Patent Application: US-48556904-A

Abstract:
the invention relates to a liposome comprising a chloroform soluble and extractable total polar lipid of mycobacterium spp , particularly a chloroform soluble extractable total polar lipid of mycobacterium spp bcg . the chloroform soluble and extractable polar lipid may comprise at least one of phosphatidylinositol , phosphatidylinositol mannoside , phosphatidylinositol dimannoside , mono and dipalmitoylated forms of pim 1 and pim 2 , phospholipid of 899 m / z , phosphatidylethanolamine and cardiolipid . the liposome may be prepared by drying chloroform soluble and extractable lipid and then hydrating said dried lipid at a temperature of 65 to 75 ° c . in water or phosphate buffered saline . the liposome may be used , for example , to activate dendritic cells to secrete cytokines and modulate an immune response in a mammal , or to direct an immune response to confer protection against a pathogen or a cancer .

Description:
liposomes are closed spherical vesicles composed of a lipid bilayer with polar headgroups exposed to inner and outer surfaces and the lipid chains forming the interior part of the bilayer . water - soluble drugs or antigens are either bound to the surface or entrapped in the fluid space within the liposome , whereas hydrophobic molecules tend to associate with the lipid layer . in the present invention the total lipids from fresh bcg cells are extracted with ambient temperature methanol / chloroform / water ( 2 : 1 : 0 . 8 , v / v ), and the polar chloroform - extractable lipids separated from neutral lipids as the cold - acetone insoluble fraction . we show for the first time that this total polar lipid fraction , and purified lipids therefrom such as pims and palmitoyl - pims , will form liposomes provided the temperature is sufficiently high and preferably 65 - 75 ° c . animals may then immunized with bcg liposomes associated with one or more protective antigens to confer protection to pathogens or cancer where a strong immune response is required , or for the production of high antibody titres for research purposes . adjuvant activity includes not only the mhc class ii mediated th2 and antibody arm of the immune response , but also mhc class i responses evident by induction of ctl responses and inf - gamma secreting cd8 + t cells in the elispot assay and by showing that a bcg liposome vaccine can protect in a mouse tumor model . there have been no previous reports to our knowledge demonstrating either liposome construction from the lipids of mycobacteria , and specifically from the polar - extractable lipids , or reports of immunomodulation by such liposomes , thus obviating the need to include additional adjuvants in a bcg liposome vaccine . because temperatures above 55 ° c . are required to prepare liposomes efficiently from bcg total polar lipids , or its purified components , it follows that at lower temperatures such as body temperatures the liposome membranes would pass from liquid crystalline phase to solid phase . in solid phase the membranes would be less leaky to entrapped antigen and stability of the liposomes would be enhanced resulting in an improved vaccine carrier . further , several of the purified bcg lipids when converted to liposomes have immunostimulatory activity , shown herein by the activation of dendritic cells , the most potent cell type for processing and presenting antigen to t cells . the active lipids are composed of a glycerol backbone linked sn - 1 , 2 with saturated fatty acyl c19 : 0 chains of tuberculosteric ( unique to mycobacteria ) and c16 : 0 palmitic acids , and a phosphoinositol headgroup at the sn - 3 position linked to 1 or 2 mannose sugar residues ( pim1 , pim2 ), sometimes mono - or dipalmitoylated . these active lipids may be obtained from bcg cells that are grown easily with high yield , or alternatively it may be appreciated they may be chemically synthesised , as their structures are known . further , the tuberculosteric acid found in mycobacteria species is a methyl - branched , long - chain , fully saturated fatty acyl chain , and as such is predicted to contribute to liposome stability . indeed , the only bcg lipid that has unsaturation is the cardiolipids fraction 8 . [ 0036 ] mycobacterium bovis ( bcg ) pasteur strain was obtained from dr . robert north ( trudeau institute , usa ) and grown aerobically in 1 - liter shake flasks containing standard complex medium . bacillus firmus was purchased from the american type culture collection ( attc 14575 ) and grown aerobically on nutrient broth 8 g / l , yeast extract 3 g / l ( difco laboratories , mich . ), urea 1 . 5 g / l and bactopeptone 1 g / l at 30 ° c . el - 4 and eg . 7 , a subclone of el - 4 stably transfected with the ova gene , were obtained from the attc , and maintained and grown as described before ( krishnan et al . 2000 ). briefly , total lipid extracts were obtained from frozen - thawed cell pastes of b . firmus , or from fresh cell paste of m . bovis by adding a one - phase solution of methanol , chloroform , and water ( 2 : 1 : 0 . 8 , v / v ) in a ratio of 15 g cell dry weight / l . after 16 h the cellular debris was collected by centrifugation and re - extracted twice more . extracts were pooled and made biphasic by addition of chloroform and water by the bligh and dyer method previously described ( sprott et al . 1995 ). polar lipids in the chloroform bottom phase were freed of neutral lipids by differential solubility in cold acetone ( sprott et al . 1995 ). polar lipids , insoluble in cold acetone , were dried and dissolved into chloroform as the chloroform - extractable total polar lipids . bcg total polar lipids dissolved in chloroform were filtered using a 0 . 45 μm nylon syringe - filter , to ensure there was no carry over of whole cells into the lipid extract . 1 ) b . subtilis liposomes — about 30 mg of total polar lipids in chloroform were dried under a nitrogen stream , and hydrated by adding 3 . 0 - ml of pyrogen - free water . hydration was allowed to proceed for 2 - 3 h at 35 ° c . with shaking prior to the addition of 10 mg ovalbumin ( ova )/ 30 mg lipid . average vesicle diameters were decreased from 80 to 100 nm in a sonic bath . preparations were then freeze - dried and re - hydrated in phosphate buffered saline ( pbs , 10 mm potassium phosphate plus 160 mm nacl , ph 7 . 1 ). ova was removed by centrifugation and three washes with pbs . the final liposome pellets were re - suspended into pbs , and liposomes filter - sterilized using syringe - driven 0 . 45 μm filters ( millipore , mass .). entrapped ova was quantified after lipid removal by the sds - lowry colour development method as described before ( krishnan et al . 2000 ) and dry weights determined . pyrogen - free sterile water was used throughout . 2 ) commercial lipids — l - α - dimyristoylphosphatidylcholine ( dmpc ), l - α - dimyristoylphosphatidylglycerol ( d ) mpg ), l - α - phosphatidylinositol ( soybean , pi ), cardiolipid ( bovine heart ), and l - α - dipalmitoylphosphatidylethanolamine ( dppe ) were purchased from sigma . liposomes were prepared as described for total polar lipids of b . subtilis , except for the omission of ova . 3 ) bcg liposomes — about 30 mg of total polar lipids in chloroform were dried under a nitrogen stream followed by 1 - h under vacuum . hydration was routinely done by adding 3 - ml of pyrogen - free water containing the antigen ( for example 10 mg ova ) and incubating for 2 - 3 h at 65 ° c . with shaking . to investigate the effect of temperature on liposome formation , hydration was allowed to occur at 35 ° c . to 75 ° c ., in 10 ° c . steps . average vesicle diameters were decreased between 80 - 100 nm in a sonic bath at 65 ° c . preparations were then freeze - dried and re - hydrated in pbs at 65 ° c . liposomes were left overnight at 4 ° c . to anneal , then any ova not associated with the liposomes was removed by ultracentrifugation and washing liposomes with pbs thrice . the final liposome pellets were re - suspended into pbs , and liposomes filter - sterilized using 0 . 45 μm filters . entrapped ova was quantified after lipid removal and dry weights determined , as above . average diameters were measured in a 5 mw he / ne laser particle sizer ( nicomp model 370 ). bcg liposomes were made from isolated lipid fractions 1 to 8 by the above method , except for bcg pe fraction 7 . pe liposomes were made by including 80 mole % dmpc , as pe lipids in general do not make liposomes in pure form . this also was the case for bcg pe . polar lipid extracts were analysed by fast atom bombardment mass spectrometry ( fab ms ) with a jeoljms - ax 505h instrument operated at 3 kv in negative ion mode . the xenon gun was operated at 10 kv . current - controlled scans were acquired at a rate of 10 - s full scale . a mixture of triethanolamine and kryptofix ® ( sigma ) was used as the matrix . staining for functional groups was done after separating the polar lipids on pre - coated 0 . 25 mm silica gel 60 thin - layer plates ( merck ) developed with an acidic solvent chloroform / methanol / acetic acid / water ( 85 : 22 . 5 : 10 : 4 , v / v ) or basic solvent chloroform / methanol / 7 n ammonium hydroxide ( 60 : 35 : 8 , v / v ). lipid spots were characterized using the phospholipid ( zinzade &# 39 ; s reagent ), glycolipid ( α - naphthol ), aminolipid ( ninhydrin ), and total lipid ( sulfiric acid char reagent ) sprays described in kates ( 1986 ). for sugar analysis lipids were first hydrolysed with 2 m trifluoroacetic acid for 2 h at 100 ° c . d - ribose was then added as an internal standard , and alditol acetate derivatives prepared for identification and quantification by gas chromatography - mass spectrometry ( gc ms ) ( 17 ). the total carbohydrate content of each lipid extract was determined by anthrone reaction using d - glucose as the standard . bone marrow derived dendritic cells were prepared as described before ( krishnan et al . 2001 ), and were consistently & gt ; 80 % cd11c + by flow cytometry . briefly , bone marrow was flushed from the femurs and tibias of c57bl / 6 mice , and single cell suspensions made . cells obtained were cultured ( 1 × 10 6 / ml ) in rmpi medium supplemented with 8 % fetal bovine serum , fbs ( r8 ) and 5 ng / ml of recombinant murine gm - csf ( id labs , london , on , canada ) for 6 - 8 days at 37 ° c . in 8 % co 2 . non - adherent cells were removed at days 2 and 4 of culture , and fresh r8 plus gm - csf was added . dendritic cells were harvested on days 6 - 8 as non - adherent cells . dendritic cells ( 10 5 ) were incubated in vitro with various concentrations of antigen - free archaeosomes or lipopolysaccharide ( lps , e . coli , sigma ), in triplicate in 96 - well tissue culture plates , for 72 h at 37 ° c ., 8 % co 2 , in a humidified atmosphere . at 72 h , activation of the cells was assessed by measurement of mtt ( dimethylthioazol diphenyltetrazolium bromide ) uptake and the supernatants were collected and il - 12 was assayed by sandwich elisa ( mosmann & amp ; fong 1989 ). tnf was assayed by a bioassay referenced in krishnan et al . 2001 . female , c57bl / 6 mice , 6 - 8 weeks of age , were immunized subcutaneously at the base of the tail at 0 and 21 days . immunizations were with 15 μg ova either with no adjuvant , or ova entrapped in liposomes prepared from the total polar lipids extracted from bcg or b . firmus . in some cases fca was included in the first immunization and freund &# 39 ; s incomplete adjuvant ( fia ) in the second at 62 % strength with 15 μg ova in pbs . sera were collected from blood obtained from the tail veins of mice and analysed for anti ova antibodies . the antibody titres were determined by indirect antigen - specific elisa . briefly , elisa plates ( eia microtitration plates , 96 - well flat bottom , icn biomedicals inc ., aurora , ohio ) were coated with antigen in pbs ( 10 μg / ml ), and serial two - fold dilutions of serum ( from individual mice ) were assayed in duplicate . hrp - conjugated goat anti - mouse immunoglobulin ( igg + igm ) revealing antibody ( caltag , san francisco , calif .) was used to determine total antibody titres of sera . the reactions were developed with abts microwell peroxidase system ( kirkegaard and perry laboratories , gaithesburg , md .) and absorbance determined at 415 nm after 15 min . antibody titres are represented as endpoint dilutions exhibiting an optical density of 0 . 3 units above background . ctl assays for ctl assays , 30 × 10 6 spleen cells were cultured with 5 × 10 5 irradiated ( 10 , 000 rads ) eg . 7 cells in 10 ml of rpmi plus 8 % fbs containing 0 . 1 ng / ml il - 2 , in 25 cm 2 tissue culture flasks ( falcon ), kept upright . after 5 days ( 37 ° c ., 8 % co 2 ), the cells recovered from the flask were used as effectors in a standard 51 cr - release ctl assay and % specific lysis against eg . 7 targets determined ( krishnan et al . 2001 ). enumeration of ifn - γ secreting cells was done by elispot assay ( vijh and pamer , 1997 ). briefly , spleen cells were incubated in anti - ifn - γ antibody coated elispot plates in various numbers ( in a final cell density of 5 × 10 5 / well using feeder cells ) in the presence of il2 ( 1 ng / ml ) and rpmi media or ova 257 - 264 ( 10 μg / ml ) for 48 h at 37 ° c ., 8 % co 2 . the plates were subsequently blocked , incubated with the biotinylated secondary antibody ( 4 ° c ., overnight ), followed by avidin - peroxidase conjugate ( room temperature for 2 h ). spots were revealed using di - amino benzidine . a murine solid tumor model was used to assess the relative protective potential of cd8 + t cells induced by bcg tpl liposomes with ova entrapped . mice were injected twice at 0 and 21 days with ova , 15 μg / 0 . 1 ml injected per mouse , given subcutaneously . eg . 7 cells ( 5 × 10 6 ) expressing ova ( in pbs plus 0 . 5 % normal mouse serum ) were injected in 0 . 1 ml in the shaved lower dorsal region , 9 weeks post first injection . from day 5 onwards , detectable solid tumors were measured using callipers . tumor size , expressed in mm 2 , was obtained by multiplication of diametrically perpendicular measurements . in this invention centrifuged cell pellets of bcg ( 10 g ) are extracted at ambient temperature by stirring for 24 h with 1 liter of 1 - phase bligh and dyer consisting of methanol / chloroform / water ( 2 : 1 : 0 . 8 , v / v ). the mixture is centrifuged at 10 , 500 × g for 15 min and supernatant and pellet fractions separated . the pellet fraction is extracted twice more as above and the three supernatants combined . upon storing at 4 ° c . a cloudy white precipitate forms and is removed by centrifuging at 4 , 100 × g for 15 min . this supernatant contains most of the chloroform extractable lipids . the small pellet removed is extracted again with 1 - phase and the supernatant from this extraction combined with the chloroform extractable lipids . a volume of chloroform and water each equal to the total volume of the chloroform extractable lipids divided by 3 . 8 is added to obtain a 2 - phase system in glass separatory funnels . the bottom chloroform phase containing the desired lipids is removed , and two 200 - ml volumes of chloroform are used to wash the upper methanol - water phase . these chloroform washes are combined with the first chloroform phase . also combined with the chloroform phases is chloroform recovered by centrifuging the milky emulsions formed at the interface of the two phases . total polar lipids are recovered from the chloroform phases concentrated by flash evaporation by precipitating upon adding 20 - volumes of ice - cold acetone . the pellet obtained is washed twice with ice cold acetone , dissolved in chloroform , and finally filtered through nylon 0 . 45 μm filters to obtain chloroform extractable total polar lipids ( tpl ). the total lipids account for about 10 . 2 % of the starting bcg cell weight , of which 65 % is total polar lipids and 35 % acetone soluble lipids . a typical fab ms spectrum of the total polar lipids is shown in fig1 . dominant lipids were assigned as pe , pi , pim 1 , palmitoyl - pim 1 , pim 2 , palmitoyl - pim 2 , and cardiolipid . dominant fatty acid carboxylate anions generated from the polar lipids during ms analysis are c16 : 0 , c19 : 0 and c18 : 1 . the signal of m / z 297 . 3 corresponds to the m . tuberculosis c19 : 0 fatty acid , 10 - methyloctadecanoate , known as tuberculosteric acid ( leopold and fisher 1993 ). a headgroup analysis shows mannose to be the major sugar present in about equal amount to inositol ( table 1 ). a hot ethanol soluble lipid fraction may be obtained from the cell pellet above , already extracted three times with 1 - phase bligh and dyer solution , by dispersing into 50 % ethanol and refluxing at 65 ° c . for 8 h . the mixture is centrifuged at 10 , 500 × g for 20 min and the supernatant flash evaporated to remove the ethanol . the remaining liquid is extracted with 1 - phase bligh and dyer solution and made 2 - phase to recover the hot ethanol lipids in the chloroform phase . hot ethanol extracted lipids were similar to tpl in mannose and inositol content and represent roughly half of the mannose and inositol recovered in tpl lipids ( table 1 ). thin layer chromatography and fab ms show the same lipids present as in tpl . thus , although only the chloroform extractable tpl is used herein , it is appreciated that tpl yield may be increased by combining , or replacing with a hot ethanol extraction . it may also be appreciated that a hot ethanol extraction may be used as an alternative to the bligh and dyer method to obtain essentially the same lipids using less costly and less toxic solvent . thin layer chromatography is used to separate tpl into 8 fractions , all of which stain positively for phosphate ( fig1 ). fraction 4 has similar mobility to standard pi and fraction 7 corresponds to a pe standard . staining reactions further define these 8 lipid fractions ( table 2 ). lipids 1 , 2 , 4 and 5 are phosphoglycolipids , 3 , 6 and 7 are phospholipids , and lipid 7 is a phosphoaminolipid . to purify components of tpl , bcg tpl is applied as a band ( 6 mg / plate ) and separated in this way into the 8 fractions by locating bands with iodine vapor and recovering the chloroform soluble lipids from the removed adsorbent . bands 3 and 4 merge and may be recovered together , then separated and recovered using another thin layer plate and an alkaline solvent . the relative abundance of each recovered fraction 1 to 8 is shown in table 2 . the 8 lipid fractions are defined structurally by fab ms analysis in fig3 to 10 . fraction 1 consists of pim 2 and an 899 . 5 m / z lipid . both have c19 : 0 and c16 : 0 chains as only these two chains are seen as carboxylate anions ( fig3 ). the 899 . 5 m / z lipid is clearly in low relative amount , as it is not seen in a spectrum of tpl ( fig1 ). an acyl - pgp lipid structure consistent with the above spectrum is shown in fig3 in which the acyl group must be 57 m / z ( either a propionic fatty acyl or butyl group ). in these structures of bcg lipids , glycerol moieties are shown in ‘ stick ’ form and tuberculosteric acid chain position is sn - 1 based on gilleron et al . ( 2001 ). lipid fraction 2 is primarily palmitoyl - pim 2 with c19 : 0 and c16 : 0 chains ( fig4 ), and fraction 3 is pure pi ( fig5 ). in the case of pi most molecules have c19 : 0 plus c16 : 0 chains , with the bulk of the remaining pi having c19 : 0 and c 15 : 0 chains . fraction 4 is a phosphoglycolipid defined by fab ms as dipalmitoyl - pim 2 of various sn - 1 , 2 chain forms ranging from c35 : 0 to c38 : 0 ( fig6 ). pi is detected also in this fraction . palmitoyl - pim 1 and pi in about equal amount comprise fraction 5 ( fig7 ) a phosphoglycolipid fraction of only 3 % abundance in bcg tpl ( table 2 ). fraction 6 is a phospholipid of m / z 899 . 1 ( fig8 ) comprising only 1 . 2 % of tpl . the most dominant pa fragment ion is 731 . 2 m / z indicating a methyl - pgp with sn - 1 , 2 tuberculosteric acid fatty acid chains . however , other pa fragment anions , and fatty acid carboxylate signals , indicate pgp molecules with other sn - 1 , 2 - chains and acyl moieties on the terminal phosphate to total a m / z of 899 . 1 , the primary signal for the molecular anion . clearly , fraction 7 is pure pe with several sn - 1 , 2 chain combinations , primarily c18 : 0 plus c16 : 0 ( c34 : 0 ). pa fragment ions at m / z 647 . 1 and 675 . 1 confirm the pe assignments and correspond to fragments from c32 : 0 and c34 : 0 , respectively . finally , mobility of thin layer plates , staining reaction , and fab ms identifies fraction 8 as a cardiolipid with mainly c18 : 1 and c16 : 0 chains and a molecular anion signal of 1403 . 2 m / z ( fig1 ). contrary to expectation bcg tpl ( total polar lipid ) did not form liposomes at the normal growth temperature of m . bovis bcg , namely 37 ° c . tlp lipids were dried from solvent and liposomes monitored by phase microscopy after addition of water , or pbs buffer , at 35 , 45 , 55 , 65 , and 75 ° c . liposomes did not form well at 55 ° c . or less , resulting in clumps of lipid , but elevating the temperature to 65 or 75 ° c . resulted in a dramatic formation especially when water was used for hydration . in this invention then , chloroform extractable bcg tpl is hydrated preferably at 65 ° c . in the presence of antigen to form multilamellar liposomes . smaller liposomes are produced , if desired , by size reduction at preferably 65 ° c . using a bath sonicator . other methods of size reduction could be used if the temperature is 65 ° c . entrapment of antigen may be improved by lyophilization and rehydration of the liposome powder in water at 65 ° c ., followed by pbs . liposomes are then annealed and any unentrapped antigen removed as described in materials and methods . average diameters were 230 ± 136 nm with ova loadings in 3 preparations ranging from of 33 to 67 μg / mg dry weight of liposomes . average diameters of bcg liposomes made from the purified lipid fractions are shown in table 3 . those skilled in the art will appreciate that the various methods described in liposome formation should apply to these bcg lipids providing care is taken to achieve the required temperature , preferably 65 ° c . activation of dendritic cells by bcg tpl liposomes and liposomes prepared from purified lipids traction 1 to 8 because dendritic cells represent the major antigen presenting cells in mammals , they are the preferred cells for in vitro adjuvant testing . bone marrow dendritic cells were cultured with zero ( r8 medium only ) to 10 μg dry weight liposomes / ml of r8 medium . liposomes tested are shown in table 3 and include several made from commercial lipids . after 72 h the numbers of viable cells were quantified by the mtt assay and secreted inflammatory cytokines assayed in the culture supernatants . liposomes at 10 μg / ml giving an mtt of & gt ; 25 % above the control r8 medium , were limited to bcg tpl liposomes , and purified bcg lipid fractions 3 ( pi ) and 6 . all liposomes made from non - bcg lipids ; namely , dmpc , dmpg , pi from soybean , dppe + dmpc , and cardiolipid from brain , were without significant activity , measured as il - 12 secretion from dendritic cells . however , pim fractions 1 and 2 , as well as fraction 6 , induced secretion of il - 12 several - fold above that induced even by bcg tpl liposomes ( at 10 μg / ml ). further , induction of il - 12 secretion required the addition of at least the complexity of 1 mannose unit to bcg pi , as purified bcg pi ( fraction 3 ) was relatively inactive in this regard . further unexpected results are seen in the case of induction of tnf ( tumor necrosis factor ) secretion from dendritic cells . bcg tpl liposomes were again active . however , contrary to the effects on il - 12 secretion , tnf secretion occurred with purified bcg pi liposomes , and not with other bcg lipid liposomes or commercial lipid liposomes , clearly showing that bcg pi is the active lipid in bcg tpl accounting for tnf secretory activity . the fact that pi from soybean ( with no tuberculosteric acid chains ) was inactive , while bcg pi was highly active points solidly to the tuberculosteric acid ( c19 : 0 ) as a key structural difference to explain active versus inactive pis . clearly , the lipids in bcg tpl have very different biological effects on activation and cytokine secretion from dendritic cells and consequently on the type of immune response obtained . also clear , preparing liposomes using various combinations of bcg polar lipids is indicated as a mechanism to direct the type of immune response obtained to an entrapped antigen . phosphatidylethanolamines ( pes ) are known generally as fusogenic lipids , capable of promoting fusion of membranes , and account for about 25 % of the lipids in bcg tpl ( table 2 ). to mount a ctl immune reaction it is first necessary to deliver antigen to the cytosol of antigen presenting cells . it follows that inclusion of this lipid in a liposome with antigen entrapped , may aid in directing the immune response to mhc class i presentation of antigen and mounting a ctl response . in a first example the adjuvant activity of bcg liposomes is compared to several other adjuvant systems . in one of these tpl liposomes from another gram positive bacterium are included . the bacterium chosen was bacillus firmus based on the observation that injections of these lipids into mice 5 days prior to infection with listeria monocytogenes resulted in some short - term protection ( mára et al . 1992 ), presumably by activating the innate immune system . the tpl lipids of this bacterium extracted by the bligh and dyer method and collected as the acetone insoluble lipids , are characterized by fab ms ( fig1 ). in the case of b . firmus polar lipid extracts , m / z signals for the molecular anion of each lipid were assigned to a cluster of pg lipids with fully saturated sn - 1 , 2 fatty acyl chains consisting of from 25 to 33 carbon atoms ( sum of both chains ) ( fig3 ). these assignments are consistent with the m / z of the carboxylate anions generated from the fatty acyl glycerochains during the analysis , which ranged from c13 : 0 to c17 : 0 . also , signals were found in the cardiolipid and lpg ( lysophosphatidylglycerol ) regions of the spectrum . an amino acid analysis confirmed the presence of small amounts of lysine in hydrolysates of b . firmus polar lipid extract , indicating the possibility of lpg and / or lysylcardiolipids ( fisher and leopold 1999 ). table 4 represents a first example of an enhanced immune cytotoxic t cell ( ctl ) response raised in an animal to an antigen entrapped in bcg liposomes . bcg liposomes served to promote an immune response to the entrapped antigen that was similar to live bcg recombinant , and superior to freund &# 39 ; s adjuvant . further , the tpl of another gram positive bacterium also with saturated glycerolipids formed liposomes with inferior properties to bcg liposomes , teaching away from the positive result with bcg liposomes . table 5 describes the humoral adjuvant activity of bcg liposomes to entrapped protein . first , injection of equivalent amounts of bcg liposomes and ova produced no adjuvant activity , whereas entrapment resulted in humoral adjuvant activity comparable to the adjuvant alum . the toxic adjuvant fca was superior in potency and in maintaining the antibody titre for longer periods after vaccination . live recombinant bcg expressing ova in vivo produced ctl as expected ( dudani et al , 2002 ), but no antibody response . finally , liposomes containing no bcg lipids gave very low antibody titres that improved as bcg lipids were incorporated at increasing amounts from 10 to 50 %. the effect of loading bcg liposomes with different amounts of antigen per mg liposomes is shown to be insignificant from the range of 15 μg antigen loaded in 0 . 22 to 1 . 8 mg ( dry weight ) of liposomes . this is shown in table 6 for the mhc class i - restricted , cd8 + t cell response . in a ) results are shown of ctl assays and in b ) numbers of ifn - gamma secreting precursor t cells in spleens of the variously immunized mice that specifically recognize the antigen in the original vaccination . both assays show good adjuvant activity but no differences based on loading of the vaccine within this range . mice given large numbers of eg . 7 tumor cells develop rapidly growing solid tumors reaching & gt ; 250 mm 2 in all 5 mice in the naive group within 12 days ( fig1 ). injections of antigen - free bcg liposomes resulted in a modest decline in tumor growth , where only 2 mice out of 5 developed tumors & gt ; 250 mm 2 after 12 days . furthermore , a clear delay in the onset of tumor growth is seen . considerably more protection was seen for mice immunized with the bcg ova liposome vaccine , where in all mice tumors were & lt ; 250 mm 2 after 12 days and remained so for the duration of the study ( for 4 mice out of 5 ). [ 0079 ] table 2 thin layer chromatography of the chloroform extractable total polar lipids from bcg cells into 8 fractions . an acidic solvent was used to separate fractions 1 , 2 and 5 to 8 . in the case of fractions 3 and 4 the lipids were recovered together and run again on a second thin layer plate using a basic solvent to achieve separation . staining reactions and recoveries are shown for each lipid fraction . % ( w / w ) phosphate abundance in fraction stain sugar stain amino stain tpl 1 + + − 8 . 6 2 + + − 12 . 4 3 + − − 14 . 1 4 + + − 10 . 4 5 + + − 3 . 0 6 + − − 1 . 2 7 + − + 25 . 9 8 + − − 24 . 4 [ 0080 ] table 3 activation of bone marrow dendritic cells by antigen - free bcg liposomes . the ability of liposomes prepared from bcg total polar lipid and bcg lipid fractions 1 to 8 are compared to liposomes made from commercial lipids . background measurements for r8 medium alone were not subtracted from the values in the table , but were 0 . 542 ( mtt ), 9 . 3 ( il - 12 ), and 0 ( tnf ). nd , not done . liposome mtt ( absorbance ) il - 12 ( ng / ml ) tnf ( pg / ml ) ( diameter nm ) 0 . 1 μg / ml 1 μg / ml 10 μg / ml 0 . 1 μg / ml 1 μg / ml 10 μg / ml 0 . 1 μg / ml 1 μg / ml 10 μg / ml bcg tpl 0 . 418 0 . 495 0 . 727 10 . 8 8 . 5 48 . 5 19 45 26 ( 414 ± 273 ) bcg 1 0 . 374 0 . 321 0 . 574 4 . 2 3 . 0 168 . 4 0 1 0 ( 53 ± 34 ) bcg 2 0 . 468 0 . 415 0 . 618 4 . 5 29 . 8 230 . 2 1 1 10 ( 48 ± 44 ) bcg 3 0 . 448 0 . 382 0 . 774 2 . 5 2 . 8 36 . 6 46 80 146 ( 53 ± 35 ) bcg 5 0 . 459 0 . 532 0 . 613 4 . 0 13 . 8 92 . 9 0 0 0 ( 162 ± 97 ) bcg 6 0 . 438 0 . 501 0 . 745 7 . 3 26 . 6 147 . 2 0 5 25 ( 138 ± 111 ) bcg 7 + dmpc 0 . 407 0 . 443 0 . 499 7 . 2 8 . 6 39 . 9 0 0 0 ( 68 ± 37 ) bcg 8 0 . 439 0 . 604 0 . 896 3 . 2 5 . 2 41 . 6 0 0 32 ( 45 ± 24 ) dmpc 0 . 431 0 . 438 0 . 394 9 . 0 7 . 9 13 . 1 0 4 2 ( 270 ± 214 ) dmpg 0 . 421 0 . 386 0 . 404 3 . 6 4 . 8 8 . 4 nd nd nd ( 94 ± 49 ) pi soybean 0 . 402 0 . 408 0 . 448 6 . 0 5 . 3 7 . 6 0 1 2 ( 29 ± 20 ) dppe + dmpc 0 . 372 0 . 388 0 . 439 7 . 6 5 . 4 3 . 4 4 12 20 ( 135 ) cardiolipid 0 . 419 0 . 429 0 . 609 6 . 8 6 . 6 10 . 2 11 14 5 ( 128 ± 73 ) lps — 0 . 564 0 . 562 — 187 . 9 182 . 7 nd 110 968 [ 0081 ] table 4 comparison of ctl activity in splenic cell cultures from mice immunized with ova in various adjuvant systems . mice were immunized subcutaneously at 0 and 21 days with 15 μg ova entrapped in bcg total polar lipid liposomes , mixed with freund &# 39 ; s adjuvant , or entrapped in liposomes prepared from the total polar lipids extracted from b . firmus ( 92 ± 48 nm diameter , loading 53 μg / mg ). in the case of live bcg cells expressing ova , only one subcutaneous injection was given containing 10 6 cells . spleens from duplicate mice were pooled for each analysis 10 weeks post first injection with exception of b . firmus taken 6 weeks post first injection . lysis of control el - 4 cells not expressing the ova peptide was always & lt ; 2 %, and % lysis of target ( t ) eg . 7 cells expressing ova by splenic effector ( e ) cells is shown in the table . bcg b . firmus e : t ratio naïve fca live bcg liposomes liposomes 100 : 1 1 ± 1 17 ± 1 . 8 39 ± 2 30 ± 2 11 ± 0 . 6 33 : 1 2 ± 3 9 ± 1 . 7 23 ± 0 . 9 15 ± 1 . 5 9 ± 4 11 : 1 0 ± 1 3 ± 0 . 5 11 ± 3 8 ± 0 . 9 2 . 5 ± 0 . 3 3 . 7 : 1 1 ± 0 0 . 6 ± 0 . 7 4 ± 0 . 9 4 ± 0 . 4 1 ± 0 . 4 [ 0082 ] table 5 comparison of anti ova antibody titres in sera of mice immunized with ova in various adjuvant systems . in all cases groups of 4 to 7 c57bl / 6 mice were immunized with 15 μg ova per injection at 0 and 3 weeks . bcg liposomes with ova entrapped were prepared from 100 % bcg tpl lipids mixed with dmpc / dmpg / cholesterol lipids from 0 to 100 %, such that 0 % bcg lipids were pure dmpc / dmpg / cholesterol liposomes . bcg ova - free liposomes were separate injections of an equivalent amount of antigen - free bcg liposomes and ova ( unentrapped ). for fca ova was mixed with fca for the first injection and fia for the second . alum was imject alum to which ova was bound . bcg live are bcg cells genetically modified to express ova ( see table 4 injection details ). blood was taken at various time points from first injection and anti ova antibody in the sera was titrated by elisa . adjuvant day 10 day 31 day 41 100 % bcg liposomes 740 ± 380 23309 ± 16863 8266 ± 5240 50 % bcg liposomes 55 ± 54 14227 ± 4301 9643 ± 4287 10 % bcg liposomes 10 ± 0 1600 ± 1062 1499 ± 978 0 % bcg liposomes 10 ± 0 1065 ± 1020 857 ± 839 bcg ova - free 0 388 ± 259 345 ± 350 liposomes fca 5156 ± 6722 41420 ± 26376 65671 ± 26080 alum 472 ± 371 8306 ± 2074 12302 ± 6848 bcg live 50 0 0 no adjuvant 0 519 ± 552 821 ± 738 [ 0083 ] table 6 effect of antigen loading in bcg liposomes on induction of an immune response in mice . 6 wk pfi a . ctl - % lysis of eg . 7 targets e : t ratio naïve 15 μg / 1 . 8 mg 15 μg / 1 . 6 mg 15 μg / 0 . 22 mg 100 : 1 0 . 9 ± 0 . 1 52 ± 2 51 ± 2 55 ± 2 33 : 1 0 ± 0 . 7 41 ± 3 36 ± 3 45 ± 3 11 : 1 0 ± 1 29 ± 3 22 ± 3 31 ± 5 3 . 7 : 1 0 ± 0 . 9 16 ± 2 10 ± 0 . 1 15 ± 2 b . elispot - number of ifn secreting colonies ova peptide naïve 15 μg / 1 . 8 mg 15 μg / 1 . 6 mg 15 μg / 0 . 22 mg + 0 23 ± 1 26 ± 3 24 ± 3 − 0 0 0 0 c . anti ova antibody titres ( 2 to 4 mice / group ) ova , no mouse number adjuvant 15 μg / 1 . 8 mg 15 μg / 1 . 6 mg 15 μg / 0 . 22 mg 1 & lt ; 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