DNA Vaccines and Applications of Recombinant Proteins

INTRODUCTION

DNA vaccination involves the direct introduction into appropriate tissues of a plasmid containing the DNA sequence encoding the antigen(s) against which an immune response is sought, and relies on
the in situ production of the target antigen.
DNA vaccines are more stable, cost-efficient, easy to manufacture and safe in handling. They are being investigated for various applications including therapy of cancer, allergies, autoimmune and infectious diseases.

Course of Action of DNA Vaccines

The classical ways for vaccine delivery are intramuscular, intradermal and subcutaneous injections. These address primarily myocytes and keratinocytes, respectively, including antigen presenting cells (APC) residing near the injection side. In case of DNA vaccines, after their internalization the DNA needs to translocate to the nucleus for transcription, followed by translation in the cytoplasm.

Challenges to DNA Vaccine Applications

1. Transfection efficiency
2. Transcription and translational efficiencies
3. Inefficient immune response
4. Immune evasion.
5. The most serious challenge for DNA vaccines intended to induce an anti-tumor immune response are caused by immune evasion strategies of the tumor. In this regard, tumor cells are often characterized by defective processing of antigens for subsequent presentation via Major histocompatibility complex MHC I and impaired expression of MHC I. In addition, within the tumor microenvironment both parenchymal and infiltrated immune cells may overexpress tolerance-promoting non-classical MHC I molecules like human leukocyte antigen (HLA-G) and HLA-E. Besides, tumor-associated macrophages (TAM) protect the tumor from immune responses by various mechanisms including the release of anti-inflammatory interleukin-10 and the expression of surface receptors like programmed death ligand 1 which inhibit the functions of activated T cells.
6. The effectiveness of DNA vaccines can also be diminished by CD4+CD25+FoxP3+ regulatory T cells (Treg) that promote cancer growth which inhibit T effector cells.
7. Moreover, both TAM- and tumor-derived mediators promote the expansion of myeloid-derived suppressor cells (MDSC) which interfere with the activation of T cells in the tumor microenvironment and attenuate T effector cells as well.

DNA Vaccines Optimization

Promoter

With regards long term expression, viral promotors are often subjected to methylation-mediated inactivation, whereas eukaryotic promoters and hybrids of eukaryotic/viral promoters remain active. Furthermore, the use of cell type-specific promotors may allow to restrict antigen expression to APC.
CD11c has been well established as a DC-specific marker in mouse. 82 integrin is expressed more broadly in human by different immune cell types. DC-STAMP is expressed in immature human and mouse DC, down-regulated in response to maturation. Dectin-2 (CLEC6A) is a C-type lectin receptor (CLR) that binds mannose-rich surface structures of various pathogens and is primarily expressed by Langerhans cells (LC) which constitute the epidermal DC population. Fascin-1 protein is highly expressed in neuronal cells, and mediates the structural integrity of axons.

Antigen

DNA vaccines provide flexibility such that different epitopes/peptides from a single antigen can be designed on a vector to promote broader immune response.
Care should be taken when choosing an antigen so as to avoid induction of autoimmune responses as the immune system will also be turned against healthy tissues expressing the antigen. • Example Unique and Shared Tumor Associated Antigen (TAA)
Unique antigens are the first choice if designing a DNA vaccine as studies have shown that effector T cell responses are more potent against mutated antigens.
Furthermore, antigens with a higher half-life have been shown to induce stronger cytotoxic T cell responses and thereby increased immunogenicity

Codon Optimization

Even if the encoded antigen is expressed at high amounts, antigen presentation/recognition still may be an obstacle to prompt a sufficient immune response. This problem is addressed by introducing epitope-specific changes in the antigen to increase MHC affinity. The design of an antigen-encoding expression unit includes the selection of several immunogenic antigens derived from one or different proteins to be presented via MHCI/II to yield parallel CD4+/CD8+ T cell activation,

Adjuvant

Use of aluminium salt-based Alum

• Transcription Factors
  • immunostimulatory CpG oligonucleotides which engage the endosomal located toll like receptor (TLR)9 to stimulate APC (intrinsic adjuvancy of plasmid DNA).
  • Co-delivery of an influenza antigen encoding DNA vaccine and an IRF-3 encoding vector by biolistic transfection of mice resulted in an increase of activated CD4+ and CD8+ T cells
• Use Stimulatory Cytokines and Chemokines
  • DNA vaccines can be co-delivered with expression constructs for soluble mediators like cytokines or chemokines that exert stimulatory/chemoattracting effects on APC and other immune cells
  • IL-12 is produced by activated APC to promote Th1 differentiation, but also stimulates APC themselves. The end result is enhanced antigen-specific CD4+ and CD8+
  • IL-15 is also released by activated DC, and exerts broad stimulatory effects on effector CD4+ and CD8+ T cells, B cells, NK cells as well as DC

Vector Backbone

1. Reduction of Vector size to encourage transfection
   • For transfection the prokaryotic part of a DNA vaccine is not necessary.
   • For DNA vaccine inversely correlated with transfection efficiency even in mitotically active cell lines.
   • To circumvent this problem, the concept to delete prokaryotic sequences of the plasmid after its propagation in bacteria was developed, yielding so-called minicircle DNA
2. Generation of Bicistronic vectors
   • Internal Ribosome Entry Site (IRES)
   • virus-derived T2A sequence
3. Use of DNA sequences to mediate nuclear translocation
   • Addition of SV40 enhancer site to the vector backbone
   • virus-derived peptides like the SV40 large T-antigen can be used to facilitate nuclear plasmid translocation by attaching these peptides either directly to the vector backbone or to DNA delivery systems

Nano-Carriers (NC)

These are particles within 1-1000nm in size.

Requirements:

• The delivery vector has to offer a sufficient capacity to efficiently package DNA/RNA, which is an obstacle especially for longer plasmid DNA, in order to enable delivery of a sufficient amount of molecules per target cell.
• The delivery system has to show stability against serum proteins that may form a protein corona around the NC and thereby affect its targeting and uptake efficiency.
• After uptake by the cell, the NC cargo has to evade endo/lysosomal degradation and to enter the cytoplasm by endosomal escape. While released mRNA is translated directly in the cytoplasm, DNA vaccines need to translocate into the nucleus for subsequent transcription.

Benefits of NC

• Nano-carriers (NC) offer the advantage to shield the DNA vaccine from degradation by Dnases and other enzymes.
• Specificity
• Surface modifications of NC with moieties like antibodies or natural ligands like carbohydrates may enable direct targeting of DC
• Improved Transfection Efficiency
• Cellular uptake and endosomal release of NC-complexed DNA is enhanced by cell penetrating peptides (CPP) which are either attached directly to DNA or to the DNA-complexing NC
• Cationic particles are internalized better by APC
• For gold NP (AuNP) it was demonstrated that spherical particles are more efficient than any other shape

Safety Concerns with NC

• Biocompatibility
• Biodistributiion and clearance
• The induction of unwanted immune reactions (against PEG used to protect the CPP against protein corona)

Routes of Vaccination

1. Oral (bacteria)
2. Pulmonary using nebuliser
3. Intravenous
4. Subcutaneous
5. Intramuscular
6. Intradermal
7. Transdermal using gene gun, microneedle

Remarks

The immunogenicity of DNA vaccines in human is still to low to yield therapeutically convincing results.
However, in the last 15 years different approaches have shown that optimization of different parameters contributes to enhanced transfection and hence immunogenicity of DNA vaccines also in human

References

1. Hobernik D, Bros M. DNA Vaccines—How Far From Clinical Use? International Journal of Molecular Sciences. 2018; 19(11):3605. Accessed October 7, 2021
2. Corr, M., D. J. Lee, D. A. Carson, H. Tighe. 1996. Gene vaccination with naked plasmid DNA: mechanism of CTL priming. J. Exp. Med. 184: 1555-1560.
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3. Barry, M. A., S. A. Johnston. 1997. Biological features of genetic immunization. Vaccine 15: 788-791.
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