A fusion protein vaccine for inhibiting group A streptococcal infection and its application

Abstract

A fusion protein vaccine for inhibiting group A streptococcal infection and its application, belonging to the field of biomedical technology. In order to solve the technical problems of high vaccine price, low safety and effectiveness of group A streptococcal vaccines in the prior art, the present invention provides a fusion protein vaccine for inhibiting group A streptococcal infection, wherein the amino acid sequence of the fusion protein in the fusion protein vaccine is shown in SEQ ID NO.1; the fusion protein vaccine can induce a high level of specific antibodies, and the antibodies have no cross-reaction with human heart, brain, and kidney tissues, and effectively protect mice against M12 type GAS infection. The fusion protein vaccine provided by the present invention can be used to prepare drugs for inhibiting group A streptococcal infection, has the advantages of targeting the Chinese epidemic strain, high safety, and simple use, and is easy to promote and use.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a vaccine for inhibiting group A streptococcal infection and its application. Background Art

[0002] Group A Streptococcus (GAS), also known as group A Streptococcus or group A Streptococcus, can cause various types of superficial infections such as tonsillitis, impetigo, erysipelas, etc. in mild cases, and severe invasive infections that can be life-threatening, such as bacteremia, toxic shock, and necrotizing fasciitis. Another important reason why GAS has attracted people's attention is that it is related to autoimmune diseases. Recurrent GAS tonsillitis can lead to autoimmune sequelae, such as acute rheumatic fever, rheumatic heart disease, glomerulonephritis, etc. Among them, rheumatic heart disease and invasive infections cause more than 500,000 deaths worldwide each year.

[0003] GAS has more than 150 serotypes, and new serotypes are constantly emerging. Infection with one serotype cannot produce effective immune protection against different serotypes. At present, the GAS vaccine targets designed by domestic and foreign researchers mainly include a variety of bacterial components and secreted toxins. The recombinant SpyCEP protein is combined with J8-DT and an aluminum salt adjuvant is added for subcutaneous immunization. The vaccine can effectively prevent and control highly toxic CovR / S mutant strains, but its single immune effectiveness is low (Pandey M, Mortensen R, Calcutt A, Powell J, Batzloff MR, Dietrich J, Good MF. Combinatorial Synthetic Peptide Vaccine Strategy Protects against Hypervirulent CovR / S Mutant Streptococci. J Immunol (Baltimore, Md.: 1950). 2016; 196 (8): 3364-3374.). The M2-type GAS recombinant SOF2 protein vaccine prepared by the researchers can produce antiserum that can opsonize and phagocytize M2, M4, and M28-type GAS that express SOF, but is ineffective against M5-type GAS that does not express SOF (Courtney HS, Hasty DL, Dale JB. Serum opacity factor (SOF) of Streptococcus pyogenes evokes antibodies that opsonize homologous and heterologous SOF-positive serotypes of group Astreptococci. Infect Immun. 2003; 71(9): 5097-5103.).Comparing the protective effects of various candidate vaccines, the recombinant M1 protein vaccine has a protection rate of up to 100% against the same serotype (Rivera-Hernandez T, Pandey M, Henningham A, Cole J, Choudhury B, Cork AJ, Gillen CM, Ghaffar KA, West NP, Silvestri G, Good MF, Moyle PM, Toth I, Nizet V, Batzloff MR, Walker MJ. Differenting Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models. mBio. 2016; 7(3): e00618-16.). M protein is an important virulence factor on the surface of GAS bacteria, which has the effects of promoting bacterial adhesion, invasion and anti-phagocytosis. The anti-M protein antibody produced after the body is infected with GAS is a protective antibody, but the antibodies produced against the intermediate fragments of the M protein have cross-reactions with tissues such as the human myocardium, which limits the application of M protein in vaccine preparation. In response to the above problems, researchers selected the N-terminal amino acid sequences of the M protein of 26 GAS serotypes, developed a 26-valent recombinant protein vaccine, and removed the fragments that cross-reacted with human tissues (Hu MC, Walls MA, Stroop SD, Reddish MA, Beall B, Dale JB. Immunogenicity of a 26-valent group A streptococcal vaccine. Infect Immun. 2002; 70 (4): 2171-2177.). Based on the 26-valent vaccine, a 30-valent recombinant protein vaccine of M protein was launched, which can induce animals to produce bactericidal antibodies and has a high intra-type protection rate, but the vaccine price is relatively high and needs to be further optimized (Pastural). McNeil SA, MacKinnon-Cameron D, YeLY, Langley JM, Stewart R, Martin LH, Hurley GJ, Salehi S, Penfound TA, Halperin S, Dale JB. Safety and immunogenicity of a 30-valent Mprotein-based group astreptococcal vaccine in healthy adult volunteers: A randomized, controlled phase I study.Vaccine.2020;38(6):1384-1392.).

[0004] Therefore, those skilled in the art have been eager to develop a vaccine that is low in price, highly protective, and highly safe and can effectively inhibit group A streptococcal infection. Summary of the invention

[0005] The present invention aims to solve the technical problems in the prior art that the Group A Streptococcus vaccine has high vaccine price, low safety and low effectiveness, and provides a fusion protein vaccine for inhibiting Group A Streptococcus infection and its application.

[0006] One of the purposes of the present invention is to provide a fusion protein vaccine for inhibiting group A streptococcal infection, wherein the fusion protein vaccine comprises a fusion protein, a KSI tag and an acceptable adjuvant.

[0007] In a preferred embodiment of the present invention, the amino acid sequence of the fusion protein is shown as SEQ ID NO.1.

[0008] In a preferred embodiment of the present invention, the adjuvant is any one of aluminum salt adjuvant, Freund's adjuvant, CPG, AS01, AS03, AS04, MF-59, Montanide ISA 720 or MPL.

[0009] In a preferred embodiment of the present invention, the mixing volume ratio of the fusion protein to the adjuvant in the fusion protein vaccine is 1:1.

[0010] In a preferred embodiment of the present invention, the fusion protein vaccine is administered by any one of intramuscular injection, subcutaneous injection or intranasal inhalation immunization.

[0011] The second object of the present invention is to provide the use of the above fusion protein vaccine in the preparation of drugs for inhibiting group A streptococcal infection.

[0012] In a preferred embodiment of the present invention, the route of infection by group A streptococci is the respiratory system or the skin.

[0013] In a preferred embodiment of the present invention, the infection is a human pharyngeal infection in the respiratory system.

[0014] Beneficial effects of the present invention: The present invention provides a fusion protein vaccine for inhibiting group A streptococcal infection and its application, wherein the amino acid sequence of the fusion protein in the fusion protein vaccine is shown in SEQ ID NO. 1. The results of the serum titer test of immunized mice showed that the above fusion protein can induce a high level of specific antibodies, and the antibodies have no cross-reaction with human heart, brain, and kidney tissues, and effectively protect mice against M12 type GAS infection; and the above fusion protein vaccine composition can enhance Th1 type immunity, and the fusion protein vaccine can exert a long-term protective effect through the increase of effector memory T cells.

[0015] The present invention provides a fusion protein vaccine for inhibiting group A streptococcal infection, which can be used to prepare a drug for inhibiting group A streptococcal infection. The fusion protein vaccine has the advantages of being superior to the Chinese prevalent strains, having high safety, being easy to use, and being easy to promote and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The expression diagram of the recombinant plasmid protein in Example 1; NPE is the supernatant, and DPE is the inclusion body;

[0017] Figure 2 Figure 1 is a diagram of the purification process of the fusion protein in Example 1; MW is the molecular weight, IN is the stock solution, FT is the flow-through solution, W is the eluent, and E1-E11 are sample collection tubes;

[0018] Figure 3 This is a verification diagram of the fusion protein in Example 1;

[0019] Figure 4 This is a graph showing the changes in body weight of immunized mice in Example 2; * indicates P < 0.05, **** indicates P < 0.0001;

[0020] Figure 5 Figure 2 is a graph showing the antibody titer detection of the immunized mice serum in Example 2; A is a graph showing the serum titer 2 weeks after the first immunization; B is a graph showing the serum titer 2 weeks after the second immunization; C is a graph showing the serum titer 2 weeks after the third immunization; D is a statistical graph showing the comparison of the three serum antibody titers; ** is P < 0.01, *** is P < 0.001, **** is P < 0.0001;

[0021] Figure 6 This is a survival curve of mice immunized subcutaneously infected with MGAS9429 in Example 2;

[0022] Figure 7 CD4 secretion of IFN-γ by spleen cells of immunized mice in Example 2 + T cell flow cytometry analysis results, AC is the second immunization results, DF is the third immunization results, G is the percentage of IFN-γ secreting cells in CD4 + T cell percentage statistics, FMO represents IFN-γ gate negative isotype control, **** means P < 0.0001;

[0023] Figure 8 Figure 2 is the flow cytometry analysis result of memory T cells in spleen cells of immunized mice in Example 2, AC is the result of the second immunization, DF is the result of the third immunization, G is a statistical graph of the proportion of effector memory T cells, FMO represents CD44 or CD62L gate negative isotype control, **** is P <0.0001. DETAILED DESCRIPTION

[0024] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0025] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments and drawings. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0026] Example 1: Preparation of fusion protein

[0027] Step 1: The 42nd to 91st amino acids (50aa) at the N-terminus of the M protein of MGAS9429 (NCBI GenBank: ABF32911.1, the amino acid sequence is shown in SEQ ID NO. 2) and the 376th to 438th amino acids (63aa) at the C-terminus of the M protein were connected using the GS (2aa) sequence, and the protein sequence shown in the amino acid sequence of SEQ ID NO. 1 was connected to the KSI tag sequence by gene synthesis to construct a pET-28b (+) recombinant plasmid;

[0028] Step 2: The recombinant plasmid obtained in step 1 is transferred into competent cells BL21 or T7 Express to obtain an activated bacterial solution;

[0029] Step 3: Take the activated bacterial solution obtained in step 2 at a ratio of 1:100, transfer it to 1.5 ml of LB medium containing 50 ug / ml kanamycin (the LB medium consists of 1% tryptone, 0.5% yeast extract and 1% sodium chloride), and place it in a shaker at 37°C and 200 rpm for cultivation and growth;

[0030] Step 4: Wait until the bacterial solution in step 3 is cultured to OD (600nm) =0.6-0.8, add the inducer 1M IPTG (final concentration 1mM) at a ratio of 1:1000, and culture in a shaker at 37°C and 200rpm for 4h to express the fusion protein (KSI-M12-N+C). Collect 3ml of the expressed bacterial solution, centrifuge at 12000rpm for 1min, add 1ml PBS to the obtained bacterial precipitate, mix well, and perform ultrasound (ultrasound conditions: ultrasound power 45%, ultrasound on 2s, ultrasound off 2s, total ultrasound time 3min). Centrifuge at 12000rpm for 1min, aspirate 200ul of the supernatant into a new EP tube, mark it as "NPE", add 200ul PBS (containing 8M urea) to the precipitate after centrifugation, mix well, and mark it as "DPE";

[0031] Step 5: Add 50uL of reducing 5× loading buffer to the NPE and DPE samples obtained in step 4, shake gently to mix, do not turn upside down, cook the samples in a metal bath at 98°C for 5 minutes, and use 12% SDS-PAGE to detect the NPE and DPE samples containing the fusion protein (KSI-M12-N+C) (the expression of the recombinant plasmid protein is shown in Figure 2). Figure 1 shown).

[0032] In this example, the KSI-M12-N+C fusion protein obtained above was purified using Ni purification resin. PBS with pH=7.5 was added to the purified KSI-M12-N+C protein to collect the target protein and dialyze it. The obtained KSI-M12-N+C protein was detected by SDS-PAGE. The protein purification process is as follows: Figure 2 As shown, the concentration of the purified KSI-M12-N+C protein is 1.1 mg / ml; the KSI-M12-N+C protein verification results are as follows Figure 3 As shown, it can be seen that the KSI-M12-N+C protein is stably expressed without degradation, and the theoretical molecular weight of the KSI-M12-N+C protein is 29.19 kDa, which is consistent with the actual electrophoresis detection band result, proving that the expression result of the KSI-M12-N+C protein is accurate.

[0033] Example 2: Preparation of a fusion protein vaccine for inhibiting group A streptococcal infection

[0034] In this example, the KSI-M12-N+C protein prepared in Example 1 and aluminum hydroxide adjuvant (ThermoFisher, 77161) were mixed in a volume ratio of 1:1 to prepare a fusion protein vaccine, which was named KSI-M12-N+C vaccine.

[0035] Effect experiment:

[0036] 1. Weight changes

[0037] The KSI-M12-N+C vaccine prepared in Example 2 was used to vaccinate 38 female 6-8 week old BALB / c mice, and the immunization injections were performed on feeding day 0 (first immunization), day 14 (second immunization) and day 28 (third immunization), respectively. Eight mice injected with KSI-Alum vaccine (the KSI-Alum control group provided KSI label to prepare the vaccine) and 58 mice injected with PBS-Alum vaccine (the PBS-Alum control group used PBS instead of KSI-M12-N+C protein to prepare the vaccine) were used as control groups, and the weight of the mice was measured before each immunization.

[0038] The results are as follows Figure 4 As shown, compared with the weight of mice before immunization, the average weight of immunized mice injected with KSI-M12-N+C vaccine before the second immunization was significantly increased; compared with the weight of mice before the second immunization, the average weight of immunized mice injected with KSI-M12-N+C vaccine before the third immunization was significantly increased, and there were significant differences. In addition, there was no death in mice injected with KSI-M12-N+C vaccine three times by muscle, and the weight could grow normally, therefore, it was proved that the KSI-M12-N+C vaccine provided by the present invention is highly safe.

[0039] 2. Serum antibody titer detection

[0040] The present invention detects the titer of serum specific antibodies induced by the prepared KSI-M12-N+C vaccine. The serum of mice immunized with the KSI-M12-N+C vaccine is taken as a sample 2 weeks after each immunization (Day 14, 28, and 42). The serum of mice immunized with the KSI-Alum vaccine and the PBS-Alum vaccine are used as a control group, and the antiserum titer is detected by an indirect ELISA method.

[0041] The results are as follows Figure 5As shown, the serum titer of KSI-M12-N+C vaccine after the second immunization was >1:32000, and the serum titer of KSI-M12-N+C vaccine after the third immunization was >1:64000, both of which were significantly higher than those of KSI-Alum vaccine and PBS-Alum vaccine groups, indicating that the KSI-M12-N+C fusion protein vaccine provided by the present invention can induce high levels of serum specific antibodies.

[0042] 3. GAS infection experiment

[0043] The present invention conducted an infection experiment on mice injected with KSI-M12-N+C vaccine 2 weeks after the third immunization. The immunized mice were subcutaneously infected with M12-type GAS (MGAS9429, ATCC No. BAA-1315, purchased), and the infection dose was 2.0×10 8 CFU, the infected mice were observed and the survival rate of mice for 15 days was calculated. The immunized mice injected with KSI-Alum vaccine and PBS-Alum vaccine were used as the control group, and the same subcutaneous infection experiment was carried out as above.

[0044] The results are as follows Figure 6 As shown, the survival rate of mice immunized in the PBS-Alum control group was 11.76%, the survival rate of mice immunized in the KSI-Alum control group was 37.5%, and the survival rate of mice immunized in the KSI-M12-N+C vaccine experimental group was 61.53%; the survival rate of the KSI-M12-N+C vaccine experimental group was about 5.23 times higher than that of the PBS-Alum control group (P=0.0091), and the statistical difference was significant; the survival rate of the KSI-M12-N+C vaccine experimental group was about 1.64 times higher than that of the KSI-Alum control group (P=0.3416); the survival rate of the KSI-Alum control group was about 3.19 times higher than that of the PBS-Alum control group (P=0.1599). It can be seen that the KSI-M12-N+C vaccine provided by the present invention can effectively inhibit M12-type GAS infection and improve the survival rate of immunized mice.

[0045] 4. Changes in induced Th1 immunity and effector memory T cells

[0046] The spleen of the immune mice injected with KSI-M12-N+C vaccine was taken 5 days after the second immunization and 5 days after the third immunization to prepare a single cell suspension. 1×10 6 The spleens of mice immunized with PBS-Alum vaccine 5 days after the second immunization and 5 days after the third immunization were used as the control group. Similarly, 1×10 6 splenocytes.

[0047] The cell immune type was analyzed by flow cytometry. Figure 7 As shown, the CD4+ cells that produced IFN-γ in the immunized mice after the third immunization were higher than those in the immunized mice after the second immunization. + The proportion of T cells increased significantly, indicating that the KSI-M12-N+C vaccine formula can promote CD4 + T cells differentiate into Th1 effector cells and regulate the immune response by increasing the production of IFN-γ.

[0048] Flow cytometry was used to detect changes in memory T cells in vaccine-immunized mice. Figure 8 As shown, in the experimental group injected with KSI-M12-N+C vaccine and the control group injected with PBS-Alum vaccine, the proportion of effector memory T cells (Tem) in the immunized mice after the third immunization was significantly increased compared with that in the immunized mice after the second immunization, indicating that the third immunization increased the effective duration of the protective effect of KSI-M12-N+C vaccine by increasing the number of Tem.

[0049] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. A fusion protein vaccine for inhibiting group A streptococcal infection, characterized in that: The fusion protein vaccine comprises a fusion protein, a KSI tag and an acceptable adjuvant; The amino acid sequence of the fusion protein is shown in SEQ ID NO.1; The adjuvant is any one of aluminum salt adjuvant, Freund's adjuvant, CPG, AS01, AS03, AS04, MF-59, Montanide ISA720 or MPL; The mixing volume ratio of the fusion protein to the adjuvant in the fusion protein vaccine is 1:1; The fusion protein vaccine is administered by any one of intramuscular injection, subcutaneous injection or intranasal inhalation immunization.

2. Use of the fusion protein vaccine according to claim 1 in the preparation of a drug for inhibiting group A streptococcal infection, characterized in that: The group A streptococcus is of type M12.

3. The use according to claim 2, characterized in that: The route of infection of group A streptococcus is respiratory system or skin.

4. The use according to claim 2, characterized in that: The infection is an infection of the human pharynx in the respiratory route.

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