Preclinical evaluation of a candidate naked plasmid DNA vaccine against SARS-CoV-2

Abstract

New generation plasmid DNA vaccines may be a safe, fast and simple emergency vaccine platform for preparedness against emerging viral pathogens. Applying platform optimization strategies, we tested the pre-clinical immunogenicity and protective effect of a candidate DNA plasmid vaccine specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The DNA vaccine induced spike-specific binding IgG and neutralizing antibodies in mice, rabbits, and rhesus macaques together with robust Th1 dominant cellular responses in small animals. Intradermal and intramuscular needle-free administration of the DNA vaccine yielded comparable immune responses. In a vaccination-challenge study of rhesus macaques, the vaccine demonstrated protection from viral replication in the lungs following intranasal and intratracheal inoculation with SARS-CoV-2. In conclusion, the candidate plasmid DNA vaccine encoding the 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.

Fig. 1. SARS-CoV-2 DNA vaccine.

a DNA vaccine construct pNTC-Spike; full length, human codon optimized SARS-CoV-2 spike sequence cloned into expression vector NTC8685-eRNA41H with significant vector features indicated. b Protein expression from the DNA vaccine candidate was confirmed by DNA lipofection into Vero E6 cells followed by western blotting using anti-S1 (left) and anti-S2 (right) detecting mouse monoclonal antibodies S1-1047 and S2-1254, respectively. Lanes: (1) SARS-CoV-2 infected Vero E6 extract (2) Spike expressing plasmid (commercial positive control), (3) pNTC-Spike, (4) NTC vector control, and (5) VERO E6 (negative control). Lower panels show the 42 kDa β-actin expression as loading control. Full-length Spike protein and Spike S1/S2-fragments are indicated with solid arrows and brackets, respectively. All blots derived from the same experiment and were processed in parallel. Raw, uncropped blots are presented in Supplementary Fig. 2. c Expression of pNTC-Spike-derived SARS-CoV-2 spike protein in Vero E6 cells, visualized by immunofluorescent labeling. Forty-eight hours after transfection, cells were incubated with either the anti-S1 mouse monoclonal antibody S1-1047 (top panel) or anti-S2 mouse monoclonal antibody S2-1254 (bottom panel) followed by a goat anti-mouse IgG Alexa Fluor 488 conjugate for detection (green). Cell nuclei were counterstained with DAPI (blue). Size scale bar: 20 µm.

Fig. 2. Humoral and cellular immune responses to pNTC-Spike in mice.

a An overview of the immunization strategy in CB6F1 mice. Animals received three immunization of either 10 µg pNTC-Spike (N = 5), 50 µg pNTC-Spike (N = 5), or 50 µg vector control (N = 5) at 2 week intervals via the intradermal route using needle-and-syringe injection. b Temporal end-point titers of IgG antibodies specific for the SARS-CoV-2 spike ectodomain and receptor binding domain (RBD). Dotted lines indicate the assay limit of quantitation. c 50% virus-neutralizing antibody (NAb) titer against a SARS-CoV-2 clinical isolate as determined in a live virus microneutralization assay. Dotted lines indicate the assay limit of quantitation. d Interferon gamma (IFNγ), interleukin 5 (IL-5), and interleukin 17a (IL-17a) levels—representing T helper (Th) 1, Th2, and Th17 responses—measured by ELISA following restimulation of immunized rabbit splenocytes with the SARS-CoV-2 spike ectodomain and RBD. Cell culture medium and concanavalin A (ConA) served as antigen negative and positive controls, respectively. e End-point titers of SARS-CoV-2 spike-specific IgG subclasses in mice after the third immunization with either 10 µg or 50 µg pNTC-Spike (week 6). The Th1/Th2 dominance determined as the ratio of IgG2a:IgG1 and IgG2c:IgG1 end-point titers. Bar graphs indicate the median with interquartile range. Mouse silhouette created with BioRender.com.

Fig. 3. Humoral and cellular immune responses to pNTC-spike vaccination in rabbits.

a An overview of the immunization strategy in New Zealand white rabbits. Animals received three immunization of 125 µg pNTC-Spike at 2 week intervals delivered either as a 100 µL intradermal dose using a needle-and-syringe (N = 3) or the needle-free PharmaJet® Tropis ID device (N = 4) or as a 500 µL intramuscular dose using the needle-free PharmaJet® Stratis IM device (N = 5). b Temporal end-point titers of IgG antibodies specific for the SARS-CoV-2 spike ectodomain and receptor-binding domain (RBD). c 50% virus-neutralizing antibody titer against a SARS-CoV-2 clinical isolate as determined in a live virus microneutralization assay. d Correlation analysis between the level of SARS-CoV-2 spike-specific binding IgG and virus neutralization. e Interferon gamma (IFNγ) responses measured by ELISA and ELISpot following restimulation of immunized rabbit splenocytes with the SARS-CoV-2 spike ectodomain and RBD. Cell culture medium and concanavalin A (ConA) served as antigen negative and positive controls, respectively. f Percentage body weight change from baseline (day 0) of animals measured daily for 4 days after each intramuscular immunization and every 2 day thereafter. g Body temperature measured on the day of each intramuscular immunization (0 and 6 h) and daily thereafter. A temperature above 40 °C constitutes a fever (dotted line). Bar graphs indicate the median with interquartile range. Rabbit silhouette created with BioRender.com.

Fig. 4. Humoral immune responses to pNTC-Spike in nonhuman primates.

a An overview of immunogenicity and virus challenge study in rhesus macaques. pNTC-Spike vaccinates (N = 6) received three immunizations of 2 mg at 2 week intervals and were challenged with 1.0 × 10⁵ TCID₅₀ (1.2 × 10⁸ RNA copies, 1.1 × 10⁴ PFU) SARS-CoV-2 (strain nCoV-WAI-2020; MN985325.1) 4 weeks after the final immunization (week 8). Sham controls (N = 2) were SARS-CoV-2 immune naïve until inoculation with the same SARS-CoV-2 viral dose at week 8. b End-point titers of binding IgG antibodies specific for the SARS-CoV-2 spike ectodomain. c Neutralizing antibody titers measures as a 50% inhibition of virus infection (PRTN₅₀) and the more stringent 90% inhibition (PRNT₉₀). d Correlation between anti-spike IgG binding antibody titers and 50% neutralization titers (PRNT₅₀) for the challenge strain. Bar graphs indicate the median and interquartile range. Dotted lines indicate the assay limit of quantitation. SARS-CoV-2 virion image created with BioRender.com.

Fig. 5. Viral load and anamnestic responses in rhesus macaques following challenge with SARS-CoV-2 by the intranasal and intratracheal route.

a Peak and temporal SARS-CoV-2 viral loads, measured as SARS-CoV-2 E gene subgenomic mRNA, in BAL of pNTC-Spike vaccinated animals (N = 6) and untreated sham controls (N = 2). b Peak and temporal viral loads in nasal swabs of pNTC-Spike vaccinated animals and untreated sham controls. c End-point titers of binding IgG antibodies specific for the SARS-CoV-2 spike ectodomain on the day of virus challenge and days 2, 4, 7, and 10 following inoculation. Bar graphs indicate the median with interquartile range; Line graphs present each animal as a separate black line and the red line the median for each time point. Dotted lines indicate the assay limit of quantitation (1.69 log₁₀ sgmRNA copies/mL).

Fig. 6. SARS-CoV-2 variant cross-neutralizing antibody responses induced by the pNTC-Spike vaccine after three immunizations (week 6).

a pNTC-Spike vaccinated rabbit sera were evaluated for the Alpha variant (lineage B.1.1.7), Beta variant (lineage B.1.351), and Delta variant (lineage B.1.617.2) in a microneutralization assay at Statens Serum Institut, Denmark. b Sera from pNTC-Spike vaccinated rhesus macaques were evaluated in a plaque reduction neutralization test at BIOQUAL, Inc., USA. The variants tested were dependent on the viral isolates available at each institute. ns: not significant.