Patent Application: US-82411292-A

Abstract:
cytokine receptors for tumor necrosis factor α which are found on microorganisms may , if bound with exogenous tnfα , enhance the response of natural killer cells activated by the microorganisms , or increase tnfα production by peripheral blood lymphocytes treated with the microorganisms . microorganisms with receptor - bound exogenous tnfα have enhanced cellular invasion ability which may change the immune response thereto . clinical and pharmaceutical applications of these discoveries including vaccines with increased efficacy are provided .

Description:
the present invention relies in part on the fact that receptors for cytokines such as tnfα are found on microorganisms such as gram - negative bacteria , gram - positive bacteria , and the yeast form of candida albicans . other aspects of the invention derive from the fact that bacteria with receptor - bound tnfα enhance nk cell activity ( table 1 ) and induce tnfα production from human pbl &# 39 ; s ( table 2 ) to a much greater extent than bacteria without receptor - bound tnfα . such bacteria are also taken up by macrophages and epithelial cells more avidly than bacteria without tnfα on their surface , viz ., a virulence property is altered by the presence of tnfα . coating bacteria with tnfα resulted in a more immunogenic antigen which resulted in higher levels of antibody being produced in mice ( table 4 ). these observed tnfα - related effects support the claimed clinical and laboratory applications in the present invention . those skilled in the art will recognize that both other cytokines and other microorganisms than those exemplified herein may be quite analogously utilized . table 1______________________________________nk cell activation by tnfα coated bacteria nk cell activity %. sup . 51 cr release e : t ratioculture conditions 3 : 1 12 : 1 50 : 1______________________________________human pbl plus : medium 3 10 32il2 15 40 60s . flexneri 6 14 38s . flexneri coated 10 25 49with tnfαs . typhimurium 8 12 30s . typhimurium coated 13 29 52with tnfα______________________________________ human pbl are incubated 18 hr with one of the following : 1 ) il2 500 u / ml , 2 ) 10 . sup . 5 cfu of s . flexneri either untreated or coated with tnfα or 3 ) 10 . sup . 5 cfu of s . typhimurium either untreated or coated with tnfα . nk cell activity is assessed using a 4 hr . sup . 51 cr release assay with k562 tumor cells . table 2______________________________________tnf production by human pbl stimulated withtnfα - coated s . flexnericulture conditions tnf u / ml______________________________________human pbl plus : medium 12s . flexneri 13s . flexneri coated with 109tnfα______________________________________ human pbl are incubated 18 hr with 1 ) medium , 2 ) s . flexneri , or 3 ) s . flexneri coated with tnfα . supernatants from these cultures are assessed for tnf bioactivity using the l929 assay . the following examples are presented to describe preferred embodiments and utilities of the present invention and are not meant to limit the present invention unless otherwise specified in the claims appended hereto . taken together , the examples illustrate the best mode of implementing the invention as it is currently understood . tnfα binding to a shigella flexneri is investigated using 125 i - labeled human recombinant tnfα and bacterium - 125 i - tnfα complexes quantitated by filtration . 125 i - labeled recombinant human tnfα ( 200 - 800 ci / mmol ) is obtainable from amersham corp ., arlington heights , ill ., or may be produced using the iodogen method with rhtnfα obtained from ubi inc ., lake placid , n . y . unlabeled recombinant human tnfα ( 2 × 10 7 units / mg ) and tnfβ ( 3 × 10 7 units / mg ) are available from genzyme , boston , mass . bacteria from overnight cultures of s . flexneri serotype 2a , strain sa100 are grown to mid - logarithmic phase , then incubated 10 min at 37 ° c . with 0 . 01 % azide in rpmi medium . four shigella flexneri strains of this serotype are available from the american type culture collection , 12301 parklawn drive , rockville , md . 20852 - 1776 . treated - bacteria ( 2 × 10 9 cfu ) are then washed and incubated in 250 μl of pbs containing 1 % heat inactivated fcs or 1 % bsa plus varying concentrations of labeled or unlabeled tnfα or tnfβ . after appropriate incubation at 37 ° c . with mixing every 10 min , bacteria - tnf mixtures are transferred to a syringe ( 10 ml ) equipped with a 0 . 45 μm nitrocellulose filter . tubes ( bsa - coated microfuge ) which contained bacteria - tnf mixtures are washed with 250 μl of rpmi , and this volume added to the syringe - filter . bacteria are then isolated by filtration and filters containing bacteria washed with 1 ml of rpmi . filters are then assessed for the amount of bound 125 i - tnfα . filters used in these experiments are pretreated with fcs . 125 i - tnfα binding to filters in the absence of bacteria is ≦ 6 % of the total cpm added to the binding mixture . this value is always subtracted from cpm obtained from 125 i - tnf - bacteria complexes isolated by filtration . non - specific binding to bacteria is assessed using ≧ 100 - fold excess of unlabeled tnfα . non - specific binding is usually about 10 % of total bound cpm . scatchard analysis is performed as described by stuart . 125 i - tnfα binding to s . flexneri varies among different commercial lots of 125 i - tnfα . this appears to correlate with the level of biological activity retained by the 125 i - labeled tnfα . little binding is detected when 125 i - tnfα has & lt ; 20 % of its biological activity as measured by the l - 929 bioassay . in this regard , over one - half of the commercially obtained lots of 125 i - tnfα have lost 80 - 90 % of their biological activity and give low levels of binding to s . flexneri . 125 i - tnfα binding to azide - treated versus untreated bacteria is identical during the first 20 min of incubation . however , untreated bacteria have a doubling time of around 30 - 40 min and consistently bind more tnfα than azide - treated bacteria at time periods longer than 20 min . data presented above and in table 1 are obtained using azide - treated bacteria when binding is assessed at 37 ° c . non - azide - treated bacteria are used when binding is assessed at 4 ° c ., which is optimal at 4 hr incubation . scatchard analysis of data obtained from binding experiments done at 4 ° c . gives a kd of 3 . 0 nm with 215 binding sites for tnfα per bacterium . as illustrated in fig1 - 3 , s . flexneri binds significant levels of 125 i - tnfα . this binding is saturable with optimal binding occurring at 40 min when binding is performed at 37 ° c . ( fig1 & amp ; 2 ). fig2 shows the time course of 125 i - tnfα binding using 0 . 1 nm 125 i - tnfα ( 3 - 4 × 10 4 cpm ). when binding is measured at 4 ° c ., optimal binding occurs at 4 hr . fig3 shows the competition of tnfα ( open circles ) and tnfβ ( solid circles ) with 125 i - tnfα for binding to s . flexneri . fig3 a shows scatchard analysis indicating a kd of 2 . 5 nm , with 276 binding sites for tnfα per bacterium . the binding of 125 i - tnfα to s . flexneri is inhibited by various concentrations of unlabeled tnfα , but unlabeled tnfβ is ineffective at competing with 125 i - tnfα . in contrast , tnfα receptors on eukaryotic cells can be occupied by both tnfα and tnfβ . thus , the bacterial receptors for tnfα appear to differ from tnfα receptors on eukaryotic cells with regard to binding specificity for tnfα versus tnfβ . the ability to bind tnfα is not exclusive to shigella flexneri . an avirulent escherichia coli and a virulent salmonella typhimurium both bind significant levels of 125 i - tnfα ( table 3 ). further , both a virulent ( sa100 ) and an isogeneic non - pathogenic s . flexneri strain ( sa100ni ) appear to bind comparable levels of 125 i - tnfα ( table 3 ). these data indicate that bacteria - tnfα binding may be common property of both virulent and avirulent gram - negative bacteria . no difference is found between the levels of tnfα bound by rough versus smooth strains of salmonella . however , heating , ( 52 ° c ./ 3 min ), formalin fixation or trypsin treatment of bacteria results in complete to partial reduction of tnfα binding ( table 3 ), indicating that bacteria - encoded protein forms at least a part of the tnfα receptor . table 3______________________________________tnfα binding to bacteria tnfα specific binding ( cpm ) at : bacterium 4 ° c . 37 ° c . ______________________________________exp . 1 s . flexneri ( sa100ni ) 6591 ± 124 5994 ± 404 e . coli 5801 ± 58 6150 ± 150 s . typhimurium 4951 ± 160 5850 ± 300exp . 2 s . flexneri ( sa100 ) untreated 7065 ± 48 5226 ± 73 formalin - fixed 5818 ± 33 4176 ± 66 heat - treated 3199 ± 91 3056 ± 8exp . 3 s . flexneri ( sa100 ) untreated 3588 ± 99 trypsin - treated 327 ± 97exp . 4 c . albicans 6053 ± 120 5410 ± 91______________________________________ . sup . 125 i - tnfα binding to different bacteria was assessed . bacteri ( 2 × 10 . sup . 9 ) are incubated with 0 . 1 nm . sup . 125 itnfα ( 3 - 4 × 10 . sup . 4 cpm ) for 40 min at 37 ° c . or for 4 hr at 4 . degree c . a laboratory strain of e . coli ( dh5α ) and a rough strain of s . typhimurium ( tml ) are grown and treated as described in fig1 for s . flexneri . candida albicans were grown to midlogarithmic phase and washed twice . . sup . 125 iinfα binding to candida albicans was assessed usin 5 × 10 . sup . 7 organisms and using assay conditions exactly as described for . sup . 125 itnfα binding to bacteria . s . flexneri are also assessed for tnfα binding following heat treatment ( 52 ° c . for 3 min ), fixation by 1 % formaldehyde , or by trypsin treatment . trypsin treatment of bacteria is achieved by incubating 4 × 10 . sup . s . flexneri ( sa100 ) in 10 ml of pbs with trypsin ( 100 μg / ml , sigma , st louis , mo ) for 30 min at 37 ° c . soybean trypsin inhibitor ( 100 μg / ml , sigma ) is then added and after 15 min at 37 ° c . the bacteria are pelleted and washed . trypsin treated or control treated bacteria are then assessed for their ability to bind . sup . 125 itnfα as described in fig1 . data presented are the mean ( sd ) of duplicated determinations . important virulence factors of s . flexneri are its ability to penetrate and replicate within epithelial cells of the intestinal mucosa , resulting in subsequent tissue damage . these factors are investigated in hela cells by pretreatment of s . flexneri sa100 with tnfα . s . flexneri ( sa100 or sa100ni ) are incubated in 1 ml of rpmi - 1640 with or without varying concentrations of tnfα . after 4 hr at 4 ° c ., bacteria ( 10 3 cfu / ml ) are pelleted by centrifugation ( 1500 xg ) and washed once with 4 ml of rpmi . bacteria ( pretreated with media vs tnfα ) are then assessed for their ability to invade hela cells . hela cell invasion is assessed by using an agarose - agar overlaying procedure as previously described . data presented are from triplicate determinations and from one representative experiment of 10 experiments . the results show a dramatic enhancement of hela cell invasion ( fig4 ). s . flexneri sa100 ( 10 3 cfu ) pretreated with 5000 u of tnfα for 4 hr at 4 ° c . and then washed twice has a 20 - fold enhancement in cellular invasion . it is apparent that enhancement of cellular invasion is dependent upon bacterial virulence factor ( s ) because non - invasive s . flexneri can not be converted to an invasive form by tnfα binding . a non - invasive isogeneic variant of s . flexneri , sa1ooni , which binds equivalent levels of 125 i - tnfα ( table 3 ) does not invade hela cells after tnfα pretreatment ( fig5 ). the mechanism ( s ) involved in the enhanced cellular invasion by tnfα - shigella complexes is unknown , but could possibly result from enhanced interaction with the cell surface . coating a pathogen with a cytokine was found to result in a more immunogenic antigen . salmonella typhimurium ( 10 8 ) were incubated with rtnfα ( 10 , 000 u ) in a volume of 250 ml . after 4 hours at 4 ° c . the bacteria - tnfα complexes were formalin - fixed , washed twice and injected ( ip ) into c57b1 / 6 mice . as a control , salmonella were treated in an identical fashion in medium with no tnf . mice were bled at 6 days post challenge . results of a representative experiment are illustrated in table 4 . table 4______________________________________enhanced antibody productionin vivo by tnf - coated s . typhimurium antibody to tmlmice immunized with elisa assay ( od units ) ______________________________________s . typhimurium ( tml ) . 6523 × . 15s . typhimurium ( tml ) 1 . 075 × . 31coated with tnfα______________________________________ c57b1 / 6 mice ( 5 ) were challenged ( ip ) with 10 . sup . 8 formalinfixed tml which had been pretreated with 10 . sup . 4 of rtnfα . serum from individual mice were obtained at 6 days and assessed for antibody to tml by elisa assay . data is mean ± sd od units from 5 mice per group . coating salmonella with tnfα resulted in an enhanced antibody response to salmonella . these data indicate that coating a pathogen with tnfα can enhance the immune response to that pathogen . these results could be applied for the preparation of vaccines for both human and animal use . improved vaccines could result from coating any pathogen with cytokines such as interleukin - 1 ( il1 ), interleukin - 2 ( il2 ) or interferon ( ifn ). the coating could be easily performed as described above or by cross - linking the cytokine onto the pathogen . additionally , deactivated microorganisms or antigenic microorganism surface components retaining bound cytokines could be used as administered material in the usual course of human or domestic animal vaccination . oral administration of cytokine - containing avirulent microorganism strains is a preferred mode of immunization , of course when the elicited immunity precludes adverse effects of analogous virulent microorganisms . the following references are incorporated in pertinent part in the specification for the reasons therein . 1 . beutler , b . and cerami , a . ann . rev . immunol . 7 , 625 - 655 ( 1989 ). 2 . sherry , b . and cerami , a . j . cell biol . 107 , 1269 - 1277 ( 1988 ). 4 . nakane , a ., minagawa , t ., and kato , k . infect . immun . 56 , 2563 - 2569 ( 1988 ). 5 . kindler , v ., sappino , a . p ., grau , g ., piquet , p . f ., and vassalli , p . cell 56 , 731 - 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364 ( 1986 ). 23 . niesel , d . w ., hess , c . b ., cho , y . j ., klimpel , k . d ., and klimpel , g . r . infect . immun . 52 , 828 - 833 ( 1986 ). changes may be made in the construction , operation and arrangement of the various microorganisms , cytokines , steps and procedures described herein without departing from the concept and scope of the invention as defined in the following claims .