CN101130071A - Vaccine - Google Patents

Abstract

The present invention relates to the field of bacterial polysaccharide antigen vaccines. In particular, the present invention relates to bacterial polysaccharides conjugated to protein D from H. influenzae.

Description

Vaccine

The application is that application number is 00807773.8 (PCT/EP00/02467), the applying date to be the dividing an application of application for a patent for invention on March 17th, 2000.

Technical field

The present invention relates to bacterial polysaccharides antigen vaccine, its production method and the application of this type of polysaccharide in medical science.

Especially, three aspects the present invention relates to be mutually related: the A-vaccine, contain a kind of pneumococcal polysaccharide antigen, pneumococal polysaccharide conjugate antigen particularly, this vaccine is added with a kind of proteantigen and a kind of optional Th1 inducing adjuvant from streptococcus pneumoniae; B-is added with the specific favourable pneumococal polysaccharide conjugate of Th1 adjuvant; And C-bacterial polysaccharides conjugate, protein D common and from Haemophilus influenzae combines.

Background technology

Pneumonia streptococcus Pseudomonas gram positive bacteria, it can cause very high sickness rate even death (especially to children age and advanced age people), and can cause such as affecting conditions and the diseases relevant with cluster such as pneumonia, bacteremia and meningitiss, as acute otitis media.In the U.S., the age is 100,000/3-8 greater than the ratio estimate that 60 years old people suffers from pneumococcal pneumonia.Wherein have 20% can cause bacteremia, other then show as meningitis, and its mortality rate is near 30%, even also are like this with antibiotic therapy.

Streptococcus pneumoniae is wrapped in the polysaccharide pod membrane of chemical crosslinking, and this can give its serotype specificity.Present known streptococcus pneumoniae serotype has 90 kinds, and pneumococcal pod membrane is the main determining factor of its virulence, because the pod membrane of these antibacterials not only can protect its inner surface to avoid the complement effect, and the immunogenicity of pod membrane itself is very weak.Polysaccharide is a T-independent antigen, can not be processed or be presented on the MHC molecule with the T cell interaction.But they can come stimulating immune system by replacement mechanism, and this mechanism relates to crosslinked with the B cell surface receptor.

Some experiments show, and are relevant strongly with the pod membrane specific antibody to the protective action of aggressive streptococcus pneumoniae disease, and this protective action has serotype specificity.

Vaccine based on polysaccharide antigen is known by this field.Being allowed to be used for human has four kinds, comprise the Vi polysaccharide of Salmonella typhi, from the PRP polysaccharide of Haemophilus influenzae, contain the tetravalence meningococcus vaccine of serotype A, C, W135 and Y, and 23 valency Pnu-Imune 23s (account at least streptococcus pneumoniae blood separator 90%) that contain the polysaccharide that is equivalent to serotype 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33.

The three kinds of vaccines in back can be used to protect the antibacterial that causes respiratory tract infection, this type of infection can cause serious sickness rate and death in the baby, but these vaccines are not allowed to be used for the age less than 2 years old child, reason is that they do not have enough immunogenicities (Peltolaet al. (1984), N.Engl.J.Med.310:156-1566) in this age group.Streptococcus pneumoniae be baby and child of tender age's aggressive bacterial disease and otitis media most commonly encountered diseases because of.Equally, advanced age, the people was to very weak (the Roghmann et al. of the reaction of Pnu-Imune 23, (1987), J.Gerontol.42:265-270), make the bacterial pneumonia sickness rate of this colony rise (Verghese and Berk, (1983) Medicine (Baltimore) 62:271-285).

The strategy that is used for overcoming this immunogenicity defective among the baby comprises, this polysaccharide is connected with the immunogenicity large protein, and this albumen can be assisted other T cell, and can induce the immunological memory at its bonded polysaccharide antigen.By evaluation, think that at present streptococcus pneumoniae glycoprotein conjugate vaccine is all safe and effective in age groups, and have immunogenicity.

A) pneumococcal polysaccharide vaccine

The clinical effectiveness of the non-binding Pnu-Imune 23 of 23 valencys (Fedson et al. (1994) the Arch Intern Med. that between 0%-81%, alters a great deal 154: 2531-2535).As if its effect with relevant by the risk population of immunity, as advanced age people, Hokdkin disease, splenectomy, drepanocytosis and agammaglobulinemia (Fine et al. (1994) Arch InternMed.154:2666-2677), and relevant with the clinical manifestation of disease.This 23 valency vaccines do not demonstrate can protect pneumococcal pneumonia (such as advanced age the people wait among some high-risk group) and otitis media disease.

Therefore, we need improved Pnu-Imune 23 compositions, particularly prevention or improve advanced age the people and child of tender age's streptococcus pneumoniae property disease (especially pneumonia) aspect more effective vaccine combination.

The present invention will provide this improvement vaccine.

B) Xuan Ding pneumococal polysaccharide conjugate+3D-MPL compositions

The antibody concentration that protection efficiency that it is generally acknowledged the non-binding Pnu-Imune 23 of commercialization induces during with inoculation has relation more or less; In fact, these 23 kinds of polysaccharide are just obtaining authentication (Ed.Williams et al.New YorkAcademy of Sciences 1995 pp.241-249) aspect the immunogenicity of every kind of polysaccharide component.Therefore; further strengthen to improve baby and the ratio that advanced age, the people utilized the antibody of protectiveness level that the polysaccharide or the polysaccharide conjugate generation of injection are first replied, and can reduce and induce necessary dosage of the protective immunity of streptococcus pneumoniae infection and frequency injection to the antibody response of pneumococal polysaccharide.

Research worker just tests to adding in a large number in the antigen to improve its immunogenic complex in vaccine combination that (summary is seen M.F.Powell ﹠amp since 20 beginnings of the century; M.J.Newman, Plenum Press, NY, " vaccine design--subunit and adjuvant method " (1995) the 7th chapters " vaccine adjuvant and excipient catalog ").Wherein have many very effective, but can cause tangible part or systemic side effects, be applied to human vaccine combination thereby hinder it.The aluminum type adjuvant (as Alumen, aluminium hydroxide or aluminum phosphate) that nineteen twenty-six describes first remains the unique immunological adjuvant that is used for human vaccine through U.S.'s allowance.

Aluminum type adjuvant belongs to carrier class adjuvant, can play a role by " storing up effect " of its attack.Antigen is adsorbed on its surface, and when the injection said composition, adjuvant and antigen can not be dispersed in the blood flow immediately--but be retained in the local environment of injection, thereby can obtain more obvious immunne response.These carrier class adjuvants also have other advantage, promptly are suitable for making easy decomposition antigen, as some polysaccharide antigen, keep stable.

3D-MPL belongs to non-carrier class adjuvant.Its full name is 3-O-deacylated tRNA base monophosphoryl lipid A (or 3 take off-O-acyl group monophosphoryl lipid A or 3-O-deacylated tRNA base-4 ' monophosphoryl lipid A), is expressed as 3D-MPL, to illustrate that the 3rd of its reducing end glycosamine is for taking off-O-acyl group form.Its preparation method can be with reference to GB2220211A.From chemically analyzing, it is 3-deacylated tRNA base monophosphoryl lipid A and 4,5 or the mixture of 6 kind of acidylate chain.It is prepared in phase early 1990s the earliest, and method at that time is that 4 ' of fat A-single phosphonyl derivatives (MPL) is carried out the 3-O-deacylation, and this method can produce more weak but the molecule that immunostimulatory activity is constant of a kind of toxicity.

At present with 3D-MPL itself as a kind of independent adjuvant, or preferably be used in combination, as aluminium hydroxide, aluminum phosphate or oil-in-water emulsion with a kind of accumulation type carrier adjuvant.The antigen that comprises in these compositionss is identical grain structure with 3D-MPL, thus more effectively antigen-presenting signal and immunostimulation signal.Studies show that 3D-MPL can further strengthen immunogenicity of antigens (Thoelen et al.Vaccine (1998) 16:708-14 that is adsorbed by Alumen; EP 689454-B1).This type of combination also is used for easy adsorption antigen (as the bacterial polysaccharides conjugate) by this field as method for optimizing, helps to make it to keep stable after these antigens are adsorbed on the Alumen.The most frequently used is sedimentary aluminum type adjuvant, because they are the unique adjuvants that use in the at present licensed human vaccine.Therefore, the vaccine that contains 3D-MPL and aluminum type adjuvant simultaneously is easy to development and can introduces market fast enjoy this field to like because of it.

MPL (non-3-deacylation) has been used as the adjuvant of several monovalent polysaccharide-conjugate vaccine antigen, and comes thus it is evaluated.MPL and normal saline injected simultaneously can strengthen serum antibody replying four kinds of monovalent polysaccharide conjugates: streptococcus pneumoniae PS 6B-tetanus toxoid, streptococcus pneumoniae PS 12-diphtheria toxin, diphtherotoxin and with bonded golden yellow Fructus Vitis viniferae ball Seedling 5 types of Pseudomonas aeruginosa exotoxin A and staphylococcus aureus 8 types (Schneerson et al.J.Immunology (1991) 147:2136-2140).Enhanced replying is considered to have antigenic specificity.There is the effect that all the time be better than in normal saline MPL with antigen in the MPL that is in the oil-in-water emulsion (carrier model adjuvant) with identical grain structure because of it, and is considered to send the preferred adjuvant system of other polysaccharide conjugate vaccine.

People such as Devi (Infect.Immun. (1991) 59:3700-7) evaluated the MPL (non-3-deacylation) that is in the normal saline Mus to the auxiliaring effect aspect the antibody response of Cryptococcus neoformans capsule-associated polysaccharide TT conjugate.When SimultaneouslyWhen using MPL and this conjugate, the IgM-of PS and IgG-specificity replied all have only slight raising; But after 2 days during with the MPL administration, the effect of MPL is much bigger in the conjugate administration.The practicality that MPL need be postponed the immunization protocol of administration with respect to antigen is worth suspecting, especially to the baby.As if MPL depend on compositions to the auxiliaring effect of polysaccharide and polysaccharide-protein conjugate.In addition, MPL is added in the suitable slow release delivery system (as using the carrier adjuvant) can obtain more persistent secondary effects, and the problem that can avoid the time allotment and postpone administration.

In a word, with present state-of-art, when being adjuvant, preferably itself and a kind of carrier adjuvant (as aluminum type adjuvant) are worked in coordination with use, so that farthest realize its immune-stimulating effect with MPL or 3D-MPL.

The inventor is surprised to find, for some pneumococal polysaccharide conjugate, the immunogenicity the when immunogenicity of its vaccine combination when antigen and 3D-MPL prepare separately will be apparently higher than antigen and 3D-MPL and the collaborative preparation of carrier adjuvant (as a kind of aluminum type adjuvant).This improvement that observes does not rely on the concentration of used 3D-MPL, also with the particular combination thing is used with the unit price composition forms or is used in combination to form the multivalence compositions it irrelevant.

C) bacterial polysaccharides-protein D conjugate

As mentioned above, known by this field based on the vaccine of polysaccharide antigen.Above-mentioned licensed polysaccharide vaccine has different actual clinical effects.According to estimates, the Vi polysaccharide vaccine is 55%-77% (Plotkin and Cam, (1995) Arch Intern Med in the effectiveness of prevention aspect the typhoid fever of cultivating affirmation 155: 2293-99).Meningococcus C polysaccharide vaccine is in next 79% the effectiveness (De Wals P, et al. (1996) Bull World HealthOrgan.74:407-411) that demonstrates of popularity.The clinical effectiveness of 23 valency Pnu-Imune 23s has very big difference (Fedson et al. (1994) Arch Intern Med. between 0%-81% 154: 2531-2535).The antibody concentration that induces when as mentioned above, protection efficiency that it is generally acknowledged Pnu-Imune 23 is with inoculation has relation more or less.

There is a problem in the method for inoculating with polysaccharide, and promptly polysaccharide is originally as weak immunogen.The strategy that is used for overcoming this immunogenicity defective comprises, polysaccharide is connected with the high immunogenicity large protein carrier that can assist other T cell.

These high immunogenicity carriers commonly used that are used for producing polysaccharide immunogens at present comprise, for example, and diphtheria toxin, diphtherotoxin (DT or CRM197 mutant), tetanus toxoid (TT), keyhole limpet homocyanin (KLH) and tuberculin purfied protein derivative (PPD).

The problem of common carrier

These common carrier all have many problems separately, comprising producing GMP conjugate aspect, also comprise the immunological characteristic aspect of these conjugates.

Although these carriers are often used, and also successfully induce the antibody response of anti-polysaccharide, they have some shortcomings.For instance, when being pre-existing in, be tetanus toxin at this carrier herein, antibody the time, antigen-specific immune response can be suppressed (epi-position inhibition) (Di John et al; (1989) Lancet, 2:1415-8).And as convention, these vaccines all are used for human inoculation, so in whole colony, the people who DT and TT all is pre-existing in immunity occupies very high ratio.For example, there is 95% child accept the DTP vaccine in Britain, wherein just contains DT and TT.Other author has described also that the epi-position to peptide vaccine suppresses problem (Sad et al, Immunology, 1991 in the animal model; 74:223-227; Schutze et al, J.Immunol.135:4,1985; 2319-2322).

In addition, for the regularly short vaccine that rises of needs, use such as TT and the contour immunogenic carrier of DT and may after injection for several times, promptly suppress replying of polysaccharide antigen.Repeatedly inoculation also can be with untoward reaction, as delayed super-reaction (DTH).

KLH is used for human clinical trials as the carrier of IgE peptide as strong immunogen.But also observe some side reactions (DTH sample reaction or IgE sensitization) and at the antibody response of antibody.

Therefore, when selecting carrier protein to be used for vaccine based on polysaccharide, need be in the necessity of using carrier (extensively MHC discerns), induce high-caliber antipolysaccharide antibody to reply and low-level anticarrier antibody is sought a kind of balance between replying applicable to all patients.

Therefore, be used for a lot of shortcomings being arranged in the past based on the carrier of the vaccine of polysaccharide.Be used in the combination-vaccine then especially obviously, this moment, then epi-position inhibition problem can especially severe if will be used for different polysaccharide antigens with a kind of carrier.In order to overcome this influence as far as possible, WO98/51339 has used variety carrier in combination-vaccine.

The invention provides a kind of new support, be used to prepare polysaccharide with above-mentioned shortcoming/based on the immunogenic conjugates of polysaccharide.

The invention provides a kind of protein D (EP059461 B1) or its fragment that derives from Haemophilus influenzae, as immunogenic composition based on polysaccharide, comprising vaccine, carrier.This carrier is beneficial in combination-vaccine especially and uses.

Summary of the invention

A) streptococcus pneumoniae polysaccharides vaccine

The invention provides a kind of vaccine combination, said composition contains a kind of streptococcus pneumoniae (Streptococcus pneumoniae) polysaccharide antigen (preferably bonded polysaccharide antigen) and a kind of streptococcus pneumoniae proteantigen or its immunologic function equivalent at least, and optionally contains a kind of Th1 adjuvant (inducing the adjuvant of Th1 immunne response).Preferably pneumoprotein and Th1 adjuvant include.Said composition of the present invention is specially adapted to people's at advanced age pneumonia treatment.

Pneumococcal polysaccharide vaccine (bonded or unconjugated) possibly can't protect advanced age the crowd must not pneumonia, the probability that this disease is suffered from by this colony is very high.Resisting pneumococcal main defense mechanism is that the opsonin phagocytosis is (by producing the incident by body fluid B cell/neutrophil cell mediation that anti-pneumococal polysaccharide antibody causes, at last antibacterial is engulfed), but relate to some part of opsonin mechanism and obstacle occurs advanced age in the human body, and promptly PMN (polymorphonuclear cell) produces the morphotropism of peroxide, other type oxygen production, PMN mobilization, PMN apoptosis, PMN.The intravital antibody response of people at advanced age also obstacle can occur.

The anti-capsular polysaccharide antibody of normal level can not be removed antibacterial effectively fully, this with the rule of generally acknowledging antithesis, its reason is that streptococcus pneumoniae can be invaded host cell to hide this branch's approach of immune system.

The inventor is surprised to find, this branch's approach of immune system (immunity cell-mediated as T) of irritation cell mediation simultaneously can obtain a kind of potentiation (or synergism) again except that this branch's approach of immunity system (immunity cell-mediated as B), and this effect can promote that the host removes streptococcus pneumoniae.This discovery helps to prevent common people's streptococcus pneumoniae sexuality to dye, and for advanced age the people pneumonia prevention most important because they are not had curative effect based on the vaccine of polysaccharide.

The inventor finds, if with together administration of pneumococal polysaccharide (preferably bonded pneumococal polysaccharide) and a kind of pneumoprotein (preferably streptococcus pneumoniae surface expression or by a kind of albumen that can process and be presented to infected mammalian cell surface in II class and MHC I class environment of its secretion or release), then two kinds of immune systems can produce synergism thus.The inventor also finds, but although pneumoprotein trigger cell mediated immunity itself, but the existence of Th1 inducing adjuvant can be assisted this immune system in the bacterin preparation, and can further strengthen the synergism between these two kinds of immune systems astoundingly.

B) Xuan Ding pneumococal polysaccharide conjugate+3D-MPL compositions

Therefore, the present invention also provides a kind of antigen composition, said composition contains one or more pneumococal polysaccharide conjugates, and be added with the 3D-MPL adjuvant and do not contain aluminum type adjuvant substantially, wherein having a kind of pneumococal polysaccharide conjugate at least is the immunogenicity of immunogenicity in the compositions that contains 3D-MPL and aluminum type adjuvant at the same time in containing the compositions of 3D-MPL.

Preferred embodiment provided by the invention is the antigen composition that contains the conjugate of following one or more pneumococcal capsular polysaccharides: serotype 4,6B, 18C, 19F and 23F.Every kind of polysaccharide in these compositionss all is the immunogenicity of immunogenicity in the compositions that contains 3D-MPL and a kind of aluminum type adjuvant at the same time in the compositions that only contains 3D-MPL.

Therefore, an embodiment of the present invention provides a kind of antigen composition, said composition contains and a kind of immunogenic protein and the bonded serotype 4 of 3D-MPL adjuvant, 6B, 18C, 19F or 23F streptococcus pneumoniae capsular polysaccharide, and wherein said composition does not contain aluminum type adjuvant substantially.

Another embodiment of the present invention provides a kind of combined antigen composition that does not contain aluminum type adjuvant substantially, two or more pneumococal polysaccharide conjugates that said composition contains the 3D-MPL adjuvant and is selected from serotype 4, serotype 6B, serotype 18C, serotype 19F and serotype 23F.

C) bacterial polysaccharides-protein D conjugate

Therefore, the invention provides a kind of polysaccharide conjugate antigen, this antigen contains protein D or the bonded polysaccharide antigen that derives from a kind of pathogenic bacterium of its protein D fragment a kind of and from Haemophilus influenzae (H.influenzae).In addition, the present invention also provides multivalent vaccine composition, wherein has one or more polysaccharide antigens to combine with protein D.

Detailed Description Of The Invention

A) pneumococcal polysaccharide vaccine

The invention provides a kind of improvement vaccine, the streptococcus pneumoniae sexuality that is used for prevention especially or improves people at advanced age (and/or baby and child) is dyed.

In the present invention, the age is equal to or greater than 55 years old patient and is considered to the people at advanced age, typically then greater than 60 years old, more generally then greater than 65 years old.

Therefore, an embodiment of the present invention provides a kind of vaccine combination that is applicable to people at advanced age (and/or baby and child), and said composition contains at least a streptococcus pneumoniae polysaccharides antigen and at least a streptococcus pneumoniae proteantigen.

Another embodiment preferred of the present invention provides a kind of vaccine (being applicable to people's at advanced age pneumonia prevention), and this vaccine contains at least a streptococcus pneumoniae polysaccharides antigen and at least a streptococcus pneumoniae proteantigen and a kind of Th1 adjuvant.

Can think that this vaccine also is applicable to other high-risk group, as baby or child, the streptococcus pneumoniae sexuality dye the treatment of (as otitis media).

The 3rd embodiment of the present invention provides a kind of vaccine combination, and said composition contains a kind of pneumococcal polysaccharide antigen and a kind of Th1 adjuvant.

Streptococcus pneumoniae polysaccharides antigen of the present invention

Streptococcus pneumoniae vaccine of the present invention all contains polysaccharide antigen (preferably bonded polysaccharide antigen) usually, and polysaccharide antigen wherein derives from the streptococcus pneumoniae of four kinds of serotypes at least.Preferred, these four kinds of serotypes comprise 6B, 14,19F and 23F.More preferably contain 7 kinds of serotypes in the said composition at least, as derive from serotype 4,6B, 9V, 14,18C, 19F and 23F.More preferably contain 11 kinds of serotypes in the said composition at least, for example the compositions in embodiment contains the capsular polysaccharide (preferably bonded polysaccharide) that derives from serotype 1,3,4,5,6B, 7F, 9V, 14,18C, 19F and 23F.Although the present invention also considers to adopt, for example, the scheme of 23 valencys (as serotype 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17 F, 18C, 19A, 19F, 20,22F, 23F and 33F), but a preferred embodiment of the present invention contains and has plenty of at least 13 kinds of polysaccharide antigens (preferably bonded polysaccharide antigen).

For people's inoculation at advanced age (as being used for preventing pneumonia), add in addition that in above-mentioned 11 valency antigen compositions serotype 8 and 12F (most preferably adding 15 and 22) have more advantage with the scheme that forms 15 valency vaccines, and add that 6A and 19A are more conducive to the baby with the scheme that forms 13 valency vaccines or the child uses (what more pay close attention to this moment is otitis media).

When preventing/improving crowd's at advanced age (+55 years old) pneumonia and baby (before 18 months) and child's's (being generally 18 months-5 years old) otitis media, the preferred embodiments of the invention are that the multivalence streptococcus pneumoniae polysaccharides described in the literary composition is combined with a kind of pneumonia streptococcus mycoprotein or its immunologic function equivalent.

Pneumoprotein of the present invention

For the purpose of the present invention, the definition of " immunologic function equivalent " is a kind of proteic peptide, and this albumen contains a kind of proteic protective epitope of the present invention that derives from least.The characteristic of these epi-positions is to be exposed to the surface, high conservative, and can in the host, cause the bactericidal properties antibody response and maybe can avoid toxic action.Preferred, this function equivalent has 15 at least and derives from proteic continuous amino acid of the present invention, preferably then is at least 30 or more.Most preferred be to use this proteic fragment, such as stride film district deletion mutation body deletants such as (promptly using zone, proteic extracellular), fusant, through chemistry or the toxicide derivant of genetic method etc., prerequisite is that they can cause and the essentially identical immunne response of native protein.

Optimization protein of the present invention is those pneumoproteins that are exposed to streptococcus pneumoniae outer surface (at least in the part-time of streptococcus pneumoniae biocycle can by host's immune system recognition), or by streptococcus pneumoniae secretion or the albumen that discharges.Most preferred albumen then is toxin, adhesin, bi-component signal transducer or the lipoprotein of streptococcus pneumoniae, or its immunologic function equivalent.

The particularly preferred albumen that can be included in the described combination-vaccine includes, but are not limited to: pneumolysin (pneumolysin) (preferably carrying out detoxification by chemical treatment or sudden change) (Mitchell et al.Nucleic Acids Res.1990 Jul 11; 18 (13): 4010 " deriving from pneumolysin gene and the proteic comparison of 1 type and 2 type streptococcus pneumoniae ", Mitchell et al.Biochim Biophys Acta 1989 Jan 23; 1007 (1): 67-72 " the pneumolysin gene expression in the escherichia coli: fast purifying and biological characteristics ", WO 96/05859 (A.Cyanamid), WO 90/06951 (Paton etal), WO 99/03884 (NAVA)); PspA and stride film district deletion mutant (US 5804193-Briles et al.); PspC and stride film district deletion mutant (WO 97/09994-Brileset al.); PsaA and stride film district deletion mutant (Berry ﹠amp; Paton, Infect Immun1996 Dec; 64 (12): 5255-62 " PsaA, to the vital a kind of 37kD supposition of the virulence of streptococcus pneumoniae adhesin, the sequence heterogeneity "); Streptococcus pneumoniae choline binding protein and stride film district deletion mutant; CbpA and stride film district deletion mutant (WO 97/41151; WO 99/51266); Glyceraldehyde-3-phosphate-dehydrogenase (Infect.Immun.199664:3544); HSP70 (WO 96/40928); PcpA (Sanchez-Beato et al.FEMS Microbiol Lett 1998,164:207-14); M sample albumen, SB number of patent application EP0837130; And adhesin 18627, SB number of patent application EP0834568.

Preferred, the albumen that the present invention uses is selected from two or more the combination in pneumolysin, PsaA, PspA, PspC, CbpA or these albumen.The present invention also comprises the immunologic function equivalent of these albumen (deciding as mentioned).

This albumen in the compositions can assist to induce cell-mediated the replying of T of anti-streptococcus pneumoniae disease--this is the special needs of protection pneumonia institute--this reply to match with immunity system can suppress pneumococcal invasion and attack, and can stimulate the opsonin phagocytosis.

Another advantage that includes this proteantigen is and can sends more antigen to the opsonin phagocytosis, and can suppress bacterial adhesion element (if using adhesin) or make toxin neutralization (if using toxin).

The present invention provides a kind of Streptococcus pneumoniae vaccine in an embodiment, this Streptococcus pneumoniae vaccine contains a kind of pneumococal polysaccharide conjugate vaccine, this pneumococal polysaccharide conjugate vaccine contains the polysaccharide antigen that derives from least 4 kinds of serotypes, preferably at least 7 kinds, be more preferably at least 11 kinds, and contain at least a pneumonia streptococcus mycoprotein, and preferably two kinds.Preferred, it is pneumolysin or PsaA or PspA or CbpA (most preferred is toxicide pneumolysin) that a kind of albumen is wherein arranged.Preferably be combined as and contain the pneumolysin or derivatives thereof at least, and PspA.

As indicated above, there is a problem in the method for inoculating with polysaccharide, and promptly polysaccharide is originally as weak immunogen.In order to overcome this point, polysaccharide can be connected with the protein carrier that can assist other T cell (bystander T-cell help).Therefore, the polysaccharide that preferably the present invention is used is connected with a kind of like this protein carrier.The examples of such carriers that is commonly used to produce polysaccharide immunogens at present comprises, for example, diphtheria class and tetanus toxoid (being respectively DT, DTCRM197 and TT), keyhole limpet homocyanin (KLH), from OMPC and the tuberculin purfied protein derivative (PPD) of Neisseria meningitidis

But all there are many problems (" problem of common carrier " joint sees above) separately in these common carrier.

The present invention provides a kind of new support in a preferred embodiment, is used to prepare the immunogen construct based on polysaccharide with these shortcomings.The preferred vector that is used for the immunogenic composition (or vaccine) based on pneumococal polysaccharide is Haemophilus influenzae protein D or its fragment (EP594610-B).The fragment that is suitable for using comprises the fragment that contains helper T cell epitope.Especially, the fragment of protein D preferably should contain the part of this albumen n end 1/3.

Another preferred vector that is used for pneumococal polysaccharide is pneumoprotein itself (" pneumoprotein of the present invention " joint is decided as mentioned).

Vaccine of the present invention preferably should be by adjuvantization.Suitable adjuvant comprises a kind of aluminum salt, as alumine hydroxide colloid (Alumen) or aluminum phosphate, but also can be a kind of calcium salt, iron salt or zinc salt, or acidylate tyrosine, or cation or the anionic derivative or the polyphosphazene of the insoluble float of acidylate sugar, polysaccharide.

Preferred, selected adjuvant should be a kind of preferred derivant that the TH1 type is replied, with the immunne response of helper cell mediation.

TH1 adjuvant of the present invention

High-caliber Th1 cytokines is beneficial to and induces given antigenic cell-mediated immune responses, and high-caliber Th2 cytokines is beneficial to and induces antigenic humoral immunoresponse(HI).

Should remember that importantly the difference of Th1 type and Th2 type immunne response is not absolute.In fact our immunne response that individuality is kept is described as that Th1 preponderates or Th2 preponderates.But can come easily the pair cell factor family to study (Mosmann to the description of Mus CD4+ve T cell clone according to Mosmann and Coffman usually, T.R.and Coffman, R.L. (1989) TH1 and TH2 cell: different lymphokine secretion modes cause different functional characteristics.Annual Review of Immunology，7，p145-173)。Usually it is relevant that the Th1 type is replied the INF-γ and the IL-2 that produce with the T lymphocyte.Normal other cytokine directly related with inducing Th1 type immunne response as IL-12, then be can't help the generation of T cell.By contrast, the Th2 type is replied relevant with the secretion of IL-4, IL-5, IL-6, IL-10.Can promote the dominant proper adjuvant of replying of Th1 to comprise that monophosphoryl lipid A or derivatives thereof, particularly 3-take off-O-acyl group monophosphoryl lipid A, and monophosphoryl lipid A, preferably 3-takes off-compositions of O-acyl group monophosphoryl lipid A (3D-MPL) and a kind of aluminum salt.

A kind ofly improve the combination that system relates to monophosphoryl lipid A and a kind of saponin derivative, the particularly combination of disclosed QS21 and 3D-MPL among the WO 94/00153, thereby or the open method of pressing WO 96/33739 with cholesterol a kind of compositions that QS21 cancellation reactionogenicity is more weak.

WO 95/17210 has described a kind of especially effectively adjuvant prescription, and this prescription relates to QS21,3D-MPL and tocopherol are added in a kind of oil-in-water emulsion, and this prescription is preferred prescription.

Preferred, can contain a kind of Saponin in addition in the vaccine, preferred then is QS21.This prescription also can comprise a kind of oil-in-water emulsion and tocopherol (WO 95/17210).

The present invention also provides a kind of method, is used to produce a kind of bacterin preparation, and this method comprises the excipient that a kind of albumen of the present invention and a kind of pharmacy are allowed, as 3D-MPL, mixes mutually.

The non-methylated CpG (WO 96/02555) that contains oligonucleotide also is to be applicable to that preferred TH1 of the present invention replys derivant.

Special preferred composition of the present invention contains one or more bonded pneumococal polysaccharides, one or more pneumoproteins and a kind of Th1 adjuvant.Use this Th1 adjuvant to help replying of inducing cell mediation by pneumoprotein (as indicated above), and help collaborative between two kinds of immune systems, thereby can obtain a kind of especially effectively vaccine, resist common people's streptococcus pneumoniae property disease, the more important thing is the pneumococcal pneumonia that can resist the people at advanced age.

On the other hand, the invention provides a kind of immunogen or vaccine as described herein, be used in medical science, using.

Again on the one hand, the invention provides a kind of compositions that contains pneumococal polysaccharide conjugate and Th1 adjuvant (preferably 3D-MPL), said composition can cause seroconversion or induce anti-polysaccharide antigen in no response colony humoral antibody is replied.

The people of the known 10-30% of having does not produce the polysaccharide immunization and replys (serotype more than 50% in the vaccine is not produced reply) (Konradsen et al., Scand.J.Immun 40:251 (1994); Rodriguez et al., JID, 173:1347 (1996)).Using the conjugate vaccine also is (Musher et al.Clin.Inf.Dis.27:1487 (1998)) like this.To high-risk group (baby, child and advanced age people) especially severe then.

The inventor is surprised to find, and bonded pneumococal polysaccharide (being easy to produce low replying in special group) is used in combination with a kind of Th1 adjuvant (" Th1 adjuvant of the present invention " sees above) can overcome this unresponsiveness.Preferably use 3D-MPL, the most preferred 3D-MPL (can obtain so better to reply) that does not contain aluminum type adjuvant that is to use.Therefore, the invention provides these compositionss, and further provide a kind of method, be used for the treatment of pneumococal polysaccharide is not produced the individuality of replying, method is with these compositions administrations, the application of Th1 adjuvant in containing the drug manufacture of bonded pneumococcal polysaccharide antigen also is provided simultaneously, and at the application in the streptococcus pneumoniae property treatment of diseases of (or protection) polysaccharide antigen unresponsiveness individuality.

The present invention provides a kind of method in an embodiment, be used for preventing or improving people's at advanced age pneumonia, this method comprises vaccine described in the literary composition of safety and effective dose to described elderly patient administration, this vaccine contains a kind of streptococcus pneumoniae polysaccharides antigen, and contains a kind of Th1 adjuvant or a kind of pneumoprotein (preferably the both comprises).

The present invention provides a kind of method in another embodiment, be used for preventing or improving baby or child's otitis media, this method comprises that a kind of vaccine with safety and effective dose is to described baby or child's administration, this vaccine contains a kind of streptococcus pneumoniae polysaccharides antigen, and contains a kind of streptococcus pneumoniae proteantigen or a kind of Th1 adjuvant (preferably the both comprises).

In said method of the present invention, preferably provide described polysaccharide antigen with GL-PP conjugate form.

Bacterin preparation of the present invention

Bacterin preparation of the present invention can be used to protection or treatment easy infection mammal, and method is through whole body or mucosal route administration with described vaccine.These medications comprise intramuscular injection, intraperitoneal injection, intradermal injection or intravenous injection; Or through mucous membrane is to oral cavity/digestive tract, respiratory tract, urogenital canal drug administration.The method for optimizing that is used for the treatment of pneumonia or otitis media is that vaccine is carried out intranasal administration (because can more effectively prevent streptococcus pneumoniae to transport through nasopharynx, thereby in its earliest stages infection being weakened).

Conjugated antigen amount in every vaccinating agent can be chosen in the amount that can cause protective immune response in the typical vaccine but not cause apparent side effect.Its numerical value will and be presented the difference of method and different according to used specific immunogen.It is generally acknowledged that every dose should contain 0.1-100 μ g polysaccharide, preferably 0.1-50 μ g is more preferably 0.1-10 μ g, and most preferred is 1-5 μ g.

Proteantigen content in the vaccine is generally 1-100 μ g, 5-50 μ g preferably, the most preferred 5-25 μ g that is generally.

The best composition content of specific vaccine can be determined that this method relates to be observed the suitable immunne response of main body by the research on standard method.Behind primary vaccination, main body can be accepted once or suitable short liter immunity blanking time for several times.

In " vaccine design ", the preparation method of vaccine has been carried out comprehensive argumentation (" subunit and adjuvant method " (eds Powell M.F.; Newman M.J.) (1995) Plenum PressNew York).4,235,877 of the United States Patent (USP)s of Fullerton have been described the method that is encapsulated in the liposome.

B) Xuan Ding pneumococal polysaccharide conjugate+3D-MPL compositions

For the purpose of the present invention, term " pneumococal polysaccharide conjugate of the present invention " is meant that those immunogenicities in the compositions that only contains 3D-MPL are higher than the immunogenic streptococcus pneumoniae capsular polysaccharide conjugate (as the conjugate of serotype 4, the conjugate of serotype 6B, the conjugate of serotype 18C, the conjugate of serotype 19F or the conjugate of serotype 23F) in the compositions that contains 3D-MPL and a kind of aluminum type adjuvant at the same time.

For the purpose of the present invention, term " does not contain aluminum type adjuvant substantially " and is meant not contain in the compositions and is enough to that pneumococal polysaccharide conjugate of the present invention is lower than only contains 3D-MPL aspect immunogenicity and do not add the aluminum type adjuvant (as aluminium hydroxide, particularly aluminum phosphate) of the same combination of aluminum type adjuvant.The adjuvant that preferred antigen composition is comprised should be essentially 3D-MPL.Preferred, the amount of the aluminum type adjuvant that adds in every dosage should be less than 50 μ g, more preferably is less than 30 μ g, more preferably is less than 10 μ g, and does not add any aluminum type adjuvant in the antigen composition most preferably of the present invention.

For the purpose of the present invention, should be by the described immunogenicity of the immunogenicity of pneumococal polysaccharide conjugate in the compositions that only contains 3D-MPL in the compositions that contains 3D-MPL and a kind of aluminum type adjuvant at the same time that determine whether of embodiment 2.As the immunogenicity of the compositions that only contains 3D-MPL higher indication whether obviously, only containing the compositions of 3D-MPL should be greater than 2 with the GMC IgG concentration ratio that contains the compositions of 3D-MPL and a kind of aluminum type adjuvant simultaneously, preferably greater than 5, be more preferably greater than 7, what be more preferably is greater than 9, and most preferably greater than 14.

In the problem relevant with the polysaccharide inoculation method, it is that polysaccharide is originally as weak immunogen that a problem is arranged.The strategy that is used for overcoming this immunogenicity defective comprises, with polysaccharide be connected with the large protein carrier that can assist other T cell (combination).Preferably pneumococal polysaccharide of the present invention is connected with a kind of protein carrier of other T cell of assisting.Spendable examples of such carriers comprises, for example, and the OMPC of diphtheria toxin, diphtherotoxin, diphtheria toxin mutation and tetanus toxoid (being respectively DT, CRM197 and TT), keyhole limpet homocyanin (KLH), tuberculin purfied protein derivative (PPD) and Neisseria meningitidis.

Most preferably protein D (EP 0594610-B) or its fragment (seeing the C trifle) with Haemophilus influenzae is used for pneumococal polysaccharide of the present invention as this immunogenic protein carrier.

In an embodiment, antigen composition of the present invention contains the pneumococal polysaccharide serotype (PS) 4 that combines with a kind of immunogenic protein, and prepares with the 3D-MPL adjuvant, and wherein said composition does not contain aluminum type adjuvant substantially.Antigen composition in other embodiment then contains PS 6B, 18C, 19F or the 23F that combines with a kind of immunogenic protein respectively, and prepares with the 3D-MPL adjuvant, and wherein said composition does not contain aluminum type adjuvant substantially.

The present invention provides a kind of combined antigen composition in another embodiment, said composition contains two or more pneumococal polysaccharide conjugates, and prepare with the 3D-MPL adjuvant, these conjugates are selected from PS 4, PS 6B, PS 18C, PS19F and PS 23F, and wherein said composition does not contain aluminum type adjuvant substantially.

Add other pneumococal polysaccharide conjugate and can obviously not influence the immunogenicity (embodiment 3) of pneumococal polysaccharide conjugate of the present invention.Therefore, as optimal way, the invention provides a kind of combined antigen composition, said composition contains one or more pneumococal polysaccharide conjugates of the present invention and one or more other pneumococal polysaccharide conjugates simultaneously, wherein said composition is prepared with the 3D-MPL adjuvant, and does not contain aluminum type adjuvant substantially.

The present invention provides combined antigen composition in another preferred embodiment, these compositionss contain a kind of in PS4,6B, 18C, 19F or the 23F pneumococal polysaccharide conjugate at least, preferred then contain wherein 2 kinds, 3 kinds, 4 kinds or all 5 kinds, other pneumococal polysaccharide conjugate that contains combination in any in addition, these compositionss are all prepared with the 3D-MPL adjuvant, but do not contain aluminum type adjuvant substantially.

Combined streptococcus pneumoniae antigen compositions of the present invention all contains polysaccharide conjugate antigen usually, polysaccharide wherein derives from 4 kinds, 7 kinds, 11 kinds, 13 kinds, 15 kinds or 23 kinds of serotypes (see above " streptococcus pneumoniae polysaccharides antigen of the present invention ", its preferred serotype compound mode depends on the disease that will treat) at least.

Preferably, antigen composition of the present invention can be used for the prevention (or treatment) that the streptococcus pneumoniae sexuality is dyed as vaccine combination, especially for advanced age people and baby and child.

Other embodiment of the present invention comprises: provide above-mentioned antigen composition to be used for medical domain; A kind of method is used for inducing the immunne response to the streptococcus pneumoniae capsular polysaccharide conjugate, and this method comprises the step of one of above-mentioned antigen composition of safety and effective dose to patient's administration; And one of above-mentioned antigen composition is in the application of the drug manufacture that is used for prevention (or treatment) streptococcus pneumoniae property disease.

When preventing/improving crowd's at advanced age (+55 years old) pneumonia and baby (before 18 months) and child's's (being generally 18 months-5 years old) otitis media, another preferred embodiment of the present invention is to combine with a kind of pneumonia streptococcus mycoprotein or its immunologic function equivalent by the multivalence streptococcus pneumoniae polysaccharides conjugate of the preparation of method described in the literary composition.Preferred albumen/protein combination can be with reference to " pneumoprotein of the present invention " joint above.

If do not use immediately, preferably with antigen composition mentioned above (and vaccine) lyophilizing, and can be before use with diluent with its instant reconstruct.More preferably under the situation that 3D-MPL exists with its lyophilizing, and with the instant reconstruct of saline solution.

Composition freeze-drying can be made its stable more (for example can avoid polysaccharide antigen to decompose).Surprisingly, this method can also make antibody higher to tiring of pneumococal polysaccharide.For the PS6B conjugate, this phenomenon is obvious especially.Therefore, another form of the present invention is a kind of freeze dried antigen composition, and said composition contains PS 6B conjugate, and with 3D-MPL adjuvant preparation and do not contain aluminum type adjuvant substantially.

The preparation of vaccine can be with reference to above " bacterin preparation of the present invention " joint.

C) bacterial polysaccharides-protein D conjugate

Tend to use the advantage of combination-vaccine to have, reduce discomfort, be convenient to the arrangement progress, and guarantee finishing of strict control excipient; But also can be attended by the danger (the relevant argumentation of excessively using carrier protein to cause epi-position to suppress sees above) that vaccine efficient reduces.Therefore, useful is formulate between the needs that can satisfy certain colony and its component not show the interferential vaccine compound mode of immunogenicity.These advantages can realize by immunogenic composition of the present invention (or vaccine), and they are particularly conducive to combined vaccine to baby, child or the high-risk crowd's administration of people etc. at advanced age.

The invention provides a kind of Haemophilus influenzae protein D or its fragment, to be used for immunogenic composition as carrier, comprising vaccine based on polysaccharide.The fragment that is suitable for using comprises the fragment that contains helper T cell epitope.Especially, the fragment of protein D preferably should contain the part of this albumen n end 1/3.

Protein D is a kind of IgD conjugated protein (EP 0594610B1) that derives from Haemophilus influenzae, and it is a kind of potential immunogen.

What the present invention considered comprises with the bonded polysaccharide of protein D, but be not limited to, the Vi polysaccharide antigen of anti-Salmonella typhi, meningococcal polysacharide (comprises the A type, the C type, W135 type and Y type, and category-B meningococcal polysacharide and modified polysaccharide), polysaccharide from staphylococcus aureus, polysaccharide from streptococcus agalactiae, polysaccharide from streptococcus pneumoniae, from the polysaccharide that props up bacillus such as the Mycobacterium tuberculosis five equilibrium (as mannose phosphoinositide trehalose, mycolic acid, the AM of band mannose medicated cap, its pod membrane and arabinogalactan), polysaccharide from neogenesis cryptococcus, the lipopolysaccharide of unfiled Haemophilus influenzae, capsular polysaccharide from Haemophilus influenzae b, the lipopolysaccharide of Moraxella catarrhalis, the lipopolysaccharide of shigella sonnei, the lipopeptid phosphoglycan (LPPG) of schizotrypanum cruzi, the Ganglioside, GD3 relevant with cancer, GD2, the mucin relevant with tumor, particularly T-F antigen and sialic acid T-F antigen, and the HIV relevant polysaccharide relevant on the structure with T-F antigen.

Polysaccharide with can adopt any known method (as the method for people's such as the United States Patent (USP) 4,372,945 of Likhite and Armor United States Patent (USP) 4,474,757) being connected of carrier protein.Preferably adopt CDAP combined techniques (WO 95/08348).

In the CDAP method, preferably use cyanidization agent 1-cyano group-dimethylamino naphthyridine Tetrafluoroboric acid ester (CDAP) to synthesize the polysaccharide-protein conjugate.This cyanidization agent can play a role under gentle relatively condition, thereby can avoid the hydrolysis of alkali sensitivity polysaccharide.This synthetic method can be used for combining with the direct of carrier protein.

Polysaccharide is solubilized into water or saline solution.CDAP is dissolved in acetonitrile, and is added in the polysaccharide solution immediately.CDAP can with the hydroxyl reaction of polysaccharide, thereby form a kind of cyano group ester.After this activation step, add carrier protein.Lysine amino can be reacted with activatory polysaccharide, thereby it is covalently bound to form a kind of isourea.

After this association reaction, add excessive greatly glycine again with the remaining activation of cancellation functional group.Then product is carried out gel permeation chromatography to remove unreacted carrier protein and residual reagent.Therefore, the invention provides a kind of method, be used for producing GL-PP D conjugate, this method comprises with the polysaccharide activation and with polysaccharide and protein D step of connecting.

The present invention provides a kind of immunogenic composition (or vaccine) preparation in a preferred embodiment, is used for preventing streptococcus pneumoniae infection.

Streptococcus pneumoniae also is not very clear to the mechanism of lung, cerebrospinal fluid and blood diffusion.Bacterial growth can be suppressed by the activate the phagocytic capacity of the environment of its relatively dry and pulmonary alveolar macrophage to the extension of normal alveolar.These defence of coordinating mutually all can cause lung that the susceptibility that infects is increased if any tissue or physiological variation occur.The cell wall of streptococcus pneumoniae has important function (Gillespie et al. (1997) I﹠amp in the production process of alveolar inflammatory reaction; I65:3936).

Streptococcus pneumoniae vaccine of the present invention generally all contains the protein D polysaccharide conjugate, and polysaccharide wherein derives from 4 kinds, 7 kinds, 11 kinds, 13 kinds, 15 kinds or 23 kinds of serotypes at least.Preferred serotype combination can be with reference to above " streptococcus pneumoniae polysaccharides antigen of the present invention ", and these compound modes depend on the disease that will treat.

The present invention provides a kind of Neisseria meningitidis vaccine in another embodiment; Particularly derive from the vaccine of serotype A, B, C, W-135 and Y.Neisseria meningitidis is one of most important cause of disease of bacterial meningitis.The saccharide pod membrane of these antibacterials can be used as the determiner of its virulence, and can serve as the effective object of protection antibody.But well-known is that saccharide is a kind of weak immunogen in child of tender age's body.The invention provides a kind of protein carrier that is specially adapted to these polysaccharide, protein D, it can provide the t cell epitope of can activated T cell replying, thereby helps the propagation and the maturation of polysaccharide antigen specific b cells, and helps the induction of immunity memory.

The present invention provides a kind of capsular polysaccharide of Haemophilus influenzae b (PRP)-protein D conjugate in an optional embodiment.

The present invention has also considered to protect the combined vaccine of a series of different pathogens.Surprisingly, the protein D carrier can be used as the effective carrier of the combined vaccine that is combined with multiple polysaccharide antigen.As mentioned above, if various polysaccharide all use identical carrier, then may cause epi-position and suppress.WO 98/51339 provides some compositions, attempts this interference is reduced to minimum, and method is that a certain proportion of polysaccharide in the compositions is combined on the DT, and remaining then is combined on the TT.

The inventor is surprised to find, and protein D is particularly suitable in combined vaccine this type of epi-position depression effect being reduced to minimum.It is useful that one or more polysaccharide in the compositions are combined on the protein D, preferred then be that all antigens in this type of combined vaccine all are combined on the protein D.

Preferred compositions comprises can protect a kind of vaccine that Neisseria meningitidis C and Y (preferably then also having A) infect, and one or more the polysaccharide antigen that wherein derives from serotype Y and C (most preferred then also have A) all is connected in protein D.

Based on the vaccine of Haemophilus influenzae polysaccharide (preferably with TT, DT or the bonded PRP of CRM197, or most preferably with the bonded PRP of protein D) available combinations thereof type vaccine prepares.

At present, there are many children's to provide with combined vaccine form, can reduce the frequency injection that the child must accept like this with vaccine.Therefore, when using vaccine as children's, available vaccine of the present invention is prepared other antigen.For instance, can use well-known " trivalent " combination-vaccine that contains diphtheria toxoid (DT), tetanus toxoid (TT) and pertussis component (being generally toxicide DT-Pa (PT) and filamentous hemagglutinin (FHA) and optional pertactin (PRN) and/or agglutinin 1+2), as contain the antigenic commercialized vaccine INFANRIX-DTPa of DT, TT, PT, FHA and PRN ^TM(SmithKlineBeechamBiologicals), or the whole cell pertussis component, as the commercialization Tritanrix of SmithKlineBeechamBiologicals s.a. ^TM, prepare vaccine of the present invention, or independent administration simultaneously.The vaccine of combination also can contain other antigen, as hbs antigen (HBsAg), poliovirus antigen (as the trivalent poliovirus of deactivation--IPV), Moraxella catarrhalis outer membrane protein, unfiled Haemophilus influenzae albumen, Neisseria meningitidis B outer membrane protein.

The example that can be included in the Moraxella catarrhalis proteantigen in the combined vaccine (particularly be used for prevention of otitis media) has: OMP106 (WO 97/41731 (Antex) ﹠amp; WO 96/34960 (PMC)); OMP21; LbpA ﹠amp; LbpB (WO 98/55606 (PMC)); TbpA ﹠amp; TbpB (WO 97/13785 ﹠amp; WO 97/32980 (PMC)); CopB (Helminen ME, et al. (1993) Infect.Immun.61:2003-2010); UspA1/2 (WO 93/03761 (University of Texas)); And OmpCD.The antigenic example of unfiled Haemophilus influenzae that can be included in the combined vaccine (particularly be used for prevention of otitis media) has: fimbrin ((US 5766608-Ohio State Research Foundation)) and contain fusant from the fimbrin peptide (as LB1 (f) peptide fusant; US 5843464 (OSU) or WO 99/64067); OMP26 (WO 97/01638 (Cortecs)); P6 (EP 281673 (State University of New York)); TbpA and TbpB; Hia; Hmw1,2; Hap; And D15.

The preferred children's that the present invention considers has with vaccine:

A) Neisseria meningitidis C polysaccharide conjugate and Haemophilus influenzae b polysaccharide conjugate, and optional Neisseria meningitidis A and/or Y polysaccharide conjugate, condition is to have a kind of polysaccharide antigen at least, preferably all polysaccharide antigens combine with protein D.

B) vaccine that contains DT, TT, pertussis component (preferably containing PT, FHA and PRN), hbs antigen and IPV (the trivalent poliovirus vaccine of deactivation) a).

C) with the bonded streptococcus pneumoniae polysaccharides antigen of protein D.

D) contain one or more antigenic vaccine c that derive from Moraxella catarrhalis and/or unfiled Haemophilus influenzae).

It is all favourable to above all combined vaccines as carrier to add protein D.Clearly, the kind of carrier that comprises in the combined vaccine many more (for example suppressing) in order to overcome epi-position, final vaccine is just expensive more and complicated more.Therefore, all or most of polysaccharide antigen of combined vaccine is combined on the protein D can brings sizable interests.

When the pneumonia of preventing crowd at advanced age (+55 years old) and baby or child's otitis media, the preferred embodiments of the invention are that the streptococcus pneumoniae polysaccharides of the multivalence described in the literary composition-protein D antigen is combined with a kind of pneumonia streptococcus mycoprotein or its immunologic function equivalent.Optimization protein/the protein composition that can comprise about this compositions can be with reference to " pneumoprotein of the present invention " joint above.

Therefore, the invention provides a kind of immunogenic composition, said composition contains a kind of streptococcus pneumoniae polysaccharides-protein D conjugate and a kind of streptococcus pneumoniae proteantigen.

Preferably in bacterin preparation of the present invention with polysaccharide-protein D conjugate antigen adjuvantization of the present invention.Suitable adjuvant comprises aluminum salt, as alumine hydroxide colloid (Alumen) or aluminum phosphate, but also can be calcium salt, iron salt or zinc salt, or the insoluble float of acidylate tyrosine, or the cation or the anionic derivative of acidylate sugar, polysaccharide, or polyphosphazene.

For being used for people's at advanced age vaccine, preferably selected adjuvant can be used as the preferred derivant that the TH1 type is replied.

Can be about specific Th1 adjuvant with reference to above " Th1 adjuvant of the present invention ".

On the other hand, the invention provides a kind of immunogen or vaccine as described herein, be used in medical science, using.

About the preparation/medication of conjugate vaccine, can be with reference to above " bacterin preparation of the present invention ".

In the vaccine of anti-otitis media, use protein D favourable too,, can produce cell-mediated anti-unfiled Haemophilus influenzae (ntHi) protective action of B because protein D itself is a kind of immunogen.NtHi can invade host cell, and hides the cell-mediated effect by the inductive B of proteantigen.The inventor is surprised to find, and has a kind of method can improve protein D is used for the otitis media vaccine as antigen (no matter be use separately or as the carrier of polysaccharide) effectiveness.Method is with the protein D adjuvantization, makes it can induce a kind of strong Th1 to reply in subject, so just can optimize the cell-mediated immune system at protein D.Surprisingly, use the freeze-dried composition that contains protein D and a kind of Th1 adjuvant (preferably 3D-MPL) can realize this point, said composition can instant reconstruct before administration.Therefore, the present invention also provides the application at otitis media kind apoplexy due to endogenous wind of this based composition, this type of method for compositions of a kind of preparation (method is to contain the mixture lyophilizing of protein D and a kind of Th1 adjuvant) and said composition.

On meaning widely, the inventor thinks that at Th1 adjuvant (seeing " Th1 adjuvant of the present invention "), preferably 3D-MPL under the situation of existence can improve the immunogen lyophilizing to this immunogenic Th1 immunne response usually.Therefore, the present invention is applicable to any immunogen that need produce strong Th1 immunne response at it.These immunogens comprise bacillary, viral and the tumprigenicity proteantigen, also comprise these albumen itself and peptide thereof.

Description of drawings

Figure 1A and 1B demonstration suppress to tire (HLI) by the ELISA IgG and the haemolysis of serum measurement after the immunity of III level.

Fig. 1 C shows that the 11 valency polysaccharide conjugate vaccines of adding PdB and AlP04+MPL adjuvant have produced very significantly protective action (p＜0.007) (black stripe is represented arithmetical average).

Embodiment

These embodiment can the present invention will be described, but do not limit the present invention.

Embodiment 1

Streptococcus pneumoniae capsular polysaccharide:

Candidate's 11 valency vaccines contain serotype 1,3,4,5,6B, 7F, 9V, 14,18C, 19F and 23F capsular polysaccharide, and its preparation is substantially as described in the EP 72513.Utilize CDAP chemicals (WO 95/08348) with every kind of polysaccharide activation and derivation, and combine with protein carrier.Except that serotype 3 (reducing size to reduce its viscosity), all polysaccharide are all with its native form combination.

Protein carrier:

Selected protein carrier is to derive from unfiled Haemophilus influenzae but at the recombiant protein D of expression in escherichia coli.

The expression of protein D

Haemophilus influenzae

Be used for the gene constructed of protein D expression

Raw material

The protein D coding DNA

Protein D high conservative all in all serotypes and non-classified Haemophilus influenzae.The carrier pHIC348 that contains whole protein D gene code DNA sequence is by doctor A.Forsgren, Department of Medical Microbiology, and University of Lund, Malm  General Hospital, Malm , Sweden provides.The DNA sequence of protein D is delivered by Jansonet al. (1991) Infect.Immun.59:119-125.

Expression vector pMG1

Expression vector pMG1 derives from pBR322 (Gross et al., 1985), has wherein introduced the regulatory factor that is derived from phage, and this regulatory factor is used to regulate and control the transcribing and translating of exogenous gene (Shatzman et al., 1983) of insertion.In addition, ampicillin resistance gene has been replaced by kalamycin resistance gene.

Coli strain AR58

Promptly obtain coli strain AR58 with deriving from the P1 phage original seed transduction N99 that grows in the bacterium (galE::TN10, LambdaKil cI857 Δ H1) at SA500 in advance.N99 and SA500 belong to e. coli k12 strain, are provided by Martin doctor's Rosenberg of National Institute of Health laboratory.

Expression vector pMG1

When producing protein D, will encode this proteic dna clone in expression vector pMG1.This plasmid uses the signal of lambda bacteriophage dna to promote external source and inserts gene transcription and translation.This carrier contains promoter PL, operon OL and 2 kinds of available sites (NutL and NutR), thereby can alleviate transcription polarity effect (Gross et al., 1985) when N albumen exists.The carrier that will contain the PL promoter is introduced a kind of escherichia coli lysogeny host, to keep the stable of plasmid DNA.The lysogeny host strain contains the replication defect type lambda bacteriophage dna (Shatzman et al., 1983) that is incorporated in the genome.Lambda bacteriophage dna in the chromosome can instruct the cI repressor proteic synthetic, and this albumen can combine with the OL repressor of carrier, combines with the PL promoter by RNA polymerase and transcribes thereby avoid inserting gene.The cI gene of expression strain AR58 contains a kind of temperature-sensitive mutation, thereby can regulate and control transcribing of PL guidance by variations in temperature, promptly improves cultivation temperature and can make the repressor inactivation, thereby begin synthetic foreign protein.This expression system can be realized the virose foreign protein of foreign protein, particularly pair cell, controlled synthetic (Shimataka ﹠amp; Rosenberg).

Coli strain AR58

The AR58 lysogeny coli strain that is used for producing the protein D carrier derives from the (F in standard N IH e. coli k12 strain N99 ^-Su ^-GalK2, lacZ ^-Thr ^-).It contains deficiency lysogeny bacteriophage lambda (galE::TN10, LambdaKil ^-CI857 Δ H1).The Kil phenotype can prevent that the host from stopping synthetic macromolecule.The cI857 sudden change can make the cI repressor have a kind of thermally sensitive damage.Δ H1 disappearance can be removed the right side operon of bacteriophage lambda and host's bio, uvr3 and chlA gene loci.With deriving from bacterium (galE::TN10, LambdaKil at SA500 in advance ^-CI857 Δ H1) the P1 phage original seed of growth transduction N99 promptly obtains coli strain AR58 in.Owing to have the TN10 transposon of coding tetracyclin resistance near the galE gene, thereby can utilize tetracycline that the deficiency lysogen is screened to the transduction situation of N99.The structure of carrier pMGMDPPrD

What the structure of pMGMDPPrD used is the pMG1 carrier (pMGNSI) that contains influenza virus S1 non-structural protein encoding gene.Utilization amplifies protein D gene through PCR by pHIC348 carrier (Janson et al.1991) at the PCR primer that 5 ' and 3 ' end contains NcoI and XbaI restriction site respectively.The NcoI/XbaI fragment is incorporated between the NcoI and XbaI of pMGNSI then, has so just created a kind of 81 amino acid whose fusion rotein of NS1 albumen n end that before PD albumen, contain.This carrier is designated as pMGNS1PrD.

On the basis of above-mentioned construct, produce and be used for the final construct that protein D is expressed.From pMGNS1PrD, remove the BamHI/BamHI fragment.This DNA hydrolysis can be removed the NS1 coding region and only keep preceding 3 residues that its N holds.Carrier is reconnected, has promptly produced the gene that a kind of codified has the fusion rotein of following N terminal amino acid sequence:

-----MDP SSHSSNMANT-----

The NS1 protein D

Protein D does not contain the N end cysteine of leading peptide or being connected with the fat chain usually.Therefore, this albumen can not be secreted in the pericentral siphon, can be by fatization yet, but be retained in the Cytoplasm with soluble form.

By 37 ℃ of heat shocks final construct pMG-MDPPrD is incorporated in the AR58 host bacterium.Screening contains the antibacterial of plasmid under the situation that kanamycin exists.With the selected isolating plasmid DNA of endonuclease enzymic digestion, therefore prove the existence of protein D coding DNA insertion sequence.The coli strain of reorganization is called ECD4.

At λ P _LPromoter/O _LProtein D is expressed in the regulation and control of operon down.Contain temperature sensitivity cI gene in the genome of host bacterium AR58, it at low temperatures can by with O _LIn conjunction with hindering P by λ _LInitial expression.In case temperature improves, O _LPromptly discharge cI, thereby protein D can be expressed.After fermentation ends, can be with cell concentration and freezing.

As described below by the cell extracting and the purifying protein D that collect.Refrigerated cell culture precipitation is melted, and be resuspended in cell pyrolysis liquid (citrate buffer solution pH6.0), make final OD ₆₅₀=60.Make flow through for the twice high-pressure homogenization instrument of P=1000bar of this suspension.By the homogenate of centrifugal clarification cell culture, and filter and remove cell debris.Purification step is that filtering lysate is splined on cation-exchange chromatography post (SP Sepharose Fast Flow) for the first time.PD can combine with gel-type vehicle by ion exchange, and can be by the ionic strength that increases elution buffer with its eluting.

Purification step can make impurity be retained in (Q SepharoseFast Flow) on the anion exchange substrate for the second time.PD does not combine with this gel, thereby can be collected in the effluent.

Twice column chromatography step all utilizes OD to monitor the fraction collection thing.With the effluent ultrafiltration and concentration of anion-exchange chromatography post, wherein contain the protein D of purification.

At last, make contain protein D the ultrafiltration retentate stream through 0.2 μ m filter membrane.

Chemical reaction:

Activation and coupling chemical reaction:

Every kind of polysaccharide has specific activation and coupling condition.These conditions are listed in table 1.Natural polysaccharide (except the PS3) is dissolved in 2M NaCl or water to be used for injection.Evaluate the best polysaccharide concentration of all serotypes.

The 100mg/ml CDAP liquid storage that will be dissolved in acetonitrile is added to (the CDAP/PS ratio is 0.75mg/mg PS) in the polysaccharide solution.1.5 add the 0.2M triethylamine after minute to obtain specific activation pH value.Under this pH and 25 ℃ of conditions, polysaccharide is activated 2 minutes.Protein D (quantity depends on initial PS/PD ratio) is added in the activatory polysaccharide, and makes coupling reaction under specific pH, continue 1 hour.To react cancellation 30 minutes with glycine then under 25 ℃, 4 ℃ are spent the night again.

With the conjugate purification, what filter use is the equilibrated Sephacryl 500HR of 0.2M NaCl solvent resistant column by gel filtration.

Measure the saccharide and the protein content of elution fraction.Conjugate is merged reuse 0.22 μ m degerming membrane filtration degerming.Measure the PS/ Protein ratios of this conjugate preparations.

Character is identified:

Identify the character of each conjugate, its result meets the described detailed description of table 2.The mensuration of polyoses content (μ g/ml) adopts the resorcinol algoscopy, and the mensuration of protein content (μ g/ml) adopts the Lowry algoscopy.Determine final PS/PD ratio (w/w) by the ratio of concentration.Residual DMAP content (ng/ μ g PS):

Cyano group can be introduced polysaccharide with the CDAP activated polysaccharide, and discharge DMAP (4-dimethylamino-pyridine).Utilize the fc-specific test FC method of SB exploitation to determine residual DMAP content.Dissociation amylase content (%):

With 4 ℃ of preservations or 37 ℃ of conjugate and α-PD antibody insulations that store 7 days, reuse ammonium sulfate is saturated, and is centrifugal then, measures the dissociation amylase content in the supernatant at last.

Utilize α-PS/ α-PS ELISA method to measure the content of free polysaccharide in the supernatant.Also utilize α-PD/ α-PS ELISA method to measure the conjugate blank in addition.The amount that reduces free polysaccharide can obtain improved conjugate vaccine.

Antigenicity:

Utilize the antigenicity of the above-mentioned conjugate of sandwich ELISA methods analyst, wherein capture antibody and detection antibody are respectively α-PS and α-PD.

Floating preteins content (%):

Measure the level of " free " residual protein D by the method for handling sample with SDS.At 100 ℃ and exist under the condition of 0.1%SDS, be splined on SEC-HPLC solvent resistant column (TSK 3000-PWXL) then with conjugate heating 10 minutes.Because protein D is a dimer, so may make the level of " dissociating " protein D by too high estimation the dissociated method of this structure with SDS.

Molecular size (K _Av):

Go up the mensuration molecular size at SEC-HPLC solvent resistant column (TSK 5000-PWXL).

Stability:

Go up the stability of measuring 4 ℃ of preservations or 7 days conjugate of 37 ℃ of storages at HPLC-SEC solvent resistant column (TSK 6000-PWXL).

Table 2 provides the characteristic of 11 valency vaccines.

Protein conjugates can be adsorbed on the aluminum phosphate, merge then, become final vaccine.Conclusion:

Promptly produced immunogenic conjugates thus, found afterwards that this conjugate can be used as a kind of composition of quite promising vaccine.All found the optimization CDAP condition that obtains high-quality final pneumococal polysaccharide conjugate at every kind 11 valency vaccine.Therefore, these pneumococal polysaccharide conjugates that obtained by the CDAP method of above-mentioned improvement (optimization) (irrelevant with used carrier albumen, but preferably protein D) can be used as another aspect of the present invention.

The improved adjuvant of embodiment 2-is to the sex research of the 11 valency streptococcus pneumoniae PS-PD immunogens of conjugate vaccine in underage rat

With 11 valency streptococcus pneumoniae PS-PD conjugate vaccine immunity underage rats, dosage is every kind of polysaccharide (pressing the method preparation of embodiment 1) 0.1 μ g, and uses following adjuvant prescription: no adjuvant, AlPO ₄, 3D-MPL, be adsorbed on AlPO ₄On 3D-MPL.

In 11 kinds of antigens, the dosage form that only contains 3D-MPL has 5 kinds than what other dosage form had more immunogenicity (the highest GMC IgG) statistically.When using high concentration and low concentration 3D-MPL all is like this.

The opsonin phagocytosis is also supported this GMC result.

Materials and methods

Immunization protocol

The teenage OFA rat in 7 day age that the female Mus of difference is bred at random carries out first immunisation.And after 14 and 28 days, carry out 2 immunity again.Get blood when the 56th day (after the immunity of III level the 28th day).All vaccines are subcutaneous injection, 10 rats of every group of vaccine immunity.

1,3,4,5,6B, 7F, 9V, 14,18C, 19F, 23F with the 11 valency streptococcus pneumoniae conjugate vaccine immunity rat that contain the following serotype polysaccharide that is combined on the protein D:.

Compound method

For detecting the different effects of improving adjuvants, it is constant that the dosage of every kind of polysaccharide conjugate is remained on 0.1 μ g, and use AlPO ₄Prepare with various dose and combining form with the 3D-MPL adjuvant, comprising not adding any adjuvant.Its result lists in table 3 with the numerical value form, for your guidance.Be adsorbed on the AlPO4

The preparation method of the concentrated univalent vaccine that is adsorbed is as follows.With 50 μ g AlPO ₄Mix with 5 μ g conjugate polysaccharide (pH 5.1).After 2 hours its pH is transferred to pH 5.1, and then placed 16 hours.Add 1500mM NaCl, make salinity finally be 150mM.After 5 minutes, add the 2-phenoxyethanol of 5mg/ml.Placed 30 minutes, and again pH was transferred to 6.1, place more than 3 days in 4 ℃ then.

The preparation of diluent

3 kinds of diluent of preparation in NaCl 150mM/5mg/ml phenoxyethanol

A：1mg/ml AlPO ₄

B: concentration be respectively 250 and 1000g/ml be adsorbed on AlPO ₄On 3D-MPL, 3D-MPL/AlPO ₄Weight ratio=5/20.

C: concentration is respectively the AlPO that is adsorbed on of 561 and 1000 μ g/ml ₄On 3D-MPL, 3D-MPL/AlPO ₄Weight ratio=50/89.

The preparation of the 11 valency vaccines that are adsorbed

11 kinds of concentrated PS-PD univalent vaccines that are adsorbed are mixed with proper proportion.A adds AlPO by diluent ₄When needs add 3D-MPL, then with aqueous solution form (not adsorption form, the method 1 that sees below) or by diluent B or C (2 multiple doses be adsorbed on AlPO ₄On 3D-MPL, the method 2 that sees below) add.

Method 1

3D-MPL is added in the combined conjugate that is adsorbed with the waterborne suspension form.Under the room temperature it is mixed 10 minutes with 11 valency vaccines, and before administration, be stored in 4 ℃.

Method 2

3D-MPL is adsorbed on AlPO in advance ₄On, add to then (diluent B and C) in the combined conjugate that is adsorbed.Under the room temperature waterborne suspension (250 or 561 μ g) of 3D-MPL and 1mg are dissolved in the AlPO of 150mM NaCl pH6.3 ₄Mixed 5 minutes, and made the 1ml diluent.Dilute this solution with NaCl pH6.1/ phenoxyethanol, again 4 ℃ of incubated overnight.

The preparation of the 11 valency vaccines that are not adsorbed

11 kinds of PS-PD conjugates are mixed, and be diluted in 150mM NaClpH6.1, phenoxyethanol with proper proportion.When needs add 3D-MPL, then add with solution form (not adsorption form).

All preparations that are used to inject all are time preparations in the 18th day before first administration.

ELISA

What the ELISA method of measurement rat IgG adopted is the scheme of WHO Workshop, and this ELISA method can be used to measure the anti-streptococcus pneumoniae capsular polysaccharide IgG antibody content in the human serum.In essence, be the capsular polysaccharide direct coated microtitration plate of using purification.With blood serum sample and total cell wall polysaccharides (C material) pre-incubation of all streptococcus pneumoniae, this cell wall polysaccharides accounts for 0.5% in the pneumococal polysaccharide according to disclosure (EP 72513 B1) purification.Utilize Jackson ImmunoLaboratories Inc. reagent to detect bonded Mus IgG then.According to the logic logarithmic equation modeling rendering of internal standard substance (monoclonal antibody) curve of tiring.Utilize SoftMax Pro software to be calculated again.Can think that these results' maximum absolute error coefficient is less than 2.Relative error then is lower than 30%.

The opsonin phagocytosis

Tire by the opsonin that CDC method (with the opsonin phagocytosis of the HL60 cell detection streptococcus pneumoniae that breaks up, 1.1 schemes) is measured serotype 3,6B, 7F, 14,19F and 23F with the people PMN and the young rabbit complement of purification.Make an amendment and comprise and use self (in house) streptococcus pneumoniae bacterial strain and replace HL60 phagocyte (having dependency highly between these phagocyte) with people's neutrophil cell PMN of purification.Add the 3mm bead in addition to strengthen mixing in microtitration plate, can make phagocyte like this: the ratio of antibacterial is reduced to 400 these recommendations.

The result

IgG concentration

Table 4-10 shows the IgG concentration geometric mean of every kind of serotype and PD.For ease of relatively, also provide before this by serotype 6B, 14, the 19F of 4 valency bacterin preparations acquisition and the result of 23F.

Table 4-10 has highlighted the highest IgG concentration.Table 11 is that 3D-MPL is with respect to 3D-MPL/AlPO ₄The statistical p value of compositions.In 11 kinds of serotypes, the highest GMC ' s that 9 kinds of serotypes are arranged is obtained by No. 4 adjuvant prescription (the not absorption conjugate that contains high dose 3D-MPL).The tool immunogenicity of another kind is a low dosage MPL prescription, accounts for 5/11.In addition, compare with the method that changes dosage, the adjuvant method can make all serotypes all obtain higher GMC ' s (data not shown), and serotype 4,6B, 7F, 18C and 23F have statistical significance (p＜0.05 that is drawn by 95%CI) on the one hand at this.

The opsonin phagocytosis

Table 4-8 shows the opsonin phagocytosis result of the combining anteserum of serotype 3,6B, 7F, 14,19F and 23F.These opsonin are tired and can further be confirmed GMCIgG to a great extent.In fact, the IgG concentration dependency of serotype 6B, 19F, 23F is greater than 85% (data not shown).Be important to note that, to have only the 3D-MPL group to induce the OA that is higher than threshold value to serotype 3.

Conclusion

In this experiment, the IgG concentration of unexpectedly using 3D-MPL to induce separately is the highest.

To compare by changing adjuvant maximum GMC IgG that obtains and the maximum GMC that obtains by change PS dosage, can find in 11 kinds of serotypes have 5 kinds to be that 3D-MPL induces obviously higher replying.

Table 11 shows, by 3D-MPL and 3D-MPL/AlPO ₄The comparison of compositions (relatively compound method and 3D-MPL dosage) can find that the immunogenicity when only preparing with 3D-MPL is apparently higher than using 3D-MPL+AlPO ₄Immunogenic polysaccharide conjugate during preparation has 5 kinds: PS4, PS 6B, PS 18C, PS 19F and PS 23F.

Embodiment 3-combined method is to PS 4, PS 6B, PS 18C, PS 19F and the sex research of the immunogen of PS 23F in adult rat

With the independent immune adult rat of pneumococcal polysaccharide-protein D conjugate vaccine, or with the immune adult rat of multivalence compositions (4 valencys, 5 valencys, 7 valencys or 10 valencys) that merges.10 rats are 1 group, every group of immunity twice, 28 days at interval, and the 28th day and the 42nd day (after the administration for the second time the 14th day) got blood and tested.

By the anti-pneumococal polysaccharide IgG antibody in the ELISA mensuration serum.The measurement result of ELISA shows that all conjugates all induce specific I gG antibody.Table 12 shows the combined method of unit price PS6B, PS 18 C, PS 19F and PS 23F protein D conjugate to its immunogenic influence in adult rat, and this result obtains by the IgG concentration of measuring after the administration for the second time the 14th day.

All samples is carried out statistical analysis, whether notable difference is arranged to determine the antibody concentration after the combination.Polyvalent vaccine with serotype PS 6B, PS 18C, PS 19F and PS 23F protein D conjugate combination in any all can obviously not change its immunogenicity.

Table 1

Specific activation/coupling/the quenched conditions of PS streptococcus pneumoniae-protein D conjugate

Serotype	1	3 (μfluid.)	4	5	6B	7F
							PS concentration (mg/ml)	2.0	3.0	2.0	7.5	5.4	3.0
The PS dissolving	NaCl 2M	NaCl2M	H 2O	H 2O	NaCl2M	NaCl2M
							PD concentration (mg/ml)	5.0	5.0	5.0	5.0	5.0	5.0
PS/PD is initially than (w/w)	1/1	1/1	1/1	1/1	1/1	1/1
							CDAP concentration (mg/mg PS)	0.75	0.75	0.75	0.75	0.75	0.75
pH a＝pH c＝pH q	9.0/9.0/9.0	9.0/9.0/9.0	9.0/9.0/9.0	9.0/9.0/9.0	9.5/9.5/9.0	9.0/9.0/9.0

Serotype	9V	14	18C	19F	23F
						PS concentration (mg/ml)	2.5	2.5	2.0	4.0	3.3
The PS dissolving	NaCl2M	NaCl2M	H 2O	NaCl2M	NaCl2M
						PD concentration (mg/ml)	5.0	5.0	5.0	5.0	5.0
PS/PD is initially than (w/w)	1/0.75	1/0.75	1/1	1/0.5	1/1
						CDAP concentration (mg/mg PS)	0.75	0.75	0.75	0.75	0.75
pH a＝pH c＝pH q	8.5/8.5/9.0	9.0/9.0/9.0	9.0/9.0/9.0	10/9.5/9.0	9.0/9.0/9.0

The detailed description (the bright serotype of first numerical table of lot number code) of table 2:11 valency streptococcus pneumoniae PS-PD vaccine

Standard	D01PDJ227	D03PDJ236	D4PDJ228	D5PDJ235	D6PDJ209
							PS/ Protein ratios (w/w) dissociation amylase content (%)＜10% floating preteins content (%)＜15% DMAP content (ng/ μ g PS)＜0.5ng/ μ g PS molecular size (Kav) stability	1/0.66 18 0.2 0.18 do not change	1/1.09 1＜1 0.6 0.13 does not change	1/0.86 7 19 0.4 0.12 do not change	1/0.86 9 21 1.2 0.11 slightly change	1/0.69 09 0.3 0.13 do not change
	D07PDJ225	D09PDJ222	D14PDJ202	D18PDJ221	D19PDJ206								D23PDJ212
						PS/ Protein ratios (w/w) dissociation amylase content (%)＜10% floating preteins content (%)＜15% DMAP content (ng/ μ g PS)＜0.5ng/ μ g PS molecular size (Kav) stability	1/0.58 18 0.1 0.14 do not change	1/0.80＜1 0.3 0.6 0.14 does not change	1/0.68＜13 0.3 0.17 does not change	1/0.62 4 21 0.2 0.10 do not change	1/0.45 4 10 0.1 0.12 change	1/0.74 0 12 0.9 0.12 do not change

The employed adjuvant prescription catalog of 11 valency streptococcus pneumoniae PS-PD that table 3. is tested in underage rat

Group number	AlPO 4	MPL	Method	Explanation
					1				Do not have
2	100			AlPO 4
					3		5		MPL is low
4		50		The MPL height
					5	100	5	Method 1	Method 1 is low
6	100	50	Method 1	Method 1 height
					7	100	5	Method 2	Method 2 is low
8	100	50	Method 2	Method 2 height

IgG concentration geometric mean, the seroconversion that table 4. carries out after the immunity of III level the 28th day serotype 6B with the 11 valency PS-PD that contain different adjuvants to underage rat on average tire with opsonin (comparing) with 4 valency vaccine immunities

Group number	AlPO 4 μg	MPL μg	Method	6B GMC IgG (μg/ml)	The 6B seroconversion	The 6B opsonin is tired *	6B GMC IgG (μg/ml)	The 6B seroconversion	The 6B opsonin is tired *
														4 valency vaccines			11 valency vaccines
1				0.047	2/10	12.5	0.004	1/10	＜6.25
										2	100			0.048	4/10	65	0.019	4/10	＜6.25
3		5					1.345	10/10	43
										4		50					4.927	10/10	192
5	100	5	1				0.042	7/10	＜6.25
										6	100	50	1				0.255	10/10	＜6.25
7	100	5	2	0.033	3/10	＜6 25	0.048	8/10	＜6.25
										8	100	50	2				0.057	8/10	＜6.25

IgG concentration geometric mean, the seroconversion that table 5. carries out after the immunity of III level the 28th day serotype 14 with the 11 valency PS-PD that contain different adjuvants to underage rat on average tire with opsonin (comparing) with 4 valency vaccine immunities

Group number	AlPO 4	MPL	Method	14 GMC IgG (μg/ml)	14 seroconversion	14 opsonin are tired *	14 GMC IgG (μg/ml)	14 seroconversion	14 opsonin are tired *
														4 valency vaccines			11 valency vaccines
1				0.046	3/10	64	0.022	3/10	＜6.25
										2	100			0.99	10/10	88	0.237	8/10	27
3		5					0.233	10/10	41
										4		50					0.676	10/10	81
5	100	5	1				0.460	9/10	67
										6	100	50	1				0.477	10/10	98
7	100	5	2	0.81	10/10	49	0.165	8/10	81
										8	100	50	2				1.611	10/10	133

IgG concentration geometric mean, the seroconversion that table 6. carries out after the immunity of III level the 28th day serotype 19F with the 11 valency PS-PD that contain different adjuvants to underage rat on average tire with opsonin (comparing) with 4 valency vaccine immunities

Group number	AlPO 4	MPL μg	Method	19F GMC IgG (μg/ml)	The 19F seroconversion	The 19F opsonin is tired *	19F GMC IgG (μg/ml)	The 19F seroconversion	The 19F opsonin is tired *
														4 valency vaccines			11 valency vaccines
1				0.04	2/10	64	0.021	2/10	＜6.25
										2	100			1.07	9/10	367	0.222	7/10	79
3		5					4.028	10/10	296
										4		50					21.411	10/10	1276
5	100	5	1				1.649	10/10	172
										6	100	50	1				2.818	10/10	208
7	100	5	2	1.09	10/10	193	0.766	10/10	323
										8	100	50	2				3.539	10/10	241

IgG concentration geometric mean, the seroconversion that table 7. carries out after the immunity of III level the 28th day serotype 23F with the 11 valency PS-PD that contain different adjuvants to underage rat on average tire with opsonin (comparing) with 4 valency vaccine immunities

Group number	AlPO 4	MPL μg	Method	23F GMC IgG (μg/ml)	The 23F seroconversion	The 23F opsonin is tired *	23F GMC IgG (μg/ml)	The 23F seroconversion	The 23F opsonin is tired *
														4 valency vaccines			11 valency vaccines
1				0.06	2/10	＜6.25	0.152	3/10	＜6.25
										2	100			0.29	10/10	70	0.56	8/10	＜6.25
3		5					2.296	9/10	389
										4		50					4.969	10/10	＞1600
5	100	5	1				0.462	5/10	17
										6	100	50	1				0.635	8/10	54
7	100	5	2	0.38	10/10	＜6.25	0.203	3/10	18
										8	100	50	2				0.501	7/10	43

Table 8. carries out after the immunity of III level the 28th day serotype 3 and IgG concentration geometric mean, seroconversion and the opsonin of 7F with the 11 valency PS-PD that contain different adjuvants to underage rat and on average tires

Group number	AlPO 4	MPL μg	Method		3 GMC IgG (μg/ml)	3 seroconversion	3 opsonin are tired *	7F GMC IgG (μg/ml)	The 7F seroconversion	The 7F opsonin is tired *
											1				0.003	1/10	＜6.25	0.040	7/10	＜6.25
2	100			0.008	6/10	＜6.25	0.25	9/10	43
										3		5		0.070	10/10	＜6.25	2.435	10/10	477
4		50		0.108	10/10	18	2.569	10/10	332
										5	100	5	1	0.015	10/10	＜6.25	0.579	10/10	54
6	100	50	1	0.027	10/10	＜6.25	0.611	9/10	59
										7	100	5	2	0.006	10/10	＜6.25	0.154	8/10	30
8	100	50	2	0.034	10/10	＜6.25	0.638	9/10	140

Table 9. carries out the 28th day serotype 1,4 and 5 IgG concentration geometric mean and seroconversion after the immunity of III level with the 11 valency PS-PD that contain different adjuvants to underage rat

Group number	AlPO 4	MPL μg	Method	1 GMC IgG (μg/ml)	1 seroconversion	4 GMC IgG (μg/ml)	4 seroconversion	5 GMC IgG (μg/ml)	5 seroconversion
										1				0.026	4/10	0.005	0/10	0.040	3/10
2	100			0.282	8/10	0.052	5/10	0.774	9/10
										3		5		1.614	10/10	3.452	10/10	7.927	10/10
4		50		2.261	10/10	7.102	10/10	13.974	10/10
										5	100	5	1	0.568	10/10	0.676	10/10	3.015	10/10
6	100	50	1	1.430	10/10	0.419	9/10	5.755	10/10
										7	100	5	2	0.478	10/10	0.267	9/10	2.062	10/10
8	100	50	2	1.458	10/10	0.423	10/10	5.009	10/10

Table 10. carries out after the immunity of III level the 28th day serotype 9V, 18C and IgG concentration geometric mean and the seroconversion of PD with the 11 valency PS-PD that contain different adjuvants to underage rat

Group number	AlPO 4	MPL μg	Method	9V GMC IgG (μg/ml)	The 9V seroconversion	18C GMC IgG (μg/ml	The 18C seroconversion	PD GMC IgG (μg/ml)	The PD seroconversion
										1				0.018	0/10	0.013	1/10	0.003	0/10
2	100			0.489	6/10	0.092	5/10	0.993	10/10
										3		5		0.482	7/10	6.560	10/10	3.349	10/10
4		50		11.421	10/10	14.023	10/10	5.446	10/10
										5	100	5	1	2.133	9/10	0.690	10/10	11.407	10/10
6	100	50	1	2.558	10/10	1.771	10/10	1.258	10/10
										7	100	5	2	1.536	10/10	0.528	10/10	1.665	8/10
8	100	50	2	2.448	9/10	0.980	10/10	5.665	10/10

Table 11: it is very high that statistical significance (p value) the .p value of the immunogenic improvement situation of the immunogenicity when some pneumococal polysaccharide conjugate is only prepared with 3D-MPL when preparing with 3D-MPL/AlPO4 is considered to significance less than 0.01. and method 1 and method 2 show compound method.

Serotype	50 μ g3D-MPL are to 3D-MPL/AlPO 4		5 μ g3D-MPL are to 3D-MPL/AlPO 4
					Method 1	Method 2	Method 1	Method 2
	1	0.3	0.05	0.079					0.11
3					0.075	0.01	0.27	0.008
	4	0.002	0.0003	0.02					0.003
5					0.04	0.002	0.1	0.12
	6B	0.001	0.0001	0.001					0.0006
7F					0.13	0.15	0.01	0.005
	9V	0.02	0.02	0.1					0.04
14					0.65	0.21	0.3	0.66
	18C	0.0008	0.0002	0.006					0.004
19F					0.0009	0.006	0.21	0.04
	23F	0.002	0.0004	0.01					0.0004

Table 12: use 1.0 μ g GL-PP D conjugates separately or use 4 valencys, 5 valencys, 7 valencys or 10 valence group to close the IgG concentration geometric mean (μ g/ml) that vaccine carried out after the administration for the second time adult rat the 14th day. these aggregation of data 5 independent result of experiment.

Serogroup vaccine	4 H	6B T	18C H	19F	23F T
						Independent	9.3	0.11	15	5.2	2.5
Combination	4	0.23	3.7	3.7	2.8
						T: be combined into 4 valencys (T) (PS 6B.14.19F, 23F), 5 valencys (T adds PS3), 7 valencys (H) (T adds PS4,9V and 18C) and 10 valencys (H add PS1,5 and 7F) combination-vaccine .H: be combined into 7 valencys (H) (T adds PS4,9V and 18C) and 10 valencys (H add PS1,5 and 7F) combination-vaccine.

Embodiment 4-interpolation pneumolysin and 3D-MPL are to the useful influence of 11 valency polysaccharide PD conjugate vaccines in the protection effect aspect the anti-mice streptococcus pneumoniae property lung cluster

Immunity shows

The ELISA consumption of pneumolysin specific serum IgG

With the 2 μ g/ml recombinant natural pneumolysins (PLY) that are diluted in PBS with the amount in 100 μ l/ holes with 37 ℃ of bags of Maxisorp Nunc immunity plate by 2 hours.With NaCl 0.9%Tween-20 0.05% buffer plate is cleaned 3 times.Add anti-PLY as 2 times of dilutions (in PBS/Tween-200.05%, 100 μ, 1/ hole) successively of standard curve (from 670ng/ml IgG) then with reference to serum and blood serum sample (beginning), 20 ℃ of vibration insulations 30 minutes by 1/10 dilution factor.Clean as previously mentioned, adding 5000 * be diluted in the peroxidase coupling mountain sheep anti-mouse igg (Jackson) of PBS/Tween-20 0.05%, 20 ℃ of vibrations are incubated 30 minutes.As above clean, (be dissolved in OPDA 0.4mg/ml and the H of 100mM citrate buffer solution pH4.5 with the developing buffer in 100 μ l/ holes under the room temperature then ₂O ₂0.05%) insulation is 15 minutes.Stop developing by adding 50 μ l/ hole HCl 1N.Utilize Emax immunity readout meter (Molecular Devices) 490 and 620nm under measuring light density.Tire with 4 parameter the mathematical approach calculating antibody with SoftMaxPro software.

Haemolysis suppresses

This assay method measurable serum antibody is to the inhibition ability of pneumolysin (PLY) hemolytic activity.In order to remove cholesterol (easy and PLY interacts), blood serum sample is carried out 2 following * processing: mix the back vibration with isopyknic chloroform and be incubated 45 minutes.Centrifugal 10 minutes of 1000rpm collects supernatant then.In 96 orifice plates (Nunc.), will remove the serum dilution of cholesterol and (use the 1mM dithiothreitol, DTT, 0.01%BSA, 15mM TRIS, 150mMNaCl, pH7.5) 2 times of dilutions successively.Every hole adds the solution that 50 μ l contain 4HU (hemolytic unit) PLY, and 37 ℃ are incubated 15 minutes.Add 100 μ l sheeps blood erythrocytes (1% solution) then, 37 ℃ are incubated 30 minutes.Centrifugal 10 minutes of 1000rpm collects supernatant (150 μ l) then to another 96 orifice plate, measuring light density under 405nm.The result who provides tires for mid point is dilution.

The chemical detoxification of pneumolysin

The natural pneumolysin (PLY) of reorganization is dialysed to the buffer of phosphate 50mM, NaCl 500mM pH7.6.Following all under the condition of 39.5 ℃ and vibration once in a while, carry out in steps.The 1st day, in PLY solution, add the phosphate buffer (3/100 v/v) that contains the phosphate buffer (1/100 v/v) of Tween-8010% (1/250 v/v), N-acetyltryptophan 57.4mM pH7.6 (3/100 v/v), glycine 2.2 M and contain 10% formaldehyde.Added 10% formaldehyde of 3/100 and 2/100 v/v ratio on the the 2nd and the 3rd day more respectively.39.5 ℃ of insulations to the are 7 days under the condition of vibration once in a while.At last with the buffer dialysis of PLY to phosphate 50mM, NaCl500mM pH7.6.Haemolysis is measured and is shown the PLY complete deactivation.

The streptococcus pneumoniae intranasal of OF1 mice is attacked

With OF1 female mice anesthesia in 7 ages in week, with 5.10 ⁵The Mus adaptability streptococcus pneumoniae serotype 6B of CFU carries out intranasal vaccination.Attack after 6 hours lung is taken out, and homogenate (Ultramax, 24000rpm, 4 ℃) is in Todd Hewith Broth (THB, Gibco) culture medium.37 ℃ of bags of culture dish that will contain THB agar and add yeast extract with the lung homogenate thing of 10 times of dilutions are successively spent the night.Measure the quantity of the CFU/ Mus of pneumococcal lung infection, be expressed as logarithmic weighted mean.Detectable limit is 2.14 log CFU/ Mus.

Embodiment 4A

The influence of 3D-MPL adjuvant aspect anti-pneumolysin immunne response

The present invention has evaluated the 3D-MPL adjuvant at anti-natural reorganization pneumolysin (PLY, by J.Paton, Children ' s Hospital, North Adelaide, Australia provides) and the influence of the immunne response aspect of chemical toxicide homologue (DPLY).The toxicide enforcement of chemistry is as mentioned below.

Per 10 6 the week age Balb/c mice be one group, at the 0th, 14 and 21 day with being included in A:AlPO4 100 μ g; Or 1 μ g PLY or DPLY among the B:AlPO4 100 μ g+5 μ g 3D-MPL (3 take off-O-acyl group monophosphoryl lipid A, are provided by Ribi Immunochem) carry out the intramuscular immunity to many groups mice.Figure 1A and 1B demonstration suppress to tire (HLI) by the ELISA IgG and the haemolysis of serum measurement after the immunity of III level.

Which kind of antigen no matter, what induce best immunne response all is animal with the preparation inoculation of adding 3D-MPL.What is interesting is, when with AlPO4+3D-MPL together during administration, the immunogenicity of DPLY is identical with PLY, and is a kind of more weak immunogen in the AlPO4 preparation.This shows that 3D-MPL has the advantage of replying of raising antibody to the detoxification pneumolysin.

In containing the compositions of pneumolysin, preferably use chemically toxicide pneumolysin and do not use with the toxicide pneumolysin of mutation method.This is because the detoxification mutant of Huo Deing is still residual up to now a toxin chemistry--and chemical toxicide pneumolysin can not.Therefore, as another aspect of the present invention, generally should use a kind of Th1 adjuvant, 3D-MPL preferably assists a ruler in governing a country and contains through the compositions of chemical detoxification with the pneumolysin (or pneumolysin mutant) that is used for vaccine.The present invention promptly provides these compositionss.A kind of method also is provided in addition, is used for improving in the immunogenic composition immunne response through the toxicide pneumolysin of chemistry, this method is included in the step of adding a kind of Th1 adjuvant (preferably 3D-MPL) in the compositions.

Embodiment 4B

Add pneumolysin attenuation mutant and 3D-MPL adjuvant to of the useful influence of 11 valency polysaccharide PD conjugate vaccines in the protection effect aspect the streptococcus pneumoniae property lung cluster of resisting the OF1 mice of attacking through serotype 6B intranasal

The present invention has evaluated and has contained 11 valency polysaccharide-protein D conjugates, pneumolysin attenuation mutant antigen (PdB, WO 90/06951) and the preventive effect of the vaccine of AlPO4+3D-MPL adjuvant, and compare with the 11 valency polysaccharide-protein D conjugate conventional formulation that are adsorbed on the AlPO4.

Per 12 4 the week age female OF1 mice be one group, at the 0th and 14 day with containing A:50 μ g AlPO4; Conjugate vaccine+50 μ the gAlPO4 of 11 valency polysaccharide of B:0.1 μ g PS/ serotype-PD); Or the 11 valencys polysaccharide-PD conjugate vaccine of C:0.1 μ g PS/ serotype+10 μ g PdB are (by J.Paton, Children ' s Hospital, North Adelaide, Australia provides)+preparation of 50 μ g AlPO4+5 μ g 3D-MPL (being provided by Ribi Immunochem) carries out subcutaneous immunity to many groups mice.And attacked at the 21st day as mentioned above.

Shown in Fig. 1 C, the 11 valency polysaccharide conjugate vaccines of adding PdB and AlPO4+MPL adjuvant have produced very significantly protective action (p＜0.007) (black stripe is represented arithmetical average).On the contrary, in animal body, do not observe tangible protective action with 11 valencys polysaccharide conjugate/AlPO4 preparation immunity.This result proves, adds the anti-pneumonia efficient that pneumolysin antigen (or even attenuation) and 3D-MPL adjuvant can strengthen 11 valency polysaccharide conjugate vaccines.

Embodiment 4C

Protective action is immune-related shown in the embodiment 4B

In order to establish the immune-related of the protective action that produces among the embodiment 4B, in 11 valency polysaccharide conjugate vaccines, add the pneumolysin mutant (PdB) and the 3D-MPL of attenuation, and mensuration as indicated above is attacked preceding serum antibody response to polysaccharide 6B and PdB.

Then antibody titer is compared with pulmonary's bacterial clump measured value of attack corresponding animal after 6 hours.On the Log/Log linear regression graph, calculate R ²

By calculating the R of anti-PdB and anti-6B antibody response ²Be respectively 0.18 and 0.02.This shows between humoral immunoresponse(HI) and this two kinds of antigenic protective actions does not have dependency.Tiring with 11 valency conjugate vaccines (GMY=0.318ng/ml) immunity or immunely with the identical vaccine (GMY=0.458ng/ml) of adding PdD and 3D-MPL do not have evident difference between on the same group of anti-6B antibody.Therefore, the protective action that is observed by formulation C is strengthened not for no other reason than that stronger to the antibody response of polysaccharide 6B.

In sum, these results show that protective action is not only by the humoral immunoresponse(HI) mediation, but also have the participation of the cell-mediated immunity that PdB antigen induces under the situation that 3D-MPL exists.This has just supported to add proteantigen and potential adjuvant more to coordinate the viewpoint that two kinds of immune systems obtain the optimized protection effect in pneumococal polysaccharide conjugate vaccine.

Embodiment 5-is with the pneumolysin active immunity and with the intravital two kinds of immune synergism of the mice of anti-streptococcus pneumoniae PS antibody passive immunity

The definite concentration that after passive administration, can prevent anti-6B polysaccharide (anti-PS) antibody of pneumonia of embodiment 5A-

Method

Vaccine group: the described group technology of according to the form below was carried out passive immunity (i.p.) with the undiluted rat anti polysaccharide of 100 μ l antiserum to 4 groups of mices at the 1st day, every group of 16 mices (totally 64 mices).

Group number	Specificity	IgG concentration in the antiserum
			G1	α-PS-6B	5μg/ml
G2	α-PS-6B	2μg/ml
			G3	α-PS-6B	0.75μg/ml
G4	Contrast	0μg/ml

Animal: from Charles River, 64 male CD-1 mices of Canada.The about 35g of weight (about 10 ages in week)

Anesthesia: add O2 (1L/min) with mouse anesthesia with isoflurane (3%).

Organism: collect streptococcus pneumoniae N1387 (serotype 6) and be suspended in the 6mlPBS from the tryptone bean peptone agar plate (TSA) of adding 5% horse blood.Before infecting, the 1ml bacterial suspension is diluted in the refrigerative Nutrient agar that dissolves of 9ml, and remains in 41 ℃.Every mice is accepted 6.0 log10 cfu approximately, volume 50 μ l.

Infect: the 0th day, as indicated above with mouse anesthesia, and with streptococcus pneumoniae N1387 (the refrigerative bacterial suspension of 50 μ l) infecting mouse, method is to be undertaken instiling in the bronchus by non-operation endotracheal intubation.Woodnut and Berry (Antimicrob.Ag.Chemotherap.43:29 (1999)) describes to some extent to this method.

Sampling: infected the back the 3rd day, and utilized excessive CO2 that 8 mices in every group are put to death, take out lung and homogenate in 1mlPBS.With PBS 10 times of dilutions successively, deposit amount of viable bacteria with calculating.(20 μ μ l) is seeded on the TSA flat board of adding 5% horse blood with sample, and triplicate, 37 ℃ of incubated overnight are evaluated then.Other mice was then put to death at the 7th day and sampling as mentioned above.

The result:

IgG concentration in the rat blood serum (μ g/ml)	Infect certain day the bacterial population (log10cfu/ lung) in back
			3	8
	5	6.7±0.7(1/7)			7.2±0.7(5/8)
2			6.5±0.7(1/7)	6.9±1.8(4/7)
	0.75	7.7±0.5(5/8)			4.8±1.4(2/8)
0			6.7±1.5(3/6)	6.3±1.5(3/9)

Numeral in the bracket is a size of animal dead before sample time.

Conclusion:

Generally speaking, between the isolated bacterial population of all processed group, there is not evident difference.This shows, until and comprise that the anti-polysaccharide antiserum of 5 μ g/ml concentration can not produce measurable protective action.

This and some human clinical trial's observed result is similar, and promptly antipolysaccharide antibody is not enough to protect the pneumococcal pneumonia of some colony.

Embodiment 5B-measure anti-pneumonia effect that the active administration by the Ply that contains or do not contain adjuvant (pneumolysin) produces and with the synergism of the anti-PS antibody of Asia-the best (sub-optimal).

Method

Animal: from Charles River, St.Constant, Quebec, 128 male CD-1 mices of Canada (being 6 ages in week during immunity, was 10 ages in week during infection).Animal nearly weighs 20gm when 6 weeks, nearly weigh 38g during 10 weeks.

Immunity: 6 groups of mices are carried out subcutaneous immunity, every group of 16 mices (totally 128 mices) with the following vaccine of 100 μ l the 22nd day and the 14th day.PdB (WO 90/06951) is by the generous donations of Australian James doctor Paton.3D-MPL is from Ribi/Corixa.

The 1st day, specific group (seeing the following form) is carried out passive immunity (i.p.100 μ l) with the mouse-anti polysaccharide antibody of 4.26 μ g/ml concentration (5 μ g/ml concentration are got 4ml+2 μ g/ml concentration and got 1.3ml).

Group number	The active volume injected	22nd, the vaccine (dosage μ g) of injection in 14 days	Passive volume injected	Passive IgG (the 1st day)
					1-1	100μls.c.	PdB/AlPO4(10/50)		None
1.2	100μls.c.	PdB/MPL/AlPO4(10/5/50)		None
					1.3	100μls.c.	PdB/AlPO4(10/50)	100μli.p.	α-PS
1-4	100μls.c .	PdB/MPL/AlPO4(10/5/50)	100μli.p.	α-PS
					1.5	100μls.c.	MPL/AlPO4(5/50)	100μli.p.	α-PS
1-6	100μls.c.	MPL/AlPO4(5/50)		None

Infect: the 0th day, with mouse anesthesia (3% isoflurane adds 1L/min O2).The preparation method of bacterial inoculum is from the streptococcus pneumoniae N1387 (serotype 6) of tryptone bean peptone agar plate (TSA) the collection growth of adding 5% horse blood and is suspended in the 6ml PBS.Before infection, be prepared into 10 times of diluents (1ml+9ml) (remaining in 41 ℃) with the refrigerative Nutrient agar that dissolves.Undertaken instiling in the bronchus by non-operation endotracheal intubation, with infecting mouse.Every mice is accepted 6.0 log10cfu approximately, volume 50 μ l.Woodnut and Berry (Antimicrob.Ag.Chemotherap.43:29 (1999)) describes to some extent to this method.

Sampling: infect back the 72nd, utilize excessive CO2 that 8 mices in every group are put to death, take out lung and homogenate in 1mlPBS.With PBS 10 times of dilutions successively, deposit amount of viable bacteria with calculating.(20 μ l) is seeded on the TSA flat board of adding 5% horse blood with sample, and triplicate, 37 ℃ of incubated overnight are evaluated then.Then execution in the 8th day and the sampling as mentioned above after infection of other mice.

Data analysis

Bacterial population in the lung of measure to infect the back the 3rd day and the 7th day, and the result of different disposal method compared.The result is expressed as cell mean and standard deviation.Carry out statistical analysis by Students t-test, p value＜0.05 be considered to have significance.

The result:

Infected the back the 72nd hour

The count of bacteria of 1-4 group is starkly lower than 1-3 group (p＜0.05).

The count of bacteria of 1-4 group is starkly lower than 1-5 group (p＜0.05).

Infected the back the 168th hour

The 8th day antibacterial number average of each group hangs down about 2 orders of magnitude than the 3rd day bacterial population, and this shows that infection disappears.

The count of bacteria of 1-2 group is starkly lower than 1-5 group (p＜0.05).

Group number	The 3rd day		The 8th day
						Log CFU/ lung	Standard deviation	Log CFU/ lung	Standard deviation
1-1	6.93	0.61	5.23	1.28
					1.2	6.59	1.25	4.08	1.34
1-3	7.09	0.8	532	1.26
					1-4	6.09	1.43	4.46	2.32
1-5	7.19	0.89	5.42	1.05
					1-6	6.68	1.14	5.01	1.48

As implied above, use antipolysaccharide antibody (group 1-5) can not produce the protective action of anti-pulmonary streptococcus pneumoniae growth separately.With AlPO4 was that the PdB of adjuvant also is so, but PdB tended to produce a kind of protective action (organizing 1-2) when being used in combination with 3D-MPL at the 8th day.

In the time of the 3rd day, use all 3 kinds of compositions, the antipolysaccharide antibody of PdB, 3D-MPL and passive administration, group 1-4 produced the most tangible protective action.Mortality rate is also supported this result.Group 1-4 has only 2/8 death, and by contrast, group 1-5 and 1-3 have 5/10 death.

Conclusion:

Because the animal of should experiment using passive immunity, so the cooperative effect that is produced by the active immunity of pneumolysin and MPL is not that antibody horizontal increase by anti-polysaccharide antigen causes in addition.

Because animal is only accepted once the passive immunity of anti-pneumococal polysaccharide, thereby in the time of the 8th day, the major part of this antibody is consumed by the host.

However, adding with pneumolysin in the group of 3D-MPL immunity, especially in add the group of antipolysaccharide antibody immunity that 3D-MPL adds passive administration with pneumolysin, still observed the protective action of tangible anti-pneumococcal pneumonia, this shows that this compound mode has cooperative effect.

If carry out anti-polysaccharide immunity with active mode (preferably using bonded polysaccharide), then effect can be more obvious because of the memory effect of B cell, the anti-PS antibody of constant level can promote that also immune synergism (can reference in addition, for example, Fig. 1 C, the multiple animal pulmonary after attack that carries out active immunity with polysaccharide and albumen that wherein shows does not have antibacterial and exists).The immunogenicity of the 11 valencys streptococcus pneumoniae that embodiment 6-3D-MPL assists a ruler in governing a country-GL-PP D conjugate vaccine in 1 years old Balb/C mice

Before mention target:

The protective action that anti-streptococcus pneumoniae sexuality is dyed is mediated by the opsonin phagocytosis by serotype specificity antibody.Can infer that the concentration that increases this antibody can make protective action stronger, thereby many effort of being done all are in order to seek to strengthen the method for humoral response.Have a kind of strategy that uses the immunostimulating adjuvant be successfully applied to clinical before the research of conjugate vaccine (see summary Poolman et al.1998, based on the bacterial vaccine of saccharide.In：Handbook ofExperimental Pharmacology eds.P.Perlmann and H.Wigsell.Springer-Verlag，Heidelberg，D)。

The data that this section provides are up-to-date result of experiment, and this experiment is adopted through design can simulate the scheme of clinical trial, and uses clinical batch of medicine.

Scheme:

With streptococcus pneumoniae-GL-PP D conjugate vaccine or 1 years old balb/c mice of the common polysaccharide vaccine immunity of 23 valencys, dosage is 1/10 of human dosage.Used vaccine is clinical crowd of medicine DSP009, DSP013 or DSP014, be equivalent to 11 valency combined vaccines of 1mcg dosage serotype 6B and 23F and all the other serotypes of 5mcg, 11 valency combined vaccines of 0.1mcg dosage respectively, or with 11 valency combined vaccines of the 0.1mcg dosage of 5mcg3D-MPL adjuvant preparation.All 11 valency combined vaccines all are added with 50 μ gAlPO in addition ₄Adjuvant.

To 20 is that one group mice carries out the intramuscular immunity.The 0th day and the 21st day the listed mice of respectively organizing of following table is injected.Get the blood test at the 35th day (for the second time administration after the 14th day).

Table: the immune timetable of using 1 years old Balb/c mice of clinical batch of medicine immunity of streptococcus pneumoniae-GL-PP D conjugate vaccine

Group number	The 0th day the 1st time vaccine administration	The 21st day the 1st time vaccine administration	Number of mice
				1	Pneumovax-23 2.5mcg	Buffer	20
2a	11-valency Pn-PD 0.1mcg	Buffer	20
				2b	11-valency Pn-PD 0.1mcg	11-valency Pn-PD 0.1mcg	20
3a	11-valency Pn-PD+MPL 0.1mcg+5mcg	Buffer	20
				3b	11-valency Pn-PD+MPL 0.1mcg+5mcg	11-valency Pn-PD+MPL 0.1mcg+5mcg	20
4a	11-valency Pn-PD 1/0.5mcg	Buffer	20
				4b	11-valency Pn-PD 1/0.5mcg	11-valency Pn-PD 1/0.5mcg	20
Contrast	Buffer	Buffer	20

Unify scheme according to CDC/WHO, promptly with cell wall polysaccharides with in the serum and after, utilize the anti-pneumococal polysaccharide IgG antibody of ELISA method test sera.Being standard with serotype specificity IgG1 monoclonal antibody proofreaies and correct the result of ELISA, is the antibody concentration of mcg/ml to obtain unit.

The statistical analysis of comparative result calculates with UNISTAT 5.0beta version.The Tukey-HSD method is adopted in the variance analysis of IgG concentration log value.The paired comparison of seroconversion rate uses the method for accurate testing of Fisher.

The result:

Following table shows immunity for the second time (administration for the second time) back the 14th day anti-11 kinds of serotypes and the GMC IgG of protein D and 95% confidence interval.What wherein can't calculate 95% confidence interval then is shown as seroconversion rate.

Group 1 shows that carrying out immune result with common polysaccharide only induces IgM in animal body.The most IgG level is lower than detection threshold; But the balb/c mice can produce IgG, especially serotype 3,19F and 14 at a small amount of pneumococal polysaccharide.

Carry out the IgG antibody that immunity can induce anti-23F all serotypes in addition with the conjugate vaccine, and can obtain high seroconversion rate.

Find have only serotype 7F and 19F to produce dose dependent and reply (group 4 is compared with group 2), but these observed results not to have statistical significance by observation.(the b group is compared with a group) that obtain to reply more by force after twice administration has serotype 3,6B, 7F and 19F and PD, and these observed results all have statistical significance under the multiple situation of all 3 kinds of preparations.

What is interesting is most the effect of 3D-MPL.Twice administration of vaccine (group 3b) of 3D-MPL preparation can be induced the highest specificity GMC IgG, this all has statistical significance to all serotypes beyond the 23F, to 23F then is to obtain obviously higher seroconversion rate (p=0.02 group 3b compares the accurate test of Fisher with group 2b).

Table: the interior specific streptococcus pneumoniae serotype of 1 years old Balb/c mice body and [IgG] geometric mean of protein D and 95% the confidence interval of carrying out after the immunity of II level the 14th day with 11 valency PS-PD conjugate vaccines

Group	1	2a	2b	3a	3b	4a	4b
								Serotype	GM[IgG] μg/ml (95％Cl)	GM[IgG] μg/ml (95％Cl)	GM[IgG] μg/ml (95％Cl)	GM[IgG] μg/ml (95％Cl)	GM[IgG] μg/ml (95％Cl)	GM[IgG] μg/ml (95％Cl)	GM[IgG] μg/ml (95％Cl)
3	0.24 (0.16-0.6)	0.18 (0.11-0.27)	0.84 (0.47-1.5)	0.72 (0.51-1.0)	4.84 (3.0-7.9)	0.22 (0.14-0.35)	0.95 (0.19-1.8)
								6B	0.02 0/20 #	0.04 8/19	0.19 (0.09-0.41)	0.14 (0.07-0.27)	0.74 (0.29-1.9)	0.09 (0.05-0.16)	0.11 (0.05-0.23)
7F	0.04 0/20 #	0.07 (0.04-0.12)	0.19 (0.10-0.39)	0.15 (0.10-0.22)	0.97 (0.49-2.0)	0.09 (0.06-0.14)	0.45 (0.20-1.02)
								14	0.15 3/20 #	4.5 (2.5-8.1)	6.2 (3.6-10.5)	12.9 (7.8-21.2)	13.6 (9.4-19.7)	4.0 (2.0-8.0)	6.9 (4.6-10.6)
19F	1.2 (0.56-2.6)	6.7 (3.6-12.5)	12.1 (7.6-19.3)	10.1 (5.5-18.5)	58.5 (42-81)	5.9 (3.5-9.9)	22.0 (16.0-30.2)
								23F	0.07 1/20 #	0.08 3/20 #	0.08 2/19#	0.07 2/10 #	0.17 9/20 #	0.06 1/18 #	0.10 4/20 #
PD ＊	0.25 1/20 #	5.2 (3.3-8.3)	11.9 (6.9-20.7)	13.5 (9.5-19.0)	98.0 (491-195.)	10.9 (6.4-18.4)	38.7 (21.3-70.3)

^*Unit is EU/ml; The # seroconversion rate is interpreted as exceeding 2 times of standard deviations than the meansigma methods of negative control.

Table is to the definition of group before please refer to.

Conclusion:

The data that provide show, add 3D-MPL and can strengthen advanced age balb/c mice to all immunne response of test sera type in 11 valencys streptococcus pneumoniae-GL-PP D conjugate vaccine.

In most of the cases, twice administration of vaccine can be induced the IgG concentration geometric mean higher than single-dose.And use common polysaccharide vaccine not observe this phenomenon, even to be used for the mankind also be so, thereby can think that this result has shown the generation of a kind of T cell dependent immune response and immunological memory.

These data have given support to the vaccine administration scheme of using bonded pneumococal polysaccharide and assist a ruler in governing a country with a kind of Th1 adjuvant (preferably 3D-MPL), this scheme is to the adjuvant vaccine administration of major general's 2 multiple doses, its interval is 1-12 week preferably, and most preferred was 3 weeks.This dosage regimen can be used as another aspect of the present invention.

The mice that this experiment is used does not produce and replys PS23 (common or bonded).What is interesting is when being adjuvant,, and no matter use which kind of vaccine combination to be not always the case, reply (seroconversion rate then obviously improves) but there is more mice that PS23 has been produced although the level of antipolysaccharide antibody is still very low with 3D-MPL.Th1 adjuvant, particularly 3D-MPL are for the application of unresponsiveness in containing the vaccine of bonded pneumococal polysaccharide of removing certain pneumococal polysaccharide in the vaccine can be used as another aspect of the present invention.The method of utilizing the doubling dose dosage regimen to remove the unresponsiveness of above-mentioned composition also can be used as one aspect of the present invention.

Embodiment 7-Neisseria meningitidis C polysaccharide-protein D conjugate (PSC-PD)

A: the expression of protein D

As embodiment 1

B: the generation of polysaccharide C

C type polysaccharide origin is in Neisseria meningitidis C11 bacterial strain.Utilize traditional fermentation technique (EP72513) with its fermentation.The polysaccharide dry powder and the Mencevax (SBBiologicals s.a.) that are used for cohesive process are identical.

A C11 bacterial strain sample is melted, get the 0.1ml suspension one added the yeast extract dialysate (10%, streak culture on Mueller Hinton culture medium plate v/v), and in steam-laden 36 ℃ of Air oscillators the insulation 23-25 hour.

Then epontic single bacterium colony is resuspended in the no bacteria fermentation culture medium, and with this suspension be seeded in one contain add the yeast extract dialysate (10%, in MuellerHinton culture medium v/v) and the Roux bottle of sterile glass beads.Insulation is 23-25 hour in steam-laden 36 ℃ of Air oscillators, and then epontic single bacterium colony being resuspended in 10ml does not have in the bacteria fermentation culture medium, this suspension of 0.2-0.3ml is seeded in other 12 Mueller Hinton culture medium Roux bottles again.

Insulation is 23-25 hour in steam-laden 36 ℃ of Air oscillators, and then epontic single bacterium colony being resuspended in 10ml does not have in the bacteria fermentation culture medium.Bacterial suspension is incorporated in the conical flask.

Under aseptic condition, this suspension is transferred in the fermentation tank then with asepsis injector.

At the fermentation tank that places the negative pressure clean room meningococcus is fermented.Fermentation was generally finished after 10-12 hour, was equivalent to about 10 ¹⁰Individual antibacterial/ml (being that stable phase is early stage) improves pH then to detect.

This phase with whole broth bouillon heat inactivation (56 ℃ 12 minutes), centrifugal then.Before deactivation and after the deactivation, from broth bouillon, take a sample, and streak culture on Mueller Hinton culture medium plate.

The C:PS purification

This purification process needs entire fermentation broth is carried out the multistep operation.At the purification initial stage,, reclaim supernatant with the inactivated culture centrifugal clarification.

The foundation of polysaccharide purification is to precipitate with quaternary ammonium salt (cetrimonium bromide/CTAB, CETAVLON R).CTAB can be according to the pI value of polyanions such as polysaccharide, nucleic acid and albumen and with the insoluble complex of its formation.Under the situation of control ion condition, this method can be used to precipitated impurities (low conductivity) or polysaccharide (high conductivity).

With kieselguhr (CELITE ^R545) for substrate with the polysaccharide precipitation that comprises in the clarified supernatant, can avoid in different precipitation/purge processes forming insoluble inert substance like this.

The purification scheme of Neisseria meningitidis polysaccharide C

Step 1: the PBC-CTAB complex is fixed on CELITE ^ROn 545, and clean to remove cell debris, nucleic acid and albumen with 0.05%CTAB.

Step 2: with 50%EtOH eluting PS.Abandon the initial muddy component that contains impurity and LPS.Utilize the flocculation method of testing to verify that there is situation in the PS in the component subsequently.

Step 3: the PS-CTAB complex is fixed on the CELITE R 545 once more, and cleans to remove the nucleic acid and the albumen of trace with 0.05%CTAB.

Step 4: with 50%EtOH eluting PS.Abandon first turbid fractions.Utilize the flocculation method of testing to verify that there is situation in the PS in the component subsequently.

Eluate is filtered, and collect the filter liquor that contains rough polysaccharide.Add ethanol to final concentration 80%, make the polysaccharide precipitation in the filter liquor.Reclaim the powdery polysaccharide of white then, vacuum drying also is stored in-20 ℃.

The combination of D:CDAP

PSC combines with PD's

When PSC combined with PD, preferred CDAP combination technology was the CNBr activation and passes through a kind of sept and the bonded classical way of carrier protein.At first use 1-cyano group-4-dimethylamino naphthyridine Tetrafluoroboric acid ester (CDAP) polysaccharide to be activated by cyanidation.CDAP is a kind of water solublity cyanidization agent, and the electrophilicity of its cyano group will be higher than CNBr, thereby can carry out cyanogenation under gentle relatively condition.Polysaccharide after the activation can directly be connected with carrier protein by its amino, and need not to introduce any spacer molecule.But the unreacted cyanate group of excessive response cancellation by glycine.Can reduce the required total number of steps of conjugate vaccine production like this, the most important thing is not exist in the end-product to have immunogenic spacer molecule.

Cyano group can be introduced polysaccharide with the CDAP activated polysaccharide, and discharge dimethylamino naphthyridine (DMAP).Cyano group can be transformed into carbamate with proteic NH2-radical reaction in integrating step subsequently.

The combination of PSC activation and PSC-PD

Activation and be combined in+carry out under 25 ℃.

In WFI, 120mg PS was dissolved 4 hours at least.

Add CDAP solution (newly being dissolved in acetonitrile with 100mg/ml concentration), making CDAP/PS (w/w) ratio is 0.75.

After 1 minute 30 seconds, add triethylamine and make pH rise to activation pH value (pH10), and keep stable until adding PD.

In the time of 3 minutes 30 seconds, add NaCl to final concentration be 2M.

In the time of 4 minutes, add the PD of purification, making the PD/PS ratio is 1.5/1; PH is transferred in conjunction with pH value (pH10) immediately.Keeping under the stable situation of pH solution placement 1 hour.Cancellation

The glycine solution of 6ml2M is added in the PS/PD/CDAP mixture.PH is transferred to cancellation pH value (pH8.8).Under operating temperature with solution stirring 30 minutes, then+2-8 ℃ spends the night and constantly slowly stir.

The purification of PS-PD

PS-PD conjugate purification cold indoor S400HR Sephacryl gel will filter (5 μ m) with gel permeation chromatography after is to remove micromolecule (comprising DMAP) and unconjugated PD; Eluting--NaCl 150mM pH6.5; Monitoring--UV 280nm, pH and electrical conductivity.

According to the different molecular size of reacted constituent, what at first elute is the PS-PD conjugate, is free PD then, is DMAP at last.To contain the component merging of conjugate according to the testing result of DMAB (PS) and μ BCA (albumen).With the component filtration sterilization (0.2m) that merges.

E: the preparation that is adsorbed in the conjugate vaccine of PSC-PD

Clean AlPO ₄

In order to make the PSC-PD conjugate be adsorbed on AlPO as far as possible perfectly ₄On, can be with AlPO ₄Cleaning is to reduce PO ₄ ^3-Concentration:

--clean AlPO with 150mM NaCl ₄, centrifugal again (4 *);

--then precipitation is resuspended among the 150 mM NaCl, filters then (100 μ m);

And

--filter liquor is heated degerming.

The AlPO of this cleaning ₄Be called as WAP (phosphate that cleans and sterilize).

Compound method

Loose PSC-PD conjugate is adsorbed on AlPO ₄On the WAP, be mixed with end-product at last.The AlPO that will contain PSC-PD under the room temperature ₄WAP stirred 5 minutes.PH is transferred to 5.1, and at room temperature with mixture restir 18 hours.Adding NaCl solution to concentration is 150mM, and at room temperature mixture is stirred 5 minutes.Adding 2-phenoxyethanol to concentration is 5mg/mL, stirs 15 minutes under the room temperature, then pH is transferred to 6.1.

The ultimate constituent/consumption

--PSC-PD 10μgPS

--AlPO ₄WAP 0.25mgAl ³⁺

--NaCl 150mM

--2-phenoxyethanol 2.5mg

--water for injection is to 0.5ml

--pH 6.1

F: the information before the clinical use

Polysaccharide conjugate is in the intravital immunogenicity of mice

The immunogenicity of PSC-PD conjugate is evaluated in the Balb/C mice at 6-8 age in week.With common (not absorption) conjugate or be adsorbed on AlPO ₄On conjugate inject as univalent vaccine.The mensuration of the anti-PSC antibody that induces adopts the ELISA method, and the sterilization method of testing is adopted in the analysis of functional antibodies, and all (USA) scheme is the basis to these two kinds of methods for disease control and prevention center, Atlanta with CDC.For variation and adjuvant with dosage are replied in evaluation

(AlPO ₄) effect and the result of these two kinds of different experiments of implementing is as follows.

The dosage range experiment

In this experiment, with twice (2 weeks at interval) injection Balb/C mice of PSC-PD.Be formulated in AlPO ₄On the using dosage of conjugate have four kinds: 0.1; 0.5; 2.5; With 9.6 μ g/ animals.The 14th day (I level immunity back 14 days), the 28th day (after the immunity of II level the 14th day) and the 42nd day (after the immunity of II level the 28th day) mice (10/organize) is got blood.Measure the geometric average concentration (GMCs) of polysaccharide C specific antibody by ELISA, and serve as with reference to the form that the result is expressed as μ gIgG/ml with the IgG of purification.Under the condition that is added with young rabbit complement, measure the bactericidal properties antibody of combining anteserum, and will tire and be expressed as to kill the form dilution reciprocal of 50% antibacterial with Neisseria meningitidis C11 bacterial strain.

Dosage-the response curve that obtains is shown as platform area since 2.5 μ g dosage places.The result shows, after back 14 days of I level immunity and the immunity of II level between the 14th day once good short liter reply.After the immunity of II level the 28th day antibody horizontal at least with the immunity of II level after the 14th day equate.Tiring of bactericidal properties antibody conforms to the concentration results of ELISA, thereby confirmed the immunogenicity of PSC-PD conjugate.

The effect of adjuvant

This experiment is to being formulated in AlPO ₄On a collection of PSC-PD conjugate evaluate, and common to inject (unconjugated) conjugate is contrast.Is that one group mice carries out subcutaneous injection twice with 2 μ g conjugates to 10, at interval 2 weeks.The 14th day (I level immunity back 14 days), the 28th day (after the immunity of II level the 14th day) and the 42nd day (after the immunity of II level the 28th day) mice is got blood and carry out ELISA and measurement that functional antibodies is tired (after only to the immunity of II level the 14th day with the immunity of II level after the test that sterilizes of the 28th day blood sample).Compare with the preparation that does not add adjuvant, the preparation that adds AlPO4 can induce the antibody titer up to 10 times.

Conclusion

Can draw following common conclusions by above-mentioned experimental result:

--the PSC-PD conjugate can induce a kind of anamnestic response, show PSC in conjunction with after become a kind of T cell dependence antigen.

--the anti-PSC antibody concentration and the tiring of bactericidal properties antibody of being measured by ELISA have good dependency, illustrate that antibody that the PSC-PD conjugate induces has the function of anti-Neisseria meningitidis serogroup C.

--about 2.5 μ g conjugates are adsorbed on the antibody response that as if can in the mice body, obtain the best on the AlPO4.

--the CDAP chemicals can be used as the preparation that a kind of suitable method is used for immunogenicity PSC-PD conjugate.

Embodiment 8-derives from the preparation of the polysaccharide-PD conjugate of Neisseria meningitidis serogroups A

With Polysaccharide A (PSA) dry powder dissolving 1 hour, making its final concentration was 8mg/ml in 0.2M NaCl solution.Make pH value be stable at 6 with HCl or NaOH then, and the temperature of solution is controlled at 25 ℃.In PSA solution, add 0.75mgCDAP/mg PSA (being dissolved in the preparation of acetonitrile with 100mg/ml concentration).1.5 add 0.2M NaOH after minute, making pH value under the situation of not regulating pH is 10.2.5 add protein D (being concentrated into 5mg/ml) after minute, make the ratio of PD/PSA be about 1.Keeping pH value in 1 hour association reaction phase process is 10.Add 10mg glycine (2M pH9.0)/mg PSA then, and pH value is transferred to 9.0,25 ℃ of maintenances 30 minutes.Then 4 ℃ of preservations of mixture are spent the night, utilize exclusion column chromatography (purchasing S400HR) to carry out purification again in the Sephacryl of Pharmacia.What at first elute is conjugate, is unreacted PD and by-product (DMAP, glycine, salt) then.Collect conjugate and on the Sartopore of Sartorius film, carry out 0.2 μ m filtration sterilization purchasing.

The vitro characteristics of embodiment 9-embodiment 7 and 8 product is identified

Key property is summarized in following table:

Numbering	The conjugate explanation	Albumen and PS content (μ g/ml)	The PS/ Protein ratios	Floating preteins (%)	Free PS (%)
						1	PSC-PD transfers pH with NaOH	PD：210PS：308	1/0.68	＜2	8-9
2	PSC-PD transfers pH with TEA	PD：230PS：351	1/0.65	＜2	5-6
						3	PSA-PD transfers pH with NaOH	PD：159PS：149	1/1.07	5	5-9

Result in the body

With the Balb/C mice is the immunogenicity of animal model test conjugate.Conjugate is adsorbed on AlPO ₄Or Al (OH) ₃Go up (500 μ g Al ³⁺Adsorb 10 μ g PS) or do not adsorb.As described below mice is injected: inject 2 times, at interval 2 weeks (2 μ g PS/ time).

Can conclude at first that by these results free PS is very big to the immunne response influence.Contain free PS and be less than 10% conjugate and obtained better result and utilize.Therefore, the above improvement to the CDAP method can be used as another aspect of the present invention.

Dosage form is also very important.In this model, AlPO ₄Seemingly optimal adjuvant.Conjugate can produce to be urged to rise effect, and when only injecting polysaccharide, this effect would not occur.

Conclusion

The final PS/ Protein ratios of gained Neisseria meningitidis A conjugate and C conjugate is respectively 1 and 0.6-0.7 (w/w).Free PS and free carrier protein are lower than 10% and 15% respectively.Polysaccharide recovery is higher than 70%.Therefore, PSA conjugate that can be obtained by above-mentioned improvement (optimization) CDAP method and PSC conjugate (do not consider carrier protein, but preferably protein D) can be used as another aspect of the present invention.

Embodiment 10-derives from the preparation of polysaccharide-PD conjugate of Haemophilus influenzae b

Haemophilus influenzae b is that child below 2 years old suffers from meningitic main diseases therefore.Haemophilus influenzae capsular polysaccharide (PRP) conjugate that is combined on the tetanus toxoid is known (utilizing the chemical method of J.Robbins exploitation to carry out combination) for people.CDAP is a kind of improved chemical method.Below be grope with PRP with, preferably, the bonded best CDAP condition of PD.

The factor that can influence association reaction is as follows:

The initial concentration of polysaccharide (can produce double influence) to the final content and the filtration sterilization step of free polysaccharide.

The initial concentration of carrier protein.

The initial ratio of polysaccharide and polysaccharide (can produce double influence) to the final content and the filtration sterilization step of free polysaccharide.

The amount of used CDAP (being generally excessive greatly).

Reaction temperature (can influence the decomposition of polysaccharide decomposition, kinetics and reactive group).

Activation and bonded pH value.

The pH value of cancellation (influencing the amount of residual DMAP).

The time of activation, combination and cancellation.

The inventor finds that 3 most critical factors optimizing the end-product quality are: polysaccharide/proteic initial ratio; The initial concentration of polysaccharide; With bonded pH value.

Therefore, be that 3 axles are drawn three-dimensional reaction coordinate with above-mentioned 3 conditions.These mid point (with the experiment value scope) is: PS/ Protein ratios-1/1 (± 0.3/l); [PS]=5mg/ml (± 2mg/ml); With in conjunction with pH value=8.0 (± 1pH unit).

Be the setting value of the lower factor of importance below: used polysaccharide is 30mg; 25 ℃ of temperature; [CDAP]=0.75mg/mg PS; PH regulator uses 0.2M NaOH; Activation pH=9.5; Activation temperature=1.5 minute; In conjunction with temperature-1 hour; [albumen]=10mg/ml; Cancellation pH=9.0; Cancellation temperature=1 hour; PS is dissolved in the dissolving of temperature in the solvent=in 2M NaCl 1 hour; Purification on Sephacryl S-400HR 150mM NaCl eluting, flow velocity 12cm/ hour; SAROLAB P20, speed 5ml/ minute are used in filtration sterilization.

For the data of setting up best product preparation condition and needing to investigate in above-mentioned three-dimensional reaction coordinate have: the maximum production after the filtration, bonded maximum protein content; And the data of product quality--final PS/ Protein ratios, free PS level, floating preteins level, minimum residual DMAP (catabolite of CDAP) level.

Output after the filtration

Initial [PS] is to influence the factor of filtering back output with the interaction that combines between pH value and the initial PS/ Protein ratios.Under the situation of low [PS], the interaction between back two kinds of factors is very little, and always can obtain good filterability (all products all reach about 95%).But under the high concentration situation, can make filterability reduce the (filtration rate of high [PS], lowest ratio, minimum pH=99% with initial ratio if improve pH; But the filtration rate of high [PS], ceiling rate and pH=19%).

Protein bound level

The proteic final ratio of PS/ and the ratio of initial ratio can be used as the criterion of joint efficiency.Under high [PS] condition, pH does not influence the ratio of two kinds of ratios.But initial ratio can influence this ratio (during low initial ratio is 1.75, is 1.26 during high initial ratio).Under low [PS] condition, the ratio of two kinds of ratios most likely reduces, but bigger (low pH low ratio time=0.96 of the influence of pH value at this moment; The low high ratio of pH time=0.8; High pH low ratio time=1.4; The high ratio of high pH time=0.92).

Final PS/ Protein ratios

Final ratio depends on initial ratio and [PS].High initial ratio combines with high [PS] and can obtain the highest final ratio.PH does not resemble low [PS] obvious to the influence of final ratio.

The level of floating preteins D

The amount minimum (being on close level 0.0) of the floating preteins D that under high pH and height [PS] condition, observes.When pH was very low, the influence of high [PS] became obvious especially.Improve initial ratio and can cause a small amount of increase of floating preteins D.

Residual DMAP

The influence of initial ratio is not obvious.But the level of DMAP can improve with the raising of [PS], and reduces with the raising of pH.

Conclusion

Therefore, most preferred as follows: in conjunction with pH=9.0 in conjunction with condition; [PS]=3mg/ml; And initial ratio=1/1.The characteristic of the end-product that is obtained by these conditions is as follows:

Final PS/ Protein ratios		PS output (%) after the filtration		The ratio of ratio		Floating preteins D (%)		DMAP level (ng/10 μ g PS)
										Value	Scope	Value	Scope	Value	Scope	Value	Scope	Value	Scope
1.10	0.91- 1.30	92.6	50- 138	1.16	1.03- 1.29	0.71	0- 10.40	4.95	2.60- 7.80

Therefore, the PRP conjugate that can be obtained by above-mentioned improvement (optimization) CDAP method (do not consider carrier protein, but preferably protein D) can be used as another aspect of the present invention.Embodiment 11: be antigen with the protein D--will how to improve the protection efficiency of its anti-unfiled Haemophilus influenzae after itself and the 3D-MPL preparation

With 11 valency pneumococcal polysaccharide-protein D conjugate vaccines female Balb/c mice (10 every group) is carried out immunity (intramuscular mode), carry out the immunity first time during 20 ages in week (D0), the immunity second time is carried out in 2 week backs (D14).The immunity back was got blood on the 7th day for the second time.According to tiring of the quantitative determination protein D antibody of IgG1 type, IgG2a type and IgG2b type antibody.

Prepare freeze dried 11 valency vaccine (no AlPO ₄), method is the lactose of adding 15.75% in conjugate, stirs 15 minutes under the room temperature, pH is transferred to 6.1 ± 0.1, and (lyophilization cycle generally from-69 ℃, transfers to-24 ℃ gradually in lyophilizing then, 3 hours consuming time, keep this temperature then 18 hours, and transferred to-16 ℃ more gradually, 1 hour consuming time, kept this temperature then 6 hours, transfer to gradually again+34 ℃, 3 hours consuming time, kept this temperature at last 9 hours).

Table 13 shows the composition of preparation and the reconstructors of lyophilized products.

Know that at present the typical method of measuring whether Th1 type cell-mediated immune responses produce is relevant with the level of IgG2a antibody.Can find by these data, if, then can make IgG2a surprising increasing substantially occur protein D and together lyophilizing of a kind of Th1 adjuvant (be 3D-MPL this moment).

Table 13: the composition and the antibody (1/10 dosage) of preparation (everyone class dosage) are tired in the intravital anti-protein D of mice

Physical state	PS(/500μl)	AIPO(/500μl)	Immunostimulant	Binding agent	Antiseptic	Reconstructors	IgG1	IgG2a	IgG2b	IgG1	IgG2a	IgG2b
													μg/ml			％
							Liquid form	1μg	500μg	Do not have	Do not have	2-PE 3							Do not have	76	0.425	0.24	99.1	0.554	0.313
Liquid form	5μg	500μg	Do not have	Do not have	2-PE	Do not have							66	0.284	0.176	99.3	0.427	0.265
							Liquid form	1μg	0μg	Do not have	Do not have	2-PE							Do not have	6.6	0.207	0.036	96.4	3.02	0.526
Liquid form	5μg	0μg	Do not have	Do not have	2-PE	Do not have							5.2	0.169	0.043	96.1	3.12	0.795
							Lyophilized form	1μg	0μg	Do not have	Lactose 3.15%	Do not have							NaCl 150 mM 1	5.2	0.147	0.046	96.4	2.73	0.853
Lyophilized form	5μg	0μg	Do not have	Lactose 3.15%	Do not have	NaCl 150 mM 1							11.1	0.11	0.168	97.6	0.967	1.477
							Lyophilized form	1μg	0μg	Do not have	Lactose 3.15%	Do not have							AlPO 4 500μg 2	45	1.86	0.075	95.9	3.96	0.160
Lyophilized form	5μg	0μg	Do not have	Lactose 3.15%	Do not have	AlPO 4 500μg 2							19	0.077	0.119	99.0	0.401	0.620
							Lyophilized form	1μg	0μg	Do not have	Lactose 3.15%	Do not have							MPL 50μg 1	45	2.6	3.5	88.1	5.09	6.849
Lyophilized form	5μg	0μg	Do not have	Lactose 3.15%	Do not have	MPL 50μg 1							135	25	5.1	81.8	15.1	3.089
							Lyophilized form	1μg	0μg	MPL(50μg)	Lactose 3.15%	Do not have							Buffer 1	43	22	5.7	60.8	31.1	8.062
Liquid form	1μg	500μg	MPL(50μg)	Do not have	2-PE	Do not have							441	7.1	9.1	96.5	1.55	1.990
							Liquid form	5μg	500μg	MPL(50μg)	Do not have	2-PE							Do not have	299	1.4	0.899	99.2	0.465	0.298

¹Before the injection; ²Before the injection ± 2 hours; ³The 2-phenoxyethanol

Claims (18)

1. immunogenic composition, said composition contains at least a streptococcus pneumoniae polysaccharides antigen and at least a streptococcus pneumoniae proteantigen or its immunologic function equivalent, and a kind of adjuvant.

2. the immunogenic composition of claim 1, proteantigen wherein is a kind of outer surface protein or secretory protein or its immunologic function equivalent of streptococcus pneumoniae.

3. claim 1 or 2 immunogenic composition, proteantigen wherein is a kind of toxin, adhesion element or the lipoprotein of streptococcus pneumoniae, or its immunologic function equivalent.

4. the immunogenic composition of claim 1-3, proteantigen wherein or its immunologic function equivalent are selected from: pneumolysin, PspA or its stride that film district disappearance variant, PspC or its are striden film district deletion mutant, PsaA or it strides film district disappearance variant, glyceraldehyde-3-phosphate-dehydrogenase, and CbpA or its are striden film district disappearance variant.

5. the immunogenic composition of claim 1-4, polysaccharide antigen wherein provides with polysaccharide-protein carrier conjugates form.

6. the immunogenic composition of claim 5, carrier protein wherein is selected from: diphtheria toxoid, tetanus toxoid, CRM197, keyhole limpet homocyanin (KLH), tuberculin protein derivatives (PPD), and Haemophilus influenzae protein D.

7. a kind of immunogenic composition of one of claim 1-6, vaccine wherein contains the pneumococcal polysaccharide antigen of four kinds of different serotypes at least.

8. a kind of immunogenic composition that requires in the literary composition, said composition contains a kind of adjuvant in addition.

9. a kind of immunogenic composition of claim 8, adjuvant wherein contains a kind of aluminum salt.

10. a kind of immunogenic composition of claim 8, adjuvant wherein is a kind of preferred derivant that TH1 replys.

11. a kind of immunogenic composition of claim 10, adjuvant wherein contain a kind of in following at least: 3D-MPL, a kind of Saponin immunologic stimulant or a kind of immunostimulating CpG oligonucleotide.

12. a kind of immunogenic composition of claim 11, adjuvant wherein contains a kind of carrier, and this carrier is selected from: oil-in-water emulsion, liposome and aluminum salt.

13. a kind of immunogenic composition that requires in claims, said composition can be used as drug use.

14. a vaccine, this vaccine contains the immunogenic composition of claim 1-12.

15. method, the streptococcus pneumoniae sexuality that is used for preventing or improves patient more than 55 years old is dyed, this method comprises a kind of vaccine administration with effective dose, and this vaccine contains a kind of streptococcus pneumoniae polysaccharides and at least a pneumonia streptococcus mycoprotein, and optionally contains a kind of Th1 inducing adjuvant.

16. the application of the pneumococcal polysaccharide antigen that is used in combination with a kind of streptococcus pneumoniae proteantigen and a kind of optional Th1 adjuvant in the drug manufacture of prevention pneumonia of patient more than 55 years old.

17. a method is used for preparing the immunogenic composition that requires in claims, the step of this method comprises:

Select one or more pneumococcal polysaccharide antigen;

Select one or more pneumoprotein antigen; And

Described polysaccharide is mixed with a kind of appropriate excipients mutually with proteantigen.

18. method, be used for preventing or improving baby's otitis media, this method comprises a kind of vaccine with safety and effective dose to described baby's administration, and this vaccine contains a kind of streptococcus pneumoniae polysaccharides antigen and a kind of streptococcus pneumoniae proteantigen, and optionally contains a kind of Th1 adjuvant.

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