China adds a new crown inactivated vaccine: led by Gao Fu and other scholars, efficient and safe

  Surging news reporter He Liping

  The new crown pandemic threatens global public health. To prevent and control the new crown epidemic, humans urgently need vaccines. The Chinese research team has developed a new crown inactivated vaccine called BBIBP-CorV, which has shown great potential in animal experiments: the vaccine is highly efficient and safe in mice, rats, guinea pigs, rabbits and non-human primates Animals (cynomolgus monkeys and rhesus monkeys) can induce high levels of neutralizing antibody titers in vivo, and 2 μg/dose can provide efficient protection against new coronavirus. At the same time, the candidate vaccine is the second important inactivated vaccine developed in China, which increases the selection of candidate vaccines in China.

  The above results come from a heavy paper published on June 6th, local time, the international authoritative academic journal "CELL": "Development of candidate inactivated vaccine BBIBP-CorV, which can provide effective protection for SARS-CoV-2" (Development of an inactivated vaccine candidate, BBIBP-CorV, with potent protection against SARS-CoV-2).

  Corresponding authors of the paper are Gao Fu, Academician of the Chinese Academy of Sciences, Director of the Center for Disease Control, Qin Chuan, Director of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences, Tan Wenjie, Director of the Emergency Technology Center of the Chinese Academy of Disease Control Center, Professor Zhizhi Lou of Tsinghua University, and China Food and Drugs Wu Guizhen, a researcher at the National Institute of Laboratory Medicine, chief expert in biosecurity at the Chinese Center for Disease Control, and party secretary of the Institute of Viral Diseases.

  The research team comes from the Beijing Institute of Biological Products Co., Ltd., the Chinese Center for Disease Control and Prevention, the National Center for Human Disease Animal Model Resources, the National Key Laboratory of Comparative Medicine for Human Diseases, the Chinese Academy of Medical Sciences Institute, Comparative Medicine Center of Peking Union Medical College, Beijing Key Laboratory of Animal Model Research on Newly Recurring Infectious Diseases, Tsinghua University, etc.

  The researchers detailed the experimental production of the SARS-CoV-2 inactivated candidate vaccine (BBIBP-CorV) in mice, rats, guinea pigs, rabbits and non-human primates (cynomolgus monkey monkeys and Heng (Rhesus monkey) can induce high levels of neutralizing antibody titers in vivo to provide protection against SARS-CoV-2. In the endotracheal immunization of rhesus monkeys, the use of 2 μg/dose of BBIBP-CorV provided effective protection against SARS-CoV-2 without detecting antibody-dependent enhancement (ADE) infection. In addition, BBIBP-CorV shows high productivity and good genetic stability in vaccine production.

  Compared with SARS-CoV and MERS-CoV, SARS-CoV-2 seems to spread faster, 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 vaccines are widely used in the prevention of emerging infectious diseases, and their development speed is faster, and they have better development prospects for the prevention of new coronaviruses. It is worth noting that the emerging evidence shows that there may be antibody-dependent enhancement (ADE) of SARS-CoV infection, suggesting that special attention should be paid to safety evaluation when developing coronavirus vaccines.

  The first inactivated new crown vaccine in China came from Sinopharm Group. Sinopharm Group China Biotech announced on April 14th that after the company's new coronavirus inactivated vaccine received clinical trials, the first phase of the clinical trial has been carried out in Jiaozuo, Henan.

  On the other hand, the adenovirus type 5 (Ad5) vector vaccine jointly developed by the team of academician Chen Wei from the Institute of Bioengineering, Academy of Military Medical Sciences and Chinese Academy of Military Sciences has made significant progress. On the evening of May 22 local time, the top medical journal "The Lancet" published a paper entitled "Safety, Tolerance and Immunogenicity of Recombinant Adenovirus Type 5 Vector COVID-19 Vaccine" : Increasing dose, open label, non-random, first human trial". Researchers such as Chen Wei reported the Phase 1 clinical data of the Ad5 carrier COVID-19 vaccine used in Chinese healthy adults within 28 days after vaccination to initially assess the vaccine's safety, tolerability, and immunogenicity.

Vaccine design and production

  The research team isolated three SARS-CoV-2 strains from the bronchoalveolar lavage samples and throat swabs of three inpatients with new crowns, and used them to establish a preclinical in vitro neutralization challenge for SARS-CoV-2 inactivated vaccine candidates. Poisonous animal model. The three new crown strains are: 19 ncov-cdc-tan-hb02 (HB02), 19 ncov-cdc-tan-strain03 (CQ01) and 19 ncov-cdc-tan-strain04 (QD01). These three strains were scattered in different positions of the phylogenetic tree, indicating that they are well representative of SARS-CoV-2.

  It is worth noting that all these strains have been isolated from Vero cells and have been certified by the World Health Organization for vaccine production. These Vero cells are infected by the patient's throat swab, but not other cell lines, to prevent mutations that may occur during virus cultivation and isolation. Efficient proliferation and high genetic stability are the keys to the development of inactivated vaccines. They first found that among the three virus strains, 88 Vero cells of HB02 strain had the best replication effect and produced the highest virus yield (Figure 1a). Therefore, the researchers chose the HB02 strain to further develop the inactivated SARS-CoV-2 vaccine (BBIBP-CorV). Comparison of the whole genome sequence of the HB02 strain and other SARS CoV-2 strains from domestic and international sources shows that the HB02 strain is homologous to other virus strains and proves that the main protective antigen (spike protein) is 100% Homology. This shows that it has the potential to widely fight against various SARS-CoV-2 strains (Supplementary Figure 1 and Supplementary Figure 2). In order to obtain a virus medium suitable for high yield, the researchers purified the HB02 strain and passaged it in Vero cells to produce the P1 virus medium. The P1 medium was adapted, passaged and expanded on Vero cells. The researchers used the 7-generation adapted strain (BJ-P-0207) as the original seed for vaccine production (BJ-P1). To evaluate genetic stability, the researchers subcultured it three times to obtain P10 virus culture medium. Through in-depth sequencing analysis, we performed whole-genome sequencing on HB02 and P10 strains, and the results showed that their sequence homology was 99.95% greater. In addition, no amino acid variation was found in the entire sequence of the P10 raw material virus, including near the Flynn cleavage site. These results indicate that the HB02 strain has high genetic stability and is conducive to further development.

  For efficient manufacturing, the researchers established a raw material production strategy for BBIBP-CorV based on a new basket reactor carrier (Figure 1b). The growth kinetic analysis of P7 medium in Vero cells showed that the virus in the medium could effectively replicate, and the highest titer exceeded 7.0 log10 CCID50 48-72 hours after infection (hpi), and the multiplicity of infection (MOI) was 0.01-0.3 (Figure 1c). To inactivate virus production, the researchers thoroughly mixed the propionolactone with the harvested virus solution at a ratio of 1:40 at 2-8°C. The three batches of inactivation of the virus eliminated the infectivity of the virus and verified the good stability and repeatability of the inactivation process (Figure 1d). Western-blotting analysis revealed that the vaccine library contains viral structural proteins (protective antigens) (Figure 1e). An electron microscope image of negative staining showed elliptical virus particles with a diameter of approximately 100 nm (Figure 1f).

Immunogenicity of BBIBP-CorV

  To evaluate the immunogenicity of BBIBP-CorV, the researchers injected BALB/c mice with different immunization protocols and different doses (2, 4, 8 μg/dose) of vaccines.

  The single-dose immunization group was administered intraperitoneally on day 0 (D0) with three doses, which were high (8 μg/dose), medium (4 μg/dose), and low dose (2 μg/dose) BBIBP-CorV. Observe the injection Neutralizing antibody (NAb) levels in the last 7, 14, 21, and 28 days. The results showed that the seroconversion rate of the high, medium and low dose groups reached 100% at 7 days after immunization, and the immunization effect was related to time (Figure 2, Supplementary Table 1). The levels of neutralizing antibodies in the low- and medium-dose groups showed significant changes on days 7, 14, and 21, but there was no significant difference between days 21 and 28. In the high-dose group, significant changes were observed only on days 7 and 14 (Figure 2a).

  The two-dose immunization group adopted different immunization protocols (day 0/day 7, day 0/day 14, day 0/day 21). The seropositivity of the high-, medium-, and low-dose groups in the double-dose immunization reached 100% 7 days after the second immunization (Figure 2b, Supplementary Table 1). The immunogenicity of the high-dose and medium-dose dual-dose immunization regimen is significantly higher than that of the single-dose immunization regimen. 7 days after the second immunization with the Day 0/Day 21 protocol, the neutralizing antibody level was the highest.

  The researchers also tested the immunogenicity of the three-dose immunization program. They received three doses of vaccine in the abdominal cavity of mice on days 0, 7 and 14 days, which were high (8 μg/dose), medium (4 μg/dose) or low (2 μg/dose) vaccines (Figure 2c). The neutralizing antibody levels of each group were measured on days 7, 14, 21, and 28. On the 7th day after the first immunization, the seroconversion rates of the three groups reached 100% (Figure 2c, Supplementary Table 1).

  The results showed that the neutralizing antibody level at day 28 of the three-dose (day 0/7 day/14 day) immunization schedule was higher than the one-dose immunization schedule (Figures 2a and 2c). In addition, they analyzed the neutralizing antibody levels of high-, medium-, and low-dose vaccines in mice vaccinated with single dose, double dose (day 0/21 day) and 3 doses (day 0/7 day/14 day), and in The level of neutralizing antibody was measured 28 days after the first immunization to maintain the same starting and ending points. The results showed that the three-dose (day 0/7 day/14 day) immunization schedule was more immunogenic than the single-dose and double-dose immunization schedules (Figure 2d).

  Next they measured the immunogenicity of BBIBP-CorV in different animal models such as rabbits, guinea pigs, rats and mice. The animals were immunized with high-dose (8 μg/dose), medium-dose (4 μg/dose)) and low-dose (2 μg/dose) vaccines using a single-dose (D0) immunization protocol, and neutralizing antibody levels were measured 21 days after immunization. The results showed that BBIBP-CorV had good immunogenicity, and the seroconversion rate reached 100% in all animal models 21 days after immunization (Figure 2e, Supplementary Table 1). In the three-dose (day 0/7 day/14 day) immunization group, cynomolgus monkeys, rabbits, guinea pigs, rats and mice were vaccinated with high (8 μg/dose) medium (4 μg/dose) or low (2 μg/dose) doses Vaccine. The seroconversion rate reached 100% in all animal models 21 days after immunization. The level of neutralizing antibodies 21 days after the first immunization showed that the three-dose (day 0/7 day/14 day) immunization program in rabbit and guinea pig models was higher than the single dose (0 Days) immunization program (Figure 2 e 2 f, Supplementary Table 1).

Protection in non-human primate models

  The research team evaluated the immunogenicity and protective effect of BBIBP-CorV on rhesus monkeys.

  All rhesus monkeys were immunized twice on day 0 (D0) and day 14 (D14). The placebo group was intramuscularly injected with saline, and the two groups of experimental groups were intramuscularly injected with low dose (2 μg/dose) or high dose (8 μg/dose) BBIBP-CorV (Figure 3a). Before the virus challenge, the NAb GMT of the low-dose group and the high-dose group reached 215 and 256, respectively (Figure 3b). At D24 (day 10 after the second immunization), all rhesus monkeys were intratracheally injected with 10 TCID50 of SARS-CoV-2 under anesthesia. From 0 to 7 days after virus infection, the body temperature of the vaccinated group and the placebo group fluctuated within a range (Figure 3c, supplementary Figure 4a).

  In addition, the serum biochemical parameters of rhesus monkeys remained unchanged after challenge (Supplementary Figure 3). This result shows that inoculation of BBIBP-CorV does not show side effects in terms of serum biochemical parameters.

  Next, the research team used RT-PCR to determine the viral load of rhesus monkey throat and anal swabs. The results showed that all placebo groups of rhesus monkeys showed and maintained high viral load throughout the evaluation period after challenge (Figures 3d, 3e, supplementary Figures 4b and 4c).

  The viral load of the rhesus swab of the rhesus monkey in the low-dose group peaked at 5 dpi (5.33 log10 copies/ml) and dropped to 1.12 log10 copies/ml at 7 dpi, which was significantly lower than that of the placebo group. It is worth noting that 3 of the 4 rhesus monkeys in the low-dose group were unable to detect viral load at 7 dpi. The viral load of 4 rhesus swabs in the high-dose group was negative. In addition, no viral load was detected in 2 anal swabs of 4 rhesus monkeys in the high-dose group.

  At 7 dpi, the research team euthanized all animals to determine the viral load in the lung tissue and performed pathological examination (Figure 3f, 3g). In the low-dose and high-dose groups, no virus load was detected in any lobes of all rhesus monkeys, which was significantly different from the results of the placebo group (Figure 3f). In the placebo group, high viral load was detected in the left lung, right lung and right secondary lung, and histopathological analysis was severe interstitial pneumonia.

  However, it is worth noting that of the 7 lung lobe sections in the placebo group, only 3 were detected for infection. The paper pointed out that this may be because the virus infection in the lung lobes changes dynamically.

  In general, after vaccinating with BBIBP-CorV vaccine, the lungs of all rhesus monkeys were normal, and a small number of lung lobes had local mild histopathological changes (Figure 3g), which shows that BBIBP-CorV vaccine can effectively block monkey SARS- CoV-2 infection.

  At 7dpi, placebo-treated rhesus monkeys produced low levels of NAb with a titer of 1:16, while the high-dose group had a NAb level of up to 1:2048 (average 1:860) and the low-dose group had a NAb level of up to 1:1024 (average 1:512) (Figure 3b). Studies indicate that these results indicate that low and high doses of BBIBP-CorV have a highly effective protective effect on rhesus monkey SARS-CoV-2, and no antibody-dependent enhancement of infection has been observed.

safety

  The research team first conducted a single intramuscular injection experiment on rats (Sprague-Dawley) to assess the acute toxicity of BBIBP-CorV. The study divided 20 rats into 2 groups (n=10, 5/sex), intramuscular injection of 3 times the dose of BBIBP-CorV (8 μg/dose, 24 μg/rat), and saline as the control group. Observation was continued for 14 days after inoculation, and euthanasia was carried out on the 15th day to evaluate systemic dissection and observation.

  Four groups of rats had no death or were about to die within 14 consecutive days after inoculation, and no obvious clinical symptoms. In addition, there was no significant difference in weight and feeding status between the experimental group and the control group (Figure 4a, supplementary Figure 4d). There was no histopathological changes in these rats after euthanasia.

  It is worth noting that the maximum tolerated dose (MTD) of a single intramuscular injection in rats is 24 μg/rat, which is equivalent to 900 times the human body. The research team believes that this indicates that BBIBP-CorV has potentially good safety for the human body.

  The research team then evaluated the systemic allergic reaction caused by BBIBP-CorV by intramuscular injection and intravenous injection of guinea pigs. Through clinical observation and measurement of guinea pig body weight, the results showed no abnormal reaction during sensitization (Figure 4b). Negative control group (physiological saline) and experimental group showed no allergic reaction symptoms in D19 and D26. The positive control group (human albumin) was highly positive for allergic reactions (1/6 animals were positive, 3/6 animals were strongly positive, and 2/6 animals were extremely strong). In sharp contrast, the low and high dose test groups D19 and D26 had no allergic reactions, and all the allergic reactions were negative.

  The research team further evaluated the long-term toxicity of BBIBP-CorV to rhesus monkeys. Forty cynomolgus monkeys (20/sex) were divided into 4 groups (5/sex/group) and were injected intramuscularly with saline (group 1) or 2 μg, 4 μg, 8 μg BBIBP-CorV (group 2-4). Three of each group were dissected at D25, and the remaining two were dissected at D36. Systemic dissection and histopathological examination were performed.

  During the experiment, there was no death or imminent death in groups 2-4, distribution of lymphocyte subsets (CD3+, CD3+CD4+, CD3+CD8+, CD20+, CD3+CD4+/CD3+CD8+), cytokines (TNF-αIFN-γ , IL-2, IL-4, IL-5 and IL-6), c-reactive protein, or body weight observations also have no significant abnormal clinical physiological and pathological indicators (Figure 4c-4e, supplementary Figure 4e).

  D25 and D36 showed no abnormalities in the system anatomy of the euthanized macaques in each dose group. Granulomatous inflammation occurred in groups 2-4 at D25 and persisted at the end of the recovery period (D36), but slightly improved compared to D25. Rhesus monkeys showed only local irritation, with mild to severe granulomatous inflammation, but no such reaction two weeks after injection. The 8 μg/dose experimental group showed unobserved adverse reaction levels (NOAEL).