The mRNA vaccine, a swift warhead against a moving infectious disease target

ABSTRACT

Introduction

The rapid development of mRNA vaccines against SARS-CoV-2 has revolutionized vaccinology, offering hope for swift responses to emerging infectious diseases. Initially met with skepticism, mRNA vaccines have proven effective and safe, reducing vaccine hesitancy amid the evolving COVID-19 pandemic. The COVID-19 pandemic has demonstrated that the time required to modify mRNA vaccines to counter new mutant strains is significantly shorter than the time it takes for pathogens to mutate and generate new variants that can thrive in vaccinated populations. This highlights the notion that mRNA vaccine technology appears to be outpacing viruses in the ongoing evolutionary race.

Areas covered

This review article offers valuable insights into several crucial aspects of mRNA vaccine development and deployment, including the fundamentals of mRNA vaccine design and synthesis, the utilization of delivery systems, considerations regarding vaccine safety, the longevity of the immune response, strategies for modifying the original mRNA vaccine to address emerging mutant strains, as well as addressing vaccine hesitancy and potential approaches to mitigate reluctance.

Expert opinion

Challenges such as stability, storage, manufacturing complexities, production capacity, allergic reactions, long-term effects, accessibility, and misinformation must be addressed. Despite these hurdles, mRNA vaccine technology holds promise for revolutionizing future vaccination strategies.

KEYWORDS:

• SARS-CoV-2
• COVID19
• mRNA vaccine
• virology
• vaccine hesitancy
• in vitro mRNA synthesis

1. Introduction

The utilization of nucleic acids in immunization has garnered significant attention within the realm of next-generation vaccines. DNA vaccine technology with the first proof of concept was made in 1990 [1] and now researchers are studying DNA vaccines to fight HIV [2] and certain cancers [3] but many factors are responsible for its poor immunogenicity in humans [4]. As of September 2021, FDA had approved the usage of DNA vaccine in certain animal diseases, such as West Nile Virus in horses [5–8] and melanoma in dogs [9], which was later withdrawn. The same 1990 publication showcased that naked RNA could also lead to the in vivo expression of encoded protein. Despite this, greater emphasis was placed on the use of plasmid DNA over mRNA, likely due to concerns about mRNA instability [10,11]. Nevertheless, DNA vaccines encounter challenges, including the theoretical possibility of triggering autoimmunity or integrating into the host genome although no solid evidence of genome integration has been reported [11]. For about a decade there seemed to be less interest in mRNA compared to DNA as a vaccine platform technology. Recently, mRNA vaccine was used to combat SARS-CoV-2 infection during COVID-19 pandemic and is now extended to other human diseases including cancer [12]. Unlike traditional vaccines, which use weakened or inactivated versions of a virus or bacteria, mRNA vaccines use synthetic mRNA to express the desired protein that triggers an immune response [13]. The COVID-19 pandemic has demonstrated the potential of mRNA vaccines as a powerful tool for combating infectious diseases. In record time, mRNA vaccines have been developed, tested, and deployed to help protect people against the SARS-CoV-2 infection.

One of the key advantages of mRNA vaccines is their remarkable speed of development. This makes them an attractive choice for responding to emerging infectious diseases, where time is of the essence in controlling an outbreak. Similar to DNA vaccines [14], it has been suggested that mRNA vaccines do not integrate into the host genome [11,15,16], obviating the palpable anxiety in end users about potential insertional mutagenesis [12]. It must be noted that mRNA integration into host genome has not been well studied, further studies are required to confirm the lack of potential insertional mutagenesis of mRNA vaccines [15]. The mRNA vaccines are manufactured in a cell- free system that allows rapid and scalable production in a cost-effective manner. For example, a bioreactor of five-liter capacity can produce almost one million doses of mRNA vaccine in a single reaction [17]. Another advantage of mRNA vaccines is their flexibility. The mRNA sequence can be easily modified to target different strains or variants of a pathogen. This could potentially make it easier to develop new vaccines for emerging strains of viruses or bacteria, without having to start from scratch each time. Incorporating multiple open reading frames encoding multiple antigens in the single mRNA, helps in strengthening the immune response against resilient pathogens [18,19] and enables the targeting of multiple pathogen variants of bacterial or viral origin with a single formulation [18,20].

However, there are also some challenges associated with mRNA vaccines. These vaccines require specialized storage and transportation conditions to maintain the stability of the mRNA sequence. Recent studies have investigated to better understand the stability and storage of mRNA with the use of lipid nanoparticles (LNP’s) [21]. For convenience in biological applications, investigators have suggested storage of LNPs at physiological pH (7.4) in PBS at 2°C for up to 160 days [22]. While the mRNA vaccine produced by Pfizer required ultralow temperatures for storage, the mRNA vaccine produced by Moderna did not require ultracold chains. Additionally, the long-term safety and efficacy of mRNA vaccines are still being studied, as this technology is relatively new [23,24]. In fact, the enthusiasm in mRNA therapeutics was previously dampened due to concerns about mRNA stability and its excessive immunostimulation. However, the recent developments in mRNA pharmacology, development of effective delivery vehicles, improved mRNA stability and controlling the mRNA immunogenicity has boosted the enthusiasm in the field of mRNA therapeutics [25,26].

Overall, during COVID-19 pandemic, mRNA vaccines have shown great promise as a rapidly produced warheads against moving infectious disease targets. With ongoing research and development, they may become an important tool in the fight against emerging infectious diseases and other illnesses impacting mankind. The mRNA vaccine design involves identifying the target antigen, generating a stable mRNA sequence that codes for the antigen, modifying the mRNA to enhance stability and reduce immune activation, formulating the mRNA into lipid nanoparticle (LNP) for delivery, administering the vaccine through injection, and triggering an immune response that generates antibodies and memory T cells. This review article focuses on the principles of mRNA design and synthesis by in vitro transcription systems, mRNA delivery vehicles, modifications of the mRNA vaccine in response to the emergence of mutant strains of the infectious pathogen, duration of immune response and safety of mRNA vaccines, the hesitancy of mRNA vaccine and potential measure to combat the hesitancy.

2. Principles of mRNA design and synthesis

The first step in designing an mRNA vaccine is to identify the specific protein or antigen that the vaccine should target. This can be done by analyzing the genetic sequence of the pathogen and identifying which proteins are most essential for its survival and infectivity. The structurally and functionally important proteins that are highly expressed during the course of infection and produce neutralizing antibodies are the preferred antigen targets for vaccine design. For example, in SARS-CoV-2 virus the surface glycoprotein is an important structural component of the virion that facilitates the virus engagement to the host cell receptor and represents a suitable target for vaccine design. Similarly, the highly conserved regions of SARS-CoV-2 nucleocapsid protein might be highly beneficial and broadly protective antigen targets of interest [27].

The next step is to synthesize an mRNA encoding the target antigen of interest using an in vitro transcription system. The mRNA encoding the antigen must structurally mimic the eukaryotic mRNA having several key structural features important for its stability and translation by the host translation apparatus. These structural features include the 5’ cap, 5’ untranslated region (UTR), open reading frame encoding the antigen (ORF), 3’ UTR and 3’ poly(A) tail (Figure 1(a)). Majority of the eukaryotic mRNAs contain a 5’ cap comprised of a modified guanine nucleotide-m (7)G which is added to the 5’ end of the mRNA through a triphosphate bridge during transcription [29]. This cap structure helps protect the mRNA from degradation by the host RNA degradation machineries and is also important for translation initiation [29]. The eukaryotic mRNA is circularized during translation by establishing an indirect contact between the 5’cap and 3’ poly(A) tail with the assistance of poly(A) binding proteins and translation initiation factors [30,31]. The circularized mRNA forms an efficient template for translation by the host translation apparatus (Figure 1). The length of the poly(A) tails plays an important role in translation initiation and mRNA turnover by the cytoplasmic RNA degradation machinery [30]. A sufficiently long poly (A) of 100–150 nucleotides is necessary for binding to the poly(A) binding proteins to generate the circularized mRNA in which 5’ cap is protected from the attack of host decapping system [30,32]. In addition, the first or second nucleotide from the 5′ end of mammalian mRNA is methylated on the 2′ hydroxyl of the ribose (2′- O- methylation). This methylation is important form immunologic standpoint as it prevents recognition by cytoplasmic sensors of viral RNA [29], and thus prevents unintended immune responses.

Figure 1. Eukaryotic mRNA engaging in translation: a eukaryotic mRNA showing five structural components; 5’ cap, 5’ untranslated region (5’UTR), open reading frame (ORF), 3’UTR, and poly(A) tail. The 5’ cap – m [7]G attached to the first nucleotide via a triphosphate bridge is shown. The 5’ cap structure along with first two nucleotides of the transcript was borrowed from [28]. Engagement of the mRNA in translation is shown on the right. The mRNA gets circularized by indirect contact between 5’ cap and 3’ poly(A) tail with the assistance of poly(A) binding protein (PABP) and other translation initiation factors, as shown. Other translation initiation factors have not been shown for simplicity.

The 5’ and 3’ UTR sequences contain regulatory elements that control mRNA translation, subcellular localization and mRNA half-life [33]. For the synthesis of an mRNA by the in vitro transcription system the naturally occurring 5’ and 3’ UTR sequences of highly expressed mRNAs such as alpha and beta globulin are preferably used [33]. Alternatively, the optimized UTR sequences for desired application and target cell can be used [34]. The goal of the optimization is to incorporate UTR sequences lacking the MicroRNA (miRNA) binding sites and avoiding the AU rich sequences in the 3’ UTR to avoid mRNA degradation. MicroRNAs (miRNAs) are small non-coding RNA molecules that play important roles in post-transcriptional gene regulation by binding to specific sites within the mRNA transcripts [35,36]. These interactions typically occur within the 3’ untranslated region (UTR) of target mRNAs, where miRNAs can influence mRNA stability and translation efficiency. However, miRNA binding sites can also be found in other regions of mRNA, including the 5“ UTR, although they are less common compared to the 3” UTR [35,36]. The miRNA interactions with the 5“ or 3” UTR of mRNA can have significant regulatory consequences for gene expression, contributing to the complexity and versatility of post-transcriptional gene regulation mediated by miRNAs [35,36]. In addition, the sequences forming the complex secondary and tertiary structures such as hair pins are eliminated especially in the 5’ UTR to avoid negative impact during ribosome scanning, a critical step in search of an AUG codon during translation initiation [34].

The open reading frame (ORF) encodes the target antigen. The ORF sequence is usually codon optimized for high expression in the desired expression systems. However, codon optimization must be carried out with caution as it can impact the protein folding [37]. Some mRNAs contain rarely used codons to slow down the rate of translation for optimal folding of the protein [38]. The biopharmaceutical company CureVac AG discovered that codons of the human mRNA rarely contain A or U at the third position and patented a strategy for the replacement of A or U at the third position in the open reading frame with G or C for the best protein expression in human cells [39]. CureVac then used this optimization strategy in the development of its SARS- CoV-2 vaccine candidate CVnCoV, which is now in phase III trials. Recently researchers have developed algorithms for optimized mRNA design while increasing stability and immunogenicity [40].

Host immune responses to unmodified single-stranded RNA trigger anti-virus reactions upon RNA entry into cells, causing mRNA instability and reducing translation efficacy [41]. Chemical modification of nucleosides in mRNA therapy suppresses innate immune activation and maintains stability, overcoming the anti-mRNA effects of unmodified RNA. Modified nucleosides, like 2-thiouridine and 5-methyl-cytidine, prevent immune responses, resulting in higher in vivo protein expression compared to unmodified RNA [42]. Several modified nucleotides that have been commonly used in mRNA vaccine technology to enhance stability, translatability, and reduce immunogenicity [43,44] are given in Table 1. All native eukaryotic mRNAs contain modified nucleotides to avoid detection by the host immune surveillance systems [45]. For example, the unmodified single-strand mRNA expressed from viral genome during infection can be recognized by pattern recognition receptors, such as Toll- like receptor 3 (TLR3), TLR7 and TLR8, and the retinoic acid- inducible gene I (RIGI) receptor, triggering type I interferon response which ultimately results into transient shutdown of mRNA translation [46]. Thus, codon optimization and the use of modified nucleotides are important to enhance the mRNA translation and avoid the mRNA detection by the host surveillance system. Both Moderna and Pfizer – BioNTech used modified nucleotides in the SARS-CoV-2 vaccine that showed more than 95% efficacy in phase III clinical trials [47].

Table 1. Common nucleotide modifications of mRNA vaccines.

Modification	Nucleotide	Effect in mRNA Vaccines
Pseudouridine (Ψ)	Uridine	Reduces innate immune response to mRNA; enhances mRNA stability and translational efficiency
7-Methylguanosine (m7G)	Guanosine	Prevents mRNA degradation, enhances translation initiation
5-Methylcytidine (m5C)	Cytidine	Improves overall stability and performance of mRNA; reduces the innate immune response
N6-Methyladenosine (m6A)	Adenosine	A common modification in cellular RNA; enhances translation efficiency and mRNA stability.
2-Thiouridine (s2U)	Uridine	Increases resistance to nucleases, improving mRNA stability.
N1-Methylpseudouridine (m1Ψ)	Pseudouridine	Improves translation efficiency, reduces immune response.
5-Methyluridine (m5U)	Uridine	Enhances mRNA stability and translation efficiency.
2’-O-Methylguanosine (m2,2’−OG)	Guanosine	Reduces activation of innate immune sensors.
2’-O-Methylcytidine (m2,2’−OC)	Cytidine	Improves mRNA stability and translation efficiency.
5-Methyl-2’-O-Methylcytidine (m5C-m2OC)	Cytidine	Enhances mRNA stability and translation.

The addition of poly (A) tail downstream of the 3’ UTR by poly (A) polymerase generates the poly(A) tails of varying lengths. To avoid the poly (A) length variation and additional reaction steps, the poly (A) tail sequence of >100 nucleotides is incorporated in the DNA segment encoding the mRNA. Since poly(A) tails of >100 nucleotides are optimal for therapeutic mRNAs [48], it is easier and cost effective to incorporate them in the DNA sequence downstream of the 3’ UTR. Since long pol(A) stretches might destabilize the plasmid DNA harboring the gene of interest, it is recommended to incorporate short UGC linkers within the poly(A) tail sequence to improve plasmid stability [48]. Using this strategy Pfizer – BioNTech vaccine BNT162b2 against SARS- CoV-2 contains a 10 bp UGC linker to produce the sequence A30(10 bp UGC linker) A70 [18]. Once the 5’UTR, codon optimized ORF, 3’UTR and poly(A) tail sequence has been selected, the encoding DNA segment is synthesized and incorporated in a plasmid downstream of the T7 promoter (Figure 2). The plasmid is amplified in E. coli, purified and used in an in vitro T7 transcription reaction for the synthesis of the desired mRNA. Alternatively, the synthetic DNA segment is PCR amplified, purified, and directly used in the in vitro T7 transcription reaction [49]. The mRNA is co-transcriptionally capped with a 2′- O-methylated cap using the clean cap system from TriLink Biotechnologies [50,51]. Clean Cap technology yields a natural Cap 1 structure via co-transcriptional capping, eliminating the prohibitive costs of legacy enzymatic methods [51]. The mRNA is modified during synthesis using the chemically modified nucleotides. After transcription is complete, the mRNA is purified by high performance liquid chromatography (HPLC) to remove the incomplete transcripts, double stranded RNA and other reaction contents. The in vitro T7 transcription system produces ~10 milligram quantities of mRNA in just 90 minutes [52]. Chromatography stands as a widely embraced purification method in the pharmaceutical industry. Ion-exchange chromatography (IEC) that uses the charge difference between target mRNA and various impurities as well as cellulose-based chromatography that uses the ability of dsRNA to bind cellulose in the presence of ethanol has also been used [53].

Figure 2. In vitro production of mRNA vaccine and elicitation of immune response through transfection of antigen presenting cells [1]: after sequencing the genome of the pathogen, the sequence encoding the antigen of interest, flanked with 5’ and 3’ UTR sequences, is cloned in the plasmid downstream of the T7 promoter [2]. The purified plasmid is used as template in the in vitro T7 transcription reaction for the synthesis of mRNA encoding the antigen [3]. The mRNA is purified by high performance liquid chromatography (HPLC) and formulated into lipid nanoparticles (LNPs) for delivery [4]. The LNPs are filtered to remove the free mRNA [5]. The purified LNPs are stored in vials and used for vaccination [6]. Endocytosis of the LNP by antigen presenting cell in vaccinated individuals [7]. The mRNA escapes the endocytosed LNP and engages into translation in the cellular cytoplasm [8]. The translated antigen is broken down into small fragments by the proteosome and the resulting protein fragments are displayed on the surface of by major histocompatibility complex (MHC) class I molecules to activate cytotoxic T cells [9]. The activated cytotoxic T cell produces toxic molecules such as perforin and granzyme that target the infected cells [10]. The secreted antigen can be the cells, degraded and presented on the surface by MHC class II proteins to activate T helper cells [11]. Helper T cells activate the B cells to produce neutralizing antibodies for the clearance of the pathogen. Helper T cells also stimulate phagocytes such as macrophages by inflammatory cytokines.

Numerous mRNA platforms, such as, self-amplifying mRNA, trans-amplifying mRNA and circular RNA have been designed to extend expression time of IVT mRNA and for large scale production [54]. Self-amplifying mRNA (SAM) is a type of mRNA technology that incorporates elements of the RNA replication machinery from viruses like alphaviruses – a group of positive-sense RNA viruses. The concept behind SAM is to leverage the viral replication machinery to amplify the production of the desired protein encoded by the mRNA. This is achieved by including not only the gene of interest (which codes for the antigen in the case of vaccines) but also additional genetic elements from the virus that facilitate efficient replication within host cells [55]. The advantages of SAM include the potential for increased protein expression and the ability to achieve a strong immune response with lower doses of mRNA. However, there are also challenges and considerations, such as the need for careful design to balance replication efficiency and safety, as well as potential concerns related to the use of viral components [55].

Exogenous circular mRNA, designed to prolong expression compared to linear IVT mRNA, utilizes a circularization strategy involving ribozymatic methods, often employing self-splicing introns [56]. This circular mRNA lacks free ends, enhancing stability and overcoming the short half-life of linear IVT mRNA. A permuted group 1 catalytic intron-based system is commonly used, involving a double transesterification reaction. Strategies include inserting internal ribosomal entry sites (IRES) and the gene of interest between exon fragments E1 and E2 in a permutated intron – exon (PIE) structure [56].

Trans-amplifying RNA (taRNA) is a vaccine platform that combines a non-replicating mRNA and a transreplicon (TR) RNA. This method ensures strong RNA self-replication and immunogenicity, potentially mitigating the need for extensive large-scale RNA production [57]. The second-generation taRNA system’s potential for vaccine development is hampered however, by the absence of suitable nanoparticle-encapsulated formulations, necessary for improved in vivo delivery and prolonged circulation [58].

3. Formulating the mRNA into lipid nanoparticle (LNP) for delivery

The ultimate goal is to deliver the purified synthetic mRNA to the cytoplasm of immune cells for translation and antigen presentation. Since the mRNA molecules are large in size and are negatively charged, they cannot pass through the polar lipid bilayer environment of the cell membrane. In addition, the mRNA must be prevented from degradation due to potential attacks from cellular nucleases. During routine cell culture studies, the mRNA can be delivered to target cells grown in dishes by electroporation or using transfection reagents. However, in vivo delivery requires mRNA-delivery-vehicles that safely transfer the mRNA to the immune cells without degradation or triggering unwanted cytotoxicity [59]. Among multiple available delivery systems, lipid nanoparticles (LNPs) are the most clinically advanced delivery system used for this purpose. Their advantages include the ease of formulation, biocompatibility, and potential to carry a large payload of synthetic mRNA. LNP delivery systems are in use for all SARS-CoV-2 mRNA vaccines currently available for vaccination (Table 2). The LNPs are composed of ionizable lipids, cholesterol, helper phospholipids and PEGylated lipids, which together encapsulate and protect the weaker mRNA core [18,59]. During LNP formulation the lipids are dissolved in ethanol and the mRNA is dissolved in aqueous citrate buffer at pH 4. Mixing the two solutions protonates the ionizable lipid generating a positive charge which then attracts the negative charged mRNA. The hydrophobic interactions due to poor aqueous solubility of lipids triggers the spontaneous self-assembly of LNPs harboring the encapsulated mRNA inside. The resulting LNPs are subjected to dialysis or filtration to remove the non-aqueous solvent and free mRNA (Figure 2), and the pH is adjusted to physiological level [18]. The automated Microfluidic mixers are now available that can be used in high throughput mode to produce large quantities of LNPs of uniform size [60]. T mixers with good manufacturing practice (GMP) can operate at the much higher flow rates (60–80 ml min⁻¹) necessary for large-scale manufacturing [60]. Although DODAP and DODMA were the first ionizable lipids used for RNA delivery, the continued research in this area has produced a variety of ionizable lipids with unique properties that can be used to generate LNPs with improved efficacy. One such ionizable lipid (SM-102) was used in the Moderna vaccine mRNA-1273 against SARS- CoV-2. Apart from efficacy there is growing interest to generate LNPs that can preferentially target immune cells, which is highly advantageous for vaccine development and immunotherapies. Lipids containing polycyclic adamantane tails, such as 11-A- M [61], or those containing cyclic imidazole heads, such as 93-O17S [62], have been designed to target T cells in vivo [18].

Table 2. List of Major SARS-CoV-2 vaccine trials⁺.

Vaccine	Developer	Phase	Status	Country/Region
Pfizer-BioNTech	Pfizer, BioNTech	Phase 3	Authorized	Multiple countries
Moderna	Moderna	Phase 3	Authorized	Multiple countries
Oxford-AstraZeneca	AstraZeneca	Phase 3	Authorized	Multiple countries
Janssen, J&J	Johnson & Johnson	Phase 3	Authorized	Multiple countries
Sinovac	Sinovac	Phase 3	Authorized	Multiple countries
Bharat Biotech	Bharat Biotech	Phase 3	Authorized	India
Sinopharm	Sinopharm	Phase 3	Authorized	Multiple countries
CanSino	CanSino Biologics	Phase 3	Authorized	Multiple countries
Novavax	Novavax	Phase 3	Authorized	Multiple countries
Covovax	Novavax	Phase 3	Authorized	Multiple countries
EpiVacCorona	Vector Institute	Phase 3	Authorized	Russia
Covishield	Serum Institute	Phase 3	Authorized	Multiple countries
Covaxin	Bharat Biotech	Phase 3	Authorized	India
Abdala	Center for Genetic Eng.	Phase 3	Authorized	Cuba
Soberana 02	Center for Genetic Eng.	Phase 3	Authorized	Cuba
RBD-Dimer	Anhui Zhifei Longcom	Phase 3	Authorized	China
Covilo	Binnopharm	Phase 3	Authorized	Russia
QazCovid-in	RISBSP^^	Phase 3	Authorized	Kazakhstan
ZF2001	Anhui Zhifei Longcom	Phase 3	Authorized	China
Razi Cov Pars	Razi Vaccine & Serum^$	Phase 3	Authorized	Iran
Minhai COVID-19	Minhai Biotechnology	Phase 3	Authorized	China
Covovax (PQ)	Novavax Serum Institute	Phase 2/3	In progress	Multiple countries
Soberana Plus	Center for Genetic Eng.	Phase 2/3	In progress	Cuba
Covovax (US)	Novavax	Phase 2/3	In progress	United States
Covigenix Vax	Genexine Consortium	Phase 2/3	In progress	South Koera
Covovax (Canada)	Novavax	Phase 2/3	In progress	Canada

^{^}Research Inst. for Biological Safety Problems, ^$ Razi Vaccine & Serum Res. Inst. ⁺ This table lists the information of vaccine trials until September 2021.

Cholesterol, another structural component of LNPS plays an important role in the stability, size and shape of LNPs and also mediates the fusion with endosomes during LNP uptake by the cells [63]. Helper lipids play a critical role in maintaining the fluidity of LNP. They also facilitate the specificity of LNP targeting to desired organs [64]. They enhance efficacy by promoting lipid phase transitions that aids the membrane fusion with the endosome [65]. The choice of helper lipid for the construction of LNP depends on both the ionizable lipid and the mRNA cargo. For example, the saturated helper lipid DSPC was used in the FDA approved SARS-CoV-2 vaccines mRNA-1273 and BNT162b2 [18]. The PEGylated lipid, another structural component of LNP is composed of polyethylene glycol (PEG) conjugated to an anchoring lipid [66]. The hydrophilic PEG plays a crucial role in the LNP stability, regulation of LNP size and an increase in the LNP half-life by regulating the nonspecific interaction with macrophages [67]. The molecular weight of PEG and size of anchoring lipid determines the circulation time and immune cell uptake of the LNP [68]. Other delivery vehicles used for mRNA vaccines are discussed in [18].

Once the mRNA is encapsulated into LNPs or any other delivery system, the free mRNA and other reaction components are removed by dialysis for filtration and the mRNA vaccine becomes ready for inoculation to the desired populations (Figure 2). The mRNA vaccine for SARS-CoV-2 were inoculated by intramuscular injection. Apart from mRNA vaccine numerous other vaccine trails were carried out for SRAS-CoV-2 during the COVID-19 pandemic (Table 2).

The innate immune response triggered by LNPs plays a crucial role in the body’s defense mechanisms against foreign substances. The innate immune system is the first line of defense and is nonspecific, meaning it responds to a wide range of pathogens without specifically targeting a particular invader [67,69]. However, there is potential for harm arising from inappropriate or excessive immune activation. Thankfully, there are approaches to mitigate these unwanted effects, such as implementing chemical alterations to the RNA, adjusting the lipid composition, or modifying the delivery routes and refining these strategies will result in safer and more efficient LNPs [67,69].

4. Duration of immune response and safety of mRNA vaccines

After inoculation, the mRNA is taken up by both immune and nonimmune cells and translated into the protein. The antigen producing cells either directly produce the antigen by the translation of the mRNA or take up a translated antigen and finally transport it to lymph nodes where the interaction between B cells, follicular helper T cells (T_FH cells) and antigen producing cells trigger the formation of a germinal center (Figure 2) [70]. Within the germinal center the B cells then proliferate, differentiate, and rearrange their genes for the production of highly specific neutralizing antibodies that bind the target antigen with specificity and high affinity [18].

For the rapid formation of germinal center and induction of T_FH cell response some delivery systems selectively transfer the mRNA cargo to antigen presenting cells for quick translation of the antigen. Such selective delivery of the mRNA cargo is achieved by either conjugating the mAbs to the LNP surface [71] or decorating the LNP surfaces with the dendritic cell specific ligands [72]. Moreover, improving the stability of mRNA molecules by nucleotide modifications extends the sustained production and availability of intact antigen to the immune system, which has been reported to produce the neutralizing antibodies that bind the target antigen with improved affinity [73]. Sustained availability of intact antigen during the germinal center reaction has been reported to enhance the antibody production by tenfold [74]. The elicitation of germinal center reaction and induction of T_FH cell response was observed in the preclinical studies of mRNA vaccines against SARS-CoV-2, HIV-1 and Zika virus [75]. The induced germinal B-cell response for at least 12 weeks by the SARS-CoV-2 vaccine (BNT162b2) produced antibodies that predominantly targeted the receptor binding site of the viral spike protein [76]. The two doses of mRNA-1273 vaccine elicited strong antibody response over a period of 6 months, there after the antibody titers started declining but the neutralizing antibodies were retained across all age groups [77]. The duration of antibody response is very complex phenomenon and will require long-term data for clear understanding [18].

The mRNA vaccines developed by Pfizer-BioNTech and Moderna have been shown to be safe and effective in clinical trials, which was further confirmed by the worldwide usage of the vaccine. Numerous studies and regulatory agencies have evaluated the safety of these vaccines and have found them to be very safe. The most common side effects reported are mild and temporary, such as pain at the injection site, fatigue, headache, and fever. These side effects typically go away on their own within a few days. In general, the safe profiles of mRNA vaccines are promising. However, some rarer and scattered incidents observed in mRNA vaccines demands further optimization and of the mRNA antigen and the structural components of the delivery vehicles used. For example, CureVac’s protamine-based rabies vaccine candidate CV7201 triggered severe adverse effects in 78% of participants [78], which prompted the use of LNPs as the preferred delivery vehicles for the next vaccine candidate CV7202 [79]. In addition, similar to other routinely used drugs, the dosage of the mRNA vaccine has profound impact upon the safety. For example, the higher dosage of vaccine candidate CV7202 (5 μg/dose) trigger unacceptable reaction in phase I clinical trials when the smaller dose of 1 μg was tolerable.

Anaphylactic reactions were rarely reported in SARS-CoV-2 mRNA vaccine. Only 4.5 and 2.5 incidents of anaphylactic reactions were reported in one million individuals vaccinated with Pfizer – BioNTech and Moderna mRNA vaccines, respectively [80]. Although these rarer incidents are negligible compared to the benefits, this indent rate is 2–4-fold higher in comparison to traditional vaccines [81]. It has been hypothesized that anaphylactic reactions are due to preexisting antibodies against PEGylated lipids used in LNP delivery systems [82]. PEG is considered safe and present a numerous consumer products including toothpaste, shampoos, and laxatives. It has been suggested that PEG activates the humeral immune response in T cell independent manner [83]. Anti-PEG antibodies have been observed in 40% of the population which might increase their risk for anaphylactic reactions due to LNP encapsulated mRNA vaccines. Thus, further attention is need in the design and formulation of mRNA vaccines, especially the delivery vehicles, to improve the safety profiles.

While mRNA vaccines primarily induce the production of neutralizing antibodies, they also elicit a robust cell mediated response, including the activation of cytotoxic T cells, helper T cells, and the generation of memory T cells [84,85]. This dual immune mechanism enhances the overall efficacy of the immune system against viral infections, providing a broader and more comprehensive protection. The significance of cell-mediated immunity becomes particularly apparent in situations where neutralizing antibodies might have limitations, such as, in the case of certain viruses like Lassa virus [86].

5. Modifications of the mRNA vaccine in response to the emergence of mutant strains of the infectious pathogen

The higher evolution rates of many viruses are attributed to the large population size, shorter replication time, and higher mutation rate [87]. Among all these factors, mutation rate is the important determinant of evolutionary rate across taxa. DNA viruses have relatively low mutation rates ranging from 10⁻⁸ to 10⁻⁶ substitutions per nucleotide site per cell infection (s/n/c). In comparison, RNA viruses, however, have higher mutation rates that range between 10⁻⁶ and 10⁻⁴ s/n/c [88,89]. The higher mutation rate of RNA viruses is due to their RNA genome replication machinery (RNA dependent RNA polymerase (RdRp)) that doesn’t have the proof-reading activity and thus fails to correct the potential errors during the replication process. However, the members of the Nidovirales family, including coronaviruses, toroviruses, and roniviruses have RdRp independent proofreading activity that relatively decreases their mutation rate. It is believed that proofreading activity of Nidovirales enables them to harbor larger genomes (~26 kb) in comparison to other RNA viruses [90]. Retroviruses also have high mutation rates because reverse transcriptase also doesn’t have the proofreading activity. In addition, single stranded viruses tend to have higher mutation rates in comparison to double stranded viruses [89], suggesting the double stranded genomes tend to create an error proof genetic system during the evolutionary process. Some single-strand DNA viruses have error rates comparable to double-strand RNA viruses [89]. While higher mutation rates in RNA viruses and retroviruses give rise to higher evolutionary rates, some DNA viruses have mutation rates comparable to RNA viruses, highlighting the role of other factors, sch as, host dynamics and cell tropism in determining the generation of evolutionary diverse species [87–89].

Mutation rate for any pathogen is important in determining the emergence of new mutant strains with a potential to evade a vaccine or a drug or have expanded their host range for infection. The loss-in-function mutations generate variants that lose fitness to survive in the given environment. In comparison, the gain-in-function mutants are relatively more fit to survive in the challenging hostile environment created in the immunized hosts or in individuals taking anti-infectives. In viral population dynamics it is critically important to determine the time it takes for a set of infected cells to produce a sufficiently diverse viral population that has gained enough fitness to survive in the new challenging environment. This is important especially in pandemics caused by lethal pathogens that can cause significant damage to the invading population. While such time frames are influenced by the mutation rate of the pathogen, we have seen the emergence of six SARS-CoV-2 variants in a time frame of ~ two years (2020–2021) that gained fitness due to mutations in the spike protein. The emergence of alpha variant in United kingdom [91], Beta variant in south Africa [92], Gama variant in Brazil [93], delta variant in India [94], Epsilon variant in California [95] and Omicron variant [96] created palpable anxiety among people.

While the emergence of a virulent pathogen strain escaping the available vaccines or anti-infectives has potential to wipe out the target population from an endemic area, the technological development in the design and production of mRNA vaccines has created a new hope to counteract such challenges. The rapid development of mRNA vaccines by Pfizer-BioNtech and Moderna against newly emerging strains of SARS-CoV-2 has confirmed the possibilities for timely modification of the parent mRNA vaccine to counteract the newly emerging mutant strains in timely fashion [97]. This has become possible by the implementation of global health surveillance policies that encourage the frequent sequencing of viral genomes using advanced sequencing tools [98]. Once the new mutants with a potential to evade the vaccine or antiviral drugs are identified, the corresponding mutations are incorporated in the mRNA encoding the viral antigen. Due to ease of incorporating mutations in the mRNA, the vaccine become available for testing in a short period of time. Based on the information from COVID-19 pandemic, the mRNA vaccine can be modified at much faster speed in comparison to the speed at which new mutants emerge. Thus, it appears that mRNA vaccines have gained advantage in the viral evolutionary race.

The evolving landscape of infectious diseases, with the potential for new variants, also underscores the importance of adaptability in vaccination strategies. Continued research and real-world data on vaccine efficacy guide recommendations for booster doses [99]. Vaccines may require boosters due to waning immunity over time, emerging variants of the targeted virus, optimization of protection, changing disease prevalence, new research findings, prolonged vaccine rollout, and the evolving nature of infectious diseases. Booster shots help reinforce and extend the effectiveness of the initial vaccination. Public health authorities regularly assess the need for boosters to align with the dynamic nature of infectious disease dynamics and maintain robust immunity in the population [100]. During COVID-19 pandemic it was observed that unlike subunit vaccines the mRNA vaccines required multiple booster doses for efficacy.

6. The mRNA vaccine hesitancy and potential counter measures

The vaccine hesitancy is rooted far back in the history [101]. Fear about vaccines stemmed first from Edmund Massey’s Sermon in London on 8 July 1722, entitled ‘A sermon against the dangerous and sinful practice of inoculation.’ The sermon appears to have objected to the inoculation of any form to prevent disease, as Massey stated in his sermon ‘Let us not sinfully attempt to alter the Course of Nature’ [102,103]. The clearer anti-vaccine propaganda was observed when the smallpox vaccine was introduced, linking the vaccine to denial of rights” and antisocialism [104]. However, the recent mRNA vaccine hesitancy refers to the reluctance or refusal to receive COVID-19 vaccines that use messenger RNA (mRNA) technology and was produced by Pfizer-BioNTech and Moderna. There are various reasons why people may be hesitant about receiving mRNA vaccines. Some concerns may include the speed of vaccine development, potential side effects, misinformation or lack of information about the vaccines, and distrust of the healthcare system or pharmaceutical companies. It is important to note that mRNA vaccines have been extensively tested in clinical trials [23,24,105–108] and have been shown to be safe and effective in preventing COVID-19. The benefits of vaccination, such as reducing the risk of severe illness, hospitalization, and death, outweigh the risks of side effects. Public health officials and healthcare providers play a critical role in addressing vaccine hesitancy and ensuring that accurate information is available to the public. This can include providing education about the vaccines, addressing concerns and questions, and building trust in the healthcare system.

The vaccine hesitant people can be classified into two main groups based on the reasons for their vaccine denial. The first group called anti-vaxxer or vaccine deniers are the people who believe vaccines are not safe and have no health benefits. They do not take vaccines and discourage others to take them. Anti-vaxxers are highly motivated to take an anti-vaccine stance and discredit evidence-based medicine [101]. They appear to be politically, culturally, and/or socially motivated to discredit the vaccine benefits. For example, a recent study of more than 1,000 demographically representative participants found that about 22% of Americans self-identify themselves as anti-vaxxers, and tend to embrace the label as a form of ‘social identity’ [109]. The second group called Vaccine hesitant people show a very slow response in accepting a freely available vaccine. Their hesitancy is mainly based on the misinformation spread by the anti-vaxxers. Vaccine hesitant people are mostly concerned about the safety of vaccines and refuse vaccines until any safety concerns are clarified [110].

Some of the genuine reasons for vaccine hesitancy include (i) Safety: During COVID-19 pandemic, it was observed that some vaccine hesitant people were concerned about the safety of mRNA vaccines and were worried that mRNA vaccine could cause harm or have long-term side effects [111]. The speedy development and first-time use of mRNA vaccine created suspicion about its safety, despite detailed explanations provided by US government about it safe development processes [111]. This concern created hesitancy for SARS-CoV-2 mRNA vaccine even among well educated people having scientific background. However, as the safety of mRNA vaccine was proven and the benefits became evident, the mRNA vaccine uptake gradually increased [112] (ii) Lack of trust: Lack of trust in government or public health authorities, or mistrust of pharmaceutical companies that produce vaccines, is the another important factor that adds to vaccine hesitancy. A recent survey revealed that broader mistrust in public health institutions is a stronger and more consistent predictor of vaccine hesitancy [112]. (iii) Religious or cultural beliefs: Some people have religious or cultural beliefs that are in conflict with vaccination. For example, objection to vaccination was found to be related to the faith in divine protection and healing for Protestants, Catholics, Jewish and Muslims [113]. A questionnaire-based study revealed that religious taboos were among the main reasons for non-vaccination among Hinduism and Sikhism believers as well [114]. (iv) Misinformation: Misinformation or conspiracy theories about vaccines are the leading causes of vaccine hesitancy [115]. For example, misinformation spread through social media that vaccines can cause autism or mRNA vaccines are not safe, could be government plots to gain political benefits without caring about the damage to the public health [115]. (v) Fear of needles: Some people may have a phobia of needles, which can make getting vaccinated a difficult experience. A recent study revealed that across the adult population, blood-injection-injury fears may explain approximately 10% of cases of COVID-19 vaccine hesitancy [116]. (vi) Convenience: Some people, especially in poor countries, may not be able to get vaccinated because of logistical or practical issues, such as lack of access to transportation or time off work. (vii) Political beliefs: In some cases, vaccine hesitancy can be tied to political beliefs. A critical factor in so many people choosing not to be vaccinated during COVID-19 pandemic is their political views. A recent study demonstrated that in counties with a high percentage of Republican voters in the United States, vaccination rates were significantly lower and COVID-19 cases and deaths per 100,000 residents were much higher [117]

Combatting vaccine hesitancy is critical for achieving widespread immunity and controlling the spread of infectious diseases. Here are some potential means to combat vaccine hesitancy: Providing accurate information: Healthcare professionals and public health authorities should provide accurate and easy-to-understand information about vaccines, including their safety, efficacy, and potential side effects.

Addressing concerns: Addressing specific concerns about vaccines can help alleviate fears and build trust. For example, providing information about the rigorous testing and approval process for vaccines can reassure people of their safety. Using trusted messengers: Using trusted messengers such as doctors, nurses, and community leaders to convey information about vaccines can be effective in building trust and addressing concerns. Empowering individuals: Providing individuals with the resources and tools to make informed decisions about their health, including vaccination, can be empowering and increase uptake. Making vaccination easy and accessible: Making vaccination easy and accessible by offering flexible scheduling, convenient locations, and reducing out-of-pocket costs can increase uptake. Combating misinformation: Public health authorities should combat misinformation and conspiracy theories about vaccines by providing accurate information and addressing false claims. Leading by example: Public figures and leaders can help combat vaccine hesitancy by publicly getting vaccinated themselves and encouraging others to do so. By taking these steps, we can help combat vaccine hesitancy and promote widespread vaccination.

7. Regulatory issues and approvals

The regulatory considerations for mRNA vaccines closely resemble those relevant to all vaccines. These encompass guaranteeing the quality of initial materials, ensuring consistency in manufacturing processes, and presenting strong evidence of safety and efficacy through pre-clinical studies, clinical trials, and post-marketing surveillance (16). For over 70 years, the World Health Organization (WHO) has been tasked with establishing worldwide norms and standards for biologicals, including vaccines, to ensure their quality, safety, and effectiveness. Currently, WHO is actively engaged in new initiatives aimed at fostering a comprehensive consensus (16). These initiatives seek to develop international guidelines for the production and quality control, as well as the nonclinical and clinical assessment, of mRNA vaccines. The goal is to facilitate global alignment in manufacturing and regulatory practices, offering essential assistance to National Regulatory Authorities in WHO member states (118–120). In the United States, FDA plays an important role and has a rigorous process in place to ensure safety, effectiveness, quality, and licensing of vaccines. Distribution of a particular vaccine lot by manufacturers is only allowed upon FDA approval. The FDA reviews the manufacturer’s test results, which usually cover vaccine sterility, purity, potency, and consistency. Additionally, confirmatory testing may be conducted by the FDA before releasing a vaccine lot (121). The Center for Biologics Evaluation and Research (CBER) is the FDA branch responsible for regulatory oversight of vaccine development and to encourage and facilitate the development and availability of a needed vaccine, an expedited approval process is also available (121). The possibility of future pandemics is a cause of concern, but outbreaks can be controlled by improved preparation by the above agencies.

8. Expert opinion

The mRNA vaccine technology is already at the forefront of the fight against infectious diseases, most notably with the rapid development of COVID-19 vaccines like Pfizer-BioNTech and Moderna. However, like any medical technology, mRNA vaccines come with their own set of challenges that can be solved to make this technology of highest priority for vaccine development in future. The fragile mRNA vaccine must be stored at ultra-low temperatures, which can be logistically challenging in certain regions, especially in developing countries. This might contribute to global inequity in vaccine distribution. Chemical modifications of the mRNA molecule to improve stability and optimization of storage conditions will be required to overcome the challenges of short shelf-life of the mRNA vaccine, which is not the case with traditional vaccines. Unlike traditional vaccines, the scaling up production rapidly in response to a pandemic or other emerging infectious diseases can be challenging due to manufacturing complexity associated with cost and requirement of sophisticated production facilities. Although severe allergic reactions are rare, monitoring and managing the potential allergy risks due to the components of mRNA vaccine, especially the lipid nanoparticles will be critical for this vaccine technology. Since mRNA vaccine technology is relatively new, the long-term effects of these vaccines are not fully understood. Continuous monitoring and research are necessary to assess any potential long-term risks. Building public trust and providing accurate information is crucial to overcome public hesitancy or misinformation surrounding the mRNA vaccine. Despite these challenges, mRNA vaccine technology represents a significant advancement in vaccine development and has shown great promise in responding to emerging infectious diseases. Ongoing research, development, and improvements in manufacturing and distribution processes will likely address many of these challenges over time.

Article highlights

• The meticulous design and synthesis of mRNA that encodes the desired target antigen are pivotal elements in the development of mRNA vaccines.

• The selection of the right lipid nanoparticle (LNP) formulation is crucial for the safe and precise delivery of mRNA vaccines to the intended target cells.

• The safety of mRNA vaccine together with the duration and memory of immune response determines the level of protection offered to the immunized host.

• The adaptability of mRNA vaccines for swift modification in response to the emergence of mutant strains of infectious pathogens is a key advantage of mRNA vaccine technology.

• Implementing effective countermeasures to address vaccine hesitancy is crucial in any vaccine distribution program.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Author contribution

Both the authors contributed equally to the conception and design of the review article and interpreting the relevant literature, and have been involved in writing the original review article and its revised version.

Additional information

Funding

This paper was not funded.