CN104474538A - Recombinant Bacillus Calmette Guerin vaccine - Google Patents
Recombinant Bacillus Calmette Guerin vaccine Download PDFInfo
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Abstract
The invention discloses a recombinant Bacillus Calmette Guerin vaccine (BCG), total bacteria amount of which is between 105CFU and 106CFU. The recombinant Bacillus Calmette Guerin vaccine comprises recombinant BCG for overexpression of 85 A antigen, recombinant BCG for overexpression of 85 B antigen and recombinant BCG for overexpression of HspX antigen. By synergism of multiple bacterial strains of the recombinant BCG vaccine, the recombinant BCG vaccine has an immunoprotection function in different development phases of tuberculosis, and negative effects of mechanisms or factors such as simultaneous overexpression of multi-antigen or fusion expression, etc. on preventive effect of the BCG itself can be avoided.
Description
Technical field
The invention belongs to biomedicine field, more specifically, relate to a kind of recombinant bacillus Calmette-Guerin vaccine.
Background technology
According to World Health Organization (WHO) (WHO), within 2012, still about have 860 Wan Xinfa tuberculosis (tuberculosis, TB) cases, comprising 500,000 affected children ranges, all the other are all adult's cases, have 1,300,000 people dead because of TB.And about there are more than 20 hundred million people's latent infection mycobacterium tuberculosis (Mycobacterium tuberculosis, M.tb) in the whole world.The particularly appearance of the popular and extensive resistant tuberculosis (XDR-TB) of multi-drug resistance tuberculosis (MDR-TB) in recent years, and the factor such as the rising of M.tb and HIV co-infection, cause TB to be still in the world today one of infectious disease of the most threatening property of the mankind.Bacillus calmette-guerin vaccine (M.bovis Bacillus Calmette-Gu é rin, BCG) is the attenuated live vaccine prepared from mycobacterium bovis BCG.Current as unique TB vaccine, the rate of vaccination of global infant every year up to more than 90%, and is applied 159 countries and regions.Epidemiology discloses low-down global childhood tuberculosis number of patients, confirms that it more efficiently pre-child-resistant can suffer from TB really.But huge latent infection crowd and a huge new adult T B case load, also show that BCG immunity is to the poor effect removing latent infection and prevention adult T B.Therefore, development of new, more efficiently vaccine replaces BCG, for preventing TB, most important to the control of TB epidemic situation.
As the pioneer field of global TB prevention and control, the candidate vaccine that development TB is novel, comprises the types such as subunit vaccine, attenuated live vaccine, inactivated vaccine and recombinant bacillus Calmette-Guerin vaccine (recombinant BCG, rBCG).Recombinant bacillus Calmette-Guerin vaccine (rBCG) is because it is on the basis of parental generation BCG; expression alien gene or knock out autogene; the immunodominance of parental generation BCG can be inherited, the deficiency of parental generation BCG can be made up again and improve protectiveness, and be easy to include existing planned immunization system in and promote the use of.Relative to the candidate vaccine of other type, recombinant bacillus Calmette-Guerin vaccine has unrivaled advantage, in widespread attention.The recombinant bacillus Calmette-Guerin vaccine of expression alien gene, by the single antigen of construction expression, to the multiple antigenic shift of construction expression.As on the basis of rBCG30 (process LAN Ag85B), the AERAS-422 (simultaneously the recombinant bacillus Calmette-Guerin vaccine of process LAN Ag85B, Ag85A and Rv3407) further developed, its antigen A g85B and Ag85A belongs to acute stage antigen, at the antigen that macrophage internal breeding is expressed after M.tb actute infection, Rv3407 belongs to antigen incubation period, participates in regulating M.tb to enter the antigen of latent infection state in vivo.In different types of zoopery, all show AERAS-422 and there is the protectiveness and immunogenicity that significantly strengthen compared with parental generation bacillus calmette-guerin vaccine.The crowd of the recombinant bacillus Calmette-Guerin vaccine immunity of setting up based on these M.tb multistage negotiation antigens, even if after exposing M.tb, its immunity produced can the M.tb of targeting different growth conditions in vivo, thus the generation prevented infections and development, to reach prophylactic object.But namely the AERAS-422 of process LAN antigen incubation period stops after carrying out I clinical trial phase, reason is the herpesvirus of latent infection in its energy activate immunity people colony, has stronger side effect.
Summary of the invention
For above defect or the Improvement requirement of prior art, the invention provides a kind of recombinant bacillus Calmette-Guerin vaccine, its object is to the bacillus calmette-guerin vaccine by selecting process LAN different phase antigen, and above-mentioned bacillus calmette-guerin vaccine is coordinated acquisition " cocktail " recombinant bacillus Calmette-Guerin vaccine according to a certain percentage, thus better immunocompetence is provided and alleviates side effect, solve existing bacillus calmette-guerin vaccine immunne response effect thus undesirable, the technical problem that existing recombinant bacillus Calmette-Guerin vaccine side effect is stronger.
For achieving the above object, according to one aspect of the present invention, provide a kind of recombinant bacillus Calmette-Guerin vaccine, it is characterized in that, its bacterium total amount of described bacillus calmette-guerin vaccine is 10
5cFU to 10
6between CFU, comprise the recombinant bacillus Calmette-Guerin vaccine of process LAN 85A antigen, the recombinant bacillus Calmette-Guerin vaccine of process LAN 85B antigen and the recombinant bacillus Calmette-Guerin vaccine of process LAN HspX antigen.
Preferably, described recombinant type bacillus calmette-guerin vaccine, comprises the bacillus calmette-guerin vaccine rBCG::X of the bacillus calmette-guerin vaccine rBCG::85A of total number of bacteria 20% to 33%, the bacillus calmette-guerin vaccine rBCG::85B and 15% to 33% of 33% to 60%.
Preferably, described recombinant type bacillus calmette-guerin vaccine, comprises the bacillus calmette-guerin vaccine rBCG::85AB of coexpression Ag85A and Ag85B of total number of bacteria 20% to 80%, and the rBCG::XB of 20% to 80% coexpression HspX and Ag85B.
In general, the above technical scheme conceived by the present invention compared with prior art, can obtain following beneficial effect:
" cocktail " recombinant bacillus Calmette-Guerin vaccine be mixed provided by the invention, be better than BCG to the protectiveness of acute M.tb infecting mouse, multiple bacterial strain can be worked in coordination with and be played a role, and recombinant bacillus Calmette-Guerin vaccine provided by the invention is the candidate vaccine with good application prospect.The protective antigen of different phase after selecting M.tb to infect; utilize the recombinant bacillus Calmette-Guerin vaccine advantage that the different monoclonal antibody of process LAN is former; form heterogeneous vaccine; comprehensively can realize combination among the strong ones; have complementary advantages; make it all have immanoprotection action at the different stages of development of TB, and the mechanism such as the former or amalgamation and expression of simultaneously process LAN multi-resistance or factor can be avoided to bring foregoing negative effect to BCG autoprotection effect.
Accompanying drawing explanation
Fig. 1 is antibody titer and the IgG2a/IgG1 ratio of each bacterial strain specific IgG, IgG1, IgG2a under different proportion in embodiment 7ABX bacterial strain; Wherein Fig. 1 a is antibody titer and the IgG2a/IgG1 ratio of rBCG::85A different proportion immune mouse Ag85A specific IgG, IgG1, IgG2a in ABX bacterial strain; Fig. 1 b is antibody titer and the IgG2a/IgG1 ratio of rBCG::85B different proportion immune mouse Ag85B specific IgG, IgG1, IgG2a in ABX bacterial strain; Fig. 1 c is antibody titer and the IgG2a/IgG1 ratio of rBCG::X different proportion immune mouse HspX specific IgG, IgG1, IgG2a in ABX bacterial strain;
Fig. 2 is that the induction of ABX vaccine immune mouse produces antigenic specificity IFN-γ level;
Fig. 3 is ABX vaccine immune response effect; Wherein Fig. 3 a is the multi-functional CD4+T cell of PPD specificity that the induction of ABX vaccine immune mouse produces; Fig. 3 b is the multi-functional CD4+T cell of Ag85A specificity that the induction of ABX vaccine immune mouse produces; Fig. 3 c is that the induction of ABX vaccine immune mouse produces the multi-functional CD4+T cell of Ag85B specificity; Fig. 3 d is that the induction of ABX vaccine immune mouse produces the multi-functional CD4+T cell of HspX specificity;
Fig. 4. the lotus bacterium amount of anti-M.tb actute infection mice lungs after dissimilar recombinant bacillus Calmette-Guerin vaccine immunity.
Detailed description of the invention
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.In addition, if below in described each embodiment of the present invention involved technical characteristic do not form conflict each other and just can mutually combine.
Recombinant bacillus Calmette-Guerin vaccine provided by the invention, its bacterium total amount is 10
5cFU to 10
6between CFU, comprise the recombinant bacillus Calmette-Guerin vaccine of process LAN Ag85A antigen, the recombinant bacillus Calmette-Guerin vaccine of process LAN Ag85B antigen and the recombinant bacillus Calmette-Guerin vaccine of process LAN HspX antigen.
Preferably, described recombinant bacillus Calmette-Guerin vaccine comprises the bacillus calmette-guerin vaccine rBCG::X of the bacillus calmette-guerin vaccine rBCG::85A of total number of bacteria 20% to 33%, the bacillus calmette-guerin vaccine rBCG::85B and 15% to 33% of 33% to 60%.Wherein, bacillus calmette-guerin vaccine rBCG::85A is the recombinant bacillus Calmette-Guerin vaccine of process LAN 85A antigen, its relative process LAN strengthens at least 2 times of (RT-qPCR technical appraisement, lower same), bacillus calmette-guerin vaccine rBCG::85B is the recombinant bacillus Calmette-Guerin vaccine of process LAN 85B antigen, its relative process LAN strengthens at least 2 times, and bacillus calmette-guerin vaccine rBCG::X is the recombinant bacillus Calmette-Guerin vaccine of process LAN HspX antigen, and its relative process LAN strengthens at least 2 times.
The construction method of rBCG::85A, rBCG::HspX and rBCG::85B is as follows:
(1) with the genome of M.tb H37Rv for template, adopt round pcr to obtain the encoding gene of M.tb target antigen, i.e. the encoding gene of Ag85A, Ag85B and HspX.
(2) encoding gene of acquisition is cloned in bacillus coli-mycobacteria shuttle expression carrier pMV261, is built into recombinant expressed matter pMVHspX, pMVAg85A and pMVAg85B respectively.
(3) by above-mentioned plasmid respectively electroporation be transformed into BCG strain in China, by kalamycin resistance and immunoblot assay qualification, confirm that the recombinant bacillus Calmette-Guerin vaccine of expressing above-mentioned antigen respectively successfully constructs.
These recombinant strain of BCG vaccine are called after respectively: the recombinant strain of BCG vaccine of process LAN 85A antigen, its Classification And Nomenclature is mycobacterium bovis BCG bacillus calmette-guerin vaccine BCG rBCG::85A, Latin name is mycobacterium bovis BCG rBCG::85A, be called for short bacillus calmette-guerin vaccine rBCG::85A, Wuhan, China is preserved on July 24th, 2014, China typical culture collection center CCTCC, deposit number is CCTCC M 2014356; The recombinant strain of BCG vaccine of process LAN 85B antigen, its Classification And Nomenclature is: mycobacterium bovis BCG bacillus calmette-guerin vaccine BCG rBCG::85B, Latin name is mycobacterium bovis BCG rBCG::85B, be called for short bacillus calmette-guerin vaccine rBCG::85B, Wuhan, China is preserved on July 24th, 2014, China typical culture collection center CCTCC, deposit number is CCTCC M 2014357; The recombinant strain of BCG vaccine of process LAN HspX antigen, its Classification And Nomenclature is: mycobacterium bovis BCG bacillus calmette-guerin vaccine BCG rBCG::X, Latin name is mycobacterium bovis BCG rBCG::X, be called for short bacillus calmette-guerin vaccine rBCG::X, Wuhan, China is preserved on July 24th, 2014, China typical culture collection center CCTCC, deposit number is CCTCC M 2014359.
Concrete construction method reference literature Enhanced protection against tuberculosis by vaccination with recombinant BCG over-expressing HspX protein.Vaccine.2010; 28 (32): 5237-44.I mmunogenicity and Protective Efficacy of a Novel Recombinant BCG Strain Overexpressing Antigens Ag85A and Ag85B.Clinical and Developmental Immunology, Volume 2012, Article ID 563838.
Preferably, described bacillus calmette-guerin vaccine comprises the bacillus calmette-guerin vaccine rBCG::85AB of coexpression Ag85A and Ag85B of total number of bacteria 20% to 80%, and the rBCG::XB of 20% to 80% coexpression HspX and Ag85B.Wherein, bacillus calmette-guerin vaccine rBCG::85AB is the recombinant bacillus Calmette-Guerin vaccine of process LAN 85A and process LAN 85B antigen, and relative process LAN strengthens at least 2 times, and bacillus calmette-guerin vaccine rBCG::XB is the recombinant bacillus Calmette-Guerin vaccine of process LAN 85B and process LAN X antigen, and relative process LAN strengthens at least 2 times.
The construction method of rBCG::85AB and rBCG::XB is as follows:
(1) with the genome of mycobacterium tuberculosis for template, adopt round pcr to obtain the different target antigen HspX of M.tb, the encoding gene of Ag85A or Ag85B.
(2) genes of interest Ag85A and Ag85B of acquisition is cloned in bacillus coli-mycobacteria shuttle expression carrier pMV261, is built into recombinant expression plasmid pMVAg85AB.Genes of interest HspX and Ag85B of acquisition is cloned in bacillus coli-mycobacteria shuttle expression carrier pMV261, is built into recombinant expression plasmid pMVAg85BX.
(3) respectively above-mentioned plasmid pMVAg85AB and pMVAg85BX is transferred to BCG strain in China, by kalamycin resistance and immunoblot assay qualification, confirms that the recombinant bacillus Calmette-Guerin vaccine of expressing above-mentioned antigen respectively successfully constructs.
These recombinant strain of BCG vaccine called afters: the recombinant strain of BCG vaccine of process LAN 85A antigen and 85B antigen, its Classification And Nomenclature is mycobacterium bovis BCG bacillus calmette-guerin vaccine BCG rBCG::85AB, Latin name is mycobacterium bovis BCG rBCG::85AB, be called for short bacillus calmette-guerin vaccine rBCG::85AB, Wuhan, China is preserved on July 24th, 2014, China typical culture collection center CCTCC, deposit number is CCTCC M 2014358; The recombinant strain of BCG vaccine of process LAN 85B antigen and HspX antigen, its Classification And Nomenclature is mycobacterium bovis BCG bacillus calmette-guerin vaccine BCG rBCG::XB, Latin name is mycobacterium bovis BCG rBCG::XB, be called for short bacillus calmette-guerin vaccine rBCG::XB, Wuhan, China is preserved on July 24th, 2014, China typical culture collection center CCTCC, deposit number is CCTCC M 2014360.
Based on the antigen that we express by the different phase characteristic of M.tb, construct a series of recombinant bacillus Calmette-Guerin vaccine, studied by a large amount of animal experiments, therefrom screen five kinds of recombinant bacillus Calmette-Guerin vaccines, be respectively rBCG::X, rBCG::85A and rBCG::85B bacterial strain of process LAN HspX, Ag85A and Ag85B, and recombinant bacillus Calmette-Guerin vaccine rBCG::85AB, co expression HspX and Ag85B and rBCG::XB of co expression Ag85A and Ag85B.These bacterial strains respectively immune C57BL/6 mice, carried out M.tb challenge viral dosage after 6 weeks, after counteracting toxic substances 4 weeks and 18 weeks, confirmed the infection protected effect of these bacterial strain inducings, was all obviously better than parental generation BCG.Analysis shows: the infection long lasting protective that these recombinant bacillus Calmette-Guerin vaccines strengthen; the IFN-γ level of secreting these antigenic specificities with vaccine immune mouse splenocyte rises closely related; also reflect that parental generation BCG immunity is to the reason of preventing M.tb latent infection and adult T B weak effect to a certain extent; may decline with its immunogenicity respectively and relevant to the weak immunne response of latent infection stage related antigen, this generation with TB clinically and evolution are confirmed mutually.
Because M.tb is after respiratory tract primary infection, and the intragroup pathogen of most people can be eliminated, and only causes the population infection of 30%-40%.In the infected, more than 90% crowd, in the presence of body immune system, forms granuloma and the progress of infection control formation latent infection; Only the infected of less than 10% causes TB because of primary infection.The latent infection crowd of 5-10%, at it in life, as immunity degradation, HIV co-infection, malnutrition or factor of waiting for a long time in year, be in body dormancy M.tb can reactivation (reactivation), namely former postoperative infection principal mode and cause adult T B---pulmonary tuberculosis.From pathogenic, M.tb after infection occurs, to I haven't seen you for ages experience active proliferation, dormancy and the different phase such as active proliferation again.In the primary infection stage, the representative gene that in body, M.tb expresses is as Ag85 complex (as Ag85A, Ag85B) etc.And simulate the anaerobic environment of latent infection in vitro, disclosing M.tb is regulated and controled by dormancy regulator gene DosR at latency stage, coding also up-regulated expression 48 genes, comprises HspX etc.Therefore, in order to play the cooperative effect of these recombinant bacillus Calmette-Guerin vaccine resisting tuberculosis infections further, characteristic antigens HspX, Ag85A or Ag85B of different phase after M.tb is infected, recombinant bacillus Calmette-Guerin vaccine rBCG::X, rBCG::85A and rBCG::85B bacterial strain of development, and recombinant bacillus Calmette-Guerin vaccine rBCGAB, the co expression HspX of co expression Ag85A and Ag85B and the recombinant bacillus Calmette-Guerin vaccine rBCGXB of Ag85B, combine according to special ratios.This combined vaccine immunity, likely targeting is in the M.tb of different conditions in vivo after removing and infecting, thus developing of preventing infections, even reach the ability controlling latent infection and prevention adult T B.The present invention's research specify that the effect that the ratio of this combined vaccine infects the impact of immune effect and its collaborative anti-M.tb.
Be below embodiment:
Embodiment 1
A kind of recombinant bacillus Calmette-Guerin vaccine, called after ABX, its bacterium total amount is 10
5cFU, comprises the bacillus calmette-guerin vaccine rBCG::85A of total number of bacteria 25%, the bacillus calmette-guerin vaccine rBCG::X of the bacillus calmette-guerin vaccine rBCG::85B and 15% of 60%.
Embodiment 2
A kind of recombinant bacillus Calmette-Guerin vaccine, called after ABX, its bacterium total amount is 5 × 10
5cFU, comprises the bacillus calmette-guerin vaccine rBCG::85A of total number of bacteria 20%, the bacillus calmette-guerin vaccine rBCG::X of the bacillus calmette-guerin vaccine rBCG::85B and 20% of 60%.
Embodiment 3
A kind of recombinant bacillus Calmette-Guerin vaccine, called after ABX, its bacterium total amount is 10
6cFU, comprises the bacillus calmette-guerin vaccine rBCG::85A of total number of bacteria 33%, the bacillus calmette-guerin vaccine rBCG::X of the bacillus calmette-guerin vaccine rBCG::85B and 33% of 34%.
Embodiment 4
A kind of recombinant bacillus Calmette-Guerin vaccine, called after ABXB, its bacterium total amount is 10
5cFU, comprises the bacillus calmette-guerin vaccine rBCG::85AB of coexpression Ag85A and Ag85B of total number of bacteria 20%, and the rBCG::XB of 80% coexpression HspX and Ag85B.
Embodiment 5
A kind of recombinant bacillus Calmette-Guerin vaccine, called after ABXB, its bacterium total amount is 5 × 10
5cFU, comprises the bacillus calmette-guerin vaccine rBCG::85AB of coexpression Ag85A and Ag85B of total number of bacteria 50%, and the rBCG::XB of 50% coexpression HspX and Ag85B.
Embodiment 6
A kind of recombinant bacillus Calmette-Guerin vaccine, called after ABXB, its bacterium total amount is 10
6cFU, comprises the bacillus calmette-guerin vaccine rBCG::85AB of coexpression Ag85A and Ag85B of total number of bacteria 80%, and the rBCG::XB of 20% coexpression HspX and Ag85B.
Embodiment 7
In bacterium total amount 10
6when CFU is constant, respectively according to the ratio of each constituent each 15%, 20%, 30% and 40%, three kinds of recombinant bacillus Calmette-Guerin vaccine rBCG::85A, rBCG::85B and rBCG::X bacterial strains are mixed, called after polyvalent vaccine ABX.Immune C57BL/6 mice respectively, take BCG as positive control, PBS is negative control.Immunity is after 12 weeks and 32 weeks, collect serum, ELISA method detects serum respectively for the antibody titer of the IgG antibody of the antigenic specificity of HspX, Ag85A and Ag85B, IgG1 antibody and IgG2a, calculates IgG2a/IgG1 ratio to evaluate effective dose proportioning and the immunogenicity of vaccine immunity.
1. animal grouping with immunity: SPF level C57BL/6 (H-2b) mice, Wuhan University's animal experimental center provides, (quality certification number: SCXK (Hubei Province) 2008-0004, NO.4200592947), female, 6-8 week age, 18-20g.Experiment point three large groups: the ABX vaccine group of various dose proportioning, BCG positive controls, PBS negative control group.Each large group be further divided into 12 weeks, 32 weeks two time points, 3.Experiment repetition twice.
ABX group: rBCG::85A, rBCG::85B and rBCG::X are according to the different proportion mixing respectively accounting for 15 ~ 40%, and subcutaneous injection, immunity 1 time, dosage is 1 × 10
6cFU/100 μ l/ only.
BCG group: subcutaneous injection, immunity 1 time, dosage is 1 × 10
6cFU/100 μ l/ only.PBS group: subcutaneous injection, immunity 1 time, dosage is 100 μ l/.
2.ELISA antigen-specific antibodies level and IgG2a/IgG1 ratio in detection serum.
(1) collect serum: latter 12 weeks, 32 weeks of immunity, pluck eyeball respectively and get blood, aseptic cuing open kills 3 mices, and after room temperature places 2h, the centrifugal 10min of 4000rpm, collects serum also frozen in-80 DEG C after subpackage.
(2) bag quilt: be buffered liquid (pH 9.6) with the carbonate bag of 0.05M and dilute different albumen to 5 μ g/ml, 100 μ l/ holes, blank control wells adds 100 μ l bags and is buffered liquid, 4 DEG C of overnight incubation.
(3) close: discard liquid in hole, 0.05%PBST washs 4 times, each 3min; Add 1%BSA-PBST afterwards to close, 150 μ l/ holes, hatch 2h for 37 DEG C; Wash 4 times.
(4) application of sample: the aseptic 1 × PBS of each group mice serum sample presses doubling dilution from 1:50 is doubly initial, and blank control wells adds bag and is buffered liquid 100 μ l, each sample does three multiple holes.Hatch 1h for 37 DEG C; Wash 4 times.
(5) add two to resist: the HRP labelling sheep anti mouse two adding fresh configuration resists, and antibody dilution is: IgG 1:5000, IgG11:10000, IgG2c 1:10000, and 100 μ l/ holes, hatch 1h for 37 DEG C; Washing, last is all over washing with distilled water.
(6) develop the color: Extemporaneous tmb substrate solution, 100 μ l/ holes, 37 DEG C of colour developing 30min;
(7) cessation reaction: every hole adds the 2M sulfuric acid solution of 50 μ l, color development stopping is reacted.
(8) result measures: microplate reader, and OD value is detected at wavelength 450nm place.
(9) result calculates: after each sample OD value is deducted blank control wells OD value, and multiple hole is added and is averaged OD value by each serum sample.Wherein the OD value of PBS matched group is set to negative control (N), and immunization experiment group is sample (P), when blood-serum P/N value >=2.1 can be judged as the positive.Antibody titer represents with the inverse of the most highly diluted multiple occurring positive findings.Every mice serum result is expressed as log2 (antibody titer).Occur that the most highly diluted multiple of positive findings calculates IgG2a:IgG1 with every mouse IgG 2a and IgG1.Finally calculate meansigma methods and the standard error of each group, result as shown in Figure 1.
(10) the immunoprotection mechanism main Th1 of dependence type according to resisting tuberculosis infection is replied.The standard evaluating the dosage range of the effective proportioning of recombinant bacillus Calmette-Guerin vaccine ABX is: ratio is greater than 1, is expressed as the response of Th1 type; Equal 1, be expressed as the response of Th1/Th2 mixed type; Be less than 1, be expressed as the response of Th2 type.
(11) result and conclusion: when constant total quantity, rBCG::85A mixes by the ratio accounting in ABX bacterial strain 15%, 20%, 30% and 40% respectively.Latter 12 weeks of immunity, Ag85A antigenic specificity IgG, IgG1, IgG2a titre that ABX various ratio mixing group produces, respectively with BCG group suitable (p > 0.05); Latter 32 weeks of immunity, each group of Ag85A specific IgG produced separately 1 (except 15% ratio mixing group), IgG2a titre, significantly increase (p<0.05) time all compared with 12 weeks, only 20%, the IgG2a antibody titer of 30% and 40% ratio mixing group, is significantly higher than BCG group (p<0.05).In the whole laboratory observation stage, the IgG2a/IgG1 ratio of each group is all greater than 1, and ratio also all raises with immunization time prolongation, but rBCG::85A is more obvious by the ratio increase of the ratio mixing group of 20%, 30% and 40%, and higher than BCG group.
Therefore, each subfraction accounts for the ratio of total polyvalent vaccine once lower than 20%, as the dosage of 15%, can not promote the generation that the Th1 type of antigenic specificity is replied.Therefore, effective dose ratio should respectively account for 20% ~ 40% of total polyvalent vaccine.
Result: when constant total quantity, rBCG::85A mixes by the ratio accounting in ABX bacterial strain 15%, 20%, 30% and 40% respectively.Latter 12 weeks of immunity, Ag85A antigenic specificity IgG, IgG1, IgG2a titre that ABX various ratio mixing group produces, respectively with BCG group suitable (p > 0.05); Latter 32 weeks of immunity, each group of Ag85A specific IgG produced separately 1 (except 15% ratio mixing group), IgG2a titre, significantly increase (p<0.05) time all compared with 12 weeks, only 20%, the IgG2a antibody titer of 30% and 40% ratio mixing group, is significantly higher than BCG group (p<0.05).In the whole laboratory observation stage, the IgG2a/IgG1 ratio of each group is all greater than 1, and ratio also all raises with immunization time prolongation, but rBCG::85A is more obvious by the ratio increase of the ratio mixing group of 20%, 30% and 40%, and higher than BCG group.
Result: when constant total quantity, the ratio that rBCG::85B accounts in ABX bacterial strain 15%, 20%, 30% and 40% respectively mixes.Latter 12 weeks of immunity, Ag85B antigenic specificity IgG, IgG2a titre that ABX various ratio mixing group produces are suitable with BCG group respectively; Only the IgG1 antibody titer of 20%, 30% and 40% ratio mixing group, is significantly higher than BCG group (p<0.05).Latter 32 weeks of immunity, each group of the Ag85B specific IgG produced separately, IgG1, IgG2a titre, significantly increase (p<0.05) time all compared with 12 weeks, it should be noted that the IgG2a antibody titer that each ratio mixing group produces is significantly higher than BCG group (p<0.05).In the whole laboratory observation stage, the IgG2a/IgG1 ratio of each group is all greater than 1, and ratio also all raises with immunization time prolongation, but rBCG::85B is more obvious by the ratio increase of the ratio mixing group of 20%, 30% and 40%, and higher than BCG group.
Result: when constant total quantity, rBCG::X mixes by the ratio accounting in ABX bacterial strain 15%, 20%, 30% and 40% respectively.Latter 12 weeks of immunity, the HspX antigenic specificity IgG, the IgG1 titre that produce by 20%, 30% and 40% ratio mixing group, be all significantly higher than BCG group (p<0.05); The IgG2a titre that each ratio group produces is suitable with BCG group respectively.Latter 32 weeks of immunity, HspX specific IgG, IgG1 titre and BCG group that each group produces separately are quite; Wherein BCG group and 15% ratio mixing group all compared with 12 weeks time significantly increase (p<0.05); It should be noted that the IgG2a antibody titer that the ratio mixing group of 20%, 30% and 40% produces is significantly higher than BCG group (p<0.05), and significantly increase (p<0.05) during than 12 weeks.In the whole laboratory observation stage, the IgG2a/IgG1 ratio of each group is all greater than 1, and ratio also all raises with immunization time prolongation, but rBCG::X is more obvious by the ratio increase of the ratio mixing group of 20%, 30% and 40%, and higher than BCG group.
Embodiment 8
1. also carry out immunity with reference to embodiment 7 mode with the ABX mixed vaccine in embodiment 3.
2. the preparation of splenocyte:
(1) latter 12 weeks, 32 weeks of immunity, aseptic cuing open kills 3 mices respectively, aseptically, takes out spleen from abdominal cavity.
(2) spleen put into labelling in advance, add lattice of the three lattice Micro-Organism Culture Dishs of 5ml pre-cooling PBS.(available sterile scissors to cut after a little opening gentle abrasion again on spleen, avoids damaging splenocyte).
(3) 200 order nylon screens are enclosed within 50ml sterile centrifugation tube.Be placed on nylon screen with aseptic tweezer spleen, with 1ml syringe piston gentle abrasion mouse spleen on 200 order nylon screens, with pasteur pipet or 5ml pipettor transferase 45 ml pre-cooling PBS limit rinse, limit grinding.
(4) after completing, remove nylon mesh net, build the lid of 50ml sterile centrifugation tube.2000rpm, 4 DEG C of centrifugal 10min.Abandon supernatant.
(5) splenocyte of resuspended every the mice of 5ml erythrocyte cracked liquid is got, incubated at room 1-2min.2000rpm, 4 DEG C of centrifugal 10min.Abandon supernatant rapidly.(the incubated at room time is limited in 3min.Overlong time has impact to splenocyte activity.
(6) 5ml pre-cooling PBS is added, 2000rpm, 4 DEG C of centrifugal 10min.Abandon supernatant rapidly.Wash 2 times altogether.Abandon supernatant.
(7) 1ml pre-cooling RPMI1640 complete medium re-suspended cell precipitation is added.Get 2 μ l cell suspension add 198 μ l culture medium mixing after, accurate counting under mirror.Make a record and labelling.
(8) with RPMI 1640 complete medium adjustment cell concentration, and every Mouse spleen cells sum is calculated.
3. specific antigen stimulates splenocyte and collects supernatant
(1) in 24 orifice plates, add the splenocyte in 5 × 106/ holes of 100 μ l.
(2) albumen stimulates: Ag85B, Ag85A, and HspX albumen, each 10 μ g/ml.
Positive control stimulus object: TB-PPD (10 μ g/ml).
Negative control stimulus object: RPMI 1640 culture medium.
(3) 1640 complete medium polishings are added again to volume 1ml.Place 37 DEG C, in 5%CO2 incubator.
(4) cultivate 72h and gather in the crops splenocyte supernatant for detecting IFN-γ.To collect in 24 orifice plates splenocyte and juice in aseptic EP pipe, 2000rpm, centrifugal 10min, collect supernatant, Care Mark group number and incubation time, carry out subpackage by each aequum that detects, be put in-80 DEG C of frozen standby inspections.
4.ELISA detects spleen cell cultures SNAg specific IFN-γ level: complete experiment flow according to mice ELISA detection kit workbook.Result calculates: according to standard curve calculation sample concentration, and every hole concentration deducts stimulates hole background values, as final numerical value with group 1640.Calculate mean and the standard deviation of each experimental group, carry out statistical analysis, as shown in Figure 2.
5. result and conclusion: latter 12 weeks or 32 weeks of immunity, no matter stimulate with PPD or different specific antigens such as Ag85A, Ag85B and HspX, PBS matched group only secretes very low-level IFN-γ, and all remarkable in BCG group or ABX vaccine group (p<0.05).In early days, the IFN-γ of the various antigenic specificities that ABX component is secreted, and be all significantly higher than BCG group (p<0.05), along with the prolongation of immunization time, significantly increase (p<0.05) when ABX group and BCG group are all than respective 12 weeks, but ABX group is still significantly higher than BCG group (p<0.05).Therefore, the vaccine-induced immune mouse of ABX, compares than BCG, produces high-caliberly as Ag85A, Ag85B and HspX specificity IFN-γ, can play collaborative anti-infectious function for not synantigen.
Embodiment 9
1. carry out immunity and separating Morr. cell with embodiment 8.
2. bed board stimulates:
(1), in aseptic 24 orifice plates, every hole adds splenocyte 100 μ l (5 × 106), bed board.
(2) every hole adds 20 μ l anti-CD28mcAb working solutions.
(3) every hole adds different types of stimulus object respectively: add Ag85B respectively, Ag85A, and HspX antigen protein (final concentration is 10 μ g/ml); PPD stimulates as positive control (final concentration is 10 μ g/ml), and 1640 as negative control.Do blank well contrast, the contrast of single mark pipe simultaneously.
(4) add into 1640 complete mediums to 1ml.Mixing.37 DEG C, CO2 incubator hatches 16h.4h after albumen stimulates, every hole adds blocker Brefeldin A+Monensin.The centrifugal 5min of 500g, abandons supernatant.
3. padding:
(1) every hole adds 100 μ l surface marker antibody, 0.5 μ l CD3+1.25 μ l CD4 antibody+98.25 μ l PBS respectively
(2) 4 DEG C of lucifuges hatch 30min.Add PBS 2ml, the centrifugal 5min of 500g, abandons supernatant.Wash 2 times, abandon supernatant.
4. fixing and perforation:
(1) often pipe adds the fixative 100 μ l of pre-cooling, and mixing, so that fully fixing.Room temperature places 30min.
(2) often pipe adds 1ml and wears film working solution, and the centrifugal 5min of 500g, abandons supernatant.Wash 2 times.
5. dyeing in born of the same parents:
(1) add 100 μ l cytokine fluorescent antibodys mixing, concrete grammar is:: 1.25 μ lIFN-γ+1.25 μ l TNF-α+1.25 μ l IL-2+96.25 μ l 1 × wear film liquid.Room temperature, lucifuge hatch 30min.
(2) often pipe adds 1ml and wears film working solution, and the centrifugal 5min of 500g, abandons supernatant, washes 2 times.
6. flow cytometer detection: add 300 μ l PBS re-suspended cells, 4 DEG C keep in Dark Place to be checked.Whirlpool concussion before upper machine.First go up blank tube, regulation voltage, detect successively and singly mark pipe, regulate fluorescence to compensate.
7. analysis strategy: first analyze IFN-γ in CD4+T cell+, TNF-α+, the total cell number of IL-2+, secondly press further Dan Yang, IFN-γ+-TNF-α+, the two positive and multi-functional T cell hypotype of IFN-γ+positive cell analysis of-TNF-α+-IL-2+ three of TNF-α+-IL-2+, IFN-γ+-IL-2+.
8. result calculates: according to dissimilar T lymphocyte proportion and every mouse boosting cell count results in total cell number of upper machine testing, calculate the lymphocytic absolute quantity of various cytokine secretion type T in single mouse spleen respectively, result as shown in Figure 3.
Latter 12 weeks of immunity, after PPD stimulates, ABX group total IFN-γ secreting type CD4+T cell number is significantly higher than BCG group (p<0.05), and total TNF-α or always IL-2 secreting type CD4+T cell number and BCG group remain basically stable.It should be noted that, with the prolongation of immunization time, the total TNF-α of ABX group or total IL-2 secreting type CD4+T cell number, all there is remarkable increase (p<0.05) than after immunity 12 weeks, and total IFN-γ, total TNF-α or total IL-2 secreting type CD4+T cell number, be all significantly higher than BCG group (p<0.05).Therefore, ABX vaccine immune mouse is induced PPD specific total IFN-γ in splenocyte, total TNF-α or total IL-2 secreting type CD4+T cell quantity significantly and is increased enduringly.
By total CD4+T cell number of splenocyte antigenic specificity secrete cytokines, further positive by single-factor, double factor is positive and 7 kinds of situations such as three factor positive are classified, further to screen the immune indexes finally being formed the enhancing of ABX group protectiveness.After immunity 12 weeks with 32 weeks, through PPD stimulation, ABX group IFN-γ+, the quantity of IL-2+ and TNF-α+three kinds of lists positive CD4+T cell and IFN-γ+-TNF-α+bis-positive CD4+T cell, be all significantly higher than BCG group (p<0.05).Extend with immunization time, BCG group only TNF-α+mono-positive CD4+T cell quantity, and ABX vaccine group comprises IL-2+ Dan Yang and TNF-α+mono-positive CD4+T cell quantity, is all significantly higher than the quantity (p<0.05) of respective 12 weeks.Therefore, PPD specific secretion IFN-γ in ABX vaccine immune mouse induction splenocyte+, the quantity of the single sun of IL-2+, TNF-α+three kinds and IFN-γ+-TNF-α+bis-positive CD4+T cell significantly and increase enduringly.
Latter 12 weeks and 32 weeks of immunity, stimulate with Ag85A, Ag85B or HspX albumen respectively, specific total IFN-γ, total TNF-α or total IL-2 secreting type CD4+T cell quantity in ABX group immune mouse induction splenocyte, compared with BCG group, all significantly increase, except early stage total IL-2 secreting type CD4+T cell quantity compared with BCG group quite except (p<0.05).Latter 32 weeks of immunity, no matter which kind of albumen stimulates, and the specific total IL-2 secreting type CD4+T cell quantity of ABX group induction is all significantly higher than 12 weeks (p<0.05); In addition, Ag85A, HspX specific total TNF-α secreting type CD4+T cell quantity is also significantly higher than 12 weeks (p<0.05).Therefore, ABX vaccine immune mouse is induced Ag85A, Ag85B and HspX specific total IFN-γ in splenocyte, total TNF-α or total IL-2 secreting type CD4+T cell quantity significantly and is increased enduringly.Latter 12 weeks of immunity, no matter use which kind of antigenic stimulus, antigenic specificity secretion of gamma-IFN+mono-sun of ABX group and the quantity of IFN-γ+TNF-α+bis-positive CD4+T cell are significantly higher than BCG group (p<0.05), importantly, the quantity of the CD4+T cell of this two type, extends to immune latter 32 weeks still higher than BCG group (p<0.05).And after immune 32 weeks, the quantity of the mono-positive CD4+T cell of IL-2+ of the different antigenic specificities that ABX group produces, is all significantly higher than BCG group (p<0.05).In addition, specific three sun of the Ag85A that ABX group produces and TNF-α+Dan Yang, the two sun of the specific IFN-γ+-IL-2+ of Ag85B, the quantity of HspX specific TNF-α+mono-positive CD4+T cell, is all significantly higher than BCG group (p<0.05).Therefore, ABX vaccine immune mouse, relative to bacillus calmette-guerin vaccine strain in China, induces the quantity of Ag85A, Ag85B, HspX specific secretion IFN-γ in splenocyte+mono-sun and IFN-γ+-TNF-α+bis-positive CD4+T cell significantly and increases enduringly.
Embodiment 10
With the ABXB vaccine of preparation in the ABX vaccine prepared in embodiment 3, embodiment 5, immune C57BL/6 mice, take BCG as positive control, PBS is negative control.Latter 9th week of immunity, with M.tb standard strain through tail Intravenous Infection, after 4 weeks, calculates lungs internal organs lotus bacterium amount to evaluate vaccine infection protective effect, confirms that the short-term protective of the anti-M.tb of vaccine ABX is better than BCG strain in China.
1. laboratory animal: SPF level C57BL/6 mice, Wuhan University's animal experimental center provides, and (quality certification number: SCXK (Hubei Province) 200-0013) is female, 6-8 week age, 18-22g.
2. experiment grouping and immunity: random point 6 groups, often organize 10.
Vaccine group: rBCG::85A, rBCG::85B and rBCG::X proportionally mix, name ABX group.Subcutaneous injection, immunity 1 time, dosage is 1 × 10
6cFU/100 μ l/ only.
Vaccine group: rBCG::85AB and rBCG::XB mixes, called after ABXB group.
Positive controls: BCG group.Subcutaneous injection, immunity 1 time, dosage is 1 × 10
6cFU/100 μ l/ only.
Negative control group: subcutaneous injection, immunity 1 time, dosage is 100 μ l PBS/.
3. experimental animal infection: latter 9th week of immunity, utilize M.tb H37Rv standard strain through tail vein injection mode infecting mouse, actual infective dose is 1.2 × 10
6cFU/ only.After 4 weeks, put to death mice.
4. lungs lotus bacterium amount
After often organizing 10 sacrifice, 75% alcohol-pickled 10min, sterilization skin.Aseptic taking-up lungs, are put in mortar, fully grind, and add aseptic PBS-T 2ml and rinse.Transfer in 10ml sterile tube, after agitator fully mixes.Get 100 μ l stock solutions to join in 900 μ l PBS-T, do doubling dilution.Agitator fully mixes, get each 100 μ l of 4 dilution bacterium liquid be coated with 7H11-OADC culture dishs (add cycloheximide during preparation, the growth of Antifungi, add TCH can Selective depression remain the growth of BCG).Three holes are coated with respectively with 1 dilution bacterium liquid.Residue stock solution is kept at-80 DEG C of refrigerators, for subsequent use for checking later.Plate is put in 37 DEG C of incubators and carries out colony counting after 3 ~ 4 weeks, according to count results, calculates the lotus bacterium amount (CFU) of the lungs of every mice respectively, carries out the component analysis of lotus bacterium with Log10.Calculate mean and the standard error of each experimental group, carry out statistical analysis.
The lotus bacterium amount of PBS negative control group lungs is the highest.Compared with PBS group, the lungs lotus bacterium amount of ABX, ABXB vaccine group and BCG group all significantly reduces (p < 0.001).It should be noted that compared with BCG group, the lungs lotus bacterium amount of ABX group mice significantly reduces (p=0.001), reduces 0.585log, illustrate that ABX vaccine can better suppress M.tb in the propagation of immune mouse lungs than BCG group.Therefore, ABX vaccine immunity significantly reduces the lotus bacterium amount of M.tb actute infection mice lungs.And the lotus bacterium amount no difference of science of statistics (p > 0.05) of ABXB vaccine group and BCG two groups of mice lungs; however; animal species difference may be the factor affecting its protected effect, does not get rid of effective effect that it is tested at large animal or human's body.
Those skilled in the art will readily understand; the foregoing is only preferred embodiment of the present invention; not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. a recombinant bacillus Calmette-Guerin vaccine, is characterized in that, its bacterium total amount of described bacillus calmette-guerin vaccine is 10
5cFU to 10
6between CFU, comprise the recombinant bacillus Calmette-Guerin vaccine of process LAN 85A antigen, the recombinant bacillus Calmette-Guerin vaccine of process LAN 85B antigen and the recombinant bacillus Calmette-Guerin vaccine of process LAN HspX antigen.
2. recombinant type bacillus calmette-guerin vaccine as claimed in claim 1, it is characterized in that, described bacillus calmette-guerin vaccine comprises the bacillus calmette-guerin vaccine rBCG::X of the bacillus calmette-guerin vaccine rBCG::85A of total number of bacteria 20% to 33%, the bacillus calmette-guerin vaccine rBCG::85B and 15% to 33% of 33% to 60%.
3. recombinant type bacillus calmette-guerin vaccine as claimed in claim 1, it is characterized in that, described bacillus calmette-guerin vaccine comprises the bacillus calmette-guerin vaccine rBCG::85AB of coexpression Ag85A and Ag85B of total number of bacteria 20% to 80%, and the rBCG::XB of 20% to 80% coexpression HspX and Ag85B.
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