CN117752779A - Coronavirus spike protein envelope replacement type carrier vaccine and construction method thereof - Google Patents

Abstract

The invention relates to a coronavirus spike protein envelope replacement type vector vaccine, which uses vesicular stomatitis virus VSV as a vector, replaces GP genes in a VSV virus genome with truncated forms of spike protein S genes of coronaviruses or fusion ECD-CA of extracellular segments of the spike protein S genes of the viruses and GP-C segments, wherein the truncated forms of the spike protein S genes are selected from genes with deletion of amino acids at C end parts, and the ECD-CA is a gene obtained by fusing transmembrane genes and intracellular segment genes of envelope proteins of the VSV viruses with the extracellular segments of the spike protein S genes of the viruses.

Description

Coronavirus spike protein envelope replacement type carrier vaccine and construction method thereof

The application is 15 days of application year 2020, 06 month, application number: 202010541093.4, the patent division application of an envelope replacement type viral vector vaccine and a construction method thereof.

Technical Field

The present invention relates to a virus vector vaccine constructed by Vesicular Stomatitis Virus (VSV) and effective in preventing coronavirus infection, particularly comprising SARS-CoV-2 coronavirus envelope for replacement, and a construction method thereof.

Background

Coronaviruses (Coronavir) belong to the order of the nest-forming viruses (order Nidovirals), the family of coronaviruses (family Coronavirade) and the genus coronaviruses (genus Coronavirus) in virological classification, the genome is single-stranded, positive-stranded RNA, the genome is between 26 and 32kb in total length, and the largest genome is currently known. Coronaviruses are very widely used in nature and common mammals such as dogs, cats, mice, pigs, cattle and poultry are susceptible.

Coronaviruses were analyzed by phylogenetic analysis of nucleic acid sequences, and the international classification committee on virology (ICTV, 2012) in a ninth report divided members of the genus coronavirus into four groups, a, b, y and delta. Human coronaviruses are mainly distributed in the alpha and beta groups. Wherein HCoV-229E and HCoV-NL63 are in group α, HCoV-OC43 and HCoV-HKU1 are in subgroup 2a of group β, MERS-CoV belongs to subgroup 2c of group β, and the latest roll global SARS-CoV-2 and SARS belong to subgroup 2b of group β.

Recent studies indicate that the S spike protein RBD segment of the virus binds to ACE2 by as much as 10 times that of SARS, and the transmission capacity is the strongest of the seven main coronaviruses known to infect humans. SARS-CoV-2 has similar structure to SARS coronavirus, and the spike protein S protein on the surface of the virus is a specific tissue structure on the virus envelope, so that a large number of spike proteins are formed on the surface of the virus, and the spike protein plays an important role in the invasion of the virus into target cells and the identification of the virus and the cells.

The new coronavirus is likely to be popular in human society for a long time like influenza virus, and the envelope replacement type viral vector has proved to be the most cost-effective, most effective and most durable disease prevention and control measure, so that it is imperative that the whole population vaccinates the new coronavirus replacement type viral vector.

Research shows that pathogens such as Human Immunodeficiency Virus (HIV), influenza virus, severe acute respiratory syndrome virus (SARS-CoV) and the like which cause serious infectious diseases invade and infect the body through mucosal surfaces (genital tract, respiratory tract and gastrointestinal tract), and the body cannot induce effective mucosal immune response to clear mucosal infectious pathogens, so that the pathogens rapidly spread into blood and invade the whole body, and damage the body, especially lung tissues.

Conventional envelope-substituted viral vectors such as inactivated, protein-envelope-substituted viral vectors, DNA-envelope-substituted viral vectors, subunit-envelope-substituted viral vectors, and the like are known to be generally incapable of inducing specific mucosal immune responses by conventional route immunization (intramuscular injection, subcutaneous, and the like). Regardless of the form of the envelope replacement viral vector, it is generally necessary to inoculate the target antigen from the mucosal site to be efficiently taken up and presented by APCs in mucosal tissue to further activate the mucosal immune system and induce an efficient and sustained mucosal immune response.

Known envelope replacement viral vectors, vesicular Stomatitis Virus (VSV) wild strains, can infect a wide variety of animals and insects in the natural environment. The natural VSV infection in livestock is horse, cow (sheep) and pig, and the active infection of vesicular stomatitis virus does not exist in the natural state of the population, so that the influence of pre-existing antibodies on the drug effect of virus vector vaccine (pre-existing neutralizing antibodies on adenovirus and poxvirus in human body) is avoided,

therefore, compared with other virus vectors, the Vesicular Stomatitis Virus (VSV) serving as an envelope replacement type virus vector has natural advantages, the S protein of the coronavirus can be completely exposed on the surface of the virus, the recombinant virus does not need to infect a host cell, transcribe and translate exogenous virus antigen proteins, can be directly recognized by an immune system outside the cell, and activate anti-virus innate and acquired specific immune responses, and shorten the anti-virus reaction time, so that the VSV serving as the virus vector can be deduced by adopting a gene editing technology to display the envelope protein S of the coronavirus on the surface of the virus, the intensity of the immune response of an organism can be obviously enhanced, and stronger acquired anti-virus responses (T cell immune responses and B cell immune responses) are induced.

The invention provides a coronavirus spike protein envelope replacement type vector vaccine and a construction method thereof by utilizing the natural advantages of VSV virus vectors, and the envelope replacement type virus vector vaccine has better prevention or treatment effects on human coronaviruses, especially SARS or novel coronaviruses (SARS-CoV-2).

Disclosure of Invention

The envelope replacement type viral vector vaccine is formed by replacing GP genes in a rhabdovirus genome with a truncated spike protein S gene of coronavirus or an extracellular segment fusion ECD-CA of the spike protein S gene of the virus, and constructing the envelope replacement type viral vector vaccine for preventing the coronavirus by utilizing a reverse genetic system, wherein the ECD-CA is defined as genes obtained by fusing extracellular segment ECD of the spike protein S gene of the virus with transmembrane and intracellular segment genes of VSV envelope proteins.

Preferably, the envelope replacement type viral vector vaccine is characterized in that the rhabdovirus vector is selected from vesicular stomatitis virus VSV, the VSV is selected from Indiana strains, the spike protein S gene is selected from SARS or SARS-CoV-2 coronavirus, the spike protein S gene truncated body is selected from genes corresponding to C-terminal amino acid deletion, and the number of the C-terminal amino acid deletion is 18-72.

Preferably, the envelope replacement type viral vector vaccine, the spike protein S gene truncated body is selected from C-terminal deleted genes, the number of C-terminal amino acid deletions is preferably 30, and the amino acid sequence corresponding to the spike protein S after modification is SEQ ID NO.1.

Preferably, the envelope replacement viral vector vaccine, the rhabdovirus vector is selected from vesicular stomatitis virus VSV, the VSV is selected from Indiana strain, the extracellular domain fusion ECD-CA of the spike protein S gene is selected from coronaviruses, the ECD-CA comprises the extracellular domain ECD of the spike protein S gene of coronaviruses, and the C-terminal of the ECD is fused with the transmembrane and intracellular domain gene CA of the envelope protein of the VSV.

Preferably, the envelope replacement type viral vector vaccine is characterized in that the ECD-CA is cloned into a coding sequence between an M gene and an L gene in a VSV genome, the ECD-CA amino acid sequence is SEQ ID NO.2, and the ECD-CA humanized codon gene sequence is SEQ ID NO.3.

Preferably, the envelope replacement type virus vector vaccine is a virus obtained by mutating amino acid of matrix protein M, wherein the amino acid mutation site in the matrix protein M is one or more of leucine at 20 th, methionine at 51 st and phenylalanine at 110 th and is a non-synonymous mutation. The amino acids corresponding to the leucine at position 20, methionine at position 51 and phenylalanine at position 110 after mutation can be the same or different from the amino acid type corresponding to the mutation in the next paragraph.

Preferably, the envelope replacement type viral vector vaccine is an attenuated vesicular stomatitis virus, wherein 3-site amino acid mutation occurs in a matrix protein M of the vesicular stomatitis virus, the 20 th site of the matrix protein M is mutated from leucine L to phenylalanine F, the 51 st site is mutated from methionine M to alanine A, the 110 th site is mutated from phenylalanine F to leucine L, and the amino acid sequence of the 3-site mutated matrix protein M is SEQ ID NO.4.

A construction method of coronavirus spike protein envelope replacement type vector vaccine comprises the following steps:

s1, obtaining an S-CN truncated gene of any coronavirus or an ECD-CA envelope gene by utilizing synthesis biology, and cloning a gene fragment into a pVSV-3M plasmid multiple cloning site by double enzyme digestion of MluI and XhoI to respectively obtain pCore-3M-S-CN or pCore-3M-ECD-CA plasmids, wherein the S-CN of the coronavirus is positioned as a gene sequence abbreviation corresponding to the coronavirus spike protein S gene after deleting N amino acids at the C end, wherein any natural number of N=18-72;

s2, transiently converting ACE2 stably expressed cells by using a plasmid pCAGGS-T7 for expressing T7-RNA polymerase; the ACE2 stably expressed cells are preferably prepared from 293T-hACE2, and the plasmids from which endotoxin is removed after 24h transfection comprise pCAGGS-P, pCAGGS-N, pCAGGS-L and pCore-3M-S-CN or pCore-3M-ECD-CA;

s3, carrying out liposome wrapping co-transfection on the four plasmids for 72 hours, filtering the supernatant through a filter membrane with 0.22um, and adding the filtered supernatant into 293T-ACE2 cells in the logarithmic phase;

s4, collecting cell supernatant when the cells are diseased, and identifying copy number of envelope replacement target genes in a viral genome by using an RT-PCR technology;

s5, performing plaque purification by using cells stably expressed by 293-ACE2, and performing western blotting identification to obtain the coronavirus envelope replacement vaccine VSV-delta G-S-CN or VSV-delta G-ECD-CA.

Preferably, in the construction method of the coronavirus spike protein envelope replacement type vector vaccine, the fluorescent probe primer pair for specifically amplifying the coronavirus S-CN gene in the RT-PCR in the step S4 is a sequence SEQ ID NO.5 and a sequence SEQ ID NO.6.

Preferably, in the construction method of the coronavirus spike protein envelope replacement type vector vaccine, the fluorescent probe primer pair for specifically amplifying the coronavirus ECD-CA gene in RT-PCR is SEQ ID NO.7 and SEQ ID NO.8.

The advantages are as follows:

compared with the traditional virus vector vaccine, the invention firstly utilizes a VSV virus packaging system, and can efficiently package coronavirus envelope replacement recombinant viruses through a large number of gene optimization and construction, the coronavirus envelope replacement recombinant viruses relate to specific envelope gene modification bodies (S-CN and ECD-CA), the coronavirus envelope is completely wrapped outside the genetic material of the VSV, and compared with the traditional virus vector vaccine (the virus antigen can be transcribed and translated after the host cell is infected), the antigen with high immunogenicity can be presented to immune cells, the reaction time of a host immune system is shortened, meanwhile, the most important antigen protein S of the coronavirus is reserved to the greatest extent, compared with the traditional replication defective virus vector vaccine (adenovirus vector), the coronavirus candidate vaccine related by the invention has a certain replication capacity (hACE 2 stable expression cell line), can be rapidly produced in a large scale, and meanwhile, the whole process of simulating coronavirus infection host cells (the VSV structural protein has no obvious toxicity to the host cell) as a non-inactivated vaccine is inoculated to the highest degree, and the novel antibody with high intensity can be activated in the immune system.

The VSV envelope replacement type virus vector vaccine further adopts an immunization mode of inoculating at a mucosa part, so that a body can be induced to generate stronger specific mucosa immune response to coronavirus, especially novel coronavirus (SARS-CoV-2) antigen protein S, when external pathogens invade through a mucosa, mucosal tissues can be activated, pathogens are rapidly cleared, further the VSV vector is known to have the characteristics that other tool vectors do not have, when the designed envelope replacement type virus vector is used for preventing enveloped viruses, the VSV virus can display the complete space structure of envelope proteins of target viruses on the surface of the viruses, the trimer protein S of the novel coronavirus (SARS-CoV-2) is fully displayed on the surface of a nucleocapsid of the recombinant viruses, and further after the envelope replacement type virus vector of the technical type is inactivated in vitro, the VSV envelope replacement type virus vector still has the characteristics of effectively activating the specific immune response of the body, fully activating host immune response, and meanwhile, after the recombinant virus replacement type virus vector is used as an inactivated vaccine, the VSV vector does not have secondary replication capacity, and the safety of the vaccine is further improved.

Description of the drawings:

FIG. 1A is a schematic diagram of construction of S-CN and ECD-CA genes of different S truncations into rhabdovirus skeleton vectors, B is a flow verification diagram of molecular biological cloning, C is a Western Blotting (Western Blotting) detection of the expression of target proteins of candidate vaccines obtained by package rescue, and D is a fluorescent diagram of recombinant envelope replacement vaccines packaged by S of different modification;

FIG. 2 shows that 2 strains of envelope replacement type candidate vaccines with highest packaging efficiency titer are screened, different vaccination modes are adopted, and the content (A) of IgA and the content (B) of specific IgG in serum are detected respectively after 21 days;

FIG. 3 serum was taken 21 days after immunization of various envelope replacement candidate novel coronavaccines, and neutralization antibody titers were compared in vitro using a simulated novel coronavirus developed based on lentiviruses as a detection tool;

FIG. 4 is a schematic representation of the modification of the envelope of a rhabdovirus vector into a coronavirus envelope.

The present disclosure is further described in detail below in connection with specific embodiments

Detailed Description

The following is an explanation of the present invention, but not a limitation, the present disclosure is mainly to construct different truncations (S-CN) or ECD-CA allosteric variants of the SARS-CoV-2 virus spike protein S gene onto VSV viral backbone vector (pCore-3M), the VSV envelope GP of the recombinant vector plasmid pCore-3M itself has been deleted by using genetic engineering technology, specific regions replace the coronavirus envelope gene S-CN or ECD-CA, and further to co-transfect and package in 293T cells stably expressed in ACE2 (human source) by the disclosed four plasmid system, further to rescue recombinant vaccine in which the coronavirus spike protein is completely displayed on the VSV virus surface, vaccinate by multiple routes, and successfully induce specific antiviral humoral immune response in healthy mice after immunization.

The reagents and consumables employed in the present 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.coli DB3.1 component Cells (Takara 9057), TIANGEN endotoxin-free small-scale kit (Tiangen DP 118-02), lipofectamine LTX (Invitrogen 15338100), PBS (Hyclone SH 30256.01), DMEM High-sugar medium (Gibco C11995500), double antibody (Gibco 15140-122), fetal bovine serum (Gibco 10091-148),i Reduced Serum Medium (Gibco 31985-070), 96-well cell culture plate (Corning 3599), 6-well cell culture plate (Corning 3516), 6cm cell culture plate (Corning 430166), 0.22um filter (Millipore SL GP 0)33 rb), T175 cell flask (Corning 431080).

Cell line:

293T-hACE 2-adherent cells were cultured in a specific culture environment (Thermo BB150 cell incubator) containing 5% CO2 at 37℃and with DMEM medium.

Mutant VSV viral vector:

in one embodiment, the modified VSV viral vector is preferably selected from the group consisting of vesicular stomatitis virus indiana strain, wherein the 20 th, 51 th and 110 th positions of the matrix protein (M) in the viral genome have amino acid mutations at the same time, and the amino acid substitutions are as follows: the 20 th position of the matrix protein M is mutated from leucine L to phenylalanine F, the 51 st position is mutated from methionine M to alanine A, and the 110 th position is mutated from phenylalanine F to leucine L.

Example 1 design and packaging of coronavirus envelope replacement vaccines Using VSV viral vectors

According to the existing research report, after humanized codon optimization is carried out on the S gene of SARS, the expression of the target gene in eukaryotic cells is more favorable (after humanized optimization is carried out on codons, the expression efficiency is improved by 10 times, and the packaging of recombinant viruses is favorable), therefore, in the embodiment, the published S amino acid sequence of SARS-CoV-2 is subjected to humanized codon optimization, the expression quantity in mammalian cells is improved, the sequence after S gene codon optimization is synthesized by Nanjing Jinshi biotechnology Co., ltd, and is further respectively synthesized on pCDNA3.1 eukaryotic expression vectors, after PCR amplification of the target gene, recovering and purifying target bands through a fragment purification kit, carrying out double enzyme digestion on the fragments and a pVSV-3M vector by using restriction endonucleases MCS1, particularly Mlul, MCS2, particularly Xhol,2 restriction endonucleases at 37 ℃ for 3 hours, carrying out glue recovery on the linear vector and the target fragments, adding T4 ligase for overnight connection, transferring the fragments and the target fragments to sensitive cells, carrying out bacterial liquid PCR screening positive cloning, enzyme digestion and sequencing verification, and identifying plasmid construction conditions (and respectively named pCore-3M-S-C19, pCore-3M-S-C30 and pCore-3M-ECD-CA) by the following specific implementation steps:

a variety of envelope replacement candidate vaccines based on VSV viral vectors were constructed according to (panel a of fig. 1);

primer synthesis and primer information: primers were synthesized by Souzhou Jin Weizhi Biotechnology Co., ltd, wherein PCR primers selected for amplifying S-C19 and bacterial liquid PCR primers were constructed as shown in Table 1:

TABLE 1S-C19 amplification primers

The PCR primers selected for amplifying S-C30 are shown in Table 2:

TABLE 2S-C30 amplification primers

Wherein the PCR primers selected for amplifying ECD-CA are shown in Table 3:

TABLE 3ECD-CA amplification primers

Acquisition of the target gene: carrying out PCR amplification on S-C19, S-C30 and ECD-CA by using pCDNA3.1 plasmid carrying target gene (new crown S and VSV-GP) sequences as templates and respectively using the primers in table 1, table 2 and table 3, wherein the ECD-CA fuses CA fragments to the C end of the ECD segment of the S gene through overlap extension PCR;

purifying the enzyme cutting product according to AxyPrepTM PCR Cleanup kit instruction, and measuring the concentration of the product by using Nano-300;

the PCR product and the vector are subjected to double digestion (digestion for 2h at 37 ℃);

electrophoresis is carried out to verify whether the PCR product is correct, gel strips are cut, the rest PCR product is recovered, and the product concentration is measured;

ligating the purified product with a carrier (ligating overnight at 16 ℃ C. At a ligation ratio of 1:10);

ligation products were transformed with reference to E.coli DB3.1 component Cells (TaKaRa) instructions;

selecting a monoclonal on an LB (Kana) plate, putting the monoclonal on a sterile 1.5mL tube with 100 mu L of LB (Kana) culture medium in advance, culturing at 37 ℃ and 250rpm for 2 hours, and then carrying out bacterial liquid PCR to screen positive clones;

after identification by agarose gel electrophoresis, positive clones were selected according to 1:200 ratio was transferred to 30mL shake flask, shake flask at 37℃and 250rpm overnight;

extracting plasmids according to the specification of a TIANGEN endotoxin-free small-extraction medium-amount kit;

double digestion identification is carried out on the positive plasmids (Xho I and Mlu I are digested for 2h at 37 ℃);

after enzyme digestion identification, selecting plasmids with correct identification for plasmid sequencing;

carrying out VSV virus packaging on the plasmid with correct sequence according to a standard method, and taking the VSV-WT plasmid as positive packaging contrast;

collecting virus supernatant 48h and infecting 500uL of 293T-hACE2 cells pre-spread on a 6-well plate, wherein the packaged viruses are named as VSV-WT, VSV-delta G-S-C19, VSV-delta G-S-C30 and VSV-delta G-ECD-CA respectively;

and collecting the cells after cytopathy for WB detection of antigen expression level.

The plasmid construction results and WB detection results are shown in FIG. 1:

according to the experimental results, after PCR amplification is carried out on each fragment, specific bands appear at corresponding positions, and the sizes of the band molecules are correct, so that the target bands are successfully amplified (B diagram in FIG. 1); the positive control fluorescence of VSV-WT after virus packaging infection has better expression intensity, obvious cytopathy, and the pathological cell fusion condition appears after the VSV-delta G-S-C19, VSV-delta G-S-C30 and VSV-delta G-ECD-CA virus infect cells (the graph C in figure 1); western Blot also detected the expression of each gene at the corresponding position (D panel in FIG. 1).

Example 2 immune response after two VSV viral vector-based envelope replacement vaccines adopt different immunization modes

By constructing envelope replacement plasmids for different truncations of the S gene, co-transfecting host packaging cells for 48h (293T-hACE 2) using a four-plasmid replicable packaging system as described in example 1, packaging efficiency of 293T cells stably expressing hACE2 was improved 100-fold compared to control, the supernatant after filtration through a 0.22um filter after packaging was further infected with 293T-hACE2 cells, cytopathic and fluorescent reporter gene expression was observed, and virus titer assays were performed to evaluate packaging of different truncations (S-CN) or alternatives (ECD-CA) viruses, and the results are shown in Table 4: according to statistical results, S-C30 and ECD-CA (namely, the extracellular segment ECD of the novel crown S gene fuses the transmembrane and intracellular regions of the VSV-GP gene at the C end) can better save the supernatant obtained by harvesting recombinant virus particles, the recombinant virus with envelope replacement can not be obtained by singly using the S full-length gene without any modification, and further when 16 or 17 or 73 or 74 amino acids are deleted at the C segment of the S gene, the same treatment mode can not still obtain the virus particles with effective envelope replacement, so that the conclusion can be obtained that the amino acid at the C end of the S gene seriously influences the expression and the exocrine efficiency of the protein, and further, the S gene modification variant is obtained, namely, the number of amino acids deleted at the C end is 18-72, especially 30 amino acids are deleted, the virus vector with the envelope replacement meeting the requirement can be obtained, the initial packaging titer reaches 5E6pfu/ml, and meanwhile, when the extracellular segment ECD of the S gene fuses the transmembrane and the intracellular segment of the V-GP gene (VSS) with the same recombinant virus with the same titer can be obtained.

TABLE 4 efficiency of envelope S Gene allosteric in rescue of recombinant viral vector vaccines

Example 3 effect of immune response in different immunization protocols of envelope replacement vaccines based on VSV viral vectors

Detection of specific sIgA (mucosal immune response), igG antibody levels in mice after different immunization protocols by indirect ELISA: after the recombinant RBD protein of SARS-CoV-2S virus is used for coating the ELISA plate, the virus vector vaccine is administrated through muscle, vein and nasal drip (fully simulating the process of infecting host cells by coronavirus), and the inactivated envelope replacement type vaccine is immunized through intramuscular injection, after the four immunization routes are administrated for one time, the mouse serum at 21d is diluted according to 1:200 and then is added into a corresponding detection hole, after incubation for 2 hours, different types of (sIgA and IgG) secondary antibodies are diluted according to 1:10000, and the specific antibody level is detected (figure 2), and the specific operation steps are as follows:

diluting the coating antigen (S-RBD) with a coating buffer solution to a final concentration of 5 mug/ml, taking an ELISA plate, sequentially adding samples (100 mug/hole) into the holes, and then placing the holes at 4 ℃ for coating overnight;

pouring the coating liquid in the sample hole in the next day, washing for 3 times by using a washing buffer solution, and buckling residual liquid in the sample hole on filter paper after each washing;

then 200. Mu.l of 5% BSA blocking solution was added to each well for blocking, and the wells were left at 37℃for 1 hour (plates were placed in sealed bags). Pouring off the sealing liquid, and washing the sample hole for 1 time by using a washing buffer solution;

diluting the serum to be tested and negative serum according to a proper proportion (1:100) by using an antibody serum diluent (1% BSA), adding the diluted serum into an orifice plate, and incubating for 2 hours at 37 ℃ per orifice of 100 ul;

pouring out the reaction liquid in the sample hole, washing the plate for 5 times by using a washing liquid for 1-3 min, and buckling the residual liquid on filter paper after each washing;

the enzyme-labeled secondary antibody (coat anti-mouse IgG HRP) was diluted 1:100 μl was added per well after 10000 dilutions, and then reacted at 37℃for 1h;

pouring out the unbound enzyme-labeled antibody, adding a washing solution, washing for 1-3 min each time for 5 times, and buckling and drying residual liquid on filter paper after each washing;

adding 100 μl of freshly prepared color development liquid (the color development liquid is prepared by mixing solution A and solution B in equal proportion) into each hole, standing at room temperature, and reacting for 20min in dark place;

100 μl of ELISA stop solution was added to each well to stop the reaction;

the 96-well plate was placed in an microplate reader and OD450nm was read. Comparing the OD450nm value of the sample to be tested with that of the negative sample under the same dilution ratio, and judging that the positive condition can temporarily take 2.1 times of the OD value of the negative sample as a positive test standard, namely: OD (positive >2.1 x OD (negative sample);

the results show that the two envelope replacement viruses adopt different immunization modes and strategies, the specific IgA and IgG antibody water in serum are obviously increased to higher levels in 21 days of primary immunization, and the expression levels of the antibodies in different immunization paths are different to a certain extent, wherein the nasal drip method immunization mainly activates mucosal immune response to generate stronger IgA specific antibodies, and meanwhile, nasal drip immunization also induces systemic antibody immune response (A diagram in figure 2), and the statistical results of the diagrams can be used for concluding that: the intravenous and intramuscular immunization routes mainly cause organisms to generate antigen-specific IgG type immune responses, can not generate effective mucosal immune responses (B diagram in figure 2), after the envelope replacement type viral vector vaccine is further treated and inactivated in a specific mode, the vaccine is given to mice through intramuscular injection, the content of specific antibodies in serum (indirect Elisa) is detected, the inactivated envelope replacement type candidate vaccine is found to generate higher specific humoral immune response levels, no significant difference exists compared with a live virus immune group, further the fact that the replicable vaccine vector with the envelope replacement does not destroy the induced humoral immune response of the vaccine after being inactivated at high temperature is proved, the spike protein S (C30) wrapped outside VSV genetic materials still maintains enough immunogenicity, the local mucosal immune responses are activated when the live virus vaccine is inoculated in a nasal drip mode, the higher IgA secretion is detected in serum, and therefore, the induced vaccine can be effectively combined with coronavirus at a new position of the host cell surface to protect the coronavirus from being infected by the coronavirus.

Example 4 immune serum neutralizing antibody detection based on coronavirus pseudovirus System

The titer of the generated neutralizing antibodies is determined through in vitro virus neutralization experiments, so as to evaluate antigen selection difference, screen out high-efficiency protective immunogens, compare the titers of the neutralizing antibodies which are generated by different groups and specifically aim at SARS or SARS-CoV-2, and determine the optimal vaccine preparation strategy and inoculation mode, and the specific operation steps are as follows:

extinguishing the fire at 56 ℃ for 30min, centrifuging 6000g for 3min, and taking the supernatant for later use;

the 293T-hACE2 cells were passaged and added to 96-well plates (200. Mu.L/well, containing 8. Mu.g/mL polybrene) at 2E4 cells/well after cell counting;

after 3h, the antibody was serially diluted (1:2) with Opti-MEM (10. Mu.L/tube) and simultaneously used as a positive control without antibody (20. Mu.L virus solution, final virus concentration 4E5 TU/mL) and a negative control without virus (20. Mu.L Opti-MEM);

pseudoviruses (a simulated system of lentiviral backbone packaging new crowns) were also serially diluted to 8e5 TU/mL;

10. Mu.L of diluted virus solution (8E 5 TU/mL) was added to the 10. Mu.L of serial diluted antibody contained in step 2 (1:1 blow mix) (at this time, the final virus concentration 4E5 pfu/mL);

after incubation at 37℃in a 5% CO2 incubator for 1 hour, the cells were added to 293T-hACE2 cells and infected for 24 hours to 48 hours, and fluorescence and lesions were observed.

And taking the serum dilution of the antibody corresponding to the hole with the final green fluorescence as the serum neutralization titer.

As shown in figure 3, in the immune group of PBS and VSV wild strains (VSV-WT), through in vitro neutralizing antibody detection test, the antibody capable of effectively neutralizing new coronavirus is not detected, compared with the control group, the neutralizing antibody level generated by the capsule replacement of the new coronavaccine VSV-delta G-S-C30 and VSV-delta G-ECD-CA is higher, the neutralizing antibody is induced by mice immunized by the capsule replacement, S spike protein is completely displayed on the surface of recombinant viruses on the surface, the specific antibody in the serum of the mice after the body immunization has the capability of in vitro neutralizing virus, in vitro detection, the administration modes of different immune paths are further adopted, the administration modes of primary immunization are consistent with the actual administration mode of the vaccine product in clinical use, the VSV-delta G-S-C30 and VSV-delta G-ECD-CA 2 live viruses (same administration amount) are completely displayed on the surface, the specific antibody in the serum of the mice can be activated, the candidate vaccine can be further amplified after the human body immunization has the capability of in vitro neutralizing virus (IgG and IgM), the candidate vaccine can be further amplified and inactivated vaccine can be further prepared after the candidate vaccine is further amplified and the candidate vaccine is further inactivated after the candidate vaccine is subjected to the preparation, the candidate vaccine can be further amplified and the candidate vaccine can be further inactivated, and the candidate vaccine can be further accelerated.

Claims (4)

1. A coronavirus spike protein envelope replacement vector vaccine, characterized in that: the envelope replacement type virus vector vaccine is formed by replacing GP genes in a rhabdovirus genome with spike protein S gene truncations of coronaviruses, wherein the envelope replacement type virus vector vaccine is constructed by utilizing a reverse genetic system and is used for preventing coronavirus infection, the rhabdovirus vector is selected from vesicular stomatitis virus VSV, the VSV is selected from Indiana strains, the spike protein S genes are selected from SARS or SARS-CoV-2 coronaviruses, the spike protein S gene truncations are selected from genes corresponding to C-terminal amino acid deletion, and the number of the C-terminal amino acid deletion is 18-72; the spike protein S gene truncated body is selected from C-terminal deleted genes, the number of C-terminal amino acid deletions is preferably 30, and the amino acid sequence corresponding to the spike protein S after corresponding modification is SEQ ID NO.1.

2. The coronavirus spike-protein envelope replacement vector vaccine of claim 1, wherein: the vesicular stomatitis virus is obtained by mutating the amino acid of a matrix protein M, wherein the amino acid mutation site in the matrix protein M is one or more of leucine at 20 th site, methionine at 51 st site and phenylalanine at 110 th site and is a non-synonymous mutation.

3. A construction method of coronavirus spike protein envelope replacement type vector vaccine comprises the following steps:

s1, obtaining an S-CN truncated gene of the coronavirus according to any one of claims 1 or 2 by utilizing synthesis biology, and cloning a gene fragment into a pVSV-3M plasmid multicloning site by double enzyme digestion of MluI and XhoI to obtain a pCore-3M-S-CN plasmid, wherein the S-CN of the coronavirus is positioned as a abbreviation of a gene sequence corresponding to the coronavirus spike protein S gene after N amino acids are deleted at the C end, wherein N=any natural number of 18-72;

s2, transiently transforming 293T-ACE2 cells with a plasmid pCAGGS-T7 for expressing T7-RNA polymerase; preparing transfection mixture by using plasmids with endotoxin removed after 24 hours, including pCAGGS-P, pCAGGS-N, pCAGGS-L and pCore-3M-S-CN plasmids;

s3, carrying out liposome wrapping co-transfection on the four plasmids for 72 hours, filtering the supernatant through a filter membrane of 0.22um, and adding the filtered supernatant into cells stably expressing ACE2 in the logarithmic phase;

s4, collecting cell supernatant when the cells are diseased, and identifying copy number of envelope replacement target genes in a viral genome by using an RT-PCR technology;

s5, performing plaque purification by using cells stably expressed by ACE2, and performing western blot identification to obtain the replicable coronavirus envelope replacement vaccine VSV-delta G-S-CN.

4. The method for constructing coronavirus spike-protein envelope replacement vector vaccine of claim 3, wherein: the fluorescent probe primer pair for specifically amplifying the coronavirus S-CN gene in the RT-PCR in the step S4 is SEQ ID NO.5 and SEQ ID NO.6.