CN1195993A - Intranasal vaccination against gastrointestinal disease - Google Patents

Intranasal vaccination against gastrointestinal disease Download PDF

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Publication number
CN1195993A
CN1195993A CN96196763A CN96196763A CN1195993A CN 1195993 A CN1195993 A CN 1195993A CN 96196763 A CN96196763 A CN 96196763A CN 96196763 A CN96196763 A CN 96196763A CN 1195993 A CN1195993 A CN 1195993A
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toxin
clostridium difficile
compositions
antigen
intranasal
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小W·D·汤马斯
T·P·莫纳思
F·托雷斯－罗佩兹
张振西
雷文德
D·M·赖尔利
J·S·莫克里夫
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Sanofi Pasteur Biologics LLC
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OraVax Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/08—Clostridium, e.g. Clostridium tetani
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • Y—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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

The invention features intranasal immunization methods for inducing immune responses in distal mucosal sites, e.g., the gastrointestinal or genitourinary tracts. The methods of the invention may be used to induce protective and/or therapeutic immune responses against pathogens (e.g., bacteria of the genus Clostridium, e.g., C. difficile) which infect these distal sites. Also included in the invention are vaccination methods in which combinations of mucosal (e.g., oral or intranasal) and parenteral (e.g., subcutaneous or intraperitoneal) routes of administration are used.

Description

The intranasal vaccination of anti-gastrointestinal disease
Background of invention
The present invention relates to be used to prevent and/or treat the intranasal vaccination method of gastrointestinal disease.
Clostridium difficile is to cause developing into serious, and be the diarrheal Gram-positive relevant with antibiotic of fatal colitis sometimes, the malicious antibacterial of the product of formation spore.When normal intestinal flora is upset by for example antibiotic or antitumor agent treatment, clostridium difficile can be settled down in colon, and it produces two kinds of high molecular toxin, toxin A and toxin B therein, this two peptide species is cytotoxin, but the tiring greater than 1000 times of toxin As of toxin B.Toxin A also is an enterotoxin, because it causes the accumulation of body fluid in by the animal intestinal loop of ligation.
Summary of the invention
We have shown intranasal and have mixed mucosa (for example mouthful or intranasal) and general (for example reaching down or intraperitoneal), inoculation method even inducing in the mucosal immune response of far-end mucosa site (for example gastrointestinal and/or reproduction urethra) also effective when the shortage adjuvant.Use a kind of in these methods to cause protecting these animals to avoid the attack of clostridium difficile afterwards with Toxin A Toxin A (Clostridium difficile clone seq5) or B (or toxoid) inoculation hamster.
Therefore, the present invention relates to a kind ofly induce that the gastrointestinal in the mammal or reproduction urethra pathogen are produced the far-end mucosal immune response (is outside the upper respiratory tract, for example at the mucosal immune response of gastrointestinal and/or reproduction urethra.In the method, in mammal, this polypeptide antigen is dissolved in the pharmaceutically acceptable diluent the non-polypeptide antigen that duplicates by intranasal administration, and can induce pathogen generation far-end immunne response.
The invention still further relates to a kind of method that pathogen is produced the far-end mucosal immune response of in mammal, inducing, comprise: (1) carries out parenteral with this antigen to mammal to antigen administration and (2) of mammal mucomembranous surface generation far-end immunne response with a kind of can inducing.The mixing mucosa and the parenteral of any order include in the present invention.For example, mucosa (for example, nose, mouthful, eye, stomach, rectum, vagina, gastrointestinal, or urethra) administration can be before parenteral (for example intravenous is subcutaneous, intraperitoneal or intramuscular) administration.Perhaps parenteral can be positioned at before the mucosa delivery as an example, can be with three weekly dose mucosals (for example muzzle), in the time of around the, can mix mucosa (for example muzzle) and parenteral (for example intraperitoneal) administration.
Its pathogen that produces mucosal immune response is also included within the method for the present invention, from it can deutero-antigen (for example, the non-polypeptide antigen that duplicates) comprises, but be not limited to the gastrointestinal disease substance, Helicobacter pylori (helicobacter pylori for example, cat Helicobacter pylori and Helicobacter pylori (H.heilmanii), Campylobacter (campylobacter jejuni), cause suffering from diarrhoea and the pathogen of colitis (clostridium (clostridium difficile for example for example, the Nuo Shi clostridium, the Soxhlet clostridium), enterotoxigenic E.Coli, shigella, vibrio cholera and salmonella typhi), with intestinal urethra pathogen (human immunodeficiency virus for example, herpes simplex virus, papillomavirus, treponema pallidum, chlamydia and Diplococcus gonorrhoeae).
The antigenic instantiation (for example, the non-polypeptide antigen that duplicates) that can be used in the inventive method includes, but are not limited to bacteriotoxin.For example, can use from clostridium (clostridium difficile for example, the Nuo Shi clostridium, the Soxhlet clostridium), for example Toxin A Toxin A (Clostridium difficile clone seq5) and/or category-B toxin, Nuo Shi clostridium alpha-toxin (BeHe etc., Toxicon29 (7): 877-887,1991), Soxhlet clostridium lethal toxin (Beffe etc. see above) and its immunogenic fragments and derivant.The antigen that is used for the inventive method can obtain by standard method known in the art, for example purification, recombinant DNA method and chemical synthesis process from the culture of aborning pathogen.
The present invention can adopt clostridium (for example clostridium difficile) toxoid as vaccine antigen, and toxoid is a kind of treated reducing the poisonous character of toxin, but still has kept antigenic toxin (or the mixture of toxin, for example, Toxin A Toxin A (Clostridium difficile clone seq5) and toxin B).Comprise that toxoid in the present invention uses standard method to prepare, include, but are not limited to chemistry (for example formaldehyde or glutaraldehyde) and handle that protease enzyme action and recombination method (for example pass through the fragment or the sudden change (for example point mutation) of output toxin.
In order to prevent or to reduce the following infection (being the inductive treatment immunne response) that is being stood by the chance of pathogenic infection (promptly inducing protective immune response) and/or treatment; can carry out method of the present invention; when being the situation of enteropathogen; can use method of the present invention to treat is in the initiation potential; but do not suffer from the diarrheal mammal that causes by pathogen (for example clostridium difficile) or suffer from the diarrheal mammal that causes by pathogen; according to the inventive method; the mammal that can be treated comprises as the people; as cattle; horse, pig, Canis familiaris L.; cat, sheep and goat.
An advantage of the inventive method is at least some pathogen (for example clostridium difficile toxin and toxoid), does not need mucosal adjuvants to come induce immune response (for example protective immune response).
By preferred embodiment and the claim of describing in detail below, other characteristics of the present invention and advantage will be conspicuous.Describe in detail
Accompanying drawing is at first described.Accompanying drawing
Fig. 1 be shown in by shown in the path, with the protection level of the anti-clostridium difficile disease in the hamster of clostridium difficile antigen immune, shown after attacking, the protection level of general (death) and intestinal (diarrhoea) disease with clindamycin.(referring to the table 1 that is used to describe immunization route).
Fig. 2 be by ELISA record behind vaccine by mode administration 3 dosage described, in hamster serum to Toxin A Toxin A (Clostridium difficile clone seq5), the average (+SE) antibody titer (table is seen the table 1 that is used to describe immunization route) of toxin B and full cellular antigens.Analyze specific I gG with the hamster serum behind the 3 dosage vaccine immunities; Tire and be defined as maximum dilution with absorption＞0.3.The meansigma methods of 5 animals of each bar rod expression (+SE).
Fig. 3 is the bioactive figure of demonstration by the hamster serum of the vaccine of administration 3 dosage of description.The cytotoxin A in the detection serum in the IMR-90 cell or the coagulation of active inhibition of cytotoxin B and clostridium difficile cell; Tire and be defined as the maximum dilution of biologically active.Meansigma methods (+SE) (referring to the table 1 that is used to describe immunization route) of 5 animals of each bar rod expression.
Fig. 4 is presented at the long-term antibody response in the hamster of intranasal intraperitoneal and subcutaneous immunity.Shown that (intranasal intraperitoneal-I and subcutaneous-I) and chlorine can be agree mycin and be attacked (the comparisons of replying between intranasal intraperitoneal-II and subcutaneous-II) in the last 140 days before clindamycin is attacked.By ELISA, detect contratoxin A in the serum, toxin B tires with full cellular antigens, and tiring is expressed as the maximum dilution with absorption＞0.3; The meansigma methods of 5 animals of each bar rod expression.
Fig. 5 shows the long-term antibody response in the hamster of intranasal intraperitoneal and subcutaneous immunity, shown chlorine can agree mycin attack before (intranasal intraperitoneal-I and subcutaneous-I) and clindamycin attack back 140 days (between intranasal intraperitoneal-II and subcutaneous-II) reply comparison, the cytotoxic inhibition in the IMR-90 cell of detection serum and the coagulation of clostridium difficile cell; Tire and be the serum of the maximum dilution of biologically active.The meansigma methods of 5 animals of every rod expression (+SE).
Fig. 6 A-6B is presented at and exists or when lacking CT, carries out the serum of the mice after the muzzle immunity with toxoid, feces, and antitoxin A in saliva and the vaginal secretions (Fig. 6 A) and antitoxin B (Fig. 6 B) IgA reply.
After Fig. 7 A-7C is presented at and with toxoid mice is carried out the muzzle immunity, serum antitoxin B cytotoxicity (Fig. 7 A), after carrying out the muzzle immunity with toxoid, serum antitoxin A cytotoxicity (Fig. 7 B) and carry out the intranasal immunity with toxoid after.The antitoxin A cytotoxicity of saliva and vaginal secretions.
Fig. 8 shows and uses the toxoid of using by oneself to carry out the serum of intranasal immune mouse, makes rat be avoided the passive protection level of toxin A by the intestinal loop of ligation.
Fig. 9 be presented at toxin A or toxin B cause death attack after, carried out the survival percent of muzzle mice immunized with toxoid.
After Figure 10 was presented at the immunity of toxoid intranasal, mice was by the toxin A intestinal toxic level in the ligation intestinal loop.
After Figure 11 A-11B showed that the approach that passes through to describe is used the GST-ARU immunity, toxin A specificity general (Figure 11 A) and mucosa (Figure 11 B) IgA replied (referring to the table 5 that is used to describe immunization route).
Figure 12 A-12B shows that the toxin A cytotoxicity of the serum that took out in back 40 days with the GST-ARU immunity suppresses level (referring to the table 5 that is used to describe immunization route).
Figure 13 A-13B shows that immune serum from the GST-ARU immune mouse is to the enterotoxication passive inhibition level of the toxin A in the rat intestinal loop.
Figure 14 is presented at after recombinant toxin A repetition (ARU) immunity, to the survival percent (referring to the table 5 that is used to describe immunization route) of lethal toxin A attack.
Figure 15 is presented at after the GST-ARU immunity, by the enterotoxication protection level of contratoxin A in the ligation mice intestinal loop.Be used for intranasal and mixing mucosa-general inoculation method at far-end site induce immune response
We have shown that nose is interior or mix mucous membrane and the general administering mode causes mucosal immune response in stomach and intestine and reproduction urethra.
Method of the present invention can be used to induce protectiveness and/or therapeutic immunization to the gastrointestinal disease substance to reply. Wherein pathogen comprises, but be not limited to helicobacter (helicobacter pylori, cat helicobacter and helicobacter (H.heilmanii), campylobacter (for example campylobacter jejuni) and cause suffering from diarrhoea and the pathogen of colitis, clostridium enterotoxigenic E.Coli for example, Shigella, comma bacillus and salmonella typhi; Or intestines urethra pathogen (human immunodeficiency virus for example, herpes simplex virus, papillomavirus, microspironema pallidum, Chlamydia and Diplococcus gonorrhoeae) the easy suitable vaccine antigen (for example polypeptide antigen) of selecting for the disease that causes wishing that the application of the invention method is prevented and/or treated of those skilled in the art. Be described below method of the present invention, it relates to the instantiation that can be used for the vaccine antigen in the inventive method from the antigen conduct of clostridium difficile (for example toxin or toxoid). Use clostridium difficile toxin and toxoid as vaccine
The clostridium difficile toxin polypeptide that can be used in the inventive method and the composition can prepare with the multiple standards method. For example, can be from the filtrate of clostridium difficile culture purified toxins (such as toxin A and/or toxin B) (referring to such as Kim etc., infection and immunity 55:2984-2992,1987; With referring to following embodiment 1).
Also but the recombinant DNA method of Application standard prepares the clostridium difficile toxin polypeptide (referring to such as Ausubel etc., compile, the modern molecular biology method, John Wiley and Sons, Inc., 1994), in these methods, with suitable host cell with the suitable expression vector of the nucleic acid fragment that comprises all or part toxin-encoding transform (referring to Dove etc., infection and immunity 58:480-488,1990 and Barroso etc., nucleic acids research 18:4004,1990, be respectively the nucleotides of Clostridium difficile toxin A and the amino acid sequence of supposition, and the nucleotide sequence of toxin B). Any in the multiple expression system all can be used to prepare recombinant toxin. For example, can be in prokaryotic hosts (for example Escherichia coli) or eucaryon host (for example yeast cells (for example Saccharomyces cerevisiae) mammalian cell (for example COSI, NIH3T3 or TEG3 cell) or arthropod cell (for example the meadow is greedy by moth (SF9) cell)) the preparation toxin polypeptide. These cells can obtain from multiple separate sources well known by persons skilled in the art, American type culture collection for example, Rockville, MD (also referring to, such as Ausubel etc., see above). As such as described at Ausubel above etc., the selection of employed transfection/method for transformation and expression vector is got the merchant and is determined in selected host system. Expression vector (such as plasmid or viral vectors) can from such as describe the cloning vector those in select: laboratory manual (Pouwels etc., 1985, supplementary issue 1987; Also referring to such as Ausubel etc., see above).
Also can prepare the clostridium difficile toxin polypeptide by chemical synthesis, especially short fragment is for example synthesized the middle method of describing, 1984 by solid-phase peptide, second edition, Stewart and Young compile, Pierce chemical company, Rockford, IL and the In Vitro Translation method by standard.
The inventive method also can be used the toxoid of clostridium difficile toxin. Toxoid is treated, and the toxicity of toxin is eliminated or reduces, but the toxin that antigenicity still is retained, but toxoid Application standard method prepares. For example, by chemistry (for example glutaraldehyde or formaldehyde) process (referring to, such as Libby etc., infection and immunity 36:822-829,1982). As mentioned above, toxoid also can suddenly change by producing in the gene of toxin-encoding, and the expression sudden change prepares in expression system. The toxin A that can be suddenlyd change and/or the zone among the toxin B comprise, the cysteine residues that for example keeps, nucleotides land, inner hydrophobic district, and/or carboxyl terminal duplicate block. The instantiation that can be used for these sudden changes in the clostridium difficile toxin of the present invention is described in such as Barroso etc., microbial pathogens 16, and 297-303 is in 1994.
Can be used for the anatoxic method of generation of the present invention and comprise toxicity essentially, but carry out chemical modification with the irrelevant amino acid of antigenicity. For example, known specificity is modified the amino acid that contains SH on this area, lysine, tyrosine, the reagent of tryptophan or histidine residues (referring to such as Cohen etc., is commented 37:683-695 bioid academic year, 1968), in addition, can use the substrate analogue that can covalently boundly arrive the nitrine connection of toxin avtive spot by ultraviolet irradiation, for example UDP-glucose produces toxoid.
Outside natural total length clostridium difficile toxin, the polypeptide fragment of toxin, or the toxin (or polypeptide fragment of toxin) that comprises sudden change (may or may be not being toxoid) can be used for the present invention, and prerequisite is that it has kept antigenicity. The fragment of clostridium difficile toxin for example is referring to such as Price etc., modern microbiology 16:55-60,1987; Lyerly etc., modern microbiology 21:29-32,1990, and the infection and immunity 60:2488-2492 such as Frey, 1992. The fragment of coding clostridium difficile toxin, and/or the gene of toxin that comprises sudden change is by standard method preparation (referring to such as Ausubel etc., seeing above). Can use the standard method of this area; for example by measuring induce (the seeing below) of mucosal immune response; the inducing of protective immunity (seeing below) or, therapeutic immunization is replied induces to screen the fragment that comprises in the present invention, derivative and anatoxic antigenicity.
Although be not essential, in the method for the invention, adjuvant can be with the vaccine administration, can use any in the multiple adjuvant well known by persons skilled in the art. For example, cholera toxin (CT), colibacillary heat labile enterotoxin (CT), or it has the fragment of adjuvanticity or derivative to can be used for mucosa delivery. Adjuvant, for example RIBI (immunochemistry company, Hamiton, MT) or aluminium hydroxide can be used for parenteral.
With for example adjuvant (CT for example, LT or its have fragment or the derivative of adjuvanticity) fusion that comprises clostridium difficile toxin (or its fragment or derivative) that merges is also included among the present invention, but and the preparation of Application standard method (referring to, such as Ausubel etc., see above). In addition, vaccine of the present invention can with adjuvant covalent coupling or crosslinked. Adjuvant and antigen covalent coupling or crosslinked method are described in such as Cryz etc., vaccine 13:67-71,1994; Liang etc., Journal of Immunology 141:1495-1501,1988; With Czerkinsky etc., infection and immunity 57:1072-1077,1989.
As mentioned above, the method according to this invention is with vaccine combination (being with or without adjuvant) intranasal administration.Also can use the administration of mixed type, for example carry out the administration of the vaccine of initial dose to mucosa (for example intranasal or mouth) surface, with can carry out enhance immunity (for example intraperitoneal or subcutaneous) through parenteral, this has produced unexpected good result in conjunction with administration.For example, can a week carry out the parenteral enhance immunity behind the mucosa delivery first.
The amount of vaccine of administration depends on concrete vaccine antigen, and no matter whether adjuvant with the vaccine antigen administration, the mode of administration and frequency and required effect (for example protect and/or treat) and these all can be determined by those skilled in the art.Usually, vaccine antigen of the present invention is with for example amount administration between 1 μ g and 100mg.If adjuvant with the vaccine administration, can use the amount that changes between for example 1ng and 1mg.If desired, but repeat administration, and this can be determined by those skilled in the art.For example, 3 enhancing dosage can be right after after amount of initiator by weekly interval.Vaccine can be able to be accepted carrier or diluent (for example, water, saline solution is phosphate buffered saline(PBS) for example) or bicarbonate solution (for example, 0.2MNaHCO on any medicine 3) middle administration, select to be used for carrier of the present invention and diluent according to the mode of administration and the drug study of approach and standard, appropriate drug carrier and diluent and the medicine that is used for pharmaceutical preparation must thing be described in the Remington ' s pharmaceutical science and USP/NF into this area canonical reference book.
The following examples are intended to explanation, but do not limit method of the present invention.The modification to condition and parameter that it will be apparent to those skilled in the art that proposes below comprises in the present invention.Embodiment
Two model systems, mice and hamster are used to estimate inoculation method of the present invention.Because hamster and human similar, relevant to antibiotic diarrhoea sensitivity, so the hamster model is used to directly estimate the protectiveness effect of inoculation opposing clostridium difficile disease.The C. difficile infection of hamster causes serious hemorrhagic cecitis, and it makes the people associate observed colitis when human disease's state.In addition, mix with clostridium difficile, clindamycin oral to hamster or general administration single dose causes serious diarrhoea, and this diarrhoea finally causes the death of animal.
When using various analysis, the hamster model also can be used to monitoring by the inductive immunne response of inoculation method of the present invention.For example, the inhibition that can be used to measure the cell in vitro toxin from serum and the mucosa sample of immune hamster.In addition, immune hamster can be used to by the ligation intestinal loop estimate by the inoculation inductive toxin A the active inhibition of intestinal toxicity.And, can monitor the cluster of clostridium difficile in hamster by stool culture, perhaps can determine the existence of toxin A in the hamster feces and/or toxin B by ELISA and/or cytotoxicity analysis.
The characteristics of mouse model help estimating by the inductive immunne response of inoculation method of the present invention.Specifically, can commercial buy the monoclonal antibody of identification mice IgA, thereby and facilitation the evaluation of mice mucosal immune response.On the contrary, these reagent can not be used to estimate the hamster mucosal immune response.Another advantage of mouse model is to have developed the method for the mice mucomembranous surface that is used to take a sample, and its makes the mucosa that can map to various immunization methods generations reply.
In case determined the vaccine originality of vaccine candidate thing by for example elisa assay, the mice serum sample can be used to detect the character of antibody that may be relevant with effective vaccine.For example, can analyze the ability that immune serum (1) suppresses the vitro cytotoxicity of toxin A and/or toxin B, or (2) use the mice that hits with the toxin A merit or rat by the enterotoxication ability of ligation intestinal loop inhibition toxin A.But also per os or they by the intestinal loop of colligation in merit hit immune mouse to determine for because the protection of death that the intestinal toxicity of toxin A causes or body fluid accumulation.At last, immune mouse being carried out general ground with the toxin that is known as lethal dosage attacks.Embodiment 1 usefulness comprises the vaccine combination immunity hamster of clostridium difficile toxin
Following method is used to analyze the effect of immunization method of the present invention in the hamster model system.The preparation of clostridium difficile toxoids vaccine
As (infecting and immune 36:822-829 1982) described preparation such as Libby and go out and control clostridium difficile culture filtrate.Briefly, clostridium difficile VPI bacterial strain 10463 (ATCC preserving number 43255) was cultivated centrifugal and filtration sterilization 3 days in the dialysis flask.1ml formaldehyde is added in the 100ml culture filtrate, and mixture is incubated 1 hour down at 37 ℃.Measure (Lyerly as ELISA, Deng infection and immune 47:349-352,1985), culture filtrate has the concentration of about 50 μ g/ml toxin As, and measure (Ehrich etc. infect and immune 28:1041-1043 1980) as the cell culture cytotoxicity analysis, for toxin B, it is 10 that cytotoxicity is tired 6By 500ml cell concentration device (Amicon, Beverly, MA) in to the 30KD membrane ultrafiltration, with the phosphate buffered saline(PBS) (PBS) of toxoid with 3 volumes, pH7.4 washing.Toxoid is concentrated 10 times, filtration sterilization, and be stored under 4 ℃ until use.Suppose not have the loss of tangible toxin protein according to the size (toxin A is 308KD, and toxin B is 269KD) of toxin during concentration step, recording the go out concentration of the toxin controlled of in 10X solution each is 500 μ g/ml.The toxoid material is without any the cellular cytoxicity activity of the cell (ATCC preserving number CCL186) of detectable anti-IRM-90.The preparation of whole-cell vaccines
Under 37 ℃ of following anaerobic conditions, clostridium difficile VPI bacterial strain 10463 (ATCC preserving number 43255) is being shown peptone yeast extract medium (PPY; Holbrook etc., applied bacteriology magazine 42:259-273 1977) cultivate 36 hours so that Sporulation is reduced to minimum.Centrifugal culture and with sedimentary cell with PBS washing 3 times.The last time after the washing, sedimentary cell is suspended in again (the volume: kept 24 hours down among the PBS of formaldehyde volume) and at 4 ℃ that comprises 1%, by removing excessive formaldehyde 3 times with the PBS washing, the clostridium difficile cell suspending liquid of formaldehyde treated is stored under 4 ℃, 37 ℃, under the anaerobic condition, will be to equal 10 9The inoculum cultivation that clostridium difficile colony-forming units (CFU) (O.D of 550nm place is 1.0 cell suspending liquid) is inoculated in the PPY culture medium did not produce any growth after 36 hours.Animal
The female gold hamster (Meso-cricefusauratus, Charles River, Kingston NY) that during with immunity is age in 6-8 week is used for all experiments.Between duration of immunity, animal is raised in cages with 5 one group.And during attacking, raise in cages separately with clostridium difficile.Immunization ways
Analyze seven kinds of different immunization methods (table 1).For muzzle (i.n) immunity, (5 μ l solution CA) mix for Calbiochem, La Jolla will and to comprise 5 μ g cholera toxins at the various types of toxin of 5 μ g (the toxin B of the toxin A of deactivation and deactivation) in the 10 μ l 10X toxoids.15 μ l antigen adjuvant mixture are dripped to the external nares of hamster with micropipet, in each nostril, drip the dosage of half.For gastric (ig) immunity, the every kind toxicity of 100 μ g is mixed with 10 μ g cholera toxins, and transferring to volume with PBS is that 1ml also uses the gavage administration, for intraperitoneal (ip) and subcutaneous (SC) immunity, with every kind of toxoid of 5 μ g and the fine RIBI adjuvant of 0.3ml (RIBI, immunochemistry company.Hamilton MT) mixes.For rectum (r.) immunity, will comprise the solution mixture of 10 μ g cholera toxins at the every kind of toxoid of 50 μ g in the 100 μ l toxoids and 1 μ l.For the rectally (WCr) of full cell with 5 * 10 8Cell mixes with every kind of toxoid of 50 μ g in the 100 μ l toxoids, adds the 1 μ l solution that comprises the 10ug cholera toxin, for rectum and full cell rectum immune group, with sample on the sample to inserting disposable 20 * 1 of rectum 3cm 1In/2 sample introduction needle, in animal, carry out intranasal, gastric, intraperitoneal and subcutaneous immunity with the slight anesthesia of different fluorane.In the animal of pentobarbital anesthesia, carry out rectum and full cell rectum immunity.The intranasal group (c.i.n) of contrast is accepted 5 μ g cholera toxins through intranasal.Through the subcutaneous RIBI adjuvant of accepting 0.3ml, 5 animals are one group and are used for all immunization methods that all groups were accepted 4 dosage vaccines (or adjuvant contrast) altogether at the 0th, 7,14 and 28 days that test subcutaneous group (c.s.c) of contrast.Table 1. method (inip=intranasal+intraperitoneal of the clostridium difficile culture immunity hamster of formalin deactivation; The in=intranasal; The ig=gastric; The r=rectum; The full cell rectum of wcr=; The ip=intraperitoneal; Sc=is subcutaneous; Cin=matched group intranasal; The csc=matched group is subcutaneous).
Antigen Immunization route (group) The dosage of each immunity Adjuvant, the dosage of each immunity
Culture filtrate toxoid 3 immunity, intranasal the 4th immunity first is intraperitoneal+intranasal (inip) 5 μ g toxin As, 5 μ g toxin B 5 μ g cholera toxins are used for the intranasal immunity; 0.3ml RIBI intraperitoneal immunity
Culture filtrate toxoid Intranasal (in) 5 μ g toxin As, 5 μ g toxin B 5 μ g cholera toxins
Culture filtrate toxoid Gastric (ig) 100 μ g toxin As, 100 μ g toxin B 10 μ g cholera toxins
Culture filtrate toxoid Rectum (r) 50 μ g toxin As, 50 μ g toxin B 10 μ g cholera toxins
The clostridium difficile cell that culture filtrate toxoid and formalin kill Rectum (wcr) 50 μ g toxin As, 50 μ g toxin B 5 * 10 8 Cell 10 μ g cholera toxins
Culture filtrate toxoid Intraperitoneal (ip) 5 μ g toxin As, 5 μ g toxin 0.3ml?RIBI
Culture filtrate toxoid Subcutaneous (sc) 5 μ g toxin As, 5 μ g toxin B 0.3ml?RIBI
Phosphate buffer Intranasal (cin) 15μl 5 μ g cholera toxins
In order to estimate immunne response, the 0th, 2, in the time of 4,7 and 36 days behind the brain of the hamster of different fluorane anesthesia hole obtain blood sampling (200-400 μ l).Under 4 ℃, allow blood coagulation spend the night, centrifugal acquisition serum.Only estimate serum antibody; Owing to lack super suitable anti-hamster IgA reagent, do not measure secretion IgA after the clostridium difficile merit is hit, every other day from the animal of survival, obtain fecal specimens, and be mixed for estimating exist (face as follows) of the degree of cluster and toxin with 2 volume PPY culture medium.Clostridium difficile is attacked
Attack all hamsters at the 38th day (back 10 days of the 4th immunity) per os stomach administration 0.5mg clindamycin, then after 3 hours, carry out 10 5The per os stomach inoculation of clostridium difficile 10463 bacterial strains (ATCC preserving number 43255) of CFU survival, wherein clostridium difficile 10463 bacterial strains wash to eliminate free toxin with the PPY culture medium, after the attack, observe diarrhoea and the disease of hamster every day.Diarrheal seriousness is recorded as: 0, do not suffer from diarrhoea; 1+, loose feces, but do not have moist afterbody; 2+ is around the anus and the tail region humidity; And 3+, afterbody and hypogastric region humidity (having the common hunch of animal of this phenomenon and inactive).The evaluation of tissue injury
The serious ill hamster of quiet execution.Clindamycin is attacked in the caecum sample stuck-at-0% neutral buffered formalin from the hamster that survives behind the hamster of peace and quiet execution and the every kind of immunization method of hanging oneself that obtained in back 8 days.The fixed organization embedding of formalin in paraffin, is cut into the section of 5 μ M, with h and E dyeing and use observation by light microscope.The histological score standard is 0, lymphocyte, plasma cell and eosinophilic minimum the infiltration; The 1+ lymphocyte, plasma cell, neutrophil(e) cell and eosinophilic slight infiltration, the slight hyperemia of adding mucosa is with or without the relevant adenoid hypertrophy of intestinal; 2+ mixes the medium infiltration of inflammatory cell, and medium hyperemia of lamina propria and edema are with or without the goblet cell hypertrophy, each superficial cell necrosis or vacuolization, and folliculus expands the serious inflammation of 3+, mucous hyperemia, edema and hemorrhage, superficial cell necrosis, or erosion or ulcer regression.The evaluation of infecting
The existence of the clostridium difficile in the feces that obtains after the research clindamycin is attacked.The dilution that will dilute 10 times in the PPY culture medium is inoculated in the selective medium that comprises cycloserine (125 μ g/ml) and CFX (8 μ g/ml), and counts clump count after under anaerobic being incubated 48 hours.(Techlab BlacksburgVA) determines the existence of toxin A in the feces as manufacturer's described use toxin A box.After 15 minutes, reading the O.D. at 450nm place with substrate, and (divide subset, Sunnyvale measures CA) to use Softmax software from the concentration that the standard curve of the toxin A that makes each flat board is measured toxin.For quantitative toxin B, centrifugal and filtration sterilization is as described below with the feces suspension, detects the cytopathic effect of the sample of 10 times of dilutions to the fibroblastic cell culture of IMP-90.The ELISA that is used for toxin A and toxin B antibody
(Corning, New York NY) are used in carbonate-bicarbonate buffer, and the toxin A of the purification among the pH9.3 or toxin B cover with the 100ng/ hole, and 40 ℃ of following incubated overnight with the microtitration flat board.Washing is dull and stereotyped also with the phosphate buffered saline(PBS) (PBS) of 2.5% defatted milk powder (NFDM), and pH7.4 seals.With 1: 500-1: the ratio that changes between 64000 adds the blood serum sample of twice dilution.Under 37 ℃, flat board was kept 1 hour.Add with the bonded anti-hamster IgA of alkali phosphatase (1: 1000, Southern Biotech, Birmingham, AL), 37 ℃ of insulations 1 hour down, and before adding the p-nitrophenyl phosphate substrate with its washing.In each flat board, comprise positive control; The hole is used in 100-0.8ng/ml changes, the toxin A or the toxin B of twice dilution cover, and react with specificity goat antitoxin (Techlab), follow and the reaction of anti-goat IgG alkaline phosphatase enzyme conjugates.Negative control is the hole with the toxin covering of purification, and reacts with anti-hamster IgG alkaline phosphatase enzyme conjugates.Read the O.D. of 405nm, the inverse of the maximum dilution that is defined as the sample that produces O.D. 〉=0.3 of tiring.The ELISA of full cell antigen antibody.
Is that 0.2 clostridium difficile suspension cover with being adjusted to of killing of 100 μ l formalins at the O.D. of 550nm place with flat board, and then is being incubated liquid in the rotation shaker of 150rpm.By keeping 2 hours at 70 ℃, with cell fixation to flat board, after the washing, with the blood serum sample of twice dilution with 1: 100-1: 12.800 scope adds, and keeps 1 hour at 37 ℃.Adding anti-hamster IgG and substrate positive control as mentioned above is included in 1: 500-1: 64.000 use (use standard method preparation is at the antiserum of VPI bacterial strain 10463) in sero-fast each hole of the full cell of mice clostridium difficile.To directly be used as negative control with the hole that full cell covers with anti-hamster IgG alkaline phosphatase enzyme conjugates reaction.Cytotoxic inhibition
(Gibco, Grand Island NY), are cultured in 96 hole flat boards and converge in containing the DMEM culture medium of 10% hyclone with the IMR-90 fibroblast.Cause cell to become 100% circular required toxin A or the minimum dose of toxin B is defined as 1 (CTU of cytotoxicity unit 100) for toxin A, 6.3ng/ml, for toxin B, 125pg/ml is defined as 1CTU 100Will be 1: 100-12 changes the hamster blood serum sample and the 4CTU of twice dilution between 800 100Every kind of toxin mix 37 ℃ of insulations 1 hour, and then mixture being joined in the cell.Goat antitoxin A and goat antitoxin B are used as positive control.Observation of cell after 24 hours is determined the ratio of round cell.The tiring of sample is defined as suppressing the inverse greater than the maximum dilution of the sample of 50% cell rounding.Agglutination
With 25 μ l hamster blood serum samples with 1: 25-1: 3,200 scope dilution.In trace flat board at the bottom of the 96 hole U-shapeds (Falcon, Oxnard, CA) preparation dilution, the O.D. that the clostridium difficile suspension that formalin is killed is adjusted at the 550nm place is 1.0, and the 25ml suspension is added in the serum dilution.The full cell antiserum of mouse anti clostridium difficile is as positive control, and PBS is as negative control.Under 4 ℃,, then count agglutination with dull and stereotyped incubated overnight.The tire inverse of maximum dilution of the serum that is defined as causing agglutination of terminal point.Western blot analysis
37 ℃, under the anaerobic condition, clostridium difficile VPI bacterial strain 10463 (ATCC preserving number 43255) and the cultivation of the hamster strain separated after clindamycin is attacked were cultivated 36 hours 5ml PPY culture medium.Centrifugal culture and with PBS with washing of precipitate three times.Precipitation is suspended among the PBS of 250 μ l3%SDS again, and (CA) electrophoresis electrophoresis under 200 volts carried out fractionated in 1 hour to lysate for Bio-Rad, Hercules by 12% preparation SDS-poly amic acid gel.150 volts down, and (LifeTechnologies, Grand Island NY) dispose 1.2 hours, and albumen is transferred on the celluloid from gel in Bethesda Research Laboratories Mini-V 8-10 chamber.The PBS solution of film with 5% degreasing dry powder was sealed 1 hour, washing also is locked in repeatedly (BioRad in screening (multiscreen) equipment, Hercules, CA) then add 1: 200 the dilution each hamster blood serum sample and be incubated 1 hour, with NBT/BCIP (Gibco, Gaithersburg MD) promotes reaction.Mouse anti clostridium difficile 10463 full cell serum are used as positive control.As mentioned above, for typical isolating clostridium difficile bacterial strain from feces.SDS bacteriolyze thing from separator is passed through the electrophoresis fractionated, is transferred on the celluloid, and reacts with full cell mouse antiserum.Statistical analysis
(Quick-STATISTICA software, StatsoftTulsa OK) study the possible significant correlation between the result of the hamster after antigenic immunne response of different clostridium difficiles and the clindamycin attack in utilization Kruskal-Wallis check.Result after the mould rope of chlorine Lincoln is attacked as a result
Attack hamster with clindamycin and clostridium difficile in the last immunity after 10 days.All false immunity, animal of contrast intranasal (cin) and contrast subcutaneous (C.S.C) immunity suffer from seriously that (3+) suffers from diarrhoea attacking in 48 hours dead great majority, in pathogen detection, find acute dispersivity necrosis hemorrhagic cecitis (grade 3+).Folliculus epithelium teratogenesis, expansible epithelium is full of the neutrophil(e) cell.Lymphocyte, plasma cell and neutrophil(e) cell are immersed lamina propria.It is also dead in first 48 hours to attack the immune hamster of failure, and great majority suffer from the diarrhoea of grade 3+ and the cecitis of grade 3+ in histopathology.The animal that survives from attack does not suffer from diarrhoea or suffers from the diarrhoea that seriousness changes at 1+ to 3+.Typhlitic seriousness during diarrheal seriousness and pathology detect is relevant.Suffering from 3+ diarrheal animal, to suffer from grade be the subacute of 2-2.5+, the suppurative cecitis of dispersivity mucosa.The neutrophil(e) cell, lymphocyte and plasma cell immerse lamina propria, and notice many focuses property folliculus abscess.Suffering from those animals of 2+ diarrheal, to suffer from grade be the subacute to chronic of 1.5-2+, medium cecitis.The animal that suffers from medium diarrhoea (+) suffers from medium lymphocyte cecitis, and grade is 1.0-1.5+.Also demonstrating grade in not suffering from the diarrheal hamster is the medium lymphocyte cecitis of 1+.
The results are shown among Fig. 1 that the clindamycin of all vaccine group is attacked.Accepted toxoid vaccine by gastric (ig) or rectum (r) approach, or the mucosal immunity animal of having accepted full cell toxoid vaccine by the rectum approach is to avoiding dead protection minimum, and all suffers from diarrhoea.Rectum and full cell rectum immunization method protected only 20% hamster, and the intranasal method has protected 40% hamster to avoid death.The animal of parenteral immunity (intraperitoneal and subcutaneous) is all protected to avoid diarrhoea.In the animal of intranasal immunity, observe similar results.(intranasal intraperitoneal group then obtains to avoid the protection of dead and diarrheal 100% when the animal of intranasal immunity is accepted the toxoid of escalated dose by the intranasal approach.The existence of clostridium difficile and toxin in the feces
Analyze clindamycin and attack the clostridium difficile of the fecal specimens of back acquisition, the existence of toxin A and toxin B.In the animal of all survivals, observe similar figure.Reach about 10 at back two days of attack clostridium difficile in feces 9CFO/ml; After this, cluster had minimizing slightly and kept about 10 at least 9 day 8CFU/ml, and exist irrelevant with diarrheal.On the contrary, the level of toxin A and toxin B reduces always, although there is the cluster that continues, in the time of the 9th day, in feces, almost do not find toxin, use is at the full cell antiserum of VPI 10463 bacterial strains (ATCC preserving number 43255), by the separative clostridium difficile bacterial strain of western blot analysis typing.Analysis is from different animals and different immune group strain separated, and finds that they are similar mutually, and this means only has a kind of bacterial strain cluster in all hamsters.Yet this bacterial strain is different from the bacterial strain VPI10463 that is used to attack.Immunne response
In from the hamster of all experimental grouies, measure at the antigenic serum antibody of clostridium difficile.In some groups, by the immunne response of contratoxin A behind the ELISA research initial immunity.Do not detect specific IgG at the 2nd, 4 and 7 day behind the initial vaccine dose in the animal by intranasal intraperitoneal and full cell rectum approach inoculation.In the animal of parenteral immunity (intraperitoneal and subcutaneous), after the 2nd and 4 day, do not show and reply, but, observe the slight increase of antibody titer at the 7th day.On the contrary, the antibody response of measuring in last vaccine dose (the 36th day) back has confirmed the seroconversion in all animals.Do not exist early stage (memory) antibody response of vaccine dose is at first shown that animal is the immunogen nature, and not contacted in the past toxin A.
Three kinds of methods are used to research to the antigenic systemic antibody response of clostridium difficile; 1) antigen that is fixed by ELISA identification; 2) the toxic inhibition of pair cell during cell culture is analyzed; With 3) agglutination of antibacterial.Toxin A, toxin B and full cell are used as antigen.Determine as ELISA, contratoxin A, the antibody response of toxin B and full cellular antigens is present in all groups (Fig. 2).By the intranasal intraperitoneal, intranasal, the hamster of gastric and the immunity of intraperitoneal method has than by rectum (r) and full cell rectum) and those higher replying at toxin A and toxin B of subcutaneous route immunity.Antibody horizontal at full cellular antigens demonstrates and observed similar figure at toxin.
The full cellular lysate of the isolated clostridium difficile bacterial strain of hamster after use is attacked from clindamycin is by the further antibody that characterizes full cellular antigens of western blot analysis.Animal in all immunization methods is produced as the antibody of the albumen (it is similar to toxin) of 70KD albumen and size 〉=2.00KD; Animal by the immunity of intranasal intraperitoneal approach has the strongest immunne response, by the intranasal intraperitoneal of using by oneself, the serum of the animal of intraperitoneal and subcutaneous route immunity has been discerned multiple other albumen, but these albumen are not obvious or not remarkable in the serum from mucosal immunity (rectum and full cell rectum group).In other is analyzed, hamster serum is carried out immunoblotting abreast at the toxin and the WCL of purification.These the analysis showed that the albumen with the lower molecular weight of hamster seroreaction is not the toxin fragment.
Serum antibody with biological function demonstrates and those the different figures (Fig. 3) that obtain by ELISA, and the antibody that obtains the toxin A in all animals suppresses cytotoxicity.The maximum antitoxin A activity of hamster generation by intranasal intraperitoneal and the immunity of intraperitoneal approach (is respectively meansigma methods+SE 22,000 ± 49,00 and 18,000 ± 2,000), and mucosal immunity animal (intranasal, gastric, rectum and full cell rectum) have than low activity (being respectively 580 ± 280,280 ± 146,1720+560 and 2760+290).In all groups, obtain high antitoxin B and reply, except that the rectum immune animal,, or accept by mixing mucosa-parenteral approach (intranasal intraperitoneal) to produce agglutinating antibody in the animal of toxoid vaccine only through parenteral (intraperitoneal and subcutaneous).The dependency of immunne response and protection
Intranasal intraperitoneal immune animal is all protected to avoid death and diarrhoea, and when considering ELISA and biological activity, has maximum seroimmunity and reply (Fig. 2 and 3).Avoiding dead protecting fully by all immunization methods that comprise parenteral vaccinate or intranasal immunity separately provides.On the contrary, the rectum immune animal has minimum protection ratio and serum antibody response, especially at the neutralizing antibody (Fig. 3) of toxin B.Although bigger protection provides (Fig. 1) by the intranasal vaccine, yet, as intranasal with immune-related shown in the similar antibody response in the gastric group (Fig. 2 and Fig. 3) be inconsistent.
Immune-related in order to define, analyze animal together from all groups, the result and the immunne response of attacking compared (table 2).Except full cell ELISA.Average antibody level in all tests in the animal of survival obviously from the dead result's of tool animal.Do not compare with suffering from those animals of diarrheal by all analyses, the hamster that suffers from serious diarrhoea (3+) has obviously lower seroimmunity and replys (table 3).Except replying suffering from the agglutinating antibody of 1+ diarrheal in those, the antibody response in the hamster that suffers from medium mitigation diarrhoea (1+ and 2+) obviously is not different from does not suffer from diarrheal those (P＜0.5).Table 2 is at the antigenic immunne response of clostridium difficile and protect the dependency that avoids between the death.
The antibody titer basis
Immunoassay Experiment sum (n=34) Dead (n=11) Survival (n=23)
Meansigma methods ± SE Meansigma methods ± SE The P value a
Toxin A ELISA 5210±960 2750±1360 6390±1180 .0290
Toxin B ELISA 8610±1760 3800±2840 10910±2050 .0063
Full cell ELISA 6560±1890 8363±5610 5704±980 .1382
Anticytotoxin A 7370±1620 1470±390 10195±2170 .0001
Anticytotoxin B 8690±1900 1790±1240 9330±2610 .0197
The agglutination of clostridium difficile 130±25 25±10 180±31 .0032
aKruskal-Wallis check table 3 is at the dependency between antigenic immunne response of clostridium difficile and the diarrhoea seriousness
Do not suffer from diarrhoea (n=10) Diarrhoea, 3+ (n=9)
Immunoassay Meansigma methods ± SE Meansigma methods ± SE The P value a
Toxin A ELISA 9400±1970 1333±160 .0003
Toxin B ELISA 17,800±3430 870±230 .0001
Full cell ELISA 9000±1660 1820±310 .0001
Anticytotoxin A 15840±3690 3220±610 .0016
Anticytotoxin B 11540±4770 670±440 .005
The clostridium difficile agglutination 265±49 55±28 .0098
For with do not suffer from the diarrhoea group relatively the time aThe P value, the Kruskal-Wallis check.Long-term protection
After clindamycin is attacked, will be from four surviving animals of intranasal intraperitoneal group with from four animal feedings of subcutaneous group 140 days.At the 140th day, obtain blood and fecal specimens, and animal reuse clindamycin is attacked.Three (75%) in every group of four animal are survived from attack once more.In two (50%) in four animals of intranasal intraperitoneal group and subcutaneous group of four animal zero are (0%) protected diarrhoea that avoids only.Immunne response before attacking once more and preceding the replying of obtaining of attack are first compared.In elisa assay, toxin A and toxin B antibody do not reduce, although obviously reduce (Fig. 4) at the level of full cellular antigens.When comparing biological activity, before attacking once more, observe the obvious minimizing (Fig. 5) of anticytotoxin activity and clostridium difficile agglutination.The example II vaccine combination immune mouse that comprises clostridium difficile toxin
Following method is used to analyze the effect of immunization method of the present invention in mouse model system.ELISA
The ELISA method of experiment is described as above below being used for.Briefly, by covering 96 hole flat boards with toxin A or toxin B, with each hole sealing, add sample with the PBS-tween solution of defatted milk powder, and detect with the link coupled reagent of anti-mice alkaline phosphatase (AP) that commerce can be buied and to measure the toxin specific immune response from mice.With flat board with Sigma104 alkali phosphatase (AP) substrate (Louis MO) cultivates for Sigma chemical company, St, from the data of these analyses to represent (face as follows) in the absorption at 405nm place.Cytotoxicity
Toxin A and toxin B mediation are at the cytotoxicity of each cell line, and cytotoxicity is represented by the rounding of fibroblast (IMR-90).The IMR-90 cell is to 10-100pg toxin A and 0.1-1.0pg toxin B sensitivity.Be used for dosage that cytotoxicity suppresses the toxin of experiment and be equivalent to cause 8 times of the required amount of the IMR-90 cell rounding of 50% the monolayer that converges.With serum or secretions suitably dilutes and at 37 ℃, mixed 1 hour with toxin A or toxin B, then toxin is added in the hole of converging of 96 porocyte culture plates and incubated overnight.Use phase contrast microscope to read flat board.Data are represented with the maximum dilution of protecting 50% monolayer to avoid rounding.Bacterin preparation
Prepare toxoid and recombinant toxin A (GST-ARO) as mentioned above.Intestinal toxicity
The ligation intestinal loop that use is attacked with toxin A is analyzed the intestinal toxicity of toxin A.Attack intestinal loop with the toxin A of serum pre-incubation or the secretions that comprises antibody and measure antibody and suppress intestinal toxicity by using.Before using rat is fastened, anesthesia is also got up the intestinal segment ligation.Each intestinal loop contains complete blood vessel and does not have feces.(1-10 μ g) adds in the chamber of each loop with toxin A, carries out and do not carry out pretreatment with immune serum.After 4 hours, remove intestinal loop and the inclusions of weighing.The intestinal loop of direct aggression mice and hamster is to determine at enterotoxication immune effect in a similar manner.In the mice intestinal loop was analyzed, the intestinal loop of PBS being handled volume and toxin A processing in the intestinal loop compared.Data are represented with the mg number of the inclusions of every cm ligation intestinal loop.General is attacked
Mice is used the LD of 10X through the various toxin of intraperitoneal administration 50Attack.Be shown in data among the figure (seeing below) and be the percent of the animal that from attack, survives.Hamster is used 2mg clindamycin and 1 * 10 7Trophophase clostridium difficile biological attack.Result's toxoid intranasal immune mouse
Be with or without 5 μ gCT as mucosal adjuvants, with toxoid (the various toxin of 15 μ g) by the intranasal approach weekly 5 female Switzerland Webster mices of immunity (Taconic FarmsGermantawa NY) be each group of one group.Immunization method is summarised in the table 4.Obtain serum after the immunity, saliva feces and vaginal secretions.After the immunity, in serum, can detect specific IgA and IgG antibody, saliva, feces and can detect specificity IgA antibody (Fig. 6 A and 6B) at toxin A and toxin B at the mucomembranous surface of toxin A and toxin B.No matter whether CT uses with toxoid, and this replys always tangible.The cytotoxicity (Fig. 7 A-7C) that also suppresses toxin A and toxin B from the serum of immune animal.Immune serum is used to the intestinal toxic action (Fig. 8) that passive protection rat intestinal loop is avoided toxin A.Use 10 LD 50Toxin A, and after a week, then use 10LD 50Toxin B attack animal.All immune animals survive from this attack, and matched group does not have (Fig. 9).At last, will be from being attacked with toxin A of immune animal by the colligation intestinal loop, this has directly proved inducing of antibody, this antibody may be mucosa IgA antibody, can suppress the body fluid accumulation and infers and can suppress suffer from diarrhoea (Figure 10).These data show that when being applied to mucomembranous surface the clostridium difficile vaccine causes strong protectiveness mucosal immune response, and it does not need mucosal adjuvants.Table 4, the mucosal immune response after using clostridium difficile toxoids intranasal immune mouse ,+/-cholera toxin (CT); The 0th, 7, carry out immunity in the time of 21,35,49 and 59 days.In the time of the 70th day, attack with toxicity A/B.Obtain sample (serum, feces saliva and vaginal secretions) on the same day.(CT=cholera toxin; The PBS=phosphate buffered saline(PBS)).
Antigen Dosage (μ g) Adjuvant The animal number of elements
Toxoid 15 5μg?CT 5
Toxoid 15 Do not have 5
PBS 0 5μg?CT 5
The EXAMPLE III vaccine combination immune mouse that comprises the GST-ARO fusion rotein
The carboxyl terminal district of Toxin A Toxin A (Clostridium difficile clone seq5) comprises and a series ofly is considered to participate in the bonded repetition aminoacid of saccharide residue unit on toxin and the target cell (referring to for example Luely etc., modern microbiology 21:29-32,1990; Frey etc. infect and immune 60:2488-2492 1992; The list of references of quoting with this paper).The fusion rotein that following structure is made up of the carboxyl terminal district of the clostridium difficile toxin that merges with glutathione s-transferase (GST).Use standard method, separate the Sau 3A fragment (referring to for example Dove etc., see above, be used for the sequence of toxin A gene) of 794 amino acid whose nucleotide of carboxyl terminal that comprise toxin-encoding A.The segmental terminal viscosity of polishing is connected the flat fragment of not holding of tool with the pGEX3X of SmaI digestion.Being cloned in the escherichia coli of plasmid that will comprise the GST-ARU that encodes cultivated, and on glutathion-agarose affinity column purification GST-ARU fusion rotein, with free glutathione eluting from post, and use the standard method dialysis to remove glutathion.
When being with or without CT (5 μ g), with 4 weekly doses, by gastric (IG as mucosal adjuvants; 100 μ g), intranasal (IN; 50 μ g); Or intraperitoneal (IP; 25 μ g) each organizes (n=5) (table 5) to approach with the immune female Switzerland Webster mice of toxin A fusion rotein (GST-ARU).Behind last dosage, obtain sample and be used for immunoassay.All approach induce the good seroimmunity at toxin A to reply.Even when not having CT, intranasal (IN) administration causes mucosa IgA to reply.As if the immunity by the IG approach also is effectively, but strengthen owing to the existence of mucosal adjuvants.Immune induction good systemic and feces by the IP approach are replied, but can not (Figure 11 A-11B) in saliva or vagina sample.Serum antibody does not have obviously to suppress the vitro cytotoxicity of toxin A; But can avoid intestinal toxicity by passive protection rat intestinal loop, this shows that the carboxyl terminal land only needs (Figure 12 A-12B and 13A-13B) in vivo in the toxicity.Avoid dead attack by the animal of IN and the immunity of IP approach is protected, but the IG immune animal there be not (Figure 14).The survivor that general is attacked has also proved exist (Figure 15) of in colligation intestinal loop enterotoxin neutralizing antibody.Even the IN immunity that does not have CT causes producing watching for animals and avoids the lethal effect of toxin A, and toxin A is to the circulating antibody of the intestinal toxic action of intestinal mucosa.Observe some protections by the approach of all tests, but the administration of IN approach be it seems in that to cause mucosa more effective in replying.Table 5.With the mucosal immune response behind reorganization Toxin A Toxin A (Clostridium difficile clone seq5) carboxyl terminal (GST-ARU) immune mouse,, carried out immunity, and used toxin A to attack in 14 and 21 days at the 35th day the 0th, 7.Be 28 to obtain sample (serum, feces, saliva and vaginal secretions).(CT=Xug cholera toxin; The IG=gastric; The IP=intraperitoneal; The PBS=phosphate buffered solution).
Antigen Dosage (μ g) Adjuvant Approach The animal number of elements
GST-ARU 100 CT IG 5
GST-ARU 100 Do not have IG 5
GST-ARU 50 CT IN 5
GST-ARU 50 Do not have IN 5
GST-ARU 25 RIBBI IP 5
GST-ARU 25 Do not have IP 5
PBS 0 CT IN 5

Claims (15)

1. a compositions comprises the non-polypeptide antigen that duplicates, and this antigen is dissolved in the pharmaceutically acceptable diluent, and when giving intranasal administration, can induce the far-end mucosal immune response to gastrointestinal or reproduction urethra pathogen in mammal.
2. the compositions of claim 1, wherein said far-end mucosal immune response is in gastrointestinal tract.
3. the compositions of claim 1, wherein said far-end mucosal immune response is in the reproduction urethra.
4. the compositions of claim 1, wherein said pathogen cause diarrhoea.
5. the compositions of claim 4, wherein said pathogen is from fusobacterium.
6. the compositions of claim 5, wherein said pathogen is a clostridium difficile.
7. the compositions of claim 1, wherein said antigen comprises toxin or its immunogenic fragments or the derivant of described pathogen.
8. the compositions of claim 7, wherein said antigen comprises Toxin A Toxin A (Clostridium difficile clone seq5), or its immunogenic fragments or derivant.
9. the compositions of claim 7, wherein said antigen comprises Clostridium difficile toxin B or its immunogenic fragments or derivant.
10. the compositions of claim 8, wherein said antigen comprises Toxin A Toxin A (Clostridium difficile clone seq5) and Clostridium difficile toxin B.
11. the compositions of claim 1, wherein said antigen comprises toxoid.
12. the compositions of claim 11, wherein said toxoid are clostridium difficile toxoids.
13. induce method to the far-end mucosal immune response of pathogen mammal for one kind, described method comprises step:
A. use a kind of antigen that can induce at the described immunne response of described mammal mucomembranous surface; With
B. to described mammal through the described antigen of parenteral.
14. the method for claim 13, wherein said mammal is in appearance, but does not suffer from the diarrheal critical days that is caused by described pathogen.
15. the method for claim 14, wherein said antigen are clostridium difficile toxoids.
CN96196763A 1995-07-07 1996-06-26 Intranasal vaccination against gastrointestinal disease Pending CN1195993A (en)

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