Novel coronavirus mucosal immune vaccine based on non-replicating vesicular stomatitis virus vector

Abstract

The invention discloses a novel coronal virus mucosal immune vaccine based on a non-replicating vesicular stomatitis virus vector. The vaccine is delivered through a mucosal immunization way; the vaccine can be used in new crown vaccines, and can also be used in combination with other respiratory tract infection viruses such as respiratory syncytial vaccines (RSV) and influenza. At present, preparation of respiratory mucosa vaccines, especially multivalent vaccines, from replication-deficient VSV is not reported yet. According to the invention, safety and immunogenicity evaluation is carried out on the constructed VSVMT-S2P non-replicating recombinant VSV new crown vaccine in healthy narrative golden hamster and CP immunosuppression hamster models, and the VSVMT-S2P vaccine is found to be very safe in healthy animals; and the protein is highly safe in CP immunosuppressed hamsters. The VSVMT-S2P vaccine is subjected to single inoculation in the body of the narrative golden hamster through intranasal and intramuscular injection respectively, and it is proved that the VSVMT-S2P vaccine can effectively stimulate comprehensive immune response.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of vaccines, and relates to a respiratory mucosal immune vaccine, in particular to a safe and efficient new coronavirus mucosal immune vaccine based on a non-replicative vesicular stomatitis virus carrier. BACKGROUND

[0002] The SARS-CoV2 virus continues to mutate, and the mutant strains show great differences in affinity with ACE2 receptors, transmission level and immune escape compared with the original strain. Among them, the branches BA.5 and XBB.1.5 of the Omicron strain have greater immune escape ability than the original strain, posing a great challenge to the effectiveness of vaccines. At present, various types of vaccines such as subunit vaccines, inactivated virus vaccines, RNA vaccines and adenovirus carrier vaccines have been approved for marketing worldwide, but they cannot effectively activate respiratory mucosal immunity and need to be inoculated repeatedly to take effect; due to the respiratory transmission characteristics of the Omicron strain, the development direction of the next generation of vaccines should be single-dose immunization and rapid local mucosal immunity.

[0003] The new coronavirus is infected through the respiratory tract, therefore, effectively activating mucosal immune antibodies such as IgA subtypes is crucial to contain its infection. At present, attenuated live vaccines and virus carrier vaccines are still the most effective vaccine strategies for stimulating mucosal immunity. Although the vaccine prepared by expressing the new coronavirus spike protein based on the adenovirus type 5 (ADV5) carrier can achieve mucosal immunity through the spray route, due to the widespread presence of ADV5 virus infection in the general population, there are pre-existing antibodies in the body, therefore, the protective efficacy of this type of vaccine in a large population still needs to be evaluated by clinical data. In view of the above situation, a safe and effective new coronavirus vaccine that activates human mucosal immunity is the focus of future research. SUMMARY

[0004] The application aims to provide a safe and efficient new coronavirus mucosal immune vaccine based on a non-replicative vesicular stomatitis virus carrier. MT The VSV MT -S2P) is constructed by knocking out and replacing the VSV genome VSV glycoprotein gene (G) with the S2P gene, and replacing it with the BA.1 and BA.5 strain S2P protein gene, which provides a theoretical and practical basis for developing safe and efficient new coronavirus mucosal monovalent and multivalent vaccines. MTThe significant advantages of S2P include at least: 1. further improved safety, which is crucial for some special groups, such as immunocompromised people; 2. the recombinant virus surface is provided with VSV-G in trans, and the recombinant virus has a more extensive tropism, because the receptor of VSV is low-density lipoprotein, which exists in most mammalian cells. This feature also helps multiple immunization routes, such as respiratory tract nasal drops / spray immunization and muscle injection immunization, because, even if the expression level of the ACE2 receptor in human muscle cells is low, VSV MT S2P expression can also effectively present to T cells.

[0005] The purpose of the present application is achieved by the following technical solutions:

[0006] <First aspect>

[0007] The present application provides a respiratory infection VSV recombinant virus plasmid based on a replication-defective vector, which is delivered by a mucosal immunization route.

[0008] As an embodiment, the respiratory infection virus includes respiratory syncytial virus, influenza virus, and new coronavirus.

[0009] As an embodiment, the replication-defective vector is a recombinant vesicular stomatitis virus with a knocked-out VSV glycoprotein gene.

[0010] As an embodiment, the recombinant vesicular stomatitis virus is a recombinant virus VSV MT with mutations at three different amino acid sites of matrix protein M; the 51st methionine of the gene protein M is knocked out, the 221st amino acid-valine is mutated to phenylalanine, and the 226th amino acid-glycine is mutated to arginine.

[0011] As an embodiment, the VSV MT glycoprotein gene G of the VSV genome is knocked out and replaced with an S2P gene to construct the VSV recombinant virus plasmid.

[0012] As an embodiment, the S2P gene is at least one of the S2P protein genes of the Omicron variant BA.1 strain, the BA.5 strain, the original strain of the new coronavirus, and the XBB subvariant strain.

[0013] <Second aspect>

[0014] The present application provides a use of the aforementioned VSV recombinant virus plasmid in the preparation of a respiratory infection mucosal immunization vaccine or in the preparation of a drug for treating a respiratory infection.

[0015] <Third aspect>

[0016] The application provides a new coronavirus mucosal immunity vaccine, which is constructed by knocking out a VSV MT The G gene in the genome of the VSV is removed and replaced with at least one of S2P protein genes of an Omicron variant BA.1 strain, a BA.5 strain, a new coronavirus ancestral strain and an XBB subtype strain.

[0017] <Fourth aspect>

[0018] The application provides a preparation method of the aforementioned new coronavirus mucosal immunity vaccine, which comprises the following steps:

[0019] S1, the S protein genes of BA.1 and BA.5 are cloned into a eukaryotic expression plasmid pIRES after being optimized according to human codons; a mutant gene is constructed by using an overlap PCR method, and the K986 and V987 residues of the S protein are replaced with two prolines to generate a stable fusion pre-conformation mutant S2P;

[0020] S2, based on a VSV MT vector, the G gene in the genome of the VSV MT is removed and replaced with an S Δ21 2P gene to construct a replication-defective recombinant virus expressing S Δ 21 2P; the S Δ21 is a S protein variant with 21 amino acids deleted from the carboxyl terminal.

[0021] As an embodiment, the G gene in the genome of the VSV MT is removed by using XhoI and MluI restriction endonucleases, and the S Δ21 2P gene of the BA.1 and BA.5 strains is used for replacement;

[0022] and / or, the S MT 2P gene of the new coronavirus ancestral strain is cloned into the genome of the VSV Δ21 by using XhoI and NheI sites existing between the G and L protein genes in the VSV MT genome;

[0023] and / or, the S MT 2P gene of the XBB subtype strain is cloned by using MluI and XhoI sites existing between the G and L protein genes in the VSV Δ21 genome.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] 1) the application constructs a new coronavirus mucosal immunity vaccine by using a VSV MTThe genome of VSV glycoprotein gene (G) was knocked out and replaced with S2P gene, and the BA.1 and BA.5 strain S2P protein gene was cloned into the genome of VSV to construct a non-replicating recombinant virus (VSV MT -S2P), which provides a theoretical and practical basis for the development of safe and efficient new coronavirus mucosal monovalent and multivalent vaccines.

[0026] 2) In the context of the new coronavirus epidemic, the effectiveness and safety of existing vaccines in immunocompromised populations also need to be studied. Cyclophosphamide (CP) is an alkylating agent, which is a commonly used immunosuppressive agent for the treatment of autoimmune diseases and tumors; in addition, CP is also commonly used to establish immunocompromised animal models to evaluate the efficacy of antiviral drugs and vaccines. The present application will evaluate the safety and immunogenicity of VSV MT -S2P non-replicating recombinant VSV new coronavirus vaccine in healthy Syrian golden hamsters and CP immunosuppressed hamster models, and the results are as follows:

[0027] a) VSV MT -S2P vaccine is very safe in healthy animals.

[0028] b) VSV MT -S2P vaccine is highly safe in CP immunosuppressed hamsters.

[0029] c) VSV MT -S2P vaccine was single-dosed in healthy Syrian golden hamsters by intranasal and intramuscular routes, respectively, and was confirmed to effectively stimulate a comprehensive immune response. Antibody and T cell responses against the spike protein were detected in healthy animals inoculated with VSV MT -S2P.

[0030] 3) The S2P genes of the new coronavirus ancestral strain and the S2P genes of the Omicron virus new strain were cloned into the VSV genome at the same time to prepare a bivalent vaccine that can express the S2P antigens of the new coronavirus ancestral strain and the new strain at the same time, which is the first time to use VSV as a carrier, especially to use non-replicating VSV MT as a carrier to prepare a multivalent new coronavirus vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0031] Other features, objects, and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments, which should be read in light of the accompanying drawings:

[0032] Figure 1 Schematic diagram of the pre-fusion stable conformation mutant protein (S2P) of the S protein of the new coronavirus;

[0033] Figure 2 Recombinant rVSV expressing fusion pre-conformation spike protein MT Schematic diagram of S2P virus construction

[0034] Figure 3 VSV MT Western Blotting identification of S2P virus; wherein, 1. Protein ladder; 2-5. Lysates of VSV MT S2P BA.1 , VSV MT -S BA.1 , VSV MT -S2P BA.5 , VSV MT -S BA.5 infected cells; 6. Lysate of VSV infected cells; 7. Control

[0035] Figure 4 Non-replicative VSV MT S2P BA.1 and VSV MT S2P BA.5 Identification of virus tropism and replication characteristics in cells

[0036] Figure 5 Grouping and immunization of healthy Syrian golden hamsters

[0037] Figure 6 Body weight changes of healthy Syrian golden hamsters after intranasal immunization with VSV 7 S2P virus at doses of 1×10 6 / 100 μL and 1×10 MT / 100 μL

[0038] Figure 7 Changes in blood WBC and W-LCR indicators of healthy Syrian golden hamsters after intranasal immunization with VSV 7 S2P virus at doses of 1×10 6 / 100 μL and 1×10 MT / 100 μL

[0039] Figure 8 Changes in blood WBC and W-LCR indicators of healthy Syrian golden hamsters after intranasal immunization with VSV 7 S2P virus at doses of 1×10 6 / 100 μL and 1×10 MTDetection of viral load in tissues after VSV-S2P infection; turbinate, lung, brain tissues were collected on day 2, 4, 6 after intranasal immunization. The tissues were grinded in a homogenizer into 1 mL trizol solution, after extraction of tissue RNA, reverse transcription into cDNA, detection of CT value of target VSV-N gene in tissues by RT-qPCR; wherein, (A) viral load in turbinate, (B) viral load in lung, (C) viral load in brain;

[0040] Figure 9 Intranasal immunization of healthy Syrian golden hamsters with 1×10 6 / 100 μL and 1×10 5 / 100 μL dose of VSV MT -S2P and VSV MT Detection of neutralizing antibody in blood after VSV-S virus; IN: intranasal immunization; IM: intramuscular injection;

[0041] Figure 10 Intranasal immunization of immunosuppressed Syrian golden hamsters with 2.5×10 7 / 100 μL and 2.5×10 6 / 100 μL dose of VSV MT -S2P after body weight change;

[0042] Figure 11 Intranasal immunization of immunosuppressed Syrian golden hamsters with 2.5×10 7 / 100 μL and 2.5×10 6 / 100 μL dose of VSV MT -S2P after serum neutralizing antibody titer. DETAILED DESCRIPTION

[0043] The present application will be described in detail below with examples. The following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of adjustments and improvements can be made. These are within the scope of the present application.

[0044] Example 1, construction and in vitro identification of VSV MT -S2P recombinant virus

[0045] 1.1. Construction of S2P gene

[0046] The S protein gene of Omicron variant BA.1 and BA.5 was optimized by human codon optimization (GenBank: OX008556.1; OP984772.1) and then cloned into the eukaryotic expression plasmid pIRES; the corresponding primers were designed, and the mutant gene was constructed by overlap PCR method to replace the K986 and V987 residues of S protein with two prolines to generate a stable fusion pre-conformation mutant (S2P).

[0047] F: 5'-ATACGACTCACTATAGGCTAGCATGTTCGTCTTCCTGGTCCTGC-3' SEQ ID NO. 5;

[0048] R1: 5'-CTCTGGAGGGTCCAGCCGGCTCAGGAT-3' SEQ ID NO. 6;

[0049] F1: 5'-GCCGGCTGGACCCTCCAGAGGCAGAGGTGCAGATCGAC-3' SEQ ID NO. 7;

[0050] R: 5'-GATGCATGCTCGACGCGTGAATTCTTAGGAGCCACAGCTACAGCAG-3' SEQ ID NO. 8;

[0051] The construction method is as follows: taking the synthesized optimized sequence as the template, F and R1 as the primers, PCR synthesizes S2P gene upper segment A. Taking F1 and R2 as the primers, PCR synthesizes S2P gene lower segment B. Taking PCR product A segment and B segment as the template, F and R as the primers, Overlap PCR synthesizes S2P gene full length. The PCR reaction conditions are as follows: pre-denaturation: 98℃ 5min, denaturation 98℃ 30s, annealing 60℃ 30s, extension 72℃ 4min, final extension 72℃ 10min. Among them, denaturation, annealing and extension are 30 cycles.

[0052] The S gene of Omicron BA.1 and BA.5 strains was designed and synthesized according to human codon optimization. At the same time, in order to ensure that the S protein can be successfully inserted into the envelope of VSV virus, the carboxy terminus of the S protein was truncated by 21 amino acids, that is, SΔ21. The amino acid sequence of SΔ21 protein of BA.1 strain of novel coronavirus is SEQ ID NO. 1; the optimized expression gene sequence is SEQ ID NO. 2. The amino acid sequence of SΔ21 protein of BA.5 strain of novel coronavirus is SEQ ID NO. 3; the optimized expression gene sequence is SEQ ID NO. 4.

[0053] Figure 1Schematic diagram of pre-fusion conformational mutant protein (S2P) of S protein of novel coronavirus. Among them, Omi: wild type S protein of novel coronavirus omicron lineage; Omi S2P : mutant S protein of novel coronavirus omicron lineage; both lysine at position 986 and valine at position 987 of S protein are mutated into two prolines.

[0054] 1.2. Recombinant rVSV MT -S2P virus construction

[0055] Based on the attenuated VSV virus vector (VSV MT ) with three-site mutation of M protein, a replication-defective recombinant virus expressing S Δ21 2P (abbreviated as S2P) was constructed. In order to ensure that S2P protein is effectively incorporated into VSV envelope, 21 amino acids at the carboxy terminus of S2P protein were deleted. Subsequently, S2P genes of BA.1 and BA.5 strains were cloned into pVSV MT -GFP plasmid, as shown in Figure 2 , the G gene in VSV genome was removed using XhoI and MluI restriction enzymes, and replaced with S or S2P gene of BA.1 and BA.5 strains, and these plasmids were named as pVSV MT -S2P BA.1 , pVSV MT -S2P BA.5 ; by using XhoI and NheI sites existing between G and L protein genes in VSV gene, S2P gene of novel coronavirus ancestral strain was cloned into VSV genome, and named as pVSV MT -S2P XBB -S2P WH , to prepare a bivalent vaccine capable of simultaneously expressing S2P antigens of novel coronavirus ancestral strain and emerging strain, which is also the first time to use VSV as a carrier, especially to use non-replicative VSV MT as a carrier to prepare a multivalent novel coronavirus vaccine. Figure 2 Recombinant rVSV MT -S2P virus construction. Residues K986 and V987 of spike protein (S) were mutated into two prolines to form S mutant; in addition, 21 amino acids at the carboxy terminus of the protein were deleted (S Δ21 2P), and the protein is referred to as S2P. VSV MTGenome encodes nucleocapsid protein (N), phosphoprotein (P), glycoprotein (G), matrix protein (M) and RNA polymerase (L); mutant M protein (MT) contains three mutations (M51 deletion, V221F and S226R). The VSV G protein gene was replaced by the S2P mutant gene of omicron variant BA.1 or BA.5 by MluI and XhoI restriction enzyme digestion; in pVSV MT -S2P XBB -S2P WH The XBB sublineage strain S2P was cloned in the plasmid between the MluI and XhoI sites, and the S2P gene of the ancestral strain of the new coronavirus was cloned between the XhoI and NheI restriction sites between the G and L genes of the VSV genome.

[0056] The above plasmids were used for virus rescue in BHK21 cells. BHK21 cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3) for 1 hour. Then the infected cells were co-transfected with the above-mentioned pVSV MT -S2P plasmid and helper plasmids pBS-N, P, L and G. After 48 hours of transfection, the viral supernatant was filtered through a filter with a pore size of 0.2 μm to remove residual vaccinia virus and passaged onto BHK-21 cells transfected with the pCAGGS-G plasmid (BHK21-G). After 48 hours, the supernatant was collected after observing the cytopathic effect. VSV MT -S2P viruses were plaque purified and then amplified in BHK-G cells, so the resulting viral particles had G protein on their surface and had broad-spectrum infective properties. The replication-deficient VSV MT -S2P viruses were titrated using BHK21-G cells.

[0057] 1.3. Identification of recombinant rVSV MT -S2P

[0058] To detect the expression of the S2P protein, VSV MT -S2P BA.1 or VSV MT -S2P BA.5 were inoculated at an infection index of MOI = 1 in VeroE6 cells, and after 24 hours the cell lysate was collected and subjected to WB with antibodies specific for SARS-CoV2 S1. Figure 3 VSV MT -S2P virus Western Blotting identification; VeroE6 cells were infected with VSV MT -S2P or VSV MT-S virus. Cells were then scraped and lysed 24 hours post-infection and identified by Western blotting using an S protein-specific antibody. Results are as follows... Figure 3 As shown, in non-replicated VSV MT -S2P BA.5 and VSV MT -S2P BA.1 Bands of S1 protein (approximately 110 kDa) and S protein (approximately 180 kDa) were detected in both lanes of the virus. Therefore, rVSV... MT The S2P protein of the non-replicating recombinant virus was successfully expressed and processed by furinase in the cell.

[0059] To support non-replicated VSV MT -S2P BA.1 and VSV MT -S2P BA.5 The viral tropism and replication characteristics in cells were identified, and VSV was... MT -S2P BA.1 or VSV MT -S2P BA.5 BHK21 cells or hACE2-BHK21 cells were infected with an infection index of MOI=1. Figure 4 Reflecting VSV MT -S2P cell tropism. rVSV MT hACE2-BHK21 or BHK21 cells were infected with S2P or rVSV-GFP seed virus (referred to as P1 generation) at MOI=1 24 hours post-infection. The supernatant was then collected (referred to as P2 generation) and inoculated into fresh hACE2-BHK21 or BHK21 cells. Cells were observed and photographed using a fluorescence microscope 24 hours post-infection. Results are as follows: Figure 4 As shown, VSV MT -S2P can infect BHK21 and hACE2-BHA21 cells. However, the virus cannot be passaged in BHK21 and hACE2-BHA21 cells.

[0060] Example 2, VSV MT Safety and immunogenicity of S2P in healthy hamsters

[0061] All animal studies were conducted in accordance with the protocol approved by the Shanghai Veterinary Research Institute (SV-20231201-02) and the ethics guidelines of Shanghai Jiao Tong University. SPF female Syrian golden hamsters were purchased from Charles Rivers. Virus inoculation was performed under anesthesia induced and maintained with ketamine hydrochloride and toluenethiazide to minimize animal suffering.

[0062] 2.1VSV MT-Safety experiment of S2P in healthy hamsters

[0063] Healthy Syrian golden hamsters were divided into groups as follows: Figure 5 As shown, select 1×10 7 / 100μL and 1×10 6 Two doses of 100μL each were administered intranasally as a VSV immunization. MT -S2P virus; investigating non-replicating VSV using daily weight changes, blood indicators, and organ viral load as indicators. MT -S2P BA.5 and VSV MT -S2P BA.1 The toxicity of the virus.

[0064] Syrian golden hamsters were immunized with nasal drops, and their weight changes were monitored daily until 28 days post-immunization. Results were as follows: Figure 6 As shown: Non-replicated VSV MT -S2P nasal drops resulted in a gradual increase in body weight. By day 28 post-immunization, the weight had increased from 100% of the initial body weight to approximately 140%, with no significant difference compared to the PBS group (p>0.05).

[0065] White blood cell count (WBC) and white blood cell line density (W-LCR) are crucial blood indicators reflecting the body's inflammation and immune status. Blood samples were collected from the orbital rim of LVG hamster on days 2, 4, and 6 after intranasal immunization to measure WBC and W-LCR levels. In healthy hamsters, the normal range for WBC is 5 × 10⁻⁶. 9 / L~15×10 9 / L. Nasal drops for non-replicating VSV MT Following S2P, the number of white blood cells (WBCs) in the blood of Syrian golden hamsters remained within the normal range, similar to the PBS group. The normal W-LCR in healthy Syrian golden hamsters ranges from 4% to 50%. Intranasal immunization with non-replicating VSV... MT Following S2P, the w-LCR in the blood of Syrian golden hamsters fluctuated between 5% and 20%, showing no significant difference from the PBS group (p>0.05). The w-LCR in other hamster groups also remained within the normal range without any abnormalities. Figure 7 ).

[0066] To further investigate the virulence of the virus, non-replicating VSV was studied. MTViral load in tissues following S2P intranasal immunization was detected. Viral load in the nasal turbinates, lungs, and brain tissues of Syrian golden hamsters was measured on days 2, 4, and 6 post-immunization using plaque assays and Real-time PCR. After collection, the tissues were directly homogenized into 1 mL of PBS, and the viral vector was detected using a plaque assay. Simultaneously, the tissues were homogenized into 1 mL of Trizol solution, and RNA was extracted, reverse transcribed into cDNA, and then subjected to RT-qPCR. The VSV-N gene primers were: upstream: 5'-TGATCGACTTTGGATTGTCTTCTAA-3', downstream: 5'-TCTGGTGGATCTGAGCAGAAGAG-3'. Results are as follows: Figure 8 As shown, no viral residue was detected in the plaque assay in any of the groups (data not shown). Viral load in each tissue was detected by real-time PCR using the VSV-N gene, with the PBS group serving as a control. There was no significant difference in CT values ​​between the experimental groups and the PBS group, indicating that nasal immunization with VSV... MT -S2P or VSV MT -S does not cause Syrian golden hamster target organ virus residue.

[0067] In summary, nasal drop immunotherapy for single-replicated VSV MT -S2P and Replicated VSV MT -S strains do not have toxic effects on healthy Syrian golden hamsters.

[0068] 2.2VSV MT Immunogenicity assay of S2P in healthy Syrian golden hamsters

[0069] First, the healthy Syrian golden hamsters were divided into groups, as shown in the following grouping diagram. Figure 5 As shown. The experiment selected 1×10 6 and 1×10 5 Healthy Syrian golden hamsters were immunized with two doses via nasal drop and intramuscular injection. Blood samples were collected every 7 days for serum neutralizing antibody testing. Animals were euthanized 28 days post-immunization, and bronchoalveolar lavage fluid was collected for lung neutralizing antibody testing. The experiment used a replicative VSV... MT -S is the control group.

[0070] Blood was collected via the orbital rim at 7, 14, 21, and 28 days post-vaccination. Serum was then centrifuged at 5000 rpm for 30 minutes at room temperature for later use.

[0071] Method for detection of neutralizing antibodies: Sera from experimental animals were heat treated at 56°C for half an hour before testing and serially diluted two-fold starting from 1 :10 in DMEM. Bronchoalveolar lavage fluid (BALF) was also serially diluted two-fold starting from 1 :2. To determine the neutralizing antibody titers, 50 μL of diluted serum or BALF sample was mixed with 50 μL of DMEM containing 100 PFU of VSV ΔG -S Δ21 -GFP prepared from the respective BA.1, BA.5 strain, the mixture was incubated at 37°C for 1 hour before adding to VeroE6 cells cultured in a 96-well plate. After one hour, the cells were washed three times with DPBS and supplemented with DMEM medium containing 2% FBS. Twenty-four hours (h.p.i.) after inoculation, the neutralizing antibody titers were calculated by fluorescent microscopy observation. Titers were recorded as the reciprocal of the highest antibody dilution that could 100% inhibit the cytopathic effect. Neutralizing antibody titers were calculated using GraphPad Prism 8 software.

[0072] Results are shown in Figure 9 Figure 2. Neutralizing antibody titers in blood of Syrian golden hamsters after a single vaccination with VSV MT -S2P. Neutralizing antibody titers in blood of Syrian golden hamsters after a single vaccination with VSV 2 -S2P by intranasal route. Neutralizing antibody titers in blood of Syrian golden hamsters after a single vaccination with VSV MT -S2P by intramuscular route. Neutralizing antibody titers in blood of Syrian golden hamsters after a single vaccination with VSV 3 -S2P by intramuscular route were higher than those induced by intranasal route.

[0073] In conclusion, non-replicative VSV MT -S2P can induce blood neutralizing antibodies in hamsters but not in BALF. In contrast, replicative VSV MT -S can induce both blood and mucosal antibodies.

[0074] Example 3, Safety and immunogenicity of VSV MT -S2P in immunosuppressed hamsters

[0075] 3.1 Safety of VSV MT -S2P in immunosuppressed Syrian golden hamsters

[0076] In the context of the COVID-19 pandemic, immunocompromised populations worldwide are a vulnerable group. Their weakened immune systems may lead to reduced responsiveness to traditional vaccines, exposing them to greater risks of infection and severe illness. Therefore, the efficacy and safety of next-generation vaccines in immunocompromised populations are of great concern. Cyclophosphamide (CP) is an alkylating agent that has been used clinically to treat cancer and autoimmune diseases. CP is also commonly used to establish immunocompromised animal models for viral infection and vaccine efficacy evaluation. Therefore, this study used a CP-induced immunocompromised Syrian golden hamster model to evaluate VSV. MT -S2P and VSV MT -Safety and immune response levels of the S virus.

[0077] The experiment first determined the optimal dosage of cyclophosphamide, a drug for immunosuppression, by administering intraperitoneal injections of different concentrations of cyclophosphamide (140 mg / kg, 100 mg / kg, and 70 mg / kg) with PBS. The level of immunosuppression was assessed using white blood cell counts, ranging from 1.0 to 2.0 × 10⁻⁶. 9 / L is set as moderate immunosuppression, with a white blood cell count below 1.0 × 10⁻⁶. 9 / L was set as severe immunosuppression. After comprehensive evaluation, cyclophosphamide at 100 mg / kg was selected for the trial to establish an immunodeficiency model to evaluate VSV. MT -S2P's safety and immunogenicity, while using VSV MT -S and VSV ΔG -S is the control group.

[0078] The experiment first divided the immunosuppressed animals into groups, as shown in Table 1, selecting 1×10 7 and 1×10 6 Two doses of nasal drops for VSV immunization MT -S2P, and with VSV MT -S and VSV ΔG -S represents the control group. Daily monitoring of weight changes and testing of blood WBC and W-LCR are used to indicate immune status.

[0079] Weight changes were monitored routinely until 28 days post-immunization, with results as follows: Figure 10 As shown, high-dose VSV MT -S2P intranasal immunization resulted in a 20% increase in body weight 28 days later, which was not significantly different from the PBS group (p>0.05). In contrast, wild-type VSV... ΔG -S immunization did not result in weight gain, which was significantly lower than in the PBS group and VSV group. MT -S2P group and VSV MT -S group hamsters (p<0.05).

[0080] 3.2 VSV MT Immunogenicity of -S2P in immunosuppressed Syrian hamsters

[0081] The experiment first grouped the immunosuppressed animals, see Table 1, and selected 1 x 105PFU of VSV 6 -S2P and 1 x 105PFU of VSV 5 -S for intranasal immunization. Blood was collected every 7 days for serum neutralizing antibody detection, and the animals were euthanized 28 days post-immunization and the lung lavage was collected for neutralizing antibody detection. MT -S2P, and VSV MT -S as control. Blood was collected every 7 days for serum neutralizing antibody detection, and the animals were euthanized 28 days post-immunization and the lung lavage was collected for neutralizing antibody detection.

[0082] The experiment tested the humoral and mucosal immune responses in CP-induced hamsters immunized with VSV MT -S2P or VSV MT -S. Blood was collected by orbital bleeding at 7, 14, 21, and 28 days post-inoculation, and then the blood was centrifuged at 5000 rpm for 30 minutes at room temperature, and the serum was collected for neutralizing antibody detection. The results showed that the serum neutralizing antibody titers were lower in CP-treated animals compared to healthy animals, and the peak of the neutralizing antibody titers was delayed Figure 11 ) compared to healthy animals. Replicating VSV MT -S induced higher levels of serum neutralizing antibodies in Syrian hamsters compared to non-replicating VSV MT -S2P. However, no neutralizing antibodies were detected in the lung lavage.

[0083] Table 1: Grouping of immunosuppressed Syrian hamsters and immunization

[0084]

[0085] The above describes specific embodiments of the present application. It should be understood that the present application is not limited to the above specific embodiments, and those skilled in the art can make various modifications or changes within the scope of the claims, which does not affect the essential content of the present application.

Claims

1. A respiratory infection VSV recombinant virus plasmid based on a replication-defective vector, which is delivered through a mucosal immunization route.

2. The VSV recombinant viral plasmid according to claim 1, wherein, The respiratory infection virus includes respiratory syncytial virus, influenza virus, and new coronavirus.

3. The VSV recombinant viral plasmid of claim 1, wherein, The replication-defective vector is a recombinant vesicular stomatitis virus with a knocked-out VSV glycoprotein gene.

4. The VSV recombinant viral plasmid of claim 3, wherein, The recombinant vesicular stomatitis virus is a recombinant virus VSV with mutations at three different amino acid sites of matrix protein M MT The 51st methionine of the matrix protein M is knocked out, the 221st amino acid-valine is mutated to phenylalanine, and the 226th amino acid-glycine is mutated to arginine.

5. The VSV recombinant viral plasmid of claim 4, wherein, The VSV MT The VSV recombinant virus plasmid was constructed by knocking out the VSV genome glycoprotein gene G and replacing it with the S2P gene.

6. The VSV recombinant viral plasmid of claim 5, wherein, The S2P gene is at least one of the S2P protein genes of the Omicron variant BA.1 strain, BA.5 strain, new coronavirus ancestral strain, and XBB subvariant strain.

7. Use of the VSV recombinant virus plasmid of any one of claims 1-6 in the preparation of a respiratory infection mucosal immunization vaccine or a drug for treating a respiratory infection.

8. A SARS-CoV mucosal immunization vaccine, characterized in that, VSV MT The VSV genome is knocked out of the VSV glycoprotein gene G and replaced with at least one of the S2P protein genes of the Omicron variant BA.1 strain, the BA.5 strain, the ancestral strain of the new coronavirus, and the XBB subvariant strain.

9. A method of preparing the novel coronavirus mucosal immunization vaccine as claimed in claim 8, wherein, The method comprises the following steps: S1, after the S protein genes of BA.1 and BA.5 are optimized according to human codons, the genes are cloned into a eukaryotic expression plasmid pIRES; a mutant gene is constructed by using an overlap PCR method to replace the K986 and V987 residues of the S protein with two prolines to generate a stable fusion pre-conformation mutant S2P. S2, based on VSV MT Vector, VSV MT G gene in the genome and replaced with S Δ21 2P gene, construct expressing S Δ21 Replication-defective recombinant virus of S2P; S Δ21 S protein variant with a 21 amino acid carboxy terminal deletion.

10. The method of claim 9, wherein the SARS-CoV mucosal immunization vaccine is prepared by, VSV MT G gene in the genome and S Δ21 2P gene replacement; And / or, via VSV MT The XhoI and NheI sites existing between the G and L protein genes in the gene will cause the S protein in the original SARS-CoV-2 ancestral strain to be affected. Δ21 2P gene cloned into VSV MT Genome; and / or, by cloning XBB sublineage strains S MT 2P gene between the Mlul and Xhol sites present between the G and L protein genes in the VSV Δ21 2P gene.

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