CN1109503A - New strain of ribrio comma and its live vaccine - Google Patents
New strain of ribrio comma and its live vaccine Download PDFInfo
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- CN1109503A CN1109503A CN94103386A CN94103386A CN1109503A CN 1109503 A CN1109503 A CN 1109503A CN 94103386 A CN94103386 A CN 94103386A CN 94103386 A CN94103386 A CN 94103386A CN 1109503 A CN1109503 A CN 1109503A
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Abstract
The invention provided a novel vibrio cholerae strain from which vaccine can be prepared, the said strain has lost the toxic factor structure group, but retains its surface antigen structure, it is non-toxic for humanbody and animal, it has effect of improving immunocompetence. It also provided oral live vaccine prepared from the said strain.
Description
The present invention relates to the new bacterial strain of a kind of vibrio cholerae, and the oral live vaccine of bacterial strain preparation thus.
Cholera is one of international quarantine deadly infectious disease of World Health Organization's regulation, is the legal category A infectious disease of China.Vibrio cholerae is made up of two kinds of biotypes (classic type and El Tor biotype) and two kinds of serotypes (coulee and rice leaf).When people were separated to vibrio cholerae from 1883, people had just begun cholera immunoprophylaxis research, over one hundred so far year.Still not having the ideal cholera vaccine comes out.Swede releases efficiently that cholera enterotoxin adds the full bacterium bacterin of cholera nineteen eighty-two, and through the observation of volunteer with on-the-spot 8 years, immune effect only 50% as a result, and 3 months time length, and will be through repeatedly oral, productive value is very high, is not approved by the World Health Organization so far.
In addition, people are from the strain selection that exists naturally or with the attenuated live vaccine of the method for physical chemistry mutagenesis or antibiotic method mutagenesis or bad because of immune efficacy, or because of blindly suddenling change, the possibility of the reverse mutation of hiding is not accepted.
In recent years, because engineered use, people develop Cholera Vaccine with new technology, the method that the cholera intoxicating factor is deducted one by one with genetic engineering means with the development of U.S. vaccine centre of development, from nineteen eighty-two so far, all because of remaining virulence is arranged, still be not World Health Organization's approval so far.
On the basis of our development vaccine more than summing up, from the vibrio cholerae El Tor biotype epidemic strain and non-epidemic strain of my institute's preservation, use the DNA recombinant means,, pick out the strain of 14 strains nature cholera enterotoxin structure gene disappearance with the gene probe of cholera enterotoxin.It is nontoxic to select a strain with immunological method again, bacterial strain IEM101 with good immune effect, this bacterial strain have lacked the structure gene of the virulence factor of existing report so far, keep its antigenic surface structure again, as lipopolysaccharides, structures such as outer membrane protein and main colonizing factor one pili.Survey poison through animal, prove nontoxicly, immune effect detects proves that good immune response is arranged, and then reaches 10 in that human body is once oral
9Cfu does not have the reaction of paying, and can settle down at people's enteron aisle, and stimulates human body to produce corresponding immune antibody, is that the ideal vaccine prepares strain.
The invention provides a kind of new vibrio cholera strain for preparing desirable vaccine.The inventor checks 402 strain EVC epidemic strains with the CT gene probe in research cholera enterotoxin originality, find 14 strains nature CT gene-deleted strain.Applying gene probe and immunological method have been selected 1 strain and have been lacked main virulence factor CT naturally from these bacterial strains, zot and ace structure gene, but the bacterial strain of TcpA and B pili is arranged, it all has better protection to laboratory animal and human body.Through identifying, this bacterial strain is a new strain in the vibrio cholerae kind, we are with this bacterial strain called after vibrio cholerae (Vibrio cholerae) IEM101, we are preserved in Chinese microorganism strains preservation management committee common micro-organisms center on January 29th, 1994 with this bacterial strain, and preserving number is CGMCC NO.0207.
The present invention also provides the oral live vaccine of the no residual hazard of a kind of safety; the bacterium liquid that obtains after the IEM101 bacterial strain cultivated in certain culture liquid; give oral immunity of human body volunteer, settle down more than 2-9 days, and can stimulate serum and enteron aisle to produce specific protective antibody in people's physical efficiency.Thereby obtained a kind of ideal vibrio cholerae live vaccine for oral use.
Hereinafter describe feature of the present invention in detail:
One, the biological character of IEM thalline
1, serotype: vibrio cholerae EVC Ogawa serotype.Phage biotype: 1a
2, biochemical reaction: glucose fermentation, maltose, N.F,USP MANNITOL, sucrose, semi-lactosi, sinistrose, dextrin and Zulkovsky starch produce not aerogenesis of acid; Tardy kefir milk sugar.Nonfermented pectinose, melampyrin, salicin, wood glue sugar, adonis amurensis and inositol.Indole and nitrous acid indole reaction negative, VP are tested positive, the hemolytic test positive.
3, TcpA and Type B pili Electronic Speculum detect and see Fig. 1.
4, antibiotic drug sensitive experiment: the IEM101 bacterial strain to tsiklomitsin, paraxin, gentamicin, acillin, kantlex, cephalosporin, Xin Meisu, carboxylic Bian penicillin, erythromycin, sulfanilamide (SN), Rifampin, how to decide 14 kinds of medicaments insensitives such as ketone acid, amikacin, PXB and Trichofuron are showed anti-medicine.
5, growth characteristics: see Fig. 2.
Two, inherited character detects
1, the chromosomal DNA endonuclease map is seen Fig. 3.Its chromosomal DNA restriction map spectrum is identical with ELTor type epidemic strain.
2, the DNA-DNA molecular hybridization the results are shown in Figure 4.
Gene probe:
ct?tcpA?RSI?hly?zot?st?ace?slt
- + + + - - - -
Three, toxicity test
1, the Vero cell is surveyed poison: press on people's (" Chinese epidemiology magazine ", No. 7, the 13(special issue in 1992) such as luxuriant forever: method 232) is carried out.IEM101 is produced malicious overnight incubation, get the culture supernatants test, do not see pathological change.
2, GM1-ELISA detects the generation of CT: the method that presses on people's (the same) such as luxuriant is forever carried out, and IEM101 strain P/N is 1.67(P/N〉2 positive).
3, suckling mouse LD50 measures: the method that presses on people's (the same) such as luxuriant is forever carried out, and the LD50 of IEM101 is 1.58 * 10
7Cfu is than low 4 orders of magnitude of strain.
4, tame rabbit intestinal ligature experiment: undertaken by the method that people (Infect Immun.1987:55:1116) such as Bleck RZ describe, IEM101 was produced the poison cultivation after 48 hours, get injection in the static culture intestines of 1ml, the hydrops amount of IEM101 bacterial strain is 0ml/cm as a result.(positive) greater than 1.0ml/cm.
Four, immune protective efficiency is measured:
Passive and the initiatively protection test of rabbit: the method that presses on luxuriant forever people's (the same) of grade is carried out, and rabbit initiatively protection test divides three kinds of methods to carry out:
(1) IEM101 lacks CT bacterial strain, oral administration 10 naturally
11The bacterium amount was used the EVC79005(coulee in back 28 days in immunity), 805(rice leaf) and 395 attacks of CVC Ogawa.The results are shown in Table 1.
Table 1 rabbit active immunity protection test-results
Annotate: numerical value is intestinal segment hydrops amount (ml/cm) in the table
(2) IME101 10
6-8The peroral immunity rabbit, oral back 14 days with EVC shore 43(coulee), Wujiang-2(rice leaf) and the CVC395(coulee) river 9(rice leaf) bacterial strain attacks, and the results are shown in Table 2.
Table 2 rabbit is the protection measurement result initiatively
Annotate: numerical value is intestinal segment hydrops amount (ml/cm) in the table
By finding out IEM101 peroral immunity 10 in the table
6-8Two kinds of biotypes of toxic bacterial strain and the two kinds of serotypes of all can attacking against each other have provide protection.
(3) IEM101 10
6-7Bacteria suspension directly injects tame rabbit intestinal epimere, and immunity is used after 14 days and attacked toadstool (coulee of ELTor biotype and rice leaf serotype and classical biotype coulee and rice leaf serotype) 10
5-7Bacterium is attacked experiment, and observe in 8-16 hour phleboedesis and expand, hyperemia, necrosis and hydrops amount (ml/cm) the results are shown in Table 3.
Table 3 IEM101 rabbit is the protection test result initiatively
Annotate: numerical value is intestinal segment hydrops amount (ml/cm) in the table
Rabbit passive protection power measurement result sees Table 4.
Immune serum: the 10th day separation immune serum behind the IEM101 immunizing rabbit
Attack toxic bacterial strain: Wujiang-2, shore 43 10
6The bacterium amount
The experiment of table 4 rabbit passive protection
Annotate: numerical value is hydrops amount ml/cm in the table
Last table result shows that IEM101 immune serum 1:2 dilution can protect strain to attack, and the 1:40 dilution still has protection.
Five, human safety and immunogenicity detect
1, immunity:
The volunteer: the healthy people of no cholera morbidity history is totally 31 people, and wherein the women 13, the male sex 18 people, age 25-60.31 people are divided into 7 groups by the immunizing dose difference, the I-V group, every group 4 people, the VI group is 5 people, VII group 6 people.The IEM101 10 of everyone oral 100 ℃ of heat-killed of I group
9Cfu, the II-VII group is everyone oral IEM101 10 alive successively
2, 10
4, 10
6, 10
8, 10
9And 10
10Cuf. these bacterium number averages are suspended from 2%NaHCO3 50ml.Before the clothes bacterium 20-30 minute, earlier clothes 2%NaHCO3 50ml with in and hydrochloric acid in gastric juice.
2, observations
(1) safety testing result: in 31 volunteers, the dead bacterium 10 of IEM101
9, viable bacteria 10
2-10
10The oral person of cfu does not all have side reaction.Have one to begin painless property diarrhoea in 40 hours in 10 volunteers, rare just in two days totally 4 times, the stool amount is no more than the self-healing of not taking medicine 100ml/ time.
Symptom behind the table 5 volunteer oral bacterium
(2) detection of discharge of bacteria time: collect each stool of volunteer and vomitus, direct inoculation selective medium-gentamycin agar (available from ward that I suffer from diarrhoea).Make of basic peptone water simultaneously and increase bacterium and cultivate, cultivate for 37 ℃ and observed gentamicin in 24-72 hour and cultivate colonial morphology and color, with the macroscopic agglutination calibrating, anti-cholera multivalence and coulee serum are available from Lanzhou Inst. of Biological Products, Ministry of Public Health.By day calculating discharge of bacteria time.As a result, the volunteer of oral various dose IEM101, discharge of bacteria time length is obviously different, and the increase discharge of bacteria time lengthening with oral dosage the results are shown in Table 6.
The table 6 volunteer discharge of bacteria time
(3) in immune leading zero sky and immunity back 7,14,28,90,180,270 and 360 days take a blood sample respectively separation of serum and peripheral blood lymphocytes (PBL), the detection of specific antibody in the serum: the ELISA method detects (Wang Shixia etc. " Chinese epidemiology magazine " 1990; No. 7,11(special issue): 222, vibriocidal antibody and o agglutination wait people (Infect Immun 1976 with reference to Svennerbolm AM; 13:735) carry out, indicator is 01 group cholera vibrio ELTor type epidemic strain 79005, and the detection of specific antibody secretion PBL-ELISA is undertaken by document [64].Detect PBL simultaneously and secrete anti-CT, LPS and full bacteria antibody, the result surveys the OD value in every hole with the enzyme-linked immunoassay instrument, calculates the P/N value, and the mouse-anti people IgA of horseradish peroxidase-labeled, IgG and IgM are all available from fundamental research institute of the Chinese Academy of Medical Sciences.
Pass through ELISA, vibriocidal antibody test and agglutination test, various antibody horizontals in the different time serum were all negative before and after the anti-CT antibody mediated immunity before and after detection was immune, and anti-LPS antibody, agglutination reaction and vibriocidal antibody various antibody horizontals before immunity are very low or approaching zero, obviously rose in the immunity back on the 7th day, vibriocidal antibody and agglutination reaction antibody horizontal reached the peak on the 14th day, and anti-LPS antibody I gA, IgG and IgM reached the peak in 28 days, descended gradually later on, be maintained to 270 days, and saw Table 7,8,9
Different time serum vibriocidal antibody titre before and after table 7 immunity
Numerical value is the geometric mean of each group antibody titers in the table
Different times serum lectin antibody titers before and after table 8 immunity
Numerical value is the geometric mean of each group antibody titers in the table
The anti-LPS antibody titers of different time serum before and after table 9 immunity
Annotate: numerical value is the geometric mean of each group antibody titers in the table
The cultivation of vibrio cholerae IEM101 bacterial strain
Vibrio cholerae IEM101 bacterial strain (CGMCC, 0207) is inoculated in the ordinary broth (PH7.4) that has added 0.5%Nacl, cultivated 2 hours in 37 ℃.
The preparation of vibrio cholerae IEM101 bacterial strain oral live vaccine
Cultivating the bacteria suspension that obtains as method as described in the embodiment 1 can directly use, and the dosage of viable bacteria immunity is 10
2-10
10The cfu bacteria containing amount is preferably 10
9Cfu bacterium amount.
The preparation of vibrio cholerae IEM101 bacterial strain oral live vaccine
With the bacteria suspension of embodiment 1 described method preparation, lyophilize according to a conventional method, resuspending is in 0.85%Nacl solution, and promptly making bacteria containing amount is 10
9The oral vaccine of cfu.
Claims (3)
1, the new bacterial strain IEM101 (CGMCC: 0207) of a kind of vibrio cholerae (Vibrio cholerae).
2, the cultural method of bacterial strain according to claim 1 is characterized in that IEM101 is inoculated in the ordinary broth (PH7.4) that has added 0.5%Nacl, in 37 ℃, cultivates 2 hours.
3, by the oral live vaccine of the described bacterial strain of claim 1 preparation, it is characterized in that the bacteria suspension of the described method preparation of claim 2 directly orally, or according to a conventional method with after this bacterium liquid freeze-drying, be suspended in the 0.85% Nacl solution and get final product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94103386A CN1058522C (en) | 1994-04-01 | 1994-04-01 | New strain of ribrio comma and its live vaccine |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94103386A CN1058522C (en) | 1994-04-01 | 1994-04-01 | New strain of ribrio comma and its live vaccine |
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| Publication Number | Publication Date |
|---|---|
| CN1109503A true CN1109503A (en) | 1995-10-04 |
| CN1058522C CN1058522C (en) | 2000-11-15 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN94103386A Expired - Fee Related CN1058522C (en) | 1994-04-01 | 1994-04-01 | New strain of ribrio comma and its live vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| ES2199944T3 (en) * | 1992-07-06 | 2004-03-01 | Avant Immunotherapeutics Inc. (Titular Al 50%) | PROCEDURE FOR THE ELABORATION OF A GENETICALLY STABLE MUTANT CEPA OF VIBRIO CHOLERAE. |
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1994
- 1994-04-01 CN CN94103386A patent/CN1058522C/en not_active Expired - Fee Related
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