WO2021254327A1 - Envelope replacement-type viral vector vaccine and construction method therefor - Google Patents

Abstract

Disclosed is an envelope replacement-type viral vector vaccine. The Vesicular stomatitis virus VSV is used as a vector, and the GP gene in the VSV virus genome is replaced with a truncated segment of a spike protein S gene of a coronavirus or a fused ECD-CA of an extracellular segment and a GP-C segment of the spike protein S gene of the coronavirus. The truncated segment of the spike protein S gene is selected from genes with some amino acid deletions at the C-terminus, and the ECD-CA is a gene formed by means of fusing the extracellular segment of the spike protein S gene of the coronavirus with the transmembrane and intracellular segment genes of the VSV viral envelope protein. Also disclosed is a method for constructing the envelope replacement-type viral vector vaccine.

Description

Envelope replacement type virus vector vaccine and construction method thereof Technical field

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.

Background technique

The World Health Organization (WHO) recently announced that the Coronavirus Disease 2019 (Covid-19) constitutes a public health emergency of international concern. As of March 24, 2020, there were 380,000 laboratory confirmed cases worldwide. In recent studies, the severity of some Covid-19 cases is similar to SARS-CoV. Given the rapid spread of Covid-19, a vaccine against the new coronavirus is imminent.

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.

According to the phylogenetic analysis of the nucleic acid sequence of coronaviruses, the International Commission for Classification of Virology (ICTV, 2012) divided the members of the genus Coronavirus into four groups in its ninth report: α group, β group, γ group and δ group. Human coronaviruses are mainly distributed in the α group and β group. Among them, HCoV-229E and HCoV-NL63 are in the α group, HCoV-OC43 and HCoV-HKU1 are in the 2a subgroup in the β group, MERS-CoV belongs to the 2c subgroup in the β group, and the latest SARS-CoV- 2 and SARS belong to the 2b subgroup in the β 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.

Studies have shown that 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.

Known conventional envelope replacement virus vectors, such as inactivated, protein envelope replacement virus vectors, DNA envelope replacement virus vectors, subunit envelope replacement virus vectors, etc., are immunized by conventional routes (intramuscular injection, subcutaneous, etc.) Usually cannot induce a specific mucosal immune response. Regardless of the form of the envelope-replaceable viral vector, to induce 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.

The known envelope-replacement virus vector-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. However, there is no active infection of vesicular stomatitis virus in the human population. There are neutralizing antibodies to adenovirus and poxvirus),

Therefore, the use of vesicular stomatitis virus (VSV) as an envelope-replaceable viral vector has a natural advantage compared with other viral vectors. It can completely expose the S protein of the coronavirus on the surface of the virus, and the recombinant virus does not need to infect the host cell. , Transcribes and translates foreign viral antigen proteins, which can be directly recognized by the immune system outside the cell, and activate the anti-viral innate and acquired specific immune response, shorten the anti-viral response time, so it can be inferred that VSV as a viral vector uses gene editing technology By displaying the envelope protein S of the coronavirus on the surface of the virus, 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.

Summary of the invention

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.

Preferably, the envelope replacement virus vector vaccine, the rhabdovirus vector is selected from vesicular stomatitis virus VSV, the VSV is selected from the Indiana strain, and 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.

Preferably, in the envelope replacement virus vector vaccine, 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.

Preferably, the envelope replacement virus vector vaccine, the rhabdovirus vector is selected from the vesicular stomatitis virus VSV, the VSV is selected from the Indiana strain, and the fusion of the extracellular segment of the spike protein S gene The ECD-CA is selected from coronaviruses, the ECD-CA includes the extracellular segment ECD of the coronavirus spike protein S gene, and the C-terminus of the ECD is fused with the transmembrane and intracellular segment gene CA of the envelope protein of VSV.

Preferably, in the envelope replacement virus vector vaccine, 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.

Preferably, 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.

Preferably, the envelope-replacement type viral vector vaccine, the viral vector is an attenuated vesicular stomatitis virus, the matrix protein M of the vesicular stomatitis virus has a 3 amino acid mutation, and 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:

S1. Obtain the S-CN truncated gene of any one of the above-mentioned coronaviruses or any one of the above-mentioned ECD-CA envelope genes by synthetic biology, and clone the gene fragment into pVSV-3M plasmid by double enzyme digestion with MluI and XhoI In the multiple cloning site, pCore-3M-S-CN or pCore-3M-ECD-CA plasmids were obtained respectively, wherein the S-CN of the coronavirus was positioned as the coronavirus spike protein S gene lacking N amino acids at the C-terminus The abbreviation of the corresponding gene sequence, where N = any natural number from 18 to 72;

S2. Transient cells stably expressing ACE2 with the plasmid pCAGGS-T7 expressing T7-RNA polymerase; the cells stably expressing ACE2 are preferably 293T-hACE2. Plasmids that remove endotoxin after 24 hours of transfection include pCAGGS-P , PCAGGS-N, pCAGGS-L and pCore-3M-S-CN or pCore-3M-ECD-CA plasmid preparation transfection mixture;

S3. After the four plasmids were encapsulated and co-transfected with liposomes for 72 hours, the supernatant was filtered through a 0.22um filter membrane and then added to the 293T-ACE2 cells in the logarithmic growth phase;

S4. If the cell has disease, collect the cell supernatant, and use RT-PCR technology to identify the copy number of the target gene in the viral genome to replace the envelope;

S5. 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.

Preferably, the method for constructing the coronavirus envelope replacement virus vector vaccine, the primer pair of fluorescent probes specifically amplifying the S-CN gene of the coronavirus in the RT-PCR in step S4 is the sequence SEQ ID NO. 5. SEQ ID NO.6.

Preferably, in the method for constructing the coronavirus envelope replacement virus vector vaccine, 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.

Excellence:

Compared with traditional virus vector vaccines, 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. Compared with 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. Compared with the traditional replication-deficient virus vector vaccine (adenovirus vector), this The 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. When a foreign pathogen invades through the mucosa, the mucosal tissue will be activated and the pathogen will be quickly eliminated. It is further known that the VSV virus vector also has characteristics that other tool vectors do not have. When the designed preventive envelope replacement virus vector is used to prevent In the case of enveloped viruses, 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. On the surface of the nucleocapsid, 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.

Description of the drawings:

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, and C is the Western Blotting detection package rescue The expression of the target protein of the candidate vaccine, 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 will be further described in detail below in conjunction with specific embodiments

detailed description

The following is an explanation but not a limitation of the present invention. 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. On the vector (pCore-3M), the VSV envelope GP of the recombinant vector plasmid pCore-3M itself has been deleted by genetic engineering technology, and the specific region is replaced by the envelope gene S-CN or ECD-CA of the coronavirus. 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. coli DB3.1 Competent Cells (Takara 9057), TIANGEN Endotoxin-Free Small Extract Medium Kit (Tiangen DP118-02), Lipofectamine LTX (Invitrogen 15338100), PBS (Hyclone SH30256.01), DMEM High Glucose Medium (Gibco C11995500) ), double antibodies (Gibco 15140-122), fetal bovine serum (Gibco 10091-148), Opti-MEM 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 GP033rb), T175 cell flask (Corning 431080).

Cell line:

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.

Mutant VSV virus vector:

In a technical solution, 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.

Example 1 Design and packaging of coronavirus envelope replacement vaccine using VSV virus vector

According to existing research reports, 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. In the present invention, 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:

According to (Figure A in Figure 1), a variety of VSV virus vector-based envelope replacement candidate vaccines were constructed;

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:

Table 1 S-C19 amplification primers

The PCR primers selected for the amplification of S-C30 are shown in Table 2:

Table 2 S-C30 amplification primers

The PCR primers selected for the amplification of ECD-CA are shown in Table 3:

Table 3 ECD-CA amplification primers

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;

Refer to the AxyPrepTM PCR Cleanup kit instructions to purify the above digested products, and use Nano-300 to determine the product concentration;

Double digestion of PCR product and vector (37℃ digestion for 2h);

Perform electrophoresis to verify whether the PCR product is correct, cut the gel band, recover the remaining PCR product, and determine the product concentration;

Connect the above-mentioned purified product to the carrier (at 16°C overnight, the connection ratio is 1:10);

Refer to the E.coli DB3.1 Competent Cells (TaKaRa) manual to transform the ligation product;

Pick the single clones on the LB (Kana) plate and put them in a sterile 1.5 mL tube pre-added with 100 μL of LB (Kana) medium. After incubating at 37°C at 250 rpm for 2 hours, perform PCR to screen positive clones;

After identification by agarose gel electrophoresis, 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;

Perform plasmid extraction according to the instructions of TIANGEN Endotoxin-Free Small Extract Medium Volume Kit;

Perform double digestion of the selected positive plasmids (Xho I and Mlu I are digested at 37°C for 2 hours);

After identification by restriction enzyme digestion, select the correct plasmid for plasmid sequencing;

Perform VSV virus packaging on the plasmid with correct sequencing according to the standard method, and take the VSV-WT plasmid as a positive packaging control at the same time;

Collect the virus supernatant at 48h and take 500uL to infect 293T-hACE2 cells pre-plated on a 6-well plate. The packaged viruses are named VSV-WT, VSV-△GS-C19, VSV-△GS-C30, VSV-△ G-ECD-CA;

After the cytopathy, the cells are collected for WB to detect the antigen expression level.

The results of the above plasmid construction and WB detection results are shown in Figure 1:

According to the experimental results, after PCR amplification of each fragment, a specific band appears at the corresponding position and the molecular size of the band is correct, indicating that the target band was successfully amplified (Figure 1 B); after virus packaging infection VSV-WT positive control has better fluorescence expression intensity and obvious cytopathic changes. The fusion of diseased cells appears after VSV-△GS-C19, VSV-△GS-C30, VSV-△G-ECD-CA viruses infect cells (Figure 1 Panel C); Western Blot also detected the expression of each gene at the corresponding position (Panel D in Figure 1).

Example 2 Immune response of two envelope replacement vaccines based on VSV virus vector after adopting different immunization methods

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. To evaluate the packaging of different truncated (S-CN) or alternative (ECD-CA) viruses, the results are shown in Table 4: According to the statistical results, S-C30 and ECD-CA (ie, the cells of the new crown S gene) The outer segment ECD is fused with the transmembrane and intracellular regions of the VSV-GP gene at the C-terminus), which can better rescue the virus titer in the supernatant harvested from recombinant virus particles, while the full-length S without any modification is used alone Recombinant viruses whose genes cannot be replaced by envelopes. Further, when 16 or 17 or 73 or 74 amino acids are missing from the C segment of the S gene, 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. It is further concluded that 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. At the same time, when the extracellular end of the ECD of the S gene is fused with the transmembrane and intracellular segment (CA) of VSV-GP, it can also be used Obtain high titer recombinant virus.

Table 4 The efficiency of enveloped S gene variants in rescuing recombinant viral vector vaccines

Name (envelope replacement gene) Package Condition Lesions after infection Virus titer S full length Unpackaged none none S-C16 Unpackaged none none S-C17 Unpackaged none none

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, strong high

Example 3 The immune response effect of the envelope replacement vaccine based on VSV virus vector under different immunization schemes

Detect specific sIgA (mucosal immune response) and IgG antibody levels in mice after different immunization schemes by indirect ELISA: After coating the ELISA plate with the recombinant RBD protein of SARS-CoV-2S virus, it will pass through muscle, vein, and nose drops. Administration of live virus vector vaccine (fully simulates the process of coronavirus infecting host cells), and the inactivated envelope replacement vaccine is immunized by intramuscular injection. After the above four immunization routes are administered once, the mice on the 21st day The serum is diluted 1:200 and added to the corresponding detection well. After 2 hours of incubation, the secondary antibodies of different types (sIgA, IgG) are diluted 1:10000 to detect the level of specific antibodies (Figure 2). The specific operation steps are as follows:

Take the coating antigen (S-RBD) and dilute it with coating buffer to a final concentration of 5μg/ml, take the microtiter plate, add samples to the wells (100μl/well) in sequence, and then place it at 4 degrees Celsius for coating overnight;

Pour out the coating solution in the sample well on the next day, wash with washing buffer 3 times, dry the residual liquid in the sample well on the filter paper after each wash;

Then add 200 μl of 5% BSA blocking solution to each well for sealing, and place it at 37 degrees Celsius for 1 hour (the plate is placed in a sealed bag). Pour off the blocking solution and wash the sample well with washing buffer once;

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;

Pour out the reaction solution in the sample well, wash with the washing solution for 1 to 3 minutes, and wash the plate 5 times. After each wash, dry the remaining liquid on the filter paper;

Dilute the enzyme-labeled secondary antibody (Goat anti-mouse IgG HRP) at 1:10000 with the diluent, add 100μl to each well, and react at 37°C for 1h;

Pour out the unbound enzyme-labeled antibody, add washing solution to wash, 1 to 3 minutes each time, a total of 5 times, dry the remaining liquid on the filter paper after each wash;

Add 100μl of freshly prepared color-developing solution (a solution and B solution are mixed in equal proportions to form a color-developing solution) to each well, put it at room temperature, and react for 20 minutes in the dark;

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

Put the 96-well plate into the microplate reader and read the OD450nm. Compare the OD450nm value of the test sample and the negative sample under the same dilution ratio, and determine the positive situation with 2.1 times the OD value of the negative sample as the positive test standard, namely: OD (positive>2.1*OD (negative sample);

The results show that the two envelope-replacement viruses adopt different immunization methods and strategies. After 21 days of immunization, 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. Among them, the nasal drip method of immunization mainly activates the mucosal immune response and produces strong IgA specific antibodies. At the same time, the nasal drip immunization also induces a systemic antibody immune response (Figure 2A). The statistical results can be seen from the figure. It is concluded that 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). Further, 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). It was found that the inactivated capsule replacement candidate vaccine also produced higher specific humoral immunity There is no significant difference in response level compared with the live virus immunization group, which further shows that 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. At the same time, when 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. Therefore, 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.

Example 4 Immune Serum Neutralizing Antibody Detection Based on Coronavirus Pseudovirus System

Determine the neutralizing antibody titers produced by in vitro virus neutralization experiments to evaluate the difference in antigen selection, screen out highly effective protective immunogens, and compare the neutralization of SARS or SARS-CoV-2 produced by different groups. To determine the optimal vaccine preparation strategy and vaccination method for the titer of the antibody, the specific operation steps are as follows:

Extinguish the serum to be tested at 56°C for 30 minutes, centrifuge at 6000g for 3 minutes, and take the supernatant for use;

Passage 293T-hACE2 cells, count the cells and add 2E4cells/well to a 96-well plate (200μL/well, containing 8μg/mL polybrene);

After 3 hours, the antibody was serially diluted (1:2) 10μL/tube with Opti-MEM. At the same time, a positive control without antibody (20μL virus solution, final virus concentration 4E5TU/mL) and a negative control without virus (20μL 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;

Take 10μL of the diluted virus solution (8E5TU/mL) and add it to the 10μL serially diluted antibody in step 2 (1:1 pipetting and mixing) (at this time, the final virus concentration is 4E5pfu/mL);

After incubating in a 37°C, 5% CO2 incubator for 1 hour, it was added to 293T-hACE2 cells and infected for 24h-48h to observe the fluorescence and pathological changes.

The serum neutralization titer is determined according to the dilution factor of the antibody serum corresponding to the hole with the last green fluorescence.

As shown in Figure 3, in the PBS and VSV wild strain (VSV-WT) immunization group, in the in vitro neutralizing antibody detection test, no antibodies that can effectively neutralize the new coronavirus were detected. Compared with the control group, the envelope was replaced The mice immunized with the new crown vaccines VSV-△GS-C30 and VSV-△G-ECD-CA both induced neutralizing antibodies. The surface of the S-spike protein was displayed on the surface of the recombinant virus intact, which could not only activate the body to produce enough Specific antibodies (IgG, IgM), and the specific antibodies in the mouse serum after immunization have the ability to neutralize the virus in vitro. In the in vitro test, 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. At the same time, the two types of VSV-△GS-C30 and VSV-△G-ECD-CA After the live virus vaccine (same dose) was inactivated at high temperature (Figure 3), 21 days after muscle immunization, the serum neutralizing antibody titer was tested, and it was found that after the candidate envelope replacement vaccine was inactivated, it still induced a sufficient amount of The neutralizing antibody further proves that the use of the candidate vaccine in this example can alleviate the safety concerns about replicable viral vectors and replication-deficient viral vectors. 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.

Claims (10)

An envelope-replacement type virus vector vaccine, which is characterized in that: the envelope-replacement type virus vector vaccine replaces the GP gene in the rhabdovirus genome with a coronavirus spike protein S gene truncation or virus spike The extracellular segment fusion ECD-CA of the protein S gene is an envelope replacement virus vector vaccine constructed by the reverse genetic system to prevent coronavirus infection. The ECD-CA is defined as the viral spike protein S gene The extracellular segment ECD fuses the transmembrane and intracellular segment genes of the VSV virus envelope protein GP. The envelope-replaceable viral vector vaccine according to claim 1, wherein the rhabdovirus vector is selected from vesicular stomatitis virus VSV, the VSV is selected from the Indiana strain, and the spike protein S gene is selected From SARS or SARS-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 envelope-replaceable viral vector vaccine according to claim 2, wherein the spike protein S gene truncation is selected from C-terminal deletion genes, the number of C-terminal amino acid deletions is preferably 30, and the corresponding modification The amino acid sequence corresponding to the latter spike protein S is SEQ ID NO.1. The envelope-replaceable viral vector vaccine according to claim 1, wherein the rhabdovirus vector is selected from vesicular stomatitis virus VSV, the VSV is selected from the Indiana strain, and the spike protein S gene is The extracellular segment fusion ECD-CA is selected from coronaviruses, the ECD-CA includes the extracellular segment ECD of the coronavirus spike protein S gene, and the C-terminus of the ECD is fused with the transmembrane and the cell of the envelope protein of VSV. Inner segment gene CA. The envelope replacement virus vector vaccine of claim 4, wherein the ECD-CA is cloned into the coding reading frame 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 coronavirus envelope replacement virus vector vaccine according to claim 2, wherein the vesicular stomatitis virus is a virus obtained by mutating amino acids of its matrix protein M, and the amino acid mutation position in the matrix protein M The point is one or more of leucine at position 20, methionine at position 51, and phenylalanine at position 110 and are non-synonymous mutations. The coronavirus envelope-replaceable viral vector according to claim 4, wherein the viral vector is an attenuated vesicular stomatitis virus, and the matrix protein M of the vesicular stomatitis virus has a mutation at position 3 of the amino acid , The 20th position of the matrix protein M was mutated from leucine L to phenylalanine F, the 51st position was mutated from methionine M to alanine A, and the 110th position was mutated from phenylalanine F to Leucine L, 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: S1, using synthetic biology to obtain the S-CN truncated gene of the coronavirus as described in any one of claims 2, 3 or 6 or the ECD-CA envelope as described in any one of claims 1, 4, 5 or 7 Genes and gene fragments were digested with Mlu I and Xho I and cloned into the pVSV-3M plasmid multiple cloning site to obtain pCore-3M-S-CN or pCore-3M-ECD-CA plasmids, respectively. S-CN is positioned as the abbreviation of the corresponding gene sequence after the S gene of the coronavirus spike protein has deleted N amino acids at the C terminal, where N = any natural number from 18 to 72; S2. Transform 293T-ACE2 cells with a plasmid pCAGGS-T7 expressing T7-RNA polymerase; plasmids that remove endotoxins after 24 hours include pCAGGS-P, pCAGGS-N, pCAGGS-L and pCore-3M-S-CN or pCore-3M-ECD-CA plasmid preparation transfection mixture; S3. After the four plasmids were encapsulated and co-transfected with liposomes for 72 hours, the supernatant was filtered through a 0.22um filter membrane and then added to cells stably expressing ACE2 in the logarithmic growth phase; S4. If the cell has disease, collect the cell supernatant, and use RT-PCR technology to identify the copy number of the target gene in the viral genome to replace the envelope; S5. Plaque purification is performed with cells stably expressing ACE2, and the reproducible coronavirus envelope replacement vaccine VSV-△G-S-CN or VSV-△G-ECD-CA is obtained after western blotting identification. The method for constructing a coronavirus envelope-replaced viral vector vaccine according to claim 8, wherein the fluorescent probe primer pair that specifically amplifies the coronavirus S-CN gene in the RT-PCR in step S4 is Sequence SEQ ID NO.5, SEQ ID NO.6. The method for constructing a coronavirus envelope-replaced viral vector vaccine according to claim 8, wherein 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.

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