Have mRNA vaccines sentenced DNA vaccines to death?

ABSTRACT

Introduction

After receiving emergency approval during the COVID-19 pandemic, mRNA vaccines have taken center stage in the quest to enhance future vaccination strategies for both infectious diseases and cancer. Indeed, they have significantly overshadowed another facet of genetic vaccination, specifically DNA vaccines. Nevertheless, it is important to acknowledge that both types of genetic vaccines have distinct advantages and disadvantages that set them apart from each other.

Areas covered

In this work, we delve extensively into the history of genetic vaccines, their mechanisms of action, their strengths, and limitations, and ultimately highlight ongoing research in key areas for potential enhancement of both DNA and mRNA vaccines.

Expert Opinion

Here, we assess the significance of the primary benefits and drawbacks associated with DNA and mRNA vaccination. We challenge the current lines of thought by highlighting that the existing drawbacks of DNA vaccination could potentially be more straightforward to address compared to those linked with mRNA vaccination. In our view, this suggests that DNA vaccines should remain viable contenders in the pursuit of the future of vaccination.

KEYWORDS:

• DNA vaccine
• mRNA vaccine
• encapsulation
• formulation
• adjuvants
• innate response
• antibody response
• T-cell response

1. Genetic vaccines: once upon a time…

Genetic vaccines show promise for immunization and therapy against infectious diseases and oncological malignancies. Both DNA and mRNA vaccines have been studied for decades, but only recently have they gained approval for human use. Both approaches involve genetic sequences encoding the antigen of interest. These vaccines can be delivered in either circular or linear forms; however, the currently approved nucleic acid vaccines consist of circular DNA plasmids and linear mRNA sequences [1–3].

In the early 1990s, experiments in mice showed that gene gun administration of DNA plasmids encoding the human growth hormone sequence did not increase mouse growth but did lead to the development of a humoral response against the human protein. Shortly after, several groups successfully reported that the administration of DNA plasmids intramuscularly, orally, and by gene gun in mice led to the development of antibodies [4]. Furthermore, mice immunized with plasmids encoding influenza antigens, whether administered intramuscularly or via gene gun, were protected against infectious virus challenges [4]. Specific CD8⁺ and CD4⁺ T-cells targeting the influenza nucleoprotein were also observed following intramuscular immunization of mice with DNA plasmids encoding the antigen’s sequence [5,6]. The success demonstrated by these early results prompted further research on DNA vaccines for numerous human pathogens. Immunization programs involving DNA vaccines against infectious agents and cancers, such as human immunodeficiency virus (HIV), Zika virus, Lyme disease, breast cancer, and cervical cancer, among others, have been ongoing for many years. Many of these vaccines have shown promising results in pre-clinical studies using rodent and non-human primate disease models. Clinical trials have been conducted for various DNA vaccination programs, and they have demonstrated safety and successful translation from pre-clinical to clinical settings [1,7]. Unfortunately, clinical trials also revealed that DNA vaccines failed to induce adequate levels of immunity for achieving full protection against pathogens and complete tumor regression. It is worth noting that many of the initially chosen targets were pathogens with a high degree of antigenic variation, such as HIV and malaria. Additionally, genetic therapeutic strategies for oncological malignancies necessitated the co-administration of adjuvants to achieve substantial tumor clearance [8]. Nonetheless, research endeavors with this platform resulted in licensed animal DNA vaccines. In 2005, DNA vaccines for salmon against pancreas disease infection and infectious hematopoietic necrosis, as well as for horses against West Nile virus, received approval. Three years later, a DNA-based therapy for breeding sows was sanctioned, involving the administration of a plasmid encoding the porcine growth hormone-releasing hormone (GHRH) sequence to prevent mortality, morbidity, and enhance the number of weaned piglets. In 2010, a DNA vaccine for dogs against melanoma became commercially available [9,10]. Concerning human applications, ZyCoV-D, developed by Zydus Healthcare Ltd., stands as the pioneer (and currently the only) DNA vaccine for humans. It was created and authorized in India as an emergency-use immunization approach against SARS-CoV-2 in 2021, exhibiting an efficacy rate of 67% [11,12]. Numerous DNA vaccines have undergone testing and are presently in clinical trials targeting a range of pathogens, including but not limited to the Zika virus [13], HIV, Ebola virus, SARS-CoV-2 [11,12,14], Andes virus [15], among others. In the realm of non-communicable diseases, DNA immunotherapies have been investigated for conditions such as head and neck cancer, breast cancer, prostate cancer, ovarian cancer, anal dysplasia, glioblastoma, and cervical dysplasia [14,16].

Concerning mRNA vaccines, their origins also trace back to the early 1990s when researchers first conducted intramuscular injections of naked mRNA in mice. This led to the successful translation of reporter proteins [17]. In the subsequent years, the concept of mRNA vaccination gained further ground as experiments involving the administration of mRNA encapsulated within liposomes and mRNA packaged into infectious suicide Semliki Forest virus particles (an early version of self-amplifying mRNA), both encoding the influenza nucleoprotein, yielded measurable T-cell responses in mice [18,19]. The next year, mRNA technology was extended into the field of cancer. By encoding a human carcinoembryonic antigen (CEA) in mRNA, an immune response against CEA-expressing tumor cells was elicited in a mouse model [20]. Six years later, the first human clinical trial involving autologous dendritic cells transfected with prostate-specific antigen mRNA as a therapeutic vaccine against metastatic prostate tumors was conducted, demonstrating the elicitation of tumor-specific T-cell responses [21]. However, synthetic mRNA tends to be naturally too immunogenic, leading to high levels of type-I interferons in the serum, which can be harmful to vulnerable patients. Furthermore, since synthetic mRNA is susceptible to in vivo degradation, the field of mRNA vaccination did not truly take off until the breakthrough discovery that the incorporation of pseudouridine into mRNA enhances translational capacity and stability while reducing immunogenicity [22]. This discovery was subsequently followed by numerous studies and additional clinical trials for both cancer and infectious diseases [23–25], and was ultimately considered fundamental in enabling the development of effective mRNA vaccines against Covid-19, resulting in the awarding of the 2023 Nobel Prize in Medicine to its inventors, Katalin Karikó and Drew Weissman [26]. A human clinical trial conducted in 2008 showed that, following intradermal injections of naked mRNA encoding melanoma antigens, both cellular and humoral responses were observed in melanoma patients. Although no clinical benefit was attained, the trial confirmed the safety of this genetic approach [27]. Five years later, clinical trials of mRNA vaccines in the field of infectious diseases were initiated, utilizing the genetic sequences of the rabies virus glycoprotein [28], followed by others, including those encoding the influenza hemagglutinin (HA) antigen [29] or the preMembrane/Membrane and envelope proteins from the Zika virus [30]. Administration of autologous dendritic cells (DCs) transfected with mRNA encoding the cytomegalovirus (CMV) pp65 antigen [31] or various HIV-1 antigens [32–35] was also tested and some induced strong cellular responses. However, it was only after more than 30 years of research, similar to DNA vaccines, that mRNA vaccines were approved for emergency use in humans in 2020 during the COVID-19 pandemic. These vaccines include Comirnaty/BNT162b2 (Pfizer/BioNTech) and mRNA-1273/Spikevax (Moderna), both of which demonstrated a significant reduction in the onset of severe disease symptoms with an efficacy of 95% and 94%, respectively [36]. Currently, mRNA vaccines are undergoing clinical trials against a wide range of pathogens and non-communicable diseases. These include infectious diseases such as influenza, HIV, respiratory syncytial virus (RSV), rabies, emerging strains of SARS-CoV-2, Nipah virus, Zika virus, and pneumococcal disease [30,37,38]. Additionally, mRNA immunotherapies are being tested for non-communicable diseases, such as familial hypercholesterolemia, recurrent central nervous system neoplasms, and various types of solid tumors (including prostate, ovarian, colon, melanoma, gastrointestinal, and hepatocellular carcinoma). Other areas of investigation include myeloma, lung cancer, digestive system neoplasms, nonalcoholic fatty liver disease, glioblastoma, cystic fibrosis, hypercholesterolemia, among others [37,38].

Following immunization, DNA vaccines transfect cells present at the administration site, including both somatic cells and professional antigen-presenting cells (APCs). The plasmid DNA, initially residing in the cell cytoplasm, must migrate to the cell nucleus. Once inside the nucleus, the host cellular machinery interprets the genetic information and synthesizes the corresponding mRNA. This mRNA then exits the nucleus and enters the cytoplasm, where it undergoes translation to produce the antigen encoded in its sequence. In contrast, mRNA vaccines do not require the genetic material to reach the cell nucleus. Upon administration, they are directly translated in the cytoplasm. Subsequently, depending on the vaccine design, the desired antigen may be secreted directly from the cell to the extracellular matrix. There, it will be captured by APCs, initiating an immune response [1], or be anchored to the external side of the antigen-producing cells by inserting a transmembrane domain in its coding sequence. In this scenario, a high concentration of the foreign protein exposed to the external cellular environment will attract the APCs. Subsequently, the APCs will engage in the process required for antigen presentation [39]. Importantly, antigen-producing cells can also process the antigen themselves through both the major histocompatibility complex (MHC) class I and MHC class II pathways, subsequently stimulating APCs and ultimately resulting in both CD8⁺ and CD4⁺ T-cell responses. Additionally, antigen-producing cells may undergo apoptosis or release exosomes containing the antigen, which can then be taken up by APCs [1].

Here, the advantages and disadvantages of both DNA and mRNA vaccines for infectious diseases and cancer are discussed. We will also explore how the mRNA strategy gained prominence over DNA vaccines, potential enhancements for both technologies, and whether such efforts could provide an advantage for DNA vaccines over mRNA vaccines.

2. Advantages and disadvantages of genetic vaccines

Since both DNA and mRNA vaccines share many traits, it is not surprising that many of their advantages and disadvantages overlap. However, each of these genetic vaccine approaches also has specific benefits over the other.

Both genetic vaccination approaches are simple to produce, making their manufacturing development an easy and cost-effective process. These platforms are versatile; once the sequence of an antigen from either a pathogen or cancer is known, it can be easily encoded in DNA or mRNA. This is particularly important during pandemics, as more aggressive strains might quickly emerge. Additionally, both approaches can be produced at a large scale [1].

Plasmid DNA is more stable than linear mRNA sequences and can be stored at relatively higher temperatures compared to mRNA and other vaccination platforms. In contrast, due to their instability, mRNA vaccines require more complex handling and storage at much colder temperatures. Consequently, the logistical distribution of mRNA vaccines is more challenging and can only be carried out in places with an adequate cold-chain system. Thus, mRNA vaccines are not a suitable option for countries that do not meet these refrigeration requirements, making this immunization strategy unfeasible for neglected tropical diseases like Dengue and rabies [1]. Also, foreign mRNA sequences are susceptible to degradation by host cell RNAses [40], whereas DNA plasmids may remain in the host cell nucleus for months [41].

Another advantage of mRNA vaccines over DNA vaccines is that they cannot integrate foreign genetic material into the host cell genome. As previously mentioned, DNA plasmids must migrate to the nucleus after administration and transfection of host cells to initiate transcription and synthesize mRNA. This mRNA then exits the nucleus and moves to the cell cytoplasm, where the antigen of interest is produced. While many clinical trials have demonstrated the safety of DNA vaccines and the rarity of integration into the host genome [1], instances of integration have been reported in mouse models [42–45], potentially leading to unintended side effects. However, several experimental DNA vaccines do not show significant levels of integration into host cellular DNA [43,44,46–48]. In cases where integration is detected, it typically occurs at rates significantly lower than the natural mutation frequency [45]. It is worth noting that modified or adjuvanted vectors, designed to enhance immunogenicity, may carry a slightly higher risk of integration, and this risk should be subject to further assessment [8]. Another concern is the possibility that an integrated vaccine could lead to insertional mutagenesis by activating oncogenes or inactivating tumor suppressor genes. Additionally, there is a theoretical risk that an integrated plasmid DNA vaccine could induce chromosomal instability by causing chromosomal breaks or rearrangements [8]. It is crucial to emphasize that none of these concerns have been observed in the preclinical or clinical evaluation of DNA vaccine products [8].

Both DNA and mRNA vaccines induce antigen-specific cellular CD8⁺ responses, including both effector and memory responses. These responses play a crucial role in clearing intracellular pathogens and promoting tumor regression. They can also be reactivated in response to subsequent infections or booster immunizations. However, it’s important to note that mRNA vaccine-induced cellular responses tend to be transient and less robust compared to those elicited by DNA vaccines [49,50].

In addition to the aforementioned advantages, the potential length of DNA that can be utilized in a vaccine is theoretically much greater when compared to mRNA vaccines. This is because DNA is a larger molecule than mRNA and possesses the capacity to accommodate longer genetic sequences, often extending to several thousand nucleotides. Therefore, DNA vaccines could be ideal for the simultaneous delivery of antigen sequences and other genetic sequences encoding proteins that can modulate immune responses [51]. In contrast, the length of mRNA vaccines has been limited, with the Comirnaty/BNT162b2 vaccine having a sequence of 4284 nucleotides [52]. The preference for shorter mRNA sequences may primarily stem from challenges related to the purification, stability, and delivery of longer mRNA molecules [53]. Consequently, shorter mRNA sequences may exhibit better translation efficiency and a reduced risk of degradation, which is crucial for vaccine production and the induction of proper immunogenicity.

3. How can genetic vaccines be improved?

3.1. Encapsulation

For both genetic vaccination approaches, the use of carriers serves to protect them from host proteases and can enhance delivery efficacy. Encapsulation of DNA in nanoparticles, such as poly(lactic-co-glycolic acid) (PLGA), either alone or in combination with polyethylenimine (PEI) and chitosan, among others, has been employed through intradermal, subcutaneous, and mucosal administration methods. This enhances uptake by APCs and subsequently improves immune responses [1,8]. In a recent mouse model, co-immunization with plasmids encoding herpes simplex virus 1 (HSV-1) gD1 protein and interleukin 29 (IL-29) encapsulated in PLGA resulted in 100% survival against challenge with infectious HSV-1 viral particles [54]. The immunogenicity of a DNA vaccine against hepatitis B virus (HBV) was also enhanced at both humoral and cellular levels by formulating it with chitosan [55]. Similarly, lipid nanoparticles (LNPs) protect DNA from enzymatic degradation and enhance the immune response elicited by DNA vaccines in animal models [12,52]. Neutralizing antibody titers increased when DNA vaccines for Lyme disease, Zika virus, and Andes virus were formulated with LNPs [12,56]. An additional advantage is that both polymers and LNPs are synthesized with a cationic charge, facilitating binding to the negatively charged external cellular membrane and subsequent entry into host cells [57]. Likewise, encapsulating mRNA with various carriers protects naked mRNA from ribonucleases and promotes its cellular uptake. Examples of carriers include cationic peptides like protamine [58], and various types of biodegradable ionizable LNPs [25,59–61]. The latter represents a significant breakthrough for mRNA vaccines because these carriers not only provide an additional layer of protection against degradation but also have demonstrated a preference for delivering mRNA to APCs. This allows APCs to carry and produce the mRNA-encoded protein directly in the vaccine-draining lymph nodes [62]. Furthermore, LNPs have exhibited efficient mRNA delivery into innate immune cells and strong adjuvant activity, inducing the production of chemokines and pro-inflammatory cytokines [63–66], a feature also observed in all mRNA-based COVID-19 vaccines [67]. It is worth noting that DNA vaccines induce immune responses with more extended duration. In contrast, mRNA-LNP vaccines often necessitate multiple booster shots to sustain immunity over time, despite the fact that these vaccines generally result in higher levels of antigen expression compared to DNA vaccines. For instance, when using mRNA-LNPs in mice to generate monoclonal antibodies against the HIV CD4 binding site, protein levels of up to 170 μg/mL were observed in the serum just 24 hours after injection (using 1.4 mg/kg of mRNA). However, weekly injections of 1 mg/kg of mRNA were necessary to sustain antibody levels above 40 μg/mL [68]. In contrast, DNA-encoded monoclonal antibodies generally result in lower peak levels of production but have greater durability. For example, an antibody against CTLA-4 was still detectable even 400 days after a single injection of mice with 400 μg of plasmid DNA [69]. In the context of vaccination, this means that mRNA vaccines can generate strong immune responses even at low vaccine doses, while higher doses of DNA vaccines may be required to achieve the same levels of antigen expression [1,70].

3.2. Adjuvants

3.2.1. Self-adjuvanticity of nucleic acid vaccines

It is known that both DNA and mRNA can act as natural adjuvants, primarily through intracellular recognition of unmethylated cytosine – guanine dinucleotide (CpG) motifs in bacterial DNA (typically found in the non-coding regions of plasmids) by Toll-like receptor (TLR)-9, and through the recognition of mRNA by TLR-3, TLR-7/-8, and acid-inducible gene (RIG-1) proteins [71–73]. However, intracellular recognition of mRNA results in a robust innate immune activation, leading to the secretion of high levels of type I interferons (IFNs) into the extracellular matrix. This creates an environment that favors Th1 responses by mimicking the milieu of viral infections, thus making mRNA vaccines particularly suitable for viral pathogens [62,74–76]. As mentioned earlier, nucleoside modifications in the mRNA sequence increase both stability and translation efficiency while simultaneously reducing the activation of innate immunity to sustainable levels [77].

3.2.2. Chemical adjuvants

Several chemical adjuvants, such as alum, Vaxfectin®, AS03, and montanide, have been shown to enhance either antibody responses, cellular immunity, or both, induced by DNA vaccines in animal models, as elegantly reviewed elsewhere [12]. For instance, in a guinea pig model, a DNA vaccine for herpes HSV-2 formulated with Vaxfectin®, a cationic lipid-based adjuvant, reduced viral replication to undetectable levels, whereas animals immunized with the DNA vaccine alone did not exhibit the same outcome [78].

3.2.3. Molecular adjuvants

Co-administration of plasmids encoding cytokines or other molecules, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α), IL-6, IL-29, Fms-related tyrosine kinase 3 ligand (FLT3L), CD40 and CD40L, IL-12, IL-2, and pyroptotic molecules, among others, has been demonstrated to enhance the immune response [1,7,12,54,79–81]. For example, GM-CSF has been shown to enhance the immunogenicity of vaccines against cervical cancer in animal models, either as a recombinant protein [82] or co-expressed with the E7 antigen [83]. Adjuvants based on the pyroptotic effect have shown promising results in mouse models of melanoma and colon cancer [81].

3.3. Dosage optimisation

Dose optimization in DNA vaccines refers to the process of determining the most effective and safe dosage of the DNA-based vaccine required to induce a robust immune response. Several factors must be considered when optimizing the vaccine dosage. The paramount consideration lies in the host’s safety, necessitating a dosage that the recipient can tolerate without adverse effects. When determining the dosage of DNA vaccines, it becomes imperative to factor in immunogenicity. This entails selecting a dose that not only can effectively provoke a robust and targeted immune response against the desired antigen but also avoids the risk of triggering an overly aggressive immune reaction or potential toxicity. An optimal dose strikes a balance between efficacy and safety. Additionally, it is important to determine the frequency and timing of vaccine administration, which are relevant considerations in dose optimization. Some vaccines may require multiple doses given at specific intervals to ensure a sustained and durable immune response. Another factor to consider is the target population, as different populations or individuals may respond differently to vaccine dosages. Dose optimization must take into account elements such as age, immune status, and underlying health conditions [84]. The effect of dose optimization in DNA vaccines can significantly impact the vaccine’s efficacy. Finding the right dosage can enhance the magnitude and duration of the immune response, resulting in improved protection against the target pathogen or disease. Furthermore, dose optimization can reduce the potential for side effects and adverse reactions, making the vaccine safer for administration [8]. Like with any vaccine development, dose optimization in DNA vaccines necessitates thorough preclinical and clinical testing to identify the most effective dose that strikes a balance between efficacy and safety. These studies play a crucial role in determining the recommended dosage for effective DNA vaccine candidates to be used in large-scale vaccination campaigns [7]. These principles are generally applicable to mRNA vaccines as well. However, distinctions must be made between dosage ranges of different types of mRNA vaccines, especially between unmodified and nucleoside-modified mRNA. As mentioned previously, mRNA itself generates a strong innate immune activation which, if overdosed, may easily lead to undesired side effects. For example, excessively high reactogenicity was observed in humans after intramuscular administration of doses as low as 5 μg of an LNP-formulated unmodified mRNA vaccine against rabies [85]. On the other hand, the nucleoside-modified Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines were generally safe at the doses used, which were 30 μg and 100 μg, respectively. However, among the potential enhancements and advancements in the mRNA vaccine field, those primarily being investigated concern the refinement of mRNA dosage and the activation of innate immunity to prevent potential common and rare side effects. Indeed, although the side effects of the mRNA-based COVID-19 vaccines were similar to those observed with other vaccines [86], there have been reports of myocarditis and pericarditis occurring in some adolescents and young adults [87]. Despite being rare, these cases of myocarditis and pericarditis occurred at a higher rate among vaccinated individuals compared to the general population [87]. An important consideration in this regard could be investing in the development of self-amplifying mRNA vaccines that have experimentally been shown to have the potential to be administered at reduced dosages compared to ‘standard’ non-amplifying mRNA vaccines [88].

3.4. Storage

Since plasmid DNA is a relatively stable molecule, DNA vaccines typically require distinct storage and stability conditions when compared to traditional protein-based and whole-organism (attenuated or inactivated) vaccines. As DNA vaccines use genetic material, their storage and stability considerations are centered around preserving the integrity of the DNA molecules. To maintain stability, DNA vaccines are typically stored at low temperatures, usually at −20°C or −80°C. Exposure to higher temperatures can lead to the degradation of DNA, which could result in a loss of vaccine efficacy. Also, DNA can be damaged by repeated freeze-thaw cycles. Therefore, it is essential to minimize the number of freeze-thaw cycles during storage and transportation to preserve the vaccine’s integrity. To enhance the stability of DNA vaccines at higher temperatures, lyophilization (or freeze-drying) can be used. Lyophilized DNA vaccines can indeed be stored at higher temperatures (e.g. 2–8°C) and reconstituted before use. This can be advantageous for distribution and stockpiling in regions with limited access to ultra-cold storage facilities. During storage and transportation, it is advised to use proper packaging, as the use of appropriate containers is crucial to prevent contamination and maintain the vaccine’s stability. An important consideration is the shelf life of DNA vaccines, which varies depending on the formulation, storage conditions, and stability of the DNA. It is essential to determine and monitor the shelf life to ensure that the vaccine remains effective throughout its intended use. It is important to note that advancements in vaccine technology and formulation are continually being made, including research to improve the stability of DNA vaccines at different temperatures and extend their shelf life. These improvements aim to make DNA vaccines more accessible and practical for use in various settings, including regions with limited cold-chain infrastructure [89,90]. On the other hand, the requirement for cold-chain storage conditions is stricter for mRNA vaccines as mRNA is inherently unstable and much more susceptible to degradation at higher temperatures compared to DNA. Therefore, developing mRNA vaccines with increased stability at higher temperatures that could reduce the need for ultra-cold storage is of high importance [91].

3.5. Delivery site

Further efforts have been made to enhance the immune responses elicited by DNA vaccines. Successful methods to increase transfection efficiency include the use of gene guns and electroporation [1,12,92,93]. Many routes can be used to administer DNA vaccines, and each route has its advantages and challenges [1,12,94]. Intramuscular injections are the most common routes for DNA vaccine administration. Intramuscular injection delivers the DNA vaccine directly into the muscle tissue, where it can be taken up by muscle cells for expression of the encoded antigen, inducing both cellular and humoral immune responses. It is a relatively straightforward and effective method, often used in clinical trials and commercial vaccines [95]. If DNA vaccines are given intradermally, i.e. into the skin’s dermal layer, where a high number of APCs reside, the latter will efficiently take up and process the antigen, leading to an effective immune response. This route requires smaller vaccine doses while still eliciting strong immune responses, which means it also has lower possibilities of provoking adverse reactions. ZyCoV-D, the only DNA vaccine approved for human use, is administered through this route [11]. Intradermal administration is, however, more challenging than the intramuscular route and requires specialized devices or techniques [96]. Another possible administration route is subcutaneous, in which the DNA vaccine is given into the layer of tissue just beneath the skin. It is a relatively simple and widely used administration route for vaccines in general and induces both cellular and humoral immune responses [96].

To enhance the DNA uptake by cells, the administration routes described above can be followed by electroporation (mostly in the context of animal research) or using jet injectors [97]. Both create temporary pores in the cell membranes, either by applying brief electrical pulses to the injection site or by means of compressed air, thus facilitating DNA entry into cells. These techniques can significantly enhance the immune response, allowing for lower vaccine doses and potential needle-free administration. Nonetheless, this requires specialized equipment, and not all vaccination centers may be supplied with such technology [96].

Administration of DNA vaccines can also be performed intranasally. The nasal mucosa contains APCs that can take up the vaccine and initiate an immune response. Unlike the routes mentioned before, it can induce mucosal immunity against respiratory pathogens. However, this route also presents its challenges, such as optimization of the correct formulation, stability in the mucosa, and efficiency of DNA uptake by APCs [94,98].

Administration of mRNA has also commonly been performed via different routes, including intramuscular, intradermal, subcutaneous, intranodal, intravenous, and intratumoral injection [70,99]. However, the fact that the approved mRNA vaccines are formulated with LNPs poses an additional advantage, as LNPs favor uptake from APCs, mostly neutrophils and monocytes, even in the context of intramuscular injection, which is often considered the easiest and most convenient route of administration [62]. Neutrophils, and especially monocytes, have indeed been shown to translate mRNA most efficiently than other cell types [62].

3.6. Design

Codon optimization has found application in numerous biological fields, particularly for enhancing the synthesis and production of foreign proteins. In the context of DNA and mRNA vaccine development, it plays a pivotal role in elevating both the expression and immunogenicity of the encoded antigens. This process entails modifying the gene sequence of interest to optimize the codons, which are sets of three nucleotides responsible for coding specific amino acids during protein synthesis. The genetic code is redundant, signifying that multiple codons can represent the same amino acid. However, the prevalence of different codons within a gene sequence can significantly affect protein expression efficiency. In many organisms, particular codons are more frequently employed for specific amino acids, a phenomenon known as codon bias. In the context of designing genetic vaccines, codon optimization seeks to align the codon usage of the gene with the codon preferences of the host organism or the target cells where the vaccine will be administered. Typically, this is accomplished by substituting infrequent codons with those used more frequently, all while preserving the encoded amino acid sequence of the protein. In the context of DNA and mRNA vaccines, they offer several advantages, such as i) enhanced protein expression, i.e. utilizing codons preferred by the target host organism, the translation machinery can efficiently generate the encoded protein, resulting in higher expression levels, ii) improved antigen presentation, i.e. codon optimization can enhance the stability and expression levels of the antigenic protein within host cells; this, in turn, improves the presentation of antigenic peptides to the immune system and strengthens the subsequent immune response, ultimately leading to enhanced vaccine efficacy, iii) cross-species applicability, i.e. codon optimization can render DNA and mRNA vaccines more effective across different species, making them suitable for broader application in both animal and human vaccinations, iv) reduced risk of undesirable immune responses, i.e. since the use of non-optimal codons could potentially trigger an immune response against the vaccine itself, codon optimization helps mitigate this risk. Codon optimization is frequently conducted through the utilization of bioinformatics tools that analyze the codon usage patterns of the target organism and propose optimized sequences. This process constitutes a pivotal step in the design and development of genetic immunization strategies, as it can profoundly impact the immunogenicity and efficacy of the vaccine [1,8,100].

The manufacturing and use of DNA plasmid vaccines come with several drawbacks, including the presence of antibiotic resistance genes, unwanted additional DNA sequences within the plasmid backbone, and the necessity to remove impurities from bacterial cultures, such as proteins, genomic DNA, RNA, and endotoxins. Moreover, long lead times for manufacturing and product supply, inefficient uptake of large plasmid DNA molecules into the cellular nucleus, and challenges in integrating plasmid production into automated Current Good Manufacturing Practices (cGMP) workflows can pose significant challenges. Therefore, there is an unmet need for innovative DNA vaccination platforms that i) feature a smaller backbone to accommodate larger coding genes, ii) yield higher production rates, iii) are manufactured through simpler processes, iv) are more cost-effective to produce, v) maintain high safety standards, and vi) enhance cellular and nuclear uptake. To address these issues, new DNA vaccine technologies have recently emerged, wherein DNA is produced in a cell-free process, eliminating the need for bacterial fermentation. The resulting vaccine possesses a structurally improved design. For instance, DNA amplicons are rapidly synthesized using a standardized process, ensuring consistent yields, with nearly 100% of the produced DNA comprising target sequences. These novel bacteria-free manufacturing platforms have already demonstrated stability and immunogenicity in preclinical models, exhibiting strong in vivo stability and immunogenicity [3,101–103]. For example, the Doggybone strategy has demonstrated its protective efficacy in a hamster animal model of severe SARS-CoV-2 disease, resulting in the generation of high levels of neutralizing antibodies against multiple emerging viral variants [104]. Additionally, DNA amplicons generated through polymerase chain reaction (PCR) have displayed the capacity to provoke a robust antitumoral immune response, specifically targeting neoantigens expressed by a colon cancer model, as well as eliciting humoral responses (both binding and neutralizing antibodies) and cellular responses against SARS-CoV-2 [3,103]. A similar approach involves the use of minicircles, which are smaller plasmids with specific sequences removed, such as resistance markers, without affecting production or altering immunogenicity. Minicircles containing the sequences of HBV antigens have demonstrated the ability to induce cellular immunity in a mouse model, achieving comparable levels to a conventional plasmid DNA vaccine encoding the same antigens [105]. However, it’s important to note that the DNA minicircle approach still necessitates bacterial fermentation and may consequently contain bacterial impurities.

As mentioned earlier, due to DNA’s stability, it is possible to incorporate lengthy genetic sequences into plasmids. This attribute also renders them suitable for encoding complex or composite proteins. An illustrative example is the Vaccibody^TM technology, where the DNA vaccine encodes a chimeric dimeric protein consisting of a targeting domain, a dimerization unit, and an antigen sequence. This approach has shown enhancements in immunogenicity and the efficacy of immune responses compared to conventional DNA vaccines. This is achieved by tailoring them to target specific immune cells, such as APCs [92,93,106]. Using this strategy, a Vaccibody molecule known as VB10.16 has been designed as an immunotherapy for both premalignant and malignant mucosal lesions caused by the human papilloma virus strain 16 (HPV-16). This DNA vaccine incorporates the human chemokine (C-C motif) ligand 3-like 1 (CCL3L1), which binds to APCs through the CCR5 receptor, and the HPV-16 mutation-inactivated E6/E7 antigens. These antigens are internalized by APCs following the CCL3L1-CCR5 interaction. As previously mentioned, APCs would then migrate to a nearby lymph node, and the E6/E7 antigens would be processed through the APCs’ MHC class I and II pathways, subsequently cross-presented to T-cells. These activated T-cells would then target and eliminate tumor cells expressing HPV16 E6/E7 antigens. In a Phase I/IIA trial, women with HPV-16-positive high-grade cervical intraepithelial neoplasia (CIN) were treated with VB10.16. It was confirmed that VB10.16 exhibited no toxic effects and resulted in a reduction in tumor size in 94% of the volunteers, along with viral clearance in 47% of patients. Additionally, a significant correlation was observed between interferon-γ (IFN-γ) producing T-cell levels and lesion size [107].

For mRNA vaccines, apart from the dosage and reactogenicity concerns mentioned earlier, another issue is the relatively short duration of protective immunity and the need for multiple doses, as observed with COVID-19 vaccines [108]. Therefore, extending the duration of protective immunity is crucial. Current efforts are focused on evaluating additional modifications to the untranslated (UTR) regions at the 5’and 3’ ends of the mRNA sequence to enhance mRNA stability and translation efficiency. These modifications have the potential to lead to reduced vaccination regimens [109].

The potential modifications to the design of genetic vaccines are depicted in Figure 1.

Figure 1. Enhancing genetic vaccine design: strategies to boost immunogenicity. several strategies can be used to elicit higher immunogenicity of genetic vaccines at the design level, such as (a) codon optimization (fine-tuning the genetic code for heightened expression), (b) compact DNA plasmid backbones (employing smaller, more efficient genetic constructs), (c) utilization of cell-free products (enhancing vaccine components), (d) encoding chimeric proteins (combining beneficial elements for a stronger response), (e) optimized UTR in mRNA sequences (improving translation efficiency), and (f) enhanced culture yields (increasing the availability of essential components for vaccine production). Figure created with BioRender.

4. Is there a future for both mRNA and DNA vaccines?

In this article, we have presented crucial information regarding the development of genetic vaccines, elucidated their mechanisms of action, highlighted their achievements, and identified potential avenues for improvement (Figure 2 and Table 1). Given the emergency human use approval and widespread administration of mRNA-based COVID-19 vaccines, it might seem reasonable to conclude that mRNA vaccines have definitively taken the lead over DNA vaccines in the quest for the future of vaccination, especially in the field of infectious diseases. However, from our perspective, this quest may not have reached its conclusion. Indeed, upon a meticulous examination of this article, one can discern strengths and weaknesses for both mRNA and DNA vaccines. A pivotal question arises: whether the transformation of weaknesses into strengths is more achievable within the mRNA or DNA vaccine platform. Equally essential is the determination of which strengths and weaknesses hold the greatest significance.

Figure 2. Summary of possible common and individual improvements that may be underway for DNA and mRNA vaccines. common improvements for genetic vaccines could be achieved by advanced codon optimization, optimized dosage, and different routes of administration. However, while DNA vaccines may potentially achieve improved safety and immunogenicity with smaller templates or minicircles, encapsulation into LNP and addition of adjuvant encoding sequences, specific improvements for mRNA vaccines may instead require technology advances and sequence modifications aimed at improving stability and at reducing levels of inflammation and potential side effects. Figure created with BioRender.

Table 1. Summary of the advantages, disadvantages, and potential improvements of genetic vaccines.

DNA vaccines			mRNA vaccines
Advantages	Disadvantages	Potential improvements	Advantages	Disadvantages	Potential improvements
Long-lasting immune responses	Limitedimmunogenicity	Genetic adjuvants	Highly immunogenic	Short-lived immune responses	Optimization of number of doses, genetic sequence, route of administration
Very safe, as shown by multiple clinical trials	Delivery challenges	Encapsulation	Generally safe, as shown by multiple clinical trials	Storage, cold-chain requirements	Uncharted technology improvements
Easiness to design and produce	Host genome integration risk	None, risk considered extremely low	Easiness to design and produce	Delivery, i.e. needs to be delivered with LNPs to protect from degradation	Modification of UTR sequences
Multivalency, i.e. the same vaccine can carry multiple antigens, co-express antigens and molecular adjuvants, etc.	Specific cellular targeting, e.g. APCs	Encapsulation, design which includes an APC-targeting molecule	Flexibility, i.e. the same platform can be applied to multiple pathogens or cancer	Limitation in the number of antigens or adjuvant molecules that can be present in the same formulation	Optimization of genetic sequence and delivery agents
Low cost of production	Regulatory issues	Lessons learned from genetic vaccines approved during SARS-CoV-2 pandemic	Rapid Development, once the target pathogen or cancer antigens are identified	Accessibility, i.e. costs associated with mRNA production and storage	Uncharted technology improvements, optimization of genetic sequence
Highly stable

Regarding mRNA vaccines, we believe that an important strength lies in their formulation into LNPs. This not only enhances stability but also facilitates simple administration through injections. Moreover, it is crucial for enhanced delivery to high protein-producing cells, such as APCs. Another significant advantage is the intracellular recognition of mRNA, triggering a highly pro-inflammatory response with the secretion of abundant type-I IFNs. This acts as a self-adjuvant, leading to elevated levels of both antibody and T-cell responses. However, this characteristic could also be a drawback since the resulting intense inflammation and innate immune activation due to mRNA recognition might occasionally be too robust and poorly tolerated by some individuals. As previously mentioned, although they remain relatively rare, certain severe side effects have been documented following the administration of COVID-19 mRNA-based vaccines. These incidents raise concerns about risk assessment in the context of future vaccines for infectious diseases with an overall low risk of complications due to infection in the general population. This highlights the pressing need to fine-tune mRNA dosages to achieve an appropriate level of innate immune activation without compromising the magnitude of adaptive immune responses. Another aspect of mRNA vaccines that can be seen as both advantageous and disadvantageous is the potency and kinetics of the immune response. These vaccines generate robust responses quickly, which can be advantageous in certain scenarios. However, their duration is limited, and the need for multiple booster doses to sustain protective immunity presents a clear drawback. Administering multiple doses of mRNA vaccines, whether for COVID-19 or other infectious diseases, raises significant potential risks that demand careful consideration. Given that mRNA vaccine technology is relatively new, the long-term safety profile remains an ongoing subject of study. It is noteworthy that adverse local or systemic reactions may become more likely and severe with repeated doses [110,111]. There is also a legitimate concern that multiple administrations of mRNA vaccines could overly stimulate the immune system, possibly leading to autoimmune reactions [112]. Public perception regarding the potential risks associated with repeated dosing could significantly impact trust in vaccines and contribute to vaccine hesitancy, thereby influencing public health outcomes. Additionally, ethical considerations, including costs, resource allocation, and equitable vaccine distribution, become paramount when deciding to administer subsequent doses. Nevertheless, the most significant disadvantages of mRNA vaccines are the inherent instability of mRNA and the requirement for cold-chain storage. This poses a major hindrance to distribution and limits the suitability of mRNA vaccines for global use.

Remarkably, DNA vaccines exhibit an almost contrasting profile when considering the points mentioned earlier. In terms of encapsulation, unlike mRNA vaccines, approved human and veterinary DNA vaccines are administered without encapsulation. However, they do require jet injection for efficient delivery, which poses a major disadvantage as delivery devices may not be globally available. The encapsulation of nucleic acids into lipid nanoparticles (LNPs) has primarily been employed with mRNA vaccines, primarily aimed at protecting them from degradation. As mentioned earlier, this approach also comes with the added benefit of facilitating delivery to APCs. This development marks a significant milestone in the evolution of mRNA vaccines. The reason why the encapsulation of plasmid DNA into LNPs has been relatively unpopular so far is related to the inherent stability of DNA and may also be discouraged by the relatively large size of plasmid DNA. However, it is worth noting that encapsulation of plasmid DNA into LNPs has not only been demonstrated to be possible but has also been shown to increase the level of neutralizing antibodies following the administration of encapsulated Andes virus and Zika virus DNA vaccines in rabbits and nonhuman primates when compared to unformulated vaccines [113]. This was also demonstrated to likely result from increased in vivo DNA delivery, potentially allowing for a 10-fold increase in protein production with a 10-fold reduction in the dosage of encapsulated DNA compared to naked DNA [113]. Additionally, as mentioned earlier, smaller DNA molecules, such as minicircles, Doggybone, and amplicons, have been successfully employed as DNA vaccines in animal models [3,101–103]. These smaller DNA molecules could be encapsulated within LNPs, addressing various concerns, including the ease of encapsulating smaller molecules, enhanced transfection of APCs facilitated by the LNPs, and the absence of bacterial contaminants, as these DNA molecules would not be produced through bacterial fermentation. From this perspective, it would be highly valuable to prioritize the optimization of DNA vaccine technology to harness the substantial advantages of convenient and improved DNA delivery, along with its inherent stability.

Aspects of DNA vaccination that can be viewed as both advantageous and disadvantageous include low immunogenicity and slow but durable kinetics of immune responses following vaccination. Low immunogenicity might correspond to reduced rates of side effects, but it may also lead to poor adaptive immune responses, whereas slow kinetics might be suboptimal for acute infections but optimal in chronic infections and cancer settings. Low immunogenicity and slow kinetics may, at least in part, result from poor intracellular recognition of DNA by muscle cells. Intracellular recognition of DNA can trigger the release of type-I interferons and other pro-inflammatory cytokines via activation of the cGAS/STING pathway [114], This would mostly occur through direct interaction between plasmid DNA and APCs. The same principle applies to the intracellular recognition of bacterial CpG motifs included in the plasmid’s non-coding regions by intracellular TLR9, which has been demonstrated to enhance antibody responses when CpG interacts directly with B cells [115].

Considering all the aforementioned points, we maintain the belief that it is indeed plausible to envision further advancements that could enhance the immunogenicity of DNA vaccines. This enhancement could potentially elevate them to levels comparable to those demonstrated by mRNA vaccines, thereby making DNA vaccines more competitive within the market. For example, a DNA vaccine composed of smaller molecules, such as amplicons, capable of being encapsulated within LNPs with fewer constraints, could facilitate optimal delivery to APCs and expedite cellular transfection. Moreover, intracellular recognition of DNA within APCs, rather than muscle cells, might also offer a degree of self-adjuvanticity similar to what is observed with mRNA vaccines. Alternatively, if necessary, an intriguing avenue could involve employing TLR3/7 agonists to initiate a similar chain of events triggered by intracellular mRNA recognition, but this time in the presence of DNA. This approach has the potential to not only provide an additional boost to the immune response following DNA vaccination but also offer a valuable opportunity to establish an optimal balance between the necessary levels of inflammation for an effective vaccine response and the occurrence of side effects. This balance could be achieved by regulating the controlled release of type-I interferons through carefully dosed amounts of such adjuvants. In simpler terms, DNA amplicons formulated within LNPs and adjuvanted with TLR3/7 agonists could theoretically yield optimal protein production by APCs, resulting in both robust and enduring immune responses while maintaining a favorable safety profile. When coupled with molecular stability and less stringent cold-chain requirements, this would significantly enhance the desirability of DNA vaccines. The widespread use of DNA vaccines also raises ethical and regulatory considerations that require careful examination. Similar to mRNA vaccines, our understanding of the long-term safety profiles of DNA vaccines remains incomplete, especially given the limited human data available, primarily from the ZyCoV-D vaccine in India [11]. One of the major concerns is centered around the theoretical, albeit minimal, risk of foreign DNA integration associated with DNA vaccines, as mentioned earlier [1]. While extensive research, rigorous risk assessments, and the utilization of non-integrating vectors all contribute to minimizing this risk [37–42,46,84], it is crucial to maintain transparency regarding this theoretical possibility when administering DNA vaccines. Such transparency might influence vaccine hesitancy. The potential advantages of DNA vaccines, including robust immune responses and potential disease prevention, must be thoughtfully weighed against the theoretical risk of genomic integration.

5. Conclusions

In essence, DNA vaccines could not only match the effectiveness of mRNA vaccines but also potentially surpass them, offering a safer profile and greater suitability for global distribution. These are objectives that currently appear challenging to achieve with mRNA vaccines.

6. Expert opinion

While it might appear that we are conveying a message suggesting that enhancing DNA vaccines will undoubtedly make them superior to mRNA vaccines, thereby rendering the latter obsolete, the reality is different. We simply believe that the success of mRNA vaccines against SARS-CoV-2, approved for emergency human use during the 2020 pandemic, has prematurely led to widespread beliefs that DNA vaccines are outdated. In this manuscript, we outline the potential improvements that could result in effective DNA vaccines. Our intention is not to emphasize competition between mRNA and DNA vaccines, but rather to present the latter as a viable option in situations where mRNA vaccines could encounter difficulties related to infrastructure and storage requirements, such as maintaining an adequate cold-chain system. Finally, we also hold the belief that these enhancements in intensifying the strength and duration of specific immune responses from both mRNA and DNA vaccines can prove beneficial in a heterologous prime/boost strategy. In this type of scheme, the initial immunization would be carried out using a DNA vaccine, followed by a subsequent boost using an mRNA vaccine. Based on the results of a clinical trial wherein volunteers received three doses of a SARS-CoV-2 DNA vaccine (Spike and ORF3), with the first two doses at weeks 0 and 8 (at either 0.6 or 1.2 mg), or at weeks 0 and 12 (at 1.2 mg), and then received the Moderna mRNA vaccine (Spike, 100 μg) at weeks 24, 36, and 48, there was a significant increase in the levels of binding and neutralizing antibodies, as well as cellular responses [116]. Additionally, this heterologous prime/boost approach using distinct nucleic acid vaccines demonstrated safety and good tolerability. Consequently, we believe that both forms of genetic vaccines hold significant potential as immunization strategies, whether used independently or in combination. One should not be prioritized over the other solely based on past achievements. Further efforts to enhance both mRNA and DNA vaccines are unquestionably essential to develop effective immunization strategies against infectious diseases and cancer.

Article highlights

• Genetic vaccines can offer greater advantages than conventional vaccines against both cancer and infectious diseases, particularly in terms of inducing T-cell responses.

• DNA vaccines induce lower levels of immunogenicity compared to mRNA vaccines, likely due to differences in intracellular recognition and mechanisms of action.

• Delivery strategies, such as lipid nanoparticles (LNPs), could enhance both mRNA and DNA vaccine delivery and immunogenicity.

• While mRNA is inherently potent as a self-adjuvant and requires modulation, the use of adjuvants, such as Toll-like receptor agonists, might enable precise shaping and refinement of the innate immune response in DNA vaccines.

• Embracing and optimizing various enhancements and techniques could potentially tilt the balance in favor of DNA vaccines.

Declaration of interest

Bruno Douradinha and Alberto Cagigi are employed by Nykode Therapeutics ASA, a biotech company developing DNA vaccines for oncology, autoimmunity and allergy, and infectious diseases. However, the opinions and ideas stated in this manuscript are solely from the authors, and do not reflect the company’s position on the issues discussed in this manuscript. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or material discussed in the manuscript apart from those disclosed.

Reviewer disclosures

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

Author contributions

Both authors have made significant contributions to the conception and design of this Perspective article, the interpretation of relevant literature, and have been involved in writing and revising the review article for intellectual content.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This paper was not funded.