Nucleic Acid‑based Vaccine Platforms Against The Coronavirus Disease 19 (COVID‑19)

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has infected 673,010,496 patients and caused the death of 6,854,959 cases globally until today. Enormous efforts have been made to develop fundamentally different COVID-19 vaccine platforms. Nucleic acid-based vaccines consisting of mRNA and DNA vaccines (third-generation vaccines) have been promising in terms of rapid and convenient production and efficient provocation of immune responses against COVID-19. Several DNA-based (ZyCoV-D, INO-4800, AG0302-COVID19, and GX-19N) and mRNA-based (BNT162b2, mRNA-1273, and ARCoV) approved vaccine platforms have been utilized for the COVID-19 prevention. mRNA vaccines are at the forefront of all platforms for COVID-19 prevention. However, these vaccines have lower stability, while DNA vaccines are needed with higher doses to stimulate the immune responses. Intracellular delivery of nucleic acid-based vaccines and their adverse events needs further research. Considering the re-emergence of the COVID-19 variants of concern, vaccine reassessment and the development of polyvalent vaccines, or pan-coronavirus strategies, is essential for effective infection prevention.

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Keywords

Coronavirus disease 19 · Nucleic acid–base vaccines · Immunity · Protection

Background

In late 2019, a novel beta coronavirus emerged in Wuhan, China, and rapidly spread worldwide. The Coronavirus disease 2019 (COVID-19) has a high potential of a pandemic due to its high contagious rate with high mortality globally (Sharma et al. 2020; Su et al. 2020; Wibawa 2021). Therefore, substantial efforts are needed to develop effective vaccines or therapies against the disease (Su et al. 2020). Symptoms of COVID-19 disease vary, including mild fu-like symptoms, pneumonia, acute respiratory distress syndrome (ARDS), and fatal outcome. Patients with cancer, diabetes, cardiovascular diseases, older adults, and even genetically predisposed individuals are at the highest risk of COVID-19 severity (Sharma et al. 2020; Su et al. 2020; Wibawa 2021; Vakil et al. 2022). As per the World Health Organization (WHO) recommendations, wearing masks, using antiviral drugs, social distancing, and adhering to vaccination procedures are crucial behaviors to control of COVID-19 pandemic around the world (Sharma et al. 2020). The scientific effort toward the development of efficient vaccines against invasive pathogens dates back many years since long (Deb et al. 2020; Zhang et al. 2020; Wibawa 2021). These vaccine platforms have also been designed against pathogenic bacteria (Farhani et al. 2019; Jafari and Mahmoodi 2021). In this regard, developing an efficient, protective, and safe vaccine is considered a pivotal preventive approach to hinder the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread (Moore and Klasse 2020). Therefore, different pharmaceutical companies and research teams worldwide competed to present a safe and efficient vaccine against COVID-19 for international community use. These efforts have developed other vaccine platforms to enter preclinical and clinical trials and some of them have been approved (Chen et al. 2021), including traditional vaccines such as live or inactivated, subunit, and nucleic acid-based vaccines as next-generation vaccines (Moore and Klasse 2020). Based on the scientific evidence, live-attenuated vaccines stimulate the innate, cellular, and humoral immune responses by inducing Toll-like Receptors (TLRs) with long-term immunity and may develop hypersensitivity. The main drawback of these vaccines is their costly safety and efficacy assessments. Inactivated viral vaccines poorly provoke cellular immune responses which mitigates their efficacy. In April 2020, an inactivated COVID-19 vaccine was manufactured by Sinovac and Wuhan Institute of Biological Products (Sinopharm) (Moore and Klasse 2020; Su et al. 2020). Subunit vaccines are safe, with some defects including low immunogenicity, booster or adjuvant requirement, and high cost (Koirala et al. 2020; Su et al. 2020). Nucleic acid-based vaccines have been developed based on sequence information. They include DNA or mRNA sequences of antigens that strongly stimulate cellular and humoral immune responses in various doses. Due to their advantages, such as fast production, and the earliest COVID-19 vaccines in clinical trials, a noticeable advantage of DNA-based vaccines is their stability in various storage conditions (Silveira et al. 2020; van Riel and de Wit 2020). RNA-based vaccines received more attention from pharmaceutical companies like Pfizer/Biotech and Moderna. In contrast to DNA vaccines, they stimulate effective humoral immune response as TLR ligands without adjuvant, and their sequence is modified to preclude mRNA degradation (Moore and Klasse 2020; van Riel and de Wit 2020; Soiza et al. 2021). This review aimed to assess recent developments in nucleic acid-based vaccines, including mRNA and DNA vaccines against COVID-19.

Main text

Coronavirus molecular mechanism of infection and immune response

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The COVID-19 genome is a positive single-stranded RNA that encodes four main structural proteins consisting of envelope (E), spike (S), membrane (M), and nucleocapsid (N) (Stadler and Rappuoli 2005), as represented in Fig. 1a. Human SARS-CoV encompasses spike (S) glycoprotein as a part of the envelope. Virus incorporation to host cells is performed by spike protein which consists of S1 and S2 subunits. They play an essential role in the attachment to host cell receptors known as Angiotensin-Converting Enzyme II (ACE2) for the infection initiation (Fig. 1b). Central region of the S1 domain binding to ACE2 with high affinity is a receptor-binding domain (RBD). In this regard, the interaction between the RBD and the ACE2 is required for human cell infection initiation (He et al. 2020). In addition, the S protein truncation is essential for viral entry into host cells; following RBD region attachment to ACE2, a host protease known as TM protease serine 2 (TMPRSS2) cleaves the S protein into S1 and S2 domains which leads to S2 domain fusion to the host cell (Huang et al. 2020). Although strong immune responses are effective against COVID-19, hypersensitivity, and cytokine storm (mostly interleukin-6-, IL-1b, GM-CSF-, interferon-γ/ IFNγ-, necrosis factor α/TNFα-, IL-10-, IL-2- and IL- 7-driven responses) must be prohibited (Chowdhury et al. 2020). Following viral attachment (spike-ACE2 interaction) and entry into respiratory cells, phagocytic alveolar macrophages and dendritic cells (DCs) present viral antigens to T cells and activate T CD4+(helper T cell) and T CD8+(cytotoxic T cell). Subsequently, proinflammatory cytokines like IL6, IL12, TNFα, and IFNγ, etc. are released to encounter the virus. However, high levels of cytokine production leading to the cytokine storm cause lung damage (Hosseini et al. 2020). According to scientific evidence, helper T cells are necessary for viral infection elimination, induction of B cells to produce antibodies, and stimulation of cytotoxic T cells (Sharma et al. 2020). COVID‑19 antigenic targets In developing a safe and protective vaccine against a pathogenic organism, identifying the best immunogenic targets is essential (Lu et al. 2020b). Adopting potential antigenic targets is critical to provoke T cell and B cell epitopes to properly induce cellular and humoral immunity (Rueckert and Guzmán 2012). The viral S protein interacts with the host cells via the RBD domain as an essential ligand. The RBD domain can induce neutralizing antibody production and T-cell immune response against COVID-19. Also, the immunogenicity of the S protein is confirmed (Pushparajah et al. 2021). The N protein has a small and highly conserved sequence compared to other viral proteins. The N protein is highly expressed during virus infection, with a significant associated humoral immune response against COVID-19 among patients. Additionally, with cellular responses against the N protein, it can be considered a proper candidate antigen in vaccine design (Dutta et al. 2020). In addition, the M protein and E protein stimulate T CD4+epitopes (Wang et al. 2005; Abdelmageed et al. 2020; Dong et al. 2020). Eliciting strong immune responses against the S protein is determining and necessary (Buchholz et al. 2004). The S protein includes the most immunogenic T cell and B cell epitopes known as preferred targets in vaccine development against COVID-19. It has been applied in all developed mRNA platform vaccines like Pfizer/BioNtech and Moderna vaccines.

Fig. 1 a The SARS-CoV-2 virion structure; major surface proteins of COVID-19 virus include Spike (S) glycoprotein, Membran (M), and Envelope (E) proteins. The S protein is the main vaccine and therapeutic target which interacts with the angiotensin-converting enzyme II (ACE2) receptor for infection initiation. b Domains of spike protein; the S protein includes S1 (NTD or non-translated domain and RBD or receptor binding domain) and S2 subunits

History of nucleic acid‑based drugs

Oligonucleotides were entered into clinical trials more than 30 years ago. The history of using nucleic acid-based therapeutic approaches dates to 1977 when Paterson et al. used nucleic acids to attune gene expression (Paterson et al. 1977). Currently, they have received more consideration. Nucleic acid-based drugs are divided into different categories, including antisense forms, ribozymes, mRNA, and DNA-based vaccines (Sharma et al. 2014). A synthetic oligodeoxynucleotide inhibited the replication of Rous Sarcoma Virus (RSV), which was complementary to the RSV mRNA and was known as antisense (Zamecnik and Stephenson 1978). The first antisense was entered into the clinic against Cytomegalovirus (CMV) (Mulamba et al. 1998). Small interfering RNA (siRNA) possesses the potential to inhibit gene expression and, for the first time, was reported in 1998. Another group of small non-coding RNAs includes microRNAs (miRNAs) possessing an indispensable role in the regulation of gene expression, with functions similar to that of siRNA and therapeutic potential (Usman and Blatt 2000; Sharma et al. 2014). As mentioned, the ribozyme class of RNA molecules acts as enzymes that target transcription. Ribozymes entered clinical trials against cancer and some viral infections such as human immunodeficiency virus (HIV) (Usman and Blatt 2000; Abera et al. 2012). In addition to mRNA- and DNA-based vaccines, they are classified in nucleic acid-based drugs introduced in 1990 and received more attention in the development of vaccines to combat COVID-19 (Le et al. 2020; Zhang et al. 2020). Major advantages of these groups of vaccines include their rapid production and high specificity against corresponding target antigens (Le et al. 2020).

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mRNA‑ based vaccines

The initial delivery of mRNA molecules into host cells included the mRNA transfection into fibroblast cells using a cationic lipid (Park et al. 2021). mRNA vaccines include pathogen antigens mRNA which produce antigenic proteins by human cells. Several advantages of mRNA vaccines include a simple production process, efficient and protective immunity, convenient manipulation and industrialization, and flexibility to respond to COVID-19 variants (Kaufman et al. 2016; Fang et al. 2022). Some approaches, such as the addition of 5ʹ-cap and Kozak sequences, are applied using 3ʹ-poly-A sequences and modification of mRNA nucleosides (Borah et al. 2021). The mRNA vaccines are translated into the host cytosol. Therefore, there is no risk to host genomes insertion, which is known as their main advantage. In this regard, mRNA-based vaccines received more attention as a safe preventive approach against cancer and infectious diseases recently (Kaur and Gupta 2020). The primary mechanism of action of mRNA vaccines has been depicted in Fig. 2.

DNA‑ based vaccines

DNA vaccines comprise several genes encoding viral antigenic peptides expressed by plasmid vectors and transmitted to cells via electroporation (EP). Compared to other vaccine platforms, DNA vaccines propose a flexible and rapid platform for developing vaccines, making it a fascinating technology to fight developing epidemics like COVID-19. Moreover, DNA vaccine antigen production occurs in target cells, which assists in recapitulating the viral antigen’s natural conformation and post-translational modification. The main disadvantage of DNA vaccines is their restricted immunogenicity. Hence, it is important to consider strategies such as adjuvant or the use of a prime-boost regimen that could increase the DNA vaccine’s potential. Also, integrating nucleic acid into the host DNA is another biosafety concern resulting in oncogenesis and mutagenesis (Rauch et al. 2018). Although according to prior studies, the risk of DNA vaccine insertion is negligible, the WHO and FDA recommend the implementation of DNA vaccine safety for integration (Wang et al. 2004; Schalk et al. 2006). DNA vaccines transfer the coronavirus genes into human cells. The principle of vaccination depends on the DNA delivery into the cell nucleus such that the antigen transcription is started and followed by a translation. DNA vaccines commonly utilize plasmids as vectors. Based on the vaccine administration route, both myocytes and keratinocytes are addressed. However, antigen-presenting cells near the injection site may also be transfected straight using DNA vaccines. In such instances, the cross-priming process represents antigens using both major histocompatibility complex (MHC-I/II) molecules (Hobernik and Bros 2018). The generated antigens are released using apoptotic bodies or exosomes which result in their recognition by antigen-presenting cells, which in turn provoke cellular and humoral immune responses. Diverse delivery strategies are utilized to produce a strong immune response (Donnelly et al. 2005; Li and Petrovsky 2016; Strizova et al. 2021). Concerning the immune regulation during COVID-19 infection, it has been revealed that patients at risk with pericardial effusion with a mis-prognosis indicate raised CD3+CD8+ T cells plus diminished CD14+HLA-DR and T regulatory (Treg) cells (Duerr et al. 2020). These results demonstrate that severe infection occurs due to an imbalanced immune response which exacerbates the disease conditions (Tay et al. 2020). COVID-19 vaccine progress aimed to develop an effective and appropriate immune response (including both arms) without progress to such imbalance (Hobernik and Bros 2018). While the human clinical trials by DNA vaccines triggered total responses, these responses are frequently inadequate to prompt acceptable clinical advantages. Additionally, basic components of plasmid DNA, for example, unmethylated CpG sequences, may cause activation of innate immune responses, increasing adaptive immune responses against the expressed antigens. Thus, DNA vaccines are more applicable in veterinary medicine (Coban et al. 2013; Silveira et al. 2017; Hobernik and Bros 2018). Due to this drawback, some research lines concentrate on the optimization and delivery of DNA vaccines, including codon optimization, promoter design, molecular adjuvants, EP application, prime-boost vaccination, or “omics” methods for advanced vaccine design (Li et al. 2012; Silveira et al. 2020). Figure 3 illustrates the main stages of DNA vaccine mechanisms in effective immune responses.

Fig. 2 mRNA vaccine formulation, administration, and mechanisms of immune provocation: an mRNA formulation with a lipid nanoparticle (LNP) maintains and enhances the sequence stability. b A saline solution of mRNA-LNP vaccine is administered by intramuscular route. c The LNP-containing mRNA is transfected into the antigen-presenting cells (APCs) through endocytosis. The mRNA is released in the cytoplasm and translated into viral proteins, then they are lysed by the proteasome and bind to the major histocompatibility complex I (MHC-I) onto the surface of the endoplasmic reticulum and presented to T cytotoxic (Tc) cells

The potential of mRNA‑ and DNA‑based vaccines

An mRNA-level vaccine structure contains elements to remediate stability and protein expression including the 3′ UTR, coding sequence, 5′UTR, 5′ cap, and poly (A) tail (Liu 2019). The 5′ and 3′ UTR elements flanking the coding sequence derived from viral or eukaryotic genes enhance the structural stability and improve the translation of mRNA which are essential factors for vaccines (Ross and Sullivan 1985; Holtkamp et al. 2006). For efficient protein translation from mRNA, a 5′ cap structure is needed (Martin et al. 1975; Ross and Sullivan 1985). The mRNA poly (A) tail also has a regulatory role in mRNA stability and translation (Holtkamp et al. 2006). Also, codon usage has a vital role in protein translation. In this regard, to increase protein translation from mRNA, rare codons are replaced with used synonymous codons that have plenteous cognate tRNA in the cytosol (Stepinski et al. 2001). Another approach for sequence optimization is the enrichment of G: C content. Several methods have developed efficient and safe candidate DNA vaccines in recent years. In a DNA-based vaccines platform, bacterial-derived plasmid vectors are applied to express those desired antigens within host cells. The bacterial plasmids must be entered into eukaryotic cells and translocated to the nucleus. The DNA is then transcripted and translated from foreign genes in the host cell’s nucleus and cytoplasm, respectively. Therefore, designing a proper plasmid with high transfection efficiency and protein expression is essential to reach a potent DNA vaccine (Malone et al. 1989). The sequence of the eukaryotic region (in DNA vaccines plasmids), the upstream of inserted gene, is composed of a promoter and a poly A signal (polyA) (AAU AAA) located at the 3′ ends of the antigenic sequence (Shan et al. 2011). Promoters are critical elements needed in DNA vaccine plasmids to provoke high expression levels of the desired antigens in the host cells (Becker et al. 2008) and result in the mRNA transcription from the inserted gene. The most commonly used promoter in DNA vaccines includes the human Cytomegalovirus (CMV) promoter. The polyA sequence signal causes mRNA stability and transfer and eukaryotic gene expression efficiently. Another critical element is a Kozak sequence (ACCATGG) which has a vital role in translation by the eukaryotic ribosome. Also, adding one- or two stop-codon sequences is necessary to avoid incorrect translation of the inserted gene in the host cell (Becker et al. 2008; Williams 2013). Comparison of DNA and mRNA vaccine platforms Although DNA and mRNA vaccines have been under development since the 1990s and recent development and the licensure of various veterinary DNA vaccines, considerable enthusiasm has turned to mRNA. Both need efforts to enhance their antigenicity, stability, and efficacy by manipulating plasmid DNA and mRNA straight or adding immunomodulators or adjuvants and formulations and delivery systems (Liu 2019). The stability duration of mRNA is lower than that of plasmid DNA. It has been unraveled that DNA vaccines generate the encoded protein quickly, being higher amounts than mRNA vaccines due to the higher intrinsic plasmid DNA stability. It has been found that plasmid DNA persists in muscle for up to 6 months in a nonintegrated mode (Ledwith et al. 2000). Although DNA and mRNA vaccines are supposed to be an expression system for the favorite protein, none are immunologically inert. Also, DNA plasmids as well as mRNA stimulate innate immunity (Campbell 2017). The DNA and mRNA-based vaccine technology might not be wholly general. mRNA is more complicated than plasmid DNA due to modified nucleosides and formulations required for delivery, stability, and the necessity to control the intrinsic immunostimulatory activity of the mRNA. Nevertheless, it benefits production that evades the demand for each cellular or animal product. The promise is that clinical achievements will be feasible after that plasmid DNA and mRNA vaccine challenges be resolved particularly by applying advanced technologies to prevent and treat diseases (Liu 2019).

Fig. 3 The process of COVID-19 DNA vaccine expression into the antigen-presenting cells (dendritic cells or DCs). The plasmid DNA enters into the DC nucleus and expresses antigens in vivo (1) Then, the antigens are presented to the T cells (T cell receptors or TCRs) via both MHC-I and MHC-II molecules. Antigen presentation occurs via the DC CD80/86 molecules and CD8+T cell CD28 molecules alongside the MHC-I-TCR and also via the DC CD40 and CD4+T cell CD40L alongside the MHC-II-TCR interactions (2a, 3 and 4) The activation and proliferation of the CD8+T cells and cytokines’ release leads to   effective immune responses such as macrophages activation (2b) Additionally, the activation of the CD4+T cells following binding to and cytokines effects from DCs is associated with the B cells activation, proliferation, and antibodies’ secretion (5)

Progress in DNA and mRNA vaccine delivery

Foreign DNA and RNA uptake by eukaryotic cells is not efficient compared to viral vectors. Many physical delivery approaches have been established to increase cellular uptake capacity (Mellott et al. 2013). Physical methods of gene transfection include delivery systems transferring genetic material via mechanical procedures, including EP and projector devices (Gulce-Iz and Saglam-Metiner 2019). Biojector devices utilize the CO2 pressure to transport therapeutics through IM (Intramuscular), ID (Intradermal), and also SC (Subcutaneous) administration without the needle requirement (Jorritsma et al. 2016), giving considerable advantages over conventional needle injection, including mitigation of adverse effects, needle cross-contamination, and needle-stick damages (Zhang et al. 2015). Relevantly, two DNA vaccines have been assessed against the Zika virus in the phase I trial, demonstrating increased cellular responses following needle-free administration compared to needle use (Gaudinski et al. 2018). Moreover, an mRNA vaccine delivery platform against rabies utilizing a projector revealed enhanced antibody responses (Alberer et al. 2017). The increased vaccine effciency through the jet injection can be ascribed to a broader distribution of vaccines, thus resulting in higher uptake using APCs (Williams et al. 2000; Alberer et al. 2017). Currently, ID or IM injections followed by EP are usually utilized for DNA vaccine delivery in clinical studies (Sardesai and Weiner 2011). EP includes pore formation within the skin cells to increase cellular uptake of the genetic material using electric pulses (Pushparajah et al. 2021). The IM EP method was primarily applied in 1998 (Aihara 1998) enhancing the penetrability of the muscle cells for the delivery of DNA vaccines (Rizzuto et al. 1999; Dupuis et al. 2000; Sokołowska and Błachnio-Zabielska 2019). Numerous reports have revealed better antigen expression and increased antigen-specific immune responses in vivo using EP (Yan et al. 2008; Yan et al. 2009). It has been reported that an HIV DNA vaccine, ADVAX, increased the immunogenicity following the transfer through the EP (Vasan et al. 2011), compared to the IM administration (Vasan et al. 2010). Nevertheless, the EP approach suffers from a cell death risk due to the utilization of high voltages (Gulce-Iz and Saglam-Metiner 2019). Novel delivery approaches, such as EP, are yet under investigation for RNA vaccine delivery.

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The EP efficacy for self-amplifying vaccine delivery has been similar to that for plasmid DNA vaccines in terms of gene expression and immune responses in mice (Cu et al. 2013). However, the EP has not enhanced the delivery efficacy of traditional non-replicating RNA vaccines (Johansson et al. 2012), possibly reducing its effectiveness in duplicating RNA vectors. Accordingly, the EP and bio injectors are advantageous for the parenteral injection of COVID-19 DNA/RNA vaccines. These approaches contribute to higher production and the delivery of DNA vaccine candidates and improve outcomes remarkably (Pushparajah et al. 2021).

Benefits and limitations of nucleic acid‑based vaccines

As novel and promising methods of immunization, nucleic acid-based vaccines provide insights into developing safe and protective vaccination (Choi and Chang 2013) as demonstrated by millions of people during the COVID-19 pandemic. Nucleic acid-based vaccines have short development cycles, facilitating rapid distribution during a pandemic. Utilization of recombinant DNA vaccines needs efficient transferring of DNA vector into the nucleus, transcription, and then translation into the desired antigen (Leitner et al. 1999). Due to convenient manipulation and low-cost production, naked plasmid DNA is a fascinating vector for presenting antigens (Williams 2013). Usually, a plasmid DNA comprises basic genetic elements, including the antigen-encoding gene, a promoter, enhancers, and transcription termination/polyadenylation sequences (Vogel and Sarver 1995). Plasmid DNA platform is a promising bio-pharmaceutical construct replicated in high levels within inexpensive prokaryotic cells though requiring purification (Prazeres et al. 1999; Ferreira et al. 2000; Suschak et al. 2017). RNA vaccines are composed of mRNA molecules containing an antigen RNA surrounded by 3′, poly-A tail, and 5′ termini and lack the requirement for transcription. (Zhang et al. 2019). Several RNA vaccines undergo self-amplification known as new technology under development. Accordingly, the RNA molecule can be replicated and translated in the host after delivery, despite the possibility of naked RNA instability, thus increasing the expression of immunogenic peptides (Pardi et al. 2018; Zhang et al. 2019; Wadhwa et al. 2020). The mRNA is degraded by ubiquitous ribonuclease enzymes (Wadhwa et al. 2020; Xu et al. 2020). The addition of a 3′ poly-A tail and a 5′–7-methylguanosine cap is critical for increasing the stability and translation of mRNA in the cytosol (Gallie 1991; Schlake et al. 2012). Strategies for increasing vaccine uptake and expression have been chiefly evaluated for DNA vaccines than for RNA vaccines, as the DNA requires crossing two cell membranes to reach the nucleus. In contrast, the RNA penetrates the cytoplasm through a single membrane (Rauch et al. 2018). The DNA structure has comparably higher stability. Over 7 years, it was found that plasmid DNA remained intact without difference with newly provided DNA kept at −20 °C (Walther et al. 2013; Pushparajah et al. 2021). Low pH and temperature are critical for maintaining DNA integrity for a long time. Conversely, the RNA is extremely temperature-sensitive and must be kept at −70 °C in the enzyme-free medium to enhance its half-life (Jones et al. 2007).

Challenges in developing nucleic acid‑based vaccines

It is well known that COVID-19 vaccines must be efficient and protective enough (Graham 2020) and elicit long-lived immunity. However, annual vaccination may be possible based on annual flu vaccine experiences (Randolph and Barreiro 2020; van Riel and de Wit 2020). Developing the COVID-19 vaccine poses challenges even with new platforms. Despite the high immunogenicity and protection of the coronavirus spike protein vaccine, the occurrence of mutations makes concerns and re-emergence of the virus. Hence, the prognosis of the time and location of disease emergence along with the accurate adoption of the target protein sequence is the forefront stage of the development process needing proper implementation of clinical trials (Lurie et al. 2020). The main challenge of DNA vaccines is eliciting relatively lower immune responses in humans and large animals than in small animal systems (Grunwald and Ulbert 2015; Suschak et al. 2017). Noticeably, further challenges remain to be addressed regarding COVID-19 vaccines such as protection durability, effectiveness in specific subgroups, preventing viral transmission, and public acceptance (Pushparajah et al. 2021). The delayed antigen expression in self-amplifying mRNA vaccines might limit these vaccines' efficacy. However, this platform gives higher yields and thus provides equivalent protection at significantly lower doses (Vogel et al. 2018; Strizova et al. 2021).

Recent advances in COVID‑19 DNA and mRNA vaccines

In vaccine development against COVID-19, numerous studies have focused on mRNA vaccine platforms (Verbeke et al. 2021) leading to the approval of several vaccines (Vitiello and Ferrara 2021) such as those from Moderna and Pfizer/ BioNtech companies. The Moderna vaccine includes the entire encoding of COVID-19 spike glycoprotein mRNA, while Pfizer/BioNtech contains the RBD mRNA sequence (Brüssow 2020). The Pfizer/BioNTech and Moderna vaccines' efficacy levels include 95% and 94.5%, respectively (Rauch et al. 2021; Widge et al. 2021). The Moderna vaccine storage temperature is between −25 °C and −15 °C, while that of the Pfzer vaccine is between −80 °C and −60 °C (Meo et al. 2021; Rauch et al. 2021). Compared to the Moderna vaccine, Pfizer/BioNTech vaccine has lower costs and side effects (Rauch et al. 2021). Another mRNA vaccine against COVID-19 was developed by Chinese researchers and used thermostable (at least one week at 25 °C) RBD-encoding mRNA (Brüssow 2020). In another survey, CureVac has been used as the engineered full-length S protein mRNA platform for COVID-19 vaccine development with substitutes in two proline residues to improve protein stability (Rauch et al. 2021). Ruklanthi de Alwis et al. (2021) developed a self-transcribing and replicating mRNA vaccine for COVID-19 using full-length S protein and a replicon (de Alwis et al. 2021) with the potential of application as an effective and safe single-dose vaccine to combat the COVID- 19 (de Alwis et al. 2021). Similarly, a self-amplifying RNA encoding the COVID-19 S protein encapsulated within a lipid nanoparticle (LNP) was employed and outlined high titers of antibody and cellular immune responses (McKay et al. 2020). Some DNA vaccine candidates such as the S, N, and M protein-based vaccines have been developed for SARS-CoV. Among these, the S protein-based DNA vaccine has effectively induced a protective effect against COVID-19 infection, possibly owing to the vital role of the S protein in receptor binding (Zhao et al. 2020). INO-4800 is a COVID-19 DNA vaccine candidate encoding the COVID-19 S protein (Sarwar et al. 2020; Smith et al. 2020). The pre-clinical results in mice and guinea pigs demonstrated humoral and cellular immune responses. In phase, I clinical studies, two doses of INO-4800 were injected via the ID path, supplemented with EP via CELLECTRA®2000 Inovio Pharmaceutical (Diehl et al. 2013; Amante et al. 2015). A complete immune response was explained based on antibody and cellular responses in 34 of the 36 participants in their phase I clinical trial. Ten reported adverse effects (AEs) were observed without any serious adverse events (SAEs) (phase). A phase I/II clinical trial was launched to evaluate the immunogenicity, safety, and tolerability of INO- 4800 (Tebas et al. 2019). The INO-4800 has similar storage conditions (Smith et al. 2020), hopeful for simpler vaccine distribution. In addition, some other groups of COVID-19 DNA vaccines have launched trials. In June 2020, a phase I and IIa clinical trial for the GX-19 launched recruitment. A DNA vaccine, AG0301-COVID-19, produced by the cooperative attempts of Osaka University (Japan), Takaro Bio, and AnGes, launched recruitment for the phase I and II clinical trials in July 2020 to assess its immunogenicity and safety (Speiser and Bachmann 2020). Phase I and II clinical trials to evaluate the immunogenicity and safety of three doses of ZyCoV-D were performed (Kumar et al. 2021). This oral vaccine encoded spike protein in the plasmid DNA, dynamically amplified in the live Bifidobacterium longum, a well-recognized intestinal probiotic bacterium. Another phase I clinical trial was launched to assess CORVax12, a DNA vaccine encoding the spike protein. The efficiency of electroporated CORVax12 alone or in combination with another plasmid encoding interleukin 12 (IL-12) was investigated. A variety of COVID-19 mRNA vaccines are under development and have unveiled promising results (Leitner et al. 1999; Croyle et al. 2001). A diverse approach to developing RQ3013-VLP (encoding S, E, and M proteins) was efficient in vivo using an mRNA cocktail. This mRNA vaccine was integrated with changed nucleosides and then packed in LNP and unraveled the capability to elicit strong cellular and humoral immune responses in mice (Le et al. 2020; Lu et al. 2020a). An engineered arginyl-glycyl-aspartic acid (RGD) domain DNA vaccine in two doses of 60 µg using electroporation improved the effects in BALB/cJ mice (Guo et al. 2021). The IM +jet injection of a DNA vaccine in a single dose (0.2 mg) could immunize the Syrian hamsters (Brocato et al. 2021). Another DNA vaccine using the S protein subjected in three doses protected the Rhesus macaques (Yadav et al. 2021). The fusion of RBD to the hepatitis B virus preS1 amino-terminal and IM injection for three doses (weeks 0, 2, and 4) in C57BL/6 mice provoked humoral and cellular immune responses (Jeong et al. 2021). Additionally, the IM injection of S protein plasmid DNA and S1 subunit (recombinant protein) in three doses at weeks 0, 2, and 8 in Rhesus macaques stimulated the neutralizing antibodies (Prompetchara et al. 2021). The IM+EP injection of S protein in three doses (weeks 0, 2, and 4) in ICR mice stimulated humoral and cellular responses (Li et al. 2021). Other DNA vaccines in clinical trials have included S protein in phases I/II in June 2022 (NCT04445389, IM route in adults aged 18–50 years old), July 2021 (NCT04463472, IM route in adults aged 20–60 years old), September 2021 (NCT04527081, IM route in adults of 20–65 years old), and phase I (NCT04336410, ID route in 18 years and older), February 2022 (NCT04334980, oral in adults with 18 years and older) and June 2021 (NCT04591184, IM route in adults 18–84 years). Additionally, mRNA vaccines in clinical trials included those in phase II in November 2021 (NCT04515147, IM, 18–60 years old), phase II-III in December 2022 (NCT04368728, IM in adults 18–85 years old), and phase I in June 2021 (NCT04566276, IM in adults 18–75 years old). COVID-eVax was an RBD-based vaccine that elicited sufficient immune responses in mice, ferrets, and rats after 38 days (Conforti et al. 2022). Two (X-19 and GX-19N) DNA vaccines encoding spike and nucleocapsid proteins were evaluated in phase I trials among adults aged 19–49 years and the binding antibodies were detectable at the second dose of vaccination. The safety and tolerability of these vaccines were confirmed, where the GX-19N induced higher levels of T cells and antibody responses (Ahn et al. 2022). An Xcl1-SARS-CoV-2 spike fusion DNA Vaccine elicited a higher rate of antibody and T cell-mediated responses compared to a plasmid-containing spike gene singly in vitro and in vivo (Qi et al. 2022). A recent baculoviral COVID-19 Delta DNA vaccine could protect 100% of mice against COVID-19 (Jang et al. 2023). A linear DNA (linDNA) encoding the SARS-CoV-2 RBD (Lin-COVIDeVax) was able to elicit antibody and T cell responses and provide safety and lacked adverse effects (Conforti et al. 2023). Table 1 represents clinical trials and approved nucleic acid-based vaccines against COVID-19. According to updated data, 229, 820, 324 individuals have received COVID-19 vaccines worldwide among whom, those from African countries have had lower rates of vaccination (https://www.usnews.com/news/coronavirus-and-vaccine-news, https://www.bloomberg.com/graph ics/covid-vaccine-tracker-global-distribution/). Accordingly, the vaccination rate is associated with the economic conditions of various regions/areas.

Potential safety concerns for COVID‑19 vaccines

Monitoring safety in every developed preventive or prophylactic vaccine is one of the essential aspects. The use of toxic chemicals or cell culture is not required in mRNA vaccine production processes, thus known as a safe platform. The short manufacturing time also reduces the risk of contamination by microorganisms (Wang et al. 2020). The most commonly reported effects include headache, fatigue, and other systemic severe events, such as fever, chills, myalgia, vomiting, abdominal pain, and rare diarrhea reports. However, no death has been observed in mRNA vaccine recipients (Chapin-Bardales et al. 2021; Skowronski and De Serres 2021). Some systemic side effects, such as anaphylaxis, are usually reported in individuals with a history of allergy and are estimated at 2.5–11.1 cases per million doses (Shimabukuro et al. 2021). Strong immune response in people younger than others may lead to high systemic events and more side effects are reported following the injection of a second vaccine dose compared to the first dose (Male 2021; Skowronski and De Serres 2021). In addition, pain at the injection site, redness, and swelling are reported as the most local side effects (Anand and Stahel 2021). Based on sufficient evidence from approved mRNA vaccines including Pfizer/BioNTech and Moderna, no risk for miscarriages in pregnancy (rate=0%) has been demonstrated (Male 2021). Noticeably, DNA vaccines are safe enough, but not always immunogenic; hence sufficient immune response needs additional doses. The humoral immune response has not been consistent in human trials, while cellular immunity seems more common. Accordingly, the safety of DNA vaccines among older and younger populations is more auspicious (Ledgerwood et al. 2011; Houser et al. 2018; Carter et al. 2019). The safety concerns hint at the possible integration of transfected DNA into germline and somatic cells of the host. In these cases, the dysregulation of gene expression possibly occurs along with multiple considerable mutations. Nevertheless, only extrachromosomal and deficient chromosomal integration plasmids are typically used in DNA vaccine development. Besides, most plasmids remain at the administration site (Schalk et al. 2006). A recent systematic review and meta-analysis demonstrated that mRNA vaccines are associated with higher adverse events compared to other platforms (Kouhpayeh and Ansari 2022). Recently, a rare case of BNT162b2 mRNA vaccine-associated myositis was observed in a 34-year-old woman (Magen et al. 2022). DNA methylation and corresponding epigenetic alterations also disturb the efficacy of DNA and mRNA vaccines (Pang et al. 2022). Noticeably, several approved nucleic acid vaccines (ZyCoV-D, DNA plasmid vaccine utilized intradermally, India), BNT162b2 (mRNA, 2 doses, Germany), mRNA- 1273 (Moderna, USA, 2 doses), ARCoV (WALVAX, China) and clinical trials including 302-COVID19 (DNA plasmid vaccine, phase II/III intramuscular, Japan), INO-4800 (DNA plasmid, phase II/III, intradermal, China), GX-19N (DNA vaccine, Genexine, phase II/III), Covigenix VAX-001 (DNA vaccine, Entos Pharmaceuticals, phase I/II, intramuscular), COVID-eVax (DNA vaccine, phase I/II, intramuscular, Rome) and bacTRL-Spike (DNA vaccine, phase I, oral, Symvivo) have been developed (Sheridan 2021; Liu and Ye 2022a). These vaccines have stimulated both humoral and cellular immunity except for GX-19N and AG0302- COVID-19. Some adverse effects of COVID-19 nucleic acid-based vaccines include pain, lymphadenopathy, erythema, redness, swelling, nausea, fatigue, arthralgia, myalgia, fever, cardiorespiratory arrest, stroke, hypersensitivity reaction, alcoholic liver disease, Bell’s palsy, paroxysmal ventricular arrhythmia and death (Norquist et al. 2012; McNeil and DeStefano 2018; Baden et al. 2021; Momin et al. 2021; Liu and Ye 2022b, 2022a).

Phenylethanol glycoside is the main active component of Cistanche deserticola

Conclusion

The rapid COVID-19 pandemic has made an unmet need for developing efficient vaccines to prevent the disease. Although DNA vaccines’ immunogenicity in animals is acceptable, clinical validation is warranted in humans. RNA vaccines might provide appropriate immunological features and considerable advantages over DNA vaccines. Problems with the unstable nature of the RNA have been addressed using appropriate storage approaches and formulations to cease its degradation. Vaccine safety is also important and could not be compromised for greater efficiency. There are approved nucleic acid-based vaccines to control the COVID-19 spread. The follow-up of participants must be continued. There is a need for learning regarding nucleic acid-based COVID-19 vaccine's side effects. Moreover, vaccine reassessment and polyvalent vaccines development, or pan-coronavirus strategies is promising considering the re-emergence of new variants of concern.

Table 1 Clinical trials and approved COVID-19 vaccines and their characteristics

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