CN101443454A - Polysaccharide-protein latticeing substance and vaccine derivatized from polyvalent meningitis cocci - Google Patents

Polysaccharide-protein latticeing substance and vaccine derivatized from polyvalent meningitis cocci Download PDF

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CN101443454A
CN101443454A CNA2005800486574A CN200580048657A CN101443454A CN 101443454 A CN101443454 A CN 101443454A CN A2005800486574 A CNA2005800486574 A CN A2005800486574A CN 200580048657 A CN200580048657 A CN 200580048657A CN 101443454 A CN101443454 A CN 101443454A
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sba
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serogroups
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R·P·赖亚尔
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Sanofi Pasteur 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
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]

Abstract

The present invention describes derivatized polysaccharide-protein conjugates, a composition comprising one or more of such derivatized polysaccharide-protein conjugates and methods of immunizing human patients with the same. The derivatized polysaccharide-protein conjugates are purified capsular polysaccharides from Neisseria meningitidis serogroups A, C, W-135, and/or Y, derivatized chemically activated and selectively attached to a carrier protein by means of a covalent chemical bond, forming polysaccharide-protein conjugates capable of eliciting long-lasting immunity to a variety of N. meningitidis strains.

Description

The polysaccharide-protein conjugate of derivatized from polyvalent meningitis cocci and vaccine
Invention field
Present invention relates in general to medical field, and more specifically relate to microbiology, immunology, vaccine and infect by immunization prevention bacterial pathogen.
Background of invention
Neisseria meningitidis (Neisseria meningitidis) is bacterial meningitis and a pyemic major cause all over the world.The sickness rate of region meningococcal disease is 1-5/100 in developed country during nearest 30 years, 000, and be 10-25/100 in developing country, 000 (Reido, F.X. wait people J.Ped.Infect.Dis., 14, the 643-657 pages or leaves [1995]).At epidemic period, the sickness rate of meningococcal disease is near 1000/1000,000.There is about 2,600 routine bacterial meningitises every year in the U.S., and is average 330,000 examples in developing country.Case fatality rate is 10-20%.
Pathogenic meningococcus is by the polysaccharide pod membrane bag quilt that is attached to biological outer membrane face.13 kinds of different meningococcus serogroupss (Frasch, C.E. wait people Rev.Infect.Dis., 7, the 504-510 pages or leaves [1985]) have been differentiated based on the immunology specificity of capsular polysaccharide.In these 13 kinds of serogroupss, 5 kinds cause most of meningococcal diseases; These comprise serogroups A, B, C, W135 and Y.Serogroups A is responsible for maximum prevailing disease.Serogroup B, C and Y cause that most of prevailing disease and part break out.
People's nose-mucous membrane of oropharynx is the natural reservoir host of unique known Neisseria meningitidis.Build the group and occur in the outside surface of mucomembranous cell and the last subcutis of nasopharynx.Meningococcal carrying can continue the several months.Meningococcal propagation is by directly contacting or taking place via the air spittle.Meningococcus by through mucous epithelium via phagocytic vacuole as the result of the endocytosis aggressive that become.The meningococcal host defense of aggressive depends on the bacteriolysis of complement-mediated.The serum antibody major part of the bacteriolysis of responsible complement-mediated is at outer capsular polysaccharide.
Vaccine based on meningococcal polysacharide has obtained describing, and it causes the immunne response at capsular polysaccharide.These antibody can carry out the meningococcal bacteriolysis of serogroups specificity of complement-mediated.Meningococcal polysaccharide vaccine is presented among children and the grownup that effectively (Peltola, H. wait people New Engl.J.Med., 297, the 686-691 pages or leaves [1997]; And Artenstein, M.S. waits people New Engl.J.Med., 282, the 417-420 pages or leaves [1970]), but in baby and young child, render a service limited (Reingold, A.L. wait people Lancet, 2, the 114-118 pages or leaves [1985]).The polysaccharide of subsequent dose causes weak or does not have booster response (Goldschneider, I. wait people J.Infect.Diseases in younger colony, 128, the 769-776 page or leaf [1973] and Gold, R., Deng people J.Infect.Diseases, 136, S31-S35[1977]).The time length of the protection that meningococcal polysaccharide vaccine causes is not persistent, and has estimated to be in grownup and the children more than 4 years old 3-5 (Brandt, B.L. and Artenstein; M.S., J.Infect.Diseases, 131; S69-S72 page or leaf [1975], Kyhty, H.; Deng people J.Infect.Diseases, 142, the 861-868 pages or leaves [1980]; and Cessey, S.J. waits people J.Infect.Diseases; 167, the 1212-1216 pages or leaves [1993]).For the 1-4 children in year, the time length of protection is less than 3 years (Reingold, A.L. wait people Lancet, 2, the 114-118 pages or leaves [1985]).
Polysaccharide can not be in conjunction with main histocompatibility complex molecule, and this is that antigen presentation is arrived the auxiliary lymphocyte of T and stimulated the auxiliary lymphocytic prerequisite of T, that is, they are not dependence antigens of T cell.Polysaccharide can need not T auxiliary lymphocytic help stimulation bone-marrow-derived lymphocyte and produce antibody.As the T of bone-marrow-derived lymphocyte dependent stimulation result not, lack memory by these antigen immunes inoculation backs and induce.Polysaccharide antigen can in the grownup, cause very effective T not dependency reply, but these T not dependency to reply in the jejune immunity system of baby and young child be faint.
By polysaccharide covalent is attached to protein molecule (" carrier " or " carrier proteins ") can with T not dependency polysaccharide antigen antigenic shift be the T dependence antigen.Can be by the special helper cell of peptide be activated in conjunction with the B cell of the polysaccharide fraction of conjugate vaccine, described peptide is the part of the carrier proteins puted together.Produce at the auxiliary antibody that is used to strengthen of replying of the T of carrier proteins at polysaccharide.
The serogroup B polysaccharide has been presented in the people colony weak to non-immunogenicity (Wyle, F.A. wait the people, J.Infect.Diseases, 126, the 514-522 pages or leaves [1972]).The immunne response of this serogroups polysaccharide and the not obvious change laboratory animal of proteinic chemical attachment (Jennings, H.J. and Lugowski, C.J., Immuno1., 127, the 1011-1018 pages or leaves [1981]).Think that shortage is because serogroup B polysaccharide and how sialylated host's glycoprotein, for example structural similarity between the N-CAM at the reason of the immunne response of this serogroups polysaccharide.
Meningococcal conjugate vaccine based on the serogroup C polysaccharide has obtained describing.This univalent vaccine causes the powerful antibody response at capsular polysaccharide, and described capsular polysaccharide is presented in the Neisseria meningitidis strain corresponding to serogroup C.This type of vaccine is uniquely can protect the vaccine that is not subjected to the bacterial disease of serogroup C.
In young child, use limited and lasting protection is not provided in the grownup based on the existing vaccine of meningococcal polysacharide.Shown and can all colonies in being in meningococcal infection danger comprise the unique meningococcus vaccine that causes lasting protection among the children; based on polysaccharide, and do not provide the protection of infecting at other serogroupss from the single serogroups of Neisseria meningitidis.Therefore, need a kind of meningococcal conjugate vaccine, it can give to protect at the children and the extensive, lasting of the meningococcal disease among the grownup that are in the meningococcal infection danger.Multivalent meningococcal polysaccharide of the present invention solves this needs by vaccine preparation is provided, and the immunogenicity polysaccharide from the main pathogenic serogroups of Neisseria meningitidis in described vaccine preparation changes the T dependence antigen into by puting together with carrier proteins.
Measure (SBA-BR) about the FDA license awarding of meningococcal polysaccharide vaccine based on the sterilization of the use children rabbit complement that those the blood sample with licensed-in vaccine immunity is carried out.Many governments and expert group have announced about measure the present requirement and the suggestion of assessment meningococcal polysaccharide vaccine at this type of.
It is the good related (Goldschneider of bactericidin level that has shown and measured by serum sterilizing the complement-mediated of (SBA) detection because of the people's immunity at meningococcal disease that license awarding is allowed, I., Deng the people, J.Exp.Med., 129:1307-1326[1969] and Goldschneider, I. waits the people, J.Exp.Med., 129:1327-1348[1969]).The definite alternative level of tiring of end user's complement (SBA-H) in mensuration at the 1:4SBA of serogroup C.Yet, for the prerequisite of the allowance of meningococcal polysaccharide vaccine based on using young rabbit complement (SBA-BR) to induce (WorldHealth Organization.1976.Requirements for meningococcalpolysaccharide vaccine.World Health Organization technical report sequence number 594.World Health Organization as what the serum sterilizing in complement source in measuring was replied, Geneva, Switzerland (WHO 1976).According to this suggestion, the antibody titer of serum from least 90% experimenter of inoculation meningococcal polysaccharide vaccine should show 4 times or bigger rising when 2-4 week is at following target bacterial strain or equivalent strains test after immunity: for the A1 of serogroups A, for the C11 of serogroup C, for the S-1975 of serogroups Y, and for the S-4383 of serogroups W-135 (WHO 1976, WHO 1981, Bureau of Biologics, Food and Drug Administration July 17,1985).BB (Bureau of Biologics) has adopted the suggestion of WHO, and combination group A and C, and the meningococcal polysaccharide vaccine of combination group A, C, Y and W-135 is allowed based on this requirement in the U.S..In order to promote by comparing between the laboratory of meningococcus vaccine inductive fungicidal activity, set up stdn SBA (SBA-BR) (the Maslanka S.E. that uses young rabbit complement by many laboratory studyes, Deng the people, Clin.Diagn.Lab.Immunol., 4:156-167 (1997).
Because the data from meningococcal conjugate C vaccine begin to become and can obtain, so begin to appear in the mensuration worry that the high SBA that uses the rabbit complement to lead to errors tires.After in March, 1999 meeting clarification and the solution problem relevant with experimental determination, described experimental determination is used for the meningococcus serogroups A and the C specific antibody of analyst's serum, WHO uses the SBA of young rabbit complement can be used for antibody response (The World Health Organization.1999.Standardization and validation of serological assays for the evaluation ofimmune responses to Neisseria meningitidis serogroup A/C vaccines.Geneva, the WHO/V﹠amp of metering needle to serogroup C about the Committee of Experts (the WHO Expert Committee onBiological Standardization) suggestion of biological standardization; B/99.19 (WHO 1999)).In the effort of the protection of avoiding too high estimated service life children rabbit complement, the WHO suggestion should be taked to study to make and measure the tire SBA that measures with end user's complement of the threshold value of measuring by the SBA that uses young rabbit complement and tire and associate.The result who has held meeting subsequently and presented supports common conclusions: use young rabbit complement＜SBA of 1:8 tire relevant with the protection that lacks at serogroup C, and use young rabbit complement=SBA of 1:128 tires and tires good relatedly with the protectiveness SBA of the 1:4 of end user's complement.Do not provide about other meningococcus serogroupss for example A, Y or W-135 or the information of tiring about the respective associated SBA-BR of polysaccharide conjugates.
Use the SBA of the 1:8-1:64 of young rabbit complement tire needn't with the protectiveness SBA of the 1:4 of end user's complement tire good related (Jodar, L. wait the people, Biologicals, 30:323-329[2002]).WHO Committee of Experts suggestion, the SBA-BR of 1:8,1:16,1:32 and 1:64 end user's complement reevaluating of tiring after the vaccination.The SBA-BR that solves 1:8,1:16,1:32 and the 1:64 4 times of risings that probabilistic other measures comprise before the assessment vaccination and back antibody SBA tires of tiring.Also provide confirmation, yet the Committee of Experts recognizes that the data available about these surrogates is insufficient or limited as the related memory of protection.
It is better mark at people's immunity of meningococcal disease that the SBA-BR that is higher than 1:8 tires, from before the immunity after the immunity the about 45 days immunity back time period SBA-BR of about 15-tire 4 times of risings or higher also be like this.
In one embodiment, the invention provides method with multivalent meningococcal polysaccharide conjugates composition immunity human patients, wherein the serum SBA-BR of human patients tires and is 1:16 or higher, preferred 1:32 or higher, and more preferably 1:64 or higher, and more preferably 1:128 or higher again.More further in the embodiment, the invention provides method with meningococcus polysaccharide conjugates composition immunity human patients, wherein said human patients before vaccination and back antibody SBA tire and raise 4 times or higher.
In an embodiment again, the invention provides by using multivalent meningococcal polysaccharide conjugates composition immunity human patients, provide method at the immunity of multiple Neisseria meningitidis serogroups, wherein said composition to comprise to human patients and be selected from Neisseria meningitidis serogroups A and W-135; Y and W-135; C and Y; C and W-135; A, C and Y; A, C and W-135; C, Y and W-135; A, Y and W-135; And two or more polysaccharide of A, C, Y and W-135.
More further in the embodiment, the invention provides by using multivalent meningococcal (purifying) polysaccharide conjugates composition immunity human patients, provide method to human patients at the immunity of multiple Neisseria meningitidis serogroups, wherein said polysaccharide derivatize is extremely less than 100,000 dalton.In one embodiment of the invention, the polysaccharide depolymerization of purifying is about 5,000-about 75,000 daltonian average polysaccharide sizes; Preferably, about 7,000-about 50,000 daltonian average polysaccharide sizes; More preferably, about 8,000-about 35,000 daltonian average polysaccharide sizes; Again more preferably, about 12,000-about 25,000 daltonian average polysaccharide sizes.In one embodiment of the invention, the average polysaccharide size in the composition is about 15, about 22,000 dalton of 000-.
Summary of the invention
The invention provides by the immune composition of using meningococcal polysacharide-protein conjugate method at the human immunity of the meningococcal disease that is caused by pathogenic Neisseria meningitidis is provided.In one embodiment of the invention, immune composition comprises two or more protein-polysaccharide conjugates, and wherein conjugate comprises the capsular polysaccharide of puting together from Neisseria meningitidis, with carrier proteins separately.In preferred embodiments, immune composition comprises the protein-polysaccharide conjugate that two or more are different, and wherein conjugate comprises the capsular polysaccharide of puting together from different Neisseria meningitidis serogroupss, with carrier proteins separately.
The method that provides at the human immunity of the meningococcal disease that is caused by pathogenic Neisseria meningitidis is provided, it comprises uses the immune composition that comprises two or more different proteins-polysaccharide conjugates, and wherein conjugate comprises the capsular polysaccharide of puting together from different Neisseria meningitidis serogroupss, with carrier proteins separately.
The method that provides at the human immunity of the meningococcal disease that is caused by pathogenic Neisseria meningitidis is provided, and it comprises uses meningococcal polysacharide-protein conjugate.The invention provides the multivalent meningococcal vaccine, it comprises the different protein-polysaccharide conjugate of 2-4 kind of immune significant quantity, each self-contained different capsular polysaccharide of puting together with carrier proteins of conjugate wherein, and wherein every kind of capsular polysaccharide is selected from capsular polysaccharide from serogroups A, C, W-135 and Y.The present invention further provides the method for inducing at the immunne response of Neisseria meningitidis capsular polysaccharide, it comprises the immune composition of the present invention of using immune significant quantity to the people.In one embodiment, the multivalent meningococcal vaccine comprises the protein-polysaccharide conjugate that 2 kinds of immune significant quantity are different, each self-contained different capsular polysaccharide of puting together with carrier proteins of conjugate wherein, and wherein every kind of capsular polysaccharide is selected from the capsular polysaccharide from serogroups A, C, W-135 and Y, more preferably, comprise capsular polysaccharide A and W-135, A and Y, C and W-135, C and Y and W-135 and Y.In one embodiment, the multivalent meningococcal vaccine comprises the protein-polysaccharide conjugate that 3 kinds of immune significant quantity are different, each self-contained different capsular polysaccharide of puting together with carrier proteins of conjugate wherein, and wherein every kind of capsular polysaccharide is selected from the capsular polysaccharide from serogroups A, C, W-135 and Y, more preferably, comprises capsular polysaccharide A, C and W-135, A, C and Y, C, Y and W-135, C, W-135 and Y, and A, W-135 and Y.In another embodiment, the multivalent meningococcal vaccine comprises the protein-polysaccharide conjugate that 4 kinds of immune significant quantity are different, wherein conjugate comprises the different capsular polysaccharide of puting together with carrier proteins separately, and wherein every kind of capsular polysaccharide is selected from capsular polysaccharide from serogroups A, C, W-135 and Y.
The present invention further provides the method for inducing at the immunne response of Neisseria meningitidis capsular polysaccharide, it comprises the immune composition of the present invention of using immune significant quantity to the human or animal.
The invention provides the multivalent meningococcal vaccine, it comprises the different protein-polysaccharide conjugate of 2-4 kind of immune significant quantity, wherein conjugate comprises the different capsular polysaccharide of puting together with carrier proteins separately, and wherein every kind of capsular polysaccharide is selected from capsular polysaccharide from serogroups A, C, W-135 and Y.
The invention provides the method that protection is infected the human or animal of susceptible to Neisseria meningitidis, it comprises the vaccine of the present invention of using immune effective dose to the human or animal.
More further in the embodiment, the invention provides and strengthen the method for replying that causes behind meningococcus vaccine such as first dose, second dose, the 3rd dose or the immunogenic composition.In some such embodiment, first dose of (or multi-agent) meningococcus vaccine or immunogenic composition comprise the capsular polysaccharide from one or more Neisseria meningitidis serogroupss (serogroups A, B, C, Y, W-135 etc.).In certain other embodiments, first dose of (or multi-agent) meningococcus vaccine or immunogenic composition comprise the capsular polysaccharide of puting together from one or more Neisseria meningitidis serogroupss (serogroups A, B, C, Y, W-135 etc.), with one or more carriers (for example carrier proteins).In preferred embodiments, carrier proteins is immunogenic and/or other treatment or other interests is provided.In some preferred embodiment, behind the meningococcus vaccine of potion or multi-agent or immunogenic composition the experimenter (for example, the people) primary response that causes in is strengthened by the vaccine or the immunogenic composition of potion or multi-agent subsequently, and described vaccine or immunogenic composition comprise from 1,2,3,4 or the capsular polysaccharide of more kinds of different Neisseria meningitidis serogroupss.Yet the invention is not restricted to use the vaccine that comprises unconjugated capsular polysaccharide or the immunogenic composition of one or many initial dose, the present invention does not expect to be limited to yet and uses one or many and comprise the capsular polysaccharide-carrier protein immunogenic composition puted together or the booster dose of vaccine.Similarly, the present invention does not expect and is limited by the timed interval of first or booster dose.
In certain embodiments, this example provides the method for using disclosed tetravalence (A, C, Y and W-135) meningococcus diphtheria toxoid conjugate composition (being formulated as for example people's vaccine) in the experimenter, described experimenter had before accepted the unit price meningococcal conjugate vaccine composition (for example, serotype A, B, C, Y and/or W-135) of potion or multi-agent.Present method is not expected and is accepted unit price meningococcal conjugate vaccine or the restriction of subject age during the disclosed tetravalence immune composition of potion or multi-agent subsequently at it.For example, in certain embodiments, the experimenter who is fit to for this application process and scheme includes, but not limited to baby, the child who has just learnt to walk, children, teenager, 13 to 19 years old teenager and grownup.In some preferred embodiment, the experimenter who is fit to comprises about 1 month-55 years old, or about 12 months-20 years old.In particularly preferred embodiments, the experimenter of Shi Heing comprised 24 months-5 years old.
The present invention provides the method that is used for using immunogenic composition to the experimenter in some preferred embodiment, it comprises, provides: experimenter, wherein said experimenter have inoculated unit price meningococcal conjugate vaccine dose; Tetravalence meningococcal polysacharide protein conjugate composition; With inoculate behind the described unit price meningococcal conjugate vaccine at least 12 months described experimenter, use described tetravalence meningococcal polysacharide protein conjugate composition for described experimenter.
In other embodiments, the invention provides the method that is used for using immunogenic composition to the experimenter, it comprises, provides: experimenter, wherein said experimenter have inoculated unit price meningococcal conjugate vaccine dose; Tetravalence meningococcal polysacharide protein conjugate vaccine; With inoculate behind the described unit price meningococcal conjugate vaccine at least 12 months described experimenter, use described tetravalence meningococcal polysacharide protein conjugate vaccine for described experimenter.
Other embodiments provide the method that is used for using immunogenic composition to the experimenter again, and it comprises, provides: experimenter, wherein said experimenter inoculating needle to the protective antigen of Neisseria meningitidis serogroup C; Tetravalence meningococcal polysacharide protein conjugate vaccine; With inoculate behind the described unit price meningococcal conjugate vaccine at least 12 months described experimenter, use described tetravalence meningococcal polysacharide protein conjugate vaccine for described experimenter.
Other embodiments of the present invention provide the method that is used for using immunogenic composition to the experimenter, and it comprises, provides: experimenter, wherein said experimenter have inoculated unit price Neisseria meningitidis serogroup C meningococcus vaccine; Tetravalence meningococcal polysacharide protein conjugate composition; With inoculate behind the described unit price meningococcal conjugate vaccine at least 12 months described experimenter, use described tetravalence meningococcal polysacharide protein conjugate vaccine for described experimenter.
An embodiment provides the method for meningococcal disease among the prevention experimenter again, it is included in described experimenter and has inoculated behind the described unit price meningococcal conjugate vaccine about 12 months, gives the tetravalence meningococcal conjugate vaccine of effective dose on described experimenter's administering therapeutic.
Except the composition and method described, the present invention further provides the method for meningococcal disease among the prevention experimenter, it is included in described experimenter and has inoculated behind the unit price Neisseria meningitidis serogroup C meningococcus vaccine about 1-120 month, gives the tetravalence meningococcal conjugate vaccine of effective dose on described experimenter's administering therapeutic.
The further again embodiment of the present invention provides the method at experimenter's moderate stimulation and/or induce immune response, it is included in described experimenter and (has for example inoculated at least potion unit price meningococcal conjugate vaccine, one or more serogroups A, C, B, Y and/or W-135) after at least 12 months, give the tetravalence meningococcal conjugate vaccine of effective dose on described experimenter's administering therapeutic.In certain embodiments; the experimenter who is fit to (for example; preferred mammal; and more preferably people) accepted the vaccine that one or more unit price polysaccharide-proteins put together or the concomitant administration of the unconjugated vaccine of polysaccharide, described vaccine comprises the protective antigen of the disease that causes at Neisseria meningitidis or by Neisseria meningitidis.
In certain embodiments, the present composition comprises the tetravalence meningococcal conjugate vaccine of the aseptic 0.5ml liquid dosages that is formulated as in acceptable application device, and described application device is syringe (for example single use), intranasal injection, spraying gun, atomizer, transcutaneous device etc. for example.
Various embodiments of the present invention provide the composition of about 0.5ml multivalence (for example tetravalence) meningococcal conjugate vaccine, and it comprises the capsular polysaccharide from the about 10 μ g purifying of about 0.01 μ g-of one or more Neisseria meningitidis serogroupss capsular polysaccharide of the purifying of one or more serogroups A, C, Y or W-135 (for example from).
Other embodiments of the present invention provide the combination-vaccine composition, and it comprises 1,2,3 or 4 kind of different meningococcal protein matter-polysaccharide conjugates and from one or more antigens or the immunogenicity component of other vaccine compositions (for example licensed-in vaccine).In other embodiment again, the invention provides immunogenic composition, it comprises 1,2,3 or 4 kind of different protein-polysaccharide conjugate and from one or more antigens or the antigen component of vaccine (for example licensed-in vaccine) composition.The present invention's expection, but be not limited to, the Neisseria meningitidis protein-polysaccharide conjugate that the 1-4 kind is different and from the combination of the immunogenicity component (for example antigen) of vaccine, described vaccine point to prevention or improve the effect of the disease that Neisseria meningitidis causes of can't help.For example, in certain embodiments, the invention provides immunogenic composition, it comprises 1,2,3 or 4 kind of different protein-polysaccharide conjugate and from one or more components of the licensed-in vaccine that generally is applied to the traveller, described vaccine comprises, but be not limited to, at typhoid fever (Typhim for example
Figure A200580048657D0012163943QIETU
), yellow jack (YF-for example
Figure A200580048657D0012163952QIETU
) vaccine, Poliomyelitis Vaccine etc.For example, in other embodiment again, the invention provides the different Neisseria meningitidis protein-polysaccharide conjugate of 1-4 kind and from the combination of the immunogenic composition of vaccine, described vaccine points to prevention or improves hepatitis (first type for example, B-mode, third type, fourth type or penta type), smallpox and cowpox, AIDS, tuberculosis, diphtheria, Haemophilus influenzae, measles, parotitis, Whooping cough, the streptococcus pneumoniae disease, poliomyelitis, rubella, tetanus, varicella, the adenovirus disease, anthrax, cholera, encephalitis (Japanese encephalitis for example, tick encephalitis etc.), the plague, rabies, typhoid fever, bleb, malaria, parasitosis, poxvirus disorders, the respiratory syncytial virus disease, rotavirus, the α virus infection (for example, Venezuela, west or eastern equine encephalitis, the chikungunya virus disease, ross river virus disease etc.), cloth Buddhist nun virus disease (for example, Rift valley fever, the Hantaan virus disease), the arenavirus disease (for example, Junin virus (Junin virus) disease), Rickettsiae disease (for example Q heat), tularemia, gibraltar fever, the pseudomonas disease, sausage poisoning, Xi Niluo disease and staphylococcal infections, and other are based on virus, the effect of bacterium and parasitic disease.In preferred embodiments, derive from licensed-in vaccine with the non-Neisseria meningitidis immunogenic composition that different Neisseria meningitidis protein-polysaccharide conjugates of the present invention are used in conjunction with (for example, following).
What the invention is not restricted to vaccine or immunogenic composition anyly specifically follows combination, also is not limited to use any this type of to follow the method for combination.In fact, the present invention also provides the different Neisseria meningitidis protein-polysaccharide conjugate of 1-4 kind and from the combination of the immunogenicity component that does not obtain permission (for example experimental) non-Neisseria meningitidis vaccines or immunogenic composition.
The Neisseria meningitidis protein-polysaccharide conjugate that the 1-4 kind is different and can follow from the using of combination of the immunogenicity component of non-Neisseria meningitidis vaccines or immunogenic composition.In certain embodiments, the Neisseria meningitidis protein-polysaccharide conjugate that the 1-4 kind is different and from the concomitant administration of the immunogenicity component of non-Neisseria meningitidis vaccines or immunogenic composition uses similar or different application processes to finish at 2 or more a plurality of position.In other these embodiments again, the Neisseria meningitidis protein-polysaccharide conjugate that the 1-4 kind is different and from the concomitant administration of the immunogenicity component of non-Neisseria meningitidis vaccines or immunogenic composition, by two or more composition physical combination being realized in single gathering composition described single gathering composition uses one or more route of administration to use at one or more positions subsequently.
The Neisseria meningitidis protein-polysaccharide conjugate that the 1-4 kind is different and from the using of the combination of the immunogenicity component of non-Neisseria meningitidis vaccines or immunogenic composition carried out on 2 different times can separating on by second, minute, hour, day equal time.The present invention does not expect that the approach of the incident of being applied and/or selection of time limit.
In other embodiment again, (for example the invention provides to the experimenter, the people) (for example uses one or more immune compositions, people's vaccine) method, thus make kind of the immune composition of winning make the experimenter prepare booster action after second kind of (or more kinds of) immune composition used in advance.In some such embodiment, first kind, second kind, the third or more kinds of immune composition comprise protein.The protein carrier that is fit to includes, but not limited to diphtheria toxin, diphtheria toxoid, CRM 197, Toxoid,tetanus, pertussis toxin, intestinal bacteria (E.coli) LT, intestinal bacteria ST, exotoxin A, outer membrane complex c (OMPC), porin, transferrin bindin, pneumolysis, streptococcus pneumoniae surface protein A (PspA), streptococcus pneumoniae adhesion protein (PsaA), ovalbumin, keyhole limpet hemocyanin (keyhole limpithemocyanin) (KLH), the tuberculin protein derivatives (PPD) of bovine serum albumin(BSA) (BSA) or purifying etc.In preferred embodiments, carrier proteins comprises diphtheria toxin, diphtheria toxoid, CRM 197, Toxoid,tetanus, exotoxin A or outer membrane complex c (OMPC).In an especially preferred embodiment, carrier proteins comprises diphtheria toxin or diphtheria toxoid.
Other embodiments provide at least a composition of the present invention's expection in the purposes for the treatment of, prevent or weakening in the mammalian diseases state.In some such embodiment, morbid state causes by infectation of bacteria, for example, and the infection that causes by one or more Neisseria meningitidis serogroupss.
Other embodiment provides the purposes that at least a composition manufacturing of the present invention's expection is used for the treatment of the medicine of application again, described treatment application examples as treatment, prevent or weaken in the Mammals by one or more Neisseria meningitidis serogroupss to as described in the morbid state that causes of mammiferous infectation of bacteria.
In other embodiment again, composition of the present invention at test kit (for example, commercial package) provides in, with the specification sheets of using and/or storing about test kit content (for example ampoule, syringe, bottle, patch, suppository, ointment etc. comprise meningococcus vaccine or meningococcus vaccine component).As used herein, term " about use the specification sheets of described immune composition (or people's vaccine) to the experimenter " comprises about using the specification sheets of the meningococcal infection disease that the composition prevention that comprises in the test kit causes by Neisseria meningitidis.In certain embodiments, specification sheets further comprises according to 21C.F.R. § 201 and following or the like, and/or those of other U.S. that are suitable for or international managing body, to the recommendation or the statement of dosage usually of the composition that comprises in the test kit.About the other information of label and be applicable to the inventive method and the explanation of composition requires can obtain on the internet webpage of U.S.F.D.A.
As used herein, term " third party " refers to be engaged in sale, puts in storage, distributes perhaps promise to sell any entity of the present composition or method.
All patents that this paper quotes, patent application and other publication integral body are incorporated herein by reference, except the invention of they and the disclosure conflicts.
The accompanying drawing summary
Fig. 1-8 has shown certain embodiments of the present invention.
Detailed Description Of The Invention
The present invention includes the immune composition of two or more different protein-polysaccharide conjugates, wherein conjugate comprises the capsular polysaccharide of puting together with carrier protein separately. Therefore, the present invention includes the composition of the capsular polysaccharide that comprises two or more different derivatizations, described capsular polysaccharide and one or more carrier proteins are puted together.
Capsular polysaccharide can prepare by standard technique well known by persons skilled in the art. In the present invention, preferably prepare capsular polysaccharide from Neisseria meningitidis serogroups A, C, W-135 and Y.
In a preferred embodiment, these meningococcus sero-group conjugates are prepared and are formulated in the single dose preparation by the method for separating. For example, the capsular polysaccharide from Neisseria meningitidis serogroups A, C, W-135 and Y is to separate purifying.
In a preferred embodiment of the invention, the polysaccharide of purifying is also activating of depolymerization before puting together with carrier protein. In a preferred embodiment of the invention, the capsular polysaccharide from Neisseria meningitidis serogroups A, C, W-135 and Y is to use gentle oxidizing condition to carry out the part depolymerization.
Natural meningococcal polysacharide is about 500,000-1,500,000 dalton. The present invention relates to the less meningococcal polysacharide of size. When the purifying natural polysaccharide, the polysaccharide of particular percentile will have less size. Yet, in order to obtain better yield, general preferred with natural meningococcal polysacharide depolymerization or derivatization to the preferred size scope, preferably be less than 100,000 dalton. In one embodiment of the invention, the polysaccharide with purifying depolymerizes to about 5,000-Yue 75,000 daltonian average polysaccharide sizes; Preferably, about 7,000-about 50,000 daltonian average polysaccharide sizes; More preferably, about 8,000-about 35,000 daltonian average polysaccharide sizes; Even more preferably, about 12,000-about 25,000 daltonian average polysaccharide sizes. In one embodiment of the invention, the average polysaccharide size in the composition is about 15,000-Yue 22,000 dalton.
The depolymerization of polysaccharide or part depolymerization can be followed activation step subsequently. " activation " refer to the chemical treatment polysaccharide with provide can with the chemical group of carrier protein reaction. Preferred activation method relates under pH5.0.+-.0.1 to be processed about 2 hours in 15-30 ℃ with the adipic dihydrazide (dihyrazide) that is dissolved in physiological saline. A kind of activation method is at U.S. Patent number 5,965, obtains describing in 714.
In case activation, capsular polysaccharide can be puted together with one or more carrier proteins subsequently. In a preferred embodiment of the invention, every kind of capsular polysaccharide is puted together separately with single carrier protein kind respectively. In preferred embodiments, put together with identical carrier protein kind respectively separately from the capsular polysaccharide of Neisseria meningitidis serogroups A, C, W-135 and Y.
Carrier protein can comprise bacteriotoxin, diphtheria toxin for example, the bacteriotoxin of deactivation, for example diphtheria toxoid, CRM197, tetanus toxoid, DT-Pa, E.coli LT, Escherichia coli ST and from the exotoxin A of pseudomonas aeruginosa (Pseudomonas aeruginosa). Also can use bacterial outer membrane protein, for example outer membrane complex c (OMPC), PFP, transferrin bindin, pneumonolysis, Pneumococal surface protein A (PspA) or pneumococcus adhesion protein (PsaA). Other protein, for example the PPD derivative (PPD) of ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purifying also can be used as carrier protein. The preferred nontoxic and non-reactionogenicity of carrier protein and can obtain the protein of q.s and purity. Carrier protein can be complied with standard and put together operation. In a preferred embodiment of the invention, from corynebacterium diphtheriae (Corynebacteria diphtheriae) culture purifying and the diphtheria toxin that uses formaldehyde chemistry detoxifcation as carrier protein. Alternative carrier protein is D albumen, and it is the protein that the Hemophilus influenzae outer membrane face exposes.
In one embodiment of the invention, the average ratio of the polysaccharide of every kind of derivatization and carrier protein is the about 1:20 of about 1:1-(w/w). In a preferred embodiment of the invention, the polysaccharide of total derivatization and the average ratio of carrier protein are the about 1:10 of about 1:2-(w/w), and the polysaccharide of every kind of derivatization and carrier protein in addition preferred average ratio be the about 1:6 of about 1:2-(w/w). In preferred embodiment of the present invention, the polysaccharide of total derivatization and the average ratio of carrier protein are about 1:(4 ± 1); More preferably, 1:(4 ± 0.5); Even more preferably, 1:(4 ± 0.25) (w/w).
After capsular polysaccharide and carrier protein were puted together, polysaccharide-protein conjugate can carry out purifying (amount about polysaccharide-protein conjugate is concentrated) by various technology. A target of purification step is to remove unconjugated polysaccharide from polysaccharide-protein conjugate. A kind of method for purifying is described in U.S. Patent number 6,146, and in 902, it relates to ultrafiltration in the presence of ammonium sulfate. Alternately, conjugate can carry out purifying from unreacted protein and polysaccharide by the standard technique of any number, and described standard technique comprises that especially, size exclusion chromatogram, density gradient centrifugation, hydrophobic interaction chromatography or ammonium sulfate classification separate. Referring to, for example P.W. Anderson waits people (1986) .J.Immunol.137:1181-1186. Also referring to H.J. Jennings and C.Lugowski (1981) J.Immunol.127:1011-1018.
After polysaccharide and carrier protein are puted together, make up to prepare immune composition of the present invention by the polysaccharide-protein conjugate with various derivatizations. Immune composition of the present invention comprises two or more different capsular polysaccharides of puting together from one or more carrier proteins. The preferred embodiments of the invention are divalence immune compositions, and it comprises the capsular polysaccharide from Neisseria meningitidis serogroups A and C, the derivatization puted together with diphtheria toxin or toxoid respectively. More preferably, the present invention is the tetravalence immune composition, and it comprises from Neisseria meningitidis serogroups A, C, W-135 and Y, the capsular polysaccharide of puting together with diphtheria toxin or toxoid respectively.
The present invention partly relates to the polysaccharide conjugates composition of multicomponent, derivatization, and the polysaccharide of the every kind of derivatization that wherein exists is the about 15 μ g/ agent of about 0.5-. Therefore, composition can comprise the polysaccharide of the total derivatization of 1 μ g-60 μ g. In preferred embodiments, the relative quantity of the polysaccharide of every kind of derivatization is interior equal approximately ± 50% in the composition; More preferably, in ± 30%; Even more preferably, in ± 20%.
The preparation of carrier protein and purposes, and various potential conjugation methods are well-known in the art. The information that obtains easily in the instruction that comprises among use the present invention and the general document can prepare conjugate of the present invention by this type of technical staff. Also can obtain to instruct from any one or all following United States Patent (USP)s, the instruction integral body of described patent is incorporated herein by reference: U.S. Patent number 4,356,170; 4,619,828; 5,153,312; 5,422,427 and 5,445,817.
Alternately, by two or more Neisseria meningitidis sero-groups being cultivated together also copurification, depolymerization, activate and are puted together polysaccharide, or by culture purified Neisseria meningitidis sero-group separately and in depolymerization, activate and put together the polysaccharide of 2 kinds of combinations before or after arbitrary step of polysaccharide or more kinds of purifying, can prepare immune composition.
Prepare immune composition of the present invention by separating preparation from the polysaccharide-protein conjugate of different meningococcus sero-groups and making up subsequently conjugate. Immune composition of the present invention can be used as vaccine. The preparation of vaccine of the present invention can use art-recognized method to finish. Vaccine combination of the present invention also can comprise one or more adjuvants. Adjuvant comprises, for example and be not limited to, aluminium adjuvant (for example aluminium salt, for example aluminium hydroxide, aluminum phosphate, aluminum sulfate or its combination), Freunds adjuvant (complete or incomplete), BAY, DC-chol, pcpp, single phosphinylidyne (monophoshoryl) lipid A, CpG, QS-21, cholera toxin and formylmethionyl peptide. Referring to, Vaccine Design for example, the Subunit and Adjuvant Approach, 1995 (M.F.Powell and M.J.Newman, eds., Plenum Press, N.Y.). The preferred aluminium adjuvant of adjuvant, for example aluminium hydroxide or aluminum phosphate.
Alternative adjuvant comprises the oil in water emulsion preparation, for example such as PCT publication number WO 90/14837) the middle MF59 that describes, SAF, comprise 10% saualane, 0.4 %Tween 80,5%pluronic block polymer L121, and thr-MDP, RibiTM adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) comprise 2% squalene, 0.2 % Tween 80, and from one or more bacterial cell wall fractions of monophosphoryl lipid A (MPL), TDM (TDM) and cell wall skeleton (CWS), preferred MPL+CWS (DetoxTM); Saponin adjuvant for example can use StimulonTM (Cambridge Bioscience, Worcester, Mass.) or by the particle of its generation ISCOMs (immunostimulating complex) for example; Cell factor is such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 etc.), interferon (for example IFN-γ), macrophage colony stimulatory factor (M-CSF), TNF (TNF).
In one embodiment of the invention, the average glycosylation ratio (ratio of polysaccharide and protein) of protein-polysaccharide conjugate is about 0.05-about 2; More preferably, average ratio is about 0.08-about 1.25; And even more preferably, average ratio is about 0.1-about 0.9. In a preferred embodiment, the average glycosylation ratio (ratio of polysaccharide and protein) of protein-polysaccharide conjugate is about 0.2-about 0.8; More preferably, average ratio is about 0.2-about 0.6; And in addition preferred embodiment in, average ratio is about 0.3-about 0.5.
As hereinafter confirming, in various animal models, cause T dependence sample immune response according to vaccine of the present invention and immune composition, and polysaccharide vaccine causes not dependence sample immune response of T. Therefore, composition of the present invention or useful research tool, the biological pathway and the process that relate to for the T dependence sample immune response of I (Neisseria meningitidis) antigens to be used for research.
The amount of the vaccine of the present invention that will use to the human or animal and application program can be determined according to pharmacy and the well-known standard technique of veterinary applications those of ordinary skill, wherein consider this type of factor, concrete antigen for example, adjuvant (if existence), concrete animal or patient's age, sex, weight, species and condition, and route of administration. In the present invention, be provided for to be the about 5 μ g/kg body weight of about 0.02 μ g-for the amount of the polysaccharide-protein carrier of the vaccinated effective dose of Neisseria meningitidis. In preferred composition of the present invention and method, dosage is about 0.1 μ g-3 μ g/kg body weight. If after for example infecting through time less, effective dose will need less antibody so, because the time less of bacterial multiplication. Bacterial load when in a similar fashion, effective dose will depend on diagnosis. Use for treatment and can consider the multiple injections used through time period a couple of days.
Multivalence conjugate of the present invention can be used as single dose or uses in a series of (that is, with " once strengthening " or " repeatedly strengthening "). For example, as advising about other vaccines of prevention CHD that at present children can accept single dose in early days at life, subsequently until use booster after 10 years old.
Booster will produce the antibody from the B cell of sensitization, i.e. anamnedstic response. In other words; when comparing with licensed-in polysaccharide vaccine; the multivalence conjugate vaccine causes that in younger colony high first (after being the vaccine single administration) functional antibodies replys; and can cause anamnedstic response (after namely strengthening using), thereby confirm that the protective immune response that is caused by multivalence conjugate vaccine of the present invention is lasting.
Composition of the present invention can comprise the liquid preparation for the aperture, such as in the mouth, nose, anus, vagina, per os, stomach, mucous membrane (such as through tongue (perlinqual), alveolus, gum, smell or respiratory mucosa) etc., use for example suspension, syrup or elixir; With, be used in parenteral, subcutaneous, intracutaneous, intramuscular, the peritonaeum or intravenous is used the preparation of (for example injectable is used), for example sterile suspensions or emulsion. Preferred intravenous and parenteral administration. Such composition can mix with suitable carrier, diluent or excipient such as sterilized water, physiological saline, glucose etc. Composition can also be freeze-drying. Depend on required route of administration and preparation, composition can comprise auxiliary substance, strengthens additive, anticorrisive agent, flavor enhancement, pigment etc. such as wetting agent or emulsifying agent, pH buffer, gelling agent or viscosity. Can be with reference to the normative document that is incorporated herein by reference, for example " REMINGTON ' S PHARMACEUTICAL SCIENCE ", 1985, or annex subsequently, preparing suitable preparation, and need not unsuitable experiment.
In one embodiment of the invention, preferred route of administration is intramuscular or subcutaneous, preferred intramuscular approach. Using can be by injection or by alternative delivery apparatus.
Composition of the present invention can be provided as liquid preparation easily, for example isotonic water solution, suspension, emulsion or cementitious compositions, and they can be buffered to selected pH. If it is preferred that digest tube absorbs, then composition of the present invention can be pill, tablet, capsule, caplet (caplet) of " solid " form etc., comprise " solid " preparation that discharges or have liquid filler according to the time, the coated liquid of gelatin for example, the gelatin dissolving is under one's belt with for delivery to intestines thus. If it is required that nose or breathing (mucous membrane) are used, composition can be certain forms and pass through squeeze spray distributor, pump dispenser or aerosol dispenser and distribute so. Aerosol pressurizes by hydrocarbon usually. Pump dispenser preferably can distribution and computation dosage or the dosage with specific granular size.
Liquid preparation is usually than gel, other cementitious compositions and the easier preparation of solid composite. In addition, fluid composition is more convenient for to a certain extent particularly by injection or is administered orally in animal, children, particularly young children and other other objects with difficulties such as swallowing pill, tablet, capsule, or in the multi-agent situation. On the other hand, it is interior to provide and mucous membrane that cementitious compositions can be formulated in suitable viscosity scope, for example the lining of stomach or schneiderian membrane, long section time of contact.
In a preferred embodiment of the invention, vaccine combination is formulated as sterile liquid, without the physiological saline of pyrogen, phosphoric acid buffer, contains or do not contain anticorrisive agent. In a preferred embodiment, every dose prescription comprises the about 1.0mg sodium phosphate of about 0.3-and the about 6.0mg sodium chloride of about 3.5-and the water that is up to 1.5mL. In a preferred embodiment, every dose prescription comprises about 0.6 ± 0.2mg sodium phosphate and 4.4 ± 0.2mg sodium chloride and the water that is up to about 0.5 ± 0.2 mL.
Significantly, suitable carrier and the selection of other additives will be depended on the character of definite route of administration and given dose form, for example the liquid dosages form (for example, no matter whether composition is formulated as solution, suspension, gel or another kind of liquid form), or the solid dosage form (for example, no matter composition whether be formulated as pill, tablet, capsule, caplet, according to time releasing pattern or liquid-filled form).
Solution, suspension and gel contain the water (preferably purifying waste water) of more amount usually except activeconstituents.Also can there be for example pH regulator agent of more a spot of other compositions (for example alkali, for example NaOH), emulsifying agent or dispersion agent, buffer reagent, sanitas, wetting agent, jelling agent (for example methylcellulose gum), pigment and/or condiment.Composition can be isoosmotic, and promptly it can have the osmotic pressure identical with blood and tear.
The required isotonicity of the present composition can use the inorganic or organic solute of sodium tartrate, propylene glycol or other to realize.In one embodiment, the preferred isotonicity of composition is obtained by sodium phosphate or sodium-chlor or its mixture.Sodium-chlor is particularly preferred for the damping fluid that comprises sodium ion.
Use pharmaceutically acceptable thickening material the viscosity of composition can be maintained selected level.Methylcellulose gum is preferred, because it can be easily and obtains economically and be easy to work.Other thickening materials that are fit to comprise, for example xanthan gum, carboxymethyl cellulose, hydroxypropylcellulose, carbomer etc.The preferred concentration of thickening material will depend on selected reagent.Importantly use the amount that will reach selected viscosity.Viscous composition is formed by formulations prepared from solutions by adding this type of thickening material usually.
Can adopt pharmaceutically acceptable sanitas to increase the storage life of composition.Phenylcarbinol can be fit to, although multiple sanitas comprises that for example parabens, Thiomersalate, trichloro-butyl alcohol or benzalkonium chloride also can adopt.The concentration of preservatives that is fit to will be based on the 0.02%-2% of gross weight, although depend on selected reagent obvious variation can be arranged.
Those skilled in the art will recognize that with regard to N.Meningitidis PsC-protein carrier conjugate, must be chosen to be at chemically is the inert composition component.
The present invention will be by further describing with reference to following illustrative, non-limiting example, and described embodiment elaborates several preferred embodiments of inventive concept.Under the situation that does not deviate from spirit of the present invention, other examples of the present invention will be conspicuous for those skilled in the art.
Following abbreviation and trade mark are: ACIP, and consultative committee (AdvisoryCommittee on Immunization Practices) is put into practice in immunity; AE, adverse events; Cetavalon TM, cetyl trimethylammonium bromide, CTAB; CFR, code of Federal Regulations (Code of FederalRegulations); CRF, case report form; DTP, the diph-tet Whooping cough; ELISA, enzyme-linked immunosorbent assay; FDA, FDA (Food and DrugAdministration); GCP, GCP (Good Clinical Practice); GMC, geometric mean concentration; GMT, geometric mean titer; IgG, immunoglobulin G; IgG1, immunoglobulin G subclass 1; IgG2, immunoglobulin G subclass 2; IgM, immunoglobulin M; ICH, international coordination meeting (International Conference onHarmonization); IND, investigational new drug; IRB, institutional review board (InstitutionalReview Board); MenA/C-Dt divalence (A and C) meningococcal polysacharide diphtheria conjugate vaccine; MenPS, meningococcus group specificity polysaccharide; ML, milliliter; Menomune TM, licensed-in meningococcus A, C, Y and W-135 polysaccharide vaccine; OD, optical density(OD); PBS, phosphate-buffered saline; SAE, serious adverse events; SBA, serum bactericidal activity; SBA-BR, the serum bactericidal activity of using young rabbit complement to carry out is measured; SBA-HC, the serum bactericidal activity that end user's complement carries out is measured; SIDS, sudden infant death syndrome (SIDS); TetraMenD, tetravalence (A, C, Y and W-135) meningococcal polysacharide diphtheria conjugate vaccine; Td, tetanus and diphtheria vaccine; UAE, unexpected unfavorable experience; URI, upper respiratory tract infection; μ g, microgram.
Embodiment
The preparation of the capsular polysaccharide powder of embodiment 1 Neisseria meningitidis serogroups A, C, W-135 and Y purifying
The preparation of thick paste
Respectively, the thaw wet refrigerated inoculum of Neisseria meningitidis serogroups A, C, W-135 and Y and recover and be planted in the Blake bottle that comprises Mueller Hinton nutrient agar by liquid Watson Scherp substratum.With Blake under 35-37 ℃ at CO 2In the atmosphere incubation 15-19 hour.Incubation is after the time period, shifts out grower and add to comprise in the 4L flask of Watson Scherp substratum from the Blake bottle.With flask under 35-37 ℃ on the platform vibrator incubation 3-7 hour.The content of 4L flask is transferred to the fermenting container that comprises Watson Scherp substratum.Fermenting container at 35-37 ℃ of following incubation 7-12 hour, is wherein controlled dissolved oxygen content and pH by replenishing charging and antigassing additive.Incubation is after the time period, and the content of fermenting container is transferred to the 500L groove, adds Cetavlon TM, and with material mixing 1 hour.Make grower that Cetavlon handles about 15,000-17, under the 000xg about 30-70 liter/hour flow velocity under centrifugal.By the Cetavlon second time TMPrecipitation precipitates Crude polysaccharides from supernatant liquor.With Cetavlon TMAdd supernatant liquor and material was at room temperature mixed 1 hour at least.Material preserve in 1-5 ℃ 8-12 hour.About 45,000-50, under the 000xg under 300-400ml/ minute flow velocity the polysaccharide of centrifugal collecting precipitation.The paste of collecting is stored in-60 ℃ or lower until further processing.
The preparation of the polysaccharide powder of purifying
The paste of deactivation is thawed and be transferred to agitator.Paste is mixed with 0.9M calcium chloride to produce homogeneous suspension.Make suspension about 10, under the 000xg centrifugal 15 minutes.Supernatant liquor passes through no waste pad impouring container as extracting for the first time.Add the 0.9M calcium chloride of volume for the second time to paste, and fusion is to produce homogeneous suspension.Suspension is centrifugal as mentioned above, and supernatant liquor is mixed with the supernatant liquor that extracts from the first time.Carry out 4 times altogether and extract, and compile supernatant liquor.The extract that compiles concentrates by using the ultrafiltration of 10-30kDa MWCO volution ultra filtration unit.
In enriched material, add magnesium chloride, and use sodium hydroxide that pH is adjusted to 7.2-7.5.In enriched material, add DNA enzyme and RNA enzyme, and mixed incubation 4 hours in 25-28 ℃.Add the concentration of ethanol to 30-50%.By 10, removed sedimentary nucleic acid and protein under the 000xg in centrifugal 2 hours.Reclaim supernatant liquor and by adding ethanol to 80% and allowing it to precipitate polysaccharide 1-5 ℃ of following standing over night.Siphon off ethanol, and with sedimentary polysaccharide 10, under the 000xg centrifugal 5 minutes.With the sedimentary polysaccharide of washing with alcohol.Use the washing with acetone polysaccharide, 10, under the 000xg centrifugal 15-20 minute.Dry polysaccharide under vacuum.Initial polysaccharide powder is dissolved in the sodium acetate solution.Add magnesium chloride and use sodium hydroxide solution that pH is adjusted to 7.2-7.5.In solution, add DNA enzyme and RNA enzyme, and mix incubation 4 hours to remove residual nucleic acid in 25-28 ℃.Behind these enzyme incubations, in polysaccharide-enzyme mixture, add isopyknic sodium acetate-phenol solution, and placing under 1-5 ℃ on the platform vibrator about 30 minutes.Mixture is 10, under the 000xg centrifugal 15-20 minute.Aqueous layer above reclaiming and preserving.Hydrotropisms's layer adds isopyknic sodium acetate-phenol solution, and extracts as mentioned above.Carry out altogether extracting for 4 times from polysaccharide soln, to remove protein and intracellular toxin.The aqueous extract of combination is up to 10 times with water for injection dilution, and at the water for injection diafiltration of 10 volumes.In the polysaccharide of diafiltration, add calcium chloride.By adding ethanol to 80% in the 1-5 ℃ of precipitation polysaccharide that spends the night.Fetch the ethanol supernatant liquor, and pass through, centrifugal 15 minutes collection polysaccharide under the 000xg 10.With the polysaccharide of purifying with washing with alcohol 2 times, and with washing with acetone 1 time.The powder of washing is dry in moisture eliminator under vacuum.The exsiccant powder is stored in-30 ℃ or lower until handling on conjugate.
The depolymerization of the capsular polysaccharide powder of embodiment 2 Neisseria meningitidis serogroups A, C, W135 and Y purifying
The material that uses in preparation comprises the capsular polysaccharide powder (according to embodiment 1 preparation) from the purifying of Neisseria meningitidis serogroups A, C, W-135 and Y, aseptic 50mM sodium acetate buffer, pH6.0, aseptic 1N hydrochloric acid (hydrocholoric acid), aseptic 1N sodium hydroxide, 30% hydrogen peroxide and stroke-physiological saline solution (0.85% sodium-chlor).
Every kind of serogroups polysaccharide depolymerization in dividing other reaction.Stainless steel tank is mounted with the capsular polysaccharide powder that is up to the 60g purifying.With the aseptic 50mM sodium acetate buffer of pH 6.0 add polysaccharide with produce the 2.5g polysaccharide/liter concentration.Allow polysaccharide soln to mix 12-24 hour down to produce solution at 1-5 ℃.Reactive tank is connected to heat exchange unit.Add the 50mM sodium acetate buffer of other pH 6.0 so that polysaccharide is diluted to the reaction density that 1.25g/ rises.With polysaccharide soln be heated to 55 ℃+-.0.1.30% hydrogen peroxide of aliquots containig is added reaction mixture to produce the reaction density of 1% hydrogen peroxide.
By monitoring that the polysaccharide molecule size is along with reaction process is monitored in the variation in past time.Every 15-20 minute, from reaction mixture, take out aliquots containig and be expelled in the HPSEC post to measure the molecular size of polysaccharide.When the molecular size of polysaccharide reaches the target molecule size, turn off heating unit and make polysaccharide soln be quickly cooled to 5 ℃ by the ice-water bath circulation.By reactive tank being connected to the ultra filtration unit that is equipped with 3000MWCO regenerated Mierocrystalline cellulose tube the polysaccharide soln of depolymerization is concentrated into the 15g/ liter.Spissated depolymerization polysaccharide soln is at stroke-physiological saline solution (0.85% sodium-chlor) diafiltration of 10 volumes.The polysaccharide of depolymerization is stored in 1-5 ℃ until next treatment step.
By measure the molecular size of depolymerization polysaccharide through the gel filtration chromatography post of selling down in trade(brand)name " Ultahydrogel.TM.250 ", described chromatographic column is used the dextran molecule size criteria and is calibrated by polygonal laser light scattering.Use Bartlet, G.R.J. (1959) Journal ofBiological Chemistry, 234, the method of 466-468 page or leaf, by the phosphorus content of serogroups A, and use Svennerholm, L. (1955) Biochimica Biophysica Acta 24, the method of 604-611 page or leaf is measured the amount of polysaccharide by the sialic acid content of serogroup C, W135 and Y.By Hesterin, S. (1949) Journal of Biological Chemistry180, the 249th page method is measured the content of O-ethanoyl.By Park, J.T. and Johnson, the method for M.J. (1949) Journal of Biological Chemistry 181, the 149-151 pages or leaves is measured reducing activity.Measure the structural integrity of depolymerization polysaccharide by protein .sup.1H and .sup.13C NMR.Measure the purity of depolymerization polysaccharide by measuring LAL (intracellular toxin) content and residual content of hydrogen peroxide.
The derivatize of embodiment 3 Neisseria meningitidis serogroups A, C, W-135 and Y depolymerization polysaccharide
The material that uses in current preparation comprises the capsular polysaccharide from Neisseria meningitidis serogroups A, C, W-135 and Y (according to embodiment 2 preparations) of hydrogen peroxide depolymerization, adipic dihydrazide, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDAC) be only for serogroups A, sodium cyanoborohydride, aseptic 1N hydrochloric acid, aseptic 1N sodium hydroxide, aseptic 1M sodium-chlor and stroke-physiological saline solution (0.85% sodium-chlor).
Every kind of serogroups polysaccharide derivatize in the reaction that separates.Stainless steel tank is mounted with the depolymerization polysaccharide of purifying, and with aseptic 0.85% physiological saline be diluted to reach the 6g polysaccharide/liter end reaction concentration.In this solution, add the spissated aliquots containig adipic dihydrazide that is dissolved in aseptic 0.85% physiological saline, to reach the reaction density that 1g/ rises.Only, add EDAC, to reach the reaction density that 1g/ rises as the spissated aliquots containig that is dissolved in aseptic 0.85% physiological saline for serogroups A.PH is adjusted to 5.0.+-.0.1, and uses aseptic 1N hydrochloric acid and aseptic 1N sodium hydroxide that this pH was kept 2 hours under room temperature (15-30 ℃).After 2 hours, in reaction mixture, add the spissated aliquots containig sodium cyanoborohydride that is dissolved in 0.85% physiological saline, to reach the reaction density that 2g/ rises.Reactant stirred 44 hours+-4 hours down in room temperature (15-30 ℃), simultaneously pH was maintained 5.5.+-.0.5.After this reaction times section, pH is adjusted to 6.0.+-.0.1, and by reactive tank being connected to be equipped with the ultra filtration unit of 3000MWCO regenerated Mierocrystalline cellulose tube, with the polysaccharide of derivatize be concentrated into the 12g polysaccharide/liter.Spissated derivatize polysaccharide is the 0.15M sodium-chlor diafiltration of 10 volumes at the 1M sodium-chlor of 30 volumes subsequently.Groove and ultra filtration unit separated and stored 7 days in 1-5 ℃.Groove reconnects with the ultra filtration unit that is equipped with 3000 MWCO regenerated Mierocrystalline cellulose tubes, and at the 1M sodium-chlor of 30 volumes, is the 0.15M sodium-chlor diafiltration of 10 volumes subsequently.
The molecular size of derivatize polysaccharide, the content of the amount of polysaccharide and O-ethanoyl is measured by the same procedure that the depolymerization polysaccharide is used.Hydrazides content passes through Snyder, S.L. and Sobocinski, and 2,4 of P.Z. (1975) Analytical Biochemistry 64, the 282-288 pages or leaves, 6-trinitro-benzene-sulfonic acid method is measured.The structural integrity of derivatize polysaccharide is passed through proton 1H and 13C NMR measures.The content of the level of the purity of derivatize polysaccharide by measuring unconjugated hydrazides, LAL (intracellular toxin) content and residual cyano group hydroborate is measured.
The preparation of embodiment 4 carrier proteinss
The thick proteic preparation of diphtheria toxoid
Freeze dried inoculum was by reconstruct and incubation 16-18 hour.To be transferred to the 0.5 liter of flask that comprises growth medium from the aliquots containig of culture, and with culture flask under 34.5-36.5 ℃ on gyrate shaker incubation 7-9 hour.To be transferred to 4 liters of flasks that comprise growth medium from the aliquots containig of culture flask, and with culture flask under 34.5-36.5 ℃ on gyrate shaker incubation 14-22 hour.Culture from 4 liters of flasks is used to inoculate the fermentor tank that comprises growth medium.Fermentor tank was at 34.5-36.5 ℃ of following incubation 70-144 hour.The content of fermentor tank enters in the collection container by deep filter.37% formaldehyde solution of aliquots containig adds in the cutting concentration to reach 0.2%.PH is adjusted into 7.4-7.6.Cutting is filled in aseptic 20 litre flasks by 0.2 micron filter tube.Bottle was 34.5-36.5 ℃ of following incubation 7 days.37% formaldehyde solution of aliquots containig adds in each 20 litre flask the concentration to reach 0.4%.The pH of mixture is adjusted into 7.4-7.6.Bottle under 34.5-36.5 ℃ on vibrator incubation 7 days.37% formaldehyde solution of aliquots containig adds in each 20 litre flask the concentration to reach 0.5%.The pH of mixture is adjusted into 7.4-7.6.Bottle is 34.5-36.5 ℃ of following 8 weeks of incubation.Test thick anatoxic detoxification.Bottle is stored in 1-5 ℃ during the test duration section.
The thick proteic purifying of diphtheria toxoid
Allow thick toxoid to heat, and the content of 20 litre flasks is combined in the purifying groove to room temperature.Anatoxic pH is adjusted to 7.2-7.4, and in thick toxoid, adds gac and mixed 2 minutes.Allow gac toxoid mixture to leave standstill 1 hour, and be filled in second purifying groove by the deep filter tube subsequently.In filtrate, add solid ammonium sulfate with reach 70% saturated.PH is adjusted to 6.8-7.2, and allows solution left standstill 16 hours.By protein that filters collecting precipitation and the 70% saturated ammonium sulphate solution washing of using pH7.0.Resolution of precipitate in sterile distilled water, and is filled into protein soln in the stainless steel collection container.PH is adjusted to 6.8-7.2, and adds the saturated of ammonium sulfate to 40%.PH is adjusted to 7.0-7.2, and allows solution left standstill 16 hours.By filtering the removal precipitation and discarding.In filtrate, add the saturated of ammonium sulfate to 60%, and pH is adjusted to 7.0-7.2.Allow mixture to leave standstill 16 hours, and by filtering the protein of collecting precipitation.Resolution of precipitate in sterile distilled water, is filtered removing undissolved protein, and at 0.85% physiological saline diafiltration.
The diphtheria toxoid of purifying is proteic to be concentrated and sterile filtration
Use 10,000 MWCO regenerated Mierocrystalline cellulose cartridge filters that protein soln is concentrated into that 15g/ rises and at 0.85% physiological saline diafiltration of 10 volumes.Spissated protein soln filters by 0.2 micron membranes and sterilizes.Protein soln is stored in 1-5 ℃ until handling on conjugate.
Protein soln is by Lowry, and the method for people such as O.H. (1951) Journal of BiologicalChemistry 193, the 265-275 pages or leaves is measured.Proteinic purity is measured by aseptic, LAL (intracellular toxin) content and residual formaldehyde content.
The preparation of embodiment 5 Neisseria meningitidis serogroups A, C, W-135 and Y polysaccharide and the proteic unit price conjugate of diphtheria toxoid
The material that uses in preparation comprises the hexanodioic acid depolymerization polysaccharide (according to embodiment 3 preparations) from Neisseria meningitidis serogroups A, C, W-135 and Y, aseptic diphtheria toxoid albumen (according to embodiment 4 preparations), EDAC, ammonium sulfate, aseptic 1N hydrochloric acid, aseptic 1N sodium hydroxide and stroke-physiological saline solution (0.85%).
Every kind of serogroups polysaccharide conjugates is by prepared in reaction separately.All 4 kinds of conjugates are all by following method preparation.Stainless steel tank be mounted with reaction density be the micromolar reaction hydrazides of 700-1000/liter the polysaccharide of hexanodioic acid derivatize of purifying and reaction density be 3.8-4.0g protein/liter the diphtheria toxoid albumen of purifying.0.85% physiological saline is used for starting material are diluted to target response concentration and pH is adjusted to 5.0.+-.0.1.The EDAC of aliquots containig is added in the polyose-protein mixture, to reach the reaction density that 2.28-2.4g/ rises.The pH of reaction is maintaining under the 5.0.+-.0.1 2 hours under 15-30 ℃.After 2 hours, use aseptic 1N sodium hydroxide that pH is adjusted to 7.0.+-.0.1, and with reactant be stored in 1-5 ℃ 16-20 hour.
Allow reaction mixture to heat, and with reaction vessel and outfit 30, the ultra filtration unit of 000MWCO regenerated Mierocrystalline cellulose tube connect to 15-30 ℃.Add solid ammonium sulfate to 60% saturated (for serogroups A, W-135 and Y) and 50% saturated (for serogroup C).The conjugation reaction mixture is 0.85% physiological saline diafiltration of 20 volumes at 60% of 20 volumes saturated ammoniumsulphate soln (for serogroups A, W-135 and Y) and 50% saturated ammoniumsulphate soln (for serogroup C) subsequently.The conjugate of diafiltration is at first by comprising the strainer capsule of 1.2 microns and 0.45 micron filter, and filters by the second kind of strainer capsule that comprises 0.22 micron filter subsequently.
By measuring the amount and the O-acetyl content of polysaccharide with the same procedure that depolymerization and derivatize polysaccharide are used.Measure proteinic amount by the Lowry method.By measure the molecular size of conjugate through the gel filtration chromatography post of selling down in trade(brand)name " TSK6000PW ", described chromatographic column uses DNA as the void volume mark, ATP as cumulative volume mark and bovine thyroglobulin as the reference mark.In addition, the molecular size of the conjugate that goes out from TKS6000PW post wash-out is measured by polygonal laser light scattering.The antigen property of conjugate is measured by using double fastener heart ELISA method to combine with anti-polysaccharide serogroups specific antibody.The purity of conjugate is measured by following method, measures the amount via not combination (not puting together) polysaccharide of hydrophobic interaction chromatography post wash-out, by the unconjugated protein of capillary electrophoresis, aseptic, LAL (intracellular toxin) content, residual EDAC content and residual ammonium ion content.
The preparation of embodiment 6 multivalent meningococcal A, C, W-135 and Y polysaccharide diphtheria toxoid conjugate vaccine
The material that uses in current preparation comprises according to polysaccharide-diphtheria toxoid conjugate of serogroups A, C, W-135 and the Y of embodiment 5 preparations, the physiological saline (0.85% sodium-chlor) of aseptic 100mM sodium phosphate buffer.
To the physiological saline that gathers the aseptic 100-500mM sodium phosphate buffer of physiological saline (0.85%) adding aliquots containig in the groove (bulking tank) at stainless steel, to produce the final vaccine concentration of 10mM sodium phosphate.In the aseptic unit price meningococcal polysacharide of the 2-4 kind-diphtheria toxoid conjugate that gathers adding aliquots containig in the groove that comprises the aseptic sodium phosphate physiological saline of 10mM each is to produce the ultimate density of every kind of serogroups polysaccharide/milliliter damping fluid of 8 μ g.The tetravalence conjugate of preparation mixed and be filled into second by 0.2 micron filter gather in the groove.
Use the high pH anion-exchange chromatography that detects with pulsed current to measure the amount of the every kind of serogroups polysaccharide that exists in the multivalence preparation by the component sugars analysis.Measure proteinic amount by the Lowry method.Use the compound electrode that is connected with pH meter to measure the pH of vaccine.By using double fastener heart ELISA method to combine the antigen property of measuring the multivalence conjugate vaccine with anti-polysaccharide serogroups specific antibody.In animal model, cause the immunogenicity of measuring the multivalence conjugate vaccine for the first time with the ability of strengthening anti-polysaccharide IgG immunne response by the every kind of conjugate that exists in the vaccine.By using the high pH anion-exchange chromatography that detects with pulsed current to measure not in conjunction with the amount of (not puting together) polysaccharide, measure aseptic, LAL (intracellular toxin) content, pyrogenicity content and Generally Recognized as safe, measure the purity of multivalence conjugate vaccine.
Embodiment 7 is the preparation of the multivalent meningococcal polysaccharide diphtheria toxoid protein conjugate of adjuvant with aluminium hydroxide
Preparation with the conjugate of aluminium hydroxide absorption.The material that uses in current preparation comprises according to polysaccharide-diphtheria toxoid conjugate, the stroke-physiological saline solution (0.85% sodium-chlor) of serogroups A, C, W-135 and the Y of embodiment 5 preparations and is dissolved in aseptic aluminium hydroxide in the physiological saline (0.85% sodium-chlor).
To the every kind of aseptic unit price meningococcal polysacharide diphtheria toxoid conjugate that adds aliquots containig in the groove that gathers that comprises physiological saline, to produce the ultimate density of every kind of serogroups polysaccharide/milliliter damping fluid of 8 μ g.Add the aseptic aluminium hydroxide of aliquots containig that is dissolved in the physiological saline (0.85% sodium-chlor) to the multivalence conjugate vaccine, to reach the ultimate density of 0.44mg aluminum ion/milliliter vaccine.
Embodiment 8 is the preparation of the conjugate of adjuvant with the aluminum phosphate
The material that uses in current preparation comprises according to polysaccharide-diphtheria toxoid conjugate, the stroke-physiological saline solution (0.85% sodium-chlor) of serogroups A, C, W-135 and the Y of embodiment 5 preparations and is dissolved in aseptic aluminum phosphate in the physiological saline (0.85% sodium-chlor).
To the every kind of aseptic unit price meningococcal polysacharide diphtheria toxoid conjugate that adds aliquots containig in the groove that gathers that comprises physiological saline, to produce the ultimate density of every kind of serogroups polysaccharide/milliliter damping fluid of 8 μ g.Add the aseptic aluminum phosphate of aliquots containig that is dissolved in the physiological saline (0.85% sodium-chlor) to the multivalence conjugate vaccine, to reach the ultimate density of 0.44mg aluminum ion/milliliter vaccine.
The material that embodiment 9 uses in people's clinical study and the general description of method
The immunogenicity of the conjugate vaccine of tetravalence derivatize
The research conjugate vaccine causes the ability of immunne response in the people under many different clinical protocol.Following research overview the result.Except as otherwise noted, material and the method for using in following each research is:
TetraMenD
The TetraMenD vaccine comprises 4 kinds of meningococcal capsular polysaccharide of serogroups A, C, Y and W-135, and every kind of polysaccharide 4 μ g are with 48 μ g diphtheria toxoid albumen covalent attachment altogether.Vaccine is prepared in the physiological saline of aseptic, no pyrogeneous substance, phosphoric acid buffer, does not contain sanitas.Prescription comprises 0.6mg sodium phosphate, 4.4mg sodium-chlor and is up to the water of 0.5mL.
Figure A200580048657D00291
Figure A200580048657D00292
Obtain permitting in 2 years old and above people in the U.S. and other country and use.Menomune is cryodesiccated preparation, and every vaccinating agent comprises every kind of A of 50 μ g, C, Y and W-135 polysaccharide as antigen, with isotonic sodium chlorrde solution diluent reconstruct, anticorrosion and give as 0.5mL dosage is subcutaneous with Thiomersalate.The vaccine of every 0.5mL dosage comprises the 2.5mg-5mg lactose as stablizer.
Figure A200580048657D00293
-A/C/Y/W-135, is used for the meningococcal polysaccharide vaccine of subcutaneous purposes at combination group A, C, Y and W-135, is the freeze-dried preparation from the group specificity polysaccharide antigen of Neisseria meningitidis group A, group C, group Y and group W-135.Thinner is a distilled water aseptic, no pyrogeneous substance.Illustrate on freeze-drying prods such as the label with after the thinner reconstruct, every 0.5mL dosage is formulated as and comprises in isotonic sodium chlorrde solution from each 50 μ g " isolating product " among serogroups A, C, Y and the W-135.The grownup is with adsorbing tetanus and diphtheria class
Figure A200580048657D0029165059QIETU
(being called Td subsequently)
It is the sterile suspensions of alum-precipitated toxoid(APT) in isotonic sodium chlorrde solution, comprises sodium phosphate buffer with control pH.Vaccine is used for intramuscularly.Be formulated as and comprise 5Lf Toxoid,tetanus, 2Lf diphtheria toxoid and 0.28mg aluminium at the most by measuring every 0.5mL dosage.Tetanus and diphtheria toxoid are renderd a service the toxinicide/mL that induces at least 2 units and 0.5 unit in the test respectively cavy.When going to a doctor for the first time, Td uses for all participants as single 0.5mL dosage, and it is by using No. 5 pin intramuscularlys of 1 in2 in the deltoid muscle of left arm.Every 0.5mL dosage comprises 5Lf Toxoid,tetanus and 2Lf diphtheria toxoid.
Serum sample
Extracting blood sample in the indicated date behind the baseline.For example, if scheme indicates 3 time points, the 0th day, the 28th day and 6th month, so before vaccination the 0th day (baseline), after the vaccination the 28th day (for assess primary immune response and after vaccination 6th month (in order to assess the life-span of immunne response) extract blood sample.On each time point, collect about 5mL whole blood from each experimenter.Whole blood collect in 4 hours centrifugal.Take out serum and be stored in-20 ℃.The blood sample of " the 28th day " after injection in the 0th day at least 28 days but take when also not having 57 days.The blood sample of " 6th month " is taked when injection in the 0th day added deduct 28 days in back 6 months.Therefore, the serum of the 28th day serum representative extraction in the 28th day the-the 56th day after the 0th day; And the serum that the representative of 6th month serum was extracted after the 0th day in the 149th day the-the 217th day.
Determination techniques
This research adopts many standard immunoassay to measure.Methodology used herein has been summarized in following description.Yet, other similar mensuration, the variation of those that comprise that this paper presents is that those skilled in the art are well-known and can use.
Measure (SBA-BR) by the serum sterilizing that uses young rabbit complement and measure meningococcemia antibody
Use serum sterilizing to measure the functional antibodies activity of metering needle to the meningococcemia antibody of serogroups A, C, Y and W-135.2 times of dilutions of preparation test sera in aseptic 96 hole microtiter plates.Add in serum dilution together with young rabbit complement serogroups specificity meningococcus and allow incubation., after the time period agar over lay substratum is added in serum/complement/bacterial mixture at this incubation, allow hardening, and subsequently in 37 ℃ and 5% CO 2Be incubated overnight.The bacterial colony that exists in the counting hole.Compare with the mean value in complement control hole, by producing the 50% serum dilution reciprocal of killing determines tiring of terminal point.Using the limit of detection of this assay method of rabbit complement is to tire 8.
IgG resists-the meningococcus TPPA
Use the IgG antibody activity of indirect ELISA metering needle to the meningococcemia antibody of serogroups A, C, Y and W-135.This method comprises the antibody that makes in the serum and excessive meningococcus group specificity polysaccharide (MenPs) antigen-reactive, and described antigen is adsorbed to the plastics microtiter well by the human serum albumin that methylates.The amount of bonded antibody is reacted by the mouse anti human IgG monoclonal antibody specific with peroxidase labelling and is measured.Use the colour developing product of the subsequent reactions generation metric measurement of peroxidase substrate.Resulting optical density(OD) (OD) is relevant with the amount of IgG antibody in the serum, and described IgG antibody combines with meningococcal polysacharide on the microtiter plate.Use 4 parameter logarithmic curve methods by relatively calculating the amount of IgG antibody subsequently with the reference with designated value (Lot CDC 1992 or Equivalent).
IgM meningococcemia TPPA
Use the IgM antibody activity of indirect ELISA metering needle to the meningococcemia antibody of serogroups A, C, Y and W-135.This method comprises the antibody that makes in the serum and excessive MenPs antigen-reactive, and described antigen is adsorbed to the plastics microtiter well by the human serum albumin that methylates.The amount of bonded antibody is reacted by the mouse anti human IgM monoclonal antibody specific with peroxidase labelling and is measured.Use the colour developing product of the subsequent reactions generation metric measurement of peroxidase substrate.Resulting OD is relevant with the amount of IgM antibody in the serum, and described IgM antibody combines with meningococcal polysacharide on the microtiter plate.Use 4 parameter logarithmic curves by relatively calculating the amount of IgM antibody subsequently with the reference with designated value (Lot CDC 1992 or Equivalent).
High avidity resists-meningococcus IgG TPPA
Use the ELISA that modifies in the high avidity IgG antibody activity of Aventis Pasteur Inc. metering needle to the meningococcemia antibody of serogroups A, C, Y and W-135.This is measured at present and also will qualify before the test clinical sample in the exploitation of Aventis Pasteur Inc..In brief, 96 hole microtiter plates are antigen coated with MenPs.Behind the plate of suction and ishing bag quilt, use phosphate-buffered saline (PBS) the serum dilution buffer liquid that comprises the 75mM ammonium thiocyanate, in plate, directly prepare the serial dilution thing of clinical serum, and allow to be incubated overnight.The amount of bonded antibody is reacted by the mouse anti human IgG monoclonal antibody specific with peroxidase labelling and is measured.Use the colour developing product of the subsequent reactions generation metric measurement of peroxidase substrate.Resulting OD is relevant with the amount of high avidity IgG antibody in the serum, and described IgG antibody combines with meningococcal polysacharide on the microtiter plate.Use subsequently 4 parameter logarithmic curves by with relatively calculate the amount of high avidity IgG antibody with reference to (Lot CDC 1992 or Equivalent).
IgG1 and IgG2 subclass meningococcus TPPA
Use the ELISA metering needle that the IgG1 and the IgG2 subclass antibody of the meningococcemia antibody of serogroups A, C, Y and W-135 are distributed.Antibody that exists in the serum serial dilution thing and the MenPs antigen-reactive that is adsorbed to micro titer plate well.The amount of bonded antibody will use anti-human IgG1 Fc or IgG2Fc specific reagent to measure.Produce the colour developing product of metric measurement with the subsequent reactions of enzyme substrates.The amount of IgG1 or IgG2 antibody is relevant in resulting OD and the serum, and described antibody combines with meningococcal polysacharide on the microtiter plate.The amount of antibody will be reported as IgG1:IgG2 ratio or IgG1 in the sample or the IgG2 concentration in the serum sample, if can obtain suitable reference.
Metabolism by the VERO cell suppresses to measure diphtheria antibody
The diphtheria antibody response is measured by the ability that test sera protection VERO cell is not attacked by diphtheria toxin.Use aseptic 96 hole microtiter plates, attack 2 times of dilutions of the test sera that begins from the 1:4 dilution and allow incubation with diphtheria toxin.Add the VERO cell subsequently, with sterile mineral oil sealing gap and incubation 6-8 days.Determine antibody horizontal by the colour-change of observing the pH in culture medium indicator subsequently, described colour-change is produced by the byproduct of cellular metabolism effect.The result relatively is reported as international unit/mL by the WHO reference serum with calibration, and by in the presence of the diphtheria toxin of challenge dose, allowing the highest serum dilution of cellular metabolism to determine.Detect lower limit and determine, and be generally 0.005IU/mL by the minimum detectable toxinicide level of reference serum and the initial dilution of test sera.
Measure anti-tetanus antibody by Elisa
(ELISA) measures the anti-tetanus antibody horizontal by indirect ELISA.This method comprises antibody that makes in the test sera and the Toxoid,tetanus reaction that is adsorbed to the plastics microtiter well.The amount of bonded antibody is measured by reacting with the anti-human IgG specific antibody of the goat that is conjugated to alkaline phosphatase.Produce the colour developing product of metric measurement with the subsequent reactions of alkaline phosphatase substrate.The amount of the antibody of the microtiter plate of conjugated antigen bag quilt is relevant in OD (optical density(OD)) and the serum dilution.By the parallel lines analytical method via with have the international man who specifies unit vol and relatively come calculating antibody concentration with reference to (WHO Lot TE-3).The result is reported as international unit/milliliter (IU/mL).The quantitative minimum level of anti-tetanus IgG ELISA is 0.01IU/mL, and the sampling report that the value that obtains is lower than this level is＜0.01IU/mL.
Adverse events (AE) is defined as and " takes place in the patient of drug administration product or clinical study experimenter and need not to have any disadvantageous medical events of causalnexus with this treatment.Therefore whether adverse events can use relevant in time, any undesirable and unexpected symptom (comprising unusual laboratory discovery), symptom or disease with medical science (research) product, no matter relevant with medical science (research) product." (ICH guilding principle, GCP (E6) § 1.2).
Serious adverse events (SAE) is " to take place, cause any following result's any unfavorable medicine to experience on any dosage: death; life-threatening unfavorable medicine is experienced; inpatient's hospital care or existing hospital care prolong; lasting or significant deformity/insufficiency, or birth defect/inborn defect.May not cause death, life-threatening or need the important medical incident of hospital care can be considered as serious unfavorable medicine experience, when based on suitable medical judgment, they may endanger patient or experimenter, and may need medical science or surgical intervention with one of the consequence of preventing to list in this definition the time.The example of this type of medical events comprise need be in emergency room or family allergy bronchospasm, the blood dyscrasia that does not cause inpatient's hospital care or convulsions or the development drug dependence or the drug abuse of powerful treatment " (21 CFR Ch.I, § 312.32 (a)).
It is unexpected that adverse events (UAE) be that " any unfavorable medicine is experienced, and its specificity or seriousness and present investigator's brochure is inconsistent; Or as revision, if when investigator's brochure is optional or obtainable, the dangerous information of describing in its specificity or seriousness and general Study plan or the elsewhere applied at present is inconsistent.”(21 CFR Ch.I，§312.32(a))。
This research is put into practice according to standard clinical and is carried out, and patient's selected or exclusion standard is in this research:
The standard that comprises about the patient:
1. determine that according to medical history and health check-up the participant is healthy.
2. the participant is when vaccination at least 11 years old but also do not have 19 years old.
Where applicable patient/guardian or participant's signed institutional review board (IRB) approval Informed Consent Form.
4. the letter of consent of where applicable participant signed institutional review board (IRB) approval.
Exclusion standard about the patient:
1. serious chronic disease (that is, heart trouble, ephrosis, neuropathy, metabolic disease, rheumatosis etc.).
2. known or suspection is damaged immunologic function.
3. in nearest 72 hours, follow or do not follow the acute medical science disease of heating, or when comprising mouth temp 〉=38 ℃ (100.4 ℉).
4. certified aggressive meningococcal disease or former meningococcus vaccination vaccine history.
5. in nearest 3 months, use immunoglobulin (Ig), other blood products, or oral or injection reflunomide or other immune tuning treatments in 6 weeks of research vaccine.The individuality that the timetable that oral steroid dosage reduces gradually continues＜7 days can be selected in test, as long as they do not accept to surpass the course of treatment once in the time period in selected preceding 2 weeks.
6. the antibiotic therapy in preceding 72 hours of vaccination.
7. in the selected preceding 28 days time period, accept any vaccine, or in the selected back 28 days time period, arrange to accept any vaccination, except other vaccination is specified in this research.
8. suspect or known super quick any vaccine component.
9. can not finish whole search time section maybe can not participate in the prescription on individual diagnosis of arrangement or comply with research method.
10. selected another clinical trial.
11., can cause any situation of health risk or the assessment of interference vaccine to the participant In the view of the investigator.
12. in the women, positive or ambiguous urine pregnancy tests during vaccination.
Embodiment 10 research A-dose studies
Research A is that non-blind property, open label, the dosage of the TetraMenD vaccine of 3 kinds of dosage levels using of the participant to 3 age groups progressively increases test.The selected Phase I of 90 normal adults (18-55 year) is also accepted the single injection of TetraMenD vaccine.30 healthy childrens (12-22 month) are selected in Phase and accept 2 injections of the TetraMenD vaccine of single dose level.90 healthy babies (6-12 week) are selected in Phase I and accept 3 injections of the TetraMenD vaccine of single dose level.
The dose study of Phase I in the 18-55 grownup in year
This clinical trial is that non-blind property, open label, the dosage of the TetraMenD vaccine of 3 kinds of dosage levels progressively increases test, and described vaccine administration is in the participant of 3 age groups.In the stage 1,90 normal adults (18-55 year) are accepted the single injection of TetraMenD vaccine.
For grownup participant, the baseline before TetraMenD uses (the 0th day) and use the serum sample that the back obtains to be used for serological analysis the 28th day the time at TetraMenD.About SBA at meningococcal polysacharide serogroups A, C, Y and W-135, and by the sample at all acquisitions of elisa assay of the IgG antibody of these identical serogroupss.SBA and IgG ELISA about all serogroupss find to be summarized in hereinafter.
The immunogenicity terminal point of a key is the ratio from the participant of 〉=4 times of baseline risings.In order to determine baseline SBA tires what effect is raise 〉=4 times ratio of SBA had, the tire baseline of the grownup that is less than 1:64 and specific antigen of the baseline of every kind of specific antigen is tired and carried out the subgroup analysis for the grownup of 1:64 at least.
The security features of TetraMenD is comparable to
Figure A200580048657D0034164804QIETU
Security features.The result of this research is summarized in the following table.
Figure A200580048657D00351
Figure A200580048657D00371
Figure A200580048657D00381
Table A-3: Phase I (grownup)-reach in the time of the 28th day in baseline and the injection back ratio (meeting scheme colony) of SBA threshold value according to the TetraMenD dosage level
Figure A200580048657D00391
N: each time point (the 0th day; The 28th day) go up appreciable participant's number
Table A-4: Phase I (grownup)-according to the TetraMenD dosage level at baseline and injection back SBA and the IgG ELISA result (meeting scheme colony) in the time of the 28th day
Figure A200580048657D00392
Table A-4: Phase I (grownup)-according to the TetraMenD dosage level at baseline and injection back SBA and the IgG ELISA result (meeting scheme colony) in the time of the 28th day
Table A-4: Phase I (grownup)-according to the TetraMenD dosage level at baseline and injection back SBA and the IgG ELISA result (meeting scheme colony) in the time of the 28th day
Figure A200580048657D00411
The 0th day: the baseline blood specimen that before vaccination, extracts.
The 28th day: the blood sample that after vaccination, extracted in 28 days.
The rising of % 〉=4 times: compared GMT 〉=4 times the adult per-cent that raises in the time of the 28th day with the 0th day.
N: appreciable participant's number.
*GMTs is for the SBA data computation; GMCs is for IgG ELISA data computation.
Table A-5 representative is according to the GMT of dosage, patient age and serogroups.
Figure A200580048657D00421
Figure A200580048657D00441
Phase is a dose study among 12 months-22 months the child who has just learnt to walk at the age
This clinical trial is that non-blind property, open label, the dosage of the TetraMenD vaccine of 3 kinds of dosage levels progressively increases test, and described vaccine administration is in the participant of 3 age groups.In Phase, 30 healthy childrens (12-22 month) are accepted 2 injections of the TetraMenD vaccine of single dose level.
For the child participant who has just learnt to walk, at the serum sample that obtains to be used for serological analysis on 3 time points: during baseline (the 0th day) before the 1st injection of TetraMenD, when being selected in back the 60th day (back 60 days of the 1st injection and tightly before the 2nd injection of TetraMenD), and when being selected in back the 90th day (back 30 days of the 2nd injection).About SBA at meningococcal polysacharide serogroups A, C, Y and W-135, and by the sample at all acquisitions of elisa assay of the IgG antibody of these identical serogroupss.SBA and IgG ELISA about all serogroupss find to be summarized in hereinafter.The result is summarized in the following table.
Figure A200580048657D00471
Figure A200580048657D00481
Figure A200580048657D00491
Figure A200580048657D00501
Figure A200580048657D00521
Table A-10: Phase (child who has just learnt to walk)-according to SBA and the IgG ELISA result of TetraMenD dosage level in the child that baseline, back 60 days of the 1st injection and the 2nd injection have just been learnt to walk in the time of back 30 days
(meeting scheme colony)
Figure A200580048657D00531
Table A-10: Phase (child who has just learnt to walk)-according to SBA and the IgG ELISA result of TetraMenD dosage level in the child that baseline, back 60 days of the 1st injection and the 2nd injection have just been learnt to walk in the time of back 30 days
(meeting scheme colony)
Figure A200580048657D00541
Table A-10: Phase (child who has just learnt to walk)-according to SBA and the IgG ELISA result of TetraMenD dosage level in the child that baseline, back 60 days of the 1st injection and the 2nd injection have just been learnt to walk in the time of back 30 days
(meeting scheme colony)
Figure A200580048657D00551
The 0th day: the baseline blood specimen that before the 1st injection, extracts.
The 60th day: at the blood sample of back 60 days of the 1st injection and extraction before the 2nd injection.
The 90th day: the blood sample that the 2nd injection extracted in back 30 days.
The rising of % 〉=4 times: the 1st injection back: compared GMT 〉=4 times the child's who has just learnt to walk the per-cent that raises in the time of the 60th day with the 0th day; The 2nd injection back: compared GMT 〉=4 times the child's who has just learnt to walk the per-cent that raises in the time of the 90th day with the 0th day.
N: appreciable participant's number.
*GMTs is for the SBA data computation; GMCs is for IgG ELISA data computation.
Table A-11 is summarized GMT according to dosage, patient age and serogroups
Figure A200580048657D00552
Figure A200580048657D00561
The dose study of Phase I in the baby
This clinical trial is that non-blind property, open label, the dosage of the TetraMenD vaccine of 3 kinds of dosage levels progressively increases test, and described vaccine administration is in the participant of 3 age groups.In Phase I, 90 healthy babies (6-12 week) are accepted 3 injections of the TetraMenD vaccine of single dose level.
Baby participant accepts the TetraMenD injection when 2 months (the 1st injection), 4 months (the 2nd injection) and 6 months (the 3rd injection).On 2 time points, obtain to be used for the serum sample of serological analysis: the 6th month (back 2 months of the 2nd injection) and 7th month (back 1 month of the 3rd injection).About SBA at meningococcal polysacharide serogroups A, C, Y and W-135, and by the sample at all acquisitions of elisa assay of the IgG antibody of these identical serogroupss.SBA and IgG ELISA about all serogroupss find to be summarized in hereinafter.The result is summarized in the following table.
Table A-14: Phase I (baby)-reach when 6 months (before the 3rd administration) and 7 months (after the 3rd the administration) ratio (meeting scheme colony) of SBA threshold value according to the TetraMenD dosage level
Figure A200580048657D00591
N: each time point (6 months; 7 months) go up obtainable participant's number
Table A-15: Phase I (baby)-according to TetraMenD dosage level SBA and IgG ELISA result (meeting scheme colony) among the baby when 6 months (before the 3rd administration) and 7 months (after the 3rd administration)
Figure A200580048657D00592
Table A-15: Phase I (baby)-according to TetraMenD dosage level SBA and IgG ELISA result (meeting scheme colony) among the baby when 6 months (before the 3rd administration) and 7 months (after the 3rd administration)
Figure A200580048657D00601
Table A-15: Phase I (baby)-according to TetraMenD dosage level SBA and IgG ELISA result (meeting scheme colony) among the baby when 6 months (before the 3rd administration) and 7 months (after the 3rd administration)
N: appreciable participant's number
*GMTs is for the SBA data computation; GMCs is for IgG ELISA data computation.
Table A-16 has presented by patient age and the generalized GMT of serogroups
Figure A200580048657D00612
The paediatrics vaccine of in the baby, following TetraMenD to use
The baby accepts conventional paediatrics vaccination according to present ACIP suggestion and local practice usually.In this research, the baby accepts to follow the vaccinated TetraMenD of paediatrics.DTacP
Figure A200580048657D00631
And Hib 2,4 with use 6 months the time.Can give IPV or OPV; IPV uses (2 with 4 months the time) with the 1st time of TetraMenD and the 2nd injection.Give hepatitis B vaccine according to the locality practice; Hepatitis B vaccine uses in the time of 2 months some participant, but uses for the participant of any 4 months or 6 months.Carrying out in the process of current baby's stage of testing, Permitted and received the ACIP suggestion of using about routine.In the acceptance during of single participant under the background of current test 4 months and 6 months
Figure A200580048657D00634
The antibody response of the paediatrics vaccine antigen that assessment is used at routine when 6 months and 7 months.The result is summarized in the table separately.
The baby who participates in current test is 2,4 and accept DTacP and PRP vaccine 6 months the time; The 3rd the back blood draw of carrying out 7th month in 1 month of injection at these vaccines.For in these vaccine antigens (diphtheria, tetanus, Whooping cough FHA, Whooping cough PT and PRP) each, observed antibody horizontal confirms there is not statistically-significant difference in 3 TetraMenD dosage groups (all p values〉0.05).(referring to Table A-17).
Under the background of current test, IPV uses when 2 months and 4 months.In the 2nd back blood draw of carrying out 7th month in 3 months of injection of IPV.For 1 type poliomyelitis and 2 type poliomyelitis, the ratio of observed GMTs, NA 〉=1:4 and the ratio of NA 〉=1:8 confirm there is not statistically-significant difference in 3 TetraMenD dosage groups (all p values〉0.05).At least 95.0% ratio by NA 〉=1:8 confirms at the poliomyelitic protection of 1 and 2 types in all 3 TetraMenD dosage groups.For 3 type poliomyelitis, the GMTs in 1 μ g, 4 μ g and the 10 μ g group is respectively 562.7,164.0 and 113.3.Difference in the 3 type poliomyelitis GMTs group be statistics significant (p=0.001, ANOVA).Yet, the ratio of all 3 TetraMenD dosage groups by NA 〉=1:8 confirm at the poliomyelitic protection of 3 types (be respectively 100.0%[22/22], 100.0%[21/21] and 94.1%[16/17]).These ratios are not (p=0.283, Fisher rigorous examination) different on the statistics.In addition, the GMTs of observed 3 poliomyelitis serotypes is present in the open scope well behind 2 doses of IPV of the IPV vaccination timetable 2 that adopts in this test and 4 months.
Behind nearest Hepatitis B vaccination vaccine, carry out 7th month blood draw minimum 5 months the time.By the observed hepatitis B surface antibody level of GMT and 〉=ratio of 10mIU/mL confirms not have statistically-significant difference (2 p value 〉=0.649) in 3 TetraMenD dosage groups.It should be noted that in current test does not have the baby to accept hepatitis B vaccine when going to a doctor in 6th month, and this is the recommended age the earliest for the administration for the third time of this vaccine.This can explain hepatitis B surface antibody why tire 〉=7 months big babies' of 10mIU/mL ratio and the initial administration of this vaccine after the announcement scope of detectable antibody consistent, but be lower than after 3 times complete vaccination series desired about the protection antibody level.The result of this research is summarized in down in the tabulation.
Table A-17: Phase I (baby)-in 7 months baby, the follow immunogenicity (meeting scheme colony) of vaccine according to the TetraMenD dosage level
Figure A200580048657D00641
Table A-17: Phase I (baby)-in 7 months baby, the follow immunogenicity (meeting scheme colony) of vaccine according to the TetraMenD dosage level
Figure A200580048657D00651
Table A-17: Phase I (baby)-in 7 months baby, the follow immunogenicity (meeting scheme colony) of vaccine according to the TetraMenD dosage level
Figure A200580048657D00661
*GMT relatively uses the F check.Per-cent relatively uses the Fisher rigorous examination.
+P value＜0.05
1 and 6 month research of embodiment 11 research B in 2-10 year children
This be 2-10 year healthy children at random, active control research, thereby the Menomune of the TetraMenD of competitive list agent and single agent.Extract blood sample when after the 0th day, the 0th day before vaccination the 28th day and 6 months.TetraMenD and Menomune overall security relatively is comparable.The result of this research is summarized in down in the tabulation.
The distribution of SBA-BR antibody titer
Table B-1 has shown when baseline, the 28th day and 6th month the frequency distribution for the SBA-BR antibody titer of every kind of serogroups.
Figure A200580048657D00663
Figure A200580048657D00671
Figure A200580048657D00681
Figure A200580048657D00691
Figure A200580048657D00701
Figure A200580048657D00711
Figure A200580048657D00721
Figure A200580048657D00741
Table B-2 summarizes geometric mean titer (GMT) by subject age and serogroups for TetraMenD
Figure A200580048657D00742
Figure A200580048657D00751
Table B-3 has shown tire participant's the number and the ratio of rising 〉=4 times from 28 days SBA-BR for serogroups A, C, Y and W-135 of baseline to the.For every kind of serogroups, these per-cents are ratio in the TetraMenD group
Figure A200580048657D0075165343QIETU
Higher in the group.The difference of ratio is: be respectively-0.0397 ,-0.0452 ,-0.1092 and-0.0562 for serogroups A, C, Y and W-135.
Table B-3: for the summary of the elementary test of hypothesis that meets scheme colony
Figure A200580048657D00752
N: than tire participant's the number of rising 〉=4 times of baseline.
N: participant's overall number in the colony that uses.
P tAnd P m: respectively from TetraMenD and
Figure A200580048657D0075165343QIETU
Tire participant's the ratio of rising 〉=4 times of the SBA after vaccination of group.
The participant's of SBA antibody titer 〉=32 ratio is summarized among the table B-3 the 28th day the time after vaccination.
Figure A200580048657D00761
*％：n/N。
Figure A200580048657D0076165638QIETU
: after vaccination, tire the 28th day the time 〉=32 participant's number.
Figure A200580048657D0076165630QIETU
: the overall number that in this group, has the participant of effective blood sample the 28th day the time.
The participant's of SBA antibody titer 〉=128 ratio is summarized among the table B-4 the 28th day the time after vaccination.
Table B-4: the participant's of SBA-BR antibody titer 〉=128 per-cent and number (meeting scheme colony) the 28th day time the after vaccination
Figure A200580048657D00771
Table B-4: the participant's of SBA-BR antibody titer 〉=128 per-cent and number (meeting scheme colony) the 28th day time the after vaccination
*％：n/N。
Figure A200580048657D0077165657QIETU
: after vaccination, tire the 28th day the time 〉=128 participant's number.
Figure A200580048657D0077165704QIETU
: the overall number that in this group, has the participant of effective blood sample the 28th day the time.
The participant's that the rising of SBA-BR antibody titer is at least 4 times ratio
The SBA antibody titer was than the participant's of 〉=4 times of baseline risings ratio when table B-5 was presented at the 28th day and 6th month.Accept behind the TetraMenD 28-56 days, most of participants experienced for the SBA-BR antibody titer of the every kind of serogroups that comprises in the vaccine 〉=4 times of risings.Table B-5: the SBA-BR antibody titer is than the participant's of 〉=4 times of baseline risings per-cent and number (meeting scheme colony) when the 28th day and 6th month
Figure A200580048657D00781
Table B-5: the SBA-BR antibody titer is than the participant's of 〉=4 times of baseline risings per-cent and number (meeting scheme colony) when the 28th day and 6th month
Figure A200580048657D00782
*％：n/N。
Figure A200580048657D0078165725QIETU
: than tire participant's the number of rising 〉=4 times of baseline.
Figure A200580048657D0078165735QIETU
: participant's overall number in the colony that uses.
In the time of the 0th day, have undetectable tiring (＜8), in the time of the 28th day, reach raise 〉=4 times participant's ratio of SBA-BR antibody titer
In 2 treatment groups and for all vaccine serogroupss, when baseline, have most of participants that undetectable (＜8) SBA-BR tires and in the time of the 28th day, reach tire rising 〉=4 times of SBA.(table B-6) SBA in the time of the 0th day tires＜and 8, the TetraMenD group, be 86.21%-98.57% from raise in 28 days 〉=4 times participant's ratio of baseline to the; And
Figure A200580048657D00791
In the group 75.00-94.64.
Table B-6: in the time of the 0th day, have undetectable tiring (＜8), reached raise 〉=4 times participant's number and per-cent of SBA-BR antibody titer at the 28th day
Figure A200580048657D00792
*N=tired in the time of the 0th day in every kind of serogroups＜8 and in the time of the 28th day, tire 〉=32 participant's number
N=tired in the time of the 0th day in every kind of serogroups＜8 participant's number
Figure A200580048657D00793
Definite 95% fiducial interval for per-cent
SBA-BR antibody GMTs and average multiple raise
SBAGMTs when table B-7 is presented at after baseline and the vaccination the 28th day and 6th month and the multiple rising of SBA GMTs.
Table B-7: the sBA-BR serology result (meeting scheme colony) after baseline, vaccination when the 28th day and 6th month
Table B-7: the sBA-BR serology result (meeting scheme colony) after baseline, vaccination when the 28th day and 6th month
Figure A200580048657D00801
Table B-7: the sBA-BR serology result (meeting scheme colony) after baseline, vaccination when the 28th day and 6th month
Figure A200580048657D00811
*Tire or the multiple rising its medium multiple rising=tiring in the time of the 28th day/tiring in the time of the 0th day
Figure A200580048657D0081165809QIETU
: the participant's who in calculating, uses overall number.
ELISA IgG for serogroups A, C, W-135 and Y
IgGGMCs and the rising of the multiple among the IgG GMCs when table B-8 is presented at after baseline and the vaccination the 28th day and 6th month.
Table B-8: the IgG serology result (meeting scheme colony) after baseline, vaccination when the 28th day and 6th month
Table B-8: the IgG serology result (meeting scheme colony) after baseline, vaccination when the 28th day and 6th month
Figure A200580048657D00821
*Tire or the multiple rising its medium multiple rising=tiring in the time of the 28th day/tiring in the time of the 0th day
Figure A200580048657D0082165828QIETU
: the participant's who in calculating, uses overall number.
After accepting the vaccination TetraMenD of this research 28-56 days, most of participants' experience raise 〉=4 times for the SBA-BR antibody titer of the every kind of serogroups that comprises in the vaccine.Generally speaking, 77% TetraMenD recipient experiences 4 times of risings of the antibody titer of crossing over all serogroupss.Observe for serogroups Y and to be compared to antibody horizontal before the higher vaccination of C or W-135.This may be with natural on this age to be exposed to serogroups Y more relevant than the more common fact of thinking in the past.The Natural Exposure that the reflection of higher circulating antibody level is nearest, and can reduce the vaccine recipient's who shows 4 times or higher antibody response ratio.This clearly is rendered as like this for serogroups Y replys when with other serogroups comparisons.4 times of risings for serogroups Y are 56.6%, Comparatively speaking, are 73.4%, are 87.7% for serogroups A for serogroup C, and be 91.0% for serogroups W-135.Also observe antibody horizontal before the high vaccination for serogroups A.This may be to be interrupted the result who is exposed to several naturally occurring cross-reacting antigens through the time period that prolongs.
Influence of tiring that is pre-existing in for further assessment and research seroconversion ratio (as raise 4 times vaccine recipient's ratio definition when the antibody titer of tiring before the vaccination for any serogroups＜reach during 1:8) carry out the analysis that separates to the participant of antibody titer＜1:8 before any vaccination in 4 kinds of serogroupss that comprise in the vaccine.By using young rabbit to measure as the SBA in complement source, tire＜1:8 be regarded as representing can not detection level circulating antibody.When the participant uses this standard to assess, to observe behind inoculation TetraMenD, the seroconversion ratio is 98.6% for serogroups A, is 87.9% for serogroup C, is 96.0% for serogroups W-135, and is 86.2% for serogroups Y.
Based on the observation in the new recruit of army, the SBA that Goldschneider proposes end user's complement source measures, and minimum tires 〉=and 1:4 is relevant with protection at the affecting conditions of serogroup C.Yet, because the shortage of the needs of bioassay standardization and people's complement reliable sources proposes young rabbit complement as alternative source.It is more responsive than people complement to young rabbit complement that meningococcus seems, thereby cause the antibody titer of higher measurement.Several authors have proposed to use the rabbit complement to measure, tire 〉=1:128 indicating and protect, and tire＜1:8 indicates at least for the serogroup C susceptible.Although this level may be suitable when the assessment polysaccharide vaccine, it may be inapplicable for conjugate vaccine.The Borrow suggestion; in the experimenter who accepts unit price C conjugate vaccine; described experimenter confirms that SBA tires after the vaccination and is 8-64; use the memory response of the meningococcal polysaccharide vaccine confirmation of the minimizing dosage (10 μ g) that gives behind the some months to show that these individualities also are shielded, its antibody horizontal reaches 〉=1:128.About accept the TetraMenD vaccine for the SBA-BR of every kind of serogroups tire 〉=experimenter's of 1:128 result is presented in the table.When every kind of serogroups that these standard application comprise in vaccine, generally speaking, SBA-BR tired 〉=1:32 after 96.2% participant who accepts TetraMenD reached vaccination, and 90.5% reaches and tires 〉=1:128.Also be used to assess the association of the SBA that uses young rabbit complement and people's complement between measuring from the subclass of the serum of this clinical study, and the result is provided in follow-up study.
Total IgG for serogroup C, Y and W-135 is replied
Figure A200580048657D0084165852QIETU
In the group apparently higher than the group of accepting TetraMenD.Yet, obviously higher in the TetraMenD group for SBA GMT level after the vaccination of serogroups A, C, Y and W-135.Table B-9 provides by serogroups comparison GMC GMT has been tired.
The IgG of the lower level of producing by conjugate produces the observation than the higher levels of fungicidal activity of polysaccharide vaccine, those that strong prompting is better than producing by unconjugated polysaccharide vaccine at the character and the avidity of the antibody response of conjugate vaccine.High-affinity antibody accompaniment functions activity and memory response.This effect is also observed in the research of several announcements.These data acknowledgements TetraMenD is a hyperimmunization originality in 2-10 year children, for each the observed GMTs in the TetraMenD group in 4 kinds of serogroupss be better than in the Menomune group observed those, and tiring of reaching indicated and protected.At last, as if the TetraMenD generation is replied for the higher affinity antibodies of every kind of serogroups that comprises in the vaccine.
In process of the test, monitoring security on 4 particular point in times: reported immediate response (in vaccination 30 minutes), the part of after vaccination, bringing out in first 7 days and systemic reaction, all adverse events and lasting AEs (from the 0th day to the 28th day) and from the 0th day 6th month serious adverse events after the vaccination in time period of 28 days after vaccination.
For all participants, the reaction of bringing out for most of parts of 2 treatment groups is reported as slight and disappears in 3 days in vaccination.The frequency of local reaction is similar for each treatment group.In accepting the group of TetraMenD, at least local reaction of 58.8% report, and accept
Figure A200580048657D00851
Group 58.3% reported identical incident.In addition, intramuscular gives teen-age unit price C CRM 197The experience of conjugate vaccine shows that the local reaction rate is with observed closely similar for TetraMenD in this research.
The AEs of great majority report is not serious, reversible and irrelevant with vaccination.In this research, do not report the new outbreak of bronchial asthma, diabetes or autoimmune disorder.
Month research of embodiment 12 research C in 11-18 year children
Research C be 11-18 year healthy children at random, active control research, since the 0th day, single agent TetraMenD was to single agent
Figure A200580048657D00852
Extract serum when before vaccination the 0th day and the 28th day and analyze, and as the further assessment described among the result serum subclass from the patient.
For all participants, organize the reaction of bringing out most of parts for 2 treatments and be reported as slight and disappear in 2 days in vaccination.The frequency of local reaction ratio in accepting the group of TetraMenD (72.4%) is being accepted
Figure A200580048657D00853
Group (34.7%) in more general.This result is likely because the characteristic rather than the route of administration (intramuscular) of conjugate vaccine (diphtheria carrier proteins).The result of this research is summarized in down in the tabulation.
When table C-1 shows baseline and the 28th day for the frequency distribution of the SBA-BR antibody titer of every kind of serogroups.
Figure A200580048657D00871
Figure A200580048657D00881
Figure A200580048657D00891
Table C-2 has summarized GMT level for TetraMenD by subject age and serogroups
Figure A200580048657D00892
Figure A200580048657D00901
Table C-3 shows tire participant's the number and the per-cent of rising 〉=4 times from 28 days SBA-BR for serogroups A, C, Y and W-135 of baseline to the.For every kind of serogroups, these per-cents are ratio in the TetraMenD group
Figure A200580048657D00902
Higher in the group.
Table C-3: from tire participant's the number and the per-cent of rising 〉=4 times of 28 days SBA-BR of baseline to the
Figure A200580048657D00903
The frequency of SBA-BR antibody titer 〉=32
The participant's of SBA antibody titer 〉=32 ratio is summarized among the table C-4 the 28th day the time after vaccination.
Table C-4: the participant's of SBA antibody titer 〉=32 per-cent and number (meeting scheme colony) the 28th day time the after vaccination
*％：n/N。
: after vaccination, tire the 28th day the time 〉=32 participant's number.
Figure A200580048657D0091170007QIETU
: the overall number that in this group, has the participant of effective blood sample during at the 28th day.
The frequency of SBA-BR antibody titer 〉=128
The participant's of SBA antibody titer 〉=128 ratio is summarized among the table C-5 the 28th day the time after vaccination.
Table C-5: the participant's of SBA antibody titer 〉=128 per-cent and number (meeting scheme colony) the 28th day time the after vaccination
Figure A200580048657D00921
*%: the n/N that is expressed as per-cent.
Figure A200580048657D0092170028QIETU
: after vaccination, tire the 28th day the time 〉=128 participant's number.
Figure A200580048657D0092170031QIETU
: the overall number that in this group, has the participant of effective blood sample during at the 28th day.
The participant's that the rising of SBA-BR antibody titer is 〉=4 times per-cent
The SBA antibody titer was than the participant's of 〉=4 times of baseline risings ratio when table C-6 was presented at the 28th day.
Table C-6: the SBA antibody titer is than the participant's of 〉=4 times of baseline risings per-cent and number in the time of the 28th day
*%: the n/N that is expressed as per-cent.
Figure A200580048657D0093170057QIETU
: than tire participant's the number of rising 〉=4 times of baseline.
Figure A200580048657D0093170103QIETU
: participant's overall number in the colony that uses.
In the time of the 0th day, have undetectable tiring (＜8), in the time of the 28th day, reach raise 〉=4 times participant's per-cent of SBA-BR antibody titer
In 2 treatment groups and for all vaccine serogroupss, when baseline, have most of participants that undetectable (＜8) SBA tires and in the time of the 28th day, reach tire rising 〉=4 times of SBA.In the time of the 0th day SBA tire＜8, be 98.17%-100.0% (table C-7) from raise 〉=4 times participant's ratio of baseline to the 28 days.
Table C-7: in the time of the 0th day, have undetectable tiring (＜8), in the time of the 28th day, reach raise 〉=4 times participant's number and per-cent of SBA-BR antibody titer.
*N=tired in the time of the 0th day in every kind of serogroups＜8 and in the time of the 28th day, tire 〉=32 participant's number
N=tired in the time of the 0th day in every kind of serogroups＜8 participant's number
Figure A200580048657D00942
Definite 95% fiducial interval for per-cent
SBA-BR antibody GMTs and average multiple raise
SBA GMTs and the multiple among the SBAGMTs when table C-8 is presented at after baseline and the vaccination the 28th day raise.
Table C-8: the SBA serology result (meeting scheme colony) after baseline and vaccination the 28th day the time
Figure A200580048657D00951
*Tire or the multiple rising its medium multiple rising=tiring in the time of the 28th day/tiring in the time of the 0th day
Figure A200580048657D0095170209QIETU
: the participant's who in calculating, uses overall number.
ELISA IgG for serogroups A, C, W-135 and Y
IgG GMCs (μ g/mL) and the rising of the multiple among the IgG GMCs when table C-9 is presented at after baseline and the vaccination the 28th day.
Table C-9: the IgG serology result (meeting scheme colony) after baseline and vaccination the 28th day the time
*Tire or the multiple rising its medium multiple rising=tiring in the time of the 28th day/tiring in the time of the 0th day
Figure A200580048657D0096170225QIETU
: the participant's who in calculating, uses overall number.
ELISA IgM for serogroups A, C, Y and W-135
IgM GMCs and the multiple among the IgMGMCs when table C-10 is presented at after baseline and the vaccination the 28th day raise.Table C-10: the IgM serology result (meeting scheme colony) after baseline and vaccination the 28th day the time.
*Tire or the multiple rising its medium multiple rising=tiring in the time of the 28th day/tiring in the time of the 0th day
Figure A200580048657D0097170245QIETU
: the participant's who in calculating, uses overall number.
Accept behind the vaccination TetraMenD of this research 28-56 days, most of participants' experience raise 〉=4 times for the SBA-BR antibody titer of the every kind of serogroups that comprises in the vaccine.Generally speaking, the 90.7%TetraMenD recipient experiences 4 times of risings of the antibody titer of crossing over all serogroupss.Observe than antibody horizontal before the higher vaccination of C or W-135 for serogroups Y.This may be relevant with the following fact: serogroups Y is that to follow the most common serogroups of aggressive meningococcal disease in this age group of the U.S. and Natural Exposure at present may be more common in this serogroups.The Natural Exposure that the reflection of higher circulating antibody level is nearest, and can reduce the vaccine recipient's who shows 4 times or higher antibody response ratio.When with other serogroups comparisons, for replying, serogroups Y it seems it is like this.4 times of risings for serogroups Y are 81.8%, Comparatively speaking, are 91.7% for serogroup C, and are 96.7% for serogroups W-135.Also observe antibody horizontal before the high vaccination for serogroups A.This may be to be interrupted the result who is exposed to several naturally occurring cross-reacting antigens through the time period that prolongs.
Influence of tiring that is pre-existing in for further assessment and research seroconversion ratio (defined as 4 times vaccine recipient's the ratio of raising when the antibody titer of tiring before the vaccination for any serogroups＜reach during 1:8) carry out the analysis that separates to the participant of antibody titer＜1:8 before any vaccination in 4 kinds of serogroupss that comprise in the vaccine.By using young rabbit to measure as the SBA in complement source, tire＜1:8 be regarded as representing can not detection level circulating antibody.When the participant uses this standard to assess, to observe behind inoculation TetraMenD, the seroconversion ratio is 100% for serogroups A, is 98.1% for serogroup C, is 98.1% for serogroups W-135, and is 98.3% for serogroups Y.
As discussing in another research in the past, based on the observation in the new recruit of army, the SBA that Goldschneider proposes end user's complement source measures, and minimum tires 〉=and 1:4 is relevant with protection at the affecting conditions of serogroup C.Yet, because the shortage of the needs of bioassay standardization and people's complement reliable sources proposes young rabbit complement as alternative source.It is more responsive than people complement to young rabbit complement that meningococcus seems, thereby cause the antibody titer of higher measurement.Several authors have proposed to use the rabbit complement to measure, tire 〉=1:128 indicating and protect, and tire＜1:8 indicates at least for the serogroup C susceptible.Although this level may be suitable when the assessment polysaccharide vaccine, it may be inapplicable for conjugate vaccine.The Borrow suggestion; in the experimenter who accepts unit price C conjugate vaccine; described experimenter confirms that SBA tires after the vaccination and is 8-64; use the memory response of the meningococcal polysaccharide vaccine confirmation of the minimizing dosage (10 μ g) that gives behind the some months to show that these individualities also are shielded, its antibody horizontal reaches 〉=1:128.About accept the TetraMenD vaccine for the SBA-BR of every kind of serogroups tire 〉=experimenter's of 1:128 result is presented in the table.When every kind of serogroups that these standard application comprise in vaccine, generally speaking, SBA-BR tired 〉=1:128 after 99.2% participant who accepts TetraMenD reached vaccination.
In participant's subclass, use standard EILSA mensuration assessment IgG and IgM to reply.After the vaccination, among the TetraMenD recipient for the mean level (ML) of the IgG antibody of every kind of serogroups 2 μ g.It all is very similar replying for the IgM of every kind of serogroups in 2 treatment arms. Replying general ratio for the IgG of serogroup C, Y and W-135 in the group accepts in the group of TetraMenD higher.Yet, in each treatment group, be very similar for SBA GMT level after the vaccination of serogroup C, Y and W-135, table C-11.
Table C-11:IgG and IgM are to the Relative Contribution of total fungicidal activity
*GMC unit is μ g/mL
The IgG of the lower level of being produced by conjugate produces the observation with the fungicidal activity of the similar level of polysaccharide vaccine, those that strong prompting is better than producing by polysaccharide at the character and the avidity of the antibody response of conjugate vaccine.High-affinity antibody accompaniment functions activity and memory response.This effect is also observed in several disclosed researchs.
These data acknowledgements TetraMenD is hyperimmunization originality in teenager colony.As if is what to equate basically for 2 kinds of vaccines for each GMTs in 4 kinds of serogroupss, and tiring of reaching indicating and protect, and TetraMenD produces and replys for the higher affinity antibodies of each serogroups that comprises in the vaccine.
Research D
This research be 18-55 year normal adults at random, active control research, since the 0th day single agent TetraMenD to single agent
Figure A200580048657D00992
Extract serum when before vaccination the 0th day and the 28th day and analyze.
Usually, the security features of TetraMenD is comparable to Menomune, particularly, report about the per-cent of the local reaction (0-7 days) of bringing out, the systemic reaction of bringing out (the 0th day-), spontaneous adverse events (0-28 days), spontaneous remarkable adverse events and the serious adverse events (the 0th day the-the 6th month) of SAEs (the 29th day the-the 6th month) all in 2-3% per-cent about the Menomune report.Result of study is provided in down in the tabulation.
The distribution of SBA-BR antibody titer
When table D-1 shows baseline and the 28th day for the frequency distribution of the SBA-BR antibody titer of every kind of serogroups.
Figure A200580048657D01011
Figure A200580048657D01021
Figure A200580048657D01031
Table D-2 provides by subject age and serogroups and has summarized geometric mean titer (GMT) for TetraMenD.
Figure A200580048657D01032
Figure A200580048657D01041
Table D-3 shows for serogroups A, C, Y and W-135 from tire participant's the number and the per-cent of rising 〉=4 times of 28 days SBA-BR of baseline to the.In TetraMenD group for the number and the per-cent of serogroups A, 1028/1278 (80.4%); C, 1131/1278 (88.5%); Y, 941/1278 (73.6%); And W-135,1142/1278 (89.4%) is comparable to
Figure A200580048657D01052
In the group those, serogroups A wherein, 929/1099 (84.5%); C, 985/1099 (89.6%); Y, 872/1099 (79.3%); And W-135,1036/1099 (94.3%).
Table D-3: tire from raise 〉=4 times participant's number and per-cent of baseline for serogroups SBA-BR *
Figure A200580048657D01061
*Null hypothesis H 0:
Figure A200580048657D01062
-P TetraMenD〉=0.10 couple of H a:
Figure A200580048657D01063
-P TetraMenD＜0.10 check
Figure A200580048657D0106170441QIETU
: n=meets the overall number of participant in the scheme colony than tire participant's the number/N=of rising 〉=4 times of baseline.
Figure A200580048657D0106170504QIETU
: from raise 〉=4 times participant's per-cent of the SBA-BR potency ratio baseline behind the TetraMenD winding kind vaccine.
Figure A200580048657D01066
From The participant's that SBA-BR potency ratio baseline rising behind the winding kind vaccine is 〉=4 times per-cent.
The frequency of SBA-BR antibody titer 〉=32
The participant's of SBA-BR antibody titer 〉=32 ratio is summarized among the table D-4 the 28th day the time after vaccination.
Table D-4: the participant's of SBA antibody titer 〉=32 per-cent and number (meeting scheme colony) the 28th day time the after vaccination
Figure A200580048657D01071
*％：＝n/N
Figure A200580048657D0107170526QIETU
: after vaccination, tire the 28th day the time 〉=number/N of 32 participant: the overall number that in this group, has the participant of effective blood sample during at the 28th day.
The frequency of SBA-BR antibody titer 〉=128
The participant's of SBA-BR antibody titer 〉=128 ratio is summarized among the table D-5 the 28th day the time after vaccination.
Table D-5: the participant's of SBA antibody titer 〉=128 per-cent and number (meeting scheme colony) the 28th day time the after vaccination
Figure A200580048657D01081
*％：n/N。
Figure A200580048657D0108170552QIETU
: after vaccination, tire the 28th day the time 〉=128 participant's number.
Figure A200580048657D0108170558QIETU
: the overall number that in this group, has the participant of effective blood sample during at the 28th day.
Table D-6 is to the therapeutic action analysis of the GMTs by baseline concomitant variable adjustment: reply difference (meeting scheme colony) from the 0th day to the 28th day tire *
Figure A200580048657D01084
Figure A200580048657D01091
*The antilogarithm of therapeutic action be calculated as 2 therapeutic action (
Figure A200580048657D01092
-TetraMenD) power.
The participant's that the rising of SBA-BR antibody titer is at least 4 times ratio
The SBA antibody titer was than the participant's of 〉=4 times of baseline risings ratio when table D-7 was presented at the 28th day.
Table D-7: the SBA antibody titer is than the participant's of 〉=4 times of baseline risings number and per-cent in the time of the 28th day
Figure A200580048657D01093
*％：n/N。
Figure A200580048657D0109170619QIETU
: than tire participant's the number of rising 〉=4 times of baseline.
Figure A200580048657D0109170627QIETU
: the number that in every kind of serogroups, extracts the participant of blood.
In the time of the 0th day, have undetectable tiring (＜8), in the time of the 28th day, reach raise 〉=4 times participant's ratio of SBA-BR antibody titer
Table D-8 had undetectable tiring (＜8) when being presented at the 0th day, reached the participant's of 〉=4 times of SBA-BR antibody titer risings ratio in the time of the 28th day.In 2 treatment groups and for all vaccine serogroupss, when baseline, have most of participants that undetectable (＜8) SBA tires and in the time of the 28th day, reach tire rising 〉=4 times of SBA.In the time of the 0th day SBA tire＜8, the TetraMenD group, be 90.7%-100.0% from raise 〉=4 times participant's ratio of baseline to the 28 days, and
Figure A200580048657D01101
In the group 96.9%-99.3%.
Table D-8: in the time of the 0th day, have undetectable tiring (＜8), in the time of the 28th day, reach raise 〉=4 times participant's ratio of SBA-BR antibody titer
Figure A200580048657D01102
*％：n/N。
Figure A200580048657D0110170646QIETU
: in every kind of serogroups, in the time of the 0th day, tire＜1:8 and in the time of the 28th day, tiring 〉=participant's of 1:32 number
Figure A200580048657D0110170657QIETU
: in every kind of serogroups, in the time of the 0th day, tire＜participant's of 1:8 number.
SBA GMTs and the multiple among the SBAGMTs when table D-9 is presented at after baseline and the vaccination the 28th day raise.
Table D-9: the multiple of summarizing antibody titer geometric mean (GMT) and GMT by serogroups raises (meeting scheme colony)
Figure A200580048657D01111
*Tire or the multiple rising its medium multiple rising=tiring in the time of the 28th day/tiring in the time of the 0th day
Figure A200580048657D0111170713QIETU
: the number that in every kind of serogroups, extracts the participant of blood. Note: a participant does not finish blood sample for the second time
Figure A200580048657D01113
Based on the approximation calculation of normal distribution 95%CI for GMT.
Accept behind the vaccination TetraMenD of this research 28-56 days, most of participants' experience raise 〉=4 times for the SBA-BR antibody titer of the every kind of serogroups that comprises in the vaccine.
The per-cent that obtains the TetraMenD recipient of 4 times of risings of antibody titer is respectively 80.4%, 88.5%, 73.6% and 89.4% for serogroups A, C, Y and W-135.Observe than antibody horizontal before the higher vaccination of C or W-135 for serogroups Y.This may be relevant with the following fact: serogroups Y is that to follow the most common serogroups of aggressive meningococcal disease in this age group of the U.S. and Natural Exposure at present may be more common in this serogroups.The Natural Exposure that the reflection of higher circulating antibody level is nearest, and can reduce the vaccine recipient's who shows 4 times or higher antibody response ratio.When with other serogroups comparisons, for replying, serogroups Y it seems it is like this.4 times of risings for serogroups Y are 73.6%, Comparatively speaking, are 88.5% for serogroup C, and are 89.4% for serogroups W-135.Also observe antibody horizontal before the high vaccination for serogroups A.This may be to be interrupted the result who is exposed to several naturally occurring cross-reacting antigens through the time period that prolongs.
Influence of tiring that is pre-existing in for further assessment and research seroconversion ratio (defined as 4 times the vaccine recipient ratio of raising when the antibody titer of tiring before the vaccination for any serogroups＜reach during 1:8) carry out the analysis that separates to the participant of antibody titer＜1:8 before any vaccination in 4 kinds of serogroupss that comprise in the vaccine.By using young rabbit to measure as the SBA in complement source, tire＜1:8 be regarded as representing can not detection level circulating antibody.When the participant uses this standard to assess, to observe behind inoculation TetraMenD, the seroconversion ratio is 100% for serogroups A, is 99.4% for serogroup C, is 96.5% for serogroups W-135, and is 90.7% for serogroups Y.
As discussing in another research in the past, based on the observation in the new recruit of army, the SBA that Goldschneider proposes end user's complement source measures, and minimum tires 〉=and 1:4 is relevant with protection at the affecting conditions of serogroup C.Propose young rabbit complement as alternative source, but meningococcus seems more responsive than people complement to young rabbit complement, thereby cause the antibody titer of higher measurement.Several authors have proposed to use the rabbit complement to measure, tire 〉=1:128 indicating and protect, and tire＜1:8 indicates at least for the serogroup C susceptible.Although this level may be suitable when the assessment polysaccharide vaccine, it may be inapplicable for conjugate vaccine.The Borrow suggestion; in the experimenter who accepts unit price C conjugate vaccine; described experimenter confirms that SBA tires after the vaccination and is 8-64; use the memory response of the meningococcal polysaccharide vaccine confirmation of the minimizing dosage (10 μ g) that gives behind the some months to show that these individualities also are shielded, its antibody horizontal reaches 〉=1:128.When all serogroupss that this standard application comprises in vaccine, for serogroups A, C, Y and W-135 accept TetraMenD reach that SBA-BR tires after the vaccination 〉=participant's per-cent of 1:128 is respectively 99.8%, 98.7%, 96.9% and 97.1%.
The Td of embodiment 13 research E in 10-18 year children strengthens research
This research is as by measured in its tetanus and diphtheria ratio of having the acceptable participant who replys in tiring separately, the booster response that has compared tetanus and diphtheria toxoid (Td) in accepting the group that experimental tetravalence meningococcus diphtheria conjugate vaccine TetraMenD follows Td is with replying in the group of accepting Td and placebo.Acceptable replying is defined as, and after vaccination 28 days, raises at least 4 times and have among the experimenter who tires before the predefined high vaccination and raise at least 2 times than baseline than baseline among the participant who tires before having predefined low vaccination.
Measured as at least 4 times participant's the ratio of raising by tiring in every kind of serogroups, relatively when following Td to use in TetraMenD for the antibody response of serogroups A, C, Y and W-135, with replying when TetraMenD used behind the Td vaccine in 28 days.
This be at random, the double blinding of revising, the multiple center trial of active control, 1024 participants assign in one of 2 treatment groups at random altogether: A and B.
The 0th day V1 The 28th day V2 The 56th day V3
Group A BS-1 Td+TetraMenD The BS-2 placebo BS-3
Group B BS-1 Td+ placebo BS-2TetraMenD BS-3
The range of age of selecting 11-17 year is to catch those individualities of the part of vaccination timetable with normally accept the Td vaccine as routine the Childhood.In addition, this range of age differentiated to for development aggressive meningococcal disease highly dangerous, and most likely in a single day the meningococcal conjugate vaccine obtains the vaccination candidate of permission.In order suitably to assess security, adopt the double blinding design of the modification of using placebo.For going to a doctor for the first time, vaccinated nurse's right and wrong blind and on each arm, use vaccine according to scheme; Be TetraMenD (IM) or placebo on the right arm and on left arm, be Td.For going to a doctor for the second time, each treatment group is accepted vaccine on left arm.The nurse of assessment is local and systemic reaction and an adverse events of unwitting monitoring.
The range of age of selecting 11-17 year is to catch those individualities of the part of vaccination timetable with normally accept the Td vaccine as routine the Childhood.In addition, this range of age differentiated to for development aggressive meningococcal disease highly dangerous, and most likely in a single day the meningococcal conjugate vaccine obtains the vaccination candidate of permission.
Extract the blood sample (5mL whole blood at least) that is used for the serology test after the 0th day (baseline) before vaccination and the vaccination for the first time the 28th day the time.The participant being carried out the 3rd time in back 28 days the 2nd prescription on individual diagnosis draws blood.On each these time point, measure meningococcus serogroups A, C, Y and the W-135 of serum, diphtheria antibody and anti-tetanus antibody.
In order to assess the antibody function among the TetraMenD recipient, measure all and can obtain the SBA (SBA-BR) of sample at the use children rabbit complement of every kind of vaccine serogroups.Immunology terminal point is tire participant's the ratio of rising 〉=4 times of SBA-BR in each treatment group.The diphtheria antibody horizontal is measured by the ability that test sera protection Vero cell is not attacked by diphtheria toxoid.The anti-tetanus antibody horizontal is measured by indirect ELISA (ELISA).
This research will as by measure from the GMTs that accepts the participant of potion TetraMenD among the early stage research research C for serogroups A, C, Y and W-135 antibody response at TetraMenD, compare with the replying of participant of the TetraMenD that accepts to follow Td and after the Td vaccination, used in 28 days.Obtain to be used for the serum mark product of serological analysis after baseline before vaccination (the 0th day) and the vaccination when the 28th day (window :+28 days) and 6 months.28 days metering needles are to the antibody titer of Toxoid,tetanus and diphtheria toxoid (Td) vaccine before vaccination and after the vaccination.
Measure before the vaccination and after the vaccination 28 days all obtainable serum samples for the SBA-BR antibody titer of Neisseria meningitidis serogroups A, C, Y and W-135.Generally speaking, the security features of group A and group B is comparable.The result of this research is summarized in down in the tabulation.
Table E-1 summarizes the GMT level by subject age and serogroups.
Table E-2 is raise participant's the number and the ratio of at least 4 times or 2 times of tetanus and diphtheria antibody when being presented at the 28th day.
Figure A200580048657D01161
Tetanus and diphtheria antibody titer and SBA antibody titer for serogroups A, C, Y and W-135
Table E-2 is raise participant's the number and the ratio of at least 4 times or 2 times of tetanus and diphtheria antibody when being presented at the 28th day.The difference of ratio is: be respectively 2.78 and-2.73 for tetanus and diphtheria.
Figure A200580048657D01171
Table E-3 when being presented at the 28th day at raise at least 4 times participant's number and ratio of the antibody titer of serogroups A, C, Y and W-135.
Figure A200580048657D01172
Table E-4 had the participant's that high diphtheria and tetanus tires number when being presented at baseline and had the participant's of 2 times of risings number and ratio in the time of the 28th day.
Figure A200580048657D01173
Table E-5 had the participant's that low diphtheria and tetanus tires number when being presented at baseline and had the participant's of 4 times of risings number and ratio in the time of the 28th day.
Figure A200580048657D01181
Table E-6 is presented at number and the ratio of the participant of tetanus and diphtheria antibody titer 〉=1.0IU/ml when following after tetanus that TetraMenD or placebo give and the diphtheria vaccination the 28th day.
Figure A200580048657D01182
Table E-7 be presented at tetanus and diphtheria vaccination (following TetraMenD or placebo to give) back in the time of the 28th day about the geometric mean antibody of tetanus and diphtheria tire (GMTs).
Figure A200580048657D01183
When table E-8 is presented at after the TetraMenD vaccination the 28th day for the SBA-BR geometric mean antibody of serogroups A, C, Y and W-135 tire (GMTs).The GMT ratio is respectively 0.92,0.42,0.39 and 0.32 for serogroups A, C, Y and W-135.
When being presented in Td+TetraMenD, the placebo after the TetraMenD vaccination the 28th day, table E-9 tires (GMTs) for the SBA-BR geometric mean antibody of serogroups A, C, Y and W-135, and from the accordingly result of research MTA02.The GMT ratio is respectively 0.48,0.38,0.34 and 0.39 for serogroups A, C, Y and W-135.
Figure A200580048657D01192
When being presented in Td+ placebo, the TetraMenD group after the TetraMenD vaccination the 28th day, table E-10 tires (GMTs) for the SBA-BR geometric mean antibody of serogroups A, C, Y and W-135, and from the accordingly result of research MTA02.The GMT ratio is respectively 0.53,0.90,0.99 and 1.05 for serogroups A, C, Y and W-135.
Figure A200580048657D01201
When table E-11 is presented at after the 0th day and the TetraMenD vaccination the 28th day according to serogroups for the SBA-BR antibody titer that meets scheme colony (the SBA-BR antibody titer＜8-1024) that distributes.When showing E-12 simultaneously and being presented at after the 0th day and the TetraMenD vaccination the 28th day according to serogroups for the SBA-BR antibody titer that meets scheme colony distribute (SBA-BR antibody titer 2048-524288).Table E-13 provide other data.
Figure A200580048657D01202
Figure A200580048657D01211
Figure A200580048657D01231
Carry out correlative study to be evaluated at security and the immunogenicity of following the MCV-4 vaccine of licensed-in Td vaccine administration among the healthy teenager of 10-17 year.In brief, 10-17 year healthy person (12.9 years old mean age) is accepted TetraMenD (MCV-4)+Td in the multicenter random test, perhaps follows (n=509) or in one month separately go to a doctor (n=512) at interval.After vaccination, collect when the 8th day and 28 days for separately or follow the safety evaluation of 2 kinds of vaccines that give.4 weeks passed through at diphtheria and tetanic antibody titer before vaccination and after the vaccination, and assessed immunne response at the serum bactericidal activity (SBA) of meningococcus serogroups.Be similar to the experimenter's that gives Td+TetraMenD about the experimenter's that gives Td separately security features.The concomitant administration of Td+TetraMenD is not disturbed the immunne response at diphtheria and tetanus toxoid.Reply at the SBA of 4 kinds of serogroupss and to be summarized among the table E-14 that shows below.
Table E-14: the SBA at 4 kinds of serogroupss replys
Figure A200580048657D01241
Originally studies show that being applied in jointly in the test subject of Td and TetraMenD is safe with well tolerable.The concomitant administration of TetraMenD+Td does not have disadvantageous effect to the immunne response at tetanus and diphtheria toxoid.When MCV-4 and Td use jointly, strengthen at the immunne response of serogroup C, Y and W135 polysaccharide.Observed enhanced immunne response is astonishing and unexpected in this research.
Embodiment 14 uses the serum sterilizing of young rabbit complement and end user's complement to measure relatively for Neisseria meningitidis serogroup C, W-135 and Y
Result that the SBA-BR for serogroup C, W-135 and Y that is used for relatively obtaining in this research from the subclass of the serum sample of research A Phase I tires and SBA-HC tires.The experimenter of selected current test is at least 2 years old but also there be not 11 years old, and everyone is assigned in one of 2 vaccine group at random.After baseline (before the vaccination) and vaccination, collect from about 5mL whole blood of each experimenter the 28th day the time.Collecting centrifugal blood sample in 4 hours from the experimenter.Serum is removed grumeleuse, be transferred in the freeze pipe of mark and be stored in the refrigerator of-20 ℃ or colder monitoring temperature.The paired sera that all samples that uses in current report analysis all obtains from first experimenter who is selected in clinical study, and it has the test planned to finish of enough serum volumes.All samples is all from the experimenter of 2 years old and 3 years old, except single 4 years old exception.In the classification of purpose treatment, 2 experimenters are arranged.In these one from TetraMenD vaccine group (the 28th day sample was collected the 24th day the time after the vaccination), also have one from
Figure A200580048657D01242
Vaccine group (the 28th day sample was collected in the time of the 9th day after the vaccination).
Screen young rabbit complement (Pel-in advance
Figure A200580048657D01243
Clinical Systems LLC, Brown Deer, WI, production code member 31038) suitability in each serogroups specific assay.Standard about suitability comprises consistent with the SBA-BR test result of using the rabbit complement batch serum sample for the qualification group (in 2 times of dilutions) that qualifies in the past.Also use the predetermined standard of tiring that satisfies reference serum and control sample.The 2.5ml rabbit complement of aliquots containig is stored in-70 ℃ or lower until preparing use.Aliquots containig is thawed once and is used or abandon.
Serum from selected experimenter screens meningococcemia polysaccharide IgG and IgM level by ELISA, and test function antibody is originated with the potential complement that discriminating is used for SBA-H in SBA-BR.The standard that is used for selecting people's complement to originate of establishing is following: (1) is when when SBA-BR measures, lack detectable antibody, when (2) in mensuration, being used as the complement source, lack the inherent fungicidal activity, (3) use separate outer Dr.Ray Borrow laboratory determine before the serum of negative test result, when originating as complement with one group of negative control, the acceptable repeatability performance of one group of 24 sample is used in acceptable performance and (4).The different experimenters of the exogenous complement source form that in every kind of serogroups specific assay, uses.Do not find that the complement source is to working above a kind of serogroups.Similarly, 3 kinds of single donor/serogroupss of complement source form that in SBA measures, use.
Serogroup C
The serum (for IgG and IgM all less than 0.5 μ g/ml) that has acceptable low ELISA value from several experimenters has confirmed fungicidal activity.
Serogroups Y
Be selected from the experimenter of selected collection scheme for the complement source of serogroups Y SBA-H.Serum from the complement source shows low-level serogroups Y IgG and IgM antibody by ELISA, and negative in the SBA-BR assay method.Serum from these sources does not show the inherent fungicidal activity when using in SBA.
Serogroups W-135
Be selected from the experimenter of selected collection scheme for the complement source of serogroups W-135 SBA-H.Serum from the complement source shows low-level serogroups W-135 IgG and IgM antibody by ELISA, and negative in SBA-BR measures.Serum from these sources does not show the inherent fungicidal activity when using in SBA.
Serum sterilizing is measured
In brief, from Center for Disease Control (Centers for Disease Control), Atlanta, GA (CDC) obtains meningococcus serogroup C, Y and W-135 bacterial strain.Serogroup C, Y and W-135 by the fresh work seed batch bottle that thaws prepare the target bacterial strain that uses in mensuration.Each bottle Thayer Martin plate that is used to rule, described plate under 37 ℃ ± 0.5 ℃ at 5% CO 2In be incubated overnight.Second day, to gather in the crops isolating bacterium colony and be used to inoculate the whole surface of fresh Thayer Martin plate with sterile swab, described plate has been heated to envrionment temperature.Plate under 37 ℃ ± 0.5 ℃ at 5% CO 2Middle incubation 4 hours, to obtain to converge a small amount of velum of bacterial growth, described velum is gathered in the crops and is suspended in Dulbecco ' the s PBS+0.1% glucose damping fluid to specified optical density(OD) (absorbancy at 600nm place) with sterile swab.Preparation has the working solution of specifying bacterial concentration, is maintained at envrionment temperature and uses in 30 minutes of preparation in Dulbecco ' sPBS+0.1% glucose damping fluid.
Specimen in 56 ℃ of thermal treatments 30 minutes with deactivation endogenous complement.In the institute of 96 hole microtiter plates is porose, add Dulbecco ' s PBS+0.1% glucose damping fluid, cross over the test sera sample that plate distributes 2 times of serial dilutions subsequently, stay last 2 row holes as complement and serum control wells.Row on each plate comprise complement row ([row 11]-serum /+complement) and serum contrast row ([be listed as 121+ serum/-complement).
The fresh complement that thaws is mixed with the bacterium of working concentration, and with mixture be assigned to microtiter plate except that the serum control wells the institute porose in.The bacterium of not adding complement is assigned in the serum control wells.Plate is capped and places oscillator plate to move on to 37 ℃ ± 0.5 ℃ CO last 1 minute subsequently 2In the incubator.The incubation time is 90 minutes for the serogroups A assay plate, and is 60 minutes for serogroup C, Y and W-135 assay plate.Behind the incubation, 100 μ l in 50 ℃ ± 1 ℃ agarose coating substratum add carefully institute porose in, avoid forming bubble.After 10 minute time period, follow the microtiter plate lid crack to avoid forming moisture at ambient temperature, plate is capped and moves to drying (not adding moisture) 5% CO 2Descended 20 ± 4 hours at 37 ℃ ± 0.5 ℃ in the incubator.Behind the incubation, count the bacterial colony number in every hole.Calculating is for fall average number and be divided into half to obtain at T of every pore fungi in complement control hole 0The time 50% the survival.
The sterilization of every kind of unknown serum tire be expressed as with at T 0The time 50% survival value compare the serum dilution final reciprocal that generation 〉=50% kills.The initial dilution of sample is the 1:8 dilution in SBA-BR.For SBA-H, be reduced to the 1:4 dilution described in initial dilution such as the original mensuration.
The SBA-BR operation of measuring about serogroups A described herein and stdn SBA operation (CDC) and as at Manchester public health laboratory (Public HealthLaboratory Services), meningococcus reference unit (Meningococcal ReferenceUnit), Manchester, the comparison of the SBA operation that UK (PHLS) carries out provides as institute among the table 14-1.
Table 14-1: the serum sterilizing assay method relatively
AvP-US CDC PHLS
Freezing original seed Greaves Soln w10% glycerine Greaves Solnw10% glycerine Freezing in glycerine (15%) nutrient solution
Sterilizing buffering liquid Dulbecco ' the s that contains 0.1% glucose Dulbecco ' the s that contains 0.1% glucose The Geys that contains 0.5% BSA
The overnight growth substratum Thayer Martin(MR0232) Brain heart infusion w/1% horse serum Blood agar w/5% horse blood
The overnight growth condition 37℃w/5％ CO2 37℃w/5％ CO2 37℃w/5％ CO2
Complement Children rabbit (Pel-Freez) Children rabbit (Pel-Freez) Children rabbit (Pel-Freez)
Bacterial strain F8238 F8238 F8238
Measure the growth medium of day Thayer Martin(MR0232) Brain heart infusion w/1% horse serum Blood agar
Measure the growth conditions of day 4 hours 37 ℃ of w/5% CO2 4 hours 37 ℃ of w/5%CO2 4 hours 37 ℃ of w/5% CO2
T 0(the every ml) that target is fixed 4000CFU/ml 4000CFU/ml 80,000CFU/ml
The initial initial dilution of serum 1：4 1：4 1：2
Serum is handled 56 ℃ 30 minutes 56 ℃ 30 minutes 56 ℃ 30 minutes
Cumulative volume during incubation step 50μl 50μl 40μl
Serum mixture (volume) as the % sum 50％(25μl) 50％(25μl) 50％(20μl)
Cell suspending liquid % (volume) 25％(12.5μl) 25％(12.5μl) 25％(10μl)
Complement % (volume) 25％(12.5μl) 25％(12.5μl) 25％(10μl)
CFU/ hole (theor.) in r ' xn mixture 50 50 800
Final initial dilution 1：8 1：8 1：4
The serum incubation conditions 37 ℃ of w/5 % CO290 minute 37 ℃ 90 minutes 37 ℃ of w/oCO2 90 minutes
The incubation method of spending the night Add-37 ℃ of w/5% CO2 of 100 μ l TSB agar over lay (in 96 orifice plates) Add 100 μ l TSB Noble agar over lay-37 ℃ w/5% CO2 (in 96 orifice plates) 10 μ l (gradient method) 37 ℃ on agar plate
T 0Condition 37 ℃ 90 minutes (being complement control mean number) 37 ℃ 90 minutes (being complement control mean number) Under 37 ℃ of w/5% CO2, be incubated overnight and paved plate in preceding 90 minutes
Terminal point is tired 50% kills 50% kills 50% kills
From Dr.George Carlone, (the CDC donor-R21654-3430107) acquisition is as the reference serum of the lyophilized powder in bottle, and described reference serum is stored in 2 ℃-8 ℃ until use for CDC.When needing, each personal 0.5ml sterilized water of bottle is aquation and be stored in-80 ℃ as 100 μ l work aliquots containig once more--and 40 ℃.In the stdn SBA-BR for serogroups A, C, Y and W-135, tiring of reference serum is 1:256 ± 12 times dilution when reconstruct under these conditions.The reference serum sample moves 2 times on the different plates of daily cage plate.
For the group-specific rabbit anti-serum of serogroups A, C, Y and W-135 as the lyophilized powder in the bottle available from Difco, it is stored in 2 ℃-8 ℃ until use.When needing, each bottle is with 1ml sterilized water aquation and be stored in-80 ℃ as 50 μ l aliquots containigs once more--and 40 ℃, in SBA, to be used as quality control sample.
It is in full force and effect for tolerance range, dilutability (linearity), specificity and limit of detection that the serum sterilizing of the young rabbit complement of use provided herein is measured the result that (SBA-BR) be used for measuring at the anti-polysaccharide fungicidal activity of the complement-mediated of Neisseria meningitidis serogroup C, Y and W-135 at clinical serum sample.Use same set of serum sample to repeat SBA-H in continuous 5 days and measure (for serogroup C) to establish the precision of measuring.
SBA-BR susceptibility and specific calculating
Use the SBA-H reference point of 1:4 and 1:8 to tire tiring of obtaining among the SBA-BR is divided into true positives (TP) (and false positive [FP]) and true negative (and false negative [FN]).Susceptibility is calculated as TP/ (TP+FN) and specificity is calculated as TN/ (TN+FP).These result calculated are expressed as per-cent.
SBA-BR tires distribution relatively to the SBA of SBA-H
Preceding and the immune back 28 days SBA for serogroup C of immunity tire and are shown in table 1 and 4, are shown in table 2 and 5 for serogroups Y, are shown in table 3 and 6 for serogroups W-135.Summary in the following trifle is before the result's (BR is to H) that relatively obtains from 2 kinds of complements source the immunity and the back SBA analysis of tiring.
The serogroup C SBA distribution of tiring
In the serum sample before 101 immunity, 63 be as SBA-H tire＜1:4 and SBA-BR tire＜the defined feminine gender of 1:8.Sample is SBA-H (＜1:4) feminine gender, but SBA-BR (〉=1:8) positive before 27 immunity.The SBA-BR of use＜1:8 is 30% by the false positive rate of tiring.False positive rate reduces when higher by tiring at SBA-BR, 1:128 when tiring less than 20%, and 1:512 when tiring less than 10%.7 SBA-H (〉=1:4) male sample be SBA-BR (＜1:8) negative.
In the serum, 48 samples are SBA-H feminine genders after immunity, and have only 11 to be the SBA-BR feminine gender.In 11 SBA-BR negative samples, 3 is the SBA-H male.In the conjugate group, in 51 immunity back samples 17 (32%) being arranged is SBA-H feminine genders, but SBA-BR (〉=1:8) male.For the polysaccharide group, in 50 immunity back samples 23 (46%) being arranged is SBA-H feminine genders, (〉=1:8) male but SBA-BR tires.With regard to the positive response in the serum after the immunity, 90 (89%) are arranged in 101 samples is SBA-BR (〉=1:8) male, is SBA-H (〉=1:4) male but have only 53 (52%) in 101.When relatively the SBA that 2 vaccine group are obtained tired (BR is to H), there was significant difference in the positive response rate.For 51 in conjugate group immunity back samples, 33 (65%) being arranged in 51 is SBA-H (〉=1:4) and SBA-BR (〉=1:8) male.SBA tire between (BR is to H) consistence the SBA-BR threshold value tire for=improve when 1:64 and Geng Gao.In in the polysaccharide group 50 the immunity back samples, 17 (34%) being arranged in 50 is SBA-H (〉=1:4) and SBA-BR (〉=1:8) male.SBA tire between (BR is to H) consistence the SBA-BR threshold value tire for=improve when 1:512 and Geng Gao.
The serogroups Y SBA distribution of tiring
Different with the serogroup C preimmune serum, have only before 9 serogroups Y immunity sample be as SBA-H tire＜1:4 and SBA-BR tire＜the defined feminine gender of 1:8.There are 52 to be SBA-H (＜1:4) feminine gender, but SBA-BR (〉=1:8) male before 61 immunity in the samples.The SBA-BR of use＜1:8 is 85% by the false positive rate of tiring.False positive rate reduces when higher by tiring at SBA-BR, 1:256 when tiring less than 15%, and when 1:512 less than 2%.2 samples are SBA-H (〉=1:4) positives, but SBA-BR (＜1:8) negative.
Do not have immunity back serum sample have＜SBA-H of 1:4 tire and＜SBA-BR of 1:8 tires.19 SBA-H (＜1:4) negative sample is the SBA-BR positive (〉=1:8).As serogroup C is pointed out, there are differences in the false negative result ratio.In the conjugate group, 5 (9%) are arranged in 48 samples is SBA-H (＜1:4) feminine genders, but SBA-BR male.In the polysaccharide group, 14 (27%) are arranged in 52 samples is SBA-H (＜1:4) feminine genders, but SBA-BR male.
In the immunity back serum for serogroups Y, tiring for 2 SBA of positive response exists good consistence between (BR is to H).For the total collection of 100 samples, all 100 immunity back samples all have 〉=SBA-BR of 1:8 tires, and have 81 in 100 and have=SBA-H of 1:4 tires.Reply as SBA for serogroup C pointed, with SBA that the polysaccharide group obtains tire (BR is to H) compare, the SBA in the conjugate group tire exist between (BR is to H) better related.In 48 immunity back samples of conjugate group, 43 (90%) is SBA-H (〉=1:4) and SBA-BR (〉=1:8) male.Have only 1 SBA-BR that has less than 1:32 to tire in 48 samples, and that sample is SBA-H (〉=1:4) male.The consistence that SBA tires between (BR is to H) in the polysaccharide group is good inadequately.Have only 38 (73%) to have immunity back in 52 〉=SBA-H of 1:4 tire and=SBA-BR of 1:8 tires.The consistence that SBA tires between (BR is to H) in the serum of the immunity back of polysaccharide group is tired at SBA-BR 〉=improve during 1:128.
The serogroups W-135SBA distribution of tiring
For serogroups W-135, it is negative 54 (54%) being arranged in 100, wherein SBA-H tire＜1:4 and SBA-BR tire＜1:8.Before immunity in the sample, there are in 81 27 to be SBA-H (＜1:4) feminine gender, but SBA-BR (〉=1:8) male.The SBA-BR of use＜1:8 is 33% by the false positive rate of tiring.False positive rate reduces when higher by tiring at SBA-BR, 1:128 when tiring less than 15%, and 1:256 when tiring less than 5%.11 samples are SBA-H (〉=1:4) positives, but SBA-BR (＜1:8) negative.
3 immunity back samples be SBA-H (＜1:4) and SBA-BR (＜1:8) feminine gender.39 immunity back samples are SBA-H (＜1:4) feminine gender, (〉=1:8) male but SBA-BR tires.In the conjugate group, 11 (23%) are arranged in 47 samples is SBA-H feminine genders, but the SBA-BR male.In the polysaccharide group, 28 (53%) are arranged in 53 samples is SBA-H feminine genders, but the SBA-BR male.
Consistence between immunity back SBA-BR and SBA-H tire is comparable to serogroup C, but compare with serogroups Y good inadequately.As for serogroup C and serogroups Y, relatively there is significant difference in the consistence that 2 SBA tire between (BR is to H) during 2 vaccine group.Compare with the polysaccharide group, better for the consistence that 2 SBA of conjugate group tire between (BR is to H).In the immunity back SBA for the conjugate group tires, have 36 (77%) to have in 47 〉=SBA-H of 1:4 tires, and all are SBA-BR (〉=1:8) male.All samples from the conjugate group all has 〉=vaccination of 1:32 after SBA-BR tire.Tire after the immunity for the polysaccharide group, 2 associations between tiring are good inadequately, have only 22 (42%) to have in 53 〉=SBA-H of 1:4 tires, and has 50 (94%) to have in 53 〉=SBA-BR of 1:8 tires.
Table 1. is tired for the SBA-BR in serogroup C comparison SBA-H seropositivity and the feminine gender
Table 2. is tired for the SBA-BR that serogroups Y compares in SBA-H seropositivity and the feminine gender
Figure A200580048657D01331
Table 3. is tired for the SBA-BR that serogroups W-135 compares in SBA-H seropositivity and the feminine gender
The summary that the serogroup C that table 4. is measured by SBA-BR and SBA-H is tired and distributed
Figure A200580048657D01341
1The total number of samples order is 101.
The summary that the serogroups Y that table 5. is measured by SBA-BR and SBA-H tires and distributes
Figure A200580048657D01351
1The total number of samples order is 100.
The summary that the serogroups W-135 that table 6. is measured by SBA-BR and SBA-H tires and distributes
1The total number of samples order is 100.
Susceptibility that SBA-BR and SBA-H tire and specificity are relatively
When carrying out the assessment of susceptibility and specificity between 2 groups are tired, comparison SBA-BR tires and 1:4 and the 1:8 protectiveness of SBA-H are tired.In this is analyzed, use before the immunity and after serum.Use the SBA-H reference point of 1:4 and 1:8 to tire, calculate specificity and susceptibility, and be summarized in the table 7,9 and 10 for all 3 kinds of serogroupss.Susceptibility and specificity analyses for every kind of serogroups are discussed successively.
For serogroup C, tire with respect to the SBA-H of 1:4 and 1:8, for the SBA-BR of 1:8,1:16 and 1:32 by tiring susceptibility greater than 80%.Yet the specificity that these SBA-BR tire is less than 60%.When SBA-BR tired to 1:64, specificity increased to and surpasses 60%, and surpasses 70% when SBA-BR tires to 1:128.Tire for these back 2 SBA-BR, specificity begins to reduce gradually.When tiring to 1:64, susceptibility is 75-78% at SBA-BR, but when SBA-BR tired to 1:128, susceptibility was reduced to 62-65%.Tire at SBA-BR during 1:64, specificity continues to improve, and is respectively 73% to being up to 83% for 1:128 and 1:256.Yet specificity is reduced to less than 20% from 43%.Having SBA-BR that the SBA-BR of the optimum balance between susceptibility and the specificity tires at 1:32-1:128 for serogroup C tires in the scope.Discovery waits the people, 2001. for the susceptibility of serogroup C and the specificity result Santos GF that safely challenges comparison with Clin.Diagn.Lab.Immunol.8:616-623 the result of Huo Deing, it is by serum sample and reagent obtain (table 8) on the same group.With regard to the result of Santos, with the SBA-H potency ratio of 1:4 and 1:8, when SBA-BR tires to 1:64-1:128, observe susceptibility and specific optimum balance.
Table 7. is for susceptibility and the specificity of serogroup C SBA-BR when the protectiveness of SBA-H is tired
Figure A200580048657D01371
Table 8. is as people such as Santos 2001. Clin.Diagn.Lab.Immunol.8:616-623 reported, for susceptibility and the specificity of serogroup C SBA-BR when the protectiveness of SBA-H is tired.
Figure A200580048657D01372
For serogroups Y, the highest for the SBA-BR of 1:8-1:64, but as expected by the susceptibility of tiring, at higher SBA-BR by reducing gradually when tiring.Compare with the result of serogroup C, the specificity result of serogroups Y begins much lower, and does not reach level greater than 50% tiring by SBA-BR of 1:128.Tire for the SBA-BR of serogroups Y susceptibility and specificity optimum balance and to be 1:64-1:256.When 1:256, it is about 55% that susceptibility is gradually reduced to, but specificity increases to about 82-83% from 30% region intermediate.
Table 9. is for susceptibility and the specificity of serogroups Y SBA-BR when the protectiveness of SBA-H is tired
For serogroups W135, the more approaching value that obtains for serogroup C of the value of susceptibility, but one-piece pattern is identical for all 3 kinds of serogroupss.When tiring to 1:8, susceptibility begins very high, and is tiring at SBA-BR 〉=reduce gradually during 1:128.Similarly, specificity is low when beginning when SBA-BR tires to 1:8, and begins to flatten when tiring to 1:256.As viewed for serogroups Y, the SBA-BR that has susceptibility and specific optimum balance for serogroups W135 is 1:64-1:256.
Table 10. is for susceptibility and the specificity of serogroups W-135 SBA-BR when the protectiveness of SBA-H is tired
Figure A200580048657D01391
Table 11 summarized for serogroup C, Y and W135 in the SBA-BR that uses young rabbit complement or people's complement tires with respect to immunity after SBA 4 times the ratio of raising of tiring.To conjugate group TetraMenD and polysaccharide group
Figure A200580048657D01392
Separately carry out this analysis, and 2 group analysis include in table 11.Relatively at 3 serogroupss and for 2 vaccine group inductive sterilization reply, in the 4-4 lift mode, have some observable difference.For every kind of serogroups and for 2 vaccine group answer-mode is discussed successively.
The tire layering compa-ratios of 4 times of risings of the meningococcal polysacharide that table 11. is measured by SBA-BR and SBA-H
Figure A200580048657D01393
This table be shown as by SBA-BR tire, vaccine and serogroups stratified as each measure (from before the immunity and the back 28 days paired samples of immunity) in the tire ratio (and per-cent) of 4 times of risings of the SBA that measures.
For serogroup C, relatively there is consistence closely in 4 times of risings for conjugate group SBA-BR and SBA-H.In this vaccine group, appear to have following trend: when for example tiring 1:32-1:128 after low immunity, 4 times of risings of SBA-H lag behind 4 times of risings among the SBA-BR.But SBA-BR tires after immunity 〉=during 1:256, the experimenter's number that reaches 4 times of risings by SBA-H seems greater than the experimenter's number that reaches 4 times of risings by SBA-BR.Relatively the difference in the rising multiple in 2 complement sources is little, and can tire owing to SBA-BR before the higher immunity, and described tiring changes the per-cent of 4 times of risings that reach by SBA-BR.For the polysaccharide group, SBA-BR is the same tight not as the conjugate group with the consistence of 4 times of risings of SBA-H comparison.Similarly, do not have the 4 times risings of remarkable trend by SBA-H when higher immunity back SBA-BR tires, to become to be compared to the conjugate group observed sensitiveer.
For serogroups Y, very tight by the consistence between 4 times of risings of SBA-BR and SBA-H for 2 vaccine group.Tire with immunity back SBA-BR and to compare, have considerably less immunity back SBA-BR to tire less than 1:32 for arbitrary vaccine group for serogroup C.For this reason, compare with serogroup C, the experimenter's ratio that reaches 4 times of risings for serogroups Y in SBA-BR and SBA-H occurs in higher immunity back SBA-BR when tiring.For serogroups Y, increase to when tiring to 1:128 for ratio SBA-BR after immunity of 4 times of risings of conjugate group=50%, and for serogroup C, tiring by SBA-BR for the conjugate group is 1:32.
Compare with other 2 kinds of serogroupss, for serogroups W135 by the consistence undertighten between 4 times of risings of SBA-BR and SBA-H.As observed, tire limited less than experimenter's number of 1:32 for SBA-BR after arbitrary vaccine group immunity for serogroups Y.Tire in (BR is to H) consistence between 4 times of risings of SBA occurs in SBA-BR and tires 〉=during 1:256.As pointed for serogroup C, 4 times of rising ratios (BR is to H) there are differences in 2 vaccine group, and this is more not obvious for serogroups Y.Compare with (BR is to H) the 4 times of risings of tiring for the SBA of other 2 kinds of serogroupss by polysaccharide vaccine, the consistence that the SBA of serogroups W135 tires between (BR is to H) 4 times of risings in the polysaccharide group is the poorest.
The serum sterilizing that uses young rabbit complement is measured (SBA-BR) to be compared with the SBA (SBA-H) of corresponding end user's complement, be used to measure from the tiring of 2-3 year experimenter's serum sample, described experimenter has inoculated licensed-in tetravalence meningococcal polysaccharide vaccine
Figure A200580048657D01401
Or experimental tetravalence meningococcal polysacharide conjugate vaccine (TetraMenD).Be used for supporting this comparison of SBA by discriminated union for people's complement source of serogroup C, Y and W135.The SBA result who obtains by this comparative study by 2 kinds of methods analysts.In a method, compile for 2 vaccine group by measure before the immunity and after SBA-BR and the SBA-H data that obtain of tiring be used for analyzing.In second method, separately analyze from before the immunity of 2 vaccine group and after tire.One of target of this research is the SBA-BR serum titer of description and negative SBA-H serum titer optimal relevance.Second target is to determine to use the tiring as the association of serogroup C protection of young rabbit complement, described tiring and the optimal relevance of tiring of the positive of end user's complement in mensuration, and extrapolation is tired for the protective fungicide of serogroups Y and W135.Other laboratories disclose the result who attempts to establish for the SBA-BR protectiveness threshold value association of serogroup C.The result of this research will compare with those disclosed results.At last, 4 times of risings of tiring for the SBA that 2 kinds of complement sources are compared before immunity and the back is measured.
Only established tire related with at the protection of disease of SBA for serogroup C.End user's complement is determined the SBA association for the protection of serogroup C in SBA measures.For other serogroupss, will make following hypothesis: will be applied to other serogroupss for the SBA-H association (SBA tires and is 1:4) of the protection of serogroup C.Tire being defined in that relevant SBA-BR tires with 1:4 SBA for serogroup C may be different between the serogroups.Tire with regard to the definition with regard to SBA-BR with negative SBA-H serum titer optimal relevance, before immunity and after serum in the SBA-BR of comparison＜1:8 tire and＜SBA-H of 1:4 tires.Part based on from WHO/CDC about the tire result of study of (BR is to H) of serogroup C SBA relatively, and based in Britain university outburst (University Outbreak)＜SBA-H of 1:4 tires and the recent findings (Jones relevant to the susceptibility of serogroup C disease, G.R., Deng the people, J.Infect.Dis., 181:1172-1175[2000]), the SBA-BR of use＜1:8 tires.
Tire (BR is to H) based on the SBA that produces in this research, the SBA-BR of use＜1:8 is 30% by the false positive rate of tiring for serogroup C.It is as follows to use higher SBA-BR by tiring false positive rate to be improved:=during 1:16, false positive rate is reduced to 26%,=during 1:32, false positive rate is reduced to 24%,=during 1:64, false positive rate is reduced to 22%,=during 1:128, false positive rate is reduced to 18%,=during 1:256, false positive rate is reduced to 14%, and=during 1:512, false positive rate is reduced to 2%.SBA-BR is enough to improve the tolerance range that definition feminine gender that the SBA-H corresponding to＜1:4 tires is tired really by the increase of tiring, however when the susceptibility of using higher SBA-BR difference positive response and feminine gender when tiring to reply much lower.The susceptibility of data presentation SBA-BR in this report is when tiring to 1:8,1:16 or 1:32 the highest (81-84%).When SBA-BR tired greater than 1:32, susceptibility was reduced to and is lower than 80%.Yet when tiring to 1:8,1:16 or 1:32, the specificity of mensuration is minimum, is 51-58%.Selecting to tire relevant ending when tiring, between susceptibility, specificity and false positive rate, carry out balance with the feminine gender during people's complement is measured.Specify 1:32 be SBA-BR cause unnecessarily repelling the true positives respondent by tiring.The result of this research shows 〉=it may be that more suitable ending tired that the SBA-BR of 1:16 tires.Based on WHO/CDC research and analyse (＜1:8) and Britain university outburst analyze (＜1:4), think that minimum 2 extent of dilution are protectiveness on this is tired.
For serogroups W135 and Y, be similar to serogroup C disease and the sterilization feminine gender of corresponding people's complement in measuring of tiring by supposition bactericidin protection and tire and derive in the mensuration protectiveness by the discriminating of tiring.With compare for 30% of serogroup C, very high for the SBA-BR of serogroups Y use＜1:8 by the false positive rate of tiring, be 85%.Yet,, increase SBA-BR and reduced false positive rate by tiring as serogroup C.When tiring to 1:16, false positive rate is reduced to 84% at SBA-BR, and when 1:32, false positive rate is 75%, when 1:64, false positive rate is 61%, and when 1:128, false positive rate is 38%, when 1:256, false positive rate is 13%, and when 1:512, false positive rate is 2%.Although compare with serogroup C, begin for the false positive rate of serogroups Y much higher, by tire into=during 1:128, the false positive rate of measuring for 2 kinds of serogroupss becomes and can compare very much.Yet this type of the high threshold value that may exaggerate for positive response of tiring of ending is tired.Based on susceptibility and specificity analyses, susceptibility is in SBA-BR maximum when tiring to 1:8-1:32, and wherein susceptibility is 95-98%.Yet as serogroup C, specificity is corresponding lower when these SBA-BR tire, and is 11-18%.When the highest inferior SBA-BR tired 1:64, susceptibility was reduced to 88% from 95%, but specificity sharply increases to 35%.In serogroups Y measures＜the best feminine gender in measuring corresponding to people's complement that seems by tiring of 1:64 tires.
For serogroups W135, be 33% at the false positive rate of SBA-BR when tiring to＜1:8, this is similar to serogroup C.As pointed for serogroup C and Y, when tiring, false positive rate is reduced to lower level at higher SBA-BR.At SBA-BR when tiring to 1:16, false positive rate is reduced to 32%, and when 1:32, false positive rate is reduced to 28%, when 1:64, false positive rate is reduced to 26%, and when 1:128, false positive rate is reduced to 12%, when 1:256, false positive rate is reduced to 4%, and when 1:512, false positive rate is reduced to 0%.When susceptibility is tired to 1:8-1:64 at SBA-BR the highest (86%-81%).As expected, specificity is minimum (46%-52%) when the SBA-BR of this scope tires.Although Y compares with serogroups, begin much lowerly for the false positive rate of serogroups W135, best feminine gender in measuring corresponding to people's complement is tired is＜1:64 by tiring.
Identified tiring that people's complement of feminine gender in measuring corresponding to to(for) 3 kinds of serogroupss tires, considered that the threshold value that the SBA-BR of positive response analysis on these levels tires tires.For serogroup C, for the threshold value of positive response tire into=1:16, for serogroups Y, for the threshold value of positive response tire into=1:64, and for serogroups W135, for the threshold value of positive response tire into=1:64.Seem to tire for the SBA-H of serogroup C with respect to 1:4 or 1:8, 〉=the threshold value SBA-BR of 1:128 tires to provide and reaches the good assurance that protectiveness is tired, and relevant with effect SBA-H for the serogroup C 1:4 that tires.This threshold value is tired and is well compared for the WHO/CDC data set of serogroup C with for the analysis that the data set of Santos carries out.As pointed in these 2 researchs, tire=1:128 highly indicating and protecting, and also can be protectiveness but tire less than the SBA-BR of 1:128.For WHO/CDC and Santos data set, the SBA-BR of 1:8,1:16,1:32,1:64 tires and is called ambiguous tiring.Because the data set that presents for this paper is tired less than the SBA-BR of 1:16 for serogroup C and is considered as feminine gender, so ambiguous the tiring of Fen Xiing is 1:16-1:64 hereto, this is the subclass of other 2 researchs.In all these were analyzed, SBA-BR tire with SBA-H (1:4 or the 1:8) that tire relatively, described SBA-H tired and nineteen sixty, to withhold the natural protected data of collection relevant.
Recently, paid go out to make great efforts to make for Britain's efficacy data of unit price C conjugate directly with SBA-BR tire related (Miller E waits the people, 2002, Vaccine20:S58-S67).In current the analysis, find 〉=1:8 with=2 SBA-BR of 1:128 tire well relatedly with the efficacy data about 15-17 year experimenter of having collected so far, described experimenter inoculates potion unit price C conjugate.Yet, when Miller and colleague organize (12-30 month big) when carrying out same analysis for the child who has just learnt to walk, they find 〉=SBA-BR of 1:8 tire and effect between very good consistence, but tire at SBA-BR=during 1:128, the consistence undertighten.Miller proposes other data in the 13rd the pathogenic Neisseria gonorrhoeae meeting in the world of in September, 2002 1-6 day Norway Oslo, wherein reaching SBA-BR in 1 month after the vaccination tires 〉=experimenter's effect of the prediction of 1:64, outside 95% fiducial interval of the observed effect of age group hereto.SBA-BR in the equivocal zone of these data help supports following idea: 1:8-1:64 tires the assurance of protection can be provided.Based on the analysis of this research, the SBA-BR of 1:16-1:64 tires may provide assurance at the protection of serogroup C equally.
The specificity that serogroups Y measures when SBA-BR tires to 1:64 has only 35%, but when increasing to 1:128 and 1:256 by tiring, specificity increases to 59% and 84% respectively.The threshold value of 〉=1:256 is tired and is provided at the good assurance of tiring for the protectiveness of 1:4 and 1:8 in people's complement mensuration.Yet, for serogroups Y susceptibility and specificity balance better when threshold value is tired to 1:128.With compare with the tire SBA-BR scope of tiring of relevant corresponding 1:16-1:64 of SBA-H protectiveness for serogroup C, be the SBA-BR of the 1:64-1:128 ambiguous scope of tiring of scope representative of tiring for serogroups Y.
Serogroups W-135 when SBA-BR tires to 1:64 measures has 52% specificity, but increases to 1:128 and 1:256 along with tiring, and specificity increases to 64 and 77% respectively.As serogroups Y, 〉=threshold value of 1:256 tires and is provided at the good assurance that people's complement is tired for the protectiveness of 1:4 and 1:8 in measuring.As serogroups Y, for serogroups W135 susceptibility and specificity balance better when threshold value is tired to 1:128.With compare with the tire SBA-BR scope of tiring of relevant 1:16-1:64 of SBA-H protectiveness in this research for serogroup C, be that the SBA-BR scope of tiring of 1:64-1:128 is represented ambiguous scope for serogroups W135.
Calculating 4 times raise and the use 2 kind mensuration of sterilization in tiring for every kind of serogroups separately analyzes according to vaccine group.Generally speaking, this show for all 3 kinds of serogroupss when higher immunity back SBA-BR tires, detect SBA tire in higher 4 times of risings (BR and H).Tire at SBA according to serogroups and vaccine group and there are differences in 4 times of risings of (BR is to H).For serogroup C, when low immunity back SBA-BR tires, to compare with tiring in the mensuration of using young rabbit complement, 4 times of risings in the mensuration of end user's complement seem lower.Yet when higher immunity back SBA-BR tired, 4 times of risings in tiring seemed higher in the mensuration of end user's complement.As if this pattern advises that the mensuration of end user's complement is more insensitive than the young rabbit complement of use when low immunity back SBA-BR tires, but when high immunity back SBA-BR tired, just in time opposite, that is, the mensuration of end user's complement became sensitiveer.When the sample measured from the polysaccharide vaccine group, this pattern is not obvious.In those samples, when end user's complement in assay method, 4 times of risings in tiring seem lower.Although do not have clear explanation for this observation between the sample type, assay method that this prompting end user complement carries out lacks the susceptibility to the serum sample that comprises the low liter fungicidal activity.
For serogroups Y, there is good consistence in 4 times of risings during SBA tires when relatively 2 complements are originated.Compare with the polysaccharide group, higher slightly for 4 times of risings that conjugate group SBA tires in (BR and H), but difference is so big not as what serogroup C was pointed out.
For serogroups W135, to compare with the result of other 2 kinds of serogroupss, the consistence of 4 times of risings during the SBA of end user's complement or young rabbit complement tires in mensuration for arbitrary vaccine group is different good.4 times of risings that SBA-BR tires are all very good for 2 vaccine group, but compare with other 2 kinds of serogroupss, and the tire ratio of 4 times of risings of SBA-H is lower.4 times of risings by SBA-H in the polysaccharide group are very low, and 4 times of risings in tiring for other 2 kinds of serogroups SBA-H are lower relatively.
4 times of risings during use BR complement SBA tires have been the reference point of the meningococcal polysaccharide vaccine of registration.Recently, paid go out to make great efforts to make the 4 times risings of SBA-BR in tiring be associated with the clinical efficacy of monitoring data after Britain is to the registration of unit price C conjugate (Borrow R waits the people, 2001, Infect.Immun.69:1568-1573).Analyze based on this, the effect of unit price C conjugate in 12-30 month big child who has just learnt to walk has been estimated as 88% (69-95%) at first dose 16 months.Age group hereto, after potion unit price C conjugate vaccine, reaching tire experimenter's the ratio of 4 times of risings of SBA-BR is 89-100%.
SBA-BR provided herein provides the sterilization valence value, and it is comparable to the value that obtains as the complement source in measuring for serogroup C end user complement.Therefore; these SBA-BR tire and determine that the original research at the substitute of the protective immunity of serogroup C meningococcal disease is relevant; and support the extrapolation of clinical effectiveness provided herein, and must go up from those of other laboratory reports for the SBA-BR potency ratio of serogroup C to protection.By with serogroup C model class ratio, the performance of the SBA of end user's complement in determining to reply at the serogroups specificity of serogroups Y and serogroups W-135 capsular polysaccharide, support is used to measure the SBA-BR dependency of tiring at the sterilization of serogroups Y and W-135.
Embodiment 15 is TetraMenD (Menomune in the grownup TM) and Typhim
Figure A200580048657D01451
Concomitant administration
This embodiment has described TetraMenD (Menactra in stage 2b TM) modification, double blinding, security and immunogenicity research in the result that obtains, described TetraMenD gives or follows licensed-in Typhim separately in U.S. 18-55 year healthy person
Figure A200580048657D01452
Vaccine gives.
Typhim
Figure A200580048657D01453
Be obtained commercially in the U.S..Every 0.5ml dosage Typhim
Figure A200580048657D01454
The Vi polysaccharide that comprises 25 μ g purifying, isoosmotic phosphate-buffered saline and 0.25% phenol, described phenol adds as sanitas.Typhim
Figure A200580048657D01461
Vaccine also comprises residual polydimethylsiloxane or fatty acid ester defoamer.Always have selected this research of 945 participants.In brief, the research participant assigns to any one in 2 treatment groups at random; Group A follows when going to a doctor for the first time and accepts Menactra TMAnd Typhim
Figure A200580048657D01462
And the second time after 28 days accept the salt solution placebo when going to a doctor; Group B accepts Typhim when going to a doctor for the first time
Figure A200580048657D01463
Accept Menactra with placebo and when going to a doctor the second time after 28 days TMAlways have 469 participants selected group A and 476 selected group B are arranged.
In this research, 2 main immunogenicity targets are arranged.First target is description and compares at potion Typhim 28 days antibody response after each study group's vaccination of back.If B reaches antibody horizontal from group〉recipient's of 1.0mg/mL ratio deducts difference from the ratio of group A less than 10%, thinks that then participant from group A is at Typhim
Figure A200580048657D01465
Antibody response be similar to from the group B at Typhim
Figure A200580048657D01466
Antibody response.Select anti-Vi antibody〉level of 1.0ug/mL is as main serology terminal point, because this antibody horizontal is considered to be protectiveness.In 2 major objectives second describes and relatively at Menactra TMAmong the recipient after the vaccination 28 days at each SBA antibody response in 4 kinds of serogroupss.The participant is evenly distributed in 2 investigators according to age, sex and race.69% participant is the women, and all experimenters' mean age is 31 years old.In group A, in meeting scheme colony, there are 432 people (92.1%) to meet and comprise standard, B is 439 people (92.2%) for group.
1. immunogenicity result
Use Typhim
Figure A200580048657D01467
After the vaccine inoculation 28 days, reach from 81.6% participant of group A with from 78.5% participant of group B the antibody horizontal of 1.0ug/mL.Result's summary is shown in the following table 1.
Table 1
At Typhim After the vaccination the 28th day time the, Typhoid Vi antibody titer〉participant's the percentage ratio (meeting scheme colony) of 1.0 μ g/mL
Group ATyphim Vi+Menactra Group BTyphim Vi+ placebo
Anti-Vi PS tires〉% of 1.0 μ g/mL (341 people of 418 philtrums) 81.6% (328 people of 418 philtrums) 78.5%
Use the bilateral I class specific inaccuracy of α=0.05 and 10% surplus, when following Menactra TMWhen using at Typhim
Figure A200580048657D01471
Antibody response be similar to Typhim
Figure A200580048657D01472
Respective acknowledgement when giving separately (Fig. 1).Work as Typhim
Figure A200580048657D01473
Give separately or when comprising that with other common traveller's vaccines polysaccharide meningococcus and Hepatitis A Vaccine give, these results are with consistent based on the expectation of the seroconversion ratio of reporting in the document.Fig. 1 has shown the result from these researchs: the Typhim Vi antibody titer of time test in the 28th day in the grownup〉1.0 μ g/mL, 95% CI of % difference (%Typhim Vi+ placebo, Menactra-%TyphimVi+Menactra, placebo).
Fig. 2 shows to come comfortable Menactra to follow Typhim
Figure A200580048657D01474
The ratio that reaches the participant of 4 times of risings of SBA antibody titer among group A when vaccine gives is comparable to Menactra at Typhim
Figure A200580048657D01475
(the meeting scheme colony) that confirms among the group B when using in 1 month after the vaccination.Use the bilateral I class specific inaccuracy of α=0.05 and 10% surplus, when following Typhim
Figure A200580048657D01476
When using at Menactra TMAntibody response be similar to Menactra TMRespective acknowledgement when giving separately (Fig. 3).
2.GMTs
The target of add observing with the participant of the security that separates of relatively more selected healthy adult philtrum and immunogenicity test at Menactra TMThe GMT that replys, with in this test from the Menactra recipient's of 2 study group GMT.The identical standard of in secondary immunogenicity hypothesis, using be applied to this relatively in.In each case, in security and immunogenicity research (MTA09) participant for the GMT of every kind of serogroups with in this test from the Menactra of group A or group B TMThe recipient advantageously compares.Do not have under a kind of situation, surpass 2 for last 97.5% confidence limit (this is equivalent to bilateral 95% confidence limit) of any serogroups GMT ratio.In addition, the participant above 95% confirms that antibody titer is on the protection level of all 4 kinds of serogroupss (Fig. 4) in 2 study group.
Monitoring research participant immediate response of 30 minutes after vaccination, and part during after the vaccination 7 days and systemic reaction originality.Predeclared adverse events comprises that local reaction (for example, erythema, swelling, scleroma and pain) and constitutional symptom is (for example, heating, headache, fatigue, shiver with cold, arthrodynia, anorexia, vomiting, diarrhoea, the epileptic seizures that the through port cavity temperature is measured, do not accommodate rash), this is assessing after each vaccination.These incidents are recorded on the diary card every day, and also collect via telephone interview by the researchist in 8 days after each vaccination.If reported rash, the investigator writes down other details at once on the case report form of separating so.After each vaccination, obtained other not serious, unexpected adverse events by telephone interview in 8 and 20 days.The duration of whole research, report and write down serious adverse events.Studies show that Menactra TMWith other vaccines Typhim for example
Figure A200580048657D01481
Follow to use and be safe and do not cause that the ratio of serious adverse effect obviously increases.
These data acknowledgements ought give or follow travelling vaccine Typhim separately When giving, Menactra TMIn grownup colony hyperimmunization originality.When these data are applied to elementary hypothesis, satisfy all standards.
Compare diphtheria toxoid conjugate vaccine and non-meningococcus control vaccine among the embodiment 16 former children that inoculate unit price meningococcal conjugate vaccine
The double blind trial that present embodiment has been described at random, revised, to be evaluated among the 2-that accepted unit price meningococcus C conjugate vaccine before the anthology test at least one year＜5 years old children, at the antibody response of tetravalence (A, C, Y and W-135) meningococcus diphtheria toxoid conjugate vaccine TetraMenD (Menactra).More specifically, it is measured that present embodiment has been described at least 4 times the participant's ratio of raising as tiring by 28 days serogroup C after the vaccination, relatively in accepting the experimenter of TetraMenD for the SBA antibody response of serogroup C, with accept licensed-in Hib conjugate vaccine (
Figure A200580048657D01482
) contrast participant group in the serogroup C antibody response.All participants have accepted potion unit price meningococcus C conjugate vaccine at least at least one year before going into the anthology test in advance.Control group is accepted Hib conjugate vaccine rather than placebo, to establish the clinical benefit for these participants.
1. comprise and exclusion standard
In order to go into the anthology test, the participant must satisfy all following standards: 1) determine that according to medical history and health check-up the participant is healthy; 2) participant is 2-＜5 years old when vaccination; 3) passed through at least one year from primary vaccination unit price meningococcus C conjugate vaccine; With 4) father and mother/guardian's signed the Informed Consent Form of Ethics Committee approval.Owing to following any reason is got rid of potential participant from test: 1) suffer from serious chronic disease (for example, heart trouble, ephrosis, neuropathy, metabolic disease, rheumatosis etc.); 2) known or suspection infringement immunologic function; 3) in nearest 72 hours, follow or do not follow the acute medical science disease of heating, or when comprising auxillary temperature 〉=37.5 ℃; 4) certified aggressive meningococcal disease history; 5) in nearest 3 months, use immunoglobulin (Ig) or other blood products, or injection or oral reflunomide or other immune tuning treatments (individual that the timetable that oral steroid dosage reduces gradually continues＜7 days can comprise in test, as long as they do not accept to surpass the course of treatment once in nearest 2 weeks before selected) in 6 weeks of research vaccine; 6) in preceding 72 hours of vaccination or the antibiotic therapy in extracting preceding 72 hours of any blood samples; 7) in the selected preceding 28 days time period, accept any vaccine, or in the process of going into the anthology test, arrange to accept any vaccine; 8) in selected the previous year, accept meningococcus C conjugate vaccine; 9) suspection or known super quick to any vaccine component; 10) can not participate in the prescription on individual diagnosis of all arrangements or comply with research method; 11) selected another clinical trial; With 12) In the view of the investigator, can cause any situation of health risk or the assessment of interference vaccine to the participant.
Before vaccination, do not give febrifugee or anodyne (for example Paracetamol, Ibuprofen BP/EP etc.) routinely.The vaccination same day and/or before use anodyne/febrifugee to obtain documentary evidence.8 days observing time, temperature raise or complaint pain or discomfort if the participant experiences, and then allows the febrifugee/anodyne of stdn dosage in the section after vaccination.Following in 8 day observing time of any other pharmacotherapy section used and obtained documentary evidence.
2. the composition of using
A.TetraMenD
The TetraMenD composition of using in current test is by 4 kinds of different meningococcal capsular polysaccharide, and serogroups A, C, Y and W135 form, and described capsular polysaccharide covalent attachment is to diphtheria toxoid albumen.Vaccine is prepared in the physiological saline of aseptic, no pyrogeneous substance, phosphoric acid buffer, does not contain sanitas.Vaccine is prepared as the liquid of clarifying to slight haze in the precharging type syringe of single agent (0.5mL).The meningococcal capsular polysaccharide that is used to prepare the purifying of TetraMenD is at Aventis Pasteur Inc., Swiftwater, and Pennsylvania, the U.S. is used to make licensed-in polysaccharide vaccine
Figure A200580048657D01491
The same material of A/C/Y/W 135.The diphtheria toxoid albumen that uses is to take from the no Thiomersalate intermediate that the U.S. obtains Licensing Methods, described method is at Aventis Pasteur Inc., Swiftwater, Pennsylvania, the U.S. is used to prepare other licensed-in intermediates that comprises the diphtheria toxoid vaccine.Every 0.5mL dosage TetraMenD comprises 16 μ g meningococcal polysacharide antigens (every kind of group-specific polysaccharide antigen [A, C, Y and W135] 4 μ g), is conjugated to 48 μ g diphtheria toxoid albumen.Non-activity composition in every 0.5mL dosage TetraMenD comprises 0.6mg sodium phosphate, 4.4mg sodium-chlor and aseptic no pyrogeneous substance water for injection.
The Hib vaccine of B. puting together (
Figure A200580048657D01492
)
Figure A200580048657D01493
Provide as the freeze dried vaccine product.After thinner reconstruct, every 0.5mL dosage
Figure A200580048657D01494
Being covalently bond to about 30 μ g Toxoid,tetanuss by poly-ribosyl ribitol (PRP) capsular polysaccharide of the Hib phosphoric acid of 10 μ g purifying forms.
Figure A200580048657D01495
Thinner comprises sodium-chlor and water for injection (as sterile saline solution [0.9%]).Explanation according to manufacturers before using prepares and reconstruct
Figure A200580048657D01496
Vaccine.
3. test design and dosage are used
In fact always have selected this research of 103 participants: 52 participants accept TetraMenD, and 51 acceptance
Figure A200580048657D0150172516QIETU
Conjugate vaccine.Qualified participant accepts the single injection of TetraMenD or Hib conjugate vaccine at random.In current test, do not use and follow vaccination.
In brief, when going to a doctor for the first time, TetraMenD uses the precharging type syringe intramuscular administration with 25mm, No. 25 pins in the deltoid muscle of selecting arm as single 0.5mL dosage.Alternately, when going to a doctor for the first time, Vaccine uses the syringe intramuscular administration with 25mm, 25 gauge needles in the deltoid muscle of selecting arm as single 0.5mL dosage.Before injection, clean the injection site with suitable preservatives.Only give specified dosage (0.5mL).Carefully avoid injection liquid is administered in blood vessel and the nerve or near.After the suction,, abandon vaccine so, and repeat this operation, on different sites, use the new dosage vaccine if blood or any suspicious stain in syringe, occur.
The several security parameters of assessment in process of the test.During time period of 30 minutes after the vaccination, monitor each participant's immediate response.In vaccination that night and every day of 7 days subsequently, the father and mother/part that legal guardian's record brings out and the existence of systemic reaction and seriousness and any other the spontaneous adverse events that changes according to participant's healthy state that father and mother/legal guardian describes.During time period of 28 days after the vaccination, differentiate that (summarizing by visit and chart) also write down all spontaneous adverse events and serious adverse events.
Before vaccination, extract the blood sample (5mL whole blood) that is used for the serology test from each participant the 28th day the time after the 0th day (baseline) and the vaccination.On the time point of the 0th day and the 28th day, collect serum and assess serum bactericidal activity and IgG antibody as described herein at meningococcus serogroups A, C, Y and W135.
Each blood sample collect in 4 hours centrifugal.Serum assigns in 3 freeze pipes subsequently and tagging scheme numbering, participant's numbering and abbreviation and sample number into spectrum (V-1 or V-2).Primary sample must have 1.0mL serum at least.Remain serum in some cases and be divided into other reservation aliquots containigs.If serum is less than 1.0mL, so all serum is placed first pipe, and be regardless of in 3 freeze pipes.Serum sample is stored in the refrigerator that maintains 20 ℃ or lower non-frostless, monitoring temperature.
Following table has presented by the generalized participant's configuration of the composition of using.
Table 1 participant disposes (all participants)
Figure A200580048657D01511
*Per-cent based on the participant's number in the security colony of each vaccine group.
Figure A200580048657D01512
In (mistake! Do not find reference source) the middle research colony that limits.ITT: purpose treatment; PP: meet scheme.
Figure A200580048657D01513
Run counter to first according to it and only to count the scheme violator one time.
Hereinafter the table 2 that presents has been summarized participant's demographic feature.The participant uses at 2 by sex, age and race's baseline profile does not have significant difference (p value: be respectively 0.326,0.696 and 0.720) in the group.Participant's age is 24-57 month (mean age: 37.3 months).51.5% the male sex and 48.5% women are arranged.Most of participants are white people's (46.6%) or Aisa people's (41.7%).
Table 2 participant's demography (all participants)
*Use the Fisher rigorous examination to calculate the p value for sex and race, and calculate the p value for the F check among the age use ANOVA
Figure A200580048657D0152172326QIETU
: the participant's who in vaccine group, satisfies condition number.The denominator of per-cent is the overall number of randomized participant in each vaccine group.
4. Immunology Lab method
When being used to assess immunogenic first standard and being after baseline (before the vaccination) and vaccination the 28th day for the SBA-BR antibody response of serogroups A, C, Y and W-135.This standard comprises: 1) from tire participant's the ratio of rising 〉=4 times of 28 days (serogroups A, Y and W-135) SBA-BR of baseline to the; 2) 28 days the distribution of tiring (all 4 kinds of serogroupss) before the vaccination and after the vaccination; 3) 28 days reverse integral distribution curve (all 4 kinds of serogroupss) before the vaccination and after the vaccination; With 4) before the vaccination and after the vaccination 28 days GMTs and from 28 days geometric mean multiple of baseline to the raise (all 4 kinds of serogroupss).When being used to assess immunogenic second standard and being after baseline (before the vaccination) and vaccination the 28th day for the total IgG antibody response (for example using indirect IgG ELISA to measure) of serogroups A, C, Y and W-135.This comprise before the vaccination and vaccination after 28 days GMTs and raise from 28 days geometric mean multiple of baseline to the.All immunologic evaluations all separately carry out for every kind of serogroups (for example, A, C, Y and W-135).
Calculating is from tire participant's the ratio of rising 〉=4 times of 28 days SBA-BR of baseline to the, and 95% CI of these ratios (use normal approximation) (Agresti A., Chap.3.4Large-Sample Confidence Intervals.In:Categorical Data Analysis.NewYork:John Wiley ﹠amp; Sons; 1990. the 54-59 page or leaf).When calculating multiple and raise, for the purpose of analyzing, will be reported as＜value before 8 the baseline vaccination changes 8 into, and be reported as＜value after 8 the vaccination changes 4 into.
When the 0th day and 28 days, draw reverse integral distribution curve for each vaccine group and serogroups.Reverse integral distribution curve shows on the x axle tires more than or equal to the given participant's who tires per-cent.
Supposing tires follows lognormal distribution, so the SBA-BRGMTs when the 0th day and 28 days and be calculated as logarithm about 95% CIs of GMTs at the bottom of 2 (the Smith W. that tires, Mean.In:Armitage P, Colton T, editors.Encyclopedia of Biostatistics, the 3rd volume, Chichester, UK:John Wiley ﹠amp; Sons; 1998. 2487-2488 page or leaf; With Ratnaparkhi M., Lognormal Distribution.In:Armitage P, Colton T, eds.Encyclopedia of Biostatistics, the 3rd volume, Chichester (UK): John Wiley﹠amp; Sons; 1998. the 2349-2351 page or leaf).When calculating GMTs, for the purpose of analyzing will be reported as＜any value of 8 is converted to 4.95% CIs of rising of the calculating geometric mean multiple that SBA-BR tired from the 0th day to the 28th day and geometric mean.
Suppose that concentration follows lognormal distribution, so the IgGGMCs when the 0th day and 28 days and be calculated as logarithm about 95% CIs of GMCs at the bottom of 2 concentration.
A. measure meningococcemia antibody by the serum sterilizing that uses young rabbit complement
SBA-BR is to use the in vitro method of young rabbit (BR) complement, and its is measured antibody-mediated, complement-dependent target bacteria and kills, and is used for the purpose of metering needle to the immunne response of Neisseria meningitidis serogroups A, C, Y or W-135 capsular polysaccharide.
By clinical immunology platform (Clinical Immunology Platform), Swiftwater, PA, the U.S. measures the functional antibodies of metering needle to Neisseria meningitidis serogroups A, C, Y and W-135 via the serum sterilizing that uses the BR complement.In brief, with serogroups specificity bacterium seed at the Thayer-Martin lining out and the incubation that spends the night.Results from the isolating bacterium colony of the plate that spends the night and with the bacterium wiping on the Thayer-Martin plate.These plates of incubation are to obtain to converge a small amount of velum of bacterial growth subsequently.According to deactivation endogenous complement thermal treatment serum.
Dulbecco ' s phosphate-buffered saline (PBS) is assigned in the hole of 96 hole microtiter plates.Cross over plate subsequently and distribute heat-inactivated serum sample, reference antisera or serogroups specific antibody (in contrast), and carry out 2 times of dilutions.Follow each sample to also have serum contrast and complement control.Allow serogroups specificity meningococcus together with young rabbit complement and serum dilution incubation.
From Thayer-Martin plate results bacterial cell and be suspended in the damping fluid.The work dilution of preparation bacterial cell also mixes with isopyknic complement.Subsequently this bacterial suspension is assigned in the 96 hole microtiter plates, bag by and placed oscillator plate last 1 minute.Plate moves to 37 ℃ of CO 2Incubator and for serogroups A incubation 90 minutes was for serogroup C, Y and W-135 incubation 60 minutes.
Behind the incubation, in each hole, add agar over lay.Plate is capped and in 37 ℃ and 5% CO 2Be incubated overnight.Bacterial colony in second day counting hole.The sterilization of every kind of unknown serum is tired to be expressed as with the mean number of the T0 colony-forming unit (cfu) in complement control hole and is compared, and produces 〉=50% serum dilution final reciprocal of killing.The contrast of serogroups specific antibody is used to differentiate tested serogroups.The limit of detection of this mensuration is to tire 8.
Obtain reference serum and be stored in-70 ℃ from CDC (donor R21654-3430107).The serogroups specific antibody for A, C, Y and W-135 that is obtained commercially contrasts as quality.The serogroups specific antibody accept 5 ℃ freezing, and with 1mL sterilized water aquation once more, aliquots containig also is stored in-70 ℃.
B. meningococcemia IgG TPPA
Generally speaking, the ELISA method uses specific serum antibody to be attached to the target antigen of solid substrate with combination.The second antibody that adding produces at human IgG is to finish Ag-Ab-antibody complex.This second antibody is conjugated to the enzyme of the color reaction of catalysis after suitably substrate adds.The ELISA reader software is drawn optical density(OD) (OD), and also drafting is about the typical curve of reference serum, and use can be calculated the antibody titer of test sera by the specified unit vol of 4 parameter logarithmic curve (4PL) methods from described curve.
By the clinical immunology platform, Swiftwater, PA, the U.S. uses the total IgG antibody activity of indirect ELISA metering needle to the meningococcemia antibody of serogroups A, C, Y and W-135.In brief, clinical, contrast or reference serum incubation (use methylate human serum albumin strengthen bag be adsorbed) on wrapping in advance by the microtiter plate of excessive serogroups specific antigens.Plate is subsequently at room temperature with the mouse anti human IgG specific antibody incubation of peroxidase labelling.Add tetramethyl benzidine (TMB) to produce the colour developing product.Reach the OD of 0.6-0.7 scope until the top side reference hole at 650nm place monitoring panel.In case reach described scope, just in each hole, add the sulfuric acid stop bath.Gained OD reads on the elisa plate reader.Use the specified value of 4 parameter logarithm (4PL) curve methods, by relatively calculating the meningococcemia polysaccharide IgG level of test sera with reference serum (LotCDC 1992).The result is with the form report of mg/m1.
Elementary reference serum is the human antiserum that converges who is obtained commercially: CDC Lot 1992 (a present NIBSC code 99/706).The elementary reference of receiving with lyophilised state is stored in 2 ℃-8 ℃ after receiving.
The serum that is used for the quality control serum is Aventis Pasteur human serum storehouse AvP Lot100B 189A.Bottle is stored in-80 ℃--and 40 ℃.The work aliquots containig is stored in-30 ℃--and 10 ℃.
5. statistical analysis
Use
Figure A200580048657D0155173008QIETU
Software version 8.2 carries out statistical analysis.Generally speaking, all data of collection all are presented in summary table and/or the data list.Present classified variable (number of the participant in each classification and ratio [and 95%CI]) by frequency distribution.Present continuous variable (number of observation [n], mean number, standard deviation [SD], median and minimum value and maximum value) by summarizing statistics.
Test has at least 85% ability, and to refuse the real difference of participant's ratio of 〉=4 times of serogroup C antibody titer risings in following hypothesis: TetraMenD group and the Hib conjugate vaccine group be≤50%.This calculating is 0.05 one-tailed test based on the error of the first kind rate, and is at least 85% based on the true ratio in following hypothesis: the TetraMenD group, and the true ratio in the Hib conjugate vaccine group is less than 10%.
Main terminal point is, compare with corresponding proportion in the control group, from the baseline to the vaccination after in the 28th day TetraMenD group at raise participant's ratio of at least 4 times of the antibody titer of serogroup C.Support elementary hypothesis, be centered around the corresponding proportion that observed ratio deducts in the control group in the TetraMenD group and make up one-sided 95% fiducial interval.If be limited under one-sided 95% fiducial interval of difference〉0.50, data are supported elementary hypothesis so.This is equivalent to use the error of the first kind rate is that null hypothesis is tested in 0.05 one-tailed test, is≤0.50 (H at the antibody titer of the serogroup C corresponding proportion that participant's ratio of 〉=4 times deducts in the control group that raises in the TetraMenD group promptly 0: p TetraMenD-P Hiberix≤ 0.50), contrast alternative hypothesis, difference is〉0.50 (H 1: p TetraMenD-P Hiberix0.50).If elementary null hypothesis is rejected, can reach a conclusion so to surpassing 50% at the antibody titer of the serogroup C ratio that participant's ratio of 〉=4 times is higher than in the control group that raises in the TetraMenD group.
If participant: 1) satisfy the standard that comprises in the above-mentioned test; 2) accept to specify vaccine; With 3) have the blood sample 1 that extracts the 0th day (before the vaccination) and after vaccination at least 28 days but the blood sample 2 that extracted in 35 days at the most, the participant is qualified for the immunogenic scheme colony that meets so.
Yet,, from meet program analysis, get rid of participant without any effective serology result for the purpose of analyzing.In this background, " effectively " refers to serum and/or result's the property assessed.The participant who gets rid of from the immunogenicity analysis that meets scheme comprises to be used to analyze those of its serum loss or its serum mass deficiency.
Meet the participant who comprises the scheme of running counter to that does not influence its immunne response or its immunogenicity property assessed in the scheme colony.Meet scheme colony and be used for all immunogenicity analyses.
, and have all participants that at least once analyze required effective serology result and form by accepting a TetraMenD or the shot of Hib conjugate vaccine about immunogenic purpose treatment colony.Carry out identical immunogenicity analysis to meeting scheme with purpose treatment colony.
Security colony is by accepting a TetraMenD or the shot of Hib conjugate vaccine, and all participants with any obtainable safety information form.Safety in utilization colony carries out safety analysis.If can not obtain data of safety for any given participant, the data report from this particular participant reaction is disappearance so.
6. immunogenicity result
Summarizing table 3 shows from raise 〉=4 times participant's number and per-cent of 28 days SBA BR antibody titers for serogroups A, C, Y and W135 of baseline to the.After the vaccination 28 days, most of TetraMenD recipients experienced SBA BR antibody titer and raise 〉=4 times than baseline, and minority
Figure A200580048657D01561
The recipient reaches this result.
Table 3 is from the participant's of 〉=4 times of 28 days SBA BR of baseline to the antibody titer risings number and per-cent (meeting scheme colony)
Figure A200580048657D01571
*N: than tire participant's the number of rising 〉=4 times of baseline.
N: number with participant of effective serology data.
Figure A200580048657D0157172456QIETU
: the n/N that is expressed as per-cent.
Table 4 shows from raise 〉=4 times participant's number and per-cent of 28 days SBA-BR antibody titers for serogroup C of baseline to the.This per-cent is ratio in TetraMenD group (93.18%)
Figure A200580048657D01572
Much higher in the group (5.56%).Difference (the P of per-cent TetraMenD-
Figure A200580048657D01573
) be 87.63%, and be limited to 78.77% under one-sided 95% fiducial interval of difference, this has surpassed 50%.Based on these results, can reach a conclusion into, using the error of the first kind rate is 0.05 one-tailed test, deducts at the tire per-cent that meets the scheme participant of rising 〉=4 times of the SBA BR of serogroup C in the TetraMenD group
Figure A200580048657D0157173223QIETU
Corresponding per-cent in the group is greater than 50%.
The elementary hypothesis of table 4 test is from raise 〉=4 times participant's number and per-cent (meeting scheme colony) of 28 days serogroup C SBA-BR of baseline to the antibody titer
Figure A200580048657D01574
*N: from tire participant's the number of rising 〉=4 times of 28 days serogroup C SBA-BR of baseline to the.
N: have overall number for the participant of the effective serology data of serogroup C.
Figure A200580048657D0157172522QIETU
In the TetraMenD group from tire participant's the per-cent of rising 〉=4 times of 28 days serogroup C SBA-BR of baseline to the.
Figure A200580048657D0158172534QIETU
Figure A200580048657D01582
In the group from tire participant's the per-cent of rising 〉=4 times of 28 days serogroup C SBA-BR of baseline to the.
SBA-BR GMTs when table 5 is presented at after baseline and the vaccination the 28th day, and the geometric mean multiple of tiring from 28 days SBA BR of baseline to the raises.In the TetraMenD group, the SBA-BR antibody titer has remarkable increase between the 0th day and the 28th day; For serogroups A, it is 70.34 that geometric mean multiple that SBA-BR tires raises, and is 136.33 for serogroup C, is 16.51 for serogroups Y, and is 159.59 for serogroups W 135.
Figure A200580048657D01583
In the group, the SBA-BR antibody titer does not have or has only a little increase between the 0th day and the 28th day.
Figure A200580048657D01591
Table 6 shows the frequency distribution for the SBA-BR antibody titer of the baseline of every kind of serogroups and the 28th day.
Fig. 5,6,7 and 8 presents the 0th day and the 28th day reverse integral distribution curve for the SBA BR antibody titer of serogroups A, C, Y and W135 respectively.
IgG GMCs when table 7 is presented at after baseline and the vaccination the 28th day, and raise from the geometric mean multiple of 28 days IgG concentration of baseline to the.In the TetraMenD group, the IgG antibody titer increases to some extent between the 0th day and the 28th day; It is 26.74 that geometric mean multiple that IgG tires raises for serogroups A, is 15.86 for serogroup C, is 12.13 for serogroups Y, and is 22.00 for W-135.
Figure A200580048657D01621
In the group, the IgG antibody titer does not have or has only a little increase between the 0th day and the 28th day.
Figure A200580048657D01631
Immunogenicity is summarized
Before the vaccination, TetraMenD is similar with SBA in the Hib conjugate vaccine group with the IgG antibody horizontal.TetraMenD used back 28 days, for the SBA and the IgG antibody horizontal of all serogroupss that comprise in the composition remarkable increase (table 5 and 7) was arranged all.Particularly, serogroup C is replied with consistent at the sort of antigenic memory response.For every kind of serogroups, reach raise 〉=4 times TetraMenD recipient's ratio (table 3) of antibody titer and be similar to and in the 2-10 year person's who accepts single agent research vaccine large-scale experiment, report.(Pichichero M.E., Deng the people, A Comparative Trial of the Safety and Immunogenicity ofQuadrivalent (A, C, Y, W-135) Meningococcal Diphtheria ConjugateVacc ine (MCV-4) Versus Quadrivalent Polysaccharide Vaccine (
Figure A200580048657D0164173418QIETU
, PS) in Children 2-10 Years of Age.Abstracts of the 43rdAnnual ICAAC, Chicago, the 286th page of Illinois.American Society for Microbiology.).For serogroups Y, this ratio is 79.6%, Comparatively speaking, is 97.7%, is 93.2% and is 97.7% (table 3) for serogroups W-135 for serogroup C for serogroups A.Observe antibody horizontal (table 5) before the highest vaccination for serogroups Y.This can be owing to Natural Exposure in this serogroups, and this may be more more common than what thought in the past.Higher circulating antibody level reflects recent Natural Exposure and can reduce the vaccine recipient's who reaches antibody horizontal 〉=4 times rising ratio.Compare with serogroup C or W-135, also observe antibody horizontal before the higher vaccination for serogroups A.This may be to be interrupted the result who is exposed to several naturally occurring cross-reacting antigens through the time period that prolongs.
In the control group of accepting the Hib vaccine, there is not significant difference between the preceding and meningitis posterior coccus antibody response.The difference that the SBA-BR of TetraMenD and Hib vaccine group tires between participant's ratio of rising 〉=4 times in 4 kinds of serogroupss each all is〉50% (table 4).In control group, reach SBA-BR to tire 〉=participant's of 4 times of risings ratio is higher than for serogroup C or Y for serogroups A and W-135.This may round-robin serogroups specific antigens causes in this colony in carrying out in research in the process by Natural Exposure.The participant of vast scale convenes from the Moslem community in London, has wherein identified the clinical case of serogroups W-135 meningococcal disease.The danger that increases is with to go to Meccah (Hajj) to go on a pilgrimage related.Although known measurement result is different between the laboratory, after using the serogroup C meningococcal conjugate vaccine of booster dose in the pretreated children of unit price conjugate vaccine infancy, in SBA and IgG antibody response, observe similar trend.(McVernon J., Deng the people, Safetyand immunogenicity of meningococcus serogroup C conjugate vaccineadministered as a primary or booster vaccination to healthy four-year-oldchildren, Ped.Infect.Dis.J., 21 (8): 747-753[2002]).
In TetraMenD group, confirm that at the magnitude (table 5) of GMTs after the vaccination of all 4 kinds of serogroupss vaccine is a hyperimmunization originality in this kind of groups.All TetraMenD recipients reach the antibody horizontal consistent with protection.(Ba1mer P. and Borrow R., Serologic correlates of protection for evaluating the response tomeningococcal vaccines, Expert Rev.Vaccines.3 (1): 77-87[2004]).
7. overall security feature
Table 8 presented the participant in 30 minutes immediate response, the part of after vaccination, bringing out in 7 days and systemic reaction and during whole search time section (0-is the 28th day after the vaccination) spontaneous adverse events and the general overview of serious adverse events.
The overall participant's security features (security colony) of table 8
Figure A200580048657D01651
*N: the participant's of at least incident of report number in this classification; N: the overall number of on each of these time points, submitting the participant of safety information to.
Security is summarized
In process of the test, reported following data of safety: part and the systemic reaction brought out in the immediate response in vaccination 30 minutes, after vaccination first 7 days, and after vaccination all spontaneous adverse events in time period of 28 days.
Do not observe immediate response in vaccination in 30 minutes.The frequency of the local reaction of bringing out in 2 vaccine group is similar.In 2 vaccine group, the local reaction that great majority bring out be reported as slight and in 3 days (median time length) disappear.Irritability and sleepiness are modal general malaises.TetraMenD recipient experiences FC in 24 hours accepting vaccine.Be also noted that he has the cough of the upper airway symptoms followed.This participant has the FC history of following respiratory infection.This report on incident is possible the SAE relevant with vaccination.During the search time section, do not report other SAEs.
Spontaneous adverse events is common, and their frequency is similar in 2 vaccine group.Wherein great majority are to be reported as the common medical science that has nothing to do with vaccination not accommodate situation.Generally speaking, TetraMenD is well tolerable and the security features that has is similar to security features with the licensed-in Hib conjugate vaccine that compares.

Claims (12)

1. method that is used for using immunogenic composition to the experimenter, it comprises,
A) provide:
I) experimenter, wherein said experimenter have inoculated unit price meningococcal conjugate vaccine dose;
Ii) tetravalence meningococcal polysacharide protein conjugate composition; With
B) after described experimenter inoculates described unit price meningococcal conjugate vaccine at least 12 months, use described tetravalence meningococcal polysacharide protein conjugate composition for described experimenter.
2. method that is used for using immunogenic composition to the experimenter, it comprises,
A) provide:
I) experimenter, wherein said experimenter have inoculated unit price meningococcal conjugate vaccine dose;
Ii) tetravalence meningococcal polysacharide protein conjugate vaccine; With
B) after described experimenter inoculates described unit price meningococcal conjugate vaccine at least 12 months, use described tetravalence meningococcal polysacharide protein conjugate vaccine for described experimenter.
3. method that is used for using immunogenic composition to the experimenter, it comprises:
A) provide:
I) experimenter, wherein said experimenter inoculating needle to the protective antigen of Neisseria meningitidis serogroup C;
Ii) tetravalence meningococcal polysacharide protein conjugate vaccine; With
B) after described experimenter inoculates described unit price meningococcal conjugate vaccine at least 12 months, use described tetravalence meningococcal polysacharide protein conjugate vaccine for described experimenter.
4. method that is used for using immunogenic composition to the experimenter, it comprises,
A) provide:
I) experimenter, wherein said experimenter have inoculated unit price Neisseria meningitidis serogroup C meningococcus vaccine;
Ii) tetravalence meningococcal polysacharide protein conjugate composition; With
B) after described experimenter inoculates described unit price meningococcal conjugate vaccine at least 12 months, use described tetravalence meningococcal polysacharide protein conjugate vaccine for described experimenter.
5. method of preventing meningococcal disease among the experimenter, it is included in described experimenter and has inoculated behind the unit price meningococcal conjugate vaccine at least 12 months, gives the tetravalence meningococcal conjugate vaccine of effective dose on described experimenter's administering therapeutic.
6. method of preventing meningococcal disease among the experimenter, it is included in described experimenter and has inoculated behind the unit price Neisseria meningitidis serogroup C meningococcus vaccine at least 12 months, gives the tetravalence meningococcal conjugate vaccine of effective dose on described experimenter's administering therapeutic.
7. method that stimulates immunne response among the experimenter, it is included in described experimenter and has inoculated behind the unit price meningococcal conjugate vaccine at least 12 months, gives the tetravalence meningococcal conjugate vaccine of effective dose on described experimenter's administering therapeutic.
8. induce among the experimenter method for one kind at the immunne response of one or more Neisseria meningitidis serogroupss, described method is included in described experimenter and has inoculated behind the unit price meningococcal conjugate vaccine at least 12 months, gives the tetravalence meningococcal conjugate vaccine of effective dose on described experimenter's administering therapeutic.
9. the method for claim 8, wherein said tetravalence meningococcal conjugate vaccine are used in the syringe at single and are formulated as the 0.5ml sterile liquid.
10. the method for claim 10, wherein the described tetravalence meningococcal conjugate of 0.5ml vaccine comprises the capsular polysaccharide of the about 10 μ g of about 0.01 μ g-from the purifying of each among Neisseria meningitidis serogroups A, C, Y and the W-135.
11. the method for claim 11, wherein the described tetravalence meningococcal conjugate of 0.5ml vaccine comprises the capsular polysaccharide of about 4 μ g from the purifying of each among Neisseria meningitidis serogroups A, C, Y and the W-135.
12. the method for claim 11, wherein the described tetravalence meningococcal conjugate of 0.5ml vaccine comprises the capsular polysaccharide of the about 5 μ g of about 0.05 μ g-from the purifying of each among Neisseria meningitidis serogroups A, C, Y and the W-135.
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CN102809656A (en) * 2012-08-26 2012-12-05 云南沃森生物技术股份有限公司 Method for detecting content of each group of free polysaccharide in meningococcus polysaccharide conjugate vaccine finished product
CN106659799A (en) * 2014-07-25 2017-05-10 碧奥辛斯有限责任公司 Glycoconjugate vaccines comprising basic units of a molecular construct expressing built-in multiple epitopes for the formulation of a broad-spectrum vaccine against infections due to enteropathogenic bacteria

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CN102327605B (en) * 2011-08-25 2013-01-30 成都康华生物制品有限公司 Preparation process of meningococcal polysaccharide vaccine
CN104383532A (en) * 2014-12-02 2015-03-04 云南沃森生物技术股份有限公司 Bacterial polysaccharide protein conjugate vaccine using hepatitis B surface antigen as carrier protein and preparation method of bacterial polysaccharide protein conjugate vaccine
AU2017321863A1 (en) 2016-09-02 2019-04-11 Sanofi Pasteur, Inc. Neisseria meningitidis vaccine

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WO2005004909A2 (en) * 2003-05-07 2005-01-20 Aventis Pasteur, Inc. Method of enhanced immunogenicity to meningococcal vaccination

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CN102809656A (en) * 2012-08-26 2012-12-05 云南沃森生物技术股份有限公司 Method for detecting content of each group of free polysaccharide in meningococcus polysaccharide conjugate vaccine finished product
CN106659799A (en) * 2014-07-25 2017-05-10 碧奥辛斯有限责任公司 Glycoconjugate vaccines comprising basic units of a molecular construct expressing built-in multiple epitopes for the formulation of a broad-spectrum vaccine against infections due to enteropathogenic bacteria
CN106659799B (en) * 2014-07-25 2020-07-17 碧奥辛斯有限责任公司 Glycoconjugate vaccines comprising a basic unit of a molecular construct expressing a built-in plurality of epitopes for the preparation of a broad spectrum vaccine against infections caused by enteropathogenic bacteria

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