The new corona world threatens global public health. In order to prevent and control the new corona epidemic, human beings urgently need vaccines. The new coronavirus vaccine, called BBIBP-CorV, developed by a Chinese research team, has shown great potential in animal experimentation: it is highly efficient and safe, and can induce high levels of neutralizing antibody titer in mice, rats, guinea pigs, rabbits and non-human primates (crab-eating monkeys and rhesus monkeys), providing high levels of neutralizing and antibody titer, and providing high protection against new coronaviruss.
(Original title: China adds new crown inactivated vaccine: led by Gao Fu and other scholars, efficient and safe)
Journalist He Liping
At the same time, the candidate vaccine is the second most important inactivated vaccine developed in China, increasing the domestic vaccine candidate options.
The results are from a critical paper published online in cell , an international authoritative academic journal , june 6 local time : Developing a candidate inactivated vaccine BBIBP-CorV to provide effective protection against SARS-CoV-2 ( Development of inactivated vaccine candidate , BBIBP-CorV with, protection of the potper SARS-CoV-2).
The authors of this paper are academicianofer of the Chinese Academy of Sciences, Director of the Chinese Academy of Medicine and Disease Control Gao Fu, Director of the Institute of Medical Experimental Animals of the Chinese Academy of Medical Sciences Qin Chuan, Director of the Emergency Technology Center of the Virus Disease Institute of the Chinese Center for Disease Control and Prevention, Tan Wenjie, Professor of Tsinghua University, Li Zhiyong, Li Changgui Researcher of the China Food and Drug Inspection Research Institute, Chief Expert of Biosecurity of the Chinese Center for Disease Control and Prevention, and Wu Guizhen, Secretary of the Party Committee of the Viral Disease Institute of the Chinese Academy of Medicine.
The research team came from Beijing Institute of Biological Products Co., Ltd., China Center for Disease Control and Prevention, the National Center for Human Disease Animal Model Resource Center, the National Health and Health Commission’s Key Laboratory of Human Disease Comparative Medicine, the Chinese Academy of Medical Sciences Medical Experimental Animal Research Institute, beijing Concord Medical College Comparative Medical Center, the New Emerging Infectious Disease Storier Animal Model Research Beijing Key Laboratory, Tsinghua University, etc.
The researchers detailed the experimental production of the SARS-CoV-2 inactivated candidate vaccine (BBIBP-CorV), which induces high levels of neutralizing antibody titer in mice, rats, guinea pigs, rabbits and non-human primates (crab monkeys and rhesus monkeys) to provide protection against SARS-CoV-2. In intra-gash immunization in rhesus monkeys, the study of bbIBP-CorV using a dose of 2 ?g/dose provided effective protection against SARS-CoV-2 without detecting antibody-dependent enhancement (ADE) infection. In addition, BBIBP-CorV exhibits high productivity and good genetic stability in vaccine production.
SARS-CoV-2 appears to spread faster than SARS-CoV and MERS-CoV, leading to an urgent need for vaccines. To date, three candidate vaccines, including an inactivated vaccine, an adenovirus vector vaccine and a DNA vaccine, have been reported to protect rhesus monkeys against SARS-CoV-2 with different effects. Inactivated vaccine is widely used in the prevention of new infectious diseases, its development speed is faster, for the prevention of new coronavirus has a better development prospects. It is worth noting that emerging evidence suggests that SARS-CoV infection may have antibody-dependent enhancement (ADE), suggesting that special safety evaluation should be given to the development of coronavirus vaccines.
The first inactivated new crown vaccine in China came from China Bio, a pharmaceutical group. China Pharmaceutical Group China Bio announced on April 14 that the company’s new coronavirus inactivated vaccine has been clinically tested, the first phase of clinical has been carried out in the jiaozhu region of Henan.
On the other hand, the adenovirus 5 (Ad5) vector vaccine, jointly developed by the team of Academician Chen Wei of the Institute of Bioengineering of the Institute of Military Medicine of the Academy of Military Sciences and CanSino of China, has also made significant progress. On the evening of May 22nd, local time, a paper published in the leading medical journal The Lancet entitled “The safety, tolerance and immunogenicity of the comorphic adenovirus type 5 vector COVID-19 vaccine: dose increase, open label, non-randomization, first human trial”. Chen Wei and other researchers reported 1 stage of clinical data on the Ad5 vector COVID-19 vaccine used in healthy adults in China within 28 days of vaccination to initially assess the vaccine’s safety, tolerability and immunogenicity.
Vaccine design and production
The team isolated three SARS-CoV-2 strains from bronchopulmonary irrigation samples and throat swabs from three new coronal patients to build a clinical pre-in vitro and venomous animal model of SARS-CoV-2 inactivated candidate vaccine. The three new cogens are: 19 ncov-cdc-tan-hb02 (HB02), 19 ncov-cdc-tan-strain03 (CQ01) and 19 ncov-cdc-tan-strain04 (QD01). The three strains were scattered in different locations of the system development tree, indicating that they were well representative of SARS-CoV-2.
It is worth noting that all of these strains are isolated from Vero cells and have been certified by the World Health Organization for vaccine production. These Vero cells are infected through the patient’s throat swab, rather than other cell lines, to prevent mutations that may occur during viral culture and separation. High-efficiency proliferation and high genetic stability are the key to the development of inactivated vaccines. They first found that 88 Vero cells of the HB02 strain replicateed best in three virus strains, producing the highest yield of the virus (Figure 1a). Therefore, the researchers selected HB02 strains to further develop inactivated SARS-CoV-2 vaccine (BBIBP-CorV). A comparison of the whole genome sequence of HB02 and other SARS CoV-2 strains from domestic and international sources shows that the HB02 strain is homogenous with other virus strains and demonstrates that the main protective antigen (protrusion protein) is 100% homologous. This demonstrates the potential for a wide range of SARS-CoV-2 strains (additional Figure 2). In order to obtain a virus culture suitable for high yield, the researchers purified the HB02 strain and produced p1 virus culture in Vero cells. The P1 medium is adapted to culture, generation and amplification in Vero cells. The researchers used seven generations of adapted strains (BJ-P-0207) as the primary seed for the vaccine production (BJ-P1). In order to evaluate genetic stability, the researchers passed on it three times and obtained p10 virus media. Through deep sequencing analysis, we sequenced the hb02 and P10 strains genome-wide, and the results showed that their sequences were 99.95 percent homogenee. In addition, no amino acid variation was found in the whole sequence of P10 apiviruses, including the Flynn split site, and the results showed that the HB02 strain had high genetic stability and was conducive to further development.
In order to manufacture efficiently, the researchers established a raw material production strategy for BBIBP-CorV based on a new basket reactor carrier (Figure 1b). Analysis of the growth dynamics of P7 media in Vero cells showed that the media virus could replicate effectively, reaching a maximum titration of more than 7.0 log10 CCID50 in 48-72 hours after infection, and with a multi-severe infection (MOI) of 0.01-0.3 (Figure 1c). In order to inactivate the virus, the researchers fully mixed valproate esters with the harvested viral solution at a ratio of 2-8 degrees C. Three batches of inactivation of the virus eliminate the infection of the virus and verify the good stability and repeatability of the inactivation process (Figure 1d). Western-blotting analysis shows that the vaccine library contains viral structural proteins (protective antigens) (Figure 1e). The negatively dyed electron microscope image shows elliptical virus particles with a diameter of approximately 100 nm (Figure 1f).
Immunogenicity of BBIBP-CorV
To assess the immunogenicity of BBIBP-CorV, the researchers injected BALB/c mice with different immunization regimens and different doses (2, 4, 8 ?g/dose).
The single-dose immune group was given in three doses of celiac on day 0 (D0), which was high (8 ?g/agent), medium (4 ?g/dose), low dose (2 ?g/dose), and BBIBP-CorV, with observed levels of antibody (NAb) 7, 14, 21, 28 days after injection. The results showed that the serum conversion rate of high, medium and low-dose groups reached 100% in the 7 days after immunity, and the immune effect was time-dependent (Figure 2, Supplement table 1). Neutral antibody levels in low, medium dose groups showed significant changes in 7, 14 and 21 days, but there was no significant difference between 21 and 28 days. Significant changes were observed only in the high-dose group (Figure 2a) only 7 or 14 days.
The two-dose immunization group uses different immunization programmes (day 0/day 7, day 0/14, day 0/day 21). The high, medium and low dose groups in the two-dose immunization group were serum-positive at 100% 7 days after the second immunization (Figure 2 b, supplementary table 1). The immunogenicity of high and medium-dose two-dose immunization programmes is significantly higher than that of single-dose immunization programmes. 7 days after the second immunization using the day 0/21, where the antibody level was the highest.
The researchers also tested the immunogenicity of three doses of the immune program. They were vaccinated in three doses of the mouse celiac for 0, 7 and 14 days, namely, high (8 ?g/agent), medium (4 ?g/dose) or low (2?g/dose) (Figure 2c). On the 7th, 14th, 21st, and 28th days, the neutralizing antibody levels of each group were measured, and on the 7th day after the first immunization, the serum conversion rate of all three groups reached 100% (Figure 2c, supplementary Table 1).
The results showed that the three doses (0 days/7 days/14 days) of the immunization programme neutralized antibody levels on the 28th day, all with a higher dose of immunization programmes (Figures 2a and 2c). In addition, they analyzed neutralizing antibody levels in single, double,twice(0 days/21 days) and 3 doses (0 days/7 days/14 days) of the vaccine in mice with high, medium and low doses of the vaccine, and neutralizing antibody levels 28 days after the first immunization to maintain the same starting and ending point. The results showed that the immunogenicity of the three-dose (0-day/7-day/14-day) immunization programme was higher than that of single-dose and two-dose immunization programmes (Figure 2d).
They then measured the immunogenicity of BBIBP-CorV in different animal models, including rabbits, guinea pigs, rats, and mice. The animals were immunized with a single dose (D0) immunization programme in high doses (8 ?g/agent), medium dose (4?g/dose)) and low dose (2?g/dose)) vaccine, and neutralizing antibody levels was measured 21 days after immunization. The results showed that BBIBP-CorV had good immunogenicity and had a serum conversion rate of 100% in all animal models 21 days after immunity (Figure 2 e, supplementary Table 1). In the three-dose (0-day/ 7-day/14-day) immune group, the vaccine was inoculated in high (8 sg/dose) or low (2 sg/dose) in crab monkeys, rabbits, guinea pigs, rats and mice. Serum conversion rate reached 100% in all animal models 21 days after immunization. Neutral antibody levels for 21 days after the first immunization showed that the high, medium and low doses of the three doses (0 days/7 days/14 days) of the immune procedure in the rabbit and guinea pig model were higher than the single dose (0 days) immune procedure (Figure 2 e 2 f, supplementary Table 1).
Protective role in the model of non-human primates
The team assessed the immunogenicity and protective effects of BBIBP-CorV on rhesus monkeys.
All rhesus monkeys are immunized 2 times on day 0 (D0) and 14th day (D14). The placebo group of muscles injected with physiological saline, two groups of experimental groups of intramuscular injection low-dose (2?g/dose) or high-dose (8?g/dose) BBIBP-CorV (Figure 3a). Before the virus attacked, NAb GMT in the low-dose group and the high-dose group reached 215 and 256, respectively (Figure 3b). On D24 (10th day after second immunization), all rhesus monkeys were injected with trachea with l06 TCID50 of SARS-CoV-2 under anaesthetic. Temperatures fluctuated in the range of the vaccination and placebo groups 0 to 7 days after the infection (Figure 3c, supplemental Figure 4a).
In addition, the serobiological and biochemical parameters of rhesus monkeys remain unchanged after the attack (additional Figure 3), which shows that the inoculation of BBIBP-CorV does not show side effects in serum biochemical parameters.
The team then used RT-PCR to determine the viral load of rhesus monkey throat and swabs. The results showed that all rhesus monkeys in the placebo group showed and maintained high viral loads throughout the assessment after the attack (Figures 3d, 3e, Supplement 4b and 4c).
The viral load of rhesus monkey throat swabs in the low-dose group peaked at 5dpi (5.33 log10 copy/ml) and dropped to 1.12 log10 copy/ml at 7 dpi, significantly lower than in the placebo group. It is worth noting that three of the four rhesus monkeys in the low-dose group were unable to detect viral loads at 7 dpi. The viral load of 4 rhesus monkey throat swabs in the high-dose group was negative. In addition, two swabs in the high-dose group of 4 rhesus monkeys did not detect viral load.
At 7 dpi, the team euthanized all animals to determine the viral load in the lung tissue and perform pathological examinations (Figures 3f, 3g). In the low-dose and high-dose groups, no viral load was detected in any of the lung lobes of all rhesus monkeys, which was significantly different from the results in the placebo group (Figure 3f). The placebo group detected high viral load in the left, right, and right secondary lungs, and the pathological histological analysis was severe interstitial pneumonia.
It is worth noting, however, that only three of the seven pulmonary leaf slices in the placebo group were detected to have an infection. The paper points out that this may be because the infection of the virus in the lung leaf is dynamic.
Overall, after the BBIBP-CorV vaccine, the lungs of all rhesus monkeys were normal, and a few lung lobes had local mild histopathological changes (Figure 3g), which showed that the BBIBP-CorV vaccine was effective in blocking SARS-CoV-2 infection in monkeys.
At 7dpi, rhesus monkeys treated with a placebo produced a low level of NAb with a titage of 1:16, while the high-dose group NAb level sylweded up to 1:2048 (average 1:860) and the low-dose group NAb level of up to 1:1024 (average 1:512) (Figure 3b). The results show that low-dose and high-dose BBIBP-CorV has an effective protective effect on rhesus monkeyS SARS-CoV-2, and no antibody-dependent-enhanced infection has been observed.
The team first conducted a single intramuscular injection experiment on the rats to assess the acute toxicity of BBIBP-CorV. The study divided 20 rats into 2 groups (n-10, 5/gender), with 3 times the dose of BBIBP-CorV (8?g/agent, 24?g/rat) with physiological saline as a control group. After inoculation, it was observed for 14 consecutive days, euthanasia was carried out on the 15th day, and systematic anatomy and observation was evaluated.
Four groups of rats did not see death or imminent death for 14 consecutive days after vaccination, and no apparent clinical symptoms were found. In addition, there was no significant difference in weight and feeding status between the experimental group and the control group (Figure 4a, Supplement 4d). After euthanasia these rats had no histopathological changes.
It is worth noting that the maximum tolerable dose (MTD) for a single intramuscular injection in rats is 24 ?g/rat, which is 900 times greater than the human body. This suggests that this suggests that BBIBP-CorV has potentially good safety for humans.
The team then assessed the systemic allergic reaction caused by BBIBP-CorV through intramuscular and intravenous injections in guinea pigs. Through clinical observation and measurement of guinea pig weight, the results showed that no abnormal reaction was observed during sensitivity (Figure 4b). Neither the negative control group (physiological saline) nor the experimental group found allergic reaction symptoms in D19 and D26. The positive control group (human hemoglobin) had a high lying condition (1/6 animals were positive, 3/6 animals were strong positive, and 2/6 animals were very positive). In contrast, the low and high-dose test groups D19 and D26 were non-allergic, and allergic reactions were negative.
The team further evaluated the long-term toxicity of BBIBP-CorV to rhesus monkeys. The 40 macaques (20/gender) were divided into 4 groups (5/gender/group) with intramuscular injectionof of physiological saline (group 1) or 2 sg, 4 sg, 8 sg BBIBP-CorV (group 2-4). Each group had 3 d25 anatomy, the remaining 2 only d36 anatomy, systematic anatomy and tissue pathology examination.
During the trial, there were no deaths or imminent deaths in the group 2-4, and the subgroup distribution of lymphocytes (CD3 plus, CD3 plus CD4 plus CD3 plus CD8 plus CD20 plus CD3 plus CD3 plus CD3 plus CD8 plus), There are no clinical physiological and pathological indicators of major abnormalities for cytokines (TNF-alpha IFN-N, IL-2, IL-4, IL-5 and IL-6), c-reactive proteins, or weight observations (Figure 4c-4e, supplemental Figure 4e).
There were no abnormalities in the anatomy of the euthanasia macaque system in each dose group at D25 and D36. At 2-4 in the D25 group, there was granulomainflammation and remained at the end of the recovery period (D36), but there was a slight improvement compared to D25. The macaques showed only local stimuli, characterized by mild to severe granuloma, but did not have this reaction for two weeks after injection. The experimental group of 8 ?g/agent showed unobserved levels of adverse reactions (NOAEL).
Supplemental Figure 2
Supplemental Figure 3
Supplemental Figure 4
Supplementary Table 1