Note: Descriptions are shown in the official language in which they were submitted.
CA 02322175 2000-08-25
WO 9~1»3~9 PCTISE99I00:77
11~UCOSAL ~!~(ICItOPART1CGE CON.1UGATE VACCINE
the present invention relates to micropanicle conjugate vaccines for
mueosal, e.g. oral, administration to a mammal, including man. The vaccines
are
directed against a eettain pathogenic microorganism, particularly an
intracellular
microorganism, such as Mycobauetlrrm ~Lbtrculosis or Salmonella enrerirldis.
The
invention also relates to a method of inducing protective immunity against
such a
microorganism, and to the use of protection-generating antigens derived from
such a
microorganism conjugated to biodegradable mieroparticles, for the production
of the
vaccines.
8ackgr4uod
Generally, vaccines today are fotmulatcd for parenteral administration,
Only a few vaccines arc used orally and then for speciFtc purposes. Thus, oral
cholera
vaccines are intcaded to produce antibodies against the B-suburut CTH of the
cholera
toxin, causing diarrhea of the infected person, by disrupting the salt and
water balance
over the gut wall. The antibodies arc supposed to inhibit the binding of the
toxin via the
CTH unit to a specific receptor (the GM1 receptor) in the epithelial wall.
Moreover,
some vaccines containing attenuated polio virus, with disputed efficacy, arc
approved to
be used in some countries. However, no carrier system far oral use with
isolated antigens
has yet been approved for use in humans.
Theta are some obvious advantages with oral vaccines. They are easier to
'~o ~ P~~~l ortea, as the adminisastion does not require professional
personnel,
like nurses, and an oral administration avoids the sa~ess caused by an
injection,
particularly in children. In addition, the manufacture of an oral product is
easier and
thereby cheaper than for a sterile, parenteral product. More important though,
are the
potentially improved affoecs of an oral vaccination over a p~k~ one in
newborns,
where the irnmunc system in the mueosal and gut regions develop earlier than
in other
' parts df the body, where the parentecal vaccines are active. Also for
elderly people the
mueosal response is probably better after otal vaccination.
An impitrtartt feature of an immure response is the memory function,
which is mediated by specific 8-cells, the differentiation and proliferation
of which are
CA 02322175 2000-08-25
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2
induced by specific antigenic structures. A well functioning set of memory
cells is
needed to give the vaccinated person a life-long protection, experimentally
identified by
the so called booster effect obtained upon a late exposure to the antigun.
Moreover, .
protection against an invading microorganism is also provided by a cellular
response,
which can be detected by the so e311ed delayed-type hypersensitivity reaction,
usually
performed in the ears and footpads of mice. These irnmunologica) responses arc
frequently seen after parenteral vaccination. It has gcneraJ,ly been assumed
that oral
vaccination gives a mucosal response, detected by the production of local
antibodies of
the subtype IgA (slgA). However, it would be desirable to obtain a mucosal,
preferably
oral, vaccine against pathogenic microorganisms which gives both a memory
function
and a cellular response in addition to a strong mucosal IgA production.
Further, since
cell-mediated immunity seems to be the most important defense against
intracellular
pathogens in s host, an eCfcicnt vaccine against such pathogens should
stimulate the T-
cell immune response.
Moreover, some experimental and epidemiological indications suggest that
a cellular immune response predominately of the Thl.type is especially
important to
withstand viral and parasitic inFectians. A Th1 response is also thought to
better mimic
the response seen aRer a natural infection and to decrease the risks of later
development
of allergy.
A few vaccination studies have been performed with particulate antigens
using the parenteral immunization route. Vordermeier e~ al, showed that a 38
kDa
protein antigers from M. tuberculosis entrapped in the particulate adjuvant
poly (DL.lactide co-glyeolido) particles induced Thl-aatigea specific httmoral
and
cellular itamune responses, which, however, did not protect against an
intravenous
challenge with M. ttrbarculosis (Vordenrteier ei al., 1995).
$arlier experimental vaccination studies with protective ttntigons_dcrivcd
frota M, nrberoWlos~s, i.e, secreted proteins, against tuberculosis have more
or less
successfully been carried out with different parenteral adjuvants e.g.
Freuad's
ineompletc adjuvants (FIA), dimethyldeoetadecylammonium chloride (pDA),
poly (DL-lacdde co-glycolidc) partidcs, liposomcs, ahunittitun hydroxide and
RIBI
~.1~~ (~'a1 and Hotwitz, 1992, Andersen, 1994x, Roberts err al. 1995,
Vordctmeier el
al., 1995, Lindblad et al., 1997 and Sinha e~ al.. 1997),
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Until recently, alum precipitates, e.g, aluminum hydroxide, are the only
adjuvants approved in the US and in Sweden for human use. In a recent study by
Lindblad er ~l. (1997), the use of alumintun hydroxide with.~ecreted antigens
from rl~.f
S rubercrrlosis in an experimental vaccine was questioned.1t induced a Th2
response,
which, indeed, increased the susceptibility of the animals to a subsequent
challenge with
M tuberculosis (Lindblad et al. 1997). This result shows that adjuvattts
available today
for human use have to be replaced by new safe adjuvants for future accllular
vaccines
against intracellular pathogens, such as M, tuberc~losls.
A new adjuvant was approved last fall consisting of syntheeic, spherical
virosomes with haemagglutinin and neuramitudasc front intluet~ virus and
inactivated
hepatitis A-virus. The adjuvant is claimed to give less adverse reactions than
the
conventional aluminum a~juvants. (Gltick R. 1995).
Biodegradable microparticles, particularly starch particles, such as cross-
1S linked starch particles, have been disclosed in the prior art. The
polyaeryl starch
microspheres.conjugated to the protective antigens used in the experimental
part of the
present description of the invention, have previously been disclosed as
parenteral
adjuvants for antigen delivery (Degling and StjBrtt)cvist, 1990. The particles
themselves
do not induce art itnmunc response, but arc weak macrophage activators,
(Artursson et
al,, 1985).
The lack of a general vaccination system for oral use is due to the problems
associated with the administration of isolated antigens of protein or
carbohydrate ntiture
and the uptake of them through the gut epithelium and transport to the cull:
of the
immune system. To start with. the antigens have to be protected against
proteolytic
2S degradation during the transport through the alimentary tract down to the
immune
competent regions in the gut.1t is essential that the relevant epitopes of the
antigehs, at
least, are preserved in order to be taken up, supposedly, by the M-cells in
the Pet'er's
patches and subsequently transported to the antigen-presenting cells in the
patches.
Therefore, the vaccine bas to be formulated in such a way that the antigen
epitapes are
protected until the antigens are taken up by the immune-competent cells.
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4 '.
Description of the iaveatipa
The present invention provides, unexpectedly, ~proteetion of antigens in the
alimentary tract of mammals, as shown in mice, by conjugation of protection-
generating
antigens derived from pathogenic microorganisms to biodegradable
microparticles, such
as starch carriers, which are porous. The antigens obviously are not available
inside the
pores for the enrymes, neither are they able to diffuse out from the pores due
to the
covalent binding. It is, moreover, the current understanding that the M-cells
and/or other
endocytosing cells of the gut wall can take up and further transport only
carriers of a
narrow size in the submicro-meter region, or close to that, and with a
specific surface
structure. Unexpectedly, the mucosal micropatticle conjugate vaccine of the
invention
seems to ba partially degraded xo such a size and structure, which is optimal
in order to
be taken up~bythe M-cells, and subsequently produce immune responses, which
are
IS protecting against a challenge of the relevant microorganism.
The invention, moreover, unexpectedly gives rise to such a cellular
response - as detected by the delayed hypersensitivity test - and a mucosal
sIgA response
as wet! as a systemic IgM/IgG response, that give protection against the chsl
lenge of a
microorganistti, even when the improved st~rbility of the antigens within the
conjugated
rnicroparticulate vaccine is considered.
More precisely, the present invention is directed to a mucosnl~ microparticle
cor;jugate vaccine agaisist a certain pathogenic microorganisru, which
comprises, as art
immunizing component, a 1-cell activating arttount of protection-generating
antigens
derived From said mictnorganism conjugated, possibly via a linker, to
biodegradable
microparticles.
The biodegradable microparticles are preferably starch particles, such as
Cross-linked.stsrch particles.
In a preferred embodiment of the invention the cross-!Inked starch particles
are polyacryt starch taicroparticles.
In another prcferrcd embodiment of the invention the mucosal vaccine is
an oral vaccine.
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WO ~9Li33.19 ~ PCT/SE99/OaZ77
S
The pathogenic microorganism is e.g as intracellular pathogenic
microorganism, which in a prcferrcd embodiment of the invention is selected
from thc.
group consisting of Mycobacterium tuberculosis and Salmonella ertterltidis.
The certain intr~cetlular pathogenic microorganism may be selected from a
wide variety of different microorganisms such as. Myeobacterhrm sp.,
Salmonella sp..
Shigella sp., Lelshrriarrla sp., virus such as Rota virus, Herpes sp.,
Vaeclrrla virus and
influenza virus, Meningococces, Bordetella pertussis, Streptococcus sp.~
enterotoilgenlc
Escherlchia eoli, Helieobacter pylori. Campylobauer jejurri, Toroplasmo
gondii,
Sclsislosoma sp., Lisrerla monocylogenes. Trypanasoma cruel and other sp,,
Clamydla
sp., NIYsp., etc.
The protection-generating antigens derived from a certe.in microorganism
may be intracellular antigens, cell-wall antigens or secreted ar<tigens.
Another aspect of the invention is directed to a method of inducing
protective immunity against a certain pathogenic microorganism in a mammal,
including
man, comprising mucosal administration to said mammal of a T-cell,
particularly of the
Thl-type, activating amount of protection-generating antigens derived from
said
microorganism conjugated, possibly via a linker, to biodegradable
microparticles, as an
immunizing component.
In a prefen~ed embodiment of the invention the mucosal administration is
2Q oral administration and the protection-generating antigens derived from
said
microorganism are secreted proteins from Mycobacterium tubererrlosis or
Salmonella
e~teritldis.
Yet another aspect of the invention is directed to the use of protection.
generating antigens derived from a certain pathogenic microorganism
conjugated,
possibly via a linker, to biodegradable microparticles for the production of a
mucoaal
rr~icroparticlc conjugate vaccine against said certain pathogen, _
In a preferred embodiment of this aspect of the invention the mucosal
vaccine is an oral vaccine, said antigens deiive iirom Mycobacterium
~uberculosls or
Saln~oaella enterltldis, and the biodegradable micmparticles are starch
particles, such as
cross-liNced starch particles, including polyacryl starch m'icropartieles.
la a most preferred embodiment of the invention the protection-generating
antigens are secreted proteins from Mycobacterium tubercalosls (TH) Harlingen
strain.
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WO ~9/~J3.19 ~. . PC'TISt;99J00~77
6
The T-cell activating amount of the cot:jugate of the invention depends on
several Factors such a physical, chemical and biological characteristics of
the antigen,
on the age and species of the individual mammal, and also the immunological
and
general physical status of the vaccinated individual. Recommended dosages will
be
given by the manufacturer based on clinical trials.
it should be understood that the conjugate of the invention may not only
activate T-cells and particularly Thl-cells (even though the amount of the
Conjugate in a
vaccine is calculated on the T-cell activation to ensure immunological memory)
, hut
may nlso give rise to a sccretod IgA and a systemic IgM/IgG response.
The possible linker between the two components of the conjugate of the
invention is used to facilitate the coupling reaction or to enhance the
antigen
presentation. The linker may be an amino-acid residue such as lysine, or an
amino-acid
sequence of a di-, tri-, or polypcptide.
The mucosal tnicropatticle conjugate vaccine according to the invention
may be presented in different pharmaceutical formulations depending on the
actual
intended route of administration, the specifte conjugate and the solubility
and stability of
the antigen or antigens.
In order to guarantee the eEfcacy of the vaccine preparation it may he
possible to do so by decreasing the degradation of the microparticle eanrier
by enzymes
and/or acidic p~I in the stomach and upper intestines, or by improving the
upt~l:e of the
v$ceine by the antigen-presenting cells, by modifying the formulation of the
vaccine in
different ways. Thus, e.g.
- the cross-linking degree of the mieroparticles can easily 6e controlled by
the
derivatization degree of the starch used in the ptnduction of the
micropa~ticles, so that
higher cross-linking will yield more resistant panicles, or _
- the size of the microparticles cart be controlled during the production by
the dispersion
of the emulsion prior to the polymerization of the acrylic groups of the
dewatized
starch, so that larger particles will give a more stable product, or
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7
- the vaccine microparticles may be dispensed in hard gelatin capsules covered
6y
gastro-resistant materials such as cellulose acetate phthalate, hydroxypropyl
methyicellulose phthalate or acrylate polymers (Eudragitn" ), so that the
vaccine is
released after the transport through the stomach and the upper intestines, or
- the vaccine microparticles may be individually courted by a gastro-resistant
shell e.g,
by coacecvation -phase separation or multioriFce-centrifusal processes with
e.g. shellac
or cellulose acetate phthalate, so that the particles are protected during the
transport
through the stomach and upper intestines and thereafter released from the
shells, or
- the vaccine microparticles may be suspended in an alkaline buffer such as
sodium
bicarbonate, neutralizing the acidic pH in the stomach and the upper
intestines, or
- the vaccine micmparticles may be compressed to $ tablet with bulking agents
such as
lactose, disintcgrants such as microcrystalline cellulose, lubricants such as
magnesium
stearate in such a way that the tablet is slowly disintegrated in the
intestines making the
vaccine micropanicles available for uptake by the antigen-presenting cells, or
- the vaccine micropaniclcs may be compressed to a tablet with bulking agents
such as
lactose, disintegrants such as microctystalline cellulose, lubricants such as
magnesium
stearate , which subsequently is covered by gastro-resistant materials such as
cellulose
acetate phthalate, hydroxypropyl methylcellulose phthalate or actylate
polymers
(Eudragitt"), or -
- the vaccine micropatticle may be covered by a gel-forming material such as
hydroxypropyl methylcellulose, which is protecting the vaccine through the
transport
Through the stomach and upper intestines.
The present invention will be illustrated more in detail with the aid of the
description of experiments and the trsults, However, the experiments should
not be
considered as limiting to the :cope of the claimed invention.
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8
Description of ezperimeots
~xneriment 1
Mice were immunized with polyaccyl starch microspheres with covalently
coupled extracellular proteins from ~~ycoboeyrium luberctrlo,ris (Harlingen
strain) to
investigate the potential of the coqjugate as an oral vaccine. The humoral and
cellular
immune responses were investigated and the protection after challenge was
determined.
Materials
?he maltodextrin~was a gift from Dr, Lars Svensson (Stsdex, Malmd,
Sweden), acrylic acid glycidyl ester was from Fluka (8uchs, Switzerland) and
front
Polysciences lnc. (pp, USA), N,N,N',N'-tetramethylechylenediamine (T~M6D) and
4-
nitrophenylphosphate disadiutn salt were from Merck (Darmstadt, Germany),
Biorad
protein assay kit and horseradish peroxidase conjugated goat anti-mouse IgG
were from
Biorad (CA, USA), Freund's incomplete adjuvant was from Difco Laboratories
(MI,
USA), 8CG vaccine was from Statens scrum instituc (Copenhagen, Denmark),
alkaline
phosphasase cotEjugated human anti-mouse IgG/IgM was from Hiosource (CA, USA),
carbonyldiimidazole, bovine serum albumin (grade V), phenylmechyl sulphonyl
fluoride, ttypsin inhibitor, alkaline phosphatase conjugated goat anti-mouse
IgA and
4-chloro-1-naphtol were from Sigma (St.iLouis, MO, USA).
Purificntioo of earacehular proteins frow M. tuberculosis
MycobeuerJum lubercLlosls (Harlingen strain) was grown for one, two and
three weeks (corresponding to pmteitt solution wl, w2 and w3) in Proskauer-
Beck
medium at Smittskyddsiastitutet in Stockholm. The three (wl,w2 and w3) protein
solutions were treated separately dating the purification process. The
bacteria were
removed by centrifugation at 5000 rpm for 30 minutes and the cultwt
supernatant was
filtered through two consecutive 0.2 Elm filters and Concentrated about 50-
fold through a
YMIO filter (Araicon, MA, USA). Ammonium sulphate (final concentration 4.24 M)
was added to the concentrate during stirring. ARer centrifugation at 8000 rpm
for 30
minutes the precipitate was dissolved in phosphate buffered saline (pH 7.0).
The proteins (solution wl, wz and w3) were dialyzed extensively in a
Spectra/Pot~
dialysis membrane (Spectrum, CA, USA) with a 3500 molecular weight cut off,
against
a buffer with 0.25 M boric acid and 0.15 M NaCI, pH 8.5. The protein
concentration was
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WO 99/0~7~9 PCT/SE99100377
9
determined with Coomassie Blue according to Bradford (Bradford, 1976). Bovine
serum
albumin was used as a standard_
The proteins were stored at -80° C until further use.
Preparation of Polyaeryl Stsreh Mieroparticles - The micropnriicles were
prepared
by polymerization of acryloylated starch in an emulsion, as previously
described
(Artursson et al., 1984 and l.aakso et al., 1986). Brielty, 500 mg of
acryloylated starch
was dissolved in 5 ml of a 0.2,M sodium phosphate buffer, pH 7.5, l mM EDTA.
. Ammonium pcroxidisulphate (200 pl) was added to give s final concentration
of 0.8 M
in the aqueous phase, which then was homogetuzed in 300 ml of
toluene:ehlorofotm
(4:l). TEMED was used to initiate the polymerization. The microparticle
composition is
characterized by the D-T-C nomenclature (Hjerten 1963 and F.dman et al.,
1980)) and
the amount of TEMED added. D represents acryt4ylated starch (g/100 mL); T is
the total
concentration of acrylic groups expressed as aerylamide equivalents (g/100
ml), and C is
the relative amount of any additional emss-linking agent (e.g., bis-
acrylamidc; % w/w).
The microparticles used in this study had a D-T-C value of 10-0.5-0 and 100 ~1
of
TEMED way added
Coupliag of E:traeellular Proteins (TB) irom J4lycobocteriurrr ~nbercWlosis to
Micraparticles - The extracellular proteins (TB) w1 and w2+w3 were coupled to
micropatticles using the CDI-method of 8ethell of al. (Hcthell et al., 1981).
Mieroparticles (5 mglml) were activated with CDI (50 mg/ml) in dry DMF for 1 h
at
room temperature. After several centrifugal washings with DMF to remove
utueacted
CDI, particles (50 mg) were suspended In 10 ml of the coupling buffer, (0.250
M boric
acid with 0.15 M NaCI, pH B.S) containing mg amount of wl or w2+w3. The
mixture
was rotated end over ettd at 4-b° C for 48 h. The TB.microparticles
were then washed to
PBS, filtered through a 10 ~ filter and stored at 4-~° C. The amount of
wt and w2+w3
coupled was determined by amino acid analysis after acidic hydrolysis of the
micropatticles.
Particle Size Determlastion The'TH-particles were dried and photoeraphcd in a
scanning electron microscope (S.E.M.) (Jeol T330) at 5000 magnification.
The particle size determined from scanning electron microscope photographs was
52
pm. In previous shrdies 98% of the panicles had a diameter Q.5 ~cm determined
with
Coulter Counter (Degling and StjBrttkvisc, 1995).
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WO 99/d33~9 ~ PGT/SE99/poZ77
Immunizations - Mice of the HALB/c ABom strain (Hotttholtg8td, Ry, Denmark),
female, 8-10 weeks old, were used. Mice (5-6/group) were immunized orally by
gastric
intubation, four times on three consecutive days, with T8-microparticles
containing wl
and w2+w3 proteins. Also groups of mice were immunizedam with TB-
microparticles
S . containing wl and w2+w3 proteins or with corresponding amount soluble wl
and
w2+w3 in physiological saline, 0. l ml. As one positive control, groups of
mice were
injected ip with wl+w2+w3 in Frcund's incomplete adjuvsnt (F1A). As the other
positive contro) mice were Immunized se wig 0.1 m) diluted (with physiological
saline)
8CG vaccine. When low doses of soluble wt+w2+w3 were administered, a carrier
10 protein BSA 0.1% was co-a~dminiscered to minimize adsorption of protein to
the
glassware.
For detailed information see Table 1-l, Immunization schedule.
Collection and Preparatio4 of Hload Samples - Blood samples were collected on
day
0, 7, l5, 34, 42, 49, 57 and 65 with hcparinized capillary tubes from orbital
plexus_ The
tubes were centrifuged and the sera collected and frozen at -20° C
until further use.
Collection and E:traction of Faeces - Faeces (4-6) from each mouse were
collected at
five consecutive days after immunization into Eherman tubes and freeze dried.
The dry
weight was determined and a solution containing 50.mM EDTA, S % dry milk, 2 mM
phenylmethylsulfonyl fluoride artd 0.1 mg soybean trypsin inhibitor/ ml
phosphate-
buffered saline (pl3S-A) was added (20 pl/mg farces). Solid matter was mashed
and
separated by centrifugation at 13000 rpm for 15 minutes atld the supernatants
were
frozen at -Z0 ° C until ftuthec use.
Detorminstioa of anti-TB IgG and IgM sad aI$A with ELISA - A protein solution,
an equal mixttue of wl,v/t and w3 proteins, was diluted (181tg/ml) with 0.05 M
sodium
bicarbonate buffer with 0.05 °Y° NaN~ (pH 9.6) and Nurse
Immunoplate Maxiaorb F96
plates were coated (100 pUwell) and incubated in a moist chamber at 4 °
C over night.
The plates were shakep dry and 1 % OVA in 1 mM PBS.A, pH 7.4, was added (200
~Uwell) and then incubated for 2 h in moist chamber at room temperature to
avoid
unspecific binding to the plates. AIler 5 washings with 0.05 % Tween 20 in
physiological saline with a Titertek microplate washer 120 (Flow Laboratories)
the
scra/faeees samples were added to the plates in series of twofold ditutions
and irtcuba_ted
for 2 h and the plates were washed as before. An alkaline phosphatase-
conjugated
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1l
secondary antibody (human anti-mouse Ig G and Ig M or goat anti-mouse Ig A)
diluted
l :1000/l:?50 in P8S-A with 0,2 % Tween 20 (PBS-T) was added (100 ~tUwell) and
the
plates were incubated for 2_S h. After washings, the substrate, 4-
ctitrophenylphosphate
(1 mg/ml, in 10 % diechanolamine buffer with 0.5 mM ~MgCh and 0.02 °/a
NaN" pH 9.8)
S was added and the absorbance was measured after 10 minutes (I2 min for Ig A)
at 405
nm with a Multiscan MCC/340 microtiter plate spectrophotometer (L,absystem).
Pooled negative serum was added to each plate (Ig Gllg M measurements) as a
negative
control. An average of the absorbance values was calculated From the first
well ( 1:20
dilution); mean=0.130, sd=0.045 ts~l9. A sample was considered to be positive
if the
value exceeded mean+3 x ad, thus above 0.265. A positive sample (senun fmm
mice
immunized with !00 Itg wlw2w3 in Frcund's incomplete adjuvant) was also added
to
each plate, as a stsutdard, and was treated in the same was as the other
samples. Titers
were given as -logs (dilution x 10).
Delayed Type Hypersensltlvlty (DTH) test - In order to evaluate whether a cell
1S mediated immune response against TB had developed, a DTH test was performed
on day
52 i.e. one week after the third immunization. The mice were given an
irttradecmal
injection (10 pl) in the left ear with the tuberculosis protein mixture wl-w3
(1 mgltnl) in
physiological saline. As a control 10 Irl physiological saline was injected in
the tight ear.
The thickness of the ears was measured with a dial thickness gauge (Mitutoyo
Scandinavia AH, Upplands VBsby,.Sweden) before antigen challenge and 24, 48
and 7?
h after. The DTH response was calculated according to (A,-HrAq)~ 100, whore
A,=
increase froth Limo 0 of the oat thickness in the ear challenged with antigen
at time t, H,--
inerease from fillet 0 of'the ear thickness in the ear challenged with
physiological saline
at tune t and Ae=ear thickness in the car challenged with antigen, before
challenge
(Degling and StjBntkvist" 1995).
Ezperimeotal iafectioo of mice - Irrtmunized truce and control trice were
challenged at
day 106 (18 days after the last itartrunization) with SxlOs CFU M.
rrrberculoris
(Flatlingen strain) iv by the tail vein.
The weight of the mitt were detetrnined before and 15 days after infection.
Determioatioo of protective immunity - At day 121 (15 days aver infcctiotl)
infected
mice were killed and the spleen end lung were removed nseptieally. CFU ofM.
rubercr~losls were dctctinincd by homogenizing each organ in PBS end serial 10
fold
CA 02322175 2000-08-25
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1?
diluting the tissue homogenates before culturing the dilutions on duplicate
plates of
7H t 0 agar. Colony farming units were counted after 3 weeks of incubation at
3 7°C. ,
SDS-PAGE and Immuaoblotting - The proteins in fraction wl, w2, w3 and an equal
mixture ofwlw2w3 were separated on a PhastSystem~ (Pharmacia, Uppsala, Sweden)
S gel electrophoresis apparatus using s l0 to l5 % SDS PhastGel~ (Phamtacia
Biotech.
Uppsala, Sweden). Gels were both silver stained arid stained with Coomassie
blue.
The separated proteins were transferred onto a vitro-cellulose membrane
(Pharmacia
Biotech, Uppsala, Sweden) end incubated far 2 h in RT in a solution containing
5 % dry
milk in PBS-T on a shaker. After washings with PBS-T, b membranes were
incubated
far 20 h in RT on a shaker, in O.S % OVA pgS-T with sera from group 1-6
(diluted
1:20). After washings with PBS-T the membranes wore incubated for 3 h in
37° C on a
shaker, with the secondary antibody (horseradish peroxidase conjugated goat
anti-mouse
lgG, diluted 1;20 000 with 0.5 % OVA in PBS-T). The substrate, 4-chloro-1-
naphtol (10
mg dissolved in 3.3 ml MeOH and added to 16.7 ml 20 mM Tris, 500 mM NaCI
buffer
with 30 ~I. Hl0= (37 %)), was added after washings with PBS-T. The reaction
was
stopped after 20 min with distilled water.
Statistics - Unpaired t-test was performed comparing mans of two independent
sapmles. A difference was considered significant if p<0.05.
RESULTS
Coupling of tuberculosis proteins to polyacryl starch microparticles - From
the first
coupling of wl protein fraction, 5.63 ~tg protein per mg micropanicle was
coupled
(corresponding to a protein coupling yield of 23 9~0) and from the subsequent
coupling
with the supernatairt 1.38 Ng wl protein per mg miccoperticle (protein yield
6.4 %) was
coupled.
An additional coupling of protein fraction w1 was performed and 3.93 itg
protein per mg micropartiele was coupled (corresponding to a pc~otein yield of
15.9 %).
From the coupling with fraction w2+w3, 4.16 pg protein per mg cnicroparticle
was
coupled (corresponding to a protein coupling yield of SS %) end from the
subsequent
coupling with the ettpetnata,rtt O.B9 pg protein w2+w3 per nticroparticle was
coupled
(protein yield 10.1 %).
Aa:lysis oltbe estraeellularM. tWberc~losis proteins by SDS-PAGE attd
immunoblattiag - The three protein fractions i.e. wl, w2 and w3. were analysed
by
CA 02322175 2000-08-25
Wp 99Lt3339 PLTISE99/00377
13
SDS-PAGE in order to determine the size of the protein in the mixtwe used in
the
immunization experiment. Several bands in the region 14.4-30 kDa and 43-94 kDa
were
observed (totally ! 2 bands) by SDS-PAGE analysis. There was no difference
between
the wl, w2 and w3 protein fractions. (Results not presented}
Delayed Type Hyperseasitivhy (DTH) - As seen in Table 1-2, there was an
increase in
the ear thickness in the group immunized orally with TB-micropartieles after
24. 48 and
72 hours, however the increase was not significantly higher than in the other
groups. The
DTH-response induced in the group immunized im with TB-microparticles was,
after 24
h, significantly higher than the control group. After 48 and 72 hours the DTH-
response
increased tv be significantly stronger than both the D?H-response in the
control group
and in the group immunized im with Gee T8-antigen in physiological saline.
After 72
hours the pTH-response in this group was also significantly higher than the
response in
the HCG group and comparative with the response in the group immunized with TB-
antigen in Freund's incomplete adjuvant.
Two mice in the control group showed a 40-SO % increase in ear thickness and
three
mice did not respond at all. This explains the high mean and standard ertnr
(SD) within
this group after 4$-72 h.
The Humoral Immune Response - The group immunized with TB-microparticles im
showed a response comparative with the group immunized with TB-proteins in
Freund's
incomplete adjuvant and the group immunized im with free T8-proteins in
physiological
saline. The respotue was also signiFtcantly higher th~ett in the control and
BCG groups.
'Ihe group immunized orally with T8-aiicroparticles did not give rise to a
hu~noral (IgG
and IgIvi~ response. ('Table 1-3)
Mucosomal (sIg A) lmmuoe respoaae - Preliminary results indicate a slgA immune
raspot~e 2 days after the third inununizatiaa in the groups given
aucroparticles orally
and im and in the group immunized with antigen in Freund's incomplete
adfuvant. The
response in the BCG group was lower. However further studies h$vc to be
performed to
confirm these results.
Protection E:perimeats -The protection level was determined by two parameters,
weight loss during infection and CFU of M. ~uberct~losis in the lung after
infection.
CA 02322175 2000-08-25
WO 99/~~3i9 PCT/SE99lOOZ77
14 '.
As seen in Table t-4, both the mice in the control group and the vaccinated
groups lose
weight during infection. The CFU of M. ~ubereulosis ih the lung after
infection is
presented in Table 1-5.
A protective immunity was rttanifested in animals immunized orally with TB-
microparticles. The reduction oFviable Jtl. tuberculosis in the lung was at
(cast 10.100
fold as compared with the unimmunized control and comparable to the effect
seen after
immunization with BCG vaccine. (Table 1-S) The protection after intramuscular
itrtmunizstion with TB-microparticles was somewhat lower than the response
after orally
administered TH-microparticles although the reduction of viable A~I.
tuberculosis in the
lung was at least IQ fold. As seen in Table l-5, no protective i,tntrttutity
was seen in
animals immunized intramuscularly with free TB-antigen in physiological saline
or
intraperitoneal ly with TH-antigen together with FIA.
CA 02322175 2000-08-25
WO 9"9Id3JJ9 ~ 5
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CA 02322175 2000-08-25
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CA 02322175 2000-08-25
WO X991433.19 . PGT/SE99/OOZ77
18
Table 1-4
Weight loss during infection with Mycobaeleriuns ~Wbercrtlosis -
(St~aio: I-Isrliagen).
Challenge dose: SzIO~ bacteria
Weight loss in percent 15 days after
Immutiizatioo with: (challenged infection with M, tubercrrlosls.
(xfSD)
T13-mieroparticles 13.015.1
oral
TB-microparticles 25.6*'1.1
im
Free TH-antigen im lS.3tl.~
FIA with TH-antigen 18314.6
ip
BCG vaccine sc 9.613.9
ControWnimmunized 10.5f1.I
CA 02322175 2000-08-25
WO 911~3~49 19 . PC1'/SE99/OaZ77
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CA 02322175 2000-08-25
'VIyO'~9/t.1349 ~ PGT/SE99/OOZ77
E=neriment 2
Extracellular proteins were isolated from SQlmonella enreriridis and
covalently
coupled to polyacryl starch mieroparticles. The immunogenicicy of the
conjugate after oral
5 administration to mice and the induced protection against a challenge with
Iivc bacteria were
followed.
Materials and Metbods
Materials
In addition to those items speciFed in Experiment l, Bacto-uyptone and Bacto-
10 yeast.cxtract were from Difco (MI, USA), alkaline phosphatase-conjugated
goat anti.mouse
IgA and mouse IgA-kappa from Sigma (MO, USA) and RPMI 1640, HEPES and
glutamine
were from i.ife Technologies LTD (Paisley, Scotland).
Purification of extrxcellulas protein from Sdhnonella enteritidir
Salmonella enrerltldls wild-type was inoculated in 2 ml Lucia-Hcrtani (L8)
l5 broth (1% Hacto-tryptone/0.5% Bacto-yeast-extracdl % sodium chloride) and
grown with
shaking, 200 rpm, ak 37°C overnight. The next day the culture was
diluted in 500 ml LB and
grown under the same conditions anti! OD = 1. After centtitugacian (I,SOOxg
for 60 min at 4
°C) the bacterial pellet was resuspended in RPMI 1640 with 20 mM HEPES
and 4 mM
glutamine. The mixture was shaken (200 rpm) at 37°C fat 2 h and
thereafter the bacteria were
!0 removed by centrifugation at 1,SOOxg for 1 h at 4 °C. T,he cultwe
supernatant was filtered
through a 0.22 pm Millipore express filter and concentrated and transferred
into coupling
buffer (0,250 M boric acid with 0.15 M NaCI, pH 8.5) by Fltering through a YM
10 000 cut off Stirred Cell Ultrafilter, Amicon (MA, USA). The protein
concentration was
dccermined with Coomassie Blue according to Bradford (Hradford,1976 ) and with
a ready
:5 prepared reagent froth Hio-cad, using bovine serum albumin as a standard.
Preparation olpolyacryl:torch taitrppaitleles, eonjugatioo ofSalmonella
aotigeo and
charaeterizatlon oit6e atttlgou-particle conJttgate _ '
The Salmonella antigen-containing mictoparticles of polyacryl starch were
prepared and characterized as described in Experiment 1.
~0 Immuafaatioa procedures
The mice, from own breeding of the 8alblc strain, were divided into S groups
(4
mice/group).
(In the challenge experiment, 6-12 mice were included in each group.) In the
first group each
mouse was immunized ip with 10.5 ~tg protein in 0,1 mI Freund's adjttvanc. The
second
CA 02322175 2000-08-25
Vi/O 9~/~JJa9 PCT/SE99/Op=77
7l
group received an im injection with 10.5 ~tg pmtein conjugated tol mg
microparticles. Mice
in the third group were immunized orally by gastric incubation, with 31.5 tsg
protein
conjugated to 3 mg microparticlcs divided in doses given on 3 consecutive
days. Group four
was an untreated control group and group five was a hyperimmunization group,
which
S received 50 pg protein itt 0.1 ml Freund's adjuvant (30 ~tg proteins as
booster dose). poosters
were given after 21 days.
Collection and handling of blood sand faeces samples
The sampling procedures used for the collection and handling of blood and
faeces arc presented in Experiment 1.
s0 Assessment of immune t'esponses
The analyses of the svstemic~ response as well e.x the mueosal leA response
were performed by conventiot'tal EIrISA techniques, which are described in
Eicperiment 1.
The cellular response was anrtlyzed by the delayed-type hypersensitivity test
(DTH-test) as
presented in Experiment l,
!5 Challenge of immtetaized mice
Challenge with Salmonella en~eriridis (3 x t 0~ CFUlmouse) was performed 6
weeks after booster. Mice were killed ? days after challenge. Liver and spleen
homogenates
were incubated an LB-agar plates overnight and the number of CFU was counted,
Results
:0 Cbaracterixatioh of the s4tigcn-tnicroparticle conjugate
The conjugated starch microparticles contained 10 mg Salnsonella antigen per
mg. All particle preparations ~ used contained more than 90 ~o particles with
a diameter~less
than 3.3 mm.
Humoral immanc lespoctses
The IgG/IgM response in serum in the group immunized orally with Salmonella
proteins coupled to polyaeryl starch microparticles was comparable with the
rcsponsp induced
in the group immunized with ptnteins in Frcund's adjuvant, but lower than the
response
induced when particles were administered im (Table 2-1). Similarly, the
specific IgA
response in Fs;eees was comparable in the group immunized orally with
Selmvne!!a proteins
0 coupled to polyactyl starch microparticles and the group immunized with
proteins in Freund's
sadjuvstrtt, shearing a peak at day 27 and 28, whereas the specific IgA
response induced in the
group immunized im was lower (Table 2-2).
CA 02322175 2000-08-25
WO 99143»9 PCTISE99/002'1~
?2
Cellular immune respopse
A relatively high, continuous increase in the eor Ihiclatess was detected in
the
group immunized orally with micropanicles. The response was lower than that
obtained with
the positive control (in Freund's adjuvant) and comparable to the response
induced in the
gmup immunized im (Table 2-3).
C6allcnge of immunized mice
The results from the challenge of the immunized tnice with live Salmonella
bacteria arc shown in Tables 2-4. and ?-5. A reduction in CFU was seen in the
groups
immunized orally with antigen-coupled microparticles, microparticles with
soluble antigen or
0 with soluble antigen alone, compared to the conkrol group. The best
protection was xen in the
groups immunized with antigens together with or conjugated to starch
rnicropanieles. This
was also seen when studying the average weight loss, which showed a 10.3%
decrease for the
control group, 4.0% decrease for the group immunized orally with soluble
antigen, 3.6%
decrease for the group immunized orally with microparticles with soluble
antigen and 1.8%
S decrease for the group immunized orally with antigen-coupled miaoparticles
(not presented in
any table),
1~"he results of this ~dv show that secreted antigens derived Pram Salmonella
conjugated to
polyactyl microparticlcs may be administered as an oral vaccine capable of
inducing both
7 local secretory and systemic immune responses. Moreover, the a strong
speciFtc IgA response
was observed in this study, although with significant interindividuat
variations.. The good
protection against a challenge was also indirectly shown by following the
weight loss after
the challenge. The group treated orally with the antigen-conjugated
microparticles lost
signiFcantly less in weight (l.8 ~/°) compared to the control group,
not treated at alt, loosing
i 10.3 % in weight after the challenge.
CA 02322175 2000-08-25
\~~9~J~33~9 ' PGT/SE99100Z77
23
Table 2,1
Spccitic humoral response in serum after immunization with S enreri~idis
antigens is
different formulations.
Titers are given as mean +/- S.E.M, (n=4).
'
Way of administration Titer
Antigen formulatioti Day 0 Day 33
0 Oral immunization '
Antigens conjugated in 0,0 ~ 5.5 +/- 0.3 .
microparcicles
Im immunization
5 Antigens conjugated in 0.0 9.0 +/- 0.4
trticcopartieles
Ip immunization
Soluble antigens in 0.0 7.0 +/- 0.0
7 Freund's adjuvant
Table 2-2.
i SpeclCe mucosal response (IgA) is faeces sifter immunization with S
tWeritidis antigens
is different formulations.
Values are given as means +!- S.E:M- (n=a).
Way of administration igA (oglmg faeces)
Antigen formulatipn , Day Z6 Day Z7 . Day ZS
Oral immunization
Antigens conjugated in I .1 +I: 0.5 2.1 +I- 0.55 2.25 +/-0.8 .
micmparticlcs -
Im immunization
Antigens conjugated in 0.4 +/- 0.2 0.9 +/- 0.35 0.6 t/- 0.2
micmparticles
Ip immunization
Soluble anageas in 1.3 +/- 0.55 !.4 +l- 0,5 2.4 +/- 0.75
Freund's adjuvant
CA 02322175 2000-08-25
WO 9t9143~49 PCT/SE99100Z77
24
Table 2-3
Cellular immune response (as test on delayed type hypersensitivihy) ,
after immuniuation with S. en~eri~idis antigens iv different formulations.
Results are given as mean °Yo increase in thickness of challenged esus,
+!- S.E.M. (n=2-4) -
Way of administration % increase, hours after cballeage
Antigen tormulatioa , 24 48 ?Z
i0
Oral immuniuttion
Antigens conjugated in 41 +I- 10 67 +I- 3 99 +!. 15
microparticles
5
lrn immunization
Antigens conjugated in 107 +/.13 138 +/-24 179 +!-12
micropanicles
0
Ip immunization
Soluble antigens in 190 +!-2z 209 +I-2S 214 +!-2l
Freund's adjuvant .
5 Non-immunized 10 +/- 1 23 +/- 4 38 +!- 2
mice (controls)
0
S
CA 02322175 2000-08-25
W,O ~99I~~349 . Pt:'('/SE99I00377
Table 2-4.
Colony forming units (CFIJ) en liver of mice immunized with S ~nteriiltidis
sntigans
sftcc c6allcnge-with 3 : 1Q' CFU.
The mice were challenged 6 weeks after booster and killed 7 days after
challenge. The livers
were homogenized and total CFU counted after incubation over night in LB.agar.
The results
nre presented as geometric mean and range; n is given in parenthesis.
~Ysy of adatioistratian CFU is livcc
Antigen fprmulation Mean Range
Oral administration
Antigens conjugated in 1.8 x 10~ (6) 2 - 3.1 ~ x 106
micropatticles
Oral administration
Soluble antigens with 0.69 x 10' (6) I - l.5 x l0'
~_0 micropa~ticles ,
Qra1 adminisrration 4.S x 10' (6) [ - 5.0 x 10'
of soluble antigens
5
Non-immtuzized mice 2.6 x 10~ (12) 1.1 x 10' - l.9 x 10'
(Controls) . .
CA 02322175 2000-08-25
Wt~99/~13.19 PCTI5~99/00~17
26
Table Z-5
Colony forming units (CFU) in spleen of mice immuaizcd with antigens from S.
exterititidir after c6alleage with 3 x IO' CFU.
The mice were challenged 6 weela after booster and killed 7 days after
challenge. The livers
were homogenized and total CFU counted after incubation over night in Lt~-
agar. The result
arc presented as geometric mean and range; n is given is parenthesis.
to .
V'Vay of administration CFU iti spleen
Antigen fora~ulstioa hlean Range
Oral administration
Antigens conjugated in 3.Z2 x l0' (6) 3 ~ 2.4$ x 10'
micropartides
Orat administration
Soluble antigens with 1.43 x 10' (6) ~ l ~ 5.70 x 10'
mieroparticles
Oral administt$tion 6.04 x 10' (6) i - 3.30 x lOs
of soluble antigens
Non-immunized mice ~ 2.32 x ! 0' (12) 2.3 x 10' - 1.5 x 10'
CA 02322175 2000-08-25
~W,0 99/a7~39 PCTISE99~00~'77
~7
Referer<ces
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infection
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Z8
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l5
Robots, A. D., Sonncnberg, M. G., Ordway, D. J., Fumey, S. K., Hrennan, P. J.,
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:0
Sinha, R. K_, Verma, I. aad Khuller, G. K. Immunobiologica) properties of a 30
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