THE three scientists who made seminal contributions to the discovery of a novel virus, the hepatitis C virus (HCV), are joint recipients of this year’s Nobel Prize for Physiology or Medicine. Before their path-breaking work, the known agents that caused hepatitis (liver inflammation) were the hepatitis A virus (HAV) and the hepatitis B virus (HBV). HAV is responsible for hepatitis transmitted through contaminated food and water (“infectious or epidemic hepatitis”), and HBV is responsible for hepatitis transmitted through blood and bodily fluids (“serum hepatitis”).
HCV is the second causative agent of blood-borne hepatitis, a major health problem that causes cirrhosis and liver cancer in several thousands of people around the world.
While the discovery of HAV and HBV marked crucial advances in controlling the burden of hepatitis around the world until about the 1970s, the majority of blood-borne hepatitis cases still remained unexplained, particularly post-blood transfusion hepatitis. During blood transfusions, it was observed that even when donor blood that tested positive for HBV was carefully excluded a lot of transfusion recipients still ended up developing chronic hepatitis with serious long-term effects.
In a statement following the announcement of the award, the Nobel Committee said: “The discovery of Hepatitis C virus revealed the cause of the remaining cases of chronic hepatitis and made possible blood tests and new medicines that have saved millions of lives.” “This virus,” observed Thomas Perlmann, the secretary of the Nobel Committee for Physiology or Medicine, in a brief post-announcement interview, “has been a plague affecting millions of people—and still is, unfortunately…. It’s hard to find something that is of such benefit to mankind as what we are awarding this year.”
Global burden of hepatic diseases
According to the 2015 Global Hepatitis Report of the World Health Organisation (WHO), the different types of viral hepatitis contribute substantially to the global burden of hepatic diseases: in the year of the report, HAV infection caused 114 million cases of acute hepatitis, while 257 million people lived with chronic HBV infection and 72 million with chronic HCV infection. Because of the chronic infections that HBV and HCV lead to, they are major causes of morbidity and mortality with 1.34 million deaths reported in 2015, a 63 per cent increase from 1990, mainly because of HCV infection. This is comparable to the deaths tuberculosis caused (1.5 million reported in 2018) and higher than the deaths due to AIDS (6,90,000 reported in 2019).
The joint winners of the Nobel Prize are 85-year-old Harvey J. Alter, chief of the infectious diseases section and Associate Director of research at the Department of Transfusion Medicine at the National Institutes of Health (NIH), Maryland, United States; 71-year-old Michael Houghton, a professor of virology at the University of Alberta in Canada; and 68-year-old Charles M. Rice of Rockefeller University in New York. They were responsible respectively for three crucial steps involved in identifying and establishing that HCV was the causative virus for what was being described as “non-A, non-B hepatitis (NANBH)” (see figure).
Even though alcohol abuse, environmental toxins and autoimmune conditions can cause hepatitis, the major burden of this disease is owing to viral infections. In the 1940s, it was already known that there were two types of infectious hepatitis, A and B. The first has a short incubation period, has little long-term impact, is self-limiting, and infection results in life-long immunity. It is now known that the RNA viruses HAV and HEV cause hepatitis A, the latter having been identified only in the 1980s. Hepatitis B leads to a chronic condition in a high proportion of the patients and comes with a high risk of developing liver cancer or cirrhosis in the long term. It is now known that this long-incubation serum hepatitis is caused by the DNA virus HBV. This infection is insidious as otherwise healthy individuals can be silently infected for many years (during which period they can transmit the disease) before serious complications arise. But there is also a slight clinical variant of this disease, the discovery of whose causative agent is the story of this year’s Nobel Prize winners.
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Discovery of HBV
In the 1960s, Baruch Blumberg identified HBV, an important discovery that led to the development of diagnostic kits and an effective vaccine against it. For his discovery of HBV and the first-generation HBV vaccine, Baruch was awarded the 1976 Nobel Prize in Physiology or Medicine. As a young doctor and researcher, Alter had worked with Blumberg and made important contributions to the identification of the so-called “Australia antigen (Au-antigen)” in 1967, which, in fact, led to the discovery of HBV. Working as a clinical scientist at the NIH’s large blood bank in the 1970s, Alter was studying the occurrence of hepatitis in post-blood transfusion patients. At that time, it was noticed that the exclusion (by serological tests) of HBV-antibody positive blood donors brought down the post-transfusion hepatitis cases only by 20 per cent; the remaining 80 per cent of the cases could not be explained and seemed unrelated to HBV infection. Blood tests showed that these cases were not caused by HAV either.
This NANBH differed from hepatitis B in its clinical manifestations: it had a short incubation period and much milder symptoms during the acute phase. While it resembled HBV in its modes of transmission, it led to chronic infection more frequently. In the late 1970s, after persistent efforts, Alter and colleagues developed a primate model of the infection. They showed that the blood from patients with acute or chronic NANBH could transmit the disease to chimpanzees, the only non-human species susceptible to the infection. Alter also demonstrated that the unknown infectious agent had the characteristics of a virus. His methodological investigations confirmed the existence of a distinct clinical form of post-transfusion hepatitis transmitted by an unknown virus.
“It’s a good story,” Alter said in his post-announcement interview to the Nobel Committee, “for a kind of non-directed research, where we have a hypothesis, but you have no idea where it’s going to go, just looking to see what caused post-transfusion Hepatitis, and initiated… a very, very, very long study that involved many people. And that was all done at NIH, and probably could not have been done anywhere else because it took so long to come up with something you didn’t really expect to find. But it was decades, and a lot of people, Bob Purcell and particularly Paul Holland and Paul Schmidt, who were in the blood bank with me… the message that I think is important is that you don’t always know where you’re going. Nowadays research is so directed, and so has to come up with a drug fast, but at NIH they allowed me to just… go my way.…”
But scientists found identification of this new virus frustratingly difficult. All the well-known methods for detecting a virus failed. It eluded isolation for over a decade. Houghton, who was working at the pharmaceutical company Chiron Corporation, took up the challenge in 1982, but the efforts of his team too proved unsuccessful. In 2018, Houghton told The Lancet that the quest to identify NANBH had driven his team to despair. “For years, everything we attempted failed,” he said. “We tried all the methods that had proven successful for other infectious agents. We could not find an antigen in the blood, we were not able to grow the virus in cell culture, and if you looked in an electron microscope, you could not see it.”
Moving to Alberta, Houghton then followed an unorthodox molecular approach. He and his co-workers Qui-Lim Choo and George Kuo created a library of DNA fragments from the nucleic acids found in the blood of an infected chimpanzee and screened it for viral DNA segments. Most of the fragments were found to be from the chimpanzee’s own genome, but the researchers kept at it as it was logical to expect that some of the fragments would be of the virus. The team then decided to try a clever and novel screening approach. Since antibodies to the virus would be present in the blood taken from NANBH patients, they used patients’ sera as the detecting tool to identify the DNA fragments encoding for the viral proteins.
The researchers transferred the library of DNA fragments of the infected chimpanzee to bacteria using a highly efficient bacteriophage system as the vehicle. (Bacteriophage is a kind of virus that infects bacteria.) The expression of viral antigens in the system was identified using sera from NANBH patients. Over two years, they screened millions of cloned bacterial colonies and finally found one colony that contained neither human nor chimpanzee DNA sequences. The years of painstaking work finally paid off: the team had found what it was looking for. Houghton and colleagues then showed that the clone was derived from a novel RNA virus belonging to the Flavivirus family, and the pathogen finally got its name, HCV. The clinching evidence came from the molecular complexes that antibodies of chronic NANBH patients formed with the proteins that the viral DNA fragments from the infected chimpanzee encoded for. The results of the experiments that led to the discovery were published in 1989.
“At the time of trying to discover Hep C in the ‘80s,” Houghton said in his post-announcement interview to the Nobel Committee, “it was a difficult task. We didn’t have the tools available then that we do now of course. So, it was a lot of effort actually, a lot of brute force, and just trying to use and apply all the methods available then. And we must have tried 30 different approaches at least over seven or eight years, and eventually we got one clone, after screening probably hundreds of millions of clones. So, yes, I work with some great people, without whom I would not have had this success. And we worked very hard, and so a lot of hard work and persistence was part of our success story, for sure. Following the identification of the virus, Houghton and associates quickly developed an assay to detect HCV-specific antibodies in the blood of a donor who had transmitted the disease to 10 recipients and in the blood of NANBH patients from Italy, Japan and the United States, thus establishing a relationship between HCV infection and NANBH.
“[Q]uickly after we discovered the virus, we developed a blood test, [which] was the most urgent need to protect the blood supply…. And then of course the two big challenges were trying to find therapeutics for the virus, and that took a long time. It took… the whole field and the pharmaceutical industry working for more than 20 years. But eventually, we’ve got these wonderful drugs now that can cure nearly everybody quite quickly and safely. But it is… an epidemic, global epidemic. It is a pandemic. HCV today kills around 400,000 people every year,” Houghton said. But there was still a missing piece in the puzzle. What Houghton and colleagues showed was not really definitive proof of a causal connection between HCV infection and the chronic hepatitis disease. What they had demonstrated was, in some sense, only a correlation between the two. The transmission of the disease by transfer of infectious blood did not exclude the involvement of other cofactors in the causation of the disease. It remained to be proved that the virus alone was causing the disease. That required isolation of a virus capable of causing all the clinical features of NANBH, including chronic liver damage and persistence of the infectious agent in the blood of the patient. Basically, it had to be shown that the cloned virus was capable of replicating in the host and causing the disease. Although the virus now had a name, it remained elusive. No one had demonstrated that it replicated in the host. It was left to the third laureate to do that.
Rice, then a researcher at Washington University in St. Louis, Missouri, along with other groups working on RNA viruses, realised that in characterising the HCV genome a sequence of about 100 bases at one of its ends (the so-called 3’ end) had been missed. He reasoned that this sequence could play an important role in viral replication. “These small RNA viruses do not carry a lot of extra baggage; you have to make sure you have everything in the right place,” he said while speaking to The Lancet after the award. Rice first constructed viral RNA genomes that had the end conserved region (the 3’ end) intact and injected them into the liver of chimpanzees and looked for evidence of viral multiplication. But, unfortunately, he could not detect any newly produced virus in the chimpanzees’ blood. Rice was also aware that there could be significant copying errors in the replication of RNA viruses and had, indeed, noted that there were significant variations in the isolated virus samples. He surmised that some of these mutations could actually hinder viral replication. Through genetic engineering, Rice then built a set of “consensus” genomes of HCV that had the conserved 3’ region and were also devoid of inactivating mutations. Injecting these genomes into the liver of chimpanzees produced evidence of replication, and productive infection was established.
“We demonstrated that you could make a molecular clone of this virus that was infectious in chimpanzees, which was really the only validated system whereby HCV activity could be assessed,” he told The Lancet. Thus, Rice’s work conclusively showed that HCV alone could cause persistent long-term hepatitis and stimulate a specific antibody response and all the clinical features of infection in humans.
To the post-announcement interviewer from Stockholm, Rice said: “I feel as though I’m just kind of a representative of the sort of molecular virologist community that contributed something to this fight against this disease…. I think it was really… just a joy actually to work in this community. I think people have been very generous… with ideas and reagents. And that, together with the input of biotech and pharma, finally sort of came to the finish line in terms of developing these drugs that are so effective, that we have today. Now we still have some challenges in terms of making sure that everybody that needs them gets them and gets treated, but it is, I think, a success story for biomedical science and team science. And we’re seeing really an amazing follow-up example of that with the pandemic and the number of groups that have stepped up to the plate to work on SARS-CoV-2.”
While the discovery of HCV paved the way for the development of effective drugs against the virus, it was not easy going. Although the full-length clones of the HCV genome Rice and colleagues created infected chimpanzees, they exhibited poor replication in cell lines, thus hindering in vitro studies of the virus’ life cycle and testing of potential antiviral drugs. Ralph Bartenschlager at the University of Heidelberg constructed the first sub-genomic clones of HCV that replicated efficiently in (infected) hepatoma (human liver cancer) cell lines.
The second obstacle was the absence of small animal models. Because the virus infected only humans and chimpanzees, the precise assessment of the pathological and immunological characteristics of the disease and clinical testing of potential drugs were not possible. This was overcome by creating genetically engineered knockout mice models. The availability of in vitro virus replication platforms and small animal models for in vivo studies led to the development of effective direct-acting antiviral (DAA) drugs that revolutionised the therapeutics against HCV infection. Chronic HCV hepatitis is now, in most cases, curable and the damage caused to tissues is often reversible.
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High cost of treatment
According to the Nobel Committee’s background note to the award-winning HCV work, short-term antiviral treatment cures more than 95 per cent of patients, including advanced cases who failed to respond to previous therapeutic regimens, and has already benefited millions of individuals worldwide. The remaining obstacles towards the eradication of viral hepatitis, the note says, are now mostly associated with the lack of broad screening campaigns—according the WHO Global Hepatitis Report 2017, fewer than 20 per cent of people with HBV- or HCV-associated hepatitis have been adequately diagnosed—and, as Rice has pointed out, the high cost of the most effective treatments, which limits their availability to patients who cannot afford them, particularly those in low- and middle-income countries.
Like in the case of HIV/AIDS, Indian pharma companies have an important role to play here by stepping in to produce generic DAA drugs and becoming a supplier to low- and middle-income countries of the Third World.