THANK YOU MESSENGER RNA

THANK YOU, MESSENGER RNA, FOR GIVING US A FRESH LEASE OF LIFE.

It all started in the year 1796 when the English doctor Edward Jenner of Gloucestershire County in England noticed that milkmaids who had gotten cowpox were protected from small pox. Jenner also knew about the cowpox pustules and guessed that exposure to cowpox could be used to protect against smallpox.

 On May 14, 1796 Jenner took the watery liquid from the pustules of a milkmaid and inoculated an 8-year-old boy, James Phipps. THIS WAS THE HISTORICAL BEGINNING OF VACCINATION.

The word vaccine is derived from the Latin word Vacca means cow. Edward Jenner coined the term vaccination in 1796 to describe inserting pus from cowpox lesions into open cuts on human patients to prevent small pox.

While inoculating Phipps with pustules watery liquid from cowpox affected milkmaids, Edward Jenner little realized that he was opening up a vast potential area to protect humanity from claws of dreadful viral diseases. Incidentally James Phipps lived a healthy 65 long years.

Since Jenner’s epoch-making discovery

Several anti-virus vaccines have been developed, notable among them is the polio vaccine which has a global relevance and acceptance. POLIO VACCINE ORIGINALLY DEVELOPED BY Janos Salk of California is a heat killed polio virus vaccine which had at the outset a thundering success. Then there were cracks in the success because some virus particles escaped heat killing caused polio in children who received Salk vaccine, Then Albert Sabin developed vaccine using attenuated polio virus. Sabin vaccine is the safest vaccine available today globally to protect young children from the wrath of dreadful polio. This type of vaccine production requires vast facility for cell culture resource intensive expensive and slow in production. The recent Covid pandemic made us to realize that vaccine should be produced fast, in large quantities with simple and cheap techniques. Thanks to the progress made in molecular biology in recent decades vaccines based on individual viral components rather than whole viruses have been developed.

Modern vaccinology is a complex multi-disciplinary science which fully includes the modem knowledge of microbiology, immunology, molecular biology, epidemiology and public health

MESSENGER RNA A PARADIGAM SHIFT IN VACCINOLOGY

In our cells genetic information encoded in DNA is transferred to messenger RNA (mRNA} which is used as a template for protein production. During the 1980s efficient methods for producing mRNA without cell culture were introduced called the INVITRO TRANSCRIPTION. This accelerated the developments in molecular biology and in turn in vaccinology. In vitro transcribed mRNA was considered unstable and challenging to deliver requiring the development of sophisticated lipid systems to encapsulate mRNA. Moreover in vitro produced mRNA gave rise to inflammatory reactions.

This years (2023) Nobel prize for Physiology or Medicine has gone to two Scientists for their work developing messenger RNA vaccine. This what the Nobel Committee in their press release said “THE NOBEL ASSEMBLY AT THE KAROLINSKA INSTITUTE HAS TODAY DECIDED TO AWARD THE 2023 NOBEL PRIZE IN PHYSIOLOGY OR MEDICINE JOINTLY TO KATALIN KARIKO AND DREW WEISSMAN FOR THEIR DISCOVERIES CONCERNING NUCLEOSIDE BASE MODIFICATIONTHAT ENABLED THE DEVELOPMENT OF EFFECTIVE mRNA VACCINES AGAINST COVID-19”.

The discoveries of the two Nobel Laureates were critical for developing effective mRNA vaccines. Katalin Kariko is a Hungarian biochemist and Drew Weissman is an American immunologist are working at the University of Pennsylvania.

COVID-19 during the pandemic that began in early 2020.Through ground breaking findings which have fundamentally changed our understanding of how mRNA interacts with our immune system the laureates contributed to the unprecedent rate of vaccine development and production during one of the greatest threats to human health in modern times.

The breakthrough came in when Kariko and Weissman noticed that dendritic cells recognize in vitro transcribed mRNA as foreign substance which led to their activation and release of inflammatory signaling molecules.

(What are dendritic cells (DC). They are found in tissue that has contact with outside environment such as lung mucosa, epithelial cells of the skin and the linings of the nose and the gastro-intestinal tract. DC is an antigen presenting cell of the mammalian immune system. A DCs main function is to process antigen (pathogen) and present it on cell surface to the T cells of the immune system. They act as messengers between the innate and adaptive immune system).  

RNA contains four bases AUGC corresponding to ATGC of DNA. Kariko and Weissman knew that nucleoside bases in RNA from mammalian cells are frequently and chemically modified while in vitro transcribed mRNA is not. To clarify this phenomenon, they produced different variants of mRNA, each with unique chemical alteration in their bases which they delivered to dendric cells. The results were striking. The inflammatory response was almost abolished when modifications were included in the mRNA. This was a paradigm change in our understanding of how cells recognize and respond to different forms of mRNA which led to the idea and field of mRNA therapy.

An mRNA can teach the body how to make a specific protein that can help our immune system to prevent or treat certain diseases. Our body contains trillions of cells, the basic unit of life and every cell contains proteins. Proteins are workhorses of our cells and our body makes more than10,0000 different kinds of proteins. Our proteins are made in a process called protein synthesis and that is where mRNA comes in. Messenger RNA or mRNA exists in all the cells as a messenger or post office in our body. It is in our body for billions of years. mRNA teaches the body how to make its own medicine. Scientists design each mRNA to give cells directions to make a particular protein.

As the name suggests mRNA is a messenger like a post office. It interacts with other cell components like ribosomes to create proteins.

Each mRNA carries instruction to make a specific protein. The instructions are like a BLUE PRINT and these are delivered to the ribosomes through triplet code, sequences amino acids to a specific protein. Once the job is done an mRNA is broken down by the body.

Scientists design each mRNA to give cells directions to make particular protein. To protect the mRNA and help to deliver it into cells the mRNA is wrapped with lipids and fats.

mRNA vaccines are given as an injection.

Once it is delivered into the body, the body takes over and makes proteins according to mRNA instructions. mRNA does not remain in the body very long one its job is done. It does not cause any permanent change or alter DNA.

Now let us see how mRNA vaccine work against cov-19 virus

First mRNA COVID-19 vaccines are injected in the upper arm muscle or thigh depending on the age of the person.

After injection the mRNA will enter the muscle cells. Once inside they use the cells machinery to produce a harmless piece of the spike protein The spike protein is found on the virus surface that causes COVID19 infection. After the protein piece is made our cells break down the mRNA and remove it. Our immune system recognize that the protein does not belong there This triggers our immune system to produce antibodies.

NOW LETUS TALK ABOUT SPIKE PROTEIN

The presence of Spike proteins on corona virus is what gives rise to spike shaped protrusions found on their surface.

Spike proteins of corona virus can be divided into two important functional sub units which include the N terminal S1 sub unit which forms the globular head of the S protein and the C terminal S2 region that forms the stalk of the protein and is directly embedded into the viral envelope.

Upon interaction with a potential host cell the S1 subunit will recognize and bind to the receptor of the host cell, whereas the S2 sub unit which is the most conserved component of the Spike protein will be responsible for fusing the envelope of the virus with host cell membrane.

HOW SPIKE PROTEINS ALOW CORONA VIRUS ENTER THE CELLS

Once the S1 subunit binds to host cell receptors two major changes must occur for the S2 subunit to complete the fusion of the virus to the cell membrane. The two components of the S2 subunit involved in the fusion are hepta peptide repeat sequence (HR1 and HR2) HR1(with 912 to 984 amino acid residues) and HR2 (with 1163 to 1213 amino acid residues)

TARGETING VIRUSES VIA THE SPIKE PROTEIN

Without the SPIKE protein corona viruses cannot interact with the cells of potential hosts like animals and human beings, to cause infection. As a result, SPIKE proteins represent ideal target for vaccine and anti-viral research. In addition to its role in penetrating the cells the Protein of corona virus is the inducer of NEUTRALIZING ANTIBODIES (NABS).

NABS are protective antibodies that are naturally produced by our humoral immune system.

To elicit their antiviral activity NABS bind to the surface epitopes of viral particles to prevent their entry into host cells. The known NABS against corona virus specifically target the S1 domain.

The sensitivity of the SPIKE of corona virus protein to NABS led may researchers including the present Nobel laureates to develop methods to block the binding and fusion of SPIKE protein of corona virus to host cells.

In the case of COVID 19 vaccines the mRNA instructs the cells to make covid virus spike protein the most important component of the virus to help immune system to recognize it and build up anti bodies to fight and neutralize the virus upon subsequent infection

Though appears simple these vaccines depend on a critical packaging made of lipid molecules also called as LIPID NANO PARTICLES (LNP) to protect and deliver the mRNA into the cells. As soon as the LNP enter the cells they release the mRNA (see the diagram).

COVID -19 mRNA VACCINE PRODUCTION

Early in the COVID -19 pandemic researchers used state of the art genomic sequencers to quickly sequence SARS COVID-2 virus genome. This sequence was quickly shared with other researchers. This allowed research workers across the world to analyze the virus and better understand how it causes the disease. The speed of this very important first step enabled immediate research development of potential vaccine candidates.

SPIKE PROTEIN

Having sequenced viral genome using cutting edge bioinformatics researchers selected the

SPIKE PROTEIN GENE as the vaccine candidate. As you have seen earlier in the diagrams, the spike protein is essential for the virus to attach itself to the host cell, thereby making it an effective antigen or target for the immune system to r ecognize and attack.

For those who are new to the word plasmid, they are small circular DNA strands found in bacteria and protozoa. They are found in the cytoplasm. Plasmids are physically separate from the chromosomal DNA and replicates independently. Plasmids are much used in the laboratory manipulation of genes. The target spike protein gene is synthetically made using cutting edge technology of molecular biology and inserted into a plasmid. Plasmids are used in mRNA vaccine production because they are easy to replicate and reliably contain the target gene sequence. Once a new sequence is selected a new plasmid can be produced within two weeks. This helps the new mRNA vaccine to be tested and distribute rapidly.

Messenger RNA is created by mimicking our own biological process. First the two strands of plasmid DNA are separated. Then the RNA polymerase the enzyme that transcribes RNA from DNA, uses the spike protein gene to create a single mRNA molecule or strand.  This is nearly identical to the way genes in our body are transcribed. Finally, only mRNA is packaged as vaccine and other molecules break down. The speed and efficiency of this process can make large amount of mRNA in a short period of time.

To consolidate the earlier paragraphs

 HOW DO COVD-19 MESSENGER RNA (mRNA) VACCINES WORK

Traditional vaccines introduce a weakened, inactivated or killed germ (bacteria viruses). messenger RNA vaccines teach our body cells how to make a protein that triggers an immune response if someone gets infected. When the vaccine is injected into the upper arm the mRNA enters the cells near the site of injection and tells the cells to start making the same protein that is found in the SPIKE protein of the virus. The immune system recognizes this protein and begins to produce antibodies that can fight the virus if the vaccinated person is later infected.

mRNA vaccines do not interact with or alter the human DNA in any way and therefore cannot cause cancer. mRNA is not like DNA, so cannot be combined with DNA to change our genetic code. The mRNA vaccine against COVID-19 is the first mRNA vaccine to be produced in a mass scale in a short span of time under the pressing global demand. mRNA induced anti bodies are more effective than natural immunity in neutralizing COVID-19 and its variants. There is now compelling evidence that the mRNA vaccine induced remarkable increase in lgG4 levels and such an increase was detected in COVID -19 uninfected individuals who received mRNA vaccination before getting infected. Neutralizing antibodies (lgG4) which bind to the spike protein increased its levels by repeated vaccination (booster doses).

mRNA vaccines need to be kept at very low temperature because RNA breaks down easily and quickly. The Pfizer vaccine is kept at -70 degrees Celsius and Moderna vaccine is kept at -20 degrees Celsius.

The first shot of the vaccine primes our immune system helping our system to recognize the COVI-19 virus. The second shot strengthens the immune response. It is very important to have both doses of the same vaccine.

Some people have mild to moderate side effects, but they do not last long, about one to three days. The most common side effects include soreness at the injection site, fatigue, headache and fever. They are more common after the second dose and a person might require extra rest.  Severe side effects are uncommon and can be treated easily with common house hold methods.

As an addendum I wish to add here that, I am tempted to compare mRNA to the great Sanyasins Ascetics, Munis Maharishis and Seekers of Truth in Hindu Mythology, Ramayana and Mahabharata. The great seekers gave the world very valuable advice and utterances which are very valid even today. They remained incognito and did not put their signature on their contributions. The famous author Lloyd C. Douglas in his famous book “MAGNIFICENT OBSESSION” projects an important character obsessed with his contention not to divulge his good deeds to others and remain incognito and in the background. Messenger RNA belongs to the class and category of these great souls.

Thank you, messenger RNA,

Professor K.A.Balasubramanian

Ph.D. (IARI-New Delhi), Ph.D. (Imperial College, London), DIC (Imperial College, London)

Austin, Texas, USA.