Construction of recombinant ebola virus vaccine based on recombinant measles virus vector, reverse genetic system and application thereof

Abstract

The present application relates to the field of vaccine development, in particular to a recombinant Ebola virus vaccine based on recombinant measles virus vector, a reverse genetic system and application thereof. Specifically, the present application uses measles virus as a vector to construct and express a recombinant vaccine of Ebola virus related antigens through reverse genetic technology. The vaccine can be used to prevent Ebola virus infection and has a wide application prospect. In addition, the present application also discloses a recombinant measles virus vector, a nucleic acid molecule, a reverse genetic system, a recombinant measles virus particle and application thereof. It comprises: the negative strand RNA coding sequence of Ebola virus glycoprotein GP and VP40 inserted into the measles virus vector. The Ebola virus vaccine based on measles virus vector provided by the present application has high immunogenicity and broad spectrum, which provides a new idea for the development of new Ebola virus vaccine. The vaccine preparation method provided by the present application is simple and can be mass-produced in a short period of time.

Description

Technical Field

[0001] This invention relates to the field of vaccine development, and in particular to the construction, reverse genetics system and application of a recombinant Ebola virus vaccine based on a recombinant measles virus vector. Background Technology

[0002] The Filoviridae family includes the genera Cuevavirus, Marburgvirus, and Ebolavirus. The Ebolavirus genus includes six viruses: Zaire, Bundibugyo, Sudan, Tayforn, Reston, and Bombali viruses. Among them, the Zaire Ebola virus has a mortality rate as high as 90%, making it the deadliest virus. In addition, Sudan virus and Bundibugyo virus cause approximately 50% and 30% mortality rates, respectively. Tayforn virus is relatively rare.

[0003] Vaccines that prevent certain types of Ebola virus disease have been used to control the spread of Ebola outbreaks, but they are not effective against different species of Ebola virus.

[0004] Measles virus belongs to the genus Measlesvirus and is a member of the family Paramyxoviridae. It is an enveloped, non-segmented, single-stranded negative-sense RNA virus with a genome of approximately 16 kilobases. This genome encodes six structural proteins: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (H), and large protein (L). In addition, the P gene encodes two non-structural proteins: V protein and C protein. The H and F proteins form heterodimers and play crucial roles in viral invasion and syncytial formation. Recombinant vaccines using measles virus as a vector can provide broader protection during immunization. Due to its good immunogenicity and safety, measles virus is an ideal viral vector for developing recombinant vaccines. As a vector, measles virus can carry antigen genes from other pathogens, inducing an immune response against these antigens in the host. Because measles virus itself can effectively infect and activate the host's immune system, recombinant vaccines can produce strong cellular and humoral immune responses.

[0005] The Schwarz, Edmonston-Zagreb, and AIK-C strains are currently the safest and most effective live measles vaccines, widely used in global immunization programs. Since Radeke et al. established the measles virus reverse genetics platform in 1995, measles virus has demonstrated immense potential as a vaccine vector. This vector system is highly flexible, capable of being modified and expressing a variety of protective antigens for the prevention of emerging infectious diseases. For example, measles virus vectors are used to express antigens of West Nile virus, Chikungunya virus, HIV-1, and Lassa virus, and some vaccines have entered clinical trials. The measles virus vector vaccine platform has several key advantages. First, measles virus vaccines have been widely used since the 1960s, exhibiting extremely high safety and mature production processes. Second, even if the recipient already has measles immunity, the vaccine can still effectively induce a strong immune response and express heterologous antigens. Furthermore, the platform can flexibly insert up to 6 kilobases of exogenous transcription units, thus adapting to antigen design for different pathogens. Most importantly, the measles virus can directly deliver antigens to dendritic cells, macrophages, and B cells, inducing a strong and long-lasting immune response.

[0006] Ebola virus belongs to the Filoviridae family. Its genome is approximately 19 kb and contains seven genes encoding a nucleoprotein (NP), a glycoprotein (GP), four small viral proteins (VP24, VP30, VP35, and VP40), and an RNA-dependent RNA polymerase (L protein). All genes are located on a single negative-sense RNA strand. GP is the only surface protein of Ebola virus, with two reading frames encoding a secreted small protein (sGP) and a full-length transmembrane GP. GP plays a crucial role in the pathogenicity of Ebola virus, promoting viral invasion by binding to host cell receptors and disrupting microvascular integrity, causing vascular leakage. GP is also a major target for inducing protective immune responses, and most Ebola virus vaccine studies use GP as the target antigen. Traditional inactivated and subunit vaccines are ineffective against Ebola virus, while vaccines based on live virus vectors, using GP as the target antigen, have shown good protective effects in animals. VP40 is the most abundant protein in the virus particle and plays a key role in the Ebola virus life cycle, influencing viral replication, budding, and pathogenicity. VP40 promotes viral budding from the host cell surface and the formation of new viral particles by interacting with host cell-related proteins such as Nedd4E3 and Tsg101. Chikungunya virus vaccine candidates, expressing the major antigen while binding virus-like particle units, have entered clinical trials.

[0007] Current Ebola virus vaccines have limitations in their preventive efficacy. Some vaccines only target specific types of Ebola virus (Zaire type) and cannot provide broad protection against other virus strains or variants. Therefore, vaccine effectiveness is unsatisfactory when dealing with various subtypes and mutants of the Ebola virus. In view of this, the present invention is proposed. Summary of the Invention

[0008] The purpose of this invention is to provide a recombinant measles virus vector, nucleic acid molecule, reverse genetics system, recombinant measles virus particle, and its application to solve the above-mentioned technical problems.

[0009] This invention is implemented as follows: In a first aspect, the present invention provides a recombinant measles virus vector expressing Ebola virus protein, comprising: inserting a coding sequence of negative strand RNA of Ebola virus glycoprotein GP onto the measles virus vector.

[0010] Secondly, the present invention also provides a nucleic acid molecule comprising: operatively linking the coding sequence of the Ebola virus glycoprotein GP negative strand RNA in the above-mentioned recombinant measles virus vector to a cDNA sequence encoding the measles virus antigenome.

[0011] Thirdly, the present invention also provides a reverse genetics system for rescuing a recombinant measles virus vector expressing Ebola virus proteins, comprising: a) an antigen expression plasmid containing a coding sequence of the negative strand RNA of the Ebola virus glycoprotein GP in the aforementioned recombinant measles virus vector operably linked to a cDNA sequence encoding the measles virus reverse genome; b) an auxiliary plasmid 1 containing a coding sequence of the measles virus nucleocapsid protein; c) an auxiliary plasmid 2 containing a coding sequence of the measles virus phosphoprotein; and d) an auxiliary plasmid 3 containing a coding sequence of the measles virus RNA polymerase.

[0012] Fourthly, the present invention also provides a method for preparing recombinant measles virus particles, comprising any one of the following methods: Method 1: Co-transfect the plasmids from the above reverse genetics system into the first cell line; Method 2: Transfect the antigen expression plasmid from the above reverse genetics system into a helper cell line.

[0013] Fifthly, the present invention also provides a recombinant measles virus particle obtained by the above-described method.

[0014] In a sixth aspect, the present invention also provides a host cell obtained by transfecting cells with the aforementioned nucleic acid molecules or the aforementioned recombinant measles virus vector.

[0015] In a seventh aspect, the present invention also provides a composition comprising the above-described recombinant measles virus particles and a pharmaceutically acceptable carrier; In a preferred embodiment of the present invention, the composition is a vaccine composition.

[0016] Eighthly, the present invention also provides the use of recombinant measles virus particles or the above-described compositions in the preparation of medicaments or vaccines for the prevention and / or treatment of Ebola virus infection.

[0017] The present invention has the following beneficial effects: This invention combines the advantages of viral vector vaccines and protein nanoparticle vaccines. It utilizes a viral vector to deliver virus-encoded antigens to host cells, allowing exogenous glycoproteins (GPs) to be expressed on the host cell surface and triggering glycoprotein budding to release virus-like particles (VLPs) that exhibit glycoprotein surface presentation. Based on this, this invention constructs and designs a broad-spectrum Ebola virus vaccine based on a measles virus vector, develops a novel virus-like particle (VLP) production technology, and combines it with a measles virus vector vaccine platform. This significantly improves the immunogenicity and broad-spectrum nature of the vaccine, providing new insights for the development of novel Ebola virus vaccines. This innovative vaccine design not only enhances the preventive effect against Ebola virus but also provides a new solution for addressing future threats from emerging viruses. Recombinant measles virus, when used as an Ebola virus disease vaccine in immunized animals, can induce a strong cellular and humoral immune response in a short period, demonstrating high efficiency. The vaccine preparation method provided by this invention is simple and can be mass-produced in a short time for responding to sudden Ebola outbreaks, contributing to improved global public health security. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 For genome segmentation strategies; Figure 2 pGFMV schwarz Plasmid map; Figure 3 T-F1 plasmid map; Figure 4 pGF-MVEBOV GP ; Figure 5 pGF-MEBOV GP+VP40 ; Figure 6 Plasmid enzyme digestion identification diagram; Figure 7 A diagram illustrating virus rescue; Figure 8 Stability identification of measles virus and recombinant measles virus; Figure 9 WB identification diagram of recombinant measles virus; Figure 10 Recombinant measles virus MV-EBOV GP+VP40 Negative staining; electron microscopy observation of ultrathin sections of infected cells. Figure 11 Schematic diagram of serum antibody levels; Figure 12 A diagram illustrating the neutralization of a fake virus; Figure 13 Flow cytometry was used to detect the secretion of IFN-γ cytokines. Figure 14 ELISPOT of cytokines; Figure 15 Curves showing changes in body weight and survival in mice after rVSV-EBOV challenge; Figure 16 Curves showing changes in body weight and survival in mice after MA-EBOV challenge; Figure 17 This is a plasmid diagram of pBlunt-F1; Figure 18 This is a vector diagram of T-F2. Detailed Implementation

[0020] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0021] In this invention, "glycoprotein GP", "EBOV GP protein" and "GP protein" are synonymous.

[0022] As used in this article, the term "negative-strand RNA," such as the negative-strand RNA of the measles virus genome, refers to RNA that cannot function as mRNA and must first synthesize a complementary strand (positive-strand RNA) as mRNA before protein translation. Correspondingly, its complementary sequence is called the positive-strand RNA sequence. The positive-strand RNA corresponding to the negative-strand RNA of the measles virus genome is also called the antigenomic positive-strand RNA.

[0023] Attenuated strains of measles virus refer to strains that have been passaged in selected cells and, possibly, adapted to other cells to produce seed strains suitable for vaccine preparation. These strains possess a stable genome that does not revert to pathogenicity or integrate into the host chromosome. Specifically, an "attenuated strain" is a strain confirmed for use in a vaccine that is suitable for this invention, meaning it meets safety, potency, quality, and reproducibility criteria after rigorous review of laboratory and clinical data.

[0024] In a first aspect, the present invention provides a recombinant measles virus vector expressing Ebola virus protein, comprising: inserting a negative strand RNA coding sequence of Ebola virus glycoprotein GP onto the measles virus vector.

[0025] This invention provides a vector for expressing the Ebola virus glycoprotein GP, ​​using measles virus as a vector. Delivery via the measles virus vector enables host cells to express the EBOV GP protein, promoting efficient assembly and release of the VLP by the host cells.

[0026] In a preferred embodiment of the present invention, the coding sequence of the negative strand RNA of the Ebola virus VP40 protein is also inserted into the measles virus vector.

[0027] By simultaneously inserting the coding sequences of the negative-strand RNA of Ebola virus GP protein and VP40 protein into a measles virus vector, the recombinant measles virus particles prepared and used as an Ebola virus disease vaccine in animals can induce a strong cellular and humoral immune response in a short period of time. Furthermore, the recombinant measles virus MV-EBOV... GP+VP40 Compared to the recombinant vaccine strain MV-EBOV GP It can stimulate host cells to produce higher levels of specific antibodies in a shorter time. Furthermore, recombinant measles virus MV-EBOV GP+VP40 Compared to the recombinant vaccine strain MV-EBOV GP It can produce higher levels of specific neutralizing antibodies against the GP protein of a broad spectrum of Ebola viruses. Studies in mouse models have shown that recombinant measles virus MV-EBOV... GP+VP40 and recombinant vaccine strain MV-EBOV GP All can achieve 100% effective protection, especially MV-EBOV. GP+VP40 The vaccine strain exhibits better protective efficacy and more stable protective effect.

[0028] In a preferred embodiment of the present invention, the coding sequence of the negative strand RNA of the Ebola virus glycoprotein GP is located in the non-coding region before the 5' end of the N gene on the measles virus vector, the non-coding region between the P gene and the M gene, or between the H gene and the L gene.

[0029] In a preferred embodiment of the present invention, the coding sequence of the negative strand RNA of the Ebola virus VP40 protein is located in the non-coding region before the 5' end of the N gene, the non-coding region between the P and M genes, or between the H and L genes on the measles virus vector.

[0030] In a preferred embodiment of the present invention, the coding sequence of the negative-strand RNA of the Ebola virus glycoprotein GP is located in the non-coding region between the P and M genes on the measles virus vector, and the coding sequence of the negative-strand RNA of the Ebola virus VP40 protein is located in the non-coding region before the 5' end of the N gene on the measles virus vector. At these insertion sites, a high protein expression level is observed.

[0031] In a preferred embodiment of the present invention, the Ebola virus glycoproteins GP and VP40 are both derived from the Zaire type Ebola virus Mayinga strain, Genebank number: AF086833.2.

[0032] In a preferred embodiment of the present invention, the coding sequences of Ebola virus glycoproteins GP and VP40 are shown in SEQ ID NO: 9 and SEQ ID NO: 10.

[0033] In a preferred embodiment of the present invention, the measles virus vector includes the measles virus N gene, P gene, M gene, F gene, H gene and L gene.

[0034] In a preferred embodiment of the present invention, the measles virus vector includes the N, P, M, F, H and L genes of an attenuated measles virus strain, the attenuated measles virus strain being Schwarz, Genebank number AF266291.1.

[0035] In a preferred embodiment of the present invention, the measles virus vector includes a sequence complementary to the negative strand RNA sequence of the measles virus strain, and the negative strand RNA sequence includes: N gene, P gene, M gene, F gene, H gene and L gene.

[0036] The coding sequence of the negative strand RNA of the N gene on the measles virus vector is shown in SEQ ID NO:6, the coding sequence of the negative strand RNA of the P gene is shown in SEQ ID NO:7, and the coding sequence of the negative strand RNA of the L gene is shown in SEQ ID NO:8.

[0037] In a preferred embodiment of the present invention, the measles virus vector includes measles virus gene fragments as shown in SEQ ID NO:1-5. The measles virus gene fragments shown in SEQ ID NO:1-5 are fragments of approximately 3kb each, representing the full-length reverse genome sequence of the attenuated measles vaccine strain divided into equal lengths. SEQ ID NO:1-5 are F1 (2451bp), F2 (3905bp), F3 (3020bp), F4 (3359bp), and F5 (3337bp), respectively. In other embodiments, those skilled in the art may group the full-length reverse genome sequence of the attenuated measles vaccine strain into fragments of other lengths as needed, and are not limited to the grouping described above.

[0038] In a preferred embodiment of the present invention, the measles virus vector further includes at least one regulatory element: a transcription promoter, a termination sequence, an enhancer, and other cis-acting elements. These regulatory elements may be homologous or heterologous to the measles virus strain used.

[0039] In a preferred embodiment of the present invention, the measles virus vector sequentially comprises DNA sequences (5' to 3') corresponding to the following gene transcription units: a polynucleotide encoding the N protein of MV, a polynucleotide encoding the P protein of MV, a polynucleotide encoding the Ebola virus GP protein, a polynucleotide encoding the M protein of MV, a polynucleotide encoding the F protein of MV, a polynucleotide encoding the H protein of MV, and a polynucleotide encoding the L protein of MV.

[0040] In a preferred embodiment of the present invention, the measles virus vector sequentially comprises DNA sequences (5' to 3') corresponding to the following gene transcription units: a polynucleotide encoding the Ebola virus VP40 protein, a polynucleotide encoding the N protein of MV, a polynucleotide encoding the P protein of MV, a polynucleotide encoding the Ebola virus GP protein, a polynucleotide encoding the M protein of MV, a polynucleotide encoding the F protein of MV, a polynucleotide encoding the H protein of MV, and a polynucleotide encoding the L protein of MV.

[0041] Secondly, the present invention also provides a nucleic acid molecule comprising: the coding sequence of the Ebola virus glycoprotein GP negative strand RNA in the above-mentioned recombinant measles virus vector and the cDNA sequence of the measles virus antigenome; and the coding sequence of the Ebola virus glycoprotein GP negative strand RNA is operatively linked to the cDNA sequence encoding the measles virus antigenome.

[0042] For example, it can be inserted into a gene intergenic region. For instance, it can be located between the N and P genes, between the P and M genes, between the M and F genes, between the F and H genes, or between the H and L genes. In some embodiments, the coding sequence of the Ebola virus glycoprotein GP negative-strand RNA and the coding sequence of the VP40 protein negative-strand RNA can each be inserted into a different site in the measles virus vector, such that the coding sequence of the Ebola virus glycoprotein GP is located between the P and M genes, and the coding sequence of the other VP40 protein is located between the H and L genes. In some preferred embodiments, the coding sequence of the Ebola virus glycoprotein GP is located in the non-coding region between the P and M genes on the measles virus vector, and the coding sequence of the Ebola virus VP40 protein is located in the non-coding region before the 5' end of the N gene on the measles virus vector.

[0043] In some cases, the recombinant measles virus vector may additionally contain negative-strand RNA sequences corresponding to multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, introns, kozak sequences, polyA sequences, selection elements, and reporter genes. These regulatory elements may be homologous or heterologous to the measles virus coding sequence.

[0044] As used herein, the term “operably linked” refers to a functional link between different polynucleotides in a vector, whereby the different polynucleotides and nucleic acid constructs are efficiently transcribed and, where appropriate, translated, particularly in cells or cell lines, especially in cells or cell lines or host cells that produce the recombinant infectious measles virus particles of the present invention as part of a rescue system.

[0045] In a preferred embodiment of the present invention, the above-mentioned nucleic acid molecule further includes: a coding sequence for the negative strand RNA of the Ebola virus VP40 protein, and the coding sequence for the negative strand RNA of the Ebola virus VP40 protein is operatively linked to a cDNA sequence encoding the measles virus antigenome.

[0046] In a preferred embodiment of the present invention, the above-mentioned nucleic acid molecule comprises, from 5' to 3', the coding sequence of the negative strand RNA of Ebola virus VP40 protein, the DNA coding sequence of measles virus N gene, the DNA coding sequence of P gene, the coding sequence of Ebola virus glycoprotein GP negative strand RNA, the DNA coding sequence of M gene, the DNA coding sequence of F gene, the DNA coding sequence of H gene, and the DNA coding sequence of L gene.

[0047] Thirdly, the present invention also provides a reverse genetics system for rescuing recombinant measles virus vectors expressing Ebola virus proteins, comprising: a) an antigen expression plasmid containing a coding sequence of the negative strand RNA of the Ebola virus glycoprotein GP in the aforementioned recombinant measles virus vector operably linked to a cDNA sequence encoding the positive strand RNA of the measles virus reverse genome; b) an auxiliary plasmid 1 containing a coding sequence of the measles virus nucleocapsid protein; c) an auxiliary plasmid 2 containing a coding sequence of the measles virus phosphoprotein; and d) an auxiliary plasmid 3 containing a coding sequence of the measles virus RNA polymerase.

[0048] Reverse genetics is a genetic research method that determines the function of a gene by studying its phenotype through site-directed mutation. In virus research, reverse genetics can express and manipulate viruses containing specific mutations in cell culture through DNA recombination, thereby assessing the effects of mutations on viral replication and transcription, pathogenicity, virus-host interactions, suppression of host cell responses, and host range or transmissibility. Various reverse genetics methods have been used to restore recombinant viruses from various viral families, including positive-sense RNA viruses such as tube viruses, picornaviruses, and flaviviruses; and negative-sense RNA viruses such as influenza viruses and arenaviruses. Reverse genetics has also been used to develop vaccines based on attenuated viral forms and to generate recombinant viruses carrying reporter genes to track viral infections. Due to its numerous advantages, such as rapid production and mutation of viruses (including rearrangements), reverse genetics is an effective tool in virology and vaccine manufacturing.

[0049] In a preferred embodiment of the present invention, the antigen expression plasmid is a eukaryotic expression plasmid.

[0050] In a preferred embodiment of the present invention, the antigen expression plasmid is an animal-animal shuttle plasmid; In a preferred embodiment of the present invention, the antigen expression plasmid and helper plasmids 1-3 are all plasmids containing the T7 promoter; In a preferred embodiment of the present invention, the attenuated measles virus strain is Schwarz, Genebank number AF266291.1.

[0051] Fourthly, the present invention also provides a method for preparing recombinant measles virus particles, comprising any one of the following methods: Method 1: Co-transfect the plasmids from the above reverse genetics system into the first cell line; Method 2: Transfect the antigen expression plasmid from the above reverse genetics system into a helper cell line.

[0052] In a preferred embodiment of the present invention, the method further includes: collecting cell supernatant after transfection to infect a second cell line and culturing it; In a preferred embodiment of the present invention, cell supernatant is collected and purified to obtain recombinant measles virus particles expressing Ebola virus protein. In a preferred embodiment of the present invention, the mass ratio of the antigen expression plasmid to helper plasmid 1, helper plasmid 2 and helper plasmid 3 is 4-4.1:2.5-2.6:2.5-2.6:0.8-1; In a preferred embodiment of the present invention, the first cell line is a cell line that stably expresses T7 RNA polymerase, and the second cell line is the Vero cell line.

[0053] Fifthly, the present invention also provides a recombinant measles virus particle obtained by the above-described method.

[0054] In a sixth aspect, the present invention also provides a host cell obtained by transfecting cells with the aforementioned nucleic acid molecules or the aforementioned recombinant measles virus vector.

[0055] In a seventh aspect, the present invention also provides a composition comprising the above-described recombinant measles virus particles and a pharmaceutically acceptable carrier.

[0056] These compositions induce an immune response against Ebola virus, particularly a protective immune response, and specifically trigger antibody production against Ebola virus antigen proteins and / or cellular immune responses against Ebola virus infection. These compositions may accordingly contain a suitable delivery vehicle for administration to a host, particularly a human host, such as a pharmaceutically acceptable delivery vehicle, and may further contain, but are not essential, adjuvants to enhance the immune response in the host.

[0057] The term "pharmaceuticalally acceptable" refers to a drug that is approved by a regulatory agency or listed in a national pharmacopoeia or other generally recognized pharmacopoeia for use in animals, especially humans. The term "carrier" refers to a diluent, adjuvant, excipient, or medium used with a pharmaceutical composition or vaccine composition (e.g., an immunogenic or vaccine formulation) for administration. Saline solutions, as well as aqueous solutions of glucose and glycerol, can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. Examples of suitable drug carriers are described in EWMartin's "Remington's Pharmaceutical Sciences." The formulation should be suitable for the route of administration.

[0058] In a preferred embodiment of the invention, the composition is an immunogenic composition, particularly a vaccine composition. The composition or vaccine is used in prophylactic therapy to prevent Ebola virus infection, and optionally, to prevent Ebola virus infection simultaneously. The vaccine composition advantageously comprises recombinant measles virus particles rescued from a reverse genetics system.

[0059] Eighthly, the present invention also provides the use of recombinant measles virus particles or the above-described compositions in the preparation of medicaments or vaccines for the prevention and / or treatment of Ebola virus infection; In a preferred embodiment of the present invention, the subject is a human being.

[0060] Vaccines can be administered to mammalian subjects, such as monkeys, apes, chimpanzees, cats, dogs, cattle, horses, mice, rats, rabbits, and humans (including adults and children).

[0061] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0062] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0063] The construction method of recombinant measles virus is summarized as follows: Reverse genetic systems used to rescue measles virus include: (1) Helper plasmid 1 (pCAGGS-N) contains the gene encoding the nucleocapsid protein of measles virus; (2) Helper plasmid 2 (pCAGGS-P) contains the gene encoding the phosphoprotein of measles virus; (3) Helper plasmid 3 (pCAGGS-L) contains the gene encoding the RNA polymerase of measles virus; (4) Recombinant measles virus plasmid; The measles virus is the attenuated measles vaccine strain Schwarz, and the transcription plasmid containing the full-length measles antigenome is the pGFMV plasmid containing the full-length measles antigenome. schwarz Among them, pGFMV schwarz The nucleotide sequence is shown in SEQ ID NO.1.

[0064] S1, Construct the full-length anti-genomic plasmid pGFMV for measles virus. schwarzThe strategy is briefly described as follows: The full-length reverse genome sequence is divided into segments of approximately 3kb each, namely F1 (2451bp), F2 (3905bp), F3 (3020bp), F4 (3359bp), and F5 (3337bp). The genome segmentation strategy is as follows: Figure 1 As shown, after synthesizing five fragments, a seamless cloning method was used to obtain the correct plasmid. The steps included are as follows: (1) Primer design: Using a plasmid containing the target fragment of the measles virus genome as a template, DNA fragments F1, F2, F3, F4, and F5 containing A were amplified by PCR. The amplified F1 fragment overlapped with the 3' end of the linearized vector and the 5' end of the amplified F2 fragment. The amplified F2 fragment overlapped with the 3' end of the F1 fragment and the 5' end of the F3 fragment. The amplified F3 fragment overlapped with the 3' end of the F2 fragment and the 5' end of the F4 fragment. The amplified F4 fragment overlapped with the 3' end of the F3 fragment and the 5' end of the F5 fragment. The amplified F5 fragment overlapped with the 3' end of the F4 fragment and the 5' end of the linearized vector. The corresponding 5 fragments were obtained by PCR amplification.

[0065] (2) Design primers and use plasmid PGF as a template to obtain linearized vector fragments PGF-1 and PGF-2 by PCR amplification.

[0066] The five fragments described in (1) and the two fragments described in (2) were ligated using seamless cloning technology to construct a plasmid containing the full-length reverse genome of measles virus, and the recombinant plasmid was named pGFMV. schwarz . S2: Insert the gene encoding the target protein into pGFMV schwarz : This invention selects to insert the Ebola virus GP protein into the non-coding region before the 5' end of the N gene, between the non-coding regions of the Ebola virus VP40, P gene, and M gene. Therefore, a repetitive non-coding region is first inserted between the P gene and M gene to reduce the impact of the foreign gene insertion on the measles virus itself, including the following steps: (1) Primer design: Using a plasmid containing repetitive sequence fragment A as a template, fragments I and II were obtained by PCR amplification. Fragment I carries the entire non-coding region of the 3' end of the measles P gene and the Pac1 restriction site; fragment II carries the entire non-coding region of the 5' end of the measles M gene and the Mul1 restriction site. (2) Design primers and use fragments I and II as templates to amplify fragment III by overlap PCR. This fragment contains the non-coding region of the 3' end of the P gene, the non-coding region of the Pac1 and Mul1 restriction sites, and the 5' end of the M gene. (3) Using plasmid T-F2 as a template, primers were designed and linearized vectors were obtained by PCR amplification. The vector sequence was formed by the 3' end of the P gene and the 5' end of the M gene at both ends. (4) The fragments obtained by the methods described in (2) and (3) are used to construct a shuttle vector with repeating interval sequences using a seamless cloning method. Subsequent clones will be constructed based on this vector.

[0067] Example 1 This embodiment provides a method for preparing an Ebola virus disease vaccine using the attenuated measles virus vaccine strain Schwarz as a vector.

[0068] The measles virus is the attenuated measles vaccine strain Schwarz, and the transcription plasmid containing the full-length measles reverse genome is pGFMVschwarz; the recombinant measles virus is a recombinant virus expressing filamentous virus antigens, named as follows: recombinant virus expressing Ebola glycoprotein pGFMV-EBOVGP, and recombinant virus expressing both Ebola glycoprotein and matrix protein pGFMV-EBOV. GP+VP40 .

[0069] Part 1: Synthesis of the measles virus genome and the Ebola GP+VP40 gene.

[0070] The measles virus used was the attenuated measles vaccine strain Schwarz (Genebank ID AF266291.1), which contained the full-length measles reverse genome sequence. The full-length reverse genome sequence was divided into segments of approximately 3kb each, namely F1 (2451bp), F2 (3905bp), F3 (3020bp), F4 (3359bp), and F5 (3337bp). The genome segmentation strategy was as follows: Figure 1 As shown, five artificially synthesized fragments have sequences as shown in SEQ ID NO:1-5. Helper plasmid 1: PCAGGS-N contains the gene encoding the nucleocapsid protein of measles virus (SEQ ID NO:6); Helper plasmid 2: PCAGGS-P contains the gene encoding the phosphoprotein of measles virus (SEQ ID NO:7); Helper plasmid 3: PCAGGS-L contains the gene encoding the RNA polymerase of measles virus (SEQ ID NO:8).

[0071] Ebola GP and VP40 are derived from the Zaire type Ebola virus Mayinga strain (Genebank number: AF086833.2).

[0072] The coding sequence of the negative strand RNA of the optimized Ebola GP gene is shown in SEQ ID NO:9, and the coding sequence of the negative strand RNA of the optimized Ebola VP40 gene is shown in SEQ ID NO:10.

[0073] Part Two: Carrier Construction.

[0074] 1. Construction of measles virus rescue vectors Constructing a full-length anti-genome plasmid of measles virus Primer design: Using plasmids containing the target fragments pBlunt-F1 / F2 / F3 / F4 / F5 as templates, DNA fragments containing F1, F2, F3, F4, and F5 were amplified by PCR. DNA fragment F1 was amplified using F1-BAC-F and F1-BAC-R, DNA fragment F2 was amplified using F2-BAC-F and F2-BAC-R, DNA fragment F3 was amplified using F3-BAC-F and F3-BAC-R, DNA fragment F4 was amplified using F4-BAC-F and F4-BAC-R, and DNA fragment F5 was amplified using F5-BAC-F and F5-BAC-R.

[0075] The amplified F1 fragment partially overlaps with the 3' end of the linearized vector pGF, and the amplified F2 fragment partially overlaps with the 5' end. The amplified F2 fragment partially overlaps with the 3' end of the F1 fragment and the 5' end of the F3 fragment. The amplified F3 fragment partially overlaps with the 3' end of the F2 fragment and the 5' end of the F4 fragment. The amplified F4 fragment partially overlaps with the 3' end of the F3 fragment and the 5' end of the F5 fragment. The amplified F5 fragment partially overlaps with the 3' end of the F4 fragment and the 5' end of the linearized vector pGF. Five corresponding fragments were obtained using PCR amplification.

[0076] Primers were designed, and plasmid PGF (a generous gift from Researcher Wang Yun of the Wuhan Institute of Virology, Chinese Academy of Sciences) was used as a template to amplify linearized vector fragments PGF-1 and PGF-2 using PCR. Specifically, plasmid PGF was amplified with pGF-F and pGF-R to obtain linearized vector fragment PGF-1, and plasmid PGF was amplified with PGF-VECTOR-F and PGF-VECTOR-R to obtain linearized vector fragment PGF-2.

[0077] The five fragments grouped in step 1 and two fragments (PGF-1 and PGF-2) were ligated using seamless cloning technology to construct a plasmid containing the full-length reverse genome of measles virus. The recombinant plasmid was named pGFMVschwarz, and its map is shown below. Figure 2 As shown in Table 1, the primers involved are listed in the table.

[0078] Table 1. Primer list for constructing the full-length measles virus plasmid.

[0079] 2. Construction of vectors related to recombinant viral vaccine strains Using measles virus as a vector, the target gene is inserted into the non-coding region before the 5' end of the N gene, the non-coding region between the P and M genes, or between the H and L genes. This study selected the non-coding region before the 5' end of the N gene and the non-coding region between the P and M genes for insertion of the target protein. To reduce the possibility of mutations in the measles genome during the construction of the recombinant measles virus plasmid and to eliminate the impact of the inserted foreign sequence in the non-coding region on the life cycle of measles, a shuttle plasmid was constructed. The insertion of a repetitive non-coding region between the P and M genes involved the following steps: 2.1 Construction of the shuttle carrier (1) Primer design: using a plasmid containing the repetitive sequence fragment pBlunt-F1 (plasmid map reference) Figure 17 (As shown) is a template. Fragments I and II were obtained by PCR amplification. Fragment I carries the entire non-coding region of the 3' end of the measles virus P gene and the Pac1 restriction site; Fragment II carries the entire non-coding region of the 5' end of the measles virus M gene and the Mul1 restriction site. (2) Design primers and use fragments I and II as templates to amplify fragment III by overlap PCR. This fragment contains the non-coding region of the 3' end of the P gene, the non-coding region of the Pac1 and Mul1 restriction sites, and the 5' end of the M gene. (3) Using plasmid T-F2 (plasmid map reference) Figure 18 Using the template shown, primers were designed, and a linearized vector was obtained by PCR amplification. The vector sequence was formed by the 3' end of the P gene and the 5' end of the M gene at both ends. (4) The fragments obtained by the methods described in (2) and (3) are used to construct a shuttle vector T-F1 with repeating interval sequences using a seamless cloning method. The map is shown below. Figure 3 As shown in Table 2, subsequent clones will be constructed based on this vector, and the primers involved are listed in Table 2.

[0080] Table 2: Primer list for shuttle plasmid construction

[0081] 2.2 Construction of recombinant viral plasmids (1) Primer design: Using plasmids containing the target gene EBOV-GP / EBOV-VP40 as templates, PCR was used to amplify DNA fragments I and II containing the target DNA fragments. DNA fragment I contains some bases of the 3' untranslated region of PacI, Ebola virus glycoprotein EBOV-GP and some bases of the 5' untranslated region of MluI. DNA fragment II contains some non-translated bases of the front end of the measles virus N gene, the Ebola matrix protein EBOV-VP40 gene sequence and some bases of the front end sequence of the measles virus N gene.

[0082] (2) Design primers (Reapeat-enzyme-F and Reapeat-enzyme-R), use T-F1 plasmid as template, and linearize the vector by PCR amplification. The linearized vector has PacI 3' end and MluI 5' end at both ends. (3) The DNA fragment I from step (1) and the linearized vector described in (2) are used to obtain the intermediate plasmid T-EBOV using seamless cloning technology. GP The list of primers involved is shown in Table 3.

[0083] (4) Design primers, the specific sequences of which are shown in Table 3, using the measles virus plasmid pGFMV. Schawarz Using a template, the plasmid was linearized into three fragments, namely pGFMV, using PCR. Schawarz -1 (AaTII-F and PGF-R), pGFMV Schawarz -2 (PGF-F and LR), pGFMV Schawarz -3 (LF and PflFI- -R).

[0084] (5) Design primers (refer to EBOV-PACI-F and EBOV-GP-MluI-R as shown in Table 3) to use the intermediate plasmid T-EBOV described in (3). GP Using a template, a DNA fragment containing the target EBOV-GP was obtained by PCR amplification. This DNA fragment includes partial nucleotides of the measles virus N gene, and partial genes of EBOV-GP and the measles virus P gene.

[0085] (6) The DNA fragments from steps (4) and (5) were recombined using seamless cloning technology to obtain the measles recombinant virus plasmid pGFMV-EBOV. GP， plasmid maps as follows Figure 4 As shown in Table 4, the primers involved are as follows.

[0086] (7) Design primers. The primer sequences are shown in Tables 3 and 5. Use the recombinant measles virus plasmid pGFMV-EBOV. GP Using a template, the plasmid was linearized into three fragments using PCR: pGFMV-EBOV. GP -1 (N-circle-F and PGF-R), pGFMV-EBOV GP -2 (PGF-F and LR), pGFMV-EBOV GP -3 (LF and N-circle-R).

[0087] (8) Recombinant measles virus pGFMV-EBOV is obtained by performing four-fragment recombination using the DNA fragment II described in (1) and the three DNA fragments described in (7) through seamless cloning technology. GP+VP40 Plasmid map as follows Figure 5 As shown in Table 5, the primers involved are as follows.

[0088] (9) The measles virus plasmid constructed based on the above description and the recombinant measles virus plasmid were identified by enzyme digestion. XhoI restriction endonuclease was selected, and 0.5 μg of plasmid was digested for identification. The band sizes were consistent, such as... Figure 6 As shown.

[0089] Rescue and identification of measles virus and recombinant virus vaccine strains Table 3: List of primers for constructing intermediate plasmids of recombinant measles virus

[0090] Table 4. List of primers for constructing intermediate plasmids of recombinant measles virus

[0091] Table 5 MV-EBOV GP+VP40 Construct a primer list

[0092] Part 3: Transfection, Preparation of Viral Particles The three helper plasmids constructed above and pGFMV schwarz / pGFMV-EBOV GP / pGFMV-EBOV GP+VP40 BSR-T7 cells were transfected with Lipofectamine 2000. A rescue diagram is shown below. Figure 7 As shown, the specific experimental method is as follows: Transfection: Helper cells BSR-T7 were seeded in six-well plates at a density of 5 × 10⁶ cells per well. 5Cells were cultured overnight in a DMEM + 10% FBS medium at 37°C in a 5% CO2 incubator. The medium was changed on the day of transfection, and the cells were cultured in fresh DMEM medium containing 10% FBS. When cell confluence reached 70-80%, the measles virus plasmid pGFMV was extracted from each transfection well. schwarz / pGFMV-EBOV GP / pGFMV-EBOV GP+VP40 Add 4 μg of helper plasmid 1, 2.5 μg of helper plasmid 2, 1.5 μg of helper plasmid 3, and 0.8 μg of helper plasmid 3 to 300 μl of OPTI-MEM, mix well, and incubate at room temperature for 5 min. Dilute 14 μL of liposomes with 300 μL of OPTI-MEM culture medium and incubate at room temperature for 5 min. Mix the two solutions well and incubate at room temperature for 20 min. Then, add the mixture evenly to the cells.

[0093] After three more days of incubation at 37°C, the cells developed cytopathic effects. Western blotting (WB) of the collected supernatant revealed the expression of the measles virus N protein, confirming successful rescue of the measles virus.

[0094] Cell culture flasks were repeatedly frozen and thawed three times in a -80°C ultra-low temperature freezer. The culture medium and cell mixture were collected, and the cell-supernatant mixture was added to Vero-E6 cells for passage and expansion culture to rescue the measles virus and recombinant measles virus vaccine strain. The rescued measles virus or recombinant virus vaccine strain showed obvious measles-specific cytopathic effects in Vero-E6 cells. The frozen-thawed cells were collected from the cell culture flasks and centrifuged at 3000g for 10-15 minutes. The supernatant of the cell lysate was collected, and a portion was used to infect and passage Vero cells confluent in the culture flasks. The remaining supernatant was frozen and stored in a -80°C ultra-low temperature freezer for later use. Example 2 This embodiment describes the identification and titer determination of purified samples of recombinant measles virus vector Ebola vaccine.

[0095] 1. Stability identification of recombinant measles virus Measles virus and recombinant measles virus were passaged for 10 generations, and the virus was collected from each generation. RNA was extracted from cell lysis using a nucleic acid extractor in accordance with the instructions of Novizan's DNA / RNA extraction kit. RT-PCR detection: Using the RNA extracted in the above steps as a template, the genomic RNA was reverse transcribed into cDNA using Novizan's HiScript III 1st Strand cDNASynthesis Kit (+gDNA wiper) with random primers, following the instructions. Primers were designed, and their sequences are shown in Table 6. Using the cDNA from the previous steps as a template, fragments I, II, and III were obtained by PCR amplification. Fragment I was amplified to obtain the nucleotides of the measles virus N gene (MNF and MNR); fragment II was amplified to obtain the nucleotides of the Ebola virus glycoprotein EBOV-GP gene (EBOV-GP-F and EBOV-GP-R); and fragment III was amplified to obtain the nucleotides of the Ebola virus matrix protein EBOV-VP40 gene (EVP40-F and EVP40-R). (Results are shown in Table 6). Figure 8 (As shown).

[0096] Table 6 Primers for Virus Stability Detection

[0097] 2. Western blot (WB) detection Measles virus and recombinant measles virus were used to infect Vero-E6 cells with an MOI of 1. Cells were collected 48 hours after infection for Western blot analysis. The proteins detected included measles virus N protein, Ebola glycoprotein EBOV-G, and internal control protein GAPDH.

[0098] The results are as follows Figure 9 As shown, NC is the blank cell sample; MV-EBOV GP It is recombinant measles virus MV-EBOV GP Infected cell samples; MV-EBOV GP+VP40 It is recombinant measles virus MV-EBOV GP+VP40 Infected cell samples; The results showed that the expression of measles virus N protein could be detected in all recombinant measles virus infected samples, and the recombinant measles virus MV-EBOV was also present. GP MV-EBOV GP+VP40 The expression of the Ebola virus protective antigen EBOV-GP could be detected in all infected cells.

[0099] 3. Titer determination The titer of recombinant measles virus was determined using IFA. The specific method is as follows: The steps are as follows: 1) Seed Vero-E6 cells at a density of 2*10^5 / ml in 24-well plates. Wait until the cells form a monolayer and the cell confluence is 80%-90% before titer determination. This ensures that the cells have grown into a confluent monolayer when titer is measured.

[0100] 2) Serially dilute the obtained measles virus sample or recombinant measles virus sample 10-fold, for a total of 8 dilutions. Aspirate the cell supernatant and add 500 μL of virus dilution to the cells, making two accessory wells for each dilution. Two hours after infection, discard the virus solution and add the cells to DMEM containing 5% FBS and 1% methylcellulose, and incubate at 32°C for 6 days.

[0101] 3) Add 4% paraformaldehyde to each well at a dose of 500 μL and fix at room temperature for 30 minutes. After washing the cells three times with PBS, add 500 μL of 2% Triton-X100 to each well, incubate on ice for 15 minutes, and then wash the cells three times with PBS.

[0102] 4) The antibody Measles-N (abcam, ab106292) was diluted with 1% BSA in PBS at a ratio of 1:500. 200 μL of the diluted antibody was added to each well and incubated at 37°C for 1 hour. 5) Discard the antibody, wash the cells 3 times with PBS, add 0.25 mL of Alex-488 labeled Rat Anti-Mouse Antibody (Invitrogen, A-11001 1:100 dilution) to each well, and incubate at 37°C for 1 hour; 6) Discard the Rat Anti-Mouse Antibody diluent and wash the cells three times with PBS. Count the green fluorophores under a fluorescence microscope.

[0103] The titer test results showed that the infection titer reached 5*10. 5 FFU / mL, calculated as follows: number of green fluorescent groups per well / virus inoculation amount per well (ml) * virus dilution.

[0104] 4. Morphological observation using electron microscopy 4.1 Negative staining To visually verify the morphological characteristics of recombinant measles virus MV-EBOV GP+VP40 It can secrete Ebola VLP into the supernatant and collect recombinant measles virus MV-EBOV. GP+VP40 Cell supernatant collected 48 hours post-infection was negatively stained for observation. The procedure is as follows: After standing for 5-10 minutes, excess liquid at the edge of the screen was blotted away with filter paper. While the screen was not completely dry, sodium phosphotungstic acid staining solution was added, and staining was performed for 3-5 minutes. Excess liquid at the edge of the screen was blotted away with filter paper, and the screen was allowed to air dry. The supernatant was then observed under a transmission electron microscope. The morphology of Ebola filamentous virus-like particles could be observed in the supernatant sample, as shown in the results. Figure 10 The attached diagram on the right shows the recombinant measles virus vector Ebola vaccine, which has a shape consistent with the measles virus.

[0105] 4.4.2 Frozen Sections Further morphological verification revealed that, after viral infection, the recombinant virus proliferated within the cells and simultaneously secreted Ebola virus VLPs into the extracellular space. In situ ultrathin sections were selected to reconstruct the cell infection process, as shown in the following steps: 1) Glutaraldehyde fixation: Prepare a monolayer of cell samples in advance in a petri dish. Multiple cell layers will affect subsequent fixation and infiltration, thus affecting the sectioning effect. For iron wall cells, remove the cell culture supernatant and add 2.5% glutaraldehyde to cover the monolayer of cells for fixation (fix at 37°C for 2 hours, at least 0.5 hours, or overnight at 4°C). 2) Osmium tetroxide fixation: Rinse three times with PBS for 15 min each time, then fix with 1% osmium tetroxide pre-cooled at 4℃ at room temperature (20℃) for 2-3 h (time may vary depending on the sample), and then rinse three times with 0.1M phosphate buffer (pH 7.4) for 15 min each time.

[0106] 3) Dehydration: The sample is dehydrated by gradient alcohol (30%, 50%, 70%, 80%, 85%, 90%, 95%, 100% twice), with the 30%-80% dehydration time being 5 minutes, the 85%-100% dehydration time being 3 minutes, and the 100% alcohol dehydration time being twice (the time can be appropriately extended for samples with high water content and thick cell membranes).

[0107] 4) Infiltration: Preheat the epoxy resin to 37℃. After the epoxy resin melts, add it to the dehydrated cells and infiltrate at 37℃ for 2 hours. Prepare the encapsulation capsules by pre-treating them, adding a label, and then adding the epoxy resin. Polymerize the capsules and samples together at 60℃. After reaching the appropriate polymerization level, invert the capsules into the samples and further polymerize at 60℃ (24-48 hours). After polymerization is complete, place the samples in liquid nitrogen and repeatedly freeze-thaw to remove the epoxy resin, thus completing sample preparation. 5) Ultrathin section: The section thickness is generally 80-100nm.

[0108] 6) Double staining: lead and uranium double staining (2% uranium acetate saturated aqueous solution, lead citrate, staining at room temperature for 15 min), drying at room temperature overnight, and observation under electron microscope.

[0109] Observation results as follows Figure 10 The middle image shows the morphology of measles virus particles and typical Ebola virus virus-like particles observed in infected cells. The electron microscopy results indicate that the recombinant measles virus MV-EBOVGP+VP40 can simultaneously express the protective antigen EBOV-GP in infected cells and secrete EBOV-VLP.

[0110] Example 3 This embodiment is based on the immunological evaluation of the measles virus-based Ebola virus disease vaccine in a mouse model.

[0111] 1. Experimental materials.

[0112] 1.1 Laboratory Animals SPF-grade female BALB / c mice (4-6 weeks old) were purchased from Vital River. The mice were housed at the Animal Center of Wuhan Institute of Virology, Chinese Academy of Sciences.

[0113] 1.2 Experimental Materials Fluorescently labeled antibodies: FITC Hamster Anti-Mouse CD3e (145-2C11), PerCP-Cy5.5 Rat Anti-Mouse CD8a (53-6.7), BV421 Rat Anti-Mouse CD4 (GK1.5), Rat Anti-Mouse IFN-γ (XMG1.2); Fixation and penetration solution: Cytofix / Cytoperm TM Fixation andPermeabilizaiton Solution, lotionPerm / Wash TM Buffer, BD TM ELISPOT set, BD TM ELISPOTAEC substrate set and blocking agent were purchased from BDPharmingen; erythrocyte lysis buffer was purchased from Solarbio; RPMI 1640 medium was purchased from Gibco; BSA was purchased from Merck; HRP-labeled anti-mouse IgG antibody was purchased from Proteintec; truncated secretory GP (46aa-364aa) was purified in our laboratory.

[0114] 2. Immunization in mice.

[0115] According to the experimental design, the purified recombinant measles virus Ebola vaccine and the recombinant measles virus vector control vaccine were diluted with physiological saline to an infection titer of 2 × 10⁻⁶. 6 FFU / mL, using a 1mL syringe, was administered via intramuscular injection into the medial side of the left hind leg, 50μL per mouse, with an immunization dose of 1×10⁻⁶ mice. 5 FFU. The mouse immunization grouping is shown in Table 7 below: Table 7: List of mouse immunization groups

[0116] 3. Detection of humoral immunity levels.

[0117] 3.1 Blood collection and serum separation After immunization, mice were venous blood collected via tail vein at specific time points. The blood was allowed to stand at room temperature for at least 1 hour. Once serum had formed, the cells were centrifuged at 5000 rpm for 10 minutes, transferred to new centrifuge tubes, and stored at -20°C for later use.

[0118] 3.2 Serum antibody level ELISA detection The day before the experiment, the ELISA strips were coated with 4 μg / mL of truncated secretory EBOV-GP or SUDV-GP and incubated overnight at 4°C. On the day of the experiment, the liquid in the wells was discarded, and the strips were washed three times with ELISA washing buffer (PBS + 1% Tween 20).

[0119] Discard the washing buffer and blot the liquid from the wells with clean absorbent paper. Add 120 μL of 2% BSA to each well and incubate at 37°C for 1 hour. Discard the blocking buffer, wash the plate three times, and add 100 μL of sample diluent (PBST + 0.2% BSA) to each well. Serum samples are serially diluted at a specific initial dilution (determined through preliminary experiments), with seven dilutions per sample. After serum dilution, add the diluted serum to ELISA plates coated with the specific antigen and incubate at 37°C for 1 hour.

[0120] Wash the ELISA plate four times with washing buffer, add 100 μL of HRP-conjugated anti-mouse IgG secondary antibody (1:10000) to each well, and incubate at 37°C for 1 hour. Wash the plate four times with washing buffer, remove the liquid from the wells, and proceed with the colorimetric reaction.

[0121] The color development process involved adding single-component TMB chromogenic solution to each well, developing the solution for 15 minutes, and then stopping the reaction with ELISA stop solution. Finally, the absorbance at 450 nm was measured using a microplate reader.

[0122] Wells with an OD450 greater than 2.1 times the reading of blank wells were designated as positive wells. The dilution factor of the highest-dilution positive well in each serum sample was recorded as the antibody titer of that sample.

[0123] Test results as follows Figure 11 As shown, recombinant measles virus MV-EBOV GP+VP40 Compared to the recombinant vaccine strain MV-EBOV GP It can generate higher levels of EBOV-GP specific antibodies in a shorter time. The activity of binding antibodies against SUDV-GP / BDBV-GP in serum 28 days post-immunization was detected by ELISA. The results showed a trend consistent with that against EOV-GP, indicating the presence of recombinant measles virus MV-EBOV. GP+VP40 Compared to the recombinant vaccine strain MV-EBOV GP It exhibits higher antibody binding rates.

[0124] 3.3 Detection of serum neutralizing antibody levels (pseudovirus neutralization experiment - methods and results) 1. Passage Vero cells into 96-well cell culture plates one day in advance, with a cell density of 1*10⁻⁶ cells / well. 5 / ml, and the cells grew into a monolayer the next day.

[0125] 2. On the day of the experiment, serum was diluted: the samples were diluted 1:10 with serum-free culture medium, followed by 4-fold serial dilutions, for a total of 7 concentrations, with 3 replicates for each dilution. Cell wells with neither serum nor virus were set as negative controls, and cell wells with only virus and no serum were set as positive controls.

[0126] 3. Take out the rVSV-EBOV virus with a known titer, thaw it, and then add it according to 1000 TCID50. 50 Calculate the required viral suspension at a volume of 50 μL / well. Add the viral diluent to the diluted serum at a dose of 50 μL / well, mix thoroughly, and incubate at 37°C for 1 hour.

[0127] 4. Remove the incubated virus and diluted serum mixture. Discard the cell supernatant, wash the cells once with PBS at 100 μL / well, and then add the mixture to the cells at a rate of 100 μL / well. Mix thoroughly and incubate at 37°C for 1 hour. Discard the supernatant, replace with 2% FBS DMEM cell culture medium, and continue culturing at 37°C.

[0128] 5. Incubate at 37℃ for approximately 24-48 hours until a suitable fluorescence ratio is achieved, then fix the sample. Discard the supernatant, add twice the volume of 10% formaldehyde per well, and fix at room temperature for 30 minutes.

[0129] 6. Discard formaldehyde, rinse with tap water, add DAPI and stain at room temperature for 15 minutes. Then scan for high content fluorescence to determine the proportion of fluorescence and perform data processing and analysis.

[0130] The results are as follows Figure 12 As shown, the titer of neutralizing antibodies against rVSV-EBOV / SUDV / BDBV in serum 28 days post-immunization was detected using a pseudovirus neutralization assay. The results showed that rVSV-EBOV... GP+VP40 Compared to the recombinant vaccine strain MV-EBOV GP It can produce higher levels of EBOV-GP / SUDV-GP / BDBV-GP specific neutralizing antibodies, demonstrating better broad-spectrum potential.

[0131] 4. Detection of cellular immunity levels.

[0132] 4.1 Isolation of splenic lymphocytes Mice were euthanized by cervical dislocation and immersed in 70% alcohol for 3 minutes. The spleen was aseptically removed from the mouse in a biosafety cabinet and placed on a 200-mesh cell sieve in a sterile petri dish. 10 mL of RPMI 1640 complete culture medium was added, and the spleen was gently ground into single cells using the plunger of a syringe. The spleen cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 800 g for 5 minutes at 4°C. The supernatant was discarded, and the cells were resuspended in 2 mL of Solarbio Science Red Cell Lysis Buffer and lysed at room temperature for 5 minutes. 13 mL of RPMI 1640 complete culture medium was added, and the cells were centrifuged at 500 g for 5 minutes at 4°C. The supernatant was discarded, and the cells were washed again with 10 mL of RPMI 1640 complete culture medium, resuspended with an appropriate amount of culture medium, filtered through a 200-mesh cell sieve into a 10 mL test tube, and 50 μL was diluted 20-fold for cell counting.

[0133] 4.2 Flow cytometry was used to detect the secretion of specific cytokines.

[0134] 4.2.1 In vitro stimulation of mouse spleen cells Take an appropriate amount of the isolated mouse spleen cells and dilute it to 1×10⁻⁶. 7 0.1 mL of cells / mL was added to each well of a 96-well plate. Spleen cells from each mouse were used to create wells with specific CTL epitope stimulation and wells without stimulation. The peptides used for specific CTL epitope stimulation were EBOV-GP and MARV-GP. As positive controls, wells were stimulated with PMA and ionomycin at concentrations of 100 ng / mL and 1 μg / mL, respectively.

[0135] Cells were cultured at 37°C in a 5% CO2 incubator for 12 hours and then stained with relevant antigens for flow cytometry detection of intracellular cytokines.

[0136] 4.2.2 Staining of cell surface antigens and intracellular cytokines After 6 hours of in vitro stimulation, spleen cells were transferred to flow cytometry tubes and centrifuged at 500g for 5 minutes at 4°C, discarding the supernatant. The appropriate amounts of fluorescently labeled antibodies (FITC Hamster Anti-Mouse CD3, PerCP-Cy5.5 Rat Anti-Mouse CD8, BV421 Rat Anti-Mouse CD4) were pre-diluted with PBS + 2% FBS according to the manufacturer's instructions and incubated at 4°C for 30 minutes. After 30 minutes, 3 mL of PBS + 2% FBS was added to each tube, and the cells were centrifuged at 500g for 5 minutes at 4°C, discarding the supernatant. Cytofix / Cytoperm™ Fixation and Permeabilizaiton Solution was added according to the manufacturer's instructions, and the cells were incubated at 4°C for 20 minutes for fixation and perforation. After 20 minutes, 1×Perm / Wash™ Buffer was added, and the cells were centrifuged at 600g for 5 minutes at 4°C, discarding the supernatant. Dilute an appropriate amount of BV786 Rat Anti-Mouse IFN-γ antibody with 1×Perm / Wash™ Buffer according to the manufacturer's instructions, mix gently, and incubate at 4°C for 30 minutes. Finally, wash each tube once with 1 mL of 1×Perm / Wash™ Buffer and once with 3 mL of PBS, discard the supernatant, resuspend in 200 μL of PBS, and then perform detection. To adjust fluorescence compensation between dyes during detection, unstained tubes, single-stained FITC Hamster Anti-Mouse CD3 tubes, single-stained PerCP-Cy5.5 Rat Anti-Mouse CD8 tubes, single-stained BV421 Rat Anti-Mouse CD4 tubes, and single-stained BV786 Rat Anti-Mouse IFN-γ tubes were used. The BV786 Rat Anti-Mouse IFN-γ single-stained tubes used positively stimulated cells.

[0137] 4.2.3 On-machine testing Flow cytometry was performed using BD. First, the appropriate voltage was adjusted, and fluorescence compensation between dyes was adjusted using single-fluorescence staining of the sample, followed by sample loading. Lymphocytes were circled using FSC and SSC, designated as gate 1. Cells emerging from gate 1 were circled using FITC and SSC to identify CD3 cells, designated as gate 2. Cells emerging from gate 2 were then analyzed using PerCP-Cy5.5. BV421 is set as gate 3 and gate 4, and CD8 is analyzed using BV786 and PerCP-Cy5.5. + The percentage of IFN-γ positive cells among total T cells (CD3 cells) was analyzed using BV786 and BV421. +The percentage of IFN-γ positive cells among total T cells (CD3 cells) was determined. The results were analyzed using FlowJo flow cytometry software.

[0138] like Figure 13 As shown, the results indicated that at 2 weeks and 22 weeks post-immunization, the level of IFN-γ secreted by CD8-positive cells in the spleen cells of mice immunized with the recombinant measles virus Ebola vaccine was significantly higher than that in the control vaccine group.

[0139] 4.3 ELISPOT Detection of Cytokines ELISPOT plates were coated with 5 μg / mL anti-mouse IFN-γ antibody and incubated overnight at 4°C. Before the experiment, the plates were blocked for 2 hours at room temperature using RPMI 1640 + 10% FBS. Before the experiment, the blocking solution was discarded, and 100 μL of RPMI 1640 + 10% FBS medium containing Ebola virus (EBOV)-GP peptide mixture was added to each well. 100 μL of isolated mouse spleen cells were added to each well as designed, resulting in a cell concentration of 2 × 10⁶ cells / well. 6 Cells / mL. 50 ng / mL PMA was added to each well of the positive control well, and a non-stimulating control was also included. Cells were cultured at 37°C in a 5% CO2 incubator for 18-24 hours. The next day, cells were discarded, and 200 μL of each well was washed twice with distilled water, then three times with washing buffer PBS + 0.1% Tween-20, incubating for 2-3 minutes each time. The washing buffer was discarded, and 100 μL of Biotinylated anti-mouse IFN-γ diluted 1:250 in PBS + 10% FBS was added to each well, incubating at room temperature for 2 hours. After 2 hours, the liquid in the wells was discarded, and the cells were washed three times with washing buffer, incubating for 2-3 minutes each time. 100 μL of streptavidin-horseradish peroxidase diluted 1:100 in PBS + 10% FBS was added to each well, incubating at room temperature for 1 hour. The liquid in the wells was discarded, and the cells were washed four times with washing buffer, then three times with PBS. A colorimetric reaction was performed using the BD ELISPOT AEC substrate set. Once the spots in the wells have grown to a suitable size, discard the chromogenic substrate and rinse thoroughly with distilled water to terminate the reaction. Allow the plate to dry and then count the spots using an ELISA dot imaging system.

[0140] The experimental data processing results are as follows: Figure 14 As shown, the amount of IFN-γ secreted by spleen cells in mice immunized with the Ebola vaccine group was significantly higher than that in the control vaccine group. Meanwhile, MV-EBOV... GP and MV-EBOV GP+VP40There was no significant difference in the amount of IFN-γ secreted by spleen cells in mice immunized with either of the two recombinant measles virus vaccines, whether at 2 weeks or 22 weeks post-immunization. This result is consistent with the findings of flow cytometry.

[0141] According to the experimental design, the recombinant measles virus and measles virus vector control vaccines were diluted to appropriate infectious titers with OPTI-MEM and administered via intraperitoneal injection (100 μL per mouse) using a 1 mL syringe. Four weeks post-immunization, a challenge protection test was performed to evaluate the vaccine's effectiveness. Weight changes, survival status, and viral clearance in tissues of mice in each immunized group were continuously monitored after challenge. The mouse groups used to evaluate the effectiveness of the recombinant measles virus vaccine against Ebola virus using rVSV-EBOV (replicating recombinant pseudovirus) and MA-EBOV (ebola virus mouse-adapted live strain) are shown in Table 8 below. Table 8: List of Mouse Challenge Groups

[0142] The weight changes and survival curves of rVSV-EBOV challenged mice are as follows: Figure 15 As shown; the weight changes and survival curves of MA-EBOV challenged mice are as follows. Figure 16 As shown. Both vaccine strains can achieve 100% effective protection against Ebola pseudovirus and live virus. Among them, the MV-EBOV recombinant measles virus vaccine strain provides better protection against MA-EBOV challenge. GP+VP40 The body weight of the mice showed a stable trend, MV-EBOV GP The vaccine strain showed a trend of first decreasing and then increasing, suggesting that MV-EBOV... GP+VP40 The vaccine strain showed better protective effects.

[0143] In summary, the Ebola recombinant measles virus vector vaccine induced highly efficient cellular and humoral immunity in mouse models. Both cellular and humoral immune responses are crucial for the body's resistance to Ebola virus infection. Recent studies in animal models have shown that, compared to cellular immune responses, the level of Ebola GP-specific IgG antibodies is more strongly correlated with the survival of challenged animals.

[0144] In summary, this invention provides a novel Ebola virus vaccine based on a measles virus vector, which has broad-spectrum and high-efficiency properties and can provide effective protection against possible future Ebola outbreaks.

[0145] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant measles virus vector expressing Ebola virus protein, characterized in that, It includes: The measles virus vector contains the coding sequences for the negative strand RNA of Ebola virus glycoprotein GP and Ebola virus VP40 protein, as shown in SEQ ID NO: 9 and SEQ ID NO:

10. The measles virus vector includes sequences complementary to the negative strand RNA sequence of the measles virus strain, and the negative strand RNA sequence includes the N gene, P gene, M gene, F gene, H gene, and L gene. The coding sequence of the negative strand RNA of the N gene on the measles virus vector is shown in SEQ ID NO:6, the coding sequence of the negative strand RNA of the P gene is shown in SEQ ID NO:7, and the coding sequence of the negative strand RNA of the L gene is shown in SEQ ID NO:

8. The coding sequence of the negative strand RNA of the Ebola virus glycoprotein GP is located in the non-coding region between the P gene and the M gene on the measles virus vector. The coding sequence of the negative strand RNA of the Ebola virus VP40 protein is located in the non-coding region before the 5' end of the N gene on the measles virus vector. The measles virus strain is the attenuated measles virus strain Schwarz, Genebank number AF266291.

1.

2. The recombinant measles virus vector according to claim 1, characterized in that, The measles virus vector further includes at least one of the following regulatory elements: transcription promoter, termination sequence, enhancer, and other cis-acting elements; The measles virus vector includes: measles virus gene fragments as shown in SEQ ID NO:1-5.

3. A nucleic acid molecule, characterized in that, It includes: The recombinant measles virus vector of any one of claims 1-2 contains the coding sequence of Ebola virus glycoprotein GP negative-strand RNA and the cDNA sequence of measles virus antigenome; wherein the coding sequence of Ebola virus glycoprotein GP negative-strand RNA is operably linked to the cDNA sequence encoding the measles virus antigenome, and the nucleic acid molecule further comprises: the coding sequence of Ebola virus VP40 protein negative-strand RNA as described in any one of claims 1-2, wherein the coding sequence of Ebola virus VP40 protein negative-strand RNA is operably linked to the cDNA sequence encoding the measles virus antigenome, and the nucleic acid molecule comprises, from 5' to 3', the coding sequence of Ebola virus VP40 protein negative-strand RNA, the DNA coding sequence of measles virus N gene, the DNA coding sequence of measles virus P gene, the coding sequence of Ebola virus glycoprotein GP negative-strand RNA, the DNA coding sequence of M gene, the DNA coding sequence of F gene, the DNA coding sequence of H gene, and the DNA coding sequence of L gene.

4. A host cell, characterized in that, It is obtained by transfecting cells with the nucleic acid molecule of claim 3 or the recombinant measles virus vector of any one of claims 1-2.

5. The use of the recombinant measles virus vector expressing Ebola virus protein as described in any one of claims 1-2 in the preparation of a medicament or vaccine for the prevention and / or treatment of Ebola virus infection; The subjects were humans.

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