Patent Application
20230127808
This application includes a sequence listing in computer readable form (a “txt” file) that is submitted herewith on an ASCII text file named Sequence Listing.txt, created on Mar. 15, 2022 and 6,399 bytes in size. This sequence listing is incorporated by reference herein.
The present invention relates to an Ad7-vectored vaccine for preventing SARS-CoV-2 infection.
Vaccines are the most economical and effective intervention measures to prevent and control SARS-CoV-2 infection. The published results obtained from the alignment of more than 100 SARS-CoV-2 virus genomes have shown that the overall mutation degree of the virus is relatively low, and no recombination phenomenon has been found. Therefore, SARS-CoV-2 vaccines, if successfully developed, will protect human from SARS-CoV-2 infection, thereby preventing the outbreak of a new epidemic.
In the virus particle structure of the SARS-CoV-2 coronavirus, S protein which constitutes the “crown” is an obvious target, and has become the research focus of most research teams. Through computer simulation of the three-dimensional structure of S protein, some research teams have successfully revealed the relationship between the S protein and an ACE2 receptor during the invasion of cells. Therefore, the S protein plays an important role in mediating the binding of a virus particle to a host cell receptor and inducing a neutralizing antibody.
The complete sequence of SARS-CoV-2 is as shown in NC 045512.2, in which the 21563..25384 nucleic acid is a coding sequence of Spike protein (S). The S protein is a major structural protein of a virus particle, and plays an important role in mediating the binding of a virus particle to a host cell receptor and inducing a neutralizing antibody. Therefore, for vaccines with S protein as an antigen, including nucleic acid vaccines, subunit vaccines and virus-vectored vaccines, the expression levels and protein structures of S proteins determine the efficacies of such vaccines.
However, experiments have shown that the expression level of the gene of Spike protein (S) of natural SARS-CoV-2 in a human kidney cells HEK293 is very low. Therefore, if the original S codons are used for expression as the antigen, the vaccine may be ineffective or have a low titer, which is not enough to resist the virus infection.
Adenoviruses are common vectors in vaccine research and gene therapies, and have been widely used in the biomedical field. Compared with other virus vectors, the adenoviruses have a low toxicity, and infection with adenoviruses only cause mild cold symptoms. At present, most adenovirus-vectored vaccines developed by research teams are based on adenovirus type 5. Preformed antibodies against adenovirus type 5 vectors have a very high proportion in the human population, which will affect the titer of an adenovirus type 5 vectored vaccines to a certain extent.
An object of the present disclosure is to provide an Ad7-vectored vaccine for preventing SARS-CoV-2 infection, in order to overcome at least one deficiency of the prior art.
The present disclosure blocks SARS-CoV-2 infection by integrating an optimized S gene onto an Ad7 vector, especially a replication-defective Ad7 vector, infecting cells via the adenovirus, presenting an novel coronavirus antigen, and producing a specific immune response in an organism after immunization.
The technical solutions used in the present disclosure are as follows:
An adenovirus vectored vaccine for preventing SARS-CoV-2 infection, comprising an Ad7 vector loaded with a nucleic acid sequence as shown in SEQ ID NO: 1.
To further improve the safety of the vaccine, in some embodiments, the Ad7 vector is a replication-defective Ad7 vector.
In some embodiments, the replication-defective Ad7 vector is a replication-defective Ad7 vector with genes in E1 and E3 regions deleted.
In some embodiments, the nucleotide sequence can be expressed as protein in a human-derived cell or the human body.
In some embodiments, in the human body, the protein is capable of:
In some embodiments, the adenovirus vectored vaccine further comprises a pharmaceutically acceptable adjuvant, vector, diluent or excipient. At least one from the group consisting of suitable adjuvant, vector, diluent or excipient may be selected according to specific needs.
In some embodiments, the adenovirus vectored vaccine further comprises at least one medicament useful for treating COVID-19.
In some embodiments, the dosage form of the adenovirus vectored vaccine includes, but not limited to common vaccine dosage forms, such as an oral preparation, an injection, an aerosol inhalant.
In some embodiments, the adenovirus vectored vaccine may also be used in combination with other vaccines.
To further improve the safety of products, in some embodiments, the Ad7 vector is capable of regulating the expression of the nucleic acid sequence as shown in SEQ ID NO: 1.
In some embodiments, the transcription direction of the nucleic acid sequence shown in SEQ ID NO: 1 is opposite to those of the other genes in the Ad7 vector. In this way, the nucleic acid sequence shown in SEQ ID NO: 1 can be better expressed.
The beneficial effects of the present disclosure are as follows:
Some embodiments of the disclosure have good safety and are convenient to use.
Experiments have shown that the vaccine is capable of producing more S protein in human cells, which is expected to be developed as an Ad7-vectored vaccine for preventing SARS-CoV-2 infection.
Some embodiments of the present disclosure may be used in combination with other vaccines, and may also be used as therapeutic vaccines for COVID-19. When a patient is vaccinated with the vaccine of the present disclosure at the initial stage of infection, the vaccine can quickly induce an immune response in the human body, thereby achieving a therapeutic effect.
The amino acid sequence of Spike (S) protein of SARS-CoV-2 is shown in YP_009724390.1, which is denoted as NB1.
The pre-mRNA transcribed by eukaryotic cells can produce various mRNA splicing isoforms by various splicing modes (by selecting different splicing site combinations), which ultimately leads to various proteins resulting from the same gene sequence. This is very unfavorable for the expression of the protein. By performing codon optimization on the wild-type natural nucleic acid sequence and removing potential variable splicing sites based on self-owned technology, the inventors ensured the uniqueness of the expression of the protein and reduced the difficulty in the subsequent purification of the protein. The optimized nucleic acid sequence is denoted as NB2, and the specific sequence is shown in SEQ ID NO: 1:
Construction of Ad7 vector for S protein expression (pAd7-NB2):
Using NB1 and NB2 as templates respectively, an NB1 fragment was obtained through PCR amplification with primers NB1-F and NB1-R, and an NB2 fragment was obtained through PCR amplification with primers NB2-F and NB2-R. A vector plasmid backbone pGK was obtained through PCR amplification by using CMV-R and BGH-F as primers and using pGA1-EGFP plasmid as a template. Then, in vitro two-fragment recombination with the NB1 and NB2 fragments was carried out, respectively, by using a homologous recombination enzyme (Exnase), to obtain pGK-NB1 and pGK-NB2. The pGK-NB2 was linearized by using Bstzl7I and SgarAI, and was subjected to homologous recombination with pAd7AE1AE3 (i.e., Ad7 empty vector), which was digested to delete the unique restriction site in the E1 region and linearized, by using competent BJ5183 to construct a pAd7-NB2 vaccine vector.
PCR conditions: 95° C. for 3 min; 95° C. for 30 s; 60° C. for 30 s; 72° C. for 2 min; 30 cycles; and 72° C. for 5 min.
Rescue and production of Ad7-NB2 vector:
1) according to a conventional method, pAd7-NB2 was linearized with AsiSI, recovered by precipitation in ethanol, and transfected into 293 cells by means of cationic lipofection;
2) 8 hours after the transfection, 2 ml of DMEM medium containing 5% fetal bovine serum was added, incubated for 7-10 days, and cytopathic effect observation was carried out; 3) after the cytopathic effect was observed, the cells and culture supernatant were collected, freeze-thawed for three times in a water bath at 37° C. and liquid nitrogen, and centrifugated to remove cell debris, and then a 10 cm-dish was infected with the supernatant;
4) after 2 or 3 days, the cells and culture supernatant were collected, freeze-thawed for 3 times, centrifugated to remove cell debris, and then 3-5 15 cm-dishes were infected with the supernatant;
5) after 2 or 3 days, the cells were collected, freeze-thawed for 3 times, and centrifugated to remove cell debris;
6) 30 15 cm-dishes were infected with the supernatant, after 2 or 3 days, the cells were collected, freeze-thawed for 3 times, and centrifugated to remove cell debris;
7) the supernatant was added to a cesium chloride density gradient centrifuge tube;, and centrifuged at 4° C., 40000 rpm, for 4 hours, then a virus band was pipetted out, desalted, and subpackaged; and
8) the virion titer was determined by means of OD260 absorbance, with the calculation formula being: virus concentration=OD260×dilution multiple×36/genome length (Kb), and the virus stock solution was cryopreserved at −80° C.
Detection of Spike Gene Expression:
According to a conventional method, 2.5 μg of pGK-NB1 and pGK-NB2 were respectively transfected using cationic liposomes, and after 48 hours, the cells were collected. The HEK293 cells were infected with Ad7-NB2 and Ad7 empty vector viruses, and after 24 hours, the cells were collected. The four samples mentioned above were processed according to the conventional Western Blot method, and were detected for the protein (
It could be seen from
Immunogenicity Evaluation of Ad7-NB2 Vaccine:
1. Immunized Mice
An immunogenicity evaluation scheme of the Ad7-NB2 vaccine in mice was designed, as shown in Table 1, and immunization was carried out according to the designed immunization scheme.
Balb/c mice, 6 to 8 weeks old, were divided into 2 groups with 5 mice in each group. The G1 group was an experimental control group and mice in the G1 group were intramuscularly injected with GT101 (the virus stock solution) at a dosage of 5×109VP/animal, while the G2 group was an experimental group and mice in the G2 group were intramuscularly injected with the Ad7-NB2 vaccine at a dosage of 5×109VP/animal. At the 28th day after immunization, blood was taken from orbit, and serum was separated. Binding antibody levels of the S protein of SARS-ncov2 were detected. From
2. Immunized Macaque Monkeys
The macaque monkeys were obtained from Guangdong Landau Biotechnology Co. Ltd. The vaccinated macaque monkeys were 2 to 3 years old. The macaque monkeys were randomly divided into 3 groups, with 2 monkeys in each experimental group, and 4 monkeys in each control group, specifically as shown in Table 2:
Macaque monkeys were immunized with the vaccine prepared from the Ad7-NB2 virus strain. Blood was taken at the 14th day and the 18th day after immunization to determine antibody binding titers by ELISA. The peripheral blood was separated and detected for a cellular immune response by ELISpot assay.
Experimental Results:
(1) Binding Antibodies
At the 14th day and the 18th day after intramuscular vaccination, significant presence of S-specific IgG and RBD-specific IgG could be detected in the sera of all the macaque monkeys intramuscularly injected with 1×1011VP, and among the two macaque monkeys in the immunization group, significant presence of anti-Ω IgG could be detected in one macaque monkey (
At the 14th day after the intranasal vaccination, among the two macaque monkeys vaccinated with 1×1011 VP in the immunization group, significant presence of S-specific IgG and RBD-specific IgG could be detected in one macaque monkey. S2-specific IgG could be detected in all macaque monkeys in the immunization group. At the 18th day after immunization, S-specific IgG and S2-specific IgG could both be detected. Among the two macaque monkeys in the immunization group, significant presence of RBD-specific IgG could be detected in one monkey (
The difference between the antibody titers induced by intramuscular injection immunization and intranasal immunization was small.
(2) Cellular Immunity
To determine whether Ad7-NB2 could also induce a cellular immune response in non-human primates (NHPs), the responses of S-specific IFN-γ secreting cells from the peripheral blood mononuclear cells (PBMCs) to S1 and S2 peptide libraries were detected.
The Results Showed that:
1) At the 18th day after the intramuscular injection vaccination, all macaque monkeys intramuscularly injected with 1×1011VP had cellular immune responses to the S1 and S2 peptide libraries (
2) At the 18th day after the intranasal vaccination, among the two vaccinated macaque monkeys, one macaque monkey showed weak cellular immune response to the S1 peptide library, and neither of them had obvious response to the S2 peptide library at the 18th day (
Therefore, in the macaque monkeys, the cellular immune response was mainly directed at the S1 region. These results indicate that the intramuscular injection immunization of the vaccine could cause systemic cellular immune response to S protein, especially to the S1 region, while the systemic cellular immune response caused by the mucosal immunization of the vaccine was relatively weak.
The above experimental results showed that the vaccine could induce cellular immunity in macaque monkeys, which may further enhance the protective effect on the body.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010182122.2 | Mar 2020 | CN | national |
| 202010293658.1 | Apr 2020 | CN | national |
| 202011031062.0 | Sep 2020 | CN | national |
The present application is a national phase entry under 35 USC § 371 of International Application PCT/CN2021/074837, filed on Feb. 2, 2021, which claims the benefit of and priority to Chinese Patent Application No. 2020101821222, No. 2020110310620 and No. 2020102936581, filed on Mar. 16, 2020, Sep. 27, 2020 and Apr. 15, 2020 respectively, the entire disclosures of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/074837 | 2/2/2021 | WO |
