Rabies virus inactivated vaccine and preparation method thereof

Abstract

The invention provides a rabies virus inactivated vaccine and a preparation method thereof, and belongs to the technical field of biological medicines. The invention provides a rabies virus inactivated vaccine. The rabies virus inactivated vaccine comprises an adjuvant and an inactivated VSV-rabies recombinant virus, the inactivated VSV-rabies recombinant virus is obtained by carrying out inactivation on a VSV-rabies recombinant live virus; the VSV-rabies recombinant live virus is prepared by replacing envelope protein G of vesicular stomatitis virus with G protein of rabies virus. The inactivated rabies virus vaccine is good in safety and high in induced antibody level, the immune effect of the inactivated rabies virus vaccine is equivalent to that of a live virus vector vaccine, and the inactivated rabies virus vaccine has a certain application value in the aspect of preventing and controlling rabies.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an inactivated rabies virus vaccine and a preparation method thereof. Background Art

[0002] Rabies is an acute infectious disease of the central nervous system caused by rabies virus infection. Rabies virus mainly invades the human body through damaged skin or mucous membranes. The clinical features are encephalomyelitis, mostly manifested as specific fear of wind, fear of water, pharyngeal muscle spasm, progressive paralysis, etc. Vaccination before or after exposure to rabies is an effective preventive intervention measure. Rabies vaccines on the market are mainly obtained by culturing rabies virus in cells or chicken embryos, and then inactivating and purifying RABV particles. The production process of these vaccines has high requirements for safety levels. In addition, although these vaccines have good protective effects, the best time to start injection is within 24 hours of being bitten. The conventional injection process is divided into 5 times, one injection each on the 1st day, 3rd day, 7th day, 14th day, and 30th day. Multiple vaccinations are required, and the procedure is cumbersome. Summary of the invention

[0003] The purpose of the present invention is to provide an inactivated rabies virus vaccine and a preparation method thereof. The inactivated rabies virus vaccine of the present invention has a simple vaccination procedure, a high level of induced antibodies, and an immune effect equivalent to that of a live virus vector.

[0004] The present invention provides an inactivated rabies virus vaccine, comprising an adjuvant and an inactivated VSV-rabies recombinant virus; the inactivated VSV-rabies recombinant virus is obtained by inactivating the VSV-rabies recombinant live virus;

[0005] The VSV-rabies recombinant live virus is obtained by replacing the envelope protein G of the vesicular stomatitis virus with the G protein of the rabies virus;

[0006] The adjuvant includes M103 adjuvant or gel02 adjuvant.

[0007] Preferably, the amino acid sequence of the rabies virus G protein is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0008] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the rabies virus G protein is shown as SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.

[0009] Preferably, the vesicular stomatitis virus is the vesicular stomatitis virus strain Indiana.

[0010] Preferably, the amino acid sequence of the G protein of the vesicular stomatitis virus is shown in SEQ ID NO.7.

[0011] Preferably, the adjuvant accounts for 15% of the volume of the inactivated rabies virus vaccine; the volume ratio of the inactivated VSV-rabies recombinant virus and the adjuvant is (3:1) to (17:3).

[0012] The present invention also provides a method for preparing the inactivated rabies virus vaccine described in the above scheme, comprising the following steps:

[0013] The envelope protein G of the vesicular stomatitis virus is replaced with the G protein of the rabies virus to obtain a recombinant virus full-length plasmid of the vesicular stomatitis virus carrying the G protein of the rabies virus;

[0014] First, the cells to be infected are infected with a poxvirus expressing T7 polymerase, and the infected cells are co-transfected with the recombinant virus full-length plasmid and the helper plasmid, and the supernatant of the diseased cells is collected after co-transfection;

[0015] The supernatant is used to infect the second cells to be infected, and after amplifying the virus, the virus-infected cell culture supernatant is collected to obtain the VSV-rabies recombinant live virus;

[0016] Inactivating the VSV-rabies recombinant live virus to obtain an inactivated VSV-rabies recombinant virus;

[0017] The inactivated VSV-rabies recombinant virus is mixed with an adjuvant to obtain an inactivated rabies virus vaccine.

[0018] Preferably, replacing the envelope protein G of the vesicular stomatitis virus with the G protein of the rabies virus comprises: cloning a nucleic acid molecule encoding the G protein of the rabies virus into the envelope protein G gene position of the vesicular stomatitis virus by homologous recombination and replacing the ORF region of the G protein gene;

[0019] Preferably, the first cells to be infected are BHK-21 cells; and the second cells to be infected are Vero cells.

[0020] The present invention also provides a neutralizing antibody, which is induced by the inactivated rabies virus vaccine described in the above scheme.

[0021] The present invention provides an inactivated rabies virus vaccine, comprising an adjuvant and an inactivated VSV-rabies recombinant virus; the inactivated VSV-rabies recombinant virus is obtained by inactivating the VSV-rabies recombinant live virus; the VSV-rabies recombinant live virus is obtained by replacing the envelope protein G of the vesicular stomatitis virus with the G protein of the rabies virus. The inactivated rabies virus vaccine of the present invention overcomes the safety problem caused by the potential neurotoxicity of the VSV live virus vector vaccine, has the advantages of good safety and high induced antibody level, is equivalent to the live virus vector vaccine, and has certain application value in the prevention and control of rabies. It has been verified that the inactivated rabies virus vaccine of the present invention adopts "0+7" two immunizations, and the number of vaccinations is small. In the mouse experiment, it can be seen that the inactivated vaccine and the live virus vector vaccine immunization can produce a comparable neutralizing antibody level after "0+7" two immunizations. Therefore, the inactivated rabies virus vaccine of the present invention has a high titer, and after vaccination, it is expected to solve the problem of the cumbersome existing vaccination procedures. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 It is a schematic diagram of the construction of the reverse genetic recombination plasmid of the VSV vector rabies recombinant virus in the present invention;

[0024] Figure 2 The VSV-CTNG and VSV-SADG recombinant viruses rescued by the reverse genetics system were used to infect Vero, Vero-E6 and BHK21 cells, respectively, and the cytopathic effects were observed after 72 hours.

[0025] Figure 3 The figure is a recombinant virus growth curve drawn by measuring the virus titer at each time point after VSV-CTNG and VSV-SADG recombinant viruses infected Vero cells for 24h, 48h, 72h and 96h respectively;

[0026] Figure 4 This is the change in the weight of mice 10 days after intracranial inoculation of VSV-CTNG and VSV-SADG recombinant viruses into mice;

[0027] Figure 5 is the survival of mice 10 days after intracranially inoculating mice with VSV-CTNG and VSV-SADG recombinant viruses;

[0028] Figure 6The inactivated VSV-CTNG was used to infect Vero cells and the cells were cultured for three generations to produce cytopathic effects. The Control group was a blank control group that was not infected with the virus.

[0029] Figure 7 The weight change of mice 14 days after immunization with the inactivated rabies vaccine provided by the present invention;

[0030] Figure 8 This is a graph showing the results of measuring the neutralizing antibody levels produced in mice on days 21 and 28 after the mice were immunized with the inactivated rabies vaccine provided by the present invention. DETAILED DESCRIPTION

[0031] The invention provides an inactivated rabies virus vaccine, comprising an adjuvant and an inactivated VSV-rabies recombinant virus; the inactivated VSV-rabies recombinant virus is obtained by inactivating a VSV-rabies recombinant live virus; the VSV-rabies recombinant live virus is obtained by replacing the envelope protein G of the vesicular stomatitis virus with the G protein of the rabies virus; the adjuvant comprises an M103 adjuvant or a gel02 adjuvant.

[0032] The inactivated rabies virus vaccine of the present invention and the adjuvant are used in combination, have good safety, induce high antibody levels, and have an immune effect equivalent to that of a live virus vector vaccine, and have certain application value in preventing and controlling rabies.

[0033]

[0034] In the present invention, the codon optimization is to optimize the base sequence of the virus according to the base preference of eukaryotic protein expression, which is convenient for the subsequent preparation of the virus on eukaryotic cells and used for production, and is more conducive to protein expression and preparation of recombinant virus stock solution, thereby improving the titer of the virus stock solution.

[0035]

[0036]

[0037] The rabies strain selected by the present invention is CTN-1 strain, SAD B19 strain or CVS11 strain. Among them, CTN-1 strain was isolated from the brain tissue of a patient who died of rabies in 1956, and is the most recent isolation among all vaccine strains; compared with other rabies vaccine strains, CTN-1 vaccine strain always maintains the highest homology with street virus variants, and the identity with the variant street virus strain decreases the slowest; considering the comprehensive time and homology with street viruses, CTN-1 strain is most suitable for use as a vaccine strain for preventing rabies. SAD B19 is also a commonly used vaccine strain at home and abroad, and CVS11 is a laboratory standard attack strain, which is specified by the World Health Organization (WHO) as a strain for rabies virus neutralizing antibody detection.

[0038]

[0039] In the specific implementation of the present invention, M103 is a nanocomposite water adjuvant and gel02 is a water adjuvant.

[0040] In the specific implementation process of the present invention, the adjuvant accounts for 15% of the volume of the inactivated rabies virus vaccine; the volume ratio of the inactivated VSV-rabies recombinant virus and the adjuvant is (3:1) to (17:3); the adjuvant can enhance the immunogenicity of the inactivated rabies virus vaccine.

[0041] The present invention also provides a method for preparing the inactivated rabies virus vaccine described in the above scheme, comprising the following steps:

[0042] The envelope protein G of the vesicular stomatitis virus is replaced with the G protein of the rabies virus to obtain a recombinant virus full-length plasmid of the vesicular stomatitis virus carrying the G protein of the rabies virus;

[0043] First, the cells to be infected are infected with a poxvirus expressing T7 polymerase, and the infected cells are co-transfected with the full-length recombinant virus plasmid and a helper plasmid, and the supernatant of the diseased cells is collected after co-transfection;

[0044] The supernatant is used to infect the second cells to be infected, and after amplifying the virus, the virus-infected cell culture supernatant is collected to obtain the VSV-rabies recombinant live virus;

[0045] Inactivating the VSV-rabies recombinant live virus to obtain an inactivated VSV-rabies recombinant virus;

[0046] The inactivated VSV-rabies recombinant virus is mixed with an adjuvant to obtain an inactivated rabies virus vaccine.

[0047] In the specific implementation process of the present invention, the G protein of VSV is replaced by the G protein of rabies virus, and the VSV-rabies recombinant live virus is obtained through virus rescue.

[0048] The invention first replaces the envelope protein G of vesicular stomatitis virus with the G protein of rabies virus to obtain a recombinant virus full-length plasmid of vesicular stomatitis virus carrying the G protein of rabies virus.

[0049] In the specific implementation process of the present invention, replacing the envelope protein G of the vesicular stomatitis virus with the G protein of the rabies virus includes: cloning the nucleic acid molecule encoding the G protein of the rabies virus into the envelope protein G gene position in the vesicular stomatitis virus by homologous recombination and replacing the ORF region of the G protein gene; the homologous recombination is performed using a homologous recombinase (Uniclone One Step Seamless Cloning Kit).

[0050] After obtaining the full-length recombinant virus plasmid, the first cells to be infected in the present invention are infected with a poxvirus expressing T7 polymerase, and the infected cells are co-transfected with the full-length recombinant virus plasmid and a helper plasmid, and the supernatant of the diseased cells is collected after co-transfection.

[0051] In the specific implementation process of the present invention, the first cell to be infected is a BHK-21 cell; the auxiliary plasmid is an auxiliary plasmid expressing VSV-N, VSV-P, VSV-L, and VSV-G.

[0052] After collecting the supernatant of the diseased cells after co-transfection, the present invention uses the supernatant to infect the second cells to be infected, amplifies the virus, collects the virus-infected cell culture supernatant, and obtains the VSV-rabies recombinant live virus.

[0053] In the specific implementation process of the present invention, the second cell to be infected is a Vero cell.

[0054] The VSV-rabies recombinant live virus of the present invention can be rescued by virus reverse genetics technology.

[0055] After obtaining the VSV-rabies recombinant live virus, the present invention inactivates the VSV-rabies recombinant live virus to obtain the inactivated VSV-rabies recombinant virus.

[0056] In the present invention, inactivating the VSV-rabies recombinant live virus includes: using β-propiolactone to inactivate the VSV-rabies recombinant live virus; the final concentration of the β-propiolactone is 0.05%; the inactivating the VSV-rabies recombinant live virus using β-propiolactone includes: mixing the VSV-rabies recombinant live virus liquid and β-propiolactone for inactivation; the present invention has no special restrictions on the inactivation conditions, and conventional conditions in the art can be used. The titer of the inactivated VSV-rabies recombinant virus obtained by the inactivation method of the present invention is greater than or equal to 10 6.0 TCID 50 / 50μL.

[0057] The inactivation method adopted by the present invention can directly act on viral nucleic acid to achieve the purpose of inactivation by changing the structure of viral nucleic acid, without directly acting on protein, because β-propiolactone is easily hydrolyzed, has no residue, and is harmless after hydrolysis. It has an ideal inactivation effect and a short inactivation time, and can shorten the vaccine production cycle. The structural integrity of rabies virus G protein is crucial to its immunogenicity, and the inactivation method can effectively ensure the structural integrity of rabies virus G protein.

[0058] After obtaining the inactivated VSV-rabies recombinant virus, the present invention mixes the inactivated VSV-rabies recombinant virus with an adjuvant to obtain an inactivated rabies virus vaccine.

[0059] In the present invention, the VSV-rabies recombinant live virus is inactivated and used in combination with an adjuvant as a vaccine, showing good safety. The inactivated VSV-rabies recombinant virus obtained by the preparation method of the present invention can further improve the immune effect, enhance the neutralizing antibody level, and enhance the protective effect after being used in combination with an adjuvant.

[0060] The present invention also provides a neutralizing antibody, which is induced by the inactivated rabies virus vaccine described in the above scheme.

[0061] In order to further illustrate the present invention, a rabies virus inactivated vaccine and a preparation method thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0062] Example 1

[0063] 1. Preparation method of pVSV-CTNG

[0064] 1. Virus Construction

[0065] Using VSV as a vector, its G protein gene was replaced with the G protein gene of rabies virus, and the VSV-rabies recombinant live virus ( Figure 1 ).

[0066] The G protein sequence of the rabies virus in this embodiment is derived from the RABV CTN-1 strain (GeneBank Accession no. AIA08921.1), the amino acid sequence is SEQ ID NO.1, and the gene synthesis is performed after codon optimization, and the nucleotide sequence after codon optimization is SEQ ID NO.4. The vesicular stomatitis virus strain is the Indiana strain (Gene Bank: J02428.1). The amino acid sequence of the G protein of VSV is shown in SEQ ID NO.7, and the nucleotide sequence of the gene encoding the G protein of VSV is shown in SEQ ID NO.8.

[0067] After gene synthesis, the above synthetic fragments and VSV vector were PCR amplified according to the instructions of Primer Star enzyme. The primer sequence information of amplification is shown in Table 1.

[0068] Table 1 Amplification primer sequences

[0069]

[0070] The coding gene sequence corresponding to the rabies virus G protein SEQ ID NO.3 was cloned into the G protein gene position in the viral vector VSV by homologous recombination (Uniclone One Step Seamless Cloning Kit) using homologous recombination enzyme (Uniclone One Step Seamless Cloning Kit) and replaced the ORF region of the G protein gene to form a VSV recombinant virus full-length plasmid carrying rabies G protein, named pVSV-CTNG. Single colonies were picked and bacterial liquid PCR was performed using universal vector primers and Taq enzyme, and the PCR products with the correct band size were sent for testing. Plasmids were extracted from the colony clones with correct sequencing.

[0071] 2. Virus rescue

[0072] The poxvirus expressing T7 polymerase was used to infect BHK-21 cells for 1 hour, followed by co-transfection of the full-length plasmid constructed in step 1 and the auxiliary plasmids expressing VSV-N, VSV-P, VSV-L, and VSV-G. After 48 hours, the cells and supernatant were collected and filtered with a 0.22 μm filter for later use. The virus stock solution was inoculated into new Vero cells (without adding trypsin), and the cultured cells were observed for lesions. If lesions were present, the cells were collected again and repeatedly frozen and thawed three times, filtered with a 0.45 μm filter, aliquoted, and frozen at -80°C to obtain the virus stock solution. The collected VSV-rabies recombinant live virus was named VSV-CTNG, and the VSV-rabies recombinant live virus titer was measured.

[0073] The Reed-Muench method was used to measure the titer of VSV-rabies recombinant live virus. The VSV-rabies recombinant live virus was diluted 10 times and inoculated into a 96-well plate with Vero cells. After 48 hours of culture, the cytopathic effect was observed, and the number of positive and negative wells was recorded, and the virus TCID was calculated. 50 After measurement, the virus titer of the VSV-rabies recombinant live virus in this example can reach 10 6.5 TCID 50 / ml and above. It can be seen that the VSV-CTNG without VSV-G envelope protein of the present invention can be rescued by virus reverse genetics technology.

[0074] 2. Preparation of pVSV-SADG

[0075] 1. Virus Construction

[0076] The G protein sequence of the rabies virus in this embodiment is derived from the SAD B19 strain (GeneBank Accession no. AAA47202.1), the amino acid sequence is SEQ ID NO.2, and after codon optimization, gene synthesis is performed, and the nucleotide sequence after codon optimization is SEQ ID NO.5. The vesicular stomatitis virus strain is the Indiana strain (Gene Bank: J02428.1). The amino acid sequence of the G protein of VSV is shown in SEQ ID NO.7, and the nucleotide sequence of the gene encoding the G protein of VSV is shown in SEQ ID NO.8.

[0077] After gene synthesis, the above synthetic fragments and VSV vector were PCR amplified according to the instructions of Primer Star enzyme. The primer sequence information of amplification is shown in Table 2.

[0078] Table 2 Amplification primer sequences

[0079]

[0080] The coding gene sequence corresponding to SEQ ID NO.4 of the above-mentioned rabies virus G protein was cloned into the G protein gene position in the viral vector VSV by homologous recombination (Uniclone One Step Seamless Cloning Kit) and replaced the ORF region of the G protein gene to form a recombinant virus full-length plasmid carrying rabies G protein in VSV, named pVSV-SADG. Single colonies were picked and bacterial liquid PCR was performed using universal vector primers and Taq enzyme, and the PCR product with the correct band size was sent for testing. Plasmids were extracted from the colony clones with correct sequencing.

[0081] 2. Virus rescue

[0082] BHK-21 cells were infected with poxvirus expressing T7 polymerase for 1 hour, and then co-transfected with the full-length plasmid constructed in step 1 and the auxiliary plasmid expressing VSV-N, VSV-P, VSV-L, and VSV-G. After 48 hours, the cells and supernatant were collected and filtered with a 0.22μm filter for standby use. The virus stock solution was inoculated into new Vero cells (no need to add trypsin), and the cultured cells were observed for lesions. If lesions occurred, the cells were collected again and repeatedly frozen and thawed three times, filtered with a 0.45μm filter, and then frozen at -80℃ to obtain the virus stock solution. The collected VSV-rabies recombinant live virus was named VSV-SADG, and the VSV-rabies recombinant live virus titer was measured.

[0083] The Reed-Muench method was used to measure the titer of VSV-rabies recombinant live virus. The VSV-rabies recombinant live virus was diluted 10 times and inoculated into a 96-well plate with Vero cells. After 48 hours of culture, the cytopathic effect was observed, and the number of positive and negative wells was recorded, and the virus TCID was calculated. 50 After measurement, the virus titer of the VSV-rabies recombinant live virus in this example can reach 10 6.5 TCID 50 / ml and above. It can be seen that the VSV-SADG without VSV-G envelope protein of the present invention can be rescued by virus reverse genetics technology.

[0084] 3. Genetic stability passage and growth curve determination of VSV-rabies recombinant live virus

[0085] 1. Stability verification

[0086] Vero cells were plated in 6-well plates, and each well was inoculated with 100 μL of virus. The virus could be continuously subcultured and replicated. It can be seen that the VSV-rabies recombinant live virus harvested in step 1 and step 2 can be stably subcultured in Vero cells. To verify the replication ability of VSV-rabies recombinant live virus in sensitive cell lines, Vero cells, Vero-E6 cells, and BHK-21 cells were plated in 12-well cell plates and cultured at 37°C and 5% CO2 for 12 to 24 hours. When the growth density reached 90% to 100% under a microscope, the recombinant virus was inoculated with a dose of MOI = 0.1. The cell pathological changes were observed after 72 hours.

[0087] The results are as follows Figure 2 As shown, compared with the control group, different forms of cytopathic effects can be observed in cells inoculated with VSV-rabies recombinant live viruses VSV-CTNG and VSV-SDAG. In Vero cells and Vero-E6 cells, the lesions are manifested as cell shrinkage and rounding, and in BHK-21 cells, the cell morphology can be observed to shrink and round from long strips. The results show that the above VSV-rabies recombinant live viruses can stably replicate in Vero cells, Vero-E6 cells, and BHK-21 cells.

[0088] 2. Growth Curve Determination

[0089] In order to verify the intracellular replication dynamics of VSV-rabies recombinant viruses VSV-CTNG and VSV-SADG, we established a virus growth curve on Vero cells. The specific steps are as follows: VSV-rabies recombinant live virus was inoculated into a 6-well cell plate of Vero monolayer cells at a dose of MOI = 0.1, and cultured at 37°C after infection. After infection, the corresponding cell plates were repeatedly frozen and thawed three times at 24, 48, 72, and 96 hours to collect the virus liquid. Frozen at -80°C. The virus liquid at each time point was diluted 10 times in a gradient manner and inoculated into a 96-well plate with Vero cells, and the virus TCID was calculated according to the Reed-Muench method. 50 , the growth curve of VSV-rabies recombinant live virus was drawn with time as the horizontal axis and virus titer as the vertical axis.

[0090] Growth kinetics curve of VSV-rabies recombinant live virus Figure 3 As shown in the figure, it can be seen that VSV-CTNG and VSV-SADG can grow at high titers on Vero cells, and the titers can reach 10 at 24h, 48h, 72h, and 96h. 8 TCID 50 / ml or more. Among them, the titer of VSV-CTNG was the highest at 72h, and the titer of VSV-SADG was the highest at 48h.

[0091] IV. Evaluation of neurotoxicity of VSV-rabies recombinant live virus

[0092] Given that the rabies virus G protein determines the neurotoxicity of rabies virus, the neurotoxicity of the VSV-rabies recombinant live virus harvested in steps 1 and 2 was studied by intracranial injection in mice. Since VSV virus also has potential neurotoxicity, wild-type VSV-WT was used as a positive control, and a negative control (injection water group) was set up. Wild-type VSV-WT, VSV-CTNG, and VSV-SADG were directly inoculated into the mouse brain at a dose of 10 5 TCID 50 / mouse, this dose of wild-type VSV inoculation can cause experimental central nervous system infection symptoms in mice. After the virus attack, the phenotypic symptoms, body weight and survival rate of the mice were recorded to evaluate the safety of the VSV-rabies recombinant live virus.

[0093] The results of the weight changes of mice after the virus attack are as follows Figure 4As shown, the results showed that, similar to the positive control VSV-WT, the mice in the VSV-CTNG and VSV-SADG groups had a significant decrease in weight after intracranial inoculation, and showed obvious symptoms of dullness, ruffled hair, curling up, and trembling. All mice in the VSV-WT group died on the second day after inoculation, while mice in the VSV-CTNG group also began to die on the third day after inoculation, with a final survival rate of only 20%; mice in the VSV-SADG group began to die on the second day after inoculation, with a final survival rate of 40% ( Figure 5 ). The negative control group showed stable signs and no mice died. Therefore, VSV-rabies recombinant live virus has strong neurotoxicity and has potential safety risks as a vaccine.

[0094] 5. Inactivation of VSV-rabies recombinant live virus

[0095] In order to eliminate the neurotoxicity of VSV-rabies recombinant live virus, VSV-rabies recombinant live virus was inactivated. β-propiolactone is easily hydrolyzed, leaving no residue and no harm after hydrolysis; it achieves the purpose of inactivation by directly destroying the viral nucleic acid structure without destroying the viral antigen protein structure. Therefore, β-propiolactone is used to inactivate and eliminate VSV-rabies recombinant live virus. The specific implementation steps are:

[0096] The VSV-rabies recombinant live virus solution was introduced into the inactivation tank, and β-propiolactone with a final concentration of 0.05% by mass volume was added, and mixed thoroughly to ensure that β-propiolactone and virus particles fully interacted, and inactivated overnight at 4°C. The inactivated virus solution was hydrolyzed at 37°C for 2h and stored at 2-8°C. The inactivated virus titer was greater than or equal to 10 6.0 TCID 50 / 50μL.

[0097] After the virus is inactivated, the inactivated virus is tested. The specific steps are as follows: take the inactivated virus solution, dilute it 10 times with DMEM culture medium containing 2% fetal bovine serum, and inoculate Vero cells that have grown into a dense monolayer. Inoculate 2 T25 square bottles for each sample, inoculate 1mL in each bottle, and discard the inoculation solution after adsorption at 37℃ for 1h. Add cell maintenance solution, continue to culture at 37℃ and observe for 4-5 days. Then blindly propagate for 3 consecutive generations to observe the occurrence of cytopathic effects. The results of the inactivated virus test are as follows: Figure 6 As shown, no pathological changes occurred in cells of all generations, proving that the virus inactivation was qualified.

[0098] 6. Adding M103 adjuvant to the inactivated VSV-rabies recombinant virus

[0099] The immunogenicity of inactivated vaccines is often weak and generally cannot induce sufficient host immune response. In order to overcome this shortcoming, an adjuvant is added to the inactivated virus to enhance the immunogenicity of the inactivated vaccine. The adjuvant used in this embodiment is M103 adjuvant.

[0100] The inactivated VSV-rabies recombinant virus and M103 adjuvant were mixed in a volume ratio of 3:1, with the volume of M103 adjuvant accounting for 15% of the total volume, and the rest was made up with sterile PBS solution. During the preparation process, the inactivated VSV-rabies recombinant virus was first added to sterile PBS, placed on a magnetic stirrer for stirring, and the M103 adjuvant was slowly added dropwise, and stirred evenly at room temperature for 30 minutes. After the preparation was completed, it was stored at 4°C.

[0101] Among them, the PBS formula is: prepare 1L PBS solution formula: 7.9g NaCl, 0.2g KCl, 0.24g KH2PO4 and 1.8g K2HPO4, dissolve in 800ml distilled water, adjust the pH value of the solution to 7.4 with hydrochloric acid, and finally add distilled water to make up to 1L.

[0102] Example 2 Adding gel02 adjuvant to inactivated VSV-rabies recombinant virus

[0103] Replace step 6 in Example 1 with the following steps:

[0104] The volume ratio of the inactivated VSV-rabies recombinant virus and gel02 adjuvant is 85:15, and the volume of gel02 adjuvant is 15% of the total volume. The gel02 adjuvant is added dropwise to the inactivated VSV-rabies recombinant virus, and stirred with a magnetic stirrer. After adding, stir at room temperature for 20 minutes. After the configuration is completed, store it at 4°C.

[0105] The rest is the same as in Example 1.

[0106] Comparative Example 1: Adding aluminum adjuvant to inactivated VSV-rabies recombinant virus

[0107] Replace step 6 in Example 1 with the following steps: the volume of aluminum gel adjuvant is 15% of the total volume, the volume ratio of the inactivated VSV-rabies recombinant virus and the aluminum gel adjuvant is 3:1 after mixing, and the rest is made up with sterile PBS solution. During the preparation process, the inactivated virus is first added to the sterile PBS, placed on a magnetic stirrer for stirring, and the aluminum gel adjuvant is slowly added dropwise, and stirred at room temperature for 30 minutes. After the preparation is completed, it is stored at 4°C.

[0108] Experimental Example 1 Safety and efficacy evaluation of inactivated VSV-rabies recombinant virus combined with adjuvant in mouse model

[0109] The inoculation reagents are inactivated vaccines prepared by respectively preparing the VSV-CTNG inactivated viruses harvested in Examples 1 and 2 and Comparative Example 1 and a plurality of different adjuvants.

[0110] 1. Safety evaluation

[0111] Balb / C mice were inoculated by intramuscular or subcutaneous injection. There were 7 experimental groups, namely:

[0112] VSV-CTNG-aluminum gel vaccine group, VSV-CTNG-M103 adjuvant intramuscular injection group, VSV-CTNG-M103 adjuvant subcutaneous injection group, VSV-CTNG-gel02 adjuvant group, VSV-CTNG pre-inactivation group, VSV-CTNG post-inactivation group and PBS control group. The inoculation dose was 10 5 TCID 50 / 100μl, the immunization program was "0+7" two immunizations, and the body weight and status of Balb / C mice were monitored for 14 consecutive days after the first immunization. The weight changes were plotted as follows: Figure 7 The results show that the weight of mice in each test group changed steadily 14 days after the first immunization. Except for a slight decrease in the weight of mice in the VSV-CTNG-M103 intramuscular injection group, the other groups did not experience a significant decrease in weight or abnormal behavior, and no mice died, indicating that the VSV-rabies recombinant live virus, after inactivation, combined with an adjuvant as a vaccine, showed good safety in small animals.

[0113] 2. Effectiveness evaluation

[0114] Balb / C mice were immunized twice by intramuscular or subcutaneous injection. The immunization procedure was "0+7" two times. The experimental grouping was consistent with the safety evaluation. Each mouse in each test group was injected with 100μl, and the stock solution titer was 10 6 TCID 50 / mL. After immunization, blood was collected from the eyeballs to obtain mouse serum on days 21 and 28 to detect the neutralizing antibody titer of the serum against the inactivated vaccine.

[0115] The specific method of the neutralizing antibody detection experiment is as follows:

[0116] 1) Cell preparation: Vero cells were plated into 96-well cell plates and used when the confluence reached 85% to 90%.

[0117] 2) Serum treatment: Inactivate the serum to be tested at 56°C for 30 min.

[0118] 3) Dilute the VSV-CTNG pseudovirus to 200 TCID using DMEM medium containing 2% FBS. 50 / 100μL, which is the virus dilution solution for detection.

[0119] 4) Dilute the serum to be tested in a 3-fold ratio, add an equal volume of virus diluent to each dilution gradient, shake to mix, and incubate in a 37°C 5% CO2 incubator for 60 minutes.

[0120] 5) Take a 96-well cell plate, wash it twice with PBS solution, remove floating cells, add the incubated serum-virus mixture to the cell plate, and make 3 control wells for each dilution gradient, 100 μl / well. At the same time, use the virus dilution as the virus control, and use 2% FBS DMEM culture medium as the negative control.

[0121] 6) The cell plate was placed in a 37°C 5% CO2 incubator and cultured for 48 hours.

[0122] 7) After the culture is completed, the percentage of cell fluorescence expression is observed under a microscope, and the neutralizing antibody titer is calculated according to the Reed-Muench method.

[0123] The results of the neutralizing antibody titers of each test group are shown in Figure 8 , it can be seen from the results that compared with the PBS control group, the mouse sera of each test group produced a certain level of neutralizing antibodies, and the neutralizing antibody level of the single inactivated virus without adjuvant group was lower. The serum neutralization titer of the VSV-CTNG-gel02 immunization group with adjuvant addition was equivalent to that of the VSV-CTNG recombinant live virus immunization group before inactivation, and the serum neutralization titer reached 2005 and 2449 at 21d and 28d after immunization. In addition, mice immunized with VSV-CTNG-M103 intramuscular injection group also produced a high level of neutralizing antibodies. From the above experimental results, it can be seen that the inactivated VSV-rabies recombinant virus obtained by the method of the present invention can further improve the immune effect, strengthen the level of neutralizing antibodies, and enhance the protective effect after being used in combination with an adjuvant.

[0124] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A rabies virus inactivated vaccine, characterized in that: It comprises an adjuvant and an inactivated VSV-rabies recombinant virus; the inactivated VSV-rabies recombinant virus is obtained by inactivating the VSV-rabies recombinant live virus; The VSV-rabies recombinant live virus is obtained by replacing the envelope protein G of the vesicular stomatitis virus with the G protein of the rabies virus; The adjuvant includes M103 adjuvant or gel02 adjuvant.

2. The inactivated rabies virus vaccine according to claim 1, characterized in that: The amino acid sequence of the rabies virus G protein is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

3.

3. The inactivated rabies virus vaccine according to claim 2, characterized in that: The nucleotide sequence of the nucleic acid molecule encoding the G protein of the rabies virus is shown as SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.

6.

4. The inactivated rabies virus vaccine according to claim 1, characterized in that: The vesicular stomatitis virus is the vesicular stomatitis virus strain Indiana.

5. The inactivated rabies virus vaccine according to claim 4, characterized in that: The amino acid sequence of the G protein of the vesicular stomatitis virus is shown in SEQ ID NO.

7.

6. The inactivated rabies virus vaccine according to claim 1, characterized in that: The adjuvant accounts for 15% of the volume of the inactivated rabies virus vaccine; the volume ratio of the inactivated VSV-rabies recombinant virus and the adjuvant is (3:1) to (17:3).

7. The method for preparing the inactivated rabies virus vaccine according to any one of claims 1 to 6, comprising the following steps: The envelope protein G of the vesicular stomatitis virus is replaced with the G protein of the rabies virus to obtain a recombinant virus full-length plasmid of the vesicular stomatitis virus carrying the G protein of the rabies virus; First, the cells to be infected are infected with a poxvirus expressing T7 polymerase, and the infected cells are co-transfected with the recombinant virus full-length plasmid and the helper plasmid, and the supernatant of the diseased cells is collected after co-transfection; The supernatant is used to infect the second cells to be infected, and after amplifying the virus, the virus-infected cell culture supernatant is collected to obtain the VSV-rabies recombinant live virus; Inactivating the VSV-rabies recombinant live virus to obtain an inactivated VSV-rabies recombinant virus; The inactivated VSV-rabies recombinant virus is mixed with an adjuvant to obtain an inactivated rabies virus vaccine.

8. The preparation method according to claim 7, characterized in that: Replacing the envelope protein G of vesicular stomatitis virus with the G protein of rabies virus comprises: cloning the nucleic acid molecule encoding the G protein of rabies virus into the envelope protein G gene position in vesicular stomatitis virus by homologous recombination and replacing the ORF region of the G protein gene.

9. The preparation method according to claim 7, characterized in that the first cells to be infected are BHK-21 cells; and the second cells to be infected are Vero cells.

10. A neutralizing antibody, characterized in that The neutralizing antibody is induced by the inactivated rabies virus vaccine according to any one of claims 1 to 6.

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