Insight

DNA vaccines are nucleotide-based genetic vaccines that utilize plasmid DNA to encode target antigens. After being delivered into host cells, the plasmids drive intracellular antigen expression and subsequently initiate immune responses. The core mechanism involves introducing antigen-encoding plasmids into the cell nucleus, where antigens are synthesized via transcription and translation. These antigens are then processed by antigen-presenting cells (APCs) and presented through major histocompatibility complex (MHC) molecules to activate CD4⁺ and CD8⁺ T cells, triggering both cellular and humoral immune responses.

Compared with protein vaccines, DNA vaccines are capable of encoding full-length multi-antigens and incorporating immunomodulatory molecules, featuring simple production procedures and mild adverse reactions. In contrast to messenger ribonucleic acid (mRNA) vaccines, DNA vaccines possess superior membrane permeability and longer half-lives, yet they carry potential risks of insertional mutagenesis and microbial contamination. As an emerging genetic immunotherapy modality, the optimization of DNA vaccines mainly focuses on four core dimensions: structural modification to enhance immunogenicity, innovative delivery strategies to improve transfection efficiency, combined therapy to amplify synergistic effects, and optimized administration routes to achieve targeted immune activation. Systematic exploration of these strategies will accelerate the translational progress of DNA vaccines from laboratory research to clinical application and provide solid technical support for future vaccine development.

Precise plasmid structural design is pivotal to boosting the potency of DNA vaccines. Antigen screening constitutes the primary step; bioinformatic tools and artificial intelligence algorithms enable the identification of highly immunogenic tumor antigens, with priority given to neoantigens to circumvent autoimmune tolerance. The nuclear entry efficiency of plasmids can be elevated by inserting nuclear localization sequences (NLS), while rational promoter selection markedly augments antigen expression, though it is necessary to address the inhibitory effect of viral promoters under inflammatory conditions.

Furthermore, codon optimization favoring high GC content and abundant transfer ribonucleic acid (tRNA) abundance, insertion of Kozak sequences and untranslated region (UTR) modification jointly facilitate transcription and translation efficiency. Epitope anchoring modification can strengthen the binding affinity between antigens and MHC molecules.

In terms of innate immune activation, the cytosine-phosphate-guanine (CpG) islands and double-stranded DNA structures can induce the secretion of type I interferons to potentiate innate immune responses. The CRISPR/Cas9 gene-editing technology allows precise manipulation of plasmid sequences, such as promoter replacement and antigen sequence optimization.

Personalized and multi-antigen vaccine design has emerged as a research hotspot for heterogeneous tumor therapy. Patient-specific mutant antigens can be screened via whole-exome sequencing, and immunodominant epitopes can be predicted using AI algorithms to construct personalized vaccines. Multi-epitope vaccines can cover clonal tumor heterogeneity, and optimizing MHC binding affinity or introducing molecular adjuvants can broaden the spectrum of immune responses. The single-chain trimer technology enables direct targeting of APCs through secreted chimeric proteins, which has been verified to inhibit tumor metastasis in preclinical mouse models.

Combination regimens of DNA vaccines with other immunotherapies can effectively reinforce anti-tumor immune responses. Cytokine adjuvants are the most commonly adopted combinatorial agents, including interleukin-2 (IL-2), interleukin-12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF). IL-2 facilitates immune cell differentiation, and its combination with HER2-targeted vaccines enhances CD8⁺ T cell-mediated immune responses in clinical settings. Local administration of IL-12 induces inflammatory reactions within the tumor microenvironment and even elicits abscopal regression of distant tumor lesions. Although GM-CSF may recruit myeloid-derived suppressor cells (MDSCs), it has been proven to enhance specific antibody responses in prostate cancer vaccine trials.

Immune checkpoint blockade (ICB) and conventional clinical therapies are mainstream combination partners. Synergistic administration of DNA vaccines and ICB agents can reverse immune tolerance. For instance, the combination of prostate cancer DNA vaccines and pembrolizumab successfully elicits antigen-specific T cell responses in patients. Chemotherapeutic agents exert dual synergistic effects; chemotherapeutics such as paclitaxel can promote the release of tumor-associated antigens. Radiotherapy amplifies vaccine efficacy by inducing local inflammatory responses, and combined application of human papillomavirus (HPV) vaccines with chemoradiotherapy achieves a high progression-free survival rate in 90% of cervical cancer patients.

Combination with hormonal therapy represents an innovative therapeutic direction. For hormone-dependent malignancies such as prostate cancer, co-administration of DNA vaccines and androgen deprivation therapy can delay disease progression, albeit with limited immune activation efficacy. Postoperative adjuvant DNA vaccination can significantly prolong patient survival, demonstrating great potential in eliminating residual metastatic lesions.

The core principle of combinatorial strategies is to remodel immunosuppressive tumor microenvironments and expand antigenic coverage. Rational matching of cytokines, ICB inhibitors, chemotherapy and radiotherapy has yielded favorable clinical benefits, whereas the balance between therapeutic efficacy and autoimmune adverse events must be strictly maintained. Optimized compatibility between personalized neoantigen vaccines and established therapies remains a key research priority.

The in vivo delivery route directly determines the final immune efficacy of DNA vaccines. Intradermal injection is widely preferred owing to abundant APCs such as Langerhans cells in the dermis, which can provoke potent immune responses with reduced vaccine dosage. Needle-free injection devices represented by thermal-driven jet injectors eliminate needle-related risks and significantly upregulate antigen expression and T cell responses in animal models.

Gene gun technology delivers DNA-encapsulated gold microparticles directly into epidermal tissues with ultra-low dosage, yet it predominantly induces Th2-biased immune responses, which is suitable for specific antigens such as HPV E7 protein. Intramuscular injection features convenient operation and mild side effects, commonly performed at well-vascularized sites including the deltoid muscle. Auxiliary electroporation can substantially improve cellular uptake of plasmids, elevate transfection efficiency and trigger local inflammatory responses to synergize immune activation.

DNA tattoo technology induces innate immune activation via microtrauma, enabling faster immune initiation compared with conventional inoculation despite relatively complicated procedures. Apart from conventional routes, intratumoral injection directly disrupts local immunosuppressive microenvironments with low systemic toxicity and minimal required dosage. Oral delivery targets the intestinal immune system and exhibits unique advantages in the treatment of gastrointestinal tumors. Appropriate delivery routes should be selected flexibly according to tumor types and immunotherapeutic objectives. Multiple booster immunizations are essential for sustained vaccine efficacy, and heterologous prime-boost strategies can simultaneously potentiate cellular and humoral immune responses.

The insufficient immunogenicity of DNA vaccines is mainly attributed to multiple physiological delivery barriers. Exogenous plasmids need to undergo cellular internalization via endocytosis, escape lysosomal and endosomal degradation, and cross the nuclear membrane to achieve intranuclear antigen expression. Nanocarriers and advanced delivery systems optimize delivery efficiency through the following mechanisms: 1) encapsulating plasmid DNA to resist nuclease degradation; 2) enhancing targeted delivery towards APCs; 3) improving cellular and nuclear membrane penetration capacity. Representative delivery systems are summarized as follows:

Polymeric carriers are widely applied in nucleic acid delivery due to excellent tunability and biosafety profiles. Positively charged chitosan can efficiently bind negatively charged DNA and stimulate type I interferon secretion to activate APCs; mannose-modified chitosan further achieves targeted delivery to dendritic cells (DCs). Polylactic-co-glycolic acid (PLGA) possesses favorable biocompatibility but fails to provide sufficient protection for nucleic acids, thus requiring composite modification with other biomaterials.

The transfection performance of polyethyleneimine (PEI) is closely correlated with molecular weight; high-molecular-weight PEI confers high transfection efficiency yet severe cytotoxicity, which can be alleviated via chemical conjugation modification for intranasal and oral delivery applications. Polyethylene glycol (PEG) prolongs the in vivo circulation time of nanoparticles but may hinder APC transfection, requiring rational dosage regulation in practical application.

Lipid nanoparticles serve as pivotal delivery vehicles for DNA vaccines, mainly including liposomes and niosomes. Composed of phospholipids and cholesterol, liposomes facilitate efficient cytoplasmic delivery of antigen-encoding DNA and exert intrinsic adjuvant effects. Surface functionalization enables targeted binding to mannose receptors on dendritic cells to enhance cellular uptake.

Niosomes constructed with cholesterol and non-ionic surfactants exhibit superior structural stability compared with traditional liposomes, and mannose conjugation further boosts their immunostimulatory activity. Despite inherent limitations in in vivo stability, lipid nanoparticles remain promising candidates for DNA vaccine delivery by virtue of controllable physicochemical properties and satisfactory transfection capacity.

Lipopolyplexes integrate the superiorities of polymers and lipid-based nanoparticles to form hybrid delivery systems. Antigen-encoding nucleic acids are encapsulated within polymeric cores, while the outer lipid bilayer shell endows the system with high transfection efficiency, biocompatibility and biodegradability, as well as prolonged in vivo stability and circulation duration. Surface PEGylation is commonly adopted to evade immune recognition and further stabilize nanoparticles.

Virus-like particles are non-infectious self-assembled nanostructures composed of viral structural proteins without viral genomic materials, which can be genetically engineered to load exogenous DNA fragments. Two mainstream DNA encapsulation strategies are established: passive internalization via incubating preformed VLPs in low-ion-strength buffer driven by internal positive electrostatic potential, and in-situ DNA integration during VLP self-assembly via electrostatic interaction.

Compared with traditional viral vectors, VLPs eliminate insertional mutagenesis and biosafety risks, while possessing prominent adjuvant potency, uniform particle morphology and excellent biocompatibility. Early VLP delivery systems were restricted to loading DNA fragments below 4 kb, whereas novel encapsulation technologies have broken through this limitation to achieve loading capacity up to 17 kb, greatly expanding their application scope.

Inorganic nanoparticles such as gold, iron and silver nanoparticles are ideal delivery candidates owing to facile surface functionalization, favorable biocompatibility and adjustable size and morphology. Gold nanoparticles display excellent lymph node targeting capability, which has been validated to deliver antigen-encoding plasmids effectively, induce potent anti-tumor immunity and prolong survival time in preclinical models.

Magnetic iron nanoparticles enable precise targeted delivery and in vivo imaging with low cost and negligible toxicity. Silver nanoparticles can act as both delivery carriers and immune adjuvants to diversify the application scenarios of inorganic delivery systems.

With outstanding programmability and favorable safety profiles, DNA cancer vaccines have become a promising research direction in tumor immunotherapy. Despite suboptimal overall immune response intensity in current clinical trials, they demonstrate tremendous developmental potential through optimized combinatorial regimens, upgraded delivery vectors and innovative administration strategies.

Advancements in personalized precision medicine create unprecedented opportunities for DNA vaccine development. High-throughput sequencing technology facilitates neoantigen identification, and multi-dimensional data including immunosuppressive cell phenotypes and cancer-associated fibroblast biomarkers within tumor microenvironments support the formulation of individualized therapeutic regimens. Such personalized strategies optimize target selection and dosage adjustment, and synergize with conventional therapies to reduce non-specific toxic side effects.

Although cost-effectiveness remains a major challenge for large-scale clinical promotion, the establishment of dynamic efficacy evaluation systems based on specific biomarkers will maximize therapeutic efficacy while minimizing adverse reactions, ultimately paving the way for the full clinical transformation and popularization of DNA vaccines.