WO2021254327A1 - Vaccin à vecteur viral de type à remplacement d'enveloppe et procédé de construction associé - Google Patents

Vaccin à vecteur viral de type à remplacement d'enveloppe et procédé de construction associé Download PDF

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WO2021254327A1
WO2021254327A1 PCT/CN2021/100107 CN2021100107W WO2021254327A1 WO 2021254327 A1 WO2021254327 A1 WO 2021254327A1 CN 2021100107 W CN2021100107 W CN 2021100107W WO 2021254327 A1 WO2021254327 A1 WO 2021254327A1
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gene
envelope
ecd
coronavirus
virus
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秦晓峰
韦治明
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睿丰康生物医药科技(浙江)有限公司
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    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • 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 present invention relates to a virus vector vaccine constructed by using vesicular stomatitis virus (VSV) to effectively prevent coronavirus infection, especially including SARS-CoV-2 coronavirus envelope for replacement, and a construction method thereof.
  • VSV vesicular stomatitis virus
  • Coronavirus is a member of the order Nidovirals, family Coronavirus, and genus Coronavirus in the virological classification.
  • the genome is a single-stranded, positive-stranded RNA with a complete genome. It is between 26-32 kb in length and is currently the largest RNA virus known in its genome. Coronavirus infections are widespread in nature, and common mammals such as dogs, cats, mice, pigs, cattle and poultry are all susceptible.
  • the International Commission for Classification of Virology (ICTV, 2012) divided the members of the genus Coronavirus into four groups in its ninth report: ⁇ group, ⁇ group, ⁇ group and ⁇ group.
  • Human coronaviruses are mainly distributed in the ⁇ group and ⁇ group.
  • HCoV-229E and HCoV-NL63 are in the ⁇ group
  • HCoV-OC43 and HCoV-HKU1 are in the 2a subgroup in the ⁇ group
  • MERS-CoV belongs to the 2c subgroup in the ⁇ group
  • the latest SARS-CoV- 2 and SARS belong to the 2b subgroup in the ⁇ group.
  • the latest research shows that the ability of the RBD segment of the S-spike protein of the virus to bind to ACE2 is 10 times that of SARS, and the transmission capacity is the strongest among the seven major human-infecting coronaviruses currently known.
  • the structure of SARS-CoV-2 is similar to SARS coronavirus.
  • the spike protein S protein embodied on the surface of the virus is a specific tissue structure on the virus envelope. A large number of spike proteins are formed on the surface of the virus, which can be used as the target of virus invasion. Cells and viruses and cells play an important role.
  • the global outbreak of the new crown epidemic has made it urgent to develop drugs for new crown pneumonia.
  • the JAMA research report in the Journal of the American Medical Association shows that some recovered patients are still carriers of the new crown virus, which has also triggered whether the new coronavirus has become a global epidemic.
  • the discussion of the new coronavirus may be like influenza virus, which has been prevalent in human society for a long time, and the envelope replacement virus vector has been proved to be the most cost-effective, effective and long-lasting disease prevention and control measure. Therefore, the whole people are vaccinated with the new coronavirus package.
  • Membrane replacement virus vectors are imperative, and the development and release of new coronary pneumonia envelope replacement virus vectors in a short period of time is a powerful means to prevent the spread of the epidemic.
  • pathogens that currently cause severe infectious diseases such as human immunodeficiency virus (HIV), influenza virus, severe acute respiratory syndrome virus (SARS-CoV), etc., all invade through mucosal surfaces (genital tract, respiratory tract, gastrointestinal tract) And the infected body, because the body cannot induce an effective mucosal immune response to clear the mucosal infectious pathogens, the pathogens quickly spread into the blood, and then invade the whole body, causing damage to the body, especially the lung tissue.
  • HCV human immunodeficiency virus
  • influenza virus influenza virus
  • SARS-CoV severe acute respiratory syndrome virus
  • envelope replacement virus vectors such as inactivated, protein envelope replacement virus vectors, DNA envelope replacement virus vectors, subunit envelope replacement virus vectors, etc.
  • conventional routes intramuscular injection, subcutaneous, etc.
  • mucosal immune response it is usually necessary to inoculate the target antigen from the mucosal site, so that it can be effectively taken up and presented by APC in the mucosal tissue, further activate the mucosal immune system, and induce effective and durable Mucosal immune response.
  • VSV envelope-replacement virus vector-vesicular stomatitis virus
  • VSV vesicular stomatitis virus
  • VSV as a viral vector uses gene editing technology
  • the strength of the body's immune response will be significantly enhanced, and a stronger acquired antiviral response (T cell immune response and B cell immune response) will be induced.
  • the present invention utilizes the natural advantages of the VSV virus vector to propose an envelope replacement virus vector vaccine and its construction method.
  • This kind of envelope replacement virus vector vaccine is effective against humans suffering from coronaviruses, especially SARS or new crown pneumonia virus (SARS-CoV- 2) It has a better preventive or therapeutic effect.
  • an envelope-replacement type virus vector vaccine which replaces the GP gene in the rhabdovirus genome with the spike protein S gene truncation of the coronavirus or the virus spike protein S gene Extracellular segment fusion ECD-CA, and the use of reverse genetic system to construct an envelope-replacement type viral vector vaccine for the prevention of coronavirus, the ECD-CA is defined as the extracellular segment ECD of the viral spike protein S gene The gene after fusion of the transmembrane and intracellular segment genes of the VSV virus envelope protein.
  • the envelope replacement virus vector vaccine is selected from vesicular stomatitis virus VSV
  • the VSV is selected from the Indiana strain
  • the spike protein S gene is selected from SARS or SARS- For CoV-2 coronavirus
  • the spike protein S gene truncation is selected from genes corresponding to C-terminal amino acid deletions, and the number of C-terminal amino acid deletions is 18 to 72.
  • the spike protein S gene truncation is selected from C-terminal deletion genes, and the number of C-terminal amino acid deletions is preferably 30, and corresponds to the modified spike protein
  • the amino acid sequence corresponding to S is SEQ ID NO.1.
  • the envelope replacement virus vector vaccine is selected from the vesicular stomatitis virus VSV
  • the VSV is selected from the Indiana strain
  • the ECD-CA is selected from coronaviruses
  • the ECD-CA includes the extracellular segment ECD of the coronavirus spike protein S gene
  • the C-terminus of the ECD is fused with the transmembrane and intracellular segment gene CA of the envelope protein of VSV.
  • the ECD-CA is cloned into the coding sequence between the M gene and the L gene in the VSV genome, and the ECD-CA amino acid sequence is SEQ ID NO. 2,
  • the ECD-CA humanized codon gene sequence is SEQ ID NO.3.
  • the envelope replacement type viral vector vaccine, the vesicular stomatitis virus is a virus obtained by mutating the amino acid of its matrix protein M, and the amino acid mutation site in the matrix protein M is the bright 20th position.
  • One or more of amino acid, methionine at position 51, and phenylalanine at position 110 are non-synonymous mutations.
  • the amino acids corresponding to the mutation of leucine at position 20, methionine at position 51, and phenylalanine at position 110 may be the same or different from the types of the mutated amino acids in the following paragraph.
  • the envelope-replacement type viral vector vaccine is an attenuated vesicular stomatitis virus
  • the matrix protein M of the vesicular stomatitis virus has a 3 amino acid mutation
  • the matrix protein M The 20th position was changed from Leucine L to Phenylalanine F, the 51st position was changed from Methionine M to Alanine A, and the 110th position was changed from Phenylalanine F to Leucine L, so
  • the amino acid sequence of the 3-point mutant matrix protein M is SEQ ID NO.4.
  • a method for constructing an envelope-replacement type viral vector vaccine includes the following steps:
  • Plaque purification is performed with cells stably expressing 293-ACE2, and the coronavirus envelope replacement vaccine VSV- ⁇ G-S-CN or VSV- ⁇ G-ECD-CA is obtained after western blotting identification.
  • the method for constructing the coronavirus envelope replacement virus vector vaccine is the sequence SEQ ID NO. 5.
  • SEQ ID NO.6 is the sequence SEQ ID NO. 5.
  • the fluorescent probe primer pair that specifically amplifies the coronavirus ECD-CA gene in RT-PCR is SEQ ID NO. 7, SEQ ID NO. 8.
  • the present invention uses the VSV virus packaging system for the first time, through a large number of gene optimization and construction, in vitro screening of recombinant viruses that can efficiently package the coronavirus envelope replacement type, involving specific envelope gene modification The body (S-CN and ECD-CA), the coronavirus envelope is completely wrapped in the genetic material of VSV.
  • the traditional virus vector vaccine the virus antigen can be transcribed and translated after the host cell is infected
  • it can be highly immunogenic.
  • the antigen is presented to immune cells, shortening the response time of the host immune system, while retaining the most important antigen protein S of the coronavirus to the greatest extent.
  • coronavirus candidate vaccine involved in the invention has a certain degree of replication ability (a cell line that stably expresses hACE2), can be produced on a large scale and quickly, and at the same time as a non-inactivated vaccine, it simulates the entire process of coronavirus infecting host cells to the highest degree ( VSV structural protein is not significantly toxic to host cells), which can activate the body's immune system to produce a high-strength neutralizing antibody response against COVID-19.
  • the VSV envelope replacement virus vector vaccine further adopts mucosal vaccination, which will induce the body to produce a stronger specific mucosal immune response against the coronavirus, especially the new coronary pneumonia virus (SARS-CoV-2) antigen protein S.
  • SARS-CoV-2 new coronary pneumonia virus
  • the mucosal tissue will be activated and the pathogen will be quickly eliminated.
  • the VSV virus vector also has characteristics that other tool vectors do not have.
  • the VSV virus can display the complete spatial structure of the envelope protein of the target virus on the surface of the virus, and fully expose the trimeric protein S of the new coronary pneumonia virus (SARS-CoV-2) on the recombinant virus.
  • SARS-CoV-2 new coronary pneumonia virus
  • the envelope-replaced virus vector of this technology type is inactivated in vitro, it still has the specific immune response that can effectively activate the body and fully activate the host immune response. After the live vaccine is inoculated, there is no secondary replication ability, which further improves the safety of the vaccine.
  • Figure 1A is a schematic diagram of the construction of the S-CN and ECD-CA genes of different S truncations into the rhabdovirus backbone vector
  • B is the process verification diagram of molecular biological cloning
  • C is the Western Blotting detection package rescue
  • D is the fluorescence image of the recombinant envelope replacement vaccine packaged by S with different modifications
  • Figure 2 Screened out the two envelope replacement vaccine candidates with the highest packaging efficiency titers, and adopted different vaccination methods. After 21 days, the serum IgA content (A) and the specific IgG content (B) were detected respectively;
  • Figure 3 The sera after 21 days of immunization with multiple envelope replacement candidate new coronavirus vaccines were taken out, and the simulated new coronavirus pseudovirus developed based on lentivirus was used in vitro as a detection tool to compare the titers of neutralizing antibodies;
  • Figure 4 is a schematic diagram of the transformation of the envelope of the rhabdovirus vector into the envelope of the coronavirus.
  • the present disclosure mainly constructs different truncated (S-CN) or ECD-CA variants of the SARS-CoV-2 virus spike protein S gene into the VSV virus backbone.
  • S-CN truncated truncated virus spike protein S gene
  • ECD-CA envelope gene
  • the disclosed four-plasmid system is co-transfected and packaged in 293T cells stably expressed by ACE2 (human origin) to further rescue the recombinant vaccine in which the coronavirus spike protein is completely displayed on the surface of the VSV virus.
  • the vaccine is vaccinated through multiple channels and after immunization Successfully induced a specific antiviral humoral immune response in healthy mice.
  • the reagents and consumables used in this disclosure are as follows: Q5 Hot start High-Fidelity DNA polymerase (NEB M0493L), Mlu I-HF (NEB R3198L), Xho I (NEB R0146S), T4 DNA Ligase Enzyme (NEB M0202L), E.
  • the 293T-hACE2 adherent cells were cultured in a special culture environment (Thermo BB150 cell incubator) containing 5% CO2 at 37°C, and cultured in DMEM medium.
  • the modified VSV virus vector is preferably derived from the Indiana strain of vesicular stomatitis virus, and the 20th, 51st and 110th positions of the matrix protein (M) in the viral genome simultaneously have amino acid mutations,
  • the amino acid substitutions are as follows: the 20th position of the matrix protein M is changed from leucine L to phenylalanine F, the 51st position is changed from methionine M to alanine A, and the 110th position is changed from phenylalanine A.
  • Alanine F was mutated to Leucine L.
  • the humanized codon optimization of SARS gene S is more conducive to the expression of the target gene in eukaryotic cells (after the humanized codon is optimized, the expression efficiency is increased by 10 times, which is beneficial to Recombinant virus packaging). Therefore, in this example, the released SARS-CoV-2 S amino acid sequence is humanized codon optimized to increase its expression in mammalian cells.
  • the S gene code The sub-optimized sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and further synthesized into pCDNA3.1 eukaryotic expression vector. After PCR amplification of the target gene, the target band was recovered and purified by a fragment purification kit.
  • Fragment and pVSV-3M vector use restriction endonuclease MCS1 specifically Mlul, MCS2 specifically refers to Xhol, two restriction endonucleases double digestion at 37°C for 3 hours, the next step is to gel and recover the linear vector and the target fragment. Then T4 ligase was added, ligated overnight, and then transferred to competent cells.
  • the bacterial liquid PCR screened positive clones, digested with restriction enzymes and sequenced to verify the plasmid construction (and named the constructed plasmids as pCore-3M-S-C19, pCore-3M-S-C30 and pCore-3M-ECD-CA), the specific implementation steps are as follows:
  • Primer synthesis and primer information The primers were synthesized by Suzhou Jinweizhi Bio-Biotechnology Co., Ltd.
  • the PCR primers selected for the amplification of S-C19 and the bacterial liquid PCR primers are shown in Table 1:
  • PCR primers selected for the amplification of S-C30 are shown in Table 2:
  • PCR primers selected for the amplification of ECD-CA are shown in Table 3:
  • Obtaining the target gene Use the pCDNA3.1 plasmid carrying the target gene (New Crown S and VSV-GP) sequence as a template, and use the primers of Table 1, Table 2, and Table 3 to perform PCR amplification S-C19, S-C30, ECD -CA, where ECD-CA fused the CA fragment to the C-terminus of the ECD segment of the S gene by overlap extension PCR;
  • the positive clones were selected and transferred to a 30 mL shake flask at a ratio of 1:200, and cultured overnight at 37°C and 250 rpm in a shaker;
  • VSV-WT VSV- ⁇ GS-C19
  • VSV- ⁇ GS-C30 VSV- ⁇ G-ECD-CA
  • the cells are collected for WB to detect the antigen expression level.
  • envelope replacement plasmids of different truncations of the S gene By constructing envelope replacement plasmids of different truncations of the S gene, using the four-plasmid replicable packaging system as described in Example 1, after co-transfecting host packaging cells (293T-hACE2) for 48 hours, use 293T stably expressing hACE2 Compared with the control, the packaging efficiency of cells will be increased by 100 times. The supernatant after packaging after 0.22um filter membrane is further infected with 293T-hACE2 cells, the cytopathic changes and the expression of fluorescent reporter genes are observed, and the virus titer is determined.
  • the same processing method still fails to obtain effective envelope-replaced virus particles, so a conclusion can be drawn
  • the C-terminal part of the amino acid of the S gene seriously affects the expression and exocrine efficiency of the protein.
  • the S gene modified variant that is, the number of amino acids deleted at the C-terminal is 18-72, especially when the deletion of 30 amino acids can be Obtain an envelope-replaceable viral vector that meets the requirements, and the initial packaging titer reaches 5E6pfu/ml.
  • CA transmembrane and intracellular segment
  • Table 4 The efficiency of enveloped S gene variants in rescuing recombinant viral vector vaccines
  • S-C18 Can be packaged Yes
  • weaker Low S-C19 Can be packaged Yes
  • weaker Low S-C28 Can be packaged Yes
  • weaker Low S-C30 Can be packaged Yes
  • strong high S-C37 Can be packaged Yes
  • weaker Low S-C53 Can be packaged Yes
  • weaker Low S-C72 Can be packaged Yes
  • weaker Low S-C73 Unpackaged None weaker none S-C74 Unpackaged None
  • weaker none ECD-CA Can be packaged Yes
  • Example 3 The immune response effect of the envelope replacement vaccine based on VSV virus vector under different immunization schemes
  • test serum Dilute the test serum and the negative serum in an appropriate ratio (1:100) with antibody serum diluent (1% BSA), add 100ul per well, and incubate at 37 degrees Celsius for 2 hours;
  • antibody serum diluent 1% BSA
  • the two envelope-replacement viruses adopt different immunization methods and strategies.
  • the levels of specific IgA and IgG antibodies in the serum significantly increased to a higher level, while the expression levels of each antibody under different immunization pathways were certain.
  • the nasal drip method of immunization mainly activates the mucosal immune response and produces strong IgA specific antibodies.
  • the nasal drip immunization also induces a systemic antibody immune response (Figure 2A). The statistical results can be seen from the figure.
  • the intravenous and intramuscular immunization methods mainly cause the body to produce an antigen-specific IgG type immune response, and cannot produce an effective mucosal immune response (Figure 2B).
  • the envelope replacement type viral vector vaccine is used After the inactivation was treated in a specific way, the mouse vaccine was given by intramuscular injection, and the content of specific antibodies in the serum was detected (indirect Elisa).
  • the inactivated capsule replacement candidate vaccine also produced higher specific humoral immunity
  • the high temperature inactivation of the replicable vaccine vector with the envelope replacement did not destroy the induced humoral immune response of the vaccine, which proves that it is encapsulated in the VSV genetic material
  • the external spike protein S (C30) still retains sufficient immunogenicity to induce the body to produce an acquired antigen-specific humoral immune response.
  • the live virus vaccine is vaccinated through the nose, no matter it is These candidate vaccines all activate the local mucosal immune response, and high secretion and expression of IgA have been detected in the serum.
  • the mucosal response induced by mucosal vaccination can effectively bind to the virus surface in the early stage of new coronavirus infection.
  • the antigenic position of the virus prevents the virus from entering the host cell, which greatly protects the body from the infection of the coronavirus.
  • the antibody was serially diluted (1:2) 10 ⁇ L/tube with Opti-MEM.
  • a positive control without antibody (20 ⁇ L virus solution, final virus concentration 4E5TU/mL) and a negative control without virus (20 ⁇ L Opti- MEM);
  • Pseudovirus (a simulation system for the spike protein of the lentiviral backbone to package the new crown) is also serially diluted to 8E5TU/mL;
  • step 2 Take 10 ⁇ L of the diluted virus solution (8E5TU/mL) and add it to the 10 ⁇ L serially diluted antibody in step 2 (1:1 pipetting and mixing) (at this time, the final virus concentration is 4E5pfu/mL);
  • the serum neutralization titer is determined according to the dilution factor of the antibody serum corresponding to the hole with the last green fluorescence.
  • the administration method of different immunization routes is further adopted. After 21 days of immunization , The level of neutralizing antibodies produced by intravenous and intramuscular immunization is higher, which is consistent with the actual administration method of this vaccine product in clinical use.
  • the candidate new crown vaccine is amplified and prepared in a replicable form during production, and is produced by GMP After purification, it can be irradiated and inactivated and filled to form a preparation, speeding up the application and popularization of this type of candidate vaccine.

Abstract

L'invention concerne un vaccin à vecteur viral de type à remplacement d'enveloppe. Le virus de la stomatite vésiculaire VSV est utilisé en tant que vecteur, et le gène GP dans le génome du virus VSV est remplacé par un segment tronqué d'un gène de protéine de spicule S d'un coronavirus ou d'un ECD-CA fusionné d'un segment extracellulaire et d'un segment GP-C du gène de la protéine de spicule S du coronavirus. Le segment tronqué du gène de la protéine de spicule S est choisi parmi des gènes ayant certaines délétions d'acides aminés à l'extrémité C-terminale, et l'ECD-CA est un gène formé par fusion du segment extracellulaire du gène de protéine de spicule S du coronavirus avec les gènes de segment transmembranaire et intracellulaire de la protéine d'enveloppe virale de VSV. L'invention concerne également un procédé de construction du vaccin à vecteur viral de type à remplacement d'enveloppe.
PCT/CN2021/100107 2020-06-15 2021-06-15 Vaccin à vecteur viral de type à remplacement d'enveloppe et procédé de construction associé WO2021254327A1 (fr)

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