Patent Application: US-201213343153-A

Abstract:
the invention relates to the use of campylobacter jejuni gene product jlpa and immunogenic fragments of jlpa as a component of a pharmaceutical formulation capable of eliciting an anti - campylobacter jejuni immune response . the invention also relates to a method of inducing an anti - campylobacter immune response by the administration of c . jejuni jlpa gene products or immunogenic fragments of such gene product to a subject .

Description:
jlpa is a campylobacter specific protein and is highly conserved among campylobacter strains ( fig1 ), which make it a valuable vaccine candidate against c . jejuni . an aspect of this invention is an immunogenic composition comprising campylobacter jejuni protein jlpa or fragments thereof . the composition can be isolated and prepared from natural c . jejuni isolates or be recombinantly produced . another aspect of this invention is a method of inducing an immune response against campylobacter by the administration of jlpa or its immunogenic fragments . expression of jlpa as a recombinant histidine tagged protein in e . coli in an exemplary embodiment of the invention , the jlpa gene from campylobacter jejuni 81 - 176 was amplified by polymerase chain reaction ( hereafter “ pcr ”) with the jlpa forward primer : the polymerase used for the amplification in this embodiment was hf2 dna polymerase ( clontech laboratories , inc . mountain view , calif .). the primers introduced ndei and bamhi sites into the amplicon . the amplicon was digested with ndei and bamhi and ligated to ndei and bamhi - digested pet19b plasmid ( novagen ®, gibbstown , n . j .). the ligation mixture was used to transform dh5 alpha cells with selection on ampicillin resulting in strain pg2604 . the dna in this plasmid was sequenced to confirm that the appropriate gene had been cloned . the plasmid in pg2604 was transformed into bl21 ( de3 ) to express recombinant jlpa with a hexahistidine tag . the confirmed dna sequence of the cloned jlpa gene with a hexahistidine tag was identified in seq id . no . 4 . the predicted protein encoded by this dna sequence had a predicted mass of 43 . 33 kda and a predicted pi of 5 . 03 . the predicted protein sequence was identified in seq id . no . 3 . study subjects were diarrhea patients enrolled in field studies during cobra gold 1999 , in thailand . the pathogens isolated are shown in the table 1 . a group of 10 subjects who participated in the cobra gold exercise in 2000 in thailand , but did not develop diarrhea were used as negative controls . plasma samples were collected on study days 7 and 60 , following presentation at the clinic . a single blood sample was collected from the control group during the exercise . samples were kept frozen until assayed for jlpa81 - 176 - specific iga or igg using elisa . mean + 2 standard deviation value of the control group was used to determine responder rates among the infected groups . mean iga and igg titers among the control group were 1 , 040 ± 1 , 032 and 3 , 800 ± 2 , 437 , respectively . responders were defined as individual having iga titer of 1 : 3 , 200 and igg titer of ≧ 1 : 9 , 000 . following infection ( in a field setting ), systemic immune response to jlpa was observed at the time points tested in 50 % of the subjects recovering from campylobacter infection . a murine study were undertaken in order to evaluate the immunogenicity and potential protective efficacy of an embodiment of the vaccine composition . in the study , adult female balb / c mice were lightly anesthetized with isoflurane and immunized intranasally with 30 μl of pbs containing recombinant jlpa protein alone or with a mucosal adjuvant , ltr192g . the experimental groups are illustrated in table 2 . three doses of the embodiment vaccine composition were delivered at 14 day intervals to the subject , on study days : 0 , 14 and 28 . seven days after the last vaccination , on study day 35 , antigen - specific secretory iga was measured from fecal pellets . antigen - specific serum igg was determined in tail blood collected 21 - 22 days after the last vaccination , on study day 49 - 50 . all antigen - specific responses were detected using elisa . on study day 58 ( 30 days post last vaccination ), all animals were challenged intranasally with 3 × 10 9 cfu of c . jejuni 81 - 176 . following the challenge , animals were followed for 6 consecutive days for the development of the infection associated illness . based on the severity of sickness a score was assigned to each animal as follows . 0 = no apparent illness 1 = ruffled fur 2 = ruffled fur and hunched back 3 = dead daily sickness indices , and then group average indices were determined and vaccine efficacy was calculated as : ( control — vaccinated )/( control )× 100 jlpa - specific serum igg responses in mice were shown in fig2 . jlpa - specific fecal iga responses in mice were shown in fig3 . data are presented as group geometric mean titer ( natural log e ) and standard deviation . as illustrated in fig2 , a robust vaccine dose dependent serum igg levels were detected in animals immunized with recombinant jlpa alone , which was further enhanced by inclusion of a mucosal adjuvant ltr192g ( fig2 ). animals immunized with pbs showed low levels of anti - jlpa igg responses ( mean + 2 standard deviations ≦ 1 : 100 ). based on this cut off , all animals in all groups immunized with or without ltr192g were categorized as igg responders . similar to serum igg response , a robust mucosal iga response was detected after vaccination . interestingly , ltr192g had a greater adjuvant effect on mucosal iga response than observed for serum igg response , with 5 to 6 fold increase when the same amount of protein was delivered with the adjuvant . animals immunized with pbs showed no detectable levels anti - jlpa fecal iga response ( mean + 2 standard deviations ≦ 1 : 4 ). based on this cut off , 100 % of the animals receiving 100 μg of vaccine alone or vaccine doses tested ( 5 μg , 25 μg , 100 μg ) with the adjuvant were categorized as responders for iga . five of 6 animals ( 83 %; sample was unavailable for one animal ) receiving 25 μg showed detectable levels of jlpa - specific iga in stool extracts . following the challenge with c . jejuni 81 - 176 , animals were observed for the development of sickness . the illness index and calculated jlpa efficacy of different vaccine formulations are presented table 3 . twenty four hours following each vaccination , animals receiving 25 μg or higher dose of vaccine with the adjuvant showed a mild transient signs of ruffled fur , which lasted for & lt ; 24 hours . no apparent signs of vaccine associated side effects were seen among animals receiving pbs or vaccine alone . animals immunized with 100 μg of the vaccine with the adjuvant showed 66 . 3 % protection . to achieve a moderate ( approximately 35 %) protection , a 100 μg dose alone or 25 μg with the adjuvant was required . other doses showed no protection against illness in mice . these studies illustrate the utility of the jlpa construct of c . jejuni 81 - 176 as components , alone or in combination with other moieties , as vaccines against campylobacter . accordingly , the inventive constructs can be used in methods for induction of protective anti - campylobacter immunity induction of the anti - c . jejuni mediated response in human a prophetic method for the induction of the anti - c . jejuni mediated response in humans contains the following steps : a . administration of immunogen comprising jlpa , or immunogenic fragments thereof , with or without a tag , such as histidine . if a boosting dose or doses are to be given this first administration of immunogen is a priming dose . the immunogen can be derived from isolated native polypeptide or recombinantly produced jlpa , or jlpa fragments . the immunogen can be administered orally , nasally , subcutaneously , intradermally , transdermally , transcutaneously intramuscularly , or rectally . the range of a unit dose of immunogen is 25 μg to 1 mg of immunogen . the immunogen is administered in any number of aqueous buffered solutions with or without carrier protein or adjuvant . the adjuvant can be any number of potential adjuvants , including but not limited to ltr 192g , aluminum hydroxide , rc529e , qs21 , e294 , oligodeoxynucleotides ( odn ), cpg - containing oligodeoxynucleotides , aluminum phosphate , mpl ® ( glaxosmithkline , middlesex , uk ) or combinations of these or other potential adjuvants . b . the inventive method also contemplates immunization with or without administration of subsequent boosting . the boosting step comprises the administration of , subsequent to a priming dose , 1 to 4 boosting doses with a unit dose range of 50 μg to 1 mg of immunogen in buffered aqueous solutions . alternative embodiments of the inventive method include inserting nucleotide constructs encoding jlpa , or fragments thereof , into an expression system capable of expression in mammalian subjects . in this embodiment , the expression system can be a plasmid , viral or dna expression vector . another alternative embodiment of the inventive method is to induce immunity by administering a live attenuated carrier strain of bacteria transformed with a suitable viral or dna expression system that contains one or more nucleic acid sequences encoding one or more of the polypeptides jlpa , or fragments thereof . the expression system contemplated is capable of expressing in the selected bacteria carrier . having described the invention , one skilled in the art will appreciate in the appended claims that many modifications and variations of the present invention are possible in light of the above teaching . it is therefore , to be understood that , within the scope of the appended claims , the invention may be practiced otherwise than as specifically described . fry , b . n ., feng , s ., chen , y ., newell , d . g ., coloe , p . j ., and korolik , v . 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