A vaccine is an accurate means to prevent and contain an epidemic or pandemic. It has been challenging to create a vaccine against viruses like HIV, SARS-Cov-2, Ebola, and Zika. This complication is due to the rapid mutations of the virus in response to its environment. Further studies and research into this project have created high hopes for the development of mRNA vaccines to combat the problem. It is a versatile approach and faster to design. mRNA vaccines are a potent immune-stimulatory agent.
What is an mRNA vaccine?
To understand an mRNA, you need to understand the central dogma of molecular biology.
- Replication: After the cell has all the nutrients it needs, the DNA replicates itself, marking the start of cell division for growth.
- Transcription: The double-stranded DNA transcribes to give single-stranded mRNA in the presence of RNA polymerases.
- In translation, the mRNA nucleotide bases code for proteins (codons) that regulate cell growth. tRNA plays a significant role in protein production.
Essentially, the central dogma is DNA → DNA (replication) → RNA (transcription) → proteins (translation)
The mature in-vivo mRNAs are flanked by untranslated regions on both ends of the strand (post-transcriptional modification) for translation efficacy. These flanks improve the half-life, stability, and expression of mRNA. Increasing the Guanine-cytosine content in the sequence can increase the stability by a considerable level, although it questions the efficiency.
In-vitro mRNA production
To create an in-vitro mRNA, they use phage RNA polymerases to transcribe the DNA template, which is the “sequence of interest”. They design it to mimic conventional and mature mRNA, all ready to start the process of translation. As soon as it enters the cell, it is prone to standard cellular translation producing a fully folded and mature protein in its respective cell compartment.
The “sequence of interest” codes for the respective viral antigens (proteins) that can trigger an immune response without expressing any virulence, i.e., without attacking the recipient system.
This activity of protein delivery of mRNA has a considerable advantage in protein replacement therapies, where they need to target a protein to the correct compartment site for expression. The nucleases finally degrade the in-vitro translated mRNA minimizing toxicity.
Additionally, they can also replace the rare codons with synonymous codons, which have abundant anticodons containing tRNAs to increase protein production. Although minor intentional changes in the mRNA can interfere with its efficacy and protein folding.
Why prefer mRNA vaccines?
Firstly, the production of mRNA can be massive, owing to the high-speed in-vitro transcription. mRNA is non-infectious. Unlike DNA-based vaccines, mRNA will not incorporate with our cells’ genetic material, thereby reducing the likeness of mutagenesis (mutation). To further increase its safety profile, researchers can also reduce its capacity of eliciting an immune response, unlike DNA vaccines.
Moreover, the typical activity of enzymes in the system can quickly degrade the mRNA, making it safer– also safe in the events of repeated administration. Since its susceptibility is also a disadvantage, it demands additional facilitating agents.
To overcome the inefficient delivery, they formulate it into a carrier molecule, which makes the uptake and expression easier for our cells. This process also allows for a prolonged-expression of mRNA. They also use various in-vivo and in-vitro transfection agents– which aids in the nucleic acid delivery into the eukaryotic cell.
Purification and optimization
mRNA is immune-stimulatory and recognized by innate receptors. This feature is both beneficial and detrimental. It can inhibit the innate immune sensing interfering the antigen expression. Besides, enzymatically produced mRNA consists of dsRNA as contaminants. dsRNA provokes a type-1 interferon immune response, activating protein kinase R, which can degrade cellular and ribosomal RNA. To overcome this complication, they purify it via HPLC and FPLC.
Apart from dsRNA, mRNA itself can activate toll-like receptors and induce type-1 interferon production. Nucleoside modification can help overcome this problem. Hence, modified and purified mRNA is most efficient. However, other studies suggest that unmodified and unpurified mRNA results in similar protein production in HeLa cells (laboratory cells used for immune studies).
Delivery of mRNA vaccines into the recipient
The mRNA should enter the cell for expression and hence must pass the lipid membrane. The two basic approaches are– (i)loading via dendritic cells, (ii)direct delivery of the naked mRNA strand.
Dendritic cells as delivery vehicles
The T and B cells can recognize an antigen only if presented with major histocompatibility cells (MHC). Dendritic cells are professional antigen-presenting cells. They can activate the CD8 and CD4 receptors for an immune response, along with B-cell activity.
First, they isolate the dendritic cells from the recipient. The mRNA for delivery is inserted into these cells via electroporation (high voltage pulse), outside the body (ex-vivo). They now transfect the dendritic cells, carrying the mRNA vaccines, back into the recipient for expression and immunogenicity. This mechanism of vaccine delivery can induce a profound cell-mediated response and hence used to treat cancer.
Naked mRNA delivery
The direct naked mRNA delivery targets the antigen-presenting cells via intranodal or intradermal injections. It can also generate robust T-cell responses.
In-vivo uptake of mRNA
Gene gun method: To increase the in-vivo uptake efficacy of mRNA, the researchers coat it with gold particles (reacts the least and keeps it stable) and use a gene gun. Gene gun aids in the microprojectile delivery method of mRNA into the target compartment. The high speed of the gene gun can easily cross the lipid barrier. This method is preferential over electroporation under a few conditions, especially when the latter is only efficient in cancer therapy.
Protamines: The cationic peptide protamine protects the mRNA from degradation. However, it is efficient in a cancer vaccine model. They resolved the complication by formulating the protamine, which acts as an immune activator rather than an expression vector.
Lipid and polymer-based: Cationic and lipid-based delivery method is preferential for cancer and primary cells. But, they exhibit low in-vivo efficacy and high toxicity. However, there has been substantial investment to make lipids and polymers the most preferential delivery vehicles.
Types of mRNA vaccines
The two major types of RNA vaccines are– (i) self-amplifying and (ii)non-replicating.
(i)Self-amplifying mRNA vaccines
Self-amplifying mRNA vaccines conserve the replication machinery but replace the structural proteins with the antigen of interest. Since it is self-amplifying, it can result in abundant protein production with a small dose– for instance, as little as 100ng replicon vaccine coding for the fusion protein results in an elicited T and B cell immune response.
For example, self-amplifying vaccine coding for a protein of influenza virus complexed with lipid nanoparticles resulted in a potent immune response conferring protection in mice. Further studies also demonstrated its immunogenicity in human cytomegalovirus, HIV-1, Hepatitis-C, Ebola, and Zika in various animal models.
(ii)Non-replicating mRNA vaccines
Non-replicating mRNA vaccines are simple and appeal to an economical administration. When complexed with liposomes, it can generate an elicited cytotoxic lymphocyte response. However, uncomplexed mRNA is also practical in various animal models and protects them from lethal viruses.
They also carried out various experiments studying its immunogenicity for HIV, influenza, etc., in different animal models. While testing its immunogenicity for influenza virus in healthy volunteers, they observed that all of them experienced mild to moderate injection site reactions, and most of them experienced the typical pattern of fever, headache, and chills.
Surprisingly, the needle syringe injections did not generate antibodies in 98% of subjects. Contrastingly, the needle-free method exhibited a tremendous immune response in almost all the subjects but failed to retain immunity for over a year. Based on the difference of immunogenicity between the two routes of administration, the trial for an accurate delivery mechanism is ongoing.
