo and Dr. George Kuo, who had great experimental qualities and ideas, and having an external collaborator, Dr. Daniel Bradley, who was a leading expert in animal transmission of HCV (then termed non-A, non-B hepatitis). In addition, focusing on the goal of HCV identification over seven long years, all the time trying numerous different approaches to crack this tough ‘nut’. Along with a contribution from ‘lady luck’ which is always important, these were all contributory factors in our success which was made in a pre-PCR and pre-deep sequencing era.” According to Dr. Matthias Götte, Chair of the Department of Medical Microbiology and Immunology at the University of Alberta where Dr. Houghton currently holds a faculty appointment, “The discovery of HCV was an amazing accomplishment that paved the way for the development of life-saving antiviral treatments.” Dr. Lorne Tyrrell, Founding Director of the Li Ka Shing Institute of Virology, where Dr. Houghton is currently the Director, proudly stated that “The discovery of hepatitis C virus as the causative agent for non-A, non-B hepatitis is one of the most important medical discoveries to benefit humankind in the last 50 years. Michael Houghton, with his colleagues, Qui-Li Choo and George Kuo, made this foundational discovery that spawned a large new field of research and the eventual cure of chronic HCV. There have been very few medical discoveries that have had such a profound impact on public health. I am particularly proud of Michael's achievement and recognition with his co-winners Charlie Rice and Harvey Alter as it is only the second time a Professor at a Canadian university has won this prestigious prize (the first being Banting and Macleod for the discovery of insulin in 1923). This is a momentous occasion for the Li Ka Shing Institute of Virology, the University of Alberta, and Canada.” Dr. Houghton and his group at Li Ka Shing have for years been intensely focused on developing a vaccine for the prevention of HCV infection, and we would all love to someday witness the codiscoverer of HCV developing a vaccine for the human pathogen he is credited with finding.
Proving This Virus Caused NANB Hepatitis
Finding a novel viral sequence associated with NANB hepatitis was the end of the first chapter in the story of HCV, but in many ways, it was also the beginning of the next chapter. At this point, the new viral sequence had not been shown to directly cause liver disease, and studying the virus and finding its weaknesses continued to require perseverance, patience, creativity, and innovation, as well as tireless effort from hundreds of researchers and their trainees. For many viruses, one simply needs to add the virus to cells and then watch it grow. That was not at all the case for HCV. Molecular virologists were immediately confronted with a dilemma—the inability to grow HCV in the laboratory. Charlie Rice and his team began to dive deep into the machinery of the virus in an effort to figure out how to cultivate this virus in the laboratory. After several years of frustrating attempts, using techniques that had worked well for classical flaviviruses, they began to wonder if they might be missing something. Alexander “Sasha” Kolykhalov, an incredible molecular biologist, tackled this challenge by modifying methods they had used for yellow fever virus and revisited the terminal sequences of the HCV genome. Along with Kunitada Shimotohno's laboratory in Japan, the Rice group found a missing piece at the 3′ end of the genome that was highly conserved among diverse HCV genotypes. However, even with the inclusion of this sequence, RNA transcripts were still not infectious in cell culture, nor when injected into the liver of the only nonhuman HCV-susceptible host, the chimpanzee. Again, worried about what might be wrong they decided to rule out sequence variation and possible lethal mistakes by determining a consensus sequence, Sasha Kolykhalov painstakingly constructed a cDNA clone that reflected this sequence. Steve Feinstone and his team at the FDA then tested these transcripts in vivo, and finally, the virus showed up! This finding provided irrefutable evidence that HCV was sufficient to cause liver disease, and that they now knew the complete sequence of a functional HCV genome. These findings were independently confirmed shortly thereafter by a team led by Jens Bukh in Bob Purcell and Sue Emerson's group at the NIH. When I asked Charlie to comment on the resilience of the field and their unwillingness to back down from the challenges that came at them, he said, “I think our contributions were driven by curiosity, frustration and the belief that we would prevail. I was fortunate to have a cast of talented and dedicated colleagues who never gave up even when each breakthrough was followed by another roadblock. This was a mindset shared by countless others in the field who contributed to this remarkable biomedical success story.”
Even with these advances made, there was still a long road ahead before tractable cell culture systems were available that could support RNA replication, which circles us back to the 1999 Science article that I mentioned earlier by Volker Lohmann and Ralf Bartenschlager reporting the first HCV subgenomic replicons. Although an animal model had been established, and the replicon systems could facilitate testing of putative antiviral compounds directed at numerous targets within the HCV replication machinery, this field was not going to stop there. Eventually, HCV pseudovirus systems would be developed to study virus entry and the efficacy of neutralizing antibodies. And finally, in 2005, the complete HCV life cycle could be studied due to the establishment of HCVcc systems based on the JFH-1 sequence identified by Takaji Wakita's group, but this is a whole other chapter for another time. This story focuses on the discovery phase of HCV as the causative agent for NANB hepatitis.
In closing, I am reminded of one time when I was in a meeting with Sue Emerson and Bob Purcell, and they asked me to do a certain experiment. I replied that I could not perform that experiment because there was no way to do it… I will never forget Sue's response and I have relayed this lesson to all of my own trainees. She said, in a tone that I can still hear, “Don't tell me that something can't be done. I spent my whole career finding ways to do things that couldn't be done. That is why we're here, to find ways to do things that can't be done.” And that is the answer to all scientific questions. HCV has been cured. COVID-19 can be cured. Effective vaccines for HCV, HIV, and SARS-CoV-2 are possible. We just have not found the way to do these things yet. That is indeed why we are in science, why we do what we love to do. To end off on a lighter note, because we all need to smile right now, I will give the third member of this Nobel team the last word. If you have ever had the pleasure to meet Harvey Alter, you will not be surprised by the fact that when I asked him for a quote for this editorial, he simply replied, “Nobel elation is transient, but emails are forever!” He offered to provide something more serious, but we both agreed that was perfect.