Part 1: Introduction to mRNA Vaccines

1.2. How are mRNA Vaccines Different?

Now that we’ve seen how normal vaccines work, it’s time to ask how messenger RNA vaccines work. These vaccines also fall under the ‘modern’ category - as you’ll see in a moment, their design requires a deep understanding of a lot of cellular processes.

Compared to other (e.g., subunit) vaccines, mRNA vaccines borrow even more from the process of building immunity against viruses. Instead of directly injecting antigens and adjuvants, mRNA vaccines deliver antigen-encoding instructions in the form of carefully packaged mRNA molecules, which function as both immunogens and adjuvants. Put another way, mRNA vaccines deliver instructions for building antigens rather than the antigens themselves to cells, with the cellular machinery responsible for executing those instructions and producing proteins that induce a strong immune response. This mechanism was tested in 1990, when researchers expressed proteins successfully by injecting mRNA into the body. The first clinical trial involving the mechanism took place 9 years later, when an mRNA antitumor vaccine was tested for the first time

What is mRNA?

mRNA was only discovered quite recently, in 1961. In any cell, messenger RNA (mRNA, for short) is produced when DNA is read and cloned in a process called transcription. While DNA molecules are used for storing information throughout a cell’s lifetime, messenger RNA (mRNA) molecules are used to transport it - their lives can be as short as a few minutes. After transcription of a DNA segment is complete, the mRNA molecule may be modified and then transported from the cell’s nucleus (where the DNA is located). Molecular machinery known as ribosomes assembles around floating mRNA strands, then reads them to ‘build’ proteins in a process called translation. Proteins are one of life’s main functional molecules - they do everything from facilitating reactions to sensing. In a sense, mRNA molecules are primarily transient carriers of information, taking it from the DNA to the place where it’s needed for protein synthesis.

Do mRNA vaccines just contain raw mRNA?

mRNA vaccines do not simply contain mRNA - naked mRNA would be inherently unstable (remember - mRNA can have a half-life of a few minutes!). Moreover, mRNA vaccine molecules are unable to cross a cell’s outer membrane due to their relatively large size (10-1,000 kDa) and negative charge. Even if they were, some molecules present in blood (nucleases) would break the molecules down (often called ‘degrading’) before they get near cells. In tissue, immune cells usually engulf or attach to mRNA molecules. For these reasons, a delivery mechanism is needed to make mRNA molecules get to cells.

Currently, the only drug delivery mechanism that has successfully been approved for human use is lipid nanoparticles (LNPs). While other mechanisms are being tested in preclinical trials, LNPs remain the established and preferred go-to delivery system thanks to their clinical validation. As the name suggests, they are very small particles that stabilize mRNA and help it enter cells. They’re relatively easy to pack, have low toxicity, and protect mRNA from being degraded by increasing stability. Their production is also scalable. mRNA vaccines are usually administered by subcutaneous or intramuscular injection, but other routes such as aerosol inhalation, eye drops, and oral administration are also being investigated.

At the structural level, LNPs are composed of 4 distinct lipid types, each with its own role. Ionizable cationic lipids are positively charged and thus interact with negatively charged mRNA molecules, forming a complex called a lipoplex. PEGylated lipids prevent the LNPs from being ingested by macrophages (immune system cells) and regulate LNP particle size. The physical properties of LNPs, such as surface charge and size, directly impact their biological activity. Along with helper lipids, they make LNPs very stable. Helper lipids also drive delivery of LNP contents into cells: the LNP outer layer fuses with the cell membrane, releasing its contents into the cell. Cholesterol maintains the stability of LNPs by filling gaps between other lipids.

Does mRNA only contain information about the protein it produces (i.e., the coding sequence)?

In Part 3, the coding sequence - the part of the mRNA that encodes the protein - will take center stage. Still, as you might imagine, it’s not the only part of the mRNA molecule. From left (also called the 5’ end) to right (3’ end), there are several parts of the molecule that work in tandem. The 5’ end contains a ‘cap’ structure, normally added in the nucleus for molecules originating in the cell (i.e., not from vaccines), which prevents the mRNA molecule from being recognized as viral and thus attacked by the immune system. The 5’ UTR (untranslated) region is next, marking the start of mRNA sequences translated from the DNA. While UTR sequences do not contain information about the protein itself, they play a pivotal role in recruiting ribosomes and completing the translation. The coding sequence - also called the open reading frame (ORF) - follows, starting with a ‘START’ sequence and followed by a signal sequence that dictates where the protein will be transported after translation, as well as the sequence coding for the protein itself. This is then followed by a 3’ UTR region and a poly(A) tail, which confer stability to the molecule. A long poly(A) tail directly prevents mRNA molecules from being degraded by special proteins inside cells, and is regularly added in the nucleus after transcription. Producing mRNA molecules at scale with these modifications began in 1983-1985, when Cap analogs and T7 RNA polymerases were commercialized, enabling in vitro transcription (IVT). Nowadays, these so-called post-transcriptional modifications can be replicated during IVT - an easily scalable process - to the point where cells are not able to differentiate between endogenous (cell-produced) and exogenous (foreign) mRNA molecules.

To understand how mRNA vaccines work, let’s follow the journey of a COVID-19 vaccine. The mRNA is packaged into lipid nanoparticles and delivered to normal muscle cells near the injection site. These packages go through the cell’s lipid bilayer (membrane) and release the mRNA into the cell. Then, the cell machinery - ribosomes - bind to the mRNA and translate it, producing antigens. The mRNA will also instruct the cell to export the antigens to the cell membrane (outside the cell). There, dendritic cells - called by the body’s scouts/intelligence network - pick up the antigens and bring them to lymph nodes where the adaptive immune system does its dance. Ultimately, about two weeks later, not only are antibodies against the antigen produced, but the adaptive immune system will also know to produce them should a real COVID virus with proteins similar to the antigen be detected.

Figure 2 - Predominant mRNA vaccine mechanism of action. Image taken from Chaudhary et al. (2021) [c8] with caption: “(1) Injected mRNA vacc — Figure 2 - Predominant mRNA vaccine mechanism of action. Image taken from Chaudhary et al. (2021) with caption: “(1) Injected mRNA vaccines are endocytosed by antigen-presenting cells. (2) After escaping the endosome and entering the cytosol, mRNA is translated into protein by the ribosome. The translated antigenic protein can stimulate the immune system in several ways. (3) Intracellular antigen is broken down into smaller fragments by the proteasome complex, and the fragments are displayed on the cell surface to cytotoxic T cells by major histocompatibility complex (MHC) class I proteins. (4) Activated cytotoxic T cells kill infected cells by secreting cytolytic molecules, such as perforin and granzyme. (5) Additionally, secreted antigens can be taken up by cells, degraded inside endosomes, and presented on the cell surface to helper T cells by MHC class II proteins. (6) Helper T cells facilitate the clearance of circulating pathogens by stimulating B cells to produce neutralizing antibodies and by activating phagocytes, such as macrophages, through inflammatory cytokines. BCR, B cell receptor; ER, endoplasmic reticulum; TCR, T cell receptor.”

By taking extra steps, mRNA vaccines come with several advantages. First, the same mRNA molecule will likely result in many antigens before it degrades. This means that mRNA vaccines require less mRNA, as they only deliver instructions that are amplified once they enter the cell. Second, producing mRNAs is just as (if not more) scalable than producing proteins. Proteins are harder to deal with because they can be unstable, prone to misfolding, and require higher production volumes (for the same number of doses).

Meanwhile, mRNA may vary in length and structure, but the manufacturing process is similar across vaccines. While protein-based vaccines require extensive testing to determine how proteins can be stabilized before injection, mRNA vaccines share the same properties, with only their stability in question (more on this in part 3). Lastly, some proteins require specialized media to maintain their shape, leading to different vaccine formulations. Meanwhile, mRNA vaccines often use the same mRNA sequence inside a lipid nanoparticle. The last two points mean that mRNA vaccines can be designed in days compared to weeks/months for purely protein-based vaccines.

Compared to protein subunit vaccines, mRNA vaccines have a much shorter production cycle and are not limited to short peptide sequences, allowing a much broader range of antigens to be targeted. Compared to DNA vaccines, mRNA vaccines do not need to enter a cell’s nucleus, which simplifies their mechanism of action while improving safety, as there is no real potential for gene insertion or subsequent mutations. Lastly, by varying the modifications of mRNA molecules or LNPs, the immunogenicity of mRNA vaccines can be controlled, allowing greater flexibility in vaccine design.

Will new advancements make mRNA vaccines even better?

Next-generation mRNA vaccine technologies are already in the works. Particularly, self-amplifying mRNA molecules offer the same benefits as normal mRNA molecules while requiring lower doses. They do this by incorporating virus-originating mechanisms to promote the translation of the mRNA molecule. Circular RNA is another technology that converts mRNA into molecules that form closed loops. These molecules have much longer half-lives than linear mRNAs thanks to more stable structures that are also resistant to degradation. This makes circRNAs stick inside the cell for longer, prolonging antigen selection. However, circRNAs still perform poorly in targeted delivery, with research still searching for the culprit. In the short term, normal IVT-produced mRNA (along with LNPs) remains the primary choice for preclinical and clinical applications, with well-established production and quality control procedures.

That being said, mRNA vaccines still have some shortcomings. While they’ve already been tested at a large scale (see next subsection), the technology is still new and should be studied carefully. Moreover, mRNA molecules are thermally unstable and thus require storage and transport at low temperatures, increasing costs. Lastly, while they allow for larger antigen sequences than subunit vaccines, large mRNA structures still have difficulty traversing tissue and cellular barriers, prompting the need for improved delivery strategies.