Patent Application: US-29725603-A

Abstract:
the present invention provides a method of immunising a subject comprising the step of administering a composition comprising an antigen and a carbohydrate polymer comprising mannose to a mucosal site of the subject , methods of use of the composition for vaccination and sterilization and use of the composition in manufacturing a medicament .

Description:
preferred embodiments of the invention will now be described with reference to the following , non - limiting examples , in which :— [ 0064 ] fig1 shows the results of intranasal versus intraperitoneal immunisation : ( cba × balb / c ) f1 mice were immunised on days 0 , 10 , 17 with 12 μg of m - llofp intranasally or intraperitoneally , and serum was obtained from individual mice on day 24 . antigen specific iga ( fig1 a ) and igg1 ( fig1 b ) were detected by elisa and optical density was measured at 450 nm . closed squares (▪) indicate intranasally immunised mice , open circles (◯) intraperitoneally immunised mice and open triangles ( δ ) the unimmunised controls . results for individual mice are shown . [ 0065 ] fig2 shows subclasses of antibody in the serum of m - llo . fp immunised mice . ( cba × balb / c ) mice were immunised intranasally with 12 μg of m - llo . fp or unconjugated llo . fp on day 0 , 10 and 17 and serum from 5 individual mice obtained on day 24 . antigen specific iga ( a ), igg1 ( b ) and igg2a ( c ) was detected by elisa . individual titres are shown as dot plots , with corresponding symbols for different antibody classes from the same mouse . in addition , titres were converted to geometric means ( heavy bar ) and standard deviations ( vertical bar ) for display . differences between groups receiving m - llo . fp or unconjugated llo . fp were significant for all classes of antibody . [ 0066 ] fig3 shows the levels of antigen specific iga in the serum and distant mucosal sites over a 41 day period : ( cba × balb / c ) f1 mice were immunised on day 0 , 10 and 17 with 12 μg of m - llofp , with 1pg of ct mixed with 12 μg of llofp or with 12 μg of llofp alone . serum ( a ), vaginal washings ( b ), and mouth washings ( c ) were collected from 5 individual mice on days 7 , 20 , 27 and 41 . antigen specific iga titres were determined by elisa and the progress in individual mice shown . [ 0067 ] fig4 shows the antigen specific iga at distant mucosal sites over a 112 day period : ( cba × balb / c ) f1 mice were immunised on day 0 , 28 and 56 with 12 μg of m - llofp , with 1 μg of ct mixed with 12 μg of llofp or with 12 μg of llofp alone . serum ( a ) and , vaginal washings ( b ) were collected from 4 individual mice on day 31 , 74 , and 112 . antigen specific iga titres were determined by elisa and the progress in individual mice shown . [ 0068 ] fig5 shows the antigen specific iga in the tears and in the lung washings . ( cba × balb / c ) f1 mice were immunised on day 0 , 10 and 17 with 121 g of m - llo . fp or with 12 μg of llo . fp alone . mice were anaesthetised on day 24 of the experiment and tears ( 111 ) collected from 5 mice per group ( a ). mice were euthanased on day 35 of the experiment and lung washings ( 0 . 5 ml ) collected ( b ). antigen specific iga titres were determined by elisa and individual titres are shown as dot plots . in addition , titres were converted to geometric means ( heavy bar ) and standard deviations ( vertical bar ) for display . differences between groups receiving m - llo . fp or unconjugated llo . fp were significant ( p & lt ; 0 . 01 ) in tear samples and ( p & lt ; 0 . 05 ) for lung washings . [ 0069 ] fig6 shows the effects of the m - llofp conjugate when in the oxidised form : ( cba × balb / c ) mice were immunised intranasally with 12 μg of m - llofp in the oxidised or reduced form on day 0 , 10 and 17 . serum from 5 individual mice was collected on day 24 and antigen specific iga ( a ), igg1 ( b ) and igg2a ( c ) was detected by elisa . individual titres are shown as dot plots , with corresponding symbols for different antibody classes from the same mouse . in addition , titres were converted to log 10 and geometric means ( heavy bar ) and standard deviations ( vertical bar ) were derived and converted back to arithmetic numbers for display . differences between groups receiving oxidised or reduced m - llo . fp were significant ( p & lt ; 0 . 02 ) for all classes of antibody ( student &# 39 ; s t test ). [ 0070 ] fig7 ( a ) shows the effects of oxidised mannan as adjuvant for mycobacterial 19 kda fp : groups of 4 c57 / b . 10 mice were immunised intranasally with 12 μg of m - 19fp or 12 μg of 19 kdafp on day 0 , 10 and 17 . serum was collected on day 24 and antigen specific iga determined by elisa . individual titres are shown as dot plots . in addition , titres were converted to log 10 and geometric means ( heavy bar ) and standard deviations ( vertical bar ) were derived and converted back to arithmetic numbers for display . differences between groups receiving m - 19fp and 19fp alone were significant by student &# 39 ; s t test ( p & lt ; 0 . 02 ). [ 0071 ] fig7 ( b ) shows elisa readings for iga titres for mice immunised with hsp65 — mannan conjugate . [ 0072 ] fig8 . comparison of intranasal and intraperitoneal immunisation with m - llo . fp . ( cba × balb / c ) f1 mice ( 4 per group ) were immunised with 12 μg of m - llo . fp i . n . or i . p . or unconjugated control on day 0 , 10 and 17 and mice sacrificed on day 24 . ( a ) proliferative response : spleen cells from all groups were cultured in the presence of antigen ( 45 μg / ml llo ( 215 - 226 ) (▪) or without stimulus (□). data represent the means and standard deviations of triplicate cultures . * p & lt ; 0 . 002 compared with m - llo . fp i . p . ( b ) il - 2 production : spleen cells from all groups were cultured in the presence of antigen ( 45 μg / ml llo ( 215 - 226 ) (▪) or llo . fp ( )) or without stimulus (□). data represent the means and standard deviations of triplicate cultures . * p & lt ; 0 . 01 ** p & lt ; 0 . 002 compared m - llo . fp i . p . ( c ) ifn - γ producing cells : spleen cells from all groups were cultured in the presence of antigen ( 45 μg / ml of llo . fp (▪)) or without stimulus (□). data represent the means and standard deviations of triplicate cultures . * p & lt ; 0 . 05 compared with mllo . fp i . p . [ 0073 ] fig9 . ifn - γ and il4 production following i . n . immunisation with oxidised or reduced m - llo . fp . ( cba × balb / c ) fl mice ( 5 per group ) were immunised i . n . with 12 μg of m - llo . fp ( oxidised ) or with m - llo . fp ( reduced ) on day 0 , 10 and 17 or infected i . v . with 1 × 10 3 listeria on day 17 . mice were sacrificed on day 24 . spleen cells from all groups were cultured in the presence of antigen ( 45 μg / ml of llo . fp (▪)) or without stimulus (□) and the frequency of ( a ) ifn - γ and ( b ) il - 4 producing cells determined . data represent the means and standard deviations of triplicate cultures . * p & lt ; 0 . 02 , ** p & lt ; 0 . 05 compared with the reduced group for ifn - γ and ila respectively . note different scales . [ 0074 ] fig1 . ifn - γ production following i . n . immunisation with m - 19 . fp . c57bu10 mice ( 4 per group ) were immunised i . n . with 12 μg of m - 19 . fp or unconjugated control on day 0 , 10 and 17 or infected i . n . with m . evium 6 weeks prior to immunisation . mice were sacrificed on day 24 and spleen cells from all groups cultured in the presence of antigen (▪) ( 19 kda . fp 23 μg / ml ), 10 7 live m . avium ( ) or without stimulus (□). data represent the means and standard deviations of triplicate wells . * p & lt ; 0 . 05 compared with the 19 . kda . fp group . [ 0075 ] fig1 : antibody titres in serum following immunisation with vp5 246 - 274 fp with and without mannan adjuvant : mice were immunised intranasally with 6 μg vp5 246 - 274 - mannan of vp5 246 - 274 alone on days 0 , 10 and 17 , and bled from the tail vein on days 24 and 35 . titres are expressed as log 10 geometric means plus or minus standard deviation for groups of 5 mice . differences p & lt ; 0 . 05 are considered significant . ** p & lt ; 0 . 02 , * p & lt ; 0 . 05 . [ 0076 ] fig1 : iga titres on mucosal surfaces following immunisation with vp5 246 - 274 fp with and without mannan adjuvant : mice were immunised intranasally with 6 μg vp5 246 - 274 - mannan of vp5 246 - 274 alone on days 0 , 10 and 17 . titres are expressed as log 10 geometric means plus or minus standard deviation for groups of 5 mice . ( a ). on day 24 tears were collected as described and iga antibody assayed by elisa . note limit of detection was { fraction ( 1 / 100 )} and no antibody was detected in mice receiving antigen alone . ( b ). on day 34 stools were collected , homogenised in 10 × volume of pbs and the extracted iga antibody assayed by elisa . note limit of detection was { fraction ( 1 / 10 )} and no antibody was detected in mice receiving antigen alone . ( c ) on day 35 the mice were sacrificed and lung washings collected . significant difference in titres , p & lt ; 0 . 02 . [ 0080 ] fig1 . protection against porphyromonas gingivalis lesions : groups of 5 c57b1 / 10 mice were immunised with cpx - mannan given intranasally ( fig1 ( a )) or cpx in incomplete freund &# 39 ; s adjuvant ( ifa ) subcutaneously ( fig1 ( b )). control mice received the respective adjuvants alone . mice given cpx - mannan intranasally showed no lesions — complete protection ( p & lt ; 0 . 001 ), while those given cpx in ifa showed incomplete protection ( p & lt ; 0 . 05 ). [ 0081 ] fig1 . serum antibody titres : groups of 5 c57b1 / 10 mice were immunised with cpx - mannan given intranasally or cpx in incomplete freund &# 39 ; s adjuvants ( ifa ) subcutaneously . control mice received the respective adjuvants alone . the mice were bled from the retro - orbital plexus after 2 or three doses and serum antibody assayed by elisa . titres on pooled sera are shown . [ 0082 ] fig1 : antibody titres in serum following immunisation with e7 . fp coupled to mannan under oxidising or reducing conditions or without mannan adjuvant ( e7 alone ): mice were immunised intranasally with 12 μg antigen ( with or without mannan ) on days 0 , 10 and 17 , and bled from the tail vein on days 24 . titres are expressed as log 10 geometric means plus or minus standard deviation for groups of 5 mice . titres of all classes of antibody in groups immunised e7 . fp plus either oxidised or reduced mannan were significantly different from the group receiving e7 . fp , alone p & lt ; 0 . 01 . mice : female 6 - 8 week old ( cba × balb / c ) f1 and c57b1 / 10 mice were bred and maintained under conventional but infection - free conditions in the animal house of the department of microbiology and immunology , university of melbourne . production of antigens : a p . bluescript plasmid containing the gene for listeriolysin o ( llo ) from listeria monocytogenes ( without its leader sequence ) was obtained from richard strugnell ( university of melbourne , australia ) and subcloned into the escherichia coli expression vector pgex - 2t [ 16 ] in the correct reading frame and orientation . the expression of an 84 kda llo glutathione - s - transferase ( gst ) fusion protein ( llo . fp ) was induced with 0 . 1 mm iptg ( sigma chemical co ., mo , usa ) at 37 ° c . for 5 hours . bacteria were collected by centrifugation ( 1 , 500 × g ) for 5 minutes , washed and lysed by sonication . the pellet was collected after centrifugation and solubilised in 8m urea ( eastman kodak co ., rochester , n . y ., usa ) overnight at 4 ° c . after further centrifugation ( 26 , 000 × g ) for 15 minutes the supernatant was dialysed in 0 . 01 m tris ( sigma chemical co ., mo , usa ) ph 8 . 0 / 1 m urea and the biocad perfusion chromatography system ( perceptive biosystems , framingham , mass ., usa ) used to purify the protein by anion exchange . the 19 kda lipoprotein from mycobacterium tuberculosis was produced using a recombinant 19 kda - plasmid construct was obtained from professor douglas young ( imperial college school of medicine , london , u . k ) and the expression of a 19 kda his tag fusion protein ( 19fp ) induced with 0 . 1 mm iptg at 37 ° c . for 5 hours . bacteria were collected by centrifugation ( 1 , 500 × g ), washed and lysed by sonication . the supernatant was collected after further centrifugation ( 26 , 000 × g ) for 15 minutes and dialysed in 0 . 01 m tris ph 8 . 0 / 300 mm nacl / 20 mm imidazole and purified under native conditions on a nickel chelate column ( qiagen , hilden , germany ) using the biocad perfusion chromatography system . rotavirus vp5 antigen was produced recombinantly using the gene for the fragment of vp5 from amino acid 248 - 475 ( vp5 246 - 274 ) [ 17 ]. this gene fragment was cloned into the pgex plasmid to create a fusion protein with glutathione s transferase . the recombinant e . coli was grown overnight at 37 ° c . in lb - ampicillin medium and subcultured at 1 : 50 dilution into fresh lb - ampicillin medium . after incubation of the bacteria at 20 ° c . until od6 %= 1 . 4 - 1 . 6 ( about 12 - 16 hours ), expression of the fusion protein was induced with 0 . 01 mm iptg for 6 - 8 hours at 20 ° c . bacteria were harvested and washed before sonication in pbs for 15 seconds each . centrifugation followed by passage through a 0 . 45 μm filter was used to collect soluble protein . the gst fusion protein was purified using glutathione beads , according to the manufacturers &# 39 ; instructions ( sigma , mo ., usa ). the eluted gstvp5 was passed through the 0 . 45 μm filter to remove traces of beads and the protein dialysed against ph 9 bicarbonate buffer . the antigen , hsp65 of mycobacterium avium was produced recombinantly according to standard methods . the cloning and expression of hsp65 of mycobacterium avium has been described by v . nagabhushanam , j . praszkier and c . cheers , immunology and cell biology ( 2001 ) in press . briefly the sequences of hsp65 genes from various mycobacterial species were obtained from genbank , aligned and used to design primers . the open reading frame of the hsp65 gene of m . avium was amplified by pcr . the purified pcr product was inserted into m13tg131b . the insert was re - cloned into m13tg130b , which facilitated sequencing of the complementary strand of the insert . the fragment containing the coding sequence the hsp65 was moved from the m13tg130b derivative into p2gex - 2t , so that the hsp65 reading frame was fused in - frame to the 3 ′ end of one of the two copies of the glutathione - s - transferase ( gst ) of schistosoma japonicum . e . coli strain bl21 , transformed with the recombinant plasmid p2gexhsp65 . expression of the gst fusion was induced by the addition of 0 . 1 mm isopropylthiogalactoside ( iptg ) ( sigma ). bacteria were harvested , washed and lysed . the cell lysate was cleared by centrifugation and the separated cell pellet and supernatant analysed on a 12 % sds - page . the supernatant , which contained the majority of the fusion protein ( gst - hsp65 ) was filtered and the gst fusion protein purified by binding to glutathione agarose beads according to manufacturers &# 39 ; instructions ( sigma ). to cleave hsp65 from gst , the beads were incubated overnight at room temperature with thrombin ( pharmacia ) ( 10 units per milligram of protein ) in 10 ml of pbs , then pelleted , the supernatant collected and the protein content estimated spectrophotometrically . the e7 antigen of human papilloma virus ( hpv ) was produced by recombinant means . the dna sequence for the e7 antigen from hpv was cloned into the pgex - 2t vectors . protein expression was induced by iptg , and purified using glutathione - sepharose column chromatography . elution of the bound protein was with a solution of 5 mm glutathione in tris 0 . 05m , ph 8 . 0 . the antigen , cpx , of porphyromonas gingivalis was prepared according to o &# 39 ; brien - simpson et al . [ 18 ]. preparation of oxidised or reduced mannan - antigen conjugates mannan ( sigma chemical co ., st louis , mo ., usa ) was coupled to the antigens under oxidising conditions . mannan at 14 mg / ml in 0 . 1 m phosphate buffer ph 6 . 0 was oxidised with 0 . 01 m sodium periodate for 60 min at 4 ° c . ten microlitres of ethandiol ( sigma chemical co ., st louis , mo ., usa ) was added to quench the reaction and the mixture incubated for 30 min at 4 ° c . this mixture was then passed through a pd - 10 column ( pharmacia biotech , uppsala , sweden )- equilibrated with bicarbonate buffer ph 9 . 0 . the oxidised mannan , eluted in the 2 ml void volume , was mixed with 700 μg of the antigen , incubated overnight at room temperature and used without any further purification . conjugation was confirmed when the conjugates were separated using 12 . 5 % sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( sds - page ) and a heterogeneous smear ( compared with the single band of uncoupled protein ) was observed using comassie blue stain . for comparison in some experiments , the oxidised conjugates were reduced with sodium borohydride ( nabh 4 ) ( aldrich , castle hill , nsw , australia ) 1 mg / ml overnight at room temperature and used without further purification . immunisation with mannan - antigen conjugates : mice were lightly anaesthetised with penthrane and 50pl of mannan - antigen conjugate ( 12 μg antigen / mouse in bicarbonate buffer ph 9 . 0 , unless otherwise specified ), placed onto the nares to be inhaled by the mouse . the same amount of non - conjugated antigen was similarly applied . unless stated otherwise , this procedure was performed on days 0 , 10 and 17 of the experiments . in some experiments mice were given 12 μg of mannan - llo . fp ( m - llo . fp ) conjugate intraperitoneally in 0 . 2 ml bicarbonate buffer ph 9 . 0 on days 0 , 10 and 17 . cholera toxin ( sigma chemical co ., st louis , mo ., usa ) was also used for comparative purposes as an adjuvant to act as a positive control in some experiments . one microgram of ct was mixed with 12 μg of llo . fp and administered intranasally ( i . n ) in a 50 μl volume on days 0 , 10 and 17 . collection of samples : serum samples were collected after mice were bled by cardiac puncture following euthanasia at the end of each experiment . for time - course experiments mice were placed on a heat box , a small incision made in a lateral vein and 200 μl of blood collected with a micropippeter . the serum was subsequently separated by centrifugation . mouth and vaginal washings were collected from anaesthetised mice , by washing with 50 or 100 μl of phosphate buffered saline ( pbs ) respectively , using a micropippetor . for the collection of faecal samples , mice were placed in cages without sawdust and 2 - 5 fresh stools were collected per mouse . one ml of pbs / sbti ( pbs containing 0 . 1 mg / ml soybean trypsin inhibitor ( sigma chemical co ., st louis , mo ., usa )) was added for every 0 . 1 gm of faeces . the samples were vortexed for 15 minutes and then microcentrifuged at 15 , 000 × g for 15 minutes at 4 ° c . phenyl sulphonyl fluoride ( pmsf ) ( sigma chemical co ., st louis , mo ., usa ) at a final concentration of 1 mm was then added to the supernatants . tears were collected by lightly anaethetising the mice with penthrane and a sliver of filter paper was briefly touched on the upper and lower conjunctiva , collecting about 1 μl of tears . the paper was then immersed in 100 μl pbs - tween to extract antibody [ 19 ]. lung washings were obtained after mice were euthanased with co 2 . lungs were washed in situ with 0 . 5 ml pbs through an opening in the trachea . all samples were stored at − 20 ° c . prior to assay . detection of antibody in the serum and mucosal sites by elisa : microtitre plates ( nunc roskilde , denmark ) were coated overnight at 4 ° c . with 5 μg / ml antigen in carbonate buffer ph 9 . 1 . the wells were then blocked with 2 % foetal calf serum ( fcs ) ( trace biosciences , castle hill , nsw , australia ) in pbs for 1 hour at 37 ° c . the plates were washed 3 times with 0 . 08 % tween 20 ( bdh laboratory supplies , poole , england ) pbs and appropriately diluted samples in 50 μl added and incubated for 2 hours at room temperature . after two more washes , antigen - specific iga was detected by the addition of an anti - mouse iga affinity purified horse radish peroxidase ( hrp ) conjugate ( southern biotechnology associates inc . birmingham , usa ) diluted { fraction ( 1 / 1000 )} in 0 . 1 % bovine serum albumin ( bsa ) ( csl , melbourne , australia ) for 1 hour at room temperature . antigen specific igg1 or igg2a was detected with the addition of a biotinylated anti - mouse igg1 or igg2a conjugate ( caltag laboratories , burlinggame , ca , usa ) diluted { fraction ( 1 / 1000 )} in 0 . 1 % bsa . the plates were washed twice more and a streptavidin peroxidase conjugate ( boehringer mannheim , mannheim , germany ) added at a { fraction ( 1 / 1000 )} dilution in 0 . 1 % bsa . following a further two washes , the antibody titres of all the subclasses tested were determined when the substrates containing either 0 . 4 g / l 3 , 3 ′, 5 , 5 ′- tetramethylbenzidine ( tmb ) ( kirkgaard and perry laboratories , gaithersburg , md ., usa ) and 0 . 02 % h 2 o 2 or 2 , 2 ′- azino - bis ( 3ethylbenthiazoline - 6 - sulphonic acid ) ( abts ) ( sigma chemical co ., st louis , mo ., usa ) and 0 . 03 % h 2 o 2 were added ( 50 μl / well ). plates were left 10 minutes for colour to develop and the reaction stopped with 2m h 2 so 4 for the tmb substrate or 0 . 2m citric acid for abts . od at 450 nm ( tmb ) or 405 nm ( abts ) was read in an elisa reader ( labsystems , helsinki , finland ). antibody titres were presented as the highest dilution which yielded an optical density at 450 nm or 405 nm & gt ; 0 . 1 od units higher than normal serum at { fraction ( 1 / 100 )} dilution . for calculation of means , the titre was converted to log 10 and a geometric mean was derived . spleen cell proliferation and cytokine production : mice were euthanased with co 2 and spleen cells prepared by passage through an 80 gauge wire mesh sieve . red blood cells were lysed using tris - nh 4 cl buffer [ 20 ]. after thorough washing , suspensions of spleen cells were cultured in flat - bottomed 96 well plates at a concentration of 2 × 10 6 / ml in 200 μl volumes with and without the mhc class 11 restricted epitope of llo ( 215 - 226 ) ( 45 μg / ml ) for 3 days at 37 ° c ., 5 % co 2 . for the last 5 hrs the cells were pulsed with 0 . 25 μci [ 3 h ]- tdr ( amersham , buckinghamshire , u . k ) before being harvested onto glass fibre filters ( packard , meridan , conn ., usa ) using the micro 96 harvester ( skatron instruments , wokingham , uk ) and β emissions counted using a packard matrix 9600 ( packard ). for il - 2 bioassay , supernatants from similarly - cultured cells were collected after 24 hour culture and il - 2 was assayed by its ability to cause the proliferation of ctll cells [ 21 ]. elispot : spleen cells were assayed for the frequency of ifn - γ and il - 4 producing cells . maxisorp 96 well plates ( nunc ) were coated overnight at 4 ° c . with 50 μl of 10 μg / ml anti - ifn - γ monoclonal antibody hb170 [ 22 ] or 100 μl of anti - 1 l - 4 monoclonal antibody 11b11 [ 23 ] in carbonate buffer ph 9 . 6 . the plates were washed with pbs and blocked with culture medium at 37 ° c . for 1 hr . cells ( 2 × 10 5 , 1 × 10 5 , 5 × 10 4 , 2 . 5 × 10 4 per well ) were added to the wells in conjunction with the appropriate antigen ( 45 μg / ml ) and incubated for 72 hrs at 37 ° c . in 5 % co 2 . the cells were removed and cytokines detected with the addition of a biotinylated secondary rat anti - mouse anti - ifn - γ antibody xmg 1 . 2 [ 24 ] or rat anti - mouse anti - il4 antibody bvd6 [ 25 ] at a { fraction ( 1 / 1000 )} dilution . following the addition of streptavidin alkaline phosphatase ( boehringer mannheim ) the spots were developed using 5 - bromo - 4 - chloro - 3 - indyl phosphate / nitrobluetetrazolium ( bcip / nbt ) ( sigma ) tablets . the spots were counted using a dissecting microscope and the frequency determined by averaging the number of spots for triplicate wells . statistics : the statistical significance of data was determined by the two - sample ranks test ( wilcoxon - white ) or by student &# 39 ; s t test based on the log 10 titre . differences with p & lt ; 0 . 05 were considered significant . a listeriolysin - mannan ( m - llo ) conjugate was prepared as described in example 1 . llo specific antibodies in serum m - llo . fp following intranasal immunisation : to determine whether the intranasal route of immunisation was superior to intraperitoneal in inducing substantial iga antibody responses , ( cba × balb / c ) f1 mice were immunised i . n or i . p . on days 0 , 10 and 17 with 12 μg m - llo . fp . serum was obtained from three mice 7 days after the final immunisation and antibody levels measured by elisa ( fig1 ). mice immunised i . n with m - llo . fp , produced antigen - specific iga to a geometric mean titre of { fraction ( 1 / 900 )} ( log 2 . 96 ± 0 . 21 ) ( fig1 a ), whereas iga antibody was not detectable after the standard i . p . immunisation . it should be noted that i . p . immunisation could lead to antibody production , as igg1 titres of { fraction ( 1 / 640 )} were detected ( fig1 b ). however , even with this isotype i . n immunisation was superior , inducing titres in excess of { fraction ( 1 / 1280 )}. to further investigate the different antibody classes induced by i . n immunisation , ( cba × balb / c ) f1 mice were immunised i . n with 12 μg of m - llo . fp conjugate on days 0 , 10 and 17 and bled on day 24 . the serum antibody levels from individual mice were measured by elisa ( fig2 ). iga serum antibody responses in m - llo . fp immunised mice ({ fraction ( 1 / 5184 )}) were higher than those obtained from mice immunised i . n with 12 μg of non - conjugated llo . fp ( 1 / 25 ) ( fig2 a ). a significant difference ( p & lt ; 0 . 01 ) in the igg1 response ( fig2 b ), was observed with titres more than 80 × higher for m - llo . fp compared with non - conjugated llo . fp . a significant difference ( p & lt ; 0 . 01 ) was observed between conjugated and non - conjugated llo . fp with igg2a titres of { fraction ( 1 / 32512 )} and { fraction ( 1 / 383 )} respectively ( fig2 c ). infection with listeria monocytogenes , an organism known for its induction of cell mediated immunity , resulted in minimal antibody responses . overall , the greatest percentage increases were noted for the iga and igg2a subclasses of antibody . the experiment was repeated with conjugates prepared on different occasions with three different batches of mannan , and proved to be a very reproducible observation . comparison between oxidised mannan and ct as mucosal adjuvants : in previous studies where ct has been used as a mucosal adjuvant in mice , the immunisation regimes adopted have varied [ 26 , 27 ]. therefore , to compare the adjuvanticity of mannan and ct the initial experiment was performed using the schedule previously found optimal for mannan [ 28 ] of administering antigen and adjuvant on days 0 , 10 and 17 . ( cba × balb / c ) f1 mice were given either 12 μg of mllo . fp or ct + llo . fp i . n . serum , vaginal washings , mouth washings and faecal samples were collected at the times shown on fig3 for titration of iga . iga titres in negative control groups , including mannan alone , ct alone and normal serum , were not more than the limit of detection and are not shown . oxidised mannan was a consistently better mucosal adjuvant than ct when administered with llo . fp . immunisation with m - llo . fp induced a peak geometric mean iga titre of { fraction ( 1 / 667 )} in serum with some titres in excess of { fraction ( 1 / 1280 )}. this compared with { fraction ( 1 / 165 )} for ct + llo . fp ( fig3 a ). llo . fp alone induced a mean titre of only { fraction ( 1 / 69 )}. the superior efficacy of m - llo . fp was also observed at distant mucosal sites . iga titres in vaginal washings ( fig3 b ) were more variable than in serum , but individual mice reached titres & gt ;{ fraction ( 1 / 320 )} ( geometric mean ={ fraction ( 1 / 195 )}) after m - llo . fp , compared with a mean of { fraction ( 1 / 55 )} for ct + llo . fp . llo . fp alone induced a mean titre of { fraction ( 1 / 37 )}. iga titres in the saliva ( fig3 c ) of m - llo . fp immunised mice were only detectable on day 41 of the experiment with a mean of { fraction ( 1 / 10 )} and a high of { fraction ( 1 / 28 )}. titres , although low , were higher than those observed for llo . fp or ct + llo . fp . in both instances , ct was quite a poor adjuvant . in a second series of experiments ( fig4 ), the immunisation schedule was extended to 0 , 28 and 56 days , with doses as before , to test whether a longer time between prime and boost would favour either adjuvant . again the response to m - llo . fp produced higher titres than ct + llo . fp . the peak geometric mean titres following m - llo . fp were { fraction ( 1 / 1040 )} in serum and { fraction ( 1 / 256 )} in vaginal washings . this compared with { fraction ( 1 / 44 )} and { fraction ( 1 / 22 )} respectively following ct + llo . fp . although differences were often not statistically significant because of the variability of individual mice , oxidised mannan was consistently the better adjuvant under two different immunisation regimes . iga titres were examined over a considerable timecourse in these experiments . in general , they did not reach substantial levels until after three injections , whether of m - llo . fp or ct + llo . fp . iga persisted at peak titre in the serum for more than three weeks after the last immunisation , particularly of m - llo . fp ( fig3 a , 4 a ). iga production in the vagina ( fig3 b , 4 b ) also required three immunisations and these titres were more variable than in - serum , and generally less sustained . to extend the investigation of mucosal iga to other sites , ( cba × balb / c ) f1 mice were immunised i . n . with m - llo . fp as before and tears and lung washings collected on days 24 and 35 respectively . mice immunised with m - llo . fp conjugate produced significantly higher titres of iga ( p & lt ; 0 . 01 ) in tears when compared with the unconjugated controls ( geometric means titres : m - llo . fp { fraction ( 1 / 121 )} compared with { fraction ( 1 / 25 )} for llo . fp alone ) ( fig5 a ). the same trend was observed in the lungs at day 35 ( fig5 b ). significantly higher titres of iga were detected in mice immunised with m - llo . fp ( 1 / 975 ) compared with llo . fp alone ( 1 / 38 ) ( p & lt ; 0 . 05 ). to investigate whether there was a need for the mannan to be conjugated to llo . fp or whether the adjuvant effect could be achieved when mannan was simply mixed with antigen , mice were immunised with the m - llo . fp conjugate or with a mannan + llo . fp mixture ( mannan and 12 μg of llo . fp mixed together ). ( cba × balb / c ) f1 mice were immunised i . n on days 0 , 10 and 17 . serum and vaginal washings were taken from individual mice on day 24 of the experiment . iga titres were subsequently determined by elisa ( table 1 ). the results showed that a conjugate provides a better adjuvant effect . the geometric mean antibody titre in the serum for conjugated llo . fp was & gt ;{ fraction ( 1 / 1000 )}. this was significantly higher than the mannan + llo . fp mixture which produced an average titre of { fraction ( 1 / 34 )}. notwithstanding this some antibody is obtained from the mannan antigen admixture . a similar pattern was seen in the antibody response at a distant mucosal site , namely the vagina , with titres of { fraction ( 1 / 195 )} for the conjugate and { fraction ( 1 / 45 )} for the mixture . all negative controls produced undetectable levels of iga with small titres of antibody observed in vaginal washings for the llo . fp alone group ({ fraction ( 1 / 14 )}). earlier experiments with mannan - conjugated proteins injected i . p . showed that conjugates produced under reduced conditions were better at inducing antibody than were oxidised conjugates [ 13 ]. therefore , ( cba × balb / c ) f1 mice were immunised i . n with 12 μg of m - llo . fp conjugate ( oxidised form ) or 12 μg of m - llo . fp conjugate ( reduced form ). the results indicated that the oxidised form of the mllo . fp conjugate given i . n induced higher titres of iga , igg1 and igg2a in the serum ( fig6 a , b and c ) compared with the reduced form . all four mice immunised with m - llo . fp ( oxidised form ) had higher titres than the corresponding four mice given the reduced form of m - llo . fp , with the exception of one high responder mouse in the group given reduced mannan . significant differences ( p & lt ; 0 . 02 ) applied to all the subclasses assayed ( geometric mean titres : iga ={ fraction ( 1 / 1380 )} for oxidised , { fraction ( 1 / 275 )} for reduced ; igg1 ={ fraction ( 1 / 3388 )} for oxidised , { fraction ( 1 / 813 )} for reduced ; igg2a ={ fraction ( 1 / 1660 )} for oxidised , { fraction ( 1 / 173 )} for reduced ). to demonstrate that the use of mannan as a mucosal adjuvant was applicable to other antigens mannan was conjugated to the 19 kda lipoprotein of mycobacterium tuberculosis and to mycobacterium avium antigen hsp65 as set forth in example 1 . c57b1 / 10 mice were immunised i . n . with 12 μg of m - 19fp conjugate on days 0 , 10 and 17 . on day 24 of the experiment the mice were euthanased and bled by cardiac puncture . mice were similarly immunised with mannan hsp65 . serum was separated and iga titres determined by elisa ( fig7 a ). the four mice immunised with m - 19fp produced significantly higher titres of iga ( geometric mean { fraction ( 1 / 1530 )}) when compared with mice given 12 μg of 19 kdafp ( titres of iga & lt ;{ fraction ( 1 / 100 )}). the results for mice immunised with mannan - hsp65 are shown in fig7 ( b ). iga titres for the immunised mice were well above that of the normal mice . the examples above present mannan as a novel mucosal adjuvant which , when administered intranasally , induced high mucosal and serum titres of iga , igg1 and igg2a specific for recombinant protein antigens with which it was administered . oxidised mannan was previously used conjugated to the breast cancer antigen muc1 and injected i . p . into mice where it induced ctl and a th1 cytokines , protecting against tumours expressing muc1 [ 12 , 13 ]. in that context it induced a poor antibody response , dominated by igg2a . it was the change to intranasal administration in the current experiments which resulted production of iga and other classes of antibody in serum and mucosal secretions . the induction of iga responses on mucosal surfaces is a highly desirable outcome in immunisation against a wide spectrum of diseases . amongst other examples , mucosal antibodies and particularly iga , have been shown to protect against influenza in the lung [ 29 ], against helicobacter pylori in the stomach [ 30 ], against haemophilis influenzae in the ear [ 31 ] and against chlamidia trachomatis in the genital tract [ 32 ]. induction of iga and other mucosal antibodies following intramuscular or subcutaneous immunisation with conventional vaccines is poor [ 33 , 33a ]. therefore oxidised mannan may be a valuable adjuvant if coupled to protective antigens from a wide variety of infectious agents . cholera toxin has become the most extensively studied mucosal adjuvant in experimental models , although its relative toxicity has so far precluded it from approval as a human adjuvant [ 34 ]. in the present experiments , immunisation with mannan - conjugated llo intranasally induced superior iga and igg2a responses in the mucosa and serum compared with ct plus llo or llo alone . the difference in the igg1 response was less marked , but still higher for the mannan adjuvant . titres of antibody following immunisation with mannan conjugates were not only higher than with ct , they were more sustained . mannan has the further advantage over ct in being non - toxic and already approved for human use and is currently involved in human trails for cancer therapy [ 14 ]. as with the present observations on mannan , a mixture of the b subunit of ct ( ctb ) with antigen resulted in higher titres of iga in the serum and mucosa when given to mice intranasally rather than intraperitoneally [ 33 ]. it is widely believed that this influence of the route of exposure is a function of the mucosal antigen presenting cells . two interesting observations were made when analysing the form of the antigen / adjuvant in the present experiments . the first showed that oxidised mannan conjugated to the antigen ( llo . fp or 19 kdafp ) facilitates the adjuvant effect . this confirms the observations with the muc 1 studies where conjugation of oxidised mannan to the muc 1 antigen induced better immune responses [ 13 ]. the second observation contrasts with the muc 1 studies because the oxidised form of the m - llo . fp induced maximum antibody production . immunisation with the oxidised form of mannan - muc 1 resulted in low antibody levels [ 13 ]. how , then , does mannan act as an adjuvant ? it has been shown that antigens bearing mannose residues are bound to the mannose receptors on antigen presenting cells ( apc ), facilitating uptake into the mhc class ii pathway for efficient antigen presentation [ 35 , 36 , 37 ]. oxidised mannan - muci induced both class i restricted ctl and a th1 response [ 12 ]. it has been suggested that oxidised mannan - muc1 , by virtue of the aldehyde residues created by the oxidation process , escapes the phagocytic pathway and is transported into the mhc class i pathway inducing ctl [ 15 ]. this pathway would not be open to reduced complexes , where aldehyde residues are reduced to alcohols by the action of boral hydrate . the exact mechanism by which aldehyde acts is unknown . curiously , the inventors did not detect the induction of class i - restricted ctl in experiments with m - llo . fp injected i . p . furthermore , the response to the inventor &# 39 ; s antigens given intranasally was not classically either th1 or th2 , since igg1 and iga ( generally considered th2 responses ) and igg2a ( th1 ) were allelevated . this was confirmed by cytokine assays where both ifn - γ and il4 were elevated . this aspect is further explored in example 5 . the mechanism by which m - llo . fp and m - 19fp induce such excellent antibody responses after i . n immunisation is unknown . others have shown preferential induction of th2 responses following intranasal instillation of leishmania antigens , even in strains of mouse which are genetically constrained to produce a typical th1 response to these organisms injected parenterally [ 38 ]. without wishing to be bound by theory it may be that alveolar macrophages or dendritic cells , which have been shown to present antigens efficiently in other systems [ 39 ], are playing a significant role in the induction of these iga responses . induction of mucosal immunity begins with the uptake of antigen by membranous ( m ) cells ( specialised epithelial cells ) on the mucosal surface . these cells either process and present antigen to underlying t - cells or b - cells themselves or transport antigen to parenchymal macrophages , dendritic cells and b - cells . once interaction of the antigen presenting cell ( apc ) with t and b lymphocytes has occurred , an immune response or mucosal tolerance may result . immune responses generally involve antibody production , with iga the predominant antibody isotype . antigen sensitised immune cells are then circulated to other systemic and mucosal sites for expansion of effector mechanisms [ 40 ]. the fact that there is a preferential circulation of t cells activated at mucosal sites to return to the same or other mucosal sites accounts for the induction of iga at remote mucosal surfaces . oxidised mannan - listeriolysin o conjugates induce th1 / th2 cytokine responses after intranasal immunisation clearance of infectious organisms does not always require polarised th1 or th2 responses . it may in fact be advantageous for both a th1 and th2 response to be elicited for effective protection against an invading pathogen . it was the aim of this example to investigate oxidised mannan as a possible th1 / th2 adjuvant . mice , production of antigens , conjugates and immunisation were the same as example 1 . spleen cell proliferation , cytokine production and elispot assays were as described in example 1 . having established that the intranasal route of administration produced better antibody responses than those measured after intraperitoneal immunisation as described in example 2 , a direct comparison between the two routes of immunisation was undertaken to determine if a similar pattern could be established for cellular responses . ( cba × balb / c ) f1 mice were immunised i . n . or i . p . on days 0 , 10 and 17 with 12 μg of m - llo . fp . on day 24 the mice were sacrificed and spleens removed . spleen cell preparations were used to establish proliferation , il - 2 and ifn - γ elispot assays . spleen cells were cultured in the presence of the mhc class ii restricted epitope of llo ( 215 - 226 ) and incorporation of [ 3 h ]- tdr measured to determine the proliferative response ( fig8 a ). mice immunised with m - llo . fp intranasally produced counts significantly higher than the group of mice which had the m - llo . fp administered by the intraperitoneal route ( 3972 ± 502 vs 983 + 145 , p & lt ; 0 . 002 ). the latter approximated background levels . llo . fp without adjuvant by either route induced background levels of proliferation only . the ability of spleen cells to produce il - 2 is shown in fig8 b . spleen cells were cultured in the presence of llo ( 215 - 226 ) or llo . fp ( 45 μg / ml ) and supernatants harvested at 24 hours . the ability of the supernatants to maintain the il - 2 dependent ctl cell line was determined by the incorporation of [ 3 h ]- tdr . mice immunised with mllo . fp intranasally produced counts of 1219 ± 268 ( llo ( 215 - 226 )) and 4523 ± 689 ( llo . fp ). these values were significantly higher ( p & lt ; 0 . 01 , p & lt ; 0 . 002 ) than the group of mice which received the m - llo . fp administered by the intraperitoneal route with geometric means of 182 ± 73 ( llo ( 2 15 226 )) and 417 ± 40 ( llo . fp ), which approximated background levels . llo . fp without adjuvant by either route induced background levels of il - 2 . importantly this pattern was also observed with the ifn - γ elispot ( fig8 c ). spleen cells were cultured in the presence of llo . fp and the frequency of ifn - γ - producing cells determined by elispot . mice immunised with m - llo . fp intranasally produced a high frequency of ifn - γ - producing cells / 10 6 spleen cells ( 58 ± 20 ), significantly higher ( p & lt ; 0 . 05 ) than the group of mice which had the mllo . fp administered by the intraperitoneal route ( 10 ± 8 ) which approximated background levels . mice given llo . fp by either route without adjuvant recalled levels of ifn - γ producing cells just above background . intranasal immunisation with 12 μg of m - llo . fp resulted in significant proliferative and th1 cytokine ( il - 2 and ifn - γ ) responses , but th1 responses are often favoured by lower doses of antigen [ 41 ]. therefore , ( cba × balb / c ) f1 mice were immunised i . n . on days 0 , 10 and 17 with 12 μg , 6 μg or 3 μg of m - llo . fp and on day 24 the mice were sacrificed and spleens removed . proliferative responses and the frequency of ifn - γ - producing cells from spleen cell preparations were measured . proliferative responses after in vitro stimulus with the mhc class ii restricted epitope of llo indicated that intranasal immunisation with the 12 μg dose was significantly higher than the 6 μg ( p & lt ; 0 . 02 ) and 3 μg ( p & lt ; 0 . 01 ) dose of m - llo . fp ( 6385 ± 1506 , 1962 ± 649 and 1630 ± 375 respectively ). cells from unconjugated doses of llo . fp produced levels of proliferation slightly above background ( results not shown ). a similar pattern of results was observed for the production of ifn - γ . the ifn - γ producing cells / 10 6 spleen cells for the 12 μg dose was significantly higher than for the 6 μg ( p & lt ; 0 . 01 ) and 3 μg ( p & lt ; 0 . 01 ) dose of m - llo . fp ( 97 ± 20 , 32 ± 8 and 12 ± 6 respectively ). mice given the unconjugated control antigen all produced fewer than 50 ifn - γ producing cells / 10 6 spleen cells ( results not shown ). earlier studies with mannan - conjugated proteins injected intraperitoneally showed that conjugates produced under oxidising conditions were better at inducing th1 responses compared to conjugates produced under reduced conditions [ 13 ] that favoured th2 responses . to test this hypothesis ( cba × balb / c ) fl mice were immunised i . n . with 12 μg of m - llo . fp conjugate ( oxidised form ) or 12 μg of m - llo . fp conjugate ( reduced form ) on days 0 , 10 and 17 and mice sacrificed on day 24 . a comparison of the two forms of conjugate for their ability to produce the th1 cytokine ifn - γ or the th2 cytokine il4 was performed using the elispot assay . spleen cells were cultured in the presence of llo . fp and the frequency of ifn - γ or il4 producing spots determined ( fig9 ). the results clearly indicated that the oxidised form of the conjugate produced significantly higher frequencies of both ifn - γ and il - 4 producing cells ( 107 ± 12 and 123 ± 12 respectively ) when compared to the reduced form ( 32 ± 10 and 57 ± 38 ) ( p & lt ; 0 . 02 , p & lt ; 0 . 05 ). there was no indication that , using this antigen and route of immunisation , that the oxidised form induced th1 and the reduced form induced th2 cytokines . however this may be dose - dependent . these experiments present oxidised mannan as an adjuvant which , when administered intranasally , induced proliferative responses , il - 2 production , ifn - γ production and il - 4 production by cultured lymphocytes . comparison of intranasal and intraperitoneal routes of immunisation revealed the superior efficacy for induction of these immune responses by the intranasal route . as most proteins administered to the mucosal surface without adjuvant fail to induce systemic or mucosal immune responses , oxidised mannan may be used as an adjuvant to induce both th1 and th2 immune responses . it offers the great advantage as an adjuvant of being non - toxic and already approved for human use in the context of breast - cancer therapy . there is evidence in the literature that suggests that variations in antigen dose can polarise immune responses , inducing either dth or antibody responses depending on antigen concentration . it is generally believed that low doses of antigen favour th1 responses and high doses of antigen favour th2 responses [ 41 ]. the results described in this study do not support this . low doses of m - llo . fp conjugate i . n . were unable to induce th1 responses . interestingly , the highest dose of m - llo . fp conjugate was responsible for increases in the th1 cytokine ifn - γ . these results contradict earlier observations where immunisation with low doses of mannan - conjugates favoured th1 responses [ 28 ]. it appears that oxidised mannan administered intranasally can be an effective adjuvant for the induction of th1 and th2 responses as seen with the two model antigens used in this example . this ability is of particular interest with infections such as c . trachomatis or h . pylon where both cell mediated and humoral responses appear necessary for control of infection . in the case of c . trachomatis , cell mediated responses alone appear to contribute to immunopathology whereas antibody responses are not sufficient to control infection [ 42 , 43 ]. it has been suggested in the case of h . pylon that th1 and th2 responses are required at different stages for control of infection . the early response is dominated by th1 and the late by th2 [ 44 ]. candidate vaccines such as those contemplated by the present invention with the ability to produce both th1 and th2 responses might therefore prove to be effective against both pathogens . mice and immunisation thereof were as described in example 1 . samples of serum , tears and / or stools were collected as described in example 1 at days 24 and 34 after immunisation . on day 35 the mice were sacrificed and lung washings were collected as shown in example 1 . detection of antibody in the samples was carried out as described in example 1 . the results are represented in fig1 and 12 . fig1 shows that in mice immunised intranasally with 6ug vp5 246 - 274 - mannan , titres of serum iga , igg1 and igg2a were significantly higher than in mice immunised intranasally with the antigen alone , at 24 and 35 days after immunisation . [ 0135 ] fig1 shows the presence of iga in tears collected 24 days after immunisation with the antigen - mannan conjugate , and in the stools collected at day 34 . no antibody was detected in mice receiving antigen alone . iga in lung washings was also significantly higher than in samples from animals injected with the antigen alone . the antigen used was the major virulence factor of porphyromonas gingivalis , the extracellular complex of arg - and lysspecific proteases and adhesions designated the rpga - kgp complex ( formerly the prtr - prtk complex and abbreviated cpx ), prepared and conjugated with mannan as described in example 1 . cpx antigen ( 12 μg ) was administered intranasally on days 0 , 10 and 17 . alternatively , the cpx antigen was administered subcutaneously in incomplete freunds adjuvant as previously described [ 18 ]. mice were infected with 8 × 10 9 cells of porphyromonas gingivalis subcutaneously in the abdomen on day 30 and lesion sizes measured over 14 days . lesion sizes were statistically analysed using the kruskal - wallis test and the mann - whitney u - wilcoxon rank sum test with a bonferroni correction . groups of 5 c57b1 / 10 mice were immunised with cpx - mannan given intranasally or cpx in incomplete freund &# 39 ; s adjuvant ( ifa ) subcutaneously . control mice received the respective adjuvants alone . results of protection against challenge with porphyromonas gingivalis are shown in fig1 . cpx linked to mannan afforded complete protection . ifa , the standard adjuvant used in studies of porphyromonas gingivalis vaccination afforded only partial protection . interestingly , the serum antibody titres , although raised in the mice immunised with cpx - mannan , was higher in the mice immunised with cpx in ifa . both had significant adjuvant effects ( p & lt ; 0 . 05 ). this emphasises the point that , particularly where inflammatory lesions are critical to disease , antibody titre may not be an accurate predictor of protection . mice were immunised with hpv - e7 conjugated to oxidised mannan , e7 conjugated to reduced mannan and with e7 alone , as per example 1 . the results ( fig1 ) show no substantial difference between the oxidised and reduced forms although this may have been due to dose levels . antigens used in accordance with the invention may also be produced by conventional peptide synthesis after selecting putative immunogenic peptides . the putative immunogenic peptides may be selected from published studies or may be peptides suspected of being immunogenic based on a skilled person &# 39 ; s knowledge of the art . the putative immunogenic peptides are then coupled to a carrier protein which is , in turn , conjugated to mannan . alternatively , plasmids containing genes encoding selected antigens are expressed in e . coli . the proteins coupled to mannan are administered and the antibody response at the site most relevant to protection is examined . examples of proteins that can be tested are as follows : protective site of organism disease antigen antibody influenza flu haemagglutinin lungs virus helicobacter stomach ulcers , recombinant stomach pylori cancer proteins , urease rotavirus gastroenteritis recombinant vp4 intestine in infants protein and its fragment vp5 and other outer capsid proteins chlamydia sexually transmitted major outer vagina , eye trachomatis disease , pelvic membrane protein inflammatory disease , non - specific urethritis , trachoma mannan - antigen complexes are encased in various timeddelivery capsules and delivered intranasally , orally or rectally . at intervals , serum , saliva , lung or vaginal washings will be collected , and elisa are used to measure igg1 , igg2a , ige and iga . in this study the inventors have shown the application of the invention to a number of antigens . in addition , they have shown that antibodies to the antigens have been detected at a number of sites including serum , lungs , vagina , lachrymal glands and colon . these results are surprising for a number of reasons . past studies have demonstrated that mice immunised by standard methods with oxidised mannan - human muc1 conjugate produced cellular immunity but little humoral immunity ( i . e . antibody ). whereas when the reduced form of the same conjugate was administered to mice by standard methods only a serum antibody response was found . other studies have shown that when various antigens ( muc1 , lysteriolysin ) conjugated to oxidised mannan were administered to humans , mice and monkeys mostly igg1 antibodies were found . there was no iga response . given this background it was extremely surprising that intranasal administration of mannan - antigen gave rise to secreted iga . the work done by the inventors indicates that intranasal administration of mannan and antigen may advance vaccine development for sexually transmitted diseases such as chlamydia or human immunodeficiency virus , gum disease , eye diseases , other mucosally acquired infections like influenza and rotavirus or even have veterinary application with uses in animal infections or pest control . the fact is that this is a novel , non - toxic adjuvant that induces systemic and mucosal antibody responses superior to the standard mucosal adjuvant ct . it is to be understood that although preferred embodiments of the invention have been described in detail , various modifications , variations and adaptations may be made by those skilled in the art without departing from the central concept of the invention . it is also to be understood that the term “ comprise ” or any of its grammatical equivalents has the same scope as the term “ include ” and its grammatical equivalents . 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