Patent Application: US-201414188065-A

Abstract:
the present invention is concerned with the development of a vaccine against aeromonas hydrophila for use especially in fish . the invention provides an immunogenic s - layer protein of approximately 50 kda of a . hydrophila for use in the development of a vaccine , as well as the nucleic acid encoding said protein and vaccines comprising said protein or nucleic acid encoding said protein .

Description:
the present invention will now be further described by way of example and with reference to the figures which show : fig1 : antibody response of common carp against different isolates of a . hydrophila determined by elisa expressed as an absorbance at 450 nm . all sera were diluted 1 : 512 . fig2 : western blot analysis of different whole cell preparations of a . hydrophila against pooled serum from common carp infected with 6 different a . hydrophila isolates . lanes : ( 1 ) standard marker ; ( 2 ) t4 ; ( 3 ) 98141 ; ( 4 ) hh ; ( 5 ) vds ; ( 6 ) catla ; ( 7 ) c24li ; ( 8 ) 2d20 ; ( 9 ) 3d14 ; ( 10 ) 2n14 ; ( 1 ) 98140 ; ( 12 ) 98139 ; ( 13 ) b2 / 12 ; ( 14 ) f1d75 ; ( 15 ) calf . fig3 : western blot analysis of different preparations of 6 a . hydrophila isolates screened with serum raised against corresponding a . hydrophila isolates in common carp . ( a ) t4 , ( b ) 98141 , ( c ) hh , ( d ) vds , ( e ) catla , ( f ) c24li . lanes : ( 1 ) standard marker ; ( 2 ) wc in vitro ; ( 3 ) wc in vivo 25 kda ; ( 4 ) wc in vivo 100 kda ; ( 5 ) omp in vitro ; ( 6 ) omp in vivo 25 kda ; ( 7 ) omp in vivo 100 kda ; ( 8 ) ecp in vitro ; ( 9 ) ecp in vivo 25 kda ; ( 10 ) ecp in vivo 100 kda . fig4 : 2d western blot analysis of whole cell preparation of a . hydrophila t4 isolate screened with antibody from common carp infected with a . hydrophila t4 isolate . fig5 : cumulative percentage of goldfish mortality in preliminary vaccination trial fig6 : maldi - tof ms spectrum showing the peptide profiles of 50 kda band fig7 : nucleic and amino acid sequences of a . hydrophila s - layer protein signal peptide sequences are highlighted ( nucleic acid in green and amino acid in yellow ). fig8 : genomic sequences of a . hydrophila isolate t4 bases different to that genome reported for the s - layer protein of a . hydrophila isolate tf7 by thomas and trust ( 1995a ) have been highlighted . fig9 : amplification of the s - layer gene of a . hydrophila isolate t4 shown on a 1 % agarose gel . lanes : ( 1 ) standard marker ; ( 2 ) s - layer protein gene ; ( 3 ) purified s - layer protein gene ; ( 4 ) pqe60 vector carrying s - layer protein gene . fig1 : expression of s - layer protein of a . hydrophila with e . coli wc protein . ( a ) 12 % sds - page stained with coomassie blue , ( b ) western blot of protein using an anti - histidine tag antibodies . lanes : ( i ) standard protein marker ; ( 2 ) wc preparation of recombinant e . coli without iptg induction ; ( 3 ) wc preparation of recombinant e . coli with iptg induction showing 5 - layer protein . fig1 : cumulative percentage mortality of carp vaccinated with recombinant s - layer protein and challenged with a . hydrophila isolates . ( a ) t4 , ( b ) hh , ( c ) 98140 , ( d ) 98141 , ( e ) vds , ( f ) b2 / 12 . four virulent strains ; t4 , 98141 , hh , vds and two avirulent strains ; carla and c24li of a . hydrophila were used ( table 1 ). common carp ( average weight 30 g ) were maintained in 6 separate glass tanks . the fish were anesthetized and injected intraperitoneally ( ip ) with 0 . 1 ml pbs containing 1 × 10 6 viable a . hydrophila . each strain was injected into 24 fish and an additional 24 fish were injected with phosphate buffered saline ( pbs ) as controls . after injection , the fish were supplied with re - circulating water that had been passed through a sedimentation tank , drum filter , biofilter and ultraviolet ( uv ) radiation . the temperature of the tank water was maintained at 20 ± 1 ° c . blood samples were taken 3 , 9 , 12 and 21 days post - injection and pre - injection bleeds were taken from six fish . blood was stored overnight at 4 ° c . and the serum collected by centrifuging at 2000 × g for 5 min . the serum was stored at − 20 ° c . until further analysis . western blot analysis was performed using fish anti - sera as per the method outlined by wiens et al . ( 1990 : with modifications ). western blot analysis was carried out for the 14 a . hydrophila isolates shown in table 1 using pooled common carp serum raised against six isolates of a . hydrophila strains ( t4 , 98141 , hh , vds , catla and c24li ). different preparations of the bacterium ( i . e . whole cell ( wc ), outer membrane protein ( omp ) and extracellular products ( ecp )) prepared from these 6 isolates of a . hydrophila grown either in vitro or in vivo were screened by western blot with each of the 6 anti - sera raised against different isolates of a . hydrophila . the bacterial preparations described above were subjected to 12 % sds - page and the resolved antigens transferred to nitrocellulose membranes (@ 60 v for 1 h ). the nitrocellulose membranes were blocked using 2 % ( w / v ) casein for 1 h at 20 - 22 ° c . the membranes were washed three times ( tris buffered saline containing 0 . 1 % ( v / v ) tween - 20 : ttbs / 5 min per wash ) and then incubated overnight in the common carp anti - serum diluted 1 / 10 in tris buffered saline ( tbs ). membranes were again washed and incubated with an anti - carp igm monoclonal antibody ( aquatic diagnostics ltd , stirling , uk ) for 2 h . the membranes were washed and incubated with anti - mouse igg - hrp ( sigma , mo ., usa ) for 1 h . the blots were developed by adding chromogen and substrate solution ( 2 ml of 4 - chloro - naphthol solution with 10 mls of pbs and 10 μl of h 2 o 2 ) until bands were observed , the antigenic profile of wc preparation of isolate t4 grown in vitro was screened using 2d sds - page and western blotting with anti - serum ( raised for t4 isolate ) from common carp . assessing the levels of protection of a 50 kda protein in goldfish against a . hydrophile challenge : preparation of the antigen . volumes ( 100 μl ) of sample were subjected to 12 % sds page . the gels were run for between 5 - 6 h at 250 v / 130 ma . on completion the 50 kda band was cut from the gel and finely chopped . these were placed in tubes containing 300 μl sds - page reservoir buffer a blotting paper disk and a porous polyethylene plug . the tip of the tube was cut and placed into a 1 . 5 ml centrifuge tube containing 300 μl of 4 × sds - page reservoir buffer . the tubes were then placed into an electroluter and subjected to 50 v at 0 . 5 ma and a reverse run at 50 v for 5 sec at the end . the eluted protein was collected and the reservoir buffer removed using a 10 , 000 mw cut - off spin concentrator . the concentration of protein was determined . the presence of the 50 kda protein was confirmed by sds - page and western blot . for preliminary vaccination , four goldfish weighing around 30 - 40 g were injected ip with 200 μl , of suspension having 12 . 3 μg of 50 kda protein in 60 μl of pbs and 140 μl of montanide adjuvant . another four fish were also injected with pbs to serve as controls . all the fish were challenged with a . hydrophila 31 days post - vaccination and sacrificed 21 days after challenge as described above . samples were taken from their kidneys . the relative percentage survival ( rps ) was calculated using the following formula ( ellis , 1988 ). in addition , two goldfish weighing 30 - 40 g were injected ip with 200 μl of antigen ( i . e . 12 . 3 μg 50 kda protein ) emulsified with freund &# 39 ; s complete adjuvant ( fca ). thirty four days later , both fish were re - vaccinated with the same suspension as described above except freund &# 39 ; s incomplete adjuvant was used in place of fca . seventeen days after the booster injection , blood was collected from one fish and the anti - serum collected . three goldfish weighing between 30 - 40 g were immunised by ip injection with 0 . 1 ml of goldfish sera raised against the 50 kda protein electro - eluted from a . hydrophila , and 3 fish were injected with control serum collected from non - vaccinated goldfish . after 24 h all the fish were challenged with 0 . 1 ml of 2 . 5 × 10 7 ml − 1 a . hydrophila t4 isolate in pbs by ip injection , but on the opposite side to the site where they had been injected with the antiserum ( lafrentz , 2003 ). kidney samples from fish which died during the experiment and surviving fish at day 21 post - challenge were streaked on tsa to confirm specific mortality . sequencing and identification of the 50 kda protein of a . hydrophila a whole cell preparation of a . hydrophila t4 isolate in sds - page sample buffer was prepared for sequencing and analysis of the 50 kda protein by maldi - tof ms . samples were in - gel reductively alkylated prior to staining with colloidal coomassie blue , then digested in 0 . 1 % of n - octyl glucoside / 20 mm ammonium bicarbonate plus 12 . 5 μg ml − 1 trypsin , and the sample ( 1 . 5 μl ) was spotted from the extract ( 30 μl ) after adding an equal volume of acetonitrile for performing maldi - tof ms analysis . recombinant protein was produced in order to have a sufficient quantity of protein for a large scale vaccination trial . all the recombinant protein work was conducted at the genomic laboratory , tokyo university of marine sciences and technology , japan . polymerase chain reaction ( pcr ) of a . hydrophila 50 kda protein gene specific primers were designed to amplify the full length of the 50 kda protein gene based on the sequence data for the s - layer gene of a . hydrophila published by thomas and trust ( 1995a : see fig8 ). restriction sites nco i and bgl ii were added to the forward and reverse primers respectively to assist its cloning into the expression vector pqe60 . the pcr was run for 32 cycles ( 95 ° c ./ 5 min ; denaturation for 95 ° c ./ 30 sec ; annealing at 55 ° c ./ 30 sec ; elongation at 72 ° c ./ 1 min and a final elongation step at 72 ° c ./ 5 min ). the primers used were as follows : forward : acatgggagttaatctggacactggtgc ; reverse : gacttgtggtacttgcgtaagtctaga the pcr products were resolved by 1 % agarose gel electrophoresis and the dna was extracted using a dna purification kit . digestion of the pcr products and the pqe 60 vector ( qiagen ) were carried out overnight at 37 ° c . both pqe 60 vectors and pcr products were purified after the digestion process and ligated by mixing 2 μl of vector with 8 μl of pcr products and adding 10 μl ligation high ( cosmo bio co ltd , tokyo ) before incubating it overnight at 16 ° c . transformation of vectors carrying 50 kda protein gene into e . coli escherichia coli , m15 ( quiagen , tokyo , japan ) was transformed with pqe 60 vectors carrying the amplified 50 kda protein gene of a . hydrophila . expression of the recombinant 50 kda protein in e . coli the clones containing the 50 kda protein gene insert identified by pcr , were inoculated into lb broth containing ampicillin ( 100 μg ml − 1 ) and kanamycin ( 25 μg ml − 1 ), and incubated overnight at 37 ° c . recombinant protein expression was induced by addition of 1 mm isopropyl - β - thiogalactoside ( iptg ) for 4 h . for large scale production , positive clones were cultured in 50 ml of antibiotic supplemented lb broth overnight at 37 ° c . with vigorous shaking . this culture was transferred to 1 l fresh lb broth and cultured at 37 ° c . with vigorous shaking . recombinant protein expression was induced by adding 1 mm iptg . sequencing of the a . hydrophila t4 isolate 50 kda protein gene the whole 50 kda protein gene of a . hydrophila isolate t4 was sequenced at the genomic laboratory , tokyo university of marine sciences and technology . vaccination of common carp with recombinant 50 kda protein recombinant 50 kda protein of a . hydrophila diluted in pbs was mixed with montanide adjuvant at a ratio of 30 : 70 ( v / v ) to a final antigen concentration of 300 μg ml − 1 . buffer ( pbs ) mixed with the adjuvant was also prepared at the same ratio as the antigen to serve as a negative control . one hundred and fifty common carp ( 30 - 40 g ) were vaccinated by ip injection with 0 . 1 ml of the vaccine preparation , and another 150 fish were injected with the pbs adjuvant mixture . all the fish were maintained for 35 days in 1 × 1 m ( diameter × depth ) tanks with recirculating water before challenging with six different isolates of a . hydrophila . each of the six virulent isolates described above were used to challenge vaccinated fish . twenty vaccinated and 20 control fish were injected ip with each strain . the concentrations of the bacteria used in the challenge were 1 × 10 8 , 2 × 10 7 , 2 × 10 7 , 5 × 10 7 , 7 . 5 × 10 6 and 2 × 10 7 bacteria ml − 1 for t4 , 98140 , 98141 , hh , b2 / 12 and vds respectively . all 40 fish within each group were placed in separate glass tank ( 90 cm length × 47 cm height × 40 cm depth ) with aeration and recirculating water . the fish were maintained for 16 days post - challenge and dead fish were removed 3 times a day . samples from the kidney of dead fish and also from surviving fish at the end of the experiment on day 16 post - challenge were streaked onto tsa . the results obtained were statically analysed using chi - square test for survival , comparing the mortality of vaccinated fish with the control group fish after challenging with bacteria . antibody response of common carp infected with different isolates of a . hydrophila the antibody levels increased after day 9 and a positive response was observed on day 12 post - infection with all the isolates , except for isolate 98141 . by day 21 post - infection , this response had increased for all the isolates , with the highest antibody response recorded against isolate t4 ( fig1 ), the wc preparations of a . hydrophila isolates grown in vitro , screened by western blot with the anti - sera from infected common carp , exhibited a distribution of bands between 20 and 160 kda ( fig2 ). carp antibodies bound to antigens ranging from 30 - 50 kda for 3 of the virulent isolates , t4 , hh and b2 / 12 . except for isolate 2d20 , one band was observed at approximately 50 kda . the antibody response against wc , omp and ecp preparations of a . hydrophila grown in vitro and in vivo , showed similar profiles among isolates t4 , 98141 and hh ( fig3 ). with all the virulent isolates ( t4 , hh , 98141 and vds ), a band was evident at around 50 kda in wc and omp preparations . this band was also present in ecp preparations from in vitro cultured bacteria . the omp preparations from isolate vds grown in vitro showed 6 bands between 25 and 50 kda . a band at 50 kda was observed in wc and omp preparations of isolate catla grown in vitro . a band at around 50 kda was seen in both wc and omp preparations from isolate c24li grown in vitro and in vivo . six bands were seen between 35 and 100 kda with ecp preparations from bacteria grown in mw cut - off tubes but the bands were weakly stained in the case of bacteria grown in the 100 mw cut off tube . the 2d western blot for a . hydrophila t4 isolate using the antibody raised against the isolate in common carp expressed three spots at approximately 50 kda with pi values between 5 and 5 . 7 ( fig4 ). vaccination and passive immunisation of goldfish with an electro - eluted 50 kda protein from a . hydrophila in the vaccination experiment , two control and one vaccinated fish died due to unknown causes before challenging them with a . hydrophila isolate t4 . the two fish remaining in the control group died on day one and day 4 post - challenge ( fig6 ). one fish from the vaccinated group was also sacrificed one week post - challenge as it was suffering from a severe lesion and a . hydrophila was isolated from swabs taken from the lesion and kidney of the sacrificed fish . the remaining two fish in the vaccinated group were healthy and sacrificed at the end of the experiment , at 21 days post - challenge . all kidney swabs taken from dead fish were positive for a . hydrophila while the samples taken from two vaccinated fish at the end of experiment were negative . though the numbers of fish used in the experiment were low , the rps value was 66 . 7 %. the fish serum raised against the 50 kda protein of a . hydrophila used to passively immunise fish , had a titre of 1 / 16 . in the trial , one fish from the control group died two days post - infection and the presence of a . hydrophila in its kidney was confirmed using an api 20e strip . no other fish died and no kidneys were positive for the bacteria when remaining fish were sampled at the end of the trial on day 21 . maldi - tof sequence of the 50 kda protein from a . hydrophila after maldi - tof analysis and sequencing , the 50 kda protein of a . hydrophila isolate t4 was identified as a 47 . 6 kda s - layer protein ( fig7 and fig9 ). six bases were found to be different in the whole s - layer genome of a . hydrophila t4 isolate ( fig9 ) compared with the s - layer genome sequence of isolate tf7 reported by thomas and trust ( 1995a : fig8 ). the amino acid sequence shown in fig8 was obtained from the ncbi database . production of a recombinant protein for the s - layer of a . hydrophila isolate t4 bands at 1353 bp on the 1 % ( w / v ) agrose gel verified that the amplification of s - layer genomic dna was successful ( fig1 ). after transformation of vectors into e . coli cells , the presence of the s - layer genome in e . coli was confirmed by pcr ( fig1 la ) and western blot ( fig1 b ). all six strains t4 , 98140 , 98141 , hh , b2 / 12 and vds were passaged twice through common carp and the bacteria were successfully recovered from both passages . during the first passage , no mortalities occurred in any of the groups of fish , while most fish died upon passaging the bacterium a second time with all strains except t4 . the values obtained in the preliminary challenge experiment in which the ld 50 dose for each strain was determined are given in table 2 . in fish challenged with isolate t4 , 75 % of control and 10 % of vaccinated fish died . a high percentage of mortalities were recorded in control fish challenged with isolate t4 compared with fish challenged with the other isolates of a . hydrophila . fifteen percent of the control group died by the first day post - challenge and 25 % had died by day 2 post - challenge . the levels of mortality decreased to 10 % by day 3 post - challenge and thereafter it varied between 5 and 10 % until the mortalities stopped by day 8 post - challenge . the mortality in the vaccinated group was 5 % on day 1 post - challenge and another 5 % had died by day 5 ( fig1 a ). a relatively high percentage survival ( rps ) value ( 87 %) was found with isolate t4 compared with other isolates ( table 3 ). mortality of 5 % was noted in the control group challenged with isolate hh on day 1 post - challenge . however , the mortality increased to 20 % by day 2 , 15 % occurred on day 5 and 10 % on day 6 . the remainder of mortalities ( i . e . 15 %) were distributed over the period after day 7 . in the vaccinated group , 5 % of mortalities were recorded on the first and third day post - challenge ( fig1 b ). the second highest kps value ( 85 %) in the trial was observed with this isolate . fifty - percentage mortality was seen with the control group challenged with isolate 98140 . thirty percent died in the control group during the first two days post - challenge and remainder died over the course of the experiment ( 16 days post - challenge ). five percent mortality was recorded in the vaccinated group during the first two days post - challenge , and no further mortalities occurred in this group leading to an 80 % rps value for this isolate ( fig1 c ). in the control group challenged with isolate 98141 , 25 % of mortality occurred over the first two days of the experiment and thereafter 15 % mortalities occurred . the mortality with the vaccinated group was similar to that of the mortality recorded with vaccinated group challenged with isolate 98140 ( fig1 d ). an rps value of 75 % was recorded with this isolate . the control group challenged with isolate vds experienced a 10 % mortality on day 1 , day 2 and day 5 post - challenge , while 5 % mortalities occurred on the third and sixth day post - challenge ( fig1 e ). a total of 15 % mortality occurred in the vaccinated group distributed over day 2 , 3 and 5 post - challenge . the rps value with this isolate was 62 . 5 %. percentage mortality in the control group rose to 30 % during the first two days after challenging the fish with isolate b2 / 12 . another 15 % mortality occurred in this group over the remainder of the experiment . the highest percentage mortality amongst vaccinated fish was recorded in the group challenged with b2 / 12 . ten percent mortality was observed in this group on the next day post - challenge and 5 % of mortality occurred on the second day and the third day post - challenge ( fig1 f ). the rps value was low ( 56 %) with this isolate compared to other isolates . all the fish that died during the experiment showed the presence of a . hydrophila in their kidneys . in contrast , a . hydrophila was not cultured from kidney swabs taken from the surviving fish except very few colonies from one fish in the vaccinated group challenged with isolate 98140 and one fish in the control group challenged with isolate 98141 . statistical analysis revealed that survival against isolates t4 , 98140 , 98141 and 1 - 111 were significant in vaccinated fish compared to control fish , while levels of survival were not statistically significant for isolates b2 / 12 and vds ( table 3 ). in this study , common carp were infected with a . hydrophila and the anti - sera produced were used to identify immunogenic components of the bacterium . the different a . hydrophila isolates examined elicited a variety of responses in common carp , as determined by elisa . an increase in antibody response against a . hydrophila was seen after day 9 post - infection for all the isolates except one ( 98141 ). antibody response peaked on day 12 post - infection for two isolates ( hh and catla ) and on day 21 post - infection for three isolates ( t4 , vds and c24li ). the antibody response of common carp did not show any differentiation between virulent and avirulent isolates of a . hydrophila . this may be due to differences in the ability of the immune system of the host to respond to foreign agents . western blot analysis using the anti - sera produced on day 21 post - infection , against different strains of a . hydrophila , showed differences in the profiles between the isolates . however , when pooled sera ( from common carp infected with 6 different isolates ) were used to examine the response against the 14 isolates of a . hydrophila ( described in table 1 ), a band at around 50 kda was observed in all the isolates grown in vitro , except for isolate 2d20 . moreover , bands from 30 - 50 kda were stained in the profiles of 3 of the virulent isolates , t4 , hh and b2 / 12 grown in vitro . a band at around 50 kda was seen with all the preparations ( wc , omp , ecp ) from the virulent isolates grown both in vitro and in vivo compared with avirulent isolates , with the exception of the ecp from bacteria grown in vivo . the 2d western blot analysis of a . hydrophila ( t4 isolate ) wc revealed 3 spots at approximately 50 kda between 5 and 5 . 7 pl range when serum raised against t4 isolates was used . the results of the 1d and 2d western blot analysis suggest that a molecule at approximately 50 kda ( ranging between 47 and 51 kda ) might be one of the major immunogenic components of a . hydrophila . the 50 kda protein of a . hydrophila was considered to be the most immunogenic and most homogenous protein , recognised on each of the a . hydrophila isolates examined . the 50 kda protein conferred protection in goldfish against a . hydrophila in the direct immunisation trial . after maldi - tof ms sequenceing , six bases were found to be different in the whole s - layer genome of a . hydrophila t4 isolate ( fig9 ) compared with the s - layer genome sequence of isolate tf7 reported by thomas and trust ( 1995a : fig8 ). this in turn could result with changes in 4 amino acids in the s - layer protein of a . hydrophila isolate t4 compared with s - layer amino acid sequence reported for isolate tf7 . a recombinant s - layer protein of a . hydrophila was produced to confirm the protection efficacy of this protein in common carp against different isolates of a , hydrophila . the recombinant s - layer protein proved reactive in western blot analysis against anti - a . hydrophila t4 common carp serum and it was used to vaccinate a number of fish . these fish were then challenged with a range of different a . hydrophila isolates . high mortality rate was observed both in the vaccinated and control group within two days post - challenge compared with the mortality from day 3 post - challenge as described in fig1 . the protection elicited by the s - layer protein in vaccinated fish indicates a potential role for this protein in the virulence of a . hydrophila . the s - layer protein antigen of a . hydrophila appears to have conferred protection against the different isolates of a . hydrophila tested , although the rps values of carp did vary between the different challenge isolates . no mortalities occurred in any of the groups of fish after day 11 post - challenge in the vaccination trial described in this chapter . moreover , no colonies of a . hydrophila grew from the kidney swabs taken from surviving fish at the end of experiment except for two fish . this suggests that most of the surviving fish in the control group had cleared the bacterium through their own immune response , as fish can produce an antibody response against different components of bacterium and clear the bacteria in blood circulatory system within seven days post - infection ( leung and stevenson , 1988b ; chandran et al ., 2002b ). asha a ., nayak d . k ., shankar k . m . and mohan c . v . 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