mRNAin vitro transcription, innate and adaptive immunity activation
AnmRNAvaccine is a type ofvaccine that uses a copy of a molecule calledmessenger RNA (mRNA) to produce an immune response.[1] The vaccinedelivers molecules ofantigen-encoding mRNA intocells, which use the designed mRNA as a blueprint to build foreignprotein that would normally be produced by apathogen (such as avirus) or by acancer cell. These protein molecules stimulate anadaptive immune response that teaches the body to identify and destroy the corresponding pathogen or cancer cells.[1] The mRNA isdelivered by a co-formulation of theRNA encapsulated inlipid nanoparticles that protect the RNA strands and help their absorption into the cells.[2][3]
Video showing how vaccination with an mRNA vaccine works
Reactogenicity, the tendency of a vaccine to produce adverse reactions, is similar to that of conventional non-RNA vaccines.[4] People susceptible to anautoimmune response may have an adverse reaction to messenger RNA vaccines.[4] The advantages of mRNA vaccines over traditional vaccines are ease of design, speed and lower cost of production, the induction of bothcellular andhumoral immunity, and lack of interaction with thegenomic DNA.[5][6] While some messenger RNA vaccines, such as thePfizer–BioNTech COVID-19 vaccine, have the disadvantage of requiringultracold storage before distribution,[1] other mRNA vaccines, such as theModerna vaccine, do not have such requirements.[7]
Timeline of some key discoveries and advances in the development of mRNA-based drug technology
The first successfultransfection of designed mRNA packaged within a liposomalnanoparticle into a cell was published in 1989.[18][19] "Naked" (or unprotected) lab-made mRNA was injected a year later into the muscle of mice.[3][20] These studies were the first evidence thatin vitro transcribed mRNA with a chosen gene was able to deliver the genetic information to produce a desired protein within living cell tissue[3] and led to the concept proposal of messenger RNA vaccines.[21][22][23]
Liposome-encapsulated mRNA encoding a viralantigen was shown in 1993 to stimulateT cells in mice.[24][25] The following year self-amplifying mRNA was developed by including both a viral antigen andreplicase encoding gene.[24][26] The method was used in mice to elicit both ahumoral andcellular immune response against a viral pathogen.[24] The next year mRNA encoding atumor antigen was shown to elicit a similar immune response against cancer cells in mice.[27][28]
BioNTech in 2008, andModerna in 2010, were founded to develop mRNA biotechnologies.[34][35] The US research agencyDARPA launched at this time thebiotechnology research program ADEPT to develop emerging technologies for theUS military.[36][37] The agency recognized the potential of nucleic acid technology for defense againstpandemics and began to invest in the field.[36] DARPA grants were seen as a vote of confidence that in turn encouraged other government agencies and private investors to invest in mRNA technology.[37] DARPA awarded at the time a $25 million grant to Moderna.[38]
The first human clinical trials using an mRNA vaccine against an infectious agent (rabies) began in 2013.[39][40] Over the next few years, clinical trials of mRNA vaccines for a number of other viruses were started. mRNA vaccines for human use were studied for infectious agents such asinfluenza,[41]Zika virus,cytomegalovirus, andChikungunya virus.[42][43]
The goal of a vaccine is to stimulate theadaptive immune system to createantibodies that precisely target that particularpathogen. The markers on the pathogen that the antibodies target are calledantigens.[48]
Traditional vaccines stimulate an antibody response by injecting eitherantigens, anattenuated (weakened) virus, aninactivated (dead) virus, or a recombinant antigen-encodingviral vector (harmless carrier virus with an antigentransgene) into the body. These antigens and viruses are prepared and grown outside the body.[49][50]
In contrast, mRNA vaccines introduce a short-lived[51]synthetically created fragment of the RNA sequence of a virus into the individual being vaccinated. These mRNA fragments are taken up bydendritic cells throughphagocytosis.[52] The dendritic cells use their internal machinery (ribosomes) to read the mRNA and produce the viral antigens that the mRNA encodes.[4] The bodydegrades the mRNA fragments within a few days of introduction.[53] Although non-immune cells can potentially also absorb vaccine mRNA, produce antigens, and display the antigens on their surfaces, dendritic cells absorb the mRNA globules much more readily.[54] The mRNA fragments are translated in thecytoplasm and do not affect the body's genomic DNA, located separately in thecell nucleus.[1][55]
Once the viral antigens are produced by the host cell, the normal adaptive immune system processes are followed. Antigens are broken down byproteasomes. Class I and class IIMHC molecules then attach to the antigen and transport it to the cellular membrane, "activating" the dendritic cell.[55] Once activated, dendritic cells migrate tolymph nodes, where theypresent the antigen toT cells andB cells.[56] This triggers the production of antibodies specifically targeted to the antigen, ultimately resulting inimmunity.[48]
mRNA
mRNA components important for expressing the antigen sequence
The central component of a mRNA vaccine is its mRNA construct.[57] Thein vitrotranscribed mRNA is generated from an engineeredplasmid DNA, which has anRNA polymerase promoter and sequence which corresponds to the mRNA construct. By combiningT7 phageRNA polymerase and the plasmid DNA, the mRNA can be transcribed in the lab. Efficacy of the vaccine is dependent on the stability and structure of the designed mRNA.[4]
Thein vitro transcribed mRNA has the same structural components as natural mRNA ineukaryotic cells. It has a5' cap, a5'-untranslated region (UTR) and3'-UTR, anopen reading frame (ORF), which encodes the relevant antigen, and a3'-poly(A) tail. By modifying these different components of the synthetic mRNA, the stability and translational ability of the mRNA can be enhanced, and in turn, the efficacy of the vaccine improved.[57]
The mRNA can be improved by using synthetic 5'-cap analogues which enhance the stability and increase protein translation. Similarly,regulatory elements in the 5'-untranslated region and the 3'-untranslated region can be altered, and the length of the poly(A) tail optimized, to stabilize the mRNA and increase protein production. The mRNAnucleotides can be modified to both decreaseinnate immune activation and increase the mRNA'shalf-life in the host cell. Thenucleic acid sequence andcodon usage impacts protein translation. Enriching the sequence withguanine-cytosine content improves mRNA stability and half-life and, in turn, protein production. Replacing rarecodons withsynonymous codons frequently used by the host cell also enhances protein production.[4]
Delivery
Major delivery methods and carrier molecules for mRNA vaccines
For a vaccine to be successful, sufficient mRNA must enter the host cellcytoplasm to stimulate production of the specific antigens. Entry of mRNA molecules, however, faces a number of difficulties. Not only are mRNA molecules too large to cross thecell membrane by simplediffusion, they are also negatively charged like the cell membrane, which causes a mutualelectrostatic repulsion. Additionally, mRNA is easily degraded byRNAases in skin and blood.[55]
Various methods have been developed to overcome these delivery hurdles. The method of vaccine delivery can be broadly classified by whether mRNA transfer into cells occurs within (in vivo) or outside (ex vivo) the organism.[55][3]
Ex vivo
Dendritic cells display antigens on theirsurfaces, leading to interactions withT cells to initiate an immune response. Dendritic cells can be collected from patients and programmed with the desired mRNA, then administered back into patients to create an immune response.[58]
The simplest way thatex vivo dendritic cells take up mRNA molecules is throughendocytosis, a fairly inefficient pathway in the laboratory setting that can be significantly improved throughelectroporation.[55]
In vivo
Since the discovery that the direct administration ofin vitro transcribed mRNA leads to the expression of antigens in the body,in vivo approaches have been investigated.[20] They offer some advantages overex vivo methods, particularly by avoiding the cost of harvesting and adapting dendritic cells from patients and by imitating a regular infection.[55]
Different routes ofinjection, such asinto the skin,blood, ormuscles, result in varying levels of mRNA uptake, making the choice of administration route a critical aspect ofin vivo delivery. One study showed, in comparing different routes, thatlymph node injection leads to the largest T-cell response.[59]
Naked mRNA injection
Naked mRNA injection means that thedelivery of the vaccine is only done in abuffer solution.[60] This mode of mRNA uptake has been known since the 1990s.[20] The first worldwide clinical studies usedintradermal injections of naked mRNA for vaccination.[61][62] A variety of methods have been used to deliver naked mRNA, such as subcutaneous, intravenous, and intratumoral injections. Although naked mRNA delivery causes an immune response, the effect is relatively weak, and after injection the mRNA is often rapidly degraded.[55]
The first time the FDA approved the use oflipid nanoparticles as a drug delivery system was in 2018, when the agency approved the firstsiRNA drug,Onpattro.[65] Encapsulating the mRNA molecule in lipid nanoparticles was a critical breakthrough for producing viable mRNA vaccines, solving a number of key technical barriers in delivering the mRNA molecule into the host cell.[65][66] Research into using lipids to deliver siRNA to cells became a foundation for similar research into using lipids to deliver mRNA.[67] However, new lipids had to be invented to encapsulate mRNA strands, which are much longer than siRNA strands.[67]
Principally, thelipid provides a layer of protection against degradation, allowing more robust translational output. In addition, the customization of the lipid's outer layer allows the targeting of desired cell types throughligand interactions. However, many studies have also highlighted the difficulty of studying this type of delivery, demonstrating that there is an inconsistency betweenin vivo andin vitro applications of nanoparticles in terms of cellular intake.[68] The nanoparticles can be administered to the body and transported via multiple routes, such asintravenously or through thelymphatic system.[65]
One issue with lipid nanoparticles is that several of the breakthroughs leading to the practical use of that technology involve the use ofmicrofluidics. Microfluidic reaction chambers are difficult to scale up, since the entire point of microfluidics is to exploit the microscale behaviors of liquids. The only way around this obstacle is to run an extensive number of microfluidic reaction chambers in parallel, a novel task requiring custom-built equipment.[69][70] For COVID-19 mRNA vaccines, this was the main manufacturing bottleneck. Pfizer used such a parallel approach to solve the scaling problem. After verifying that impingement jet mixers could not be directly scaled up,[71] Pfizer made about 100 of the little mixers (each about the size of aU.S. half-dollar coin), connected them together with pumps and filters with a "maze of piping,"[72][73] and set up a computer system to regulate flow and pressure through the mixers.[71]
Another issue, with the large-scale use of this delivery method, is the availability of the novel lipids used to create lipid nanoparticles, especially ionizable cationic lipids. Before 2020, such lipids were manufactured in small quantities measured in grams or kilograms, and they were used for medical research and a handful of drugs for rare conditions. As the safety and efficacy of mRNA vaccines became clear in 2020, the few companies able to manufacture the requisite lipids were confronted with the challenge of scaling up production to respond to orders for several tons of lipids.[70][74]
Advantages and disadvantages of different types of vaccine platforms
mRNA vaccines offer specific advantages over traditionalvaccines.[4][5] Because mRNA vaccines are not constructed from an active pathogen (or even an inactivated pathogen), they are non-infectious. In contrast, traditional vaccines require the production of pathogens, which, if done at high volumes, could increase the risks of localized outbreaks of the virus at the production facility.[5] Another biological advantage of mRNA vaccines is that since the antigens are produced inside the cell, they stimulatecellular immunity, as well ashumoral immunity.[6][79]
mRNA vaccines have the production advantage that they can be designed swiftly. Moderna designed theirmRNA-1273 vaccine for COVID-19 in 2 days.[80] They can also be manufactured faster, more cheaply, and in a more standardized fashion (with fewer error rates in production), which can improve responsiveness to serious outbreaks.[4][5]
The Pfizer–BioNTech vaccine originally required 110 days to mass-produce (before Pfizer began to optimize the manufacturing process to only 60 days), which was substantially faster than traditional flu and polio vaccines.[72] Within that larger timeframe, the actual production time is only about 22 days: two weeks for molecular cloning of DNA plasmids and purification of DNA, four days for DNA-to-RNAtranscription and purification of mRNA, and four days to encapsulate mRNA in lipid nanoparticles followed byfill and finish.[81] The majority of the days needed for each production run are allocated to rigorous quality control at each stage.[72]
DNA vaccines
In addition to sharing the advantages of theoreticalDNA vaccines over established traditionalvaccines, mRNA vaccines also have additional advantages over DNA vaccines. ThemRNA istranslated in thecytosol, so there is no need for the RNA to enter thecell nucleus, and the risk of being integrated into the hostgenome is averted.[3]Modified nucleosides (for example,pseudouridines, 2'-O-methylated nucleosides) can be incorporated to mRNA to suppressimmune response stimulation to avoid immediate degradation and produce a more persistent effect through enhanced translation capacity.[31][82][83] Theopen reading frame (ORF) anduntranslated regions (UTR) of mRNA can be optimized for different purposes (a process called sequence engineering of mRNA), for example through enriching theguanine-cytosine content or choosing specific UTRs known to increase translation.[52] An additional ORF coding for areplication mechanism can be added to amplify antigen translation and therefore immune response, decreasing the amount of starting material needed.[84][85]
Disadvantages
Storage
Because mRNA is fragile, some vaccines must be kept at very low temperatures to avoid degrading and thus giving little effective immunity to the recipient. Pfizer–BioNTech'sBNT162b2 mRNA vaccine has to be kept between −80 and −60 °C (−112 and −76 °F).[86][87] Moderna says theirmRNA-1273 vaccine can be stored between −25 and −15 °C (−13 and 5 °F),[88] which is comparable to a home freezer,[87] and that it remains stable between 2 and 8 °C (36 and 46 °F) for up to 30 days.[88][89] In November 2020,Nature reported, "While it's possible that differences in LNP formulations or mRNA secondary structures could account for the thermostability differences [between Moderna and BioNtech], many experts suspect both vaccine products will ultimately prove to have similar storage requirements and shelf lives under various temperature conditions."[79] Several platforms are being studied that may allow storage at higher temperatures.[4]
Recent
Before 2020, no mRNA technology platform (drug or vaccine) had beenapproved for therapeutic use in humans, so there was a risk of unknown effects.[79] The 2020 COVID-19 pandemic required faster production capability of mRNA vaccines, which made them attractive to national health organisations, and led to debate about the type of initial authorization mRNA vaccines should get (includingemergency use authorization orexpanded access authorization) after the eight-week period of post-final human trials.[90][91]
Side effects
Reactogenicity is similar to that of conventional, non-RNA vaccines. However, those susceptible to anautoimmune response may have an adverse reaction to mRNA vaccines.[4] The mRNA strands in the vaccine may elicit an unintended immune reaction – this entails thebody believing itself to be sick, and the person feeling as if they are as a result. To minimize this, mRNA sequences in mRNA vaccines are designed to mimic those produced by host cells.[5]
Strong but transient reactogenic effects were reported in trials of novel COVID-19 mRNA vaccines; most people will not experience severe side effects which include fever and fatigue. Severe side effects are defined as those that prevent daily activity.[92]
Efficacy
The COVID-19 mRNA vaccines from Moderna and Pfizer–BioNTech had short-term efficacy rates of over 90 percent against the original SARS-CoV-2 virus. Prior to mRNA, drug trials on pathogens other than COVID-19 were not effective and had to be abandoned in the early phases of trials. The reason for the efficacy of the new mRNA vaccines is not clear.[93]
Physician-scientistMargaret Liu stated that the efficacy of the new COVID-19 mRNA vaccines could be due to the "sheer volume of resources" that went into development, or that the vaccines might be "triggering a nonspecific inflammatory response to the mRNA that could be heightening its specific immune response, given that themodified nucleoside technique reduced inflammation but hasn't eliminated it completely", and that "this may also explain the intense reactions such as aches and fevers reported in some recipients of the mRNA SARS-CoV-2 vaccines". These reactions though severe were transient and another view is that they were believed to be a reaction to the lipid drug delivery molecules.[93] In June 2021, the USFDA added a warning about the possibility of increased risk of myocarditis and pericarditis for some people.[94]
There is misinformation implying that mRNA vaccines could alter DNA in the nucleus.[95] mRNA in thecytosol is very rapidly degraded before it would have time to gain entry into the cell nucleus. In fact, mRNA vaccines must be stored at very low temperature and free fromRNAses to prevent mRNA degradation.Retrovirus can be single-stranded RNA (just as manySARS-CoV-2 vaccines are single-stranded RNA) which enters the cell nucleus and usesreverse transcriptase to make DNA from the RNA in the cell nucleus. A retrovirus has mechanisms to be imported into the nucleus, but other mRNA (such as the vaccine) lack these mechanisms. Once inside the nucleus, creation of DNA from RNA cannot occur without areverse transcriptase and appropriate primers, which both accompany a retrovirus, but which would not be present for other exogenous mRNA (such as a vaccine) even if it could enter the nucleus.[96]
Amplification
mRNA vaccines use either non-amplifying (conventional) mRNA or self-amplifying mRNA.[97] Pfizer–BioNTech and Moderna vaccines use non-amplifying mRNA. Both mRNA types continue to be investigated as vaccine methods against other potential pathogens and cancer.[32]
Non-amplifying
Mechanism of non-amplifying and self-amplifying mRNA vaccines
The initial mRNA vaccines use a non-amplifying mRNA construct.[64] Non-amplifying mRNA has only oneopen reading frame that codes for the antigen of interest.[97] The total amount of mRNA available to the cell is equal to the amount delivered by the vaccine. Dosage strength is limited by the amount of mRNA that can be delivered by the vaccine.[98] Non-amplifying vaccines replaceuridine withN1-Methylpseudouridine in an attempt to reduce toxicity.[99]
Self-amplifying mRNA (saRNA) vaccines replicate their mRNA after transfection.[100] Self-amplifying mRNA has twoopen reading frames. The first frame, like conventional mRNA, codes for the antigen of interest. The second frame codes for anRNA-dependent RNA polymerase (and its helper proteins) which replicates the mRNA construct in the cell. This allows smaller vaccine doses.[100] The mechanisms and consequently the evaluation of self-amplifying mRNA may be different, as self-amplifying mRNA is a much bigger molecule.[3]
SaRNA vaccines being researched include amalaria vaccine.[101] The first saRNA Covid vaccine authorised was Gemcovac, in India in June 2022.[102] The second wasARCT-154, developed by Arcturus Therapeutics. A version manufactured by Meiji Seika Pharma was authorised in Japan in November 2023.[103]
GSK began aphase 1 trial of an saRNA COVID-19 vaccine in 2021.[104]Gritstone bio started also started a phase 1 trial of an saRNA COVID-19 vaccine in 2021, used as abooster vaccine, with interim results published in 2023.[105] The vaccine is designed to target both the spike protein of theSARS‑CoV‑2 virus, and viral proteins that may be less prone to genetic variation, to provide greater protection against SARS‑CoV‑2 variants.[106][107] saRNA vaccines must use uridine, which is required for reproduction to occur.[99]
^May M (31 May 2021)."After COVID-19 successes, researchers push to develop mRNA vaccines for other diseases".Nature.Archived from the original on 13 October 2021. Retrieved31 July 2021.When the broad range of vaccines against COVID-19 were being tested in clinical trials, only a few experts expected the unproven technology of mRNA to be the star. Within 10 months, mRNA vaccines were both the first to be approved and the most effective. Although these are the first mRNA vaccines to be approved, the story of mRNA vaccines starts more than 30 years ago, with many bumps in the road along the way. In 1990, the late physician-scientist Jon Wolff and his University of Wisconsin colleagues injected mRNA into mice, which caused cells in the mice to produce the encoded proteins. In many ways, that work served as the first step toward making a vaccine from mRNA, but there was a long way to go—and there still is, for many applications.
^Patent:WO1990011092Archived 14 October 2021 at theWayback Machine; Inventors: Philip L. Felgner, Jon Asher Wolff, Gary H. Rhodes, Robert Wallace Malone, Dennis A. Carson; Assignees: Vical Inc., Wisconsin Alumni Research Foundation; Title: "Expression of Exogenous Polynucleotide Sequences in a VertebrateArchived 9 December 2021 at theWayback Machine"; (Quote: "The present invention relates to introduction of naked DNA and RNA sequences into a vertebrate to achieve controlled expression of a polypeptide. It is useful in gene therapy, vaccination, and any therapeutic situation in which a polypeptide should be administered to cells in vivo"; Example 8: mRNA vaccination of mice to produce the gpl20 protein of HIV virus); Priority date: 21 March 1989; Publication date: 4 October 1990.
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