WO2021185310A1 - Vecteur de virus mvsv, vaccin à vecteur viral associé et nouveau vaccin contre la pneumonie à coronavirus basé sur la médiation par mvsv - Google Patents

Vecteur de virus mvsv, vaccin à vecteur viral associé et nouveau vaccin contre la pneumonie à coronavirus basé sur la médiation par mvsv Download PDF

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WO2021185310A1
WO2021185310A1 PCT/CN2021/081524 CN2021081524W WO2021185310A1 WO 2021185310 A1 WO2021185310 A1 WO 2021185310A1 CN 2021081524 W CN2021081524 W CN 2021081524W WO 2021185310 A1 WO2021185310 A1 WO 2021185310A1
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gene
mvsv
virus
vaccine
cov
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秦晓峰
韦治明
权海峰
龙丽梅
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睿丰康生物医药科技(浙江)有限公司
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    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
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Definitions

  • the present disclosure relates to the technical field of genetic engineering, specifically the mVSV virus vector and its virus vector vaccine, and a new coronary pneumonia vaccine mediated by mVSV.
  • 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 and 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. In recent years, multiple types of coronaviruses have been isolated from beluga whales, camels, and especially bats.
  • human coronavirus 229E HCV-229E
  • HCoV-OC43 human coronavirus 229E
  • SARS-CoV that appeared in 2003
  • HCoV-NL63 that was isolated in the Netherlands in 2004
  • HCoV-HKU1 identified in Hong Kong in 2005
  • MERS Middle East respiratory syndrome virus
  • the spike protein (S protein) of the virus first recognizes the receptor protein ACE2 (angiotensin-converting enzyme 2) on the cell membrane, and then mediates and promotes the fusion of the viral envelope and the cell membrane. , And finally make the virus invade the host cell.
  • ACE2 angiotensin-converting enzyme 2
  • Coronavirus can infect the respiratory tract, digestive tract, liver, kidney and nervous system of the body, causing various degrees of pathological damage, and even death in severe cases.
  • 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.
  • SARS-CoV-2 coronavirus Since the outbreak of the novel coronavirus (COVID-19) pneumonia, it has caused more than 80,000 infections across the country and nearly 4,000 deaths; and it has caused more than 50,000 infections and thousands of deaths worldwide.
  • a highly transmissible coronavirus The difference between SARS-CoV-2 coronavirus and traditional coronavirus is that it is susceptible to everyone. It can not only infect the upper respiratory tract, causing fever, cough, laryngitis and other common cold symptoms, but also infect the lower respiratory tract, causing bronchitis and pneumonia. And other acute respiratory symptoms.
  • SARS-CoV-2 The structure of SARS-CoV-2 is similar to SARS coronavirus. It is an enveloped single-stranded positive-stranded RNA virus.
  • the spike protein S protein reflected on the surface of the virus is a specific organization structure on the virus envelope. A large number of spike proteins are formed on the surface of the virus, which play an important role when the virus invades the target cell and recognizes the virus and the cell.
  • the SARS S protein vaccine can produce high-titer anti-SARS-CoV virus neutralizing antibodies, which can effectively prevent SARS-CoV infection. Therefore, in view of the high similarity of the three-dimensional structure of SARS-CoV-2 and SARS S protein
  • the S antigen of the new coronavirus is usually used as the main target.
  • 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 the mucosal surface (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
  • the bottleneck of conventional mucosal vaccination with traditional antigens is that the frequent physical oscillations of the mucosal cilia can quickly remove foreign antigens; there is a large amount of acidic solution in the mucosal area, which is rich in hydrolytic enzymes, DNA enzymes, etc., which can quickly degrade foreign antigens. Therefore, the traditional antigen inoculated on the mucosal site is rapidly cleared and degraded, and cannot stay in the mucosal area effectively, so that it is not enough to be presented by APC and cannot induce an effective mucosal immune response. Even if induced, the degree of response is very low.
  • VSV vesicular stomatitis virus
  • the known vaccine carrier vesicular stomatitis virus (VSV) wild strain can infect a variety of animals and insects in the natural environment. There are horses, cattle (sheep), and pigs that are naturally infected with VSV in domestic animals. In the natural state of the population, there is almost no active infection of vesicular stomatitis virus, and the infection to humans will not cause obvious symptoms. Therefore, vesicular stomatitis Virus (VSV) as a viral vector vaccine has natural advantages compared with other vectors. Therefore, VSV as a viral vector chimerizing or fusing target antigens will enhance the body's immune response strength. VSV viral vector vaccines further adopt mucosal vaccination.
  • VSV vesicular stomatitis virus
  • the VSV virus vector also has characteristics that other tool vectors do not have.
  • the designed preventive vaccine is used to prevent enveloped viruses, the VSV virus can completely display the envelope protein of the target virus on its own envelope protein GP, and fully expose the antigen protein on the surface of the recombinant virus. After inactivated in vitro, this type of viral vector vaccine still has the specific immune response to effectively activate the body.
  • the foreign viral envelope protein further enhances the immunogenicity of the antigen and fully activates the host immune response through specific fusion with GP.
  • the recombinant virus vaccine does not have the ability to replicate twice, and its safety is significantly enhanced.
  • the present disclosure proposes a VSV-modified viral vector mVSV, a mVSV viral vector and a mVSV-mediated new coronary pneumonia vaccine, which has a better preventive or therapeutic effect on patients infected with the new coronary pneumonia virus.
  • the present disclosure provides a vector mVSV, a viral vector for vaccine development, and a new coronavirus vaccine based on the mVSV viral vector that can be quickly developed through the mVSV-Vac platform that can be widely used in a variety of viruses that are raging in today's society.
  • the mVSV virus vector, the vesicular stomatitis virus Indiana strain Indiana VSV gene M encoded amino acid has multiple site modification mutations, the site mutation occurs at the 51st position of the VSV M protein methionine M is replaced with benzene Alanine F, phenylalanine F at position 110 were replaced by alanine A, and Isoleucine I at position 225 was replaced by leucine L, which was defined as mVSV.
  • a mVSV viral vector vaccine comprising the above-mentioned mVSV viral vector, the heterologous antigen gene of the target virus is integrated in the mVSV, and the heterologous antigen gene is fused or chimeric at the N-terminus of the mVSV envelope GP gene or C-terminal, the target virus vaccine formed after fusion or chimeric antigen is defined as an attenuated vesicular stomatitis virus vaccine.
  • the DNA of the heterologous antigen gene is a codon-optimized sequence, and the antigen gene contains the encoding A full-length or partial truncation of the spike protein S gene of the mentioned virus envelope.
  • the full length of the spike protein S gene of the target virus envelope includes SEQ ID NO: 1 or a gene sequence having an amino acid that is at least 98% identical to that of SEQ ID NO: 2, and is defined as a chimeric antigen Gene A;
  • the partial truncation of the spike protein S gene of the target virus envelope includes the base sequence of SEQ ID NO: 3 or a gene sequence that has at least 98% identity with the amino acid encoding SEQ ID NO: 4, definition It is a chimeric combinatorial gene B.
  • heterologous antigen gene DNA is integrated into the envelope GP gene coding sequence or adjacent non-coding sequence in the mVSV vector gene fragment.
  • the heterologous antigen gene when fused at the N-terminus or C-terminus of the envelope GP gene, the 5'-end fusion of the envelope GP gene occurs after the envelope GP gene signal peptide. The 3'end fusion of the gene occurs before the stop codon of the envelope GP gene.
  • the antigen gene of the mVSV envelope GP fusion is selected from the RBD segment of the spike protein of the new coronavirus SARS-CoV-2, and the envelope GP gene is present in any of the vaccine vector pmVSV-Core backbone vectors corresponding to the target virus.
  • the heterologous antigen gene of the envelope GP fusion comprises an RBD gene or an RBD truncated gene encoding the S protein of the target virus, and the heterologous antigen gene comprises SEQ ID NO: 5 or encoding SEQ ID NO: 6
  • the gene sequence of amino acids with at least 98% identity is defined as the fusion antigen gene C.
  • a vaccine based on mVSV-mediated novel coronavirus pneumonia wherein the above-mentioned target virus is a novel coronavirus pneumonia virus, and the mVSV virus vector is chimeric or fused with SARS-CoV-2 antigen genes, and the antigen genes are selected from SARS-CoV-2.
  • the predominant epitope of the spike protein S of the CoV-2 pathogen includes a chimeric antigen gene A, a chimeric assembly gene B, or a fusion antigen gene C.
  • the antigen gene includes the full length of the spike protein S gene and the corresponding dominant antigen genes in different S gene truncations, and the dominant antigen gene is selected from those corresponding to the receptor binding domain RBD encoding the spike protein S Full-length gene or corresponding truncation gene truncation.
  • the antigen gene of SARS-CoV-2 is selected from codon-optimized synthetic genes that encode one or more of the receptor binding domain RBD of the spike protein of human neocorona pneumonia virus, wherein The receptor binding domain RBD contains one or more antigen genes of different mutant strains of new coronavirus pneumonia.
  • Inoculation and immunization can be carried out by intramuscular injection, intravenous, nasal drip or oral administration.
  • Viral vector vaccine with a dose of 10 6 -10 8 pfu (mVSV-A, B/C)
  • the present disclosure selects live viruses as vaccine vectors, chimeric or fuses specific target antigen genes, and utilizes the replication ability of vesicular stomatitis virus in cells to express target antigens efficiently and quickly,
  • a specific administration method can significantly enhance the specific mucosal immune response;
  • the modified VSV proposed in the present disclosure patent mVSV
  • mVSV virus has lower toxicity, higher antigen loading capacity, and more stable virus genome.
  • the new coronary pneumonia vaccine involved in this disclosure will cause a strong innate immune response, activate the body’s immune system, and act like an adjuvant.
  • the target antigen carried by the virus will be fully discovered, and the antigen that is different from ordinary vaccines is unstable and easily degraded.
  • the target antigen of the viral vector of the present disclosure will be expressed in the cytoplasm in large quantities along with the replication of the virus, and will be fully presented to the DC cells to cause a specific immune response of the body.
  • the vaccine When the vaccine is administered to the mucosal site, the body will be induced Produce a locally acquired mucosal immune response.
  • the mVSV-Vac vector system of the present disclosure can be used for mucosal delivery of preventive or therapeutic vaccines for various transmucosal infectious pathogens.
  • the vaccine of the present disclosure can effectively induce the production of specific mucosal SIgA and systemic IgG against SARS-CoV-2.
  • the new coronary pneumonia vaccine mediated by the viral vector (mVSV) provided in the present disclosure can be administered via intramuscular injection, intravenous, nasal drip, oral administration and other immunization routes, which can solve the problem that traditional vaccines in the prior art cannot induce high-intensity immune responses (
  • the low titers of neutralizing antibodies make up for the inability of antigens carried by traditional vaccines to stay in the mucous membranes effectively, and cannot be fully presented to immune cells by APCs.
  • the activated immune response is weak and antibody titers are low.
  • the mVSV new crown vaccine involved in this disclosure can be used in combination with other vector vaccines.
  • the first shot uses mVSV new crown vaccine to immunize first
  • the second shot uses the second viral vector vaccine (adenovirus vector vaccine, poxvirus vector vaccine) for the second shot.
  • the second stimulation will further activate the acquired immune response against the new crown antigen, which greatly improves the response rate of vaccination.
  • the mVSV vector-mediated new coronary pneumonia vaccine involved in this disclosure has the following three characteristics:
  • Its core is a virus vaccine-mediated vector, which encodes the coronavirus spike protein spike protein S and different truncated bodies, preferably the antigen protein sequence is mainly derived from the SARS-CoV-2 strain.
  • the recombinant attenuated vesicular stomatitis virus is packaged through a specific modified plasmid (low copy) packaging system, that is, the vesicular stomatitis virus recombination subsystem.
  • the immune effect of mVSV vaccines is significantly improved, and the increased systemic IgG neutralizing antibodies and the number of sIgA antibodies in the mucosa are further induced in the body.
  • the fusion candidate vaccine mVSV-C will further activate the body's anti-viral T cell immunity while inducing a specific humoral immune response, forming a permanent memory, and producing a life-long protective effect.
  • the mVSV viral vector involved in the present disclosure can be used in the research of virus vaccines for purposes other than the currently rampant new coronavirus; in addition, it is also possible to administer immunologically effective amounts of the recombinant new coronavirus vaccine vector and the recombinant new coronavirus vaccine to patients with new coronavirus
  • Different immune adjuvant compositions have obvious curative and preventive effects; the immune response is the induction of anti-S protein serum antibodies and induces a specific protective immune response against S protein, and the induced specific neutralizing antibody titer exceeds 1 International units/ml.
  • the present disclosure provides the new coronary pneumonia vaccine and its adjuvant composition one or more times according to the patient's clinical performance, and even then provides the recombinant new coronary vaccine to the patient within weeks, months or years of the first providing step Or a vaccine combination combined with a target viral vector and a composition containing an adjuvant.
  • Patients who are provided with a new coronavirus vaccine recombinant new coronavirus vaccine or its adjuvant composition include: the individual exhibits one or more symptoms of SARS-CoV-2 The individual lacks any symptoms of SARS-CoV-2, the individual has been exposed to SARS-CoV-2, the individual has been in contact with an individual suffering from SARS-CoV-2, the individual is a child, an elderly person People who are exposed to or at risk of biological weapons are members of the military or health care workers.
  • mVSV virus vector wherein the mVSV virus vector includes mVSV virus, and the mVSV virus is a virus obtained by mutating the amino acid of the matrix protein M of the vesicular stomatitis virus Indiana strain, and the mutation occurs in the matrix protein.
  • M methionine at position 51 was mutated to phenylalanine
  • phenylalanine at position 110 was mutated to alanine
  • isoleucine at position 225 was mutated to leucine.
  • a vaccine comprising the mVSV viral vector described in (1), wherein a heterologous antigen gene of the target virus is integrated into the gene of the mVSV viral vector.
  • the SARS-CoV-2 antigen gene of the new coronavirus pneumonia virus is a codon-optimized sequence, and the codon-optimized sequence includes a full-length or partial truncated body of the new coronavirus pneumonia virus SARS-CoV-2 spike protein S gene.
  • a method for treating or preventing COVID-19 wherein the method includes the step of administering the vaccine according to any one of (2) to (11) to the patient.
  • FIG. 1 Schematic diagram of pmVSV-Core-A and pmVSV-Core-B plasmid construction, virus packaging and identification;
  • Figure 6 is a schematic diagram of VSV-spike protein S recombinant virus vaccine packaging through genetic recombination plasmid;
  • Figure 7 relates to the toxicity comparison between different point mutants of VSV and wild strains, including MTT toxicity detection in MEF cells (A), replication of different mutant strains (B), and safety comparison of different mutant strains in mouse models (C ).
  • the term "about” means: a value includes the standard deviation of the error of the device or method used to determine the value.
  • the selected/optional/preferred “numerical range” includes both the numerical endpoints at both ends of the range and all natural numbers covered by the numerical endpoints relative to the aforementioned numerical endpoints.
  • the experimental techniques and experimental methods used in this example are conventional techniques and methods unless otherwise specified.
  • the experimental methods for which specific conditions are not indicated in the following examples usually follow conventional conditions such as Sambrook et al., Molecular Cloning: Experiment The conditions described in the room manual (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the conditions recommended by the manufacturer.
  • the materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through formal commercial channels.
  • the present disclosure mainly constructs the SARS-CoV-2 virus protective epitope onto the VSV virus backbone vector (pCore).
  • VSV virus backbone vector pCore
  • the reagents and consumables used in this disclosure are as follows: Q5 Hot start High-Fidelity DNA polymerase (NEB M0493L), Nhe I-HF (NEB R3131L), Xho I (NEB R0146S), T4 DNA Ligase Enzyme (NEB M0202L), E.
  • the 293T and 293T-hACE2 adherent cells were cultured in a special culture environment (Thermo BB150 cell culture incubator) containing 5% CO2 at 37°C, using DMEM high-sugar complete medium for culture.
  • Attenuated mVSV virus vector Attenuated mVSV virus vector:
  • a three-site mutant of the matrix protein (M) of the recombinant vesicular stomatitis virus Indiana strain selected by the attenuated mVSV virus vector
  • the mutated amino acid positions preferably have amino acid substitutions from the 51st, 110th and 225th positions at the same time.
  • the amino acid substitution mode is: the 51st methionine M is replaced by phenylalanine F, and the 110th phenylalanine Acid F was replaced with Alanine A, and Isoleucine I at position 225 was replaced with Leucine L.
  • Example 1 The toxicity of the M protein three-site mutation against the Indiana strain of VSV is greatly reduced
  • the M protein can induce the apoptosis of host cells. It is the main factor for the VSV wild strain to infect cloven-hoofed animals.
  • the best way for wild VSV strains is to carry out genetic engineering mutations in the M gene.
  • Existing studies have shown that non-synonymous mutations at the 51st amino acid of the M protein will reduce the neurotoxicity of wild VSV. Therefore, the technique of the present disclosure is implemented Firstly, a single point mutation comparison was performed. At position 51, methionine was mutated to phenylalanine, alanine, leucine and arginine (control).
  • M51F-F110 (A/R/L), found that the toxicity of M51F-F110A has been further reduced in the 2 mutant strains, but in the follow-up (Figure 7C), the VSV wild strain sensitive Balb/c mice were dropped Nasal administration (simulation of nervous system infection), it was found that high-dose E9pfu administration of M51F-F110A still caused some mice (1/4 ratio) to lose weight, although only a transient phenomenon occurred within 5 days after vaccination. However, it still shows that the attenuation modification for the M gene has not reached the best way. Further, based on the positions of the above 2 mutations, the present disclosure has found a third mutation site that can significantly reduce the toxicity of the M protein, that is, the 225th amino acid.
  • the amino acid at position L was mutated from Leucine I to Leucine L, and the attenuation effect was the most significant (part of the comparison results are not shown in Figure 7). Further, as shown in Figure 7B, at the cellular level, it passes through the three positions of the M protein. The point mutant mVSV (M51F-F110A-I225L) did not detect any damage to normal MEF fibroblasts (multiplicity of infection rose to 10, and no significant cytopathic changes were observed). Similarly, different mutant strains were used in animal models The challenge experiment is shown in Figure 7C.
  • mice Balb/c In 8-week-old mice Balb/c, the E9pfu mutant strain virus was instilled into the nose for preliminary safety evaluation (intravenous administration was also verified by mouse model administration, the same dose, each There were 6 mice in the group. Except for the control group, the wild strain of VSV produced significant adverse reactions. The mice with other mutant strains had milder symptoms). The statistical results showed that the virus with and only 3 mutations did not cause the weight of the mice. However, other mutant strains showed a significant decrease in body weight and then recovered. In addition, after the early administration, the VSV wild strain showed a toxic reaction of abnormally increasing body temperature. At the same time, the VSV wild strain and mVSV-M51R in the control group showed small symptoms. In the phenomenon of nerve paralysis in the hind legs of mice, only the three mutant strains administered to mice did not produce any adverse reactions, which proves that the attenuated strain is highly safe to normal mice, has no toxic side effects, and does not have potential neurotoxicity.
  • the S gene sequence of SARS-CoV-2 released by NCBI is codon-optimized to facilitate its expression in the cell (named antigen gene A), and multiple potential epitopes predicted based on the SARS-CoV-2 sequence are combined into
  • the new antigen gene (named as antigen gene B), the sequence of antigen gene A and antigen gene B are handed over to Nanjing GenScript Biotech to synthesize into pCDNA3.1 and pUC57 vectors respectively, after PCR amplification of the target gene, the fragment purification reagent Recover and purify the target band from the cassette, use restriction endonucleases, MCS1 (Xhol) and MCS2 (Nhel) to digest the fragment and pmVSV-GFP vector at 37°C for 3 hours, and then perform the ligation reaction after the vector and the target fragment are recovered by the gel.
  • Primer synthesis and primer information The primers were synthesized by Suzhou Jinweizhi Bio-Biotech Co., Ltd.
  • the PCR primers and bacterial liquid PCR primers selected for the amplification of A gene were constructed as shown in Table 1:
  • Obtain the target gene use the pCDNA3.1 plasmid with the target gene sequence as a template to PCR amplify the A gene with the primers in Table 1; use the pUC57 plasmid with the target gene sequence as the template to PCR amplify the B gene with the primers in Table 2;
  • Double-enzyme digestion of the above purified product and vector 37°C digestion for 3h
  • the VSV virus was packaged according to the standard method for the correctly sequenced plasmid, and the pmVSV-GFP plasmid was taken as a positive packaging control; the virus supernatant was collected once at 48h and 72h, and 300uL was used to infect the 293T cells pre-plated on the 6-well plate.
  • the packaged viruses were named mVSV-GFP, mVSV-A, mVSV-B; the cells were collected after cytopathic disease to detect the antigen expression level by WB.
  • 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 C fragment was fused to the C-terminus of the VSV-G envelope gene on the pVSV-GFP vector by overlap extension PCR (product number is GP-C), or fused to the N-terminus of the VSV-G envelope gene (product number is ( C-GP).
  • the second round of PCR takes the two target fragments of the first round of PCR as templates, and uses the upstream primers and fragments of fragment 1 respectively.
  • the downstream primer of 2 carries out the amplification of the fusion fragment, the fragment and pVSV-GFP vector are digested with restriction endonucleases (MCS1(MluI) and MCS2(XhoI)) at 37°C for 3h, and the vector and the target fragment are recovered by the gel
  • MCS1(MluI) and MCS2(XhoI) restriction endonucleases
  • Primer synthesis and primer information The primers were synthesized by Suzhou Jinweizhi Bio-Biotechnology Co., Ltd., and the specific primer information is shown in the following table:
  • viruses Collect the virus supernatant once at 48h and 72h each, and take 300uL to infect the 293T cells pre-plated on the 6-well plate and package them.
  • the viruses are named mVSV-(GP-C) and mVSV-(C-GP) respectively;
  • the cells are collected for WB to detect the antigen expression level.
  • 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 antibody was serially diluted (1:2) 10 ⁇ L/tube with Opti-MEM, and a positive control without antibody (20 ⁇ L virus solution, final virus concentration 4E5TU/mL) and a negative control without virus (20 ⁇ L) Opti-MEM);
  • the pseudovirus is also serially diluted to 8E5TU/mL;
  • step 5 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 where the green fluorescence appears last.
  • mVSV-C In the intravenous administration group, the most neutralizing antibodies were induced, which indirectly indicates that the specific and preferred neocorona antigen RBD will be fused with VSVG (high immunogenicity) (N-terminal fusion) to produce neutralizing antibodies against neocorona, indirectly supporting evidence
  • VSVG high immunogenicity
  • N-terminal fusion N-terminal fusion
  • the fusion of foreign dominant antigens to the N-terminus of mVSV vector gene GP can enhance the body's recognition of antigens by enhancing the display of dominant antigens in the recombinant virus envelope, and induce the production of neutralizing antibodies and antiviral immune memory.
  • mVSV-mediated SARS-CoV-2 vaccine series products include mVSV-A/B and mVSV-C.
  • the antigen gene of the new coronavirus is integrated into the non-enveloped core region of the virus, preferably the chimeric position is in the coding region of the envelope GP and polymerase L.
  • This viral vector candidate must be a live strain at the time of vaccination due to the antigen gene It needs to be transcribed and translated into protein by infected host cells in the body before it can be presented by the immune cell DC.
  • the antigen gene can express the foreign antigen in the body with the replication of the virus, and activate the body to produce specific immunity against the new crown.
  • the design strategy of another mVSV-C candidate vaccine is completely different.
  • the technical solution is to integrate the truncated antigen gene of the new coronavirus into the envelope GP gene of mVSV, preferably integrated into the N of the GP gene. From the results of Examples 3 and 4, it can be found that the RBD protein integrated at the N-terminus is expressed in fusion with the GP protein. At the protein level, it is proved that the candidate antigen protein of the mVSV-C-GP vaccine is displayed on the surface of the envelope. Type vaccines can be inactivated (irradiation or high temperature) and then inoculated.
  • the safety is better, although in the examples, it is not further elaborated whether such candidate vaccines will be inactivated after inactivated vaccination.
  • the live operation reduces the specific immune response induced by the antigen.
  • the reduction of immunogenicity can improve the response efficiency by increasing the vaccination dose during clinical vaccination, and make up for the disadvantage of weak immunogenicity caused by inactivation.

Abstract

L'invention concerne un vecteur viral recombinant mVSV de la protéine M du virus de la stomatite vésiculaire (VSV). Le vecteur viral recombinant comprend des souches mutantes de virus obtenues par la mutation de trois sites M51F, F110A et I225L de la protéine M d'une souche sauvage du VSV Indiana. L'invention concerne en outre un nouveau vaccin contre la pneumonie à coronavirus formé par la chimérisation d'un vecteur de virus mVSV ou la par la fusion d'un vecteur de virus mVSV avec un domaine de liaison au récepteur de la protéine spike S des pathogènes du SARS-CoV-2.
PCT/CN2021/081524 2020-03-20 2021-03-18 Vecteur de virus mvsv, vaccin à vecteur viral associé et nouveau vaccin contre la pneumonie à coronavirus basé sur la médiation par mvsv WO2021185310A1 (fr)

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