Fig. 2

Intravenous immunization with CW-rBCG::RBD rapidly induces anti-RBD Nabs responses in mice. (a) Schematic overview of the immunization protocols, including the timeline and grouping for vaccination and monitoring of antibody responses across four groups of vaccinated mice. Eight-week-old BALB/c mice were immunized with indicated dose and route of CW-rBCG::RBD at week 0, or followed by a booster at week 4. Blood samples were collected at week 4 and 8 to evaluate RBD-specific IgG titers and neutralizing titers, respectively. (b-c) ELISA analysis of RBD-specific IgG titers in pooled sera from each group at weeks 4 (b) and 8 (c). CW-rBCG::RBD groups are labeled as “rBCG::RBD”, with “10⁶” indicating the standard-dose group (10⁶ cfu/ml) and “10⁵” representing a 10-fold lower dose. Mice were immunized intravenously (i.v.), subcutaneously (s.c.), or intramuscularly (i.m.) as specified. Prime denotes the initial immunization, while Boost1 refers to the first booster dose. RBD-specific IgG titers were defined as the serum dilution factor at which the absorbance at 450 nm was twice the value of the maximum dilution from PBS-immunized controls. The dotted line represents the detection limit. All experiments were performed at least twice, with data presented as the geometric mean ± SEM from two independent experiments, using six mice per group in each. (d) Neutralizing antibody titers (NT₅₀) values at week 8 post-immunization were measured using a SARS-CoV-2 RBD pseudovirus (PsV) neutralization assay. Sera from six mice per group were pooled in equal volumes. Data are presented as geometric mean ± SEM from two independent experiments (with two replicates in experiment 1). The right panel shows percent inhibition curves used to calculate NT₅₀ values (50% inhibition of PsV infection), analyzed by non-linear regression in GraphPad Prism. Statistical significance was assessed by an one-way ANOVA. Statistical significance is indicated as   ****p < 0.0001.