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Nature Series Review: Technological Innovation and Research Progress of mRNA Vaccines.
Vaccines are the most effective public health intervention to prevent the spread of infectious diseases. Successful vaccination eradicates many life-threatening diseases, such as smallpox and polio, and the World Health Organization estimates that vaccines prevent 2-3 million deaths each year from tetanus, whooping cough, influenza, and measles.
However, despite their apparent success, conventional vaccines are not effective against pathogens that evade immune surveillance, such as Plasmodium, Hepatitis C and HIV. In addition, they need to be modified regularly to deal with rapidly mutating pathogens, such as influenza viruses.
mRNA-based nucleic acid vaccines were proposed more than 30 years ago in hopes of producing vaccines that are safe, versatile and easy to manufacture.
mRNA vaccines have many advantages over traditional vaccines: Unlike some viral vaccines, mRNA does not integrate into the genome, avoiding concerns about insertional mutations; mRNA vaccines can be manufactured in a cell-free manner, allowing for rapid, cost-effective, and efficient production.
Furthermore, a single mRNA vaccine can encode multiple antigens, enhance immune responses against adaptive pathogens, and be able to target multiple microbial or viral variants in a single formulation.
Initially, mRNA therapy was not taken seriously due to concerns about its stability, inefficiency, and excessive immune stimulation.
However, in the past ten years, the field of mRNA therapeutics has undergone rapid changes, including the in-depth study of mRNA pharmacology, the development of effective vectors and the control of mRNA immunogenicity, which has brought the clinical application of mRNA vaccines into a new stage.
mRNA design and synthesis
In vitro transcribed ( IVT ) mRNA mimics the structure of endogenous mRNA and has five sections ranging from 5ʹ to 3ʹ including: 5ʹcap, 5ʹ untranslated region ( UTR ), an antigen-encoding open reading frame, 3ʹUTR and a PolyA tail.
Like native eukaryotic mRNAs, the 5′ cap structure contains a 7-methylguanosine nucleoside, which sterically protects the mRNA from exonuclease degradation and cooperates with translation initiation factor proteins , recruits ribosomes to initiate translation. The length of the PolyA tail indirectly regulates mRNA translation and half-life.
The 5′ and 3′ UTRs flanking the coding region regulate mRNA translation, half-life and subcellular localization, and native UTRs from highly expressed genes such as α- and β-globin genes are the first choice for synthetic mRNAs. In addition, UTRs can be optimized according to cell type, such as by removing miRNA-binding sites and AU-rich regions in the 3′ UTR to minimize mRNA degradation.
The open reading frame of an mRNA vaccine is the most critical component. Although the open reading frame is not as malleable as the non-coding regions, codon optimization can be used to increase translation without changing the protein sequence.
For example, CureVac AG found that human mRNA codons rarely have an A or U in the third position, thus replacing the A or U in the third position of the open reading frame with a G or C. and applied this optimized strategy to its SARS-CoV-2 vaccine, CVnCoV, which is currently in Phase III trials.
To maximize translation, mRNA sequences often contain modified nucleosides, such as pseudouridine, N1-methylpseudouridine, or other nucleoside analogs.
The use of modified nucleosides, especially modified uridines, prevents recognition by pattern recognition receptors, ensuring that the translation process produces sufficient levels of protein.
Both Moderna and Pfizer–BioNTech SARS-CoV-2 vaccines contain nucleoside modifications.
Another strategy to avoid detection of pattern recognition receptors, pioneered by CureVac, uses sequence engineering and codon optimization to deplete uridine by increasing the GC content of vaccine mRNAs.
In addition to improvements in mRNA sequences, significant progress has been made in streamlining mRNA production.
Synthetic mRNAs used clinically are transcribed in vitro from DNA plasmids using bacteriophage RNA polymerase T7 ( T3 and SP6 polymerases can also be used ).
Additionally, purification steps are simplified using CleanCap’s co-transcriptional capping technology.
mRNA vaccine vector
Since mRNAs are large ( 10 4 –10 6 Da) and negatively charged, they cannot pass through the anionic lipid bilayer of the cell membrane. Furthermore, in vivo, it is engulfed by cells of the innate immune system and degraded by nucleases.
Therefore, in vivo applications require the use of mRNA delivery vectors that transfect immune cells without causing toxicity or unnecessary immunogenicity. At present, many solutions based on innovative materials have been developed for this purpose.
Lipid Nanoparticles (LNPs)
Lipid nanoparticles are the most clinically advanced mRNA carriers. As of June 2021, all SARS-CoV-2 mRNA vaccines under development or approved for clinical use use LNPs.
LNPs offer many benefits for mRNA delivery, including formulation simplicity, modularity, biocompatibility, and large mRNA payload capacity.
In addition to RNA drugs, LNPs typically include four components, ionizable lipids, cholesterol, helper phospholipids, and pegylated lipids, which together encapsulate and protect fragile mRNAs.
Ionizable lipids form nanoparticles with mRNA in an acidic buffer, which positively charges the lipid and attracts the RNA.
Furthermore, they are positively charged in the acidic environment of endosomes, which facilitates their fusion with the endosomal membrane, releasing them into the cytoplasm.
DODAP and DODMA were the first ionizable lipids for RNA delivery. The efficacy of DODMA was improved by design, resulting in DLin-MC3-DMA.
This is the first FDA-approved drug formulation to use an ionizable lipid: the siRNA drug patisiran ( Onpattro ). In addition to the efficient and safe delivery of siRNA, DLin-MC3-DMA was also used for mRNA delivery.
Currently, many groups in academia and industry use combinatorial reaction protocols to synthesize potential delivery materials, and this approach yields a number of potent lipids, including C12-200, 503O13, 306Oi10, OF-02, TT3, 5A2-SC8 , SM-102 ( for the Moderna vaccine mRNA-1273 against SARS-CoV-2 ) and ALC-0315 ( for the Pfizer vaccine BNT162b2 ).
In addition to the quest to improve efficacy, there is an increasing focus on improving the specificity of drugs, especially for vaccines and immunotherapies.
The lipid 11-A-M58, which contains a polycyclic adamantane tail, and the lipid 93-O17S59, which contains a cyclic imidazole head, have been designed to target T cells in vivo.
Although the mechanism remains unclear, the cyclic groups of these lipids are critical for targeting T cells.
Although ionized lipids are arguably the most important components of LNPs, three other lipid components ( cholesterol, helper lipids, and pegylated lipids ) also contribute to nanoparticle formation and function.
Cholesterol, a naturally occurring lipid, enhances nanoparticle stability by filling the voids between lipids and facilitates fusion with endosomal membranes during uptake into cells.
Helper lipids regulate nanoparticle fluidity and enhance efficacy by promoting lipid phase transitions that facilitate membrane-endosome fusion.
The choice of the optimal helper lipid depends on the ionizable lipid material and the RNA carrier. For example, for lipid-like materials, saturated helper lipids ( such as DSPC ) are best for the delivery of short RNAs ( such as siRNA ), while unsaturated lipids ( such as DOPE ) are best for the delivery of mRNAs.
DSPC has been used in FDA-approved SARS-CoV-2 vaccines mRNA-1273 and BNT162b2.
The pegylated lipid component of LNPs is composed of polyethylene glycol ( PEG ) combined with anchor lipids such as DMPE or DMG .
Hydrophilic PEG can stabilize LNPs, modulate nanoparticle size by limiting lipid fusion, and increase nanoparticle half-life by reducing nonspecific interactions with macrophages.
Both mRNA-1273 and BNT162b2 SARS-CoV-2 vaccines contain pegylated lipids.
Polymers and Polymer Nanoparticles
Although not as clinically advanced as LNPs, polymers have similar advantages to lipids for efficient mRNA delivery. Cationic polymers condense nucleic acids into complexes of different shapes and sizes that can enter cells by endocytosis.
Polyethyleneimine is the most widely studied nucleic acid delivery polymer. Despite its excellent efficacy, its toxicity limits applications due to its high charge density. In addition, several less toxic biodegradable polymers have been developed. For example, poly( β-aminoester ) excels in mRNA delivery, especially to the lung.
Recently, a new class of lipid-containing polymers, called charge-altered releasable transporters ( CARTs ), have been developed, which can effectively target T cells, which are very difficult to manipulate. Therefore, CARTs are extremely Attractive delivery material with great potential in mRNA vaccines and gene therapy.
Other delivery systems
In addition to lipid and polymer carriers, peptides can also deliver mRNA into cells thanks to cationic or amphiphilic amine groups ( such as arginine ) in their backbone and side chains, which The base electrostatically binds to the mRNA and forms a nanocomplex.
For example, membrane fusion cell-penetrating peptides containing a repeating arginine-alanine-leucine-alanine ( RALA ) motif.
The arginine-rich protamine peptide, which is positively charged at neutral pH, can also concentrate mRNA and facilitate its delivery.
The complex of protamine with mRNA activates the Toll-like receptor ( TLR7, TLR8 ) pathway that recognizes single-stranded mRNA, therefore, it can also be used as an adjuvant for vaccine or immunotherapy applications.
CureVac AG is evaluating RNActive, a protamine-containing delivery platform, for clinical trials in melanoma, prostate cancer and non-small cell lung cancer.
Finally, mRNA can also be delivered by cationic nanoemulsions based on squalene, which consist of oily squalene cores. Some squalene formulations, such as Novartis’ MF59, are used as adjuvants in FDA-approved influenza vaccines. MF59 causes cells at the injection site to secrete chemokines, thereby recruiting antigen-presenting cells, inducing monocytes to differentiate into dendritic cells, and enhancing antigen uptake by antigen-presenting cells. The mechanism by which squalene-based cationic nanoemulsions escape from endosomes and deliver mRNA into the cytoplasm is unclear.
mRNA vaccine technology innovation
The most important innovations in mRNA vaccine technology are: mRNA sequence design; development of methods for simple, rapid and large-scale production of mRNA; development of efficient and safe mRNA vaccine delivery materials.
A recent study used a cell culture-based systematic selection process to identify novel UTRs that significantly increased protein expression of IVT mRNA. These sequences induce approximately 3-fold more protein from the relevant transcripts compared to the human β-globin 3’UTR.
In addition, an interesting new vaccine format was recently reported that utilizes mRNA encoding an alphavirus RNA-dependent RNA polymerase plus a second mRNA encoding an antigen to enable replication in the cytoplasm.
This system can effectively induce protective immune responses in mice at very low doses ( 50ng ). These findings are particularly attractive because the use of low doses reduces the cost of vaccine production.
The delivery-free material further reduces costs, simplifies manufacturing, and increases the likelihood of vaccine lyophilization and storage at ambient temperatures.
In terms of production, in addition to the cleancap technique, a simple method for purifying mRNA by adsorption of double-stranded RNA contaminants to cellulose, an inexpensive and abundant polysaccharide , has recently been developed.
This highly scalable and inexpensive method was shown to be as effective as high-performance liquid chromatography in removing dsRNA contaminants from IVT mRNA samples.
In terms of delivery materials, similar to the polymer-based CART platform, mRNA-LNPs also achieved selective T cell targeting. A new platform, called ASSET ( Anchored Secondary Targeting Single Chain Antibody ), in which T cell-specific monoclonal antibodies are linked to LNPs to target T cells.
This flexible platform also has great potential for mRNA vaccines and other applications.
Another lipid complex preferentially targets dendritic cells after systemic administration. Selectively targeting DCs with mRNA vaccines to induce robust immune responses is a potentially key finding, and this platform has already demonstrated its promise in clinical trials.
Research progress of mRNA vaccines in infectious diseases
mRNA-based therapies represent a relatively novel and highly potent class of drugs. Several recently published studies have highlighted the potential efficacy of mRNA vaccines in the treatment of different types of malignancies and infectious diseases where traditional vaccine strategies fail to elicit protective immune responses.
By the end of 2019, 15 candidate mRNA vaccines against infectious diseases had entered clinical trials, at a time when mRNA vaccines were thought to be at least 5-6 years away from regulatory approval. But those expectations were upended when the COVID-19 pandemic hit the world in early 2020.
Over the next few months, the development, manufacture and deployment of mRNA vaccines all entered a phase of leaps and bounds.
Most SARS-CoV-2 vaccine candidates mount an immune response to the spike protein on the virus surface. Spike protein binds to its receptor angiotensin-converting enzyme 2 on the surface of its host cells.
The attached Spike protein is then cleaved by the cell’s transmembrane serine protease 2, which induces a conformational change that exposes the Spike protein’s fusion peptide and facilitates fusion with the cellular or endosomal membrane.
Typically, the antigen encoded by vaccine mRNA is either the full-length spike protein or the receptor-binding domain of the spike protein.
As of June 18, 2021, 185 CVID-19 vaccine candidates were in preclinical development and another 102 had entered clinical trials. In clinical trials, 19 were mRNA vaccines. On December 11, 2020, Pfizer’s BNT162b2 vaccine received emergency authorization from the FDA, becoming the first mRNA drug approved for use in humans.
A week later, Moderna’s vaccine, mRNA-1273, was also authorized for use in the United States. Ultimately, they were the first SARS-CoV-2 vaccines to be licensed in the US, UK, Canada and several other countries.
Pfizer and BioNTech have jointly developed five mRNA vaccine candidates that encode variants of the spike protein antigen. The two lead drug candidates, BNT162b1 and BNT162b2, use Acuitas Therapeutics’ ionizable lipid ALC-0315 and nucleoside-modified mRNA in which all uridines are replaced by N1 methyl pseudouridines to enhance mRNA translation. BNT162b1 encodes the receptor-binding domain of the trimeric secreted spike protein, while BNT162b2 encodes the full-length SARS-CoV-2 spike glycoprotein with two proline substitutions in the S2 subunit, locking the protein in the prefusion conformation .
In phase 1 trials of both vaccines, high titers of neutralizing antibodies were induced and strong CD4+ and CD8+ responses were induced, with mild to moderate adverse effects. Both vaccine candidates were well tolerated and effective, but only the BNT162b2 vaccine entered Phase II/III trials due to its milder systemic and local adverse effects. In Phase 3 trials, BNT162B2 showed 95% overall prevention and 90-100% efficacy.
Moderna partnered with the National Institutes of Health to develop mRNA-1273. The vaccine uses the ionizable lipid SM-102 to prepare LNPs that encapsulate N1-methylpseudouridine-modified mRNA. This sequence encodes the SARS-CoV-2 spike protein with two proline substitutions conferring a prefusion conformation.
In Phase 1 clinical trials, mRNA-1273 was very effective and well tolerated. In a phase III trial involving 30,420 volunteers, two 100-μg doses of the vaccine had a prevention rate of 94.1%, with local pain at the injection site being the most common side effect. After the second dose, half of the volunteers reported moderate to severe systemic side effects ( eg, fatigue, muscle pain, joint pain ) that resolved within 48 hours.
Although the vaccines produced by Pfizer and Moderna have proven efficacy and safety profiles, their need for cold chain storage poses significant difficulties in safeguarding. mRNA-1273 can be stored at 4-8°C for one month, while BNT162b2 needs to be stored at -60°C.
CureVac’s vaccine candidate, CVnCoV, is stable for 3 months at 5°C. CVnCoV uses ionizable lipids from Acuitas Therapeutics ( probably ALC-0315 ) and unmodified mRNA encoding a full-length spike protein with two proline substitutions. In a phase 1 clinical trial, the volunteers produced neutralizing antibodies similar to those in CVID-19 convalescent patients and were well tolerated. Unfortunately, in a phase III clinical trial involving 40,000 people, CVnCoV showed only 47% efficacy. Interim analyses suggest that the lower efficacy of CVnCoV is attributable to emerging SARS-CoV-2 variants.
Currently, CureVac is working with GSK to develop a second-generation drug candidate, CV2CoV, optimized to enhance translation and immunogenicity relative to CVnCoV. CV2CoV uses the 5′UTR from the human hydroxysteroid 17-β-dehydrogenase 4 gene and the 3′UTR from the human proteasome 20S subunit β3 gene. In preclinical studies, CV2CoV showed 1.8-fold higher protein expression than CVnCoV in vitro and induced high titer crossovers against B.1.1.7, B.1.1.298 and B.1.351 variants in rats and antibodies.
Another heat-resistant vaccine candidate, ARCoV, was developed by the Chinese Academy of Military Sciences in collaboration with Walvax Biotechnology and is stable at 25°C for a week. ARCoV encodes the receptor-binding domain of the spike protein. In preclinical studies, high SARS-CoV-2-specific IgG antibodies and strong virus neutralization titers were induced in cynomolgus monkeys. Although the reasons behind the thermostability of CVnCoV and ARCoV are unclear, mRNA secondary structure, smaller mRNA size, GC content, and lipids may be important factors.
Several other mRNA vaccine candidates are also in development. LNP-nCoVsaRNA, developed in collaboration with Imperial College London and Acuitas Therapeutics, encodes a full-length spike protein. Currently, a Phase I clinical trial is being evaluated using a 0.1–1 µg dose-escalation regimen ( ISRCTN17072692 ), which used the lowest RNA dose of all candidate mRNA vaccines.
Another self-amplifying mRNA vaccine candidate, ARCT-021 ( also known as LUNAR-COV19 ), was developed by Arcturus using its proprietary LUNAR lipid carrier and self-transcribed and replicating RNA ( STARR ) platform. It encodes the full-length prefusion spike protein.
Flu virus vaccine
Worldwide, between 290,000 and 650,000 people die each year from influenza viruses. Current vaccines target the viral hemagglutinin protein that facilitates viral entry. Traditional influenza vaccines are inactivated influenza viruses grown in eggs, which take a long time to produce and are difficult to purify. Additionally, viruses mutate in eggs for optimal growth, sometimes rendering them ineffective in humans.
Therefore, there is a real need for alternative antigen targets and production methods. Synthetic mRNAs transcribed in vitro could meet this need and ensure rapid vaccine production should novel influenza strains emerge. For example, in 2013, a self-amplifying mRNA vaccine based on LNP ( DLinDMA ) was rapidly developed within 8 days of the H7N9 outbreak in China, however, unfortunately, clinical trials could not be conducted due to the lack of GMP facilities for mRNA manufacturing at that time.
There are also efforts to develop a universal flu vaccine that does not require annual revisions. This vaccine provides immunity against several strains and subtypes of influenza virus. Influenza mRNA vaccine first demonstrated in 2012, three intradermal injections induced homologous and heterologous immunity against H1N1 and H5N1 strains in mice, respectively.
Notably, the conserved handle region of hemagglutinin, which is less prone to mutation, has recently emerged as a novel universal vaccine target. Yet another study used LNPs to deliver a 50 ng dose of mRNA encoding three conserved influenza proteins: neuraminidase, nucleoprotein and matrix-2 ion channel protein and the hemagglutinin handle region. Incredibly, this tiny mRNA dose produced a broad range of protective antibodies.
Zika virus vaccine
Zika virus infection was first identified in 1947, and patients with Zika virus are often asymptomatic or have mild symptoms such as fever, rash and muscle pain. However, Zika virus became a global health crisis during the 2015-2016 epidemic in the Americas, which caused severe fetal neurological malformations and fetal death during pregnancy. Membrane and envelope protein ( prM-E ) is a common antigenic choice for mRNA vaccines against Zika virus, as neutralizing antibodies against prM-E prevent viral fusion.
In collaboration with Washington University School of Medicine, Moderna has developed an improved prM-E mRNA that contains a mutated fusion loop epitope in the E protein. Two 10 µg doses of mRNA protected mice from Zika virus challenge and reduced dengue-enhancing antibody production. These encouraging preclinical results prompted a Phase I trial, with interim results showing that vaccine mRNA-1893 induced 94-100% seroconversion within 10 days and was well tolerated.
Additionally, another study used a passive immunization approach to deliver mRNA encoding ZIKV neutralizing antibodies using squalene-based nanocarriers. This is an attractive approach for immunocompromised patients whose immune systems are compromised and unable to synthesize autoantibodies.
Globally, HIV currently affects 38 million people and is expected to affect as many as 42 million people by 2030. Despite 30 years of research, no effective vaccine has yet been developed, largely because of the remarkable antigenic diversity of HIV envelope proteins and the ‘glycan barrier’ that hides key envelope protein epitopes.
Several preclinical studies have used a variety of carriers, including cationic nanoemulsions, DOTAP/DOPE liposomes, polymers, and ionizable LNPs, which have seen some effect to varying degrees. These studies suggest that, in addition to effective carriers, new antigens are critical for effectively targeting HIV.
Respiratory syncytial virus vaccine
Respiratory syncytial virus is the leading cause of acute lower respiratory tract infections worldwide. Every year, an estimated 60,000 children under the age of 5 die, and more than 14,000 people over the age of 65 die.
Current RSV vaccine candidates primarily target the highly conserved F protein. Although some candidates failed clinical trials due to insufficient neutralizing antibody titers, new findings on the F protein conformation suggest that vaccination against the prefusion conformation produces superior neutralizing antibody responses.
The discovery promises to improve future vaccine designs.
Moderna is evaluating three single-dose vaccine candidates encoding the prefusion F protein: mRNA-1172 and mRNA-1777 for adults, and mRNA-1345 for children.
In a phase I clinical trial, mRNA-1777 induced a strong humoral response to RSV neutralizing antibodies and CD4+ T cell responses to RSV F protein without serious adverse events.
The sequence of mRNA-1345 has been further designed and codon optimized to enhance translation and immunogenicity relative to mRNA-1777.
One month after vaccination, mRNA-1345 produced approximately eight-fold higher titers of neutralizing antibodies than mRNA-1777.
Ultimately, Moderna aims to integrate mRNA-1345 with its pediatric human metapneumovirus/parainfluenza virus type 3 ( hMPV/PIV3 ) vaccine candidate, mRNA-1653, and vaccinate children against three different pathogens in a single formulation.
Ebola virus vaccine
Ebola virus ( EBOV ) was first identified as the causative agent of the Ebola outbreak in 1976. The viral haemorrhagic fever claimed more than 11,000 lives in the 2014-2016 Ebola outbreak in West Africa. In 2019, the FDA approved a recombinant vesicular stomatitis virus ( VSV )-based Ebola vaccine ( rVSV-EBOV ). Although rVSV-EBOV was 97.5% effective in preventing Ebola transmission compared with no vaccination, clinical trials noted some safety concerns ( eg, acute arthritis and rash ).
mRNA vaccines against EBOV may be safer than this virus-based vaccine because they do not replicate in the body. An mRNA vaccine encoding the EBOV glycoprotein has demonstrated efficacy in mice. The vaccine protects animals from lethal viruses by inducing strong expression of glycoprotein-specific IgG antibodies and IFN-γ and IL-2 by CD8+ and CD4+ T cells.
Rabies is a zoonotic disease characterized by neurological symptoms with a mortality rate of nearly 100%. More than 50,000 people die each year from rabies despite the vaccines being approved, underscoring the need for more effective and cheaper vaccines.
To meet this need, CureVac utilized its RNActive platform to screen for CV7201, an unmodified mRNA vaccine encoding the rabies virus glycoprotein. In a preclinical study, CV7201 induced high neutralizing antibody titers in mice and pigs and elicited antigen-specific CD4+ and CD8+ T cell responses.
However, in a phase I clinical trial, it was found that although the route of administration did not affect the immune response, the drug delivery device only produced a transient humoral immune response with an intradermal syringe.
This weak drug delivery effect and high incidence of adverse events suggest the need for further optimization of the drug delivery platform.
Subsequently, CureVac used a proprietary LNP produced by Acuitas Therapeutics as a carrier for its new vaccine, CV7202. In a preclinical study, CV7202 induced strong antibody titers and CD8+ and CD4+ T cell responses. Phase I clinical trial results showed that two 1µg doses produced high titers of neutralizing antibodies and strong adaptive immune responses, which were well tolerated.
While the vast majority of mRNA vaccines in development are aimed at preventing viral infections, there are efforts to prevent other infectious diseases. Malaria, caused by unicellular eukaryotic parasites, ranks first in morbidity and lethality.
Each year, malaria afflicts more than 200 million people worldwide and takes the lives of more than 400,000 patients.
The production of antimalarial vaccines has been difficult due to the lack of surface antigens and the complex life cycle of the malaria parasite.
Fortunately, studies of the body’s natural immune response to Plasmodium infection have identified potential non-surface antigen targets.
For example, the Plasmodium-secreted cytokine macrophage migration inhibitory factor ( PMIF ) has been shown to prevent T cells from producing long-term memory. Based on this finding, a vaccine was prepared from a squalene-based cationic nanoemulsion loaded with self-amplifying mRNA encoding PMIF. Two 15 µg doses improved helper T cell development and elicited anti-Plasmodium IgG antibodies and memory T cell responses.
Another mechanistic study of malaria infection identified Plasmodium falciparum glutamate-rich protein ( PfGARP ) as a potential mRNA vaccine target. A nucleoside-modified mRNA vaccine encoding the PfGARP antigen is in development using Acuitas Therapeutics’ proprietary LNP. Preclinical studies have shown that the vaccine can reduce the response of animals to Plasmodium infection.
Key Issues with mRNA Vaccines
At present, there are still many doubts about the safety of mRNA vaccines. In addition, mRNA vaccines have only been approved for the prevention of SARS-CoV-2, and their efficacy in other aspects remains to be further studied. There are many problems that need to be solved. Let’s discuss some key issues that mRNA vaccines may face in the future.
Duration of antigenic response
Following vaccination, antigens are taken up by antigen-presenting cells and transported to lymph nodes, where interactions between B cells, antigen-presenting cells, and follicular helper T cells ( Tfh ) promote the formation of germinal centers. Within the germinal center, B cells then proliferate, differentiate and mutate their antibody genes to produce high-affinity neutralizing antibodies against the attacking antigen. Germinal center responses and Tfh cell induction are critical for durable antibody responses that will protect patients for months or years.
To enhance this first step in the immune response process, some delivery systems target antigen-presenting cells and translate mRNA. Several promising strategies to actively target antigen-presenting cells include binding monoclonal antibodies to the LNP surface and modifying the LNP surface with dendritic cell-specific ligands. Alternatively, modulating the physical properties of LNPs, such as surface charge, has been used to improve cancer vaccine efficacy.
Furthermore, altering vaccine pharmacokinetics by prolonging translation of antigenic mRNAs has emerged as an exciting tool for enhancing antibody responses. Sustained antigen availability during germinal center responses has been shown to increase antibody production by approximately 10-fold. A study in mice showed that nucleoside-modified mRNAs circulated longer and induced stronger Tfh cell and germinal center B cell responses than unmodified mRNAs.
In clinical trials, two doses of mRNA-1273 also elicited durable antibody responses over six months. Although antibody titers declined slightly over the study period, neutralizing capacity remained high in all age groups. These results are promising, however, the duration of antibody responses is a complex phenomenon that varies across antigens and requires longer-term data to fully understand.
Mutations against viruses
Mutations in viral genomes are common during replication. While most mutations have little or no effect on virus function, some can enhance immune evasion and hinder vaccine development. For example, the rapid mutation of HIV has hindered the development of an effective vaccine for more than 30 years, while the mutation of the influenza virus requires annual revisions of vaccine formulations to target the dominant strain.
Emerging SARS-CoV-2 variants have also raised concerns about the efficacy of mRNA vaccine cross-mutation. The B.1.351 and P.1 variants have a glutamate ( E ) to lysine ( K ) mutation at position 484 ( E484K ) of the spike protein receptor-binding domain , which promotes immune evasion.
Fortunately, the FDA-approved mRNA vaccines BNT162b2 and mRNA-1273 produced cross-neutralizing antibodies against B.1.351 and P.1, among other variants, suggesting that they could provide protection against them.
However, the cross-neutralization effect has been significantly reduced compared to the original virus. In addition, in a phase IIb/III trial of CureVac’s candidate CVnCoV, 57% of the 124 COVID-19 cases sequenced were mutants, including B.1.351 and P.1 variants.
If these variant strains become dominant over time, variant-specific mRNA enhancers may be required. Currently, Moderna is evaluating the original mRNA-1273 vaccine and the latest version of the vaccine as a third dose booster: mRNA-1273.351, which encodes a spike protein from the B.1.351 variant, and mRNA-1273.211, an mRNA-1273 1:1 mixed vaccine with mRNA-1273.351.
Pan-coronavirus vaccines that provide protection against SARS-CoV-2 and future coronavirus outbreaks will be more beneficial in the long run. As with HIV and influenza, new structural insights are expected to facilitate the discovery of conserved sites in coronaviruses, accelerating antigen discovery and vaccine design.
Overall, the mRNA vaccine has a favorable safety profile, with only mild or moderate adverse events occurring in clinical trials. However, there are individual events that require further optimization of mRNA antigen and delivery vehicle components.
For example, CureVac’s protamine-based rabies candidate CV7201 caused serious adverse events in 78% of participants, prompting CureVac to adopt LNPs as the delivery platform of choice for its follow-on rabies candidate CV7202.
As with most drugs, adverse effects of mRNA vaccines tend to increase with dose. For example, in a phase 1 trial of CV7202, the 5 μg dose had unacceptable toxicity, while 1 μg was the highest dose that was well tolerated.
In addition, in the Phase 1 trial of the Moderna H10N8 influenza vaccine, serious adverse events occurred in patients at the 400ug dose, so the trial continued with the 100ug dose.
Allergic reactions were observed in approximately 4.7 and 2.5 parts per million vaccinated with Pfizer–BioNTech and Moderna’s COVID-19 vaccines, respectively, which is approximately 2-4 times higher than with conventional vaccination.
One theory is that allergic reactions are due to pre-existing antibodies in the population against PEGylated lipids in LNPs. These antibodies are thought to react to PEG present in many consumer products such as toothpaste, shampoos, and laxatives .
Anti-PEG antibodies have been reported in 40% of the population, which may increase the risk of allergic reactions in some individuals and hinder the efficacy of vaccines.
Currently, the CDC recommends that individuals with a history of hypersensitivity to any component of the Pfizer–BioNTech or Moderna vaccines should not use the mRNA vaccine.
Clearly, we need a better understanding of how mRNA vaccine formulations cause allergic reactions so that formulations can be redesigned to improve safety.
Vaccinations for specific groups of people
Most vaccines, whether conventional or mRNA, are developed for children or healthy adults. However, some populations may benefit from alternative vaccination strategies or respond differently to vaccination due to differences in immune systems.
The dynamic nature of the immune system during pregnancy increases a person’s susceptibility to infectious diseases, which can have catastrophic effects on maternal health and fetal development.
To address these challenges, maternal vaccination has emerged as a tool to improve maternal health and reduce neonatal morbidity.
Maternal IgG antibodies can easily cross the placenta and enter the fetal circulation by binding to the neonatal Fc receptor, protecting the fetus from pathogens.
In several studies, maternal vaccination with mRNA-loaded LNPs prevented fetal Zika virus transmission in pregnant mice and protected neonatal mice from herpes and streptococci.
Although vertically transferred maternal antibodies protect against neonatal infection, they also hinder the effects of vaccination on infants later in life, the mechanism by which is unclear. Prolonged antigen availability may promote a more robust germinal center response, resulting in a robust infant immune response in the presence of maternal antibodies.
An mRNA vaccine against SARS-CoV-2 has also been shown to be immunogenic in pregnant and lactating populations, and neutralizing antibodies have been detected in cord blood and human milk.
Preliminary data suggest that mRNA-1273 and BNT162b2 elicit similar adverse events in pregnant and non-pregnant populations and that the vaccine does not increase the incidence of neonatal death or congenital anomalies.
However, further longitudinal studies are needed to assess the impact of mRNA vaccines on maternal and neonatal health.
Effective vaccines are urgently needed for this group, as many infectious diseases affect older adults. For example, 70 to 90 percent of flu-related mortality occurs in patients over 65 years of age, while the death rate from COVID-19 is 62 times higher in patients over 65 than in younger patients.
Older adults are more difficult to vaccinate because aging adversely affects both innate and adaptive responses of the immune system. Reduced expression of Toll-like receptors prevents monocytes and macrophages from secreting cytokines and chemokines and limits crosstalk with the adaptive immune system.
Adaptive immune responses during infection are often inadequate due to impaired cytokine signaling and physiological and cellular changes.
These changes include thymus involution, reduction of naive B and T cells, reduced diversity of T cell receptors, higher susceptibility to T cell apoptosis, and reduced expression of key receptors such as CD28 on cytotoxic CD8+ T cells.
Fortunately, there is growing evidence that mRNA vaccines may have robust efficacy across all age groups.
For example, in a phase III trial, Pfizer–BioNTech’s vaccine candidate BNT162b2 was more than 93% effective across all treatment arms.
Moderna’s vaccine candidate, mRNA-1273, was also highly effective, showing 86.4% efficacy in volunteers ≥65 years old, compared to 95.6% in volunteers 18-65 years old.
The design of the delivery vehicle is important for improving vaccine efficacy in older adults. mRNA vectors can act as inflammatory adjuvants, amplifying vaccine responses by enhancing the recruitment of antigen-presenting cells to the injection site.
In a preclinical study, CureVac’s RNAVAC activated TLR7 and produced durable immune responses against lethal influenza in mice.
Novartis’ emulsion MF59 has been used as an mRNA delivery vehicle and also as an adjuvant. MF59 enhances the immunogenicity of the influenza vaccine and has been approved for use in the elderly.
Access to vaccines
Access to vaccines is the greatest challenge in achieving widespread prevention of infectious diseases, especially in low-income countries. Cold storage requirements for currently approved SARS-CoV-2 mRNA vaccines further limit vaccine access. Portable and reusable Arktek freezers enable rapid deployment of millions of doses of vaccine during pandemics.
However, the COVID-19 virus needs to inoculate billions of people, which requires a heat-resistant vaccine. Currently, there are two SARS-CoV-2 vaccine candidates that are heat-resistant at room temperature, and if these heat-resistant vaccine candidates can show good results in clinical trials, they may simplify global access to mRNA vaccines in the near future .
Vaccines are only effective after they have been given, and the data supporting their safety and efficacy is abundant, and vaccines have eradicated several infectious diseases in parts of the world, saving countless lives. However, due to misinformation and the anti-vaccine movement, public distrust has grown, threatening the maintenance of herd immunity and putting our most vulnerable populations at risk.
Declining vaccination coverage could lead to a resurgence of life-threatening diseases. For example, measles, which was eradicated from the United States in 2000, infected more than 1,200 people in 2019 due to poor vaccine adherence in multiple communities. For COVID-19, the current 56–75% acceptance may not be sufficient to achieve at least 80–90% coverage in the United States, a threshold considered necessary for herd immunity against SARS-CoV-2.
While much of the burden of increasing vaccine coverage falls on governments and public health officials, the scientific community can also help by improving the efficacy and safety of mRNA vaccines. Improving efficacy would reduce the acceptance needed for herd immunity, and improving safety would deter media coverage of adverse events, thereby reducing fear of vaccination.
Over the decades, advances in mRNA design and nucleic acid delivery technology, coupled with the discovery of neoantigen targets, have made mRNA vaccines an extraordinary tool for combating emerging and existing infectious diseases. Two mRNA vaccines against SARS-CoV-2, developed at revolutionary speed and offering superior protection rates, promise to end the COVID-19 pandemic.
In addition, these vaccines elevate LNP and RNA treatments from niche products to prophylactic treatments that are successfully implemented in large populations. The result is a wealth of safety and efficacy data, as well as successful regulatory approvals. We can be optimistic that mRNA therapy will have the potential to transform modern medicine in terms of vaccination, cancer immunotherapy, and protein replacement therapy.
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2. Recent advances in mRNA vaccine technology. Curr Opin Immunol. 2020 Aug;65:14-20.
Technological Innovation and Research Progress of mRNA Vaccines
(source:internet, reference only)