A vaccine against wetland virus glycoprotein Gc and its application

Abstract

The present invention discloses a vaccine of wetland virus glycoprotein Gc and its application, which belongs to the field of biomedical technology. The wetland virus glycoprotein Gc is a recombinant wetland virus glycoprotein Gc, and the wetland virus glycoprotein Gc is a truncated glycoprotein truncated at the C-terminus; the amino acid sequence of the truncated wetland virus glycoprotein Gc is the protein shown in positions 1-525 of SEQ ID No. 1. After immunizing mice with the vaccine of the present invention, high-titer antibodies against the truncated wetland virus glycoprotein Gc can be produced, and the authentic WELV virus can be neutralized; after boosting immunization of mice with the vaccine prepared by the present invention, 100% of the mice can be protected from death threat under the fully lethal dose of WELV; the recombinant truncated WELV glycoprotein Gc subunit candidate vaccine prepared by the present invention has good safety, can effectively stimulate the immune response, and can completely protect mice from death threat.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a vaccine of wetland virus glycoprotein Gc and its application. Background Art

[0002] Tick-borne virus infections are a global health problem associated with significant morbidity and mortality. In the past decade, several new tick-borne viruses associated with human diseases have been discovered, such as severe fever with thrombocytopenia syndrome virus, Heartland virus, etc. Within the Bunyaviridae family, the genus Orthonairovirus contains the main species causing human infections, including Crimean-Congo hemorrhagic fever, Dugbe virus, Songling virus, etc., all of which are transmitted by tick vectors. Except for the pathogenic Crimean-Congo hemorrhagic fever virus (CCHFV), the diseases caused by other infections are relatively mild; however, due to their broad host range and wide geographical distribution, they have public health significance.

[0003] WELV particles are enveloped, spherical and oval, with a diameter ranging from 80 - 160 nm. WELV has a negative-sense single-stranded tripartite RNA genome: small (S), middle (M) and large (L), encoding nucleoprotein (NP), glycoprotein precursor (GPC) and RNA-dependent RNA polymerase (RdRP), respectively. The L, M and S segments have typical inverted complementary sequences at their termini, with UCUCAAAGA at the 5'-end and AGAGUUUCU at the 3'-end. The M segment contains an open reading frame encoding a 1436-amino acid glycoprotein precursor. Two cleavage sites of subtilisin-kexin isozyme 1 / site 1 protease (SKI-1 / S1P) have been identified at positions RRLM 306↓ and RKLL 791↓. Generally, the replication cycle of orthonairoviruses starts with the binding of the virus to receptors on the cell surface, which is mediated by membrane proteins. However, the specific mechanisms by which membrane proteins are involved in virus adsorption, internalization and fusion are still unclear, and the cellular receptors required for orthonairovirus invasion have not been discovered yet.

[0004] WELV and CCHFV both belong to the Bunyaviridae family. The case fatality rate of Crimean-Congo hemorrhagic fever (CCHF) varies by region but is generally between 30% and 50%. Humans are generally susceptible to CCHFV, and there are no significant differences in susceptibility by age or gender. Currently, there are no effective preventive or treatment measures for CCHF infection, which seriously threatens people's physical health. The World Health Organization (WHO) has listed CCHFV in the list of research and development blueprints for infectious pathogens. The surface glycoprotein of the WELV virus can specifically recognize the receptor on the surface of host cells. By binding to specific receptors, it initiates the invasion process of the virus into host cells. The virus's own proteins are usually one of the main targets recognized by the immune system and can stimulate the host's immune response, including the production of antibodies and the activation of cellular immunity. These antibodies can neutralize the virus and prevent it from infecting host cells, while cellular immunity can directly attack virus-infected cells. These proteins of the virus itself are one of the important targets for vaccine design. By inducing the body to produce antibodies against these protein antigens, virus infection can be effectively prevented. In recent years, some studies have explored different types of CCHFV candidate vaccines, such as DNA candidate vaccines, recombinant attenuated live vesicular stomatitis virus vaccines, recombinant human adenovirus type 5 CCHFV candidate vaccines, etc. However, so far, no CCHF vaccine that combines high efficiency, safety, and low cost has been developed, and no vaccine has been approved for market. The wetland virus WELV was first announced in 2024, and the corresponding vaccine and drug research is even blank. There is an urgent need to develop a WELV vaccine that combines high efficiency, safety, and low cost. Summary of the Invention

[0005] Based on the problems existing in the prior art, the present invention provides a vaccine of wetland virus glycoprotein Gc and its application, specifically, the wetland virus (WELV) glycoprotein Gc, an immune composition containing this recombinant protein Gc, and its application.

[0006] According to the first aspect of the technical solution of the present invention, a wetland virus glycoprotein Gc is provided. The wetland virus glycoprotein Gc is a recombinant wetland virus glycoprotein Gc, and the wetland virus glycoprotein Gc is a C-terminal truncated glycoprotein truncate.

[0007] Preferably, the wetland virus glycoprotein Gc truncate is any of the following proteins:

[0008] A1, a protein comprising the amino acid sequence shown in positions 1-525 of SEQ ID No.1;

[0009] A2, a protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence defined by A1 and having the same function;

[0010] A3, a protein having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the amino acid sequence defined by A1 or A2 and having the same function;

[0011] A4, a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined by any one of A1 - A3.

[0012] According to the second aspect of the technical solution of the present invention, a biological material is provided, and the biological material is any one of the following:

[0013] C1, a nucleic acid molecule encoding the wetland virus glycoprotein Gc described above;

[0014] C2, an expression cassette containing the nucleic acid molecule described in C1;

[0015] C3, a recombinant vector containing the nucleic acid molecule described in C2, or a recombinant vector containing the expression cassette described in C2;

[0016] C4, a recombinant microorganism containing the nucleic acid molecule described in C1, or a recombinant microorganism containing the expression cassette described in C2, or a recombinant microorganism containing the recombinant vector described in C3);

[0017] C5, a recombinant cell containing the nucleic acid molecule described in C1, or a recombinant cell containing the expression cassette described in C2, or a recombinant cell containing the recombinant vector described in C3.

[0018] According to the third aspect of the technical solution of the present invention, a product containing the above-mentioned wetland virus glycoprotein Gc is provided, and the product is an immune composition or a pharmaceutical composition. The immune composition is a vaccine, and the vaccine further contains an adjuvant. The adjuvant is an aluminum adjuvant or a CpG adjuvant. The aluminum adjuvant includes an aluminum hydroxide adjuvant; the CpG adjuvant is any one of the following:

[0019] (1) CpG2006 with a nucleotide sequence of SEQ ID No.5 or its thiolated product;

[0020] (2) CpG1018 with a nucleotide sequence of SEQ ID No.6 or its thiolated product;

[0021] (3) CpG684 with a nucleotide sequence of SEQ ID No.7 or its thiolated product;

[0022] The thiolated product is a full-chain thiolated modification product.

[0023] Further, in the product, the vaccine is composed of a C-terminal truncated glycoprotein truncate, an aluminum adjuvant, and a CpG adjuvant; wherein, the mass ratio of the C-terminal truncated glycoprotein truncate, the aluminum adjuvant, and the CpG adjuvant is 10(2.5): 100 : 50 by mass; the amount of the aluminum adjuvant is calculated based on the content of aluminum therein.

[0024] According to the fourth aspect of the technical solution of the present invention, a preparation method of the wetland virus glycoprotein Gc as described above is provided, and the method comprises the following steps:

[0025] Step S1, expressing the truncated wetland virus glycoprotein Gc in yeast;

[0026] Expressing the truncated wetland virus glycoprotein Gc in Pichia pastoris genetically engineered in the N-glycosylation modification pathway to obtain recombinant yeast cells; the Pichia pastoris genetically engineered in the N-glycosylation modification pathway is Pichia pastoris with inactivated α-1,6-mannosyltransferase;

[0027] Expressing the truncated wetland virus glycoprotein Gc in the Pichia pastoris genetically engineered in the N-glycosylation modification pathway is achieved by introducing a nucleic acid molecule encoding the truncated wetland virus glycoprotein Gc into the Pichia pastoris genetically engineered in the N-glycosylation modification pathway;

[0028] Step S2, fermentatively culturing the recombinant yeast cells, and purifying the truncated wetland virus glycoprotein Gc from the supernatant of the fermentative culture;

[0029] Step S3, expressing the truncated wetland virus glycoprotein Gc in the Expi-293F mammalian cell expression system; expressing the truncated wetland virus glycoprotein Gc in the Expi-293F mammalian cell expression system is achieved by introducing a nucleic acid molecule encoding the truncated wetland virus glycoprotein Gc into the Expi-293F mammalian cell expression system;

[0030] Step S4, performing suspension culture on the Expi-293F mammalian cells, and purifying the truncated wetland virus glycoprotein Gc from the supernatant of the suspension cell culture; purification includes the step of subjecting the supernatant of the suspension cell culture to nickel ion affinity chromatography.

[0031] Further, in the preparation method, the nucleic acid molecule encoding the truncated wetland virus glycoprotein Gc is any one of the following:

[0032] a1, a DNA molecule with a nucleotide sequence as shown in SEQ ID No.2 or the 1st to 1575th positions of SEQ ID No.2;

[0033] a2, a DNA molecule that hybridizes with the DNA molecule defined by a1 under stringent conditions and encodes a truncated form of the wetland virus glycoprotein Gc;

[0034] a3, a DNA molecule that has an identity of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the DNA sequence defined by a1 or a2 and encodes a truncated form of the wetland virus glycoprotein Gc.

[0035] According to the fifth aspect of the technical solution of the present invention, there is provided an application of the wetland virus glycoprotein Gc as described above in the preparation of an immune product capable of inducing an antigen-specific antibody in an animal, or an application of the above biological material in the preparation of an immune product capable of inducing an antigen-specific antibody in an animal. In the said application, the immune product is a vaccine. The vaccine is immunized by intramuscular injection or subcutaneous injection.

[0036] Compared with the prior art, the present invention has at least the following beneficial effects:

[0037] 1. The present invention discloses a method for constructing an expression vector of a truncated form of WELV glycoprotein Gc (truncated Gc subunit). The constructed expression vector can express the truncated form of WELV glycoprotein Gc (truncated Gc subunit) in mammalian cells and yeast cells, and has the characteristics of fast growth and high safety.

[0038] 2. The truncated form of the wetland virus (WELV) glycoprotein Gc (truncated Gc subunit) obtained by recombinant expression of the present invention does not have the problem of over-glycosylation. After immunizing mice with it, high-titer specific IgG antibodies against the truncated form of WELV glycoprotein Gc (truncated Gc subunit) can be produced. After booster immunization, the mice can be protected from lethal attacks by WELV. Therefore, the recombinant prepared truncated WELV Gc subunit sub-unit candidate vaccine can provide a good protective response and provide a reference for the development of WELV subunit vaccines. Brief Description of the Drawings

[0039] Figures 1A - 1B They are respectively schematic diagrams of the structure of the recombinant WELV glycoprotein Gc subunit truncated form (truncated Gc subunit); among them, Figure 1A is a schematic diagram of the structure of the truncated Gc subunit of WELV, Figure 1B is the three-dimensional structure prediction of the truncated Gc subunit of WELV.

[0040] Figures 2A - 2C They are respectively the identification results of the protein expression of the recombinant WELV glycoprotein Gc subunit truncated form changing with time in the Expi 293F expression system; among them, Figure 2A is the curve of the cell viability changing with time after transfection; Figure 2BSDS-polyacrylamide gel electrophoresis (SDS-PAGE) showing protein expression over time Figure 2C Results detected by Anti-His tag monoclonal antibody (Sigma, A7058).

[0041] Figures 3A - 3C Identification of nickel affinity chromatography, SDS-PAGE and WB of the truncated recombinant WELV glycoprotein Gc subunit samples, respectively; among them, Figure 3A is the nickel ion affinity chromatography chromatogram; Figure 3B is the SDS-PAGE of the samples during the purification process; Figure 3C is the WB identification of the samples during the purification process.

[0042] Figure 4 SDS-PAGE of the purified truncated recombinant WELV glycoprotein Gc subunit samples

[0043] Figure 5 Detection of antibody titer in mice immunized intramuscularly with the truncated recombinant WELV glycoprotein Gc subunit protein

[0044] Figure 6 Survival curve for evaluating the protective effect of the truncated recombinant WELV glycoprotein Gc subunit protein in a lethal WELV model in mice immunized intramuscularly

[0045] Figure 7 Weight changes in mice immunized intramuscularly with the truncated recombinant WELV glycoprotein Gc subunit protein after challenge with a lethal dose of WELV

[0046] In each quantitative result graph in the above figures, ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001. Detailed implementation manners

[0047] The present invention will be further described in detail below in conjunction with specific implementation manners. The provided examples are only for clarifying the present invention and not for limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way.

[0048] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0049] The present invention provides a vaccine against wetland virus glycoprotein Gc and its application. Wetland virus (WELV) belongs to a new species of the genus Orthonairovirus in the family Nairoviridae. The wetland virus glycoprotein Gc is a recombinant wetland virus glycoprotein Gc. Further, the present invention also provides a vaccine against wetland virus (WELV), wherein the antigen, wetland virus (WELV) glycoprotein Gc, is a truncated form. The amino acid sequence of the truncated form of the glycoprotein Gc of wetland virus (WELV) is shown in SEQ ID No.1. After immunizing mice with the vaccine of the present invention, high-titer antibodies against the truncated form of wetland virus (WELV) glycoprotein Gc can be produced, and the true virus of WELV can be neutralized. After booster immunization of mice with the vaccine prepared by the present invention, at the fully lethal dose of wetland virus (WELV), the mice can be 100% protected from the threat of death. The recombinant WELV glycoprotein Gc truncated form subunit candidate vaccine prepared by the present invention has good safety, can effectively stimulate the immune response, and can completely protect mice from the threat of death. The present invention can provide a reference for the development of a subunit vaccine against wetland virus (WELV).

[0050] In one embodiment, the present invention provides a recombinant glycoprotein Gc of wetland virus (WELV). Further, the antigen in the vaccine against wetland virus (WELV) is a truncated form of the glycoprotein Gc of WELV. The recombinant glycoprotein Gc comprises a polypeptide having the amino acid sequence shown in SEQ ID NO.1.

[0051] The truncated form of the glycoprotein Gc of wetland virus (WELV) is any of the following proteins:

[0052] A1, a protein having the amino acid sequence shown in SEQ ID No.1 or the amino acid sequence of positions 1-525 of SEQ ID No.1;

[0053] A2, a protein obtained by substituting and / or deleting and / or adding one or several amino acid residues in the amino acid sequence defined by A1 and having the same function;

[0054] A3, a protein having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the amino acid sequence defined by A1 or A2 and having the same function;

[0055] A4, a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in any of A1-A3.

[0056] In the above-mentioned protein, the tag refers to a polypeptide or protein that is expressed by fusion with the target protein using in vitro DNA recombination technology, so as to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag can be a Flag tag, His tag, MBP tag, HA tag, Myc tag, GST tag, and / or SUMO tag, etc.

[0057] In the above-mentioned protein, the identity of the amino acid sequence can be determined using an identity search site on the Internet, such as the BLAST web page on the NCBI homepage website. For example, in Advanced BLAST 2.1, by using Blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gapexistence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0058] In the above-mentioned protein, the identity of more than 95% can be at least 96%, 97%, or 98% identity. The identity of more than 90% can be at least 91%, 92%, 93%, or 94% identity. The identity of more than 85% can be at least 86%, 87%, 88%, or 89% identity. The identity of more than 80% can be at least 81%, 82%, 83%, or 84% identity.

[0059] In another embodiment, a vaccine containing the recombinant wetland virus (WELV) glycoprotein Gc is provided, and the wetland virus (WELV) vaccine further includes an adjuvant. Among them, the adjuvant can be an aluminum adjuvant and / or a CpG adjuvant.

[0060] Furthermore, the aluminum adjuvant can be an aluminum hydroxide adjuvant.

[0061] Furthermore, the CpG adjuvant can be any one of the following:

[0062] (1) CpG2006 with the nucleotide sequence of SEQ ID No.5 or its thiolated product;

[0063] (2) CpG1018 with the nucleotide sequence of SEQ ID No.6 or its thiolated product;

[0064] (3) CpG684 with the nucleotide sequence of SEQ ID No.7 or its thiolated product;

[0065] Among them, the thiolated product can be a full-chain thiolated modification product.

[0066] In a specific embodiment of the present invention, the Wetland virus (WELV) vaccine is composed of the truncated Wetland virus (WELV) glycoprotein Gc, the aluminum adjuvant, and the CpG adjuvant; wherein, the mass ratio of the WELV glycoprotein Gc truncation, the aluminum adjuvant, and the CpG adjuvant is (2.5 - 20) : 100 : 50. Wherein, the amount of the aluminum adjuvant is calculated based on the aluminum content therein.

[0067] In another specific embodiment of the present invention, the Wetland virus (WELV) vaccine is composed of the truncated Wetland virus (WELV) glycoprotein Gc and the aluminum adjuvant; wherein, the mass ratio of the WELV glycoprotein Gc truncation and the aluminum adjuvant is (2.5 - 20) : 100.

[0068] Further, the mass ratio of the WELV glycoprotein Gc truncation and the aluminum adjuvant is 10 : 100.

[0069] Wherein, the amount of the aluminum adjuvant is calculated based on the aluminum content therein.

[0070] According to the third aspect of the technical solution of the present invention, a method for preparing the recombinant WELV glycoprotein Gc truncation as described above is provided, and the method comprises the following steps:

[0071] Step S1, expressing the truncated Wetland virus glycoprotein Gc in Pichia pastoris.

[0072] Step S2, expressing the WELV glycoprotein Gc truncation in an Expi-293F mammalian cell expression system to obtain an expression supernatant.

[0073] Further, expressing the WELV glycoprotein Gc truncation in the Expi-293F mammalian cell and Pichia pastoris expression systems is achieved by introducing a nucleic acid molecule encoding the WELV glycoprotein Gc truncation into the Expi-293F mammalian cell expression system and Pichia pastoris.

[0074] Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.

[0075] Even further, the nucleic acid molecule encoding the WELV glycoprotein Gc truncation can be any of the following:

[0076] a1, a DNA molecule with a nucleotide sequence as shown in positions 1 - 1575 of SEQ ID No.2;

[0077] a2, a DNA molecule that hybridizes with the DNA molecule defined by a1 under stringent conditions and encodes the truncated WELV glycoprotein Gc;

[0078] a3, a DNA molecule that has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the DNA sequence defined by a1 or a2 and encodes the truncated Gc protein of WELV.

[0079] Among the above nucleic acid molecules, the stringent conditions may be as follows: Hybridize at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5 M Na3PO4 and 1 mM EDTA, and wash in 2×SSC, 0.1% SDS at 50°C; It may also be: Hybridize at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and wash in 1×SSC, 0.1% SDS at 50°C; It may also be: Hybridize at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and wash in 0.5×SSC, 0.1% SDS at 50°C; It may also be: Hybridize at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and wash in 0.1×SSC, 0.1% SDS at 50°C; It may also be: Hybridize at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and wash in 0.1×SSC, 0.1% SDS at 65°C; It may also be: Hybridize in a solution of 6×SSC, 0.5% SDS at 65°C, and then wash the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

[0080] Among the above nucleic acid molecules, the identity of the nucleotide sequence is determined using an identity search site on the Internet, such as the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gapexistence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search to calculate the identity of a pair of nucleotide sequences, and then the identity value (%) can be obtained.

[0081] In the above nucleic acid molecule, the identity of more than 95% may be at least 96%, 97%, 98% identity. The identity of more than 90% may be at least 91%, 92%, 93%, 94% identity. The identity of more than 85% may be at least 86%, 87%, 88%, 89% identity. The identity of more than 80% may be at least 81%, 82%, 83%, 84% identity.

[0082] Step S3: Ferment and culture the recombinant yeast cells, and purify the truncated glycoprotein Gc of WELV from the supernatant of the fermentation culture.

[0083] Furthermore, during the fermentation culture process, the expression of the truncated glycoprotein Gc of WELV is induced by methanol. In a specific embodiment of the present invention, the recombinant yeast cells are inoculated into BMGY medium and cultured on a shaker at 25°C and 200 rpm. After 24 - 48 hours, 0.5% (V / V) methanol is added every 12 hours for induction, and the induction lasts for 48 hours.

[0084] Step S4: Suspension culture the transfected Expi-293F mammalian cells, and purify the truncated glycoprotein Gc of WELV from the culture supernatant.

[0085] Furthermore, during the transfection culture process, the expression of the truncated glycoprotein Gc of WELV is carried out. In a specific embodiment of the present invention, the cells are cultured on a shaker at 37°C and 125 rpm, and samples are collected after 48 hours.

[0086] Furthermore, the purification includes subjecting the transfection culture supernatant to nickel ion affinity chromatography (Chelating FF column).

[0087] In another embodiment, there is provided an application of the recombinant truncated glycoprotein Gc of WELV as described above in the preparation of a product capable of inducing an antigen-specific antibody in an animal, and the recombinant WELV glycoprotein Gc is immunized by intramuscular injection or subcutaneous injection.

[0088] In another embodiment, there is provided an immunization vaccine, which comprises the recombinant truncated glycoprotein Gc of WELV as described above, or the composition containing the recombinant truncated glycoprotein Gc of WELV as described above.

[0089] In another embodiment, there is provided a pharmaceutical composition, which comprises the truncated glycoprotein Gc of WELV as described above, or the composition containing the truncated glycoprotein Gc of WELV as described above.

[0090] In another embodiment, there is provided a biological material, which is any one of the following:

[0091] C1, a nucleic acid molecule encoding the truncated WELV glycoprotein Gc described above;

[0092] C2, an expression cassette containing the nucleic acid molecule described in C1;

[0093] C3, a recombinant vector containing the nucleic acid molecule described in C2, or a recombinant vector containing the expression cassette described in C2;

[0094] C4, a recombinant microorganism containing the nucleic acid molecule described in C1, or a recombinant microorganism containing the expression cassette described in C2, or a recombinant microorganism containing the recombinant vector described in C3;

[0095] C5, a recombinant cell containing the nucleic acid molecule described in C1, or a recombinant cell containing the expression cassette described in C2, or a recombinant cell containing the recombinant vector described in C3.

[0096] In another embodiment, the present invention provides any of the following applications:

[0097] D1, the application of the above-mentioned protein or the above-mentioned biological material in the preparation of the above-mentioned WELV vaccine;

[0098] D2, the application of the above-mentioned WELV vaccine or the above-mentioned protein or the above-mentioned biological material in the preparation of a product for preventing and / or treating Congo hemorrhagic fever / WELV;

[0099] D3, the application of the above-mentioned WELV vaccine or the above-mentioned protein or the above-mentioned biological material in the preparation of a product for relieving the symptoms of hemorrhagic fever / WELV;

[0100] D4, the application of the above-mentioned WELV vaccine or the above-mentioned protein or the above-mentioned biological material in the preparation of a product for anti-WELV infection;

[0101] D5, the application of the above-mentioned WEL vaccine or the above-mentioned protein or the above-mentioned biological material in the preparation of a product for neutralizing WELV.

[0102] In the present invention, the WELV strain can be obtained from "A New Orthonairovirus Associated with Human Febrile Illness. N Engl J Med. 2024;391:821-31" in the New England Journal of Medicine.

[0103] The pCAGGS mammalian expression vector was purchased from OriGene, catalog number HG-VPA0057.

[0104] The Expi-293F mammalian expression system was purchased from Thermo Fisher Scientific Inc., catalog number A14635CN.

[0105] The Anti-His tag mouse monoclonal antibody was purchased from Sigma, catalog number A7058.

[0106] Chelating Sepharose FF chromatography medium was from Cytiva, product catalog number 17057502.

[0107] BALB / c mice (strain code: 211) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0108] In the following examples, the virus is specifically the Wetland virus (WELV), recorded in the literature "NCBI (National Center for Biotechnology Information, (https: / / www.ncbi.nlm.nih.gov / ): A New Orthonairovirus Associated with Human Febrile Illness. N Engl J Med. 2024 Sep 5;391(9):821-831. doi: 10.1056 / NEJMoa2313722". The public can obtain this biological material from the applicant in accordance with the relevant regulations on biosafety. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0109] SEQ ID No.1, the amino acid sequence of the Gc truncation (525AA) is as follows:

[0110] FFKGLNSAASRMLNAHKLMTSVSIDAPWGAIQVESTYKPKLPVSNIELAWNSIEEQGDKIILSGKSTSILKLEERTGVQWSLGAESASEEKRLLVSVLDYTQVYSSTFQYITGDRTISEWPKATCTGDCPDRCACRTSTCLFKSWPHSRNWRCNPTWCWGVGTGCTCCGVDIERPFNKYFAVKWSTEYVRTDALVCVELTDLERHCDVVEAGSQFVIGPVRVVVSDPQNVQSKLPQEVLTVQKLDKHQHLDLMHVTNIISAKNACKLQSCTHGSPGDMQILHTDNLILNSHDDGANLAESIPEVNTTWMSWEGCDLDYYCTTGSWPSCTFTGVNTENTESFENLLNTEANLIDRYHFHSKRIYAQGATLQMDLKGRPKSGGGELTVLVDIKGLELHSKRVVLKGLEIKALSCTGCYSCSSGMSCTVDVRIEKPDEFTVHLRSTDPNTAISEGSIMARKLSGGPQSKVRAFTALKVSEVCVEIVEKNYCPTCKETDTKKCAKVDLQPPKDILLEHKGTLIKSQNDS。

[0111] SEQ ID No. 2, the coding gene sequence corresponding to the Gc truncation (1575 bp) is as follows:

[0112]

[0113] SEQ ID No. 3, the amino acid sequence of the Gc truncation + HIS tag (1 - 536 AA) is as follows:

[0114] FFKGLNSAASRMLNAHKLMTSVSIDAPWGAIQVESTYKPKLPVSNIELAWNSIEEQGDKIILSGKSTSILKLEERTGVQWSLGAESASEEKRLLVSVLDYTQVYSSTFQYITGDRTISEWPKATCTGDCPDRCACRTSTCLFKSWPHSRNWRCNPTWCWGVGTGCTCCGVDIERPFNKYFAVKWSTEYVRTDALVCVELTDLERHCDVVEAGSQFVIGPVRVVVSDPQNVQSKLPQEVLTVQKLDKHQHLDLMHVTNIISAKNACKLQSCTHGSPGDMQILHTDNLILNSHDDGANLAESIPEVNTTWMSWEGCDLDYYCTTGSWPSCTFTGVNTENTESFENLLNTEANLIDRYHFHSKRIYAQGATLQMDLKGRPKSGGGELTVLVDIKGLELHSKRVVLKGLEIKALSCTGCYSCSSGMSCTVDVRIEKPDEFTVHLRSTDPNTAISEGSIMARKLSGGPQSKVRAFTALKVSEVCVEIVEKNYCPTCKETDTKKCAKVDLQPPKDILLEHKGTLIKSQNDSGGGGSHHHHHH。

[0115] SEQ ID No.4, the coding gene sequence corresponding to the Gc truncation + HIS tag (1608bp) is as follows:

[0116]

[0117] The following are the CpG sequences of SEQ ID No. 5 - SEQ ID No. 7:

[0118] SEQ ID No.5, CpG2006 is as follows: tcgtcgtttt gtcgttttgt cgtt;

[0119] SEQ ID No.6, CpG1018 is as follows: tgactgtgaa cgttcgagat ga;

[0120] SEQ ID No.7, CpG684 is as follows: tcgacgttcg tcgttcgtcg ttc.

[0121] In the following examples, unless otherwise specified, total gene synthesis, nucleotide synthesis, primer synthesis, sequencing, etc. were carried out by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. and Beijing Tsingke Biotechnology Co., Ltd.

[0122] In the following examples, unless otherwise specified, the first nucleotide of each nucleotide sequence is the 5'-terminal nucleotide of the corresponding DNA, and the last nucleotide is the 3'-terminal nucleotide of the corresponding DNA.

[0123] Example 1. Construction of a mammalian expression vector for the truncated Gc subunit.

[0124] (1) Obtaining the gene of the truncated WELV glycoprotein Gc (truncated Gc subunit) and constructing a mammalian expression vector.

[0125] According to the M fragment sequence of WELV published in GenBank: OR860408, the amino acids at positions 1 to 525 (F1 - S525) of the Gc protein were selected, and a codon DNA sequence was artificially designed, with a GGGGS linker peptide and 6 HIS tags added. It is a DNA fragment with the sequence shown in SEQ ID No.4. Beijing Tsingke Biotechnology Co., Ltd. was commissioned to synthesize the relevant DNA fragment (from the 5' end to the 3' end, in turn, the EcoRI cleavage site, BM40 signal peptide, the codon DNA sequence of the fragment shown in SEQ ID No.4, and the NotI cleavage site). The synthesized fragment was inserted between the EcoRI and NotI cleavage sites of the pCAGGS vector to obtain the recombinant expression vector pCAGGS - truncated Gc subunit, that is, the truncated Gc subunit expression vector.

[0126] The structure of the recombinant expression vector pCAGGS-Gc subunit truncated body is described as: a recombinant plasmid in which the DNA fragment between the EcoRI and NotI recognition sequences of the pCAGGS vector is replaced with the DNA fragment shown in SEQ ID No. 4. SEQ ID No. 4 is the coding gene sequence obtained by artificial codon optimization based on the Gc subunit truncated body (F1-S525), encoding the Gc subunit truncated body protein shown in SEQ ID No. 1 carrying 6 His tags and a GGGGS linker peptide. The 1st to 536th positions of SEQ ID No. 3 are the Gc subunit truncated body protein. Figure 1A It is a schematic diagram of the structure of the Gc subunit truncated body (Gc subunit truncated body) of WELV (the green part represents the extracellular domain, the gray part represents the transmembrane region, and the white part represents the intracellular domain). Figure 1B It is the three-dimensional structure prediction of the Gc subunit truncated body of WELV (the blue part represents the part involved in the present invention, and the gray part represents the truncated part).

[0127] (2) Extraction of the recombinant expression vector pCAGGS-Gc subunit truncated body plasmid (Tiangen endotoxin-free mini midiprep kit, Beijing, DP118-02).

[0128] Example 2: Expression and purification of the recombinant Gc subunit truncated body glycoprotein.

[0129] (2.1) Transfection of Expi293F suspension cells.

[0130] Pick the plasmid identified in Example 1 (i.e., pCAGGS-Gc subunit truncated body) and transfect it into Expi293F suspension cells with a cell density of 3×10 6 cells / mL. The transfection system is shown in Table 1 below (taking a transfection volume of 100 mL as an example, and the other volumes are scaled up proportionally).

[0131] Table 1 Transfection system

[0132]

[0133] After transfection, culture at 37°C, 8% CO2, 125 rpm. Monitor the cell viability every 12 h and take a 200 μL sample for protein expression process monitoring. Stop protein expression when the cell viability drops below 60%.

[0134] The results are as Figures 2A - 2C shown, Figure 2A indicating the change in cell viability over time after cell transfection; Figure 2B indicating the SDS-PAGE of the target protein expression over time, Figure 2C indicating the WB detection of the target protein expression over time. Figure 2AIt is shown that 60 hours after cell transfection, the cell viability has decreased to 60%-70%, and at this time, protein expression needs to be stopped. Electrophoresis was performed on the process samples after cell transfection, and the results Figure 2B and Figure 2C showed that the expression of Gc protein increased with the prolongation of time. Considering factors such as cell viability and protein yield, it was decided to select 60 hours after cell transfection as the protein expression endpoint.

[0135] (2.2)Purification of Gc subunit truncation.

[0136] 1. Nickel ion affinity chromatography.

[0137] The culture supernatant induced for 60 hours in step one was dialyzed overnight to replace the system, and the replacement system was 20 mM pH 7.5 Tris-HCl + 500 mM NaCl pH 7.5.

[0138] A: 20 mM pH 7.5 Tris-HCl + 500 mM NaCl + 5 mM imidazole pH 7.5;

[0139] B: 20 mM pH 7.5 Tris-HCl + 500 mM NaCl + 500 mM imidazole pH 7.5.

[0140] After the loading was completed, it was equilibrated with A, and then eluted with B at 5%-10%-25%-50%-100% respectively.

[0141] The purified sample is as Figures 3A - 3C shown. Figure 3A represents the nickel ion affinity chromatography chromatogram; Figure 3B represents the SDS-PAGE of the sample during the purification process; Figure 3C represents the WB identification of the sample during the purification process. Figure 3B and Figure 3C The results showed that the target protein was eluted when the imidazole concentration was 125 and 250 mM, and the separation of the target protein from the miscellaneous proteins was good (no obvious miscellaneous proteins were seen in the lanes). The fractions containing the target protein (25%B, 50%B) were ultrafiltered and concentrated to replace the system and then the next experiment could be carried out. Figure 4 is the SDS-PAGE of the pure Gc protein obtained by purification.

[0142] Example 3. Mouse immunization experiment.

[0143] The specific immunization method is as follows: 40 female BALB / C mice aged 6-8 weeks were randomly divided into the following 4 groups:

[0144] Immune group 1 (n = 6): 100 μl of the vaccine was intramuscularly injected on days 0 and 21 respectively. The vaccine used was 2.5 μg truncated Gc subunit / 100 μg Al(OH)3 (CRODA, Denmark, the same below) / 50 μg CpG2006. Among them, the truncated Gc subunit was the recombinant WELV glycoprotein Gc subunit truncated body expressed by Expi-293F prepared above. The vaccine was formulated with physiological saline according to 10 μg of the truncated Gc subunit, 100 μg of Al(OH)3 and 50 μg of CpG 2006 in a volume of 100 μl.

[0145] Immune group 2 (n = 6): 100 μl of the vaccine was intramuscularly injected on days 0 and 21 respectively. The vaccine used was 10 μg truncated Gc subunit / 100 μg Al(OH)3 / 50 μg CpG2006. Among them, the truncated Gc subunit was the recombinant WELV glycoprotein Gc subunit truncated body expressed by Expi-293F prepared above. The vaccine was formulated with physiological saline according to 2.5 μg of the truncated Gc subunit, 100 μg of Al(OH)3 and 50 μg of CpG2006 in a volume of 100 μl.

[0146] Immune group 3 (n = 6), namely the adjuvant control group: 100 μl of the vaccine was intramuscularly injected on days 0 and 21 respectively. The adjuvant used was 100 μg Al(OH)3 + 50 μg CpG2006. The vaccine was formulated with physiological saline according to 100 μg of Al(OH)3 and 50 μg of CpG2006 in a volume of 100 μl.

[0147] Immune group 4 (n = 6), namely the blank control group: 100 μl of physiological saline was intramuscularly injected on days 0 and 21 respectively.

[0148] The nucleotide sequence of the above CpG2006 is SEQ ID No.5 and is fully phosphorothioated. If necessary, CpG2006 can also be replaced with any of the following: CpG1018 with the nucleotide sequence of SEQ ID No.6 or its phosphorothioated product; CpG684 with the nucleotide sequence of SEQ ID No.7 or its phosphorothioated product (such as full-chain phosphorothioation).

[0149] Blood was collected from the infraorbital venous plexus of the immunized mice in each group before immunization, two weeks after the first immunization, and two weeks after the second immunization.

[0150] The specific IgG antibody titer against the truncated Gc subunit in the sera of the mice in each group was measured by indirect ELISA. The operation steps refer to the Compendium of Molecular Biology Experimental Guidelines [M]. Science Press, 2008.

[0151] The results are asFigure 5 As shown, the antibody titers of the once-three-week and twice-three-week immunization groups were extremely significantly higher than those of the adjuvant control group ( P <0.01). Different doses of Gc antigen did not show statistically significant differences in specific antibody titers after each immunization. However, there were significant differences in specific antibody titers in mice after three weeks of the first immunization and three weeks of the second immunization with a 2.5 μg Gc antigen dose ( P <0.05), and there were extremely significant differences in specific antibody titers in mice after three weeks of the first immunization and three weeks of the second immunization with a 10 μg Gc antigen dose ( P <0.01). At the same time, after three weeks of the second immunization, the specific antibody titers of all antigen immunization groups were greater than 1:100000, and the subunit vaccine effectively stimulated the immune response in mice.

[0152] Example 4: True virus challenge test.

[0153] 1) Vero cells containing 10% FBS DMEM medium were mixed, counted, and seeded into 24-well plates at 1×10 5 cells / well. After the cells adhered for 12 h, they were inoculated with the virus;

[0154] 2) Discard the cell culture medium, dilute the serum of the test mice (serum collected three weeks after the third immunization obtained in Example 3) and the WELV virus with DMEM medium. Mix 100 μL / well of the diluted virus (preferably, the titer is 5×10 3 FFU / mL) and 100 μL / well of the diluted serum, and incubate at 37°C for 1.5 h; The process disclosed in "A New Orthonairovirus Associated with Human Febrile Illness. N Engl J Med. 2024;391:821 - 31" can be used;

[0155] 3) Discard the virus solution, add PBS, slowly shake well to wash the cell surface, then aspirate it out. Add DMEM medium containing 1.25% methyl cellulose, seal the cell plate with a sealing film, and culture at 37°C for 3.5 days;

[0156] 4) Add 3.7% formaldehyde, place it on a horizontal shaker for 30 min, aspirate and discard the formaldehyde and medium mixture, add formaldehyde, shake well and aspirate it out, repeat 2 - 3 times;

[0157] 5) After cleaning the methyl cellulose, add formaldehyde, place it on a horizontal shaker and gently shake for 30 min to fix the cells, and wash the formaldehyde 4 - 5 times with 1×PBST;

[0158] 6) Discard the formaldehyde, add PBS blocking and permeabilization solution containing 0.3% Triton-X100 and 5% skim milk, and gently shake on a horizontal shaker for 1.5 h;

[0159] 7) Discard the blocking and permeabilization solution, wash with 0.05% PBST, gently shake on a horizontal shaker for 5 min, and repeat 3 times;

[0160] 8) Anti-rabbit WELV NP antibody. After immunizing rabbits with WELV NP protein, incubate with the antiserum against NP protein (preferably diluted with 0.05% PBST at a dilution of 1:2000) for 1.5 h, and wash three times with 0.05% PBST;

[0161] 9) Incubate with anti-rabbit HRP-IgG antibody (preferably diluted with 0.05% PBST at a dilution of 1:1000) for 1 h, and wash three times with 0.05% PBST;

[0162] 10) Develop color with DAB chromogenic solution, discard the chromogenic solution, wash 3 times with 0.05% PBST, air-dry the plate, and perform spot counting.

[0163] The results are as Figure 6 、 Figure 7 shown. Figure 6 is the survival curve of mice in each group after virus challenge. Under the lethal dose challenge, all the mice in the normal saline + double adjuvant group died on the 2nd day after virus challenge, and the mice in the normal saline group began to die on the 2nd day after virus challenge and all died on the 4th day (n = 6). Both the 2.5 μg Gc dose group and the 10 μg Gc dose group could completely protect mice under the WELV lethal dose challenge. Figure 7 is the body weight change curve of mice in each group after virus challenge. The results showed that compared with the 2.5 μg Gc dose group, the body weight of mice in the 10 μg Gc dose group fluctuated less, less than 5%. It is suggested that high-dose immunization with Gc antigen can better protect mice under WELV challenge.

[0164] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In general, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. A wetland virus glycoprotein Gc, characterized in that: The wetland virus glycoprotein Gc is a recombinant wetland virus glycoprotein Gc, and the wetland virus glycoprotein Gc is a C-terminally truncated wetland virus glycoprotein Gc truncation; the wetland virus glycoprotein Gc truncation is a protein whose amino acid sequence is shown in positions 1-525 of SEQ ID No.

1.

2. The wetland virus glycoprotein Gc according to claim 1, characterized in that: The wetland virus glycoprotein Gc truncate is a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in the amino acid sequence of positions 1-525 of SEQ ID No.

1.

3. A biomaterial, characterized in that: The biological material is any of the following: C1, a nucleic acid molecule encoding the wetland virus glycoprotein Gc according to any one of claims 1-2; C2, an expression cassette containing the nucleic acid molecule described in C1; C3, a recombinant vector containing the nucleic acid molecule described in C1, or a recombinant vector containing the expression cassette described in C2; C4, a recombinant microorganism containing the nucleic acid molecule described in C1, or a recombinant microorganism containing the expression cassette described in C2, or a recombinant microorganism containing the recombinant vector described in C3; C5, a recombinant cell containing the nucleic acid molecule described in C1, or a recombinant cell containing the expression cassette described in C2, or a recombinant cell containing the recombinant vector described in C3.

4. A product containing the wetland virus glycoprotein Gc according to any one of claims 1-2.

5. The product according to claim 4, characterized in that: The product is an immune composition or a pharmaceutical composition.

6. The product according to claim 5, characterized in that: The immunological composition is a vaccine.

7. The product according to claim 6, characterized in that: The vaccine also comprises an adjuvant.

8. The product according to claim 7, characterized in that: The adjuvant is an aluminum adjuvant or a CpG adjuvant.

9. The product according to claim 8, characterized in that: The aluminum adjuvant comprises aluminum hydroxide adjuvant; or the CpG adjuvant is any one of the following: (1) CpG2006 or its thiolated product whose nucleotide sequence is SEQ ID No.5; (2) CpG1018 or its thiolated product whose nucleotide sequence is SEQ ID No.6; (3) CpG684 or its thiolated product whose nucleotide sequence is SEQ ID No. 7; The thiolated product is a full-chain thiolated modified product.

10. The product according to claim 8, characterized in that: The vaccine is composed of a C-terminally truncated wetland virus glycoprotein Gc truncate, an aluminum adjuvant and a CpG adjuvant; wherein the mass ratio of the C-terminally truncated wetland virus glycoprotein Gc truncate, the aluminum adjuvant and the CpG adjuvant is 10:100:50 or 2.5:100:50 by mass; the amount of the aluminum adjuvant is calculated based on the aluminum content therein.

11. A method for preparing wetland virus glycoprotein Gc as claimed in claim 1, characterized in that: The method comprises the following steps: Step S1, expressing the wetland virus glycoprotein Gc truncated body in yeast, wherein the wetland virus glycoprotein Gc truncated body is a protein with an amino acid sequence as shown in positions 1 to 525 of SEQ ID No. 1; The wetland virus glycoprotein Gc truncation is expressed in Pichia pastoris genetically modified by N-glycosylation modification pathway to obtain a recombinant yeast cell; the Pichia pastoris genetically modified by N-glycosylation modification pathway is Pichia pastoris with inactivated α-1,6-mannosyltransferase; The expression of the wetland virus glycoprotein Gc truncation in the Pichia pastoris genetically modified by the N-glycosylation modification pathway is achieved by introducing a nucleic acid molecule encoding the wetland virus glycoprotein Gc truncation into the Pichia pastoris genetically modified by the N-glycosylation modification pathway; Step S2, fermenting and culturing the recombinant yeast cells, and purifying the Gc protein truncation of the wetland virus from the fermentation culture supernatant.

12. A method for preparing wetland virus glycoprotein Gc as claimed in claim 1, characterized in that: The method comprises the following steps: Step S3, expressing the wetland virus glycoprotein Gc truncate in the Expi-293F mammalian cell expression system; expressing the wetland virus glycoprotein Gc truncate in the Expi-293F mammalian cell expression system is achieved by introducing a nucleic acid molecule encoding the wetland virus glycoprotein Gc truncate into the Expi-293F mammalian cell expression system; Step S4, culturing the Expi-293F mammalian cells in suspension, and purifying the wetland virus glycoprotein Gc truncation from the suspension cell culture supernatant; the purification includes the step of subjecting the suspension cell culture supernatant to nickel ion affinity chromatography.

13. The preparation method according to claim 11 or 12, characterized in that: The nucleic acid molecule encoding the wetland virus glycoprotein Gc truncation is any of the following: a1, a DNA molecule with a nucleotide sequence as shown in SEQ ID No.2 or positions 1 to 1575 of SEQ ID No.2; a2, a DNA molecule that hybridizes with the DNA molecule defined by a1 under stringent conditions and encodes the truncated form of the wetland virus glycoprotein Gc; a3, a DNA molecule that has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the DNA sequence defined by a1 or a2 and encodes the wetland virus glycoprotein Gc truncation.

14. Use of the wetland virus glycoprotein Gc as described in any one of claims 1 to 2 in the preparation of an immune product that can induce animals to produce antigen-specific antibodies, or use of the biological material as described in claim 3 in the preparation of an immune product that can induce animals to produce antigen-specific antibodies.

15. The use according to claim 14, characterized in that: The immune product is a vaccine.

16. The use according to claim 15, characterized in that: The vaccine is administered by intramuscular or subcutaneous injection.

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