Understanding mRNA vaccine technologies

Vaccination is one of the most effective public health interventions to prevent, control and even eradicate infectious diseases. It is believed that, with the exception of clean water and sanitation, vaccination has had the greatest contribution of all health interventions to human health and life expectancy​[1]​.

Vaccines can be broadly classified as ‘live’, containing attenuated strains of a microorganism, or ‘non-live’, containing either killed microorganisms or components of a pathogen​[2]​. Many types of vaccine exist, with the strategy for each presenting its own advantages and disadvantages (see Table 1).

Despite the success of conventional vaccines, significant challenges are still faced at all stages of the research and development process. Prominent challenges have traditionally been the length of time required for, and the financial cost of, each stage of the process​[5,6]​. When responding to fast-developing infectious disease outbreaks, it is the amount of time required for conventional vaccine development, not the lack of efficacy, which puts conventional vaccines in a disadvantaged position compared with RNA and DNA vaccines​[7]​.

Conventional vaccine constructs (attenuated strains of pathogen or components thereof) are achieved through extensive cell or animal cultures​[8]​. Therefore, manufacturing attenuated strains inherently involves replication in a foreign host, where the pathogen needs to accumulate mutations to impair its virulence to human hosts​[2]​. Not only can this process be time-consuming and yield poorly attenuated strains, but when dealing with a virus with high frequency of recombination, such as SARS-CoV-2, it can also lead to recombination of a fully virulent strain​[2,9]​.

Emergent alternatives to traditional vaccines, which use recombinant bacteria or viruses, include genetic vaccines that consist of DNA (as plasmids) or RNA (as messenger RNA, also known as mRNA). These vaccines are taken up by cells and translated into proteins, which then act as intracellular antigens and stimulate an immune response​[10,11]​. Although the use of mRNA vaccines is only recently — in 2020, following the outbreak of COVID-19 — the first report of successful RNA transfection (introduction of nucleic acid into animal cells) was in 1989 (see Figure 1).

Today, mRNA-based vaccines are a promising technology platform for the development of many therapeutic and prophylactic vaccines. Their manufacturing process is cell-free, safe and time-saving, and avoids the need to grow highly pathogenic organisms on a large scale, reducing the risks of contamination or release of dangerous pathogens. Furthermore, the flexible nature of the mRNA vaccine platform negates the need for a dedicated product-specific production facility. During manufacturing, changes to the encoded protein only alter the sequence of the RNA molecule, meaning the physiochemical characteristics of a mRNA sequence remain largely unaffected. This allows for a more streamlined manufacturing process, accelerating cost-effective mRNA vaccine development and mass production​[11]​. For many emerging infectious diseases, the main challenge of traditional vaccination techniques is obtaining a stockpile in a short timeframe. Therefore, mRNA vaccines are particularly helpful in a pandemic scenario, where time is critical and vaccines are desperately needed; this has been evidenced in the recent worldwide rollout of the Pfizer-BioNTech and Moderna COVID-19 vaccines, in an unprecedented acceleration of vaccine production​[6]​.  

The successful introduction of mRNA vaccines in response to the COVID-19 pandemic has increased interest and focus in mRNA platforms, with many mRNA-based vaccine candidates already in pre-clinical and clinical stages​[23]​. With the emergence of new mRNA vaccines approved for use, pharmacists have a significant role in educating the public and advocating the uptake of vaccines. Furthermore, pharmacists play a vital role, not only in vaccine distribution but also administration in several countries, such as the UK. This article outlines how mRNA vaccines work, their clinical uses, and how pharmacists can educate and advise other healthcare professionals and patients.

Immunological mechanisms of vaccination

The principle of vaccination is to activate an effective immune response without causing the associated disease. Vaccines do this by inducing active immunity to provide immunological memory. Immunological memory enables the immune system to recognise and respond rapidly to exposure to the same pathogen later on. To achieve this, vaccines contain natural or synthetic antigens, which either consist of, relate to or are derived from the pathogen​[24]​. A schematic representation of vaccine-induced immunity is shown in Figure 2.

Following immunisation, the vaccine components are taken up by phagocytic cells, such as macrophages and dendritic cells that live in the peripheral tissue. Antigen-presenting cells (APCs) take up antigens and become activated and migrate towards nearby lymph nodes. Inside the lymph nodes, the APCs present the processed antigen to T- and B-lymphocytes, which, under the right conditions (e.g. recognition of the antigen and appropriate co-stimulatory signals), results in the activation of these lymphocytes. These antigen-specific B- and T-cells then clonally expand to produce multiple progenitors that recognise the same antigen. In addition, memory B- and T-cells are formed to provide long-term, sometimes lifelong, protection against infection with the pathogen​[24]​.

Pharmacology of mRNA vaccines

mRNA is the intermediate step between the translation of protein-encoding DNA and the production of proteins by ribosomes in the cytoplasm. The concept behind mRNA vaccines is simple: once the antigen of choice from the pathogen is identified and the gene is sequenced, mRNA is synthesised in vitro from a DNA template​[26]​. Once inside the cell, the mRNA sequence is translated into the corresponding antigen protein, eliciting cellular and humoral immunity (see Figure 3)​[22,27]​. Synthetic mRNA is engineered to resemble mature eukaryotic mRNA molecules and generally consists of an open reading frame encoding the target antigen, flanking untranslated regions (UTRs), a five-prime (5’) cap and a terminal poly(A) tail (see Figure 3)​[28,29]​. The 3’ and 5’ non-coding sequence enhances translational efficacy and boosts mRNA stability​[29]​. The poly(A) tail also plays a significant regulatory role and prevents degradation of the molecule​[30]​.

Two general classes of mRNA are currently used in vaccinology: non-replicating mRNA and self-amplifying RNA; current COVID-19 mRNA vaccines belong to the former category. Vaccines based on non-replicating mRNA only encode the antigenic protein of interest, whereas self-amplifying RNA amplifies the immune response by encoding the antigen of interest as well as the viral replication machinery that enables intracellular RNA amplification and abundant protein expression​[28,30,31]​.

Administration routes and delivery strategies for mRNA vaccines

Major administration routes 

Several factors influence vaccine efficacy, which can be broadly divided into three categories:

1. Vaccine features (i.e. design, formulation, use of adjuvant)
2. Individual variation amongst recipients (i.e. age, sex, comorbidity)
3. Administration-related parameters (i.e. route of administration and delivery vehicle)​[32]​.

The anatomical and physiological properties of the administration site can affect the safety profile of the vaccine​[33]​. The choice between systemic or local administration strategies is governed by the antigen expression localisation requirements of mRNA vaccines; for example, intraperitoneal and intravenous routes are employed when systemic expression of the antigen is desirable for therapeutic purposes, whereas intramuscular, subcutaneous and intradermal injections are commonly used routes for mRNA vaccines against infectious diseases (see Table 2)​[29]​.

Optimisation of mRNA stability and delivery

One of the major limitations of in vitro transcribed mRNA vaccines is their high innate immunogenicity, where a robust immune response resembling viral invasion is induced, which can produce toll-like receptor-mediated inflammation​[41,42]​. To negate this problem, modified nucleotides capable of escaping immune system surveillance have been introduced in the mRNA sequence​[43]​. Indeed, replacement of the nucleosides cytidine, uridine and adenosine with modified analogues, such as 5-methylcytidine, pseudouridine and N⁶‐methyladenosine, has reduced immunogenicity while enhancing the stability and translation efficiency of the molecule​[41]​.

A major challenge impeding the successful translation of mRNA therapeutics is in vivo delivery. The large size and negative charge of the mRNA molecule hinder membrane permeability. Furthermore, mRNAs are easily degraded by ubiquitous extracellular ribonucleases​[44,45]​. Encapsulation of mRNA into a carrier is therefore vital to protect the molecule against digestion and confer efficient cellular uptake. Injection of naked mRNA without an additional carrier has been used successfully to target APCs via intranodal injections​[46]​. However, in the absence of a delivery vehicle, mRNA absorption is severely diminished and the half-life of mRNA is around seven hours.

Ex vivo loading of the dendritic cells with mRNA vaccine allows precise cell-specific delivery and high transfection efficacy, but this labour-intensive costly approach is not a pragmatic technique to vaccination​[47]​. Delivery systems can generally be divided into viral and non-viral vectors. A summary of advantages and limitations of current delivery systems can be seen in Figure 4. An ideal delivery vehicle protects mRNA from enzymatic digestion, confers cell uptake and triggers endosomal escape and release of the cargo in the cytoplasm​[29]​.

Lipid-based vectors, such as liposomes and lipid nanoparticles (LNPs), are the most widely used mRNA delivery tool​[47,53,54]​. LNPs consist of four main components:

1. Ionisable cationic lipids, which mediate self-assembly, facilitate membrane fusion and promote endosomal escape;
2. Lipid-anchored polyethylene glycol (PEG), which increases half-life of the formulation, prolongs circulation time and prevents aggregation by acting as a steric barrier;
3. Cholesterol, which acts as a stabilising agent;
4. Neutral phospholipids, which support the bilayer structure​[55]​.

Clinical use, safety, limitations and prospects of mRNA vaccines

mRNA vaccines have a promising safety profile. Advances in mRNA design, nucleic acid delivery technologies and the cell-free manufacturing process have minimised the risks associated with nucleic acid-based vaccines​[56]​. However, rare cases of severe local and systemic reactions have been reported​[57,58]​. Moreover, further studies are needed to investigate biodistribution and persistence of immunogen​[47]​. Induction of potent type I interferon responses associated with inflammation and autoimmunity have been reported in a few nucleic acid-based vaccine studies​[59–61]​.

Another potential safety issue stems from the presence of extracellular RNA leading to an increase in the permeability of endothelial cells, which can contribute to oedema. Furthermore, extracellular RNA can serve as promoters of blood coagulation and pathological thrombus formation​[62]​. Therefore, longitudinal studies are required to monitor and assess the safety profile of these vaccines, and understand the durability of immunity​[63]​.

A higher incidence of anaphylactic reactions has been reported with the Pfizer-BioNTech COVID-19 vaccine (around 4.7 per 1 million vaccinations) and the Moderna COVID-19 vaccine (around 2.5 per 1 million vaccinations) which is about two- to four-fold more than traditional vaccines​[63,64]​. It has been hypothesised that PEGylated lipid used in the nanoparticles of these vaccines is the reason, owing to the presence of pre-existing antibodies against PEG in some individuals as a result of exposure to PEG present in many consumer products, such as toothpastes, shampoos and laxatives​[65]​. Table 3 lists commonly reported side effects for COVID-19 mRNA vaccines.

Challenges around mRNA vaccines are interlinked and exacerbated in a pandemic situation. Some of these challenges are:

• Cold-chain storage, which affects distribution of the vaccines worldwide, particularly in warm countries or those without reliable cold-chain storage. This also impacts stability and degradation of the mRNA vaccine. One strategy to address this may be changing the formulation, paying special consideration to mRNA size, guanine-cytosine and lipid content;
• Emerging viral variants, where some mutations in the viral genome can enhance immune evasion and reduce cross-variant efficacy of mRNA vaccines, resulting in the need for variant-specific mRNA boosters;
• Duration of antibody response, where a decline in antibody titres have been witnessed over time. The durability of the response will vary from one antigen to another and warrants longer-term data collection;
• Vaccine acceptance, where public mistrust fuelled by misinformation can jeopardise maintenance of herd immunity when a rapid response is needed to eradicate the viral infection of interest. Pharmacists have an important role in improving vaccine acceptance by addressing myths around vaccination, especially around mRNA vaccines (see Figure 5 for myths around mRNA vaccines and facts that can be used as counselling points)​[56]​.

Many publications have explored the application of mRNA vaccines against viral and tumoral targets​[41]​. According to data analytics company GlobalData, there are currently 23 out of 44 ongoing mRNA vaccine trials related to infectious diseases​[69]​. Although clinical evaluation is still limited, development of mRNA vaccines against a wide range of infectious diseases, such as zika virus, influenza, rabies, Ebola, HIV and respiratory syncytial virus is currently being explored​[56,70]​.

The SARS-CoV-2 pandemic gave prominence to mRNA-based vaccines. The fast manufacturing pace, minimalist nature, high immunogenicity and ease of modification resulted in an unprecedented speed in vaccine development, where clinical testing of the first mRNA vaccine candidate began only 66 days after SARS-CoV-2 sequencing data was made public​[71]​.

mRNA-based cancer vaccines hold great promise for the treatment of malignancies where tumour-associated and tumour-specific neoepitopes unique to cancerous cells can be targeted. The majority of cancer vaccines are therapeutic rather than prophylactic and seek to shrink and destroy tumours by simulating cell-mediated responses​[72]​. Furthermore, they are non-oncogenic, well-tolerated and capable of delivering large amounts of patient-specific antigens, making them an attractive form of cancer immunotherapy​[73]​.

A limited number of studies have applied mRNA vaccine technology towards combating bacterial and parasitic diseases. However, the structural and immunological complexities that stem from multiplex bacterial composition and parasites’ reproduction cycles complicate the choice of vaccine antigen. Furthermore, the majority of bacterial and parasitic infections are treatable using low-cost antibiotics and antiparasitic drugs, diminishing the need for vaccine development​[41]​.

Summary

With the recent worldwide approval of mRNA vaccines against COVID-19, the future for mRNA vaccines is undoubtedly bright. Nevertheless, further insights into their mechanism of action, potency and long-term safety are needed. Continued advancement in mRNA formulation and delivery should accelerate mRNA vaccine development.