Development of an inactivated vaccine candidate for SARS-CoV-2

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. Because of the novelty of the virus, there are currently no SARS-CoV-2-specific treatments or vaccines available. Therefore, rapid development of effective vaccines against SARS-CoV-2 are urgently needed. Here, we developed a pilot-scale production of PiCoVacc, a purified inactivated SARS-CoV-2 virus vaccine candidate, which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats, and nonhuman primates. These antibodies neutralized 10 representative SARS-CoV-2 strains, suggesting a possible broader neutralizing ability against other strains. Three immunizations using two different doses, 3 or 6 micrograms per dose, provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without observable antibody-dependent enhancement of infection. These data support the clinical development and testing of PiCoVacc for use in humans.

Fig. 1. Characterization of the SARS-CoV-2 vaccine candidate, PiCoVacc.

(A) The SARS-CoV-2 maximum likelihood phylogenetic tree. The SARS-CoV-2 isolates used in this study are depicted with black lines and labeled. Viral strains were isolated in infected patients who traveled from the continents as indicated (B) Growth kinetics of PiCoVacc (CN2) P5 stock in Vero cells. (C) Flowchart of PiCoVacc preparation. (D) Protein composition and purity evaluation of PiCoVacc by NuPAGE 4-12% Bis-Tris Gel. (E) Representative electron micrograph of PiCoVacc. White scale bar = 100 nm.

Fig. 2. PiCoVacc immunization elicits neutralizing antibody response against ten representative SARS-CoV-2 isolates.

BALB/c mice and Wistar rats were immunized with various doses of PiCoVacc or control (adjuvant only as the sham group) (n=10). Serums from recovered COVID19 patients (RECOV) and non-infected (NI) individuals were used as positive and negative controls, respectively. The antibody responses were analyzed in mice (A), humans (B) and rats (C). Top: SARS-CoV-2-specific IgG responses as measured by ELISA; bottom: neutralizing antibody titer determined by microneutralization assay. The spectrum of neutralizing activities elicited by PiCoVacc was investigated in mice (D) and rats (E). Neutralization assays against the other nine isolated SARS-CoV-2 strains was performed using mouse and rat serums collected 3 weeks post-vaccination. Data points represent mean +/− SEM of individual animals and humans from five to ten independent experiments; error bars reflect SEM;; dotted lines indicate the limit of detection; horizontal lines indicate the geometric mean titer (GMT) of EC₅₀ for each group.

Fig. 3. Immunogenicity and protective efficacy of PiCoVacc in nonhuman primates.

Macaques were immunized three times through the intramuscular route with various doses of PiCoVacc or adjuvant only (sham) or placebo (n=4). SARS-CoV-2-specific IgG response (A) and neutralizing antibody titer (B) were measured. Data points represent mean +/− SEM of individual macaques from four independent experiments; error bars reflect SEM; dotted lines indicate the limit of detection; horizontal lines indicate the geometric mean titer (GMT) of EC₅₀ for each group. Protective efficacy of PiCoVacc against SARS-CoV-2 challenge at week 3 after immunization was evaluated in macaques (C-F). Viral loads of throat (C) and anal (D) swab specimens collected from the inoculated macaques at day 3, 5 and 7 pi were monitored. Viral loads in various lobes of lung tissue from all the inoculated macaques at day 7 post-infection were measured (E). RNA was extracted and viral load was determined by qRT-PCR. All data are presented as mean ± SEM from four independent experiments; error bars reflect SEM. Asterisks represent significance: *P < 0.05 and **P < 0.01. Histopathological examinations (F) in lungs from all the inoculated macaques at day 7 post infection. Lung tissue was collected and stained with hematoxylin and eosin.

Fig. 4. Safety evaluation of PiCoVacc in nonhuman primates.

Macaques were immunized three times at day 0, 7 and 14 through the intramuscular route with low dose (1.5 μg per dose) or high dose (6 μg per dose) of PiCoVacc or adjuvant only (sham) or placebo. (A and B) Hematological analysis in all four groups of macaques (n=4). Lymphocyte subset percents (A), including CD3⁺, CD4⁺ and CD8⁺ were monitored at day -1 (1 day before vaccination), 18 (3 days after the second vaccination) and 29 (7 days after the third vaccination). Key cytokines (B), containing TNF-α, IFN-γ and IL-2 were examined at day -1, 1 (the day of the first vaccination), 4, 18 and 29 after vaccination. Data points show mean ± SD from four independent experiments; error bars reflect SD. (C) Histopathological evaluations in lungs from four groups of macaques at day 29. Lung tissue was collected and stained with hematoxylin and eosin.