Figures and data
Growth dynamics of the vaccine candidate strain at different temperatures
Vero cells were infected with the wild-type parent B-1 (D614G) or the BK2102 vaccine candidate strains at a multiplicity of infection (MOI) = 0.01, and virus titers in the supernatants were determined for samples harvested every day, after incubating at 32 °C (A) or 37 °C (B). Infectious virus titers were determined using the TCID50 method. Symbols indicate the average of three independent experiments, and error bars represent the SD. Days post-infection are indicated on the x-axis.
Immunogenicity of the vaccine candidate in hamsters
(A) Spike-specific IgG in the sera of BK2102-inoculated hamsters and mock-treated hamsters. Spike-specific IgG in sera obtained four weeks post-inoculation was detected by ELISA. Symbols depict data of individual hamsters, and bars correspond to the median value. The limit of dilution is indicated in the x-axis.
(B) Neutralizing antibodies in the sera were induced by BK2102-inoculated hamsters and mock-treated hamsters. Neutralizing antibodies in the sera were measured at day 28 post-inoculation using the following authentic SARS-CoV-2 strains: wild-type D614G (left), Delta (middle), and BA.5 (right). Symbols represent titers of individual animals, and the bars indicate the median. The dotted line represents the assay’s limit of detection (LOD).
(C) Immune response in monkeys. Neutralizing antibodies in the sera of BK2102-inoculated monkeys was measured at the indicated time points post-inoculation. The data for individual monkeys are shown.
(D) Neutralizing antibodies persist in hamsters for at least 364 days. The neutralizing antibody titer against the authentic D614G wild-type strain was measured periodically in the sera of hamsters inoculated with BK2102 (once or twice at the indicated doses) for about a year. Symbols represent the mean of 9 to 10 animals, and error bars represent the SD.
(E and F) Evaluation of the cellular immune response in BK2102-inoculated hamsters. Splenocytes were collected at two weeks post-inoculation and were stimulated in vitro with spike or nucleocapsid peptide pools. IFN-γ (E) and IL-4 (F) in the supernatants were measured with commercially available ELISA kits (MABTECH AB and FineTest, respectively). Symbols represent titers of individual animals, and bars indicate the median. For statistical analysis, one-way ANOVA with Tukey’s multiple comparison test was performed (ns, not significant; *, p < 0.05; **, p < 0.01).
BK2102 induces protective immunity.
(A and B) BK2102 protects hamsters against homologous and heterologous virus challenges. Hamsters that received a full vaccination protocol with the indicated doses of BK2102 were challenged with wild-type D614G (A) or BA.5 (B) strains, and their body weight was monitored for four days. Body weight is expressed as a percentage of the initial weight. Two-way ANOVA with Tukey’s multiple comparison test was performed for statistical analysis (**, p < 0.01; ****, p < 0.0001).
(C, D, F and G) The infectious virus titer in the lungs and nasal wash specimens taken on day four post-challenge was measured via a plaque assay for the wild-type D614G strain (C and D) and for the BA.5 strain (F and G). One-way ANOVA with Dunnett’s multiple comparison test was performed for statistical analysis (ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001).
(E and H) Lung inflammation scores were determined via H&E staining of D614G- (E) and BA.5-challenged (H) hamsters. The percentage of the disrupted area in the entire visual field was classified as 0: not remarkable (< 10%); 1: minimal (10–50%); and 2: mild (50–70%). One-way ANOVA with Tukey’s multiple comparison test was performed for statistical analysis (ns, not significant; *, p < 0.05).
(I) Weight changes after the challenge assay one year post-inoculation with BK2102. Hamsters inoculated with BK2102 were challenged with the wild-type D614G strain at 3×105 PFU on 420 days. Nine-month-old elder hamsters were used as the naïve group. The symbols represent the average weight of the hamsters, and error bars indicate the mean SD. Two-way ANOVA with Tukey’s multiple comparison test was performed for statistical analysis (****, p < 0.0001).
BK2102 caused localized tissue damage and posed as low risk of transmission.
(A) Scheme for the evaluation of tissue damage in acute infection with BK2102 in a hamster model. The wild-type D614G strain was used as a positive control.
(B) Inflammation score of nasal cavity sections and lungs determined via H&E. The percentage of the disrupted area in the entire visual field was classified as 0: not remarkable (< 10%); 1: minimal (10–50%); 2: mild (50–70%), respectively.
(C) SARS-CoV-2 spike protein staining in the nasal cavity sections and lungs determined via immunohistochemistry using a SARS-CoV-2 spike RBD-specific antibody. The proportion of positive cells in the entire visual field was classified as 0: not remarkable (< 10%); 1: minimal (10–50%); and 2: mild (50–70%), respectively.
(D) Scheme for the evaluation of BK2102 transmission via in vivo passage in hamsters. The TS-strain A50-18 was used as a positive control.
(E) Ct values obtained for the RT-PCR performed using RNA extracted from the nasal wash specimens.
BK2102 showed a highly safe phenotype in Tg mice
(A and B) Survival rate of Tg mice infected with the wild-type D614G, B-1 ΔFCS, L50-33, and A50-18 TS strains (A) and BK2102 (B).
SARS-CoV-2 strains
Primer list
Genetic variations of viruses passaged in vivo
