An Interview with Manuel and Ann Varela: Who was Jennifer McKimm-Breschkin ?—Virus Fighter!

Aug 16, 2021 by

We enjoy working together…and it’s natural that we talk about science at home.”

—Jennifer McKimm-Breschkin

Michael F. Shaughnessy

1) Outstanding female scientists come from all over the world—One “Virus Fighter” came from Australia (down under, as they say). When was she born, and in what locale in Australia?

Dr. Jennifer McKimm-Breschkin is best known for her efforts in discovering the antiviral medicine called zanamivir, a drug that fights the dread influenza-causing virus. She has enjoyed a long and productive career as a prominent virologist.

Jennifer McKimm was born in 1953 in Melbourne, Australia. She was fascinated with science-related subjects from a young age, but initially, she was interested in becoming a physician. One opportune chance meeting, nonetheless, made her alter her plans. After she submitted an application to the medical school, a faculty member told her, “Medicine is a b**** of a career for a woman.” McKimm thought that if that was the mindset of the medical school professors, they were not going to be very supportive of female students. Hence, she decided to register for courses concentrating on the science curriculum with the end goal of teaching. In line with the standard practice of the day, females majoring in science ultimately lead to a teaching career. After earning her bachelor’s degree, however, she changed her mind about teaching science and decided to go on for an honors degree that included a one-year research project. McKimm discovered how much she enjoyed working in the laboratory and decided to become a researcher.

McKimm graduated from Monash University in Melbourne, Australia, in 1974 with a first-class honors degree. After that, she earned a Fulbright scholarship at the Hershey Medical Center of Pennsylvania State University in the United States as a graduate student, which ultimately led to her Ph.D. in virology in 1978. At Hersey, she met Alan Breschkin, an American postdoc with a Ph.D. from Vanderbilt University. They spent seven days a week in the laboratory together until they completed their program requirements at Pennsylvania State University, after which they married and relocated to Australia.

McKimm-Breschkin returned to the Melbourne University Microbiology department as a Queen Elizabeth II Post-doctoral fellow in 1979 with research focused on the newly discovered rotaviruses. She completed another postdoctoral position at the Walter and Eliza Hall Institute of Medical Research as a Colin Syme Junior Fellow working in virology and cellular immunology. McKimm-Breschkin and her husband had two children during this time. For a brief period, she worked part-time, but the Australian child-care facilities made it possible for her to continue working and eventually return to full-time status. Reportedly, her husband has been very supportive of her career.

McKimm-Breschkin worked on animal viruses at the Australian Commonwealth Health Department for two years. From 1987-2016, Breschkin worked for the health department of the Australian Commonwealth Scientific and Industrial Research Organization (CSIRO).

2) Apparently, McKimm-Breschkin earned a Ph.D. in virology—how common is this?

Overall, the numbers of Ph.D. degrees in virology granted every year, a rate of around two to three dozen per year in the U.S., are relatively fewer than most in the general scientific workforce. Nevertheless, the demand for expertise in virology at the doctorate level is increasing with each passing year. Along with the smallpox virus, measles virus, and poliovirus, one of the driving forces for this current demand can be seen in the light of the COVID-19 pandemic, with countless morbidity and mortality numbers due to the emerged causative agent SARS-CoV-2 virus. Thus, the need for scientists who are trained in virology is in demand as never before.

Historically, viruses have had significant impacts on the number of disease cases and deaths. Another etiological agent of the disease, the influenza virus, responsible for periodic outbreaks of the flu, also known as influenza, is an additional mainspring in need for people with virological expertise in the workforce. Ailments like the common cold are endemic in many areas of the world. The viruses that cause these seemingly mild respiratory infections are, as a group, responsible for loss of economic productivity, as patients cannot work or they miss school, not to mention the misery associated with the common cold. Many of these sorts of virological microbes constantly mutate, potentially becoming more dangerous than before. There are several hundred individual viral causative agents of the common cold, and an old coronavirus species was one of them.

In addition to respiratory diseases, specific viruses cause gastrointestinal disorders, liver disease, brain infections, and even cancer. Viruses are pathogenic to all other living beings on Earth. Thus, it is essential to understand these tiny microbes to prevent, control, and develop treatments of the diseases they cause.

It seems that for millennia, the influenza virus has wreaked havoc on human populations in massive proportions. The influenza virus microbe has been a thorn in the sides of humanity as a continual endemic presence and occasional severe pandemics. The flu pandemic of 1918-1919 comes to mind. At the time, the microbe was estimated to account for somewhere between 30 and 50 million deaths. The name influenza provides a clue to the seriousness of the microbe. This flu virus has greatly “influenced” many aspects of the daily life of human populations since recorded history began.

Another historically significant human viral disease is measles, also called rubeola, caused by the measles virus, known by its scientific name of the genus Morbillivirus, a member of the Paramyxoviridae family of viruses. Measles represents one of the most contagious of the known infectious diseases. When the vaccine was introduced widely in the late 1960s and early 1970s, disease incidence levels exhibited a dramatic drop in numbers afterward. Thus, the study of viruses and the discovery of vaccines against them are promising avenues for addressing viral disease prevention.

In graduate school, McKimm-Breschkin studied the measles virus. In one of her thesis projects, she examined the ability of the measles virus to elicit the production of a protein called interferon. See Figure 1. Typically, the innate immunity aspect of the overall immune system responds to viral infection by producing interferon, which interferes with viral replication.

Figure 1. Ribbon structure of interferon-gamma.

File:Interferon Gamma.png

https://commons.wikimedia.org/wiki/File:Interferon_Gamma.png

McKimm-Breschkin studied a so-called temperature-sensitive mutant of the wild-type measles virus, the mutant of which failed to elicit the interferon production. She also examined a so-called revertant, one in which the mutant reverts to an original form of the parental virus. The revertant also failed to produce interferon in host cells harboring the viruses. Furthermore, when McKimm-Breschkin co-infected the host cells with both mutant and revertant measles viruses, the interferon failed to manifest itself. Even when McKimm-Breschkin co-infected host cells with a mixture of parental and mutant measles viruses, there was no sign of a control system that might have regulated interferon such that its production might still be turned on. In their scientific article, published in the peer-reviewed journal called Virology in early 1977, McKimm-Breschkin and her co-author Fred Rapp concluded that the interferon-producing mechanism in hosts’ cells was lost when the virus underwent its mutation process, even when the revertants appeared. The possibility also seemed that the wild-type virion lacked the machinery to induce interferon, suggesting an independent process was responsible.

In a follow-up study, McKimm examined whether mutagenesis or selection of viral progeny unable to induce interferon was responsible for the lack of induction levels. When she studied three distinct strains of the measles virus, McKimm found that a specific subpopulation of viruses in the progeny showed interferon production while another sub-set of the progeny did not have the interferon-inducing ability. Thus, she demonstrated that not all individuals in the viral progeny production could genetically inherit the interferon induction process. The results were intriguing in scientific circles because it strongly suggested that interferon induction by the measles virus, an imperative characteristic, was under the genetic control of the mature virion. It was a notable finding, and the study was published in July of 1977 in the high-status Proceedings of the National Academy of Sciences, sponsored by none other than John Enders, a 1954 Nobel Laureate who had successfully cultured poliovirus. Thanks to the pioneering work of McKimm, in modern times, we know that measles viruses are excellent inducers of interferon. We understand that these same viruses harbor specific mechanisms that upset the actions of the interferons.

3) Virology and immunology—how are they alike, and what is the relationship between these two things?

The field of virology considers the microbes called viruses, while immunology studies how the body monitors and fights the viruses and other microbes or cancer and foreign cells, tissues, and organs. Virology and immunology are similar because they both fall under the umbrella of microbiology, the discipline devoted to the study of microbes. In a way, viruses are the microbial aggressors causing disease and misery, and the immune system represents the body’s natural protective defenses, which have evolved to confer immunity against these and other sorts of dangerous microbes. Thus, virology and immunology are interrelated in that one field examines the devastating destruction that viruses bring upon living organisms while immunology has mechanisms meant to protect and provide immunity to viral pathogens.

Viruses are microbes, with each virion (an intact mature viral microbe) consisting of a nucleic acid genome protected by a protein shell or a coat called a capsid. A biological membrane called an envelope sometimes accompanies these basic structures of viral genomes and capsids. While not all microbes are dangerous to humans and other living beings, virtually all viruses are somehow pathogenic to something living. For almost all living creatures, some viruses may attack them to cause illness or even death. With the one exception of the multicellular parasite worms, the same sort of antagonistic relationship is not true when considering the relationships between the other categories of microbes and other living creatures on Earth. Most bacteria, fungi, algae, and protozoa, all of which are microbes in microbiology, are not considered pathogenic. Thus, the vast majority of the viruses (and parasitic worms) are unabashedly harmful to other organisms.

The immune system has evolved in living organisms over billions of years of life on Earth to protect from attack by antigens like microbes (and cancer or non-self-material). One hallmark of immunity is memory. The immunity mechanisms have developed into a molecular and cell system that protects us from future attacks from the same antigens. If an individual survives and recovers, the primary antigens are remembered the second time they are encountered. The immune system entails a cycle of exposure to the antigens, illness, recovery, and then immunity. A sort of biological memory process characterizes this immune status. For instance, the original offending microbe is recognized a second time an individual is exposed to the pathogen. This memory is the basis of the immunity status. The individual who has suffered an illness and has recovered is essentially immune to the infecting microbe, should the individual become exposed to the pathogen again.

The relationships between viruses and the immune system are a giving and taking process. Each entity evolves to gain a kind of predominance over the other. The primary purpose of viruses is to reproduce to ensure their continual presence on Earth. These microbes do so at the expense of the hosts they infect. Viruses have not all evolved yet to minimize pathology, which would help their survival cause. Incapacitating or killing a patient would prevent viral spread and survival. In some cases, the infecting virus manages to keep a patient well enough to permit viral spread before ultimately killing the unfortunate victim.

Conversely, a prime purpose of the immune system is to defend living beings from the harsh effects of viral infection. Thus, as a virus changes its genome and structure, the infection process enhances viral reproductive efficiency. However, the immune system is in place to anticipate and adapt to viral changes such that pathogenicity is thwarted or minimized. Sometimes the virus wins out, and the infected individual succumbs to the exposure, suffering illness and perhaps even death. Individuals can often recognize the virus for what it is, a potentially dangerous antigen, and respond by mounting an effective immune response characterized by a viral-neutralizing process and an establishment of memory to respond vigorously to a second encounter with the virus. Here, the infected individual recovers from the viral infection and is immune to the viral antigen.

On occasion, the virus will have evolved specific ways to evade the immunity process. There are several ways that viruses perform this immune system evasion. One notable tactic used by viruses is called antigenic variation. The so-called antigenic variation process can change the viral structure, perhaps slightly or possibly even considerably. In any case, the relationship between viruses, their changing antigens, and the immune system with its sophisticated cell and molecular machinery is a dynamic one. The viruses and the immune system are continually adapting to each other. Sometimes the virus succeeds, and sometimes the immune system prevails.

4) Apparently, McKimm-Breschkin was part of a team that developed zanamivir (trade name Relenza). What is essential about this drug?

The antiviral agent zanamivir has been used as a means of medical treatment for the flu. Also known by the colloquial name Relenza, the zanamivir medicine targets an essential influenza virus protein called neuraminidase, an enzyme. Zanamivir is an inhibitor of the neuraminidase molecule, which serves several essential roles for the influenza virion to survive.

One critical role of viral neuraminidase is to hydrolyze the protective mucous that surrounds host cells of, for instance, the respiratory tract in the airway of an individual. Mucous is a natural immunity barrier that traps microbes and other antigens and sweeps them up using cell cilia to purge them from the respiratory and gastrointestinal tracts. The neuraminidase helps the influenza virus in the airway overcome this innate mucous physical barrier. A hydrolytic process accomplishes the destruction of mucous by neuraminidase. Thus, neuraminidase helps the infecting influenza virus acquire access to the host’s airway cells because the mucous barrier is destroyed. With this innate immunity-based mucous now compromised or removed, the virus can reach the host cells directly.

A second essential role of neuraminidase is to prevent specific viral glycoproteins from aggregating on the host cell’s surface. The neuraminidase removes sialic acid, a cell receptor for the hemagglutinin molecule of the influenza virus. Thus, neuraminidase allows the removal of potentially confounding sialic acid receptors, which might have otherwise allowed viral clumping outside the cell. See Figure 2. This aggregation prevention function by neuraminidase helps the influenza virus to infect a cell efficiently. Once inside a cell, the virus has access to the needed cell machinery for viral reproduction. An intracellular location for a rouge microbe like the influenza virus is a highly desirable one.

Figure 2. Influenza virus entry into the cell by endocytosis.

File:Sialic acid and the influenza virus.jpg

https://commons.wikimedia.org/wiki/File:Sialic_acid_and_the_influenza_virus.jpg

A third significant role of viral neuraminidase is to permit the assembled and multiplied mature virions inside the cell to become released from that infected cell. During assembly of the virion inside the cell, the nascent virus incorporates glycoproteins into the envelope structure to make an intact mature virion. Thus, neuraminidase helps the intracellularly replicated influenza virus assemble and then escape from the insides of host cells. With neuraminidase, viral release readily occurs. The glycoproteins in their proper viral place, the envelope, permit viral escape from the infected cell and viral binding to enter another host cell ultimately.

Before it was named zanamivir (or Relenza), the potential antiviral candidate was called by the scientific name 4-guanidino-2,3-didehydro-2-deoxy-N-acetylneuraminic acid (also called 4-guanidino-Neu5Ac2en). The three roles played by neuraminidase are disrupted by zanamivir. As such, zanamivir is an effective anti-influenza medicine. The neuraminidase inhibition by zanamivir prevents sialic acid cleavage. The result is that viral hemagglutinin binds to the excess sialic acid molecules, allowing clumping of viruses to occur, which prevents proper viral entry, assembly, and ultimately, the release of the virus from a cell. Therefore, zanamivir inhibition of neuraminidase provides antiviral chemotherapy for influenza patients by blocking access of the virus to, and release from, host cells.

The amount of work performed by McKimm-Breschkin on the biology of the influenza virus and antiviral therapy is extensive. McKimm-Breschkin’s involvement in one area of investigation began with her work on neuraminidase, starting in the early 1990s. She had developed a new and rapid method to purify the heads of the neuraminidase molecules from virally infected host cells in the laboratory. McKimm-Breschkin also determined the structure of the neuraminidase molecule that was bound to the sialic acid receptor. The molecular structure of the complex between sialic acid and the active site of the neuraminidase was studied closely by McKimm-Breschkin’s laboratory. She realized that the enzyme-bound sialic acid molecule had an empty pocket. The sialic acid pocket had a hydroxyl group with a negative charge, a place that she astutely realized could be used to attach new chemical moieties, perhaps to make a modified form that might bind neuraminidase more tightly. The hydroxyl group of the empty pocket was replaced with a new moiety, called 4-guanidino, and attached to the base platform called DANA (2,3-didehydro-2-deoxy-N-acetylneuraminic acid), a transition state intermediate, to make the zanamivir. See Figure 3. The new variant molecule had a 4-guanidino group in place of the hydroxyl.

Figure 3. Chemical structure of zanamivir.

File:Zanamivir.png

https://commons.wikimedia.org/wiki/File:Zanamivir.png

Next, McKimm-Breschkin tested the so-called IC50 character of the zanamivir molecule on the viral neuraminidase enzyme activity. The IC50 is defined as the inhibiting concentration of a drug at which the enzyme’s activity is reduced by 50% compared to control groups lacking the drug. Amazingly, the zanamivir drug had IC50 properties at the nanomolar level. Zanamivir was shown to be effective against both influenza types A and B viruses. Since the early to mid-1990s, zanamivir has been used clinically to treat the flu with remarkable success.

McKimm-Breschkin studied a group of neuraminidase inhibitors, such as zanamivir, and other drug candidates like oseltamivir (Tamiflu), peramivir, and laninamivir (a derivative of zanamivir), and their relationships to influenza virus mutants, which showed various levels of resistance to the antiviral agents. McKimm-Breschkin mapped out the specific places of the mutations within the influenza virus proteins, identifying precise amino acids altered in the new viral variants. She had found that many of the affected amino acids were within the enzyme’s active site, and many others were of a non-active site nature. However, very little resistance to zanamivir as an effective medicine has emerged in much of the influenza virus clinical isolates.

5) Now, the influenza virus has two very different, distinct types of spikes or protuberances. Why is this important? Moreover, does the influenza virus have anything to do with the coronavirus?

The influenza virus structure has several key features that enable host infection in human respiratory tract cells. Two of these viral features are called spikes. The two types of spikes are forms of proteins with sugars attached to them and are called glycoproteins. The two spike glycoproteins are called neuraminidase (NA) and hemagglutinin (HA). The NA protein spike of the influenza virus is used to access a cell by hydrolyzing mucous, virus assembly, and viral release from infected cells. The influenza virus uses the HA protein to bind to the cell’s surface receptor sialic acid for virion binding and cellular entry of the flu microbe.

Various strains of influenza viruses are denoted with variant spikes and are given numbers to indicate variations in their structures. For instance, the so-called swine flu and the 1918-1919 pandemic strain were called H1N1. See Figure 4. The “H” denotes the hemagglutinin, and the number one denotes an antigenic character, of which about 18 HA variants are known. Likewise, the “N” stands for neuraminidase, with about 11 unique antigenic NA types that are known. The H1N1 strain has been inaccurately called the “Spanish flu.” Other strains are H2N2 (colloquially called the Asian flu), H3N2 (Hong Kong flu), and H5N1 (avian or bird flu). In modern times, virologists and other investigators have attempted to avoid associating geographical locations with various microbial agents of disease. One reason is inaccuracy. For instance, the Spanish flu did not originate in Spain—instead, we know it to have emerged from a military post in Kansas, in the U.S. Another reason holds that people who live in the regions named often suffer retribution by other individuals who are ignorant of the complicated nature of disease outbreaks and their modes of microbial transmission.

Figure 4. H1N1 influenza virus.

File:H1N1 Influenza Virus Particles (8411599236).jpg

https://commons.wikimedia.org/wiki/File:H1N1_Influenza_Virus_Particles_(8411599236).jpg

The influenza virus infection process has a complicated multistep reproductive cycle in a cell. See Figure 5. First, the mature influenza virion binds to the sialic acid receptor on permissive host cells. The bound virion then enters the cell by endocytosis using the viral-bound receptor to do so. This cell entry process is called receptor-mediated endocytosis. The viral entry results in the formation of a membrane-enclosed virus inside an internal structure called an endosome. Since the influenza virus is already enclosed in an envelope, the endocytosis encases the virion inside two membranes. Next, inside the cell within an endosome, the internalized virus becomes unpacked, releasing the viral genome, a set of eight single-stranded, negative-sense RNA molecules, which enter the cell’s nucleus as a series of ribonucleoprotein complexes.

Figure 5. Influenza virus particle and life cycle.

File:12035 2012 8320 Fig5 HTML.webp

https://commons.wikimedia.org/wiki/File:12035_2012_8320_Fig5_HTML.webp

In the nucleus, the viral RNA is replicated for genome production. The RNA is also prepared for translation and moved to the cell cytoplasm where ribosomes, i.e., protein-making machinery, reside. Viral proteins are made, usually at the expense of the cell’s health and the host overall. The newly made viral proteins are processed in the cell’s endoplasmic reticulum and Golgi apparatus. Some of this processing involves proteolytic cleavage and attachment of sugars to make various viral glycoproteins. The various components of the virion are assembled, and the replicated genomic RNA molecules are packaged with so-called nucleocapsid proteins that protect them and then further packaged inside a protein matrix structure. As the virus assembles, it also escapes by a budding process, taking some host cell membrane with it, becoming the mature virion’s envelope. The newly released mature virion is now ready to start the infection cycle again by binding to another cell using its sialic acid receptor.

The influenza viruses and the coronaviruses are both RNA-based enveloped virions that cause respiratory illness. Nevertheless, the influenza virus differs from the coronavirus in several fundamental ways. First, the influenza virus is a member of the so-called Orthomyxoviridae family of viruses. There are three genera or types of influenza viruses, called types A, B, and C. The influenza virus genome is segmented, meaning that it comprises several pieces, usually eight individual RNA molecules, each anti-sense and single-stranded. The glycoprotein spikes of the influenza virus differ from coronavirus in terms of sequence, glycosylation, and protein structure.

The coronaviruses belong to a separate family called Coronaviridae. The genome of the coronaviruses consists of a single molecule of a single-stranded positive-sense RNA. There are three distinct genera, called alphacoronavirus, betacoronavirus, and gammacoronavirus. The betacoronavirus is the genus of current interest in that one of its species, SARS-CoV-2, is responsible for the historic 2019-2021 pandemic of COVID-19. The term SARS-CoV-2 stands for severe acute respiratory syndrome coronavirus number two. The SARS-CoV-1 species was responsible for several outbreaks of SARS in 2002 and 2003. Before that earlier incident, coronavirus was merely a common-cold causing virus, harmless and temporary. However, the emergence of SARS-CoV resulted from an antigenic change into a more severe, lethal form. In 2012, another new coronavirus had emerged, called Middle East respiratory syndrome coronavirus (MERS-CoV). The club-shaped coronavirus spikes give these virions their name “corona” because they are reminiscent of the sun’s corona seen during eclipses. See Figure 6.

Figure 6. This image is a computer-generated representation of COVID-19 virions (SARS-CoV-2) under an electron microscope.

File:Corona virus Covid-19 Single Virion.png

https://commons.wikimedia.org/wiki/File:Corona_virus_Covid-19_Single_Virion.png

6) Viruses seem to mutate easily and quickly—what challenges does this present to scientists?

Soon after introducing neuraminidase inhibitors, mutants of the influenza virus had emerged with resistance to many of the antivirals. The viruses had changed their genomic sequences, making new viral proteins with different shapes to their structures and making the inhibitors bind less well than before. The main reason why these viruses mutate so quickly is that they are RNA-based. The viral RNA-making machinery is the same sort that makes host cell RNA. The RNA polymerase enzymes make RNA, but they do so with a higher mutation rate than DNA-making machinery. The infecting viruses do not harbor many genome-making proteins, making them reliant upon stealing the needed nucleic acid synthesis machinery from the host cell. The host cell unwittingly provides the necessary genome synthesis proteins. Some viruses do package in their virion structures specific proteins for specialized functions, like making sense-RNA or anti-sense RNA or double-stranded RNA, but more often, these functions are merely a kick-start for them. These viruses need more cell machinery, such as translational equipment like ribosomes to make proteins or proteolytic processing enzymes or enzymes for post-translational modification.

In particular, RNA-based viruses exhibit more mutations than DNA-based microbes. This propensity for mutation in RNA viruses is due mainly to the inclination of stolen RNA polymerases that make RNA but produce errors when forming chains of ribonucleotides on the RNA molecules. Thus, the RNA viruses have high mutation rates and can be troublesome concerning producing drug-resistant mutants and more pathogenic variants with severe outcomes in a disease. On the other hand, DNA viruses that steal and use DNA-making cell machinery do not exhibit many mutations. This more accurate, i.e., less error-prone ability of DNA polymerases, is due to the exact nature of the proofreading ability of specific sub-units within the DNA polymerases.

Specific microbes, especially influenza viruses, undergo two sorts of mutation called antigenic drift and antigenic shift. In the antigenic drift system, the RNA genome of the influenza virus, for instance, undergoes minor or subtle changes in ribonucleotide sequences that encode neuraminidase and hemagglutinin, producing viral spike structural components that are altered only slightly. The viral RNA sequence drifting is minor, producing minor changes which can be addressed with a functional immune system and by producers of vaccines. Sometimes, molecular biologists and vaccine makers can predict, using computer modeling programs, which and how many antigenic drift variants will predominate in the immediate future, allowing vaccine production efforts to address these variants effectively. Much of the time, these antigenic drift predictions are accurate. If the predictions are inaccurate, then outbreaks of the flu can be possible.

Antigenic shift is an entirely different type of mutation, especially concerning particular influenza virus type A strains. In this genomic sequence shifting process, also called reassortment, significant changes occur in the genes of different virus strains, even between strains that affect animals and humans. The antigenic shift can occur when infection involves more than one strain of the influenza A virus. One good example is genetic recombination, where a large section of the RNA genome from one strain moves to an RNA segment of a different strain, producing a recombined virus with a significant change in sequence pattern and a resulting considerable variation in viral structure. The antigens of reassorted influenza virus strains exhibit significant alterations. Such reassorted viral variants can evade the immune system and become extremely difficult to predict, making such antigenically shifted viruses difficult to stem and readily prevent with vaccines. These recombined variant viruses cannot easily be predicted with current molecular technology. Thus, reassorted viruses can potentially cause new outbreaks, if not full-blown pandemics, such as was seen in the great influenza pandemic of the early 20th century, not to say the COVID-19 pandemic of this century.

7) The treatment of influenza—seems to be via either pill or oral inhalation—how exactly does oral inhalation work, and what are the mechanisms?

In order to relieve the symptoms of influenza, which consist of fever, headache, aches, pains, fatigue, and weakness, several hundred million dollars are typically spent on antihistamines, acetaminophen, and similar medicines. However, there are varieties of specific medicines that target the influenza virus during the clinical treatment of severe cases.

The antiviral medicines of choice for influenza include neuraminidase inhibitors. These medicines include zanamivir (Relenza), which McKimm-Breschkin helped to develop, plus oseltamivir (Tamiflu) and peramivir (trade name Rapivab). They all target influenza viruses A and B. These neuraminidase inhibitors bind to the enzyme and stop its activity. The result is that the neuraminidase enzyme of the infecting influenza virus binds to sialic acid on other glycoproteins, permitting additional viruses to form worthless clumps of viruses or to stick on the internal membrane surfaces, thereby preventing the release of the virions from the cell. Either way, neuraminidase inhibitors break the influenza virus infectious reproductive cycle. The disease can be treated if medical therapy is provided within the first two days after illness onset.

Oseltamivir is in pill form and taken orally. Zanamivir is taken to the respiratory tract via oral inhalation by the patient. An inhaler device provides the oral, respiratory administration of zanamivir. A “rotadisk” device loads the medicine from individual blisters into the inhaler apparatus as the patient inhales the drug. As the medication is taken in, the inhaler device breaks the blister, releasing the zanamivir into an airstream that the inhaling patient creates. The flu treatment dosage is typically two inhalations per day for about five days, with each inhalation about five to 10 mg of the drug per blister.

8) Currently, McKimm-Breschkin is continually developing new drugs that can treat influenza—why is this currently important? 

New anti-influenza drugs must be developed continually. One important reason is that specific strains of the virus can alter their genome sequences, producing drug resistance and, thus, potential treatment failures. A related reason is that antigenic shift, reassortment, may produce variants with devastating pandemic-causing potential. Therefore, the flu and its causative agent, the influenza virus, is a constantly moving target. Another reason to discover new medicines is that those who need them, potentially, may not always follow the necessary prevention and control measures, thus allowing the spread of viral transmission, the emergence of flu disease outbreaks, and enhanced mortality.

McKimm-Breschkin had focused some of her molecular structure studies on neuraminidase, the protein that the influenza virus needs for successful infection in human respiratory cells. McKimm-Breschkin continued to focus her efforts on the active site of the viral enzyme to develop newer and better inhibitors that might be approved someday for treatment of the flu, especially in cases where antiviral drug resistance is a problem.

In 2018, she astutely understood that the so-called head structure of the neuraminidase protein attached to a tail had never been determined in molecular detail. It had been known that the intact protein consisted of two main sections: a globular head and a stalk. In turn, the intact stalk had three sub-regions: the stalk region, a transmembrane domain, and a cytoplasmic region. McKimm-Breschkin made crystals of the neuraminidase head with and without an artificial stalk and used X-ray crystallography to elucidate their molecular shapes in high-resolution detail. It had been the first time in history that the neuraminidase head structure attached to a tail had become known. The groundbreaking result was published in early 2019.

At about the same time, McKimm-Breschkin reported on newly developed DFSA and its derivatives and their ability to act as potent inhibitors of neuraminidase. The DFSA term stands for the chemical name difluorosialic acid. One of these DFSA derivatives, called 5-N-(acetylamino)-2,3,5-trideoxy-2,3-difluoro-D-erythro-β-L-manno-2-nonulopyranosonic acid, showed that the neuraminidase activity and viral replication were inhibited. McKimm-Breschkin mapped out the molecular contacts between the inhibitor and the enzymatic active site of the neuraminidase glycoprotein. She showed that a carbon atom of the inhibitor made chemical contact with a tyrosine at position number 406 of the neuraminidase enzyme, which effectively inhibited the activity.

Then, in another study, McKimm-Breschkin made a group of deoxygenated derivatives of DFSA with each of their hydroxyl groups missing and determined which ones had the most considerable impacts on binding affinities and antiviral activities. She had found that the hydroxyls at numbers eight and nine positions of the various deoxygenated DFSA molecules were the critical missing hydroxyl groups. Thus, McKimm-Breschkin determined critical molecular contacts between the neuraminidase enzyme and its new potent inhibitors. Such molecular-based work shows promise to garner new and upcoming antiviral agents for treating the flu, especially drug-resistant strains of influenza viruses.

9) What have I neglected to ask about the treatment of influenza and the work of this great female scientist?

We discussed above how McKimm-Breschkin was critically involved in significant zanamivir studies and viral resistance to this clinically helpful therapeutic. Treatment of influenza involves other distinct drug types. Another class of antiviral medicines for the flu include amantadine and rimantadine, both of which are inhibitors of the influenza A virus component called M2, a membrane-bound ion channel protein. Typically, during the influenza viral infection, the virus enters the cell by endocytosis, enclosing the virions inside a vesicle called an endosome within the cytoplasm of the infected host cell. The M2 ion channel protein resides in the endosomal membranes of newly internalized virions. The prime substrate of the M2 channel is specific for protons, and it allows these ions to enter the vesicle, thereby permitting acidification of the endosome. As the protons enter the endosome where the virus is located, the acidified vesicle permits the virion to be unpackaged from its encasement. Amantadine and rimantadine prevent this proton endosome entry and thus prevents acidification of the vesicle, trapping the virion in the internal endosome and preventing uncoating of the RNA genome. This endosome trapping thwarts the viral life cycle and the path of the influenza infection of a host cell.

The third class of anti-influenza drugs targets the virus’s RNA polymerase enzyme sub-unit called PB2. A fully infectious influenza virus has an RNA polymerase composed of three sub-units: PB1, PB2, and PA. The regular function of the PB2 protein serves as an endonuclease to steal a so-called 5’ cap of a person’s cellular mRNA and use it as a primer starter for viral mRNA molecules! Recently approved by the FDA, the new drug called baloxavir marboxil (trade name Xofluza) inhibits PB2 of influenza viruses A and B. The baloxavir marboxil drug is an analog of nucleosides, and it fails to work when bound to PB2. Inhibition of PB2 by baloxavir marboxil prevents the mRNA cap-stealing endonuclease activity. The result of PB2 inhibition is that viral transcription is prevented, making viral RNA unavailable for making influenza proteins and building intact influenza virions. Incompletely built influenza viruses cannot continue the cellular infection.

These classes of anti-influenza drugs suffer from viral variants that may be drug-resistant. It was found that prophylactic use of these and other antivirals led to resistant influenza virus strains. Some strains are naturally drug-resistant and may be selected with drug use, eliminating susceptibility but unable to thwart naturally resistant viral strains, which can predominate and confound flu treatment. Additionally, as we have learned from people who cannot (or will not) wear masks or socially distance in a practical manner, it is almost impossible to reduce the airborne transmission of viruses, such as influenza virus or even SARS-CoV-2. Thus, the best method to control virally caused respiratory disease transmission is prevention through vaccination.

Presently, there are three classes of influenza vaccines in use for prevention. The first is called toxoid or inactivated subunit influenza vaccine. Purifying or extracting neuraminidase and hemagglutinin proteins from viral preparations and treating them with chemicals prepare these types of influenza vaccines. The immune system will detect the foreign proteins and produce a protective immune response.

The second type of influenza vaccine contains viruses that are “killed” or inactivated with formaldehyde, a chemical used to preserve cadavers. In a 37% solution called formalin, however, the chemical denatures the virion and makes it useless for cellular infection but quite helpful in mounting an effective immune response.

The third type of flu vaccine is characterized as a “live” but attenuated influenza virus vaccine. In this case, the influenza virus is a mixture of reassorted forms that are not infectious but quite antigenic. This vaccine can be available as a nasal spray. It can mount an immune characterized by both humoral and cellular mediated immunity.

Annual vaccination for influenza is highly recommended because of a yearly incidence of five million cases and a death rate of over 80,000 people in the U.S. Vaccination every year would reduce these stunning numbers.

Print Friendly, PDF & Email