WO2021209060A1

WO2021209060A1 - Inactivated vaccine for sars-cov-2 and preparation thereof

Info

Publication number: WO2021209060A1
Application number: PCT/CN2021/087957
Authority: WO
Inventors: Qiang Gao; Lin Wang; Yurong LI; Zhidong YAO; Jiling SHANG; Yanhui YIN; Yajing LI; Zhe LV; Zhuofa LI; Cien SHAN; Xiaoqin Ge; Zhiwei Wang; Yangqi SUN; Zengbing GUO; Feng Xue
Original assignee: Sinovac Research & Development Co., Ltd.
Priority date: 2020-04-17
Filing date: 2021-04-17
Publication date: 2021-10-21
Also published as: CN115836124A; CO2022016537A2; CL2022002832A1; BR112022019601A2

Abstract

Provided is an inactivated vaccine for SARS-Cov-2, and the preparation thereof. Also provided is a method for treatment or prevention or immunization against diseases associated with SARS-Cov-2 viruses infection, comprising administrating to a subject a pharmaceutical composition or a vaccine comprising a pharmaceutically effective amount of the inactivated SARS-Cov-2 virus and a pharmaceutically acceptable carrier.

Description

Inactivated Vaccine for SARS-CoV-2 and the Preparation thereof

Introduction

The World Health Organization declared that the outbreak of coronavirus disease (C OVID-19) is a Public Health Emergency of International Concern on 30 January 2020, and a pandemic on 11 March 2020. It is reported that about 80%of COVID-19 patients exhibit mild-to-moderate symptoms, while about 20%of them develop serious manifestations such as severe pneumonia, acute respiratory distress syndrome (ARDS) , sepsis and even death. The number of COVID-19 cases has increased at a staggering rate globally. As of 10 April, 2020, the total confirmed caseshave reached 1,623,173 and the death toll has risen to 97,236 all over the world. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , the causative agent of the ongoing pandemic, belongs to the genus Betacoronavirus (β-CoV) of the family Coronavirdae. SARS-CoV-2 along with the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle Eastern respiratory syndrome-related coronavirus (MERS-CoV) , constitute the three species of coronaviruses most life-threatening human beings. SARS-CoV-2 harbors a linear single-stranded positive sense RNA genome, encoding 4 structural proteins [including spike (S) , envelope (E) , membrane (M) , and nucleocapsid (N) ] , wherein S is a major protective antigen that elicits highly potent neutralizing antibodies (NAbs) , 16 non-structural proteins (nsp1-nsp16) and several accessory proteins. No specific antiviral drugs or vaccines against the newly emerged SARS-CoV-2 are currently available. Therefore, urgency in the development of vaccines is of vital importance to curb the pandemic and prevent future viral outbreaks.

Multiple SARS-CoV-2 vaccine types, such as DNA-or RNA-based formulations, recombi nant-subunits containing viral epitopes, adenovirus-based vectors and purified inactivated virus are under development. Purified inactivated viruses have been traditionally used for vaccine development and such vaccines have been found to be safe and effective for the prevention of diseases caused by viruses like influenza virus and poliovirus. However, since SARS-CoV-2 is a newly emerging virus, and our understanding on their biological nature, transmission, and pathogenesis is limited, and developing a vaccine against a virus has been a great challenge, let alone a newly emerging virus.

Summary of the Invention

The present invention provides an inactivated vaccine against SARS-CoV-2 virus, the preparation and the use thereof. The inactivated vaccine of the present invention induced SARS-CoV-2-specific neutralizing antibodies in animals, in particular mammals, including mice, rats, dogs, cats, non-human primates and human beings. These antibodies potently neutralized at least ten kinds of representative SARS-CoV-2 strains all over the world, indicative of a possible broader neutralizing ability against SARS-Cov-2 strains worldwide. Therefore, the inactivated vaccine of the present invention can be used to protect animals against SARS-CoV-2 challenge, wherein the animals include but are not limited tomice, rats, dogs, cats, non-human primates and human beings. In particular, the inactivated vaccine of the present invention fails to induce antibody-dependent enhancement. In other words, the inactivated vaccine of the present invention induces potent humoral responses devoid of immunopathology. In addition, the inactivated vaccine of the present invention is proved to be safe via systematic evaluation, involving monitoring clinical signs, hematological and biochemical index, and histophathological analysis.

In one aspect, the present invention provides an inactivated SARS-Cov-2 virus (SARS-Cov-2) , or derivative or relative thereof, wherein the infectivity of SARS-Cov-2 is undetectable, and wherein the inactivated SARS-Cov-2 induces an immune response against SARS-Cov-2 virus when administrated to a patient.

In some embodiments, the SARS-Cov-2 virus or derivative or relative thereof is inactivated with a virus inactivating agent, preferably, β-propiolactone and/or formalin. Preferably, the obtained inactivated SARS-Cov-2 virus or derivative or relative thereof is followed by purification with Ion-exchange Chromatography (IEC) and/or Size Exclusion Chromatography (SEC) . Preferably, prior to Size Exclusion Chromatography (SEC) , the inactivated SARS-Cov-2 virus is treated with a non-restriction endonuclease.

Preferably, the volume ratio of β-propionolactone to virus harvesting solution is 1: 4000-1:6000, and the inactivation time is 16-72 hours, preferably 20-24 hours at 4 ℃. Further preferably, the inactivation time is 24 hours.

In some embodiments, the SARS-Cov-2 virus or derivative or relative thereof comprises a spike protein (S) having an amino acid sequence encoded by the nucleotides 21508-25356 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

In some embodiments, the SARS-Cov-2 virus or derivative or relative thereof comprises a nucleocapsidprotein (N) having an amino acid sequence encoded by the nucleotides 28246-29505 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

In some embodiments, the SARS-Cov-2 virus or derivative or relative thereof comprises a membrane protein (M) having an amino acid sequence encoded by the nucleotides 26495-27163 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

In some embodiments, the SARS-Cov-2 virus or derivative or relative thereof comprises ORF1a having a nucleotide sequence of the nucleotides 238-13455 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

In some embodiments, the SARS-Cov-2 virus or derivative or relative thereof comprises ORF1ab having a nucleotide sequence of the nucleotides 13740-21527 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

In some embodiments, the SARS-Cov-2 virus or derivative or relative thereof comprises ORF3a having a nucleotide sequence of the nucleotides 25365-26192 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

Preferably, the SARS-Cov-2 virus or derivative or relative thereof comprises ORF1a having a nucleotide sequence of the nucleotides 238-13455 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or ORF1ab having a nucleotide sequence of the nucleotides 13740-21527 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a spike protein (S) having an amino acid sequence encoded by the nucleotides 21508-25356 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or ORF3a having a nucleotide sequence of the nucleotides 25365-26192 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a membrane protein (M) having an amino acid sequence encoded by the nucleotides 26495-27163 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a nucleocapsid protein (N) having an amino acid sequence encoded by the nucleotides 28246-29505 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

Preferably, the SARS-Cov-2 virus or derivative or relative thereof comprises ORF1a having a nucleotide sequence of the nucleotides 238-13455 of SEQ ID NO: 1; ORF1ab having a nucleotide sequence of the nucleotides 13740-21527 of SEQ ID NO: 1; a spike protein (S) having an amino acid sequence encoded by the nucleotides 21508-25356 of SEQ ID NO: 1; ORF3a having a nucleotide sequence of the nucleotides 25365-26192 of SEQ ID NO: 1; a membrane protein (M) having an amino acid sequence encoded by the nucleotides 26495-27163 of SEQ ID NO: 1; and a nucleocapsid protein (N) having an amino acid sequence encoded by the nucleotides 28246-29505 of SEQ ID NO: 1.

Preferably, the isolated SARS-CoV-2 virus, or derivative or relative thereof, has a genome, comprising a RNA sequence having its reverse complement sequence shown by any one of SEQ ID NOs: 1-3, or a sequence which is at least 95%identical therewith.

Preferably, the SARS-CoV-2 virus, or derivative or relative thereof has a genome encoded by the sequence shown by any one of SEQ ID NOs: 1-3, or a sequence which is at least 95%identical therewith. Preferably, the SARS-CoV-2 virus, or derivative or relative thereof has a genome encoded by the sequence shown by any one of SEQ ID NOs: 1-3, or has a genome, comprising a RNA sequence having its reverse complement sequence shown by any one of SEQ ID NOs: 1-3. The SARS-Cov-2 virus or derivative or relative thereof of the present invention exhibits high-yielding in Vero cells.

Preferably, before being inactivated with β-propiolactone, the SARS-Cov-2 virus is cultured with Vero cells at a cell density of 2×10 ⁶ cell/mL to 10×10 ⁶ cell/mL, for 3-7 days in a bioreactor at 35-39℃ to obtain virus harvesting solution.

In another aspect, the present invention provides a pharmaceutical composition or a vaccine comprising a pharmaceutically effective amount of the said inactivated SARS-Cov-2 virus, or derivative or relative thereof and a pharmaceutically acceptable carrier. The composition or the vaccine is either in a liquid state or in a frozen state, for example, in a lyophilized state.

In some embodiments, the dose of the pharmaceutical composition or the vaccine is in the range of 0.5-8μg by the weight of protein, preferably, in the range of 3-6μg by the weight of protein. In other words, 0.5μg-8μg by the weight of protein per dose of the vaccine produce complete protection against SARS-CoV-2 challenge, preferably, 3μg-6μg by the weight of protein per dose of the vaccine produce complete protection against SARS-CoV-2 challenge.

In some embodiments, the SARS-Cov-2 virus is inactivated with β-propiolactone and/or formalin. Preferably, the inactivated SARS-Cov-2 virus is followed by purification with Ion-exchange Chromatography (IEC) and/or Size Exclusion Chromatography (SEC) . Therefore, preferably, the pharmaceutical composition or the vaccine comprises the inactivated SARS-Cov-2 virus, which is purified with Ion-exchange Chromatography (IEC) and/or Size Exclusion Chromatography (SEC) after being inactivated. Preferably, prior to Size Exclusion Chromatography (SEC) , the inactivated SARS-Cov-2 virus is treated with non-restriction endonucleases.

Preferably, the volume ratio of β-propionolactone to virus harvesting solution is 1: 4000-1:6000, and the inactivation time is 16-72 hours, preferably 20-24 hours at 4℃. Preferably, the inactivation time is 24 hours.

The pharmaceutically acceptable carrier may be Al (OH) ₃ adjuvant and/or CpG oligodeoxynucleotide. In some embodiments, inactivated SARS-Cov-2 viruses are filtered through a membrane before mixing with Al (OH) ₃ adjuvant and/or CpG oligodeoxynucleotide. Preferably, the membrane has a pore diameter of 0.2-0.25μm , preferably, 0.22μm.

In some embodiments, the CpG oligodeoxynucleotide comprises at least two CpG units with a length of at least 20bp, and all nucleotides in the CpG oligodeoxynucleotide are thio-modified.

Preferably, the SARS-CoV-2 virus, or derivative or relative thereof has a genome encoded by the sequence shown by any one of SEQ ID NOs: 1-3, or a sequence which is at least 95%identical therewith.

Preferably, the SARS-CoV-2 virus, or derivative or relative thereof has a genome encoded by the sequence shown by any one of SEQ ID NOs: 1-3, or has a genome, comprising a RNA sequence having its reverse complement sequence shown by any one of SEQ ID NOs: 1-3.

The SARS-Cov-2 virus of the present invention exhibits high-yielding in Vero cells.

In particular, the pharmaceutical composition or the vaccine of the present invention confers complete protection in non-human primates against SARS-CoV-2 strains circulating worldwide by eliciting potent humoral responses devoid of immunopathology.

The pharmaceutical composition or the vaccine of the present invention 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, suggest that the pharmaceutical composition or the vaccine of the present invention is capable of eliciting more effective antibody responses

Previous experiences with the development of SARS and MERS vaccine candidates had raised concerns about pulmonary immunopathology, either directly caused by a type 2 helper T-cell (Th2) response or as a result of (ADE) . Although T-cell responses elicited by many vaccines have been demonstrated to be crucial for acute viral clearance, protection from subsequent coronavirus infections is largely mediated by humoral immunity. The “cytokine storm” induced by excessive T-cell responses have been actually shown to accentuate the pathogenesis of COVID19. Therefore, T-cell responses elicited by SARS-CoV-2 vaccine have to be well controlled in order to avoid immunopathology. In context with this, we systematically evaluated safety of the vaccine of the present invention in macaques by recording a number of clinical observations and biological indices. Neither fever nor weight loss was observed in any macaque after the immunization, and the appetite and mental state of all animals remained normal. Hematological and biochemical analysis, including biochemical blood test, lymphocyte subset percent (CD3+, CD4+ and CD8+) and key cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-5 and IL-6) showed no notable changes in vaccinated groups when compared to the sham and placebo groups. In addition, histopathological evaluations of various organs, including lung, heart, spleen, liver, kidney and brain, demonstrated that the vaccine of the present invention did not cause any notable pathology in macaques. No antibody-dependent enhancement of infection (ADE) was observed for all vaccinated macaques.

The present invention demonstrates the safety of the vaccine in macaques; neither infection enhancement nor immunopathological exacerbation was observed. The present invention also demonstrate a complete protection against SARS-CoV-2 challenge with a 6μg per dose of the vaccine of the present invention in macaques.

In another aspect, the present invention provides a method of preparing an inactivated SARS-Cov-2 composition or vaccine, comprising an inactivating SARS-Cov-2 virus using a virus inactivating agent, preferably, β-propiolactone and/or formalin.

In some embodiments, the SARS-Cov-2 viruses are cultured in large-scale bioreactor, for example, vero cells factories or fermentor, in order to to obtain virus harvesting solution, and inactivated with β-propiolactone for 24 hours. Preferably, the volume ratio of β-propionolactone to virus harvesting solution is 1: 4000-1: 6000, and the inactivation time is 16-72 hours, preferably 20-24 hours at 4℃. Preferably, the inactivation time is 24 hours.

In some embodiments, the volume ratio of formalin to virus harvesting solution is 1: 1000-1: 4000, and the inactivation time is 3-72 hours, preferably 3-13 hours at 4℃.

Preferably, the method further comprises the step of purifying the inactivated SARS-Cov-2 viruses with Ion-exchange Chromatography (IEC) and/or Size Exclusion Chromatography (SEC) . Preferably, prior to Size Exclusion Chromatography (SEC) , the inactivated SARS-Cov-2 virus is treated with a non-restriction endonuclease.

Preferably, the method further comprises the step of mixing the inactivated SARS-Cov-2 viruses with Al (OH) ₃ adjuvant. In some embodiments, inactivated SARS-Cov-2 viruses are filtered through a membrane before mixing with Al (OH) ₃ adjuvant and/or CpG oligodeoxynucleotide. Preferably, the membrane has a pore diameter of 0.2-0.25μm , preferably, 0.22μm.

In some embodiment, to produce vaccine, the SARS-Cov-2 viruses are propagated in a 50-1000-liter culture of Vero cells, for example, 50, 100, 200, 300, 400, 500, 600, 700 liter culture of Vero cells, using bioreactorand inactivated by using β-propiolactone and/or formalin. The virus is purified using centrifugation or depth filtration, and two optimized steps of chromatography, yielding a highly pure preparation of the vaccine. 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.

Preferably, the isolated SARS-CoV-2 virus, or derivative or relative thereof, has a genome, comprising a RNA sequence having its reverse complement sequence shown by SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

Preferably, the SARS-CoV-2 virus, or derivative or relative thereof has a genome encoded by the sequence shown by any one of SEQ ID NOs: 1-3, or has a genome, comprising a RNA sequence having its reverse complement sequence shown by SEQ ID NO: 1.

The SARS-Cov-2 virus of the present invention exhibits high-yielding in Vero cells. In another aspect, the present invention provides a method of treating or immunizing against diseases associated with SARS-Cov-2 viruses infection, comprising administrating to a subject a pharmaceutical composition or a vaccine comprising a pharmaceutically effective amount of the inactivated SARS-Cov-2 virus and a pharmaceutically acceptable carrier. In some embodiments, the route of administration of the pharmaceutical composition or the vaccine is intradermal, subcutaneous, intramuscular, oral or nasal vaccination. Preferably, the pharmaceutical composition or the vaccine is administrated via intramuscular injection into the upper deltoid.

Preferably, the diseases include but are not limited to pneumonia, severe acute respiratory infection, renal failure, heart failure, adult respiratory distress syndrome (ARDS) , liver injury, intestinal disease or severe acute respiratory syndrome.

The present invention demonstrates the safety of the vaccine in macaques; neither infection enhancement nor immunopathological exacerbation was observed. The present invention also demonstrate a complete protection against SARS-CoV-2 challenge with a 0.5-8μg by the weight of protein per dose of the vaccine of the present invention in macaques, preferably, the dose of the pharmaceutical composition or the vaccine is in the range of 2-6μg by the weight of protein, preferably, in the range of 3-6μg by the weight of protein.

In some embodiments, the SARS-Cov-2 virus is obtained from hospitalized patients. In some embodiments, the SARS-Cov-2 virus has the genome sequence shown in 2019-nCoV-BetaCoV/Wuhan/WIV04/2019 or EPI_ISL_412973 respectively, or having at least 95%identity to the sequence shown in 2019-nCoV-BetaCoV/Wuhan/WIV04/2019 and EPI_ISL_412973 respectively, preferably, having 96%, 97%, 98%, 99%, 99.5%, 99.9%identity to the sequence shown in 2019-nCoV-BetaCoV/Wuhan/WIV04/2019 or EPI_ISL_412973 respectively.

In another aspect, the present invention provides an isolated SARS-CoV-2 virus, or derivative or relative thereof, comprising a spike protein (S) having an amino acid sequence encoded by the nucleotides 21508-25356 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

Preferably, the SARS-CoV-2 virus, or derivative or relative thereof has a genome encoded by the sequence shown by any one of SEQ ID NOs: 1-3, , or has a genome, comprising a RNA sequence having its reverse complement sequence shown by any one of SEQ ID NOs: 1-3,.

The isolated SARS-Cov-2 virus of the present invention exhibits high-yielding in Vero cells.

The SARS-Cov-2 virus of the present invention exhibits high-yielding in Vero cells. And the SARS-Cov-2 virus of the present invention after being inactivated exhibits potently neutralizing activity in at least ten kinds of representative SARS-CoV-2 strains all over the world, indicative of a possible broader neutralizing ability against SARS-Cov-2 strains worldwide, and induces potent humoral responses devoid of immunopathology.

In another aspect, the present invention provides an isolated nucleic acid having the nucleotide sequence of the nucleotides 21508-25356 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

In another aspect, the present invention provides an isolated nucleic acid having the nucleotide sequence of the nucleotides 28246-29505 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

In another aspect, the present invention provides an isolated nucleic acid having the nucleotide sequence of the nucleotides 26495-27163 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

In another aspect, the present invention provides an isolated nucleic acid having the nucleotide sequence of the nucleotides 238-13455 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

In another aspect, the present invention provides an isolated nucleic acid having the nucleotide sequence of the nucleotides 13740-21527 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

In another aspect, the present invention provides an isolated nucleic acid having the nucleotide sequence of the nucleotides 25365-26192 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

In another aspect, the present invention provides an isolated nucleic acid comprising the nucleotide sequence of the nucleotides 238-13455 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or the nucleotide sequence of the nucleotides 13740-21527 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or the nucleotide sequence of the nucleotides 21508-25356 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or the nucleotide sequence of the nucleotides 25365-26192 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or the nucleotide sequence of the nucleotides 26495-27163 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or the nucleotide sequence of the nucleotides 28246-29505 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.

In another aspect, the present invention provides an isolated nucleic acid comprising the nucleotide sequence of the nucleotides 238-13455 of SEQ ID NO: 1; the nucleotide sequence of the nucleotides 13740-21527 of SEQ ID NO: 1; the nucleotide sequence of the nucleotides 21508-25356 of SEQ ID NO: 1; the nucleotide sequence of the nucleotides 25365-26192 of SEQ ID NO: 1; and the nucleotide sequence of the nucleotides 26495-27163 of SEQ ID NO: 1; the nucleotide sequence of the nucleotides 28246-29505 of SEQ ID NO: 1.

In another aspect, the present invention provides an isolated nucleic acid having the nucleotide sequence of any one of SEQ ID NOs: 1-3, or a sequence which is at least 95%identical therewith.

In another aspect, the present invention provides an isolated nucleic acid having the nucleotide sequence of any one of SEQ ID NOs: 1-3, or its reverse complement.

“at least 95%” means at least 96%, at keast97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.99%, and so on.

The sequence shown in SEQ ID NO: 2 corresponds to the virus strain P1_CADDE-CD1716_hCoV-19/Brazil/L70_CD1716/2020|Brazil_AM_Manaus|2020-12-16 ; and the sequence shown in SEQ ID NO: 3 corresponds to the virus strain ACC_20SF18530_GZ-GD_South America_2020-12-08.

Brief Description of the Drawings

Figure 1 shows characterization of PiCoVacc. (A) SARS-CoV-2 maximum likelihood phylogenetic tree. The SARS-CoV-2 isolates used in this study are depicted with black lines and labeled. The continents where the virus strains were from are colored differently. (B) Growth kinetics of PiCoVacc (CN2) P5 stock in Vero cells. (C) Protein composition and purity evaluation of PiCoVacc by NuPAGE 4-12%Bis-Tris Gel. (D) Representative electron micrograph of PiCoVacc.

Figure 2 shows that PiCoVacc immunization elicits neutralizing antibody response against 10 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 people (NI) act 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 response 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 9 isolated SARS-CoV-2 strains were performed using mouse and rat serums collected 3 weeks post vaccination. Points represent individual animals and humans; dotted lines indicate the limit of detection; horizontal lines indicate the geometric mean titer (GMT) of EC50 for each group.

Figure 3 showsimmunogenicity 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. Points represent individual macaques; 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 lobe 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. 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.

Figure 4 shows 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 day after the second vaccination) and 29 (7 day after the third vaccination) . Key cytokines (B) , containing TNF-α, IFN-γ and IL-2 were examined at day -1, 1 (the day for the first vaccination) , 4, 18 and 29 after vaccination. Values are mean ± s.d. (C) Histopathological evaluations in lungs from four groups of macaques at day 29. Lung tissue was collected and stained with hematoxylin and eosin.

Figure 5 shows SARS-CoV-2 amino acid sequence comparisons. 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 1) .

Figure 6 shows purification of S, RBD and N protein. (A) SDS-PAGE analysis of the S, RBD and N protein. Lane 1: molecular weight ladder, with relevant bands labeled; lane 2: the recombinant S protein; lane 3: the recombinant RBD protein; lane 4: the recombinant N protein. (B) Size-exclusion chromatogram of the affinity-purified S, RBD and N protein. (Left) Data of S protein from a Superose 6 10/300 column are shown in red. (Middle) Data of RBD protein from a Superdex 200 10/300 column are shown in red. (Right) Data of N protein from a Superdex 200 10/300 column are shown in black.

Figure 7 shows that body weight and body temperature are monitored to evaluate the safety 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. Body weight and body temperature are monitored at different time points.

Figure 8 shows that hematological indices are monitored to evaluate the safety 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 number of hematological indices are measured at different time points. ALT (Alanine aminotransferase) , AST (Aspartate aminotransferase) , ALP (Alkaline phosphatase) , TBil (Total bilirubin) , GGT (γ-glutamyltranspeptidase) , TP (Total protein) , Alb (Albumin) , Glb (Globulin) , A/G (Albumin/globulin ratio) , UREA (Blood urea) , Cre (Creatinine) , CK (Creatine kinase) , Glu (Glucose) , LDH (Lactate dehydrogenase) , CHO (Total cholesterol) , TG (Triglycerides) , Ca (Calcium) , P (Phosphorus) , Na+ (Sodium ion) , K+(Potassium ion) , Cl- (Chloride ion) .

Figure 9 shows that key cytokines are monitored to evaluate the safety 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. IL4, IL5 and IL6 are measured at different time points.

Figure 10 shows that histopathological evaluations are performed to evaluate the safety 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. 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.

Description ofParticular Embodiments of the Invention

The descriptions of particular embodiments and examples are provided by way of illustration and not by way of limitation. Those skilled in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

Examples

Materials and Methods

Facility and ethics statements

All experiments with live SARS-CoV-2 viruses were performed in the enhanced biosafety level 3 (P3+) facilities in the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) approved by the National Health Commission of the People’s Republic of China. All experiments with mice, rats and macaques were carried out in accordance with the Regulations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China.

Virus titration

SARS-CoV-2 virus titer was determined by microdose cytopathogenic efficiency (CPE) assay. Serial 10-fold dilutions of virus contained samples were mixed withVero cells, and then plated in 96-well culture plate. After 3-7 days culture in a 5%CO ₂ incubator at 36.5℃, cells were checked under a microscope for the presence of CPE. Virus titer was calculated by the method of Karber.

Vaccine preparation

SARS-CoV-2 coronavirus (CN2) , isolated from a COVID-19 infected patient, was provided by Zhejiang Provincial Center for Disease Control and Prevention. Viruses were cultured in bioreactors, and inactivated with β-propiolactone for 24 hours, followed by purification with Ion-exchange Chromatography (IEC) and Size Exclusion Chromatography (SEC) method. Prior to Ion-exchange Chromatography (IEC) and Size Exclusion Chromatography (SEC) , the inactivated SARS-Cov-2 viruses are treated with non-restriction endonucleases. The purified viruses were mixed with Al (OH) ₃ adjuvant and served as SARS-CoV-2 vaccine candidate. Tnactivated SARS-Cov-2 viruses are filtered through a membrane before mixing with Al (OH) ₃ adjuvant. Preferably, the membrane has a pore diameter of 0.22μm. The obtained vaccine is named as PiCoVacc, which has a genome encoded by the sequence shown by SEQ ID NO: 1; or has a RNA sequence having its reverse complement sequence shown by SEQ ID NO: 1.

RT-PCR

Total RNA was extracted from organs as described previously with the RNeasy Mini Kit (Qiagen) and the PrimerScript RT Reagent Kit (TaKaRa) . The forward and reverse primers targeting against the envelope (E) gene of SARS-CoV-2 used for RT-PCR were 5’-TCGTTTCGGAAGAGACAGGT-3’and 5’-GCGCAGTAAGGATGGCTAGT-3’, respectively. RT-PCR was performed at the reaction conditions of 50℃ for 30 min, followed by 40 cycles of 95℃for 15 min, 94℃ for 15 s, and 60℃ for 45s.

Vaccine immunogenicity analysis

Balb/c mice, wistar rats were randomly divided into three groups and immunized intraperitoneally and intramuscularly with the trial vaccine at three doses (1.5μg, 3 μg, 6 μg/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.

Neutralizing assay

Serum samples collected from immunized animals were inactivated at 56℃ 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℃ in a 5%CO ₂incubator. Vero cells were then added to the serum-virus mixture, and the plates were incubated for 5 days at 36.5℃ 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.

Enzyme linked immunosorbant assay (ELISA)

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 μg of purified S protein, M protein, N protein individually at 2-8℃ overnight, and blocked with 2%BSA for 1h at room temperature. Diluted sera (1: 100) were applied to each well for 2h at 37℃, followed by incubation with goat anti-mouse antibodies conjugated with HRP for 1h at 37℃after 3 times PBS wash. The plate was developed using TMB, following 2M H ₂SO ₄ addition to stop the reaction, and read at 450/630nm by ELISA plate reader for final data.

Vaccine safety evaluation

SARS-CoV-2 trial vaccine’s safety was evaluated in macaques. Four groups monkeys (5 female and 5 male monkeys/group) were immunized with high dose (6μg /dose) , low dose (1.5μg /dose) vaccine, Al (OH) ₃ adjuvant and physiological saline individually for three times at

days

0, 7 and 14. Datasets of many safety related parameters were collected during and after immunization, including clinical observation, body weight, body temperature. Analysis of lymphocyte subset percent (CD3+, CD4+ and CD8+) , key cytokines (TNF-α, IFN-γ, 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.

Challenge assay of rhesus macaques

Rhesus macaques (3-4 years old) were divided into four groups and injected intramuscularly with high dose (6 μg/dose) , medium dose (3 μg/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.

Protein expression and purification

To express the prefusion S ectodomain, a gene encoding residues 1-1208 of COVID-19 S (GenBank: MN908947) with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682-685) and a 2×StrepTag was synthesized and cloned into the mammalian expression vector pCAGGS. To express the COVID-19 RBD, residues 319-591 of COVID-19 S were cloned upstream of a 2×StrepTag into the mammalian expression vector pCAGGS.

To express the N protein, residues 1-419 (GenBank: QHW06046.1) was cloned into vector pET-28a containing a C-terminal 6×His. S ectodomain and RBD were used to transiently transfect HEK Expi 293F cells (Thermo Fisher) using polyethylenimine. Protein was purified from filtered cell supernatants using StrepTactin resin (IBA) before being subjected to additional purification by size-exclusion chromatography using either a Superose 6 10/300 column (GE Healthcare) or a Superdex 200 10/300 Increase column (GE Healthcare) in 20mM Tris pH 8.0, 200 mMNaCl. The N protein was produced in BL21 (DE3) upon the introduction of IPTG. After ultrasonication, the supernatant was loaded over Ni-NTA as manual described (GE Healthcare) and eluted with elution buffer (20 mMTris-HCl, 500 mMNaCl, 200 mM imidazole, pH8.0) , and then execute the size-exclusion chromatography using a Superdex 200 10/300 Increase column (GE Healthcare) .

Phylogenic tree analysis

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 with default parameters. The inferred maximum likelihood tree is plotted using ggtree.

Cryo-EM sample preparation

For cryo-EM sample preparation, a 3 μL aliquot of purified viral particles was applied to a glow-discharged C-flat R2/1 Cu grid. Grids were manually blotted for 3s in 100%relative humidity for plunge-freezing (Vitrobot; FEI) in liquid ethane, as descripted previously. All samples were examined on a Titan Krios microscope (FEI) .

Example 1. Characterization of PiCoVacc and the prepration of PiCoVacc.

To develop preclinical in vitro neutralization and challenge models for a candidate SARS-CoV-2 vaccine, SARS-CoV-2 strains were isolated from the bronchoalveolar lavage fluid (BALF) samples of 11 hospitalized patients (including 5 ICU patients) , among which 4 are from China, 4 from Italy, 1 from Switzerland, 1 from UK and 1 from Spain (Table 1) . 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. 5) . Strain CN2 was chosen for purified inactivated SARS-CoV-2 virus vaccine development (PiCoVacc) and other 10 strains (termed as CN1, CN3-CN5 and OS1-OS6) were chosen as preclinical challenge strains. A number of strains amongst these, including CN1, OS1, which are closely related to 2019-nCoV-BetaCoV/Wuhan/WIV04/2019 and EPI_ISL_412973, respectively, cause severe clinical symptoms, including respiratory failure, requiring mechanical ventilation. The whole genome DNA sequence of the strain PiCoVacc is shown in SEQ ID NO: 1, which corresponds to the RNA sequence of the strain PiCoVacc. The nucleotides 238-13455 of SEQ ID NO: 1 encodes ORF1a of the strain PiCoVacc; the nucleotides 13740-21527 of SEQ ID NO: 1 encodes ORF1ab of the strain PiCoVacc; the nucleotides 25365-26192 of SEQ ID NO: 1 encodes to ORF3a of the strain PiCoVacc; the nucleotides 26495-27163 of SEQ ID NO: 1 encodes the membrane protein (M) of the strain PiCoVacc; the nucleotides 28246-29505 of SEQ ID NO: 1 encodes the nucleocapsid protein (P) of the strain PiCoVacc; the nucleotides 21508-25356 of SEQ ID NO: 1 encodes the spike glycoprotein (S) of the strain PiCoVacc.

Table 1Virus strain information

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 4 days post infection (dpi) at multiplicities of infection (MOI) of 0.0001-0.01 at temperatures between 33℃-37℃ (Fig. 1B) . To evaluate the genetic stability of 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-T49I) , occurred in P5 and P10 stocks (Table 2) , suggesting that PiCoVacc CN2 strain possesses excellent genetic stability without any S mutations that might potentially alter the NAb epitopes. To produce PiCoVacc, the virus was propagated in a culture of Vero cells using bioreactor and inactivated by using β-propiolactone. The virus was purified using centrifugation and two optimized steps of chromatography, yielding a highly pure preparation of PiCoVacc (Fig. 1C) . Therefore, for producing the vaccine, the virus was propagated in a culture of Vero cells using bioreactor and inactivated by using β-propiolactone, then the virus was purified using centrifugation and two optimized steps of chromatography. Additionally, cryo-electron microscopy (cryo-EM) analysis showed intact oval-shaped particles with diameters of 90-150nm, which are embellished with crown-like spikes, representing a prefusion state of the virus (Fig. 1D) .

Table 2 Genetic stability analysis of PiCoV ₂. The sequence initially determined for PiCoV ₂ P1 stock was used as a reference sequence.

Exmaple2 PiCoVacc immunization elicits neutralizing antibody response against 10 representative SARS-CoV-2 isolates.

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 μg per dose, 0 μg 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. 6) . SARS-CoV-2 S-and RBD-specific immunoglobulin G (IgG) developed quickly in the serum of vaccinated mice and peaked at the titer of 819, 200 (>200 μg/ml) and 409, 600 (>100 μg/ml) , respectively, at week 6 (Fig. 2A) . RBD-specific IgG 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 IgG 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) , suggest that PiCoVacciscapable 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 IgG 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) .

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

The immunogenicity and protective efficacy of PiCoVacc were evaluated in rhesus macaques (Macacamulatta) , a non-human primate species that shows a COVID-19-like disease caused by SARS-CoV-2 infection. Macaques were immunized three times via the intramuscular route with medium (3 μg per dose) or high doses (6 μg per dose) of PiCoVacc at

day

0, 7 and 14 (n=4) . S-specific IgG and Nab (neutralizing antibody) were reduced at week 2 and rose to ～12,800 and ～50, respectively at week 3 (before virus challenge) in both vaccinated groups, whose titers are similar to those of serum from the recovered COVID-19 patients (Fig. 3A and 3B). Unexpectedly, NAb titer (61) in medium dose immunized group was ～20%more than that observed (50) in high dose vaccinated group at week 3, due to individual differences in the ability of one animal in medium dose group in eliciting ～10-fold higher titer when compared to the other three (Fig. 3B) . Excluding this exception, NAb titer in medium dose group would drop down to 34, ～40%lower than that in high dose group. Subsequently, we conducted a challenge study by a direct inoculation of 10 ⁶ TCID ₅₀ of SARS-CoV-2 CN1 into the animal lung through intratracheal route at day 22 (one day after the third immunization) in vaccinated and control macaques to verify the protective efficacy. Expectedly, all control (sham and placebo) macaques showed excessive copies (10 ⁴-10 ⁶/ml) of viral genomic RNA in pharynx, crissum and lung by day 3-7 post-inoculation (dpi) and severe interstitial pneumonia (Fig. 3C-3F) . By contrast, all vaccinated macaques were largely protected against SARS-CoV-2 infection with very small histopathological changes in lung, which probably were caused by a direct inoculation of 10 ⁶ TCID ₅₀ of virus into the lung through intratracheal route, that needed longer time (more than one week) to recover completely (Fig. 3F) . Viral loads decreased significantly in all vaccinated macaques, but increased slightly in control animals from day 3-7 after infection (Fig. 3C-3E) . All four macaques that received the high dose, had no detectable viral loads in pharynx, crissum and lung at day 7 after infection. In medium dose immunized group, we indeed partially detected the viral blip from pharyngeal (3/4) , anal (2/4) and pulmonary (1/4) specimens at day 7 after infection, whilst viral loads presented a ～95%reduction when compared to the sham groups (Fig. 3C-3E) . Interestingly, NAb titer in vaccinated groups decreased by ～30%by 3 days post infection to neutralize viruses, then rapidly increased from day 5-7 after infection to maintain its potent neutralization efficacy. In comparison with high dose vaccination group (titer of ～145) , higher NAb titers observed in medium dose vaccinated group at day 7 after infection (～400 for 4 macaques; ～300 for 3 macaques, if the one outlier is discarded) might have resulted from relatively low level of viral replication, suggesting a requirement of longer time for complete viral clearance. No antibody-dependent enhancement of infection (ADE) was observed for any vaccinated macaques despite the observation that relatively low NAb titer existed within the medium dose group before infection, offering partial protection.

Example 4 Safety evaluation of PiCoVacc in nonhuman primates.

Previous experiences with the development of SARS and MERS vaccine candidates had raised concerns about pulmonary immunopathology, either directly caused by a type 2 helper T-cell (Th2) response or as a result of (ADE) . Although T-cell responses elicited by many vaccines have been demonstrated to be crucial for acute viral clearance, protection from subsequent coronavirus infections is largely mediated by humoral immunity. The “cytokine storm” induced by excessive T-cell responses have been actually shown to accentuate the pathogenesis of COVID19. Therefore, T-cell responses elicited by SARS-CoV-2 vaccine have to be well controlled in order to avoid immunopathology. In context with this, we systematically evaluated safety of PiCoVacc in macaques by recording a number of clinical observations and biological indices. Two groups of macaques (n=10) were immunized by intramuscular injection with low (1.5 μg) or high doses (6 μg) and another two groups of macaques (n=10) were immunized with adjuvant (sham) and physiological saline (placebo) for three times at

day

0, 7 and 14. Neither fever nor weight loss was observed in any macaque after the immunization of PiCoVacc, and the appetite and mental state of all animals remained normal (Fig. 7) . Hematological and biochemical analysis, including biochemical blood test, lymphocyte subset percent (CD3 ⁺, CD4 ⁺and CD8 ⁺) and key cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-5 and IL-6) showed no notable changes in vaccinated groups when compared to the sham and placebo groups (Fig. 4A-4B and Fig. 8-9) . In addition, histopathological evaluations of various organs, including lung, heart, spleen, liver, kidney and brain, from the 4 groups at day 29 demonstrated that PiCoVacc did not cause any notable pathology in macaques (Fig. 4C and Fig. 10) .

The serious pandemic of the current COVID19 and the precipitously increasing numbers of death worldwide necessitate the urgent development of a SARS-CoV-2 vaccine, requiring a new pandemic paradigm. The safety and efficacy are essential for vaccine development at both stages of preclinical studies and clinical trials. Rhesus macaques that mimic COVID-19-like symptoms after SARS-CoV-2 infection appear quite promising animal models for studying the disease. We provide extensive evidences for the safety of PiCoVacc in macaques; neither infection enhancement nor immunopathological exacerbation was observed in our studies. Our data also demonstrate a complete protection against SARS-CoV-2 challenge with a 6μg per dose of PiCoVacc in macaques.

Claims

An inactivated SARS-Cov-2 virus or derivative or relative thereof, wherein the infectivity of the inactivated SARS-Cov-2 is undetectable, and wherein the inactivated SARS-Cov-2 induces an immune response against SARS-Cov-2 virus when administrated to a subject.
The inactivated SARS-Cov-2 virus or derivative or relative thereof of claim 1, wherein the inactivated SARS-Cov-2 virus is obtained by inactivating the SARS-Cov-2 virus with a virus inactivating agent, preferably, β-propiolactone and/or formalin.
The inactivated SARS-Cov-2 virus or derivative or relative thereof of claim 1 or 2, wherein the inactivated SARS-Cov-2 virus is further purified via ion-exchange chromatography and/or size exclusion chromatography.
The inactivated SARS-Cov-2 virus or derivative or relative thereof of claim 1 or 2, wherein the inactivated SARS-Cov-2 virus is treated with a non-restriction endonuclease prior to size exclusion chromatography.
The inactivated SARS-Cov-2 virus or derivative or relative thereof of claim 1 or 2, wherein, before being inactivated with β-propiolactone, the SARS-Cov-2 virus is cultured with Vero cells at a cell density of 2×10 ⁶ cell/mL to 10×10 ⁶ cell/mL, for 3-7 days in a bioreactor at 35-39℃ to obtain virus harvesting solution.
The inactivated SARS-Cov-2 virus or derivative or relative thereof of claim 1 or 2, wherein the volume ratio of β-propionolactone to virus harvesting solution is 1: 4000-1: 6000, and the inactivation time is 16-72 hours, preferably, 20-24 hours at 4℃, further preferably, the inactivation time is 24 hours.
The inactivated SARS-Cov-2 virus or derivative or relative thereof of claim 1 or 2, wherein the SARS-Cov-2 virus or derivative or relative thereof comprises: ORF1a having a nucleotide sequence of nucleotides 238-13455 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or ORF1ab having a nucleotide sequence of nucleotides 13740-21527 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a spike protein S having an amino acid sequence encoded by nucleotides 21508-25356 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or ORF3a having a nucleotide sequence of nucleotides 25365-26192 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a membrane protein M having an amino acid sequence encoded by nucleotides 26495-27163 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a nucleocapsid protein N having an amino acid sequence encoded by nucleotides 28246-29505 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
The inactivated SARS-Cov-2 virus or derivative or relative thereof of claim 1 or 2, wherein the SARS-Cov-2 virus or derivative or relative thereof comprises: ORF1a having a nucleotide sequence of nucleotides 238-13455 of SEQ ID NO: 1; ORF1ab having a nucleotide sequence of nucleotides 13740-21527 of SEQ ID NO: 1; a spike protein S having an amino acid sequence encoded by nucleotides 21508-25356 of SEQ ID NO: 1; ORF3a having a nucleotide sequence of nucleotides 25365-26192 of SEQ ID NO: 1; a membrane protein M having an amino acid sequence encoded by nucleotides 26495-27163 of SEQ ID NO: 1; and a nucleocapsid protein N having an amino acid sequence encoded by nucleotides 28246-29505 of SEQ ID NO: 1.
The inactivated SARS-Cov-2 virus or derivative or relative thereof of claim 1 or 2, wherein the SARS-CoV-2 virus or derivative or relative thereof has a genome, comprising an RNA sequence having a reverse complement sequence shown by any one of SEQ ID NOs: 1-3, or a sequence which is at least 95%identical therewith.
The inactivated SARS-Cov-2 virus or derivative or relative thereof of claim 1 or 2, wherein the SARS-CoV-2 virus or derivative or relative thereof has a genome encoded by the sequence shown by any one of SEQ ID NOs: 1-3, or a sequence which is at least 95%identical therewith.
The inactivated SARS-Cov-2 virus or derivative or relative thereof of claim 1 or 2, wherein the SARS-CoV-2 virus or derivative or relative thereof has a genome encoded by the sequence shown by any one of SEQ ID NOs: 1-3, or has a genome comprising an RNA sequence having a reverse complement sequence shown by any one of SEQ ID NOs: 1-3.
A pharmaceutical composition or a vaccine, comprising a pharmaceutically effective amount of the inactivated SARS-Cov-2 virus or derivative or relative thereof, and a pharmaceutically acceptable carrier.
The pharmaceutical composition or the vaccine of claim 12, wherein the inactivated SARS-Cov-2 virus or derivative or relative thereof is obtained by inactivating the SARS-Cov-2 virus with a virus inactivating agent, preferably, β-propiolactone and/or formalin.
The pharmaceutical composition or the vaccine of claim 12, wherein the pharmaceutical composition or the vaccine is either in a liquid state or in a frozen state, preferably, in a liquid state or a lyophilized state, further preferably, the dose of the pharmaceutical composition or the vaccine is in the range of 0.5-8μg by the weight of protein, preferably, in the range of 3-6μg by the weight of protein.
The pharmaceutical composition or the vaccine of claim 12, wherein the pharmaceutically acceptable carrier is Al (OH) ₃ adjuvant and/or CpG oligodeoxynucleotide.
The pharmaceutical composition or the vaccine of claim 12, wherein the inactivated SARS-Cov-2 virus or derivative or relative thereof is purified via ion-exchange chromatography and/or size exclusion chromatography after being inactivated.
The pharmaceutical composition or the vaccine of claim 16, wherein the inactivated SARS-Cov-2 virus or derivative or relative thereof is treated with a non-restriction endonuclease prior to size exclusion chromatography.
The pharmaceutical composition or the vaccine of claim 16, wherein, before being inactivated with β-propiolactone, the SARS-Cov-2 virus or derivative or relative thereof is cultured with Vero cells at a cell density of 2×10 ⁶ cell/mL to 10×10 ⁶ cell/mL, for 3-7 days in a bioreactor at 35-39℃ to obtain virus harvesting solution.
The pharmaceutical composition or the vaccine of claim 16, wherein the volume ratio of β-propionolactone to virus harvesting solution is 1: 4000-1: 6000, and the inactivation time is 16-72 hours, preferably 20-24 hours at 4℃, further preferably, the inactivation time is 24 hours.
The pharmaceutical composition or the vaccine of claim 16, wherein the inactivated SARS-Cov-2 virus or derivative or relative thereof is filtered through a membrane before mixing with Al (OH) ₃ adjuvant and/or CpG oligodeoxynucleotide, the membrane has a pore diameter of 0.2-0.25μm, preferably, 0.22μm.
The pharmaceutical composition or the vaccine of claim 12, wherein the SARS-Cov-2 virus or derivative or relative thereof comprises: ORF1a having a nucleotide sequence of nucleotides 238-13455 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or ORF1ab having a nucleotide sequence of nucleotides 13740-21527 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a spike protein S having an amino acid sequence encoded by nucleotides 21508-25356 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or ORF3a having a nucleotide sequence of nucleotides 25365-26192 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a membrane protein M having an amino acid sequence encoded by nucleotides 26495-27163 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a nucleocapsid protein N having an amino acid sequence encoded by nucleotides 28246-29505 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
The pharmaceutical composition or the vaccine of claim 12, wherein the SARS-Cov-2 virus or derivative or relative thereof comprises: ORF1a having a nucleotide sequence of nucleotides 238-13455 of SEQ ID NO: 1; ORF1ab having a nucleotide sequence of nucleotides 13740-21527 of SEQ ID NO: 1; a spike protein S having an amino acid sequence encoded by nucleotides 21508-25356 of SEQ ID NO: 1;ORF3a having a nucleotide sequence of nucleotides 25365-26192of SEQ ID NO: 1; a membrane protein M having an amino acid sequence encoded by nucleotides 26495-27163 of SEQ ID NO: 1; and a nucleocapsid protein N having an amino acid sequence encoded by nucleotides 28246-29505 of SEQ ID NO: 1.
The pharmaceutical composition or the vaccine of 12, wherein the SARS-CoV-2 virus or derivative or relative thereof has a genome which comprises an RNA sequence having a reverse complement sequence shown by any one of SEQ ID NOs: 1-3, or a sequence which is at least 95%identical therewith.
The pharmaceutical composition or the vaccine of claim 12, wherein the SARS-CoV-2 virus or derivative or relative thereof has a genome encoded by the sequence shown by any one of SEQ ID NO: 1, or a sequence which is at least 95%identical therewith.
A method of preparing an inactivated SARS-Cov-2 composition or vaccine, comprising inactivating SARS-Cov-2 virus with a virus inactivating agent, preferably, β-propiolactone and/or formalin.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 25, wherein the inactivated SARS-Cov-2 virus is further purified via ion-exchange chromatography and/or size exclusion chromatography after being inactivated.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 25, wherein the inactivated SARS-Cov-2 virus is treated with a non-restriction endonuclease prior to size exclusion chromatography.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 25, wherein, before being inactivated with β-propiolactone, the SARS-Cov-2 virus is cultured with Vero cells at a cell density of 2×10 ⁶ cell/mL to 10×10 ⁶ cell/mL, for 3-7 days in a bioreactor at 35-39℃ to obtain virus harvesting solution.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 28, wherein the volume ratio of β-propionolactone to virus harvesting solution is 1: 4000-1: 6000, and the inactivation time is 16-72 hours, preferably 20-24 hours at 4℃, further preferably, the inactivation time is 24 hours.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 28, wherein the volume ratio of formalin to virus harvesting solution is 1: 1000-1: 4000, and the inactivation time is 3-72 hours, preferably, 3-13 hours at 4℃.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 25, wherein the inactivated SARS-Cov-2 virus is filtered through a membrane before mixing with Al (OH) ₃ adjuvant and/or CpG oligodeoxynucleotide, the membrane has a pore diameter of 0.2-0.25μm, preferably, 0.22μm.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 25, further comprising a step of mixing the inactivated SARS-Cov-2 viruses with Al (OH) ₃ adjuvant and/or CpG oligodeoxynucleotide.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 25, wherein the inactivated SARS-Cov-2 virus is filtered through a membrane before mixing with Al (OH) ₃ adjuvant and/or CpG oligodeoxynucleotide, wherein the membrane has a pore diameter of 0.20-0.25μm, preferably, the membrane has a pore diameter of 0.22μm.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 25, wherein the SARS-Cov-2 viruses are propagated in a 50～1000-liter culture of Vero cells using bioreactor and inactivated by using β-propiolactone.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 25, wherein the viruses are purified using depth filtration or centrifugation and two optimized steps of chromatography.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 25, wherein the SARS-Cov-2 virus comprises: ORF1a having a nucleotide sequence of nucleotides 238-13455 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or ORF1ab having a nucleotide sequence of nucleotides 13740-21527 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a spike protein S having an amino acid sequence encoded by nucleotides 21508-25356 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or ORF3a having a nucleotide sequence of nucleotides 25365-26192 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a membrane protein M having an amino acid sequence encoded by nucleotides 26495-27163 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a nucleocapsid protein N having an amino acid sequence encoded by nucleotides 28246-29505 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 25, wherein the SARS-Cov-2 virus comprises: ORF1a having a nucleotide sequence of nucleotides 238-13455 of SEQ ID NO: 1; ORF1ab having a nucleotide sequence of nucleotides 13740-21527 of SEQ ID NO: 1; a spike protein S having an amino acid sequence encoded by nucleotides 21508-25356 of SEQ ID NO: 1; ORF3a having a nucleotide sequence of nucleotides 25365-26192 of SEQ ID NO: 1; a membrane protein M having an amino acid sequence encoded by nucleotides 26495-27163 of SEQ ID NO: 1; and a nucleocapsid protein N having an amino acid sequence encoded by nucleotides 28246-29505 of SEQ ID NO: 1.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 25, wherein the SARS-CoV-2 virus has a genome, comprising an RNA sequence having a reverse complement sequence shown by any one of SEQ ID NOs: 1-3, or a sequence which is at least 95%identical therewith.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 25, wherein the SARS-CoV-2 virus has a genome encoded by the sequence shown by any one of SEQ ID NOs: 1-3, or a sequence which is at least 95%identical therewith.
The method of preparing the inactivated SARS-Cov-2 composition or vaccine of claim 25, wherein the SARS-CoV-2 virus has a genome encoded by the sequence shown by any one of SEQ ID NOs: 1-3, or has a genome, comprising an RNA sequence having a reverse complement sequence shown by any one of SEQ ID NOs: 1-3.
A method of treating or immunizing against a disease associated with SARS-Cov-2 infection in a subject, comprising administrating to the subject a pharmaceutically effective amount of the pharmaceutical composition or the vaccine of any one of claims 12-24.
The method of treating or immunizing against a disease associated with SARS-Cov-2 infection in a subject of claim 41, wherein the pharmaceutical composition or the vaccine is administrated via intradermal, subcutaneous, intramuscular, oral or nasal vaccination, preferably, via intramuscular injection into the upper deltoid of the subject.
The method of treating or immunizing against a disease associated with SARS-Cov-2 infection in a subject of claim 41, wherein the disease comprises, but is not limited to pneumonia or pneumonia syndrome, severe acute respiratory infection, renal failure, heart failure, adult respiratory distress syndrome (ARDS) , liver injury, intestinal disease or severe acute respiratory syndrome.
The method of treating or immunizing against a disease associated with SARS-Cov-2 infection in a subject of claim 41, wherein the subject is an animal, preferably, a mammal, including mice, rats, dogs, cats, non-human primates and human beings.
The method of treating or immunizing against a disease associated with SARS-Cov-2 infection in a subject of claim 41, wherein the pharmaceutical composition or the vaccine confers complete protection in non-human primates against SARS-CoV-2 infection by eliciting a potent humoral response devoid of immunopathology.
The method of treating or immunizing against a disease associated with SARS-Cov-2 infection in a subject of claim 41, wherein the pharmaceutical composition or the vaccine do not produce antibody-dependent enhancement of infection.
The method of treating or immunizing against a disease associated with SARS-Cov-2 infection in a subject of claim 41, wherein the pharmaceutical composition or the vaccine in a dose of 0.5-8μg produces complete protection against SARS-CoV-2 challenge in a subject, preferably, the vaccine in a dose of 2-6μg produces complete protection against SARS-CoV-2 challenge in a subject.
The inactivated SARS-Cov-2 virus of any one of claims 1-11, the pharmaceutical composition or the vaccine of any one of claims 12-25, the method of preparing the inactivated SARS-Cov-2 composition or vaccine of any one of claims 26-40, the method of treatting or immunizing against a disease associated with SARS-Cov-2 infection of any one of claims 41-47, wherein the SARS-Cov-2 virus has the genome sequence shown in 2019-nCoV-BetaCoV/Wuhan/WIV04/2019 or EPI_ISL_412973 respectively, or having at least 95%identity to the sequence shown in 2019-nCoV-BetaCoV/Wuhan/WIV04/2019 or EPI_ISL_412973 respectively, preferably, having 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%or 99.9%identity to the sequence shown in 2019-nCoV-BetaCoV/Wuhan/WIV04/2019 or EPI_ISL_412973 respectively.
An isolated SARS-CoV-2 virus, or derivative or relative thereof, comprising a spike protein S having an amino acid sequence encoded by nucleotides 21508-25356 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
The isolated SARS-CoV-2 virus, or derivative or relative thereof of claim 49, further comprising a nucleocapsid protein N having an amino acid sequence encoded by nucleotides 28246-29505 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
The isolated SARS-CoV-2 virus, or derivative or relative thereof of claim 49, further comprising a membrane protein M having an amino acid sequence encoded by nucleotides 26495-27163 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
The isolated SARS-CoV-2 virus, or derivative or relative thereof of claim 49, further comprising ORF1a having a nucleotide sequence of nucleotides 238-13455 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
The isolated SARS-CoV-2 virus, or derivative or relative thereof of claim 49, further comprising ORF1ab having a nucleotide sequence of nucleotides 13740-21527 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
The isolated SARS-CoV-2 virus, or derivative or relative thereof of claim 49, further comprising ORF3a having a nucleotide sequence of nucleotides 25365-26192 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
The isolated SARS-CoV-2 virus, or derivative or relative thereof of claim 49, wherein the isolated SARS-CoV-2 virus, or derivative or relative thereof comprises ORF1a having a nucleotide sequence of nucleotides 238-13455 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or ORF1ab having a nucleotide sequence of nucleotides 13740-21527 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a spike protein S having an amino acid sequence encoded by nucleotides 21508-25356 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or ORF3a having a nucleotide sequence of nucleotides 25365-26192 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a membrane protein M having an amino acid sequence encoded by nucleotides 26495-27163 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or a nucleocapsid protein N having an amino acid sequence encoded by nucleotides 28246-29505 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
The isolated SARS-CoV-2 virus, or derivative or relative thereof of claim 49, wherein the isolated SARS-CoV-2 virus, or derivative or relative thereof comprises ORF1a having a nucleotide sequence of nucleotides 238-13455 of SEQ ID NO: 1; ORF1ab having a nucleotide sequence of nucleotides 13740-21527 of SEQ ID NO: 1; a spike protein S having an amino acid sequence encoded by nucleotides 21508-25356 of SEQ ID NO: 1; ORF3a having a nucleotide sequence of nucleotides 25365-26192 of SEQ ID NO: 1; a membrane protein M having an amino acid sequence encoded by nucleotides 26495-27163 of SEQ ID NO: 1; and a nucleocapsid protein N having an amino acid sequence encoded by nucleotides 28246-29505 of SEQ ID NO: 1.
The isolated SARS-CoV-2 virus, or derivative or relative thereof of claim 49, wherein the isolated SARS-CoV-2 virus, or derivative or relative thereof has a genome, comprising an RNA sequence having its reverse complement sequence shown by any one of SEQ ID NOs: 1-3, or a sequence which is at least 95%identical therewith; or has a genome encoded by the sequence shown by any one of SEQ ID NOs: 1-3, or a sequence which is at least 95%identical therewith.
An isolated nucleic acid, comprising a nucleotide sequence of nucleotides 21508-25356 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
An isolated nucleic acid, comprising a nucleotide sequence of nucleotides 28246-29505 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
An isolated nucleic acid, comprising a nucleotide sequence of nucleotides 26495-27163 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
An isolated nucleic acid, comprising a nucleotide sequence of nucleotides 238-13455 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
An isolated nucleic acid, comprising a nucleotide sequence of nucleotides 13740-21527 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
An isolated nucleic acid, comprising a nucleotide sequence of nucleotides 25365-26192 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
An isolated nucleic acid, comprising a nucleotide sequence of nucleotides 238-13455 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or the nucleotide sequence of nucleotides 13740-21527 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or the nucleotide sequence of nucleotides 21508-25356 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or the nucleotide sequence of nucleotides 25365-26192 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or the nucleotide sequence of nucleotides 26495-27163 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith; and/or the nucleotide sequence of nucleotides 28246-29505 of SEQ ID NO: 1 or a sequence which is at least 95%identical therewith.
An isolated nucleic acid, comprising a nucleotide sequence of nucleotides 238-13455 of SEQ ID NO: 1; a nucleotide sequence of nucleotides 13740-21527 of SEQ ID NO: 1; a nucleotide sequence of nucleotides 21508-25356 of SEQ ID NO: 1; a nucleotide sequence of nucleotides 25365-26192 of SEQ ID NO: 1; a nucleotide sequence of nucleotides 26495-27163 of SEQ ID NO: 1; and a nucleotide sequence of nucleotides 28246-29505 of SEQ ID NO: 1.
An isolated nucleic acid, comprising a nucleotide sequence of any one of SEQ ID NOs: 1-3, or a sequence which is at least 95%identical therewith, or its reverse complement.