A linear SARS-CoV-2 DNA vaccine candidate reduces virus shedding in ferrets

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused more than 760 million cases and over 6.8 million deaths as of March 2023. Vaccination has been the main strategy used to contain the spread of the virus and to prevent hospitalizations and deaths. Currently, two mRNA-based vaccines and one adenovirus-vectored vaccine have been approved and are available for use in the U.S. population. The versatility, low cost, and rapid production of DNA vaccines provide important advantages over other platforms. Additionally, DNA vaccines efficiently induce both B- and T-cell responses by expressing the antigen within transfected host cells, and the antigen, after being processed into peptides, can associate with MHC class I or II of antigen-presenting cells (APCs) to stimulate different T cell responses. However, the efficiency of DNA vaccination needs to be improved for use in humans. Importantly, in vivo DNA delivery combined with electroporation (EP) has been used successfully in the field of veterinary oncology, resulting in high rates of response after electrochemotherapy. Here, we evaluate the safety, immunogenicity, and protective efficacy of a novel linear SARS-CoV-2 DNA vaccine candidate delivered by intramuscular injection followed by electroporation (Vet-ePorator™) in ferrets. The linear SARS-CoV-2 DNA vaccine candidate did not cause unexpected side effects. Additionally, the vaccine elicited neutralizing antibodies and T cell responses on day 42 post-immunization using a low dose of the linear DNA construct in a prime-boost regimen. Most importantly, vaccination significantly reduced shedding of infectious SARS-CoV-2 through oral and nasal secretions in a ferret model.

Fig. 1

Experimental design and body temperature after vaccination with the linear SARS-CoV-2 DNA vaccine candidate. A A total of fifteen 12- to 16-month-old ferrets (Mustela putorius furo) were divided into three groups with three males and two females per group, corresponding to the vaccine regimen to be administered: control sham-immunized (G1), single dose (G2), and prime + booster (G3). B Body temperature following intramuscular vaccination with the linear SARS-CoV-2 DNA vaccine candidate was measured on the days of vaccine administration and on the subsequent 2 days as indicted in the graphic. Data are presented as the mean ± standard error.

Fig. 2

Serological and cellular responses to the linear SARS-CoV-2 DNA vaccine candidate assessed by bead-based multiple, virus neutralization, and ELISpot assays. A Antibody responses following immunization were measured by bead-based multiplex assay to quantify IgG anti-SARS-CoV-2 spike receptor-binding domain (RBD) in serum samples collected on day 42 post-immunization (pi). Results are presented as fold change from day 0 (pre-immunization). B Neutralizing antibody (NA) responses to SARS-CoV-2 in serum samples collected on day 42 pi. Neutralizing antibody titers represent the reciprocal of the highest dilution of serum that completely inhibited infection with 100–200 TCID₅₀ of SARS-CoV-2. C The SARS-CoV-2 spike receptor-binding domain (RBD)-specific T cell response elicited by the linear DNA vaccine was assessed by ELISpot assay for IFN-γ. Proliferation of peripheral blood mononuclear cells (PBMCs) in samples from all of the ferrets was measured after stimulation with RBD pool peptides on day 42 post-immunization (pi). * = p < 0.05; ** = p < 0.01. Data are presented as the mean ± standard error.

Fig. 3

Experimental design and clinical observations following intranasal challenge with 5 × 10⁵ PFU of a SARS-CoV-2 Alpha variant of concern (isolate NYC853-21). A Black squares represent the collection/measure time points for each sample type/parameter described. Clinical parameters, including temperature, body weight, activity, and signs of respiratory disease, were monitored daily after challenge. Oropharyngeal (OPS), nasal (NS), and rectal swab (RS) and blood samples were collected at various times points (black squares). Animals were humanely euthanized on day 10 pc. B Body temperature and (C) body weight following intranasal viral challenge were recorded throughout the experimental period. Data are presented as the mean ± standard error. Body weight was normalized to day 0, which represents 100%.

Fig. 4

Viral genomic and subgenomic RNA in nasal and oropharyngeal secretions and feces after SARS-CoV-2 challenge. Detection of viral RNA in A oropharyngeal swab (OPS), B nasal swab (NS), and C rectal swab (RS) samples from ferrets challenged with a SARS-CoV-2 Alpha variant of concern. Samples were tested for the presence of SARS-CoV-2 RNA by real-time reverse transcription PCR (rRT-PCR) (genomic and subgenomic viral RNA load). Detection of subgenomic SARS-CoV-2 RNA (sgRNA) in (D) OPS, (E) NS, and (F) RS samples from challenged ferrets. Day 0 represents swab samples collected prior to challenge (day 42 post-immunization). Data are presented as the mean ± standard error. * = p < 0.05; ** = p < 0.01.

Fig. 5

Titer of infectious virus in oropharyngeal secretions and feces and titers of neutralizing antibodies following SARS-CoV-2 challenge. A, B Shedding of infectious virus in oropharyngeal and B nasal swab samples from ferrets challenged with a SARS-CoV-2 Alpha variant of concern. C Titers of neutralizing antibodies in the animals after SARS-CoV-2 challenge. Day 0 represents swab samples collected prior to challenge (day 42 post-immunization). Data are presented as the mean ± standard error. * = p < 0.05; ** = p < 0.01; *** = p < 0.005; **** = p < 0.001.