Unlike traditional vaccines that rely on inactivated pathogens or recombinant proteins, nucleic acid vaccines deliver DNA or RNA molecules encoding antigens to directly synthesize target proteins in host cells and activate precise immune responses.

This path has shown great potential in the COVID-19 pandemic - it took only 11 months from sequence design to approval for mRNA vaccines, setting a new record in the history of drug development. It not only verifies the rapid response advantage of nucleic acid technology, but also promotes a profound reconstruction of the global biomedical industry.

However, despite the advantages of high efficiency, rapidity, and safety, this technology still faces challenges such as delivery efficiency, reliance on low-temperature storage, and long-term safety to be verified. This article aims to sort out nucleic acid vaccine technology, including concepts, principles, challenges, and future trends, in order to increase readers' understanding and promote industrial development.

 

Industry Overview               

Nucleic acid vaccines, also known as gene vaccines, are to directly introduce exogenous genes encoding a certain antigen protein into animal cells, synthesize antigen proteins with the help of the host cell expression system, and then induce the host to produce an immune response to the antigen protein to achieve the purpose of preventing and treating diseases.

According to the main components, nucleic acid vaccines are divided into DNA vaccines and mRNA vaccines. Nucleic acid vaccines have changed the traditional vaccine model of relying on pathogen treatment. Starting from the genetic level, they have opened up a new path to use human cells to produce immune stimulants, bringing new ideas and methods to disease prevention and treatment.

 

Compared with traditional vaccines, nucleic acid vaccines have many advantages:

• First, the protective efficacy is enhanced. Nucleic acid vaccines can stimulate cellular immunity and humoral immunity at the same time. Compared with traditional vaccines that often trigger a single immune response, such as inactivated vaccines that mainly activate humoral immunity, the dual activation mechanism of nucleic acid vaccines provides stronger immune protection.

 

• Second, the preparation is simple, saving time and effort. Nucleic acid vaccines can be amplified in large quantities, the purification process is relatively simple, and they can be combined with a variety of recombinant plasmids encoding different antigen genes to prepare multivalent vaccines, reducing the consumption of manpower, material resources, and financial resources.

 

• Third, cross-protection between the same species and different strains. Nucleic acid vaccine expression vectors can flexibly transform the target genes they carry and select antigenic determinants in a targeted manner, thereby achieving cross-protection against pathogens of the same species and different strains.

 

• Fourth, it is safer to use. Protein antigens are expressed in host cells, and there is no risk of causing disease due to virulence reversion or residual virulence, nor will it cause serious adverse reactions in the body. The fifth is a lasting immune response. Nucleic acid vaccines have strong immune persistence and can obtain long-term immunity after one vaccination. The sixth is the expansion of treatment areas. Traditional vaccines are mainly used in the field of infectious disease prevention. Nucleic acid vaccines have successfully expanded their application scope to new fields such as tumor treatment, breaking the application limitations of traditional vaccines.

 

Nucleic acid vaccine technology research and development process

The research and development process of nucleic acid vaccines includes six core steps:

• The first is antigen design and sequence screening. Based on the pathogen gene data, the target antigen (such as the new coronavirus S protein) is determined, the coding sequence is optimized through bioinformatics, the 5' cap structure, UTR region and codon preference of mRNA are adjusted, or a DNA plasmid containing a strong promoter is designed.

 

• The second is nucleic acid synthesis. DNA vaccines are amplified and purified by Escherichia coli fermentation plasmids, while mRNA vaccines use in vitro transcription technology to synthesize modified RNA (such as pseudouridine substitution) and remove impurities such as double-stranded RNA.

 

• The third is the construction of a delivery system. DNA vaccines rely on electroporation or chemical carriers to penetrate the cell membrane and nuclear membrane, and mRNA vaccines are encapsulated by lipid nanoparticles (LNPs), and nanoparticles are homogenized by microfluidics technology.

 

• Fourth, in vitro validation. Transfect candidate vaccines into cell lines to detect antigen expression efficiency and innate immune activation (ELISA to measure cytokines)

 

• Fifth, animal model evaluation, testing immunogenicity (neutralizing antibodies, T cell responses) and safety (local/systemic toxicity) in mice or non-human primates. Sixth, production process development. Establish modular production lines (such as continuous flow LNP synthesis), optimize purification processes (HPLC to remove impurities), and formulate quality control standards (nucleic acid integrity, sterility, LNP particle size detection) to lay the foundation for large-scale production.

 

Technical Challenges

Insufficient efficiency and targeting of delivery systems

• Limited delivery efficiency For DNA vaccines, they must not only pass through the cell membrane, but also cross the nuclear membrane to enter the cell nucleus to function. Although electroporation technology can improve its efficiency of entering cells to a certain extent, this technology will cause damage to local tissues, making it necessary to use it with caution in clinical applications.

 

• Although mRNA vaccines do not need to enter the cell nucleus, the current mainstream lipid nanoparticle (LNP) delivery system has a low cell uptake rate and an unsatisfactory intracellular release efficiency, which cannot fully stimulate the immune response.

 

•  Poor organ targeting The current LNP delivery system has obvious organ targeting defects. It is more likely to be enriched in the liver in the body, making it difficult to achieve precise targeted delivery to other organs, such as the lungs expected by respiratory virus vaccines, and organs such as the spleen and lymph nodes that play a key role in the immune response.

 

• For example, when developing mucosal immune vaccines against respiratory viruses, the vaccine needs to be accurately delivered to the lungs to stimulate an effective immune response, but it is difficult to achieve this goal with existing technologies.

 

Stability and storage and transportation bottlenecks

• The low-temperature dependence dilemma of mRNA vaccines mRNA molecules are unstable and will be rapidly degraded as long as RNA enzymes are present in the environment, which makes mRNA vaccines extremely demanding on storage conditions and usually need to be stored in ultra-low temperature environments of -20°C to -70°C.

 

• Although freeze-drying technology, such as the technology used in CureVac's second-generation vaccine, can improve this situation to a certain extent and increase the storage temperature to 2-8°C, the protective agent used in this technology is complex and expensive, which increases the production cost of the vaccine.

 

• Contradiction between DNA vaccine stability and expression efficiency Compared with mRNA vaccines, plasmid DNA has better stability at room temperature and can be stored for a long time.

 

• However, the expression efficiency in vivo is much lower than that of mRNA vaccines. In order to improve the delivery and expression efficiency of DNA vaccines, physical methods such as electroporation or gene guns are often required, but these methods are complicated to operate, have high requirements for equipment and operators, and face many difficulties in large-scale application, making it difficult to meet the needs of large-scale production and vaccination.

 

Long-term safety controversy mRNA vaccines have exposed some potential risks in actual applications, such as the induction of myocarditis and pericarditis.

 

 

Although the incidence is low, about 0.003%, the specific mechanism of occurrence is still unclear. It is speculated that it may be related to the activation of innate immune responses by nucleic acid molecules or the induction of cross-immunity. As for DNA vaccines, although there is no clinical evidence to show that there is a risk of genomic integration, theoretically, this risk does exist.

• Challenges of large-scale production and cost control 1. Process bottleneck of LNP production The key component of LNP, ionizable lipids, such as ALC-0315, has an extremely complex synthesis process and requires ultra-low temperature reaction conditions during the synthesis process.

 

• This not only places extremely high requirements on the reaction equipment, but also has huge equipment investment costs, with a single production line costing more than $100 million. In addition, the PEG lipids in LNP may cause allergic reactions in actual applications.

 

• Although alternative materials, such as PEGylated phospholipids, are being actively developed, the feasibility and effectiveness of these alternative materials in actual production processes still need to be further verified. 2. mRNA purification technology threshold In the process of in vitro transcription to generate mRNA, impurities such as double-stranded RNA will be produced, which will affect the safety and effectiveness of the vaccine.

 

• At present, high-performance liquid chromatography (HPLC) technology is mainly used to purify mRNA, but this technology has obvious limitations. On the one hand, the purification efficiency of HPLC equipment is directly related to the quality of mRNA.

 

• On the other hand, the production capacity of global HPLC equipment is limited. When producing nucleic acid vaccines on a large scale, it cannot meet the demand for rapid and large-scale purification of mRNA, which has become the main bottleneck restricting the expansion of nucleic acid vaccine production capacity.