key: cord-1035309-n2405b3k authors: Wang, Hui; Zhang, Yuntao; Huang, Baoying; Deng, Wei; Quan, Yaru; Wang, Wenling; Xu, Wenbo; Zhao, Yuxiu; Li, Na; Zhang, Jin; Liang, Hongyang; Bao, Linlin; Xu, Yanfeng; Ding, Ling; Zhou, Weimin; Gao, Hong; Liu, Jiangning; Niu, Peihua; Zhao, Li; Zhen, Wei; Fu, Hui; Yu, Shouzhi; Zhang, Zhengli; Xu, Guangxue; Li, Changgui; Lou, Zhiyong; Xu, Miao; Qin, Chuan; Wu, Guizhen; Gao, George Fu; Tan, Wenjie; Yang, Xiaoming title: Development of an inactivated vaccine candidate, BBIBP-CorV, with potent protection against SARS-CoV-2 date: 2020-06-06 journal: Cell DOI: 10.1016/j.cell.2020.06.008 sha: f3ecff5dd787f8e4069093af1d38463d9f1f5258 doc_id: 1035309 cord_uid: n2405b3k Summary 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. To assess the immunogenicity of BBIBP-CorV, BALB/c mice were injected with 121 different immunization programs and various doses (2, 4 or 8 µg/dose) of vaccine 122 mixed with aluminium hydroxide adjuvant. In the one-dose immunization group, mice 123 were intraperitoneally administered a high (8 µg/dose), middle (4 µg/dose) or low (2 124 and middle-dose groups show significant variation, while no significant variation 130 between 21 and 28 days was observed. In the high-dose group, a significant 131 variation only was observed between 7 and 14 days (Figure 2a) . 132 In the two-dose immunization group, we tested different immunization programs 133 (D0/D7, D0/D14 and D0/D21 intervals) in which two immunizations at days 0/7, days 134 0/14, and days 0/21, respectively, were administered. The seropositivity of the high-, 135 medium-, and low-dose groups from all three immunization programs reached 100% 136 at 7 days after the second immunization (Figure 2b, Supplementary Table 1 ). The 137 immunogenicity of the two-dose immunization program was significantly higher than 138 that of the one-dose immunization program in the high-and middle-dose groups. 139 Moreover, use of the D0/D21 interval obtained the highest NAb level at 7 days after 140 the second immunization. 141 We also tested the immunogenicity of a three-dose immunization program, in 142 which the mice were intraperitoneally administered a high (8 µg/dose), middle (4 143 µg/dose) or low (2 µg/dose) dose of vaccine at days 0, 7 and 14 ( whole evaluation period after virus challenge by both throat and anal swabs ( Figure 193 3d and 3e, Supplementary Figure 4b and 4c ). In contrast, the viral load in the throat 194 swabs of the low-dose group peaked (5.33 log 10 copies/ml) at 5 dpi and then 195 decreased to 1.12 log 10 copies/ml at 7 dpi, which was significantly lower than that of 196 the placebo group. In particular, among the 4 macaques in the low-dose group, 3 197 showed a nondetectable viral load at 7 dpi. The throat swabs of all 4 macaques in 198 the high-dose group were negative for viral load. Moreover, no viral load was 199 detected in the anal swabs of 2 (out of 4) macaques in the high-dose group. 200 At 7 dpi, all animals were euthanized to determine the viral load in the lung 201 tissue and for pathological examination (Figure 3f The long-term toxicity of BBIBP-CorV was further evaluated in cynomolgus 252 monkeys. Forty cynomolgus monkeys (20/gender) were divided into 4 groups 253 saline injection, group 1) or 2, 4 or 8 µg of BBIBP-CorV (groups 2 to 4) in a volume of 255 0.5 ml. The animals were injected once a week for 3 continuous weeks (4 times in 256 total). A total of 3/5 animals of each sex in each group were dissected on D25, and 257 the remaining 2/5 animals of each sex in each group were dissected on D36. The 258 gross anatomy was evaluated, and histopathologic examination was administered. immunization programs, respectively, and the NAb levels at 28 days after the first 380 immunization were checked by the microtitration method (n = 10). (e) Rabbits (n = 381 5), guinea pigs (n = 10), rats (n = 10) and mice (n =10) were immunized with high (8 382 immunization, and the NAb levels at 21 days after the first immunization were tested 384 by the microtitration method. (f) Cynomolgus monkeys (n = 10), rabbits (n = 5), 385 guinea pigs (n = 10), rats (n = 10) and mice (n = 10) were immunized with high (8 386 µg/does), middle (4 µg/dose) and low (2 µg/dose) doses of vaccine by three-dose 387 (D0/D7/D14) immunization, and the NAb levels at 21 days after the first 388 immunization were tested by the microtitration method. For all panels, error bars 389 reflect the geometric SD. 390 Figure 454 3c, 3d and 3e). Individual body weight of (d) rats (n = 5) and (e) cynomolgus 455 monkeys (n = 10) in safety evaluation (Related to Figure 4a and 4c) . 456 Further information and requests should be directed to and will be fulfilled by the 462 Cynomolgus monkeys, rabbits, guinea pigs, rats and mice were randomly 551 divided into three groups (5 rabbits in each group, and for other species, 10 animals 552 in each group) and immunized intraperitoneally and intramuscularly with the trial 553 vaccine at three doses (2 µg/inoculation, 4 µg/inoculation, 8 µg/inoculation). Blood 554 was collected from each model animal before immunization, and the serum was 555 isolated the next day as a control. Each animal was inoculated with 0.5 mL of the test 556 sample (equivalent to 1 human dose). 557 The serum of the animal to be tested was diluted 1:4 in advance and inactivated 558 in a 56 °C water bath for 30 minutes. Serum was successively diluted 1:4 to the 559 required concentration by a 2-fold series, and an equal volume of challenge virus 560 solution containing 100 CCID 50 virus was added. After neutralization in a 37 °C incubator for 2 h, a 1.0~2.5×10 5 /ml cell suspension was added to the wells (0.1 562 ml/well) and cultured in a CO 2 incubator at 37 °C for 4 days. The Karber method 563 (Ramakrishnan, 2016) by observing the CPE was used to calculate the neutralization 564 endpoint (convert the serum dilution to logarithm), which means that the highest 565 dilution of serum that can protect 50% of cells from infection by challenge with 100 566 CCID 50 virus is the antibody potency of the serum. A neutralization antibody potency 567 <1:4 is negative, while that ≥ 1:4 is positive. 568 569 Twenty rats (10/gender) were divided into 4 groups (5/gender/group) and 571 intramuscularly injected with 24 µg/rat of BBIBP-CorV or sodium chloride as a 572 control. Thirty-six male guinea pigs were evenly divided into 4 groups: 1/4 were 573 injected with sodium chloride as a negative control, and 1/4 were injected with 574 human blood albumin as a positive control. The other two groups were injected with 575 different doses and used as the low-and high-dose groups. Sensitization was 576 carried out on D1, D3, and D5 by intramuscular injection. Three guinea pigs from 577 each group were selected for intravenous excitation via the foot, with secondary 578 excitation of the remainder of the guinea pigs in each group performed at D26. Forty 579 cynomolgus monkeys (20/gender) were divided into 4 groups (5/gender/group) and 580 intramuscularly injected with a control solution (sodium chloride injection, group 1) or 581 injected once a week for 3 continuous weeks (4 times in total). Three of the five 583 animals of each sex in each group were dissected on D25, and the remaining 2/5 584 animals of each sex in each group were dissected on D36. Collection of data on 585 safety-related parameters (body weight and body temperature) and clinical 586 observation were carried out during and after immunization. Analysis of lymphocyte 587 subset percentages (CD3 + , CD3 + CD4 + , CD3 + CD8 + , CD20 + , CD3 + CD4 + /CD3 + CD8 + ) 588 and key cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-5 and IL-6) and biochemical blood 589 tests of the collected blood samples were also carried out. Sixty percent of the 590 monkeys were euthanized at day 25 postimmunization, and the remaining 40% were 591 euthanized at day 36. 592 593 Challenge assay in rhesus macaques 594 Rhesus macaques (3-4 years old) were divided into three groups: 2 in the 595 placebo group, the animals in which were intramuscularly injected with physiological 596 saline; 4 in the low-dose vaccine group, the animals in which were intramuscularly 597 injected with 2 µg/dose vaccine; and 4 in the high-dose vaccine group, the animals in 598 which were intramuscularly injected with 8 µg/dose vaccine. All macaques were 599 immunized at days 0 and 14. A challenge study was conducted 10 days after the 600 second immunization by direct inoculation of l0 6 TCID 50 of SARS-CoV-2 virus 601 through the intratracheal route under anesthesia. The general symptoms of the 602 animals were observed and recorded every day during the experiment, along with the animal's body temperature before and after challenge. Peripheral blood was 604 collected on days 0, 7, 14 and 21 before immunization; and day 7 postinoculation 605 (dpi), and a neutralizing antibody test and routine blood biochemical test were 606 conducted. Throat and anal swabs were collected 3, 5, and 7 days after challenge 607 and used to determine the viral load. Seven days after challenge, all animals were 608 euthanized, the viral load in the lung tissue was detected, and a pathological 609 examination was conducted. For quantification of lymphocyte percentage by flow cytometry (Figures 4d-4e) , 672 values represent the positive cell population percentage. Statistical analyses were 673 performed using a double-tailed analysis with the inspection level at 5% or P≤0.05. 674 The mean number and standard deviations of all data results are calculated in the 675 Provantis system (SAS 9.2 statistic software). 676 677 The data were analyzed with SPSS (version 17.0) software. Student's t-test was 679 used to determine the statistical significance of the differences. See Method Details 680 section "Serum biochemical evaluation" for more details. 681 • An inactivated SARS-CoV-2 vaccine candidate, BBIBP-CorV, is developed • BBIBP-CorV induces high levels of neutralizing antibodies titers in animal models • Two-dose immunization with 2 µg/dose BBIBP-CorV efficiently protects rhesus macaques • BBIBP-CorV is efficiently produced, genetically stable and seems to be safe in animals Wang et al. report the development, characterization, and preclinical evaluation of an inactivated SARS-CoV-2 vaccine candidate for COVID-19 that safely induces high levels of neutralizing antibodies in multiple mammalian species and protective efficacy against SARS-CoV-2 challenge in rhesus macaques. 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