a detoxified lt , e . g ., containing a mutated lt a , is a desired vaccine adjuvant due to its high immunogenecity . the mutated lt a of this invention , designed to achieve this purpose , is made by introducing an amino acid substitution in an lt a at a position corresponding to the position 61 of seq id no : 5 . lt a produced by different enterotoxigenic e . coli strains are highly homologous . thus , by comparing the amino acid sequence of an lt a with seq id no : 5 , a skilled artisan can find out which position in this lt a corresponds to position 61 of seq id no : 5 . just like the lt a of seq id no : 5 , almost all wild - type lt a s include a serine at the position corresponding to position 61 of seq id no : 5 . an amino acid that is different from serine in , e . g ., size or polarity , can be used as a substitutent . examples of the substitutent amino acids include hydrophobic amino acid residues ( e . g ., i and l ), charged amino acid residues ( e . g ., d , e , k , r , and h ), or amino acid residues with bulky side chains ( e . g ., n , q , y , and w ). proline also can be used as a substitutent as it generally alters the local structure of a polypeptide . these substitutent amino acids include non - naturally occurring amino acids , e . g ., d - amino acids or α - amino acids . the lt a mutant of this invention can be prepared by methods well known in the art . for example , the mutant is produced by a recombinant method as follows . a cdna that encodes a wild - type lt a is isolated from an enterotoxigenic e . coli strain and subjected to site - directed mutagenesis to produce a cdna encoding the desired lt a mutant . see ho et al ., gene , 77 : 51 - 59 , 1989 . the cdna bearing the mutation is then inserted into an expression vector for transforming cells . finally , the lt a mutant produced in the transformed cells is purified and assembled with lt subunit b to form whole lt protein . the toxicity of an lt containing an lt a mutant described above can be assessed using assays such as the y - 1 adrenal cell assay . see cheng et al ., vaccine , 18 : 38 - 49 , 2000 . the lt a mutant of this invention can be used as an adjuvant in a vaccine . the vaccine ( i . e ., a human vaccine or a veterinary vaccine ) can contain an antigen and the lt a mutant itself or an lt containing the lt a mutant . the antigen can be derived from a bacterium , e . g ., streptococcus pyogenes , streptococcus pneumoniae , neiseria gonorrhoea , neisseria meningitides , corynebacterium diphtheriae , clostridium botulinum , clostridium perfringens , clostridium tetani , haemophilus influenzae , klebsiella pneumoniae , klebsiella ozaenae , staphylococcus aureus , vibrio cholerae , escherichia coli , pseudomonas aeruginosa , campylobacter jejuni , aeromonas hydrophila , bacillus cereus , yersinia enterocolitica , yersinia pestis , salmonella typhimurium , treponema pallidum , borrelia vincentii , borrelia burgdorferi , mycobacterium tuberculosis , pneumocystis carinii , mycoplasma spp ., rickettsia prowazeki , chiamydia spp ., helicobacter pylori . it also can be derived from a virus , e . g ., influenza , herpes simplex virus , human immunodeficiency virus , cytomegalovirus , hepatitis c virus , delta hepatitis virus , poliovirus , hepatitis a virus , hepatitis b virus , epstein - barr virus , varicella zoster virus , respiratory syncytial virus , enterovirus or japanese encephalitis virus . the vaccine can further contain a pharmaceutically acceptable carrier such as phosphate buffered saline or a bicarbonate solution . the carrier is selected on the basis of the mode and route of administration , and standard pharmaceutical practice . suitable pharmaceutical carriers and diluents , as well as pharmaceutical necessities for their use , are described in remington &# 39 ; s pharmaceutical sciences . methods for preparing vaccines are generally well known in the art , as exemplified by u . s . pat . nos . 4 , 601 , 903 ; 4 , 599 , 231 ; 4 , 599 , 230 ; and 4 , 596 , 792 . vaccines may be prepared as injectables , as liquid solutions or emulsions . the antigen and the lt a mutant or the lt containing it may be mixed with physiologically acceptable excipients , which may include , water , saline , dextrose , glycerol , ethanol , and combinations thereof . the vaccine may further contain minor amounts of auxiliary substances such as wetting or emulsifying agents , or ph buffering agents to enhance the effectiveness of the vaccines . vaccines may be administered parenterally , by injection subcutaneously or intramuscularly . alternatively , other modes of administration including suppositories , oral , or topical formulations may be desirable . for suppositories , binders and carriers may include , for example , polyalkalene glycols or triglycerides . oral formulations may include normally employed incipients such as , for example , pharmaceutical grades of saccharine , cellulose , magnesium carbonate and the like . these compositions take the form of solutions , suspensions , tablets , pills , capsules , sustained release formulations or powders . the vaccine is administered in a manner compatible with the dosage formulation , and in an amount that is therapeutically effective , protective and immunogenic . the quantity to be administered depends on the subject to be treated , including , for example , the capacity of the individual &# 39 ; s immune system to synthesize antibodies , and if needed , to produce a cell - mediated immune response . precise amounts of active ingredient required to be administered depend on the judgment of the practitioner . however , suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of micrograms of the polypeptide of this invention . suitable regimes for initial administration and booster doses are also variable , but may include an initial administration followed by subsequent administrations . the dosage of the vaccine may also depend on the route of administration and varies according to the size of the host . the specific examples below are to be construed as merely illustrative , and not limitative of the remainder of the disclosure in any way whatsoever . without further elaboration , it is believed that one skilled in the art can , based on the description herein , utilize the present invention to its fullest extent . all publications cited herein are hereby incorporated by reference in their entirety . a 1 . 8 - kb dna fragment of an lt gene , including both subunit a and subunit b , was isolated from human entero - toxigenic e . coli h10407 and cloned into pbluescript ii ks (−) vector ( pbluescript - lthwt ). the nucleotide sequence ( seq id no : 6 ) of the lt gene ( encoding both subunits a and b ) and the amino acid sequence ( seq id no : 5 ) of subunit a of this lt are shown below : nucleotide sequence ( seq id no : 6 ) of lt ( subunit a : 1 - 777 ; amino acid sequence ( seq id no : 5 ) of mature lt a various lt a mutants were constructed using a site - directed mutagenesis method ( ho et al ., 1989 , gene 77 , 51 - 59 ). more specifically , lt a mutants , including lt a ( s61k ), lt a ( s61r ), lt a ( s61h ), lt a ( s61y ) and lt a ( s61f ) were constructed by replacing the serine at position 61 of seq id no : 5 with k , r , h , y , and f , respectively . the following oligonucleotide primers were used to construct these mutants : lt a ( s61k ) [ 5 ′ tct caa act aag aga agt ttt aac ata tcc gtc atc ata 3 ′]( seq id no : 11 ), lt a ( s61r ) [ 5 ′ act tct caa act aag aga agt tct aac ata tcc gtc atc 3 ′]( seq id no : 12 ), lt a ( s61f ) [ 5 ′ act tct caa act aag aga agt gaa aac ata tcc gtc atc 3 ′]( seq id no : 13 ), lt a ( s61y ) [ 5 ′ act tct caa act aag aga agt ata aac ata tcc gtc atc 3 ′]( seq id no : 14 ), lt a ( s61h ) [ 5 ′ act tct caa act aag aga agt atg aac ata tcc gtc atc 3 ′]( seq id no : 15 ). the nucleotide sequences and amino acid sequences of these lt a are shown below : amino acid sequence ( seq id no : 2 ) of lt a ( s61k ) amino acid sequence ( seq id no : 4 ) of lt a ( s61r ) amino acid sequence ( seq id no : 8 ) of lt a ( s61h ) amino acid sequence ( seq id no : 10 ) of lt a ( s61y ) for comparison , another lt mutant , ltp ( s63k ), derived from ewd299 ( dallas et al ., 1979 , j . bacteriol . 139 , 850 - 859 ) was also constructed by replacement of serine at amino acid position 63 of subunit a with lysine . pbluescript ii ks (−) vectors containing native or mutant lt genes , including genes for both subunit a and subunit b , were transformed into e . coli hb101 . the native and mutant lt &# 39 ; s were purified from cultures grown overnight in a 3 - liter flask containing l - broth supplemented with 100 μg of ampicillin per ml . the cells were harvested by centrifugation , resuspended in tean buffer ( 0 . 2 m nacl , 50 mm tris , 1 mm edta and 3 mm nan 3 , ph 7 . 4 ), and lysed with microfluidizer ( microfluidics corporation , usa ). after the lysates were clarified by centrifugation , the lt was fractionated by adding solid ammonium sulfate to 65 % saturation . the preparation was then suspended in tean buffer , dialyzed thoroughly against the same buffer and used as the crude lt . the crude lt was subjected to chromatography on immobilized d - galactose ( pierce , rockford , ill .) columns equilibrated with tean buffer at 4 ° c . ( uesaka et al ., 1994 , microbial pathogenesis 16 : 71 - 76 ). native and mutant lt &# 39 ; s were eluted with 0 . 3 m galactose in tean . each purified toxin was dialyzed against pbs buffer for biological and immunological assays . the purified wild - type and mutant lt were separated by sds - page , wherein the molecular weight of lt subunit a was about 28 to 29 kda , and the molecular weight of lt subunit b was about 12 to 13 kda . the yields of the entire lt containing lt a ( s61k ), i . e ., lth ( s61k ), and lt a ( s61r ), i . e ., lth ( s61r ) were similar to the lt containing the lt a of seq id no : 5 . ab 5 heterohexamers and b 5 pentamers of the lt were separated by ph 3 - 10 isoelectric focusing gel ( invitrogen ). intensity of ab5 and b5 protein bands was assayed by uvib and software ( uvitec limited ) to calculate the percentage of the ab 5 heterohexamers and the b 5 pentamers ( the isoelectric point ( pi ) value of ab 5 was between 8 . 0 and 7 . 8 , and the pi value of b 5 was between 8 . 3 and 8 . 1 ), the results are listed in table 1 . caco - 2 cells ( atcc htb - 37 ) were maintained in mem - α medium supplemented with 20 % fbs in 24 - well plate at a concentration of 5 × 104 cells per well , grown to near confluency , and incubated in mem - α containing 1 % fbs and 1 mm 3 - isobutyl - 1 - methylxanthine ( ibmx ) for 30 min in 5 % co2 prior to addition of toxins ( grant et al ., 1994 , infection and immunity 62 : 4270 - 4278 ). native or mutant lt was added to each well and incubated for 4 hours . the cells were then washed twice with cold pbs . intracellular camp was extracted by adding 200 μl of 0 . 1 n hcl to each well and incubated at room temperature for 15 minutes . supernatant of cell lysates were collected following addition of 0 . 1 n naoh to each well . ( cheng et al ., 2000 , vaccine 18 : 38 - 49 ; park et al ., 1999 , experimental and molecular medicine 31 : 101 - 107 ). camp was measured with a camp enzyme immunoassay kit ( assay designs ; correlate - eia ). results obtained from this example show that lth ( s61k ) did not increase the concentration of intracellular camp . determining toxicity of lt containing mutated lt a using y1 adrenal tumor cell assay in this study , lt samples , including wild - type lt , lt active site mutant ( h61k ), and lt subunit b complex , b5 , were evaluated for enterotoxic effect . mouse y - 1 adrenal tumor cells ( atcc ccl - 79 ) maintained in ham &# 39 ; s f12 media supplemented with 15 % horse serum , 2 . 5 % fetal bovine serum , 2 mm l - glutamine , and 1 . 5 g / l sodium bicarbonate were seeded in 96 - well flat - bottom plates at a concentration of 2 × 104 cells per well ( 200 ul / well ) at 37 ° c . in 5 % co2 for 48 hrs . cells in 96 - well flat - bottom plates were washed twice with 1 × pbs ( ph 7 . 4 ) and then treated with serially diluted lt samples ( 10 μg / 200 μl ˜ 10 - 10 μg / 200 μl ) at 37 ° c . in 5 % co2 overnight . cells were examined by light microscopy for typical cell rounding 24 hrs after toxin treatment . activity is defined as the minimum concentration of the toxin required to initiate cell rounding ( eci ) or the toxin concentration required for 50 % cell rounding ( ec50 ). see david et al ., 1975 , infection and immunity , 11 : 334 - 336 ; cheng et al ., 1999 , vaccine 18 : 38 - 49 . the toxicity of lth ( s61k ), lth ( s61r ), lth ( s61h ) is significantly lower compared to wild - type lt , i . e ., 10 − 6 versus 1 . see table 2 below . the toxicity of lth ( s61f ) is also lower ( i . e ., 10 − 5 ), but the reduction is not as significant as the other mutants . the assay was performed as previously described ( giannelli et al ., 1997 , infection and immunity 65 : 331 - 334 ; giuliani et al ., 1998 , j . exp . med . 187 : 1123 - 1132 ). the new zealand adult rabbits , ˜ 2 . 5 kg each , were used for this assay . loops , each 5 cm long , were made by starting at the end of the rabbit &# 39 ; s small intestines and moving toward the stomach . 0 . 5 ml samples with various amounts of lt or lt mutants were injected intro each loop and then the abdomen was closed . after 18 hours , the liquid accumulated in each loop was collected and measured . the experiment was performed three times and the results expressed in milliliters per centimeter . results from this experiment show that 500 μg of lth ( s61k ) only accumulated 1 ml fluid in the rabbit ileal loop , and the volume of the accumulated fluid was similar to native control . when 0 . 1 μg of wild - type lt was used , the volume of accumulated fluid of wild - type lt was considerably larger than that of lth ( s61k ). in addition , the fluid accumulation of other ltp ( s63k ) also considerably larger than that of lth ( s61k ). determining adjuvant effect of lt containing mutated lt a in intranasal immunization deactivated influenza virus , a / puerto rico / 8 / 34 ( h1n1 ) ( pr8 ) ( atcc vr - 95 ), was used in this example . the viral particles were prepared as previously described in ( aitken et al ., 1980 , eur . j . biochem . 107 : 51 - 56 ; gallagher et al ., 1984 , j . clin . microbiol . 20 : 89 - 93 ; johansson et al ., 1989 , j . virol . 63 : 1239 - 1246 ). briefly , the virus was propagated in the allantoic cavity of 10 - day - old embryonated hen &# 39 ; s eggs at 35 ° c . for two days . the allantoic fluid from eggs infected with pr8 was first centrifuged at low - speed and then centrifuged at 96 , 000 × g for 1 hour to precipitate viral particles , which were resuspended in phosphate - buffered saline ( pbs ). these particles were loaded onto a 30 - 60 % sucrose density gradient and centrifuged for 5 hours at 96 , 000 × g . the fraction containing the virus was collected and diluted with pbs . the virus was further pelleted at 96 , 000 × g for 1 hour and resuspended in pbs . purified virus was treated with 0 . 025 % formalin at 4 ° c . for a week . protein concentration was measured and standardized based on the optical density , bio - rad protein assay , and the haemagglutinin ( ha ) content was determined by sds - polyacryamide gel electrophoresis ( oxford et al ., 1981 , j . biol . stand . 9 : 483 - 491 ). female balb / c mice , between 6 and 8 weeks old , were obtained from taiwan national laboratory animal center . groups consisting of five of mice each were immunized intranasally with 25 μl pbs containing 20 μg of inactivated influenza virus ( flu . ag ) alone or in combination with 8 μg of native or mutant lt under anesthesia . the mice were re - immunized 3 weeks later . control mice were given pbs under the same condition . two weeks after the final immunization , mice in each group were sacrificed to obtain serum and conduct haemagglutination inhibition ( hi ) assay . significantly enhanced hi titers were detected in mice intranasally immunized with the inactivated virus vaccine in combination with lth ( s61k ) or ltp ( s63k ) when compared with mice intranasally immunized with inactivated virus vaccine alone . the hi titer of lth ( s61k ) in combination with the inactivated virus was substantially increased . the hi titer of lth ( s61k ) in combination with the virus is 813 , similar to that of lth ( s63k ) in combination with the virus but larger than that of the virus alone . determining adjuvant effect of lt containing mutated lt a in intramuscular immunization the procedure carried out in example 6 was repeated except that the immunization utilizes intramuscular delivery . intramuscular vaccines were prepared in 50 μl pbs containing 10 μg of inactivated virus vaccine alone or in combination with 4 μg of native or mutant lt and were injected into the posterior thigh muscle . the immunization and sampling programs were performed as described . the hi titer of lth ( s61k ) in combination with the inactivated virus was substantially increased . the hi titer of lth ( s61k ) in combination with the virus was 512 , which is significantly higher than that of wild type lt in combination with the virus or that of the virus alone . all of the features disclosed in this specification may be combined in any combination . each feature disclosed in this specification may be replaced by an alternative feature serving the same , equivalent , or similar purpose . thus , unless expressly stated otherwise , each feature disclosed is only an example of a generic series of equivalent or similar features . from the above description , one skilled in the art can easily ascertain the essential characteristics of the present invention , and without departing from the spirit and scope thereof , can make various changes and modifications of the invention to adapt it to various usages and conditions . thus , other embodiments are also within the scope of the following claims .
this invention relates to a mutant e . coli heat - labile enterotoxin subunit a that can be used as an adjuvant . this subunit a mutant contains an amino acid substitution at a position corresponding to position 61 of a wild - type lt . an lt containing this mutated subunit a exhibits reduced toxicity compared to its wild type counterpart .