by Antonino Napoleone, Giuseppe Scarlata, Maria Chiara Rosace | November 28, 2020.

A shining light that seems to give a glimmer of hope and positivity at the end of the year that has been certainly troubled: two of the most promising vaccine candidates against COVID-19 have proved effective in phase 3 studies and have achieved the necessary criteria to continue in the final stages of clinical trials, before being launched on the market. In the race for the winning vaccine (citing our article) there are other candidates in the testing and study phase, using different technological platforms, some already tested in other vaccines (like the GSK and Sanofi candidate) and others completely innovative. The last ones include the mRNA vaccine, which is the result of a biotechnological process that has never been validated before in the field of vaccines against infectious diseases and which has enabled the Moderna and Pfitzer/BioNtech pharmaceutical companies to develop their respective mRNA-1273 and BNT162b2 candidates.

mRNA vaccine

The purpose of the vaccine is to expose the body to viral antigens, which are treated artificially in order to cause no disease but to trigger an appropriate immune response capable of blocking the pathogen, eliminating it and preventing a possible infection over time. Ideally, this is an irreproachable strategy, and over time various systems have been exploited to achieve this goal, varying in degree of safety, efficacy and route of administration. The strategy adopted by the two pharmaceutical companies exploits a technology that is based on a slightly different logic, and for this reason unprecedented. First of all, it is necessary to understand how proteins are formed and which role mRNA plays within the process known as the “dogma of biology”: this process takes place inside the cell and it explains how the genetic information contained in the DNA, is transferred into the messenger RNA (mRNA), through a process called transcription, and then translated into the synthesis of proteins, through a process called translation (figure 1).

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Figure 1. The molecular process leads to the synthesis of proteins from the genetic information contained in DNA and delivered by the mRNA inside cells.


Proven strategies for developing a vaccine generally involve reproducing the proteins or antigens of the pathogen in an attenuated or inactivated form in the laboratory, so that they are injected into the individual without triggering the infection process, but only the immune response. These strategies have proven to be historically effective (as in the case of smallpox and polio eradication), but in some cases, they can have enormous disadvantages, especially for immunosuppressed individuals, to whom it is highly inadvisable to administer vaccines based on viral vectors, inactivation or attenuation of the pathogen. The respective vaccines developed by Moderna and Pfitzer/Biontech are instead composed of molecular instructions of the virus in the form of mRNA, which, once supplied to human cells by intramuscular injection, allows them to reproduce the coronavirus antigens so that they become targets of the immune system of vaccinated subjects.

Both vaccine candidates proved to be safe and promising in early clinical trials in humans. To date, data published in the literature report mild to moderate side effects, such as fatigue, headache, nausea and pain at the injection site (Table 1). For phase 3 clinical trials, 44,000 individuals were recruited from Pfitzer/Biontech and 30,000 from Moderna, whose data are still being monitored and reviewed by the Food and Drug Administration (FDA) and European Medicine Agency (EMA).

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Table 1. Systemic and Local Adverse Events from the mRNA1273 vaccine by Moderna. The severity of solicited adverse events was graded as mild, moderate, or severe.

This strategy is surprising and would allow each individual receiving the vaccine to reproduce SARS-CoV-2 (Spike protein) antigens within his or her own system, and develop a specific immune response. Moreover, this system would also allow coverage in immunocompromised individuals, because no direct use is made of infectious viral proteins, but only of mRNA molecules necessary to convey information to the immune system, which in itself has no virulence. Another important point that needs to be clarified is the inability of mRNA vaccines to alter genetic information at the DNA level of vaccinated individuals, as many no-vax sceptics have argued. In fact, as also confirmed by regulatory authorities, this is by no means a gene therapy approach, so there is no danger of generating toxicity or modification at the genetic level. In addition, this system has a transient nature, i.e. mRNA is expressed for a limited period of time, which makes it possible not to repeat the production of viral antigens in vaccinated subjects, nor to over-stimulate the immune system. It remains to be assessed whether this approach is capable of ensuring a protracted immune response over time through the development of an adequate degree of immunological memory.
The first results are surprising, especially in terms of the immune defences that they manage to trigger from the first dose. After the second dose, the highest peak of immune response is reached with the development of a response mediated by T lymphocytes and neutralising antibodies directed against SARS-CoV-2 (figure 2).

Graphic
Figure 2. General description of how the mRNA vaccine will work. After the mRNA is delivered into the cells, coronavirus antigens will be produced and the two main protagonists of the immune response, T lymphocytes and neutralising antibodies will start to trigger a protective effect against the virus.
What are mRNA vaccines made of?

The most difficult challenge related to the development of this type of vaccines concerns the targeted administration of mRNA within human target cells in order to fully exploit the ability to trigger an immune response against SARS-CoV-2. mRNA is a particularly sensitive and unstable molecule (it is highly susceptible to changes in pH and temperature and to degradation by enzymes), so it requires special conditions of treatment, but above all of encapsulation and administration to preserve all its biological properties. This is the main reason why the logistical planning of transport and storage of the vaccine will be a crucial aspect when global distribution will start (Figure 3)

Vaccine comparison
Figure 3. Comparison of different types of vaccines. When compared to the influenza vaccine (inactivated virus vaccine) and the measles/parotitis/varicella vaccine (attenuated virus vaccine), the candidates developed by Moderna and Pfizer/Biontech consist of genetic virus information in the form of mRNA. Since mRNA is a very sensitive and easily degradable molecule, it requires very low temperatures for storage and transport. They can only remain at room temperature for a few hours only, just before administration.
So what was the strategy adopted by Moderna and Pfizer/Biontech to overcome this limitation?

Once the potential of an mRNA vaccine was understood, all that was left was to develop an effective strategy to encapsulate and stabilise the mRNA molecules and to deliver them to the target human cells. Both in the laboratory and in vivo, attempts were made to inject mRNA molecules directly, but the results were almost unsatisfactory, as the molecule was immediately degraded by enzymes (called RNases) present in the environment outside the target cells. So the only solution to overcome this limit was to find a system to protect and transport the mRNA using one of the available molecular transport or “carrier” technologies, including the use of polymers, lipids, microparticles, inorganic nanoparticles or lipid nanoparticles (LNP). LNPs have proven to be the most effective in stabilising and delivering mRNA molecules, and this is where the race for the winning vaccine between Moderna and Pfizer/Biontech started, to perform studies and clinical trials until the resounding announcement:

Moderna’s COVID-19 Vaccine Candidate Meets its Primary Efficacy Endpoint in the First Interim Analysis of the Phase 3 COVE Study.

Moderna Press Releases, November 16, 2020.

Pfizer and Biontech conclude phase 3 study of COVID-19 vaccine candidate, meeting all primary efficacy endpoints.

Pfizer Press Releases, November 18, 2020.

What are lipid nanoparticles?

Lipid nanoparticles (LNP) are envelopes less than 200 nm in size consisting of an aqueous core containing the molecules to be delivered (in this case mRNA), surrounded and protected by a double-layer membrane of different lipids, such as phospholipids and cholesterol. The lipid composition membrane performs crucial biological functions to ensure stability in an extracellular environment and the functionality of nanoparticles in transporting mRNA molecules into the target cells. In addition, it has been observed that LNPs would be able to trigger an immune response, providing an adjuvant effect on the vaccine. This system constitutes a substantial technological platform for mRNA vaccines (Figure 4) developed by Moderna and Pfizer/Biontech.

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Figure 4. mRNA molecules can be encapsulated in lipid nanoparticles (LNPs) to reach target cells safely and efficiently. By injecting only mRNA molecules, there is a process of degradation by RNase enzymes in the extracellular environment.
Other applications

This technology is not totally new in the scientific world, in fact, before understanding its potential for the development of prophylactic vaccines against infectious diseases, other mRNA vaccines have already been developed and in clinical trials for the treatment of genetic diseases and for anti-tumour immunotherapy applications. Also in these applications, molecular carriers based on lipid nanoparticles have been used to encapsulate mRNA, with very promising results.

Conclusions

The potential of mRNA applications has recently emerged in the therapeutic field, especially in the field of immunotherapy, and it is surprising how, in such a short time, such progress and results have been achieved also in the field of prophylactic vaccines. This technology opens an infinite number of horizons to the world of pharmaceutical and biomedical sciences, especially in view of the current pandemic. The monitoring of the clinical trials of Moderna and Pfizer/Biontech is still ongoing, especially to investigate the level of protection to SARS-CoV-2 that the respective vaccines offer in vaccinated subjects and the duration of the immunological memory developed after administration. The scenario is certainly promising, we just have to wait for the final results. It will be interesting to find out if the efficacy and safety of the mRNA vaccine technology platform will be demonstrated, especially in comparison with the other types of vaccines against SARS-CoV-2 being tested. If these results will be confirmed, it will be possible to conceive in a different way the field of Vaccinology, providing a launchpad for technological innovation related to antigens, adjuvants and molecular carriers such as lipid nanoparticles, for the development and formulation of new vaccines to combat other infectious diseases. It is necessary to wait for the final results, and start planning awareness and information campaign to convince and reassure the general population, an undertaking no less complicated than the development of the vaccine itself. So it is necessary to give a stronger message to have and share faith in medical sciences rather than the media.

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