Vector Design for Improved DNA Vaccine Efficacy, Safety and Production

Abstract

DNA vaccination is a disruptive technology that offers the promise of a new rapidly deployed vaccination platform to treat human and animal disease with gene-based materials. Innovations such as electroporation, needle free jet delivery and lipid-based carriers increase transgene expression and immunogenicity through more effective gene delivery. This review summarizes complementary vector design innovations that, when combined with leading delivery platforms, further enhance DNA vaccine performance. These next generation vectors also address potential safety issues such as antibiotic selection, and increase plasmid manufacturing quality and yield in exemplary fermentation production processes. Application of optimized constructs in combination with improved delivery platforms tangibly improves the prospect of successful application of DNA vaccination as prophylactic vaccines for diverse human infectious disease targets or as therapeutic vaccines for cancer and allergy.

Keywords: DNA vaccination; adjuvant; antibiotic-free; fermentation; immunization; innate immunity; non-viral; plasmid.

Figure 1

(a) DNA vaccine vector production and application flowchart. Stages 1 and 4 are very sensitive to vector changes and must be optimized coordinately since vector modification to enhance one parameter can have multiple undesired effects on other parameters. Stages 2 and 3 are largely generic; (b) Insert design flowchart.

Figure 2

DNA vaccine vectors. (a,b) 1st; (c) 2nd; and (d) 3rd; generation DNA Vaccine vectors; (e) 2nd and 3rd generation vectors increase in vivo expression compared to first generation vector gWIZ. 5 µg muSEAP vectors delivered intramuscularly with EP to mice on day 0, serum muSEAP assayed on indicated days. 3rd generation vector NTC9385R has significantly higher expression than gWIZ or 2nd generation vector NTC8385 (p-value = 0.05; Mann-Whitney rank-sum test); (f) 3rd generation vectors dramatically increase in vivo expression, compared to 2nd generation. 50 µg muSEAP vectors in 50 µL saline delivered intradermally to mice with EP on day 0, muSEAP assayed on indicated days. 3rd generation vector NTC9385R has significantly higher expression than 2nd generation vector NTC8685 (p-value = 0.05; Mann-Whitney rank-sum test). NTC8685 is a 2nd generation vector similar to NTC8385. The NTC8385 1,518 basepair (bp) bacterial region (spacer region) is reduced to 855 bp in NTC8385-min and 454 bp in NTC9385R. This compares to 2,678 bp for gWIZ, and 1,970 bp for pVAX1.

Figure 3

RNA selectable marker DNA vaccine plasmids. Purple arrow in bacterial region is pUC replication origin, brown arrow in panels (a) and (c) is the RNA selection marker. Eukaryotic region promoter, transgene and polyA are depicted with orange arrow, blue arrow and green box, respectively. (a) NTC8385 plasmid borne RNA-OUT RNA binds a chromosomally encoded constitutively expressed mRNA that contains the RNA-IN target sequence in the leader. This prevents translation of the downstream levansucrase (sacB), allowing growth on sucrose media; (b) pMINI pUC origin encoded RNAI binds a chromosomally encoded constitutively expressed mRNA that contains the RNAII target sequence in the leader. In the murselect-system, an essential gene (murA) is modified to contain a repressor binding site in the promoter and the RNAII target sequence is incorporated into the repressor mRNA leader. RNAI binding to RNAII prevents repressor translation, allowing expression of the essential gene; (c) pFAR4/pCOR plasmid borne suppressor tRNA allows read-through translation of an amber nonsense codon in a chromosome encoded essential gene. Adapted from Oliveira and Mairhofer, 2013 [48].

Figure 4

Molecular mechanisms of DNA vaccines. Transfected B DNA (the most common double helical DNA structure) is sensed in the cytoplasm (cyto) by DNA receptors interferon-inducible protein 16 (IFI16) and DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41) activating the cGAMP synthase (cGAS) [98] /STING/TBK1 pathway to induce type 1 interferon production and NF-κB. An additional cytoplasmic innate immune pathway activated by transfected DNA is the cytoplasmic AIM2 inflammasome. IFI16, DDX41 and AIM2 detect DNA generically and are not sequence specific although IFI16 may preferentially recognize DNA that forms cruciforms or is negatively supercoiled [99]. By contrast, specific CpG motifs in DNA vaccines are sensed by the endosomal (endo) TLR9 innate immune receptor. To improve innate immune activation, addition of optimized immunostimulatory CpG motifs in the vector backbone may be used to increase TLR9 activation while immunostimulatory RNA expressed from the vector may be utilized to activate alterative RNA sensing innate immune receptors such as RIG-I (plasmid backbone adjuvant). Due to limited transgene expression after DNA vaccination in large animals, vector modifications and deliveries that improve transgene expression also improve adaptive immunity. Certain delivery modalities such as EP that improve gene transfer efficiency also activate innate immunity through tissue damage [100,101,102]. EP conditions need to be carefully optimized, since the optimal EP conditions for DNA vaccination are not necessarily those with the highest gene expression [103] and optimal delivery parameters vary between strains [100].