WO2021185310A1

WO2021185310A1 - Mvsv virus vector and virus vector vaccine thereof, and novel coronavirus pneumonia vaccine based on mvsv mediation

Info

Publication number: WO2021185310A1
Application number: PCT/CN2021/081524
Authority: WO
Inventors: 秦晓峰; 韦治明; 权海峰; 龙丽梅
Original assignee: 睿丰康生物医药科技(浙江)有限公司
Priority date: 2020-03-20
Filing date: 2021-03-18
Publication date: 2021-09-23
Also published as: CN111088283B; CN111088283A

Abstract

Provided is a recombinant viral vector mVSV of the vesicular stomatitis virus (VSV) M protein. The recombinant viral vector includes mutant virus strains obtained by the mutation of three sites M51F, F110A and I225L of the M protein of a wild Indiana strain VSV. Further provided is a novel coronavirus pneumonia vaccine formed by the chimerization of a mVSV virus vector or fusion of a mVSV virus vector with a receptor binding domain (RBD) of the spike protein S of SARS-CoV-2 pathogens.

Description

mVSV virus vector and its virus vector vaccine, a new coronary pneumonia vaccine mediated by mVSV

Technical field

The present disclosure relates to the technical field of genetic engineering, specifically the mVSV virus vector and its virus vector vaccine, and a new coronary pneumonia vaccine mediated by mVSV.

Background technique

Coronavirus is a member of the order Nidovirals, family Coronavirus, and genus Coronavirus in the virological classification. The genome is a single-stranded, positive-stranded RNA with a complete genome. It is between 26 and 32 kb in length and is currently the largest RNA virus known in its genome. Coronavirus infections are widespread in nature, and common mammals such as dogs, cats, mice, pigs, cattle and poultry are all susceptible. In recent years, multiple types of coronaviruses have been isolated from beluga whales, camels, and especially bats. There are currently six human coronaviruses known, namely, human coronavirus 229E (HCoV-229E) and HCoV-OC43 discovered in the 1960s, SARS-CoV that appeared in 2003, and HCoV-NL63 that was isolated in the Netherlands in 2004 , HCoV-HKU1 identified in Hong Kong in 2005 and the new Middle East respiratory syndrome (Middle East respiratory syndrome virus, MERS) coronavirus MERS-CoV that appeared in the Middle East in 2012. The genes of SARS-CoV-2 and SARS (severe acute respiratory syndrome) have a high degree of homology. In the process of 2019-nCoV infecting host cells, the spike protein (S protein) of the virus first recognizes the receptor protein ACE2 (angiotensin-converting enzyme 2) on the cell membrane, and then mediates and promotes the fusion of the viral envelope and the cell membrane. , And finally make the virus invade the host cell. Coronavirus can infect the respiratory tract, digestive tract, liver, kidney and nervous system of the body, causing various degrees of pathological damage, and even death in severe cases.

According to the phylogenetic analysis of the nucleic acid sequence of coronaviruses, the International Commission for Classification of Virology (ICTV, 2012) divided the members of the genus Coronavirus into four groups in its ninth report: α group, β group, γ group and δ group. Human coronaviruses are mainly distributed in the α group and β group. Among them, HCoV-229E and HCoV-NL63 are in the α group, HCoV-OC43 and HCoV-HKU1 are in the 2a subgroup in the β group, MERS-CoV belongs to the 2c subgroup in the β group, and the latest SARS-CoV- 2 and SARS belong to the 2b subgroup in the β group.

Since the outbreak of the novel coronavirus (COVID-19) pneumonia, it has caused more than 80,000 infections across the country and nearly 4,000 deaths; and it has caused more than 50,000 infections and thousands of deaths worldwide. A highly transmissible coronavirus. The difference between SARS-CoV-2 coronavirus and traditional coronavirus is that it is susceptible to everyone. It can not only infect the upper respiratory tract, causing fever, cough, laryngitis and other common cold symptoms, but also infect the lower respiratory tract, causing bronchitis and pneumonia. And other acute respiratory symptoms. The latest research shows that the ability of the RBD segment of the S-spike protein of the virus to bind to ACE2 is 10 times that of SARS, and is the strongest among the seven major coronaviruses known to infect humans. The structure of SARS-CoV-2 is similar to SARS coronavirus. It is an enveloped single-stranded positive-stranded RNA virus. The spike protein S protein reflected on the surface of the virus is a specific organization structure on the virus envelope. A large number of spike proteins are formed on the surface of the virus, which play an important role when the virus invades the target cell and recognizes the virus and the cell. A number of studies have shown that the SARS S protein vaccine can produce high-titer anti-SARS-CoV virus neutralizing antibodies, which can effectively prevent SARS-CoV infection. Therefore, in view of the high similarity of the three-dimensional structure of SARS-CoV-2 and SARS S protein In the development of a new coronavirus vaccine, the S antigen of the new coronavirus is usually used as the main target.

The global outbreak of the new crown epidemic has made it urgent to develop drugs for new crown pneumonia. The results of the JAMA study in the Journal of the American Medical Association show that some recovered patients are still carriers of the new crown virus, which has also triggered whether the new coronavirus has become a global epidemic In the discussion, the new crown virus may be like the flu virus, which has been prevalent in human society for a long time, and the vaccine has been proved to be the most cost-effective, effective and long-lasting disease prevention and control measure. Therefore, it is imperative for all people to be vaccinated against the new crown. The short-term development and release of a new crown pneumonia vaccine is the most powerful means to prevent the spread of the epidemic.

Studies have shown that pathogens that currently cause severe infectious diseases such as human immunodeficiency virus (HIV), influenza virus, severe acute respiratory syndrome virus (SARS-CoV), etc., all invade through the mucosal surface (genital tract, respiratory tract, gastrointestinal tract) And the infected body, because the body cannot induce an effective mucosal immune response to clear the mucosal infectious pathogens, the pathogens quickly spread into the blood, and then invade the whole body, causing damage to the body, especially the lung tissue.

It is known that conventional vaccines such as inactivated, protein vaccines, DNA vaccines, subunit vaccines, etc. usually cannot induce specific mucosal immune responses through conventional immunization (intramuscular injection, subcutaneous, etc.). Regardless of the form of the vaccine, to induce mucosal immune response, it is usually necessary to inoculate the target antigen from the mucosal site before it can be taken up and presented by APC in the mucosal tissue, further activate the mucosal immune system and induce a strong mucosal immune response.

The bottleneck of conventional mucosal vaccination with traditional antigens is that the frequent physical oscillations of the mucosal cilia can quickly remove foreign antigens; there is a large amount of acidic solution in the mucosal area, which is rich in hydrolytic enzymes, DNA enzymes, etc., which can quickly degrade foreign antigens. Therefore, the traditional antigen inoculated on the mucosal site is rapidly cleared and degraded, and cannot stay in the mucosal area effectively, so that it is not enough to be presented by APC and cannot induce an effective mucosal immune response. Even if induced, the degree of response is very low.

The known vaccine carrier vesicular stomatitis virus (VSV) wild strain can infect a variety of animals and insects in the natural environment. There are horses, cattle (sheep), and pigs that are naturally infected with VSV in domestic animals. In the natural state of the population, there is almost no active infection of vesicular stomatitis virus, and the infection to humans will not cause obvious symptoms. Therefore, vesicular stomatitis Virus (VSV) as a viral vector vaccine has natural advantages compared with other vectors. Therefore, VSV as a viral vector chimerizing or fusing target antigens will enhance the body's immune response strength. VSV viral vector vaccines further adopt mucosal vaccination. In this way, it will induce the body to produce a stronger specific mucosal immune response. When a foreign pathogen invades through the mucosa, the mucosal tissue will be activated to quickly clear the pathogen. It is further known that the VSV virus vector also has characteristics that other tool vectors do not have. When the designed preventive vaccine is used to prevent enveloped viruses, the VSV virus can completely display the envelope protein of the target virus on its own envelope protein GP, and fully expose the antigen protein on the surface of the recombinant virus. After inactivated in vitro, this type of viral vector vaccine still has the specific immune response to effectively activate the body. The foreign viral envelope protein further enhances the immunogenicity of the antigen and fully activates the host immune response through specific fusion with GP. At the same time, the recombinant virus vaccine does not have the ability to replicate twice, and its safety is significantly enhanced.

The present disclosure proposes a VSV-modified viral vector mVSV, a mVSV viral vector and a mVSV-mediated new coronary pneumonia vaccine, which has a better preventive or therapeutic effect on patients infected with the new coronary pneumonia virus.

Summary of the invention

The present disclosure provides a vector mVSV, a viral vector for vaccine development, and a new coronavirus vaccine based on the mVSV viral vector that can be quickly developed through the mVSV-Vac platform that can be widely used in a variety of viruses that are raging in today's society.

The mVSV virus vector, the vesicular stomatitis virus Indiana strain Indiana VSV gene M encoded amino acid has multiple site modification mutations, the site mutation occurs at the 51st position of the VSV M protein methionine M is replaced with benzene Alanine F, phenylalanine F at position 110 were replaced by alanine A, and Isoleucine I at position 225 was replaced by leucine L, which was defined as mVSV.

A mVSV viral vector vaccine, comprising the above-mentioned mVSV viral vector, the heterologous antigen gene of the target virus is integrated in the mVSV, and the heterologous antigen gene is fused or chimeric at the N-terminus of the mVSV envelope GP gene or C-terminal, the target virus vaccine formed after fusion or chimeric antigen is defined as an attenuated vesicular stomatitis virus vaccine.

Further, when the heterologous antigen gene is chimerized at the N-terminus or C-terminus of the mVSV envelope GP gene, the DNA of the heterologous antigen gene is a codon-optimized sequence, and the antigen gene contains the encoding A full-length or partial truncation of the spike protein S gene of the mentioned virus envelope.

Further, the full length of the spike protein S gene of the target virus envelope includes SEQ ID NO: 1 or a gene sequence having an amino acid that is at least 98% identical to that of SEQ ID NO: 2, and is defined as a chimeric antigen Gene A; the partial truncation of the spike protein S gene of the target virus envelope includes the base sequence of SEQ ID NO: 3 or a gene sequence that has at least 98% identity with the amino acid encoding SEQ ID NO: 4, definition It is a chimeric combinatorial gene B.

Further, the heterologous antigen gene DNA is integrated into the envelope GP gene coding sequence or adjacent non-coding sequence in the mVSV vector gene fragment.

Further, when the heterologous antigen gene is fused at the N-terminus or C-terminus of the envelope GP gene, the 5'-end fusion of the envelope GP gene occurs after the envelope GP gene signal peptide. The 3'end fusion of the gene occurs before the stop codon of the envelope GP gene.

Further, the antigen gene of the mVSV envelope GP fusion is selected from the RBD segment of the spike protein of the new coronavirus SARS-CoV-2, and the envelope GP gene is present in any of the vaccine vector pmVSV-Core backbone vectors corresponding to the target virus. Position, wherein the heterologous antigen gene of the envelope GP fusion comprises an RBD gene or an RBD truncated gene encoding the S protein of the target virus, and the heterologous antigen gene comprises SEQ ID NO: 5 or encoding SEQ ID NO: 6 The gene sequence of amino acids with at least 98% identity is defined as the fusion antigen gene C.

A vaccine based on mVSV-mediated novel coronavirus pneumonia, wherein the above-mentioned target virus is a novel coronavirus pneumonia virus, and the mVSV virus vector is chimeric or fused with SARS-CoV-2 antigen genes, and the antigen genes are selected from SARS-CoV-2. The predominant epitope of the spike protein S of the CoV-2 pathogen, the predominant epitope includes a chimeric antigen gene A, a chimeric assembly gene B, or a fusion antigen gene C.

Further, the antigen gene includes the full length of the spike protein S gene and the corresponding dominant antigen genes in different S gene truncations, and the dominant antigen gene is selected from those corresponding to the receptor binding domain RBD encoding the spike protein S Full-length gene or corresponding truncation gene truncation.

Further, the antigen gene of SARS-CoV-2 is selected from codon-optimized synthetic genes that encode one or more of the receptor binding domain RBD of the spike protein of human neocorona pneumonia virus, wherein The receptor binding domain RBD contains one or more antigen genes of different mutant strains of new coronavirus pneumonia.

A preparation method of mVSV-mediated new coronary pneumonia vaccine is as follows. Firstly, 293T cells are cultured in a 10cm petri dish, and 10ul-100ul poxvirus (MOI=1-50) are incubated with 293T cells in serum-free DMEM medium 1 -12h, use specific transfection reagent to recombine the mVSV reverse genetic plasmid pmVSV-core-A/B/C(1-15ug), pP(0.1-5ug), pL(0.1-5ug), pN(0.1-5ug) ) And the five-plasmid system of pVSVG (0.1-5ug) according to the specific optimal transfection ratio (the optimal ratio needs to be further optimized according to specific application cases), after the plasmid gene is transfected into 293T cells, 48h-72h The cell supernatant was collected and centrifuged at a high speed of 25000 rpm, and the virus concentrated by the centrifugation was further divided into 100 ul of virus preservation solution and stored at 4°C or -80°C.

1) Inoculation and immunization can be carried out by intramuscular injection, intravenous, nasal drip or oral administration.

2) Only 1 immunization

3) Viral vector vaccine with a dose of 10 ⁶ -10 ⁸ pfu (mVSV-A, B/C)

Technical effect: Compared with the prior art, the present disclosure selects live viruses as vaccine vectors, chimeric or fuses specific target antigen genes, and utilizes the replication ability of vesicular stomatitis virus in cells to express target antigens efficiently and quickly, The properties of attracting T cells to the mucosal site where the vaccine is administered, promoting CD8 ⁺ T cell response, etc., a specific administration method (mucosal administration route) can significantly enhance the specific mucosal immune response; in addition, the modified VSV proposed in the present disclosure patent (mVSV) Compared with VSV in the prior art, mVSV virus has lower toxicity, higher antigen loading capacity, and more stable virus genome. Due to the specificity of the virus itself, the new coronary pneumonia vaccine involved in this disclosure will cause a strong innate immune response, activate the body’s immune system, and act like an adjuvant. As the virus is discovered, recognized and eliminated by the immune system, The target antigen carried by the virus will be fully discovered, and the antigen that is different from ordinary vaccines is unstable and easily degraded.

The target antigen of the viral vector of the present disclosure will be expressed in the cytoplasm in large quantities along with the replication of the virus, and will be fully presented to the DC cells to cause a specific immune response of the body. When the vaccine is administered to the mucosal site, the body will be induced Produce a locally acquired mucosal immune response. The mVSV-Vac vector system of the present disclosure can be used for mucosal delivery of preventive or therapeutic vaccines for various transmucosal infectious pathogens. The vaccine of the present disclosure can effectively induce the production of specific mucosal SIgA and systemic IgG against SARS-CoV-2.

The new coronary pneumonia vaccine mediated by the viral vector (mVSV) provided in the present disclosure can be administered via intramuscular injection, intravenous, nasal drip, oral administration and other immunization routes, which can solve the problem that traditional vaccines in the prior art cannot induce high-intensity immune responses ( In particular, the low titers of neutralizing antibodies make up for the inability of antigens carried by traditional vaccines to stay in the mucous membranes effectively, and cannot be fully presented to immune cells by APCs. The activated immune response is weak and antibody titers are low. At the same time, The mVSV new crown vaccine involved in this disclosure can be used in combination with other vector vaccines. The first shot uses mVSV new crown vaccine to immunize first, and the second shot uses the second viral vector vaccine (adenovirus vector vaccine, poxvirus vector vaccine) for the second shot. The second stimulation will further activate the acquired immune response against the new crown antigen, which greatly improves the response rate of vaccination.

In summary, the mVSV vector-mediated new coronary pneumonia vaccine involved in this disclosure has the following three characteristics:

1) Its core is a virus vaccine-mediated vector, which encodes the coronavirus spike protein spike protein S and different truncated bodies, preferably the antigen protein sequence is mainly derived from the SARS-CoV-2 strain.

2) The recombinant attenuated vesicular stomatitis virus is packaged through a specific modified plasmid (low copy) packaging system, that is, the vesicular stomatitis virus recombination subsystem.

3) By optimizing the design of immune pathways, immune procedures, immune doses, and immune positions, the immune effect of mVSV vaccines is significantly improved, and the increased systemic IgG neutralizing antibodies and the number of sIgA antibodies in the mucosa are further induced in the body. The fusion candidate vaccine mVSV-C will further activate the body's anti-viral T cell immunity while inducing a specific humoral immune response, forming a permanent memory, and producing a life-long protective effect.

It should be noted that the mVSV viral vector involved in the present disclosure can be used in the research of virus vaccines for purposes other than the currently rampant new coronavirus; in addition, it is also possible to administer immunologically effective amounts of the recombinant new coronavirus vaccine vector and the recombinant new coronavirus vaccine to patients with new coronavirus Different immune adjuvant compositions have obvious curative and preventive effects; the immune response is the induction of anti-S protein serum antibodies and induces a specific protective immune response against S protein, and the induced specific neutralizing antibody titer exceeds 1 International units/ml. The present disclosure provides the new coronary pneumonia vaccine and its adjuvant composition one or more times according to the patient's clinical performance, and even then provides the recombinant new coronary vaccine to the patient within weeks, months or years of the first providing step Or a vaccine combination combined with a target viral vector and a composition containing an adjuvant. Patients who are provided with a new coronavirus vaccine recombinant new coronavirus vaccine or its adjuvant composition include: the individual exhibits one or more symptoms of SARS-CoV-2 The individual lacks any symptoms of SARS-CoV-2, the individual has been exposed to SARS-CoV-2, the individual has been in contact with an individual suffering from SARS-CoV-2, the individual is a child, an elderly person People who are exposed to or at risk of biological weapons are members of the military or health care workers.

According to the knowledge of professionals in the field, SARS-CoV-2 and SARS virus antigen S-induced antibodies have cross-reactivity, but the homology between the two is only 78%, which further proves that the new crown antigen S gene and its truncated body are identical. When it is less than 98%, the body will also induce the body to produce specific antibodies. Therefore, the antigenic amino acid homology of the new crown vaccine developed based on the mVSV vector is not restricted by the ratio of 98%. Based on the mVSV vaccine vector chimerization or fusion specific antigen table All candidate vaccines are bound by this disclosure.

This disclosure records the following technical solutions:

(1) mVSV virus vector, wherein the mVSV virus vector includes mVSV virus, and the mVSV virus is a virus obtained by mutating the amino acid of the matrix protein M of the vesicular stomatitis virus Indiana strain, and the mutation occurs in the matrix protein. M methionine at position 51 was mutated to phenylalanine, phenylalanine at position 110 was mutated to alanine, and isoleucine at position 225 was mutated to leucine.

(2) A vaccine comprising the mVSV viral vector described in (1), wherein a heterologous antigen gene of the target virus is integrated into the gene of the mVSV viral vector.

(3) The vaccine according to (2), wherein the heterologous antigen gene is chimeric or fused in the gene of the mVSV viral vector, and the chimeric or fused position is mVSV viral vector envelope G and L Between genes.

(4) The vaccine according to (2) or (3), wherein the target virus is the new coronavirus SARS-CoV-2.

(5) The vaccine according to any one of (2) to (4), wherein the SARS-CoV-2 antigen gene of the novel coronavirus pneumonia virus is chimerized at the N-terminus or C-terminus of the GP gene of the mVSV viral vector envelope, The SARS-CoV-2 antigen gene of the new coronavirus pneumonia virus is a codon-optimized sequence, and the codon-optimized sequence includes a full-length or partial truncated body of the new coronavirus pneumonia virus SARS-CoV-2 spike protein S gene.

(6) The vaccine according to any one of (2) to (5), wherein the full length of the new coronavirus SARS-CoV-2 spike protein S gene comprises the sequence shown in SEQ ID NO:1 or Contains a gene sequence encoding an amino acid that is at least 98% identical to SEQ ID NO: 2; the partial truncation of the new coronavirus SARS-CoV-2 spike protein S gene comprises the sequence shown in SEQ ID NO: 3 or contains A gene sequence encoding an amino acid that is at least 98% identical to SEQ ID NO: 6.

(7) The vaccine according to any one of (2) to (6), wherein the SARS-CoV-2 antigen gene of the new coronavirus pneumonia is chimeric into or adjacent to the mVSV viral vector envelope GP gene coding sequence Non-coding sequence.

(8) The vaccine according to any one of (2) to (7), wherein the SARS-CoV-2 antigen gene of the new coronavirus pneumonia is fused to the N-terminus or C-terminus of the mVSV viral vector envelope GP gene, so The 5'end fusion of the envelope GP gene occurs after the signal peptide of the envelope GP gene, and the 3'end fusion of the envelope GP gene occurs before the stop codon of the envelope GP gene.

(9) The vaccine according to any one of (2) to (8), wherein the fused new coronavirus SARS-CoV-2 antigen gene comprises the RBD of the new coronavirus SARS-CoV-2 spike protein S Segment or RBD truncated corresponding gene.

(10) The vaccine according to any one of (2) to (9), wherein the fused novel coronavirus SARS-CoV-2 antigen gene comprises the sequence shown in SEQ ID NO: 5 or contains the sequence shown in SEQ ID NO: 5 or contains the sequence shown in SEQ ID NO: 5 or ID NO: 6 A gene sequence of amino acids with at least 98% identity.

(11) The vaccine according to any one of (2) to (10), wherein the RBD segment or RBD truncated body of the new coronavirus SARS-CoV-2 spike protein S is derived from the new coronavirus SARS-CoV- 2 different mutants.

(12) A method for treating or preventing COVID-19, wherein the method includes the step of administering the vaccine according to any one of (2) to (11) to the patient.

Description of the drawings:

Figure 1. Schematic diagram of pmVSV-Core-A and pmVSV-Core-B plasmid construction, virus packaging and identification;

Figure 2. Detection of serum specific antibodies in mice immunized with mVSV-A and mVSV-B virus vector vaccines;

Figure 3. The construction, virus packaging and identification of pmVSV-Core-C plasmid;

Figure 4. Detection of serum antibodies (IgG, IgA) after multi-channel immunization of mVSV-C new crown vaccine;

Figure 5. Detection of neutralizing antibody content in mouse serum after multi-channel administration of mVSV-A, mVSV-B and mVSV-C candidate new crown vaccines;

Figure 6 is a schematic diagram of VSV-spike protein S recombinant virus vaccine packaging through genetic recombination plasmid;

Figure 7 relates to the toxicity comparison between different point mutants of VSV and wild strains, including MTT toxicity detection in MEF cells (A), replication of different mutant strains (B), and safety comparison of different mutant strains in mouse models (C ).

The following specific embodiments will further illustrate the embodiments described in the present disclosure in conjunction with the above-mentioned drawings.

Detailed ways

definition

When used in conjunction with the term "comprising" in the claims and/or specification, the word "a" or "an" can mean "a", but can also mean "one or more", "at least One" and "one or more than one".

As used in the claims and specification, the words "include", "have", "include" or "contain" mean inclusive or open-ended, and do not exclude additional, unquoted elements or methods step.

Throughout the application documents, the term "about" means: a value includes the standard deviation of the error of the device or method used to determine the value.

Although the disclosed content supports the definition of the term “or” only as an alternative and “and/or”, the term “or” in the claims refers to the term “or” in the claims unless it is clearly stated that only alternatives or alternatives are mutually exclusive. "and / or".

When used in the claims or specification, the selected/optional/preferred “numerical range” includes both the numerical endpoints at both ends of the range and all natural numbers covered by the numerical endpoints relative to the aforementioned numerical endpoints.

The experimental techniques and experimental methods used in this example are conventional techniques and methods unless otherwise specified. For example, the experimental methods for which specific conditions are not indicated in the following examples, usually follow conventional conditions such as Sambrook et al., Molecular Cloning: Experiment The conditions described in the room manual (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through formal commercial channels.

The following is a further detailed description of the present disclosure with reference to specific examples. The description is an explanation and not a limitation of the present disclosure. The present disclosure mainly constructs the SARS-CoV-2 virus protective epitope onto the VSV virus backbone vector (pCore). , Through synthetic biology technology, designed a reverse genetic operation rapid rescue system for the attenuated rhabdovirus VSV, obtained a viral vector vaccine that can stably express the candidate antigen, and obtained serum after immunization in mice, and passed the biological Scientific experiments identify the acquired immune response and the production of neutralizing antibodies against the virus.

The reagents and consumables used in this disclosure are as follows: Q5 Hot start High-Fidelity DNA polymerase (NEB M0493L), Nhe I-HF (NEB R3131L), Xho I (NEB R0146S), T4 DNA Ligase Enzyme (NEB M0202L), E. coli DB3.1 Competent Cells (Takara 9057), TIANGEN endotoxin-free small extraction medium kit (Tiangen DP118-02), Lipofectamine LTX (Invitrogen 15338100), PBS (Hyclone SH30256.01), DMEM high glucose medium (Gibco C11995500), double Anti-(Gibco 15140-122), Fetal Bovine Serum (Gibco 10091-148), Opti-MEM I Reduced Serum Medium (Gibco 31985-070), 96-well cell culture plate (Corning 3599), 6-well cell culture plate (Corning 3516) ), 6cm cell culture plate (Corning 430166), 0.22um filter (Millipore SL GP033rb), T175 cell flask (Corning 431080).

Cell line:

The 293T and 293T-hACE2 adherent cells were cultured in a special culture environment (Thermo BB150 cell culture incubator) containing 5% CO2 at 37°C, using DMEM high-sugar complete medium for culture.

Attenuated mVSV virus vector:

In a technical solution, a three-site mutant of the matrix protein (M) of the recombinant vesicular stomatitis virus Indiana strain (gene sequence NC_001560.1, version on August 13, 2018) selected by the attenuated mVSV virus vector The mutated amino acid positions preferably have amino acid substitutions from the 51st, 110th and 225th positions at the same time. The amino acid substitution mode is: the 51st methionine M is replaced by phenylalanine F, and the 110th phenylalanine Acid F was replaced with Alanine A, and Isoleucine I at position 225 was replaced with Leucine L.

Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although representing specific embodiments of the present disclosure) are given for explanatory purposes only, because after reading the detailed description, they are made within the spirit and scope of the present disclosure. Various changes and modifications will become obvious to those skilled in the art.

Example 1 The toxicity of the M protein three-site mutation against the Indiana strain of VSV is greatly reduced

It is known that the most important pathogenic gene of the VSV wild strain is M. At the same time, the M protein can induce the apoptosis of host cells. It is the main factor for the VSV wild strain to infect cloven-hoofed animals. The best way for wild VSV strains is to carry out genetic engineering mutations in the M gene. Existing studies have shown that non-synonymous mutations at the 51st amino acid of the M protein will reduce the neurotoxicity of wild VSV. Therefore, the technique of the present disclosure is implemented Firstly, a single point mutation comparison was performed. At position 51, methionine was mutated to phenylalanine, alanine, leucine and arginine (control). As shown in Figure 7A, some of the results are It is found that mVSV-M51F has weaker cytotoxicity in normal fibroblasts compared with mVSV-M51R, but M51F still has certain cytotoxicity. Based on this, we continue to synthesize biological mutations based on M51F and get 2 mutations. M51F-F110 (A/R/L), found that the toxicity of M51F-F110A has been further reduced in the 2 mutant strains, but in the follow-up (Figure 7C), the VSV wild strain sensitive Balb/c mice were dropped Nasal administration (simulation of nervous system infection), it was found that high-dose E9pfu administration of M51F-F110A still caused some mice (1/4 ratio) to lose weight, although only a transient phenomenon occurred within 5 days after vaccination. However, it still shows that the attenuation modification for the M gene has not reached the best way. Further, based on the positions of the above 2 mutations, the present disclosure has found a third mutation site that can significantly reduce the toxicity of the M protein, that is, the 225th amino acid. The amino acid at position L was mutated from Leucine I to Leucine L, and the attenuation effect was the most significant (part of the comparison results are not shown in Figure 7). Further, as shown in Figure 7B, at the cellular level, it passes through the three positions of the M protein. The point mutant mVSV (M51F-F110A-I225L) did not detect any damage to normal MEF fibroblasts (multiplicity of infection rose to 10, and no significant cytopathic changes were observed). Similarly, different mutant strains were used in animal models The challenge experiment is shown in Figure 7C. In 8-week-old mice Balb/c, the E9pfu mutant strain virus was instilled into the nose for preliminary safety evaluation (intravenous administration was also verified by mouse model administration, the same dose, each There were 6 mice in the group. Except for the control group, the wild strain of VSV produced significant adverse reactions. The mice with other mutant strains had milder symptoms). The statistical results showed that the virus with and only 3 mutations did not cause the weight of the mice. However, other mutant strains showed a significant decrease in body weight and then recovered. In addition, after the early administration, the VSV wild strain showed a toxic reaction of abnormally increasing body temperature. At the same time, the VSV wild strain and mVSV-M51R in the control group showed small symptoms. In the phenomenon of nerve paralysis in the hind legs of mice, only the three mutant strains administered to mice did not produce any adverse reactions, which proves that the attenuated strain is highly safe to normal mice, has no toxic side effects, and does not have potential neurotoxicity.

Example 2 Construction and identification of two chimeric vaccines based on VSV virus vectors

The S gene sequence of SARS-CoV-2 released by NCBI is codon-optimized to facilitate its expression in the cell (named antigen gene A), and multiple potential epitopes predicted based on the SARS-CoV-2 sequence are combined into The new antigen gene (named as antigen gene B), the sequence of antigen gene A and antigen gene B are handed over to Nanjing GenScript Biotech to synthesize into pCDNA3.1 and pUC57 vectors respectively, after PCR amplification of the target gene, the fragment purification reagent Recover and purify the target band from the cassette, use restriction endonucleases, MCS1 (Xhol) and MCS2 (Nhel) to digest the fragment and pmVSV-GFP vector at 37°C for 3 hours, and then perform the ligation reaction after the vector and the target fragment are recovered by the gel. Then transfer to competent cells, screen positive clones by PCR and verify the plasmid construction by restriction enzyme digestion and sequencing (and name the constructed two plasmids pmVSV-Core-A and pmVSV-Core-B respectively), and the specific implementation steps as follows:

1) Construct two chimeric vaccines based on VSV virus vector according to (Figure 1A);

2) Primer synthesis and primer information: The primers were synthesized by Suzhou Jinweizhi Bio-Biotech Co., Ltd. The PCR primers and bacterial liquid PCR primers selected for the amplification of A gene were constructed as shown in Table 1:

Table 1 A gene amplification and detection primers

3) The PCR primers selected for the amplification of B gene and the PCR primers of bacterial liquid are shown in Table 2:

Table 2 B gene amplification

Obtain the target gene: use the pCDNA3.1 plasmid with the target gene sequence as a template to PCR amplify the A gene with the primers in Table 1; use the pUC57 plasmid with the target gene sequence as the template to PCR amplify the B gene with the primers in Table 2;

Refer to the AxyPrepTM PCR Cleanup kit instructions to purify the above digested products, and use Nano-300 to determine the product concentration;

Double-enzyme digestion of the above purified product and vector (37°C digestion for 3h);

Use 1% Agarose gel for electrophoresis, use the corresponding DNA maker as a control to verify whether the PCR product is correct, cut the gel band, recover the remaining PCR product, and use Nano-300 to determine the product concentration;

Connect the above-mentioned purified product to the carrier (at 16°C overnight, the connection ratio is 1:5);

Refer to the E. coli DB3.1 Competent Cells (TaKaRa) instruction to transform the ligation product;

Pick the single clones on the LB (Kana) plate into a sterile 1.5 mL tube pre-added with 200 μL of LB (Kana) medium, culture at 37°C, 250 rpm, and culture for 3 hours, then perform Colony PCR to screen positive clones;

After identification by agarose gel electrophoresis, select positive clones and transfer them to 15 mL shake flasks at a ratio of 1:500, and incubate them on a shaker at 37°C and 250 rpm for 14 to 16 hours;

Perform plasmid extraction according to the instructions of TIANGEN Endotoxin-Free Small Extract Medium Amount Kit;

Perform double digestion of the selected positive plasmids (Xho I and Nhe I are digested at 37°C for 3 hours);

After identification by restriction enzyme digestion, select the correct plasmid for plasmid sequencing;

The VSV virus was packaged according to the standard method for the correctly sequenced plasmid, and the pmVSV-GFP plasmid was taken as a positive packaging control; the virus supernatant was collected once at 48h and 72h, and 300uL was used to infect the 293T cells pre-plated on the 6-well plate. , The packaged viruses were named mVSV-GFP, mVSV-A, mVSV-B; the cells were collected after cytopathic disease to detect the antigen expression level by WB.

The results of the above plasmid construction and WB detection results are shown in Figure 1:

According to the experimental results, after PCR amplification of antigen gene A and antigen gene B, specific bands appeared at the corresponding positions and the molecular size of the bands was correct, indicating that the target band was successfully amplified (Figure 1B); virus packaging After infection, the mVSV-GFP positive control fluorescence expression intensity is better, and the mVSV-A and mVSV-B virus infected cells also have lesions (Figure 1C); Western Blot also detected the expression of A and B genes in the corresponding position (Figure 1D) ).

Example 3 Immune response effects of two chimeric vaccines based on VSV virus vectors under different immunization schemes

Detection of specific IgA and IgG antibody levels in mice after different immunization protocols by indirect ELISA: After coating the ELISA plate with the recombinant RBD protein of SARS-CoV-2S virus, it will be immunized by three immunization methods: muscle, intravenous, and nasal drip The mouse serum at 21 days after one time was diluted 1:200 and added to the corresponding well. After incubating for 2 hours, the secondary antibodies of different types (IgA, IgG) were diluted 1:10000 to detect the level of specific antibodies (Figure 2). The operation steps are as follows:

1) Take the coating antigen (S-RBD) and dilute it with coating buffer to a final concentration of 5μg/ml, take the ELISA plate, add samples to the wells (100μl/well) in turn, and then place it at 4 degrees Celsius for coating overnight;

2) Pour out the coating solution in the sample well the next day, wash with the washing buffer solution 3 times, dry the residual liquid in the sample well on the filter paper after each wash;

3) Then add 200 μl of 5% BSA blocking solution to each well for sealing, and place it at 37 degrees Celsius for 1 hour (the plate is placed in a sealed bag). Pour off the blocking solution and wash the sample well with washing buffer once;

4) Dilute the test serum and the negative serum in an appropriate ratio (1:100) with antibody serum diluent (1% BSA), add 100ul per well, and incubate at 37 degrees Celsius for 2 hours;

5) Pour out the reaction solution in the sample well, wash with the washing solution for 1 to 3 minutes, wash the plate 5 times, and dry the remaining liquid on the filter paper after each wash;

6) Dilute the enzyme-labeled secondary antibody (Goat anti-mouse IgG HRP) at 1:10000 with the diluent, add 100 μl to each well, and react at 37°C for 1 hour;

7) Pour out the unbound enzyme-labeled antibody, add washing solution to wash, 1 to 3 minutes each time, a total of 5 times, after each wash, dry the remaining liquid on the filter paper;

8) Add 100μl of freshly prepared color-developing liquid (a liquid and liquid B are mixed in equal proportions to form a color-developing liquid) to each well, put it at room temperature, and react for 20 minutes in the dark;

9) Add 100μl of ELISA stop solution to each well to stop the reaction;

10) Put the 96-well plate into the microplate reader and read the OD450nm. Compare the OD450nm value of the test sample and the negative sample under the same dilution ratio, and determine the positive situation with 2.1 times the OD value of the negative sample as the positive test standard, namely: OD (positive>2.1*OD (negative sample);

The results showed that when mVSV-A virus, mVSV-B virus, and mVSV-GFP virus were immunized with different immunization methods for 21 days, the level of specific IgA and IgG antibodies in the serum rose to a significant level, and the expression levels of each antibody under different immunization pathways were quite different. Among them, the nasal drip method mainly activates IgA mucosal immunity (Figure 2A), and veins and muscles mainly cause IgG type immune responses (Figure 2B).

Example 4 Construction and identification of fusion vaccine based on VSV virus vector

The C fragment was fused to the C-terminus of the VSV-G envelope gene on the pVSV-GFP vector by overlap extension PCR (product number is GP-C), or fused to the N-terminus of the VSV-G envelope gene (product number is ( C-GP).

After the first round of PCR amplifies the two target genes, the target bands are recovered with the gel recovery kit. The second round of PCR takes the two target fragments of the first round of PCR as templates, and uses the upstream primers and fragments of fragment 1 respectively. The downstream primer of 2 carries out the amplification of the fusion fragment, the fragment and pVSV-GFP vector are digested with restriction endonucleases (MCS1(MluI) and MCS2(XhoI)) at 37℃ for 3h, and the vector and the target fragment are recovered by the gel After that, the ligation reaction is carried out, and then transferred to competent cells, the positive clones are screened by PCR, and the plasmid construction is verified by restriction enzyme digestion and sequencing. The specific implementation steps are as follows:

1) According to (Figure 4A), construct the envelope fusion type VSV virus vector vaccine;

2) Primer synthesis and primer information: The primers were synthesized by Suzhou Jinweizhi Bio-Biotechnology Co., Ltd., and the specific primer information is shown in the following table:

Table 3 Two forms of fusion C gene amplification

3) Obtain the target gene: use pVSV-GFP (amplified VSV-G) with the target gene sequence and pCDNA3.1 plasmid containing the target gene as templates to amplify the target fragment;

4) Use 1% Agarose gel for electrophoresis, use the corresponding DNA maker as a control to verify whether the PCR product is correct, cut the gel band, recover the remaining PCR product, and use Nano-300 to determine the product concentration;

5) Take a certain amount of the products recovered from the PCR gel according to the cloning sequence in the primer table and perform the second round of PCR to amplify the final fusion envelope gene fragment;

6) Use 1% Agarose gel for electrophoresis, use the corresponding DNA maker as a control to verify whether the PCR product is correct, cut the gel band, recover the remaining PCR product, use Nano-300 to determine the product concentration, purified product and vector Perform double digestion (Mlul and Xhol, digestion at 37°C for 3 hours);

7) After the digestion is completed, refer to the AxyPrepTM PCR Cleanup kit instructions to purify the above digestion products, and use Nano-300 to determine the product concentration;

8) Connect the above-mentioned purified product to the carrier (at 16°C overnight, the connection ratio is 1:5);

9) Refer to the E.coli DB3.1 Competent Cells (TaKaRa) manual to transform the ligation product;

10) Pick a single clone on the LB (Kana) plate into a sterile 1.5 mL tube pre-added with 200 μL of LB (Kana) medium, culture at 37°C, 250 rpm, and 3 hours, then perform Colony PCR to screen positive clones;

11) After identification by agarose gel electrophoresis, select positive clones and transfer them to 15 mL shake flasks at a ratio of 1:1000, and culture them on a shaker at 37°C and 250 rpm for 14-16 hours;

12) Perform plasmid extraction according to the instructions of the TIANGEN endotoxin-free small extraction medium-volume kit;

13) Perform double restriction digestion of the selected positive plasmids (MluI and XhoI digestion at 37°C for 3h).

14) After restriction enzyme digestion and identification, select the correct plasmid for plasmid sequencing;

15) Pack the plasmids with correct sequencing according to standard methods for VSV virus packaging, and at the same time take pVSV-GFP plasmid as a positive packaging control;

16) Collect the virus supernatant once at 48h and 72h each, and take 300uL to infect the 293T cells pre-plated on the 6-well plate and package them. The viruses are named mVSV-(GP-C) and mVSV-(C-GP) respectively;

17) After the cytopathy, the cells are collected for WB to detect the antigen expression level.

The results of the above plasmid construction and WB detection results are shown in Figure 3:

According to the experimental results, after one round of PCR amplification, specific bands appeared at the corresponding positions and the molecular sizes of the bands were correct. After the second round of fusion PCR, the two fragments were successfully fused together (Figure 3B); virus packaging infection After the mVSV-GFP positive control, the fluorescence expression intensity was better. The mVSV-(GP-C) gene and mVSV-(C-GP) virus also showed lesions after infecting the cells (Figure 3C); Western blot also detected VSVG in the corresponding position Expression (Figure 3D).

Example 5 Immune response effects of fusion vaccine based on VSV virus vector under different immunization schemes

Detection of specific IgA and IgG antibody levels in mice after different immunization protocols by indirect ELISA: After coating the ELISA plate with the recombinant RBD protein of SARS-CoV-2S virus, it will be immunized by three immunization methods: muscle, intravenous, and nasal drip Once, the mouse serum at 21d was diluted 1:200 and added to the corresponding well. After incubating for 2 hours, the secondary antibodies of different types (IgA, IgG) were diluted 1:10000 to detect the level of specific antibodies (Figure 4). The operation steps are as follows:

1) Take the coating antigen (S-RBD) and dilute it with coating buffer to a final concentration of 5μg/ml, take the microtiter plate, add samples to the wells (100μl/well) in turn, and then place it at 4 degrees Celsius for coating overnight;

9) Add 100μl of ELISA stop solution to each well to stop the reaction;

The results showed that when immunized with mVSV-(GP-C) virus, mVSV-(GP-C) virus, and mVSV-GFP with different immunization methods for 21 days, the level of specific IgA and IgG antibodies in the serum rose to a significant level, and under different immunization routes The expression levels of various antibodies are quite different. Among them, the nasal drip method mainly activates the IgA mucosal immunity (Figure 4B), and the vein and muscle mainly cause the IgG type immune response (Figure 4B).

Example 6 Immune serum neutralizing antibody detection based on pseudovirus system

Determine the neutralizing antibody titers produced by in vitro virus neutralization experiments to evaluate the difference in antigen selection, screen out highly effective protective immunogens, and compare the neutralizing antibody titers specific to SARS-CoV-2 produced by different groups To determine the optimal vaccine preparation strategy and vaccination method, the specific operation steps are as follows:

1) Extinguish the serum to be tested at 56°C for 30 minutes, centrifuge at 6000g for 3 minutes, and take the supernatant for use;

2) Passage 293T-hACE2 cells, count the cells and add 2E4cells/well to a 96-well plate (200μL/well, including 8μg/mL polybrene);

3) After 3 hours, the antibody was serially diluted (1:2) 10μL/tube with Opti-MEM, and a positive control without antibody (20μL virus solution, final virus concentration 4E5TU/mL) and a negative control without virus (20μL) Opti-MEM);

4) The pseudovirus is also serially diluted to 8E5TU/mL;

5) Take 10μL of the diluted virus solution (8E5TU/mL) and add it to the 10μL serially diluted antibody in step 2 (1:1 pipetting and mixing) (at this time, the final virus concentration is 4E5pfu/mL);

6) After incubating in a 37°C, 5% CO2 incubator for 1 hour, add it to 293T-hACE2 cells for 24h-48h and observe the fluorescence and pathological changes.

7) The serum neutralization titer is determined according to the dilution factor of the antibody serum corresponding to the hole where the green fluorescence appears last.

The results show that as shown in Figure 5, the mVSV-A, mVSV-B and mVSV-C new crown vaccines were given different administration methods. When immunized for 21 days, the serum neutralizing antibody level increased significantly compared with the control group, and different immunization routes There are some differences in the expression levels of neutralizing antibodies. Among them, the level of neutralizing antibodies induced by intravenous immunized mouse serum is the highest. There is no significant difference between intramuscular injection and intranasal administration. Among the three different viral vector vaccines, mVSV-C In the intravenous administration group, the most neutralizing antibodies were induced, which indirectly indicates that the specific and preferred neocorona antigen RBD will be fused with VSVG (high immunogenicity) (N-terminal fusion) to produce neutralizing antibodies against neocorona, indirectly supporting evidence The fusion of foreign dominant antigens to the N-terminus of mVSV vector gene GP can enhance the body's recognition of antigens by enhancing the display of dominant antigens in the recombinant virus envelope, and induce the production of neutralizing antibodies and antiviral immune memory.

As further shown in Figure 6, mVSV-mediated SARS-CoV-2 vaccine series products include mVSV-A/B and mVSV-C. Two different design strategies are disclosed in this disclosure. The antigen gene of the new coronavirus is integrated into the non-enveloped core region of the virus, preferably the chimeric position is in the coding region of the envelope GP and polymerase L. This viral vector candidate must be a live strain at the time of vaccination due to the antigen gene It needs to be transcribed and translated into protein by infected host cells in the body before it can be presented by the immune cell DC. The antigen gene can express the foreign antigen in the body with the replication of the virus, and activate the body to produce specific immunity against the new crown. As shown in the figure, the design strategy of another mVSV-C candidate vaccine is completely different. The technical solution is to integrate the truncated antigen gene of the new coronavirus into the envelope GP gene of mVSV, preferably integrated into the N of the GP gene. From the results of Examples 3 and 4, it can be found that the RBD protein integrated at the N-terminus is expressed in fusion with the GP protein. At the protein level, it is proved that the candidate antigen protein of the mVSV-C-GP vaccine is displayed on the surface of the envelope. Type vaccines can be inactivated (irradiation or high temperature) and then inoculated. Compared with live attenuated virus vector vaccines, the safety is better, although in the examples, it is not further elaborated whether such candidate vaccines will be inactivated after inactivated vaccination. The live operation reduces the specific immune response induced by the antigen. However, the reduction of immunogenicity can improve the response efficiency by increasing the vaccination dose during clinical vaccination, and make up for the disadvantage of weak immunogenicity caused by inactivation.

The above-mentioned embodiments only express several implementation manners of the present disclosure, and their descriptions are more specific and detailed, but they should not be interpreted as limiting the scope of the present disclosure. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present disclosure, several modifications and improvements can be made, and these all fall within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to the appended claims.

Claims

mVSV virus vector, wherein the mVSV virus vector includes mVSV virus, and the mVSV virus is a virus obtained by mutating the amino acid of matrix protein M of the Indiana strain of vesicular stomatitis virus, and the mutation occurs in the 51st matrix protein M The methionine position was mutated to phenylalanine, the 110th phenylalanine was mutated to alanine, and the 225th isoleucine was mutated to leucine.
A vaccine comprising the mVSV viral vector of claim 1, wherein the heterologous antigen gene of the target virus is integrated into the gene of the mVSV viral vector.
The vaccine of claim 2, wherein the heterologous antigen gene is chimeric or fused in the gene of the mVSV viral vector, and the position of the chimerization or fusion is between the G and L genes of the mVSV viral vector envelope .
The vaccine of claim 2 or 3, wherein the target virus is SARS-CoV-2, a new coronavirus pneumonia virus.
The vaccine according to claim 4, wherein the SARS-CoV-2 antigen gene of the new coronavirus pneumonia is chimeric at the N-terminal or C-terminal of the GP gene of the mVSV viral vector envelope, and the new coronavirus SARS-CoV-2 The antigen gene is a codon-optimized sequence, and the codon-optimized sequence includes a full-length or partial truncated body of the new coronary pneumonia virus SARS-CoV-2 spike protein S gene.
The vaccine of claim 5, wherein the full length of the new coronavirus SARS-CoV-2 spike protein S gene comprises the sequence shown in SEQ ID NO: 1 or contains a sequence that is at least 98% to that of SEQ ID NO: 2. % Identical amino acid gene sequence; the partial truncation of the new coronavirus SARS-CoV-2 spike protein S gene includes the sequence shown in SEQ ID NO: 3 or contains the code and SEQ ID NO: 6 at least 98% Gene sequence of identical amino acids.
The vaccine of claim 4, wherein the SARS-CoV-2 antigen gene of the novel coronavirus pneumonia virus is chimeric into the coding sequence of the mVSV viral vector envelope GP gene or adjacent non-coding sequences.
The vaccine of claim 4, wherein the SARS-CoV-2 antigen gene of the new coronavirus pneumonia is fused to the N-terminus or C-terminus of the envelope GP gene of the mVSV virus vector, and the 5'-end fusion of the envelope GP gene occurs After the envelope GP gene signal peptide, the 3'-end fusion of the envelope GP gene occurs before the stop codon of the envelope GP gene.
The vaccine of claim 4, wherein the fused novel coronavirus SARS-CoV-2 antigen gene comprises the RBD segment of the new coronavirus SARS-CoV-2 spike protein S or the gene corresponding to the RBD truncated body.
The vaccine according to claim 9, wherein the fused novel coronavirus SARS-CoV-2 antigen gene comprises a sequence shown in SEQ ID NO: 5 or a sequence that is at least 98% identical to SEQ ID NO: 6 Gene sequence of amino acids.
The vaccine according to claim 9, wherein the RBD segment or RBD truncated body of the new coronavirus SARS-CoV-2 spike protein S is derived from different mutant strains of the new coronavirus SARS-CoV-2.