key: cord-0812168-m1bvurwi
authors: Gao, Qiang; Bao, Linlin; Mao, Haiyan; Wang, Lin; Xu, Kangwei; Yang, Minnan; Li, Yajing; Zhu, Ling; Wang, Nan; Lv, Zhe; Gao, Hong; Ge, Xiaoqin; Kan, Biao; Hu, Yaling; Liu, Jiangning; Cai, Fang; Jiang, Deyu; Yin, Yanhui; Qin, Chengfeng; Li, Jing; Gong, Xuejie; Lou, Xiuyu; Shi, Wen; Wu, Dongdong; Zhang, Hengming; Zhu, Lang; Deng, Wei; Li, Yurong; Lu, Jinxing; Li, Changgui; Wang, Xiangxi; Yin, Weidong; Zhang, Yanjun; Qin, Chuan
title: Rapid development of an inactivated vaccine for SARS-CoV-2
date: 2020-04-19
journal: bioRxiv
DOI: 10.1101/2020.04.17.046375
sha: e69e56b6b1a3ab44870df23cb07617d82788b4fb
doc_id: 812168
cord_uid: m1bvurwi

The COVID-19 pandemic caused by SARS-CoV-2 has brought about an unprecedented crisis, taking a heavy toll on human health, lives as well as the global economy. There are no SARS-CoV-2-specific treatments or vaccines available due to the novelty of this virus. Hence, rapid development of effective vaccines against SARS-CoV-2 is urgently needed. Here we developed a pilot-scale production of a purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc), which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats and non-human primates. These antibodies potently neutralized 10 representative SARS-CoV-2 strains, indicative of a possible broader neutralizing ability against SARS-CoV-2 strains circulating worldwide. Immunization with two different doses (3μg or 6 μg per dose) provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without any antibody-dependent enhancement of infection. Systematic evaluation of PiCoVacc via monitoring clinical signs, hematological and biochemical index, and histophathological analysis in macaques suggests that it is safe. These data support the rapid clinical development of SARS-CoV-2 vaccines for humans. One Sentence Summary A purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc) confers complete protection in non-human primates against SARS-CoV-2 strains circulating worldwide by eliciting potent humoral responses devoid of immunopathology

Switzerland, 1 from UK and 1 from Spain (table. S1). These patients were infected with SARS-CoV-2 during the most recent outbreak. The 11 samples contained SARS-CoV-2 strains are widely scattered on the phylogenic tree constructed from all available sequences, representing, to some extent, the circulating populations ( Fig. 1A   and fig. S1 ). We chose strain CN2 for purified inactivated SARS-CoV-2 virus vaccine development (PiCoVacc) and other 10 strains (termed as CN1, CN3-CN5 and OS1-OS6) as preclinical challenge strains. A number of strains amongst these, including CN1 and OS1, which are closely related to 2019-nCoV-BetaCoV /Wuhan/WIV04/2019 and EPI_ISL_412973, respectively, have been reported to cause severe clinical symptoms, including respiratory failure, requiring mechanical ventilation (9, 10) .

To obtain a viral stock adapted for efficient growth in Vero cells for PiCoVacc production, the CN3 strain was firstly plaque purified and passaged once in Vero cells to generate the P1 stock. After this another four passages were performed to generate the P2-P5 stocks. Growth kinetics analysis of the P5 stock in Vero cells showed that this stock replicated efficiently and reached a peak titer of 6-7 log 10 TCID 50 /ml by 3 or PiCoVacc, 5 more passages were performed to obtain the P10 stock, whole genome of which, together with those of the P1, P3 and P5 stocks were sequenced. Compared to P1, only two amino acid substitutions, Ala → Asp at E residue 32 (E-A32D) and Thr → Ile at nsp10 residue 49 (nsp10-T32I), occurred in P5 and P10 stocks (table. S2), suggesting that PiCoVacc CN2 strain possesses excellent genetic stability without any S mutations that might potentially alter the NAb epitopes. To produce pilot scale PiCoVacc for animal studies, the virus was propagated in a 50-liter culture of Vero cells using cell factory and inactivated by using β -propiolactone (Fig. 1C) . The virus was purified using depth filtration and two optimized steps of chromatography, yielding a highly pure preparation of PiCoVacc (Fig. 1D) . Additionally, cryo-electron microscopy (cryo-EM) analysis showed intact oval-shaped particles with diameters of 90-150 nm, which are embellished with crown-like spikes, representing a prefusion state of the virus (Fig. 1E ).

To assess the immunogenicity of PiCoVacc, groups of BALB/c mice (n=10) were injected at day 0 and day 7 with various doses of PiCoVacc mixed with alum adjuvant (0, 1.5 or 3 or 6 μ g per dose, 0 μ g in physiological saline as the sham group). No inflammation or other adverse effects were observed. Spike-, receptor binding domain (RBD)-, and N-specific antibody responses were evaluated by enzyme-linked immunosorbent assays (ELISAs) at weeks 1-6 after initial immunization ( fig. S2 ).

SARS-CoV-2 S-and RBD-specific immunoglobulin G (Ig G) developed quickly in the serum of vaccinated mice and peaked at the titer of 819,200 (>200 μ g/ml) and 409,600 (>100 μ g/ml), respectively, at week 6 ( Fig. 2A) . RBD-specific Ig G accounts for half of the S induced antibody responses, suggesting RBD is the dominant immunogen, which closely matches the serological profile of the blood of recovered COVID-19 patients ( Fig. 2A and 2B ) (11) . Surprisingly, the amount of N-specific Ig G induced is ~30-fold lower than the antibodies targeting S or RBD in immunized mice. Interestingly, previous studies have shown that the N-specific Ig G is largely abundant in the serum of COVID-19 patients and serves as one of the clinical diagnostic markers (11) . It's worthy to note that PiCoVacc could elicit much higher S-specific antibody titers than those of the serum from the recovered COVID-19

patients. This observation coupled with the fact that the antibodies targeting N of SARS-CoV-2 do not provide protective immunity against the infection (12) , suggest that PiCoVacc is capable of eliciting more effective antibody responses ( Fig. 2A and   2B ). Next, we measured SARS-CoV-2-specific neutralizing antibodies over a period of time using microneutralization assays (MN50). Similar to S-specific Ig G responses, the neutralizing antibody titer against the CN1 strain emerged at week 1 (12 for high dose immunization), surged after the week 2 booster and reached up to around 1,500

for low and medium doses and 3,000 for high dose at week 7, respectively ( Fig. 2A ).

In contrast, the sham group did not develop detectable SARS-CoV-2-specific antibody responses ( Fig. 2A and 2B ). In addition, immunogenic evaluations of PiCoVacc in Wistar rats with the same immunization strategy yielded similar results -the maximum neutralizing titers reached 2,048-4,096 at week 7 (Fig. 2C ). To investigate the spectrum of neutralizing activities elicited by PiCoVacc, we conducted neutralization assays against the other 9 isolated SARS-CoV-2 strains using mouse and rat serums collected 3 weeks post vaccination. Neutralizing titers against these strains demonstrate that PiCoVacc is capable of eliciting antibodies that possibly exhibit potent neutralization activities against SARS-Cov-2 strains circulating worldwide ( Fig. 2D and 2E ). 

Balb/c mice, wistar rats were randomly divided into three groups and immunized intraperitoneally and intramuscularly with the trial vaccine at three doses (1.5 μ g, 3 μ g, 6 μ g/dose)，respectively. All grouped animals were immunized for two times (at day 0 and 7). The control group was injected with physiological saline. Animals were bled from the tail veins, followed by antibody neutralizing assay to analyze vaccines immunogenicity.

Serum samples collected from immunized animals were inactivated at 56°C for 0.5h

and serially diluted with cell culture medium in two-fold steps. The diluted serums were mixed with a virus suspension of 100 TCID 50 in 96-well plates at a ratio of 1:1, followed by 2 hours incubation at 36.5°C in a 5% CO 2 incubator. 1-2 × 10 4 Vero cells were then added to the serum-virus mixture, and the plates were incubated for 5 days at 36.5°C in a 5% CO 2 incubator. Cytopathic effect (CPE) of each well was recorded under microscopes, and the neutralizing titer was calculated by the dilution number of 50% protective condition.

SARS-CoV-2 antibody titer of serum samples collected from immunized animals was determined by indirect ELISA assay. 96-well microtiter plates were coated with 0.1 μ g of purified S protein, M protein, N protein individually at 2-8°C overnight, and blocked with 2% BSA for 1h at room temperature. Diluted sera (1:100) were applied to each well for 2h at 37°C, followed by incubation with goat anti-mouse antibodies conjugated with HRP for 1h at 37°C after 3 times PBS wash. The plate was developed using TMB, following 2M H 2 SO 4 addition to stop the reaction, and read at 450/630nm by ELISA plate reader for final data.

SARS-CoV-2 trial vaccine's safety was evaluated in macaques. Four groups monkeys (TNF-α, IFN-γ, IL-2, IL-2、 IL-4、IL-5、IL-6) and biochemical blood test are also performed in collected blood samples. 60% of monkeys were euthanized at day 18 post immunization, and the left 40% were euthanized at day 29. Organs of lung, heart, spleen, liver, kidney and brain were collected for pathologic analysis.

Rhesus macaques (3-4 years old) were divided into four groups and injected intramuscularly with high dose (6 μ g/dose), medium dose (3 μ g/dose) vaccine, Al(OH) 3 adjuvant and physiological saline respectively. All grouped animals were immunized at three times (days 0, 7 and 14) before challenged with 10 6 TCID 50 /ml SARS-CoV-2 virus by intratracheal routes. Macaques were euthanized and lung tissues were collected at 7 days post inoculation (dpi). At day 3, 5, 7 dpi, the throat, and anal swabs were collected. Blood samples were collected on 0, 7, 14, and 21 days post immunization, and 3, 5, 7 dpi for hematological analysis and neutralizing antibody test of SARS-CoV-2. Lung tissues were collected at 7 dpi, and used for RT-PCR assay and histopathological assay.

To express the prefusion S ectodomain, a gene encoding residues 1−1208 of 

Fasta sequences for the COVID-19 were retrieved from the GISAID (https://www.gisaid.org/), NCBI and BIGD (https://bigd.big.ac.cn/ncov) database.

After quality control (removing sequences with low quality or short sequence length), 455 sequences were retained for the phylogenetic analysis. By combining 9 target sequences with the sequences from the public databases, we conducted sequence alignment using MAFFT and performed phylogenic reconstruction using IQtree (21) with default parameters. The inferred maximum likelihood tree is plotted using ggtree (22).

For cryo-EM sample preparation, a 3 μ L aliquot of purified viral particles was applied to a glow-discharged C-flat R2/1 Cu grid. Grids were manually blotted for 3 s in 100%

relative humidity for plunge-freezing (Vitrobot; FEI) in liquid ethane, as descripted previously (23). All samples were examined on a Titan Krios microscope (FEI).

Number of and percentage of amino acid differences in S are shown for the following SARS-CoV-2 isolates used in this study (Detailed information on these strains is descripted in table S1). Macaques were immunized three times at day 0, 7 and 14 through the intramuscular route with low dose (1.5 μ g per dose) or high dose (6 μ g per dose) of PiCoVacc or adjuvant only (sham) or placebo. Histopathological evaluations in brain, spleen, kidney and heart from four groups of macaques at day 29. Tissues were collected and stained with hematoxylin and eosin. Italy xxx Table S2 Genetic stability analysis of PiCoVacc. The sequence initially determined for PiCoVacc P1 stock was used as a reference sequence.

Additional mutations identified in genome genome nt position amino acid genome nt position amino acid P1

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Supplementary Materials for