Note: Descriptions are shown in the official language in which they were submitted.
CA 02207246 1997-06-06
,,, ,~ , . , . - . 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
CA 02207246 1997-06-06
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
CA 02207246 1997-06-06
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
CA 02207246 1997-06-06
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.
_
CA 02207246 1997-06-06
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.
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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
CA 02207246 1997-06-06
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
CA 02207246 1997-06-06
~ , .
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.
CA 02207246 1997-06-06 --
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.
CA 02207246 1997-06-06
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
CA 02207246 1997-06-06
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
~; :
CA 02207246 1997-06-06
,
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.
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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
CA 02207246 1997-06-06
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
CA 02207246 1997-06-06
(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
16
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
CA 02207246 1997-06-06
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
,
CA 02207246 1997-06-06
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
-
CA 02207246 1997-06-06
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
CA 02207246 1997-06-06
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
CA 02207246 1997-06-06
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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
. ~ = --
CA 02207246 1997-06-06
,
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
CA 02207246 1997-06-06
23
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.
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