Rabies virus G protein carrier, rabies virus vaccine as well as preparation method and application of rabies virus G protein carrier and rabies virus vaccine

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a rabies virus G protein carrier, a rabies virus vaccine and a preparation method and application of the rabies virus G protein carrier and the rabies virus vaccine. The rabies virus G protein vector pscAAV-G is obtained by inserting a coding gene of rabies virus G protein into a coding region between inverted terminal repetitive sequences at two ends of a double-chain adeno-associated virus vector, so that the problem of expression delay of single-chain AAV (ssAAV) is solved, and rapid and efficient expression of the rabies virus G protein is realized; furthermore, an endogenous nucleic acid adjuvant is embedded into a non-coding region of the pscAAV-G, so that the rabies virus G protein carrier pscAAV-G-Adj is prepared, and Th1 type immune response is remarkably improved. According to a rabies virus vaccine prepared by using the rabies virus G protein carrier, the antigen expression efficiency and immunogenicity of an AAV carrier vaccine are remarkably improved, long-acting protection of single inoculation is realized, and the defects that an existing rabies vaccine is short in immune cycle and needs to be inoculated for multiple times are overcome.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a rabies virus G protein vector, a rabies virus vaccine, and a preparation method and application thereof. Background Art

[0002] Rabies is a zoonotic disease with a mortality rate approaching 100%, resulting in approximately 59,000 deaths worldwide annually. Currently, rabies vaccines in clinical use primarily consist of inactivated virus vaccines and purified cell-culture-based vaccines. While these vaccines offer effective protection, they suffer from limitations such as a short immune cycle, the need for multiple booster doses, and high production costs. For example, some have adopted multi-dose vaccination strategies (e.g., a 0 / 3 / 7 / 14 / 28-day schedule for inactivated vaccines) or developed sustained-release formulations (e.g., microsphere-encapsulated antigens), but these efforts have not addressed the rapid decline in antibody titers after a single vaccination. In recent years, genetic vaccines have become a research hotspot due to their potential to induce long-lasting immune responses. Adeno-associated virus (AAV) vectors, with their low pathogenicity, strong tissue targeting, and long-term, stable expression of exogenous genes, have been widely used in gene therapy and vaccine development.

[0003] Existing AAV-based rabies vaccine research mostly uses single-stranded DNA (ssAAV) vectors to deliver rabies virus G protein. However, due to the limitation that the single-stranded AAV genome requires host cell DNA polymerase-mediated second-strand synthesis, its protein expression efficiency and immunogenicity still need to be improved. Although researchers have partially improved expression levels by optimizing single-stranded AAV (ssAAV) promoters (such as CMV and CAG strong promoters) or codon optimization of the rabies virus G protein gene, they still cannot break through the inherent biological bottleneck of ssAAV.

[0004] Traditional vaccine design has insufficient ability to regulate the type of immune response, especially the difficulty in effectively inducing Th1 cellular immunity (an immune response closely related to viral clearance). In studies on regulating the type of immune response, some have added aluminum adjuvants (Th2-biased) or TLR agonists (such as CpG ODN, Th1-biased) to vaccines, but the systemic addition of exogenous adjuvants may cause inflammatory side effects and have limited synergistic effects with AAV vectors. In addition, adjuvant integration strategies are still immature in adjuvant integration design studies. Some researchers have inserted cytokine genes (such as IL-12, IFN-γ) into vector gene expression cassettes to enhance immune responses, but such schemes may interfere with the stability of antigen expression and have the problem of complexity in gene expression regulation.

[0005] Although double-stranded AAV (scAAV) can bypass the second strand synthesis step to directly express antigens, which can improve the efficiency of antigen expression, its application potential in rabies vaccine has not been fully developed, and there are still key gaps. In addition, how to further enhance the efficacy of the vaccine through vector engineering (such as integrating an immune adjuvant system) is still a technical bottleneck that needs to be broken through in this field.

[0006] Therefore, there is an urgent need in the art to develop a new rabies vaccine scheme based on scAAV vector engineering, to break through the existing technical bottlenecks by endogenous adjuvant synergistic design, to achieve single vaccination, long-lasting Th1 type immune protection. SUMMARY

[0007] The technical problem to be solved by the first aspect of the present application is that in the prior art (1) when traditional single-stranded AAV (ssAAV) vectors deliver rabies virus G protein, they need to rely on the DNA polymerase of the host cell to complete the second strand synthesis, which leads to delayed and low-efficiency antigen expression, limiting the immune effect of the vaccine; (2) although traditional inactivated vaccines are effective, they need to be boosted multiple times to maintain antibody levels, increasing the cost of vaccination and the burden of compliance; (3) the existing vaccine design cannot effectively regulate the type of immune response, and the Th1 type immune response (characterized by IgG2a antibodies and cellular immunity) is crucial for viral clearance, and its deficiency may limit the long-term protection efficacy; (4) the existing AAV vector vaccine lacks synergistic design with nucleic acid adjuvants (such as dsRNA), and cannot further activate the innate immune signaling pathway to amplify the adaptive immune response, and first provides a rabies virus G protein vector pscAAV-G.

[0008] The technical problem to be solved by the second aspect of the present application is to provide a preparation method of the rabies virus G protein vector pscAAV-G.

[0009] The technical problem to be solved by the third aspect of the present application is to provide another rabies virus G protein vector pscAAV-G-Adj.

[0010] The technical problem to be solved by the fourth aspect of the present application is to provide a recombinant adeno-associated virus capable of expressing rabies virus G protein.

[0011] The technical problem to be solved by the fifth aspect of the present application is to provide the application of the two rabies virus G protein vectors (pscAAV-G, pscAAV-G-Adj) and the recombinant adeno-associated virus in the preparation of a rabies virus vaccine.

[0012] The technical problem to be solved by the sixth aspect of the present application is to provide a rabies virus vaccine.

[0013] The technical problem to be solved by the seventh aspect of the present application is to provide a preparation method of the rabies virus vaccine.

[0014] The technical problems of the present invention are caused by the following reasons:

[0015] (1) Inherent limitations of single-stranded AAV vectors: The linear single-stranded genome of ssAAV requires a slow second-strand synthesis process in host cells, resulting in delayed antigen expression (usually taking several weeks), making it difficult to quickly stimulate a high-intensity immune response. At the same time, in the early development of ssAAV-G vaccine in our laboratory, Western blot detection found that the expression of rabies virus G protein was delayed until 14 days later, and the neutralizing antibody titer was insufficient (1:320), which is speculated to be related to the single-stranded vector's dependence on the host's second-strand synthesis.

[0016] (2) Technical path dependence of traditional vaccines: The production of inactivated vaccines relies on virus amplification and purification, which is a complex process and expensive; while existing gene vaccines mostly focus on antigen delivery and ignore the active regulation of immune response types.

[0017] (3) Lack of synergistic design between vectors and adjuvants: Although double-stranded AAV (scAAV) can bypass second-chain synthesis and directly express antigens, its integration strategy with immune adjuvants (such as dsRNA) is not yet mature, resulting in the adjuvant's immune-promoting effect failing to fully cooperate with the vector characteristics.

[0018] (4) Insufficient regulation of Th1 / Th2 immune balance: Rabies virus is an intracellular pathogen that relies on Th1 immune response for effective clearance. However, traditional vaccine design lacks the molecular mechanism to induce Th1 dominant response (such as specific adjuvant or carrier modification), resulting in immune protection biased towards Th2 type (mainly IgG1 antibodies), which reduces the antiviral efficacy.

[0019] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0020] In a first aspect, the present invention provides a rabies virus G protein vector pscAAV-G, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0021] In a second aspect, the present invention provides a method for preparing a rabies virus G protein vector pscAAV-G, wherein the rabies virus G protein vector is obtained by inserting the coding gene of the rabies virus G protein into the coding region between the inverted terminal repeat sequences at both ends of a double-stranded adeno-associated virus vector.

[0022] The rabies virus G protein encoding gene is a codon-optimized RABV-G encoding gene, and its nucleotide sequence is shown in SEQ ID NO.4.

[0023] In some embodiments of the invention, the double-stranded adeno-associated virus vector is a pAAV-CMV-SV40 plasmid.

[0024] The rabies virus G protein vector contains a minimal CMV promoter and an SV40 poly (A) signal, and the minimal CMV promoter, the SV40 poly (A) signal and the rabies virus G protein coding gene constitute a complete rabies virus G protein expression cassette.

[0025] In a third aspect, the present invention provides another rabies virus G protein vector pscAAV-G-Adj, which is obtained by embedding an endogenous nucleic acid adjuvant dsRNA into the non-coding region of the rabies virus G protein vector described in the first aspect.

[0026] Wherein, the endogenous nucleic acid adjuvant is expressed by driving the H1 promoter.

[0027] Specifically, the H1 promoter is artificially designed, and its nucleotide sequence is shown in SEQ ID NO.6.

[0028] Specifically, the endogenous nucleic acid adjuvant dsRNA is SLR14, and its nucleotide sequence is shown in SEQ ID NO.7.

[0029] Specifically, the dsRNA can activate IFN-α / β secretion through the RIG-I / MDA5 pathway and promote Th1 immunity.

[0030] Among them, the nucleotide sequence of the other rabies virus G protein vector pscAAV-G-Adj is shown in SEQ ID NO.3.

[0031] In a fourth aspect, the present invention provides a recombinant adeno-associated virus capable of expressing rabies virus G protein.

[0032] Specifically, the recombinant adeno-associated virus contains the rabies virus G protein vector pscAAV-G, or contains another rabies virus G protein vector pscAAV-G-Adj.

[0033] In some embodiments of the present invention, the recombinant adeno-associated virus can efficiently express rabies virus G protein, and the virus titer can reach 1×10 13 GC / mL and above, meeting the requirements of subsequent animal experiments.

[0034] In a fifth aspect, the present invention provides the use of the rabies virus G protein vector pscAAV-G, or another rabies virus G protein vector pscAAV-G-Adj, or the recombinant adeno-associated virus in the preparation of rabies virus vaccine.

[0035] In a sixth aspect, the present invention provides a rabies virus vaccine.

[0036] Specifically, the rabies virus vaccine contains the rabies virus G protein vector pscAAV-G, or another rabies virus G protein vector pscAAV-G-Adj, or the recombinant adeno-associated virus.

[0037] Specifically, the rabies virus vaccine is administered to domestic animals such as dogs and cats by intramuscular injection or intranasal instillation. It can also be developed into an oral dosage form (such as a bait vaccine), and the stability of the AAV vector at room temperature can be used to achieve large-scale field deployment to block the virus transmission chain. It can also be used in combination with other pathogens, such as integrating the rabies virus G protein with other pathogen antigens (such as canine distemper virus and rabies-related lyssavirus) into the same scAAV vector to develop a "one-shot, multiple-protection" vaccine; or it can be used in combination with existing adjuvanted vaccines (such as mRNA vaccines) to form a primary immunization-boosting strategy to quickly increase the antibody level of high-risk populations.

[0038] In some embodiments of the present invention, by immunizing BalB / C mice, it was found that pscAAV-G can exhibit better immunogenicity and neutralizing antibody titers than pssAAV-G. Subsequent further research results found that the scAAV-G-Adj vaccine, which adds a dsRNA nucleic acid adjuvant to pscAAV-G, further enhanced the immunogenicity of the scAAV-G rabies vaccine in a mouse model and increased the neutralizing antibody titer of the vaccine. In addition, through analysis of mouse serum IgG subtypes (IgG1 and IgG2a), it was found that the immune response type of mice induced by the scAAV-G-Adj vaccine was more inclined to the Th1 type.

[0039] Specifically, in the neutralizing antibody test: 4 weeks after immunization, the serum neutralizing antibody titer (microtiter method) of the scAAV-G-Adj group reached 1:8192, which was 2 times higher than that of the scAAV-G group (1:4096) and 4 times higher than that of the ssAAV-G group (1:2048); in the Th1 / Th2 balance analysis: the IgG1 / IgG2a ratio of the scAAV-G-Adj group was significantly lower than that of the scAAV-G group, proving that it successfully induced a Th1 type immune dominant response.

[0040] In a seventh aspect, the present invention provides a method for preparing the rabies virus vaccine, comprising the following steps:

[0041] (1) co-transfecting the rabies virus G protein vector pscAAV-G, or the rabies virus G protein vector pscAAV-G-Adj and a helper plasmid into a host cell;

[0042] (2) collecting the cells transfected in step (1) and obtaining crude recombinant adeno-associated virus by solid-liquid separation;

[0043] (3) Purifying the crude virus obtained in step (2) to obtain the rabies virus vaccine.

[0044] Wherein, the auxiliary plasmids are pAAV2 / 1 serotype capsid plasmid and pHelper plasmid.

[0045] Wherein, the host cell is HEK293FT cell.

[0046] In some embodiments of the present invention, the purification is performed using iodixanol gradient centrifugation.

[0047] Beneficial effects:

[0048] (1) The present invention utilizes the self-complementary genome characteristics of double-stranded scAAV to bypass the second-strand synthesis step of host cells and directly initiate antigen expression, thereby constructing a rabies virus G protein vector pscAAV-G, which solves the problem of delayed expression of single-stranded AAV (ssAAV) and achieves rapid and efficient expression of rabies virus G protein.

[0049] (2) The present invention further uses dsRNA sequences as nucleic acid adjuvants and constructs a rabies virus G protein vector pscAAV-G-Adj by embedding the dsRNA sequence into the non-coding region of pscAAV-G. The embedded dsRNA activates the RIG-I / MDA5 pathway, enhances the innate immune signal, and coordinates the synchronous delivery of antigens, which can drive the Th1 type immune response (marked by IgG2a antibodies), significantly improve the Th1 type immune response (IgG2a / IgG1 ratio is 3 to 5 times higher than that of traditional vaccines), enhance the vaccine's ability to induce Th1 type immune response, and avoid systemic side effects.

[0050] (3) The present invention utilizes the rabies virus G protein vectors pscAAV-G and pscAAV-G-Adj to prepare rabies virus vaccines, which significantly improves the antigen expression efficiency and immunogenicity of AAV vector vaccines. The protein vector continuously expresses the antigen, and combined with the adjuvant to enhance immune memory, it achieves long-term protection after a single vaccination, overcoming the defects of the existing rabies vaccine, which has a short immunization cycle and requires multiple vaccinations. Among them, the scAAV-Adj vaccine, through the synergistic effect of continuous antigen expression and adjuvant activation, achieved a neutralizing antibody titer of ≥1:4000 after a single vaccination in a mouse model, far exceeding the immune persistence of inactivated vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The present invention will be further described below in detail with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more apparent.

[0052] Figure 1This is the plasmid construction diagram of the recombinant plasmid pssAAV-G.

[0053] Figure 2 This is the plasmid construction diagram of the recombinant plasmid pscAAV-G.

[0054] Figure 3 This is the plasmid construction diagram of the recombinant plasmid pscAAV-G-Adj.

[0055] Figure 4 Schematic diagram of the design of recombinant plasmid pssAAV-G, recombinant plasmid pscAAV-G, and recombinant plasmid pscAAV-G-Adj.

[0056] Figure 5 Expression and purification of RABV-G protein in 293F cells. A shows SDS-PAGE (Coomassie Brilliant Blue staining). Lane M shows protein standard; Lane 1 shows purified RABV-G (61 kDa). B shows Western blot analysis. Lane M shows protein standard; Lane 1 shows pseudovirus RABV-CVS11 (61 kDa).

[0057] Figure 6 Figure 5. Antibody responses induced by the ssAAV-G and scAAV-G vaccines. Figure A shows the endpoint titer of serum rabies virus-specific IgG antibodies in BALB / c mice on day 30 after immunization. Figure B shows the neutralization titer of the two vaccines induced by the microneutralization assay.

[0058] Figure 7 Figure 2 shows the antibody responses induced by the scAAV-G and scAAV-G-Adj vaccines. Figure A shows the endpoint titer of serum rabies virus-specific IgG in BALB / c mice on day 30 after immunization. Figure B shows the neutralization titer of the two vaccines induced by the microneutralization assay.

[0059] Figure 8 Figure 1 shows the IgG subtype responses induced by the ssAAV-G, scAAV-G, and scAAV-G-Adj vaccines. Figure A shows the serum IgG1 subtype antibody level after immunization of BALB / c mice; Figure B shows the serum IgG2a subtype antibody level after immunization of BALB / c mice; and Figure C shows the IgG1 / IgG2a ratio analysis. DETAILED DESCRIPTION

[0060] The present invention will be further described below in conjunction with specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0061] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0062] In the following examples, the female BALB / c mice aged 6 to 7 weeks were purchased from Shanghai Slake Laboratory Animal Co., Ltd.

[0063] All vaccinations, feedings, and experiments were performed at the Jiangsu Provincial Laboratory Animal Center and followed the guidelines of the Institutional Animal Care and Use Committee (IACUC-2410007).

[0064] Example 1: Vector construction

[0065] 1. Construction of recombinant plasmid pssAAV-G

[0066] The pAAV-CMV-SV40 plasmid (preserved by this laboratory for a long time) was used as the basic vector and linearized by EcoRI and NotI double enzyme digestion. The coding sequence of the rabies virus G protein gene RABV-G (the coding sequence is shown in SEQ ID NO.4 and the amino acid sequence is shown in SEQ ID NO.5) after codon optimization for adaptation to mammalian cells was amplified by PCR and digested with EcoRI / NotI, and then inserted into the linearized basic vector to form a complete expression cassette containing a strong CMV promoter, optimized RABV-G and SV40 poly (A) signal. After transformation, positive clone screening and verification, the recombinant plasmid pssAAV-G was constructed, and its plasmid construction diagram is shown in FIG. Figure 1 The nucleotide sequence is shown in SEQ ID NO.1.

[0067] 2. Construction of recombinant plasmid pscAAV-G

[0068] Using the pAAV-CMV-SV40 plasmid (preserved in our laboratory for a long time) as the base vector, we deleted the single-stranded DNA replication origin (f1ori) and inserted a complete expression cassette "minimal CMV-RABV-G-SV40 poly(A) signal" containing the target gene (the codon-optimized rabies virus G protein gene RABV-G) between the mutated AAV inverted terminal repeats (ITRs). By utilizing the palindromic mutation of the ITRs, the viral genome forms a self-complementary double-stranded structure (scAAV), which can directly initiate the expression of rabies virus G protein without relying on the host cell to complete second-strand synthesis. The recombinant plasmid pscAAV-G was constructed, and its plasmid construction diagram is shown in the figure. Figure 2 The nucleotide sequence is shown in SEQ ID NO.2.

[0069] 3. Construction of the scAAV-G-Adj vector (recombinant plasmid pscAAV-G-Adj) containing the endogenous nucleic acid adjuvant dsRNA

[0070] An artificially designed H1 promoter (sequence shown in SEQ ID NO.6) and a dsRNA sequence (i.e., SLR14, sequence shown in SEQ ID NO.7) were embedded in the non-coding region of the scAAV-G vector. This sequence forms a stable dsRNA structure during the transcription process of the vector, acting as an endogenous nucleic acid adjuvant. The recombinant plasmid pscAAV-G-Adj was constructed, and its plasmid construction diagram is shown in FIG. Figure 3 The nucleotide sequence is shown in SEQ ID NO.3.

[0071] The design schematic diagrams of the recombinant plasmids pssAAV-G, pscAAV-G and pscAAV-G-Adj are shown in FIG. Figure 4 shown.

[0072] Example 2: Preparation and purification of recombinant AAV virus (i.e., AAV rabies virus vaccine)

[0073] 1. Preparation of recombinant AAV virus

[0074] The constructed AAV recombinant plasmid (pssAAV-G, pscAAV-G or pscAAV-G-Adj recombinant plasmid) was mixed with two auxiliary plasmids (pAAV2 / 1 serotype capsid plasmid and pHelper plasmid) in a mass ratio of 1:1:1 and co-transfected into HEK293FT cells using the PEI method for virus packaging. The cells were collected 72 hours after transfection and lysed to obtain crude virus.

[0075] 2. Purification of recombinant AAV virus

[0076] The crude virus obtained in step 1 was purified by iodixanol gradient centrifugation and titered by qPCR to obtain a titer ≥ 1 × 10 13 ssAAV-G, scAAV-G, and scAAV-G-Adj viral particles at GC / mL are different AAV rabies virus vaccines carrying the RABV-G gene. Add 0.001% Tween-80, aliquot, and store at -80°C in the dark.

[0077] In summary, the in vitro transfection experiments verified that the three recombinant AAVs could efficiently express rabies virus G protein, and the virus titer quantified by qPCR reached 1×10 13GC / mL and above, meeting the requirements of subsequent animal experiments.

[0078] Example 3: Preparation of rabies virus CVS11 pseudovirus RABV-CVS11

[0079] The BHK21-T7 stable cell line has been maintained in our laboratory for a long time. By transfecting 2 μg of pmCCCG plasmid carrying the CVS11 strain G protein and structural protein N, P, and L genes into BHK21-T7 cells, the rabies virus CVS11 pseudovirus RABV-CVS11 was successfully rescued and long-term passage was achieved. The above preparation process was carried out according to the method in reference document “A simplified method forming neutralizing antibodies against rabies virus. J Virol Methods” (Wu G, McElhinney LM, Goharriz H, Amaya-Cuesta J, Fooks AR, Banyard AC. A simplified method for measuring neutralizing antibodies against rabies virus. J Virol Methods. 2023 Sep; 319: 114769. doi: 10.1016 / j.jviromet.2023.114769. Epub 2023 Jun 28. PMID: 37391076.).

[0080] Example 4: High-efficiency expression and purification of antigen protein (RABV-G protein)

[0081] To obtain highly purified rabies virus G protein (RABV-G), recombinant protein expression was performed in human embryonic kidney suspension cells (293F). Specifically, the CVS11 strain G protein was cloned into the His-tagged pCDNA3.4 vector. Polyethylenimine (PEI)-mediated transient transfection was used to transfect 293F cells that had been revived and passaged three times with 2 μg of the plasmid carrying the RABV-G gene (with an 8× His tag fused to the C-terminus) for expression. The supernatant was collected after 72 hours of culture.

[0082] Then NTA beads are used for purification. Specifically, the Ni-NTA affinity column is balanced first, and the Ni-NTA affinity column is balanced with PBS buffer to ensure that the column is completely washed and adapted to the buffer environment. Then the protein supernatant is loaded, and the supernatant containing the target protein is added to the gravity-flow Ni-NTA affinity column, and the supernatant is allowed to flow naturally, and gravity is used to complete the binding of the protein. After that, non-specific binders are removed, and the column is washed with a low concentration of 20mM imidazole solution to remove miscellaneous proteins and non-specific binding substances. Finally, a high concentration of 500mM imidazole solution is used to elute the His-tagged fusion protein bound to the affinity column. The eluate containing the target protein is collected for subsequent analysis or concentration. SDS-PAGE analysis showed that the purified RABV-G protein presented a single main band under reducing conditions, with a molecular weight of approximately 61kDa ( Figure 5 A in Figure 1) is consistent with the theoretical molecular weight (approximately 61 kDa). Western Blot further verified the specificity of the target protein ( Figure 5 The final concentration of the purified protein was 7 mg / mL (determined by BCA method), which met the requirements of subsequent immunogenicity analysis.

[0083] Example 5: Animal immunization and effect evaluation

[0084] 1. Animal immunization and serum collection

[0085] 6-7 week old SPF female BALB / c mice were selected and injected with scAAV-G vaccine (1×10 11 GC / ), scAAV-G-Adj vaccine (1×10 11 GC / ) or control ssAAV-G vaccine (1×10 11 GC / each).

[0086] On day 30 after immunization, blood was collected from the tail vein (200 μL / mouse, anticoagulated with sodium heparin), incubated at 4°C for 1 hour, centrifuged at 4000 rpm for 15 minutes to separate the serum, and aliquoted into 50 μL / tubes. The serum was then frozen and thawed once at -80°C (the serum was used directly for ELISA, while the serum for neutralization experiments required a 30-minute incubation at 56°C to inactivate complement).

[0087] 2. Western Blot

[0088] (1) Sample preparation: 40 μL of pseudovirus RABV-CVS11 was added to 8 μL of 5× loading buffer, mixed, and then boiled at 100°C for 10 minutes. The loading buffer was formulated as follows: 250 mM Tris-HCl (pH 6.8), 10% SDS (w / v), 50% glycerol (v / v), 0.05% bromophenol blue (w / v), and 5% β-mercaptoethanol (v / v).

[0089] (2) Gel electrophoresis: Perform SDS-PAGE gel electrophoresis of RABV-G protein.

[0090] (3) Transfer: After electrophoresis, remove the gel and cut off the concentrated gel. Soak the separation gel in transfer buffer (prepare 1 L of 10× storage solution: 30.3 g Tris base (final concentration 250 mM), 144.2 g glycine (final concentration 1.92 M), 10 g SDS (final concentration 1%, w / v), dissolve and dilute to 1 L with deionized water. The transfer buffer is 1× storage solution, diluted with deionized water). Soak the prepared PVDF membrane in formaldehyde for activation. Soak two pieces of filter paper in transfer buffer and stack them neatly on the positive electrode in the order of filter paper, PVDF membrane, gel, and filter paper. Cover with the negative electrode plate for transfer.

[0091] (4) Blocking: After the transfer is completed, remove the PVDF membrane, place it in a blocking dish, add 30 mL of 5% skim milk, and block at RT for 2 hours.

[0092] (5) Primary antibody incubation: discard the blocking solution, wash with PBST five times, 5 min each time, add serum harvested from mice immunized with RABV-G protein as the primary antibody at a concentration of 1:2000, and incubate at 4°C overnight.

[0093] (6) Secondary antibody incubation: discard the primary antibody, wash with PBST 5 times, 5 min each time, add goat anti-mouse HRP secondary antibody at a concentration of 1:10000, and incubate at RT for 2 h.

[0094] (7) ECL chemiluminescence color development: Wash with PBST 5 times, 5 min each time, evenly cover the prepared ECL reagent on the PVDF membrane, develop the membrane by luminescence, press and expose in dark room conditions, exposure time 8 s, develop, and fix.

[0095] 3. Enzyme-linked immunosorbent assay (ELISA)

[0096] To test antibody affinity, a 96-well plate was coated with purified RABV-G protein at a concentration of 100 ng / well and incubated overnight at 4°C. The plates were then blocked with 5% skim milk powder in PBS-Tween-20 (PBST) and incubated at 37°C for 2 hours. Next, mouse serum was diluted 2-fold, dispensed into each well, and incubated at 37°C for 1 hour. Subsequently, HRP-labeled goat anti-mouse IgG secondary antibody (HS201-01; Beijing Tiangen Biotechnology Co., Ltd.) was added at a concentration of 1:10,000 and incubated at 37°C for 30 minutes. After color development, the absorbance of each well was measured at 450 nm using a microplate reader.

[0097] Antigen-specific IgG antibody subtype detection followed the same protocol as described above. Briefly, after blocking, mouse serum was diluted twofold and incubated at 37°C for 2 hours. Subsequently, HRP-conjugated goat anti-mouse IgG1 (ab97240; Abcam) and HRP-conjugated goat anti-mouse IgG2a (ab97245; Abcam) were added at a concentration of 1:2000 and incubated at 37°C for 1 hour. Color development was performed using TMB substrate. After completion of the reaction, the reaction was terminated with 0.5 M H2PO4, and the absorbance was measured at 450 nm using an ELISA reader.

[0098] 4. Neutralization experiment

[0099] The neutralization activity of mouse antisera against pseudovirus RABV-CVS11 was determined by cytopathic effect (CPE) neutralization assay. The neutralization titer was defined as the highest serum dilution that completely inhibited the appearance of CPE.

[0100] Specifically, 2 × 10 4 BHK21 cells were plated and incubated at 37°C, 5% CO2 for at least 4 hours. Subsequently, 2-fold serial dilutions of immune serum (50 μL per well) ranging from 1:128 to 1:16384 were mixed with 100 TCID50 / 50 μL of RABV-CVS11 at a volume ratio of 1:1, mixed thoroughly, and incubated at 37°C for 1 hour. Finally, the mixture was added to the cell culture plate and incubated at 37°C, 5% CO2 for 5 days until cytopathic effect (CPE) appeared in the positive control well (containing 100 TCID50 virus alone). The final endpoint titer was calculated by the CPE observed in duplicate culture wells.

[0101] 5. Effect evaluation

[0102] (1) Immunogenicity and neutralizing antibody responses of ssAAV-G and scAAV-G vaccines

[0103] To evaluate the immunogenicity of the recombinant AAV vector rabies vaccine, BALB / c mice were immunized and serum samples were collected at specific time points for the determination of the endpoint titers of rabies virus-specific IgG antibodies using ELISA. The results showed that the IgG antibody endpoint titers of the scAAV-G immunized mice were significantly higher than those of the ssAAV-G immunized mice, indicating that the scAAV-G vaccine had stronger immunogenicity than the ssAAV-G vaccine (A of FIG. 6). Figure 6 In addition, the neutralizing antibody titers induced by the two vaccines were detected using a microneutralization test. The results showed that the neutralizing antibody titers in the sera of the mice in the scAAV-G immunized group were significantly higher than those in the ssAAV-G immunized group (B of FIG. 6), further verifying the advantage of scAAV-G in inducing neutralizing antibodies. Figure 6 These results indicate that scAAV-G can more effectively induce a humoral immune response against the rabies virus compared to ssAAV-G.

[0104] Since further optimization of the AAV vector vaccine can enhance its immune effect, an adjuvant optimization scheme based on the scAAV-G vaccine (scAAV-G-Adj) will be evaluated in subsequent experiments to explore whether the immunogenicity and neutralizing antibody titers of the vaccine can be further improved based on scAAV-G.

[0105] (2) Immunogenicity and neutralizing antibody response of scAAV-G and scAAV-G-Adj vaccines

[0106] In previous studies, the scAAV-G vaccine showed stronger immunogenicity than ssAAV-G. To further enhance the immune response, a dsRNA nucleic acid adjuvant was added to the scAAV-G genome to construct the scAAV-G-Adj vaccine, and its immunogenicity was evaluated.

[0107] First, the rabies virus-specific IgG endpoint titers of the sera of BALB / c mice after immunization were detected by ELISA. The results showed that the IgG endpoint titers of the mice in the scAAV-G-Adj group were significantly higher than those in the scAAV-G group, indicating that the dsRNA adjuvant could enhance the antibody response (A of FIG. 7). Figure 7

[0108] Subsequently, the neutralizing antibody titers induced by the two vaccines were detected using a microneutralization test. The results showed that the neutralizing antibody titers in the sera of the mice in the scAAV-G-Adj group were significantly higher than those in the scAAV-G group (B of FIG. 7), indicating that the addition of the adjuvant further enhanced the humoral immune response induced by the vaccine. Figure 7

[0109] ​​(3) Analysis of IgG subtype responses induced by scAAV-G and scAAV-G-Adj vaccines

[0110] To further analyze the effect of scAAV-G-Adj vaccine on the type of immune response, we detected the levels of IgG1 and IgG2a in the serum of mice after immunization and calculated the ratio of IgG1 / IgG2a. The results showed that the level of IgG2a in the serum of mice in the scAAV-G-Adj group was significantly higher than that in the scAAV-G group, while the level of IgG1 did not differ significantly between the two groups ( Figure 8 IgG2a is a marker of Th1 cell-mediated immune response. This result suggests that dsRNA adjuvants can effectively enhance Th1-biased humoral immune responses. In addition, IgG1 / IgG2a ratio analysis showed that the IgG1 / IgG2a ratio of the scAAV-G-Adj group was significantly lower than that of the scAAV-G group ( Figure 8 C), indicating that dsRNA adjuvants can effectively promote Th1 immune responses while inhibiting Th2-related immune responses, thereby optimizing the type of immune effect induced by vaccines.

[0111] In summary, scAAV-G-Adj significantly promoted a Th1-biased immune response pattern by enhancing IgG2a levels and reducing the IgG1 / IgG2a ratio, which may further enhance the cellular immune protection efficacy of the vaccine and provide an important basis for the optimization of AAV vector vaccines.

[0112] The present invention provides a rabies virus G protein vector, a rabies virus vaccine, and methods for preparing and applying the same. While there are numerous methods and approaches for implementing this technical solution, the foregoing description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A rabies virus G protein vector, characterized in that The rabies virus G protein vector has a nucleotide sequence as shown in SEQ ID NO.

2.

2. The method for preparing the rabies virus G protein vector according to claim 1, characterized in that: The rabies virus G protein vector is obtained by inserting the coding gene of the rabies virus G protein into the coding region between the inverted terminal repeat sequences at both ends of the double-stranded adeno-associated virus vector.

3. The rabies virus G protein vector according to claim 1, characterized in that The nucleotide sequence of the gene encoding the rabies virus G protein is shown in SEQ ID NO.

4.

4. A rabies virus G protein vector, characterized in that The rabies virus G protein vector according to claim 1 is obtained by embedding an endogenous nucleic acid adjuvant into the non-coding region.

5. The rabies virus G protein vector according to claim 4, characterized in that The endogenous nucleic acid adjuvant is expressed by driving the H1 promoter; Wherein, the nucleotide sequence of the H1 promoter is shown as SEQ ID NO.6; the nucleotide sequence of the endogenous nucleic acid adjuvant is shown as SEQ ID NO.

7.

6. The rabies virus G protein vector according to claim 4, characterized in that The rabies virus G protein vector has a nucleotide sequence as shown in SEQ ID NO.

3.

7. A recombinant adeno-associated virus capable of expressing rabies virus G protein, characterized in that: The recombinant adeno-associated virus contains the rabies virus G protein vector according to any one of claims 1 to 3, or contains the rabies virus G protein vector according to any one of claims 4 to 6.

8. Use of the rabies virus G protein vector according to any one of claims 1 to 3, or the rabies virus G protein vector according to any one of claims 4 to 6, or the recombinant adeno-associated virus according to claim 7 in the preparation of a rabies virus vaccine.

9. A rabies virus vaccine, characterized in that: The rabies virus vaccine contains the rabies virus G protein vector according to any one of claims 1 to 3, or the rabies virus G protein vector according to any one of claims 4 to 6, or the recombinant adeno-associated virus according to claim 7.

10. A method for preparing the rabies virus vaccine according to claim 9, characterized in that: The steps include: (1) co-transfecting the rabies virus G protein vector according to any one of claims 1 to 3, or the rabies virus G protein vector according to any one of claims 4 to 6, and a helper plasmid into a host cell; (2) collecting the cells transfected in step (1) and obtaining crude recombinant adeno-associated virus by solid-liquid separation; (3) Purifying the crude virus obtained in step (2) to obtain the rabies virus vaccine.

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