Recombinant protein and vaccine of middle east respiratory syndrome coronavirus

Abstract

The application discloses a recombinant protein and vaccine of a novel Middle East respiratory syndrome coronavirus, wherein the amino acid sequence of the recombinant protein comprises a sequence of two, three or four 367-602 amino acid fragments of a S protein RBD region of the Middle East respiratory syndrome coronavirus connected in series, or a sequence with more than 95% identity with the sequence of the amino acid fragments and having the same or similar biological activity. The recombinant Middle East respiratory syndrome (MERS) coronavirus vaccine prepared by mixing the target antigen with an adjuvant can produce a high-titer antibody level after immunizing an organism, and can be used for treating and / or preventing Middle East respiratory syndrome (MERS) coronavirus infection and / or disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular, to a recombinant protein and vaccine of Middle East Respiratory Syndrome coronavirus. BACKGROUND

[0002] Middle East Respiratory Syndrome (MERS) is a respiratory disease caused by Middle East Respiratory Syndrome coronavirus (MERS-CoV), which is manifested as severe viral pneumonia and respiratory failure, and can cause a variety of serious complications. The virus was first discovered in a sputum sample of a patient with severe pneumonia of unknown cause in 2012, and in the following years, only sporadic cases occurred worldwide, until a serious MERS outbreak occurred in South Korea in 2015, which caused global public health concern. With the increasing frequency of international exchanges, the risk of global spread of MERS has always existed. Therefore, the development of a safe and effective vaccine against MERS-CoV is of great significance for controlling the epidemic.

[0003] MERS-CoV is a zoonotic virus belonging to the C subgroup of the beta coronavirus genus, similar to other coronaviruses, and its morphological structure is round or oval, with a spike-like structure composed of glycoproteins outside the viral capsid. The virus particle is about 60-220 nm in diameter, is a single-stranded positive-sense RNA virus, and has a total length of 30.1 kb, containing 10 open reading frames, which encode replicase polyprotein, surface spike glycoprotein (Spike, S), small envelope protein, outer membrane protein, nucleocapsid protein, and five non-structural proteins. Among them, the S protein exists in the form of a trimer, and mainly performs the functions of virus and host receptor recognition and mediates the fusion of the viral envelope with the cell membrane or endocytic vesicle membrane. Human dipeptidyl peptidase 4 (dipeptidyl peptidase 4, DPP4, also known as hCD26) is the receptor of MERS-CoV. DPP4 is mainly expressed on the surface of epithelial cells in the lung, kidney, liver, small intestine, pancreas, and activated lymphocytes, and MERS-CoV interacts with the cell surface receptor DPP4 through the RBD on its S protein, mediates the adsorption of the virus to the cell, and then fuses with the cell membrane and enters the cell, initiating viral infection. The S protein and some domains such as S1 protein or RBD are the main targets for vaccine development.

[0004] The source of MERS-CoV transmission was investigated from multiple aspects, and it was found that the virus may originate from bats. From the perspective of molecular evolution, MERS-CoV has similar relationship with the coronaviruses in bats in Asia, Europe, South Africa, Ghana, etc., especially the flat-headed bat coronavirus HKU4 and the pipistrelle bat coronavirus HKU5. However, there is not much contact between humans and bats, so it is speculated that there is another intermediate host between bats and humans. In some countries and regions, single-humped camels are used for racing and tourism, and humans have very close contact with them. Therefore, single-humped camels were studied, and it was found that MERS-CoV neutralizing antibodies could be detected in serum samples, while no MERS-CoV neutralizing antibodies were detected in serum samples of farmed sheep, goats, and cattle, and single-humped camels in northern Europe. After that, MERS-CoV was successfully isolated from camel, nasal swab, fecal sample and camel milk, and its whole genome sequence and the sequence of the virus isolated from human body have 99.9% similarity. Therefore, the interspecies transmission of MERS-CoV is most likely to be bat-camel-human. The infection rate of MERS-CoV is relatively low, which shows limited human-to-human transmission. Infection in healthy populations is not very common, and most confirmed patients have underlying diseases before infection, such as immunodeficiency, diabetes, chronic pneumonia and kidney disease, etc. And the reported cases are mostly clustered, occurring among family members or between patients and medical staff. The incubation period of MERS-CoV in the human body is relatively short, ranging from 2 days to 14 days, usually about 5 days. Most patients show respiratory symptoms such as fever, cough, and dyspnea, and a small number have gastrointestinal symptoms, including diarrhea, nausea, and vomiting. Although the infection rate of MERS-CoV is not as terrible as imagined, its mortality rate is much higher than that of the new coronavirus (SARS-CoV-2).

[0005] Vaccine prevention is still the most economical and effective means to control the spread of the epidemic. So far, there are 11 MERS-CoV vaccines in research worldwide, 4 of which have entered clinical research, and 3 of which have completed phase I clinical trials. The main types of vaccines are inactivated vaccines, attenuated live vaccines, subunit vaccines, nucleic acid vaccines, and viral vector vaccines, and there is no approved vaccine on the market.

[0006] Similar to other coronaviruses, Middle East respiratory syndrome coronavirus (MERS-CoV) infects host cells through its surface glycoprotein, spike (S) protein, and the receptor-binding domain (RBD) of the S protein is directly involved in the binding of host cell receptors and plays an important role in the process of viral infection of host cells. At present, most of the highly neutralizing antibodies against MERS-CoV are targeted to RBD, and therefore, RBD, as an important immunodominant region, is one of the preferred target antigens for vaccine development. The native S protein is in the form of a homotrimer, and the RBD, as a domain of the S protein, also forms a trimer in the native structure. In order to maximize the simulation of the trimeric natural structure form of RBD, the current strategy is to construct a trimeric form of RBD as a vaccine candidate antigen, and at the same time, trimerization can increase the size of the antigen, achieve regular repetitive arrangement of the antigen, enhance B cell receptor cross-linking, and thus improve its immunogenicity. In order to realize the trimerization of the MERS-CoV S protein RBD region protein antigen, the common strategy is to realize it through the increase of an exogenous trimerization motif (such as T4 foldon) or the introduction of an exogenous linker. However, the introduction of exogenous sequences may cause unintended immune responses and pose a certain safety risk. Therefore, it is necessary to find a MERS-CoV S protein RBD region protein to solve the above problems. SUMMARY

[0007] Technical problems to be solved :

[0008] An aspect of the present application is to solve the problem that the trimerization of the RBD region of the S protein of the Middle East respiratory syndrome coronavirus in the prior art introduces an exogenous linker, thereby increasing the risk of unintended immune responses, and provides a recombinant protein and a vaccine of the Middle East respiratory syndrome coronavirus.

[0009] Specifically, the present application realizes the trimerization of RBD without introducing any exogenous sequences by analyzing the spatial structure of the S protein of MERS-CoV, constructs a homotrimeric RBD antigen molecule, and shows good immunization effect, which can be used as an ideal candidate vaccine antigen.

[0010] The technical scheme provided by the present application is :

[0011] A recombinant protein of Middle East Respiratory Syndrome Coronavirus (MERS-CoV), wherein the amino acid sequence of the recombinant protein comprises a sequence consisting of two, three, or four consecutive tandem segments of amino acid fragments from amino acid position 367 to 602 of the RBD region of the MERS-CoV S protein, or a sequence having more than 95% identity with the sequence consisting of said amino acid fragments and having the same or similar biological activities.

[0012] The inventors were surprised to find that the spatial structure of the S protein RBD region has the following structural features: (1) The RBD is relatively independent in spatial structure and there is almost no strong interaction with other domains of the S protein. Therefore, the folding of the RBD is independent and does not require the assistance of other domains; (2) The RBD has a compact spatial conformation. Its core structure is composed of multiple Beta sheets, which has high structural stability. RBD trimerization is not expected to affect or destroy the core structure of the RBD; (3) The two ends of the RBD are connected to other domains through long loop structures. Its own loop structure has high flexibility and can act as the connecting arm for RBD trimerization, avoiding the introduction of exogenous flexible connecting arms. At the same time, the N-terminal and C-terminal loop structures of the RBD are close to each other in space, which is conducive to the regular arrangement of the RBD during trimerization. There are no large space barriers and will not affect the core structure of each RBD. Based on further research into the structural features described above, the inventors extracted the RBD domain formed between amino acid residues 367 to 602 of the MERS-CoVS protein. This portion fully preserves the loop structures at both ends of the RBD as flexible linkers for trimerization. Furthermore, the N-terminal and C-terminal interfaces of the extracted RBD are ensured to be spatially close to facilitate the regular arrangement of the RBD during trimerization. The extracted RBDs are covalently linked end-to-end to form a new protein molecule (i.e., C05F12) as a candidate antigen for vaccines.

[0013] Preferably, in some embodiments of the present invention, the amino acid sequence of the recombinant protein comprises a sequence consisting of amino acid fragments from positions 367 to 602 of three consecutive tandemly linked RBD regions of the Middle East Respiratory Syndrome Coronavirus S protein, or a sequence having more than 95% identity with the sequence consisting of said amino acid fragments and having the same or similar biological activities.

[0014] More preferably, in one embodiment of the present invention, the amino acid sequence of the recombinant protein is as shown in SEQ ID No. 1, or a sequence that has more than 95% identity with the sequence composed of the amino acid fragment and has the same or similar biological activity.

[0015] In the above embodiments, the amino acid sequence of the recombinant protein can be substituted, deleted, or inserted with one or more amino acids to obtain a new amino acid sequence, and the new protein consisting of the new amino acid sequence has the same or substantially the same biological activity as the protein consisting of the original amino acid sequence. The new amino acid sequence is also considered to be within the scope of the present application. The biological activity includes, but is not limited to, immunological activity, binding activity of antigen and antibody, binding activity of ligand and receptor, etc.

[0016] Further, the above sequence having 95% or more identity thereto refers to an amino acid sequence having 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the recombinant protein of the Middle East respiratory syndrome coronavirus or the fusion protein. Those skilled in the art can randomly or engineerically mutate the amino acid sequence of the fusion protein described in the present specification in a suitable manner, for example, to obtain better affinity and / or dissociation properties, to improve expression performance, etc., and the sequence can have the same or substantially the same biological activity as the Middle East respiratory syndrome coronavirus or the fusion protein, and the mutated amino acid sequence is also considered to be within the scope of the present application. The biological activity includes, but is not limited to, immunological activity, binding activity of antigen and antibody, binding activity of ligand and receptor, etc.

[0017] The trimerization form of the recombinant protein of the Middle East respiratory syndrome coronavirus can also be introduced with certain foreign trimerization motifs, for example, T4 bacteriophage fibritin. However, the safety thereof is weaker than that of the recombinant protein of the Middle East respiratory syndrome coronavirus in the present application, and can also cause the occurrence of an unintended immune response.

[0018] Another aspect of the present application provides a fusion protein comprising the recombinant protein of the Middle East respiratory syndrome coronavirus.

[0019] As preferred, in certain embodiments of the present application, the fusion protein can further comprise one or more selected from a signal peptide, a tag, or an immunopotentiating peptide. The signal peptide can be used to facilitate the expression of the protein, and the tag can be, for example, a Flag tag, an enhanced green fluorescent protein (eGFP), a glutathione S-transferase (GST), etc., and can be used for detection, purification, separation, etc. The functional sequences can be used in any combination.

[0020] Another aspect of the present application provides an isolated polynucleotide encoding the recombinant protein of the Middle East respiratory syndrome coronavirus or encoding the fusion protein.

[0021] As preferred, the isolated polynucleotide can be codon-optimized.

[0022] More preferably, in an embodiment of the present application, the sequence of the isolated polynucleotide is as shown in SEQ ID No. 2, or a sequence having 95% or more identity to the sequence and having the same or similar biological activity.

[0023] Another aspect of the present application is to provide a vector comprising the above-mentioned isolated polynucleotide.

[0024] In the present application, the above-mentioned vector can be a linear vector or a circular vector. It can be a non-viral vector such as a plasmid, or a viral vector (e.g., an adenovirus vector, a measles virus vector, a mumps virus vector, a rubella virus vector, a varicella virus vector, a polio virus vector, a yellow fever virus vector), or a vector using a transposon. The vector can contain a regulatory sequence such as a promoter, a terminator, and a marker sequence such as a drug resistance gene, a reporter gene, etc.

[0025] As preferred, in an embodiment of the present application, the above-mentioned vector is an expression vector for the isolated polynucleotide of the present application, for expressing a recombinant protein or a fusion protein of the severe acute respiratory syndrome coronavirus of the present application.

[0026] The sequence having 95% or more identity thereto means a nucleotide sequence having 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide sequence.

[0027] The above-mentioned method for preparing the polynucleotide can be prepared by a known technique such as chemical synthesis or PCR amplification based on the above-mentioned nucleotide sequence. Generally, the codons encoding the amino acids of the above-mentioned domains can be optimized to optimize the expression in a host cell. The information of the above-mentioned base sequence can be obtained by searching a known literature or a database such as NCBI (https: / / www.ncbi.nlm.nih.gov / ).

[0028] Another aspect of the present application is to provide a host cell comprising the above-mentioned polynucleotide or the above-mentioned vector.

[0029] As preferred, in an embodiment of the present application, the above-mentioned host cell is an E. coli, a yeast cell, an insect cell, or a mammalian cell.

[0030] More preferably, in an embodiment of the present application, the above-mentioned host cell is a CHO cell.

[0031] Another aspect of the present application is to provide a method for preparing the recombinant protein of the MERS coronavirus or the fusion protein, comprising the following steps:

[0032] Step A) preparing the polynucleotide, constructing the expression vector, transforming or transfecting the expression vector into the host cell;

[0033] Step B) using the product of step A) to express the protein;

[0034] Step C) purifying the expression product obtained in step B) to obtain the recombinant protein of the MERS coronavirus or the fusion protein.

[0035] In step A), the polynucleotide encodes the recombinant protein of the MERS coronavirus or the fusion protein.

[0036] As a preferred embodiment of the present application, the polynucleotide sequence is shown in SEQ ID No. 2, or a sequence having more than 95% identity with the sequence and having the same or similar biological activity.

[0037] The polynucleotide can be prepared according to the polynucleotide sequence described in the present specification using any suitable molecular biology method.

[0038] In step A), the polynucleotide sequence can be constructed in the corresponding expression vector of the host cell using any suitable method.

[0039] The expression vector is then transformed or transfected into the host cell. As a preferred embodiment of the present application, the inventors transfected the constructed CHO cell expression vector into HEK293FT cells or CHO cells to construct a recombinant cell strain.

[0040] In step B), the protein expression can be performed according to the different expression systems used. Further, as a preferred embodiment of the present application, the inventors screened the cell strain capable of stably secreting and expressing the recombinant protein or the fusion protein of the MERS coronavirus by limiting dilution method.

[0041] In step C), the purification can be any suitable method. For example, salting-out method, precipitation method, dialysis or ultrafiltration, molecular sieve chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, etc. As a preferred embodiment of the present application, the recombinant protein or the fusion protein of the MERS coronavirus is purified by ion exchange and hydrophobic chromatography.

[0042] Of course, according to the prior art, a collection process of the target protein should be included before the purification step, for example, collection of a cell culture supernatant enriched with the target protein; and a process of disrupting the host cell after expression of the target protein, using any suitable disruption method such as ultrasonic disruption, repeated freeze-thaw disruption, chemical treatment, etc. The above collection process of the host cell should also be understood as included in the scope of the purification.

[0043] Another aspect of the present application provides a use of the above-mentioned recombinant protein, the above-mentioned fusion protein, the above-mentioned isolated polynucleotide, the above-mentioned vector, or the above-mentioned host cell in the preparation of a medicament for treating and / or preventing a Middle East respiratory syndrome coronavirus infection and / or a disease caused by a Middle East respiratory syndrome coronavirus.

[0044] Another aspect of the present application provides a use of the above-mentioned recombinant protein or the above-mentioned fusion protein in the detection of an antibody against a Middle East respiratory syndrome coronavirus.

[0045] Another aspect of the present application provides a use of the above-mentioned recombinant protein, the above-mentioned fusion protein, the above-mentioned isolated polynucleotide, the above-mentioned vector, or the above-mentioned host cell in the preparation of a medicament for boosting immunity in a population that has been vaccinated with a Middle East respiratory syndrome coronavirus vaccine.

[0046] As a preferred embodiment, the Middle East respiratory syndrome coronavirus vaccine is a Middle East respiratory syndrome coronavirus vaccine inactivated vaccine.

[0047] Another aspect of the present application provides a recombinant protein vaccine, which comprises the above-mentioned recombinant protein or the above-mentioned fusion protein of a Middle East respiratory syndrome coronavirus, and an adjuvant.

[0048] Any suitable adjuvant can be included in the vaccine of the present application. As a preferred embodiment, the adjuvant of the present application is aluminum hydroxide, aluminum phosphate, MF59, or CpG. More preferably, the adjuvant of the present application is aluminum hydroxide.

[0049] As a preferred embodiment, the recombinant protein vaccine of the present application further comprises a delivery system in some embodiments of the present application.

[0050] Another aspect of the present application provides a method for preparing the above-mentioned vaccine, which comprises mixing the above-mentioned recombinant protein or the above-mentioned fusion protein of a Middle East respiratory syndrome coronavirus, which is purified, with the adjuvant.

[0051] Another aspect of the present application provides a genetically engineered vector vaccine, which comprises the above-mentioned isolated polynucleotide, or the above-mentioned vector.

[0052] Another aspect of the present application provides a nucleic acid vaccine comprising the isolated polynucleotide or the vector described above.

[0053] Another aspect of the present application provides a pharmaceutical composition comprising the recombinant protein vaccine, the genetic engineering vector vaccine or the nucleic acid vaccine described above, and a pharmaceutically acceptable carrier.

[0054] The pharmaceutically acceptable carrier can be any pharmaceutically acceptable additive, such as physiological saline, cell culture medium, glucose, water for injection, glycerol, amino acids and combinations thereof, stabilizers, surfactants, preservatives, isotonic agents, etc.

[0055] The pharmaceutical composition described above can also be used in combination with other drugs for treating and / or preventing MERS-CoV infection and / or diseases caused by MERS-CoV at an effective and safe dose.

[0056] Another aspect of the present application provides a method for inducing an immune response against MERS-CoV or treating MERS-CoV in a subject, comprising administering to the subject an effective dose of the recombinant protein vaccine, the genetic engineering vector vaccine, the nucleic acid vaccine or the pharmaceutical composition described above.

[0057] The subject described above can be a human or other animals.

[0058] The administration described above can be intramuscular injection, intraperitoneal injection or subcutaneous injection.

[0059] Advantages of the present application :

[0060] The present application provides a recombinant protein of MERS-CoV in a trimeric form. The recombinant MERS-CoV vaccine prepared by mixing the recombinant protein as a target antigen with an adjuvant can produce high titer antibody levels after immunizing the body, and can be used for treating and / or preventing MERS-CoV infection and / or diseases.

[0061] Sequence Description

[0062] SEQ ID No. 1 is the amino acid sequence of the recombinant protein of MERS-CoV in the embodiments of the present application;

[0063] SEQ ID No. 2 is the nucleotide sequence encoding the recombinant protein of MERS-CoV in the embodiments of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1A construction mode diagram of a recombinant Middle East Respiratory Syndrome (MERS) coronavirus RBD trimerization protein in an embodiment of the present application;

[0065] Figure 2 A SDS-PAGE detection result diagram of a recombinant Middle East Respiratory Syndrome (MERS) coronavirus RBD trimerization protein in an embodiment of the present application, wherein lanes 1 and 2 are molecular weight markers, and lanes 3 to 6 are proteins of different concentrations;

[0066] Figure 3 A Western-blot detection result diagram of a recombinant Middle East Respiratory Syndrome (MERS) coronavirus RBD trimerization protein in an embodiment of the present application, wherein lane 1 is a molecular weight marker, lanes 2 to 5 are proteins of different concentrations, and lane 6 is a negative control;

[0067] Figure 4 A transmission electron microscope observation result diagram of a recombinant Middle East Respiratory Syndrome (MERS) coronavirus RBD trimerization protein in an embodiment of the present application;

[0068] Figure 5 A curve diagram of the binding activity of a recombinant Middle East Respiratory Syndrome (MERS) coronavirus RBD trimerization protein to T46 antibody in an embodiment of the present application;

[0069] Figure 6 A curve diagram of the binding activity of a recombinant Middle East Respiratory Syndrome (MERS) coronavirus RBD trimerization protein to T62 antibody in an embodiment of the present application;

[0070] Figure 7 A curve diagram of the binding activity of a recombinant Middle East Respiratory Syndrome (MERS) coronavirus RBD trimerization protein to RP01 antibody in an embodiment of the present application;

[0071] Figure 8 A curve diagram of the binding activity of a recombinant Middle East Respiratory Syndrome (MERS) coronavirus RBD trimerization protein to R723 antibody in an embodiment of the present application;

[0072] Figure 9 A serum specific IgG antibody detection result diagram of mice immunized with different doses of a recombinant Middle East Respiratory Syndrome (MERS) coronavirus vaccine in an embodiment of the present application;

[0073] Figure 10 A serum neutralizing antibody detection result diagram of mice immunized with different doses of a recombinant Middle East Respiratory Syndrome (MERS) coronavirus vaccine in an embodiment of the present application;

[0074] Figure 11Figure 2 shows the detection results of specific IgG antibodies in the serum of mice immunized with the recombinant MERS coronavirus vaccine of different immunization doses according to an embodiment of the present application;

[0075] Figure 12 Figure 3 shows the detection results of neutralizing antibodies in the serum of mice immunized with the recombinant MERS coronavirus vaccine of different immunization doses according to an embodiment of the present application. DETAILED DESCRIPTION

[0076] The present application discloses a recombinant protein and vaccine of Middle East respiratory syndrome coronavirus, and those skilled in the art can refer to the content herein, and appropriately improve the process parameters to achieve. It needs to be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are regarded as included in the present application, and the relevant personnel can obviously change or appropriately change and combine the content described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0077] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise explicitly stated, throughout the specification and claims, the term "comprise" or its variants such as "contain" or "include" and the like will be understood to include the stated element or component, without excluding other elements or components. The terms "such as", "for example", and the like are intended to indicate exemplary embodiments, and are not intended to limit the scope of the present disclosure.

[0078] The following explains some terms appearing in the present application.

[0079] The term "continuous series" in the present disclosure refers to the primary structure of the three subunits of the recombinant protein of Middle East respiratory syndrome coronavirus, which is the order of the subunits from N-terminal to C-terminal.

[0080] The term "fusion protein" refers to an expression product of one, two or more genes after recombination by DNA recombination technology. The fusion protein technology is a purposeful gene fusion and protein expression method for obtaining a large amount of standard fusion protein. Using the fusion protein technology, a target protein with multiple functions can be constructed and expressed.

[0081] The term "isolated" molecule (e.g., an isolated nucleic acid or protein or cell) means that it has been identified and separated and / or recovered from a component of its natural environment.

[0082] The term "vector" is a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is said to be an expression vector. Vectors can be introduced into host cells by transformation, transduction or transfection, and are replicative elements of genetic material in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcriptional initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors can contain a replication origin.

[0083] The term "host cell" is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

[0084] The term "treatment" refers to the reduction in the likelihood of disease pathology, the reduction in the occurrence of disease symptoms, e.g., a subject has a longer survival or reduced discomfort to some extent. Treatment can refer to the ability of a therapy to reduce disease symptoms, signs, or causes when the therapy is administered to a subject. Treatment also refers to palliating or reducing at least one clinical symptom and / or inhibiting or delaying progression of a disorder and / or preventing or delaying onset of a disease or condition.

[0085] The term "subject" refers to any human or other animal to whom prevention, treatment, or diagnosis is provided. Other mammals can include, for example, dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, guinea pigs, mice, and the like.

[0086] In order to make the skilled in the art better understand the technical solutions of the present application, the present application will be further described in detail below in combination with specific embodiments.

[0087] Embodiments

[0088] Example 1: Middle East Respiratory Syndrome (MERS) coronavirus RBD trimer protein designed based on protein structure and computational biology

[0089] Similar to other coronaviruses, Middle East respiratory syndrome coronavirus (MERS-CoV) infects host cells through its surface glycoprotein, spike (S) protein, and the receptor-binding domain (RBD) of the S protein is directly involved in the binding of host cell receptors and plays an important role in the process of viral infection of host cells. At present, most of the highly efficient neutralizing antibodies against MERS-CoV are targeted to RBD, and therefore, RBD, as an important immunodominant region, is one of the preferred target antigens for vaccine development. The native S protein is a homotrimer, and RBD, as a domain of the S protein, also forms a trimer in the native structure. Therefore, in order to maximize the simulation of the trimeric natural structure form of RBD, a trimeric form of RBD is constructed as a vaccine candidate antigen, and at the same time, the trimerization can increase the size of the antigen, realize the regular repetitive arrangement of the antigen, enhance the cross-linking of B cell receptors, and thus improve the immunogenicity.

[0090] A common strategy to achieve trimerization of protein antigens is to introduce an exogenous trimerization motif (e.g., T4 foldon) or an exogenous linker. However, the introduction of exogenous sequences can cause unintended immune responses, posing certain safety risks. By analyzing the spatial structure of the S protein of MERS-CoV, trimerization of the RBD was achieved without introducing any exogenous sequences, and a homotrimeric RBD antigen molecule was constructed, which showed good immunization effect and can be used as an ideal candidate vaccine antigen. Spatial structure analysis of the S protein showed that the RBD has the following structural characteristics: (1) The RBD is relatively independent in spatial structure and there is almost no strong interaction between the RBD and other domains of the S protein, so the folding of the RBD is independent and does not require the assistance of other domains; (2) The RBD has a tight spatial conformation, and its core structure is composed of multiple beta sheets, which has high structural stability, and the trimerization of the RBD is expected not to affect and destroy the core structure of the RBD; (3) The two ends of the RBD are connected to other domains through long loop structures, and the loop structure of the RBD itself has high flexibility and can serve as a flexible linker for RBD trimerization, avoiding the introduction of an exogenous flexible linker. At the same time, the N-terminal and C-terminal loop structures of the RBD are close to each other in space, which is conducive to the regular arrangement of the RBD during trimerization and does not affect the core structure of the RBD. Based on the above structural characteristic analysis, the RBD domain composed of amino acids 367 to 602 of the MERS-CoV S protein was intercepted, the loop structures at both ends of the RBD were fully retained as flexible linkers for trimerization, and the N-terminal and C-terminal interfaces of the intercepted RBD were ensured to be close in space to facilitate the regular arrangement of the RBD during trimerization. The intercepted RBD was covalently linked at the head and tail to form a new protein molecule (i.e., C05F12) as a vaccine candidate antigen.

[0091] Example 2: Expression, purification and identification of recombinant protein

[0092] According to the codon bias of the CHO cell expression system, the nucleotide sequence encoding the recombinant protein C05F12 (the amino acid sequence is shown as SEQ ID NO. 1) was codon-optimized, and the optimized nucleotide sequence is shown as SEQ ID NO. 2. After constructing a CHO cell expression vector, it was transfected into 293FT cells or CHO cells to construct a recombinant cell strain. A cell strain capable of stably secreting and expressing the recombinant protein C05F12 was obtained by limiting dilution method. After cell culture, the supernatant was harvested, and the recombinant protein C05F12 with a purity of ≥95% was obtained after serial chromatography purification. The SDS-PAGE detection result is shown as Figure 2 The molecular weight of the protein is 70-100 kD, and there are also some product-related substances such as dimeric protein and monomeric protein.

[0093] The purified C05F12 protein was subjected to SDS-PAGE electrophoresis and then electrotransferred to a PVDF membrane, and Western-blot identification was performed using MERS-RBD protein specific antibody (manufacturer: Beijing Yiqiao God States Technology Co., Ltd.; item number: 40069-T62; dilution: 1000 times) (results shown in Figure 3 The results showed that C05F12 protein could bind to MERS-RBD protein specific antibody and had good biological activity. The purified C05F12 protein was subjected to molecular exclusion chromatography analysis using a TSKgel G2500PW gel chromatography column, and the protein purity was greater than 90%. Transmission electron microscopy was used to observe the C05F12 protein, and the protein particle size was small, about 5-10 nm.

[0094] Example 3: Detection of physicochemical properties and biological activity of C05F12 protein

[0095] The purified C05F12 protein and MERS-RBD monomer protein (manufacturer: Beijing Yiqiao God States Technology Co., Ltd.; item number: 40071-V08B1) were subjected to 10-fold gradient serial dilution using coating solution starting from 10 µg / ml, 100 µl / well, coated on a 96-well enzyme-labeled plate at 4°C for 8-12 h, with a blank well as a negative control. After washing the plate with PBST solution, blocking solution was added, and the plate was incubated at 37°C for 2 h. After washing the plate with PBST solution, 1 µg / ml of antibody was added, including T46 antibody (manufacturer: Beijing Yiqiao God States Technology Co., Ltd.; item number: 40069-T46), T62 antibody (manufacturer: Beijing Yiqiao God States Technology Co., Ltd.; item number: 40069-T62), RP01 antibody (manufacturer: Beijing Yiqiao God States Technology Co., Ltd.; item number: 40069-RP01), and R723 antibody (manufacturer: Beijing Yiqiao God States Technology Co., Ltd.; item number: 40069-R723), 100 µl / well, 37°C incubation for 1 h. After washing the plate with PBST solution, diluted horseradish peroxidase-labeled goat anti-mouse or goat anti-rabbit IgG antibody was added, 100 µl / well, 37°C incubation for 1 h. After washing the plate with PBST solution, color developing solution A and B were added in sequence, color development was performed at room temperature for 5-10 min, and stop solution C was added. The double-wavelength (OD450nm and 630nm) reading was performed on an enzyme-labeled instrument to determine the cut-off value, and the protein concentration-absorbance value curve was drawn. The results of the binding activity with T46 antibody are shown in Figure 5 The results of the binding activity with T62 antibody are shown in Figure 6 The results of the binding activity with RP01 antibody are shown in Figure 7 The results of the binding activity with R723 antibody are shown in Figure 8As shown, the results show that the C05F12 protein can bind to various MERS S protein or RBD protein specific antibodies, and has good biological activity.

[0096] Example 4: Preparation of recombinant Middle East Respiratory Syndrome (MERS) coronavirus vaccine

[0097] The purified recombinant protein C05F12 was diluted to 2 times the target antigen concentration, mixed with 1.2 mg / ml aluminum hydroxide adjuvant at a ratio of 1:1 (w / w) for adsorption, and stirred on a magnetic stirrer at a speed of 200-300 rpm for 40-120 min. The supernatant residual protein content should be less than 10% of the total protein content. The semi-finished product was sterilely divided into 0.5 ml aliquots, and the vaccine was obtained.

[0098] Example 5: Evaluation of the immunological effect of different immunization doses of recombinant Middle East Respiratory Syndrome (MERS) coronavirus vaccine in mice

[0099] The recombinant Middle East Respiratory Syndrome (MERS) coronavirus vaccine prepared in Example 4 was formulated into three different doses of 2.0 µg / dose / 0.5 ml, 0.5 µg / dose / 0.5 ml, and 0.125 µg / dose / 0.5 ml, with aluminum adjuvant as a negative control. BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., SPF level, female, 6-8 weeks old) were intraperitoneally immunized, 10 mice per group. Specifically, the mice were immunized with different doses of recombinant Middle East Respiratory Syndrome (MERS) coronavirus vaccine at 0w, 1w, and 3w, and blood was collected at 4w to separate serum. MERS-RBD monomer protein (manufacturer: Beijing Yiqiao God States Technology Co., Ltd.; catalog number: 40071-V08B1) was used as a detection reagent, and ELISA was used to detect the serum specific IgG antibody titers of each group of mice. The detection results are shown in Table 1 and Figure 9 As shown, the results show that the C05F12 protein can bind to various MERS S protein or RBD protein specific antibodies, and has good biological activity. Figure 10 As shown, the results show that the C05F12 protein can bind to various MERS S protein or RBD protein specific antibodies, and has good biological activity.

[0100] Table 1. Detection results of serum specific IgG antibodies in mice with different immunization doses

[0101]

[0102] Table 2. The detection results of serum neutralizing antibodies of mice with different immunization doses

[0103]

[0104] Example 6: In vivo evaluation of immunological effects of different immunization doses of recombinant Middle East Respiratory Syndrome (MERS) coronavirus vaccine

[0105] The recombinant Middle East Respiratory Syndrome (MERS) coronavirus vaccine prepared in Example 4 was formulated into 0.5 μg / dose / 0.5 ml, and BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., SPF level, female, 6-8 weeks old) were immunized intraperitoneally according to two immunization schedules of 0w-1w-3w and 0w-3w, 10 mice per group, and blood was collected at 4w to separate serum. The MERS-RBD monomer protein (manufacturer: Beijing Yiqiao God States Technology Co., Ltd.; item number: 40071-V08B1) was used as a detection agent, and ELISA was used to detect the specific IgG antibody titers of each group of mice, and the detection results are shown in Table 3 and Figure 11 It can be seen that two immunization doses can stimulate the production of high-titer antibodies in mice, and there is no statistical difference with three immunization doses. The neutralizing antibody of the post-immunization serum of mice against MERS pseudovirus was detected by pseudovirus neutralization test method, and the detection results are shown in Table 4 and Figure 12 It can be seen that two immunization doses can stimulate the production of high-titer antibodies in mice, and there is no statistical difference with three immunization doses. The neutralizing antibody of the post-immunization serum of mice against MERS pseudovirus was detected by pseudovirus neutralization test method, and the detection results are shown in Table 4 and

[0106] Table 3. The detection results of specific IgG antibodies of serum of mice with different immunization doses

[0107]

[0108] Table 4. The detection results of neutralizing antibodies of serum of mice with different immunization doses

[0109]

[0110] The above only describes the preferred embodiments of the present application, and it should be noted that those of ordinary skill in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A recombinant protein of Middle East Respiratory Syndrome Coronavirus, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID No.

1.

2. A fusion protein, characterized in that, The amino acid sequence of the fusion protein consists of the amino acid sequence of the recombinant protein as described in claim 1, as well as a signal peptide and / or a tag.

3. An isolated polynucleotide, characterized in that, The isolated polynucleotide encodes the amino acid sequence of the recombinant protein as described in claim 1, or the fusion protein as described in claim 2.

4. The isolated polynucleotide according to claim 3, characterized in that, The isolated polynucleotides were codon-optimized.

5. The isolated polynucleotide according to claim 3, characterized in that, The sequence of the isolated polynucleotide is shown in SEQ ID No.

2.

6. A carrier, characterized in that, The carrier comprises the isolated polynucleotide as described in claim 4 or 5.

7. A host cell, characterized in that, The host cell comprises the isolated polynucleotide as described in claim 4 or 5, or the vector as described in claim 6.

8. The host cell according to claim 7, characterized in that, The host cell is Escherichia coli, yeast cell, insect cell, or mammalian cell.

9. The host cell according to claim 8, characterized in that, The host cell is a CHO cell.

10. A method for preparing the recombinant protein as described in claim 1 or the fusion protein as described in claim 2, characterized in that, Includes the following steps: Step A) Prepare the polynucleotide as described in any one of claims 3 to 5, construct the expression vector of the polynucleotide, and transform or transfect the expression vector into a host cell; Step B) Use the product of step A) to express the protein; Step C) Purify the expression product obtained in step B) to obtain the recombinant protein or the fusion protein.

11. Use of the recombinant protein of claim 1, the fusion protein of claim 2, the isolated polynucleotide of any one of claims 3 to 5, the vector of claim 6, or the host cell of claim 7, 8, or 9 in the preparation of a medicament for the treatment and / or prevention of Middle East Respiratory Syndrome Coronavirus infection and / or disease caused by Middle East Respiratory Syndrome Coronavirus.

12. Use of the recombinant protein of claim 1 or the fusion protein of claim 2 in the detection of antibodies against Middle East Respiratory Syndrome Coronavirus; The intended use is not for diagnostic purposes.

13. Use of the recombinant protein of claim 1, the fusion protein of claim 2, the isolated polynucleotide of any one of claims 3 to 5, the vector of claim 6, or the host cell of claim 7, 8, or 9 in the preparation of a medicament for boosting immunization of a population previously vaccinated with Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

14. The use according to claim 13, characterized in that, The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) vaccine is an inactivated MERS-CoV vaccine.

15. A recombinant protein vaccine, characterized in that, The recombinant protein vaccine comprises the recombinant protein as described in claim 1 or the fusion protein as described in claim 2, and an adjuvant.

16. The recombinant protein vaccine according to claim 15, characterized in that, The adjuvant is aluminum hydroxide, aluminum phosphate, MF59, CpG, polyinosinic-polycytidylic acid, or a mixture of the above adjuvants.

17. The recombinant protein vaccine according to claim 16, characterized in that, The adjuvant is aluminum hydroxide.

18. The recombinant protein vaccine according to claim 15, characterized in that, The recombinant protein vaccine also includes a delivery system.

19. A genetically engineered vector vaccine, characterized in that, The genetically engineered vector vaccine comprises isolated polynucleotides as described in any one of claims 3 to 5, or a vector as described in claim 6.

20. A nucleic acid vaccine, characterized in that, The nucleic acid vaccine comprises isolated polynucleotides as described in any one of claims 3 to 5, or a vector as described in claim 6.

21. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a recombinant protein vaccine as described in any one of claims 15 to 18, a genetically engineered vector vaccine as described in claim 19, or a nucleic acid vaccine as described in claim 20, and a pharmaceutically acceptable vector.

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