Development of an Inactivated Vaccine Candidate, BBIBP-CorV, with Potent Protection against SARS-CoV-2

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. Here, we report the pilot-scale production of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2 μg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addition, BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manufacture. These results support the further evaluation of BBIBP-CorV in a clinical trial.

Keywords: BBIBP-CorV; SARS-CoV-2; inactivated vaccine.

Graphical abstract

Figure S1

SARS-CoV-2 Maximum Likelihood Phylogenetic Tree Related to Figure 1 The SARS-CoV-2 isolates used in this study are indicated with black arrows and labeled. Viral strains were isolated from infected patients who traveled from the indicated continent/area.

Figure 1

Characterization of the SARS-CoV-2 Vaccine Candidate BBIBP-CorV (A) Viral titers of three strains of different generations. (B) Flowchart of BBIBP-CorV preparation. (C) Culture conditions. The left panel shows the effect of cell culture time on BBIBP-CorV stock virus titer, the middle panel shows the growth kinetics of the Vero cells for BBIBP-CorV stock culture, and the right panel shows the effect of inoculation MOI on BBIBP-CorV stock virus titer. (D) Inactivation kinetics of three batches of virus supernatant. (E) The protein composition of BBIBP-CorV were evaluated by incubating with antibodies targeting N protein (left panel) and S protein (middle panel) and incubation with convalescent patient sera (right panel). h, harvest; c, concentrated viral solution; p, purified viral solution. (F) Representative electron micrograph of BBIBP-CorV. Scale bar: 100 nm.

Figure S2

Neutralization of SARS-CoV-2 Strains HB02, CQ01, and QD01 by the Sera of Mice Vaccinated with BBIBP-CorV, Related to Figure 1 Mice were intraperitoneally injected with 8 μg/does of BBIBP-CorV at one time, and the ability of their sera to neutralize three SARS-CoV-2 strains was tested (n = 5) day 14 day after inoculation.

Figure S3

Serum Biochemical Parameters in Rhesus Macaques after Vaccination and Challenge with Living Virus, Related to Figure 3 Rhesus macaques were intramuscularly immunized twice on days 0 and 14, and live virus challenge was conducted on day 24 (the dotted line). Blood was collected, and serum biochemical parameters were monitored at different time points. Glu (glucose), T-Bil (total bilirubin), ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), γ-GT (γ-glutamyl transpeptidase), TP (total protein), Alb (albumin), TG (triglycerides), TC (total cholesterol), CREA (creatinine), UA (uric acid), UREA (blood urea), CK (creatine kinase), LDH (lactate dehydrogenase). The data are presented as the mean ± SD. ^∗p < 0.05 and ^∗∗∗p < 0.001 versus the placebo group (n = 2 in the placebo group, n = 4 in the 2 μg/dose and 8 μg/dose groups). The dotted line on the y axis indicates the normal upper and lower lines of the data.

Figure 2

BBIBP-CorV Immunization Elicits a Neutralizing Antibody Response in Different Animals with Different Doses and Immunization Programs (A) Mouse neutralization antibody (NAb) levels with one-dose (D0) immunization. Mice were injected intraperitoneally with high (8 μg/dose), middle (4 μg/dose), or low (2 μg/dose) doses of vaccine, and the NAb levels at 7 days, 14 days, 21 days, and 28 days after the first immunization were tested by the microtitration method (n = 10). (B) NAb levels with different immunization interval programs via two-dose immunization. Mice were injected intraperitoneally by using two-time immunization (D0/D7; D0/D14; D0/D21), and the NAb levels at 7 days after the second immunization were tested by the microtitration method (n = 10). (C) Mouse neutralization antibody levels with three-dose (D0/D7/D14) immunization. Mice were inoculated intraperitoneally with high (8 μg/dose), middle (4 μg/dose), or low (2 μg/dose) doses of vaccine at 0, 7, and 14 days, and NAb levels at 7, 14, 21, and 28 days after the first immunization were tested by the microtitration method (n = 10). (D) Mouse NAb levels with different immunization programs. Mice were injected intraperitoneally with high (8 μg/dose), middle (4 μg/dose), or low (2 μg/dose) doses of vaccine by using one-dose (D0), two-dose (D0/D21), and three-dose (D0/D7/D14) immunization programs, respectively, and the NAb levels at 28 days after the first immunization were checked by the microtitration method (n = 10). (E) Rabbits (n = 5), guinea pigs (n = 10), rats (n = 10), and mice (n = 10) were immunized with high (8 μg/dose), middle (4 μg/dose), or low (2 μg/dose) doses of vaccine by one-dose (D0) immunization, and the NAb levels at 21 days after the first immunization were tested by the microtitration method. (F) Cynomolgus monkeys (n = 10), rabbits (n = 5), guinea pigs (n = 10), rats (n = 10), and mice (n = 10) were immunized with high (8 μg/does), middle (4 μg/dose), and low (2 μg/dose) doses of vaccine by three-dose (D0/D7/D14) immunization, and the NAb levels at 21 days after the first immunization were tested by the microtitration method. For (A–F), error bars reflect the geometric SD.

Figure 3

Immunogenicity and Protective Efficacy of BBIBP-CorV in Nonhuman Primates (A) Experimental strategy. (B) Macaques were immunized twice with 2 μg/dose (n = 4) or 8 μg/dose (n = 4) of BBIBP-CorV or placebo (n = 2). The NAb titers were measured. Data are presented as geometric mean with geometric SD. (C–G) The protective efficacy of BBIBP-CorV against SARS-CoV-2 challenge at 10 days after second immunization was evaluated in macaques. Changes in clinical signs (temperature, C) were recorded. Viral loads in throat (D) and anal (E) swabs obtained from macaques at 3, 5, and 7 days post inoculation. (F) Viral loads in all seven lung lobes collected from all macaques at day 7 post inoculation were determined by real-time PCR. All data are presented as mean ± SEM from four independent experiments for the BBIBP-CorV groups and two independent experiments for the placebo group; error bars reflect the SEM. Data points represent the individual macaques. Asterisks indicate significance: ^∗∗p < 0.01. Dotted lines indicate the limit of detection. (G) Histopathological changes in lungs of macaques at day 7 post inoculation. All macaques received vaccination showed normal lung with focal mild interstitial pneumonia in few lobes. Scale bars are indicated in the panels.

Figure S4

Individual Data for Temperature, Viral Load, and Body Weight of Animals in Efficacy and Safety Evaluation, Related to Figures 3C–3E Individual (A) temperature, viral load in (B) throat and (C) anal swab data in nonhuman primate efficacy evaluation. Individual body weight of (D) rats (n = 5) and (E) cynomolgus monkeys (n = 10) in safety evaluation (Related to Figures 4A and 4C).

Figure 4

Safety Evaluation of BBIBP-CorV in Rats, Guinea Pigs, and Nonhuman Primates (A) Body weight analysis of rats in the experimental group and control group (n = 5). Male and female rat weight mean are used in this plot. Plot with individual data is presented in Figure S4D. (B) Body weight analysis of guinea pigs in the experimental groups (0.1 × dose/guinea pig, 1 × dose/guinea pig) and negative control and positive control groups (n = 9). (C) Cynomolgus monkeys were intramuscularly injected four times on days 1, 8, 15, and 22 with low (2 μg/dose), middle (4 μg/dose), and high (8 μg/dose) doses of BBIBP-CorV or placebo. Body weight analysis of cynomolgus monkeys (n = 10) in all four groups. Male and female cynomolgus monkey weight mean are used in this plot. Plot with individual data is presented in Figure S4E. (D) Hematological analysis of cynomolgus monkey in all four groups (n = 10). The percentages of the lymphocyte subsets CD3⁺, CD3⁺CD4⁺ (labeled CD4⁺), CD3⁺CD8⁺ (labeled CD8⁺), CD20⁺, and CD3⁺CD4⁺/CD3⁺CD8⁺ (labeled CD4⁺/CD8⁺) were monitored at day −1 (1 day before vaccination), day 25 (3 days after the third vaccination), and day 36 (14 days after the fourth vaccination). (E) The key cytokines TNF-α, IFN-γ, IL-2, IL-4, IL-5, and IL-6 were examined at days −1, 1 (the day for the first vaccination), 4, 15, 22, 25, and 36, respectively. For (A–E), values are shown as the mean ± SD