An Interview with Manuel and Ann Varela: Who is Katalin Karikó?

May 17, 2021 by

When I am knocked down, I know how to pick myself up. But I always enjoyed working. I imagined all of the diseases I could treat.” 

— Katalin Karikó

The important thing in science is not so much to obtain new facts as to discover new ways of thinking about them.”

—Sir William Bragg

Michael F. Shaughnessy

1) Katalin Karikó is obviously a different name—so she must be from a foreign country. Where and when was she born?

Karikó was born in central Hungary, more specifically, Szolnok, Hungary, on January 17, 1955, but was raised in the small town of Kisujszallas. She began her higher education career in 1973 and completed courses at the University of Szeged, where she earned a Ph. D. in 1972. After that, she filled a postdoctoral fellow position at Szeged’s Biological Research Center until 1985. When the research funding was depleted in 1985, Karikó and her family moved to the United States. She was in her twenties. It was difficult for her to obtain a permanent position in any one lab at the University of Pennsylvania due to not obtaining grants to study seemingly far-fetched ideas. Apparently, when one’s ideas are progressive or perhaps even eccentric, it is difficult to embark on new paths of discovery.

Karikó had accepted a postdoctoral position at Temple University in Philadelphia, where she remained until 1988. It was perhaps courageous for her and her husband Bela Francia and two-year-old daughter, Susan, to make this cross-continental move. Because the government in Hungary only permitted its citizens to take a small amount of money out of the country, one hundred dollars, Karikó and her husband performed monetary transplant surgery on their daughter’s teddy bear, at which point they sewed £900 into the stuffed toy. Incidentally, Susan became an exceptional rower on the U.S. National Team. She won two gold medals, one in 2008 with the Olympic Team in Beijing, China, and the other in 2012 with the Olympic Team in London, England.

Karikó’s life story is one of persistence. She persevered for decades and is now being celebrated for her contribution towards developing a COVID-19 vaccine.

2) Where did Karikó go to school and ultimately receive her doctorate?

After Karikó earned her Ph.D. at the University of Szeged in 1982, she carried on with her research and postdoctoral studies at the Institute of Biochemistry, Biological Research Center of Hungary until 1985, the Temple University Department of Biochemistry until 1988, and the Uniformed Services University of the Health Science in Bethesda, Maryland until 1989.

Karikó accepted a research assistant professorship in the department of medicine, Perelman School of Medicine, at the University of Pennsylvania from 1989 to 1995. She worked in the laboratory of Dr. Elliot Barnathan, a cardiologist, and the two designed experiments to insert mRNA into cells, thus stimulating them to make new proteins. To their surprise, the gamma counter indeed found radioactive molecules in their samples, which suggested that mRNA could be used to instruct any cell to make any protein at will. Though Karikó and Barnathan generated many creative ideas relating to using this breakthrough, Barnathan accepted another position at a biotech company and left the University of Pennsylvania, which left Karikó with no lab or funding to continue her research projects.

Fortunately, Karikó’s reputation and diligent work ethic made it possible for her to continue her work at the university in Dr. David Langer’s laboratory. After numerous failed attempts at using mRNA on isolated blood vessels used to study strokes, Langer left the university. Once again, Karikó found herself without a lab or funding. However, she formed a new partnership with Dr. Drew Weissman, who was looking for a vaccine against H.I.V. Karikó said she could make anything with mRNA. Unfortunately, it was one thing to instruct cells in Petri dishes to make a given protein and quite another to instruct them in living mice. Karikó was determined to unravel this mystery, and she did! Apparently, the immune system responds with inflammation when it recognizes attacking microbes by sensing their mRNA.

From 1995-2009, she was promoted to senior research investigator in the department of neurosurgery at the same institution. Presently, Karikó holds the title of adjunct associate professor at the University of Pennsylvania.

In 2013, Karikó took a position to lead the mRNA-based replacement program at BioNTech RNA Pharmaceuticals, a biotech company located in Germany. The company later collaborated with Pfizer to make the first COVID-19 vaccine. Together with Weissman, the idea for the vaccine was to present mRNA into the body that would briefly initiate human cells to produce the spike protein of the Coronavirus. On November 8, 2020, the initial results of the Pfizer-BioNTech study were revealed, showing that the mRNA vaccine put forward effective immunity to the new virus.

3) Her main thrust of research—to what did it lead?

Dr. Karikó’s scientific research has dealt primarily with the biochemistry of RNA. Her focus has been the development of specific RNA molecules for the therapy of human disease. Karikó’s research led to the development of stable mRNA molecules to treat disorders involving the immune system and the development of the recent COVID-19 RNA vaccines.

Karikó’s story with RNA began in 1985 with her first publication on the topic in the journal Biochemical and Biophysical Research Communications. Karikó and her colleague János Ludwig studied individual ribonucleosides that made up ribosomal RNA (rRNA). They produced a variant of ATP by attaching a chemical group called n-decyl-amine to the end of the ATP. The chemically modified ATP became resistant to the degradative effects of an enzyme called phosphatase. Before, the phosphatase destroyed the rRNA. However, after Karikó and Ludwig made the chemical variant, they introduced the new compound to HeLa cells in a culture treated with interferon to activate an RNA-destroying enzyme called RNase L. The RNase L enzyme has the scientific name 2’,5’-oligoadenylate-dependent endoribonuclease. The primary function of the RNase L enzyme is to degrade RNA. Remarkably, their modified rRNA was also resistant to the RNA-degrading enzyme.

Karikó’s laboratory synthesized similar chemical variants in a follow-up experiment called phosphorothioate analogs of the 2’,5’-oligoadenylate compound, a type of nucleic acid. They then used the new variant compounds to enhance the activation of the RNase L protein. The result was enhanced degradation of rRNA. These early studies permitted Karikó to acquire the skills needed to modify RNA molecules. This expertise would turn out to be a tremendous benefit for a significant number of the human population on a worldwide scale. Her ability to modify RNA molecules would solve a serious problem with these nucleic acids and the immune system. Karikó’s work resulted in developing more stable RNA molecules that would be used to create two of the world’s first COVID-19 vaccines.

4) Messenger RNA or mRNA—why is this important, and what did Karikó have to do with this?

Molecules of mRNA contain a type of gene code called codons. The mRNA carries the genetic message to the cellular machinery for making proteins. As such, the mRNA molecule functions like a template upon which the genetic message is read and translated into the production of proteins. RNA differs from DNA in that the sugar part of RNA is ribose, whereas, in DNA, the sugar is deoxyribose. See Figure 1. Ribose sugars lack a so-called hydroxyl chemical group on one of their carbon atoms.

Figure 1. The difference in structure between ribose and deoxyribose is the presence of a 2’ OH.

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There are, in general, three types of RNA molecules in living cells: the messenger RNA (mRNA) that you spoke of, ribosomal RNA (rRNA), and transfer RNA (tRNA), as shown in Figure 2. Each type of these RNA molecules functions during translation, a protein synthesis process found in all living creatures. Every one of the biological organisms on Earth uses mRNA as a carrier of DNA’s genetic information. As such, after DNA, RNA is a central molecule of life. The critical nature of RNA for life is so great that even for viruses, regardless of their type, shape, origin, or range of hosts, all viruses must present an RNA message to the cell’s internal workings to propagate themselves. Without RNA, viruses cannot reproduce.

Figure 2. Simplified diagram of three types of RNA: Messenger RNA, Transfer RNA, and Ribosomal RNA.

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The mRNA molecule is inspected by the ribosome, a key protein-making machine that connects various amino acids into long chains called polypeptides. The gene codons run along a given mRNA in particular sequences of bases, called ribonucleotides. Each of the codons is three-ribonucleotide bases long, called triplets, and they specify the nature of the amino acids that go into the new proteins.

The rRNA molecules participate as structural components of the ribosomes and function as an enzyme to make peptide bonds between amino acids and link them together in long chains called polypeptides. A part of the rRNA makes up the enzyme part of the ribosome called peptidyl transferase. Interestingly, this peptidyl transferase is RNA and is thus called a ribozyme. The newly made polypeptides fold into their functional shapes. The proteins are sometimes biochemically processed akin to a post-translational modification.

The tRNA molecules function by transferring a specific amino acid from the cytoplasm of the cell to the translational apparatus. As such, the tRNA molecule serves as a sort of adaptor, making the various amino acids ready for protein making. Each tRNA harbors an anti-codon sequence, which is specific to a given amino acid. Thus, each amino acid has a specific tRNA with a specific anti-codon. The anti-codon of the tRNA-amino acid complex binds to the codon of mRNA during protein production in the cell.

All three types of RNA play integral roles in protein synthesis, a cellular process called translation. The various rRNA molecules come together with protein subunits to form intact ribosomes. The mRNA binds the ribosome. The tRNA binds its specific amino acid, becoming “charged.”

The ribosome reads the mRNA code message, and the charged tRNA-amino acid molecule binds to the mRNA at the codon. Another charged tRNA with an attached amino acid comes into the translation assembly. The rRNA peptidyl transferase part of the ribosome catalyzes a peptide bond formation between the first and second amino acids.

The process starts over again. The translational machine permits another charged tRNA to arrive at the ribosome. According to the mRNA codon sequence, the rRNA’s ribozyme forms another peptide bond to link another amino acid to the protein chain. This translation elongation process continues until a so-called “stop” codon on mRNA is reached. At this termination point, the translation machine falls apart, and the newly made protein is released. The protein folds into its correct shape and is perhaps biochemically modified, and the protein becomes functional.

Karikó’s work helped to solve two significant problems that were encountered when working with RNA. The first main difficulty was the stability of RNA. Outside of its natural environment, RNA tends to degrade, as it is chemically sensitive to high pH conditions and susceptible to hydrolysis. The second big problem was that the immune system got rid of any therapeutic RNA before conducting its restorative work. She solved these two complications by chemically altering the bases in RNA molecules. The newly modified RNA variants were both stable and able to survive the onslaught of the immune system.

Karikó’s chemically altered RNA made it possible to package stable RNA inside artificial lipid bubbles for easy delivery to the insides of cells, where they could be used to make protein by translation. Once the stable RNA molecules are inside a cell, the immune system cannot get to them. The antibodies and immune cells of the body cannot enter a person’s cells. Thus, any foreign antigen, like Karikó’s modified RNA, located inside a living cell can evade degradation or elimination by the immune system. Karikó’s research investigations led to the ability of the modified mRNA molecules to avoid detection by the immune system just long enough to find a way into the cell. Thus, once inside a living cell, the mRNA could be used by the cell’s translational machinery to produce the desired protein that was not made before, such as a vaccine antigen.

5) Katalin Karikó worked with Drew Weissman, an immunologist—and figured out a way to fool the immune system and effectively used mRNA—what happened?

According to scientific history lore, Drs. Karikó and Weissman met at the University of Pennsylvania School of Medicine while making copies at a photocopier machine. Their collaboration would ultimately prove beneficial to developing the first RNA vaccines in history to be used on humans.

At the time they met, however, both scientists struggled with debilitating problems in their research programs. Though Karikó could coax mRNA to produce protein in cells, she was, nevertheless, having difficulties getting an RNA treatment to work in preventing blood clotting during neurosurgery. When she added mRNA coding for nitric oxide production to blood vessels that had been isolated from stroke-prone rabbits, the mRNA failed to elicit the nitric oxide that was needed to dilate clotted blood vessels. Weissman, meanwhile, had been struggling in his efforts to produce a vaccine against HIV. This human immunodeficiency virus produces AIDS, acquired immunodeficiency syndrome, and depletes the adaptive immune response. Weissman was having trouble coaxing immune cells in the lab to produce antigens for the HIV vaccine.

At the photocopier, Karikó was reported to have told Weissman that she could produce HIV vaccine antigens with her RNA method. They began a collaboration and tried using mRNA to make proteins in living laboratory mice. When Karikó and Weissman prepared mRNA and introduced it into immune cells called dendritic cells, they observed that the cells produced HIV proteins, which then had an immune response in cell culture. However, the first attempt failed to get the mice to produce the desired HIV protein. Instead, the injected mice became ill, and their immune system rejected the artificial mRNA. The laboratory mice had countered the foreign mRNA with an inflammatory response. Thus, the immune system thwarted efforts to use mRNA to produce a protein that could have been used as a vaccine. The immune system also disabled their attempts to use RNA for therapies.

If RNA were to be used therapeutically, the inhibitory effect of the immune system had to be overcome first. However, surmounting the immune system would not be easy. The molecular and cellular mechanisms that function in the immune system are vast and complicated. Karikó and Weissman had first to learn which of the many components of the immune system played a role in preventing RNA from being used as a vaccine or a therapeutic agent.

In their subsequent study, Karikó and Weissman discovered that the immune system component responsible for the response to RNA was an innate immunity molecule called Toll-like receptors (TLRs). See Figure 3. The term “Toll” is German for “fantastic.” The word was used to describe fruit fly genes in which mutations caused the formation of bizarre-looking (fantastic!) flies. However, the functions of Toll-like proteins of humans and flies are different. In humans, the Toll-like proteins take part in innate immunity, whereas in flies, the proteins play a role in embryonic development.

Figure 3. A computer-generated model of a binding domain for a TLR protein.

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These sorts of TLR molecules distinguish between “self” antigen molecules on an individual versus foreign antigens on a microbe in humans. The TLRs belong to a group of so-called pattern recognition receptors. When foreign antigens bind the TLRs on cells, the interactions signal the inflammatory response, eliminating the foreign antigens. These non-self-foreign antigens from microbes that TLRs recognize are called pathogen-associated molecular patterns, or PAMPs.

Karikó and Weissman had succeeded in identifying that part of the immune system responsible for destroying RNA. Thus, if that RNA-eliminating part of the immune system were to be somehow circumvented, RNA could be used to make novel vaccines and therapies. When Karikó and colleagues looked more closely at the relationship between mRNA and the immune system, they found those mRNA molecules formed a bonding association with the TLRs. Karikó discovered that mRNA was a so-called ligand for the receptor on the immune cells. In other words, foreign mRNA functioned like a PAMP. Foreign mRNA, but not native mRNA, was recognized as a PAMP by TLR. A complex formed between external mRNA and TLR, and it would set off a protective inflammatory response and production of an anti-viral substance called interferon.

Currently, about ten TLRs are known in humans. Each TLR type has been given a specific number. Karikó and Weissman discovered that when the foreign mRNA (a PAMP) bound to TLR number three, TLR3, the receptor became activated. The mRNA turned on the TLR3 molecule on specialized immune cells called dendritic cells. Activation of the TLR3 on the cell’s membrane caused the activated immune cell to secrete various cytokine molecules, most of which were dedicated to the activation of the immune system, including inflammation and interferon production.

After the failed HIV mRNA experiment in the mice, Karikó made a groundbreaking realization. A control experiment provided an insight. Karikó found that tRNA did not provoke the immune system as mRNA had. She thought, therefore, that since rRNA did not generate an RNA-rejecting immune response, then perhaps the modified bases in tRNA somehow permitted the evasion of the immune response. Karikó hypothesized that if specific ribonucleoside bases of mRNA were similarly changed as was known with the tRNA, then mRNA molecules altered again could evade the immune system. The new insight and the consequent research investigation by Karikó would prove highly successful for RNA as a vaccine.

With the new understanding at hand, Karikó and Weissman altered ribonucleoside bases and placed them into newly made mRNA. The base alterations took the form of pseudouridine and 5-methyluridine. Naturally-occurring tRNA molecules contain pseudouridine and 5-methyluridine. When Karikó and Weissman added their newly modified mRNA molecules made with pseudouridine, the immune system could not detect their mRNA variant! The modification permitted the unconventional form of the mRNA now inside the cells to produce new proteins using the translational machine of the cell.

The discovery by Karikó meant that a foreign mRNA molecule with ribonucleosides that were made to look like the kind of bases found in tRNA could circumvent the TLR-mediated immune response activation. The modified mRNA would not stimulate an immune response. The immune system evasion property of the new tRNA-mimicking mRNA molecule meant that a protein encoded by the new RNA could serve as a template for making a new protein.

Perhaps the gene product encoded by the mRNA could be an HIV protein for a vaccine. The mRNA code could, in theory, specify just about any desired protein. Any viral protein could be encoded on the modified immune system evading mRNA! The potential of mRNA for use in new therapeutics and vaccine production was immense, thanks to the immune system escaping mRNA molecules with their freshly incorporated substitute bases. The novel mRNA discovery by Karikó would prove to help save the world from one of the worst viral pandemics of the 21st century.

6) Treatment of brain ischemia—how does her work fit into this realm?

The medical term ischemia means that blood flow through the circulation is poor. Ischemia of the brain means that there is a lack of a good blood supply to the brain. Thus, an ischemic brain is a pathological condition because without sufficient blood to provide needed oxygen, the brain’s tissue could suffer damage (brain damage) and possibly die, a process called tissue necrosis. See Figure 4.

Figure 4. Perfusion MRI for a patient with left MCA occlusion.

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Another consequence of brain ischemia is an immune system-based condition called acute inflammation. In general, inflammation represents a so-called second-line process of the natural or innate immune system defenses in tissues of the body. In general, whether the inflammation is acute or chronic, the progression can involve redness of affected skin, increased heat to the involved tissue, swelling due to enhanced vascular permeability, infiltration of immune cells, and pain from stimulation of sensory nerves. Acute inflammation of the nervous system can worsen the extent of brain damage during an ischemic condition. The acute inflammation process in the brain involves so-called pro-inflammatory cytokines, which are small proteins that regulate the various physiological pathways during inflammation.

Karikó became involved with this brain ischemia system by discovering that mRNA is bound to the Toll-like receptors (TLRs), which are proteins regulated by pro-inflammatory cytokines. It seemed that the inflammation process that occurs during brain ischemia worked through a cell signaling mechanism involving the TLRs. During an ischemic event in the brain, the inflammation process is turned on. When brain tissue or cells are damaged, they release new molecules that bind to the TLRs, which in turn activate the production of inflammatory cytokines.

Thus, Karikó reasoned that if TLRs could somehow be regulated, inhibiting their function could reduce the production of inflammatory cytokines and thus reduce the brain-damaging ischemia. Prevention of tissue-damaging ischemic episodes in parts of the body, like the brain, is called ischemic tolerance. One cellular mechanism for inducing ischemic tolerance is called cortical spreading depression. This mechanism involves a slowly propagating wave of neuronal depolarization, followed by activity suppression in the brain’s vasculature. Thus, the brain is pre-conditioned by cortical spreading depression to undergo ischemic tolerance.

Karikó became interested in the effects of cortical spreading depression and its induction of ischemic tolerance on the levels of individual mRNA molecules. Karikó was drawn to specific mRNA molecules that encoded proteins that were protective against the onset of brain ischemia. In the brains of laboratory rats, Karikó experimentally induced the cortical spreading depression system and measured their levels of mRNA that encoded specific neuroprotective proteins. Karikó’s laboratory discovered that upon induction of the cortical spreading depression, the rats showed enhanced levels of mRNA molecules encoding a brain-derived neurotrophic factor (BDNF), a tissue plasminogen activator (tPA), and a transcriptional activator called c-Fos. The BDNF protein is known to regulate the cellular maintenance and development of neurons.

Interestingly, the tPA is a blood clot-dissolving enzyme that helps prevent heart muscle damage after a heart attack and decreases nerve damage during brain ischemia. The transcription activator c-Fos is a well-studied proto-oncogenic protein and is known to turn on the gene expression programs of a wide variety of proteins. Karikó published her novel findings in 1998 in the Journal of Cerebral Blood Flow & Metabolism.

In another study, the laboratory of Karikó found that salt solutions added to the brains of lab rats induced the production of mRNA molecules encoding inhibitors of inflammation. The immune system inhibitors included a cytokine called tumor necrosis factor (TNF), a protein called suppressor of cytokine signaling number three (SOCS3), and a cytokine called ciliary neurotrophic factor (CNTF), among others. TNFs are known inflammation regulators that are secreted by immune cells and function to kill cancer cells. The SOCS3 protein plays a role in regulating cytokine signaling systems in immune cells like macrophages and T-cells. The CNTF cytokine is an interleukin-6 type of immune system communication molecule which gets produced during tissue injury.

These sorts of studies by Karikó made it possible to identify mRNA molecules that encode inhibitors of inflammation. Efforts have been centered on finding new ways to turn on these inflammatory inhibitors during brain ischemia. Another area of research investigation by Karikó included identifying new targets for regulating inflammation in the brain during ischemia. One of these targets included Toll-like receptor number four (TLR4). The Karikó laboratory had created transgenic mice in which the gene for TLR4 was deleted to study this area. Then, the transgenic mice were subjected to an artificially induced stroke in which intracerebral hemorrhage was produced. The transgenic mice lacking the ability to make TLR4 showed less brain inflammation than normal mice with the TLR4 protein. Thus, thanks to the work of Karikó, therapeutics that somehow reduce the effects of TLR4 may show promise during brain inflammation caused by brain injury.

7) How does mRNA convince cells to make their own medication or medicine?

Karikó’s discovery that modified mRNA molecules could get around the immune system by acting like tRNA molecules provided the ability to introduce mRNA encoding desired medicines into the cell. Once the new mRNA molecules were inside the cell’s cytoplasm, the translational machinery could act upon them to produce the selected proteins. Such medicinal proteins could include immune system regulators, anti-viral proteins, and proteins that serve as good vaccines.

There are several ways to introduce foreign mRNA molecules encoding therapeutic proteins to the cells to make their new medicines. One method is called transfection, in which naked mRNA molecules are added to competent host cells, and the foreign nucleic acid-based therapeutics are taken up by the cells. Transfection can be performed by electrical means using a technique called electroporation. In contrast, host cells can take up nucleic acids using a chemical means of transfection.

Thirdly, infectious methods may be used to introduce mRNA to cells using viral vectors. These viruses are altered to take out their pathogenic genes and proteins. Then, therapeutic genes are inserted into the emptied viral genomes and used for infection to introduce the foreign mRNA into the cell.

The mRNA may be inserted using lipid vesicles, which are circular bubble-like structures encasing the nucleic acid. The mRNA-containing lipid vesicles fuse with the cell membrane and present the new RNA into the cytoplasm. One recent development is represented by the so-called nanoparticles, such as proteins or synthetically-made polymers, which can be used to deliver mRNA molecules to the cell cytoplasm. Once the mRNA is brought to the inside of the cell, it can make protein medicines.

Essential cellular machinery involved in protein synthesis, a process called translation, includes the mRNA molecule as a template, the ribosome, tRNA, and amino acids, Figure 5. The translation activity is a protein-making process in which the ribosome uses the base sequence of the genetic information that is carried by mRNA to connect specific amino acids to construct protein chains. The events of translation are separated into three general phases: initiation, elongation, and termination.

Figure 5. Players of translation: A ribosome, an mRNA, and tRNAs (each bound to an amino acid) interact to produce a peptide (or a protein) molecule. The amino acids (aa) are denoted with numbers to show the sequence along the polypeptide chain.

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During the initiation phase of translation, the two large subunits of the ribosome assemble onto mRNA, the template. The first codon, a base triplet AUG, is read by the ribosome, which then recruits a tRNA-methionine complex to arrive and bind its anti-codon to the starting codon on mRNA, a site on the ribosome called P, for peptidyl. Interestingly, a molecule of GTP is hydrolyzed as a source of energy for translation initiation.

The second translation phase, elongation, involves several new steps. First, a second tRNA molecule with an attached amino acid, specific for the second codon of mRNA, connects to the aminoacyl (A site) of the ribosome and sits next to the first tRNA-methionine complex from the initiation process. This second tRNA-amino acid complex represents the code of the second codon on mRNA. The next step involves the ribozyme undergoing a life-giving formation of a peptide bond between the first and second amino acids. It forms a dipeptide, two amino acids attached by a peptide bond. Then, the ribosome moves over one codon notch to the next codon along the mRNA string. The first tRNA falls away during the next step. The fallen-away tRNA is empty without its amino acid, and a third tRNA-amino acid complex arrives and attaches to mRNA. A new peptide bond is now formed between the second and third amino acids, creating a tripeptide.

The elongation steps then undergo a repeating process of ribosome movement, a new tRNA-amino acid attachment, a spent tRNA falling off, and another peptide bond formation to add amino acids to the growing polypeptide chain. This repeating elongation step series occurs until the termination phase.

The termination phase involves the translational machinery reaching a termination codon on mRNA. This termination site has a codon triplet base composition on mRNA of UAA, UAG, or UGA. When either of these termination codons on mRNA is reached, the translational players disassemble, and the protein-making process ends. After the termination phase of translation arrives, the nascent polypeptide folds into a three-dimensional shape and may be post-translationally modified to regulate its protein activity.

Though the phases of translation, initiation, elongation, and termination are similar in prokaryotes and eukaryotes, they are not identical. Eukaryotes have a membrane-bound nucleus, whereas prokaryotes lack such nuclei. In eukaryotes, the mRNA is extensively processed in the nucleus. At first, as mRNA is made, introns and exons are together. Nevertheless, before mRNA is sent to the cytoplasm, introns are removed, keeping only the exons, the expressed forms of the gene. This process is called splicing. In prokaryotes, mRNA molecules lack introns and exons. The 5’ ends of the mRNA are capped with modified bases and phosphates. That process is called capping. To the 3’ end of mRNA is attached a series of adenine bases, forming a so-called poly-A tail. This latter process is called polyadenylation.

In eukaryotes, a given processed mRNA molecule is used for expressing only one polypeptide. In prokaryotes, an mRNA molecule could express more than one polypeptide. In eukaryotes, an mRNA molecule is not used for translation to protein until it has been fully processed and delivered to the cell’s cytoplasm. Thus, in eukaryotes, transcription (RNA synthesis) and translation (protein synthesis) never co-occur. Transcription occurs in the cell’s nucleus, and translation occurs in the cytoplasm. In prokaryotes, transcription and translation can appear simultaneously because the machinery for the synthesis of both RNA and protein resides in the cytoplasm.

8) How does Karikó’s work relate to the current COVID-19 situation? And Moderna?

The work of Karikó led to the historical and unprecedented use of RNA molecules as vehicles for vaccine development. Before the COVID-19 pandemic, RNA had never before been used in human vaccination. Karikó is a pioneer of synthetic RNA technology for therapy and vaccine discovery.

Before the RNA vaccines for human use were developed, Karikó discovered that foreign mRNA bound to Toll-like receptors, acting as a sort of ligand by inducing inflammation, which would quickly eliminate the foreign mRNA molecules. Thus, the mRNA could not be used for translation to make protein antigens that might have provided immunity as a vaccine against COVID-19. It was a big obstacle, and Karikó solved the problem with her particular insights and expertise. To circumvent the unwanted immunogenicity of foreign mRNA molecules, Karikó modified the ribonucleoside bases of mRNA to make it act more like a non-immunogenic tRNA. When modified mRNA was used, it evaded the immune response! She had gotten the new mRNA to act like a non-immune provoking antigen. Thus, the stage was set for these modified mRNA molecules with a gene sequence for the Coronavirus spike protein to be used for the first time in humans!

The two leading Coronavirus vaccines are from Moderna and Pfizer. The Moderna vaccine is called “mRNA-1273,” and Pfizer’s vaccine is called “BNT162b2.” Both vaccines have a lipid vesicle enclosing a modified mRNA molecule that codes for the Coronavirus spike protein. See Figure 6.

Figure 6. Coronavirus spike protein.

The lipid vesicle nanoparticle with the internally located modified spike-encoding mRNA fuses with the vaccinated person’s cells. The mRNA is released to the inside of the cytoplasm, where the needed translational machinery is located. The mRNA does not produce an intact virus, but it does specify the genetic material to make the spike protein by translation. The spike proteins then insert into our cells’ membranes by a process called antigen presentation. The spike protein of the Coronavirus is detected by cells of our immune system, known as immune cells.

A type of immune cell called helper T-cells binds to the presented spike proteins on the human cell’s surface in a vaccinated person. These attached helper T-cells turn on B-cells and cytotoxic T-cells, all of which are immune cells. The B-cells morph into plasma cells to generate antibodies specifically against spike proteins during the immune response. These anti-spike protein antibodies have viral neutralizing properties and thus protect us against the Coronavirus microbe. The vaccine-generated antibodies can save us from a natural Coronavirus infection if exposed to the COVID-19-causing microbe. The cytotoxic T-cells can readily attack and kill any host cells that are infected with the Coronavirus.

Further, memory B cells are made as a result of the RNA vaccination. These patrolling immune cells remember a second exposure to anything with a spike protein, like an actual Coronavirus, producing a more vigorous immune response that protects us from a real infection.

Take note of the fact that the mRNA vaccines do not contain infectious or intact Coronavirus. See Figure 7. The mRNA does not mess with our genomes, as the RNA molecules cannot enter our cellular nuclei where our genomes are stored. As you know from the description of cellular biology above, RNA cannot edit DNA. The mRNA is broken down after its translated into spike protein; so, the mRNA does not hang around afterward.

According to Dr. Anthony Fauci, vaccination is key to acquiring herd immunity, a consensus of the world’s leading infectious disease experts. The vast majority of the human population can be immune through widespread vaccination. Reading herd immunity by massive vaccination programs is one of our biggest hopes towards stemming the flow of the COVID-19 pandemic.

Figure 7. A vial of the COVID-19 vaccine.

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9) What have I neglected to ask?

The Coronavirus microbe is a membrane-covered virion whose genome is a (+) sense single-stranded RNA molecule enclosed by a capsid protein. The term Corona was invoked to describe a feature of its structure. Some scientists hold that the spike proteins make the virion look like a crown, and still, others contend that these spikes look like halos, reminiscent of the sun’s corona during an eclipse. The RNA genome is 30 thousand bases long and forms a linear nucleic acid molecule. The hosts of Coronavirus include birds, like pigeons, rodents, like mice, and mammals, like bats and humans. The Coronaviridae family can be sub-divided into three Coronavirus genera types: alpha, beta, and gamma. The beta Coronaviruses include the so-called SARS-CoV-1 and SARS-CoV-2 virions.

After the virus was first discovered in 1965, it was called B814, and it was known to cause mild respiratory diseases like the common cold. Then, in 2002, the Coronavirus evolved by mutation into a severe form, causing a dangerous ailment called the severe acute respiratory syndrome (SARS), which could be deadly. For the first time in its history, a common cold-causing virus could change its form into a lethal type!

The SARS form of Coronavirus, SARS-CoV-1, had caused over 8,000 infections and killed over 700 people during the 2013 outbreak, occurring in about 20 different countries, causing an alarming pandemic. The nature of the SARS-CoV-1 mutation was centered on the spike protein. In 2012, another variation emerged, called MERS, for the Middle East respiratory syndrome.

Because of the 2013 SARS pandemic, some developed countries, including the U.S., initiated a pandemic policy of screening by testing, isolating, or quarantining exposed individuals, and in many cases, wearing masks and keeping a safe distance when in public places. These latter practices had been instituted because of the lessons learned during the influenza pandemic of 1918 and 1919. Shortly after that devastating flu pandemic, we had been able to stem outbreaks, keeping the flu incidences and prevalence numbers from reaching high pandemic levels, despite the influenza virus’s yearly propensity for mutation and evolution. Much of these lessons learned were forgotten or ignored in early 2020. The containment and prevention policies were not effectively followed, leading to enhanced morbidity and mortality rates and the onset of a historical out-of-control COVID-19 pandemic.

The SARS-CoV-2 structure, see Figure 8, has several components to it. The spike protein (S) is a trimer of a single polypeptide with 1273 amino acids in length. It has sugar molecules attached to it, and hence, it is a type of glycoprotein. The trimer of the spike forms two sub-units, called S1 and S2. The S1 subunit has a receptor-binding domain that allows the virus to be attached to human receptors called ACE-2 for angiotensin-converting enzyme number two. The S2 subunit of the spike protein permits the bound virus to enter host cells by fusing with the membrane of the human cells in the nose and lungs.

The cells in the nose are known to harbor a large concentration of ACE-2 receptors for the SARS-CoV-2 virus. The S2 part of the spike protein on SARS-CoV-2 binds to the nose’s ACE-2 cell receptors. Thus, the nose is a potent source of COVID-19 emission and transmission to others who breathe the emitted air droplets containing secreted SARS-CoV-2 viruses. Thus, it is critical to place one’s face mask over the mouth and nose if one wishes to lessen disease transmission.

The effectiveness of mask-wearing has been known since the start of the 20th century. We learned public health lessons from the 1918-1919 flu pandemic as scientific studies definitively demonstrated about the efficacy of facemasks at the time. The body of evidence demonstrating mask effectiveness in disease transmission prevention has since been overwhelming. The facemask must cover the nose if the disease transmission to other humans is to be stemmed.

Figure 8. Structure of the SARS-CoV-2 virion.

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https://commons.wikimedia.org/wiki/File:3D_medical_animation_coronavirus_structure.jpg

The M-protein is embedded in the membrane envelope of the virion. The function of the M-protein is to help assemble a matrix support structure and determine the envelope shape of the virus. Other investigators have named M-protein as E1 membrane glycoprotein. The hemagglutinin-esterase (HE) protein of the SARS coronavirus number two resides in the envelope of the mature virion and functions to bind particular sialic acid receptors and acting as enzymes to destroy them (i.e., the receptors).

The envelope is a biological membrane that is stolen from the human host cell. The viral envelope helps to protect the internal contents, such as the RNA genome. The envelope protein (E protein) of the virus is thought to participate in the entire virus assembly and help the mature virion escape from the infected cell in a process called budding.

The N protein is also referred to as the nucleocapsid or ribonucleoprotein and sometimes simply an “RNA protein.” The N protein binds to the RNA genome to protect it, but proteases must process it to allow the virion to mature. The N protein has also been shown to be necessary for packaging the RNA genome and releasing the mature virus from the infected cell. However, N protein has been a target for vaccines by some investigators.

The discovery of the mRNA molecules as vehicles for therapy and vaccines for the COVID-19 pandemic has its origins going back as far as the early 1960s. See Figure 9. In the timeline, written by Karikó and colleagues Ugur Sahin and Özlem Türeci in 2014, critical investigations along the way were necessary to the ultimate production and use of the Moderna and Pfizer mRNA vaccines for COVID-19. Notable discoveries include that of mRNA itself, the evidence for its presence in 1961 by François Jacob, Sydney Brenner, and Matthew Meselson. We had written about Jacob in chapter 26 of our 2018 book “Inventions and Discoveries by the World’s Most Famous Scientists.” Likewise, we discussed at length the lives and science of Brenner and Meselson in chapters 5 and 14, respectively, in our 2021 book “The World of Molecular Biology.”

Discoveries like these and others in the timeline provided the necessary foundations for Karikó to make her innovative discoveries about the immunity of mRNA and its potential towards circumventing it for COVID-19 vaccine delivery to humans. Indeed, Fauci, who is the director of the National Institute of Allery and Infectious Diseases at the National Institutes of Health, directly attributed the pioneering work of Dr. Karikó to the successful development of the COVID-19 mRNA vaccines. Without the essential contributions of Karikó, these first Moderna and Pfizer vaccines might never have transpired.

Figure 9. Important discoveries and advances in the development of mRNA as a drug technology.

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

For additional information regarding COVID-19, see: https://www.cdc.gov/

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