CA2207246A1 - Vaccine development - Google Patents
Vaccine developmentInfo
- Publication number
- CA2207246A1 CA2207246A1 CA 2207246 CA2207246A CA2207246A1 CA 2207246 A1 CA2207246 A1 CA 2207246A1 CA 2207246 CA2207246 CA 2207246 CA 2207246 A CA2207246 A CA 2207246A CA 2207246 A1 CA2207246 A1 CA 2207246A1
- Authority
- CA
- Canada
- Prior art keywords
- cell
- antigen
- adjuvant
- vaccine
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/385—Haptens or antigens, bound to carriers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6056—Antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
- A61K2039/625—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier binding through the biotin-streptavidin system or similar
Abstract
The invention relates to a novel antigen that is capable of stimulating the T-cell lymphocyte surface receptor CD40. The adjuvant is a ligand adapted for this purpose. The invention also relates to a novel vaccine incorporating the aforementioned adjuvant and also ideally an antigen that is either a T-cell dependent antigen or a T-cell independent antigen.
Description
,,, ,~ , . , . - . 1 ~
NOVEL ~ACCINE DE~ELOPMENT
~he invention relates to ~ method of m~nllf~ctllre and a system for the production of a novel hllm~n or animal Yaccine; and also a novel human or 5 animal vaccine.
It is lmown that the immlme system works on the basis of recognition and thus the ability to distinguish between self and non-self. Recognition of non-self, or invading material, is followed by a sequence of steps that are designed to kill or elir~lin~te the non-self material. As knowledge of the 10 immlme system grows and molecular biological techniques advance it has become possible to advantageously manipulate the various steps in an imm~lne response in order to enhance the nature of that response. Thus, for example, it has become possible to manufacture a wide range of vaccines using recombinant material and thus manufacture a range of vaccines which 15 were not previously available either because the relevant material was not obtainable or had not before been produced.
The specific immlme system is made up of lymphocytes which are able to recognise specific antigens. B lymphocytes recognise antigens in their native conformation through surface immllnoglobulin receptors, and T lymphocytes 20 recognise protein antigens that are presented as peptides along with self molecules known as MHC, on the surface of antigen presenting cells. There are a variety of antigen presenting cells including B lymphocytes. T
lymphocytes may be further subdivided into cytotoxic T lymphocytes, which are able to kill virally infected "target" cells, and T helper lymphocytes. T
25 "helper" lymphocytes are able to help B lymphocytes to produce speci~lc antibody, or to help macrophages to kill intracelllllar pathogens.
Bacterial infections caused by encapsulated bacteria are a major world health problem. The species Streptoccocus pneumoniae, Haemophilus influenzae and Neisseria meni~gitidis are difficult to vaccinate against due to the thymus 5 independent nature of the major sur~ace antigens~ the capsular polysacch~ 7e~
O
T-cell independent antigens present particular problems regarding the development of effective vaccines. Antibody production is low and is not normally boosted by re-imnlnni.c~tion. The ~ntibody isotypes are restricted 10 to the IgM and other isotypes and are generally of a low affinity for a specific antigen.
A major problem lies in the response of young children to T-cell independent ~accines. These individuals are amongst the most vulnerable to the aforementioned bacterial infections. Over 80% of chiklhood pneumococcal 15 infections occur in infants under the age of two. Coincidentally this age group responds most poorly to T-cell independent antigens.
T-cell dependent antigens are much more effective at eliciting high titre, high affinity antibody responses. This comes about because T lymphocyte help B
lymphocytes is elicited during the immline response to these antigens. B
20 lymphocytes binds to antigen through their speci~lc antigen receptors which leads to partial activation. If the antigen is a protein the B lymphocytes take up and process the antigen to peptides which are expressed on the cell surface along with MHC class II molecules. The MHC class II/peptide complex is then recognised by specific T lymphocytes. Upon this recognition the T
lymphocytes give "help" to the B lymphocytes, and this "help" along with the initial signal through the antigen receptor results in increased B lymphocyte proliferation, isotype switching and possibly also to increased aLr~ y antibody being eventually produced ~rough somatic hyperrmlt~tion in the antigen receptor genes. T-cell indepe.n(lent antigens are invariably not proteinin composition and cannot therefore be processed and presented by B-lymphocytes via MHC molecules. This failure in antigen presentation results in low T-cell recognition of the antigen thereby resulting in no T-cell help.
T-cell help to B-cells has two components which together with signals through the antigen receptor lead to ~-lymphocyte proliferation and antibody production.
1. Cell-cell mediated activation.
NOVEL ~ACCINE DE~ELOPMENT
~he invention relates to ~ method of m~nllf~ctllre and a system for the production of a novel hllm~n or animal Yaccine; and also a novel human or 5 animal vaccine.
It is lmown that the immlme system works on the basis of recognition and thus the ability to distinguish between self and non-self. Recognition of non-self, or invading material, is followed by a sequence of steps that are designed to kill or elir~lin~te the non-self material. As knowledge of the 10 immlme system grows and molecular biological techniques advance it has become possible to advantageously manipulate the various steps in an imm~lne response in order to enhance the nature of that response. Thus, for example, it has become possible to manufacture a wide range of vaccines using recombinant material and thus manufacture a range of vaccines which 15 were not previously available either because the relevant material was not obtainable or had not before been produced.
The specific immlme system is made up of lymphocytes which are able to recognise specific antigens. B lymphocytes recognise antigens in their native conformation through surface immllnoglobulin receptors, and T lymphocytes 20 recognise protein antigens that are presented as peptides along with self molecules known as MHC, on the surface of antigen presenting cells. There are a variety of antigen presenting cells including B lymphocytes. T
lymphocytes may be further subdivided into cytotoxic T lymphocytes, which are able to kill virally infected "target" cells, and T helper lymphocytes. T
25 "helper" lymphocytes are able to help B lymphocytes to produce speci~lc antibody, or to help macrophages to kill intracelllllar pathogens.
Bacterial infections caused by encapsulated bacteria are a major world health problem. The species Streptoccocus pneumoniae, Haemophilus influenzae and Neisseria meni~gitidis are difficult to vaccinate against due to the thymus 5 independent nature of the major sur~ace antigens~ the capsular polysacch~ 7e~
O
T-cell independent antigens present particular problems regarding the development of effective vaccines. Antibody production is low and is not normally boosted by re-imnlnni.c~tion. The ~ntibody isotypes are restricted 10 to the IgM and other isotypes and are generally of a low affinity for a specific antigen.
A major problem lies in the response of young children to T-cell independent ~accines. These individuals are amongst the most vulnerable to the aforementioned bacterial infections. Over 80% of chiklhood pneumococcal 15 infections occur in infants under the age of two. Coincidentally this age group responds most poorly to T-cell independent antigens.
T-cell dependent antigens are much more effective at eliciting high titre, high affinity antibody responses. This comes about because T lymphocyte help B
lymphocytes is elicited during the immline response to these antigens. B
20 lymphocytes binds to antigen through their speci~lc antigen receptors which leads to partial activation. If the antigen is a protein the B lymphocytes take up and process the antigen to peptides which are expressed on the cell surface along with MHC class II molecules. The MHC class II/peptide complex is then recognised by specific T lymphocytes. Upon this recognition the T
lymphocytes give "help" to the B lymphocytes, and this "help" along with the initial signal through the antigen receptor results in increased B lymphocyte proliferation, isotype switching and possibly also to increased aLr~ y antibody being eventually produced ~rough somatic hyperrmlt~tion in the antigen receptor genes. T-cell indepe.n(lent antigens are invariably not proteinin composition and cannot therefore be processed and presented by B-lymphocytes via MHC molecules. This failure in antigen presentation results in low T-cell recognition of the antigen thereby resulting in no T-cell help.
T-cell help to B-cells has two components which together with signals through the antigen receptor lead to ~-lymphocyte proliferation and antibody production.
1. Cell-cell mediated activation.
2. Cytokine activation.
In vitro experiments have shown that resting B-cells can be stim~ ted to proliferate after exposure to isolated membranes from activated T-cells. The basis for this phenomenon has been detennin~d. Following T-cell activation a 39kDa (CD 154) T-cell speci~lc cell surface protein is induced. This ligand has been identified as the target of the B-cell cell surface receptor CD40 and binding of CD154 to CD40 is the major component of T lymphocyte help to B lymphocytes.
Further evidence for the involvement of CD40 and CD154 comes from experiments in which host cells transfected with the cDNA encoding the CD154 protein can induce proliferation of B-cells in the presence of added cytokines. In addition, patients with the congenital (1ise~e X-linked hyper IgM syndrome, who fail to switch antibody isotypes have been shown to have various mutations in the gene encoding the CD 154 protein resulting in failure to activate the B-cells via CD40. The CD40-CD154 inter~ction has also been 5 shown to be an important elernent in ~ R responses to T-cell dependent antigens in 'knock-out' mice.
The other important element in B-cell activation via T-cell help involves cytokine function. Although isolated membranes from activated T-cells can induce B-cell proliferation this effect can be enhanced by the presence of 10 cytokines. Furthermore cytokines have a major role in switching of antibody isotypes. In particular IL4, intelÇeloll y and transforming growth factor beta (TGF ,B) are of importance. IL4 induces IgGl and IgE, IFNy induces IgG2a and TGFB induces IgA and IgG2b. In addition IFN~ is probably responsible for the switching to IgG3 which is seen naturally in responses to T-cell 15 independent antigens. However ligation of CD40 does not induce appreciable Ig secretion on its own, but CD40 ligation (including via T-cell membranes) seems to prepare cells for differentiation which can be induced efficiently by ILA and IL5.
Finally T-cell help has a major influence on somatic hypermllt~tion which 20 results in the selection of B-cell clones that produce high affinity antibodies.
From this description it may be surmised that T-cell independent production of antibodies by B-cells is compromised due to the lack of help offered by T-helper lymphocytes through activation via CD40 and through the influence of cytokines produced by the T-helper cell.
_ It is therefore an object of this invention to provide a means of activating B-cells to proliferate and produce the full range of antibody isotypes of high titre in response to T-cell indepenflent as well as T-cell dependent antigens.
It is a further object of this invention to use T-cell indepen(lent and/or 5 dependent antigens to produce effective vaccines that offer high titre, high affinity antibodies to protect individuals from infection.
It is yet a further object of the invention to provide a safe i~ logical adjuvant for use in a vaccine and also for use in enhancing the immllne response to T-cell independent and/or dependent antigens.
10 It is yet a further object still of the invention to provide a method for the production of a vacciné of the invention.
It is a further object of the invention to provide a system for the production of the vaccine of the invention.
In its broadest aspect the invention concerns the provision of a means for 15 a~;tiva~ g the CD40 receptor on a B-lymphocyte, ideally the means compri~ing an adjuvant which is adapted to activate said receptor, either directly or indirectly. More preferably the invention concerns a ligand which binds to the CD40 receptor on a B-lymphocyte and brings about the activation of same.
20 According to a first aspect of the invention there is therefore provided an adjuvant which is adapted to stimulate a B-lymphocyte cell surface receptor, CD40.
According to a second aspect of the invention there is provided a vaccine suitable for enh~ncing T-cell independent and T-cell dependent im~ ",ily comI ricing a T-cell dependent and/or independent antigen, or part(s) thereof, and an associated adjuvant which is adapted to stimul~te a B-lymphocyte cell 5 surface receptor, CD40.
Reference herein to the term vaccine is intended to include a wide variety of vaccines inclll~1ing, but not limite~1 to, contr~ceptive vaccines, il~ otherapy vaccines and prophylactic or therapeutic vaccines.
Reference herein to T-cell independent immunity includes reference to an 10 immune response which operates wholly or largely independently of T-cells, for example, because existing T-cells are not activated; or because existing T-cells are not functional or immllne suppressed through ~lise~se or exposure to chemicals, radiation or any other means.
To by-pass or mimic the effects of T-cell help we propose a vaccine which 15 ensures that all B-cells receiving a signal through their specific antigen receptors also receive a signal through CD40, mimicking or improving upon that which would be received during natural T-cell help. This would be achieved, ideally, by ensuring that a CD40 binding moiety were closely associated with the vaccine antigen. This could be through co-~mini.~tration 20 of the CD40 stimlll~ting moiety with the appropriate T-cell independent and/or dependent antigen, or preferably through covalent linkage, or co-entrapment on/in a carrier system.
The vaccine involves ideally the conjugation of the antigen to a CD40 ligand such as an anti CD40 antibody, or part thereof, followed by immlmis~tion of a human or ~nim~l It should be apparent to those skilled in the art that this methodology may also be applied to any antigens, but in the in~t~nce of T-cell dependent antigens could be of particular relevance to those individuals that are i..~",l,e suppressed and therefore lack T-helper lymphocytes (e.g.
5 AIDS patients).
~a preferred emborliment of the invention said antigen is soluble and ideally a protein or a polysaccharide.
.
Ideally stimulation of CD40 is via binding of said adjuvant, or part thereo~, to at least a part of CD40. In a preferred embodime~t of the invention said 10 antigen and adjuvant are bound or cross-linked together.
More preferably said adjuvant is an antibody, either polyclonal or monoclonal, but ideally monoclonal, which is adapted to bind to said CD40.
More ideally still said antibody is hum~ni.~ed.
In a preferred aspect of the invention said antibody may be whole or, 15 alternatively, comprise only those domains which are effective at binding CD40 and in particular selected parts of CD40.
In another embodiment of the invention, said adjuvant is a n~tur~l ligand of CD40, the T-cell specific CD154 cell surface antigen, ideally produced as a recombinant protein, or a CD40 binding portion of the CD154 protein, or 20 indeed any other ligand, or part thereof, that binds CD40 or part thereof.
In a further embodiment, the CD40 ligand may not be a naturally occurring CD40 ligand but represent an agent that due to its biochemicaI characteristics , r ~ , .
has an affinity for CD40.
In its broadest context, reference herein to the term adjuvant includ~s reference to any string of amino acids or ligand which is selected so as to bind to at least a part of CD40.
5 In a preferred aspect the recombinant vaccine antigen (when a polypeptide) and the adjuvant will be produced as a ~him~ric fusion protein.
It will be apparent to those skilled in the art that the said antigen may be a T-cell independent antigen and thus any antigen which is capable of eliciting a T-cell independent response.
10 ~Itern~tively, the antigen may be a T-cell dependent antigen and thus any antigen that is capable of eliciting a T-cell response.
It is apparent from the above that any antigen may be selected for use in the vaccine of the invention - the precise nature of which will depend on the "rli~e~e" that an individual is to be immuni.ced against and/or in some 15 circllmst~nces, the immnn~ status of an individual to be vaccinated.
Ideally said antigen and/or adjuvant is in the form of an immunostimulating complex, or liposomes or biodegradable microspheres, so increasing the association between antigen and CD40 binding moiety.
Alternatively said vaccine comprises an emulsion of the antigen and adjuvant 20 ideally in oil.
9 ~ -;
In a preferred embo-liment of the invention at least one selected cytokine may be included in andlor co~lmin.~tered in/with said vaccine.
According to a third aspect of the invention there is provided an adjuvant for enhancing T-cell independent immlmity wherein said adjuvant comprises an 5 agent adapted to stimlll~te a B-lymphocyte surface receptor, CD40.
Preferably said stimlll~tion of said CD40 is via binding of said adjuvant, or part thereof, thereto.
Ideally, said adjuvant is an antibody, either polyclonal or monoclonal, but ideally monoclonal, which is adapted to bind to said CD40. More ideally still 10 said antibody is hllm~ni.sed.
In a preferred aspect of the invention said antibody may be whole or, ~ltern~tively, comprise only those domain~ which are effective at binding CD40, and in particular selected parts of CD40.
In this aspect of the invention said adjuvant is co-~llmini.~tered with either 15 said T-cell independent antigen that is effective at eliciting a T-cell independent immune response or a T-cell dependent antigen that is effective at eliciting a T-cell response. This will be dependent upon the nature of the "disease" against which the individual is to be immlmi.~ed and/or the immlm~
status of the individual.
20 More p~fel~bly further still said adjuvant is cojoined to said T-cell independent antigen or said T-cell dependent antigen.
In a yet further preferred embo-liment said adjuvant in co-~(1mini~tered with at least one cytokine.
According to a fourth aspect of the invention there is provided a method for the manufacture of a novel vaccine capable of enhancing T-cell independent 5 immunity or T-cell dependant immlmity which methods comprises the selection of a suitable T-cell dependant and/or independent antigen, or part(s) thereof, and association or combination of said antigen with an adjuvant wherein said adjuvant is adapted to stim~ te a B-lymphocyte receptor, CD40.
According to a fifth aspect of the invention there is provided a method for the 10 manufacture of a novel vaccine capable of enhancing T-cell independent imm~lnity which method comprises the selection of a suitable T-cell dependent and/or independent antigen, or part(s) thereof, and association or combination of said antigen with an adjuvant wherein said adjuvant is adapted to stim~ te a B-lymphocyte receptor, CD40.
15 In yet a further preferred method of the invention said adjuvant is recombinantly manufactured.
In yet a further preferred embodiment of the method of the invention said antigen and adjuvant are bound or cross-linked theretogether.
The major T-independent antigens used in vaccines are b~cteti~l capsular 20 polysaccharides. In a preferred embodiment or method of the invention one will therefore purify polysaccharide antigens and crosslink them to a CD40 binding moiety. A commonly used technique for the cross linking of polysaccharide to protein is carbodiimide coupling. However a number of heterobifunçtion~l cross-linking agents are commercially available for both protein-protein and protein-carbohydrate cross-linking Heterobifilns~tion~l cross-linking agents have the advantage that they favour protein-carbohydrate cross-links thereby m~imising the yield of adjuvant coupled to antigen.
5 Preferably said stim~ tion of said CD40 is via binding of said adjuvant, or part thereof, thereto.
Ideally, said adjuvant is an antibody, either polyclonal or monoclonal, but ideally monoclonal, which is adapted to bind to said CD40. More ideally said antibody is hllm~ni~ed.
10 In a preferred aspect of the invention said antibody may be whole or, ~ltern~tively, comprised only those domains which are effective at binding CD40, and in particular selected parts of CD40.
In a preferred method of the invention one adds at least one cytokine to said vaccine.
15 According to a further aspect of the invention there is provided a system forthe manufacture of a vaccine capable of enh~ncing T-cell independent or T-cell dependent immunity which system comprises a cell expressing a selected T-cell dependent and/or independent antigen, or part(s) thereof, and also an adjuvant capable of stimulating a B-lymphocyte receptor, CD40.
20 According to a yet further aspect of the invention there is provided a systemfor the manufacture for a vaccine capable of enh~ncing T-cell independent immnnity which system comprises a cell expressing a selected T-cell ~; :
, dependant or independent antigen, or part(s) thereof, and also an adjuvant capable of stimlllating a B-lymphocyte receptor, CD40.
More preferably still both said antigen (when a polypeptide) and said adjuvant are adapted so as to be secreted from said cell. This may be 5 undertaken by providing both the antigen and adjuvant with secretion ~ign~l~
or providing for the production of a single piece of m?~tetiZIl comprising both the antigen and the adjuvant and having a single secretion signal associated therewith. It will be evident that in the former instance the said antigen and adjuvant will be found in associated or unbound or uncross-linked m~nn~r in 10 the supern~tant of the system and in the latter instance said antigen and adjuvant will be cojoined in the sup~rn~tant of the system.
Preferably said s~im~ tion of said CD40 is via binding of said adjuvant, or part thereof, thereto.
Ideally, said adjuvant is an antibody, either polyclonal or monoclonal but 15 ideally monoclonal, which is adapted to bind to said CD40. More ideally said antibody is h~lm~ni.ced.
In a preferred aspect of the invention said antibody may be whole or, alternatively comprise only those domains which are effective at binding CD40, and in particular selected parts of CD40.
20 It will be apparent from the above that the invention is based upon the re~lic~tion that immune responses, whether to a T-cell independent or a T-cell dependent antigen, can be enhanced by stimlll~ting the B-cell CD40 receptor using any suitable means.
According to a yet filrther aspect of the invention there is provided a n~l~leicacid molecule encoding any one or more of the aforementioned embo-limP,nts of the invention.
~ this last aspect of the invention said nucleic acid molecule may be 5 ~/lmini.~tered, conventionally, to an individual or animal to be treated so that the adjuvant and ideally also the antigen of the vaccine may be manufactured i71 VIVO.
An embo~lim~nt of the invention will now be described by way of example only with reference to the following ~lgures wherein:-10 Figure 1: Shows CD40 antibody induced enhanced, class switched antibodyresponses to PS3 (type 3 pneumococcal polysacch~ri(le) (A) and increased total serum immlmoglobulin (B). BALB/c mice (6-10 weeks old) were injected i.p with 20ng of PS3 and 500,ug of lC10, 4Fl 1 (anti-mouse CD40) or isotype control antibody GLl 17. Sera were obtained days 7, 14 and week 15 14 after injection. Ihe IgM and IgG isotype mean log~rithmic titres are shown when they were m~im~l, respectively, day 7 and day 14 after injection. All negative results were given a logarithmic titre of 20, the lowestdilution used. * indicates statistical signi~lcance compared with the relevant GLl 17 control (Student's T test p<0.05).
20 Figure 2: Shows antibody responses to other pneumococcal polysaccharides are also enhanced by CD40 antibody. IgM and IgG responses to types 8, 4, 12 and 19 S. pneumoniae capsular polysaccharides in mice immlmised with the 23 capsular polysaccharides in Pneumovax II (Merck Sharp and Dohme, USA) and either the CD40 antibodies 4Fll, lC10 (anti-mouse CD40) or control antibody GL117. Groups of ~lve BALB/c mice, were injected i.p with either 500,ug of lC10, 4Fl l or GL117, and 1/25th of the recommen~lçd hllm~n dose of Pneumovax II (commercial 23-valent pneumococcal polysaccharide vaccine, l,ug each of the 23 polysacch~n~les present). Sera 5 were obtained on day 10 after injection. All negative results were given a lop~ l""ic titre of 20, the lowest dilution used. All the lC10 responses were significantly dirre~ from the GL117 responses (Student's T test p<0.05).
Figure 3: Shows that the mech~ni.~m of lC10 action is CD4~ cell independent. PS3 specific antibody logarithmic titres induced in CD4 depleted BALB/c mice treated i.p with 20 ng of PS3 and 500,ug of lC10, 4Fll or control antibody GL117. These mice failed to respond to co-s~lmini~tered keyhole limpet haemocyanin nor were any CD4+ splenocytes discernable on FACS by FITC anti CD4 (data not shown). Sera were obtained on day 14 after injection. All negative results were given a 15 log~ ic titre of 20, the lowest dilution used. All lC10 responses were significantly different from the relevant GL117 control (Student's t test p<O.OS).
Figure 4: Shows CD40 antibodies induce responses to PS3 in normally unresponsive xid mice (A). Enhanced responses in BALB/c mice provide 20 protection against S. pneumoniae challenge 9 months after tre~mPnt (B). (A) PS3 specific antibody responses in CBA/N(xid) mice injected with 20ng of PS3 and lC10, GL117 and/or control CBA/ca mice with lC10 and GL117.
The IgM and IgG isotype logarithmic titres shown are when they were m~im~l, respectively, day 7 and day 14 after injection. All negative results 25 were given a log~rithmic titre of 20, the lowest serum dilution used. *
indicates statistical signi~lcance compared with the relevant GL117 control (Student's T test p<0.05). B) Percentage ~uLvival in BALB/c mice challenged with S. pheumoniae type 3, but ~1ministered 9 monthe previously with 20ng PS3 and 500,ug of lC10, GL117 or PBS. Survival in the lC10 group w~
significantly enhanced compared to the control groups (p<0.05 %2 test).
5 Figure 5: Shows primary antibody responses to avidin conjugated to biotinylated CD40 antibodies are enhanced. BALB/c mice were immllni7:ed with either 10,ug of control IgG2a, 10,ug of avidin conjugated to anti CD40 monoclonal antibody 4Fll, 10,ug of a combination of avidin conjugated to anti CD40 antibodies 4F11 and lC10 or 10,ug of non-conjugated avidin.
10 Antibody responses against avidin were measured by ELISA at 10 days post-immlmi~tion.
Figure 6: Shows secondary antibody response to avidin alone following primary immunisation with avidin conjugated to anti CD40 antibodies 4F11 and lC10. Experiment~l details are essentially as described in Figure 5, 15 except that mice received an immllni.~tion with 10 ,ug avidin alone one monthafter primary immllni.~tion as in Figure 5, mice were bled 10 days after this second injection and antibody responses measured by ELISA.
Methods M;ce and Materials 20 The mice used were BALB/c mice (in house), CBA/ca and CBA/N (xid) mice (Harlan-Olac). They were 6-12 weeks old at the start of the experiments. The pneumococcal capsular polysaccharides type 1, 3, 4, 8, 12, 13, 19 and 23 were obtained from ATCC, USA, pneumococcal cell wall CA 02207246 l997-06-06 polysaccharide from Statens Serum Tn.stitllte, Denm~tk and Pneumovax II
vaccine from Merck Sharp and Dohme, USA. Avidin was purchased from Sigma (Poole, Dorset). Biotinylated and non-biotinylated anti-CD40 antibodies were purified from hybridoma supern~t~nt~ in house and 5 biotinylated in house where necessary using st~nll~rd reagents (Pierce).
Immunisation Protocols - Mice were treated with 500,ug of either lC10, 4Fll or GL117 and 20ng of PS3 i.p. except those receiving Pneumovax II. BALB/c mice receiving Pneumovax II were injected i.p. with either 500,ug of lC10 or GL117 and 10 l/25th of the recommended hllm~n dose of Pneumovax II. This equates to l~ug of each of the 23 polysacch~ri~es present in the vaccine. At least S
mice were used for each experimental group. ~ expPriments where mice were immllni~e-l with avidin conjugated to biotinylated anti-CD40, avidin at lmg/ml and biotinylated antibody at lmg/ml were mixed together at a 1:1 15 ratio and left on ice for 30 minll~es. The conjugates were then diluted in PBS
to give a total of lOIlg antibody and lO,ug avidin in 0.2ml PBS, which was then injected intraperitoneally. In cases where avidin alone was used it was pre-mixed with an equal volume of PBS and left on ice for 30 minlltes before dilution and injection.
20 Experiment in CD4 depleted mice BALB/c mice, 6-10 weeks old, were depleted of CD4 cells 5 days before the experiment start. 500,ug of depleting anti CD4 antibody ~TS 191.1 was injected intravenously and again the next day intraperitoneally. The percentage of CD4+ splenocytes in the depleted mice as detected by flow cytometry had dropped to undetectable levels when the antibody and PS3 were injected. There was no antibody response to 50,ug to keyhole limpet haemocyanin, a T dependent antigen~ co-~1mini.ctered with the PS3 (data not shown).
5 Measuremen~s of polys~c~h~ride antibodies and total serum immunoglobulin by ELISA
96 well ELISA plates (Costar, UK) were coated overnight with lO,ug/ml polysaccharide or with a 1/200 dilution of anti mouse Ig serum (Sigma, UK).
Individual sera were titrated on the plates and the various isotypes detected 10 by HRP conjugated mouse isotype specific sera (Southern Biotechnology Associates, USA). Sera obtained from mice injected with Pneumovax II were absorbed against S. pneumoniae cell wall polysaccharide as described previously. Antibodies to cell wall polysaccharide, a cont~min~nt of all capsular polysaccharide pr~ ion~s might have created false positive results.
15 Total serum immllnoglobulin concçntr~tions were calculated with reference to calibrated mouse serum (Sigma, UK). With the polysaccharide results end point titres for each mouse were a~ssessed against normal mouse serum and then geometric mean titres and standard deviation calculated.
Measurement of anti-avidin responses by F~
20 96 well ELISA plates (Costar, UK) were coated overnight with lO,ug/ml avidin (Sigma) in PBS. After blocking for l hour with 3% bovine serum albumin individual sera were titrated on the plates, incubated at room temperature for 1 hour, and following washing, antibody was detected using HRP conjugated anti-mouse immunoglobulin (Southern Biotechnology , Associates USA), and substrate (OPD Sigma). End point titres for each mouse were ~sse~sed ~in.~ norm~l mouse serum, and then geometric mean titres and st~n-l~rd deviation calculated.
Challenge with S. pneumoniae 5 BALB/c mice were immnni.~ed 9 month.e before ch~llenge with 20ng PS3 and 500,ug lC10 i.p. Challenge was 105 colony forming units of encapsulated S.
pneumaniae type 3 (ATCC) given i.p.. Final numbers surviving were ascertained 2 weeks after ch~llellge.
Results and Discussion 10 The development of vaccines ~g~inst encapsulated bacteria, such as Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis, is centred on their distinctive capsular polysaccharides.
Unfortunately, the inability of antigen presenting cells (APC) to process and present polysaccharides with MHC class II means that these antigens cannot 15 stim~ te T-cells. Polys~cchzlride specific B-cells receive no direct help from their T-cells and, therefore, these antigens are considered T independent (TI-II). Due to this lack of help, TI-II antibody responses are of low titre, low average affinity, and are predomin~ntly of the IgM class with no boosting on second or later exposures to antigen. The T-cell help provided during 20 immlme responses to TD antigens induces high titre and isotype switched antibody responses. The major stimulus to B-cells is provided by CD154 (formerly CD40 ligand or gp39), which is expressed de novo on activated T-cells. The CD154 molecule binds the CD40 antigen, which is ct~n~titlltively expressed on B-cells, and their interactions provide key signals as immune -t, , .. 19 , responses develop. CD40 activation is important for the initi~tion of B-cell proliferation, i~ loglobulin class switching, germin~l centre responses, and the production of memory B-cells and plasma cells. B-cells responding to TI-II antigens lack T-cell derived cytokines and CD40 ligation and produce, as 5 a result, the poor antibody response ch~r~cteristic of TI-II antigens. We have investigated in vivo whether the ~-lmini~ttation of pneumococcal polysaccharide with anti-mouse CD40 antibody could provide a sllbstitllte for CD154 mediated CD40 ligation. The two antibodies used were lC10 and 4Fl l, chosen they are both rat IgG2a anti-mouse CD40 antibodies but possess 10 markedly different in vitro propettiP~s Intraperitoneal immllni~tion of BALB/c mice with type 3 pneumococcal capsular polysaccharide (PS3) alone induced weak IgM and IgG3 responses ~g~in~t the antigen (Figure lA). This is typical of the response to TI type II
antigens in mice (hllnl~ns produce IgM and IgG2). Administration of 15 antibodies lC10 or 4Fll with PS3 induced small but significant rises in specific IgM and IgG3, while remarkably, lClO induced signific~nt polysaccharide specific IgGl, IgG2a and IgG2b responses. These isotypes are not normally seen in response to TI II antigens. lC10 would appear to have successfully mimicked T-cell help by inducing high antibody titres and 20 isotype switching in vivo. The anti-polysacch~ride response was extremely persistent, with antibody being detected at high titres 14 weeks after the single immunisation (Figure lA). No memory response against the polysaccharide was induced as a second injection of polysaccharide alone failed to boost antibody responses (data not shown).
25 S. pneumoniae has over 80 different capsular polysaccharide types and any vaccination would be expected to induce protective immllnity against a number of the more common serotypes. A current pneumococcal vaccine, Pneumovax II (Merck, Sharp and Dohme), consists of 23 different polysaccharides. Mice were immllni.sed with this 23-valent vaccine and lC10. Figure 2 shows that inclusion of the CD40 antibody successfully 5 generated strong IgG responses ~g~inct r~ndomly chosen polysac~h~ride types 4, 8, 12 and 19. Such isotype switched responses were also gener~ted against the two other antigens we ex~mined, types 3 and 14 (data not shown).
Therefore, lC10 enhances responses to TI-II antigens other than just PS3.
Given that ~dmini~tr~tion of CD40 antibody mixed with polysacch~rkle would 10 not restrict or even target CD40 ligation to antigen specific B-cells, we anticipated polyclonal activation of B-cells with a resultant rise in total serum immlmoglobulin levels. ~deed lC10 and PS3 induced some splenomegaly and 2-4 fold rises in total serum immllnoglobulin levels (Figure lB). This, however, should be con~r~ted with up to 5-fold rises in specific antibody 15 levels, indicating that polysaccharide specific antibody production was preferentially enhanced. This skewing towards specific antibody is also not unexpected as it reflects in vitro fin(1in~s. In vitro, while lC10 could induce B-cell proliferation in the absence of stimul~tion through the antigen receptor,proliferation was synergistically enhanced by such co-stimlll~tion. 4F11, 20 which largely lacks agonist activity in vitro, did not enhance responses as efficiently as lC10, demonstrating an association between adjuvant activity in vivo and B-cell activation in vitro.
CD40 ligation is necessary for switching to IgG isotypes during a T
dependent response, but various cytokines also play important roles. It was, 25 therefore, intriguing that such isotype switched responses were obtained without the addition of exogenous cytokines. l~his suggests either that CD40 -and antigen receptor ligation may be sufficient to induce isotype switehing or that bystander cells may provide sufficient cytokines to switch the activated B-cells in vivo. We considered that the CD40 antibodies might be stim~ ting T-cell production, either directly through ligation of CD40 on T- cells or 5 indirectly through induction of co-stiml-l~tory molecules on B-cells or other APCs. The action of 4F11 showed T-cell dependency as it failed to augment polysaccharide specific responses in CD4 depleted mice (Figure 3).
However, lC10 and PS3 ~t1ministration induced a pronounced, isotype switched response in CD4 depleted mice (Figure 3) with IgG responses to 10 polysaccharide being better than those induced in normal mice, demonstrating a CD4 independent action. Similar results were obtained when athymic nude mice were used inste.~-l of CD4 depleted mice (data not shown).
Most vaccines under development for use against encapsulated bacteria are protein-polysaccharide conjugates which aim to provide T-cell help for the 15 anti-polysacch~ri-le response through T-cell recognition of epitopes on the protein. By their nature such conjugates are not as effective in CD4 deffcient patients such as those with AIDS. In contrast the use of a CD40 stimul~tor would not only avoid the high cost of conjugate production, but as we have shown, gen~r~te responses unaffected by a CD4 deficiency.
20 The major fault with capsular polysaccharide only vaccines is that infants and young children, whilst reacting norm~lly to TD antigens, respond poorly to TI-II antigens. Indeed children under two years old fail to respond at all to many TI-II antigens. The inability of their immune systems to act ~g~inst bacterial capsules correlates with increased susceptibility to infection. They 25 are the group most in need of effective vaccines. CBA/N (xid) mice have an X-linked immunodeficiency rendering them, like infants, unable to respond . ~ = --, to TI-II antigens. Although one report has stated otherwise, in our hands these mice react normally to CD40 ligation in vitro (and unpublished data A.H.). We immllni.~ed groups of xid mice with lClO plus PS3 and successfully generated IgG2a and IgG2b responses ~g~in~st PS3 (Figure 4A).
5 Thus, the B-cell defect in these mice was successfully by-passed by ~,~ministering the CD40 antibody as an adjuvant along with antigen.
Using the mouse model system, we have shown that CD40 sim~ tors can enhance the antibody response to pneumococcal polysacch~rides, producing greater antibody levels and the production of IgG isotypes. Similar to 10 protein-polysacch~n-le conjugates, lC10 can induce polysacch~ride specific responses in xid mice, which like infants are unable to respond to polysaccharide only based vaccines. Unlike protein-polysaccharide conjugates, the adjuvant action of lC10 is CD4 cell independent, which is a definite advantage for the vaccination of patients with CD4 deficiencies, for 15 example AIDS sufferers.
While lC10 ~rlministered with PS3 clearly enhances speci~lc antibody responses, the measure of a vaccine is whether it provides long-term protection against disease. We challenged mice, immunised 9 months previously, with 105 CFU of S. pneumoniae type II (Figure 4B). Of the 20 BALB/c mice ~lmini~tered with PS3 and lClO five of eight survived ch~llenge, whereas only one of six and none of eleven mice survived in the groups receiving, respectively PS3 with GLl 17 and PS3 alone (p~0.05%2test).
Finally, the induction of polyclonal antibody responses, as previously described in Figure lB, may increase the risk of auto antibody production.
25 We have investigated this problem by reducing the need to ~(lminister elevated doses of anti CD40 antibody by conjllg~ting biotinylated anti CD40 antibody with avidin (a n~tur~l ligand of biotin). By physically linking the adjuvant and antigen we have been able to reduce adjuvant levels by approximately 50-fold. Figure 5 shows the primary antibody responses of 5 BALB/c mice to a combination of biotinylated 4F11 and lC10 conjugated with avidin, to biotinylated 4F11 conjugated to avidin or to avidin alone. The primary antibody response to avidin is comp~r~ble to the response to avidin plus biotinylated IgG2a control antibody. However significant enhancement of antibody levels to avidin is achieved in response to immlmi.c~tiQn with a 10 biotinylated anti CD40/avidin conjugate. Figure 6 shows secondary antibody responses. Clearly the physical linkage of antigen to adjuvant leads to enhanced antibody responses to avidin with a reduction in the amount of adjuvant required. This methodology may also be applied to T-cell independent antigens like the capsular polysacch~ri~les of S. pneumoniae.
15 Techniques for conjugating polysacr~h~rides to protein do exist and will allow this strategy to be further developed.
It is evident that CD40 simulators, such as antibodies, recombinant soluble CD154, or molecular mimics of CD154, have consitlerable potential as immnnological adiuvants for T-cell dependentl1ndependent ~ntigene.
-
In vitro experiments have shown that resting B-cells can be stim~ ted to proliferate after exposure to isolated membranes from activated T-cells. The basis for this phenomenon has been detennin~d. Following T-cell activation a 39kDa (CD 154) T-cell speci~lc cell surface protein is induced. This ligand has been identified as the target of the B-cell cell surface receptor CD40 and binding of CD154 to CD40 is the major component of T lymphocyte help to B lymphocytes.
Further evidence for the involvement of CD40 and CD154 comes from experiments in which host cells transfected with the cDNA encoding the CD154 protein can induce proliferation of B-cells in the presence of added cytokines. In addition, patients with the congenital (1ise~e X-linked hyper IgM syndrome, who fail to switch antibody isotypes have been shown to have various mutations in the gene encoding the CD 154 protein resulting in failure to activate the B-cells via CD40. The CD40-CD154 inter~ction has also been 5 shown to be an important elernent in ~ R responses to T-cell dependent antigens in 'knock-out' mice.
The other important element in B-cell activation via T-cell help involves cytokine function. Although isolated membranes from activated T-cells can induce B-cell proliferation this effect can be enhanced by the presence of 10 cytokines. Furthermore cytokines have a major role in switching of antibody isotypes. In particular IL4, intelÇeloll y and transforming growth factor beta (TGF ,B) are of importance. IL4 induces IgGl and IgE, IFNy induces IgG2a and TGFB induces IgA and IgG2b. In addition IFN~ is probably responsible for the switching to IgG3 which is seen naturally in responses to T-cell 15 independent antigens. However ligation of CD40 does not induce appreciable Ig secretion on its own, but CD40 ligation (including via T-cell membranes) seems to prepare cells for differentiation which can be induced efficiently by ILA and IL5.
Finally T-cell help has a major influence on somatic hypermllt~tion which 20 results in the selection of B-cell clones that produce high affinity antibodies.
From this description it may be surmised that T-cell independent production of antibodies by B-cells is compromised due to the lack of help offered by T-helper lymphocytes through activation via CD40 and through the influence of cytokines produced by the T-helper cell.
_ It is therefore an object of this invention to provide a means of activating B-cells to proliferate and produce the full range of antibody isotypes of high titre in response to T-cell indepenflent as well as T-cell dependent antigens.
It is a further object of this invention to use T-cell indepen(lent and/or 5 dependent antigens to produce effective vaccines that offer high titre, high affinity antibodies to protect individuals from infection.
It is yet a further object of the invention to provide a safe i~ logical adjuvant for use in a vaccine and also for use in enhancing the immllne response to T-cell independent and/or dependent antigens.
10 It is yet a further object still of the invention to provide a method for the production of a vacciné of the invention.
It is a further object of the invention to provide a system for the production of the vaccine of the invention.
In its broadest aspect the invention concerns the provision of a means for 15 a~;tiva~ g the CD40 receptor on a B-lymphocyte, ideally the means compri~ing an adjuvant which is adapted to activate said receptor, either directly or indirectly. More preferably the invention concerns a ligand which binds to the CD40 receptor on a B-lymphocyte and brings about the activation of same.
20 According to a first aspect of the invention there is therefore provided an adjuvant which is adapted to stimulate a B-lymphocyte cell surface receptor, CD40.
According to a second aspect of the invention there is provided a vaccine suitable for enh~ncing T-cell independent and T-cell dependent im~ ",ily comI ricing a T-cell dependent and/or independent antigen, or part(s) thereof, and an associated adjuvant which is adapted to stimul~te a B-lymphocyte cell 5 surface receptor, CD40.
Reference herein to the term vaccine is intended to include a wide variety of vaccines inclll~1ing, but not limite~1 to, contr~ceptive vaccines, il~ otherapy vaccines and prophylactic or therapeutic vaccines.
Reference herein to T-cell independent immunity includes reference to an 10 immune response which operates wholly or largely independently of T-cells, for example, because existing T-cells are not activated; or because existing T-cells are not functional or immllne suppressed through ~lise~se or exposure to chemicals, radiation or any other means.
To by-pass or mimic the effects of T-cell help we propose a vaccine which 15 ensures that all B-cells receiving a signal through their specific antigen receptors also receive a signal through CD40, mimicking or improving upon that which would be received during natural T-cell help. This would be achieved, ideally, by ensuring that a CD40 binding moiety were closely associated with the vaccine antigen. This could be through co-~mini.~tration 20 of the CD40 stimlll~ting moiety with the appropriate T-cell independent and/or dependent antigen, or preferably through covalent linkage, or co-entrapment on/in a carrier system.
The vaccine involves ideally the conjugation of the antigen to a CD40 ligand such as an anti CD40 antibody, or part thereof, followed by immlmis~tion of a human or ~nim~l It should be apparent to those skilled in the art that this methodology may also be applied to any antigens, but in the in~t~nce of T-cell dependent antigens could be of particular relevance to those individuals that are i..~",l,e suppressed and therefore lack T-helper lymphocytes (e.g.
5 AIDS patients).
~a preferred emborliment of the invention said antigen is soluble and ideally a protein or a polysaccharide.
.
Ideally stimulation of CD40 is via binding of said adjuvant, or part thereo~, to at least a part of CD40. In a preferred embodime~t of the invention said 10 antigen and adjuvant are bound or cross-linked together.
More preferably said adjuvant is an antibody, either polyclonal or monoclonal, but ideally monoclonal, which is adapted to bind to said CD40.
More ideally still said antibody is hum~ni.~ed.
In a preferred aspect of the invention said antibody may be whole or, 15 alternatively, comprise only those domains which are effective at binding CD40 and in particular selected parts of CD40.
In another embodiment of the invention, said adjuvant is a n~tur~l ligand of CD40, the T-cell specific CD154 cell surface antigen, ideally produced as a recombinant protein, or a CD40 binding portion of the CD154 protein, or 20 indeed any other ligand, or part thereof, that binds CD40 or part thereof.
In a further embodiment, the CD40 ligand may not be a naturally occurring CD40 ligand but represent an agent that due to its biochemicaI characteristics , r ~ , .
has an affinity for CD40.
In its broadest context, reference herein to the term adjuvant includ~s reference to any string of amino acids or ligand which is selected so as to bind to at least a part of CD40.
5 In a preferred aspect the recombinant vaccine antigen (when a polypeptide) and the adjuvant will be produced as a ~him~ric fusion protein.
It will be apparent to those skilled in the art that the said antigen may be a T-cell independent antigen and thus any antigen which is capable of eliciting a T-cell independent response.
10 ~Itern~tively, the antigen may be a T-cell dependent antigen and thus any antigen that is capable of eliciting a T-cell response.
It is apparent from the above that any antigen may be selected for use in the vaccine of the invention - the precise nature of which will depend on the "rli~e~e" that an individual is to be immuni.ced against and/or in some 15 circllmst~nces, the immnn~ status of an individual to be vaccinated.
Ideally said antigen and/or adjuvant is in the form of an immunostimulating complex, or liposomes or biodegradable microspheres, so increasing the association between antigen and CD40 binding moiety.
Alternatively said vaccine comprises an emulsion of the antigen and adjuvant 20 ideally in oil.
9 ~ -;
In a preferred embo-liment of the invention at least one selected cytokine may be included in andlor co~lmin.~tered in/with said vaccine.
According to a third aspect of the invention there is provided an adjuvant for enhancing T-cell independent immlmity wherein said adjuvant comprises an 5 agent adapted to stimlll~te a B-lymphocyte surface receptor, CD40.
Preferably said stimlll~tion of said CD40 is via binding of said adjuvant, or part thereof, thereto.
Ideally, said adjuvant is an antibody, either polyclonal or monoclonal, but ideally monoclonal, which is adapted to bind to said CD40. More ideally still 10 said antibody is hllm~ni.sed.
In a preferred aspect of the invention said antibody may be whole or, ~ltern~tively, comprise only those domain~ which are effective at binding CD40, and in particular selected parts of CD40.
In this aspect of the invention said adjuvant is co-~llmini.~tered with either 15 said T-cell independent antigen that is effective at eliciting a T-cell independent immune response or a T-cell dependent antigen that is effective at eliciting a T-cell response. This will be dependent upon the nature of the "disease" against which the individual is to be immlmi.~ed and/or the immlm~
status of the individual.
20 More p~fel~bly further still said adjuvant is cojoined to said T-cell independent antigen or said T-cell dependent antigen.
In a yet further preferred embo-liment said adjuvant in co-~(1mini~tered with at least one cytokine.
According to a fourth aspect of the invention there is provided a method for the manufacture of a novel vaccine capable of enhancing T-cell independent 5 immunity or T-cell dependant immlmity which methods comprises the selection of a suitable T-cell dependant and/or independent antigen, or part(s) thereof, and association or combination of said antigen with an adjuvant wherein said adjuvant is adapted to stim~ te a B-lymphocyte receptor, CD40.
According to a fifth aspect of the invention there is provided a method for the 10 manufacture of a novel vaccine capable of enhancing T-cell independent imm~lnity which method comprises the selection of a suitable T-cell dependent and/or independent antigen, or part(s) thereof, and association or combination of said antigen with an adjuvant wherein said adjuvant is adapted to stim~ te a B-lymphocyte receptor, CD40.
15 In yet a further preferred method of the invention said adjuvant is recombinantly manufactured.
In yet a further preferred embodiment of the method of the invention said antigen and adjuvant are bound or cross-linked theretogether.
The major T-independent antigens used in vaccines are b~cteti~l capsular 20 polysaccharides. In a preferred embodiment or method of the invention one will therefore purify polysaccharide antigens and crosslink them to a CD40 binding moiety. A commonly used technique for the cross linking of polysaccharide to protein is carbodiimide coupling. However a number of heterobifunçtion~l cross-linking agents are commercially available for both protein-protein and protein-carbohydrate cross-linking Heterobifilns~tion~l cross-linking agents have the advantage that they favour protein-carbohydrate cross-links thereby m~imising the yield of adjuvant coupled to antigen.
5 Preferably said stim~ tion of said CD40 is via binding of said adjuvant, or part thereof, thereto.
Ideally, said adjuvant is an antibody, either polyclonal or monoclonal, but ideally monoclonal, which is adapted to bind to said CD40. More ideally said antibody is hllm~ni~ed.
10 In a preferred aspect of the invention said antibody may be whole or, ~ltern~tively, comprised only those domains which are effective at binding CD40, and in particular selected parts of CD40.
In a preferred method of the invention one adds at least one cytokine to said vaccine.
15 According to a further aspect of the invention there is provided a system forthe manufacture of a vaccine capable of enh~ncing T-cell independent or T-cell dependent immunity which system comprises a cell expressing a selected T-cell dependent and/or independent antigen, or part(s) thereof, and also an adjuvant capable of stimulating a B-lymphocyte receptor, CD40.
20 According to a yet further aspect of the invention there is provided a systemfor the manufacture for a vaccine capable of enh~ncing T-cell independent immnnity which system comprises a cell expressing a selected T-cell ~; :
, dependant or independent antigen, or part(s) thereof, and also an adjuvant capable of stimlllating a B-lymphocyte receptor, CD40.
More preferably still both said antigen (when a polypeptide) and said adjuvant are adapted so as to be secreted from said cell. This may be 5 undertaken by providing both the antigen and adjuvant with secretion ~ign~l~
or providing for the production of a single piece of m?~tetiZIl comprising both the antigen and the adjuvant and having a single secretion signal associated therewith. It will be evident that in the former instance the said antigen and adjuvant will be found in associated or unbound or uncross-linked m~nn~r in 10 the supern~tant of the system and in the latter instance said antigen and adjuvant will be cojoined in the sup~rn~tant of the system.
Preferably said s~im~ tion of said CD40 is via binding of said adjuvant, or part thereof, thereto.
Ideally, said adjuvant is an antibody, either polyclonal or monoclonal but 15 ideally monoclonal, which is adapted to bind to said CD40. More ideally said antibody is h~lm~ni.ced.
In a preferred aspect of the invention said antibody may be whole or, alternatively comprise only those domains which are effective at binding CD40, and in particular selected parts of CD40.
20 It will be apparent from the above that the invention is based upon the re~lic~tion that immune responses, whether to a T-cell independent or a T-cell dependent antigen, can be enhanced by stimlll~ting the B-cell CD40 receptor using any suitable means.
According to a yet filrther aspect of the invention there is provided a n~l~leicacid molecule encoding any one or more of the aforementioned embo-limP,nts of the invention.
~ this last aspect of the invention said nucleic acid molecule may be 5 ~/lmini.~tered, conventionally, to an individual or animal to be treated so that the adjuvant and ideally also the antigen of the vaccine may be manufactured i71 VIVO.
An embo~lim~nt of the invention will now be described by way of example only with reference to the following ~lgures wherein:-10 Figure 1: Shows CD40 antibody induced enhanced, class switched antibodyresponses to PS3 (type 3 pneumococcal polysacch~ri(le) (A) and increased total serum immlmoglobulin (B). BALB/c mice (6-10 weeks old) were injected i.p with 20ng of PS3 and 500,ug of lC10, 4Fl 1 (anti-mouse CD40) or isotype control antibody GLl 17. Sera were obtained days 7, 14 and week 15 14 after injection. Ihe IgM and IgG isotype mean log~rithmic titres are shown when they were m~im~l, respectively, day 7 and day 14 after injection. All negative results were given a logarithmic titre of 20, the lowestdilution used. * indicates statistical signi~lcance compared with the relevant GLl 17 control (Student's T test p<0.05).
20 Figure 2: Shows antibody responses to other pneumococcal polysaccharides are also enhanced by CD40 antibody. IgM and IgG responses to types 8, 4, 12 and 19 S. pneumoniae capsular polysaccharides in mice immlmised with the 23 capsular polysaccharides in Pneumovax II (Merck Sharp and Dohme, USA) and either the CD40 antibodies 4Fll, lC10 (anti-mouse CD40) or control antibody GL117. Groups of ~lve BALB/c mice, were injected i.p with either 500,ug of lC10, 4Fl l or GL117, and 1/25th of the recommen~lçd hllm~n dose of Pneumovax II (commercial 23-valent pneumococcal polysaccharide vaccine, l,ug each of the 23 polysacch~n~les present). Sera 5 were obtained on day 10 after injection. All negative results were given a lop~ l""ic titre of 20, the lowest dilution used. All the lC10 responses were significantly dirre~ from the GL117 responses (Student's T test p<0.05).
Figure 3: Shows that the mech~ni.~m of lC10 action is CD4~ cell independent. PS3 specific antibody logarithmic titres induced in CD4 depleted BALB/c mice treated i.p with 20 ng of PS3 and 500,ug of lC10, 4Fll or control antibody GL117. These mice failed to respond to co-s~lmini~tered keyhole limpet haemocyanin nor were any CD4+ splenocytes discernable on FACS by FITC anti CD4 (data not shown). Sera were obtained on day 14 after injection. All negative results were given a 15 log~ ic titre of 20, the lowest dilution used. All lC10 responses were significantly different from the relevant GL117 control (Student's t test p<O.OS).
Figure 4: Shows CD40 antibodies induce responses to PS3 in normally unresponsive xid mice (A). Enhanced responses in BALB/c mice provide 20 protection against S. pneumoniae challenge 9 months after tre~mPnt (B). (A) PS3 specific antibody responses in CBA/N(xid) mice injected with 20ng of PS3 and lC10, GL117 and/or control CBA/ca mice with lC10 and GL117.
The IgM and IgG isotype logarithmic titres shown are when they were m~im~l, respectively, day 7 and day 14 after injection. All negative results 25 were given a log~rithmic titre of 20, the lowest serum dilution used. *
indicates statistical signi~lcance compared with the relevant GL117 control (Student's T test p<0.05). B) Percentage ~uLvival in BALB/c mice challenged with S. pheumoniae type 3, but ~1ministered 9 monthe previously with 20ng PS3 and 500,ug of lC10, GL117 or PBS. Survival in the lC10 group w~
significantly enhanced compared to the control groups (p<0.05 %2 test).
5 Figure 5: Shows primary antibody responses to avidin conjugated to biotinylated CD40 antibodies are enhanced. BALB/c mice were immllni7:ed with either 10,ug of control IgG2a, 10,ug of avidin conjugated to anti CD40 monoclonal antibody 4Fll, 10,ug of a combination of avidin conjugated to anti CD40 antibodies 4F11 and lC10 or 10,ug of non-conjugated avidin.
10 Antibody responses against avidin were measured by ELISA at 10 days post-immlmi~tion.
Figure 6: Shows secondary antibody response to avidin alone following primary immunisation with avidin conjugated to anti CD40 antibodies 4F11 and lC10. Experiment~l details are essentially as described in Figure 5, 15 except that mice received an immllni.~tion with 10 ,ug avidin alone one monthafter primary immllni.~tion as in Figure 5, mice were bled 10 days after this second injection and antibody responses measured by ELISA.
Methods M;ce and Materials 20 The mice used were BALB/c mice (in house), CBA/ca and CBA/N (xid) mice (Harlan-Olac). They were 6-12 weeks old at the start of the experiments. The pneumococcal capsular polysaccharides type 1, 3, 4, 8, 12, 13, 19 and 23 were obtained from ATCC, USA, pneumococcal cell wall CA 02207246 l997-06-06 polysaccharide from Statens Serum Tn.stitllte, Denm~tk and Pneumovax II
vaccine from Merck Sharp and Dohme, USA. Avidin was purchased from Sigma (Poole, Dorset). Biotinylated and non-biotinylated anti-CD40 antibodies were purified from hybridoma supern~t~nt~ in house and 5 biotinylated in house where necessary using st~nll~rd reagents (Pierce).
Immunisation Protocols - Mice were treated with 500,ug of either lC10, 4Fll or GL117 and 20ng of PS3 i.p. except those receiving Pneumovax II. BALB/c mice receiving Pneumovax II were injected i.p. with either 500,ug of lC10 or GL117 and 10 l/25th of the recommended hllm~n dose of Pneumovax II. This equates to l~ug of each of the 23 polysacch~ri~es present in the vaccine. At least S
mice were used for each experimental group. ~ expPriments where mice were immllni~e-l with avidin conjugated to biotinylated anti-CD40, avidin at lmg/ml and biotinylated antibody at lmg/ml were mixed together at a 1:1 15 ratio and left on ice for 30 minll~es. The conjugates were then diluted in PBS
to give a total of lOIlg antibody and lO,ug avidin in 0.2ml PBS, which was then injected intraperitoneally. In cases where avidin alone was used it was pre-mixed with an equal volume of PBS and left on ice for 30 minlltes before dilution and injection.
20 Experiment in CD4 depleted mice BALB/c mice, 6-10 weeks old, were depleted of CD4 cells 5 days before the experiment start. 500,ug of depleting anti CD4 antibody ~TS 191.1 was injected intravenously and again the next day intraperitoneally. The percentage of CD4+ splenocytes in the depleted mice as detected by flow cytometry had dropped to undetectable levels when the antibody and PS3 were injected. There was no antibody response to 50,ug to keyhole limpet haemocyanin, a T dependent antigen~ co-~1mini.ctered with the PS3 (data not shown).
5 Measuremen~s of polys~c~h~ride antibodies and total serum immunoglobulin by ELISA
96 well ELISA plates (Costar, UK) were coated overnight with lO,ug/ml polysaccharide or with a 1/200 dilution of anti mouse Ig serum (Sigma, UK).
Individual sera were titrated on the plates and the various isotypes detected 10 by HRP conjugated mouse isotype specific sera (Southern Biotechnology Associates, USA). Sera obtained from mice injected with Pneumovax II were absorbed against S. pneumoniae cell wall polysaccharide as described previously. Antibodies to cell wall polysaccharide, a cont~min~nt of all capsular polysaccharide pr~ ion~s might have created false positive results.
15 Total serum immllnoglobulin concçntr~tions were calculated with reference to calibrated mouse serum (Sigma, UK). With the polysaccharide results end point titres for each mouse were a~ssessed against normal mouse serum and then geometric mean titres and standard deviation calculated.
Measurement of anti-avidin responses by F~
20 96 well ELISA plates (Costar, UK) were coated overnight with lO,ug/ml avidin (Sigma) in PBS. After blocking for l hour with 3% bovine serum albumin individual sera were titrated on the plates, incubated at room temperature for 1 hour, and following washing, antibody was detected using HRP conjugated anti-mouse immunoglobulin (Southern Biotechnology , Associates USA), and substrate (OPD Sigma). End point titres for each mouse were ~sse~sed ~in.~ norm~l mouse serum, and then geometric mean titres and st~n-l~rd deviation calculated.
Challenge with S. pneumoniae 5 BALB/c mice were immnni.~ed 9 month.e before ch~llenge with 20ng PS3 and 500,ug lC10 i.p. Challenge was 105 colony forming units of encapsulated S.
pneumaniae type 3 (ATCC) given i.p.. Final numbers surviving were ascertained 2 weeks after ch~llellge.
Results and Discussion 10 The development of vaccines ~g~inst encapsulated bacteria, such as Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis, is centred on their distinctive capsular polysaccharides.
Unfortunately, the inability of antigen presenting cells (APC) to process and present polysaccharides with MHC class II means that these antigens cannot 15 stim~ te T-cells. Polys~cchzlride specific B-cells receive no direct help from their T-cells and, therefore, these antigens are considered T independent (TI-II). Due to this lack of help, TI-II antibody responses are of low titre, low average affinity, and are predomin~ntly of the IgM class with no boosting on second or later exposures to antigen. The T-cell help provided during 20 immlme responses to TD antigens induces high titre and isotype switched antibody responses. The major stimulus to B-cells is provided by CD154 (formerly CD40 ligand or gp39), which is expressed de novo on activated T-cells. The CD154 molecule binds the CD40 antigen, which is ct~n~titlltively expressed on B-cells, and their interactions provide key signals as immune -t, , .. 19 , responses develop. CD40 activation is important for the initi~tion of B-cell proliferation, i~ loglobulin class switching, germin~l centre responses, and the production of memory B-cells and plasma cells. B-cells responding to TI-II antigens lack T-cell derived cytokines and CD40 ligation and produce, as 5 a result, the poor antibody response ch~r~cteristic of TI-II antigens. We have investigated in vivo whether the ~-lmini~ttation of pneumococcal polysaccharide with anti-mouse CD40 antibody could provide a sllbstitllte for CD154 mediated CD40 ligation. The two antibodies used were lC10 and 4Fl l, chosen they are both rat IgG2a anti-mouse CD40 antibodies but possess 10 markedly different in vitro propettiP~s Intraperitoneal immllni~tion of BALB/c mice with type 3 pneumococcal capsular polysaccharide (PS3) alone induced weak IgM and IgG3 responses ~g~in~t the antigen (Figure lA). This is typical of the response to TI type II
antigens in mice (hllnl~ns produce IgM and IgG2). Administration of 15 antibodies lC10 or 4Fll with PS3 induced small but significant rises in specific IgM and IgG3, while remarkably, lClO induced signific~nt polysaccharide specific IgGl, IgG2a and IgG2b responses. These isotypes are not normally seen in response to TI II antigens. lC10 would appear to have successfully mimicked T-cell help by inducing high antibody titres and 20 isotype switching in vivo. The anti-polysacch~ride response was extremely persistent, with antibody being detected at high titres 14 weeks after the single immunisation (Figure lA). No memory response against the polysaccharide was induced as a second injection of polysaccharide alone failed to boost antibody responses (data not shown).
25 S. pneumoniae has over 80 different capsular polysaccharide types and any vaccination would be expected to induce protective immllnity against a number of the more common serotypes. A current pneumococcal vaccine, Pneumovax II (Merck, Sharp and Dohme), consists of 23 different polysaccharides. Mice were immllni.sed with this 23-valent vaccine and lC10. Figure 2 shows that inclusion of the CD40 antibody successfully 5 generated strong IgG responses ~g~inct r~ndomly chosen polysac~h~ride types 4, 8, 12 and 19. Such isotype switched responses were also gener~ted against the two other antigens we ex~mined, types 3 and 14 (data not shown).
Therefore, lC10 enhances responses to TI-II antigens other than just PS3.
Given that ~dmini~tr~tion of CD40 antibody mixed with polysacch~rkle would 10 not restrict or even target CD40 ligation to antigen specific B-cells, we anticipated polyclonal activation of B-cells with a resultant rise in total serum immlmoglobulin levels. ~deed lC10 and PS3 induced some splenomegaly and 2-4 fold rises in total serum immllnoglobulin levels (Figure lB). This, however, should be con~r~ted with up to 5-fold rises in specific antibody 15 levels, indicating that polysaccharide specific antibody production was preferentially enhanced. This skewing towards specific antibody is also not unexpected as it reflects in vitro fin(1in~s. In vitro, while lC10 could induce B-cell proliferation in the absence of stimul~tion through the antigen receptor,proliferation was synergistically enhanced by such co-stimlll~tion. 4F11, 20 which largely lacks agonist activity in vitro, did not enhance responses as efficiently as lC10, demonstrating an association between adjuvant activity in vivo and B-cell activation in vitro.
CD40 ligation is necessary for switching to IgG isotypes during a T
dependent response, but various cytokines also play important roles. It was, 25 therefore, intriguing that such isotype switched responses were obtained without the addition of exogenous cytokines. l~his suggests either that CD40 -and antigen receptor ligation may be sufficient to induce isotype switehing or that bystander cells may provide sufficient cytokines to switch the activated B-cells in vivo. We considered that the CD40 antibodies might be stim~ ting T-cell production, either directly through ligation of CD40 on T- cells or 5 indirectly through induction of co-stiml-l~tory molecules on B-cells or other APCs. The action of 4F11 showed T-cell dependency as it failed to augment polysaccharide specific responses in CD4 depleted mice (Figure 3).
However, lC10 and PS3 ~t1ministration induced a pronounced, isotype switched response in CD4 depleted mice (Figure 3) with IgG responses to 10 polysaccharide being better than those induced in normal mice, demonstrating a CD4 independent action. Similar results were obtained when athymic nude mice were used inste.~-l of CD4 depleted mice (data not shown).
Most vaccines under development for use against encapsulated bacteria are protein-polysaccharide conjugates which aim to provide T-cell help for the 15 anti-polysacch~ri-le response through T-cell recognition of epitopes on the protein. By their nature such conjugates are not as effective in CD4 deffcient patients such as those with AIDS. In contrast the use of a CD40 stimul~tor would not only avoid the high cost of conjugate production, but as we have shown, gen~r~te responses unaffected by a CD4 deficiency.
20 The major fault with capsular polysaccharide only vaccines is that infants and young children, whilst reacting norm~lly to TD antigens, respond poorly to TI-II antigens. Indeed children under two years old fail to respond at all to many TI-II antigens. The inability of their immune systems to act ~g~inst bacterial capsules correlates with increased susceptibility to infection. They 25 are the group most in need of effective vaccines. CBA/N (xid) mice have an X-linked immunodeficiency rendering them, like infants, unable to respond . ~ = --, to TI-II antigens. Although one report has stated otherwise, in our hands these mice react normally to CD40 ligation in vitro (and unpublished data A.H.). We immllni.~ed groups of xid mice with lClO plus PS3 and successfully generated IgG2a and IgG2b responses ~g~in~st PS3 (Figure 4A).
5 Thus, the B-cell defect in these mice was successfully by-passed by ~,~ministering the CD40 antibody as an adjuvant along with antigen.
Using the mouse model system, we have shown that CD40 sim~ tors can enhance the antibody response to pneumococcal polysacch~rides, producing greater antibody levels and the production of IgG isotypes. Similar to 10 protein-polysacch~n-le conjugates, lC10 can induce polysacch~ride specific responses in xid mice, which like infants are unable to respond to polysaccharide only based vaccines. Unlike protein-polysaccharide conjugates, the adjuvant action of lC10 is CD4 cell independent, which is a definite advantage for the vaccination of patients with CD4 deficiencies, for 15 example AIDS sufferers.
While lC10 ~rlministered with PS3 clearly enhances speci~lc antibody responses, the measure of a vaccine is whether it provides long-term protection against disease. We challenged mice, immunised 9 months previously, with 105 CFU of S. pneumoniae type II (Figure 4B). Of the 20 BALB/c mice ~lmini~tered with PS3 and lClO five of eight survived ch~llenge, whereas only one of six and none of eleven mice survived in the groups receiving, respectively PS3 with GLl 17 and PS3 alone (p~0.05%2test).
Finally, the induction of polyclonal antibody responses, as previously described in Figure lB, may increase the risk of auto antibody production.
25 We have investigated this problem by reducing the need to ~(lminister elevated doses of anti CD40 antibody by conjllg~ting biotinylated anti CD40 antibody with avidin (a n~tur~l ligand of biotin). By physically linking the adjuvant and antigen we have been able to reduce adjuvant levels by approximately 50-fold. Figure 5 shows the primary antibody responses of 5 BALB/c mice to a combination of biotinylated 4F11 and lC10 conjugated with avidin, to biotinylated 4F11 conjugated to avidin or to avidin alone. The primary antibody response to avidin is comp~r~ble to the response to avidin plus biotinylated IgG2a control antibody. However significant enhancement of antibody levels to avidin is achieved in response to immlmi.c~tiQn with a 10 biotinylated anti CD40/avidin conjugate. Figure 6 shows secondary antibody responses. Clearly the physical linkage of antigen to adjuvant leads to enhanced antibody responses to avidin with a reduction in the amount of adjuvant required. This methodology may also be applied to T-cell independent antigens like the capsular polysacch~ri~les of S. pneumoniae.
15 Techniques for conjugating polysacr~h~rides to protein do exist and will allow this strategy to be further developed.
It is evident that CD40 simulators, such as antibodies, recombinant soluble CD154, or molecular mimics of CD154, have consitlerable potential as immnnological adiuvants for T-cell dependentl1ndependent ~ntigene.
-
Claims (22)
1. An adjuvant which is adapted to stimulate a B-lymphocyte cell surface receptor, CD40.
2. A vaccine including the adjuvant according to Claim 1.
3. A vaccine according to Claim 2 wherein said vaccine comprises a T-cell dependent and/or T-cell independent antigen, or part(s) thereof.
4. A vaccine according to Claim 2 or 3 wherein said adjuvant is a CD40 ligand, or part thereof.
5. A vaccine according to Claims 2 to 4 wherein said adjuvant is an antibody raised against said CD40 receptor, or a part thereof.
6. A vaccine according to Claim 5 wherein said antibody is monoclonal.
7. A vaccine according to Claim 5 or 6 wherein said antibody is humanized.
8. A vaccine according to Claims 2 to 7 wherein said antigen is soluble.
9. A vaccine according to Claims 2 to 8 wherein said antigen is a protein.
10. A vaccine according to Claims 2 to 8 wherein said antigen is a polysaccharide.
11. A vaccine according to Claims 2 to 10 wherein said adjuvant and antigen are joined theretogether.
12. A vaccine according to Claims 2 to 11 wherein said antigen is a protein or part thereof, and it is fused to said adjuvant so as to provide a fusion protein.
13. A vaccine according to Claims 2 to 12 comprising at least one cytokine.
14. A vaccine according to Claims 2 to 13 suitably formulated from administration to an individual or animal to be vaccinated.
15. A method for the manufacture of a novel vaccine capable of enhancing immunity which method comprises the selection of a suitable T-cell dependent and/or T-cell independent antigen, or part(s) thereof, and association or combination of said antigen with an adjuvant wherein said adjuvant is adapted to stimulate B-lymphocyte receptor, CD40.
16. A method according to Claim 15 wherein said vaccine is capable of enhancing T-cell independent immunity.
17. A system for the manufacture of a vaccine capable of enhancing T-cell independent or T-cell dependent immunity which system comprises a cell expressing a selected T-cell dependent and/or T-cell independent antigen, or part(s) thereof, and also an adjuvant capable or stimulating a B-lymphocyte receptor, CD40.
18. A system according to Claim 17 wherein said vaccine is capable of enhancing T-cell independent immunity.
19. A system according to Claim 17 or 18 wherein one or both of said antigen and adjuvant is provided with a secretion signal whereby expression of one or both of said antigen or adjuvant results in secretion of one or both of said antigen or adjuvant from said cell.
20. A system according to Claims 17, 18 or 19 wherein the expression of said antigen and adjuvant is adapted such that a single fusion protein is manufactured by said cell.
21. A system according to Claim 20 wherein said single fusion protein is adapted for secretion from said cell.
22. A nucleic acid molecule encoding an adjuvant according to Claim 1 or a vaccine according to Claims 2 to 14.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2207246 CA2207246A1 (en) | 1997-06-06 | 1997-06-06 | Vaccine development |
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| Application Number | Priority Date | Filing Date | Title |
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| CA 2207246 CA2207246A1 (en) | 1997-06-06 | 1997-06-06 | Vaccine development |
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| CA2207246A1 true CA2207246A1 (en) | 1998-12-06 |
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| CA 2207246 Abandoned CA2207246A1 (en) | 1997-06-06 | 1997-06-06 | Vaccine development |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001015728A1 (en) * | 1999-08-27 | 2001-03-08 | University Health Network | Method for activating cytotoxic t-lymphocytes (ctls) in vivo : composition comprising antibody anti cd40 (or cd40l or cd40 binding protein) and an antigen |
| EP1469881A2 (en) * | 2002-01-28 | 2004-10-27 | Adjuvantix Limited | Vaccine adjuvant based on a cd40 ligand |
-
1997
- 1997-06-06 CA CA 2207246 patent/CA2207246A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001015728A1 (en) * | 1999-08-27 | 2001-03-08 | University Health Network | Method for activating cytotoxic t-lymphocytes (ctls) in vivo : composition comprising antibody anti cd40 (or cd40l or cd40 binding protein) and an antigen |
| EP1469881A2 (en) * | 2002-01-28 | 2004-10-27 | Adjuvantix Limited | Vaccine adjuvant based on a cd40 ligand |
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