Novel vaccine based on adenovirus vector and application thereof

Abstract

The invention provides a novel vaccine based on an adenovirus vector and application thereof. According to the present invention, the suitable monkey pox virus gene is selected for the first time, and is efficiently integrated with the chimpanzee virus vector (AdC68) to prepare the ideal virus vaccine, and the ideal virus vaccine is suitable for nasal cavity immunization and muscle immunization, and has good nasal cavity immunization effect. The novel monkey pox vaccine can be effectively used for prevention and control of monkey pox virus and vaccinia virus infection, and the two types of viruses are considered.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering and immunology, and more particularly, the present application relates to a novel vaccine based on an adenovirus vector and its use. BACKGROUND

[0002] Monkeypox (MPX) is a disease caused by Monkeypox Virus (MPV, MPXV, hMPXV), with symptoms similar to smallpox. In 1958, the Monkeypox Virus was first isolated in monkeys, so it was named Monkeypox.

[0003] Monkeypox virus belongs to the Orthopoxvirus family and the Orthopoxvirus genus. The Orthopoxvirus family includes the Orthopoxvirus genus, the Vaccinia virus genus, the Parapoxvirus genus, the Avipoxvirus genus, the Capripoxvirus genus, the Leporipoxvirus genus, and the Suipoxvirus genus. The Orthopoxvirus family is the largest class of DNA viruses with complex structures, with brick-shaped or oval-shaped virions. It has a core, a lateral body, and an envelope. The core contains virus DNA combined with proteins. The DNA is linear and double-stranded, with low guanine and cytosine base content. There are more than 30 structural proteins and several enzymes in the virion, and the core protein contains a DNA-dependent RNA polymerase. The virus proliferates in the cytoplasm, forming inclusion bodies, and the virions are released by microvilli or cell lysis. The Orthopoxvirus family includes two subfamilies of vertebrate poxviruses and insect poxviruses. Monkeypox virus, along with smallpox virus and cowpox virus, belongs to the Orthopoxvirus family and is a double-stranded DNA virus. Monkeypox virus is transmitted through close contact and droplets, with a latent period of 7-21 days. The symptoms of monkeypox infection are similar to those of smallpox, and can include fever, headache, muscle aches, fatigue, and so on. The difference is that monkeypox infection can cause lymph node enlargement, which is not a symptom of smallpox. The mortality rate and infectivity of monkeypox are lower than those of smallpox virus. Monkeypox virus is divided into two branches of the phylogenetic spectrum, the West African strain and the Congo Basin strain, with a mortality rate of about 3.6% for the West African strain and about 10.6% for the Congo Basin strain. Similar to smallpox, the mortality rate for children is higher.

[0004] Following the first outbreak of the monkeypox virus in 2022, the virus has a high infectivity and strong pathogenicity, and is showing a global pandemic trend. Many countries have reported indigenous monkeypox virus infections. The World Health Organization has declared the monkeypox outbreak an international public health emergency. Research has found that the smallpox vaccine has some protective effect against monkeypox virus infection, but since 1980, when the World Health Organization declared the eradication of smallpox virus, newborns have not been vaccinated against smallpox, resulting in a general lack of immunity to smallpox and monkeypox virus among people born after 1980. This monkeypox outbreak spreads quickly and poses a significant health threat to young adults, teenagers, and children. Therefore, the prevention and control of the monkeypox outbreak is a top priority.

[0005] There is currently no effective drug for monkeypox, so there is an urgent need to develop a safe and effective new monkeypox vaccine to protect people's health and safety and maintain social stability.

[0006] Furthermore, in this field, there are relatively many vaccines suitable for intramuscular immunization, but very few suitable for nasal immunization with ideal efficacy. Summary of the Invention

[0007] The purpose of this invention is to provide a novel vaccine based on an adenovirus vector and its application.

[0008] In a first aspect of the present invention, a method for preparing a viral vaccine is provided, comprising: (1) providing a recombinant viral vector, the recombinant viral vector comprising: a backbone vector and an expression cassette; the backbone vector being a chimpanzee adenovirus vector; the expression cassette comprising operatively linked coding sequences for the following monkeypox virus proteins: H3L, E8L and B6R;

[0009] (2) The recombinant viral vector of (1) is transfected into viral production cells, thereby packaging to obtain a recombinant virus carrying genes from monkeypox virus and ORF6 / 7 genes from the human AdHu5 E4 region, wherein ORF6 / 7 significantly increases the yield of recombinant adenovirus vector vaccine.

[0010] (3) A viral vaccine was prepared using the recombinant virus from (2), which has immune protection against monkeypox virus.

[0011] In one or more embodiments, the chimpanzee adenovirus vector is AdC68XY3.

[0012] In one or more embodiments, the expression cassette is inserted into the chimpanzee adenovirus E1 region (original E1 region location) of the backbone vector.

[0013] In one or more embodiments, for AdC68, the E1 region in the AdC68XY3 carrier is completely deleted.

[0014] In one or more embodiments, for AdC68, the E3 region 27615-31335bp (corresponding to AC_000011.1) in the AdC68XY3 carrier is deleted.

[0015] In one or more embodiments, for AdC68, the deletion region of the E4 sequence in the AdC68XY3 vector is 33518bp-34671bp (corresponding to AC_000011.1); the insertion region of the E4 sequence in AdHu5 is 32914bp-34077bp (corresponding to AC_000008.1). The ORF1-4 sequences of AdC68 are retained.

[0016] In one or more embodiments, the expression cassette further includes the coding sequences for the following proteins: A29L, A35R, and M1R.

[0017] In one or more embodiments, the expression box includes, in the order of 5'→3', the following:

[0018] A29L, H3L, E8L and B6R (MPVX-4);

[0019] A29L, H3L, E8L, B6R and M1R (MPVX-6);

[0020] H3L, E8L and B6R (MPVX-1);

[0021] H3L, E8L, B6R and A35R (MPVX-2);

[0022] H3L, E8L, B6R, and M1R (MPVX-3); or

[0023] A29L, H3L, E8L, B6R and A35R (MPVX-4).

[0024] In one or more embodiments, the expression cassette further includes: a promoter located upstream of the coding sequence of the protein, and / or a terminator located downstream of the coding sequence of the protein.

[0025] In one or more embodiments, the promoter is a compositional promoter, preferably a CMV.

[0026] In one or more embodiments, the terminator is BGH.

[0027] In one or more embodiments, the amino acid sequence of the H3L is as shown in SEQ ID NO:1.

[0028] In one or more embodiments, the amino acid sequence of the E8L is shown in SEQ ID NO:2.

[0029] In one or more embodiments, the amino acid sequence of the B6R is shown in SEQ ID NO:3.

[0030] In one or more embodiments, the amino acid sequence of A29L is shown in SEQ ID NO:4.

[0031] In one or more embodiments, the amino acid sequence of the A35R is shown in SEQ ID NO:5.

[0032] In one or more embodiments, the amino acid sequence of the M1R is shown in SEQ ID NO:6.

[0033] In one or more embodiments, the coding sequences of the various proteins are linked by 2A peptide coding sequences; preferably, the 2A peptides include P2A peptides.

[0034] In another aspect of the present invention, a recombinant viral vector is provided, the recombinant viral vector comprising: a backbone vector and an expression cassette; the backbone vector is a chimpanzee adenovirus vector AdC68XY3; the expression cassette comprises operatively linked coding sequences for the following monkeypox virus proteins: H3L, E8L, and B6R.

[0035] In one or more embodiments, the chimpanzee adenovirus vector is AdC68XY3; preferably, the expression cassette is inserted into the chimpanzee adenovirus E1 region (original E1 region location) of the backbone vector.

[0036] In one or more embodiments, the expression cassette further includes the coding sequences for the following proteins: A29L, A35R, and M1R.

[0037] In another aspect of the invention, the use of any of the recombinant viral vectors described above is provided for assembling a recombinant virus (vaccine), the recombinant virus carrying genes from monkeypox virus and ORF6 / 7 genes from the human AdHu5 E4 region, wherein ORF6 / 7 significantly improves the yield of the recombinant adenovirus vector vaccine; and the recombinant virus is capable of inducing the body to produce immune protection against monkeypox virus and / or vaccinia virus.

[0038] In another aspect of the invention, a recombinant virus (vaccine) is provided, said recombinant virus (vaccine) being prepared by any of the methods described above.

[0039] In another aspect of the invention, the recombinant virus is provided for use in the preparation of vaccine compositions for the prevention and treatment of monkeypox virus and / or cowpox virus.

[0040] In another aspect of the invention, a recombinant viral vaccine composition is provided, the vaccine composition comprising: the recombinant virus (vaccine); and a pharmaceutically acceptable carrier or excipient.

[0041] In another aspect of the invention, a medicine box is provided, the medicine box containing the recombinant virus (vaccine) or the recombinant virus vaccine composition.

[0042] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0043] Figure 1The design of recombinant adenovirus vector monkeypox vaccines involved codon optimization of different monkeypox protein genes, followed by tandem synthesis using P2A or F2A (A), and cloning into the E1 region of a chimpanzee adenovirus vector. Six recombinant adenovirus vector monkeypox vaccines were rescued by transfection into HEK293 cells. At the cellular level, the mRNA transcription levels of various proteins in the recombinant adenovirus vector monkeypox vaccines were detected using different concentrations of the recombinant adenovirus vector monkeypox vaccine (10... 6 vp, 10 7 vp, 10 8 HEK293 cells (500,000 cells) were infected in 12-well plates using vp. Total RNA was extracted from the cells 24 hours after infection using an RNA extraction kit, and the transcriptional level (BG) of the corresponding protein-coding genes was detected by real-time PCR.

[0044] Figure 2 Six recombinant adenovirus vector monkeypox vaccines and a control vaccine were compared using a 5E10 antibody-binding reaction induced by nasal immunization. 10 Mice (C57BL / 6, female, 6-8 weeks old, 5 mice per group) were immunized intranasally at the VP dose. A booster immunization using the same strategy was administered four weeks after the initial immunization. Mice serum was collected every two weeks (+2 and +4 represent 2 or 4 weeks after the booster immunization). The immune responses induced by each protein in the six recombinant adenovirus vector monkeypox vaccines were detected using ELISA. VVT is an inactivated vaccinia virus; ***p<0.001, **p<0.01, *p<0.05.

[0045] Figure 3 The recombinant adenovirus vector monkeypox vaccine induces a binding antibody response via the muscle immune pathway. Six recombinant adenovirus vector monkeypox vaccines and a control vaccine were compared using a 5E10 assay. 10 Mice (C57BL / 6, female, 6-8 weeks old, 5 mice per group) were immunized intramuscularly at the VP dose. A booster immunization using the same strategy was administered four weeks after the initial immunization. Mice serum was collected every two weeks (+2 and +4 represent 2 or 4 weeks after the booster immunization). The immune responses induced by each protein in the six recombinant adenovirus vector monkeypox vaccines were detected using ELISA. VVT is inactivated vaccinia virus; ***p<0.001, **p<0.01, *p<0.05.

[0046] Figure 4 Neutralizing antibody responses induced by recombinant adenovirus vector monkeypox vaccines in mouse models. Five recombinant adenovirus vector monkeypox vaccines and a control vaccine were compared using a 5E10 assay. 10Mice (C57BL / 6, female, 6-8 weeks old, n=5 per group) were immunized via nasal and intramuscular routes at the VP dose, and mouse serum was collected every two weeks. Neutralizing antibody responses following immunization with the five recombinant adenovirus vector monkeypox vaccines were detected using the plaque reduction assay. ***p<0.001,**p<0.01,*p<0.05.

[0047] Figure 5 T lymphocyte responses induced by recombinant adenovirus vector monkeypox vaccine in a mouse model. Four weeks after intranasal immunization of mice with six different recombinant adenovirus vector monkeypox vaccines, T lymphocytes were isolated from the lungs and spleen of the mice for analysis. Flow cytometry was used to analyze the activation of CD4+ T and CD8+ T lymphocytes in the T cells. ***p<0.001,**p<0.01,*p<0.05.

[0048] Figure 6 The immunoprotective effect of a single dose of recombinant monkeypox vaccine on mice was compared between various recombinant adenovirus vector monkeypox vaccines and control viruses at a ratio of 5E10. 10 Mice were immunized with vaccinia virus at VP doses via nasal and intramuscular injections, respectively. Six weeks after a single immunization, mice were challenged with a lethal dose of vaccinia virus, and changes in body weight and survival rate were recorded.

[0049] Figure 7 The immunoprotective effect of AE and double-dose recombinant monkeypox vaccine on mice was compared with that of various recombinant adenovirus vector monkeypox vaccines and control virus at 5E10. 10 Mice were immunized with vaccinia virus (VP) at doses administered intranasally and intramuscularly. Four weeks after the first immunization, mice were given a booster immunization, and four weeks after the booster, they were challenged with a lethal dose of vaccinia virus. Changes in body weight and survival rate were recorded.

[0050] Figure 8 A single intranasal immunization with recombinant monkeypox vaccine significantly reduced viral titers and pathological damage in the lungs of mice. Recombinant adenovirus vector monkeypox vaccines and control viruses were compared using 5E10... 10 Mice were immunized intranasally with a dose of vaccinia virus (VP). Six weeks after a single immunization, mice were challenged with a lethal dose of vaccinia virus. On the fifth day after challenge, mice were dissected, and lung sections were taken for pathological examination and HE staining. The residual viral titer in the lungs and throat of the mice was detected by quantitative real-time PCR. Detailed Implementation

[0051] Through in-depth research, the inventors have disclosed a method for preparing a viral vaccine, comprising: (1) providing a recombinant viral vector, wherein the recombinant viral vector comprises: a backbone vector and an expression cassette; the backbone vector is a chimpanzee adenovirus vector; the expression cassette comprises the coding sequences of the following monkeypox virus proteins operatively linked: H3L, E8L, and B6R; preferably, it also comprises the coding sequences of the following proteins: A29L, A35R, and M1R. (2) Transfecting virus-producing cells with the recombinant viral vector of (1) to package and obtain a recombinant virus carrying proteins from monkeypox virus and proteins from chimpanzee adenovirus; (3) Preparing a viral vaccine with the recombinant virus of (2), which has immunoprotective efficacy against monkeypox virus. In this invention, a suitable monkeypox virus gene was selected for the first time and efficiently integrated with a chimpanzee virus vector (AdC68) to prepare an ideal viral vaccine, which is suitable for nasal and muscle immunization, and the nasal immunization effect is very good. This is closely related to the use of an appropriate viral vector (chimpanzee adenovirus vector AdC68XY3) in this invention, which produced a very good effect. The novel monkeypox vaccine of this invention can be effectively used for the prevention and control of monkeypox virus infection. More particularly, the monkeypox virus of this invention can also be used for the prevention and control of vaccinia virus, thus achieving excellent results in the prevention and control of both types of viruses.

[0052] As used herein, “object” or “individual” or “patient” refers to any target requiring diagnosis or treatment, especially mammalian objects, particularly humans. Of particular concern are objects susceptible to or already infected with monkeypox virus and / or cowpox virus.

[0053] As used herein, “immunogenicity” or “immunogenicity” refers to the ability of a natural, recombinant, or synthetic peptide to induce a specific humoral and / or cellular immune response in a mammal. The term “antigen” as used herein refers to a peptide that elicits an immune response in a mammal, whether alone or in fusion, or in combination with an accessory molecule (such as a class I or II major histocompatibility antigen molecule).

[0054] As used herein, “immune response” includes cellular and / or humoral immune responses that are sufficient to suppress or prevent infection; or to prevent or suppress the onset of disease caused by microorganisms (especially pathogenic microorganisms).

[0055] As used herein, a “promoter” refers to a nucleic acid sequence, typically located upstream (5' end) of the coding sequence of a target gene, that guides the transcription of the nucleic acid sequence into mRNA. Generally, a promoter or promoter region provides recognition sites for RNA polymerase and other factors necessary for proper transcription initiation. In this document, the promoter or promoter region includes active variants of the promoter, which can be naturally occurring allelic variants or non-naturally occurring variants. These variants include substitution variants, deletion variants, and insertion variants.

[0056] As used herein, an "expression cassette" refers to a gene expression system containing all the necessary elements required to express a target polypeptide, typically including the following elements: a promoter, a gene sequence encoding the polypeptide, and a terminator; additionally, it may optionally include a signal peptide coding sequence, etc. These elements are operatively linked.

[0057] As used herein, "operable linkage" refers to the functional spatial arrangement of two or more nucleic acid regions or sequences. For example, a promoter region is placed at a specific position relative to the nucleic acid sequence of a target gene, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby "operable linkage" of the promoter region to the nucleic acid sequence.

[0058] As used herein, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between nucleic acids from different sources and host cells. For example, if the combination of nucleic acids and host cells is not typically naturally occurring, then the nucleic acids are heterologous to that host cell. A particular sequence is "exogenous" to the cell or organism in which it is inserted.

[0059] As used herein, “containing,” “having,” or “including” includes “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0060] As used herein, "effective amount" or "effective dose" means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals, as used in this invention.

[0061] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0062] Monkeypox virus has a large genome with numerous and complex coding genes. Furthermore, it exists in two live virus particle forms: the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV). The surface antigens of these two forms differ, posing a challenge to the development of novel monkeypox vaccines. In vitro purified inactivated virus is primarily in IMV particle form, which cannot induce neutralizing antibodies against EEV. This is why inactivated poxvirus vaccines often fail to induce sufficient protective efficacy. Therefore, selecting suitable antigen combinations is crucial for success. Moreover, more antigens or a more comprehensive range of antigens are not necessarily better; inducing high antibody titers in vivo and achieving good protective effects are more important.

[0063] Through screening and comparison, and considering the neutralizing antigenic epitopes on the surfaces of IMV and EEV, as well as the exogenous gene carrying capacity of the adenovirus vector, the inventors of this invention have combined H3L, E8L, and B6R, preferably also including A29L, A35R, or M1R, and efficiently integrated them with the chimpanzee virus vector (AdC68) to obtain a novel recombinant monkeypox virus vaccine that can produce ideal immune effects and is suitable for both nasal and intramuscular immunization. Specifically, M1R, A29L, H3L, and E8L are located on the surface of mature viral particles (IMV) and are neutralizing epitopes on the IMV particle surface; A35R and B6R are located on the surface of enveloped viral particles (EEV). Antibodies against A35R and B6R can inhibit the flow of the viral outer envelope and suppress intercellular diffusion. Specifically, M1R may be involved in viral adhesion to the host cell surface and is associated with the invasion / fusion complex (EFC), serving as a protein required for viral fusion and penetration of host cells; A29L is a membrane fusion protein on the viral surface that binds to heparan sulfate on the cell surface; H3L protein is crucial for the maturation of viral particles within cells and viral infection; E8L is a surface membrane protein that binds to chondroitin sulfate on the cell surface and participates in viral adsorption to the cell surface; A35R plays an important role in the intercellular transmission of viral particles; studies have found that antibodies against A33R (monkeypox A35R) do not have a neutralizing effect in vitro, but can provide protection in experimental mice in vivo; B6R plays a role in the disintegration of the outermost membrane of enveloped viruses, allowing viral particles to enter host cells, and antibodies against B6R have neutralizing activity.

[0064] In a preferred embodiment, a 2A linker was selected between different genes to ensure that the antigen, after expression, can be cleaved into individual antigens rather than fusion proteins. The inventors detected B6R protein expression using Western blotting and measured the transcriptional level of individual antigen mRNA in the vaccine using quantitative real-time PCR. Antibody responses and specific T-cell responses induced by various immunogens were verified in small animal models. The immunoprotective effect of the recombinant monkeypox vaccine was verified by nasal challenge with a lethal dose of vaccinia virus. The results demonstrate that the novel monkeypox vaccine of this invention can effectively induce a monkeypox-specific immune response and can serve as a candidate vaccine for novel smallpox (vaccinia) and monkeypox vaccines.

[0065] In embodiments of this invention, the inventors used a unique chimpanzee adenovirus vector to express multiple combinations of monkeypox proteins as novel monkeypox vaccines, constructing recombinant adenovirus vector monkeypox vaccines MPXV-1, MPXV-2, MPXV-3, MPXV-4, MPXV-5, and MPXV-6. The mRNA transcription levels of different proteins in the vaccines were detected at the cellular level, and the expression of one protein, B6R, was detected using Western blotting. The immunoprotective effect of the recombinant adenovirus vector monkeypox vaccines was then evaluated in small animal models, and the optimal immunization route and efficacy were compared and assessed. Experimental results showed that, as verified in animal models, the recombinant adenovirus vector vaccines successfully induced antibody and T-cell immune responses in the body through both intramuscular and intranasal immunization, and could protect the body against lethal doses of viral infection, including both monkeypox virus and vaccinia virus. One vaccine is effective against both viruses, which brings greater clinical benefits.

[0066] The chimpanzee adenovirus has a relatively large genome. The inventors used a modified viral vector, including modifications to E1 and E3 in its genome, and made appropriate deletions, which is conducive to loading more exogenous genes. This is in line with the present invention's scheme for accommodating multiple monkeypox virus antigens and is suitable for accommodating the protein combination from monkeypox virus of the present invention.

[0067] The recombinant virus described in this invention can be used to prepare vaccine compositions for preventing and treating monkeypox virus and / or cowpox virus infection. The composition comprises: an effective amount of the recombinant virus described in this invention (e.g., virus particles), and a pharmaceutically acceptable vector.

[0068] The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in compositions may contain liquids such as water, saline, glycerin, and sorbitol. Additionally, these carriers may contain auxiliary substances such as lubricants, flow aids, wetting agents or emulsifiers, pH buffers, and stabilizers such as albumin.

[0069] In use, a safe and effective amount of the recombinant virus (such as viral particles) described in this invention is administered to the subject. This safe and effective amount is typically at least about 0.01 micrograms per kilogram of body weight, and in most cases does not exceed about 10 milligrams per kilogram of body weight. Preferably, the dose is between about 1 microgram per kilogram of body weight and about 1 milligram per kilogram of body weight. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0070] The present invention also provides a medicine box containing the recombinant virus (vaccine) or vaccine composition described herein. Preferably, the medicine box may further include an instruction manual explaining the method of using the recombinant virus (vaccine) or vaccine composition to guide those skilled in the art in its application.

[0071] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0072] Experimental Materials and Methods

[0073] Experimental materials

[0074] Restriction endonucleases and T4 DNA ligase (TAKARA), seamless cloning kit (Suzhou Xinsaimei), DNA ladder (GenStar Biosolutions), plasmid mini-preparation kit (Beijing Tiangen), gel extraction kit (Promega Corporation), plasmid intermediate extraction kit (MACHEREY-NAGEL), RNA extraction kit (Suzhou Xinsaimei), reverse transcription kit, and quantitative real-time PCR kit (TAKARA). Replication-defective chimpanzee adenovirus vector AdC68XY3-empty (AdC68XY3, see patent 201910777937.2).

[0075] The AdC68XY3 vector: E1 / E3 are deleted, and ORF6 / 7 and ORF6 in the E4 region are replaced with AdHu5: the deleted region of the AdC68 E4 sequence is 33518bp-34671bp (corresponding to AC_000011.1); the inserted region of the AdHu5 E4 sequence is 32914bp-34077bp (corresponding to AC_000008.1). The AdC68 ORF1-4 sequences are retained.

[0076] Human embryonic kidney cells HEK293, chicken fibroblasts DF-1, African green monkey kidney cells CV-1, and human thymkinase-deficient cells 143TK were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; transfection reagent Lipofectamine 2000 (Invitrogen); HRP-labeled anti-human and anti-mouse IgG (Sigma-Aldrich); ultrafiltration tubes (BECKMAN); cesium chloride; desalting column; C57BL / 6 female mice (Shanghai Vital River Co., Ltd.); ELISA plates (Corning); cell culture consumables; Nanodrop spectrophotometer; multi-functional microplate reader; CO2 incubator; -80℃ ultra-low temperature freezer (Thermo Scientific); etc.

[0077] Vector construction of recombinant adenovirus vector monkeypox vaccine

[0078] First, the selected monkeypox vaccine antigen was codon-optimized and synthesized (Qingke Biotechnology Co., Ltd.). Then, the synthesized antigen gene was combined in different ways using molecular cloning technology. The ligation between genes was selected using the 2A self-cleavage signal sequence, and the gene was cloned into the E1 region of the chimpanzee adenovirus vector AdC68XY3 to obtain the vector plasmid of the chimpanzee adenovirus monkeypox vaccine. Finally, the recombinant adenovirus plasmid DNA was digested and identified using different restriction endonucleases, including Bgl II, Xho I, and Mfe I, and the gene sequence was sequenced to verify the results.

[0079] Packaging and amplification of recombinant adenovirus vector monkeypox vaccine

[0080] First, the recombinant adenovirus monkeypox vaccine vector plasmid DNA was digested with Pac I to obtain a linearized plasmid. This linearized plasmid was then transfected into HEK293 cells at 80% confluence using Lipofectamine 2000. Cytopathic effects were observed daily. Cells were collected when significant cytopathic effects were observed and 60% of cells showed plaques. The cells were then subjected to three freeze-thaw cycles between room temperature and -80°C, centrifuged at 3500 rpm for 5 min, and the supernatant was collected. This supernatant was used to infect HEK293 cells. Twenty-four hours after infection, the virus was harvested using the same method. After 3-4 amplification cycles, 10 ml of serum-free medium resuspended in the supernatant containing the recombinant adenovirus monkeypox vaccine was finally harvested.

[0081] Purification and titer detection of recombinant chimpanzee adenovirus

[0082] The supernatant of the recombinant adenovirus was collected and concentrated using cesium chloride density gradient centrifugation. After desalting via a desalting column, a high concentration of recombinant adenovirus vector monkeypox vaccine was obtained. The absorbance of the recombinant adenovirus vector vaccine at A260 nm was measured using a Nanodrop 2000, and the measured absorbance was multiplied by 1.1 × 10⁻⁶. 12 The recombinant chimpanzee adenovirus titer (vp / ml) was obtained. The recombinant chimpanzee adenovirus was added to glycerol, and the final concentration of glycerol was 10%. Finally, the recombinant adenovirus vector monkeypox vaccine was aliquoted and stored in an ultra-low temperature freezer at -80°C.

[0083] Western blot detection of antigen expression

[0084] Take different concentrations (10 8 vp, 10 9 vp, 10 10 Recombinant adenovirus vector monkeypox vaccine (vp) was used to infect 6-well HEK293 cells. Twenty-four hours after infection, cells were collected and lysed using RIPA. An appropriate amount of protein supernatant was mixed with 5× reducing loading buffer and subjected to SDS-PAGE electrophoresis. After electrophoresis, the proteins on the gel were transferred to a PVDF membrane. After transfer, the membrane was blocked with 5% skim milk for 2 hours, washed three times with TBST, and incubated with the corresponding primary antibody at room temperature for 2 hours. After washing three times with TBST, the membrane was incubated with the corresponding HRP-labeled secondary antibody at room temperature for 1 hour. After washing three times with TBST, an appropriate amount of ECL chemiluminescent agent was added, and Western blot results were obtained using a LAS4000 scanning analyzer.

[0085] Detection of antigen mRNA transcription levels in recombinant adenovirus vector monkeypox vaccine

[0086] Take different concentrations of recombinant adenovirus vector monkeypox vaccine (10 6 vp, 10 7 vp, 10 8 HEK293 cells (500,000 cells) were infected with vp in 12-well plates. Total RNA was extracted from the cells 24 hours after infection using an RNA extraction kit, and the transcription level of the corresponding antigen gene was detected by real-time PCR.

[0087] The primers for quantitative real-time PCR are as follows:

[0088] H3-qp-F: AGCAGCCATCGACCAAATCA (SEQ ID NO:7),

[0089] H3-qp-R:CCCTCAGTTGCCTCCTTGTT (SEQ ID NO:8).

[0090] rtE8L-F: CCACATCTACTGGGGCAAGG (SEQ ID NO:9),

[0091] rtE8L-R: CTGTCCAGCTGGTTCACGAT (SEQ ID NO: 10).

[0092] A35R-Qpcr-F:TGTACGGCGACAAGATCCAG (SEQ ID NO:11),

[0093] A35R-Qpcr-R: TCTGTGTGTGCTAGAAGCGG (SEQ ID NO: 12).

[0094] B6R-Qp-F: CACCATGAACAACGCCAAG (SEQ ID NO: 13),

[0095] B6R-Qp-R: GCAGGGTTCTCGTACTTCC (SEQ ID NO: 14).

[0096] M1R-Qp-F: CCAGACCTCCGTGAATAACCG (SEQ ID NO: 15),

[0097] M1R-Qp-R: GTTTGTAGGAGAGCCGGGAG (SEQ ID NO: 16).

[0098] A29L-Qp-F: GAACCCCGAGACCAAGAGAG (SEQ ID NO: 17),

[0099] A29L-Qp-R: TTGTGCTTGCAGCACTTCTC (SEQ ID NO: 18).

[0100] The internal reference gene is the intracellular GAPDH gene, and the primers are:

[0101] hGAPDH-F: GGACGAGATCCCTCCAAAAT (SEQ ID NO: 19),

[0102] hGAPDH-R: GGCTGTTGTCATACTTCTCATG (SEQ ID NO: 20).

[0103] Recombining antibody response induced by recombinant adenovirus vector monkeypox vaccine

[0104] The recombinant adenovirus vector candidate vaccines and the control virus 5E10 were compared. 10 Mice (C57BL / 6, female, 6-8 weeks old, 5 mice per group) were immunized via nasal and intramuscular injections at the VP dose. Serum was collected from mice every two weeks, and bronchoalveolar lavage fluid was collected at week 4. The specific antibody responses induced by various antigens in serum and bronchoalveolar lavage fluid were detected using an indirect enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with various antigens. After milk blocking, the test samples were added, initially serially diluted 1:20 in a 2-fold series, and finally HRP-labeled anti-mouse secondary antibody was added. After TMB color development, the reaction was stopped with 2M sulfuric acid. The absorbance at OD450 was read using a microplate reader to determine the antibody titer binding to the test sample.

[0105] Neutralizing antibody response induced by recombinant adenovirus vector monkeypox vaccine

[0106] The plaque reduction assay was used to detect the neutralizing antibody response in serum. First, serum samples were serially diluted 1:20 from one well to two wells in a 96-well plate. Then, 100 PFU of Western Reserve strain vaccinia virus was added to each well. After incubation at 37°C for 1 hour, the samples were transferred to 48-well plates, and 60,000 CV-1 cells were added to each well. The 48-well plates were incubated for 48 hours, and the reduction in plaques was calculated.

[0107] T-cell immune response induced by recombinant adenovirus vector monkeypox vaccine

[0108] Four weeks after mice were immunized intranasally with a recombinant adenovirus vector monkeypox vaccine, T lymphocytes from the lungs and spleen of the mice were isolated and analyzed. Flow cytometry was used to analyze the activation of CD4+ T and CD8+ T lymphocytes in the T cells.

[0109] Immunoprotective effect of recombinant adenovirus vector monkeypox vaccine on mice

[0110] Six weeks after a single immunization or four weeks after a booster immunization, mice were challenged intranasally with a lethal dose of vaccinia virus WR strain to evaluate vaccine efficacy. Post-challenge assays included weight loss over two consecutive weeks, survival rate, and viral titer and pathological changes in the lungs and trachea on day five after infection, to assess vaccine effectiveness. Viral titer was detected using quantitative real-time PCR. First, the genome was extracted from the tissue, then the E9L gene of the virus was amplified using the following primers:

[0111] rt-E9L-F2:GCTTCGGCTAAGAGTTGCAC (SEQ ID NO: 21),

[0112] rt-E9L-R2:ACACGGAGTCGGTATCTCCA (SEQ ID NO:22).

[0113] The internal reference gene is GAPDH, and the primer sequences are as follows:

[0114] rt-mGAPDH-F: CAAGTCACTGTCACACCAGAG (SEQ ID NO: 23),

[0115] rt-mGPADH-R: CTCTGGAAAGCTGTGGCTG (SEQ ID NO: 24).

[0116] Tissue sections were stained with hematoxylin-eosin to determine pathological damage.

[0117] Statistical analysis

[0118] All statistical analyses in this paper were performed using Graphpad Prism 7. Differences between groups were analyzed using the Student T-test or One-way ANOVA, and p < 0.05 was considered statistically significant.

[0119] Sequence information

[0120] H3L (324aa; nucleotide 972bp; SEQ ID NO:1):

[0121] MAAAKTPVIVVPVIDRPPSETFPNVHEHINDQKFDDVKDNEVMQEKRDVVIVNDDPDHYKDYV

[0122] FIQWTGGNIRDDDKYTHFFSGFCNTMCTEETKRNIARHLALWDSKFFIELENKNVEYVVIIENDN

[0123] VIEDITFLRPVLKAIHDKKIDILQMREIITGNKVKTELVIDKDHAIFTYTGGYDVSLSAYIIRVTTA

[0124] LNIVDEIIKSGGLSSGFYFEIARIENEMKINRQIMDNSAKYVEHDPRLVAEHRFETMKPNFWSRIG

[0125] TVAAKRYPGVMYTFTTPLISFFGLFDINVIGLIVILFIMFMLIFNVKSKLLWFLTGTFVTAFI

[0126] E8L(304aa;912bp;SEQ ID NO:2):

[0127] MPQQLSPINIETKKAISDTRLKTLDIHYNESKPTTIQNTGKLVRINFKGGYISGGFLPNEYVLSTIHI

[0128] YWGKEDDYGSNHLIDVYKYSGEINLVHWNKKKYSSYEEAKKHDDGIIIIAIFLQVSDHKNVYFQ

[0129] KIVNQLDSIRSANMSAPFDSVFYLDNLLPSTLDYFTYLGTTINHSADAAWIIFPTPINIHSDQLSKF

[0130] RTLLSSSNHEGKPRYITENYRNPYKLNDDTQVYYSGEIIRAATTSPVRENYFMKWLSDLREACFS

[0131] YYQKYIEGNKTFAIIAIVFVFILTAILFLMSQRYSREKQN

[0132] B6R(317aa;951bp;SEQ ID NO:3):

[0133] MKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETD

[0134] KWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCP

[0135] NAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSL

[0136] SNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLS

[0137] KLSKDVVQYEQEIESLEATYHIIIMALTIMGVIFLISIIVLVCSCDKNNDQYKFHKLLP

[0138] A29L (110 amino acids; 330 base pairs; SEQ ID NO:4):

[0139] MDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNEETLKQRLTNLEKKITNITTKF

[0140] EQIEKCCKHNDEVLFRLENHAETLRAAMISLAKKIDVQTGRRPYE

[0141] A35R (182 amino acids; 546 base pairs; SEQ ID NO:5):

[0142] MMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSAFLIVRLNQCMSA

[0143] NEAAITDSAVAVAAASSTHRKVASSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAA

[0144] ESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT

[0145] M1R (251 amino acids; 753 base pairs; SEQ ID NO:6):

[0146] MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQL <​​​​​​​​​​​​The inventors used chimpanzee adenovirus vectors to express various combinations of monkeypox antigens as novel monkeypox vaccines, and constructed a total of 6 vaccines: MPXV-1, MPXV-2, MPXV-3, MPXV-4, MPXV-5 and MPXV-6.

[0152] like Figure 1 As shown in A, different monkeypox antigen genes were synthesized with codon optimization and then tandemly linked using P2A or F2A. CMV was used as the promoter and BGH as the terminator, and the genes were cloned into the E1 region of the chimpanzee adenovirus vector (replacing the E1 region of the original chimpanzee virus vector). HEK293 cells were then transfected to rescue six recombinant adenovirus vector monkeypox vaccines.

[0153] To verify whether the recombinant monkeypox vaccine constructed in this invention can efficiently express multiple different antigens, the inventors detected the mRNA transcription level of the antigen genes. HEK293 cells in 6-well plates (500,000 cells) were infected with different doses of the recombinant vaccine (10^8 vp, 10^7 vp, 10^6 vp). Cells were harvested after 24 hours, RNA was extracted and reverse transcribed into cDNA, and the transcription level of each target gene was subsequently detected using quantitative real-time PCR.

[0154] The results showed that the same recombinant adenovirus vector monkeypox vaccine could efficiently express multiple different monkeypox antigen genes. Figure 1 BG).

[0155] Therefore, vaccines expressing surface antigens selected from IMV (H3L, E8L, A29L, M1R) and EEV (B6R, A35R) were obtained.

[0156] Example 3: Efficacy Analysis of Recombinant Adenovirus Vector Monkeypox Vaccine

[0157] To evaluate the effectiveness of the recombinant adenovirus vector monkeypox vaccine constructed in this invention, the inventors validated it on a mouse animal model.

[0158] Female C57BL / 6 mice were immunized for 6-8 weeks using the same dose of recombinant adenovirus vector vaccine via intranasal and intramuscular injection, with blood samples collected every two weeks for serum separation. At week 4, mice were boosted with the same immunization route. Blood samples were then collected every two weeks thereafter for serum separation. ELISA was used to detect immune responses induced by different antigens in the serum.

[0159] Figure 2 The results shown are from nasal immunotherapy. Figure 3The results presented are from intramuscular immunization. The results show that single doses of MPXV-2 and MPXV-5 induced high-titer antibody responses against A35R, regardless of whether immunization was performed intramuscularly or intranasally. Six recombinant adenovirus vector monkeypox vaccines—MPXV-1, MPXV-2, MPXV-3, MPXV-4, MPXV-5, and MPXV-6—induced high-titer antibody responses against B6R and E8L.

[0160] MPXV-4, MPXV-5, and MPXV-6 can induce A29L antibodies after two nasal immunizations, while two intramuscular immunizations still fail to induce an A29L antibody response, with antibody titers below the detection level.

[0161] Example 4: Neutralizing antibody reaction

[0162] Neutralizing antibody titer is an important indicator for evaluating the efficacy of monkeypox vaccines. To evaluate the efficacy of the recombinant adenovirus vector monkeypox vaccine constructed in this invention, the inventors tested the production of neutralizing antibodies in mice after a single intranasal immunization.

[0163] Five recombinant adenovirus vector monkeypox vaccines and a control vaccine were compared using the 5E10 method. 10 Mice (C57BL / 6, female, 6-8 weeks old, 5 mice per group) were immunized via nasal and intramuscular routes at the VP dose, and mouse serum was collected every two weeks. Neutralizing antibody responses following immunization with the five recombinant adenovirus vector monkeypox vaccines were detected using the plaque reduction assay.

[0164] The results showed that all vaccines, whether administered via nasal or intramuscular immunization, could induce significantly higher levels of neutralizing antibodies compared to the control group.

[0165] With increasing immunization time, the neutralizing antibody titer increased significantly. Figure 4 ).

[0166] Example 5: Analysis of induced T lymphocyte response

[0167] To verify whether the recombinant adenovirus vector monkeypox vaccine constructed in this invention can effectively activate T-cell immune responses, the inventors conducted the verification on a mouse model.

[0168] The inventors first compared the T-cell immune responses induced by monkeypox vaccine through different immunization routes and found that the T-cell immune response induced by intramuscular injection was lower than that induced by nasal injection. Therefore, the inventors mainly compared and evaluated the T-lymphocyte immune responses in mice 4 weeks after nasal immunization.

[0169] Four weeks after a single intranasal immunization, mice were euthanized, and lymphocytes from the lungs and spleen were isolated. These cells were stimulated with a monkeypox antigen-specific epitope library, stained with anti-mouse cytokine fluorescent antibodies, and finally analyzed by flow cytometry. The results showed a strong T-cell immune response in the mouse lungs. Figure 5 As shown, all six vaccines administered via nasal immunization induced strong CD8+ T and CD4+ T lymphocyte responses, showing significant differences compared to the control group. The candidate vaccine MPXV-4 was particularly superior.

[0170] Example 6: Analysis of the effect of in vivo induced immune protection

[0171] To evaluate the immunoprotective effect induced by the recombinant adenovirus vector monkeypox vaccine in mice, the inventors validated it in a mouse model.

[0172] The inventors first verified the immunoprotective effect of a single-dose vaccine immunization. Eight weeks after vaccination, the virus was challenged via the nasal route with a lethal dose of the WR strain of vaccinia virus. Nasal challenge is a more brutal method of infection, facilitating viral replication and spread within the body. The results were as follows... Figure 6 As shown, a single intranasal immunization can induce 100% immune protection, while a single intramuscular immunization can only provide partial protection. Among the six recombinant vaccines, the single intramuscular immunization groups of MPXV-4 and MPXV-6 have an 80% immune protection effect, while the other four vaccine groups have relatively poor effects.

[0173] The inventors further verified the immunoprotective effect of double-dose immunization. Four weeks after the initial immunization, mice were boosted with the same immunization method and dose. Four weeks after the booster immunization, the mice were challenged with a lethal dose of vaccinia virus WR strain. The results showed that both intramuscular injection and intranasal immunization induced 100% protection. Figure 7 As shown in AE.

[0174] Although two intramuscular injections induced 100% protection, it was evident that mice in the double-intramuscular immunization group still experienced significant weight loss (ranging from 15% to 20%) for a period after challenge (Day 3-Day 8). In contrast, the mice in the intranasal immunization group maintained a relatively stable weight after challenge and showed no symptoms of disease. Therefore, this indicates that intranasal immunization is the optimal route of administration for monkeypox vaccine.

[0175] The inventors further analyzed the viral titer and lung pathological damage in mice challenged after nasal immunization with the vaccine. For example... Figure 8 As shown, mice were challenged with a lethal dose of vaccinia virus 6 weeks after a single injection. On the fifth day after challenge, the mice were dissected, and the lungs and trachea were taken for viral titer determination and lung tissue sections.

[0176] The results showed that all six recombinant adenovirus vector monkeypox vaccines significantly reduced viral titers and lung pathological damage after a single nasal immunization.

[0177] Research Summary

[0178] The recombinant adenovirus vector monkeypox vaccine of this invention can serve as a novel candidate vaccine for preventing smallpox (smallpox vaccine is the same as cowpox vaccine; the vaccine of this invention provides protection against both cowpox and smallpox) and monkeypox, preventing infection by smallpox virus and monkeypox virus. Furthermore, the single-dose intramuscular injection of candidate vaccines MPXV-4 and MPXV-6 shows superior protective efficacy compared to other candidate vaccines and can be considered as reserve vaccines for preventing monkeypox and smallpox, with candidate vaccine MPXV-4 being the most outstanding.

[0179] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method for preparing a viral vaccine, comprising: (1) A recombinant viral vector is provided, wherein the recombinant viral vector comprises: a backbone vector and an expression cassette; The backbone vector is a chimpanzee adenovirus vector. The expression cassette includes operatively linked coding sequences for the following monkeypox virus proteins: H3L, E8L, and B6R; (2) The recombinant viral vector of (1) is transfected into viral production cells, thereby packaging to obtain a recombinant virus carrying genes from monkeypox virus and ORF6 / 7 genes from the human AdHu5 E4 region, wherein ORF6 / 7 significantly improves the yield of recombinant adenovirus vector vaccine. (3) A viral vaccine was prepared using the recombinant virus from (2), which has immune protection against monkeypox virus.

2. The method as described in claim 1, characterized in that, The chimpanzee adenovirus vector is AdC68XY3; preferably, the expression cassette is inserted into the chimpanzee adenovirus E1 region of the backbone vector.

3. The method as described in claim 1, characterized in that, The expression cassette also includes the coding sequences for the following proteins: A29L, A35R, and M1R.

4. The method as described in claim 1 or 3, characterized in that, The expression box, in the order of 5'→3', includes: A29L, H3L, E8L, and B6R; A29L, H3L, E8L, B6R, and M1R; H3L, E8L, and B6R; H3L, E8L, B6R, and A35R; H3L, E8L, B6R, and M1R; or A29L, H3L, E8L, B6R, and A35R. Preferably, the coding sequences of each protein are linked by a 2A peptide coding sequence; preferably, the 2A peptide includes a P2A peptide.

5. A recombinant viral vector, said recombinant viral vector comprising: Skeleton carrier and expression box; The backbone vector is the chimpanzee adenovirus vector AdC68XY3; The expression cassette includes the coding sequences of the following monkeypox virus proteins that are operatively linked: H3L, E8L, and B6R.

6. The recombinant viral vector as described in claim 5, characterized in that, The chimpanzee adenovirus vector is AdC68XY3; preferably, the expression cassette is inserted into the chimpanzee adenovirus E1 region of the backbone vector; Preferably, the expression cassette further includes the coding sequences for the following proteins: A29L, A35R, and M1R; More preferably, the expression box includes, in the order of 5'→3', the following: A29L, H3L, E8L, and B6R; A29L, H3L, E8L, B6R, and M1R; H3L, E8L, and B6R; H3L, E8L, B6R, and A35R; H3L, E8L, B6R, and M1R; or A29L, H3L, E8L, B6R, and A35R.

7. Use of the recombinant viral vector according to any one of claims 5 to 6, for assembling to obtain a recombinant virus, wherein the recombinant virus carries a gene from monkeypox virus and an ORF6 / 7 gene from the human AdHu5 E4 region, wherein the ORF6 / 7 significantly increases the yield of the recombinant adenovirus vector vaccine; and wherein the recombinant virus can induce the body to produce immune protection against monkeypox virus and / or vaccinia virus.

8. A recombinant virus, wherein the recombinant virus is prepared by the method according to any one of claims 1 to 4.

9. Use of the recombinant virus according to claim 8 for the preparation of a vaccine composition for the prevention and treatment of monkeypox virus and / or cowpox virus.

10. A recombinant virus vaccine composition, said vaccine composition comprising: The recombinant virus as described in claim 8; And pharmaceutically acceptable carriers or excipients.

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