mRNA Vaccines – Understanding how they work and are manufactured

mRNA-vaccine

mRNA Vaccines – Understanding how they work and are manufactured

To start understanding mRNA vaccines we need to begin with talking about viruses. Although viruses fall under the field of microbiology, they are not considered as true living organisms in the conventional sense, as they cannot exist without a living host cell.

Our school Biology textbooks define living things as displaying seven characteristics:

  • Respiration
  • Movement
  • Sensation
  • Growth
  • Reproduction
  • Nutrition
  • Excretion

Viruses on the other hand only have a two phase “lifecycle”:

  • Dispersal of virions to infect host cells
  • Metabolise the hosts cell to make more virions

The distinction between living and not living is blurred however, because viruses contain genes (DNA or RNA) and proteins just as living cells do.

We are all familiar with the image of a double helix or twisted ladder of DNA but what is RNA ?

DNA-strand
mRNA-strand

RNA or Ribonucleic acid is very similar to DNA but is single-stranded i.e. one side of the ladder is missing. It is thought that the earliest life forms were based on RNA (“RNA World Hypothesis”). One key difference between DNA and RNA is that RNA has one additional oxygen atom that makes them less stable than DNA. That instability can be thought of as flexibility and can be used to the advantage of viruses to mutate in response to the host’s immune system.

So what is mRNA?

mRNA or “messenger RNA” is used as a temporary biological communication of DNA information or instructions from genes in the nucleus of cells to the synthesis part of the cell. Transfer molecules form around the mRNA to form a ribosome which translates the genetic information (analogous to a punched tape code reader). The ribosome converts this information into amino acids that then become various types of proteins for different functions in the body such as haemoglobin. The mRNA is short lived and once the message has reached the destination part of the cell, it breaks down.

Viral Infection

At this point it’s worth making some definitions of terms.

Pathogen – is an organism that causes disease

Antigen – is a piece of protein from a pathogen that triggers the body’s immune system responds to the pathogen by making antibodies. The antigen is like the “Wanted” poster of a known criminal.

Antibodies – are Y-shaped proteins that fight the disease by binding to the invading cells.

When a virus hijacks a host cell, it uses messenger RNA to instruct that cell to make copies of the virus. Fortunately, after a few days, our body’s immune system recognises that that the virus is a threat and generates antibodies which attach to the virus spike protein and act as markers. The body then targets these marked viruses with T-cells to destroy them.

Types of Vaccines

Vaccines use various methods to trigger an immune system response against the target pathogen.

Attenuated Vaccines: These were the first type of vaccines developed and use either a weakened version of the live virus or a similar version of the target virus but from another species that doesn’t cause serious illness. For example, the AstraZeneca Covid-19 vaccine uses the adenovirus which is a form of coronavirus (common cold) in chimpanzees. Another familiar example would be the MMR (measles, mumps & rubella). In the case of using a weakened virus, the virus is repeatedly cultured in chicken eggs where it becomes increasingly effective in replication within the host chicken egg and less effective at replication in human cells but is still recognised as a threat by the human immune system. There is a small risk that the attenuated virus can mutate in the human body from the weakened state and become a more virulent strain.

Inactivated Vaccines: Some vaccines use an inactivated or killed form of the virus where the virus’ DNA/RNA is denatured though heat or a chemical process such that the virus cannot replicate but the virus remains intact. Although these vaccines have no risk of the virus mutating from the weakened state and becoming more virulent, these vaccines tend to have a shorter period of immunisation.

Subunit Vaccines: Are a class of vaccines where only part of the pathogen is used. In the case of viruses, this would typically be the spike protein. The protein itself has no viral genetics but is recognisable to the immune system to provoke a response. mRNA vaccines fall under this category of vaccines.

mRNA Vaccines

mRNA vaccines are a new type of subunit vaccine that program the body’s own cells to act as micro-factories to synthesise the spike protein only. The vaccine has no interaction with the cell’s nucleus. The spike protein is expressed onto the cell membrane and activate the body’s immune system by activating B cells and T cells that both attack the invading pathogen as well as generate memory cells that provide the body with long term resistance to the pathogen.

Advantages of mRNA vaccines

One advantage of an mRNA vaccine is that it only copies the spike protein. It does not contain other parts or a weakened or inactivated virus to trigger the immune response, so we don’t suffer symptoms of the virus following vaccination.

mRNA vaccine development is a paradigm shift into vaccine development. We no longer need a sample of the virus. To make mRNA vaccines, all we need is the genetic sequence of the DNA or RNA to make a synthetic vaccine. In essence, we are seeing the digitisation of vaccines development.

From the outbreak of Covid-19 the University of Queensland published their discovery of the genetic sequence online. In less than a month, the first vaccine candidate was developed ready to begin pre-clinical testing.

They don’t need Adjuvants to enhance the immune system’s response of the vaccine and attract the right T cells. This saves time in selecting the right adjuvant.

mRNA vaccines can be thought of as programmable therapies and are an exciting development at exactly the right time in history. They can also be tailored to individual patients.

How do we make mRNA vaccines?

To make traditional inactivated virus vaccines such as the seasonal flu jab the virus is first isolated. The virus is then allowed to replicate in a bioreactor (e.g. egg or steel vessel). The live virus is then inactivated (killed) and the virus antigen purified prior to preparation of the final dosage form (vial or syringe).

With rapidly emerging mRNA vaccine technologies the production strategy is somewhat different in that the mass production of virus particles is not required. Instead the approach mass produces a stable formulation of mRNA that encodes the COVID spike protein.

Whilst there is more than one way to produce mRNA vaccines, the currently preferred approach for mass production during the COVID pandemic is based on methods such as those developed by Pfizer/BioNTech and Moderna. The vaccine production process can be broken down into 4 stages of production – mass production of the DNA template, mass production of mRNA using the DNA template, stabilisation of the mRNA by encapsulation within a lipid nanoparticle and finally preparation of the finished product for delivery (e.g. vial for delivery via syringe).

Figure 1: mRNA vaccine production stages. Further breakdown on the steps is outlined in Table 1.

mRNA-Vaccine-Production-Stages-v2

In the first Stage of production, a genetically engineered host cell (e.g. E. coli) containing DNA for the spike protein is mass produced in a bioreactor and the culture is subsequently subject to a series of recovery and purification steps that produce a sterile, pure intermediate that contains the DNA sequence for the spike protein. The second stage of production converts this DNA into mRNA via an in vitro transcription reaction which requires the addition of reagents and an enzyme that converts the DNA sequence into an RNA sequence. A pure mRNA intermediate is then prepared by digesting the spent DNA, performing various purification operations and passing the liquid through a sterile filter.

The next stage of manufacturing is to create a delivery system which involves encapsulating the purified mRNA within lipid-based nanoparticles. The purified mRNA is subjected to turbulent mixing with lipids to create lipid-based nanoparticles that serve to protect the mRNA from enzymatic degradation inside the recipient and also assists with passage across cell membranes into the recipient’s cells.

For the final stage of production, the vaccine is filled under aseptic conditions via a sterilising grade filter (0.2μm) into final containers, typically glass vials. The finished product must then be stored at very low temperatures from the end of the manufacturing process until use at the destination.

The key steps (only) for generic mRNA vaccine production can be otherwise summarised by the table below:

Table 1: Generic mRNA Vaccine Production Stages (Key Steps Only)

StageStage DescriptionStepStep DescriptionJustification
1DNA Synthesis1Cell CultivationMass production of DNA template required for mRNA production
2HarvestRecover cell mass (containing DNA)
3Cell lysisBreak open cells to release DNA
4PurificationPurification of DNA
5Sterile filtrationSterilise intermediate to prevent microbial growth
2mRNA Synthesis6mRNA synthesisMass production of mRNA (using DNA) encoding COVID spike protein
7DNA digestionDegrade DNA to assist purification
8PurificationPurification of mRNA
9Sterile filtrationSterilise intermediate to prevent microbial growth
3mRNA Encapsulation10Jet mixingLipid coating protects mRNA from degradation and assists uptake
11Sterile filtrationSterilise intermediate to prevent microbial growth
4Finished Product12FormulationPrepare final product formulation
13Fill vialsDispense formulated product into vial of other storage device
14Packaging

Conclusion

mRNA vaccines have been researched for ~50 years and the first FDA approved mRNA vaccine came along as recently as 2018. With the emergence of Covid-19 as a world wide pandemic mRNA technology is being tested in humans on a scale never seen before, fast tracking our understanding and development of this novel vaccine technology.

There have been 3 coronaviruses in the last 20 years so it is foreseeable that others will emerge. mRNA technology provides us with a rapid delivery method without the delay needed to culture the virus. We have seen that researchers are able to respond quickly to map the genetic sequence and similarly, vaccines can be developed rapidly when researchers, manufacturers and regulators collaborate through clinical trials and production qualification.

However, it is crucial that we have facilities ready to respond to future outbreaks particularly if they are more virulent or have a higher mortality rate than Covid-19. It can be expected that vaccine developments for any future pandemic may be even more rapid than demonstrated with Covid-19. We must have sterile production facilities including the supply chain of materials ready to avoid manufacturing capacity being the limiting factor for the role-out of vaccines to the population. Of course there are other needle free vaccine technologies in development, soon we won’t have crying babies.

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