Fusion protein IFN alpha-gE-Fc and application thereof in preparation of recombinant varicella-herpes zoster vaccine

Abstract

The invention belongs to the technical field of biology, and particularly relates to a fusion protein IFN alpha-gE-Fc and application of the fusion protein IFN alpha-gE-Fc in preparation of a recombinant varicella-herpes zoster vaccine. The IFN alpha-gE-Fc fusion protein is constructed, gE is dimerized through the Fc segment of human IgG, the stability and size of the protein can be improved, and effective LN targeting and dendritic cell capture are promoted. In order to further increase the treatment and presentation of DCs on antigens, cell factors IFN alpha are fused at the N end of gE-Fc to induce strong cellular immune response and humoral immunity at the same time, no exogenous sequence is introduced, cellular immune response can be effectively enhanced, the defects of traditional adjuvants are overcome, and the immunologic adjuvant has a good application prospect. A candidate vaccine is provided for developing a safe and effective herpes zoster vaccine.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a fusion protein IFNα-gE-Fc and application thereof in preparing a recombinant varicella-zoster vaccine. Background Art

[0002] Varicella-zoster virus (VZV) is a member of the alphaherpesviridae family of the human herpesvirus genus. It is an enveloped virus with high species specificity. Its natural infection only occurs in humans and gorillas. The VZV genome is a linear double-stranded DNA molecule that encodes at least 71 open reading frames. The linear VZV genome is encapsidated by a protein capsid with a lipoprotein-rich envelope outside the capsid. There are 9 glycoproteins on the envelope, namely gB, gC, gE, gH, gI, gK, gL, gM and gN. These glycoproteins play an important role in the replication and packaging of the virus. Among them, gE is encoded by ORF68, contains 623 amino acids (AA), is a type I transmembrane protein, and can be divided into the extracellular region (1-538), the transmembrane region (539-559) and the intracellular region (560-623). gE is the most abundant protein in the viral envelope and infected cells, and is closely related to the infection and pathogenicity of the virus. It has strong immunogenicity and can induce strong cellular and humoral immune responses in the body during primary infection and latent reactivation infection. VZV enters the body through the respiratory mucosa during primary infection and causes chickenpox. After chickenpox is cured, the virus lurks in the ganglia. When the body's resistance is reduced, VZV is reactivated and causes herpes zoster. Patients may have obvious pain and discomfort, and the symptoms may last for weeks or months, leading to a decrease in quality of life. The most common serious complication is postherpetic neuralgia, which occurs in 10%-30% of patients with herpes zoster. The pain may last for months or even years, seriously affecting the quality of life of patients. Currently, vaccination is the main measure to prevent shingles and reduce the burden of the disease.

[0003] Given that the gE glycoprotein of VZV has strong immunogenicity and its antigenic epitope is quite conservative, it is a more suitable candidate antigen for subunit vaccines. Shingrix, a subunit vaccine for herpes zoster developed by GSK, is based on recombinant gE protein supplemented with a new adjuvant AS01b, which can significantly enhance cellular immune responses; clinical studies have shown that this subunit vaccine can significantly reduce the risk of herpes zoster in the elderly and can effectively reduce the risk of subsequent postherpetic neuralgia, indicating that recombinant subunit vaccines can effectively prevent the occurrence of herpes zoster. It was launched in the United States in 2017 and in China in 2020. The adjuvant AS01b used in Shingrix is ​​a powerful new adjuvant. While significantly enhancing cellular immunity, the side effects of the vaccine are also more obvious; secondly, QS21, one of the main ingredients in AS01b, is a natural ingredient extracted from soap tree species. The cost of artificial synthesis is high, and it also faces problems such as short-term supply shortages.

[0004] Therefore, it is urgent to develop a safe and effective vaccine with independent intellectual property rights. The new herpes zoster vaccine can be based on subunit vaccines. It is necessary to develop new adjuvants that can stimulate the body to produce cellular immunity, or antigens that can stimulate a stronger immune response to obtain an ideal immune effect. The present invention is committed to improving the deficiencies of traditional adjuvants through the rational design of antigen gE, reducing dependence on powerful adjuvants, and thus reducing adverse reactions to vaccination.

[0005] Type I interferon is a protein with multiple biological activities secreted by different cells. It is an important antiviral cytokine and also has immunomodulatory effects. IFNα can strongly promote antigen presentation and activate adaptive immunity. The main cell group on which it plays a role is dendritic cells DC cells. After acting on immature DC cells, it stimulates DC cell activation by promoting the expression of various co-stimulatory molecules, chemokine receptors and MHC molecules on the surface of DC cells, thereby activating T cells. More and more studies have shown that IFNα can be used as an immune adjuvant molecule. Its biological effects are species-specific and play an important role in the transition between innate immunity and adaptive immunity. It can further enhance the activity and potency of the vaccine. It has been used in the recombinant subunit vaccine for the new coronavirus.

[0006] The fusion of antigens with Fc segments can greatly extend the half-life of antigens. Fc receptors are expressed on the surface of many innate immune cells. Fc fusion proteins can bind to Fc receptors expressed on the surface of APC cells, thereby promoting the presentation and processing of antigens by APCs. Currently, a variety of human IgG-Fc fusion protein drugs have been approved by the FDA for clinical use. Summary of the invention

[0007] In response to the needs of the prior art, the present invention constructs an IFNα-gE-Fc fusion protein, in which gE dimerizes through the Fc segment of human IgG to increase protein stability and size, so as to promote effective LN targeting and dendritic cell capture, while improving purification efficiency. In order to further increase the processing and presentation of DCs to antigens, cytokine IFNα is fused to the N-terminus of gE-Fc to simultaneously induce strong cellular immune responses and humoral immunity, without introducing any exogenous sequences, which can effectively enhance cellular immune responses, improve the deficiencies of traditional adjuvants, and provide candidate vaccines for the development of safe and effective varicella or herpes zoster vaccines.

[0008] The present invention provides a fusion protein IFNα-gE-Fc, characterized in that it comprises interferon α, VZV glycoprotein gE and human IgG1Fc domain, wherein IFNα can be from human or mouse, preferably mouse IFN-α4, human IFN-α2, and specifically its amino acid sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2; VZV glycoprotein gE is preferably its extracellular domain, specifically amino acids 31 to 538, as shown in SEQ ID NO: 3; human IgG1Fc domain is preferably CH2-CH3; Among them, interferon α was connected to the N-terminus of the extracellular segment of VZV glycoprotein gE, and the human IgG1 Fc domain was connected to the C-terminus of the extracellular segment of VZV glycoprotein gE, and they were directly connected in series to obtain the fusion protein IFNα-gE-Fc Furthermore, the signal peptide of IL-10 is also connected, and the specific amino acid sequence thereof is shown in SEQ ID NO:5.

[0009] Specifically, the immunoglobulin Fc fragment is selected from the immunoglobulin Fc fragment of human, mouse, rabbit, cow, goat, pig, mouse, rabbit, hamster, rat, or guinea pig; further, the immunoglobulin Fc fragment is selected from the Fc fragment of IgG, IgA, IgD, IgE or IgM; preferably, the immunoglobulin Fc fragment is selected from IgG1 Fc fragment, IgG2 Fc fragment, IgG3 Fc fragment, or IgG4 Fc fragment; particularly preferably, the immunoglobulin Fc fragment is a human IgG Fc fragment, most preferably the amino acid sequence shown in SEQ ID NO.4.

[0010] Preferably, the fusion protein comprises the amino acid sequence shown in SEQ ID NO.6 and SEQ ID NO.7.

[0011] The present invention provides a nucleic acid encoding the fusion protein; Preferably, the nucleic acid coding sequence of mouse IFN-α4 is as shown in SEQ ID NO: 8 or its degenerate sequence; the nucleic acid coding sequence of human IFN-α2 is as shown in SEQ ID NO: 9 or its degenerate sequence; the nucleic acid coding sequence of gE extracellular segment is as shown in SEQ ID NO: 10 or its degenerate sequence; the nucleic acid coding sequence of immunoglobulin Fc fragment is as shown in SEQ ID NO: 11 or its degenerate sequence; More preferably, the sequence is codon-optimized according to mammalian cell expression preferences.

[0012] Preferably, the nucleotide sequence is as shown in SEQ ID NO: 12, SEQ ID NO: 13 or a degenerate sequence thereof.

[0013] The present invention provides a recombinant vector and a recombinant cell encoding the nucleic acid.

[0014] The present invention also provides the use of the fusion protein IFNα-gE-Fc in the preparation of a varicella recombinant subunit vaccine or a herpes zoster recombinant subunit vaccine.

[0015] The present invention further provides a varicella recombinant subunit vaccine or a herpes zoster recombinant subunit vaccine, comprising the fusion protein IFNα-gE-Fc according to any one of claims 1 to 4; without or with an adjuvant, specifically Alum adjuvant, MnJ adjuvant, AS01b adjuvant. Specifically, the fusion protein is obtained by chemical synthesis or genetic recombination.

[0016] In a mouse immunization experiment, the IFNα-gE-Fc fusion protein constructed by the present invention can induce the production of higher levels of gE binding antibodies, neutralizing antibodies and cellular immunity compared with the gE prototype when there is no exogenous adjuvant or when the Alum adjuvant is used; when the MnJ adjuvant is used, both IFNα-gE-Fc and gE can induce the production of high levels of neutralizing antibodies, but the cellular immunity induced by IFNα-gE-Fc is stronger; when the AS01b adjuvant is used, IFNα-gE-Fc can induce the production of higher levels of neutralizing antibodies, but the cellular immunity level is weaker than that of AS01b / gE, indicating that the fusion protein immunogen has a significant effect in inducing VZV-specific immune response, which improves the weak immunogenicity of gE and the shortcomings of traditional adjuvants, and provides a candidate vaccine for the development of a safe and effective varicella or herpes zoster vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the molecular sieve peak diagram for IFNα-gE-Fc protein purification.

[0018] Figure 2 It is a specific binding antibody for the serum of the mouse immunization experiment without adjuvant.

[0019] Figure 3 It is the neutralizing antibody in the serum of the mouse immunization experiment without adjuvant.

[0020] Figure 4 It is the specific cellular immune response of the mouse immunization experiment without adjuvant.

[0021] Figure 5 This is the specific binding antibody of the serum in the Alum / MnJ / AS01b adjuvant comparison experiment.

[0022] Figure 6 This is the serum neutralizing antibody from the Alum / MnJ / AS01b adjuvant comparison experiment.

[0023] Figure 7 This is a comparison experiment of specific cellular immune response between Alum / MnJ / AS01b adjuvants. DETAILED DESCRIPTION

[0024] 1. Gene sequence origin and composition The varicella-zoster virus IFNα-gE-Fc fusion protein described in the present invention is prepared by directly fusing IFNα to the N-terminus of gE (31-538aa) and directly fusing the Fc segment to the C-terminus of gE, with a total of 890 amino acids. The gene sequence was synthesized by entrusting Nanjing GenScript Biotechnology Co., Ltd. and connected to the pCAGGS vector. The synthesized gene includes restriction sites, signal peptides, target genes and termination codons to obtain its full-length sequence.

[0025] Among them, the relevant sequences used are as follows: SEQ ID NO.1: Mouse IFN-α4 amino acid sequence CDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKE.

[0026] SEQ ID NO.2: Human IFN-α2 amino acid sequence CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE。

[0027] SEQ ID NO.3: Amino acid sequence of VZV gE (31 - 538) SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRY。

[0028] SEQ ID NO.4: Amino acid sequence of IgG1 Fc (CH2 CH3) PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0029] SEQ ID NO.5: Amino acid sequence of IL10-SP MHSSALLCCLVLLTGVRA。

[0030] SEQ ID NO.6: mIFNα-gE-Fc MHSSALLCCLVLLTGVRACDLPHTYNLGNKRALTVLEEMRRLPPLSCLKDRKDFGFPLEKVDNQQIQKAQAILVLRDLTQQILNLFTSKDLSATWNATLLDSFCNDLHQQLNDLKACVMQEPPLTQEDSLLAVRTYFHRITVYLRKKKHSLCAWEVIRAEVWRALSSSTNLLARLSEEKESVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0031] SEQ ID NO.7:hIFNα-gE-Fc MHSSALLCCLVLLTGVRACDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKESVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0032] SEQ ID NO.8: Mouse IFN-α4 nucleic acid sequence TGTGATCTGCCACATACCTACAACCTGGGCAACAAGAGGGCTCTGACAGTGCTTGAAGAGATGCGCAGACTGCCTCCTCTGAGCTGCCTGAAGGATAGAAAGGACTTCGGCTTCCCTCTGGAAAAGGTGGACAACCAGCAAATCCAGAAGGCCCAGGCCATCCTGGTGCTGCGGGACCTGACCCAGCAGATCCTGAACCTGTTTACCAGCAAGGACCTGTCTGCCACCTGGAATGCCACACTGCTGGACAGCTTCTGCAACGACCTGCACCAGCAGCTGAATGATCTCAAGGCCTGCGTGATGCAGGAGCCCCCCCTGACCCAAGAGGACTCCCTGCTGGCCGTGCGGACCTACTTCCACAGAATCACCGTCTACCTGAGAAAGAAAAAGCACAGCCTGTGCGCCTGGGAAGTGATCAGAGCCGAGGTGTGGCGGGCCCTGTCCAGCTCTACAAACCTGCTGGCTAGACTGAGCGAGGAAAAAGAG。

[0033] SEQ ID NO.9: Human IFN-α2 nucleic acid sequence TGTGATCTGCCTCAGACCCACAGCCTGGGCAGCAGACGGACCCTGATGCTGCTGGCTCAGATGCGGAGAATCAGCCTGTTTTCCTGCCTGAAGGATAGACACGACTTCGGCTTCCCCCAGGAGGAGTTCGGCAATCAGTTCCAGAAGGCCGAAACCATCCCAGTGCTGCACGAGATGATTCAACAGATCTTCAACCTGTTCTCCACCAAGGACAGCAGCGCCGCTTGGGACGAGACACTGCTGGACAAGTTCTACACCGAGCTGTATCAGCAGCTGAACGACCTGGAAGCCTGCGTGATCCAGGGCGTGGGCGTCACAGAGACACCTCTGATGAAGGAAGATAGCATCCTGGCCGTGCGAAAATACTTCCAAAGAATCACCCTGTACCTGAAAGAGAAGAAGTACAGCCCTTGCGCCTGGGAGGTGGTGCGGGCCGAGATCATGAGATCTTTTTCTCTCTCTACCAACCTGCAGGAGAGCCTTAGAAGCAAGGAA。

[0034] SEQ ID NO.10: Nucleic acid sequence of the extracellular domain of gE

[0035] SEQ ID NO.11: IgG1 Fc nucleic acid sequence CCTTGCCCCGCCCCTGAGCTGCTGGGAGGCCCCTCCGTCTTCCTGTTCCCCCCCAAGCCTAAGGACACTCTGATGATCTCTAGAACACCTGAGGTGACCTGCGTGGTGGTGGACGTGTCTCACGAGGATCCTGAAGTCAAATTCAACTGGTACGTGGATGGCGTTGAAGTGCACAACGCCAAGACCAAGCCCCGGGAAGAGCAGTACAACTCCACATACAGAGTGGTGAGCGTGCTGACAGTGCTCCACCAGGACTGGCTGAATGGTAAGGAATACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAACCTAGAGAGCCTCAGGTTTACACACTGCCTCCATCTCGGGACGAGCTGACAAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTTCTGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACAACACCTCCTGTGCTGGATAGCGACGGCAGTTTCTTCCTGTACAGCAAGCTGACCGTCGACAAAAGCCGCTGGCAGCAGGGCAATGTGTTCAGTTGCTCCGTGATGCATGAGGCACTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG。

[0036] SEQ ID NO.12: mIFNα-gE-Fc nucleic acid sequence

[0037] SEQ ID NO.13: hIFNα-gE-Fc nucleic acid sequence

[0038] 2. Transformation of IFNα-gE-Fc gene expression plasmid Add 100 ng of the plasmid containing IFNα-gE-Fc provided by GenScript Biotechnology Co., Ltd. to the competent E. coli DH5α, mix well, ice bath for 20 minutes, heat shock at 42°C for 90 seconds, then add 800ul of non-antibiotic LB, shake at 37°C, 180rpm for 2 hours, spread the bacterial solution on ampicillin-resistant LB solid medium, and incubate at 37°C for 12 hours. Pick a single clone colony and inoculate it in 300 mL LB (Amp+) medium, culture at 37°C, 180rpm for 16 hours, take 1ml of bacterial solution and send it to the sequencing company for sequencing and identification, and use the large plasmid extraction kit (Beijing) to extract the plasmid pCAGGS-IFNα-gE-Fc from the remaining bacterial solution.

[0039] 3. Expression and purification of target product 293F cells were cultured in a culture flask at 37°C, 5% CO2, and 180 rpm / min on a shaker. The cell density was adjusted to 2-3 × 10 cells / mL on the day of transfection. 6 / ml, 500ml / bottle; add 500ug of plasmid DNA expressing IFNα-gE-Fc fusion protein to 12.5ml150mM saline, and add 1500ugPEI to 12.5ml saline at the same time, so that the ratio of DNA to PEI reaches 1:3, and stand at room temperature for 5min. Mix the PEI mixture with the plasmid DNA, stand at room temperature for 20min, and add 25ml of the transfection mixture to 293F cells. Add the feed solution 24h and 72h after transfection. On the 5th day after transfection, collect the cell culture medium, centrifuge at 8000rpm / min for 2h to remove cells and cell debris, collect the culture supernatant, and purify.

[0040] The cell supernatant expressing IFNα-gE-Fc was filtered through a 0.22 μm filter membrane to further remove impurities, and the cell supernatant was adsorbed through a Protein A affinity column at 4°C and washed with buffer A (Na 3 PO 4 , pH 7.4) to remove non-specific binding proteins. Then, the target protein was eluted from Protein A with buffer B (Glycine, pH 3.0), and Tris pH 9.0 was immediately added to adjust the buffer pH. The eluate was concentrated more than 40 times with a 30kD concentrator to replace the buffer A, with a final volume of less than 1 ml. The target protein was further purified by molecular sieve chromatography using a Superose Increase 10 / 300GL column (GE Healthcare). The molecular sieve chromatography buffer was PBS buffer (8 mM Na 2 HPO4 , 136 mM NaCl, 2 mM KH 2 PO 4 , 2.6 mM KCl, pH 7.4). After molecular sieve chromatography, there was an elution peak at about 14 ml. SDS-PAGE analysis showed that the protein was about 260 kDa under non-reducing conditions (without DTT) and about 130 kDa under reducing conditions (with DTT). Figure 1 ).

[0041] 4. Evaluation of fusion protein immunogenicity To detect the immunogenicity of the immunogen, we immunized C57 mice with the purified protein. The C57 mice used were purchased from Vital River Company, all female, 6-8 weeks old. The mouse immunization experiment set gE and IFNα-gE-Fc as immunogens, PBS as a negative control, and immunized mice with an equimolar mass of gE, 6 mice per group, by intramuscular injection. All mice received three vaccinations on days 0, 21, and 42, with an inoculation volume of 100 μl each time. Blood was collected and centrifuged on days 19, 33, and 56, and the serum was stored in a refrigerator at -80°C for subsequent binding antibody and neutralizing antibody detection. Splenocytes were taken on day 56 and immediately subjected to Elispot experiments to determine T cell immune responses.

[0042] The specific binding antibody titer against VZV gE in mouse serum was detected by enzyme-linked immunosorbent assay (ELISA): the recombinant VZV gE-his protein (51-538aa, obtained by artificial expression and purification) was diluted to 3ug / ml with ELISA coating solution (Solabo, C1050), and the diluted gE protein solution was added to the coating plate (Corning, 3590), 100ul / well, and coated at 4℃ overnight. The next day, the coating solution was discarded, washed twice with PBS, the water was removed, and 5% skim milk prepared in PBS was added, 100ul / well, and blocked at room temperature for 1h. In the gap between blocking, dilute the mouse serum with 5% skim milk in a round-bottom 96-well plate (Corning, 3799), with the initial dilution of 20 times, followed by a 4-fold gradient dilution. Specifically, add 7.5ul serum to 142.5ul skim milk in the first well, mix well, and complete a 1:20 dilution; take 37.5ul serum from the first well and add 112.5ul skim milk and mix well to complete a 4-fold dilution in the second well, and so on. After blocking is completed, discard the blocking solution, wash with PBST 3 times, deduct the water, add the diluted mouse serum, 100ul / well, and incubate at 37℃ for 2h. After incubation, wash with PBST 4 times, deduct the water, and dilute HRP-conjugated goat anti-mouse secondary antibody (Bio-Edge, BE0102) at 1:3000 with 5% skim milk, add 100ul to each well, and incubate at 37℃ for 1.5h. After incubation, wash 5 times with PBST, remove water, add 60ul TMB colorimetric solution (Biyuntian, P0209-500ml), react for appropriate time, and stop colorimetric solution (2M HCl) after colorimetric development. Use a microplate reader to read the OD value of OD450. A value greater than 2.5 times the blank control value is considered positive, and the antibody titer is the corresponding dilution multiple at this time. Figure 2 As shown, after one, two, and three immunizations, the fusion protein IFNα-gE-Fc of the present application can induce a high level of gE-specific IgG antibody titer after immunizing C57 mice as an immunogen, and the level of gE-specific IgG antibody induced by it is significantly higher than that of the control group gE (****, p <0.0001). The results show that: when the fusion protein IFNα-gE-Fc of the present application is used in a vaccine, without adding an exogenous adjuvant, it achieves an unexpected technical effect in eliciting specific binding antibodies against VZV gE.

[0043] Serum neutralizing antibody titer determination: The frozen serum was taken out, thawed at room temperature, and inactivated in a 56°C water bath for 30 min. The serum was then diluted with DMEM / F12 medium in a 96-well plate (Corning, 3799). The initial dilution was 1:2, followed by a 2-fold serial dilution, with a total of 8 dilutions. Similarly, the VZV virus was diluted to 100 TCID using DMEM / F12 medium. 50 / 50ul; then, the diluted serum and virus were mixed in a volume ratio of 1:1 and incubated at 37°C for 30min; then, the serum-virus mixture was transferred to a 96-well plate (Corning, 3599) filled with ARPE-19 cells at 100ul / well and incubated at 37°C for 2h; then, the cells were washed once with PBS, and the medium was changed to 2% FBS DMEM / F12 for continued culture to observe cell pathology. After 5 days, fix the cells overnight with fixative solution. The next day, discard the fixative solution, wash 3 times with PBST, and block with 100ul / well of 5% skim milk prepared with PBST at room temperature for 2h; dilute the primary antibody (303-1A8) to 1ug / ml with skim milk, add the primary antibody at 100ul / well, and incubate at room temperature for 2h; wash 4 times with PBST, dilute the secondary antibody (Goat anti-human IgG HRP, dilution concentration 1:3000) with skim milk, 100ul / well, and incubate at room temperature for 1h; wash 4 times with PBST, deduct the water, add 60ul TMB colorimetric solution for color development for 10-15min (negative wells have no color, positive wells have color), and stop the color development with 60ul stop solution (2M HCl). Use an enzyme reader to read the OD value of OD450. A value less than 2 times the blank control value is considered negative, and the neutralizing antibody titer is the corresponding dilution multiple at this time. Figure 3 As shown, after immunizing C57 mice with the fusion protein IFNα-gE-Fc of the present application as an immunogen, neutralizing antibody titers against VZV virus can be induced, and the neutralizing antibody level is significantly higher than that of the control group gE. The results show that when the fusion protein IFNα-gE-Fc of the present application is used in vaccines without adding exogenous adjuvants, it achieves unexpected technical effects in eliciting neutralizing antibodies against VZV.

[0044] The specific cellular immunity of mice was detected by enzyme-linked immunosorbent spot (Elispot) assay: two weeks after the last immunization, the mice were killed by dislocating the neck after blood was taken from the eyeballs. The mice were dissected in a clean bench and the spleen was removed. The spleen was ground to prepare a single cell suspension and the cell concentration was adjusted to 1×10 7 / ml, 5×10 5splenocytes, gE peptide library was added to the experimental wells for stimulation, culture medium was added to the negative wells, and PMA was added to the positive wells. After static culture in a 37°C incubator for 30 hours, the spots were developed and read using an Elispot plate reader. Figure 4 As shown, after immunizing C57 mice with the fusion protein IFNα-gE-Fc of the present application as an immunogen, it can stimulate VZV gE-specific cellular immune response in mice, and the induced cytokines IFN-γ and IL-2 are significantly higher than those of the control group gE. In vaccine applications, the fusion protein IFNα-gE-Fc of the present application achieves unexpected technical effects in inducing specific cellular immunity against VZV without adding exogenous adjuvants.

[0045] 5. Comparison of different adjuvants Comparing Alum adjuvant, MnJ adjuvant and AS01b adjuvant, we selected the adjuvant that can induce higher level of cellular immune response and combined it with fusion immunogen. After two immunizations, ELISA was used to detect the level of specific binding antibodies, serum neutralization was used to detect the neutralizing antibody titer, and Elispot was used to detect its cellular immunity level.

[0046] Elisa was used to detect the level of gE-specific binding antibodies in the serum of immunized mice. The serum was initially diluted 1:200 and then diluted 4 times in a gradient. Figure 5 As shown, when using Alum adjuvant and AS01b adjuvant, IFNα-gE-Fc can produce higher binding antibody levels than gE after one immunization, but there is no significant difference in antibody levels after the second immunization.

[0047] For neutralizing antibody testing, serum was diluted initially at 1:4 and then 2-fold. Figure 6 The figure shows the serum neutralizing antibody titer after the second immunization. When using Alum / AS01b adjuvant, the neutralizing antibody level produced by IFNα-gE-Fc is significantly higher than that of gE. When used in combination with MnJ adjuvant, both IFNα-gE-Fc and gE can induce the production of high levels of neutralizing antibodies.

[0048] Elispot results showed that when using Alum adjuvant or MnJ adjuvant, IFNα-gE-Fc immunogen could induce more IFN-γ and IL-2 secretion, among which MnJ adjuvant was more effective than Alum adjuvant ( Figure 7 ), but the cellular immunity enhancement effect of IFNα-gE-Fc was not obvious when it was used with AS01b adjuvant, and its cellular immunity intensity was significantly lower than that of AS01b / gE.

[0049] The experimental results show that the IFNα-gE-Fc fusion protein constructed by the present invention can induce the production of higher levels of gE binding antibodies, neutralizing antibodies and cellular immunity in a mouse immunization experiment compared with the gE prototype when there is no exogenous adjuvant or when the Alum adjuvant is used; when the MnJ adjuvant is used, both IFNα-gE-Fc and gE can induce the production of high levels of neutralizing antibodies, but the cellular immunity induced by IFNα-gE-Fc is stronger; when the AS01b adjuvant is used, IFNα-gE-Fc can induce the production of higher levels of neutralizing antibodies, but the cellular immunity level is weaker than that of AS01b / gE.

Claims

1. A fusion protein IFNα-gE-Fc, characterized in that: The invention comprises interferon α, VZV glycoprotein gE and human IgG1 Fc domain, wherein IFN α can be from human or mouse, preferably mouse IFN-α4, human IFN-α2, and its amino acid sequence is specifically as shown in SEQ ID NO: 1 or SEQ ID NO: 2; VZV glycoprotein gE is preferably its extracellular domain, specifically amino acids 31 to 538, as shown in SEQ ID NO: 3; human IgG1 Fc domain is preferably CH2-CH3; Among them, interferon α is connected to the N-terminus of the extracellular segment of VZV glycoprotein gE, and the human IgG1Fc domain is connected to the C-terminus of the extracellular segment of VZV glycoprotein gE, and they are directly connected in series to obtain the fusion protein IFNα-gE-Fc.

2. The fusion protein IFNα-gE-Fc according to claim 1, characterized in that The signal peptide of IL-10 is also connected, and its specific amino acid sequence is shown in SEQ ID NO:

5.

3. The fusion protein according to claim 1, characterized in that The immunoglobulin Fc fragment is selected from the immunoglobulin Fc fragment of humans, mice, rabbits, cows, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs; further, the immunoglobulin Fc fragment is selected from the Fc fragment of IgG, IgA, IgD, IgE or IgM; preferably, the immunoglobulin Fc fragment is selected from IgG1 Fc fragment, IgG2 Fc fragment, IgG3 Fc fragment, or IgG4 Fc fragment; particularly preferably, the immunoglobulin Fc fragment is a human IgG Fc fragment, most preferably the amino acid sequence shown in SEQ ID NO.

4.

4. The fusion protein according to claim 3, characterized in that The fusion protein comprises the amino acid sequence shown in SEQ ID NO.6 and SEQ ID NO.

7.

5. A nucleic acid encoding the fusion protein according to any one of claims 1 to 4; Preferably, the nucleic acid coding sequence of mouse IFN-α4 is as shown in SEQ ID NO: 8 or its degenerate sequence; the nucleic acid coding sequence of human IFN-α2 is as shown in SEQ ID NO: 9 or its degenerate sequence; the nucleic acid coding sequence of gE extracellular segment is as shown in SEQ ID NO: 10 or its degenerate sequence; the nucleic acid coding sequence of immunoglobulin Fc fragment is as shown in SEQ ID NO: 11 or its degenerate sequence; More preferably, the sequence is codon-optimized according to mammalian cell expression preferences.

6. The encoding nucleic acid according to claim 5, characterized in that The nucleotide sequence is shown in SEQ ID NO: 12, SEQ ID NO: 13 or their degenerate sequences.

7. A recombinant vector or recombinant cell containing the encoding nucleic acid according to claim 5.

8. Use of the fusion protein IFNα-gE-Fc according to any one of claims 1 to 4 in the preparation of a recombinant subunit vaccine for varicella or a recombinant subunit vaccine for herpes zoster.

9. A recombinant subunit vaccine for varicella or herpes zoster, characterized in that: It comprises the fusion protein IFNα-gE-Fc according to any one of claims 1 to 4; it does not include an adjuvant or includes an adjuvant, specifically Alum adjuvant, MnJ adjuvant, AS01b adjuvant.

10. The varicella recombinant subunit vaccine or herpes zoster recombinant subunit vaccine according to claim 9, characterized in that: The fusion protein is obtained by chemical synthesis or gene recombination.

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