Bar graphs indicate the median and interquartile range

Bar graphs indicate the median and interquartile range. SARS-CoV-2 spike protein is immunogenic in different models and confers protection against lung infection in nonhuman primates. Further evaluation of this DNA vaccine candidate in clinical trials is warranted. Subject terms:DNA vaccines, SARS-CoV-2, DNA vaccines == Introduction == Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged GUB in Wuhan, China around December 2019. It has since caused a global pandemic that has to date, resulted in over 160 million confirmed infections and 3.5 million deaths (WHO COVID-19 Weekly Epidemiological Update; 1 June 2021), although the numbers are likely underestimated1. Developing safe and effective vaccines and testing new vaccine platforms to control the pandemic are paramount. The simplicity and stability of plasmid DNA vaccines make it an attractive immunization platform for emerging viral threats. The DNA vaccines can be designed and produced quickly once the genetic sequence is known and adapted rapidly to new emerging viral variants of concern. Clinically, the DNA vaccine modality is generally regarded as safe and is immunogenic in many different mammalian species including man2,3. Inducing both broad antibody and cellular immune responses, DNA vaccines have the potential to reduce both infection and disease. Intrinsically, the DNA vaccines are stable and can be freeze-dried, allowing for long-term storage at ambient temperature4. The plasmid DNA does not induce vector-specific antibodies, thus permitting multiple booster vaccinations including mixed modality prime-boost strategies5. Historically, first-generation DNA vaccines performed poorly in primates. This was compounded by the application of the Lapatinib (free base) platform to complex pathogens where the correlates of protection are undefined and where other traditional vaccines have similarly failed, such as human immunodeficiency virus (HIV). However, continued platform optimization has seen to the improved performance of DNA vaccines in nonhuman primates and man. For example, a candidate Zika virus DNA vaccine protected rhesus macaques against viremia following Zika virus challenge and induced neutralizing antibody titers >300 when delivered intramuscularly with the needle-free Stratis Device (PharmaJet)6,7. A different flavivirus DNA vaccine, targeting West Nile virus, is FDA approved for horses but also induced T cell and neutralizing antibody responses in humans8. Furthermore, an influenza trivalent DNA vaccine conferred protection against influenza challenge in a phase 1b clinical trial9and, through cell-mediated immunity, a human papillomavirus DNA vaccine aided the regression of lesions and viral clearance in cervical intraepithelial neoplasia-3 patients10. Considering the success of mRNA vaccines and DNA delivered by recombinant viruses Lapatinib (free base) and the intrinsic advantages and recent improved performances of plasmid DNA vaccines, an evaluation of the plasmid DNA platform is warranted in the ongoing SARS-CoV-2 pandemic. Here we describe the pre-clinical evaluation of a candidate DNA vaccine that targets the spike protein of SARS-CoV-2. Using platform optimization strategies to improve safety, antigen expression, potency, and immunogenicity, we address shortcomings associated with first-generation DNA vaccines. These optimization strategies include using: (i) a vector that lacks any antibiotic resistance genes11; (ii) an optimally reduced size vector12; (iii) vaccine antigen codon optimization13; (iv) co-expression of an immune stimulatory Retinoic-acid-inducible gene I (RIG-I) agonist that facilitates a type 1 interferon response14; (v) high yield antibiotic-free production in a current Good Manufacturing Practice process11,15; and (vi) needle-free jet administration to the skin or muscle16. Multiple SARS-CoV-2 vaccines are being developed at an unprecedented speed. Lapatinib (free base) To ensure thorough evaluation of the safety risks, potential autoimmune or hyper-immune reactions, and enhanced infection and/or disease, for as many different platforms as possible, all vaccines need to be thoroughly assessed for safety and immunogenicity and protection from viral challenge in animal models prior to clinical evaluation. Here we describe the evaluation of the immunogenicity of an optimized DNA plasmid vaccine candidate in mice, rabbits, and nonhuman primates, as well as an assessment of the protective effect in rhesus macaques, the most extensively used model for evaluation of SARS-CoV-2/COVID-19 vaccine protection. == Results == == The DNA vaccine candidate == The DNA vaccine candidate hereafter referred to as pNTC-Spike, expresses an unmodified, wild-type full-length SARS-CoV-2 spike protein derived from the Wuhan-hu-1 reference strain. The human Lapatinib (free base) codon optimized nucleotide sequence was subcloned into the NTC8685-eRNA41H (Fig.1a), a nano-plasmid eukaryotic expression vector approved for clinical use17. Considerations for the vector design are described in detail elsewhere12. Notable features of this vector backbone include: (i) the lack of any antibiotic resistance.