Patent Application
20230080694
The invention belongs to the technical field of biomedicine, and more specifically relates to a Helicobacter pylori ferritin-based novel coronavirus (tentatively known as SARS-CoV-2, also known as 2019-nCoV) S protein double-region subunit nanovaccine.
Since December 2019, a series of pneumonia cases of unknown cause have occurred in Wuhan Hubei, China, which clinical manifestations are very similar to those of viral pneumonia; the main clinical manifestations are fever, fatigue, dry cough, etc. In severe cases, shock, sepsis, respiratory failure may occur, causing death. Deep sequencing analysis of nine cases of lower respiratory tract samples was utilized to reveal presence of a novel coronavirus, tentatively known as SARS-CoV-2 (also known as 2019-nCoV). As of February 19, more than 70,000 patients have been confirmed in China, and there are still more than 5,000 suspected cases, resulting in more than 1,600 deaths, and hundreds of cases have also been confirmed in Japan, Thailand, South Korea, the United States, and many countries in Europe, having a momentum of spreading in China and even the world. Due to unclear source and pathogenesis of the novel coronavirus pneumonia, and lack of specific antiviral drugs, it has brought great difficulties to clinical diagnosis and treatment and control of the epidemic, resulting in a serious social burden and crisis.
At present, humans still lack an effective vaccine against SARS-CoV-2. Under this severe situation, developing a safe and effective vaccine against SARS-CoV-2 as soon as possible to protect susceptible population is of great significance to our people's health and national security.
For development of vaccines, structure of the virus must be understood first. Coronaviruses are a class of enveloped single positive-stranded RNA viruses that can widely exist in humans and other mammals as well as birds, and cause respiratory, digestive, liver and nervous system diseases. Before this outbreak, six coronaviruses have been known to cause disease in humans. Among them, four coronaviruses 229E, OC43, NL63 and HKU1 basically only cause common cold symptoms in immunocompromised people, while the other two, well known as SARS-CoV and MERS-CoV, can cause severe infectious diseases. Length of a single-stranded positive RNA genome at a 5′ end of the coronavirus is between 26.2 and 31.7 kb, which is the longest among all RNA viruses. Its genome has six to ten open reading frames (ORF). The first ORF contains two thirds of the genome and encodes and reproduces enzyme proteins, while the last third contains a fixed-order structural protein gene: (HE)-S-E-M-N. There are multiple ORFs encoding accessory proteins between these genes. The genome is packaged into a helical nucleocapsid surrounded by a host-derived lipid bilayer. This viral membrane contains at least three viral proteins, that is, spike protein (S), membrane protein (M) and envelope protein (E).
Among them, M protein and E protein are mainly involved in an assembly of the virus, while S protein mediates the virus to bind to receptors on host cell membrane and fuse with the host cell membrane. Therefore, the S protein plays an important role in tissue tropism, cell fusion and virulence of the virus, and is a main neutralizing antigen of the coronavirus. A receptor binding domain (RBD) of S protein of MERS-CoV and SARS-CoV is considered to be the most important antigen target region for inducing neutralizing antibodies in body. As a vaccine, RBD can make the neutralizing antibodies produced by stimulation of the body more focus on the receptor binding against the virus, which can improve immunogenicity and immune efficiency of the vaccine. MERS-CoV invades cells through RBD binding to a host cell receptor (CD26, also known as DPP4), and SARS-CoV enters cells through its RBD binding to a host cell receptor ACE2. As a core of the vaccine, it can make the neutralizing antibodies produced by stimulation of the body more focus on the receptor binding against the virus, thereby improving the immunogenicity and and neutralization efficiency of the vaccine. However, in previous studies, after vaccination in animal models, RBD monomer vaccine derived from MERS-CoV and SARS-CoV only elicited low levels of pseudovirus neutralizing antibodies.
Therefore, it is urgent to develop vaccines with high immunogenicity and neutralization efficiency against coronaviruses, especially SARS-CoV-2.
The technical problem to be solved by the present invention is to overcome the deficiencies of existing therapeutic drugs and vaccines against novel coronavirus, and to develop a safe and effective vaccine against SARS-CoV-2 as soon as possible to protect susceptible population. In the present invention, taken receptor binding domain (RBD) and fusion peptide (FP) of the virus jointly as double antigen fragments, and based on Helicobacter pylori multimeric protein (Helicobacter pylori_Ferritin, Ferritin), an antigen multimerization is realized and an RBD-FP antigen multimeric complex is constructed and developed. Specifically, both a receptor binding domain (RBD) and a fusion peptide (FP) of a virus are taken as double antigen fragments and are connected with a Helicobacter pylori multimeric protein (Helicobacter pylori_Ferritin, Ferritin(HP)) to form a fusion protein RBD-FP-HP_Ferritin, so that antigen multimerization is realized. At the same time, a signal peptide and a purification tag are added, and a self-assembled RBD-FP-HP_Ferritin protein is expressed through a plasmid transfection eukaryotic cell expression system (such as 293F cells), RBD-FP-HP_Ferritin monomers can be assembled into a spherical 24-mer nanoparticle through self-assembly of Ferritin (HP), displayed on surface of nanoparticle, which overcomes shortcomings of insufficient immunogenicity of RBD monomers, and can effectively cause a stronger immune response and produce antibodies neutralizing SARS-CoV-2 pseudovirus invading target cells. The vaccine of the present invention can significantly improve a neutralizing antibody level of the host against SARS-CoV-2; and the vaccine preparation method of the present invention is simple, the protein contains a His tag and is easy to purify, safety of Ferritin antigen as a carrier of nanovaccine has been proved in clinical trials registered by NIH, and the vaccine can be applied to clinical trials more quickly.
An objective of the present invention is to provide a 24-multimerized subunit novel coronavirus antigen constructed based on a receptor binding region of novel coronavirus (SARS-CoV-2) and a bacterial multimer.
Another objective of the present invention is to provide an application of the novel coronavirus antigen in preparation of novel coronavirus vaccine and anti-novel coronavirus medicament.
Another objective of the present invention is to provide a method for preparing the novel coronavirus antigen.
Another objective of the present invention is to provide a nucleotide sequence, a vector or a transgenic cell line that encodes and expresses the novel coronavirus antigen.
The above-mentioned objectives of the present invention are achieved through the following technical solutions.
The present invention first provides a method for improving antigen immunogenicity. The method is to take both a receptor binding domain (RBD) and a fusion peptide (FP) of a virus as double antigens, which are used as an antigen after fusion.
Further preferably, the method is to combine the receptor binding domain (RBD) and the fusion peptide FP of the virus with Helicobacter pylori multimeric protein (Helicobacter pylori_Ferritin, Ferritin (HP)) to form a new fusion protein RBD-FP-HP_Ferritin, as an antigen.
As a self-assembled globular protein, Ferritin has an amino terminal spacing of about 4.5-7.5 nm for every two adjacent subunits on its surface, which is suitable for loading antigens on an outer surface. Using such a characteristic that HP_Ferritin, a ferritin derived from Helicobacter pylori, enables to spontaneously form multimerization, and after the surface is loaded with antigens, it can induce strong humoral immune response and cellular immune response, it is a very ideal carrier, and can increase the number of antigens that can be carried by a single immunization, solving a disadvantage of weak immunity caused by RBD monomer vaccine.
In the solution for improving antigen immunogenicity of the present invention, taken receptor binding domain (RBD) and fusion peptide (FP) of the virus jointly as double antigen fragments, and based on Helicobacter pylori multimeric protein (Helicobacter pylori_Ferritin, Ferritin), an antigen multimerization is realized, which can overcome shortcomings of insufficient immunogenicity of RBD monomers, can effectively cause a stronger immune response, and can significantly improve the level of neutralizing antibodies of a host against SARS-CoV-2.
In the past antigen research, especially the SARS research, only immunogenicity of a certain segment, e.g. RBD region is focused, but the current research and development of related vaccines have all failed, so we consider using double segments for antigen immunization. The reasons for choosing RBD and FP are: (1) RBD is the region that binds to the receptor; (2) FP is the region that fuses with the receptor cell membrane. “Binding” and “fusion” constitute the two most critical and earliest steps for a virus to invade a cell. Using two domains to construct a fusion protein for immunization has not been reported in previous studies of single-segment vaccines. In addition, we also carried out multimerization of HP_Ferritin on the antigen fragments. Using a characteristic that HP_Ferritin (a ferritin derived from Helicobacter pylori) can spontaneously form multimerization, double antigens are aggregated together to form a nanoparticle, which further increase the number of antigens carried in a single immunization, so it can more fully and stably contact immune cells in the human body to stimulate the production of antibodies. The “double antigen+multimer” strategy of the present invention can achieve the effect of stimulating the body to produce an effective immune response more effectively, rapidly and stably in terms of quality (RBD+FP double antigen) and quantity (multimerization).
Preferably, the above-mentioned antigen of the present invention is preferably suitable for a coronavirus antigen, and the receptor binding domain RBD and the fusion peptide FP of the virus are a receptor binding domain RBD and a fusion peptide FP of a coronavirus.
Preferably, a novel coronavirus SARS-CoV-2 antigen is included, and the receptor binding domain RBD and the fusion peptide FP of the coronavirus are a receptor binding domain RBD and a fusion peptide FP of a novel coronavirus SARS-CoV-2.
More specifically, preferably it means that the novel coronavirus SARS-CoV-2 antigen is a surface spike protein (S protein) neutralizing antigen of novel coronavirus SARS-CoV-2, the receptor binding domain RBD and the fusion peptide FP of the coronavirus are a receptor binding domain RBD and a fusion peptide FP of a novel coronavirus SARS-CoV-2.
Specifically, an amino acid sequence of the RBD of the novel coronavirus SARS-CoV-2 is shown in SEQ ID NO: 1; an amino acid sequence of the FP is shown in SEQ ID NO: 2.
SEQ ID NO: 1 and SEQ ID NO: 2 can be directly linked to obtain a fusion protein RBD-FP.
Alternatively, SEQ ID NO: 1 and SEQ ID NO: 2 are linked by a hinge region Linker to form a new fusion protein RBD-FP. As an alternative preferred solution, the Linker may be GGSGGSGGSGGSGGGG. When the Linker is GGSGGSGGSGGSGGGG, an amino acid sequence of RBD and FP of the novel coronavirus SARS-CoV-2 are shown in SEQ ID NO: 3.
In addition, an amino acid sequence of Ferritin (HP) is shown in SEQ ID NO: 4.
SEQ ID NO: 3 and SEQ ID NO: 4 can be directly linked to obtain a new fusion protein.
Alternatively, SEQ ID NO: 3 and SEQ ID NO: 4 are linked by a hinge region Linker to form a new fusion protein RBD-FP-HP_Ferritin. As an alternative preferred solution, the Linker may be GSG. When the Linker is GSG, an amino acid sequence of the resulting fusion protein RBD-FP-HP_Ferritin is shown in SEQ ID NO: 5.
Further preferably, as an alternative embodiment, the method for improving antigen immunogenicity described in the present invention is to combine receptor binding domain (RBD) and fusion peptide FP of a virus with Helicobacter pylori multimeric protein (Helicobacter pylori_Ferritin, Ferritin(HP)) to form a fusion protein RBD-FP-HP_Ferritin, then add a signal peptide and a purification tag, and express an antigen through a eukaryotic expression system.
Preferably, the signal peptide is a secretory signal peptide (SP). Preferably, the purification tag is a His tag (His-tag). The signal peptide and the purification tag are added to an amino acid N-terminal of the RBD.
After adding the signal peptide and the purification tag, an amino acid sequence of a fusion of SP, His-tag, RBD and FP of new coronavirus SARS-CoV-2 is shown in SEQ ID NO: 6; the amino acid sequence of Ferritin (HP) is shown in SEQ ID NO: 4.
The SEQ ID NO: 6 and SEQ ID NO: 4 can be directly linked.
Alternatively, SEQ ID NO: 6 and SEQ ID NO: 4 are linked by a hinge region Linker to form a new fusion protein RBD-FP-HP_Ferritin. As an alternative preferred solution, the Linker may be GSG.
When the Linker is GSG, an amino acid sequence of the resulting fusion protein RBD-FP-HP_Ferritin is shown in SEQ ID NO: 7 (as shown in
That is, the present invention provides a SARS-CoV-2 antigen with improved immunogenicity containing a signal peptide and a purification tag, the antigen is a protein RBD-FP-HP_Ferritin using Helicobacter pylori ferritin to self-assemble proteins which have not been 24-multimerized (as shown in
The Helicobacter pylori multimeric protein (Helicobacter pylori_Ferritin, Ferritin (HP)) is a bacterial complex ferritin, and the bacterial complex ferritin forms a globular protein present in bacterium, which primarily acts to control a rate and location of polynuclear ferric oxide formation, via transport of hydrated iron ion and proton to and from a mineralized core. A globular form of ferritin is composed of a monomeric subunit protein (Ferritin), which is a polypeptide with a molecular weight of about 17-20 kD. The sequence of one such monomeric ferritin subunit is shown in SEQ ID NO: 4. These monomeric ferritin subunit proteins self-assemble into a globular ferritin protein containing 24 monomeric ferritin subunit proteins.
The fusion protein RBD-FP-HP_Ferritin can assemble RBD-FP-HP_Ferritin monomers into a spherical 24-mer nanoparticle through self-assembly of Ferritin (HP), displayed on surface of the nanoparticle, which can effectively elicit a stronger immune response from the receptor, producing antibodies that neutralize SARS-CoV-2 pseudovirus invading target cell. The 24-multimerized RBD-FP-HP_Ferritin of the present invention can overcome shortcoming of insufficient immunogenicity of RBD monomer, and significantly improve production of neutralizing antibodies of the receptor against SARS-CoV-2.
The present invention also provides a coronavirus antigen with improved immunogenicity, specifically a new self-assembled and 24-multimerized fusion protein RBD-FP-HP_Ferritin constructed by the above method.
The amino acid sequence of the novel coronavirus SARS-CoV-2 antigen (a new fusion protein RBD-FP-HP_Ferritin) is shown in SEQ ID NO: 5 (constructed by linking SEQ ID NO: 1 and SEQ ID NO: 2 with a hinge region GGSGGSGGSGGSGGG to obtain SEQ ID NO: 3, and then linking SEQ ID NO: 3 and SEQ ID NO: 4 by a hinge region GSG); or the amino acid sequence formed by adding a signal peptide and a purification tag is shown in SEQ ID NO: 7 (constructed by linking SEQ ID NO: 6 and SEQ ID NO: 4 by a hinge region GSG).
That is, as an alternative preferred embodiment of the present invention, the novel coronavirus SARS-CoV-2 antigen (a new fusion protein RBD-FP-HP_Ferritin) contains a signal peptide and a purification tag disclosed herein, RBD protein, FP protein, and self-assembled subunit protein Ferritin are linked in sequence, wherein the RBD-FP-HP_Ferritin protein can self-assembly into a nanoparticle that displays an immunogenic portion of the RBD-FP protein on its surface. After further safety and efficacy studies in animal models, the RBD-FP-HP_Ferritin vaccine has a potential to protect SARS-CoV susceptible population.
Therefore, an application of the coronavirus antigen in preparation of anti-coronavirus medicaments, specifically including an application in preparation of medicaments against novel coronavirus SARS-CoV-2, which is also within protection scope of the present invention.
As an alternative embodiment, an anti-SARS-CoV-2 vaccine can be prepared by using RBD-FP-HP_Ferritin protein in combination with a SAS adjuvant.
In addition, as an alternative embodiment, the application also includes an application in preparation of a kit; the kit contains the protein antigen, or a DNA molecule encoding the antigen, or a recombinant vector/expression kit/transgenic cell line/recombinant bacterium expressing the antigen.
In addition, the present invention further provides a recombinant vector, expression kit, transgenic cell line or recombinant bacterium expressing the above-mentioned antigen (fusion protein RBD-FP-HP_Ferritin).
Finally, the present invention further provides an alternative preparation method of the above antigen, specifically, at a 3′ end of a nucleotide sequence corresponding to amino acids as shown in direct linking or hinge linking of SEQ ID NO: 3 and SEQ ID NO: 4, or a nucleotide sequence corresponding to amino acids as shown in direct linking or hinge linking of SEQ ID NO: 6 and SEQ ID NO: 4, or a nucleotide sequence corresponding to amino acids as shown in SEQ ID NO: 5, or a nucleotide sequence corresponding to amino acids as shown in SEQ ID NO: 7, adding a translation terminator codon, cloned into an eukaryotic expression vector (as shown in
As an alternative embodiment, the eukaryotic expression system includes, but is not limited to, HEK293T cell, 293F cell, CHO cell, sf9 and other cell strains and cell lines that can be used to express eukaryotic proteins. Protocols for introducing corresponding proteins into the eukaryotic expression system include, but are not limited to, transfection, infection, transposition protocols, and the like.
As an alternative embodiment, the purification method is filtering the supernatant of cells expressing the antigen to remove cell debris, and then passing through a 10K ultrafiltration tube (Millipore) for preliminary purification, and then passing through a HisTrap HP nickel column (GE), Lectin column (GE) to capture the target protein, and finally purifying by molecular sieve chromatography using Siperose6 Increase10/300 GL column (GE) to obtain a high-purity target protein (as shown in
As an alternative embodiment, a buffer for ultrafiltration elution is: PBS buffer at pH 7.4.
As an alternative embodiment, a buffer for nickel column elution is: PBS at pH 7.4, containing 500 mM Imidazole.
As an alternative embodiment, a packing material of Lectin column (GE) is: Concanavalin A (Con A), Wheat germ agglutinin (WGA), and an eluent for column elution is: methyl-α-D-mannopyranoside, GlcNAc.
As an alternative embodiment, a buffer for molecular sieve chromatography is: PBS buffer at pH 7.4.
The nanovaccine obtained in the present invention is a purified 24-multimerized RBD-FP-HP_Ferritin protein; size of the 24-multimerized RBD-Ferritin protein is about 48 Kd under a non-reducing condition (without DTT added).
Finally, a nucleotide sequence encoding and expressing the above-mentioned antigen of the present invention, as well as a vectors or a transgenic cell line containing the nucleotide sequence, encoding and expressing the antigen, shall also fall within the protection scope of the present invention.
The present invention has the following beneficial effects.
In the present invention, a receptor binding domain (RBD) and a fusion peptide (FP) of a virus are taken together as a double-antigen fragment, and combined with Helicobacter pylori multimeric protein (Helicobacter pylori_Ferritin, Ferritin (HP)) to form a fusion protein RBD-FP-HP_Ferritin, so that antigen multimerization is realized, at the same time, a signal peptide and a purification tag are added, and a self-assembled RBD-FP-HP_Ferritin protein is expressed through a plasmid transfection eukaryotic cell expression system (such as 293F cells), RBD-FP can be assembled into a 24-multimerized nanovaccine through self-assembly of HP_Ferritin. This solution can overcome shortcomings of insufficient immunogenicity of RBD-FP monomers, and the resulting vaccine can significantly increase a level of neutralizing antibodies against SARS-CoV-2 in the host. In the present invention, the experiment of immunizing Balb/c mice with RBD-FP-HP_Ferritin nanoantigen has confirmed that the antibody produced has an ability to strongly block SARS-CoV-2 pseudovirus from invading target cells.
In addition, the vaccine preparation method of the invention is simple, the protein contains a His tag and is easy to purify, and the safety of Ferritin antigen as a carrier of nanovaccine has been proved in clinical trials registered by NIH, and the vaccine can be quickly applied to clinical trials.
The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form.
Unless otherwise specified, reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.
The schematic diagram of self-assembly of RBD-FP-HP_Ferritin fusion proteins into a nanoparticle, and the schematic diagram of structure is as shown in
Specifically, construction and preparation method of fusion protein RBD-FP-HP_Ferritin is as follows:
1. Preparation of Vector Expressing RBD-Ferritin Antigen
A translation terminator codon was added at a 3′ end of a nucleotide sequence of RBD-FP-HP_Ferritin (as shown in SEQ ID NO: 4), which was then cloned and added between Xho I and Xba I enzyme cleavage sites in an Intron and WPRE expression-enhanced expression vector (pcDNA3.1-Intron-WPRE), and an expression vector pcDNA3.1-Intron-WPRE-RBD-FP-Ferritin(HP)-IRES-GFP (as shown in
The recombinant plasmid was transformed into DH5a competent cells, cultured at 37° C. overnight, and positive clones were screened and identified by PCR. An endotoxin-depleted plasmid was extracted, then after enzyme cleavage and verification by sequencing, it was used for expression of nanoantigen protein (as shown in
2. Purification of RBD-FP-HP_Ferritin Nanoantigen
The supernatant of cells expressing RBD-FP-HP_Ferritin was filtered through a 0.22 μm filter to remove cell debris. After ultrafiltration through a 10K ultrafiltration tube, the filtered cell supernatant was combined with Histrap-excel at 4° C. for 30 minutes, and a HisTrap excel nickel column was used for crude purification.
Afterwards, firstly 50 ml was washed with PBS (pH 7.4) buffer and low-concentration imidazole buffer (PBS, 50 mM Imidazole, pH 7.4) to remove flow-through impurity protein. Thereafter, target protein was eluted by high imidazole-containing buffer (PBS, 500 mM Imidazole, pH 7.4). Subsequently, the target protein was enriched using a Lectin Agarose column (GE) packed with Con A and WGA at a ratio of 1:1.
Elution peaks of RBD-FP-HP_Ferritin 24-mer were collected and combined, and finally purified by molecular sieve chromatography using a Siperose6 Increase10/300 GL column (GE) to obtain a 24-multimerized RBD-FP-HP_Ferritin protein with a purity greater than 99% (as shown in
The RBD-FP-HP_Ferritin antigen obtained in Embodiment 1 was diluted with physiological saline to 100 μg/ml according to Table 1, and emulsified in groups with an equal volume of adjuvant SAS. 6-8 week-old Balb/C mice were then immunized in groups. The immunization strategy was as shown in
1. Preparation of Pseudovirus:
According to a sequence published by NCBI, Spike protein of SARS-CoV-2 was synthesized and inserted into a pcDNA3.1 expression vector. 293T cells were co-transfected by the expression vector of SARS-CoV-2 Spike protein with pHIV-luciferase and psPAX2 plasmid. After 5 hours of transfection, cells were washed twice with PBS, and then continued to culture with replaced serum-free DMEM medium. After 48 hours, a supernatant was collected and centrifuged to remove cell debris. After dissolving with a small volume of serum-free DMEM, HIV-luc/SARS-CoV-2-S pseudovirus was obtained.
The pseudovirus can effectively simulate a process of wild-type SARS-CoV-2 invading cells. When it infects production cells or target cells, expression of luciferase reporter gene carried by SARS-CoV-2 pseudovirus can accurately reflect results of virus infection, so that results of the experimental system can be read accurately and quickly, which can be used as an excellent antibody neutralization titer monitoring system (as shown in
2. Pseudovirus TCID 50 Assay
The virus solution collected in the previous step was diluted 5-fold and added to HEK293T cells in a 96-well plate. After 4 hours of infection, the virus solution was discarded, cells were washed twice with PBS, replaced with DMEM complete medium containing 10% serum. After 48 hours, the medium was discarded, washed twice with PBS, added with a cell lysis buffer, and lysed by shaking for 30 minutes. After freeze-thawing once at −80° C., 30 μl of each well was taken to detect a luciferase activity value using GloMax 96 (Promega). TCID 50 was calculated by Reed-Muech method.
3. Neutralization Test
The purified antibody was diluted 2-fold, mixed with pseudovirus of TCID 50 final concentration, and co-incubated at 37° C. for 1 hour. The mixture was added to HEK293T cells with a density of about 70% in a 96-well plate. After 48 hours, culture medium was discarded, cells were washed twice with PBS, cell lysis buffer was added, and the luciferase activity value was detected.
4. Result Analysis
Results are shown in
The results show that combination of RBD-FP-HP_Ferritin of the present invention and SAS adjuvant can stimulate humoral immunity of mice 10 days after once immunization, which is less than neutralizing antibody titer stimulated by 24-mer group as a parallel control, and there is a significant difference.
The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, and simplifications shall be equivalent replacement modes, which are all included in the protection scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010113700.7 | Feb 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/077676 | 3/4/2020 | WO |
