Viruses, Pandemics, and Immunity

by Arup K. Chakraborty

  Françoise Barré-Sinoussi and Luc Montagnier in France were the first to identify HIV, followed by Robert Gallo in the United States. Their key insight was that the virus could be grown in a subset of T cells, the same cells that the virus infects to cause disease. They knew that they had identified the disease-causing virus because the tests they developed showed that the virus they grew in the laboratory was the same as the one present in all infected patients. HIV was identified about two years after the first reports of a new disease. This was a stunning achievement because it took decades to identify disease-causing viruses like polio.

  Modern technologies have revolutionized the speed with which a virus can be identified. In 2003, about 6 months after reports of the disease, the virus that caused SARS was identified. In 2020, the genomic sequence of SARS-CoV-2, the virus that causes COVID-19, was identified about a month after the first reports of the disease. Quick identification of new viruses is made possible by new methods for rapid isolation of viruses and fast methods to sequence their genomes.

  The Life Cycle of Viruses Defines Targets for Antiviral Drugs

  In chapter 3, we learned how viruses function. Let us briefly review some aspects that are relevant for the development of antiviral therapies. Viruses have to replicate rapidly so that they can infect many cells in a person and many people in a population. Viruses have only the proteins that are absolutely essential for their functions. They do not even have most of the proteins that they need to replicate, which means that to multiply they need to hijack proteins from the cells they infect. Viruses can multiply rapidly because only a few proteins need to be replicated to form new virus particles. Viruses are like experienced travelers who pack very light, carrying only items that they absolutely need and that are not provided by hotels. Because viruses have only the proteins that are essential for their function, any drug that can inhibit the function of any viral protein is a potential antiviral drug.

  All viruses have a similar life cycle. Viruses enter the body through the skin, eyes, mouth, nose, rectum, vagina, or other vulnerable body part. Insect bites or hypodermic needles allow viruses to directly enter the bloodstream. Once inside the body, they enter our cells. Entry into a cell is mediated by the virus’s spike binding to a receptor on the surface of our cells. For example, the HIV spike binds to a receptor on the surface of a subtype of T cells, and SARS-CoV-2 binds to the ACE2 receptor present abundantly on lung, heart, and kidney cells. Once the virus’s spike is attached to a cell’s receptor, the next step is to force its way into the cell. This is a complex step that often involves our own cell’s proteins and a change in shape of the proteins that make up the virus spike. Once inside the cell, the viral genome is released. Our own cell’s machinery is then hijacked and the cell becomes a factory for replicating the virus’s genome to make many copies of its few proteins. The proteins are then assembled into many new virus particles. At this point, the newly assembled viruses are still trapped in the cell and need a way out. Viruses have developed many ways to escape from the cell. Some viruses rupture the cell in order to exit, while others can just bud out of the cell’s soft membranous wall. Once outside the cell, the newly produced viruses infect other cells, and the cycle is repeated until the infected person dies or the immune system controls the virus.

  Antiviral therapies aim to block one or more of the steps in the viral lifecycle: viral entry, replication, assembly, and release from the cell.

  Blocking Viral Entry

  Blocking viral entry into the cell is a proven antiviral strategy. In fact, this is a strategy that our immune system uses effectively. Some antibodies generated in response to the virus attach to the virus’s spike and block its ability to bind to receptors on human cells. This prevents the virus from infecting new cells. This is why for over a hundred years antibodies have been used to treat disease.

  As we described in chapter 4, von Behring and Kitasato showed that antibodies injected into patients could treat diphtheria. Later, physicians used blood (plasma) from patients who recovered from disease to treat viral infections that include influenza, measles, SARS, MERS and Ebola. The idea that antibodies in blood can be curative is why plasma from patients who have recovered from COVID-19 is being tested as a therapy.

  While many antibodies are generated in response to the virus, only some are really potent in preventing viral entry. Identifying the potent antibodies and using these as drugs can be an effective form of therapy. Many companies and scientists have developed rapid ways to generate these desired antibodies. For example, a clinical trial for Ebola virus infection tested a combination of three such antibodies specific to Ebola that were produced by the biotech company Regeneron. The trial was so successful that it was stopped early. Similar approaches are being used to identify effective antibodies for the treatment of COVID-19. These therapies induce a transient immunity. Questions about whether only one or multiple types of antibodies are required, and which particular antibody types (IgG or IgA) are needed remain unclear.

  Finding the right antibody can be a challenge, as it can be a bit like looking for a needle in a haystack. A way to take a short cut is to use the cell receptor that a virus’s spike attaches to as the “antibody.” For example, ACE2 is the cell surface receptor that the SARS-CoV-2 virus binds to enter the cell. One strategy might be to manufacture ACE2 and use it as a decoy. Once injected into a patient, the synthetic ACE2 receptor would bind to the virus and prevent it from binding to the ACE2 receptor on the cell. The synthetic ACE2 receptor drug could also be engineered to have one end that is like the stem of an IgG antibody. Then the mechanism that helps the body dispose of antibody-bound viruses could get rid of the virus–drug complex.

  Antibodies and receptor decoys are large molecules, in a class of drugs called biologics. These types of drugs are expensive, partly because they are more complicated to manufacture compared with drugs that are small molecules. They require injection or intravenous administration, making them difficult to deploy in large scale. Their use is probably more appropriate for treating the severely ill, either to suppress geographically localized outbreaks or to protect family members of an exposed individual. Administration of antibody therapy to everybody in a neighborhood with an outbreak would, for example, temporarily provide immunity for the community and suppress spread of the infection. A single dose would likely be sufficient since injected antibodies persist in the blood for some time.

  Once bound to a receptor on the cell, viruses need a way to force their way in and enter the cell. Viruses have figured out many ways to do this. In general, upon binding to the receptor on the cell surface, the viral spike protein can dramatically change its shape, which via complicated mechanisms generates a force that allows the virus to push its way in. Blocking this step with a drug can prevent the virus from entering the cell even if it is attached to the appropriate cell surface receptor.

  SARS-CoV-2 uses a protein already present on the surface of lung cells, called a protease, to cut the viral spike protein into two pieces. Cutting the viral spike protein acts like a spring being released, and the resulting force allows the virus to push its way in through the cell wall. A drug that could block the action of the protease that cuts the spike could prevent viral entry into the cell. Since the protease is present on many cells in different parts of the body, a drug blocking it could have wide-spread and serious adverse side effects. Preventing such side effects is a major challenge for drug development.

  Blocking Viral Replication

  As noted above, viruses have only the proteins that are essential for their function, and which they cannot steal from our cells. As we saw in chapter 3, RNA viruses have an RNA genome. So, they cannot make their proteins following the DNA to RNA to protein route that we use (recall the central dogma). So, all RNA viruses have their own polymerase, which allows them to copy their genome and replicate. Some DNA viruses also have their own polymerase that copies their genome. For retroviruses, like HIV, the polymerase is called revers
e transcriptase. It converts the viral RNA genome to DNA, which is then converted to RNA and proteins using our cell’s machinery. A drug that could specifically inhibit the viral polymerase would be efficacious and safe. It would be efficacious because it would prevent viral replication, and safe because the ideal drug would not act on our polymerase, which is distinct from that of the virus. The trick is how to find such a drug.

  A solution was provided by Gertrude Elion in the late 1970s. After graduating from high school at the age of 15, Elion attended Hunter College of the City College of New York. Despite graduating with top honors in chemistry in 1937, because of gender discrimination, her applications to graduate school were rejected multiple times. She could only find employment as a secretary and then as a food quality supervisor at a grocery store chain before finally finding a job as an assistant to George Hitchings at the Burroughs Wellcome pharmaceutical company. Hitchings was developing antibiotics, and he thought that blocking an organism from replicating its DNA might be a good strategy to stop it from multiplying. Recall from chapter 3 that DNA and RNA are chains of units called bases. These bases are G, T, A, and C for DNA and G, U, A, and C for RNA. Polymerases take these units and string them together in the specific order that encodes the genome of a particular cell or organism. Elion and Hitchings synthesized molecules that looked very similar to the usual bases. They are similar enough that the polymerase is fooled into inserting it into a growing genome. Using these molecules, they succeeded in developing antibiotics and drugs for cancer and autoimmunity.

  After Hitchings retired, Elion continued their work. In the late 1970s, she began to work on developing a drug that could specifically inhibit the replication of the herpes virus (a DNA virus). Her strategy was to synthesize artificial bases that could be inserted by the viral polymerase into a growing copy of the viral genome. Since these drugs are not true bases, once inserted, they block the polymerase from growing the chain any further. Thus, the genome stops growing before it is completely copied. This defective genome no longer encodes information about all the necessary proteins, and so the virus cannot replicate. Using this strategy, she identified the first antiviral drug, acyclovir, which the Herpes virus’s polymerase acts on, but our own polymerase does not. Early in the HIV pandemic, her team used this strategy to identify the first drug used to treat HIV, azidothymidine (AZT, zidovudine). Remdesivir, a drug being used to treat COVID-19 is also a polymerase inhibitor. It was originally designed to inhibit the Ebola virus’s polymerase but was found to also have activity against the polymerase of SARS-COV-2.

  Elion, who was not allowed to go to graduate school, shared the 1988 Nobel Prize with Hitchings for their work. Only after this would she be awarded honorary doctorates from NYU and Harvard, and elected a member of numerous honorary scientific societies.

  For HIV, we now have newer strategies to block its replication. To be as compact as possible, HIV has evolved to have only a few genes, each of which encodes information on multiple proteins. Large polyproteins, containing many individual proteins, are first made. The polyproteins are then cut into the individual proteins that HIV needs to function. Like the protease on our lung cells cuts the spike of SARS-CoV-2 to enable it to enter our cells, HIV has its own protease, which cuts its polyproteins into the right constituent proteins. Without this protease, HIV’s proteins, including its polymerase, cannot be separated from the corresponding polyprotein, and so would not function. Drugs called protease inhibitors were developed to block the function of the HIV protease, and this prevents virus replication.

  Blocking Assembly of the Virus

  Once the virus’s component proteins have been made in the infected cell, these parts need to be assembled into complete viruses. This is another step in the viral lifecycle that can be interrupted by drugs.

  Hepatitis C virus (Hep C) is transmitted through the blood and causes a liver infection. The immune system is unable to eradicate this virus in most patients. Thus, if untreated, Hep C causes a chronic, lifelong infection in most people. About 2–3 percent of the world’s population is thought to be infected with Hep C. Patients are generally asymptomatic during the early stages after infection, but the virus slowly destroys the liver. Hep C is now one of the most common causes of liver failure.

  Until the virus was identified about 30 years ago, it was a mysterious infectious agent that contaminated the blood supply. Receiving a blood transfusion was a bit like Russian roulette, and transfusions were a relatively common way to contract the virus. The identification of Hep C was a key moment because not only did it provide a way to screen for the virus and clean up the blood supply, but it also allowed the search for effective treatments.

  The first antiviral drugs used to treat Hep C had been developed in the 1970s and 1980s. One of these is called ribavirin. Ribavirin is similar to the drugs developed by Elion and Hitchings in that it resembles one of the bases that make up a virus’s RNA genome. When it is added to the growing RNA genome as it is being copied, it has several effects that include slowing chain growth. The base-like drug is also such that wrong bases can add to it. This introduces so many mutations into the copied RNA that the resulting virus is dysfunctional.

  The second drug developed to treat Hep C was interferon. Interferon is a hormone made by immune cells that creates an environment that is very inhospitable for viruses. This drug was made using recombinant DNA technology, which was invented in the 1970s by Paul Berg, Stanley Cohen, and Herbert Boyer. This method allowed the gene for interferon to be identified and isolated in the laboratory. This gene was then inserted into the DNA of another organism, which then produced “synthetic” interferon encoded by the inserted gene. This synthetic product was administered as an anti-viral treatment. Producing interferon in this way was a landmark event for the biotechnology industry. But the combination of ribavirin and interferon that was used to treat Hep C infections was effective in only about 50 percent of patients.

  To improve upon antiviral drugs like ribavirin and interferon, the search for Hep C–specific drugs began. Drugs that specifically inhibited Hep C’s polymerase and protease were developed, and they significantly increased the cure rates for the disease and reduced the need for liver transplants later. These drugs shortened the treatment period for Hep C patients from about a year to only six weeks and cured the disease in a significant proportion of patients. These drugs are convenient, requiring patients only to swallow pills. But the initial cost of the treatment, over $1,000 per pill (for Solvadi), stirred great controversy.

  An important breakthrough in the treatment for Hep C was the development of drugs that block the assembly of the virus. These drugs inhibit the action of a Hep C protein called NS5A. We do not know exactly what the function of NS5A is, but the use of NS5A inhibitors in combination with protease or polymerase inhibitors is one of the most effective antiviral therapies ever developed.

  Blocking Release of the Virus

  Once new viruses are assembled, they need to exit the cell. This is another step that drugs can block. Two influenza drugs, oseltamivir (Tamiflu) and zanamivir (Relenza), interfere with the release of viruses from the cell. As you recall, viruses need to bind to a receptor to enter the cell. But if a new virus particle binds to its receptor before exiting the cell, it will get trapped in the cell. After new viruses are assembled, the influenza virus deploys a protein that destroys its receptor, allowing newly assembled viruses to slip out of the cell without getting stuck. Oseltamivir and zanamivir block this function, thus trapping the virus in the cell. Since these drugs block only the last step of replication, they are less effective than other types of drugs. This is because the virus has already assembled many copies of itself and some can still escape from the cell. Oseltamivir and zanamivir just shorten the length of the illness by one or two days.

  Combination Therapies

  Like a stunt where Houdini miraculously escapes from a straitjacket, viruses have an uncanny ability to develop resistance to drugs. Recall from chapter
3 that the replication of viral polymerases is relatively inaccurate, and every time the virus replicates, mutations can arise in its genome. After a few cycles of replication, different strains of the same virus may emerge. When confronted with a drug, viral strains that have a mutation that allows them to function without being impaired by the drug will have an advantage over those that are affected by the drug. These mutant strains then thrive, and the drug is no longer effective. The development of drug resistance by this mechanism is very common for highly mutable viruses like HIV.

  Pharmaceutical companies have developed drugs that inhibit the functions of distinct proteins of a virus. If a combination of these drugs is used simultaneously, a drug-resistant viral strain would need to acquire multiple mutations at the specific targets of all these drugs. This is harder to do because viral polymerases make mistakes randomly, and so mutations arise in viral genomes at random spots. The emergence of multiple specific mutations in a viral strain by this random process takes a while. It’s like if the virus was playing roulette, to win against one drug it would have to guess one number correctly, while to win against a drug cocktail it would have to guess multiple numbers correctly to win. Moreover, each mutation likely results in some impairment of the virus’s functions—many would make the virus even more dysfunctional. For these reasons, combination drug therapy has been very successful for treating highly mutable viruses. For example, modern HIV and Hep C treatments typically employ a cocktail of drugs.