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Table of Contents
REVIEW ARTICLE
Year : 2022  |  Volume : 1  |  Issue : 4  |  Page : 217-233

Nucleic acid vaccines: A rising antidote for the future


Department of Biotechnology and Bioinformatics, Faculty of Life Sciences, JSS Academy of Higher Education and Research, Mysore, Karnataka, India

Date of Submission12-Aug-2022
Date of Decision01-Oct-2022
Date of Acceptance19-Oct-2022
Date of Web Publication5-Dec-2022

Correspondence Address:
Charukesi Rajulu
Department of Biotechnology and Bioinformatics, Faculty of Life Sciences, JSS Academy of Higher Education and Research, Mysore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpdtsm.jpdtsm_85_22

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  Abstract 


DNA vaccines, a type of nucleic acid vaccine, have emerged as one of the recent developments in immunology and recombinant DNA technology, offering great potential in terms of ease of manufacture, maintenance, and safety compared to conventional vaccines. Since their discovery, DNA vaccines have evolved immensely, resulting in the employment of new techniques such as gene guns, in vivo electroporation, and nanoparticle-based carriers to enhance the delivery of vaccines into the cells. Starting from the failures of the first-generation DNA vaccines to the near-success second-generation vaccines, several strategies including codon optimization, antigen design, and heterologous prime-boost have greatly helped in strengthening the vaccine's immunogenicity. The purpose of developing these third-generation vaccines is primarily to solve existing medical complications like cancer, along with therapeutic uses, to address health problems, and to aid the rapid eradication of sudden global outbreaks of infectious diseases including Ebola and COVID-19. In this review, we focus on the recent developments and strategies employed to improve the efficacy of DNA vaccines and discuss their mechanism of action, potential concerns, progress achieved, and a brief update on its clinical applications.

Keywords: DNA vaccines, immunogenicity, nucleic acid vaccines, RNA vaccines, vaccination


How to cite this article:
Devi W R, Kammar SS, Logesh S, Dsouza GL, Mohan TC, Rajulu C. Nucleic acid vaccines: A rising antidote for the future. J Prev Diagn Treat Strategies Med 2022;1:217-33

How to cite this URL:
Devi W R, Kammar SS, Logesh S, Dsouza GL, Mohan TC, Rajulu C. Nucleic acid vaccines: A rising antidote for the future. J Prev Diagn Treat Strategies Med [serial online] 2022 [cited 2023 Jan 29];1:217-33. Available from: http://www.jpdtsm.com/text.asp?2022/1/4/217/362829




  Introduction Top


Vaccines play a very important role in the lives of every individual as a preventive measure against serious outbreaks of infectious diseases. Vaccination has been believed to be the main reason that has increased the average life span of humans in the 21st century.[1] Dating back to the year 1796 which evidenced the astonishing discovery of the first vaccine, it's been more than two centuries since mankind has started enjoying the privileges of vaccines, the first one being Edward Jenner's (1749–1823) smallpox vaccine that drastically decreased the spread of the calamitous disease. The famous experiment in which Jenner successfully immunized a boy, James Phipps, against smallpox by a prior introduction to the cowpox virus successfully provided immunity to the smallpox virus.[2] It was a remarkable progression in public health that led to the discovery of a plethora of different vaccines. In this review, we aim to provide an introduction to the different vaccine types currently in use, the mechanism of action, and their limitations. We present the current status of DNA vaccines (a type of nucleic acid-based vaccines), with emphasis on their mechanism of action, methods of delivery, potential issues, clinical applications, and prospects.

What are Vaccines?

A vaccine is a biologically prepared mimic of a pathogen usually made from a killed, weakened pathogen or using one or more proteins or polysaccharides extracted from the pathogen. Vaccines, like pathogens, can produce antigenicity and induce an immune response but lack the virulent properties of the pathogen to cause diseases. Physiologically, vaccines elicit an immune response and activate both innate and adaptive immunity leading to the production of antibodies and memory cells, escalating immune response in future encounters.[3]

Vaccine types

Broadly, conventional vaccines are classified into 5 major categories-live attenuated vaccines (LAVs), inactivated or killed vaccines, Toxoids, subunit vaccines, and viral vector vaccines. Live, attenuated, or killed vaccines constitute the first-generation vaccines (e.g., polio, smallpox, chickenpox, measles, etc.). LAVs that use the attenuated or weakened form of the pathogen possess only the antigenic property of the disease-causing pathogen, lacking the virulence attributes. As this vaccine type naturally mimics the pathogen, merely 1–2 doses are sufficient to elicit strong and long-lasting protection against the pathogen. Killed/inactivated vaccines (e.g., hepatitis A, rabies, typhoid, etc.) involve the killed form of the disease-causing pathogen; these do not provide an immune response as strong as live vaccines, therefore, need many booster doses to achieve long-term immunity. The subunit vaccines, which are the products of the developments of immunological studies and the discoveries of the antigenic properties of the pathogen, comprise second-generation vaccines. As this vaccine type employs specific fragments of the pathogen (such as polysaccharide, protein, and capsid), it elicits an effective and specific immune response against the pathogen (e.g., hepatitis B-protein subunit vaccine, pneumococcal vaccine-polysaccharide subunit). Toxoids are a type of vaccine where exotoxins secreted by bacteria are purified to produce anti-toxoid antibodies that neutralize the deleterious effects of the bacterial toxins. This strategy is specifically used for an illness that is caused by secreted toxins (e.g., Diphtheria, Tetanus). Viral vector vaccines utilize an altered version of a disease-causing virus (vector) that carries the genetic instructions for the host cells. The non-virulent antigenic fragments of the virus produced by the host cells trigger the immune responses in the body (e.g., Ebola viral vaccine and Johnson and Johnson Janssen viral vector vaccine for COVID-19).[4] Third-generation vaccines that have gained popularity and experimental success rates in recent decades' clinical trials include the nucleic acid vaccines (DNA and RNA vaccines) wherein the genetic material is administered as a vaccine to produce the desired antigenic protein target inside the recipient's body.

Mechanism of action of vaccines

Most mechanisms of immunization by vaccines begin with exposure to an antigen. The encountered antigen will be ingested and degraded by the macrophages, a type of professional antigen-presenting cells (APCs) of the host immune system. The degraded fragments are then displayed (presented) on the membrane of the macrophages or the APCs by the major histocompatibility complexes (MHC) present on the cell membrane. This process is called antigen presentation [Figure 1]. The antigenic fragments presented by the MHCs are recognized by the T-lymphocytes or T-cells via T-cell receptors. There are two main classes of MHCs; MHC class I and MHC class II. MHC class I present the fragments of intracellular antigens (those generated in the cells because of infections) and are recognized by the cytotoxic T-cells. MHC class II presents the extracellular antigens and these are recognized by the helper T-cells, which in turn activate the B-cells to differentiate into plasma and memory cells.[1] The plasma cells are responsible for the production of antibodies that bind to the antigens for destruction by the phagocytes. The antibody-mediated immunity, triggered against the extracellular antigenic fragments is also called humoral immunity. The memory cells retain a memory of the encounter with the specific antigen for a long time postinfection, to develop a quick immune response against that antigen during future encounters. The cytotoxic T-cells that recognize the intracellular antigens presented by MHC class I mediate the cell-mediated immunity [Figure 1].
Figure 1: Mechanism of action of a vaccine: (a) Vaccine antigens in form of injectors are administered through intradermal or intramuscular routes. (b) Once inside the human body antigens are processed and presented by the MHCs on the surface of APCs. (c) The antigens presented by the MHCs are recognized by the T-cell receptors. (d) T-cells activate B-cells which get differentiated into plasma and memory cells. (e) B cell differentiated plasma cells produce antibodies against the specific antigens. (f) B-cell differentiated memory cells provide a second level of protection on re-exposure to the same antigen. MHCs: Major histocompatibility complexes, APCs: Antigen-presenting cells, T-cells: Thymus cells, and B-cells: Bone marrow cells, Ag: Antigens

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The majority of vaccines induce both humoral and cell-mediated immunity to protect the host against infection. However, they differ in their efficacy in stimulating an immune response. The strength of the immunization outcome is directly influenced by the vaccine type. Among all vaccine types, LAVs trigger both innate and acquired immunity through the activation of many pattern recognition receptors, a component of innate immunity, and produce higher antigen levels through replication. Closely resembling the original pathogen, live vaccines possess the ability to trigger strong humoral and cell-mediated immune responses that offer immunity for decades even with a single dose.[5] Inactivated vaccines usually require the supplementation of adjuvants to elicit a strong immune response and often require booster doses as the primary antibodies produced as a result of the first dose of nonlive vaccines eventually wane.[6] Nonprotein antigens (e.g., polysaccharide vaccine) on the other hand elicit only B-cell responses; lack the capacity to induce T-cells, and therefore are designated as T-cell-independent and cannot provide long-lasting immunity. Viral vectored vaccines are potent inducers of both B-cells and cytotoxic T-cells that get rid of the virus-infected host cells and contribute to controlling the infection.

Conventional vaccines and their limitations

Conventional vaccines have played a crucial role in eradicating and restricting the incidences of diseases such as polio, tetanus, diphtheria, and measles worldwide. However, the use of conventional vaccines is not always the preferred choice especially with the recent outbreaks of novel pathogenic strains because of its limitations. Despite the strong and lasting immunogenicity provided by the live vaccines, the expensive techniques used for the purification and the maintenance of cold storage conditions remain the major drawbacks, limiting its safe usage. In developing and under-developed countries, transportation of these types of vaccines to remote areas could be a problem and may sometimes fail to maintain the proper storage conditions. The recipient of such vaccines is at risk because of the high chance of the attenuated pathogen in the vaccine reverting to its virulent form, causing the disease instead of just evoking an immune response.[7] The polio vaccine, being a good example, is a LAV. The weakened pathogen is mutated (mostly point mutations) to alter the expression of the virulent genes. There are three strains of the virus used in the vaccine construction: Types 1, 2, and 3.[8] Out of these, one is just two and another strain is three mutations away from reverting to the virulent form.[9] The combination of several live-attenuated strains to produce combination vaccines may also cause competition between individual vaccine strains, causing an undesirable increase in virulence or a decrease in immunogenicity.[10] This could be considered one of the main reasons for the resurgence of eradicated diseases.[11] One such example is the recent polio outbreak in central and eastern African countries in 2019, which questions the safety of the historically successful live vaccines.[12] Additionally, the production of conventional vaccines is restricted to limited laboratories as it requires a biosafety level 2 or higher. Inactivated vaccines, on the other hand, exhibit limited immune response and require the fusion of immunostimulants or adjuvants to escalate the response.

The second-generation subunit vaccines are harmless in this regard; they are made up of the extracts of antigenic fragments-proteins, or polysaccharides.[13] However, alterations or denaturation of subunit vaccines during purification or handling may lead to vaccine failure. This is because the denatured proteins have immunogenic epitopes different from those on native proteins, hence recognized variably by different antibodies.[14] These technical challenges faced by conventional vaccines and the emergence/outbreak of novel pathogens in recent decades have necessitated the development of alternative, less risk-prone new vaccine types. Scientists have found that nucleic acid-based vaccines that involve the use of genetic material to stimulate an immune response are versatile strategies to combat infectious and noninfectious diseases. The limitations of attenuated and subunit vaccines could be overcome by the use of nucleic acid vaccines, which are easy to develop, inexpensive, and could be stored at room temperature. Nucleic acid vaccines are effective in inducing both B-cells and cytotoxic T-cell responses similar to LAVs; administering the pathogen's genetic material produces endogenous pathogenic proteins with native conformation that mimic the antigen of the natural infection.[15] The first observation of DNA vaccine-induced immunity was in the 1990s when plasmid DNA coding for influenza A nucleoprotein resulted in the activation of T-lymphocyte responses.[16] This initial study was followed by a series of several investigations involving animal models that demonstrated the effectiveness of DNA vaccines against infectious diseases, autoimmunity, and cancer.[17],[18],[19] In a parallel study when a gene encoding the mRNA sequence was administered to mice, researchers were able to successfully detect the produced protein.[20] These studies inspired the attention of the scientific community to develop these third-generation vaccines, one of the potential and versatile strategies to combat clinical problems in modern medicine.


  Nucleic Acid Vaccines – A Serendipitous Scientific Discovery? Top


In the autumn of 1992, at a conference, “Modern Approaches to New Vaccines,” in Cold Spring Harbor, New York, the concept of DNA vaccine technology was first publicized. Protein expression from the DNA insert was discovered in the early 1990s when a group of US researchers conducted a study on the detection of lipids that allowed the transmission of naked DNA into the muscles of the skeletal system of animals. In their experiment, naked DNA was used as a control to observe the action of the lipids. Apart from their main objective, interestingly, they observed that the naked DNA that was taken up by the skeletal muscles resulted in protein production. They experimentally confirmed their findings by injecting DNA isolated from the influenza virus into Mus musculus (laboratory mice). The DNA in the animal's system directed the synthesis of the viral proteins which were recognized as foreign particles by the animal's immune cells, thereby triggering an immune reaction. This retained the immunity of the animal against the influenza virus, hence giving protection from later encounters with the virus or infection.[20] The pioneering works of the researchers in the 1990s, demonstrated the successful expression of antigenic proteins by introducing plasmids with pathogenic gene inserts into a mammalian system,[21] followed by years of numerous experiments that focused on demonstrating the principles of DNA vaccines for many different diseases caused by different pathogens, including viruses, bacteria, and other parasites.[22],[23],[24] While techniques relating to the production and purification of nucleic acid vaccines are economical, simple, and rapid, the demonstration of the efficacy of the built vaccine in clinical trials remains the key challenge.[25]

What are DNA vaccines and how are they made?

A DNA vaccine is primarily a recombinant plasmid with foreign gene inserts that code for a specific protein/antigen. The recombinant plasmids are inserted into a bacterial host for replication and then purified for administration as vaccines.[26] These plasmids, harboring the pathogenic gene insert, regardless of their autonomous replicating properties, cannot replicate in eukaryotic cells, hence limiting their autonomous replication merely to bacterial cells. DNA vaccines are constructed in a manner to ensure the expression of the inserted foreign gene only after being administered to a eukaryotic animal. This is accomplished by using an expression vector (plasmids) that has all the regulatory elements essential for gene expression in animal cells [Figure 2].
Figure 2: Schematic representation of a recombinant plasmid used in DNA Vaccines: DNA vaccine construct is composed of bacterial plasmid having an origin of replication, antibiotic-resistant gene, eukaryotic promoter, and antigen-specific gene insert

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The choice of a gene insert may depend on the target pathogen or the antigen. By altering the gene of interest, the proteins synthesized can be manipulated. So, DNA vaccines can be produced for several pathogens or different antigens of the same pathogen, making it a possible therapy in treating diseases.[27],[28],[29] Infectious diseases are of major concern and are highly contagious in developing countries due to poor hygiene and sanitation. Due to difficulties in transportation and lack of adequate healthcare services in remote areas, residents in the remote region fail to get the proper dose of conventional vaccines that require the administration of multiple doses to provide immunity.[30] For instance, DNA vaccine candidates (VRC319 and VRC 320) administered as single and split doses against the Zika virus were safe, well-tolerated, and elicited detectable positive antibody responses.[31] Needle-free intramuscular immunization of synthetic VIU-1005 candidate DNA vaccine with only two doses of 25 μg of the vaccine, elicited significantly higher levels of severe acute respiratory syndrome coronavirus (SARS-CoV) Spike1-specific total IgG in mice.[32] Therefore, DNA vaccines could be one of the best alternative remedies because of their ability to produce endogenous antigens (effective in inducing strong T-cell and B-cell immune responses), ease of production, handling, storage, and potential to render long-term immunity with a minimum of two immunization doses.[21] After administration, DNA vaccines express pathogen-specific antigenic proteins that are recognized as foreign by the cells of the host immune system. In DNA vaccines, the antigen/protein is synthesized inside the host cell and it is also expressed as a soluble secretory protein in the extracellular fluid, thereby presented by both class I and II MHCs[33] stimulating both CD4 and CD8 T-cells.[34] Due to this advantage and its potential to elicit an immune response against a variety of infectious agents including bacteria, viruses, and parasites, DNA vaccines could be used as a suitable strategy for tackling emerging diseases.

The initial steps in the construction of a DNA vaccine involve the selection of a suitable vector with a strong eukaryotic promoter and a gene from the pathogen.[35] The steps involved in the synthesis of a DNA vaccine include: (a) The isolation of the gene encoding the target antigenic protein from the pathogen; the target gene shall be obtained either by polymerase chain reaction or by direct synthesis. (b) Cloning the gene of interest into a nonreplicating plasmid (expression vector) at a site downstream to the promoter.[36] (c) Transformation of the resultant recombinant into a bacterial host either by treatment with chemicals, heat-shock, or electroporation (EP). (d) Selection and culturing of the recombinant cells transformed with the plasmid possessing antibiotic resistance and (e) isolation and purification of the recombinant plasmids from the cultured cells. These resultant recombinant plasmids are used as DNA vaccines [Figure 3].
Figure 3: Construction of DNA vaccines: (a) Isolation of the antigenic fragment from the pathogen. (b) A eukaryotic expression plasmid is used as a vector. (c) Cloning the gene of interest (of the pathogen) into the plasmid. (d) Replication of the plasmid inside the host. (e) Protein expression and purification

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What are RNA vaccines?

RNA vaccines, another type of nucleic acid vaccine consists of an mRNA strand that codes for a pathogen-specific antigen. mRNA is a molecule that carries the information of DNA from the nucleus to the cytoplasm where it is translated to proteins by ribosomes. Once inside the host cell, the cells use the instructions present in the mRNA to synthesize the protein which subsequently triggers an immune response. Two major types of RNA vaccines include nonreplicating mRNA and self-amplifying RNA. Nonreplicating mRNA vaccine codes for only the antigen once inside the host cells but the latter type encodes both the antigen and viral replication machinery that enable RNA amplification and greater protein production.[37] Though the process of mRNA vaccine production is faster, cost-effective, and less laborious, the major disadvantage of this approach is its susceptibility to the host's RNase attack. Research studies are focused on developing efficient and safer delivery systems for administering mRNA vaccines. Synthetic functional mRNA vaccines are produced in the laboratory using a cell-free system. Engineering an mRNA vaccine requires only the sequence of the mRNA transcript that codes for the target protein/antigenic fragment and a delivery system. Steps involved in the manufacture of an mRNA vaccine involve (a) synthesis of pDNA (plasmid DNA) with antigen-specific protein linked to a bacteriophage protomer, (b) Synthesis of mRNA molecule by in vitro transcription, and (c) Purification of synthetic mRNA followed by encapsulation with a suitable delivery system.[38] In brief, RNA vaccine production is rapid and cost-effective compared to conventional vaccines and is a safer approach as it involves non-infectious pathogenic fragments as the antigen. As the production of these vaccines is completely laboratory-based, the manufacturing process can be standardized and used for rapid large-scale synthesis to combat sudden outbreaks and pandemics.

RNA vaccines have gained a lot of attention since the COVID-19 (coronavirus disease of 2019) outbreak. Currently, the world is amidst the COVID-19 outbreak which was declared a pandemic on March 11, 2020.[39] The causal agent of the disease, SARS-CoV-2 was reported to be rapidly mutating and constantly evolving, thereby challenging the global scientific community against developing a vaccine.[40],[41] After whole genome sequencing and structural study of SARS-CoV-2, the gene coding for the spike protein was selected for the development of mRNA-based vaccine candidates. Spike proteins are the glycoproteins, present on the envelope of the virus comprising different sets of domains. In Dec 2020, the first mRNA-based vaccines developed by Pfizer-BioNTech and Moderna were approved for use against COVID-19. Both the Pfizer-BioNTech and Moderna's mRNA vaccines developed against SARS-CoV-2 contains the mRNA transcripts enclosed in lipid nanoparticles (LNPs) based delivery system.[42] These two mRNA-based vaccines have been authorized by the US Food and Drug Administration (FDA) for emergency use against COVID-19 prevention. The mRNA vaccine BNT162b2 was developed by Pfizer, Inc., and BioNTech for administration to individuals above age 16, and the second mRNA vaccine mRNA-1273 was developed by ModernaTX, recommended for people 18 years and above. While mRNA vaccines for COVID-19 are currently in use, their status of use for other viral-based outbreaks caused by Influenza A, Zika, Chikungunya virus, etc., is still in different phases of clinical trials.[43]


  Comparison of DNA and RNA Vaccine Delivery and Mechanism of Action Top


DNA vaccine mechanism

Whilst the mechanism of action of DNA vaccines is not completely understood, three possible mechanisms of action have been proposed. DNA vaccines consist of a eukaryotic promoter driving the expression of an antigen/protein-coding gene. The first mechanism is the transfection of skin cells and muscle cells, but the presentation of antigen by these cells is not effective and the underlying mechanism is still unknown.[34] The second mechanism is the transfection of dendritic cells by the plasmid DNA vaccine. As the promoter harbored by the plasmid is activated, the corresponding antigen-specific gene is expressed and presented on the cell surface of the dendritic cells by the MHC molecules and is recognized and processed by T-cells.[44] Since DNA vaccines are made up of bacterial plasmids that have an unmethylated CpG motif that can function as adjuvant, Toll-like receptors present on dendritic cells can also identify them. These motifs are recognized by the immune cells as a pathogen-associated molecular pattern that helps improve immunogenicity. The third mechanism is the phagocytosis of transfected cells by the macrophages (phagocytes), presenting the antigens to the T-cells [Figure 4].[34] The proposed mechanisms will produce both humoral (also known as antibody-mediated immunity) as well as cell-mediated immune responses. Humoral response helps in generating memory that provides rapid and long-lasting immunity during reinfections.
Figure 4: The possible mechanisms for DNA vaccine- Transfection of DNA vaccine into somatic cells (a) or dendritic cell (b), express the antigen (coded by the gene insert) and the antigen presented by MHC is recognized by T-cell and in case of an encounter with macrophages, the latter phagocytize the transfected somatic cell and presents the antigen to the T-cells. (c) In case of an encounter with Macrophages, the latter phagocytize the transfected somatic cell and present the antigen to the T cells. MHCs: Major histocompatibility complexes

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The APCs, found in a variety of tissue types are the targets of DNA vaccines, which makes the skin a preferable route of administration. The simplest methods of DNA vaccine administration are intradermal or intramuscular injection of recombinant plasmids, dissolved in phosphate buffer saline (PBS), using a hypodermic syringe injection, or using particle bombardment technique.[45] Particle bombardment (also known as gene gun or biolistics) injects metal particles coated with DNA penetrating the skin with high force resulting from the expansion of compressed helium. Jet injectors use high-pressure steam released through a small exit creating a force strong enough to penetrate the epidermis thereby delivering the DNA molecules into the cell. Intradermal injection of DNA vaccines by tattooing is another method of DNA vaccination that has been proven to be effective in both small and large animal models.[46] The major challenge in achieving effective immunity through DNA vaccines is that after internalization, the administered DNA needs to enter the nucleus for transcription followed by translation in the cytoplasm.[47]

RNA vaccine mechanism

RNA molecules are highly unstable and susceptible to the host's RNase enzyme; hence, delivery by encapsulation involving LNPs, polymers, peptides, and dendritic cell-based mRNA vaccines has so far proved successful.[48] Among these delivery vehicles, LNPs have proved to be a preferred choice for this vaccine type; SARS-CoV2 is currently approved for use employing LNPs because of their ease of formulation, biocompatibility, and vaccine payload ability.[43] The vaccine component upon reaching the cell gets penetrated through receptor-mediated endocytosis and the RNA molecules are released into the cytoplasm. The RNA molecules are translated by the ribosomes into proteins that serve as an antigen. These proteins are cleaved into small protein units by the proteasomes and presented by the MHC class I and MHC class II complexes.[49] Some protein units are released to the extracellular environment by the secretive pathway and recognized by APCs. While the antigen presented by MHC class I is recognized by the CD8 receptor of cytotoxic T-cells, MHC class II antigen presentation is recognized by the CD4 receptor of helper T-cells which in turn activates the B-cells to produce antibodies. An example of the SARS-CoV-mRNA vaccine mechanism using LNPs as a delivery system into the muscle cells is shown in [Figure 5]. Knowledge gained from the working mechanism of mRNA COVID-19 vaccines shall be employed in developing mRNA vaccines for other viral infections.
Figure 5: Illustration of mRNA vaccine mechanism targeted against the spike protein of SARS-CoV2. The mRNA is enclosed with lipid nanoparticles and delivered intramuscularly to the muscle cells. Through receptor-mediated endocytosis, the vaccine gets entry into the cell. mRNA is translated into protein by ribosomes and cleaved into small protein units by proteasomes. These small proteins are released extracellularly by secretory pathways. These small spike protein units are recognized by APCs by cross-presentation and immune cells trigger an immune response against the spike protein through cell-mediated and humoral immunity. S protein: Spike protein, LNP: Lipid nanoparticle, MHC: Major histocompatibility complex, DC: Dendritic cell, Tc: Cytotoxic T-cells, TH: helper T-cells, Ag: Antigen, SARS-CoV2: Severe acute respiratory syndrome coronavirus 2

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  Advantages and Limitations of DNA Vaccines over Other Vaccine Types Top


The recent decade has seen significant progress in the field of DNA vaccines because of their broad range of benefits. The striking feature of DNA vaccines is the production of endogenous antigens. The antigenic protein is produced inside the host cells thereby avoiding the problems of improper folding and ensuring the long-term expression of the antigen resulting in prolonged immunity.[50] Conventional vaccines that are composed of proteins, like the subunit vaccines, have a higher probability of vaccine failures because of the possibility of alterations in protein folding during the isolation and purification of antigens from pathogens. Because of the conformational differences between the native antigen and the isolated antigen, the host immune cells that are immunized with the altered antigen may fail to recognize the pathogen's native antigen. But in DNA vaccines, failure due to misfolding is minimal as the pathogen's protein/antigenic fragment is synthesized inside the target host cells in its native form. For example, Hepatitis DNA vaccine can be administered epidermally (particle-mediated) to human subjects who were non-responsive to conventional subunit vaccines. These epidermal vaccines contain plasmid-DNA encoding Hepatitis B surface antigens. When administered as 3 doses of 4 micrograms each at 56days interval, these vaccines were found to elicit higher levels of antibody production.[51] DNA vaccines are as effective as LAVs in triggering both humoral and cellular mediated immunity and importantly safer and less dangerous compared to the former as they lack the virulence attributes. In addition, DNA vaccine production involves simple purification steps as compared to the expensive purification of mRNA vaccines[52] and is more stable as they do not require chain refrigeration during transportation or distribution to maintain viability. The cold chain process which is a prerequisite for protein-based vaccines contributes to 80% of the cost spent on vaccines in developing countries.[53] On the contrary, DNA vaccines can be stored in lyophilized or aqueous forms at room temperature. Hence, it is cost-effective and economical. Also, all kinds of DNA vaccines share the same feasible techniques of production and purification. Another major advantage of DNA vaccines over conventional vaccines is co-inoculation, which involves the administration of multiple plasmids into the recipient. The expression of the gene inserts in the co-inoculated plasmids will result in the synthesis of different antigens of the same pathogen. This technique is important in the treatment of infectious diseases such as malaria, acquired immunodeficiency syndrome (AIDS), and tuberculosis, in which exposure to a single antigen is not enough for complete protection against the infection. Also, the plasmid vector can carry inserts of multiple genes which will enable the expression of different genes inserted in the same plasmid. Such DNA plasmid vaccines co-expressing many genes are considered a promising anti-tumor therapy.[54]

Despite having a broad range of benefits, DNA vaccines still struggle with the biggest limitation of inadequate immunogenicity in humans. The first human trial of a DNA-based vaccine for HIV containing env and rev genes was safe and well-tolerated but exhibited low immunogenicity in the 30 μg dose group. Human subjects in 100 μg and 300 μg dose groups displayed rises in geometric mean titer by 36 weeks and importantly there was no increase in cytotoxic T-lymphocyte activity at the highest dose group of 300 μg[55] Other initial studies involving DNA vaccines targeted against diseases such as cancer and hepatitis were well tolerated but in general, suffered from poor immunogenicity. A study that administered the hepatitis B DNA vaccine by gene gun delivery system induced a booster response, but the vaccine at the low dose (0.25 μg DNA) failed to induce primary immune responses.[56] Also, DNA vaccines cannot be used for immunization against polysaccharide-based antigens (e.g., Pneumococcal polysaccharide antigen), restricting their usage.[50] The major challenge in making a DNA vaccine a promising success is achieving its delivery to the nucleus where the transcription of the gene insert occurs using the host's transcription machinery. The knowledge of the mechanism by which the DNA vaccines reach the nucleus to undergo transcription is not yet completely understood. Recent developments targeted to enhance the efficacy of DNA vaccines are discussed in section 6.


  Major Concerns in using DNA Vaccines Top


Years after the work of the pioneers in the field that led to the discovery of the DNA vaccine, many researchers followed their path resulting in improvements in the succeeding years developing a promising tool for fighting against diverse diseases.[57] The construction and development of DNA vaccines have been proven to be much easier and more cost-effective as compared to traditional vaccines. Despite its promising potency to solve the major clinical problems of the future, DNA-based vaccines tend to create uncertainties, in the mind of the general public questioning their safety.

Insertional mutation

An insertional mutation is one of the major concerns in DNA vaccine technology. Upon administration of a DNA vaccine to a eukaryotic animal system, “does the plasmid from the vaccine integrate into the genome?,” continues to be an unanswered question in the minds of young enthusiasts/scientists in the field. To answer this question, studies have been conducted to detect the integration of these plasmids into the cellular DNA of Mus musculus (mice) after intramuscular injection of three different DNA vaccines. The study involved an integration assay with a sensitivity of one plasmid per 1 microgram of DNA and carried out integration studies of three different DNA vaccines containing either influenza matrix, HIV gag gene, or influenza hemagglutinin. This study concluded that the frequency of plasmid-based DNA vaccine (pDNA vaccines) insertion into the host genome was approximately 1–8 integrations per 1, 50, 000 nuclei, which was three times less than the natural mutation rate.[58],[59] Therefore, the risk of insertional mutations due to intramuscular DNA vaccines is negligible. Another study revealed that DNA vaccines developed using human fetal cell lines contain more concentration of DNA fragment contaminants. These contaminants can undergo insertional mutations and cause cancer and or neurodevelopment diseases like autism in children. And, these mutations can increase DNA breakage and rearrangement.[60] Hence, to minimize the undesired effects of DNA vaccines, FDA has set certain approved guidelines for the production of pDNA vaccines, i.e., pDNA must be supercoiled more than 80% for preventing it from integrating with the host genome.[61]

Induction of antibodies against DNA

Another concern of DNA vaccine safety other than integration is the risk of induction of anti-DNA antibodies. These types of antibodies are produced against a foreign DNA molecule entering a host. Experimental studies have proved that the administration of the DNA vaccine using a gene gun could be effective in reducing the risk of anti-DNA antibody production. This is because a gene gun is efficient in delivering small amounts of DNA vaccine.[36] Laboratory studies in animal models have so far shown that double-stranded and purified DNA does not elicit antibody production.[25] Normal mice with multiple doses of DNA vaccine administered for 16 months did not show any clinical or serologic evidence of autoimmune disease. Similarly, in lupus (autoimmune disease) vulnerable mice, the onset or the seriousness of the disease was not affected by DNA vaccination, showing that regular DNA vaccination does not induce any harmful immune response.[62] However, the improvement of DNA vaccines to increase immunogenicity might increase the chances of integration and induction of anti-DNA antibodies. However, it remains unknown whether the DNA vaccination will evoke antibodies against DNA in humans. This essentializes the investigation of safety concerns before the initiation of clinical trials.[25],[63]

Environmental hazard

The improper waste management system of biomedical wastes from research laboratories that develop, or construct DNA vaccines could be a potential threat to the environment. Intentional or accidental release of the plasmids to the environment during the vaccination process could pose a greater risk. This is because the recombinant plasmids of the DNA vaccines possess antibiotic-resistance genes that are used as markers for the selection of recombinant plasmids. When the plasmids with these genes are released into the environment, it could transform the microflora of the environment, inducing antibiotic resistance to the species that have taken up the plasmids from the surrounding. This could be an escalation of the already problematic state of antibiotic resistance. However, it is least likely to develop antibiotic resistance in the vaccine recipients because the antibiotic resistance gene is induced only by the origin of replication in the bacterial genome (bacterial promoters), also the antibiotics in use are limited to those rarely used in treating human infections.[64] As the pDNA vaccines inside a eukaryotic cell are nonreplicative and nonintegrative, the integration risk of DNA vaccine to the host genome is negligible.


  Achievements and Progress in DNA Vaccine Research Top


To overcome the setbacks faced by DNA vaccines, tremendous efforts have been channelized towards optimizing the delivery system to achieve improved immunogenicity. The following are some of the key methodologies employed to improve the efficacy of DNA vaccines.

New approaches in DNA vaccine development

The early approaches to DNA vaccine trials were a failure because of the lack of information demonstrating vaccine-specific immunity. But it was the failure of the early first-generation DNA vaccines that paved the way to novel strategies and developments of much improved and effective second-generation DNA vaccines. The different DNA vaccine developmental strategies are discussed below.

Antigen design

It involves the use of in silico tools to study the amino acid sequences of an isolated antigenic protein as a reference to design the DNA vaccine. This involves studying a group of closely related pathogens to trace the most common (conserved) amino acid sequence among them. With the construction of the phylogenetic tree of the pathogen, the amino acid sequence is selected from the root of the tree. This sequence information is used to develop DNA vaccines for immunization against multiple pathogens or multiple antigens of the same pathogen. Based on this, a trial was conducted using structural proteins isolated from the hepatitis C virus and demonstrated the successful expression of the viral protein by the DNA vaccine designed using complementary DNA of the protein.[65] In a similar approach, chikungunya viral structural polyprotein was analyzed using bioinformatic tools to predict the conserved T- and B-cell epitopes and was subsequently used for DNA vaccine design.[66] The research group was also successful in designing a DNA vaccine using epitopes predicted from the consensus sequence of Zika virus polyprotein.[67] This approach has also proved successful in developing DNA vaccine candidates for bacterial pathogens; A recent study developed a peptide-base vaccine, MP3RT, against increased levels of interferon- γ and CD3+ T-cells, following TB infection in humanized mice.[68]

Vaccine adjuvants

In this technique, vaccine adjuvants are administered along with the DNA vaccine to increase immunogenicity.[69] Adjuvant studies were pioneered by Ramon as “substances that are used in association with a particular antigen to produce an immune response stronger than the antigen without adjuvants.” Two main groups of adjuvants are associated with DNA vaccine studies: (i) classical adjuvants (chemical compounds) and (ii) genetic adjuvants (proteins encoded by DNA plasmids). The adjuvants exhibit two mechanisms of action: (i) act as immunostimulants and interact directly with the immune system and (ii) act as carriers and stimulate delivery of the DNA vaccine to the cells of the immune system.[70] In one of the recent studies by Li et al., 2019, lymphocyte activation gene-3 (LAG3)-interfering oligonucleotide (LIO) adjuvant increased the immune response triggered by recombinant protein and inactivated influenza vaccines. LAG3, a transmembrane protein present on T-cells inhibits the T-cell-mediated activation of B-cells.[71] Using LIO, a type of antisense oligonucleotide as an adjuvant effectively inhibited the action of LAG3 thereby enhancing the immune responses of the vaccine [Figure 6].
Figure 6: Schematic representation of the mechanism of ASO as an adjuvant. (a) APCs presenting stable MHC II binding with LAG-3 transmembrane protein inhibit T-cell activation and proliferation in turn decreasing the immune response. (b) ASO against LAG-3 is synthesized and enclosed with liposomes. These ASO and liposome complex enters the cell by endocytosis and releases ASO in the cytoplasm. ASO binds to complimentary LAG-3 mRNA. RNase H then degrades the ASO and mRNA hybrid inhibiting the production of LAG-3 transmembrane protein resulting in the activation and proliferation of T-cells. ASO: Antisense oligonucleotide, APCs: Antigen-presenting cells, TH: helper T-cells, MHC II: Major histocompatibility class II molecules, CD4: Cluster of differentiation 4 receptors, TCR: T-cell receptor, LAG-3: Lymphocyte activation gene-3

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Codon optimization

It involves the application of genetic engineering techniques to change the codons of the antigen-coding gene inserted in the DNA vaccine. This increases the expression of antigenic proteins in the target organism.[72] The most frequently used codons in Homo sapiens could be determined using the codon usage database provided by GenBank.[73] The codon optimization is built on the synonymous properties of the codon and the degenerate nature of the genetic code. The antigenic polypeptide chains are synthesized by using different synonymous codons to increase the amount of antigen expressed, thereby increasing immunogenicity.[72]

DNA vaccine delivery methods

Efficient delivery of DNA vaccines in vivo is a prerequisite for achieving effective immunogenicity. The biggest challenge for DNA vaccines is to cross two membrane barriers (plasma membrane and nuclear membrane) to enter the nucleus where its transcription can take place. Therefore, several methods have been developed to improve vaccine delivery and immunogenicity attributes. Similar to conventional vaccines, DNA vaccines can be administered through intradermal, intramuscular, and transdermal routes. But due to needle injection-based delivery, DNA vaccines fail to elicit a strong immune response in humans. Currently, physical methods such as gene guns or EP have offered greater success in vaccine delivery and immunogenicity.

Electroporation

In vivo EP involves vaccine delivery using an electric pulse onto the specific area of the skin epidermis layer. The EP technique induces the interactivity of the applied electric field with the lipid regions of the cell membrane, increasing the permeability of the cell membrane. This is due to the emergence of pores in the plasma membrane, facilitating gene transfer to host cells.[36] This technique increases the uptake of plasmid DNA and enhances antigen presentation.[74] EP-delivered DNA vaccines induce high antigen-specific T-cell response.[75] EP increases the efficacy and immunogenicity by increasing the vaccine delivery up to a thousandfold greater compared to other delivery systems/methods.[76] Many attempts have been made to deliver nucleic acid vaccines through EP to increase the vaccine's immunogenicity. HIV vaccines administered through a combination of EP to skin and muscles provided an increased immune response.[77] In one of Phase I human trials of the multigenic HIV-1 DNA vaccine, the patient was injected with the vaccine intramuscularly by in vivo EP. It proved safe and was accepted as a prophylactic vaccine.[78] To date, in vivo EP has shown a significant impact on immunogenicity when combined with different types of administration routes. Some of the recent in vivo EP devices that have been successful are listed in [Table 1].
Table 1: Novel devices used for in vivo electroporation

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Nanocarrier-based chemical delivery

DNA vaccine efficacy can also be improved significantly by the use of chemical delivery methods involving nanocarriers. Biomimetic nanoparticles (BNPs), viral nanoparticles (VNPs), and biomagnetic nanoparticles (BMPs) are some of the recently preferred nanocarriers for delivering DNA vaccines.[82],[83] BNPs are a type of nanoparticles that integrate the flexibility of synthetic material and the functionality of biological membranes hence achieving better navigation in biological systems.[84] VNPs differ from BNPs only in their outer layer; BNP comprises enclosing the vaccine linked with nanoparticle carrier within the biological membrane whereas VNP uses viral capsid as the outer layer. BMPs, an interesting type of nanocarrier, are extracted from naturally occurring magnetotactic bacteria, which can orient their direction according to the magnetic field applied. The binding efficiency of the DNA vaccine to these BMPs was higher in PBS at pH 4–5; negative charges of DNA also facilitated the formation of the DNA-BMP complex.[83] A schematic representation of BNP and BMP delivery systems is illustrated in [Figure 7]a and [Figure 7]b.
Figure 7: (a) – Illustration depicting the synthesis of BNP and VNP as a delivery system. BNPs and VNPs consist of two layers for targeted delivery – vaccine payload present in the core is enclosed with nanoparticles and then layered with the cell membrane. These are synthesized using different methods. BNPs consist of the body or immune cell's cell membrane with specific receptors whereas VNPs consist of viral protein capsid as the outer layer. In the case of BNP, the DNA vaccine with the nanoparticle carrier enclosed within the cell membranes is used as the delivery vehicle; viral capsid loaded with DNA vaccine acts as the vehicle in VNP. BNP: Biomimetic nanoparticles, VNP: Viral nanoparticles, DC: Dendritic cell, RBC: Red blood cell, P: Platelets, M: Muscle cell, B: Bacteriophage, TMV: Tobacco mosaic virus, AV: Adenovirus. (b) Illustration showing the mechanism of BMP as a delivery system. BMPs linked with DNA enclosed with liposomes are injected intravenously to the targeted cell through the bloodstream. Once inside the cell through endocytosis vaccine payload is released into the cytoplasm. Under the influence of magnetic force, the payload can be delivered to the nucleus by a process called magnetofection. BMP: Biomagnetic particles

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DNA formulations

The development of different formulations and adjuvants has augmented the delivery and immunogenicity of DNA vaccines. Formulating DNA vaccines in liposomes or microparticles has increased the transfection efficiency of DNA plasmid vaccines both in animal models and humans.[64] Influenza DNA vaccine formulated with lipid compounds[85] and AS03 (oil-in-water adjuvant)[86] effectively induced the upregulation of T-cell responses. Another promising method to improve the immunogenicity of DNA vaccines is the inclusion of molecular adjuvants also known as genetic adjuvants.[87] Unlike conventional adjuvants, Molecular adjuvants such as cytokines, chemokines, and immune signaling molecules are regulatory substances that activate the immune system, facilitate antigen presentation, and augment the maturation of lymphocytes and dendritic cells. Molecular adjuvants can either be administered as individual plasmids along with the gene vaccines or as a vaccine cocktail that harbors gene vaccine and adjuvant encoding gene on the same plasmid.[69],[88] Co-administration of interleukin (IL) 12 or IL-15 with plasmid DNA vaccine was shown to increase the activity of memory T-cells in nonhuman primate models.[89] Chemokines (also known as chemotactic cytokines) are a family of small secreted proteins whose expression at the site of gene vaccine injection led to the activation and differentiation of effector T-cells.[90],[91]

Prime-boost strategy

Prime-boost strategy/regimen involves administering the vaccine in two doses, a prime dose, and a booster. It can be either homologous (both prime and booster comprise the same vaccine) or heterologous wherein the immunogen given during prime and booster doses are different or the same antigen is given with different types of vaccines.[92] This strategy is usually used to trigger higher levels of immunity compared to the immune response elicited by a single vaccination. Prime-boost also helps in achieving long-lasting immunity by activating both B-cell and T-cell responses. The outcome of prime-boost modalities is determined by several factors including the nature of the antigen, vector type, delivery routes and methods, adjuvants, order of vector administration, the intervals between the doses, etc.[93] The heterologous prime-boost regimen that included primary vaccination with DNA vaccine followed by booster dose with recombinant poxvirus harboring the same immunogen proved to be very effective in eliciting cytotoxic T-cell responses against HIV, malaria, and Cancer.[94] Compared to the homologous prime-boost vaccines, heterologous prime-boost regimes are effective in generating memory T-cells and can overcome anti-vector immunity against many viral infections.[95] For instance, the prime-boost DNA vaccine tissue antigen-cervical intra-epithelial neoplasia trial against human papillomavirus conducted in mice resulted in high antigen-specific T-cell induction after two prime administrations followed by a protein booster.[96] RV144 that targets HIV is an example of prime-boost vaccine. The vaccine was developed from a combination of ALVAC (as prime) and AIDSVAX (as booster) vaccines. Clinical trials have proven the higher efficacy of RV144 as compared to the individual vacvines, ALVAV and AIDSVAX.[97] Likewise, specific to Ebola infection, two independent studies employing heterologous prime-boost immunization modalities have shown success in nonhuman primates.[98] To fight the recent COVID-19 outbreak, many variants of heterologous prime-boost vaccines were developed that showed success in animal studies. AZD1222 vaccine developed by Oxford and AstraZeneca induced robust humoral, CD4, and CD8 T-cell responses in mice and rhesus macaques with a prime-boost regimen against COVID-19-related pneumonia.[99] Collectively, improvements in genetic engineering techniques, modification of plasmid construction, improved delivery methods, and prime-boost regulations would meet the requirements for upgrading the DNA vaccine technology.[100]

Resources available for DNA vaccine development

The DNAVaxDB is a web-based database that stores information related to DNA vaccine research as well as the plasmids and the antigen used in the DNA vaccine development. DNAVaxDB comprises details on the DNA vaccines that have been verified as effective in at least one experimental animal model.[101] VIOLIN (http://www.violinet.org) is a web-based comprehensive vaccine database and analysis system.[102] Vaxjo and VaximmutorDB are two web-based databases that are used for the identification of molecular adjuvants and to understand vaccine-induced immune mechanisms respectively.[102],[103]


  An Update on the Clinical Applications of DNA Vaccines Top


Phase-I trials of first-generation DNA vaccines were initiated almost 20 years back with HIV type I as the clinical target testing its prophylactic and therapeutic efficiencies followed by trials targeting influenza, human papillomavirus, hepatitis, and melanoma.[104] The first-generation DNA vaccine was a failure because of poor immunogenicity. It resulted in poor antibody production and low cell-mediated immunity.[105] Improvements in the delivery methods of first-generation DNA vaccines such as optimizing the vectors and the encoded antigens, and the development of novel strategies and adjuvants to enhance and control the host immune response led to the development of the second-generation DNA vaccines which elicited a stronger immune response resulting in higher antibody production.[104]

The database ClinicalTrials.gov, provided by the U.S. National Library of Medicine, records 406 DNA vaccine trials, out of which 237 are completed (as of April 16, 2020). These vaccines target several conditions including AIDS, different types of cancer, and infectious diseases among which 173 of the total DNA vaccine trials targeted HIV (https://clinicaltrials.gov). Phase I clinical trial of DNA vaccine immunization against the Ebola virus demonstrated the safety and induction of Ebola virus-specific antibodies.[106] DNA vaccines against cancer are constructed using the tumor-promoting genes that encode tumor-antigen. The antigens induce a tumor-specific immune response. This is the basis of the new strategies for the development of DNA vaccines for cancer treatment.[107] Specific to COVID-19, 28 studies are currently subjected to clinical trials as per ClinicalTrials.gov out of which INO-4800 from Inovio Pharmaceuticals, DNA vaccine by Takara Bio and Osaka University, and ZyCov-D by Zydus Cadila were in phase III clinical trials.[108]

DNA-based vaccines have also seen success as a safe and efficient option for veterinary use. Few DNA vaccines have already achieved a foothold in the commercial veterinary market. For instance, DNA vaccines for infectious hematopoietic necrosis virus (in salmon), West Nile virus (in horses), and melanoma (in dogs) have been approved for veterinary medicinal products.[109] Interestingly, a study by Choi et al., 2020 has shown the protective efficacy of DNA vaccine (POWV-SEV) against Powassan virus in murine models with single immunization.[110] Until the outbreak of COVID-19, no DNA-based vaccine saw approval for human use. The current status of some of the COVID-19 DNA vaccines is listed in [Table 2]. To date, India's ZyCoV-D is the first DNA-based vaccine approved for people in the world which has been shown to exhibit an efficacy of 66.6%.[4]
Table 2: Current status of coronavirus disease of 2019 DNA vaccines under clinical trials

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  The Future of DNA Vaccines Top


A DNA vaccine is an ideal vaccine that would be much safer than other vaccine types. A DNA vaccine is the hope of the under-developed and developing countries to deal with infectious diseases and clinical conditions whose treatments are expensive. The low immunogenicity of the DNA vaccine has been a limiting factor. This could be solved by using the prime-boost strategy. Advanced immunological studies and a better understanding of the response of each cell type to an antigen would help in deciphering the action of DNA vaccines. Upgrading the engineering techniques for DNA vaccine plasmid construction that would allow targeting a specific cell would increase the efficiency and uptake of the plasmids by the cells. Combining the techniques of Genetic Engineering and Nanotechnology could render new nanodelivery methods for DNA vaccine administration.[115] This would provide targeted-controlled vaccine delivery to the antigen-presenting cells for efficient immune response.

For each vaccine developed, it is essential to determine the risk-benefit ratio and safety. Similarly, DNA vaccines should be subjected to more careful examinations and evaluations regarding their safe uses, focusing on clinical and preclinical studies. Before the initiation of works related to DNA vaccines, awareness of the potential risks of accidents in the laboratories should be inculcated among health professionals to prevent antibiotic resistance. The limitations in the production of the human DNA vaccine could be overcome by further developments and improvements in the years to come.


  Conclusions Top


Nucleic acid vaccines are potential ideal vaccines with simple construction, easy transportation, and production for large-scale applications. The quick approach of DNA vaccine construction that requires just the gene sequence of the target antigen makes this technology more rapid than any conventional vaccine development technology. It is also one of the emerging vaccine technologies with a promising ability to combat widespread contagious infections. Although several experimental studies have demonstrated rapid vaccine development as well as the safety and potency of DNA vaccines for human use, the dependence on associated techniques such as gene guns, injection devices, or EP for greater immunogenicity remains a limitation of this technology. However, ongoing research progresses and upgraded understanding of the human immune system and vaccine mechanisms altogether could improve this vaccine technology and make DNA vaccines a success, which could be an antidote to many existing medical complications as well as an emergency solution to tackle any future pandemics or large-scale outbreaks.



Acknowledgments

We thank the JSS Academy of Higher Education and Research, Mysuru, India for providing the support and research facilities.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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