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The application prospect of mRNA vaccine in cardiovascular diseases
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The application prospect of mRNA vaccine in cardiovascular diseases.
mRNA vaccine is a relatively safe new nucleic acid vaccine, which has made great progress in the field of tumor and infectious diseases in recent years.
The occurrence and development of cardiovascular diseases are often accompanied by complex immune responses.
mRNA vaccines may benefit patients with cardiovascular disease by inducing immune tolerance, modulating T lymphocyte subsets, and reducing nonspecific inflammatory responses.
This article reviews the possible roles of mRNA vaccines in atherosclerosis, ischemic heart disease, and viral myocarditis, and forecasts their application prospects in the cardiovascular field.
As early as 1990, mRNA showed its potential as a vaccine. Wolff et al. [ 1] injected the constructed DNA and RNA expression vector into the skeletal muscle of mice, and the specific protein encoded by it could be directly detected in the skeletal muscle. It provides new ideas for the development of vaccines.
However, due to the poor stability and easy degradation of mRNA, the development of mRNA vaccines and drugs has been slow for many years.
In December 2020, clinical trials confirmed that the protection rate of the mRNA novel coronavirus pneumonia (COVID-19) vaccine BNT162b2 reached 95% [ 2] , making the mRNA vaccine stand out and attract widespread attention again.
However, the current research on mRNA vaccines is mostly limited to the field of infectious diseases and tumors, and its role in cardiovascular diseases is rarely reported.
This paper introduces the molecular design of mRNA vaccines and the induced immune responses, analyzes the possible roles of mRNA vaccines in cardiovascular diseases, and looks forward to their future clinical applications.
Overview of mRNA vaccines
1. Molecular design of mRNA vaccines:
Traditional mRNA vaccines usually consist of 5 parts, namely cap structure (Cap), 5′ untranslated region (5′ UTR), open reading frame (ORF), 3 ‘Untranslated region (3’ UTR) and tail containing at least 30 adenosine residues [poly(A) tail].
Caps can exist in different conformations in mRNA. Abnormally capped (Cap0) or uncapped (5′ ppp or 5′ pp) mRNAs are recognized by pattern recognition receptors (PRRs), triggering their destruction by type I interferons (IFNs) role [ 3] .
The cap structure plays an important role in mRNA translation, which can recruit translation initiation factors and enhance the closed-loop model of RNA [ 4] . There are complex interactions between 5′ UTR and 3′ UTR and RNA-binding proteins, which regulate the stability and translation efficiency of mRNA [ 5] .
The sequence of the open reading frame encodes the target protein, and often encodes the antigen in mRNA vaccines.
The translation efficiency can be improved by codon optimization.
Poly(A) tails are critical for maintaining mRNA stability and successful translation. In dendritic cells (dendritic cell, DC), compared with the short poly-A tail, the poly-A sequence of 120 nucleotides can provide more stable mRNA and more efficient translation [ 6] .
2. Immune response induced by mRNA vaccine: mRNA vaccine can induce innate and adaptive immunity.
Pathogen-associated molecular patterns (PAMPs) on mRNA are recognized by PRRs on the cell surface [ 7] , and the binding of ligand-receptor complexes transfers signals into cells and further initiates a series of signal transduction pathways.
Toll-like receptors (TLRs) can recognize single-stranded (TLR-7, 8) [ 8] or double-stranded RNA (TLR-3) [ 9] , and regulate the activation of IFN pathways as well as cytokines and chemokines. secretion.
The mRNA that enters the cell is translated into the corresponding protein, which can be degraded into peptide fragments, bound to the major histocompatibility complex (MHC) class I molecules, and presented to CD8 + T lymphocytes.
Translated proteins will also be secreted from transfected cells, taken up by antigen-presenting cells (APCs), and presented to CD4 + T lymphocytes on MHC class II molecules.
The secreted protein can also activate antigen-specific B lymphocytes to produce corresponding antibodies [ 10] .
The possible role of mRNA vaccines in cardiovascular disease
Inflammation plays an important role in the occurrence and development of cardiovascular disease [ 11] , and more and more scholars hope to reduce the risk of cardiovascular disease by improving the inflammatory response.
However, the application of traditional anti-inflammatory strategies in cardiovascular disease is less than satisfactory.
After entering the body, mRNA vaccines can induce complex immune responses including innate and adaptive immunity, and may improve cardiovascular disease through immunomodulatory effects. However, little research has been done in this field.
In 2020, the phase III clinical trial of the small interfering RNA (siRNA) drug inclisiran confirmed its efficacy and safety [ 12] . ) levels and benefit patients with cardiovascular disease.
As a “close relative” of siRNA drugs, how far mRNA vaccines are from cardiovascular disease is a question worth exploring.
1. mRNA vaccine and atherosclerosis:
Some scholars believe that atherosclerosis is a chronic inflammatory disease with autoimmune components [ 13] .
Atherosclerosis is traditionally considered to be a cholesterol accumulation disease caused by the retention of lipoproteins including low density lipoprotein (LDL) in the arterial intima.
Under the endothelium, oxidized LDL (ox-LDL) modified by reactive oxygen species can bind to TLR to induce arterial wall inflammation [ 14] , and after being taken up by macrophages, it can promote the proliferation of in situ macrophages and further mobilize bone marrow cells [ 15] ] .
In addition, the extensive interaction of CD4 + T lymphocytes CD11c + APC in plaques confirmed the role of antigen presentation and adaptive immunity in atherosclerosis [ 16] .
LDL as an autoantigen was first proposed in 1959, Gero et al [ 17] found that LDL immunized rabbits can inhibit the formation of atherosclerotic plaques.
The apolipoprotein (Apo) B component in ox-LDL has antigenic epitopes, which can be processed into antigenic peptides and combined with MHC molecules and presented to CD4 + T lymphocytes [ 18] .
In addition to LDL/ApoB, heat shock proteins (HSP) have also been proposed as atherosclerosis-related antigens [ 19].
The discovery of an autoimmune component of atherosclerosis sparked the idea of preventing atherosclerosis through vaccine immunization.
Studies have found that immunizing mice with peptides P101, P102, P103, P210, P265 and P295 of ApoB can protect against atherosclerosis [ 20, 21, 22, 23] , which may be related to the induction of leukocytes. Interferon (IL)-10 + ApoB-specific regulatory T lymphocytes (regulatory T lymphocytes, Tregs) related [ 18,20] .
However, the safety and efficacy of vaccination strategies have yet to be tested. On the one hand, traditional vaccines and their adjuvants are prone to cause non-specific inflammation [ 24, 25] .
It is unclear whether the prevention of atherosclerotic plaques is effective in established plaques.
In January 2021, Ugur Sahin’s team at the University of Mainz, Germany, demonstrated that 1-methylpseudouridine-modified mRNA (m1Ψ mRNA) formulated with nanoparticles delivers disease-related autoantigens to CD11c + APCs in the spleen , capable of antigen presentation without causing an inflammatory response, thereby inducing antigen-specific immune tolerance.
Mice with experimental autoimmune encephalomyelitis (EAE) induced a higher proportion of Tregs and low levels of Th1 and Th17 cells, Th1 effector IFN-γ, tumor necrosis after immunization with m1Ψ mRNA vaccine Factor (TNF)-α, IL-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion decreased, and did not inhibit the normal immune function of mice [ 26] .
This important breakthrough extends the focus of mRNA vaccines to autoimmune diseases, changing the traditional perception of their “pro-inflammatory” effects.
This finding also provides a new idea for applying mRNA vaccine to atherosclerosis—designing mRNA encoding ApoB autoantigen components and introducing nucleoside analog modification during in vitro transcription, which can weaken TLR signaling [ 27] , Relieve non-specific inflammatory response; at the same time, after mRNA enters the body, it is translated to produce self-antigen peptides, which are presented to CD4 + T lymphocytes by APC, which can induce high levels of Tregs cells and reduce antigen-specific inflammatory responses.
It is worth noting that the increasing content of bone marrow cells and lymphocytes in plaques may lead to thinning of the fibrous cap and the formation of unstable plaques, causing clinical complications such as plaque rupture and thromboembolism [ 28] .
Mechanistically, activation of CD4 + T lymphocytes and increased secretion of IFN-γ can inhibit collagen fiber synthesis, and activated macrophages can secrete cathepsin to decompose collagen and elastic fibers [ 29] , thereby destroying plaque stability.
Th1 cells are considered to have a pro-atherosclerotic effect, and the effector factors IFN-γ, TNF-α, and IL-2 secreted by Th1 cells can activate macrophages and T lymphocytes, and aggravate the plaque inflammatory response [ 30] .
Therefore, mRNA vaccines may reduce the secretion of inflammatory cytokines such as IFN-γ by inhibiting the activity of Th1 cells, and at the same time may inhibit the interaction between Th1 cells and macrophages, thereby reducing plaque inflammation and improving plaque stability from multiple aspects. sex.
In conclusion, mRNA vaccines show great potential in the prevention and treatment of atherosclerosis, which can not only prevent the formation of new atherosclerotic plaques, but also prevent the rupture of old plaques and prevent clinical complications such as coronary embolism.
2. mRNA vaccines and ischemic heart disease:
Acute myocardial ischemia leads to an initial pro-inflammatory response that aims to clear necrotic cell debris from the myocardial infarction area.
Myocardial reperfusion after percutaneous coronary intervention (PCI) can exacerbate this proinflammatory response and lead to myocardial cell death and myocardial injury, manifested within 6–24 h after reperfusion. T
he initial pro-inflammatory response is followed by an anti-inflammatory repair phase aimed at allowing wound healing and scarring, thereby preventing heart rupture.
The transition between these two phases is driven by components of the heart itself (cardiomyocytes, endothelial cells, fibroblasts, and stroma) and immune cells (neutrophils, monocytes, macrophages, dendritic cells, and Lymphocytes) are regulated by complex and delicate interactions [ 31] .
Considering the detrimental effects of an early excessive and persistent pro-inflammatory response in acute myocardial ischemia, and the beneficial efficacy of the subsequent anti-inflammatory repair phase, a potential therapeutic strategy to limit infarct myocardial size and prevent adverse left ventricular remodeling is to inhibit the initial pro-inflammatory response. response, and promote the subsequent anti-inflammatory repair response.
Immune cells act differently at different stages. Studies have shown that CD4 + T lymphocyte infiltration can aggravate reperfusion injury in the early stage of myocardial ischemia.
The myocardial infarct size of mice deficient in CD4 + T lymphocytes was significantly smaller than that of the control group [ 32] .
In the late stage of myocardial infarction (healing stage), the formation of collagen matrix in the infarcted myocardium of CD4 + T lymphocyte-depleted mice was severely disturbed, the left ventricular dilatation was aggravated, and the rate of cardiac rupture and mortality were higher than those of wild-type mice [ 33] .
In addition, CD4 + CD25 + FOXP3 + Tregs may play an anti-inflammatory (immunosuppressive) role in acute myocardial infarction by secreting anti-inflammatory cytokines (such as TGF-β, IL-10) [ 34] .
Not only can inhibit the recruitment of inflammatory cells (neutrophils, monocytes, CD4 + T lymphocytes) [ 35] , but also promote the polarization of macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype [ 36] , inhibit the differentiation of fibroblasts into myofibroblasts, thereby preventing adverse ventricular remodeling [ 37] .
Given the important role the immune response plays in ischemic heart disease, we cannot help wondering whether mRNA vaccines could benefit such patients. mRNA vaccines are thought to play an important role in immune regulation.
Mays et al. [ 38] constructed a chemically modified mRNA expressing Tregs cell transcription factor FOXP3, which could up-regulate the expression of FOXP3 after delivery to the lung, regulate the recruitment of immune cells and maintain the relationship between Tregs, Th2, and Th17 through an IL-10-dependent pathway. balance, thereby preventing the occurrence of asthma.
If this strategy can be applied in the early stage of myocardial ischemia, it may alleviate the initial pro-inflammatory response and reduce myocardial injury by promoting the proliferation of CD4 + CD25 + FOXP3 + Tregs cells and the secretion of IL-10.
In addition, upon myocardial injury, antigen-presenting cells may receive potent maturation signals of injury-related molecular patterns released by necrotic tissue, and induce activation of T and B lymphocytes by presenting cardiomyocyte autoantigens [ 39] .
Cardiac troponin I antibody (cTnI-Ab) was detected in patients with acute myocardial infarction, and it can prompt the clinical prognosis of patients [ 40] , indicating that myocardial injury caused by ischemia can break autoimmune tolerance. As mentioned above, mRNA vaccines modified with nucleoside analogs can alleviate non-specific inflammatory responses during antigen presentation, induce immune tolerance to self-antigens and high levels of Tregs, and inhibit Th1 activity and the secretion of effector factors. [ 26] .
Therefore, mRNA vaccines may be able to attenuate autoimmune damage after myocardial ischemia. Notably, CD4 +The role of T lymphocytes and other immune cells (such as M2 macrophages) in the anti-inflammatory repair stage of late myocardial ischemia is thought to be beneficial, and mRNA vaccines with anti-inflammatory effects may affect scarring in this stage.
At this time, the characteristics of easy degradation and short action time of mRNA can be fully utilized, and appropriate methods can be taken to shorten its half-life, so that its effect is limited to the early stage of pro-inflammatory response. In conclusion, the mRNA vaccine has broad application prospects in the prevention and treatment of ischemic heart disease.
If it can be used preventively in the high-risk population of acute coronary syndrome, it may reduce the morbidity and mortality of myocardial infarction and improve the quality of life of patients.
3. mRNA vaccine and viral myocarditis:
The pathogenesis of viral myocarditis is not very clear, mainly caused by coxsackie virus B3 (coxsackie virus, CVB3), adenovirus, echo virus and other infections [ 41, 42] , Recent studies have shown that COVID-19 can also cause myocarditis [ 43, 44] .
Several studies have demonstrated the effectiveness of vaccination against CVB3 in animal models, strategies including inactivated vaccines, live attenuated vaccines, DNA vaccines, and virus-like particle (VLP) vaccines.
Park et al. [ 45] replaced 2 tyrosines with phenylalanine in the conserved sequence of the C-terminal region of CVB3 VP2, and synthesized a highly attenuated mutant virus strain YYFF, which could induce high titers of neutralization after immunizing mice.
Antibody and CD8 + T lymphocyte responses against CVB3. Kim et al. [ 46] constructed recombinant CVB3 plasmids expressing VP1 and VP3.
After immunizing mice, neutralizing antibodies to the corresponding proteins were induced in vivo, and the survival rate of mice was improved for 46 days.
The recombinant baculovirus constructed by Zhang et al. [ 47] containing the complete coding region of CVB3 also proved to have a protective effect on myocarditis in mice, and the antibody titer was comparable to that induced by the attenuated CVB3 vaccine.
However, due to safety and cost issues, no vaccine has yet been put into clinical use.
Attenuated and inactivated virus vaccines have the risk of restoring original virulence and incomplete inactivation.
DNA vaccines are integrated into the host genome, leading to insertional mutations and potential carcinogenic risks, and the cost of large-scale production of some vaccines is too high.
In short, many factors limit the clinical application of vaccines, and there are still many bottlenecks that have not yet been broken through.
Notably, there may be an autoimmune response during the development of CVB3-induced myocarditis.
Various cardiac in situ cells, such as cardiomyocytes, phagocytes, and fibroblasts, may induce acute inflammatory responses by secreting cytokines, such as IL-1, IL-6, TNF-α, and IL-18, after virus infection [ 48] .
Antigen-specific lymphocyte responses are induced when adaptive immune cells begin to respond to viral antigens.
Antibodies produced by B lymphocytes help to neutralize the virus, and T lymphocytes can secrete cytokines such as IFN-γ to inhibit virus replication [ 49] .
However, the CVB3 proteome may have sequences similar to cardiac antigens, and this mimotope can generate cross-reactive T lymphocytes and autoantibodies [ 50] .
In addition, due to the cytolytic properties of the virus, intracellular and surface cardiac antigens may be released, inducing the formation of autoreactive T lymphocytes and antibodies.
This autoimmune disorder may be an important factor leading to chronic myocarditis and dilated cardiomyopathy [ 51] , and it also adds new challenges to vaccine research.
As a new type of nucleic acid vaccine, mRNA vaccine has many unique advantages and is expected to play an important role in the prevention and treatment of viral myocarditis.
The mRNA sequence is designed for viral antigenic epitopes, and the corresponding protein can be expressed in vivo.
Proteins can be good substitutes for inactivated or attenuated vaccines because their degradation products are only amino acids, which are less toxic; they also contain well-defined, purified antigens that can effectively induce cellular and humoral immune responses.
Since mRNA is translated in the cytoplasm without passing through the nuclear membrane, intracellular expression is more flexible, more efficient and safer than DNA vaccines, and avoids the risk of insertional mutations.
The natural RNA of CVB3 can also provide a reference sequence for the synthesis of mRNA vaccines, which is helpful for the large-scale production of vaccines. In addition, mRNA vaccines also show unique advantages in dealing with antigenic cross-reactions.
In 2017, Cell reported an improved mRNA vaccine encoding the mutant prM-E gene designed by the Richner team, which destroyed the conserved fusion loop epitope in the E protein of Zika virus (ZIKV), which could protect mice.
Avoiding ZIKV infection reduces the generation of antibodies that cross-react with dengue virus induced by traditional vaccines [ 52] .
If this research result can be realized in the CVB3 vaccine, it can not only produce a large number of neutralizing antibodies to control the virus infection, reduce the early acute inflammatory response, but also weaken the autoimmune response and reduce myocardial damage.
Prospects for clinical application of mRNA vaccines
Although mRNA vaccines have many advantages, they also have some disadvantages.
The first is instability, due to the ubiquitous RNases in tissues and cells, causing mRNA to be easily degraded, thereby affecting the efficiency of antigen expression.
In addition to instability, another disadvantage of mRNA is its high immunogenicity.
Exogenous mRNA itself has an immunostimulatory effect because it can be recognized by a variety of cell surface and cytoplasmic PRRs [ 53] .
Although in some cases, this property of mRNA may provide adjuvant activity to promote DC maturation, thereby triggering robust T and B lymphocyte immune responses.
However, mRNA overstimulation of the innate immune system can increase the secretion of type I IFN, thereby inhibiting the translation of mRNA vaccines and antigen-specific immune responses [ 5,54] .
In addition, as a large molecular weight and negatively charged molecule, naked mRNA is almost impossible to pass through the cell membrane of the same negatively charged phospholipid bilayer structure.
How to deliver mRNA into cells is one of the difficulties in vaccine preparation. . Therefore, the development of efficient liposome delivery systems is critical.
To apply mRNA vaccines to the clinic, a series of modifications are still needed to solve the problems of poor stability, high immunogenicity, and low translation efficiency.
Studies have shown that the 5′ cap structure modified with locked nucleic acid (LNA) can increase the stability of mRNA by 1.61 times [ 55] .
Appropriate introduction of stabilizing elements in the 3’UTR can also enhance mRNA stability [ 56] .
In response to the problem of high immunogenicity, the current strategy is to incorporate nucleoside analogs such as pseudouridine (Ψ), 5-methylcytidine (m5C) and 2-thiouridine (s2U) into mRNA molecules, It can weaken the TLR signal [ 57] .
The mRNA capped with anti-reverse cap analogue (ARCA) will have higher translation efficiency [ 58] .
At the same time, the 5’UTR sequence should avoid the formation of stable secondary structure, otherwise it will hinder ribosome binding and reduce translation efficiency [ 59] .
In addition, the smooth delivery of mRNA vaccines into cells in vivo is the basic guarantee for their functioning.
Lipid nanoparticles (LNP) are currently the most widely used delivery systems [ 60] , which can not only prevent mRNA from being degraded by nucleases, but also have good biocompatibility and are easy to fuse with receptor cell membranes [ 61] .
A liposome delivery system-based mRNA vaccine (mRNA-LPX) developed by Kranz et al. [ 62] is able to deliver mRNA-encoded antigens to lymphoid tissue-resident CD11c +APC, thereby effectively activating antigen-specific immune responses.
Adverse reactions that may be caused after mRNA vaccination are also issues that need to be considered.
JAMA has released data from 3.644 million COVID-19 mRNA vaccines (including BNT162b2 and mRNA-1273) vaccinated by self-reported data.
The results show that the top five most common adverse reactions after the second dose of vaccination are injection site pain ( 72.3%), fatigue (53.9%), headache (46.7%), myalgia (44.0%) and chills (31.3%), but the proportion of the population with the above adverse reactions dropped rapidly to less than 10% 5 days after vaccination [ 63 ] .
It is suggested that the COVID-19 mRNA vaccine is relatively safe.
Two randomized double-blind phase I clinical trials of H10N8 and H7N9 influenza virus mRNA vaccines showed that only local symptoms and mild symptoms such as myalgia, fatigue, and headache occurred after vaccination [ 64] .
Other clinical trials of mRNA vaccines against rabies virus [ 65] and respiratory syncytial virus [ 66] also showed good safety. It suggested that the adverse reactions of mRNA vaccines were within an acceptable range.
However, most of the mRNA vaccine products are still in the clinical trial stage, and are mainly concentrated in the fields of tumors and infectious diseases [ 61,67] .
The application of mRNA vaccines to cardiovascular diseases requires a lot of considerations in terms of molecular design and delivery systems in addition to sufficient theoretical basis, and there is still a long way to go.
Considering the characteristics of mRNA itself, the induced immune response, the favorable conditions for large-scale vaccine production, etc., mRNA vaccines have incomparable advantages over other vaccines, and related research on the application of mRNA vaccines to the prevention and treatment of different types of diseases is also expanding.
In conclusion, mRNA vaccines have broad prospects for clinical application and can be expected in the future.
The application prospect of mRNA vaccine in cardiovascular diseases
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