CA2322175A1 - Mucosal microparticle conjugate vaccine - Google Patents

Abstract

Mucosal, particularly oral, microparticle conjugate vaccines against certain pathogenic microorganisms, especially intracellular pathogenic microorganisms, are disclosed. An immunizing component of such a vaccine comprises protectiongenerating antigens derived from a certain pathogenic microorganism, such as Mycobacterium tuberculosis or Salmonella enteritidis, conjugated, possibly via a linker, to biodegradable microparticles, particularly starch microparticles, such as cross-linked starch microparticles, e.g. polyacryl starch microparticles. Further, a method of inducing protective immunity against a certain pathogenic microorganism in a mammal, and the use of protectiongenerating antigens derived from a certain pathogenic microorganism conjugated, possibly via a linker, to biodegradable microparticles for the production of a mucosal microparticle conjugate vaccine, are described.

Description

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 W0 49Li33.i9 : PCT/SE99IODIT7 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), ' W0~99/43349 : PCfISt:99140Z77 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.

WO 99/J33J9 :, PCT/SE99I110Z77 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.

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.

WO ~9/~J3.19 ~. . PC'TISt;99J00~77 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$ceine by the antigen-presenting cells, by modifying the formulation of the vaccine in different ways. Thus, e.g.
- the cross-linking degree of the mieroparticles can easily 6e controlled by the derivatization degree of the starch used in the ptnduction of the micropa~ticles, so that higher cross-linking will yield more resistant panicles, or _ - the size of the microparticles cart be controlled during the production by the dispersion of the emulsion prior to the polymerization of the acrylic groups of the dewatized starch, so that larger particles will give a more stable product, or WO 9$L133.i9 , PGT/SE99/OO-77 - 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.

WO 991J3~i9 ~ PCT/St:99100:77 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 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 WO 99/0~7~9 PCT/SE99100377 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 pm. In previous shrdies 98% of the panicles had a diameter Q.5 ~cm determined with Coulter Counter (Degling and StjBrttkvisc, 1995).

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 WO 991J37J9 : Pt.T'/SE99latiZ'r7 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 WO 99/.t»~9 PGT/SE99/OOZ77 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 Wp 99Lt3339 PLTISE99/00377 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.

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.

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a ~ > w V o w ~ w c '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 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).

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.

\~~9~J~33~9 ' PGT/SE99100Z77 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 WO 9t9143~49 PCT/SE99100Z77 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 lrn immunization Antigens conjugated in 107 +/.13 138 +/-24 179 +!-12 micropanicles 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) S

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 Non-immtuzized mice 2.6 x 10~ (12) 1.1 x 10' - l.9 x 10' (Controls) . .

Wt~99/~13.19 PCTI5~99/00~17 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' ~W,0 99/a7~39 PCTISE99~00~'77 ~7 Referer<ces Andersen, P., Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mi~cttue of secreted mycobacterial proteins. Inject. lmmr~n..
63 (1994x) 2~36 2544.
Axtursson, P., Edman, P., Laakso. T. and Sjbholrn L, Characteri~tion of polyacryl starch micropartictes as carrier for proteins and drugs. J. Pharrr~_ Sci., 73 ( 1984) 1507-1513.
Artursson, P., Edman. P. and Sjbholm, L. l3iodcgradablc microspheres iI:
immune respons to J a hetecologous and an autologous protein entrapped in polyactyl starch mieroparticles. J.
Pharmacol. f~rp. Ther., ?34 (1985) 255-259.
8ethell, G.S., Ayers, J.S., Hearn, M. T. W. and Hancock, W.S, Investigation of the activation of various insoluble polysaccarides with l,l'-carbonyldiinudazole and of the properities of the activated matrices, J. Chron~atagraphy. 219 (1981) 361-372, Bradford, M.M., A rapid and sensitive method for the quantitatian of microgram quantities of protein utilizing the principle of protein-dye bindning. Anal. Bioclre»t., 72 (1976) 24B-254.
?0 Degling, L. and Stjarnkvist, P., l3iodegradable microspheres XVIII: the adjuvant effect of polyacryl starch microparticles with conjugated human serum albumin, Vaccine., 13 (1995) 629~63b.
Edman, P., Ekman, H. and Sj~halm, I. Immobilization of proteins in micrQspheres of :5 biodegradable polyaeryldextran. J. Pharm. Sci: 69 (1980) 83 8-842_ -Gltlck, R., Liposomal presentation of antigen for human use. In Vaccine Design: The subunit and adjuvant approach. Edited by Michael F. Powell utd Mark 1, Newman. Plenum Press 0 New york 1995. pp 325-345.

~V1~Q ~l9/~133~9 PLTISE99100~77 Hjerten, S. Molecular sieve chromatography on polyacrylamide gels prepared according to a simplified method. ,~rcir. Biochem. Biophys, suppl.l ( 196Z) 147-151.
Laakso, T., Artursson, P. and Sjt~hohn, L, Biodegradable Microspheres IV:
Factors affecting the distribution and degradation of polyacryl starch micropariiclcs.1. Phorm.
Sci., 75 ( 1986) 962-967.
Lind6lad, tr. B., Elhay, M. J., Silva, R., Appelberg, R. and Andersen, P.
Adjuvant modulation of immune responses to tuberculosis subunit vaccines. Infection Immunity, 65,(1997) 623-629.
Pal. P.G, and Hotzvitz, M.A., Immunization wish extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis, Inject Immun., 60 (1992) X781-479?.
l5 Robots, A. D., Sonncnberg, M. G., Ordway, D. J., Fumey, S. K., Hrennan, P. J., Belisle, J, T.
and Otme 1. M. Characteristics of protective immunity engendered by vaccination of mite with purified culture filtrate protein antigens of rVlycobacrerium tuberculosis. Immunology 85 (1995) 503-508.
:0 Sinha, R. K_, Verma, I. aad Khuller, G. K. Immunobiologica) properties of a 30 lcDa secretory protein of Mycobec~erlum tuberculosis H37 Ra. Yaecine 15 (I997) 6$9-699.
Vordermeier. H. M.. Cootnbes, A. G. A., lenkins, P., McGee,1. P., O'Hagan, D. T., Davis, S. S. and 5ingh, M. Synthetic delivery system for tuberculosis vaccines:
imsztunological evaluation of the ~I. ~ubertulosis 38 lcDa protein entrapped in biodegradable PLG microspheres. Yeccine 13 (1995) 1576-1582.

Claims (10)

1. Mucosal microparticle conjugate vaccine against a certain pathogenic microorganism, which comprises, as an immunizing component, a T-cell activating amount of protection-generating antigens derived from said microorganism conjugated, possibly via a linker, to biodegradable micropacticles.

2. Vaccine according to claim 1, wherein the biodegradable microparticles are starch particles, including cross-linked starch particles.

3. Vaccine according to claim 2, wherein the cross-linked starch particles are polyacryl starch microparticles.

4. Vaccine according to any one of claims 1 - 3, wherein the mucosal vaccine is an oral vaccine.

5. Vaccine according to any one of claims 1 - 4, wherein the pathogenic microorganism is an intracellular pathogenic microorganism.

6. Vaccine according to claim 5, wherein said intracellular pathogenic microorganism is selected from the group consisting of klycobacterium tuberculosis and Salmonella enteritidis.

7. Method of inducing protective immunity against a certain pathogenic microorganism in a mammal, including than, comprising mucosal administration to said mammal of a T-cell activating amount of protection-generating antigens derived from said microorganism conjugated, possibly via a linker, to biodegradable microparticles, as an immunizing component.

8. Method according to claim 7 , wherein the mucosal administration is oral administration and the protection-generating antigens derived from said microorganism are secreted proteins from Mycobacterium tuberculosis or Salmonella enteritidis

9. Use of protection generating antigens derived from a certain pathogenic microorganism conjugated, possibly via a linker, to biodegradable mieroparticles fog the production of a mueosal mitropaeticle conjugate vaccine against said certain pathogen.

10. Use according to claim 7, wherein the mucosal vaccine is sn oral vaccine, said antigens derive from Mycobacterium tuberculosis or Salmonella enteritidis, and the biodegradable microparticles are starch particles, including cross-linked starch particles and polyacryl starch microparticles.