Patent Application
20230293679
The invention belongs to the field of genetic engineering and biomedical technology, and specifically relates to vaccines, for example, a vaccine comprising a fusion protein containing an interferon-target antigen-immunoglobulin Fc region (antibody) as framework. The vaccine of the present invention can be used as a vaccine platform for preventing hepatitis B virus (HBV) infection, human papilloma virus (HPV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), severe acute respiratory syndrome coronavirus 2 (SARA-COV2), influenza virus infection and the develop of HPV, EBV-related tumors and for treating chronic hepatitis B (CHB) infection and HBV, HPV, and EBV-related tumors.
There are about 257 million chronic HBV infections in the world, and about 887,000 people die each year from end-stage liver diseases caused by HBV, including liver failure, liver cirrhosis, and hepatocellular carcinoma[1-3]. About 30% of liver cirrhosis is caused by HBV, and about 40% of hepatocellular carcinoma (HCC) is caused by HBV[4]. HBV infection remains a major public health problem worldwide. However, there is still no effective treatment strategy for chronic hepatitis B. The existing HBV treatment methods mainly include antiviral drugs (nucleoside/nucleotide analogs) and interferon. Although they have certain therapeutic effects, they usually cannot induce an effective immune response, so that HBV infection cannot be completely eliminated; moreover, long-term dosing may lead to significant side effects, and antiviral drugs will also lead to drug resistance. Chronic HBV infection is one of the main diseases that threaten human health. It is imminent to explore effective immunotherapy strategies for chronic hepatitis B. The development of therapeutic vaccines for chronic hepatitis B has very important social and economic significance.
Seasonal influenza causes severe illness in 1-4 million persons and kills 200,000-500,000 persons annually[5]. The best way to prevent and control influenza is through the vaccination, which reduces the incidence of illness and reduces the severity of infection, especially in young children and the elderly, who are at high risk of complications from influenza. Even though currently approved flu vaccines confer good protection effect in healthy young adults, there are still some issues that need to be addressed. For example, the production of some vaccines depends on chicken embryos, such as inactivated influenza vaccines and attenuated influenza vaccines. A disadvantage of these vaccines is that if the prevailing virus strains are of poultry origin, the epidemic of the disease will lead to an increase in demand for vaccines and chicken embryos, and thus leading to lack of chicken embryo supply[6]. Another disadvantage is that the production of these vaccines requires enormous amount of time. Elderly people are more prone to severe syndromes of influenza virus, and standard vaccines are generally not effective for the elderly, whose immune system gradually weakens with age[7]. In view of the problems encountered by current influenza vaccines, for the prevalence of influenza viruses, there is an urgent need for an influenza vaccine that has a strong immunogenicity, does not depend on chicken embryos, and can be produced quickly.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pathogen that caused the pandemic of 2019 coronavirus disease (COVID-19). The clinical symptoms caused by SARS-CoV-2 mainly include asymptomatic infection, mild flu-like symptoms, pneumonia and severe acute respiratory distress syndrome, which in severe cases may cause death in infected patients. At present, there is no specific medicine against SARS-CoV-2, and the vaccine is the basic countermeasure to control and end the SARS-CoV-2 pandemic[9]. In addition, the emergence of SARS-CoV-2 mutants poses new challenges to the existing candidate vaccines and the control of epidemic[10]. Therefore, powerful vaccines that are also effective for SARS-CoV-2 mutants are urgently needed in the current epidemic situation.
The linkage of an antigen to Fc region of an immunoglobulin will significantly increase the half-life of the antigen, and the Fc region of the immunoglobulin can bind to Fc receptors on the surface of antigen-presenting cells to promote the processing and presenting of the antigen by antigen-presenting cells[11-13]. Type I interferon has many biological activities as an antiviral cytokine, which includes the stimulation of immune cells[14]. IFNα can strongly induce the differentiation and activation of human DC cells[15]. Upon acting on immature DCs, type I interferon can promote the expression of MHC molecules and co-stimulatory molecules on the surface of DCs, such as MHC class I, CD80 and CD86, thereby enhancing the ability of DCs to activate T cells[16-18]. It has been reported that type I interferon can promote the antigen-presenting ability of DCs after infection with vaccinia virus and Lymphocytic ChorioMeningitis Virus (LCMV)[19-21]. In addition, type I interferon can promote the migration of DCs to lymph nodes by up-regulating the expression of chemokine receptors after acting on DCs, thereby promoting the activation of T cells[22, 23]. Recently, more and more studies have shown that type I interferon can be used as an immune adjuvant. The study by Le Bon et al. showed that when mice were immunized with a weak immunogen, type I interferon exhibited a strong immune adjuvant effect in mice and induced long-lasting antibodies and immune memory[24], the author also found that the main cell populations in which type I interferon exerted its effect were DC cells. At the same time, antibodies are used to targeted deliver vaccines to DCs to stimulate DC activation and cross-presentation functions, which will further enhance the activity and potency of the vaccines.
There is a need for the present invention to provide a vaccine platform that enhances the response to viruses, bacteria or tumor antigens.
Vaccines are an effective way to prevent and control major outbreaks of infectious diseases. There are various types of vaccines, one of which is protein subunit vaccines. In general, simple protein subunit vaccines generally have poor immunogenicity, which often limits the use of protein subunit vaccines. Therefore, a universal protein subunit vaccine platform is urgently needed. According to the impact of immunoglobulin Fc region and type I interferon on the immune system, the inventors propose an interferon alpha-viral antigen, bacteria or tumor-immunoglobulin Fc region fusion protein vaccine platform to enhance the immune response to viruses, bacteria or tumor antigens. The present invention provides a type I interferon-protein antigen-immunoglobulin Fc vaccine platform, wherein the type I interferon can promote antigen-presenting cells to allow maturation and migration so as to better play the role in antigen presentation and T cell activation. On the other hand, the Fc moiety of the vaccine platform can bind to the Fc receptors on the surface of antigen-presenting cells to enhance the uptake of antigens by antigen-presenting cells, thereby further enhancing antigen-presenting cells to function. The present inventors propose that the fusion of Th cell helper epitopes can further enhance the immune response effect of the vaccine of type I interferon-protein antigen-immunoglobulin Fc, and thus the Th cell helper epitope is an important element of the vaccine. The present inventors propose that anti-PD-L1 and other antibodies can be used to replace Fc, and the vaccine can be delivered to DCs to stimulate DC activation and cross-presentation, which will further enhance the activity and potency of the vaccine. As a novel vaccine platform, the vaccine platform of the present invention can be used as a prophylactic and therapeutic vaccine for diseases such as viral infections, bacterial infections or tumors.
In some embodiments, the present invention provides a vaccine comprising a fusion protein containing an interferon-target antigen-immunoglobulin Fc region (or antibody) (and an additional Th epitope). In some embodiments, the present invention also provides use of the fusion protein containing an interferon-target antigen-immunoglobulin Fc region (or antibody) (and an additional Th epitope) for the preparation of prophylactic or therapeutic compositions or kits (such as medicaments or vaccine compositions or kits). The vaccine of the present invention can be produced by eukaryotic cell expression systems, and inoculated by means of subcutaneous/muscular or intranasal or other immunization routes. For the fusion polypeptide of the present invention, the antibody (Ab for short) as a structural unit is not particularly limited, and may include, for example, a complete antibody or a fragment of antibody, such as an antibody heavy chain and light chain, or a single-chain antibody, and may be antibodies for DC targeting activation, including anti-PD-L1, anti-DEC205, anti-CD80/86 and other antibodies.
In some embodiments, the target antigen described herein is not particularly limited and may be any appropriate antigen. In some embodiments, the target antigens described herein can be, for example, tumor antigens and/or pathogen antigens (e.g., viral or bacterial antigens). In some embodiments, the target antigen described herein may be, for example, a tumor antigen, such as a protein molecule highly expressed by tumor cells, for example, human epidermal growth factor receptor 2 (HER2/neu), epidermal growth factor (EGFR).
In some embodiments, the target antigen used in the vaccine of the present invention can be, for example, a mutated target antigen that is different from the wild type. In some embodiments, the target antigen described herein can be, for example, mutants of tumor antigens and/or pathogen antigens such as viral or bacterial antigens. In some embodiments, the target antigen can be, for example, full length or S1 region of the S protein of SARS-COV-2 virus, for example, the target antigen can be the antigen as shown in SEQ ID NO. 76 or SEQ ID NO. 77. Herein, the wild-type target antigen refers to viruses or other infectious agents encoded by wild-type genes or immunogenic proteins expressed by tumors (the wild-type gene refers to the prevalent allele in nature, and is often used as a standard control gene in biological experiments), for example, Spike protein (S protein) derived from original wild-type strain of SARS-CoV-2. Herein, the mutated target antigen (mutant) refers to mutated viral proteins expressed by mutant virus strains and encoded by mutated gene derived from the wild-type genes, for example, the point mutations of S protein in different mutant SARS-CoV-2 that have been found include: 69-70 deletion, Y144 deletion, 242-244 deletion, L18F, D80A, D215, R246I mutation in NTD region, and K417, E484, N501Y mutation, L452R mutation, T478K mutation, D614G, H655Y mutation in RBD region. For example, these point mutations exist in different combinations in British SARS-CoV-2 B.1.1.7 (Alpha) mutant strains, South Africa B.1.351 (Beta) mutant strains, Brazil P1 (Gamma) mutant strains, India B.1.617, B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.617.3 mutant strains, California B.1.429 mutant strains and other mutant strains. In some embodiments, mutated target antigens may include for example natural point mutation/deletion mutation/addition mutation/truncation, artificial point mutation/deletion mutation/addition mutation/truncation, any combination of natural or artificial mutations, subtypes generated by mutations, wherein the target antigen may be a tumor antigen, a pathogen antigen, such as a virus (e.g., SARS-COV-2) or a bacterial antigen. In some embodiments, the target antigen used in the vaccine of the present invention is a mutated viral antigen, for example, the mutated viral antigen can be a mutant of SARS-COV-2, including for example natural point mutation/deletion mutation/addition mutation/truncation, artificial point mutation/deletion mutation/addition mutation/truncation, any combination of natural or artificial mutations, subtypes generated by mutations, derived from SARS-COV-2 protein (such as one or more of S protein, N protein, M protein, E protein); for example, the mutated viral antigen can be a mutant of the full length of S protein, the S1 region, and the RBD region; for example, the mutated viral antigen may include one or more of the following mutations in S protein of SARS-COV-2: 69-70 deletion, Y144 deletion, 242-244 deletion, L18F, D80A, D215, R246I mutation in NTD region, K417, E484, N501Y mutation, L452R mutation, T478K mutation, D614G, H655Y mutation in RBD region; for example, the mutated viral antigen may include mutations present in the British B.1.1.7 (Alpha) mutant strain, the South Africa B.1.351 (Beta) mutant strain and the Brazil P1 (Gamma) mutant strain, India B.1.617, B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.617.3 mutants, and California B.1.429 mutant; for example, the mutated viral antigen may contain a mutant shown in any one of SEQ ID NO. 79, SEQ ID NO. 80, and SEQ ID NO. 81; the mutated viral antigen may contain a mutant comprising a sequence shown in any one of SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, and SEQ ID NO. 84. Herein, unless otherwise clearly stated or clearly limited by the context, the target antigen herein generally includes wild-type target antigens and mutant target antigens.
The object of the present invention is to provide a vaccine platform, which consists of an interferon (IFN) and a tumor, bacterium or virus antigen (hepatitis B virus Pres1 antigen, SARS-COV2 RBD antigen, influenza HA antigen, human papillomavirus HPV E7 antigen, hepatitis B virus surface antigen (HBsAg) antigen or peptide fragment, herpes zoster virus (VZV) gE antigen, Epstein-Barr virus (EBV) EBNA1/LMP2/gp350, herpes simplex virus 2 (HSV-2) gD antigen, HIV gp120 antigen-immunoglobulin Fc region (or antibody) (and an additional Th epitope). The fusion protein can be a homodimeric or heterodimeric protein. In the case that the fusion protein is in the form of a dimer, the interferon, the target antigen, and the immunoglobulin Fc region (or antibody Ab) as structural units can exist in the first polypeptide chain and/or the second polypeptide chain, and the existence of each structural unit is not particularly limited, for example, they can all exist in one chain, or any one or more structural units can exist in one chain, while other one or more structural units can exist in another chain. The interferon of the present invention can be selected from type I interferon, type II interferon and type III interferon, such as IFN-α, IFN-β, IFN-γ, IFN-λ1 (IL-29), IFN-λ2 (IL-28a), IFN-λ(IL-28b) and IFN-ω; the IFN can be derived from human or mouse; preferably type I interferon IFN-α (SEQ ID NO. 1, SEQ ID NO. 21, SEQ ID NO. 22).
The immunoglobulin Fc region of the present invention can be selected from the constant region amino acid sequences of IgG1, IgG2, IgG3 and IgG4/or IgM, preferably IgG1 (SEQ ID NO. 2, SEQ ID NO. 23, SEQ ID NO. 24).
The fusion polypeptide of the present invention may also optionally comprise one or more Th cell helper epitopes and/or linking fragments (linkers). For example, when the fusion protein is in the form of a dimer, optionally the fusion protein can also comprise one or more Th cell helper epitopes and/or linking fragments in any one or two chains of the homodimer or heterodimer (i.e. the first polypeptide chain and/or or the second polypeptide chain). As known to those skilled in the art, the various structural units of the fusion protein can be connected by appropriate linking fragments (linkers). The linking fragments that can be used in the vaccine of the present invention are not particularly limited, and can be any suitable peptide fragments known in the art. The linking fragments of each structural unit in the present invention can be flexible polypeptide sequences, and can be linking fragments 1 and 2, for example as shown in the amino acid sequences of SEQ ID NO. 4 and SEQ ID NO. 25.
The N-terminal of the polypeptide sequence composed of each structural unit in the present invention contains a corresponding signal peptide capable of promoting protein secretion, for example as shown in the amino acid sequence of SEQ ID NO. 5.
Preferred antigens described in the present invention include hepatitis B Pres1 antigen, including ad subtype (SEQ ID NO. 6), ay subtype (SEQ ID NO. 26), HBV HBsAg antigen (various subtypes and peptide fragments), including adr subtype (SEQ ID NO. 7), adw subtype (SEQ ID NO. 27), ayw subtype (SEQ ID NO. 28), SARS-COV2 RBD antigen (SEQ ID NO. 8), influenza virus HA antigen (SEQ ID NO. 9), HPV E7 antigen (SEQ ID NO. 10); herpes virus VZV-gE antigen (SEQ ID NO. 91), EBV-gp350 antigen (SEQ ID NO. 92), HSV-2-gD antigen (SEQ ID NO. 93).
The homodimeric protein described in the present invention comprises a first polypeptide and a second polypeptide, and the first polypeptide and the second polypeptide are completely identical. The order of the elements from N-terminal to C-terminal in the first polypeptide and the second polypeptide is IFN-tumor or virus antigen (hepatitis B Pres1 antigen, SARS-COV2 RBD antigen, influenza HA antigen, HPV E7 antigen, HBsAg antigen, VZV-gE antigen, EBV EBNA1/LMP2/gp350, HSV-2-gD antigen, HIV gp120 antigen)-immunoglobulin Fc region; or a polypeptide containing a Pan epitope. The homodimeric protein of the present invention comprises the sequences as shown in SEQ ID NO. 11, 12, 13, 14, 29, 30, 31, 32, 38, 39, 40, 47, 48, 49, 50, 51, 56, 57, 59, 58, 65, 66, 67, or 68.
The heterodimer of the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not identical; the first polypeptide, from the C terminal to the N terminal, is respectively IFN-immunoglobulin Fc region, and comprises an amino acid sequence as shown in SEQ ID NO. 15, 33, 42, 51, 60, or 69; the second polypeptide, from the C terminal to the N terminal, is respectively a tumor or virus antigen (Hepatitis B Pres1 antigen, SARS-COV2 RBD antigen, influenza HA antigen, HPV E7 antigen, VZV-gE antigen, EBV EBNA1/LMP2/gp350, HSV-2-gD antigen, HIV gp120 antigen)-immunoglobulin Fc region, and comprises an amino acid sequence as shown in SEQ ID NO. 16, 17, 18, 19, 34, 35, 36, 37, 43, 44, 45, 46, 52, 53, 54, 55, 61, 62, 63, 64, 70, 71, 72, or 73.
The present invention also provides a nucleotide sequence encoding the above IFN-tumor or virus antigen (hepatitis B Pres1 antigen, HBsAg antigen or peptide, SARS-COV2 RBD antigen, influenza HA antigen, HPV E7 antigen, VZV-gE antigen, EBV EBNA1/LMP2/gp350, HSV-2-gD antigen, HIV gp120 antigen)-immunoglobulin Fc vaccine platform.
The present invention also relates to a nucleotide fragment encoding the vaccine platform and fusion protein.
The present invention also relates to a preparation method of the fusion protein or vaccine platform, for example, the preparation method includes the following steps:
The present invention also includes the application of the vaccine platform; the vaccine platform can be used as a prophylactic vaccine for hepatitis B, a therapeutic vaccine for hepatitis B, a prophylactic vaccine for influenza, a prophylactic vaccine for SARA-COV2, influenza, HPV, VZV, EBV, HSV-2, and HIV, and a prophylactic vaccine for HPV and EBV-related tumors.
The present invention includes adjuvants used in the vaccine platform, wherein the adjuvants include aluminum adjuvant (Alum), Toll-like receptor 4 activator ligand MPLA, Toll-like receptor 9 ligand, M59, oligodeoxy Nucleotides (CpG-ODN) and Freund's adjuvant.
The present invention includes the clinical use of the vaccine platform as an HBV therapeutic vaccine in combination with hepatitis B virus envelope protein HBsAg vaccine in the treatment of chronic hepatitis B virus infection.
The present invention includes the clinical use of the vaccine platform as an HBV therapeutic vaccine in combination with nucleoside or nucleotide analogues in the treatment of chronic hepatitis B virus infection.
The present invention includes combined application of the vaccine platform as a prophylactic or therapeutic vaccine for HBV, influenza, SARS-COV2, HPV, VZV, EBV, HSV-2, and HIV in combination with antiviral drugs and other therapies; as a prophylactic or therapeutic vaccine for HBV, HPV, and EBV-related tumors in combination with antiviral and antitumor drugs and therapies.
The present invention comprises multivalent combination vaccine consisting of the vaccine platform and other virus or pathogen or tumor vaccines.
Any fusion protein vaccine comprising the vaccine platform of the present invention can be inoculated with the adenovirus vaccine, mRNA vaccine, inactivated vaccine or DNA vaccine for the same virus, pathogen or tumor in sequence or simultaneously.
The present invention includes the full-length sequence and any truncation sequence of the vaccine platform antigen, such as SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78.
The present invention comprises any possible mutants of said fusion protein vaccine antigen, including natural point mutation/deletion mutation/truncation, any combination of natural sit mutations, subtypes generated by mutations, and mutated sequences comprising artificial point mutation/deletion mutation/truncation constructed for the purpose of enhancing the effect of the vaccine, such as SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84.
The present invention provides a multivalent combination vaccine consisting any vaccine of the present invention as a component of the vaccine and another vaccine of the present invention or other vaccines different from the vaccine of the present invention such as other virus or pathogen or tumor vaccines, for example, a multivalent vaccine comprising the SARS-CoV-2 fusion protein vaccine of the present invention in combination with influenza vaccine or other vaccines; for example, any vaccine of the present invention and the adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine for the same virus, pathogen, or tumor can be inoculated in sequence or simultaneously; for example, the SARS-COV-2 fusion protein vaccine can be inoculated with the adenovirus vaccine, or mRNA vaccine, or inactivated vaccine or DNA vaccine for SARS-COV-2 in sequence or simultaneously; for example, the sequence for immunization may be as follows: 1) firstly immunization with the SARS-COV-2 fusion protein vaccine of the present invention, and then immunization with the adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of SARS-COV-2; 2) firstly immunization with the adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of SARS-COV-2, followed by immunization with the SARS-COV-2 fusion protein vaccine; 3) the SARS-COV-2 fusion protein vaccine and the adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine are inoculated simultaneously. As known in the art, in the case of combination use, the vaccines can be prepared as a convenient kit.
The present invention includes but not limited to the following advantages over the prior art:
Sequences involved in the present invention:
Compared with IFN-preS1-Fe, the IFN-Pan-preS1-Fc could significantly enhance the immunogenicity of antigen molecules. C57/BL6 (n=8/group) mice were subcutaneously immunized with hepatitis B Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins without aluminum adjuvant, and the level of Pres1-specific antibody in serum was detected by ELISA at specified time.
In order to make the objective, technical solution and advantages of the present invention more clear, the present invention is described in detail below with reference to the examples and the accompanying drawings. The Examples are only illustrative of the present invention and are not intended to limit the scope of the present invention, and the Examples are only a part of the present invention, and do not represent all embodiments of the present invention. The scope of the invention is defined by the appended claims.
The vaccine platform of interferon-target antigen-immunoglobulin Fc (or antibody) consists of three structural units, wherein the first structural unit is interferon, the second structural unit is immunoglobulin Fc region (or antibody), and the third unit is target antigen. In the process of construction, the three structural units could be arbitrarily arranged and combined, and the target antigen could be connected to a Th cell helper epitope through a linker 2. The representative designs were as follows:
Next, the inventors tried to connect the target antigen to a cell helper epitope by a linking fragment 2, and then combine it with other two vaccine platform components. The representative designs were as follows:
The expression and production of the vaccine platform were described by taking hepatitis B virus Pres1 and coronavirus SARS-CoV-2 RBD protein homodimer as an example.
1. Vector Construction, Host Cell Transfection and Induced Expression
1.1. The vaccine structural units were constructed on PEE12.4 vector by molecular cloning to obtain a plasmid expressing the fusion protein, which was then transiently transfected into 293F cells, the culture supernatant was collected, and finally the protein of interest was purified by Protein A affinity chromatography.
Vector Construction (Taking HBV preS1 Antigen as an Example)
Linkers between each fragment of fusion protein were as follows:
1.2. Rapid Expression of Protein of Interest by Transient Transfection:
(1) Cell thawing: Freestyle 293F cells were frozen in CD OptiCHO™ media (containing 10% DMSO) at a concentration of 3×107 cells/ml. The cells were taken out from liquid nitrogen, and then dissolved quickly in a 37° C. water bath, added into a 15 ml centrifuge tube containing 10 ml OptiCHO™ media, and centrifuged at 1,000 rpm for 5 min. The supernatant was discarded, and the cell pellet was suspended and cultured in 30 ml OptiCHO™ media at 37° C., 8% CO2, 135 rpm. After 4 days, the cells were subjected to extended culture, and the concentration should not exceed 3×106 cells/ml during the extended culture.
(2) Two days before transfection, the suspension cultured 293F cells were prepared for transient transfection (200 ml) with an inoculum density of 0.6-0.8×106 cells/ml.
(3) Two days later, the suspension of cells to be transfected was counted, and the estimated cell density was 2.5-3.5×106 cells/ml, then the cell suspension was centrifuged at 1,000 rpm for 5 min, and the supernatant was discarded.
(4) Cells were resuspended with 50 ml of fresh Freestyle 293 media, and centrifuged again at 1,000 rpm for 5 min, and the supernatant was discarded.
(5) 293F cells were resuspended with 200 ml Freestyle 293 media.
(6) 600 g plasmids were diluted with 5 ml of Freestyle 293 media, and filtered by a 0.22 μM filter for sterilization.
(7) 1.8 mg of PEI was diluted with 5 ml of Freestyle 293 media and filtered with a 0.22 M filter for sterilization. Immediately thereafter, 5 ml of the plasmid and 5 ml of PEI were mixed, and allowed to stand at room temperature for 5 minutes.
(8) The plasmid/PEI mixture was added to the cell suspension, cultured in a 37° C., 8% CO2, 85 rpm incubator, and meanwhile supplemented with growth factor 50 μg/L LONG™ R3IGF-1.
(9) After 4 hours, 200 ml EX-CELL™ 293 media medium and 2 mM Glutamine were supplemented, and then the cells were continued in culture at 135 rpm.
(10) 24 hours later, 3.8 mM of cell proliferation inhibitor VPA was added; 72 hours later, 40 ml medium D was added, and then the cells were continued in culture; 6-8 days after transfection (the cell survival rate is less than 70%), the supernatant was collected for the next step of purification.
1.3. Collection, Purification and Electrophoresis Verification of Fusion Protein
2. Purification of protein of interest by using Protein A:
(1) Sample preparation: the cell culture suspension was transferred to a 500 ml centrifuge bucket, and centrifuged at 8,000 rpm for 20 min; precipitate was discarded; and supernatant was filtered by a 0.45 μM filter to remove impurities, and then a final concentration of 0.05% NaN3 was added to prevent bacterial contamination during purification.
(2) Assembly of chromatographic column: An appropriate amount of Protein A Agarose (the amount was calculated by purifying 20 mg of human Fc fusion protein per 1 ml of Protein A) were mixed well, added to the chromatographic column, left at room temperature for about 10 minutes; after separation of Protein A and 20% ethanol solution, the outlet at the bottom was opened to allow the ethanol solution to flow out slowly by gravity.
(3) The chromatographic column was washed and equilibrated with 10 column volumes of distilled water and Binding buffer (20 mM sodium phosphate+0.15M NaCl, pH 7.0), respectively.
(4) The sample was loaded by a constant flow pump at a flow rate of 10 column volumes/hour, and flow-through was collected; and the sample was repeatedly loaded twice.
(5) The column was rinsed with more than 10 column volumes of Binding buffer to remove impurity proteins until no protein was detected in the effluent.
(6) The column was eluted by Elution Buffer (0.1 M Glycine, pH 2.7); eluent was collected in separate tubes, 1 tube for 1 ml eluent; and elution peaks were observed with a protein indicator solution (Bio-Rad protein assay). The collection tubes for the eluted peaks were mixed and added with an appropriate amount of 1 M Tris, pH 9.0 (to adjust the pH to 6-8, which should be more than 0.5 different from the isoelectric point of the purified protein).
(7) The protein of interest was substituted into required buffer by using Zeba desalting spin column or concentrating spin column (please be noted that the pH of the buffer should be adjusted to avoid the isoelectric point of the protein). BSA was used as a standard, and protein concentration was determined by SDS-PAGE electrophoresis and NanoDrop2000.
(8) After elution, the column was washed with 20 column volume of distilled water, and then with 10 column volume of 20% ethanol. Finally, the gel medium should be immersed in ethanol solution and stored at 4° C.
3. The SDS-PAGE electrophoresis map of the protein was shown in
Materials: C57BL/6 male mice (5-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Beijing Kangwei Biology Technology Co., Ltd.; 96-well ELISA assay plate was purchased from Corning Costa; ELISA chromogenic solution was purchased from eBioscience; microplate reader SPECTRA max PLUS 384 was purchased from Molecular Company of the United States. The aluminum adjuvant was purchased from SIGMA.
Methods:
(1) The mice were immunized by Pres1 fusion protein; specially, 80 pmol IFN-Pres1-Fc or 80 pmol Pres1-Fc or Pres1 protein was mixed with aluminum adjuvant and subcutaneously administered to mice. At the designated time points, the serum of the mice was collected by taking blood from the orbit for antibody detection.
(2) The antibody produced by IFNα-Pres1-Fc had extensive neutralizing effect on different genotypes of HBV virus. 5-week-old male C57BL/6 mice were infected with 1×1011 vg of AAV-HBV 1.3 (with HBV genotypes B, C, and D) through tail vein. After 6 weeks, mice with sustained and stable expression of HBV antigen were selected for the test. The selected mice (4 mice/group) were injected intravenously with serum from IFNα-Pres1-Fc immunized mice at 200 ul/mouse. After 12 hours, the serum of the mice was collected, and the changes of the Pres1 antigen in the mice before and after the injection of the antiserum were detected by ELISA.
(3) Anti-Pres1 specific antibody in serum was detected by ELISA. Pres1 (2 g/ml) coating solution was added to the ELISA plate (Corning 9018) at 50 ul per well, and the plate was coated at 4° C. overnight. The plate was washed once with PBS, 260 ul per well. The plate was blocked with 5% blocking solution (5% FBS) for two hours at 37° C. Serum samples were diluted with PBS (1:10, 1:100, 1:1000, 1:10000), added to the blocked ELISA plate at 50 ul per well and incubated at 37° C. for 1 hour. The plate was washed 5 times with PBST (260 ul for each time), added with enzyme-labeled secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted by PBS) at 50 ul per well, and incubated at 37° C. for 1 hour. The plate was washed 5 times with PBST (260 ul for each time), added with substrate TMB 100 ul/well, incubated at room temperature in the dark until color development; 50 ul stop solution (2N H2SO4) was added to each well to stop color development, and the plate was read with a microplate reader, at OD450-630.
Results: The immunogenicity of free Pres1 was weak, and the immunogenicity was greatly improved when the Pres1 was fused with IFNα and Fc moiety to form IFNα-Pres1-Fc fusion protein, which was shown in
Materials: C57BL/6 (6-8 weeks old) male mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and HBsAg detection kit was purchased from Shanghai Kehua Bio-Engineering Co., Ltd. AAV-HBV 1.3 virus was purchased from Guangzhou PackGene Biotech Co., Ltd. Other experimental materials were the same as those used in Example 3.
Methods:
(1) Mice were immunized subcutaneously with 80 pmol of different forms of Pres1 vaccines, including Pres1, Pes1-Fc, and IFNα-Pres1-Fc proteins. At day 28 after immunization, mice serum was collected and mice were infected with 1×1011 vg AAV-HBV 1.3 virus, after that, mouse serum was collected every week for four weeks to detect anti-Pres1 antibody, HBsAg, and Pres1 antigen in the serum. At the third week, peripheral HBV-DNA levels of the mice were detected.
(2) ELISA detection of Pres1-specific antigen in serum. Antigen coating: Pres1 antibody XY007 (4 g/ml) coating solution was added to the ELISA plate (Corning 9018) at 50 μl per well, and coated overnight at 4° C. The plated was washed once with PBS, 260 μl per well. The plate was blocked with 5% blocking solution (5% FBS) for two hours at 37° C. Serum samples were diluted with PBS (1:10, 1:100), added to the blocked ELISA plate at 50 μl per well (wherein, two duplicate wells were set for each dilution) and incubated at 37° C. for 1 hour. The plate was washed 5 times with PBST (260 μl for each time), added with 50 μl enzyme conjugate (obtained from Kehua HBsAg Detection Kit) per well, and incubated at 37° C. for 1 hour. The plate was washed 5 times with PBST (260 μl for each time), added with substrate TMB 100 μl/well, incubated at room temperature in the dark until color development; 50 μl stop solution (2N H2SO4) was added to each well to stop color development, and the plate was read with a microplate reader, at OD450-630.
Results: The mice in the IFNα-Pres1-Fc immunized group could produce a high level of Pres1 antibody before inoculation with the virus, and the antibody continued to maintain a high level during the virus infection, as shown in
Materials: C57BL/6 male mice (4 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. AAV-HBV 1.3 was purchased from Guangzhou PackGene Biotech Co., Ltd. HBsAg detection kit was purchased from Shanghai Kehua Bio-Engineering Co., Ltd., and other experimental materials were the same as in those in Example 4.
Methods:
(1) Screening of HBV Carrier mice: 4-week-old HBV C57BL/6 mice were injected with 1×1011 vg AAV-HBV 1.3 virus through tail vein, and HBV antigen HBsAg was detected in 1-6 weeks to screen mice with stable expression of HBsAg which were used as HBV carrier mice for experiments.
(2) The screened mice were subcutaneously injected with 80 pmol of different forms of Pres1 protein, once every two weeks for a total of three immunizations. The mouse serum was collected 14 days after immunization, and then collected once a week, and the levels of anti-Pres1 antibody, HBsAG, and Pres1 antigen in the mouse serum were detected by ELISA. HBV-DNA content in the peripheral blood of the mice was detected after the last blood collection.
Results: We detected the preS1 antigen in the serum of Carrier mice immunized with IFN-Pres1-Fc vaccine, as well as the changes of Pres1 antibody and HBsAg in the serum. The results showed that after IFNα-Pres1-Fc vaccine immunization, high level of anti-Pres1 antibody in mice was produced, as shown in
MATERIALS: The Same as Those in Example 3
Methods:
(1) the mice were immunized by Pres1 fusion proteins, specially, 80 pmol IFN-Pan-Pres1-Fc containing Pan epitope or 80 pmol IFN-Pan-Pres1-Fc, Pres1-Fc, Pres1 protein were subcutaneously inoculated in mice. At the designated time points, the serum of the mice was collected by taking blood from the orbit for antibody detection.
(2) ELISA detection of anti-Pres1 specific antibody in serum, the same as that in Example 3.
Results: Compared with fusion protein vaccines such as IFN-preS1-Fc, the IFN-Pan-preS1-Fc could significantly enhance the immunogenicity of antigen molecules and induce the production of broad-spectrum neutralizing antibodies. C57/BL6 (n=8/group) mice were subcutaneously immunized with hepatitis B Pres1, Pres1-Fc, and IFNα-Pres1-Fc proteins without aluminum adjuvant, and the level of Pres1-specific antibody in serum was detected by ELISA at specified time.
Materials: C57BL/6 male mice (4 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. AAV-HBV 1.3 was purchased from Guangzhou PackGene Biotech Co., Ltd. HBsAg detection kit was purchased from Shanghai Kehua Bio-Engineering Co., Ltd., and other experimental materials were the same as in those in Example 4.
Methods:
(1) Screening of HBV Carrier mice: 4-week-old HBV C57BL/6 mice were injected with 1×1011 vg AAV-HBV 1.3 virus through tail vein, and HBV antigen HBsAg was detected in 1-6 weeks to select mice with stable expression of HBsAg which were used as HBV carrier mice for experiments.
(2) The selected mice were subcutaneously injected with 80 pmol of different forms of Pres1 protein, once every two weeks for a total of three immunizations. The mouse serum was collected 14 days after immunization, and then collected once a week, and the levels of anti-Pres1 antibody, HBsAg, and Pres1 antigen in the mouse serum were detected by ELISA. HBV-DNA content in the peripheral blood of the mice was detected after the last blood collection.
Results: We detected the preS1 antigen in the serum of Carrier mice immunized with IFNα-Pan-Pres1-Fc vaccine, as well as the changes of Pres1 antibody and HBsAg in the serum. The results showed that after IFN-Pan-Pres1-Fc vaccine immunization, the mice produced a high level of anti-Pres1 antibody, as shown in
Materials: C57BL/6 male mice (4 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. AAV-HBV 1.3 was purchased from Guangzhou PackGene Biotech Co., Ltd. HBsAg detection kit was purchased from Shanghai Kehua Bio-Engineering Co., Ltd., and Anti-HBsAg kit was purchased from Beijing Wantai Biological Pharmacy Co., Ltd. Commercial HBsAg vaccine was purchased from Amy Hansen Vaccine (Dalian) Co., Ltd. Other experimental materials were the same as those used in Example 7.
Methods:
(1) Screening of HBV Carrier mice: 4-week-old HBV C57BL/6 mice were injected with 1×1011 vg AAV-HBV 1.3 virus through tail vein, and HBV antigen HBsAg was detected in 1-6 weeks to select mice with stable expression of HBsAg which were used as HBV carrier mice for experiments.
(2) The selected HBV Carrier mice were immunized with 80 pmol IFNα-pan-Pres1-Fc and 2 μg of commercial HBsAg vaccine at the same time for two consecutive times with an interval of 14 days between each time. The mouse serum was collected 14 days after the first immunization, and the mouse serum was collected every week thereafter, and the changes of anti-Pres1, Pres1, anti-HBsAg, and HBsAg in the serum were detected. And when the mouse serum was collected for the last time, the level of HBV-DNA in the serum was detected.
RESULTS: We found that the combination of IFNα-Pan-Pres1-Fc with commercial HBsAg as a strategy for the treatment of chronic hepatitis B could eventually break HBsAg tolerance. The immune response generated in HBV-tolerant mice could completely clear the preS1 antigen in the serum, as shown in
Materials: Balb/c male and female mice (6-8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the SARS-CoV-2 RBD protein was purchased from Beijing KEY-BIO Biotech Co., Ltd. 293-hACE2 cells were provided by Professor Zhang Zheng (Shenzhen Third People's Hospital). Luciferase Reporter detection kit was purchased from Promega.
Other experimental materials were the same as those used in Example 3.
Methods:
(1) Mice were immunized with IFNα-RBD(SARS-Cov-2)-Fc fusion protein; specially, the mice were subcutaneously immunized with 10 μg IFNα-RBD-Fc, RBD-Fc or 10 μg RBD protein mixed with aluminum adjuvant. At 28 days after immunization, the serum of the mice was collected by taking blood from orbit for detection of SARS-Cov-2-specific antibodies.
(2) Detection of serum SARS-cov2 RBD antibody Antigen coating: RBD (1.5 g/ml) coating solution was added to the ELISA plate (Corning 9018) at 1001 per well, and coated overnight at 4° C. The plated was washed once with PBS, 260 μl per well. The plate was blocked with 100 μl of 5% blocking solution (5% FBS) for two hours at 37° C. Serum samples were diluted with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000 . . . ), added to the blocked ELISA plate at 100 μl per well and incubated at 37° C. for 1 hour. The plate was washed 5 times with PBST (260 μl for each time), added with enzyme-labeled secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted by PBS) at 100 μl per well, and incubated at 37° C. for 1 hour. The plate was washed 5 times with PBST (260 μl for each time), added with substrate TMB at 100 μl/well, incubated at room temperature in dark for 15 minutes, waiting for the substrate to develop color. 50 μl of stop solution (2N H2SO4) was added to each well to stop the color development, and the plate was read with a microplate reader, at OD450-630. Calculation of titer: the maximum dilution factor that was positive was selected, and the dilution factor was multiplied (X) by the OD value/Cutoff value (0.1) corresponding to the dilution factor, and the obtained value was the antibody titer corresponding to the serum.
(3) in vitro neutralization experiment of SARS-CoV-2 S protein pseudovirus. Antiserum was diluted by 1:3 and added to a 96-well plate, and 50 μl pseudovirus particles with luciferase spike protein were added to the wells, the mixture of virus and antibody was left at 37° C. for 1 hour, and 10{circumflex over ( )}4 293-hACE2 cells per well were added to the 96-well plate. the 96-well plate was left in a 37° C. cell culture incubator, and the activity of luciferase was detected after 48 hours.
Results: The immunogenicity of free SARS-CoV-2 RBD was weak, and the immunogenicity thereof was greatly improved when the IFNα and Fc were added to the SARS-CoV-2 RBD polypeptide protein region to form a IFNα-RBD-Fc fusion protein, as shown in
Materials: Balb/c male and female mice (6-8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the RBD protein of original SARS-CoV-2 strain was purchased from Beijing KEY-BIO Biotech Co., Ltd. The RBD protein of the South Africa mutant strain of SARS-CoV-2 was purchased from Beijing Sino Biological Technology Co., Ltd.
Other experimental materials were the same as those used in Example 3.
Methods:
(1) the construction and expression of IFNα-Pan-RBD (original strain)-Fc and IFNα-RBD (SARS-CoV-2 South Africa mutant strain)-Fc protein were the same as those in Example 2.
(2) the mice were immunized with IFNα-Pan-RBD (original strain)-Fc and IFNα-Pan-RBD (SARS-CoV-2 South Africa mutant)-Fc fusion protein; specially, 10 g of IFNα-Pan-RBD (original strain)-Fc or IFNα-Pan-RBD (SARS-CoV-2 South Africa mutant strain)-Fc protein was mixed with aluminum adjuvant and subcutaneously inoculated in mice. At 14 days after immunization, the serum of the mice was collected by taking blood from orbit for detection of SARS-Cov-2-specific antibodies.
(3) Antibody response analysis by ELISA was the same as that in Example 9.
Results: The results of SDS-PAGE showed correct band size of IFNα-Pan-RBD (SARS-CoV-2 original strain)-Fc, indicating that the mutant SARS-CoV-2 IFNα-RBD (SARS-CoV-2 original strain)-Fc vaccine protein was successfully constructed, expressed and purified (
Materials: C57BL/6 female mice (6-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The SARS-CoV-2 RBD protein used in ELISA was purchased from Beijing KEY-BIG Biotech Co., Ltd.; Mouse IFNα-RBD-Fc, Mouse IFNα-Pan-RBD-Fc, Human IFNα-RBD-Fc, and Human IFNα-Pan-RBD-Fc proteins were produced in-house and other experimental materials were the same as those in Example 3.
Methods:
(1) The fusion protein design, plasmid construction and protein purification methods were the same as those in Examples 1 and 2.
(2) Immunization of mice with vaccine proteins. 10 μg Mouse IFNα-RBD-Fc, Mouse IFNα-Pan-RBD-Fc or 10 μg Human IFNα-RBD-Fc, Human IFNα-Pan-RBD-Fc vaccine proteins were mixed with 20 μg aluminum adjuvant overnight, and then inoculated to mice through muscle immunization, and a booster immunization was carried out 14 days after the initial inoculation. The mouse serum was collected on the 7th, 14th, and 28th day after immunization, and the level of RBD-specific antibody in the mouse serum was detected by ELISA.
(3) Detection of serum SARS-cov2 RBD antibody. Antigen coating: RBD (1.5 g/ml) coating solution was added to the ELISA plate (Corning 9018) at 100 μl per well, and coated overnight at 4° C. The plated was washed once with PBS, 260 μl per well. The plate was blocked with 100 μl of 5% blocking solution (5% FBS) for two hours at 37° C. Serum samples were diluted with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000 . . . ), added to the blocked ELISA plate at 100 μl per well and incubated at 37° C. for 1 hour. The plate was washed 5 times with PBST (260 μl for each time), added with enzyme-labeled secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted by PBS) at 100 μl per well, and incubated at 37° C. for 1 hour. The plate was washed 5 times with PBST (260 μl for each time), added with substrate TMB at 100 μl/well, incubated at room temperature in dark for 15 minutes, waiting for the substrate to develop color. 50 μl of stop solution (2N H2SO4) was added to each well to stop the color development, and the plate was read with a microplate reader, at OD450-630. Calculation of titer: the maximum dilution factor that was positive was selected, and the dilution factor was multiplied (X) by the OD value/Cutoff value (0.1) corresponding to the dilution factor, and the obtained value was the antibody titer corresponding to the serum.
Result:
As shown in
As shown in
Materials: C57BL/6 female mice (6-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the SARS-CoV-2 RBD protein used in ELISA was purchased from Beijing KEY-BIO Biotech Co., Ltd. The Human IFNα-RBD-Fc and Human IFNα-Pan-RBD-Fc proteins used for immunization were all produced in-house. Other experimental materials were the same as those used in Example 3.
Methods:
(1) Human IFNα-RBD-Fc and Human IFNα-Pan-RBD-Fc proteins were used to immunize mice. 10 μg Human IFNα-RBD-Fc or Human IFNα-Pan-RBD-Fc protein was mixed with aluminum adjuvant overnight, as a vaccine sample containing aluminum adjuvant; for another group, 10 μg Human IFNα-RBD-Fc or Human IFNα-Pan-RBD-Fc protein was diluted with PBS as a vaccine sample without adjuvant. In the presence or absence of aluminum adjuvant, mice were inoculated with 10 μg Human IFNα-RBD-Fc or Human IFNα-Pan-RBD-Fc proteins by intramuscular immunization, and then 14 days after inoculation a booster immunization was given. The mouse serum was collected on the 7th, 14th, and 28th day after immunization, and the level of RBD-specific antibody in the mouse serum was detected by ELISA.
(2) Detection of serum SARS-cov2 RBD antibody. Antigen coating: RBD (1.5 g/ml) coating solution was added to the ELISA plate (Corning 9018) at 100 μl per well, and coated overnight at 4° C. The plated was washed once with PBS, 260 μl per well. The plate was blocked with 100 μl of 5% blocking solution (5% FBS) for two hours at 37° C. Serum samples were diluted with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000 . . . ), added to the blocked ELISA plate at 100 μl per well and incubated at 37° C. for 1 hour. The plate was washed 5 times with PBST (260 μl for each time), added with enzyme-labeled secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted by PBS) at 100 μl per well, and incubated at 37° C. for 1 hour. The plate was washed 5 times with PBST (260 μl for each time), added with substrate TMB at 100 μl/well, incubated at room temperature in dark for 15 minutes, waiting for the substrate to develop color. 50 μl of stop solution (2N H2SO4) was added to each well to stop the color development, and the plate was read with a microplate reader, at OD450-630. Calculation of titer: the maximum dilution factor that was positive was selected, and the dilution factor was multiplied (X) by the OD value/Cutoff value (0.1) corresponding to the dilution factor, and the obtained value was the antibody titer corresponding to the serum.
Result:
As shown in
Materials:
The experimental animals C57BL/6 mice of 6-8 weeks old were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the animal certificate number as No. 110011200106828974; the RBD protein for immunization was purchased from Beijing KEY-BIO Biotech Co., Ltd.; RBD-Fc, IFNα-RBD-Fc and IFN-pan-RBD-Fc proteins were produced in-house; all adjuvants were purchased from SERVA, Germany; horseradish peroxidase (HRP) labeled goat anti-mouse IgG was purchased from Beijing Kangwei Biology Technology Co., Ltd.; 96-well ELISA assay plate was purchased from Corning Costar; ELISA chromogenic solution was purchased from eBioscience; the microplate reader SPECTRA max PLUS 384 was purchased from Molecular Company of the United States; tissue homogenizer was purchased from Beijing Heros Technology Co., Ltd.
Methods:
Mice of 6-8 weeks old were divided into 5 groups, with 10 mice in each group, and were immunized with 10 g of IFNα-pan-RBD-Fc or the same molar amount of RBD, RBD-Fc, and IFNα-RBD-Fc proteins by intranasal immunization, and the intranasal dose was 10 uL per mouse. Mice were immunized on day 0 and day 14 using two immunization procedures. The mouse serum was collected on the 7th, 14th, 21st, 28th, 35th, and 42nd days after immunization, and ELISA method was used to detect the content of SARS-CoV-2 RBD-specific antibodies in the serum of each group; the 28-day serum was collected for SARS-CoV-2 pseudovirus neutralization experiment in vitro.
Result:
As shown in
Materials:
The materials were the same as those in Example 10.
Methods:
Mice of 6-8 weeks old were divided into 4 groups, with 5 mice in each group, and were immunized with 10 g of IFNα-pan-RBD-Fc or the same molar amount of RBD, RBD-Fc, and IFNα-RBD-Fc proteins by intranasal immunization, and the dose was 10 uL per mouse. Mice were immunized on day 0 and day 14 using two immunization procedures. On the 28th day after immunization, the nasal mucosal supernatant and lung lavage fluid of the mice were collected, and the serum levels of SARS-CoV-2 RBD-specific antibodies in each group were detected by ELISA method, and the SARS-CoV-2 pseudovirus neutralization test was used to detect the neutralization experiment of SARS-CoV-2 pseudovirus in serum and nasal mucosal supernatant. Obtaining of the supernatant of nasal mucosa and alveolar lavage fluid of mice used in immunization experiments: After the mice were killed in rest, the nasal mucosa of the mice was collected and crushed with a tissue homogenizer. The homogenized liquid was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was taken as the nasal mucosa supernatant (NMDS). For the lung of mice, a 1 ml syringe was used to draw about 0.8 ml of HBSS+100 uMEDTA, injected into the endotracheal tube, blown and inhaled gently and repeatedly for three times, then the liquid was sucked out, collected into a centrifuge tube; the steps were repeated three times, and finally about 2 ml of lung lavage fluid was obtained. The mouse lung lavage fluid was centrifuged at 500 g for 5 minutes, wherein the supernatant was the mouse lung lavage fluid (BALF), and the precipitate was the lymphocytes in the mouse lung, which could be further analyzed.
Result:
As shown in
As shown in
Her2 belongs to the HER family of type I transmembrane growth factor receptors and consists of an extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine kinase domain. Once the ligand binds to the extracellular domain, the HER protein will dimerize and trans-phosphorylate its intracellular domain. The phosphorylated tyrosine residues can bind to a variety of intracellular signaling molecules, activate downstream signaling pathways, and regulate gene transcription. Most of the regulated genes are related to cell proliferation, survival, differentiation, angiogenesis, invasion and metastasis. The extracellular segment of Her2 protein is relatively large, with more than 600 amino acids, and can be divided into four domains, namely domains I, II, III, and IV. The currently approved Trastuzumab mainly binds to domain IV, Pertuzumab mainly binds to domain II, and the polypeptide vaccine E75, which is undergoing clinical trials, targets domain III. It shows that there are some important sites in different domains, which may mediate the anti-tumor effect. In order to study the vaccine platform for the prevention and treatment of tumors, the inventors selected the tumor antigen Her2 as a target, constructed IFN-Her2-Fc and IFN-Pan-Her2-Fc fusion protein vaccines, and then analyzed the anti-tumor activities and immunological activities of the vaccines in vivo.
Materials and Methods:
Materials:
BALB/c female mice (6-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; TUBO cells were obtained from TCGA; other materials were the same as those in Example 3.
Methods:
(1) The fusion protein design, plasmid construction and protein purification methods were the same as those in Examples 1 and 2.
Firstly, expression plasmids were constructed for domains III and IV of the extracellular domain of mouse Her2 (respectively denoted as: IFNα-3-Fc, IFNα-pan-3-Fc, IFNα-pan-4-Fc and IFNα-4-Fc), and then related proteins were expressed and purified in human 293F cell line. The protein size and purity were identified by SDS-PAGE and Coomassie brilliant blue staining.
(2) Analysis of Direct Antitumor Activity of IFNα-3-Fc and IFNα-Pan-3-Fc
TUBO was a breast cancer cell line derived from BALB-NeuT mice, and was used to study the growth and treatment of Her2-positive breast cancers. Antitumor activity of IFNα in proteins was detected by using TUBO tumors. TUBO breast cancer model mice were constructed, 5*105 TUBO cells were subcutaneously inoculated into BALB/C mice. The treatment was given once a week for a total of 3 times when the tumor size was 50-80 mm3. The dosage of IFNα-3-Fc was 10 g/mouse, and other drugs were administered in equimolar amounts, and CpG was used as an adjuvant. The tumor size was measured, and the tumor growth curve was drawn.
(3) Analysis of the Enhancement of Immunogenicity of Her2 Vaccine by IFNα and Pan
BALB/C female mice aged 6-8 weeks were inoculated subcutaneously with HER2 domain V fusion protein vaccines 4-Fc, IFNα-4-Fc and IFNα-pan-4-Fc without adjuvant, once a week, 3 times in total. The immunization dosage was 10 g/mouse for IFNα-4-Fc, and other proteins were inoculated in equimolar amounts. Venous blood was collected at day 14 and day 21 after immunization, and the antibody level of Her2-specific IgG was detected by ELISA method.
Result:
(1) As shown in
(2) As shown in
(3) As shown in
Materials: BALB/c female mice (6-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; HA1 (A/PR8) protein used in ELISA was purchased from Beijing Sino Biological Technology Co., Ltd.; HA1 protein for immunization (A/PR8) was purchased from Beijing Sino Biological Technology Co., Ltd.; IFNα-HA1-Fc was produced in-house; H1N1 (A/PR8) influenza viruses used to infect mice were produced in-house; other experiments materials were the same as those in Example 3.
Methods:
(1) The IFNα-HA1-Fc protein design, plasmid construction and protein purification were the same as those in Examples 1 and 2.
(2) Immunization of mice by HA1 and IFNα-HA1-Fc proteins. 10 μg IFNα-HA1-Fc or the same molar amount of HA1 protein was respectively mixed with 20 μg aluminum adjuvant overnight, and then inoculated to mice through muscle immunization, and a booster immunization was carried out 14 days after the initial inoculation. The mouse serum was collected on the 28th day after immunization, and the level of HA1-specific antibody in the mouse serum was detected by ELISA.
(3) Detection of serum HA1 antibodies. Antigen coating: HA1 (2 g/ml) coating solution was added to the ELISA plate (Corning 9018) at 100 μl per well, and coated overnight at 4° C. The plated was washed once with PBS, 260 μl per well. The plate was blocked with 100 μl of 5% blocking solution (5% FBS) for two hours at 37° C. Serum samples were diluted with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000 . . . ), added to the blocked ELISA plate at 100 μl per well and incubated at 37° C. for 1 hour. The plate was washed 5 times with PBST (260 μl for each time), added with enzyme-labeled secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted by PBS) at 100 μl per well, and incubated at 37° C. for 1 hour. The plate was washed 5 times with PBST (260 μl for each time), added with substrate TMB at 100 μl/well, incubated at room temperature in dark for 15 minutes, waiting for the substrate to develop color. 50 μl of stop solution (2N H2SO4) was added to each well to stop the color development, and the plate was read with a microplate reader, at OD450-630. Calculation of titer: the maximum dilution factor that was positive was selected, and the dilution factor was multiplied (X) by the OD value/Cutoff value (0.1) corresponding to the dilution factor, and the obtained value was the antibody titer corresponding to the serum.
(4) At day 42 after immunization, the mice were anesthetized and infected with 1000 PFU A/PR8 influenza virus by nasal dripping. From the third day after infection, the mice were observed and weighed every two days.
Result:
As shown in
Materials and Methods:
The designs, plasmid constructions and protein purifications of IFNα-Pan-VZV-gE-Fc, IFNα-Pan-EBV-gp350-Fc, and IFNα-Pan-HSV-2-gD-Fc proteins were the same as those in Examples 1 and 2.
Result:
As shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010623708.8 | Jul 2020 | CN | national |
| 202110353488.6 | Mar 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/103931 | 7/1/2021 | WO |
