Recombinant live virus vector rabies vaccine without neurotoxicity

Abstract

The invention discloses a neurotoxicity-free recombinant live virus vector rabies vaccine, which is prepared by replacing G protein of vesicular stomatitis virus with G protein of HEP-Flury rabies virus strain or G protein mutant of HEP-Flury rabies virus strain, and saving recombinant virus VSV-HEP-Flury by using a reverse genetic manipulation technology. The recombinant live virus vector rabies vaccine has no neurotoxicity after being inoculated. The method is free of neurotoxicity, high in safety, simple in inoculation procedure, good in immune effect and high in productivity.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine production, and particularly relates to a recombinant live virus vector rabies vaccine without neurotoxicity. Background Art

[0002] Rabies is an acute zoonotic infectious disease of the central nervous system caused by the rabies virus. Once symptoms appear, the fatality rate is almost 100%. The only effective method against rabies is to inoculate the rabies vaccine.

[0003] Currently, due to the transformation of the lifestyle and concepts of Chinese residents, the market scale of the pet industry in the Chinese market has been continuously expanding. Therefore, both human and veterinary rabies vaccines are increasing rapidly every year, and the market demand is extremely huge.

[0004] Currently, the main vaccines against rabies are inactivated vaccines. The virus inactivation usually uses β-propiolactone as the inactivating agent. From the source of cultured cells, they can be divided into Vero cell vaccines, human diploid cell vaccines (HDCV), primary hamster kidney (PHK) cell vaccines, and chicken embryo cell vaccines. The Vero cell rabies vaccine has a large output and controllable quality, and is the main product of rabies vaccines in China. However, the inactivated vaccine has a relatively short immune duration, requires multiple inoculations to maintain the immune effect, and has a relatively slow production speed and may have a relatively high cost. Although the inactivated vaccine can enhance the immune response by adding adjuvants, the adjuvants usually have certain side effects.

[0005] Compared with inactivated vaccines, live vector rabies vaccines usually only need to be inoculated once to quickly trigger sufficient immune responses, have a fast production speed, low cost, do not require adjuvants, and avoid the side effects brought by adjuvants. However, the neurotoxicity of the rabies virus is the biggest challenge in the development of live virus vector rabies vaccines. The G protein of the rabies virus is located on the surface of the virus particle, and is the determinant structure of the neurotropic pathogenicity of the rabies virus and also the determinant structure for binding to cell receptors. At the same time, the G protein is the only antigen of the rabies virus that can continuously induce the production of virus-neutralizing antibodies. Some key amino acid sites of the rabies G protein play an important role in the virulence of the virus. It is related to the neuroinvasive ability and trans-synaptic transmission ability, and the substitution of different amino acids can change the speed of virus diffusion in the nervous system. How to overcome the neurotoxicity of live virus vector rabies vaccines has become an urgent problem to be solved.

[0006] CN 113559254 A discloses a rabies virus vaccine and its preparation method, which replaces the envelope protein G of vesicular stomatitis virus with the rabies virus envelope protein RABVG. It can effectively induce neutralizing antibodies against rabies virus in mice and significantly improve the ability of immunized mice to resist virus infection. The rabies virus envelope protein RABVG used is the PV strain (GeneBank Accession no. AAA47218.1), and the formed rabies virus vaccine still has strong neurotoxicity after inoculation. Summary of the Invention

[0007] The purpose of the present invention is to provide a recombinant live virus vector rabies vaccine without neurotoxicity, which has no neurotoxicity, high safety, a simple inoculation procedure, good immune effect, and high production capacity.

[0008] The technical solution adopted by the present invention to solve its technical problems is as follows: A recombinant live virus vector rabies vaccine without neurotoxicity is obtained by replacing the G protein of the HEP-Flury rabies strain or the G protein mutant of the HEP-Flury rabies strain with the G protein of vesicular stomatitis virus and rescuing the recombinant virus VSV-HEP-Flury by reverse genetic manipulation technology; the recombinant live virus vector rabies vaccine has no neurotoxicity after inoculation.

[0009] In order to solve the problem of neurotoxicity after injection of the live virus vector rabies vaccine, the inventors conducted a large number of studies. Accidentally, it was found that after replacing the G protein of vesicular stomatitis virus with the G protein of the HEP-Flury rabies strain, the neurotoxicity of the obtained recombinant virus after injection disappeared. However, after replacing the G protein of vesicular stomatitis virus with the G protein of the LEP-Flury strain, which is also a rabies attenuated strain, the neurotoxicity of the obtained recombinant virus after injection still exists. In addition, the present invention further conducted research on mutagenesis to reduce toxicity and found that although the G protein of the CTN-1 strain was attenuated, the neurotoxicity of the obtained recombinant virus after injection was enhanced instead. It can be seen that the effect of the modification is completely different from the theoretical expectation and cannot be predicted. Further, the present invention performed multi-point mutagenesis on the G protein of the HEP-Flury rabies strain, and the obtained recombinant virus still has no neurotoxicity after injection, and at the same time, the cell growth ability is greatly improved, increasing the yield.

[0010] The present invention studies the neurotoxicity of rabies strains from multiple dimensions. The obtained recombinant live virus vector rabies vaccine can be mass-produced at the cell level, has no neurotoxicity after intracranial inoculation of mice, and has high safety. After immunizing mice with the "0 + 7" two-dose immunization program, it can produce a high level of neutralizing antibodies and has a good immune effect.

[0011] The amino acid sequence of the G protein of the HEP-Flury rabies virus strain is shown in SEQ ID No. 1.

[0012] The G protein mutant of the HEP-Flury rabies virus strain is a multi-site mutant, and the mutant sites include: S34R: The amino acid S at position 34 is mutated to R. V183E: The amino acid V at position 183 is mutated to E. L202P: The amino acid L at position 202 is mutated to P. And A305V: The amino acid A at position 305 is mutated to V.

[0013] The amino acid sequence of the G protein mutant of the HEP-Flury rabies virus strain is shown in SEQ ID No. 2.

[0014] The vesicular stomatitis virus strain is the Indiana strain.

[0015] The preparation method is specifically as follows: (1). Construct the VSV-RABVG plasmid: First, insert the VSV genome onto the BAC vector, and then replace the gene sequence of the G protein on the VSV genome with the gene sequence of the rabies virus G protein to obtain the pBAC-VSV-RABVG plasmid. (2). Rescue the recombinant virus: Infect the first cell to be infected with a poxvirus expressing T7 polymerase. After infection, co-transfect the cells with the pBAC-VSV-RABVG plasmid, pN, pP, pL, and pG plasmids. Collect the supernatant of the diseased cells 48 hours after transfection, infect the second cell to be infected with the supernatant, and collect the supernatant of the diseased cells after amplifying the virus to obtain the recombinant live virus vector rabies vaccine. The first cell to be infected is the BHK21 cell. The second cell to be infected is the Vero cell. When co-transfecting the pBAC-VSV-RABVG, pN, pP, pL, and pG plasmids, the mass ratio is 10:3:5:1:3.

[0016] The gene sequence of the rabies virus G protein is the gene sequence of the G protein of the HEP-Flury rabies virus strain or the G protein mutant of the HEP-Flury rabies virus strain.

[0017] The beneficial effects of the present invention are: The present invention has no neurotoxicity, high safety, simple vaccination procedure, good immune effect, and high production capacity. Description of the Drawings

[0018] Figure 1 It is a predicted diagram of the binding structure of the rabies G protein and the nAChR receptor. Figure 2It is a predicted diagram of the binding structure between rabies G protein and mGluR2 receptor; Figure 3 It is a predicted diagram of the binding structure between rabies G protein and p75NTR receptor; Figure 4 It is a schematic diagram of the molecular construction of pBAC-VSV vector rabies recombinant virus; Figure 5 It is the growth kinetic curve of each recombinant rabies vaccine candidate strain; Figure 6 It is the graph of the body weight change of mice after being challenged with each recombinant rabies vaccine candidate strain; Figure 7 It is the graph of the survival rate change of mice after being challenged with each recombinant rabies vaccine candidate strain; Figure 8 It is the comparison graph of the neutralizing antibody titers of VSV-HEPG and VSV-mHEPG. Detailed implementation manners

[0019] The technical solutions of the present invention will be further specifically described below through specific embodiments.

[0020] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0021] In order to develop a recombinant live virus vector rabies vaccine strain without neurotoxicity, we first used structural simulation to predict the binding interactions between the G proteins of various existing rabies vaccine strains and nerve receptors, and constructed the predicted G proteins of rabies strains onto the pBAC-VSV vector, mainly including: PV rabies strain, SAD-B19 rabies strain, CTN-1 rabies strain, CVS strain (CVS-11), HEP-Flury strain, LEP-Flury strain.

[0022] In addition, in order to explore the influence of different amino acid sites on the neurotoxicity of rabies strains, we made exploratory mutation modifications to certain amino acid sites of the G protein of CTN-1 strain and the G protein of HEP-Flury rabies strain, including the double-site mutation modifications of Q352A and G368E in CTN-1 strain (modifications to theoretically reduce toxicity at the sites), and the multi-site mutation modifications of S34R, V183E, L202P, A305V in HEP-Flury strain.

[0023] After rescuing the virus through viral reverse genetics, we detected the growth characteristics of these viruses. We found that compared with the unmutated HEP-Flury G protein, the growth ability of the strains with multiple-site mutations in the G protein of HEP-Flury was significantly improved on cells. Subsequently, we detected the neurotoxicity of these candidate vaccine strains by intracranial inoculation of mice. The results showed that the virulence of the strain with double-site mutations in CTN-1 was further enhanced, indicating that the modification method that theoretically could reduce toxicity might have the opposite effect. Although the LEP-Flury strain is a attenuated strain, the recombinant virus formed by constructing its G protein into the pBAC-VSV vector still has neurotoxicity. Among all the strains, only the recombinant viruses formed by constructing the HEP-Flury G protein and the G protein with multiple-site mutations in HEP-Flury into the pBAC-VSV vector are non-neurotoxic. We injected these two non-neurotoxic strains into mice by intramuscular injection and detected the neutralizing antibody levels in the mouse sera. We found that both the recombinant viruses formed by constructing the HEP-Flury G protein and the G protein with multiple-site mutations in HEP-Flury into the pBAC-VSV vector could induce high levels of neutralizing antibodies in mice.

[0024] Example 1. Prediction of the Neurotoxicity of a Rabies Vaccine Strain Rabies virus enters the body through the bites or saliva of infected animals. Its G protein binds to various receptors on nerve cells, including nicotinic acetylcholine receptor (nAChR), neural cell adhesion molecule (NCAM), p75 neurotrophin receptor (p75NTR), and metabotropic glutamate receptor 2 (mGluR2), etc., invades and spreads to the nervous system, and ultimately causes severe neurological symptoms. These receptors play a key role in the neurotoxicity of the virus. To study the neurotoxicity of rabies virus strains, we used structural simulation to study the binding interactions of the G proteins of several existing rabies vaccine strains, including the PV strain (GeneBank Accession no. AAA47218.1), SAD-B19 strain (GeneBank Accession no. AAA47202.1), CVS strain (GeneBank Accession no. AF085333.1), CTN-1 strain (GeneBank Accession no. AIA08921.1), HEP-Flury strain (GeneBank Accession no. AAA47213.1), and LEP-Flury strain (GeneBank Accession no. BDO24562.1) with the three nerve cell receptor proteins nAChR, p75NTR, and mGluR2. The structural prediction results showed that these strains interacted with nAChR ( Figure 1) and mGluR2( Figure 2 ) protein binding surfaces are almost identical with no significant difference. However, in the protein binding surface with p75NTR( Figure 3 ), because there are amino acid differences within this binding region. Among them, the binding domains of the PV strain and the SAD strain are asparagine (Asn, N), and the binding domains of the other strains are histidine (His, H). Asparagine can form intramolecular hydrogen bonds with other amino acids of the G protein, making the binding of the G protein to p75NTR more stable. While histidine cannot form intramolecular hydrogen bonds, and due to the steric hindrance effect, it affects the binding of the G protein to p75NTR. Therefore, the prediction results indicate that the PV strain and the SAD strain have strong neurotoxicity, while the CVS strain, the CTN-1 strain, the HEP-Flury strain, and the LEP-Flury strain have weak neurotoxicity.

[0025] Example 2. Preparation of Recombinant Live Rabies Viruses with VSV Vector (Virus Construction and Rescue) To verify the above results, we first used the BAC vector as the backbone vector and, by means of homologous recombination, replaced the sequence of the lacZa and its adjacent region of the BAC vector with the T7-HDV sequence (TAATACGACTCACTATAGGGGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTG GGCAACATTCCGAGGGGACCGTCCCCTCGGTAATGGCGAATGGGACCTAGCATAACCCC TTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG, SEQ ID NO.26). Then, the VSV genome was inserted between the T7 promoter and the HDV gene sequence. Subsequently, the G protein genes of each rabies virus strain in Example 1 were used to replace the G protein gene of VSV. Each candidate strain of the VSV live vector rabies vaccine was constructed using reverse genetics technology( Figure 4 ).

[0026] The G protein sequences in this example are derived from the RABV PV strain (GeneBank Accession no. AAA47218.1), the SAD-B19 strain (GeneBank Accession no. AAA47202.1), the CVS strain (GeneBank Accession no. AF085333.1), the CTN-1 strain (GeneBank Accession no. AIA08921.1), the HEP-Flury strain (GeneBank Accession no. AAA47213.1, SEQ ID No.1), and the LEP-Flury strain (GeneBank Accession no. BDO24562.1).

[0027] Some key amino acid sites of the rabies G protein play an important role in virus virulence. It is related to neuroinvasiveness and the ability of trans-synaptic transmission. Substitution of different amino acids can change the speed at which the virus spreads in the nervous system. To explore the effects of different amino acids on neurotoxicity, we made mutant modifications to some amino acid sites of the G proteins of CTN-1 strain and HEP-Flury strain to explore the effects of these mutations on neurotoxicity. Among them, double-site mutant modifications of Q352A and G368E were made to the CTN-1 strain (the amino acid sequence of the mutated G protein corresponds to SEQ ID NO.3), while multi-site mutant modifications of S34R, V183E, L202P, and A305V were made to HEP-Flury (the amino acid sequence of the mutated G protein corresponds to SEQID NO.2).

[0028] SEQ ID NO.1: MVPQVLLFAPLLVFPLCFGKFPIYTIPDKLGPWSPIDLHHLSCPNNLVVEDEGCTNLSGFSY MELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKM AGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVTDLDPYDKSLHSRVFPGGNCSGITV SSTYCSTNHDYTIWMPENLRLGTSCDIFTHSRGKRASKGDKTCGFVDERGLYKSLKGACKL KLCGVLGLRLMDGTWVAMQTSDETKWCPPGQLVNLHDFRSDEIEHLVEEELVKKREECL DALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVQTWNEIIPSKGCLR VGERCHPHVNGVFFNGIILGSDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKD GDEVEDFVEVHLPDVHKQVSGVDLGLPKWGKYVLMIAGALIALMLIIFLMTCCRRVNRPESTQSNLGGTGRNVSVPSQSGKVISSWESYKSGGETRL。

[0029] SEQ ID NO.2: MVPQVLLFAPLLVFPLCFGKFPIYTIPDKLGPWRPIDLHHLSCPNNLVVEDEGCTNLSGFSY MELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKM AGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVTDLDPYDKSLHSRVFPGGNCSGITE SSTYCSTNHDYTIWMPENPRLGTSCDIFTHSRGKRASKGDKTCGFVDERGLYKSLKGACKL KLCGVLGLRLMDGTWVAMQTSDETKWCPPGQLVNLHDFRSDEIEHLVEEELVKKREECL DVLESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVQTWNEIIPSKGCLR VGERCHPHVNGVFFNGIILGSDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKD GDEVEDFVEVHLPDVHKQVSGVDLGLPKWGKYVLMIAGALIALMLIIFLMTCCRRVNRPESTQSNLGGTGRNVSVPSQSGKVISSWESYKSGGETRL。

[0030] SEQ ID NO.3: MIPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYM ELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRSAYNWKMAG DPRYEESLHNPYPDYHWLRTVKTTKESVVIISPSVADLDPYDESLHSRVFPRGKCSGITVSS AYCSTNHDYTIWMPENPRLGTSCDIFTNSRGKRASKGSKTCGFVDERGLYKSLKGACKLK LCGVLGLRLMDGTWVAIQTSNETKWCPPDQLVNLHDFHSDEIEHLVVEELVKKREECLDA LESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVATWNEIIPSKGCLRVG ERCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLKSSVIPLMHPLADPSTVFKDGD EVEDFVEVHLQDVHKQVSGVDLGLPNWGKDVLMGAGVLTALMLMIFLMTCCRRTNRAESIQHSLGETGRKVSVTPQSGRVISSWESYKSGGETKL。

[0031] The sequence of the vesicular stomatitis virus strain used in this example is the Indiana strain gene sequence.

[0032] For the construction of each plasmid of pBAC-VSV-PVG, pBAC-VSV-SADG, pBAC-VSV-CVSG, pBAC-VSV-CTNG, pBAC-VSV-HEPG and pBAC-VSV-LEPG, after the corresponding G protein gene was synthesized, the synthesized gene fragments were respectively subjected to PCR amplification according to the Primer Star enzyme instruction manual, and the primer sequence information for amplification is shown in Table 1.

[0033] For the construction of pBAC-VSV-mCTNG and pBAC-VSV-mHEPG plasmids (mutants), the successfully constructed pBAC-VSV-CTNG and pBAC-VSV-HEPG plasmids were respectively used as templates, and amplified with the corresponding primers in Table 1.

[0034] Table 1 Primer sequences for amplification The corresponding amplified gene fragments of the G protein of the above rabies virus were respectively cloned into the position of the G protein gene in the pBAC-VSV vector by homologous recombination (Uniclone OneStep Seamless Cloning Kit) and replaced the ORF region of the G protein gene to form a recombinant virus full-length plasmid of VSV carrying the rabies G protein.

[0035] The construction process of each specific plasmid is as follows: First, use DNA polymerase (Primer Star) to perform PCR amplification to obtain the corresponding fragments. Second, use homologous recombination enzyme (Uniclone One Step Seamless Cloning Kit) to recombine each fragment and transform it into competent cells. Third, pick single colonies and perform colony PCR using universal vector primers and Taq enzyme, and send the PCR products with the correct band size for sequencing. Fourth, extract plasmids from the colonies with correct sequencing results.

[0036] The virus rescue method is as follows: Infect BHK-21 cells with the vaccinia virus expressing T7 polymerase. Subsequently, co-transfect the infected BHK-21 cells with the full-length plasmids constructed above (pBAC-VSV-PVG, pBAC-VSV-SADG, pBAC-VSV-CVSG, pBAC-VSV-CTNG, pBAC-VSV-HEPG, pBAC-VSV-LEPG, pBAC-VSV-mCTNG, and pBAC-VSV-mHEPG) and the helper plasmids expressing VSV-N, VSV-P, VSV-L, and VSV-G (pN, pP, pL, and pG. The helper plasmids were constructed by inserting the corresponding VSV protein coding sequences into the pBluscript vector using homologous recombination). When co-transfecting the full-length plasmids, pN, pP, pL, and pG plasmids, the mass ratio is 10:3:5:1:3. After 48 hours, collect the cells and supernatant, filter through a 0.22 μm filter, and take the supernatant for standby. Inoculate the virus stock solution into new Vero cells, observe whether cytopathic effects occur in the cultured cells. If cytopathic effects occur, collect the cells again and freeze-thaw them three times repeatedly, filter through a 0.45 μm filter, aliquot, and store at -80 °C to obtain the virus stock solution. Name the collected recombinant viruses as VSV-PVG, VSV-SADG, VSV-CVSG, VSV-CTNG, VSV-HEPG, and VSV-LEPG respectively, and name the two mutant-modified strains as VSV-mCTNG and VSV-mHEPG respectively, and measure the titers of the recombinant viruses.

[0037] The Reed-Muench method was used to measure the titers of the recombinant viruses. Dilute the virus in a 10-fold gradient and inoculate it into a 96-well plate paved with Vero cells. Observe cytopathic effects after culturing for 48 hours, record the number of positive and negative wells respectively, and calculate the virus TCID 50 . After measurement, the titers of the recombinant viruses in the embodiments of the present invention can all reach 10 7 TCID 50 / ml or above after the recombinant viruses are passaged stably.

[0038] Example 3. Determination of the growth kinetic curve of the recombinant virus To verify the replication kinetics of the rescued recombinant virus in Example 2 in cells, we established a virus growth curve on Vero cells. The specific steps are as follows: Inoculate the recombinant virus at a dose of MOI = 0.01 into a 6-well cell culture plate of Vero monolayer cells. After infection, place it in an incubator at 37°C. At 24, 48, 72, and 96 h post-infection, repeatedly freeze-thaw the corresponding cell culture plate three times, and collect the virus solution. Store it at -80°C. Dilute the virus solution at each time point by a factor of 10 in gradient and inoculate it onto a 96-well plate seeded with Vero cells. Calculate the virus TCID 50 using the Reed-Muench method, and plot the virus growth curve with time on the x-axis and virus titer on the y-axis.

[0039] The growth kinetic curve of the recombinant virus is as Figure 5 shown. Each recombinant virus can grow at a high titer on Vero cells, and the highest titer is greater than 10 7 TCID 50 / ml. Among them, the growth titer of VSV-mHEPG is higher than that of other strains, and the highest titer can reach 10 9 TCID 50 / ml at 48 h.

[0040] Example 4. Neurotoxicity evaluation of VSV-rabies recombinant virus To study the neurotoxicity of these VSV-rabies recombinant virus vaccine candidates, we evaluated them by intracranial injection of the virus into mice. Since VSV virus also has potential neurotoxicity, we used wild-type VSV-WT as a positive control and set up a negative control (water for injection group). Directly inoculate wild-type VSV-WT and each recombinant rabies vaccine candidate strain, including the recombinant rabies mutant strain VSV-mCTNG and VSV-mHEPG, into the brains of mice 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. Record the phenotypic symptoms, body weight, and survival rate of mice after virus challenge to evaluate the safety of the rabies recombinant virus.

[0041] The results of the changes in mouse body weight after virus challenge are as Figure 6 shown. The results show that except for the negative control, VSV-HEPG, and VSV-mHEPG groups, the body weight of mice in all other candidate virus groups decreased significantly after intracranial inoculation. In addition, these mice showed symptoms similar to those of the positive control VSV-WT mice after virus challenge, showing obvious dullness, piloerection, curling up, and tremors, which are typical symptoms of nerve infection. Among them, compared with the mice in the VSV-CTNG group, the mortality rate of mice in the VSV-mCTNG group was the highest after intracranial inoculation, and the final survival rate was only 16.7% (Figure 7 )。However, no obvious neurotoxic symptoms appeared in the mice of the VSV-HEPG group and the VSV-mHEPG group after virus inoculation. The body weights of the mice remained at a relatively stable level, and no mice died. The physical signs of the mice in the negative control group were stable and no mice died. Therefore, VSV-HEPG and VSV-mHEPG have no neurotoxicity and good safety.

[0042] Example 5. Immunogenicity evaluation of VSV-HEPG We selected two groups with good safety in Example 4, VSV-HEPG and VSV-mHEPG, to verify their immunogenicity. Six-week-old female Balb / C mice were randomly divided into two groups, a PBS negative control group and VSV-HEPG and VSV-mHEPG immunization groups. The Balb / C mice were immunized intramuscularly twice, and the immunization schedule was two immunizations of "0 + 7". Each mouse in each test group was injected with 100 μl, and the immunization dose was 10 6 TCID 50 / mouse. After immunization, blood was collected from the eyeballs of the mice on the 14th, 21st, and 28th days, and the neutralizing antibody titers in the mouse sera were detected.

[0043] The specific method for the neutralizing antibody detection experiment is as follows: 1. Cell preparation: Seed Vero cells into a 96-well cell plate and use them when the confluence reaches 85%-90%.

[0044] 2. Serum treatment: Inactivate the test serum at 56 °C for 30 min.

[0045] 3. Dilute the VSV-CVSG pseudovirus to 200 TCID 50 / 100 μL with DMEM medium containing 2% FBS to obtain the virus dilution for detection.

[0046] 4. Dilute the test serum and the standard serum (titer 0.5 IU / ml) by 3-fold serial dilution. Add an equal volume of the virus dilution to each dilution gradient, mix well by shaking, and incubate in a 37 °C 5% CO2 incubator for 60 min.

[0047] 5. Take a 96-well cell plate, wash it twice with PBS solution to remove floating cells, add the incubated serum-virus mixture into the cell plate, and set up 3 control wells for each dilution gradient, 100 μl / well. At the same time, use the virus dilution as the virus control and DMEM medium containing 2% FBS as the negative control.

[0048] 6. Incubate the cell plate in a 37 °C 5% CO2 incubator for 48 h.

[0049] 7. After the cultivation, observe the percentage of cell fluorescence expression under the microscope and calculate the neutralizing antibody titer according to the Reed-Muench method.

[0050] 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 VSV-HEPG and VSV-mHEPG strains can produce relatively high neutralizing antibody levels after immunizing mice. Among them, the neutralizing antibody level produced by VSV-mHEPG is higher than that of VSV-HEPG. From the above experimental results, it can be known that the recombinant live virus vector rabies vaccine obtained by the method of the present invention has high safety and good immunization effect.

[0051] The above-described embodiments are only a preferred solution of the present invention and do not impose any form of limitation on the present invention. There are other variations and modifications without exceeding the technical solutions recorded in the claims.

Claims

1. A recombinant live virus vector rabies vaccine without neurotoxicity, characterized in that: The G protein of the HEP-Flury rabies strain or a mutant of the G protein of the HEP-Flury rabies strain replaces the G protein of the vesicular stomatitis virus, and the recombinant virus VSV-HEP-Flury is rescued by reverse genetic manipulation technology; the recombinant live virus vector rabies vaccine has no neurotoxicity after injection.

2. A non-neurotoxic recombinant live virus vector rabies vaccine according to claim 1, characterized in that: The amino acid sequence of the G protein of the HEP-Flury rabies strain is shown in SEQ ID No.

1.

3. A recombinant live virus vector rabies vaccine without neurotoxicity according to claim 1, characterized in that: The G protein mutant of the HEP-Flury rabies strain is a multi-site mutant, and the mutation sites include: S34R: The 34th amino acid S mutated to R, V183E: The amino acid V at position 183 mutates to E. L202P: The amino acid L at position 202 mutated to P, And A305V: the amino acid A at position 305 mutated to V.

4. A non-neurotoxic recombinant live virus vector rabies vaccine according to claim 1, characterized in that: The amino acid sequence of the G protein mutant of the HEP-Flury rabies strain is shown in SEQ ID No.

2.

5. A recombinant live virus vector rabies vaccine without neurotoxicity according to claim 1, characterized in that: The vesicular stomatitis virus strain was the Indiana strain.

6. A non-neurotoxic recombinant live virus vector rabies vaccine according to claim 1, characterized in that: The preparation method is specifically as follows: (1) Construction of VSV-RABVG plasmid: First, insert the VSV genome into the BAC vector, and then replace the gene sequence of the G protein on the VSV genome with the gene sequence of the rabies virus G protein to obtain the pBAC-VSV-RABVG plasmid; (2) Saving the recombinant virus: the first cells to be infected are infected with a poxvirus expressing T7 polymerase, the infected cells are co-transfected with pBAC-VSV-RABVG plasmid, pN, pP, pL and pG plasmids, the supernatant of the diseased cells is collected 48 hours after transfection, the supernatant is used to infect the second cells to be infected, the supernatant of the diseased cells is collected after the virus is amplified, and the recombinant live virus vector rabies vaccine is obtained; The first cells to be infected are BHK21 cells; The second cell to be infected is Vero cell; The mass ratio of pBAC-VSV-RABVG, pN, pP, pL and pG plasmids during co-transfection was 10:3:5:1:

3.

7. A non-neurotoxic recombinant live virus vector rabies vaccine according to claim 6, characterized in that: The gene sequence of the rabies virus G protein is the gene sequence of the G protein of the HEP-Flury rabies strain or a mutant of the G protein of the HEP-Flury rabies strain.

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