Recombinant chimeric viruses expressing chikungunya virus structural proteins based on a getah virus backbone, and methods of making and using the same

Abstract

The application discloses a recombinant chimeric virus expressing chikungunya virus structural proteins based on a getah virus skeleton, a preparation method and application thereof. The recombinant chimeric virus contains nsP1 with an amino acid sequence as shown in SEQ ID NO. 7, nsP2 with an amino acid sequence as shown in SEQ ID NO. 8, nsP3 with an amino acid sequence as shown in SEQ ID NO. 9, nsP4 with an amino acid sequence as shown in SEQ ID NO. 10, and Cap-E3-E2-6K-E1 with an amino acid sequence as shown in SEQ ID NO. 11. Due to the genetic relationship and structural homology between GETV and CHIKV, the chimeric virus maintains the antigen conformation of the natural particle and can induce a high-efficiency neutralizing antibody response against CHIKV; the chimeric vaccine constructed based on the GETV skeleton generally has good safety and immunogenicity; and the recombinant virus can exhibit good safety and immunogenicity in a mouse body.

Description

Technical Field

[0001] This invention relates to a recombinant chimeric virus expressing chikungunya virus structural proteins based on the Getavirus backbone, its preparation method, and its application, belonging to the field of chikungunya virus infection treatment. Background Technology

[0002] Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has spread locally in more than 110 countries and regions worldwide, exposing more than 2 billion people to the risk of transmission. As of the end of 2024, 119 countries and regions had reported cases of local transmission. CHIKV infection can cause acute high fever, rash, and severe polyarthritis. Approximately 30%–40% of patients develop chronic arthritis lasting for months or even years, severely impacting quality of life. A small number of severe cases can lead to neurological complications or even death. Although two CHIKV vaccines have been approved in Europe and the United States, one of the live attenuated vaccines has recently had its license suspended due to safety concerns; and there are currently no approved specific antiviral treatments globally. Therefore, the development of a safe and effective CHIKV vaccine remains a core issue that urgently needs to be addressed in the field of public health.

[0003] Following alphavirus infection, humoral immunity plays a crucial role in viral control, and neutralizing antibodies have been proven to play a key role in controlling alphavirus infection. Chikungunya virus (CHIKV) structural proteins (especially the E1 and E2 glycoproteins) are the main antigens inducing neutralizing antibody production; therefore, subunit or virus-like particle (VLP) vaccines based on these proteins can induce good seroconversion. However, a recombinant vaccine strategy that is both safe and efficient, effectively delivering CHIKV structural proteins to induce comprehensive immune protection, is currently lacking. Alphaviruses share similar genomic structures and replication mechanisms. Using reverse genetics, exogenous antigen genes can be inserted into the viral backbone to construct chimeric viruses, thereby developing novel genetically engineered vaccines. Currently, recombinant vaccine development against Chikungunya virus (CHIKV) largely focuses on chimeric strategies based on the backbones of alphavirus members (such as SFV and VEEV). While such strategies can induce neutralizing antibodies by leveraging the conservation of structural proteins, existing approaches still have some limitations. First, not all alphavirus backbones are compatible with the insertion of CHIKV structural proteins. For example, chimerism between the SFV backbone and certain regions of CHIKV can lead to viral non-viability. Furthermore, there is an imbalance between the safety and immunogenicity of recombinant viruses. For instance, while chimeras based on attenuated VEEV strains exhibit strong immunogenicity, they are limited by BSL-3 laboratory operating procedures and the potential risk of virulence reversion. Therefore, given the current bottleneck of chimeric vaccine strategies—the difficulty in simultaneously achieving backbone compatibility, safety, and immunogenicity—there is an urgent need to explore novel recombinant viral vectors or innovative antigen presentation platforms to achieve efficient and stable presentation of CHIKV structural proteins, thereby inducing durable and broad-spectrum protective immunity while mitigating biosafety risks. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a recombinant chimeric virus based on the Geta virus backbone expressing the structural protein of Chikungunya virus, its preparation method, and its application.

[0005]

[0006]

[0007]

[0008] The present invention also provides a method for preparing the recombinant chimeric virus, comprising the following steps: replacing nucleotides 7527 to 11285 in the genome of GETV with the nucleotide sequence of CHIKV with GenBank accession number KC488650.1, thereby constructing a recombinant chimeric virus with GETV as the backbone and expressing CHIKV structural proteins.

[0009] This includes the following steps: (1) Using pAC-GETV plasmid as a template, the G1 and G2 fragments were amplified respectively; the C1 fragment was amplified using the S1 fragment as a template, and the C2 fragment was amplified using the S2 fragment as a template. (2) The G1 and C1 fragments were amplified by overlap PCR to obtain the GC1 fragment; the C2 and G2 fragments were amplified by overlap PCR to obtain the CG2 fragment; the GC1 and CG2 fragments were amplified by overlap PCR to obtain the GC1-CG2 fragment; (3) Link the GC1-CG2 fragment to the vector.

[0010] The primers for amplifying the G1 fragment in step (1) are shown in SEQ ID NO.12 and SEQ ID NO.13, and the primers for amplifying the G2 fragment are shown in SEQ ID NO.18 and SEQ ID NO.19.

[0011] In step (1), the primers for amplifying the C1 fragment are shown in SEQ ID NO.14 and SEQ ID NO.15, and the primers for amplifying the C2 fragment are shown in SEQ ID NO.16 and SEQ ID NO.17.

[0012] In step (2), when the G1 and C1 fragments are amplified by overlap PCR to obtain the GC1 fragment, the primers used are shown in SEQ ID NO.12 and SEQ ID NO.15; when the C2 and G2 fragments are amplified by overlap PCR to obtain the CG2 fragment, the primers used are shown in SEQ ID NO.16 and SEQ ID NO.19.

[0013] In step (2), when the GC1 fragment and the CG2 fragment are amplified by overlap PCR to obtain the GC1-CG2 fragment, the primers used are shown in SEQ ID NO.12 and SEQ ID NO.19.

[0014] The present invention also provides a vaccine containing a passaged virus of the recombinant chimeric virus.

[0015] The passaged virus is obtained by passage in Vero cells at least five times consecutively.

[0016] The present invention also provides the use of the recombinant chimeric virus or the vaccine in the preparation of medicaments or pharmaceutical compositions for the treatment and / or prevention of chikungunya virus infection.

[0017] This invention utilizes reverse genetics to create a recombinant chimeric virus by combining Getah virus (GETV), an important mosquito-borne virus belonging to the alphavirus genus (SFV and CHIKV), with a GETV backbone expressing CHIKV structural proteins. The process involves replacing the corresponding region in the GETV genome with the complete CHIKV structural protein gene, constructing a recombinant chimeric virus with a GETV backbone expressing CHIKV structural proteins, as shown in SEQ ID NO. 2. This technical approach focuses on the construction and validation of GETV as a vaccine expression vector platform, providing a new candidate strain for CHIKV vaccine development, and offering a referable technical path for vaccine development of other members of the alphavirus genus.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. Due to the kinship and structural homology between GETV and CHIKV, the chimeric virus maintains the antigenic conformation of the natural particles, achieving efficient and stable presentation of CHIKV structural proteins; 2. The recombinant virus can effectively induce the production of neutralizing antibodies against CHIKV in mice, exhibiting good immunogenicity; 3. This strategy allows for precise control of the virus's safety at the design level, providing a scalable technical platform for future expansion to the development of vaccines against other mosquito-borne viruses. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the amplified fragment; Figure 2 This is a schematic diagram of the recombinant virus genome structure; Figure 3 This is a schematic diagram of the construction of the pAC-GETV(nsP)-CHIKV(sp) recombinant plasmid; Figure 4 This refers to CPE generated after Vero infection with rGETV(nsP)-CHIKV(sp); Figure 5 PCR identification of rGETV(nsP)-CHIKV(sp); Figure 6 Indirect immunofluorescence identification of recombinant viruses; Figure 7 Western blotting identification of recombinant viruses; Figure 8 The growth curve of rGETV(nsP)-CHIKV(sp); Figure 9 This is a schematic diagram of mice immunized with recombinant virus. Figure 10 For the determination of neutralizing antibody titer. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0021] 1. Experimental Materials The infectious clone plasmid pAC-GETV of GETV (GenBank: MF741771.1) was constructed and preserved in our laboratory (Zhou Jingye, Meng Hui, Lu Tianyuan, et al. Construction of infectious clone of porcine gettavirus and virus rescue [J]. Journal of Yangzhou University (Agricultural and Life Sciences Edition), 2023, 44(6):62-69.). Hamster kidney cells (BHK21) and African green monkey kidney cells (Vero) were preserved in our laboratory. T-Vecor pMD 19 (Cat: 3271) was purchased from TaKaRa; Chikungunya virus E1 antibody (Cat: GTX135187) was purchased from GeneTex; DyLight 488, Goat Anti-Rabbit IgG (Cat: A23220) was purchased from Abbkine; HRP-conjugated Goat anti-Rabbit IgG (H+L) (Cat: AS014) was purchased from Abclonal; β-propiolactone was purchased from Sigma-Aldrich; vaccine adjuvants HS802 (Cat: 20250512), M107 (Cat: 20240908) and HS302 (Cat: 20250601) were purchased from Zhejiang Hongsheng Biotechnology Co., Ltd.; 6-8 week old female BALB / c mice were purchased from the Experimental Animal Center of the School of Comparative Medicine, Yangzhou University.

[0022] 2. Gene synthesis and primer design Based on the structural protein gene sequence of the CHIKV-JC2012 strain (GenBank: KC488650.1), it was divided into two gene segments, S1 and S2 (S1 segment: nucleotides 7487-9337 of the CHIKV-JC2012 sequence; S2 segment: nucleotides 9338-11233 of the CHIKV-JC2012 sequence). Figure 1As shown in Table 1, primers were designed by Jiutian Gene Technology (Tianjin) Co., Ltd. based on the pAC-GETV(nsP)-CHIKV(sp) sequence map, and primers were synthesized by Nanjing Qingke Biotechnology Co., Ltd.

[0023] Table 1 Primer Usage Table for Recombinant Plasmid Construction

[0024] 3. Construction of the infectious clonal plasmid pAC-GETV(nsP)-CHIKV(sp) Figure 1 and Figure 2 ) (1) RT-PCR amplification: Using pAC-GETV plasmid as template, and Swa I-F and 26S-R as upstream and downstream primers, the G1 fragment was amplified; using pAC-GETV plasmid as template, and 3'UTR-F and Mlu I-R as upstream and downstream primers, the G2 fragment was amplified; using S1 fragment as template, and CHIKV-F1 and CHIKV-R1 as upstream and downstream primers, the C1 fragment was amplified; using S2 fragment as template, and CHIKV-F2 and CHIKV-R2 as upstream and downstream primers, the C2 fragment was amplified. The amplification system and procedure are shown in Table 2.

[0025] Table 2 PCR amplification system and procedure

[0026] (2) Overlap PCR amplification: Using Swa I-F and CHIKV-R1 as upstream and downstream primers, the G1 and C1 fragments were amplified by overlap PCR to obtain the GC1 fragment. The amplification system and program are shown in Table 3. Using CHIKV-F2 and Mlu I-R as upstream and downstream primers, the C2 and G2 fragments were amplified by overlap PCR to obtain the CG2 fragment. The amplification system and program are shown in Table 4. Finally, using Swa I-F and Mlu I-R as upstream and downstream primers, the GC1 and CG2 fragments were amplified by overlap PCR to obtain the GC1-CG2 fragment. The amplification system and program are shown in Table 5.

[0027] (3) Ligation: The GC1-CG2 fragment was ligated to a homologous recombination kit using a ligation method. Swa I and Mlu I. In the double-digested pAC-GETV vector.

[0028] (4) Sequencing: The constructed recombinant plasmid was sent to Nanjing Qingke Biotechnology Co., Ltd. for Sanger sequencing to confirm whether the plasmid was successfully constructed.

[0029] Table 3 PCR amplification system and procedure

[0030] Table 4 PCR amplification system and procedure

[0031] Table 5 PCR amplification system and procedure

[0032] 4. Rescue of Recombinant Viruses (1) Transfection: BHK21 cells in good growth condition were evenly seeded into 6-well plates. When the cell density reached 70%-80%, the pAC-GETV(nsP)-CHIKV(sp) recombinant plasmid constructed above was transfected according to the Lipofectamine® 3000 reagent instructions. The transfected cells were placed in an incubator and cultured for 48 hours to observe cytopathic effects.

[0033] (2) Virus passage: When obvious cell rounding and detachment occur, we determine that new virus particles are produced. After collecting and centrifuging the supernatant, 10 μL of the supernatant is inoculated into Vero cells. Subsequently, Vero cells can produce obvious cytopathic effect (CPE). Figure 4 Then, the virus was passaged continuously. Each generation of virus solution was labeled and stored at -80°C.

[0034] 5. Identification of recombinant viruses 5.1 Recombinant Viral Genome Sequencing: P5 generation viral fluid was used to extract recombinant viral RNA using an RNA extraction kit, following the manufacturer's instructions. Reverse transcription was then performed to obtain cDNA. Using this cDNA as a template, the PCR amplification system and procedure were the same as in Table 2. CHIKV-F1 and CHIKV-R1 were used as upstream and downstream primers to amplify the C1 fragment; CHIKV-F2 and CHIKV-R2 were used as upstream and downstream primers to amplify the C2 fragment. The recombinant viral gene fragments C1 and C2 were amplified, and PCR identification confirmed the successful amplification of the C1 and C2 fragments. Figure 5

[0035] Based on genome sequencing and CPE results, we have successfully rescued a recombinant chimeric virus, rGETV(nsP)-CHIKV(sp), which expresses chikungunya virus structural proteins with a gettavirus backbone. Figure 3 ).

[0036] 5.2 Indirect immunofluorescence identification of recombinant virus: After infecting Vero cells with the recombinant virus for 24 hours, the cells were fixed and detected using a monoclonal antibody against the CHIKV-E1 protein. The specific steps are as follows: (1) The Vero cells in good growth condition were evenly spread into a 12-well plate. When the cell density reached 90%, 10 μL of P5 generation rescue virus solution was inoculated into the cell monolayer. At the same time, wells without virus solution were set as controls. The cells were incubated at 37°C and 5% CO2 for 24 hours.

[0037] (2) 24 hours after viral infection, the supernatant was discarded, the cell monolayer was washed twice with PBS, fixed with pre-cooled 4% paraformaldehyde at room temperature for 10 minutes, and washed twice with PBS.

[0038] (3) Add pre-cooled PBS solution containing 0.1% Triton X-100 and 2% BSA, break the membrane at room temperature, block for 30 minutes, and wash twice with PBS.

[0039] (4) Add Chikungunya virus E1 antibody diluted 1:5000, incubate at 37°C for 1 hour, and wash twice with PBS.

[0040] (5) Add DyLight 488, Goat Anti-Rabbit IgG diluted 1:1000, incubate at 37°C in the dark for 1 hour, and wash twice with PBS.

[0041] (6) Add DAPI staining solution diluted 1:10, incubate at room temperature for 5 minutes, wash twice with PBS, add an appropriate amount of PBS, observe and take pictures under an inverted fluorescence microscope.

[0042] After infection with the recombinant virus, Vero cells exhibited specific green fluorescence, while the negative control wells did not show green fluorescence. Figure 6 ).

[0043] 5.3 Western Blot Identification of Recombinant Virus (1) Seed Vero cells into 12-well plates in advance and culture them until the cell density reaches 90%.

[0044] (2) Vero cells were infected with the recombinant virus at MOI=0.1 for 24 h. The uninfected group was set up as a negative control.

[0045] (3) Discard the culture supernatant, wash twice with PBS buffer, add 100 μL of RIPA lysis buffer to each well, and collect the protein sample.

[0046] (4) The collected protein samples were identified by Western blot. The primary antibody used was Chikungunya virus E1 antibody, and the secondary antibody used was HRP-conjugated Goat anti-Rabbit IgG (H+L).

[0047] The results are as follows Figure 7 As shown, the expression of E1 protein can be detected in the virus-infected group.

[0048] Indirect immunofluorescence and Western blot results showed that the chimeric virus maintained the antigenic conformation of the native particles, achieving efficient and stable presentation of CHIKV structural proteins.

[0049] 6. Recombinant virus growth curve (1) Vero cells were pre-seeded in 6-well plates and infected with the virus when the cell density reached 90%. The supernatant was collected at 12, 24, 36 and 48 hours after infection.

[0050] (2) The collected virus solution was continuously diluted 10 times. The diluted virus was inoculated into 96-well microculture plates. Each dilution was inoculated into 4 parallel wells, with 100 µl inoculated into each well. Normal cells were set up as controls.

[0051] (3) Observe and record the results daily, count the number of infected wells, and calculate the 50% tissue culture infection dose (TCID) for each sample using the Kärber method. 50 ).

[0052] like Figure 8 As shown, the viral titer of rGETV(nsP)-CHIKV(sp) can reach 10 after 48 hours. 7.25 TCID 50 / mL.

[0053] 7. Animal testing To investigate the immunogenicity of rGETV(nsP)-CHIKV(sp) and the advantages of the GETV backbone as a CHIKV vector, we set up an inactivated virus group (virus inactivation method: treatment with 0.2% β-propiolactone at 4℃ for 24 h, followed by two blind passages to confirm inactivation) as a control. 100 μL of 10 6 TCID50 / mL rGETV(nsP)-CHIKV(sp) was mixed with three different vaccine adjuvants: HS802 (rGETV(nsP)-CHIKV(sp) to HS802 volume ratio 2:1), M107 (rGETV(nsP)-CHIKV(sp) to M107 volume ratio 8:1), and HS302 (rGETV(nsP)-CHIKV(sp) to HS302 volume ratio 10:1). Mice were immunized subcutaneously. Initial and booster immunizations were performed on day 1 and day 14, respectively. Blood samples were collected on day 14 and day 28 post-infection, and serum was separated to detect neutralizing antibody titers. Specific experimental protocols (Table 6) and procedures (…) are detailed below. Figure 9 )as follows: Table 6 Animal Experimentation Protocol

[0054] 8. Determination of neutralizing antibodies in mouse serum (1) One day in advance, seed Vero cells into 96-well plates and carry out the experiment when the cell density reaches 90%.

[0055] (2) The collected serum was inactivated at 56°C for 30 minutes and filtered in a clean bench with a 0.22 μm filter.

[0056] (3) The serum was serially diluted 2-fold with DMEM medium. For each dilution, 250 μL of diluted serum was mixed with 250 μL of diluted rGETV(nsP)-CHIKV(sp) virus solution (100 TCID50). 50 Mix thoroughly (100 μL) and incubate at 37°C for 1 hour.

[0057] (4) After incubation, discard the culture medium in the 96-well plate, add 100 μL of serum-virus mixture to the cell wells, perform 4 replicates for each dilution, and set up a positive control well (add only 100 μL TCID3). 50 The virus solution and negative control wells (containing only DMEM medium) were incubated for 48 hours in a 5% CO2, 37°C incubator, and the neutralizing titer of the immunized mouse serum was calculated. The calculation formula is: lg (neutralizing antibody titer) = L + d(S - 0.5) (L: logarithm of the highest serum dilution; d: group interval, i.e., dilution factor, which is -0.3 in a 2-fold serial dilution; S: sum of the ratios of the number of wells with CPE to the number of inoculated wells in each group).

[0058] We used a virus neutralization assay to detect the level of anti-CHIKV neutralizing antibodies in the serum of immunized mice, such as... Figure 10As shown, mice immunized with rGETV(nsP)-CHIKV(sp) produced neutralizing antibody titers against CHIKV of 2. 5 Compared to inactivated viruses, live viruses can induce higher levels of neutralizing antibodies; with the addition of vaccine adjuvants, the neutralizing antibody titer can reach 2. 6.6 .

Claims

1. A recombinant chimeric virus, characterized in that, The amino acid sequence contains nsP1 (SEQ ID NO. 7), nsP2 (SEQ ID NO. 8), nsP3 (SEQ ID NO. 9), nsP4 (SEQ ID NO. 10), and Cap-E3-E2-6K-E1 (SEQ ID NO. 11).

2. The recombinant chimeric virus according to claim 1, characterized in that, The nucleotide sequence contains nsP1 with SEQ ID NO.2, nsP2 with SEQ ID NO.3, nsP3 with SEQ ID NO.4, nsP4 with SEQ ID NO.5, and Cap-E3-E2-6K-E1 with SEQ ID NO.

6.

3. A method for preparing the recombinant chimeric virus according to claim 1 or 2, characterized in that, The steps include: replacing nucleotides 7527-11285 in the genome of GETV with the nucleotide sequence of CHIKV (GenBank accession number KC488650.1) to construct a recombinant chimeric virus with GETV as the backbone that expresses CHIKV structural proteins.

4. The method according to claim 3, characterized in that, Includes the following steps: (1) Using pAC-GETV plasmid as a template, amplify G1 and G2 fragments respectively; using S1 fragment as a template, amplify C1 fragment, and using S2 fragment as a template, amplify C2 fragment; (2) The G1 and C1 fragments were amplified by overlap PCR to obtain the GC1 fragment; the C2 and G2 fragments were amplified by overlap PCR to obtain the CG2 fragment; the GC1 and CG2 fragments were amplified by overlap PCR to obtain the GC1-CG2 fragment; (3) Link the GC1-CG2 fragment to the vector.

5. The method according to claim 4, characterized in that, The primers for amplifying the G1 fragment in step (1) are shown in SEQ ID NO.12 and SEQ ID NO.13, and the primers for amplifying the G2 fragment are shown in SEQ ID NO.18 and SEQ ID NO.

19.

6. The method according to claim 4, characterized in that, The primers for amplifying the C1 fragment in step (1) are shown in SEQ ID NO.14 and SEQ ID NO.15, and the primers for amplifying the C2 fragment are shown in SEQ ID NO.16 and SEQ ID NO.

17.

7. The method according to claim 4, characterized in that, In step (2), when the G1 and C1 fragments are amplified by overlap PCR to obtain the GC1 fragment, the primers used are shown in SEQ ID NO.12 and SEQ ID NO.

15.

8. A vaccine, characterized in that, It contains a passaged virus of the recombinant chimeric virus as described in claim 1 or 2.

9. The vaccine according to claim 8, characterized in that, The passaged virus was obtained by passaged at least five times consecutively in Vero cells.

10. The use of the recombinant chimeric virus of claim 1 or 2 in the preparation of a medicament, pharmaceutical composition or vaccine for the treatment and / or prevention of chikungunya virus infection.

Images