Source | Xiao Yao Shuo Yao
vaccine
It is the most effective public health intervention measure to prevent the spread of infectious diseases. Successful vaccination has eradicated many life-threatening diseases, such as smallpox and polio. The World Health Organization estimates that vaccines can prevent 2-3 million people from dying of tetanus, whooping cough, influenza and measles every year. However, despite the obvious success of conventional vaccines, they can not effectively deal with pathogens such as plasmodium, hepatitis C and HIV that escape immune surveillance. In addition, they need to be modified regularly to cope with rapidly changing pathogens, such as influenza virus.
on the basis ofmRNAOur nucleic acid vaccine was put forward more than 30 years ago, hoping to produce a safe, multifunctional and easy-to-produce vaccine. Compared with traditional vaccines, mRNA vaccines have many advantages: Unlike some viral vaccines, mRNA will not be integrated into the genome, thus avoiding the concern about insertion mutation; MRNA vaccine can be made in a cell-free way, so as to realize rapid, economical and efficient production. In addition, a single mRNA vaccine can encode multiple antigens, enhance the immune response against adaptive pathogens, and can target multiple microorganisms or virus variants with a single formula.
At first, mRNA therapy was not paid attention to because of concerns about its stability, inefficiency and excessive immune stimulation. However, in the past decade, the field of mRNA therapy has changed with each passing day, including the in-depth study of mRNA pharmacology, the development of effective vectors and the control of mRNA immunogenicity, which has made the clinical application of mRNA vaccine enter a brand-new stage.
Design and synthesis of mRNA
In vitro transcription (
IVT
) mRNA mimics the structure of endogenous mRNA and has five parts, from 5 to 3, including: 5?cap, 5 untranslated regions (
UTR
), an open reading frame encoding antigen, 3?UTR and a PolyA tail.
Like natural eukaryotic mRNAs, the 5′ end cap structure contains a 7- methylguanosine nucleoside, which will protect mRNA from being degraded by nucleic acid exonuclease in space, and cooperate with translation initiation factor protein to recruit ribosomes to start translation. The length of PolyA tail indirectly regulates mRNA translation and half-life.
5 ′ and 3 ′ UTR on both sides of the coding region regulate mRNA translation, half-life and subcellular localization, and are derived from highly expressed genes (
Such as alpha-and beta-globin genes.
) is the first choice for synthesizing mRNAs. In addition, UTR can be optimized according to the cell type, for example, by removing miRNA binding sites and AU-rich regions in 3 ′ UTR, the degradation of mRNA can be minimized.
The open reading frame of mRNA vaccine is the most critical component. Although the open reading frame is not as plastic as the non-coding region, the translation can be increased without changing the protein sequence by optimizing the codon. For example, CureVac AG found that human mRNA codons rarely have A or U in the third position, thus replacing A or U in the third position of the open reading frame with G or C.. This optimization strategy is applied to its SARS-CoV-2 vaccine CVnCoV, which is currently in the phase III trial stage.
In order to maximize translation, mRNA sequences usually contain modified nucleosides, such as pseudouridine, N1- methyl pseudouridine or other nucleoside analogues. The use of modified nucleosides, especially modified uridine, prevents the recognition of pattern recognition receptors and ensures that the translation process produces a sufficient level of protein. Both Moderna and Pfizer–Biontech SARS-CoV-2 vaccines contain nucleoside modifications. Another strategy to avoid pattern recognition receptor detection, pioneered by CureVac, uses sequence engineering and codon optimization to consume uridine by increasing GC content of vaccine mRNA.
Besides the improvement of mRNA sequence, great progress has been made in simplifying mRNA production. The synthetic mRNA used in clinic is synthesized by using phage RNA polymerase T7 (
T3 and SP6 polymerases can also be used.
) transcribed from DNA plasmid in vitro. In addition, the co-transcription capping technology of CleanCap is used to simplify the purification steps.
Vector of mRNA vaccine
Due to the large mRNA (
10four–10sixDa
) and is negatively charged, so it cannot pass through the anionic lipid bilayer of the cell membrane. In addition, in vivo, it will be swallowed by cells of innate immune system and degraded by nuclease. Therefore, in vivo application requires the use of mRNA delivery vectors, which transfect immune cells without causing toxicity or unnecessary immunogenicity. At present, many solutions based on innovative materials have been developed.
Lipid nanoparticles (LNP)
Lipid nanoparticles are the most advanced mRNA carriers in clinic. As of June 2021, all SARS-CoV-2 mRNA vaccines being developed or approved for clinical use have adopted LNPs. LNP provides many benefits for mRNA delivery, including simple preparation, modularization, biocompatibility and large mRNA payload capacity. In addition to RNA drugs, LNP usually consists of four components: ionizable lipid, cholesterol, accessory phospholipid and polyethylene glycol lipid, which together encapsulate and protect fragile mRNA.
Ionizable lipid and mRNA form nanoparticles in acidic buffer, which makes lipid positively charged and attracts RNA. In addition, they are positively charged in the acidic environment of endosome, which promotes their fusion with endosome membrane and releases them into cytoplasm.
DODAP and DODMA are the first ionizable lipids for RNA delivery. Through the design to improve the efficiency of DODMA, DLin-MC3-DMA was produced. This is the first ionizing lipid used in FDA-approved pharmaceutical preparations: siRNA drug patisiran (
Onpattro
）。 In addition to the effective and safe delivery of siRNA, DLin-MC3-DMA is also used for the delivery of mRNA.
At present, many groups in academia and industry use combinatorial reaction schemes to synthesize potential delivery materials. This method produces many effective lipids, including C12-200, 503O13, 306Oi10, OF-02, TT3, 5A2-SC8, SM-102 (
Moderna vaccine mRNA-1273 for anti-SARS-CoV-2
) and ALC-0315 (
BNT162b2 for Pfizer vaccine
）。
In addition to seeking to improve the curative effect, people pay more and more attention to improving the specificity of drugs, especially for vaccines and immunotherapy. Lipid 11-A-M58 containing polycyclic adamantane tail and lipid 93-O17S59 containing cyclic imidazole head have been designed to target T cells in vivo. Although the mechanism is not clear, the cyclic groups of these lipids are essential for targeting T cells.
Although ionized lipid can be said to be the most important component of LNP, the other three lipid components (
Cholesterol, auxiliary lipids and polyglycolized lipids
) also promoted the formation and function of nanoparticles. Cholesterol is a naturally occurring lipid, which enhances the stability of nanoparticles by filling the gaps between lipids, and helps to fuse with endosomal membranes during uptake into cells.
Auxiliary lipid can adjust the fluidity of nanoparticles and enhance the efficacy by promoting the lipid phase transition which is helpful to the fusion of membrane and inclusion body. The choice of the best auxiliary lipid depends on the ionizable lipid material and RNA vector. For example, for lipid-like materials, saturated auxiliary lipids (
Such as DSPC
) is most suitable for delivering short RNA (
Such as siRNA
), while unsaturated lipids (
Such as DOPE
) is most suitable for delivering mRNA. DSPC has been used in the SARS-CoV-2 vaccines mRNA-1273 and BNT162b2 approved by FDA.
The PEGylated lipid component of LNPs consists of polyethylene glycol (
PEG
) and anchored lipids (
Such as DMPE or DMG
) combined. Hydrophilic PEG can stabilize LNP, adjust the size of nanoparticles by limiting lipid fusion, and increase the half-life of nanoparticles by reducing nonspecific interaction with macrophages. Both mRNA-1273 and BNT162b2 SARS-CoV-2 vaccines contain PEGylated lipids.
Polymers and polymer nanoparticles
Although the clinical progress is not as good as LNP, polymers have similar advantages to lipids and can effectively deliver mRNA. Cationic polymers concentrate nucleic acids into complexes with different shapes and sizes, which can enter cells through endocytosis.
Polyethylenimine is the most widely studied nucleic acid delivery polymer. Although its efficacy is excellent, its toxicity limits its application because of its high charge density. In addition, several biodegradable polymers with less toxicity have been developed. For example, poly (
β-amino ester
) is excellent in mRNA transmission, especially for lung.
Recently, a new type of lipid-containing polymer, called charge-changing releasable transporter (
CARTs
), it can effectively target T cells, and it is very difficult to manipulate T cells. Therefore, CART is an attractive delivery material with great potential in the fields of mRNA vaccine and gene therapy.
Other delivery systems
In addition to lipids and polymer carriers, peptides can also deliver mRNA to cells, thanks to cations or amphiphilic amine groups in their main and side chains (
Such as arginine
), these cations or amphiphilic amino groups electrostatically combine with mRNA and form nanocomposites. For example, amino acids containing repeated arginine-alanine-leucine-alanine (
RALA
) motif of membrane fusion cell penetrating peptide.
Protamine peptide rich in arginine is positively charged at neutral pH, which can also concentrate mRNA and promote its transmission. Protamine-mRNA complexes activate Toll-like receptors that recognize single-stranded mRNA (
TLR7，TLR8
) pathway, therefore, it can also be used as an adjuvant for vaccine or immunotherapy applications. CureVac AG is evaluating a protamine-containing delivery platform RNActive for clinical trials of melanoma, prostate cancer and non-small cell lung cancer.
Finally, cationic nanoemulsions based on squalene can also deliver mRNA, and these nanoemulsions are composed of oily squalene cores. Some squalene preparations, such as Novartis MF59, are used as adjuvants in FDA-approved influenza vaccines. MF59 makes the cells at the injection site secrete chemokines, thereby recruiting antigen presenting cells, inducing monocytes to differentiate into dendritic cells, and enhancing antigen uptake by antigen presenting cells. The mechanism of squalene-based cationic nanoemulsion escaping from endosome and transporting mRNA to cytoplasm is not clear.
Technical innovation of mRNA vaccine
The most important innovations of mRNA vaccine technology are: mRNA sequence design; Develop a simple, rapid and large-scale method to produce mRNA; To develop efficient and safe mRNA vaccine delivery materials.
A recent study used a systematic selection process based on cell culture to identify new UTRs, which significantly increased the protein expression of IVT mRNA. Compared with human β -globin 3’UTR, these sequences can induce the protein of related transcripts to be about 3 times.
In addition, an interesting new vaccine form was recently reported, which used mRNA encoding RNA-dependent RNA polymerase of α virus plus a second mRNA encoding antigen to make it replicate in cytoplasm. This system can be used at very low doses (
50ng
) can effectively induce protective immune response in mice. These findings are particularly attractive because the use of low doses reduces the cost of vaccine production. No delivery material further reduces the cost, simplifies the manufacture, and improves the possibility of freeze-drying and storage of vaccines at ambient temperature.
On the production side, in addition to the cleancap technology, a method for adsorbing double-stranded RNA pollutants to cellulose (
A cheap and abundant polysaccharide
) to purify mRNA. It has been proved that this highly scalable and cheap method is as effective as high performance liquid chromatography in removing dsRNA pollutants from IVT mRNA samples.
In terms of delivery materials, mRNA-LNPs also achieves selective T cell targeting, similar to CAR-T platform based on polymer. A new platform called ASSET (
Anchored secondary targeting single chain antibody
), in which T cell-specific monoclonal antibodies are linked to LNP to target T cells. This flexible platform also has great potential in mRNA vaccine and other applications.
Another lipid complex preferentially targets dendritic cells after systemic administration. It is a potential key discovery to selectively target DC with mRNA vaccine to induce strong immune response, and the platform has proved its prospect in clinical trials.
Research progress of mRNA vaccine in infectious diseases
MRNA-based therapy represents a relatively novel and efficient drug class. Recently, several published studies have emphasized the potential efficacy of mRNA vaccine in the treatment of different types of malignant tumors and infectious diseases, in which traditional vaccine strategies cannot cause protective immune response.
By the end of 2019, 15 candidate mRNA vaccines against infectious diseases had entered clinical trials. At that time, it was thought that it would take at least 5-6 years for mRNA vaccines to be approved by the regulatory authorities. However, when COVID-19 pandemic swept the world in early 2020, these expectations were subverted. In the next few months, the research, development, manufacture and deployment of mRNA vaccine have entered a stage of rapid leap.
SARS-CoV-2 vaccine
Most candidate vaccines of SARS-CoV-2 have an immune response to spinous proteins on the virus surface. Spike protein binds to the receptor angiotensin converting enzyme 2 on the surface of its host cell. Then, the cell’s transmembrane serine protease 2 cleaves the attached spike protein, which induces conformational changes, exposes the fusion peptide of spike protein and promotes the fusion with the cell or endosomal membrane. Usually, the antigen encoded by vaccine mRNA is either the full-length spike protein or the receptor binding domain of spike protein.
As of June 18th, 2021, 185 CVID-19 vaccine candidates are in the pre-clinical development stage, and another 102 have entered clinical trials. In clinical trials, 19 kinds are mRNA vaccines. On December 11th, 2020, Pfizer’s BNT162b2 vaccine obtained the emergency authorization from FDA, becoming the first mRNA drug approved for human body. A week later, the Moderna vaccine mRNA-1273 was also authorized for use in the United States. In the end, they were the first SARS-CoV-2 vaccines authorized in the United States, Britain, Canada and several other countries.
Pfizer and BioNTech jointly developed five mRNA candidate vaccines, which encode variants of spike protein antigen. Two main candidate drugs, BNT162b1 and BNT162b2, use ionized lipid ALC-0315 of Acuitas Therapeutics and nucleoside modified mRNA, in which all uridine is replaced by N1 methyl pseudouridine to enhance mRNA translation. BNT162b1 encodes the receptor binding domain of trimeric secretory spinous protein, while BNT162b2 encodes the full-length SARS-CoV-2 spinous protein, and two proline substitutions in S2 subunit lock the protein in the pre-fusion conformation.
In the first trial of the two vaccines, both of them can induce neutralizing antibodies with high titers, and produce strong CD4+ and CD8+ reactions, accompanied by mild to moderate adverse reactions. The two candidate vaccines were well tolerated and effective, but only BNT162b2 vaccine entered the phase II/III trial because of its mild systemic and local adverse reactions. In the third phase of the trial, BNT162B2 showed 95% overall prevention and 90 ~ 100% curative effect.
Moderna cooperated with the National Institutes of Health to develop mRNA-1273. The vaccine uses ionized lipid SM-102 to prepare LNP, which encapsulates the mRNA modified by N1 methyl pseudouridine. This sequence encodes SARS-CoV-2 spinous protein, with two proline substitutions, giving it a pre-fusion conformation.
In phase 1 clinical trials, mRNA-1273 was very effective and well tolerated. In the phase III trial involving 30,420 volunteers, the prevention rate of two 100μg vaccines was 94.1%, and local pain at the injection site was the most common side effect. After the second dose, half of the volunteers reported moderate to severe systemic side effects (
Such as fatigue, muscle pain, joint pain
), these side effects disappeared within 48 hours.
Although the vaccines produced by Pfizer and Moderna have been proved to have good efficacy and safety, their demand for cold chain storage has brought great difficulties to guarantee. MRNA-1273 can be stored at 4-8℃ for one month, while BNT162b2 needs to be stored at -60℃.
CVnCoV, the candidate vaccine of CureVac, can be stored stably for 3 months at 5℃. CVnCoV uses Acuitas Therapeutics (
It may be ALC-0315
) and unmodified mRNA encoding full-length spike protein with two proline substitutes. In the first phase of clinical trial, the neutralizing antibody produced by volunteers was similar to that of patients with CVID-19 in recovery period and was well tolerated. Unfortunately, however, in phase III clinical trials involving 40,000 people, CVnCoV only showed 47% efficacy. Mid-term analysis shows that the low curative effect of CVnCoV is attributed to the new SARS-CoV-2 mutation.
At present, CureVac is cooperating with GSK to develop the second generation candidate drug-C2CoV, which has been optimized to enhance its translation and immunogenicity compared with CVnCoV. CV2CoV uses 5’UTR from human hydroxysteroid 17-β- dehydrogenase 4 gene and 3’UTR from human proteasome 20S subunit β3 gene. In preclinical studies, the protein expression of CV2CoV was 1.8 times higher than that of CVnCoV in vitro, and high-titer cross-neutralizing antibodies against B.1.1.7, B.1.1.298 and B.1.351 variants were induced in rats.
Another heat-resistant candidate vaccine, ARCoV, was developed by PLA Academy of Military Sciences in cooperation with Walvax Biotechnology Company, and can be stable at 25℃ for one week. ARCoV encodes the receptor binding domain of spike protein. In preclinical studies, high SARS-CoV-2 specific IgG antibody and strong virus neutralization titer can be induced in cynomolgus monkeys. Although the reasons behind the thermal stability of CVnCoV and ARCoV are not clear, the secondary structure of mRNA, smaller mRNA size, GC content and lipid may be important factors.
Other mRNA candidate vaccines are also under development. LNP-nCoVsaRNA was jointly developed by Imperial College London in London and Acuitas Therapeutics, encoding the full-length spike protein. At present, the phase I clinical trial evaluation is being carried out using the dose escalation scheme of 0.1–1g (
ISRCTN17072692
), which uses the lowest RNA dose among all candidate mRNA vaccines.
Another self-amplified mRNA vaccine candidate ARCT-021 (
Also known as LUNAR-COV19
) by Arcturus company using its proprietary LUNAR lipid carrier and self-transcription and replication RNA (
STARR
) platform development. It encodes a full-length fusion pre-spike protein.
Influenza virus vaccine
About 290,000 to 650,000 people worldwide die from influenza virus every year. At present, the vaccine targets the viral hemagglutinin protein that promotes the entry of the virus. The traditional influenza vaccine is an inactivated influenza virus growing in eggs, which takes a long time to produce and is difficult to purify. In addition, viruses mutate in eggs for optimal growth, sometimes rendering them ineffective in humans.
Therefore, alternative antigen targets and production methods are really needed. Synthetic mRNAs transcribed in vitro can meet this demand and ensure the rapid production of vaccines when new influenza strains appear. For example, in 2013, based on LNP (
DLinDMA
) was successfully developed within 8 days after the outbreak of H7N9 in China, but unfortunately, clinical trials could not be conducted because there was no GMP facility for mRNA manufacturing at that time.
Some people are also committed to developing a universal influenza vaccine that does not need to be revised every year. This vaccine can produce immunity to several influenza virus strains and subtypes. The influenza mRNA vaccine, which was first displayed in 2012, induced homologous and heterologous immunity against H1N1 and H5N1 strains in mice by three intradermal injections.
It is worth noting that the conserved region of hemagglutinin, which is not easy to mutate, has recently become a new universal vaccine target. Another study used LNPs to deliver 50ng dose of mRNA, encoding three conserved influenza proteins: neuraminidase, nucleoprotein and matrix -2 ion channel protein, and hemagglutinin stem region. Incredibly, this tiny dose of mRNA produced a wide range of protective antibodies.
Zika virus vaccine
Zika virus infection was first discovered in 1947. Patients infected with Zika virus usually have no symptoms or mild symptoms such as fever, rash and muscle pain. However, Zika virus became a global health crisis during the American epidemic in 2015-2016, which caused severe fetal neural malformation and fetal death during pregnancy. Membrane and envelope protein (
prM-E
) is a common antigen selection for mRNA vaccine against Zika virus, because neutralizing antibody against prM-E can prevent virus fusion.
Moderna cooperated with Washington University School of Medicine to develop an improved prM-E mRNA, which contains the mutant fusion circular epitope in E protein. Two 10μg doses of mRNA can protect mice from Zika virus attack and reduce the production of dengue enhanced antibodies. These encouraging preclinical results promoted the phase I trial, and the interim results showed that the vaccine mRNA-1893 induced 94-100% seroconversion within 10 days, which was well tolerated.
In addition, another study used passive immunization method to deliver mRNA encoding ZIKV neutralizing antibody using squalene-based nanocarriers. This is an attractive method for immunocompromised patients whose immune system is damaged and unable to synthesize autoantibodies.
HIV vaccine
Globally, HIV currently affects 38 million people and is expected to affect as many as 42 million people by 2030. Although the research has been carried out for 30 years, no effective vaccine has been developed, mainly because of the significant antigenic diversity of HIV envelope proteins and the hidden "glycan barrier" of key envelope protein epitopes. Several preclinical studies have used a variety of carriers, including cationic nanoemulsion, DOTAP/DOPE liposome, Polymers and ionizable LNP, and they have seen some effects in different degrees. These studies show that in addition to effective vectors, new antigens are crucial for effectively targeting HIV.
Respiratory syncytial virus vaccine
Respiratory syncytial virus is the main cause of acute lower respiratory tract infection in the world. Every year, it is estimated that 60,000 children under the age of 5 die and more than 14,000 people over the age of 65 die.
At present, RSV candidate vaccines mainly target highly conserved F protein. Although some candidates failed to pass the clinical trial because of insufficient neutralizing antibody titer, the new discovery of F protein conformation shows that vaccination against pre-fusion conformation can produce excellent neutralizing antibody reaction. This discovery is expected to improve the future vaccine design.
Moderna is evaluating three single-dose candidate vaccines encoding pre-fusion F protein: mRNA-1172 and mRNA-1777 for adults, and mRNA-1345 for children. In the phase I clinical trial, mRNA-1777 induced a strong humoral response of RSV neutralizing antibody, and CD4+T cells reacted to RSV F protein without serious adverse events. The sequence of mRNA-1345 has been further designed and codon optimized to enhance the translation and immunogenicity relative to mRNA-1777. One month after inoculation, the titer of neutralizing antibody produced by mRNA-1345 was about eight times that of mRNA-1777. Finally, the goal of Moderna is to combine mRNA-1345 with pediatric human metapneumovirus/parainfluenza virus type 3 (
hMPV/PIV3
) Candidate vaccine mRNA-1653 was integrated, and children were vaccinated against three different pathogens with a single formula.
Ebola virus vaccine
Ebola virus (
EBOV
) was first identified as the pathogen that caused the Ebola outbreak in 1976. This viral hemorrhagic fever claimed more than 11,000 lives in the Ebola epidemic in West Africa in 2014-2016. In 2019, FDA approved a recombinant vesicular stomatitis virus (
VSV
) Ebola vaccine (
rVSV-EBOV
）。 Although the effectiveness of rVSV-EBOV in preventing the spread of Ebola is 97.5% compared with that without vaccination, clinical trials have noticed some safety problems (
Such as acute arthritis and rash.
）。
Anti-EBOV mRNA vaccines may be safer than this virus-based vaccine because they will not replicate in vivo. An mRNA vaccine encoding EBOV glycoprotein has been proved to be effective in mice. The vaccine can induce the strong expression of glycoprotein-specific IgG antibody, IFN-γ and IL-2 through CD8+ and CD4+T cells, which can protect animals from deadly virus.
Rabies virus vaccine
Rabies is a zoonotic disease, characterized by neurological symptoms, with a mortality rate of nearly 100%. Although vaccines have been approved, more than 50,000 people still die of rabies every year, which highlights the need for more effective and cheaper vaccines.
In order to meet this demand, CureVac used its RNActive platform to screen the unmodified mRNA vaccine CV7201 encoding rabies virus glycoprotein. In a preclinical study, CV7201 can induce high neutralizing antibody titers in mice and pigs, and induce antigen-specific CD4+ and CD8+T cell responses. However, in the phase I clinical trial, it was found that although the route of administration did not affect the immune response, only the intradermal syringe produced a short-term humoral immune response. This weak drug delivery effect and high incidence of adverse events indicate that the drug delivery platform needs to be further optimized.
Subsequently, CureVac used the proprietary LNP produced by Acuitas Therapeutics as the carrier of its new vaccine CV7202. In a preclinical study, CV7202 induced strong antibody titers and CD8+ and CD4+T cell responses. The results of phase I clinical trials show that two 1g doses can produce high titer neutralizing antibodies and strong adaptive immune response, which is well tolerated.
Malaria vaccine
Although the vast majority of mRNA vaccines under development are aimed at preventing virus infection, there are also efforts to prevent other infectious diseases. Malaria is caused by unicellular eukaryotic parasites, and its incidence and lethality rank first. Every year, malaria afflicts more than 200 million people around the world and kills more than 400,000 patients. Due to the lack of surface antigen and the complex life cycle of plasmodium, the production of antimalarial vaccine has been difficult. Fortunately, the study of human natural immune response to plasmodium infection has identified potential non-surface antigen targets.
For example, the cytokine macrophage migration inhibitory factor secreted by plasmodium (
PMIF
) has been proved to prevent T cells from producing long-term memory. According to this discovery, a vaccine was prepared from squalene-based cationic nanoemulsion loaded with self-amplified mRNA encoding PMIF. Two doses of 15μg can improve the development of helper T cells and induce anti-plasmodium IgG antibody and memory T cell response.
Another study on the mechanism of malaria infection found that Plasmodium falciparum is rich in glutamate protein (
PfGARP
) is a potential target of mRNA vaccine. A nucleoside modified mRNA vaccine encoding PfGARP antigen is being developed, which uses LNP proprietary to Acuitas Therapeutics. Preclinical studies show that the vaccine can reduce the reaction of animals infected with plasmodium.
Key problems of mRNA vaccine
At present, there are still many doubts about the safety of mRNA vaccine. In addition, the mRNA vaccine has been approved only in the prevention of SARS-CoV-2, and its efficacy in other aspects needs further study. Many problems need to be solved. Let’s discuss some key problems that mRNA vaccine may face in the future.
Antigen reaction duration
After vaccination, antigen is absorbed by antigen presenting cells and transported to lymph nodes, where B cells, antigen presenting cells and follicular helper T cells (
Tfh
) promote the formation of germinal center. In the germinal center, B cells then proliferate, differentiate and mutate their antibody genes to produce high affinity neutralizing antibodies against aggressive antigens. Germinal center reaction and Tfh cell induction are very important for lasting antibody response, which will protect patients for months or years.
In order to enhance the first step of this immune response process, some delivery systems target antigen-presenting cells and translate mRNA. Several promising strategies for actively targeting antigen presenting cells include binding monoclonal antibodies to LNP surface and modifying LNP surface with dendritic cell-specific ligands. Alternatively, regulating the physical properties of LNP, such as surface charge, has been used to improve the efficacy of cancer vaccines.
In addition, changing vaccine pharmacokinetics by prolonging the translation of antigen mRNA has become an exciting tool to enhance antibody response. Continuous availability of antigen during germinal center reaction has been proved to increase antibody production by about 10 times. A study in mice showed that compared with unmodified mRNA, nucleoside-modified mRNA had a longer circulating time and induced a stronger reaction between Tfh cells and B cells in germinal center.
In clinical trials, two doses of mRNA-1273 can also trigger a lasting antibody response within 6 months. Although the antibody titer decreased slightly during the study period, all age groups maintained high neutralization ability. These results are promising, however, the duration of antibody reaction is a complex phenomenon, and different antigens will be different, which requires longer data to fully understand.
Mutations against viruses
Mutations in viral genomes are common in the process of replication. Although most mutations have little or no effect on the function of the virus, some mutations can enhance immune evasion and hinder the development of vaccines. For example, for more than 30 years, the rapid mutation of HIV has hindered the development of effective vaccines, while the mutation of influenza virus needs to modify the vaccine formula every year to target the dominant strains.
The emerging SARS-CoV-2 mutation has also attracted attention to the effectiveness of cross-mutation of mRNA vaccine. Variants B.1.351 and P.1 are located at position 484 of spinous protein receptor binding domain (
E484K
) glutamic acid exists (
E
) to lysine (
K
) mutation, which promotes immune escape. Fortunately, FDA-approved mRNA vaccines BNT162b2 and mRNA-1273 produced cross-neutralizing antibodies against B.1.351 and P.1 and other variants, which indicated that they could provide protection against them. However, compared with the original virus, the cross neutralization effect has been significantly reduced. In addition, in the 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 mutant strains gradually become major variants over time, variant-specific mRNA enhancers may be needed. At present, Moderna is evaluating the original mRNA-1273 vaccine and the latest version as the third booster: mRNA-1273.351, which encodes spinous protein from variant B.1.351, and mRNA-1273.211, a 1:1 mixed vaccine of mRNA-1273 and mRNA-1273.351.
In the long run, it will be more beneficial to provide a pan-coronavirus vaccine to protect SARS-CoV-2 and future coronavirus outbreaks. Like HIV and influenza, new structural insights are expected to promote the discovery of conserved sites of coronavirus and accelerate antigen discovery and vaccine design.
security
Generally speaking, mRNA vaccine has good safety, and only mild or moderate adverse events occurred in clinical trials. However, there are also individual events that require further optimization of mRNA antigens and vehicle components. For example, CureVac’s protamine-based rabies candidate drug CV7201 caused serious adverse events in 78% of the participants, prompting CureVac to adopt LNPs as the first choice for its subsequent rabies candidate drug CV7202.
Like most drugs, the adverse reactions of mRNA vaccine often increase with the increase of dose. For example, in the first phase test of CV7202, 5μg dose has unacceptable toxicity, while 1μg is the highest dose with good tolerance. In addition, in the first phase trial of Moderna H10N8 influenza vaccine, serious adverse events appeared in patients with a dose of 400μ g, so the trial continued with a dose of 100μg g..
When using the COVID-19 vaccines of Pfizer-BioNTech and Moderna, allergic reactions were observed in about 4.7 parts per million and 2.5 parts per million, respectively, which was about 2-4 times that of the traditional vaccination. One view is that allergic reactions are attributed to pre-existing antibodies against PEGylated lipids in LNP. These antibodies are considered to be effective against many consumer products (
Such as toothpaste, shampoo and laxatives.
) has a reaction with PEG. It is reported that 40% people have anti-PEG antibodies, which may increase the risk of allergic reactions in some individuals and hinder the effectiveness of vaccines. At present, CDC recommends that individuals who have a history of allergy to any component of Pfizer-BioNTech or Moderna vaccine should not use mRNA vaccine. Obviously, we need to better understand how the mRNA vaccine formula causes allergic reactions, so that we can redesign the formula to improve safety.
Vaccination of specific population
Most vaccines, whether traditional vaccines or mRNA vaccines, are developed for children or healthy adults. However, due to different immune systems, some people may benefit from alternative vaccination strategies or have different responses to vaccination.
Maternal/neonatal vaccination
The dynamic characteristics of the immune system during pregnancy increase a person’s susceptibility to infectious diseases, and infection will have a disastrous impact on maternal health and fetal development.
In order to meet these challenges, maternal vaccination has become a tool to improve maternal health and reduce neonatal morbidity. Maternal IgG antibody can easily cross the placenta and enter the fetal circulation by binding to the neonatal Fc receptor, thus protecting the fetus from pathogens. In several studies, maternal inoculation with mRNA-loaded LNPs can prevent the spread of fetal Zika virus in pregnant mice and protect newborn mice from herpes virus and streptococcus infection.
Although vertically transferred maternal antibodies can prevent neonatal infection, they will also hinder the effect of vaccination for infants in later life, and the mechanism is still unclear. Prolonging the availability of antigen may promote a stronger germinal center response, thus producing a strong infant immune response in the presence of maternal antibodies.
The anti-SARS-CoV-2 mRNA vaccine has also been proved to be immunogenic to pregnant and lactating people, and neutralizing antibodies have been detected in cord blood and human milk. Preliminary data show that mRNA-1273 and BNT162b2 cause similar adverse events in pregnant and non-pregnant people, and the vaccine will not increase the incidence of neonatal death or congenital abnormalities. However, further longitudinal studies are needed to evaluate the impact of mRNA vaccine on maternal and neonatal health.
the aged
This group is in urgent need of effective vaccines, because many infectious diseases affect the elderly. For example, 70 to 90% of influenza-related mortality occurs in patients over 65 years old, while the mortality rate of COVID-19 is 62 times that of young patients over 65 years old.
It is more difficult for the elderly to be vaccinated, because aging has an adverse effect on both innate and adaptive responses of the immune system. The decrease of Toll-like receptor expression will prevent monocytes and macrophages from secreting cytokines and chemokines, and limit the crosstalk with adaptive immune system. The adaptive immune response during infection is usually insufficient due to impaired cytokine signals and physiological and cellular changes. These changes include thymus degeneration, the decrease of naive B cells and T cells, the decrease of T cell receptor diversity, the higher susceptibility to T cell apoptosis, and the decrease of the expression of key receptors such as CD28 on cytotoxic CD8+T cells.
Fortunately, more and more evidence shows that mRNA vaccine may have strong efficacy in all age groups. For example, in the phase III trial, the efficacy of BNT162b2, a candidate vaccine of Pfizer-BioNTech, exceeded 93% in all treatment groups. The candidate vaccine of Moderna, mRNA-1273, is also very effective, showing 86.4% effectiveness in volunteers over 65 years old, compared with 95.6% in volunteers aged 18-65 years old.
The design of delivery tools is very important to improve the efficacy of vaccines for the elderly. MRNA vector can be used as an inflammatory adjuvant to amplify the vaccine response by enhancing the recruitment of antigen-presenting cells to the injection site. In a preclinical study, CureVac’s RNAVAC activated TLR7 and produced a lasting immune response against deadly influenza in mice. Novartis emulsion MF59 has been used as mRNA delivery carrier and adjuvant. MF59 enhances the immunogenicity of influenza vaccine and has been approved for the elderly.
Acquisition of vaccine
Obtaining vaccines is the biggest challenge to achieve widespread prevention of infectious diseases, especially in low-income countries. The cold storage requirements of the approved SARS-CoV-2 mRNA vaccine further limit the acquisition of the vaccine. Portable and reusable Arktek freezer can realize the rapid deployment of millions of doses of vaccine during an epidemic.
However, the COVID-19 virus needs to vaccinate billions of people, which requires a heat-resistant vaccine. At present, there are two candidate vaccines of SARS-CoV-2 with heat tolerance at room temperature. If these candidate vaccines can show good results in clinical trials, they may simplify the global acquisition of mRNA vaccines in the near future.
Acceptance of vaccine
Vaccines are only effective after vaccination, and there are abundant data supporting the safety and effectiveness of vaccines. Vaccines have eradicated several infectious diseases in some parts of the world and saved countless lives. However, due to misinformation and anti-vaccine campaigns, public distrust has intensified, threatening the maintenance of group immunity and putting our most vulnerable people at risk.
The decline in vaccination coverage may lead to the re-emergence of life-threatening diseases. For example, measles eradicated from the United States in 2000 infected more than 1,200 people in 2019 because of poor vaccine compliance in many communities. For COVID-19, in the United States, the current acceptance rate of 56-75% may not be enough to reach at least 80-90% coverage, and this threshold is considered to be necessary for SARS-CoV-2 population immunization.
Although most of the burden of improving vaccine coverage falls on the government and public health officials, the scientific community can also help by improving the effectiveness and safety of mRNA vaccines. Improving the curative effect will reduce the acceptance of group immunization, and improving safety will prevent the media from reporting adverse events, thus reducing the fear of vaccination.
summarize
In the past decades, the progress of mRNA design and nucleic acid delivery technology, coupled with the discovery of new antigen targets, has made mRNA vaccine an extraordinary tool to fight emerging epidemics and existing infectious diseases. Two mRNA vaccines against SARS-CoV-2 were developed at revolutionary speed and provided excellent protection rate, which is expected to end the COVID-19 pandemic.
In addition, these vaccines promote LNP and RNA therapy from niche market products to preventive treatment successfully implemented in a large number of people. As a result, a large number of safety and effectiveness data, as well as successful regulatory approval. We can be optimistic that mRNA therapy will probably change the vaccination methods, cancer immunotherapy and protein replacement therapy in modern medicine.
References:
1. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat RevDrug Discov. 2021 Aug 25 : 1–22.
2. Recent advances in mRNA vaccine technology. Curr Opin Immunol. 2020 Aug; 65:14-20.
