IMMUNOGEN FOR BROAD-SPECTRUM INFLUENZA VACCINE AND APPLICATION THEREOF
20220118077 · 2022-04-21
Assignee
Inventors
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2710/24134
CHEMISTRY; METALLURGY
C12N2760/16134
CHEMISTRY; METALLURGY
C12N2760/16122
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C12N2795/10343
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
A61K2039/545
HUMAN NECESSITIES
C12N2710/10043
CHEMISTRY; METALLURGY
C12N2710/24143
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure relates to a novel influenza immunogen with broad-spectrum anti-influenza virus effect and the immunization method thereof. The present disclosure provides a novel anti-influenza immunogen whose sequence comprises the amino acid sequence shown in SEQ ID No: 1 and SEQ ID No: 2, or an immunogenic fragment thereof, or a combination thereof. In addition, the present disclosure also provides use of the recombinant vector vaccine using said immunogen in the anti-influenza vaccine, and the immunization method of the recombinant vector vaccine using said immunogen. Through the sequential administration of multiple vector vaccines expressing the novel influenza immunogen, and the combined use of systemic administration and local administration, a high-level T cell immune response is induced in the local respiratory tract, which can produce broad-spectrum protection against multiple influenza virus infections.
Claims
1. An anti-influenza vaccine immunogen, wherein the immunogen comprises the sequences shown in SEQ ID No: 1 and SEQ ID No: 2 or an immunogenic fragment thereof, or a combination thereof.
2. The anti-influenza vaccine immunogen according to claim 1, wherein the immunogen comprises internal conserved proteins of influenza virus, or immunogenic fragments of the conserved proteins.
3. The anti-influenza vaccine immunogen according to claim 1, wherein the internal conserved proteins of influenza virus include influenza virus matrix protein (M1, M2), nucleoprotein (NP), alkaline polymerase (PB1, PB2) and acid polymerase (PA).
4. The anti-influenza vaccine immunogen according to claim 1, wherein the immunogen is derived from recombinant proteins of all influenza virus subtypes, or recombinant proteins of shared sequences thereof, or a combination thereof; and the influenza virus subtypes include H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18 subtypes, and type B influenza virus.
5. An anti-influenza vaccine, which is a recombinant vector vaccine expressed and constructed in multiple different vectors by using the anti-influenza vaccine immunogen of claim 1.
6. The anti-influenza vaccine according to claim 5, wherein the recombinant vector vaccine comprises a recombinant protein vaccine, a recombinant DNA vaccine, a recombinant virus vector vaccine, a recombinant bacterial vector vaccine, a recombinant yeast vector vaccine or a recombinant virus-like particle vaccine.
7. The anti-influenza vaccine according to claim 5, wherein the virus vector comprises an adenovirus vector, a poxvirus vector, an adeno-associated virus vector, a herpes simplex virus vector, and a cytomegalovirus vector.
8. (canceled)
9. A method for constructing a recombinant influenza vaccine for immunization, comprising the step of sequential administration with different recombinant vector vaccines according to claim 5 for each immunization, wherein each recombinant vector vaccine is administered at least once, and the vaccination process at least involves one respiratory tract administration and one systemic administration.
10. The method according to claim 9, wherein a recombinant vaccine derived from a different vector is used for each shot during the vaccination process.
11. The method according to claim 9, wherein the vaccination is performed by means of “primary-boost-re-boost” with each recombinant vaccine administered at least once, and the vaccination process at least involves one respiratory tract administration and one systemic administration.
12. The method according to claim 9, wherein the mode of systemic administration includes intramuscular injection, subcutaneous administration, and intradermal administration.
13. The method according to claim 9, wherein the mode of respiratory tract administration includes atomization and nasal drop.
14. The method according to claim 9, wherein the vaccination process is as follows: primary vaccination with recombinant DNA vaccine via intramuscular injection, boosting with recombinant adenovirus vector vaccine via the respiratory tract, and reboosting with recombinant poxvirus vaccine via intramuscular injection.
15. The method according to claim 9, wherein the recombinant poxvirus vaccine is used as the last shot for the vaccination process.
16. The method according to claim 9, wherein the interval between every two shots is at least 1 week.
17. (canceled)
18. The method according to claim 9, wherein the vaccine and vaccination technique can be used to vaccinate poultry to prevent the spread of avian influenza to human.
19. The method according to claim 9, wherein the vaccine and vaccination technique can be used to vaccinate human to reduce the pathogenicity of human infection with avian influenza.
20. The method according to claim 9, wherein the vaccine and vaccination technique can be used to vaccinate human to reduce the pathogenicity of human infection with human influenza.
21. The method according to claim 9, wherein the vaccine and vaccination technique can be used to vaccinate human to prevent human-to-human transmission of influenza.
22-27. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0036]
[0037]
[0038]
[0039]
[0040] The present disclosure will now be specifically described by way of the following examples.
DETAILED DESCRIPTION
[0041] Other aspects of the present disclosure are described in detail below. These and other features and advantages of the present disclosure will become apparent upon reading the detailed description of the embodiments disclosed below and the appended claims.
[0042] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.
Example 1: Design and Preparation of Anti-Influenza Vaccine Immunogen
[0043] The GenBank database is a gene sequence database established by the National Center for Biotechnology Information (NCBI), through which the gene sequences of about 40,000 strains of influenza virus can be retrieved.
[0044] The amino acid sequences of M1, M2, NP, PB1, PB2, and PA proteins interior to the above-mentioned about 40,000 strains of influenza viruses were computationally analyzed, and the amino acid with the highest frequency at each position of the amino acid sequence was regarded as the shared amino acid at that position. The shared amino acids at individual sites constitute the shared amino acid sequence of the protein, thus resulting in the shared amino acid sequences of M1, M2, NP, PB1, PB2, and PA proteins.
[0045] The online CD8 T cell epitope prediction software was used to analyze the shared amino acid sequences of PB1, PB2 and PA obtained above. The online software used is derived from http://tools.immuneepitope.org/main/tcell/ and http://www.syfpeithi.de/. The common CD8 T cell epitopes predicted by the two software programs were set aside, and then joined to form the amino acid epitope sequences of PB1, PB2 and PA.
[0046] Based on the resulting amino acid sequences above, the vaccine sequence was designed. The PA and PB1 amino acid epitope sequences obtained by epitope joining were combined with shared amino acid sequence of M1 protein to obtain an vaccine amino acid sequence, named SEQ ID No: 1. The PB2 amino acid epitope sequence obtained by epitope joining was combined with shared amino acid sequences of NP and M2 proteins to obtain an another vaccine amino acid sequence, named SEQ ID No: 2.
[0047] The above amino acid sequences SEQ ID No: 1 and SEQ ID No: 2 are translated into nucleic acid sequences, and the nucleic acid sequence is optimized for eukaryotic codon via http://www.jcat.de/ online software, resulting in the nucleic acid sequences of SEQ ID No: 1 and SEQ ID No: 2, which were synthesized by Suzhou GENEWIZ Biotechnology Co., Ltd. The synthesized sequences was sequenced by Suzhou GENEWIZ Biotechnology Co., Ltd and verified to be the sequences SEQ ID No: 1 and SEQ ID No: 2 of the present disclosure.
Example 2: Construction of Vaccine Based on Anti-Influenza Vaccine Immunogen
[0048] The immunogen of the present disclosure was used to construct a recombinant DNA vector vaccine, a recombinant adenovirus vector vaccine and a recombinant poxvirus vector vaccine.
[0049] The immunogen SEQ ID No: 1 or SEQ ID No: 2 of the present disclosure was inserted into the pSV1.0 vector (preserved by Shanghai Public Health Clinical Center) to construct a recombinant DNA vector vaccine, named pSV1.0-SEQ ID No: 1 and pSV1.0-SEQ ID No: 2 respectively.
[0050] The immunogen SEQ ID No: 1 or SEQ ID No: 2 was inserted into the AdC68 adenovirus vector (purchased from Institut Pasteur of Shanghai, Chinese Academy of Sciences) and transfected into 293a cells (purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) to construct a recombinant adenovirus vector vaccine, named AdC68-SEQ ID No: 1 and AdC68-SEQ ID No: 2 respectively.
[0051] Immunogens SEQ ID No: 1 and SEQ ID No: 2 were linked using cleavage peptide p2a, inserted into pSC65 vector (preserved by Shanghai Public Health Clinical Center), and transfect into TK143 cells (purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) to construct a recombinant poxvirus vector vaccine named TTV-SEQ ID No: 1/2.
[0052] The expression of anti-influenza vaccine immunogen was detected by Western blotting and the specific steps are as follows:
[0053] (1) Preparation of Experimental Samples
[0054] PSV1.0-SEQ ID No: 1 or pSV1.0-SEQ ID No: 2 was respectively transfected into 293T cells (purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), and 293T cells were collected 48 hours later. The collected cells were resuspended with 75 microliters of cell lysate followed by addition of 25 microliters of protein loading buffer. Samples were prepared in a water bath at 100° C. for 10 minutes.
[0055] AdC68-SEQ ID No: 1 or AdC68-SEQ ID No: 2 was respectively transfected into 293A cells, and 293A cells were collected 24 hours later. The collected cells were resuspended with 75 microliters of cell lysate followed by addition of 25 microliters of protein loading buffer. Samples were prepared in a water bath at 100° C. for 10 minutes.
[0056] TTV-SEQ ID No: 1/2 was transfected into TK143 cells, which were collected 48 hours later. The collected cells were resuspended with 75 microliters of cell lysate followed by addition of 25 microliters of protein loading buffer. Samples were prepared in a water bath at 100 degrees Celsius for 10 minutes.
[0057] (2) Western blotting: 8% polyacrylamide separation gel was prepared and left at room temperature for 30 minutes. 10% polyacrylamide concentrated gel was added immediately followed by gentle insertion of a comb. The resulting gel was left for 30 minutes until solidified, followed by loading into electrophoresis tank. The electrophoresis buffer was poured into the tank, with slow removal of the comb. The samples prepared as above were loaded sequentially. The electrophoresis was run for half an hour at 70V voltage and then at 90V for an additional 1.5 hours. After activating the polyvinylidene fluoride membrane in methanol for 30 seconds, the sponge, filter paper and polyvinylidene fluoride membrane were soaked with the transfer buffer, and were set up in sequence. The gel and ice bag were placed into the transfer tank which was then filled with the pre-cooled transfer buffer. The transfer was run at a constant current of 200 mA for 2.5 hours. Upon completion, the polyvinylidene fluoride membrane was removed and blocked in 5% skimmed milk powder for 1 hour. The influenza matrix protein 1 antibody (purchased from Abcam (Shanghai) Trading Co., Ltd.) at a dilution of 1:1000 and matrix protein 2 antibody at a dilution of 1:250 (Santa Cruz Biotechnology (Shanghai) Co., Ltd.) were added respectively. After incubating for 2 hours at room temperature on a shaker, the membrane was washed with Tween-20 in phosphate buffer for 3×5 minutes. Then, the horseradish peroxidase-labeled goat anti-mouse IgG antibody at a dilution of 1:5000 was added. After incubating for 1 hour at room temperature on a shaker, the membrane was washed with 5×5 minutes. The developer solution was prepared and then covered onto the polyvinylidene fluoride membrane for luminescence detection.
[0058] The immunogen expression results detected by Western blotting were shown in
Example 3: Detection of Immunogenicity of Anti-Influenza Vaccine Immunogen-Based Vaccine
[0059] As described in Example 2, the immunogen of the present disclosure was used to construct DNA vaccines, adenovirus vector vaccines, and poxvirus vector vaccines. The recombinant influenza vaccine was used to immunize mice, and four weeks after completion of the vaccination, the immunogenicity of the recombinant influenza vaccine was evaluated.
[0060] The 6-week-old C57BL/6 mice were randomly divided into 3 groups, named control group, adenovirus group and poxvirus group. The specific vaccination procedures are shown in Table 1. The mode of administration was intramuscular injection. The administration dose was 100 micrograms for pSV1.0, 10.sup.11 virus particles for AdC68, 50 micrograms for each of pSV1.0-SEQ ID No: 1 and pSV1.0-SEQ ID No: 2, 5×10.sup.10 virus particles for each of AdC68-SEQ ID No: 1 and AdC68-SEQ ID No: 2, while 10.sup.7 plaque forming units for TTV-SEQ ID No: 1/2. The interval between two shots was two weeks.
TABLE-US-00001 TABLE 1 Mouse experiment based on anti-influenza vaccine immunogen Group/week 0 week 2 weeks 4 weeks Control pSV1.0 pSV1.0 AdC68 group Adenovirus pSV1.0-SEQ ID No.: 1 pSV1.0-SEQ ID No.: 1 AdC68-SEQ ID No.: 1 group pSV1.0-SEQ ID No.: 2 pSV1.0-SEQ ID No.: 2 AdC68-SEQ ID No.: 2 Poxvirus pSV1.0-SEQ ID No.: 1 pSV1.0-SEQ ID No.: 1 TTV-SEQ ID No.: 1/2 group pSV1.0-SEQ ID No.: 2 pSV1.0-SEQ ID No.: 2
[0061] The immunogenicity of recombinant influenza vaccines was tested in mouse spleen cells using enzyme-linked immunospot assay (ELISpot) and intracellular staining of cytokines (ICS) method.
[0062] According to the epitope prediction for SEQ ID No: 1 and SEQ ID No: 2, and the reported common influenza T cell epitopes, 16 epitope monopeptides were selected to stimulate the T cell immune response in mouse, designated as: M1-1, M1-2, M1-3, M2, NP-1, NP-2, NP-3, PB1-1, PB1-2, PB1-3, PB2-1, PB2-2, PB2-3, PA-1, PA-2, and PA-3 respectively.
[0063] (1) The Procedures for Enzyme-Linked Immunospot Assay are as Follows:
[0064] One day before the experiment, mouse interferon gamma protein was diluted to a final concentration of 5 μg/ml, added 100 μl per well to the assay plate, and coated overnight at 4° C. The next day, the coating solution was discarded. Wells were washed once with 200 microliters of complete medium for each well. Then, 200 microliters of complete medium was added for blocking at room temperature for 2 hours. Upon completion, the concentration of mouse spleen cells was adjusted to 4×10.sup.6 cells per milliliter. Each well was added 50 microliters of spleen cells, then 50 microliters of 10 μg/ml monopeptide, for incubation in an incubator for about 20 hours. Upon completion, wells were washed twice with 200 microliters of distilled water for each well, and then washed 3 times with 200 microliters of Tween-20 in phosphate buffer. The anti-mouse interferon gamma biotin was diluted to a final concentration of 2 g/ml, added 100 microliters each well for incubation at room temperature for 2 hours. Upon completion, wells were washed 3 times with 200 microliters of Tween-20 in phosphate buffer for each well. The horseradish peroxidase fluorescent substrate was diluted 1:100, added 100 microliters each well for incubation at room temperature for 1 hour. Upon completion, each well was washed 4 times with 200 microliters of Tween-20 in phosphate buffer, and then washed twice with 200 microliters of phosphate buffer. The developer solution was prepared and added 100 microliters each well, allowing to react at room temperature for about 15 minutes in the dark. When clear red spots occurred, the plate was gently rinsed with tap water for 5 minutes to stop the chromogenic reaction. After drying at room temperature, the plate was placed into the enzyme-linked immunospot plate reader for reading and the number of positive spots was counted.
[0065] (2) The Procedures for Intracellular Factor Staining are as Follows:
[0066] The mouse spleen cells were diluted to 2×10.sup.7 cells per milliliter. Each well was added 150 microliters of cells and 150 microliters of peptide library, then 1 microliter of CD107a antibody. After incubating for 1 hour, each well was added 0.3 microliters protein transport blocking agent for incubation in an incubator for 6 hours. Upon completion, cells were collected into a flow tube, and then centrifuged at 800 rpm for 3 minutes. The cells were washed with 800 μl staining buffer per tube and centrifuged at 800 rpm for 3 minutes. The supernatant was discarded. CD3, CD8, cell viability/cytotoxicity staining antibody mixture was prepared. Each tube was added 40 microliters of the antibody mixture and stained for 20 minutes at room temperature in the dark. Upon completion, each tube was washed twice with 800 microliters of staining buffer, centrifuged at 800 rpm for 3 minutes. The washing solution was discarded, followed by the addition of 150 microliters of fixative per tube for fixation at room temperature for 20 minutes in the dark. Each tube was washed with 800 microliters of staining buffer, centrifuged at 800 rpm for 3 minutes. The supernatant was discarded. The interferon gamma and tumor necrosis factor alpha staining antibody mixture was prepared. Each tube was added 40 microliters of the antibody mixture and stained for 20 minutes at room temperature in the dark. Each tube was washed with 800 microliters of staining buffer, centrifuged at 1200 rpm for 3 minutes. After the supernatant was discarded, the cells were resuspended in 250 microliters of staining buffer and detected by flow cytometry. Statistical results were analyzed.
[0067] The results of the vaccine immunogenicity test are shown in
[0068] The results of the enzyme-linked immunospot assay showed that for the control group mice, spot-forming cells were not seen with no influenza-specific T cell immune response; for the adenovirus group mice, more spot-forming cells against the two epitopes of NP-2 and PB2-1 were seen with a higher level T cell immune response; while for the poxvirus group mice, spot-forming cells were seen against NP-2, NP-3, PB1-1, PB1-3, PA-3 and other epitopes with a higher T cell immune response.
[0069] Intracellular factors interferon gamma, tumor necrosis factor alpha, and CD107a staining were used to detect influenza-specific immune response level in mouse spleen cells. The results showed that there were no T cells expressing interferon gamma, tumor necrosis factor alpha, and CD107a in the control group; while T cells expressing interferon gamma, tumor necrosis factor alpha, and CD107a were found in both the adenovirus group and the poxvirus group, thus demonstrating influenza-specific T cell immune response.
[0070] This Example confirmed that the expression of anti-influenza vaccine immunogens SEQ ID No: 1 and SEQ ID No: 2 through different vaccine vectors can induce significant T cell immune responses.
Example 4: Evaluation of the Challenge-Protection Based on Anti-Influenza Immunogen
[0071] As described in Example 2, the immunogen of the present disclosure was used to construct DNA vaccines, adenovirus vector vaccines, and poxvirus vector vaccines. As described in Example 3, the recombinant influenza vaccine was used to immunize mice, and four weeks after completion of the vaccination, the protective effect of the recombinant influenza vaccine upon challenge was evaluated.
[0072] The H1N1 and H7N9 influenza challenge models were used to evaluate the protective effect of the immunogen. The H1N1 influenza challenge experiment was carried out in the biosafety level-2 laboratory, and the H7N9 influenza challenge experiment was carried out in the biosafety level-3 laboratory.
[0073] Each mouse was anesthetized by intraperitoneal injection of 50 microliters of 10% chloral hydrate, and each mouse was challenged with 50 microliters nasal drops of influenza virus. The challenge dose for H1N1 influenza virus was 500 TCID.sub.50 (median tissue culture infective dose) per mouse. The challenge dose for H7N9 influenza virus was 100 TCID.sub.50 per mouse. On the 5th day after the challenge, 5 mice in each group were sacrificed, and the lungs were taken for virus load determination.
[0074] The results of the challenge-protection results are shown in
[0075] After a lethal dose challenge of H1N1 influenza virus, mice in the control group continued to lose weight and all reported death on the 12.sup.th day. The mice in the adenovirus group began to recover on the 9.sup.th day and all survived to 14 days. For the poxvirus group, the weight loss of the mice significantly slowed down; the body weight began to rise on the 9.sup.th day; and all mice survived to 14 days.
[0076] After a non-lethal dose challenge of H7N9 influenza virus, mice in the control group lost nearly 20% of their body weight and recovered on the 9.sup.th day. Mice in the adenovirus group and poxvirus group lost less than 10% of their body weight, and their body weight recovered rapidly on the 7.sup.th day.
[0077] This Example confirmed that the expression of anti-influenza vaccine immunogens SEQ ID No: 1 and SEQ ID No: 2 through different vaccine vectors can produce cross-protective effects against H1N1 and H7N9 influenza viruses, that is, the immunogen of the present disclosure has a broad-spectrum protective effect against different subtypes of influenza virus.
Example 5: Immunogenicity Test of Influenza Vaccine Based on Different Immunization Methods
[0078] As described in Example 2, the immunogen of the present disclosure was used to construct DNA vaccines, adenovirus vector vaccines, and poxvirus vector vaccines. The immunization method of the present disclosure is used to immunize mice. Four weeks after completion of the vaccination, the immunogenicity test was performed according to the method described in Example 3.
[0079] The 6-week-old C57BL/6 mice were randomly divided into 5 groups, designated as control group 1, control group 2, control group 3, experimental group 1, and experimental group 2 respectively, in which experimental group 1 and experimental group 2 adopted the immunization method of the present disclosure. The specific vaccination procedures are shown in Table 2. The administration dose was 100 micrograms for pSV1.0, 10.sup.11 virus particles for AdC68, 50 micrograms for each of pSV1.0-SEQ ID No: 1 and pSV1.0-SEQ ID No: 2, 5×10.sup.10 virus particles for each of AdC68-SEQ ID No: 1 and AdC68-SEQ ID No: 2, while 10.sup.7 plaque forming units for TTV and TTV-SEQ ID No: 1/2. The interval between two shots was two weeks.
TABLE-US-00002 TABLE 2 Mouse vaccination experiment based on different immunization methods Group/week 0 week 2 weeks 4 weeks Control intramuscular intramuscular intramuscular group 1 injection with pSV1.0 injection with injection with TTV AdC68 Control Intramuscular Intramuscular intramuscular group 2 vaccination with vaccination with injection with TTV- pSV1.0-SEQ ID No.: 1 AdC68-SEQ ID SEQ ID No.: 1/2 pSV1.0-SEQ ID No.: 2 No.: 1 AdC68-SEQ ID No.: 2 Control Intramuscular Intramuscular intramuscular group 3 vaccination with vaccination with injection with pSV1.0-SEQ ID No.: 1 TTV-SEQ ID AdC68-SEQ ID pSV1.0-SEQ ID No.: 2 No.: 1/2 No.: 1 AdC68-SEQ ID No.: 2 Experimental Intramuscular Nasal dropping of Intramuscular group 1 vaccination with AdC68-SEQ ID vaccination with pSV1.0-SEQ ID No.: 1 No.: 1 TTV-SEQ ID pSV1.0-SEQ ID No.: 2 AdC68-SEQ ID No.: 1/2 No.: 2 Experimental Intramuscular Intramuscular Nasal dropping of group 2 vaccination with vaccination with AdC68-SEQ ID pSV1.0-SEQ ID No.: 1 TTV-SEQ ID No.: 1 pSV1.0-SEQ ID No.: 2 No.: 1/2 AdC68-SEQ ID No.: 2
[0080] The results of the vaccine immunogenicity test are shown in
[0081] The results of the enzyme-linked immunospot assay showed that in the spleen cells of mice, for the control group mice, spot-forming cells were not seen with no influenza-specific T cell immune response; for each single peptide in the control group 2, 3 and experimental group 1 and 2, spot-forming cells were seen with a high level of T cell immune response. In mouse lung lavage fluid, no spot-forming cells were seen in the control group 1, 2 and 3, and influenza-specific immune response could not be established in the lung; more spot-forming cells were seen in the experimental group 1 and 2, demonstrating that experimental group 1 and experimental group 2 using the vaccination method of the present disclosure showed a very high level of influenza-specific T cell immune response.
[0082] Intracellular factors interferon gamma, tumor necrosis factor alpha, and CD107a staining were used to detect influenza-specific immune response level in mouse spleen cells. The results showed that there were no T cells expressing interferon gamma and tumor necrosis factor alpha in control group 1, while T cells expressing interferon gamma and tumor necrosis factor alpha were found in control group 2, 3 and experimental group 1 and 2, thus exhibiting influenza-specific T cell immune response.
[0083] This Example confirmed that through the sequential administration of different recombinant vector vaccines, and the combination of the respiratory tract and systemic immunization, the experimental group 1 and the experimental group 2 using the vaccination method of the present disclosure can effectively establish a high level of influenza-specific immune response in both the whole body system and the local lung, which is superior to that of the control group.
Example 6: Evaluation of the Challenge-Protection Based on Different Immunization Methods
[0084] According to the method described in Example 5, the immunization method of the present disclosure was used to immunize mice, and four weeks after the last shot for the mouse, H1N1 and H7N9 influenza challenge models were used to evaluate the protective effect of the immunogen. The H1N1 influenza challenge experiment was carried out in the biosafety level-2 laboratory, and the H7N9 influenza challenge experiment was carried out in the biosafety level-3 laboratory.
[0085] Each mouse was anesthetized by intraperitoneal injection of 50 microliters of 10% chloral hydrate, and each mouse was challenged with 50 microliters nasal drops of influenza virus. The challenge dose for H1N1 influenza virus was 500 TCID.sub.50 (median tissue culture infective dose) per mouse. The challenge dose for H7N9 influenza virus was 500 TCID.sub.50 per mouse. On the 5th day after the challenge, 5 mice in each group were sacrificed, and the lungs were taken for virus load determination.
[0086] The results of the challenge-protection results are shown in
[0087] After the H1N1 influenza virus challenge, all mice in the control group 1 died on the 13.sup.th day, while the control groups 2 and 3 showed partial protective effects, in which 80% and 60% of the mice survived to the 14.sup.th day, respectively. The weight of mice in experimental group 1 and experimental group 2 using the vaccination method of the present disclosure recovered on the 10.sup.th day, and all survived to the 14.sup.th day, in which the viral load of the experimental group 2 was significantly reduced, showing an excellent protective effect.
[0088] After the H7N9 influenza virus challenge, the weight of mice in experimental group 1 and experimental group 2 using the vaccination method of the present disclosure quickly recovered on the 10.sup.th day, and all the mice survived to the 14.sup.th day, showing an excellent protective effect. No apparent protective effect was seen in other groups of mice.
[0089] This Example confirmed that through the sequential administration of different recombinant vector vaccines, and the combination of the respiratory tract and systemic immunization, experimental group 1 and experimental group 2 using the vaccine immunization method of the present disclosure showed excellent cross-protective effects against H1N1 and H7N9 influenza viruses; and its protective effect is superior to that of control group 2 and control group 3 which merely use one route of intramuscular injection. Moreover, when the recombinant poxvirus vector vaccine was used as the last shot of vaccine, the protective effect of the vaccine is optimal.
Example 7: Evaluation of the Enhanced Protective Effect by Additional Nasal Drop Vaccination During Challenge with Influenza Virus in the Experimental Group Mice
[0090] According to the method described in Example 5, the immunization method of the present disclosure was used to immunize mice. Four weeks after the last shot for the mouse, the H1N1 and H7N9 influenza challenge models were used to evaluate the protective effect of the immunogen. The specific procedures for influenza virus attack are described in Example 6. Throughout the challenge process, the mice were continuously offered with drinking water containing 2 μg/ml FTY720. FTY720 is an immunosuppressant that can effectively reduce the number of peripheral circulating lymphocytes and retain the lung colonization of tissue in situ memory T cells established by nasal inoculation. FTY720 was continuously used during the challenge with a lethal dose of H1N1 and H7N9 influenza viruses in order to evaluate whether the nasal inoculation showed a strengthening effect.
[0091] The experimental results are shown in
[0092] Upon H1N1 and H7N9 influenza virus challenge, the experimental group 1+FTY720 and the experimental group 2+FTY720 both showed partial protection. The weight of the mice began to rise on the 11.sup.th day and survived to the 14.sup.th day with a reduction in viral load. The protective effect in the experiment groups is superior to that of the control group 1+FTY720.
[0093] This Example confirmed that the administration mode via respiratory tract can effectively enhance the protective effect of the vaccine against H1N1 and H7N9 influenza.
[0094] The present disclosure is not limited to the above-mentioned embodiments, and those skilled in the art will understand that various modifications, additions, and substitutions can be made without departing from the scope and spirit of the present invention disclosed in the appended claims.