WO2024017103A1 - Vaccin de rappel contre la covid-19 basé sur le virus sars1 - Google Patents

Vaccin de rappel contre la covid-19 basé sur le virus sars1 Download PDF

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WO2024017103A1
WO2024017103A1 PCT/CN2023/106896 CN2023106896W WO2024017103A1 WO 2024017103 A1 WO2024017103 A1 WO 2024017103A1 CN 2023106896 W CN2023106896 W CN 2023106896W WO 2024017103 A1 WO2024017103 A1 WO 2024017103A1
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vaccine
virus
sars1
booster
seq
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黃建東
朱軒
张寳中
胡叶凡
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貝灣生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a new coronavirus booster immunity vaccine based on SARS1 virus.
  • the protection provided by the vaccine will decline over time, and the protection will be significantly weakened after a few months; on the other hand, repeated vaccination with vaccines based on the ancestor strain will also be difficult to effectively protect against mutant strains.
  • VOC variant strains of concern
  • BA.2, BA.4, BA.5 and BA.2.75 Omicron descendant lineages
  • the existing vaccines based on the founder strain are difficult to provide effective protection, and there is an urgent need to develop better booster vaccines to prevent infection, that is, to continue to vaccinate with other vaccines after standard vaccination.
  • pan-coronavirus vaccine candidates multivalent vaccines, inducing mucosal immune responses, inducing stronger T cell responses, and heterologous boosting strategies.
  • the classic strategy is to develop booster vaccines based on recently emerged variants.
  • vaccine strains are selected each year based on circulating influenza viruses.
  • several vaccines have been developed against the Beta and Omicron variants.
  • Most of these vaccines showed significant increases in neutralizing titers against multiple variants.
  • initial exposure to the imprint of the ancestor coronavirus resulted in the production of more neutralizing antibodies against the ancestor strain, rather than more neutralizing antibodies against the new variant.
  • the increased neutralizing antibody titers are still mainly directed against the ancestor new coronavirus strain, and less neutralizing antibodies against the new mutant strain.
  • the evolution of COVID-19 shows a new pattern that requires a vaccine design strategy to design booster vaccines with broad-spectrum protection against emerging COVID-19 variants.
  • Influenza virus-like evolution is more traditional, producing one or two continuously evolving major lineages through antigenic drift, and new mutations often evolve from previous major lineages.
  • the evolution of seasonal coronaviruses also mainly follows this pattern, such as HCoV-229E and HCoV-OC43.
  • the evolutionary pattern of the new coronavirus which has intermittent accelerated mutations, is different from this.
  • the mutational evolution of the new coronavirus proceeds through multiple mutation jumps, which may lead to significant immune escape, that is, the immunity established by the ancestor new coronavirus strain cannot prevent new strains.
  • This phenomenon has occurred many times with the new coronavirus.
  • Delta B.1.617.2 and AY.*
  • Omicron B.1.1.529 and BA.*
  • BA.* Omicron
  • B.1.1.529 and BA.* is not a sublineage of Delta (B.1.617.2 and AY.*). Therefore, rational selection of optimized booster vaccine strains is important to provide adequate protection against emerging VOCs.
  • the "antigenic distance hypothesis” can explain the immune imprinting phenomenon caused by repeated vaccination with different influenza vaccine strains. Previous exposure to the first strain of the virus (or a vaccine based on the first strain) may weaken the immune response to the second vaccine strain, a phenomenon known as immunoblotting. The phenomenon of immune imprinting may be caused by the following mechanism: the first vaccine induces a pre-existing cross-reaction, which partially eliminates the antigens in the second vaccine that are similar but different from the first strain. To overcome immunoblotting, one strategy is to select a vaccine seed strain that is not too close to the previous vaccine seed strain. The proximity of different vaccine strains can be defined by antigenic distance. Previous papers and preprints mapped the antigenicity of major circulating new coronavirus variants, promoting the application of this strategy in the development of new coronavirus booster vaccines.
  • the first purpose of the present invention is to provide a new coronavirus booster immunity vaccine based on SARS1 virus.
  • the additional booster shot of SARS1 virus is superior to multiple new coronavirus strains (including the ancestor strain, Delta and Omicron). It is more effective in terms of neutralizing potency and neutralizing spectrum.
  • the novel coronavirus booster immunity vaccine based on the SARS1 virus of the present invention is a vaccine of the SARS1 virus, for example, the SARS1 virus or its subunit vaccine, virus-like particles, and nucleic acid vaccine based on the SARS1 virus are used as severe acute respiratory syndrome coronavirus 2.
  • Booster vaccine against SARS-CoV-2 is a vaccine of the SARS1 virus, for example, the SARS1 virus or its subunit vaccine, virus-like particles, and nucleic acid vaccine based on the SARS1 virus.
  • the second object of the present invention is to provide the application of SARS1 virus vaccine in preparing a booster vaccine for severe acute respiratory syndrome coronavirus 2 SARS-CoV-2.
  • the SARS1 virus vaccine is a SARS1 virus or a subunit vaccine based on SARS1 virus, a virus-like particle vaccine, a nucleic acid vaccine, an inactivated vaccine or an attenuated vaccine.
  • the booster vaccine contains the nucleotide sequence of SEQ ID No. 1 or a nucleotide sequence that is at least 90% similar to it, or the amino acid shown in SEQ ID No. 2, or codes for SEQ ID No. 1
  • the booster vaccine is a recombinant protein vaccine or mucosal vaccine containing an amino acid sequence as described in SEQ ID No. 2.
  • the mucosal vaccine includes a mucosal auxiliary domain of bacterial toxins connected to the SARS1 virus spike receptor binding domain.
  • connection is through a connection sequence, and the connection sequence is: GS linker: GSGS or cleavable linker: LLSVGG.
  • the mucosal auxiliary domain of the bacterial toxin can be Corynebacterium diphtheria toxin CRM197, Clostridium difficile toxin A/B receptor binding domain, Staphylococcus aureus fibronectin binding protein, Vibrio cholerae toxin B subunit, Escherichia coli heat-labile enterotoxin B subunit.
  • amino acid sequence of SEQ ID No.3 is connected to the mucosal auxiliary domain shown in SEQ ID No.4
  • the booster vaccine is the nucleotide sequence of SEQ ID No. 1 or a predicted epitope sequence encoded by a nucleotide sequence having at least 90% similarity with it or encoding an amino acid sequence such as SEQ ID No. 2. of peptide vaccines.
  • the booster vaccine contains the nucleotide sequence of SEQ ID No. 1 or a nucleotide sequence that is at least 90% similar to it, or encodes a nucleotide sequence with an amino acid sequence as shown in SEQ ID No. 2 Sequenced mRNA vaccines.
  • the booster vaccine contains the nucleotide sequence of SEQ ID No. 1 or a nucleotide sequence that is at least 90% similar to it, or encodes a nucleotide sequence with an amino acid sequence as shown in SEQ ID No. 2 Sequenced DNA vaccines.
  • the virus-like particle vaccine is a nucleic acid sequence containing the nucleotide sequence of SEQ ID No. 1 or a nucleotide sequence that is at least 90% similar to it, or a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID No. 2. nucleotide sequence.
  • the booster vaccine contains the nucleotide sequence of SEQ ID No. 1 or a nucleotide sequence that is at least 90% similar to it, or encodes a nucleotide sequence with an amino acid sequence as shown in SEQ ID No. 2 Sequence vector vaccine.
  • rVSV vesicular stomatitis virus
  • SARS1 virus Middle East respiratory syndrome coronavirus
  • MERS virus Middle East respiratory syndrome-related coronavirus
  • a booster vaccine candidate for severe acute respiratory syndrome coronavirus 2 SARS-CoV-2.
  • Figure 1 shows the use of antigenic distance theory to select more suitable new coronavirus vaccine seed strains.
  • a the evolutionary relationship of coronaviruses in terms of spike amino acid sequences.
  • the small picture shows the relationship between different descendant strains of the new coronavirus, including the ancestor strain, the Delta strain and the Omicron strain.
  • b Antigen map and protection scope of two doses of BNT162b2 vaccine.
  • c-e The scope of protection after the third dose of the vaccine using the ancestor strain, Delta strain and Omicron strain of the new coronavirus.
  • f Range of protection after the third dose of vaccine using SARS1 virus and MERS virus.
  • 1 AU that is, 1 relative unit, represents 1 unit of antigen distance and represents a 2-fold change in neutralizing titer.
  • Figure 2 shows the relationship between antigenic distance and protection against new coronavirus infection.
  • the average neutralization level reported in phase 1 or 2 studies of different vaccines (Table 1) and the protective potency or effectiveness reported in phase 3 trials or real-world studies (Table 2).
  • the antigenic distance refers to the average neutralizing titer of the vaccinated divided by the average titer of the corresponding convalescent patients, and the unit is log 2.
  • the solid line represents the logistic model, and the shading represents the 95% confidence interval for that model.
  • b third dose Protection after vaccination.
  • c After two doses of vaccine, neutralization titers decreased and protection decreased due to immune escape of Delta and Omicron.
  • d After three doses of vaccination, neutralizing titers decreased and protection decreased due to immune escape of Delta and Omicron.
  • the red line represents the logistic model and the red shading represents the 95% confidence interval of the model.
  • FIG. 3 shows the antibody response after various vaccination strategies.
  • a experimental design.
  • b according to the standard two-dose BNT162b2 vaccination schedule, the changes in receptor-binding domain (RBD) and spike protein (Spike)-specific immunoglobulin G (IgG) responses were measured.
  • enzyme-linked immunosorbent (ELISA) was used to measure the optical density (OD) value of the serum dilution at 450 nm, and the area under the curve (AUC) value was calculated as the IgG level.
  • ELISA enzyme-linked immunosorbent
  • OD optical density
  • AUC area under the curve
  • d. Dynamics of pseudovirus neutralization titers against the ancestor strain, Delta strain and Omicron strain of the new coronavirus according to the standard two-dose BNT162b2 vaccination schedule.
  • e. Compared with the standard two-dose BNT162b2 vaccination schedule, after the additional administration of viral vector booster vaccines based on different new coronavirus variants, SARS1 virus and MERS virus, the effectiveness of the new coronavirus ancestor, Delta and Omi Pseudovirus neutralizing potency of Kron strain with two doses of BNT162b2 vaccine.
  • Figure 4 shows correlation analysis and neutralizing activity of sera from mice vaccinated with two doses of BNT162b2 or additional boosters.
  • sVNT Surrogate virus neutralization test
  • sVNT analysis after boosting the third dose of rVSV vector vaccine based on different new coronavirus variants, SARS1 virus and MERS virus compared with the standard two-dose BNT162b2 vaccination. All sera were tested at dilutions of 1:2500 against Progenitor COVID-19, 1:500 against Delta, and 1:100 against Omicron.
  • Figure 5 shows the protective immune response after challenge with Omicron BA.2.
  • a the design of the Omicron BA.2 challenge experiment.
  • b Comparison of qRT-PCR results between the RdRp gene of the new coronavirus and GAPDH in the mouse nasal cavity.
  • c qRT-PCR results of mouse lungs. ns, no significant difference; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001; ****, p ⁇ 0.0001.
  • d Representative images of immunofluorescence (IF) staining illustrating SARS-CoV-2 infection in nasal bones. Image at 10x magnification with black scale bar (100 ⁇ m). The cell nucleus is marked in blue, and the nucleocapsid protein (NP) of the new coronavirus is marked in green.
  • e Representative images of hematoxylin and eosin (H&E) staining illustrating the extent of lung inflammation. Image at 10x magnification, scale bar is 100 ⁇ m.
  • Figure 6 shows the long-term immune response after various vaccination strategies at week 18.
  • a experimental design for long-term immune response detection at week 18.
  • b according to the standard two-dose BNT162b2 vaccine Kinetics of progenitor RBD and Spike-specific IgG expressed as area under the curve (AUC) using ELISA for this plan, additional administration of viral vector booster vaccines based on different new coronavirus variants, SARS1 virus and MERS virus.
  • AUC area under the curve
  • IFN- ⁇ ELISpot results based on spots per 10 6 mouse splenocytes after boosting vaccines with additional rVSV vectors based on different new coronavirus variants, SARS1 virus and MERS virus compared with standard two-dose BNT162b2 vaccination Form unit calculations.
  • d Pseudovirus neutralization titers against progenitor coronavirus and Omicron (BA.2.12.1 and BA.4/BA.5) at week 18.
  • e True virus neutralizing titers against progenitor COVID-19, Delta and Omicron (BA.1) at week 18.
  • Student's t test is used. ns, no significant difference; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001; ****, p ⁇ 0.0001.
  • Figure 7 shows the long-term immune response after various vaccination strategies at week 27.
  • a experimental design of long-term immune response detection at week 27.
  • b According to the standard two-dose BNT162b2 vaccination schedule, an additional viral vector booster vaccine based on the ancestor new coronavirus and SARS1 virus was administered.
  • c Neutralizing titers of pseudoviruses against progenitor SARS-CoV-2 and Omicron (BA.2.12.1, BA.4/BA.5 and XBB) at week 27. Student’s t test was used.
  • d Log-linear fit of RBD-specific IgG following a standard two-dose BNT162b2 vaccination schedule (left panel).
  • the half-lives of RBD-specific IgG for each group are listed.
  • the graph on the right details the RBD-specific IgG decay rate.
  • Student’s t test is used. ns, no significant difference; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001; ****, p ⁇ 0.0001.
  • Figure 8 is a mucosal vaccine containing SARS1 virus spike receptor binding domain and mucosal auxiliary domain.
  • the mucosal auxiliary domain can be connected to the N-terminal or C-terminal of the SARS1 virus spike RBD.
  • b List of mucosal accessory domains according to bacterial toxins.
  • c Predicted structure of mucosal vaccine bound to mucosal cell fibronectin via fragment A of Staphylococcus aureus fibronectin-binding protein.
  • the spike amino acid sequences of 58 coronaviruses other than the new coronavirus were obtained from the UniProt database, and the 1524 spike protein amino acid sequences of different mutant strains of the new coronavirus were obtained from the GISAID database on January 31, 2022. All sequences were analyzed using the Nextstrain tool to construct an unrooted evolutionary tree based on amino acid sequences (Fig. 1a). Here, we used MAFFT to align sequences and then used IQ-TREE to construct a maximum likelihood phylogenetic tree based on the BLOSUM62 substitution model.
  • mutant strains with no data we used the RBD amino acid sequence for prediction.
  • H the mean neutralizing titer in vaccinated persons divided by the corresponding mean titer in convalescent patients, that is, the antigenic distance to convalescent patients, expressed as log 2.
  • H50 is the distance at which an antigenic individual will have 50% protective potency/effectiveness.
  • the MERS virus it is difficult to estimate the antigenic distance from the SARS1 virus or the new coronavirus, because compared with the SARS1 virus, the RBD of the MERS virus spike protein has 150 amino acid changes and 151 changes compared with the new coronavirus. This suggests that there is no cross-reactivity between MERS viruses and SARS-related viruses. In this way, we can assume that there is no cross-reactive protection range (Fig. 1g).
  • rVSV vesicular stomatitis virus
  • Recombinant vesicular stomatitis virus (rVSV) vector vaccine (rVSV vector vaccine for short) is a pseudovirion constructed in HEK293T cells. These pseudovirions can also be used in neutralization tests.
  • the recombinant vesicular stomatitis virus (rVSV) vector vaccine was constructed based on the ancestor new coronavirus spike protein (Spike) plasmid (reference sequence: EPI_ISL_402124) purchased from eEnzyme (SCV2-PsV-001), and the new coronavirus mutant strain Delta (B.1.617) .2)
  • the spike protein (Spike) plasmid (CB-97100-161) and Omicron (B.1.1.529) spike protein (Spike) plasmid (CB-97100-167) were purchased from Codex BioSolutions, SARS1 virus (The nucleotide sequence encoding the spike protein of SARS1 virus is shown in SEQ ID No.
  • spike protein (Spike) encoding plasmid of MERS virus is based on the microbial system. Construction of spike protein (Spike) sequencing results of SARS1 virus clinical isolate strain GZ50 and MERS virus clinical isolate strain EMC/2012. We unified the spike protein (Spike) plasmid sequence into the pCMV vector to construct a series of vectors, including the ancestor new coronavirus vector pCMV-SARS2, delta vector pCMV-Delta, Omicron vector pCMV-Omicron, and SARS1 virus Vector pCMV-SARS1 and MERS virus vector pCMV-MERS.
  • the above plasmids were transfected into 10 cm culture dishes using polyetherimide (PEI) to prepare HEK293T cells, which were cultured in Dulbecco's modified Eagle medium (DMEM; Life Technologies). The cell density reached approximately 80% on the day of transfection. 48 hours after transfection, G* ⁇ G-luciferase recombinant vesicular stomatitis virus was added. 24 hours later, the supernatant was collected and centrifuged at 2000 rpm for 10 minutes. Cell debris was then removed through a 0.45 ⁇ m filter, concentrated to 10 8 PFU/mL, and Store at -80 °C for further use.
  • PEI polyetherimide
  • DMEM Dulbecco's modified Eagle medium
  • VNT Pseudovirus neutralization test based on recombinant vesicular stomatitis virus (rVSV) vector vaccine
  • Pseudovirus neutralization assay was performed in HEK293T-hACE2 cells.
  • the construction of pseudovirions is similar to the construction of rVSV vector vaccine mentioned above.
  • the spike protein (Spike) plasmid of the new coronavirus mutant strain Omicron BA.2.12.1 and BA.4/BA.5 was purchased from Codex BioSolutions. For the rest of the construction process, see rVSV vector vaccine construction.
  • pseudovirions of the rVSV vector were obtained, including the original strain of the new coronavirus, Delta (B.1.617.2) and Omicron (B.1.1.529, BA.2.12.1 and BA.4/BA.5 ) and pseudovirions of SARS1 virus and MERS virus.
  • the final volume of DMEM culture medium is about 50 ⁇ L, and culture until the cells adhere to the wall.
  • the serum is serially diluted in 10 8 PFU/mL pseudovirus solution. Generally, for mouse serum, it is diluted 4 times starting from 1:40 to 1:10240. Finally, it is quantified to 50 ⁇ L, incubated at 37°C for 60 minutes, and then added to Remove the original culture medium from the HEK293T-hACE2 cells in the 96-well plate. At the same time, set up a control well and add 50 ⁇ L of pseudovirus solution. After 42 hours of incubation at 37°C, firefly luciferase activity was measured using the Steady-Glo Luciferase Assay System (Promega, E2520), which indicated pseudovirus infection. Fluorescence signals were acquired by a Varioskan LUX multi-mode microplate reader (Thermo Scientific) and exported by Thermo Scientific SkanIt software.
  • the signal intensity of the control well is 100%. If the serum contains neutralizing antibodies, the signal intensity of the serum and pseudovirus mixture well is less than 100%. This is also the percentage of infection rate. , so that it can be diluted with serum Degree is the abscissa, infection rate is the ordinate, and a curve of serum dilution relative to infection rate is obtained. The serum dilution when the infection rate is 50% is selected as the measured neutralizing antibody titer.
  • RBD detection and spike-specific IgG detection were performed using enzyme-linked immunosorbent assay (ELISA).
  • RBD or spike protein was coated on an ELISA plate (BIOFIL, #FEP100096) at a final concentration of 0.5 ⁇ g/mL in 50 mM coating buffer (pH 9.6 Na 2 CO 3 /NaHCO 3 ) and incubated. Incubate overnight at 4°C. Plates were blocked with 5% skim milk (Bio-Rad, cat. no. 1706404) in TBST (Thermo, cat. no. 28360) for 3 hours at room temperature and then washed 3 times in TBST. Human serum was serially diluted in TBST containing 3% skim milk and added to the wells and incubated for 1 hour.
  • HRP horseradish peroxidase
  • TMB Trimethylborane developer solution
  • Absorbance was measured at 450 nm using an ELISA reader (Varioskan Flash 4.0, Thermo) and analyzed by SkanIt Software 2.4.5 RE for Varioskan Flash (Thermo). All data were analyzed and plotted in R version 3.6.6.
  • mice were anesthetized with ketamine and xylazine, and then intranasally inoculated with 20 ⁇ L of virus liquid, 1 ⁇ 10 5 PFU of novel coronavirus Omikoron (BA.2) per mouse 49 , during viral challenge After 48 and 96 hours, mice were euthanized and organs harvested for viral replication detection and histological analysis.
  • CULATR 5440-20 and 5370-20 Animal Ethics Committee
  • Viral RNA was extracted from mouse lungs and nasal cavities using the RNeasy Mini kit (Qiagen, cat. no. 74106). Quantification of SARS-CoV-2 gene copies targeting RNA-dependent RNA polymerase (RdRp) and the internal gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the QuantiNova Probe RT-PCR Kit (Qiagen, Cat. No. 208354) . Primer and probe sequences are available upon request.
  • RdRp RNA-dependent RNA polymerase
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • T cell responses were detected by mouse IFN- ⁇ enzyme-linked immunosorbent spot (ELISpot) kit (Mabtech, catalog number 3321-4HST-2) according to the manufacturer's protocol. After harvesting spleen cells from the cysts of mice, the cells were cultured overnight at 37°C on multiscreen filter plates pre-coated with anti-IFN- ⁇ antibody. By interacting with the Spike peptide library (GLBiochem Ltd) containing 130 15-mer peptides covering the original SARS-CoV-2 spike protein with 5 residues overlapping at 0.05 g/mL of each peptide.
  • ELISpot enzyme-linked immunosorbent spot
  • SARS1 virus strain GZ50 is an archived clinical isolate in the Department of Microbiology. All experiments involving live COVID-19 and SARS1 viruses followed standard operating procedures approved by the Department of Microbiology’s biosafety level 3 facility. Isolates were cultured in FRhK-4 cells (ATCC) with or in serum-free minimal DMEM supplemented with 100 U/mL penicillin and 100 ⁇ g/mL streptomycin at 37°C with 5% carbon dioxide. 2-3 days after virus inoculation, the cultured virus liquid is inactivated with 1:1000-1:4000 formalin solution for 3-12 hours. The inactivation step was repeated three times. Then, centrifuge the inactivated virus liquid at 2000-4000g centrifugal force at 2-8°C for 10-35 minutes. The supernatant is separated to obtain virus ultrafiltrate to develop inactivated vaccine.
  • each dot represents a coronavirus or variant of the coronavirus.
  • a 50% protection circle indicates that immune protection against COVID-19 is 50% effective sex. Points outside this coverage indicate that this variant will be neutralized by a vaccination strategy that is less than 50% effective. Calculate the diameter of each coverage area using previous neutralization data ( Figure 2).
  • Figure 2 we analyzed the extent of protection provided by the progenitor SARS-CoV-2 vaccine in the form of two doses of BNT162b2 vaccine (Fig. 1b). Our results indicate that two doses of BNT162b2 vaccine can barely cover Omicron BA.1 (B.1.1.529.1) in terms of 50% protection coverage. This was set as the baseline for protective efficacy, which is consistent with recent studies.
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • rVSV vesicular stomatitis virus
  • C57BL/6J mice were injected intramuscularly with a clinically relevant primary regimen of two doses of BNT162b2 vaccine (5 ⁇ g at weeks 0 and 3). At week 9, they were boosted with intramuscular injection of rVSV vector vaccine (1 ⁇ 10 6 PFU). Neutralizing and antigen-specific antibody responses were assessed at weeks 3, 6, 9 and 10 (Fig. 3a).
  • Vaccination-induced immune responses in the nasal cavity and lungs may play a major role in the first step of suppressing COVID-19 replication. Therefore, we extended our analysis to peripheral antibodies beyond the protective activity in C57BL/6J mice, as they are susceptible to infection by novel coronavirus variants, including Omicron (B.1.1.529) and its subtypes Line (BA.*) carries the spike protein N501Y mutation. Mice received two doses of BNT162b2 vaccine at weeks 0 and 3, were boosted with the vaccine at week 9, and received BNT162b2 at week 10. Mixron BA.2 attack (Fig. 5a).
  • mice were euthanized, and nasal bones and lungs were collected for viral load analysis (Fig. 5a).
  • Vaccination cannot effectively control the replication of the new coronavirus in the nasal cavity of vaccinated animals.
  • Viral RNA was readily detected in the nasal bones of C57BL/6J mice vaccinated with BNT162b2 twice after infection with Omicron BA.2 (Fig. 5b).
  • the third dose of vaccination based on any variant/coronavirus further reduced viral replication.
  • the SARS1 virus-based booster vaccine showed the most effective inhibition of SARS-CoV-2 replication in the nasal cavity of C57BL/6J mice after 48 and 96 hours of infection (Figure 5b).
  • Vaccination effectively limits the replication of COVID-19 in the lungs of vaccinated animals compared to the nasal cavity.
  • Omicron sublineages, including BA.2 replicate poorly in the lungs of infected animals.
  • two doses of BNT162b2 vaccination have effectively limited the replication of Omicron BA.2 in the lungs, while additional booster vaccinations based on any variant/coronavirus did not further reduce the virus Replication (Fig. 5c).
  • mice in each group were euthanized 48 h after Omicron (BA.2) or viral challenge (Fig. 5d,e).
  • the other three groups were euthanized after 96 hours.
  • the new coronavirus antigen can only be detected in the nasal cavity of C57BL/6J mice vaccinated with BNT162b2 vaccine twice ( Figure 5d).
  • Figure 5d In terms of lung inflammation, there were no differences between all groups (Fig. 5e), which is consistent with our qRT-PCR results.
  • the persistence of vaccination-induced protective immunity as immunity wanes is another factor in developing the next generation of COVID-19 vaccinations. a key factor.
  • the SARS1 virus booster group showed the highest levels of RBD-specific IgG and neutralizing antibodies among all groups.
  • mice vaccinated with two doses of BNT162b2 and an additional booster vaccine based on SARS1 virus or SARS-CoV-2 were sampled four months after booster vaccination (week 27, Figure 7a).
  • SARS1 virus booster vaccination group showed longer-lasting and higher levels of antibodies, especially against the ancestor new coronavirus and the epidemic Omicron sublineage (BA.2.12.1, BA.4/BA.5 and XBB) neutralizing antibody titers (Fig. 7b, c), and importantly, its decline rate was significantly slower than that of other groups (Fig. 7d).
  • viral antigens should enter nasopharyngeal and oral mucosal cells and then stimulate local immunity (Fig. 8a).
  • bacterial toxins can be used, including Corynebacterium diphtheria toxin CRM197, Clostridium difficile toxin A/B receptor binding domain, Vibrio cholerae toxin B subunit (CTB), and E. coli heat-labile enterotoxin subunit B base (LTB) (Fig. 8b).
  • FnBP Staphylococcus aureus fibronectin-binding protein

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Abstract

L'invention concerne un vaccin de rappel contre la COVID-19 basé sur le virus SARS1. Le vaccin de rappel contre la COVID-19 est un vaccin contre le virus SARS1, et est un vaccin de rappel contre le coronavirus du syndrome respiratoire aigu sévère 2 (SARS-CoV-2). Les résultats d'expérience sur animaux montrent qu'une injection de rappel supplémentaire contre le virus SARS1 est supérieure à des vaccins contre une variété de souches de SARS-CoV-2 (y compris la souche virale, la souche Delta et la souche Omicron), et a un meilleur effet en termes de titre de neutralisation et de spectre de neutralisation. Par conséquent, le vaccin contre le virus SARS1 peut être utilisé en tant que vaccin de rappel contre le SARS-CoV-2.
PCT/CN2023/106896 2023-06-29 2023-07-12 Vaccin de rappel contre la covid-19 basé sur le virus sars1 WO2024017103A1 (fr)

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