Patent Document:

the mice were 6 to 8 week old balb / c and dba / 2 females . the antigens were klh and ovalbumin ( ova ) marketed by sigma chemical ( st louis , usa ). the trinitrophenylated hemocyanin ( tnp4 - klh ) was prepared as previously described ( shutze et al ., j . immunol . ( 1989 ) 142 : 2635 ). poly ( acrolein ) microparticles with diameter between 0 . 25 and 1 . 5 μm , marketed by polysciences inc . ( washington pa .) were coupled to ovalbumin or klh as previously described ( rembaum et al ., immunol . ( 1982 ) 52 : 341 ; ziegler et al ., eur . j . immunol . ( 1987 ) 17 : 1287 ). 1 ml of these microparticles was washed twice in pbs and resuspended in 1 ml of klh or ovalbumin ( 5 mg / ml in pbs ). after 3 hours &# 39 ; incubation at ambient temperature , the microparticles were washed twice in pbs and resuspended in 2 ml of pbs containing 15 of bovine serum albumin ( bsa ) and antibiotics . the microparticles thus obtained were stored at 4 ° c . until used . the microparticles carrying the tnp - ova or tnp - klh antigens were prepared by incubation of microparticles carrying ova or klh with tnbs ( trinitrobenzene sulfonate ). 2 ml of the microparticles which had been coupled to klh or ovalbumin were washed twice in pbs and resuspended in 2 ml of cacodylate buffer containing 10 mg / ml of tnbs . the microparticles were incubated for 30 minutes in darkness at ambient temperature and washed three times in pbs . they were resuspended in 2 ml of pbs containing 1 % bsa and antibiotics and stored at 4 ° c . 50 μl of microparticles were washed twice in pbs containing 1 % of bsa and incubated for 40 minutes at 4 ° c . with mouse anti - klh or anti - tnp serum . after two washes the microparticles were incubated with goat antibody coupled to fitc ( fluoroisothiocyanate ) directed against mouse immunoglobulins ( biosys , compiegne , france ) for 40 minutes at 4 ° c . after four washes the microparticles were resuspended in 1 ml of pbs containing 1 % of bsa . the fluorescence intensity was measured by use of a facsan flow cytometer ( becton dickinson , mountain view , calif .). the lymphocytes were cultured in rpmi 1640 ( seromed , munich , frg ) complemented with 2 mm l - glutamine , 10 % of fcs ( fetal calf serum ) inactivated by heat , 50 μm of 2 - me and antibiotics . this cell line was established and maintained according to the method described by taylor et al . ( irl press , new york ) and galelli et al . ( j . immunol . ( 1990 ) 145 : 2397 ). inguinal ganglion cells ( 4 10 6 / ml ) from dba / 2 mice which 8 days before removal of the cells had received an injection at the base of the tail of 100 μg of klh in emulsion in complete freund &# 39 ; s adjuvant were cultured for 4 days in the culture medium in the presence of klh ( 100 μg / ml ). the cultures were incubated in a humid atmosphere under 7 . 5 % of co 2 at 37 ° c . a cell line was established from this initial culture by serial passage of t cells purified on ficoll ( 2 . 10 5 / ml ) in the presence of dba / 2 mouse spleen cells which had been irradiated ( 3000 rads ) for 6 to 8 days ( rest period ) or with irradiated spleen cells plus klh ( 100 μg / ml ) for 4 days ( stimulation period ). the t cells used in these experiments were collected 8 to 10 days after their last contact with klh . cultures in triplicate containing 5 . 10 4 t cells purified on ficoll , and 5 . 10 4 purified and irradiated ( 900 rad ) tnp - specific memory b cells , or 5 . 10 5 irradiated ( 3000 rad ) entire spleen cells , or 10 5 irradiated ( 3300 rad ) adherent spleen cells , or 10 5 irradiated ( 3300 rad ) a20 b cell lymphoma cells positive for class ii mhc ( kim et al ., j . immunol . ( 1979 ) 122 : 549 ), or 10 5 tnp - specific virgin b cells activated by lps as source of the cells presenting the antigens , and different concentrations of antigen were incubated in flat - bottomed microculture plates ( corning , cambridge , mass .) under a total volume of 0 . 2 ml / well of complete medium . the t cell proliferation was estimated by incorporation of tritiated thymidine during the final eight hours of 3 days &# 39 ; culture . the results are expressed as the geometric mean of three cultures , after elimination of background noise . the standard deviation was less than 15 % of the mean . the tnp - specific b cells from normal mice were purified by adsorption and elution on tnp8 - gelatin according to the method described by haas and layton j . e ., j . exp . med . ( 1975 ) 141 : 1004 . this method was modified in order to obtain populations enriched in tnp - specific memory b cells from spleens from previously immunized mice , as described previously ( galelli et al ., j . immunol . ( 1990 ) 145 : 2397 )). the tnp - specific memory b cells were selected on the gelatin carrying a hapten ( tnp2 - gelatin ), by testing the affinity of tnp receptors by comparison with virgin b cells , and the capacity to secrete large quantities of anti - tnp immunoglobulin g in the presence of low antigen concentrations . 10 8 spleen cells containing neither erythrocytes nor dead cells were suspended in 3 ml of hepes ( 50 mm ) buffered with dmem ( seromed , munich , germany ) and incubated in plastic petri dishes covered with tnp2 - gelatin . the dishes were gently agitated for 15 minutes at 4 ° c ., then washed 10 times with dmem at ice temperature . the adherent cells were eluted by the addition of 5 ml of dmem reheated to 37 ° c . and the bonded tnp - gelatin was eliminated by digestion with collagenase ( clsiii collagenase from worthington biochemicals , freehold , n . j ., 100 u / ml ) for 15 minutes at 37 ° c . this method gives an overall yield , expressed as a percentage of the original number of spleen cells , of 0 . 3 to 0 . 6 % of cells bonding to tnp from the immunized mouse spleen . the cells were cultured overnight , before the addition of other cells and reagents , in order to enable the reexpression of surface immunoglobulins modified by the treatment with the collagenase . the presence of free tnp receptors on the cells was evaluated from their capacity to bind erythrocytes carrying tnp on their surface . 55 to 76 % of the cells obtained from the immunized mice formed rosettes with the mouse spleen b cells modified by the tnp . these cells did not proliferate in response to concanavalin a but were 20 times enriched , for the cells which secreted anti - tnp immunoglobulin g after stimulation by tnp - lh , by comparison with non - fractionated spleen cells . tnp - specific virgin b cells from non - immunized mice were purified by adsorption and elution on tnp8 gelatine as described previously . these cells were cultured to a density of 2 . 10 6 per ml in a medium containing 50 μg / ml of lps ( salmonella enteriditis , difco laboratories , detroit , mich .) for 3 days . the non - adherent lymphoblasts were purified by use of ficolle - hypaque ( pharmacia , piscataway , n . j . ), then washed and used as secondary cells . the macrophages were obtained from non - immunized spleen cells by adhesion for 4 hours at 37 ° c . followed by washing of the cells in order to eliminate the non - adherent cells as previously described ( kakiochi et al ., j . immunol . ( 1983 ) 131 : 109 ). the klh was covalently bonded to polyacrolein microparticles with diameter between 0 . 25 and 1 . 5 μm . the coupling of the klh to the microparticles was checked by flow cytofluorimetric analysis using anti - klh mouse serum . the results obtained with 1 . 5 μm microparticles are shown in fig1 . the 1 . 5 μm microparticles were coupled to ovalbumin ( b ova ) or klh ( b - klh ). the tnp - ova or tnp - klh microparticles ( designated respectively b ( tnp - ova ) and b ( tnp - klh )) were prepared by incubation of microparticles carrying ova or klh with tnbs . the cytofluorimetric analysis was carried out on microparticles incubated in the presence of pbs or anti - klh or anti - tnp mouse serum . after washing , the microparticles were incubated with goat antibodies bonded to fitc directed against mouse immunoglobulins and were analyzed by flow cytometry . control microparticles coupled to ovalbumin were not recognized by the anti - klh serum . 2 . 2 comparison of the ability of different splenocyte populations to present soluble or particulate antigens . the ability of non - fractionated splenocytes , macrophages , and tnp - specific virgin b cells was compared for presentation of soluble or particulate klh and tnp - klh to klh - specific t cells . in these experiments the splenocyte populations were prepared from non - immunized mice . after purification , the tnp - specific b cells were activated for three days by lps ; it is known that the lymphoblasts induced by lps are extremely efficient for antigen presentation ( kakiochi et al ., j . immunol . ( 1983 ) 131 : 109 ). the results are illustrated in fig2 for which 5 . 10 8 irradiated splenocytes , 10 5 adherent cells or 10 5 tnp - specific virgin b cells activated by lps were cultured with 5 . 10 4 klh - specific t cells in the presence of different quantities of soluble klh ( a ), soluble tnp - klh ( b ), or fixed on microparticles ( b klh ) ( c ), or ( b tnp - klh ) ( d ). the t cell proliferation was estimated on day 3 . as shown in fig2 ( 2a and 2b ) the macrophages and the b cells activated by lps efficiently stimulated the t cells when they were incubated with soluble klh or tnp - lh . in contrast to these results , only the macrophages , and not the lps - activated tnp - specific b cells , were able to stimulate the klh - specific t cells ( fig2 c and d ) when the microparticles carrying klh or tnp - klh were used . these results show that the macrophages are responsible for the activity of spleen cell antigen presentation when particulate antigens are used . the inability of the tnp - specific b cells to present the particulate antigen has thus been demonstrated . induction of a lysozyme specific cd4 + t - proliferative response in vivo and in vitro by lysozyme - coupled microparticles . the lysozyme ( lyso ) and the limulus hemocyanin ( lh ) were from sigma laboratories . the soluble antigen was made particulate by coupling to microparticles ( polysciences ) of between 0 . 2 and 1 μm diameter . two coupling methods were used : the polyacrolein beads or microparticles possess aldehyde groups capable of spontaneous reaction with the amine functions of the proteins . 1 ml of beads were washed 4 times in pbs and then taken up in 1 ml of antigen at 5 mg / ml concentration . after 3 hours &# 39 ; incubation at ambient temperature , the beads were washed 3 times in pbs and incubated for 30 minutes in 1 ml of pbs - 1 % human albumin in order to saturate the free reactive groups on the beads . after washing , the particles were then taken up in 2 ml of pbs - 1 % human albumin - 1 % antibiotic and stored at + 4 ° c . the antigen was coupled to polystyrene beads by glutaraldehyde , which was capable of forming a schiff &# 39 ; s base with the protein amine groups . 0 . 5 ml of beads were washed 3 times in pbs and taken up in 0 . 5 ml of 8 % glutaraldehyde . after 6 hours &# 39 ; incubation at ambient temperature , the beads were washed twice and then taken up in 1 ml of antigen at concentration 400 μg / ml . after incubation overnight at ambient temperature , the beads were washed and incubated with 1 ml of 0 . 2m ethanolamlne for 30 minutes in order to block the free aldehyde functions of the glutaraldehyde . after a final washing , the particles were taken up in 1 ml of pbs - 1 % human albumin - 1 % antibiotic then stored at + 4 ° c . this coupling method enabled the quantity of proteins coupled to the microparticles to be determined by spectrophotometry . the absorbances of the 400 μg / ml protein solution and the supernatant obtained after incubation of the beads with this protein solution were measured at 280 nm . given the number of beads used for the coupling , the difference between the quantity of protein before coupling and the residual quantity after coupling could be used to estimate the quantity of lysozyme coupled per particle . balb / c females , haplotype h - 2 d , aged 6 to 9 weeks ( reared in the institut pasteur ) were used . immunization by intra - peritoneal route : 100 μg of lysozyme with 1 mg of alum were injected , or different quantities of antigen coupled to beads without adjuvant , immunization by subcutaneous route : 100 μg of lysozyme in emulsion with complete freund &# 39 ; s adjuvant were injected at the base of the tail , or different quantities of antigen coupled to beads . the serum of each mouse was sampled 7 to 14 days after injection . the antibody strength of the serum was measured by the elisa assay . the cell proliferative response was measured on inguinal ganglions and / or on the spleen , sampled 7 and / or 14 days after each injection . the antigen ( lysozyme ) was incubated at a concentration of 5 μg / ml in 50 mm ph 9 . 6 carbonate buffer in the microplates ( nunc ) for one night at 4 ° c . after washing with a 0 . 01 % pbs - tween 20 buffer , the different serum dilutions to be tested , in 1 % bsa buffer , were incubated for 1 hour at 37 ° c . after washing , 100 μl of a mouse anti - ig conjugate ( complete anti - ig supplied by diagnostics pasteur and specific anti - ig by sigma ) were placed in each well , marked with goat peroxidase ; this was incubated for 1 hour at 37 ° c . after washing , a substrate solution was added freshly prepared as follows : 0 . 5 mg / ml of orthophenylenediamine ( sigma ) in a 0 . 1m citric acid - 0 . 2m disodium phosphate buffer , ph 5 , to which was added h 2 o 2 to 1 / 2500 . a yellow coloration revealed the presence of specific antibodies ; the enzyme reaction was stopped 8 minutes later by the addition of 50 μl of 11 . 5 % h 2 so 4 . the absorbance of each well was measured at 492 nm by an optical density reader ( dynatech ). the negative control was made with 1 : 100 serum from non - immunized balb / c mice . the results are expressed either in od × 1000 from measured absorbance , corrected for the absorbance in absence of serum , or by the antibody titer calculated from the linear regression based on the absorbance obtained with the serum from the non - immunized balb / c mice . when the antigen was in particulate form , the elisa assay was carried out in tubes . the serum dilutions to be tested were incubated directly with the antigen coupled to the beads ( 8 . 10 8 particles / ml ). washings were made by centrifuging in 0 . 1 % pbs - tween 20 buffer . when the enzyme reaction had finished , 200 μl from each tube was transferred onto a microplate and the absorbance then measured . the elisa assay measured the fixation of specific antibodies present in the serum of the immunized balb / c mice by the lysozyme . this fixation was reduced if the serum was preincubated ( before the elisa assay ) with the antigen : soluble lysozyme or lysozyme coupled to beads , which then behaved as an inhibitor . the anti - lysozyme serum was preincubated with soluble lysozyme or lysozyme coupled to beads for 1 hour at 37 ° c ., then for 1 night at 4 ° c . ; the reaction was carried out in the tubes . the fixation of antibodies not bonded to the inhibitor was evaluated by the elisa assay ( triplicates ) on microplates , in which the wells were covered with 5 μg / ml of lysozyme . the absorbance of each well was measured at 492 nm , and corrected for the absorbance in the absence of serum . the negative control was carried out with 1 : 100 serum from non - immunized balb / c mice . the absorbance without inhibitor during the preincubation of the serum corresponded to the maximum anti - lysozyme antibody fixation . results are expressed as a percentage of the inhibition of the antibody fixation and calculated according to the ratio : ## equ1 ## the graphical representation of the soluble lysozyme concentration necessary for 50 % inhibition , together with the number of beads coupled to lysozyme , enabled estimation of the quantity of lysozyme fixed per particle . a t hybridoma was produced by immunization of balb / c mice with lysozyme . it specifically recognized peptide 108 - 116 of lysozyme , in combination with molecules of the class ii i - e d major histocompatibility complex . 10 5 t hybridoma cells were stimulated by increasing antigen concentrations : lysozyme or coupled beads , in the presence of different cells presenting the antigen : 5 . 10 5 irradiated splenocytes ( 3000 rad ) of balb / c mice or 10 5 cells of b lymphoma a20 , restricted by class ii mhc molecules . the cells were cultured ( in triplicate ) in a complete rpmi medium ( seromed ) supplemented with 10 % decomplemented fetal calf serum , 50 μm β - mercaptoethanol , 2 mm glutamine , 100 ui / ml penicillin and 100 μg / ml streptomycin , on flat - bottomed microplates ( corning 25860 ). the positive control was performed by stimulation of the hybridoma by the t lymphocyte mitogen : concanavalin a at 5 μg / ml . the supernatant was removed after 24 h culture at 37 ° c . ( 7 . 5 % co 2 ), then frozen to - 20 ° c . for a minimum of 16 h . the stimulation of the hybridoma was measured by the il2 concentration of the supernatant in a ctl - l cell proliferation test . standard deviations have not been given as the error was lower than 10 % of the mean of the triplicates . the ctl - l line is dependent on interleukin 2 and interleukin 4 ; it was maintained in culture in complete medium enriched with 20 % of rat splenocyte supernatant , incubated 36 h with 2 . 5 μg / ml of concanavalin a . after thawing , the culture supernatants ( tested 1 / 2 ) were incubated in the presence of 2 . 25 . 10 4 ctl - l cells , previously washed three times in rpmi 1640 medium , for 3 days at 37 ° c . ( 7 . 5 % co 2 ). the cell proliferation was measured by the addition of tritiated thymidine with specific activity 1 ci / mmole , at a level of 2 μci / ml of culture , for the last 16 hours of culture . the cell dna was recovered after cell lysis and filtration using a &# 34 ; skatron &# 34 ;. radioactivity incorporation was counted by scintillation using a beta counter . the results are expressed in cpm based on the mean of the triplicates , corrected for the radioactivity incorporated in the absence of antigen . the spleen and / or the inguinal ganglions were removed under sterile conditions 7 or 14 days after immunization of the mice ( see immunization protocol ). 8 . 10 5 cells were incubated in the presence of different concentrations of antigen , soluble or coupled to beads . the cells were cultured ( in triplicate ) in rpmi 1640 medium ( seromed ) supplemented with 1 . 5 % decomplemented fetal calf serum , 0 . 5 % normal mouse serum , 50 μm β - mercaptoethanol , 2 mm glutamine , 100 ui / ml penicillin and 100 μg / ml streptomycin , on microplates ( corning 25860 ) for 4 days at 37 ° c . ( 7 . 5 % co 2 ). the cell proliferation was measured by the incorporation of tritiated thymidine with specific activity 25 ci / mmole , at a level of 2 μci / ml of culture , for the last 16 hours of culture . the cell dna was recovered after cell lysis and filtration using a skatron . radioactivity incorporation was counted by scintillation using a beta counter . the results are expressed in cpm based on the mean of the triplicates , corrected for the radioactivity incorporated in the absence of antigen . 2 . 1 . stimulation of ganglion cells from mice immunized with lysozyme by lysozyme coupled to microparticles . in the tests illustrated by fig3 a and 3b , balb / c mice were immunized by subcutaneous injection at the base of the tail of soluble lysozyme complemented with freund &# 39 ; s adjuvant ( cfa ). after 14 days , the inguinal ganglions were removed , and the proliferative response of these cells was tested in vitro against different concentrations of lysozyme or against different concentrations of microparticles coupled to lysozyme . the results are expressed in cpm corrected for the value obtained without antigen . soluble lysozyme induced substantial proliferation of cells from mice immunized by this antigen in freund &# 39 ; s adjuvant ( 3a ). the in vitro stimulation of these cells by lysozyme - microparticles revealed that the latter are able to induce a very strong cell proliferation ( fig3 b ). the microparticles with very large diameter , 0 . 81 and 0 . 96 μm ( spontaneous coupling ), were very effective . fig4 a and 4b correspond to the results for stimulation of lysozyme - specific t hybridoma by soluble lysozyme ( 4a ) or lysozyme coupled to microparticles ( 4b ). the degree of stimulation of the hybridoma was measured by the level of il - 2 / il - 4 produced . in the presence of irradiated splenocytes , the t hybridoma was strongly stimulated by soluble lysozyme ( fig4 a ). in the presence of these cells , the large lysozyme - microparticles ( 0 . 81 and 0 . 96 μm ) also caused substantial production of il - 2 / il - 4 ( fig4 b ), in contrast to the 0 . 5 and 0 . 25 μm microparticles which were not able to stimulate the specific t hybridoma . 2 . 3 . inability of b lymphoma a20 cells to present lysozyme coupled to beads to lysozyme - specific t hybridoma . it is known that b cell tumors carrying ia receptors can be used as antigen - presenting cells for antigens which do not react with the ig receptor but which are fixed by b cell tumors by nonspecific mechanisms ( walker et al ., j . immunol . ( 1982 ) 128 : 2164 ; glimcher et al . j . exp . med ( 1981 ) 155 : 445 ; mackean et al . j . exp . med . ( 1981 ) 154 : 1419 ). the capacity of one of these b cell tumors , the a20 line , to present lysozyme in soluble or particulate form was thus tested . the presentation of soluble or particulate lysozyme was compared using two sources of antigen - presenting cells : either a heterogenous source , irradiated entire splenocytes , or b cells from the a20 lymphoma . when the antigen was in soluble form ( fig5 a ), it could stimulate the t hybridoma equally well in the presence of splenocytes as of a20 b cells . however , particulate lysozyme was presented only by splenocytes and not by a20 b cells ( fig5 b ). these results confirm that splenocytes can present an antigen to t cells , either in soluble or particulate form . however , b lymphocytes were not able to present an antigen rendered particulate by coupling to a bead of a size of the order of a micron . 2 . 4 induction of t proliferative responses by injection of lysozyme coupled to microparticles to mice . the in vivo immunogenicity of the antigen coupled to microparticles was analyzed by immunizing balb / c mice with lysozyme in complete freund &# 39 ; s adjuvant or with this antigen coupled to polyacrolein beads . after 14 days , cells from draining ganglions of these animals were stimulated in vitro by different concentrations of soluble lysozyme . in the presence of soluble lysozyme , the ganglion cells proliferated strongly , whether originating from mice immunized with soluble lysozyme or lysozyme - microparticles ( fig6 a ). this shows that in both cases lysozyme - specific t cells were sensitized in vivo . after injection of lh - microparticles to mice , representing the specificity control , the ganglion cells of these animals were not able to proliferate in response to stimulation by soluble lysozyme in vitro ( fig6 b ). the cellular response in vivo is thus specific to the protein antigen coupled to microparticles , used during immunization of the mice . the proliferative response of the cells sensitized by 10 9 lysozyme - microparticles ( corresponding to 1 μg of lysozyme ), in the absence of adjuvant , was as high as that of cells from animals immunized with 100 μg of lysozyme in freund &# 39 ; s adjuvant ( cfa ) ( fig6 a ). in order to confirm and clarify this result , proliferative responses of ganglion cells from animals having received different doses of lysozyme in cfa or different concentrations of coupled microparticles were compared , after in vitro stimulation by soluble lysozyme . in the case of fig7 a and 7b , the mice had been immunized by subcutaneous injection at the base of the tail of soluble lysozyme and complete freund &# 39 ; s adjuvant ( cfa ) ( fig7 a ) or beads coupled to antigen without adjuvant ( fig7 b ). after 14 days , the inguinal ganglions were removed , and the proliferative response of these cells was tested in vitro against different lysozyme concentrations . the results are expressed in cpm corrected for the value obtained without antigen . in fig7 b , it should be noted that the designations 10 9 , 10 8 , 10 7 and 10 6 b - lyso correspond respectively to weights of 1 ; 0 . 1 ; 0 . 01 and 0 . 001 μg of lysozyme . these results show that the ganglion cells from animals immunized with lysozyme - carrying microparticles proliferate in vitro after contact with lysozyme , thus demonstrating sensitization of the t cells specific for this antigen . comparison of the concentration effects ( fig7 ) shows that 1 μg of lysozyme coupled to beads gives a response quasi - equivalent to that of 1 μg of antigen injected in cfa . fig8 represents the proliferative response of cells from mice immunized with lysozyme in complete freund &# 39 ; s adjuvant ( cfa ) or in pbs with microparticles coupled to lh . the addition of lh beads to lysozyme did not lead to induction of high proliferative responses , which shows that the lysozyme must be covalently coupled to the microparticles to induce t - proliferative responses . 2 . 5 -- induction of t - proliferative responses by injection of mice with hemoglobin or ovalbumin coupled to microparticles mice were immunized with hemoglobin or ovalbumin in complete freund &# 39 ; s adjuvant , or with these proteins covalently coupled to the same type of particles as in the previous examples ( polystyrene , 1 μm diameter ). the ganglion cells from these animals were restimulated in vitro by the soluble proteins and the cell proliferation was measured . the results obtained for hemoglobin ( hb ) are shown in fig9 while fig1 shows the results obtained for ovalbumin ( ova ). these results overall show that these proteins coupled to microparticles are able to sensitize cd4 + t lymphocytes specific to these proteins in vivo , in the absence of adjuvant . 2 . 6 . 1 -- t epitope from region c3 of the vp1 protein the t epitope of the c3 region ( c3 : t , 103 - 115 ) of the poliovirus protein was synthesized and covalently coupled to 1 μm beads . these beads were injected into balb / c mice . the results in fig1 clearly show that the t epitope coupled to the beads ( b - c3 : t ) induced a strong t - proliferative response for quantities of the order of 10 9 beads injected per mouse . the pre - s : t peptide ( 120 - 132 ) of the hbs antigen was synthesized and covalently coupled by glutaraldehyde to beads of 1 μm diameter . fig1 shows that the injection of 10 9 beads to dba / 1 mice induced a strong t - proliferative response , stronger than that obtained with the peptide in cfa . the injection of beads not containing the b epitope did not induce a proliferative response , showing the specificity of the response . the materials and methods were similar to those of example 2 . for fig1 a and 13b , balb / c mice were immunized by intra - peritoneal injection with 100 μg of soluble lysozyme in adjuvant ( alum ) or with beads coupled to antigen : lysozyme or limulus hemocyanin ( lh ), without adjuvant . the injections were carried out at d0 , d21 , d42 , the serums were taken at d20 , d31 , d40 and d52 and assayed by elisa for their antibody content . the results are expressed in log10 of the titer of anti - lysozyme antibody ( fig1 a ) and anti - klh antibody ( fig1 b ). three antigen injections were performed i . p . at days 0 , 21 and 42 . the lysozyme - microparticles gave very good antibody responses while no antibody response was induced by the lh microparticles . these microparticles moreover very efficiently stimulated t responses . one of the differences between lh and lysozyme is their molecular weights ( 14500 for lysozyme and 71000 for lh ). at equal concentrations of coupled antigen , the density of lh molecules on the beads is thus about 5 times lower . this could explain the absence of stimulation of antibody responses if these are due to t - independent direct stimulation by the antigen present at high density on the microparticles . mice were immunized with soluble antigen in alum adjuvant or with the same antigen in particulate form , in the absence of adjuvant . antibody appearance was then monitored over several weeks . in the case of hemoglobin ( hb ), the mice were immunized with 100 μg of protein or 10 9 beads coupled with the protein at different densities ( 2 . 10 4 and 2 . 10 5 molecules / μm 2 ). the beads carrying ovalbumin ( ova ) were tested at two densities , 7 . 10 3 and 7 . 10 4 molecules / μm 2 . an initial injection was carried out , followed by two more injections on the 21st and 40th days . serums were taken at the 20th day , the 31st day , the 41st day and the 52nd day , then assayed by elisa for their igg antibody levels . the results are expressed in log of the antibody titer . the results in fig1 show that hemoglobin coupled to beads did not induce an antibody response . for ovalbumin ( fig1 ) antibodies were detectable after several injections if the antigen was coupled at high density , but these responses were weak . these results show that proteins of high molecular weight such as hemoglobin are not able to induce an antibody response , even if they are coupled at a high density on the beads . these results , similar to those obtained with lysozyme and limulus hemocyanin , confirm that beads carrying high - molecular - weight proteins induce t - proliferative responses in the absence of any antibody production . likewise , proteins of low or medium molecular weight ( less than 50 000 ) can induce the appearance of antibodies if they are coupled to the beads at high densities . the peptides pre - s : tb ( 120 - 145 ) and pre - s : b corresponding to the portions of the hbs antigen containing respectively a t epitope and a b epitope or only the b epitope were covalently coupled to 1 μm beads with glutaraldehyde ( b - pre - s : tb and b - pre - s : b ). the antibody response induced by these beads was compared with that induced by 10 μg of soluble pre - s : tb peptide in alum adjuvant . the results in fig1 show that the beads coupled to the tb peptide , containing a t epitope and a b epitope , induced strong antibody responses , which confirms that antigens of low molecular weight coupled to beads are able to induce an antibody reaction in the absence of adjuvant . it may be noted that these responses are as good as those obtained with the free peptide in the presence of alum adjuvant . the materials and methods used were similar to those for example 2 . the immunogenicity of beads coupled to lysozyme with different numbers of molecules on their surface was tested in experiments illustrated in fig1 and 18 . for fig1 a , balb / c mice were immunized by subcutaneous injection of 100 μg of lysozyme in cfa . after 14 days , the inguinal ganglions were removed and the cells tested in vitro against beads carrying different densities of lysozyme ( from 1100 to 950 000 molecules of lysozyme on 1 μm diameter beads ). the results are expressed in cpm corrected for the value obtained without antigen . for fig1 b , balb / c mice were immunized by subcutaneous injection at the base of the tail with soluble lysozyme with adjuvant ( cfa ) or 10 9 beads carrying different densities of lysozyme without adjuvant . after 14 days , the inguinal ganglions were removed and the proliferative response of these cells was tested in vitro against different concentrations of lysozyme or beads . the results are expressed in cpm corrected for the value obtained without antigen . the proliferation of ganglion cells originating from animals immunized by soluble lysozyme in cfa was tested in vitro after stimulation by different lysozyme - microparticles . the proliferative response of these cells increased as the density of lysozyme on the microparticle surface increased . no proliferation of the ganglion cells was obtained after stimulation by microparticles with a density of 1100 lysozyme molecules per microparticle ( fig1 a ). in the experiment shown in fig1 b , the immunogenicity of these microparticles was tested in vivo . balb / c mice were immunized by the different microparticles , without adjuvant , and the ganglion cells of these animals were stimulated in vitro by different concentrations of soluble lysozyme . the proliferation of ganglion cells originating from animals immunized by microparticles coupled to lysozyme at high density ( 950 000 and 210 000 ) was high , and comparable to the response of cells sensitized by 100 μg of lysozyme in cfa . after immunization by microparticles carrying a medium density of lysozyme ( 45 000 ), the cells proliferated in response to lysozyme in vitro at concentrations from 10 - 1 μg / ml . lower - density microparticles did not sensitize t cells in vivo , since no proliferation was observed in the presence of lysozyme , even at high concentration ( fig1 b ). it should be noted that 10 9 microparticles coupled to lysozyme at high density correspond to 23 μg ( 1 - 950 000 - g ) and 5 μg ( 1 - 210 000 - g ) of coupled lysozyme , nevertheless the cell proliferation was as high as that after injection with 100 μg of lysozyme in cfa . for fig1 , balb / c mice were immunized by subcutaneous injection of lysozyme with adjuvant ( cfa ) or of 10 9 microparticles carrying different densities of lysozyme ( 950 000 ; 210 000 , 45 000 and 1100 molecules respectively on a 1 μm diameter microparticle ). after 14 days , serums were taken and assayed by elisa for their level of anti - lysozyme antibody . the results are expressed in log10 of the antibody titer . the humoral response of the mice immunized by these microparticles with different lysozyme densities were studied . injection of 100 μg of lysozyme in cfa induced a high level of anti - lysozyme antibody ( fig1 ). fourteen days after immunization , the beads coupled to the highest density of lysozyme ( 950 000 ) had induced significant antibody production , while beads of lower density had not stimulated the induction of a significant anti - lysozyme antibody response . in particular it should be noted that the beads with density 210 000 , which had induced an excellent specific proliferation of the ganglion cells , did not stimulate antibody production . these results show that t cell proliferation is induced with lysozyme densities of between 45 000 and 950 000 molecules per microparticle , while antibody production requires a high density of protein coupled to the microparticles . within the meaning of the present description , the expression &# 34 ; microparticles &# 34 ; refers to particles which may have various geometric and spatial configurations . in practice , they are preferentially microspheres or beads , such as are obtained by conventional polymer manufacturing techniques . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 5 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 13 ( b ) type : amino acid ( c ) strandedness : unknown ( d ) topology : unknown ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 1 : metglntrpasnserthrthrphehisglnthrleu510gln ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 14 ( b ) type : amino acid ( c ) strandedness : unknown ( d ) topology : unknown ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 2 : glnaspproargvalargglyleutyrpheproala510glygly ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 13 ( b ) type : amino acid ( c ) strandedness : unknown ( d ) topology : unknown ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 3 : lysleuphealavaltrplysilethrtyrlysasp510thr ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 11 ( b ) type : amino acid ( c ) strandedness : unknown ( d ) topology : unknown ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 4 : aspasnproalaserthrthrasnlysasplys510 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 40 ( b ) type : amino acid ( c ) strandedness : unknown ( d ) topology : unknown ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 5 : ileasncysthrargproasnasnasnthrarg510lysserileargileglnargglyproglyarg1520alaphevalthrileglylysileglyasnmet2530argglnalahiscysasnile3540__________________________________________________________________________

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