Patent Application: US-46753309-A

Abstract:
recombinant chimeric antigens comprising unmodified and modified reactive polypeptide fragments of expressed product of the recombinant 56 kda proteins of multiple strain of scrub typhus , such as karp , kato , gilliam , and ta763 . the invention is useful for detecting prior exposure to a number of strains of scrub typhus , based on the strength of reaction toward the chimeric protein and as a component in vaccine formulations and production of immune globulins for passive prophylaxis and immunity in subjects against heterologous infections .

Description:
western blot analysis of whole cell lysates with scrub typhus patient sera has identified at least four protein antigens of o . tsutsugamushi with molecular weights of 22 kda , 47 kda , 56 kda and 110 kda ( 30 ). among them , the 56 kda antigen is the naturally dominant protein antigen , which accounts for 10 - 15 % of the total cell protein . almost every clinically diagnosed patient serum reacts with 56 kda antigen , but not every patient serum reacts with 22 kda , 47 kda or 110 kda antigens ( 31 , 32 ). in one study , only 15 % ( 2 / 13 ) patient sera recognized the 47 kda antigen ( 31 ). recombinant 56 kda protein ( r56 ) has been shown to be protective in mice against homologous challenge ( 33 - 35 ). high titers of antibodies to o . tsutsugamushi were also detected in mouse sera . a dose - dependent pattern of lymphocyte proliferation and levels of ifn - gamma and il - 2 production ( cytokine profile of th1 cells ) was observed in spleen mononuclear cells from immunized mice ( 33 ). the 56 kda protein plays a role in the adhesion and internalization of o . tsutsugamushi into host cells ( 25 ). both polyclonal and monoclonal antibodies against this antigen can block rickettsial infection of fibroblasts ( 36 - 38 ). all these results suggest that the 56 kda protein is an ideal candidate for vaccine development . a recombinant protein from karp stain ( kp r56 ) has been developed , which has shown 60 - 100 % protection from homologous challenge in an outbred mouse model . moreover , it has been shown to be safe and immunogenic in the scrub typhus non - human primate model using cynomolgus monkeys . this vaccine candidate has recently been evaluated for protection from heterologous challenge with 5 non - karp strains of o . tsutsugamushi . various degree of protection was observed in cd - 1 mice challenged with kato ( 56 %), ta763 ( 45 %), th1812 ( 39 %), th1814 ( 33 %), citrano ( 11 %). co - administration of karp ( kp ) r56 and kato ( kt ) r56 resulted in similar protections against karp , ta263 and th1814 but increased somewhat for kato , th1812 and citrano ( karp 67 %, kato 78 %, ta763 33 %, th1812 67 %, th1814 33 %, citrano 45 %). it has also been shown that mouse sera raised against ta 763 r56 ( seq id no . 4 ) reacted with many heterologeous strains ( 39 ) and ta763 r56 reacts with sera of many serotypes , which suggests its broad reactivity . recently , additional r56 antigens have been added to the kp r56 to produce a multivalent vaccine ( kpktgm r56 ). this vaccine has shown some , but not complete heterologous protection . therefore , it is an objective of this invention to design a chimeric antigen using reactive polypeptide fragment from more than one strain of o . tsutsugamushi , including but not limited to karp , kato , gilliam , ta763 , th1811 , th1812 , th1814 , afc27 , 18032460 , 18032404 , woods , citrano , mak119 and mak243 . such recombinant chimeric antigen will offer broad cross reactivity among different stains and may be used in detection assays or as part of a vaccination composition against scrub typhus . in an embodiment of the present invention , recombinant chimeric antigens 1 , 2 and 3 are designed by making modifications to the variable domains of the 56 kda protein antigens of the karp and ta763 strains . this is an attempt to generate proteins that have potentially broad reactivity toward multiple strains of o . tsutsugamushi . the cross reactivity of these chimeric antigens were evaluated with strain - specific mouse sera and compared to the reactivity of r56s from karp and ta763 strains . it is demonstrated that the chimeric protein antigens reacted very well with as many as 14 disparate strains of o . tsutsugamushi . the results suggested the possibility of combining minimum number of r56s to provide cross reactivity with the most strains of o . tsutsugamushi , and also indicating that the combination of these r56s in a vaccine formulation may provide a better protection for heterologous challenge . the 56 kda protein sequences from karp and ta763 strains of o . tsutsugamushi were used as the building blocks for the chimeric proteins . chimeric antigen 1 ( c1 ) was designed to use the karp 56 kda protein sequence as the backbone while replacing its variable domain 1 ( fig2 a ) with the variable domain 1 of the 56 kda protein antigen of the ta763 stain with a slight modification ( fig2 b ). the final sequence of c1 is set forth in seq id no . 1 ( fig3 a ). similar strategy was used in the design of chimeric antigen 2 ( c2 ). sequence of the 56 kda protein of the ta763 strain ( fig2 b ) was used as the backbone for the chimeric antigen 2 with its variable domain 3 replaced with a slightly modified variable domain 3 of the 56 kda protein antigen of the karp strain ( fig2 a ). the sequence of c2 is set forth in seq id no . 2 ( fig3 b ). chimeric antigen 3 ( c3 ) comprising the modified variable domains 1 , 2 and 3 from karp and ta763 strains . variable domain 1 and 2 are based on the same domains of the 56 kda protein antigen of the ta 763 stain but slightly modified ( fig2 b ), and variable domain 3 of c3 is based on variable domain 3 of the 56 kda protein antigen of the karp stain also with slight modification ( fig2 a ). the sequence of c3 is set forth in seq id no . 3 ( fig3 c ). the dna was synthesized by bioclone ( san diego , calif .) and cloned into pet29a vector ( novagen ®, gibbstown , n . j .) with built - in ndei and xhoi restriction sites . the resulting sequence was confirmed . specific design strategy is shown in table 1 ( appendix a ) cloning of 56 kda protein genes from karp and ta763 strains of o . tsutsugamushi the primers with built - in bamh1 and ndei restriction sites for the 56 kda protein gene ( amino acid 80 - 456 of the open reading frame ) were designed based on the available dna sequences of the karp and ta763 strains in ncbi database . respective strain of o . tsutsugamushi was purified from infected l929 cells using renografin gradient ( 52 ). after gradient , the pure organisms were resuspended in water and freezed at − 80 ° c . until use . dna of each strain of o . tsutsugamushi was extracted using qiaamp dna mini kit ( qiagen inc ., valencia , calif .) following the manufacturer &# 39 ; s description . the purity of dna was determined spectrophotometrically and the value of od 260 / od 280 was greater than 1 . 8 . the extracted dna was then used as template in combination with appropriate primers for each specific strain in a polymerase chain reaction ( pcr ). the amplicons was cloned into a pet24a vector ( novagen ®, gibbstown n . j .). colonies were selected and the presence of amplicons was verified . sequences of the amplicons were confirmed . the plasmid was transformed into bl21 ( de3 ) cells for protein expression . bl21 ( de3 ) transformants containing correct amplicons were selected , grew in lb in the presence of 50 μg / ml kanamycin in a 37 ° c . shaker and agitated at 200 rpm . the cells were induced with 1 mm iptg when od 600 reached 0 . 8 - 1 . 0 . after induction for 19 hours , the cells were centrifuged at 4000 rpm for 30 minutes in ss34 rotor to separate cells from lb medium . the wet weight of cell pellet was determined . at least 100 μl each of cell suspension prior to and post induction was removed to examine the level of induction . samples with confirmed induction were processed for protein purification . extraction and solubilization of inclusion bodies containing the recombinant 56 kda protein three gram of cell pellet was resuspended in 15 ml of 2 % deoxycholate ( doc ) in 20 mm tris - hcl ( ph 7 . 5 ) at a w / v ratio of 1 : 5 and dispersed well to ensure complete dissolution . cell suspension was sonicated 6 times on ice at output setting of 3 for 10 seconds each . cells were then spun at 10 , 000 rpm for 30 minutes at 4 ° c . the supernantant was removed and 2 m urea solution was added to the pellet . the pellets were dispersed well by up - down suction using a 10 - ml pipette , and put it on a shaker for 30 min . the suspection was centrifuged and the surpernatant was discarded and 4 m urea in 20 mm tris - hcl ( ph 7 . 5 ) with the same volume as 2 % doc was to the pellet . the pellet was dispersed well and incubated for 30 min with gentle rocking . centrifuged the solution at 10 , 000 rpm for 30 min at 4 ° c . and poured out the supernatant . added 10 ml of 6 m urea in 20 mm tris - hcl , ph 7 . 5 to the pellet and dispersed the pellet well and incubated for 30 min with gentle rocking . the solution was centrifuged again under the same procedure and the supernatant was discarded . added 10 ml of 8 m urea in 20 mm tris - hcl , ph 7 . 5 to the pellet and dispersed the pellet well . the pellet was incubated for 30 min with gentle rocking on a shaker . the distribution of protein in each fraction was examined using sds - page . the supernatant ( approx 10 ml ) containing majority of chimeric proteins was in 6m - urea . this supernatant was collected in a 15 - ml tube and stored in 4 ° c . before further purification . the 6m urea supernatant containing recombinant protein ( r56 ) was subjected to anion - exchange chromatography ( deae , 21 . 5 × 15 cm ) using waters 2195 hplc . the buffers used were 6 m urea in 20 mm tris - hcl ( final ph = 8 . 0 ) containing 1 mm each of dtt and edta ( solvent a ), 500 mm nacl in solvent a ( elution buffer ) and 2 m nacl in solvent a ( ending buffer ). the deae column was equilibrated with starting buffer ( solvent a ) for at least column volume at 4 ml / min . the 6m urea supernatant was loaded onto the column through pump at 1 ml / min . once the sample was loaded onto the column , the gradient started at 100 % starting buffer for 25 minutes then in 10 minutes slowly increased to 6 % elution buffer . the concentration of elution buffer was slowly ramped up to 14 % in 50 minutes . chimeric proteins were eluted within this period and collected at 0 . 15 minutes per fraction ( 0 . 6 ml ). the deae column was further cleaned by using a 40 minutes run at 100 % ending buffer to remove any residual amount of bound protein . the purity of protein in each fraction was accessed by running sds - page . the pure fractions were pooled together . if the purity of protein was not satisfactory (& gt ; 95 % purity as evidenced on an overloaded sds - page ), a 2 nd run of deae chromatography was carried out after dialysis of the pooled fractions to remove nacl . the procedure for the 2 nd deae was similar to the first deae purification but the gradient was modified so that elution buffer was slowly increased from 6 % to 12 % in 50 minutes . if necessary , 6m urea supernatant containing chimeric protein can be first purified by two gel filtration columns connected in tandem using solvent a as the buffer . the fractions containing chimeric protein can be pooled together and be purified by anion exchange chromatography as described . the final fractions containing chimeric protein were evaluated for protein purity using sds - page . the dialysis tubing ( spectrum ® chemical and laboratory products , gardena , calif .) was boiled in sodium carbonate solution , rinsed with water and then stored in water containing 0 . 02 % nan 3 in 4 ° c . the volume of dialysis buffer and sample is 20 : 1 ( i . e . 10 ml of sample in 200 ml dialysis buffer ). the pure chimeric protein was in solvent a containing nacl . dialysis was done in a step - wise manner to remove nacl and lower the concentration of urea from 6 m to none . first , the purified pooled fractions were transferred into the prepared dialysis tubing and dialyzed against 4m urea in solvent a for 30 minutes twice in cold room under gentle stirring . at the end of 2 nd dialysis against 4m urea , replaced the 4m urea with 2m urea and dialyzed the sample the same way . then replaced 2m urea with 1m urea and dialyzed for the same period using the same dialysis procedure . finally , replaced 1m urea with 20 mm tris - hcl ( ph7 . 5 ) and dialyzed for the same period . all the r56s were purified to greater than 95 % purity ( fig1 a ). in all cases , protein was over loaded in each lane to ensure purity . same amount of each r56 was loaded onto the gel for western blot . fig1 b shows that all r56s reacted with the specific antibody against the 56 kda protein from karp strain . this result suggests that there are cross reactive epitopes in ta763r56 , c1 , c2 and c3 , which can be recognized by antibody raised specifically against kpr56 . the dialyzed chimeric r56s was analyzed by sds - page to examine the purity . gel was transferred onto pvdf membrane using standard procedure . the pvdf membrane was stained with codeblue staining solution to visualize protein bands . the single band between 37 and 50 kda was cut out of the membrane and analyzed by protein n - terminal sequencer ( applied biosystems 490 , applied biosystems , foster city , calif .) to confirm the expressed protein was the designed 56 kda protein . additional gel was transferred onto nitrocellulose membrane . the nitrocellulose membrane was blotted by 10 % milk in tbs - tween20 ( tbst ) for 1 h with gentle agitation . membrane was then washed with tbst for 5 minutes 3 times with agitation . primary antibody against the 56 kda protein antigen was added to 5 % milk in tbst and incubated with membrane for 1 h at room temperature with gentle agitation . the membrane was then washed three times at 5 minutes each . hrp conjugated anti - mouse igg antibody was the 2 nd antibody and was incubated with membrane in 5 % milk in tbst for 1 hour . the membrane was then washed and incubated with peroxidase substrate ( bio - rad laboratories , hercules , calif .) for up to 30 minutes for development . the presence of a band between 37 and 50 kda indicated the positive reactivity between the chimeric protein and antibody against 56 kda protein ( fig1 b ). analysis of reactivity of various r56s protein and sera from mice infected by various strains of o . tsutsugamushi the cross reactivity of the three chimeric r56s were analyzed using strain - specific mouse sera . these strains ( table 2 ) are reported in regions within the scrub typhus endemic area . in order to know whether the design of chimeric protein has improved cross reactivity , the parent r56s ( kpr56 and ta763r56 ) were also analyzed using the same set of sera . the concentration of each protein antigen ( kpr56 , ta763r56 , c1 , c2 and c3 ) was determined by bradford method . a preliminary study had established that the amounts of antigen required to coat the plate was 0 . 3 μg / well ( total volume was 100 μl ). therefore , the concentration of each chimeric protein antigen was adjusted to 0 . 3 μg / 100 μl by 0 . 2 × pbs . the plate was coated at 4 ° c . overnight . on the day of experiment , plates were first washed 3 times with 1 × pbs containing 0 . 1 % tween ® 20 ( 1 × pbst ). the plates were then blocked with 200 μl / well of 10 % milk in 1 × pbst and were incubated for 1 hour at room temperature . primary antibody ( mouse sera ) was diluted in 5 % milk in 1 × pbst . the primary antibody was diluted 1 : 100 for screening purpose . for titration purpose , primary antibody was prepared for a serial dilution differed by a factor of 4 ( 1 : 100 , 1 : 400 , 1 : 1600 etc ). after blocking , plates were washed 3 times with 1 × pbst and 100 μl / well of diluted primary antibody were added . plates were incubated for 1 hour at room temperature . after incubation , plates were washed 3 times with 1 × pbst . hrp conjugated goat anti - mouse igg was used as the secondary antibody . the antibody was diluted to 1 : 4000 in 5 % milk in 1 × pbst and 100 μl / well of diluted secondary antibody was applied and plates were incubated for 1 hour at room temperature . at the end of incubation , plates were washed 3 times with 1 × pbst . a 1 : 1 ratio of abts ® peroxidase substrate solution a and peroxidase substrate solution b ( kirkegaard & amp ; perry laboratories , gaithersburg , md .) was prepared and added at 100 μl / well . plates were incubated for 15 - 30 minutes in a dark drawer . plates were read after 30 minutes incubation at 405 nm - 650 nm on an uvmax kinetic microplate reader ( molecular devices , philadelphia , pa .). among these strains , immunization by r56 was not protective to subsequent challenged by mak119 and 243 in the mouse model . therefore , serum collected from the patients who were infected with these two strains was used for the evaluation . all the tested sera when diluted 100 folds reacted with every r56 . the titer of each serum was determined for the 5 r56s as a measurement of reactivity . there is reactivity differences among the r56s . kpr56 and ta763r56 had the highest titers with sera from respective homologous infection challenge ( table 3 ), confirming the expected hypothesis that these antigens are most reactive toward its own antibodies . in addition to the reactivity with homologous serum , kpr56 was the most reactive antigen for sera from th1812 , 18032460 , 18032404 , woods , citrano infected mice . ta763r56 was the most reactive antigen for th1811 , th1812 , th1814 , afc - 27 , 18032404 , woods , citrano and mak243 ( table 3 ). chimeric antigens c1 , c2 and c3 , behaved similarly for any of the given serum ( table 4 ). a elisa was performed as described in materials and methods . all the sera were from mice with the exception of mak119 and mak243 which were from human . the positive cutoff was at least the average od 405 for negative sera + 2 s . d . in the case of mouse sera , the cutoff was 0 . 1 . for mak119 and mak243 , the cutoff was 0 . 6 . b the titers highlighted in red are those sera showed highest titers with kpr56 and the titers highlighted in blue are those sera showed highest titers with ta763r56 . a elisa was performed as described in materials and methods . all the sera were from mice with the exception of mak119 and mak243 which were from human . the positive cutoff was at least the average of od 405 for negative sera + 2 s . d . in the case of mouse sera , the cutoff was 0 . 1 . for mak119 and mak243 , the cutoff was 0 . 6 . b the bold strains are those with the same titers in at least one of chimerics and the parent kpr56 and / or ta763r56 . c the titers highlighted in red are those sera showed highest titers among the chimerics with c1 , in blue are those sera with c2 and in green are those with c3 . furthermore , for most tested sera with the exception of karp , ta763 , th1811 and th1812 , the chimeric antigens ( c1 , c2 and c3 ) were as reactive as either kpr56 or ta763r56 . these results suggested that although the sequence modifications were made on the parent 56 kda protein , these chimeric antigens still retained similar reactivity with most sera . therefore raise the potential to use one of the three chimeric antigens as substitution of both kpr56 and ta763r56 . when comparison was made only among the chimeric antigens , c1 appeared to be the best antigen as it had the highest titers against all 14 sera . in fact , the titers with c1 were as high as those for kpr56 or ta763r56 for the seven tested sera . although it is hard to correlate the titer against a specific antigen with the overall stimulation of immune responses , measurement of titers does provide information about the reactivity of certain antigen to antibodies . it is thus plausible that these chimerics can be used as reagents for diagnostic purpose as well as vaccine candidates . cd1 female mice were immunized with r56s and subsequently challenged to evaluate the protective efficacy provided by individual r56 . ten mice were immunized with 25 μg of each protein antigen subcutaneously with cpg ( aligo 1826 ) and montanide as adjuvants according to experiment design . challenge was performed by intraperitoneal injection of live o . tsutsugamushi and the mice were monitored for additional 21 days . previous experiments have demonstrated that one or two immunizations using kpr56 offers poor heterologous protection . therefore , the mice were given three immunizations in this study at four - week interval . homologous challenges were performed to compare the protective effect of these chimeric proteins to their parental proteins ( i . e . kpr56 and ta763r56 ). we found that chimeric antigen 1 and 2 provided similar or better protection than kpr56 against karp strain challenge . although none of the chimeric provided better protection than ta763r56 against ta763 challenge , partial protection was observed by all chimerics . the results demonstrated that these chimeric protein antigens can provide excellent homologous protection against karp strain and partial protection against heterologous ta763 strain . further studies are needed to compare the protective efficacy of ta763r56 or kpr56 against heterlogous challenges with that of chimeric proteins . the combination of chimeric proteins may provide a broader protection . because these newly made chimeric proteins were similarly reactive with sera as the parent proteins , one can use one of the three chimerics , particularly c1 to substitute the parent proteins for use in diagnosis of o . tsutsugamushi infection or as vaccine candidates to improve the broad protective efficacy . an embodiment of the invention is an assay for detecting antibody to scrub typhus comprising : a . obtaining a sample from a subject ; b . exposing the sample to a recombinant chimeric antigen in assay equipment selected from the group consisting of elisa plates , dot - blot matrices , and hand held chromatographic and flow through assay devices ; wherein said chimeric antigen containing unmodified and modified reactive polypeptide fragments from more than one strains of orientia tsutsugamushi , such as the chimeric antigen proteins c1 , c2 and c3 as described in previous sections . it is possible that similar approach can be made to generate more chimerics based on 56 kda protein sequences from different strains in order to minimize the number of proteins included in the final vaccine formula yet still provide a very broad protection against most number of strains possible . 1 . brown g w , robinson d m , huxsoll d l , ng t s , lim k j , sannasey g . scrub typhus : a common cause of illness in indigenous populations . trans r soc . trop med hyg . 1976 ; 70 : 444 - 8 . 2 . kawamura a , tanaka h , tamura a . 1995 . tsutsugamushi disease . tokyo : university of tokyo press . 3 . kumar k , saxena v k , thomas t g , lal s . outbreak investigation of scrub typhus in himachal pradesh ( india ). j commun dis . 2004 december ; 36 ( 4 ): 277 - 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