Patent Application: US-51999107-A

Abstract:
infectious pancreatic necrosis virus , the etiologic agent of infectious pancreatic necrosis in salmonid fish , causes significant losses to the aquaculture industry . the gene for the viral capsid protein was cloned into a yeast expression vector and expressed in saccharomyces cerevisae . expression of the capsid gene in yeast resulted in formation of approximately 20 nanometer sub - viral particles composed solely of vp2 protein . anti - ipnv antibodies were detected in rainbow trout vaccinated either by injection of purified vp2 - subviral particles or by feeding recombinant yeast expressing rvp2 - svp . challenge of rvp2 - svp immunized trout with a heterologous ipnv strain and subsequent viral load determination showed that both injection and orally vaccinated fish had lower ipnv loads than naive or sham - vaccinated fish . this study demonstrates the ability of rvp2 - svps to induce a specific immune response and the ability of immunized fish to reduce the viral load after an experimentally induced ipnv infection . the invention is not limited to ipnv , and is applicable to other similar viruses for which svps can be made and administered to fish .

Description:
the disclosure relates to production and care of fish , including immunization of fish against viral pathogens . here , we report cloning of the ipnv - vp2 gene into a yeast expression vector , pesc - ura . expression of the vp2 protein resulted in formation of approximately 20 nanometer sub - viral particles ( svps ) in yeast , as detected by electron microscopy . purified recombinant vp2 svps ( rvp2 - svps ) were used to vaccinate fish by both injection and oral routes and their antigenicity in rainbow trout evaluated by immunoassay . an ipnv challenge trial was also carried out and the effect of vaccination on viral load evaluated . an ideal vaccine for ipnv must induce long lasting protection at an early age , prevent carrier formation , and be effective against a large number ipnv serotypes . injection cannot be used for small fish , therefore either oral delivery or immersion are more preferred routes for early vaccination . these attributes of an ideal ipnv vaccine must be met either by a recombinant subunit vaccine or by an inactivated viral vaccine , as a live attenuated vaccine could potentially lead to carrier formation . the yeast expression system has potential value for oral vaccine development , since yeast is already a component of feeds and is generally regarded as safe . this contrasts with bacterial expression in escherichia coli , which generates pyrogens that would need to be removed before use of any crude preparation as an oral vaccine ( 22 ). the use of yeast is also attractive because production is economical and , through well - developed genetic systems , can be engineered to provide an abundant supply of the protein or proteins of interest . in fact , pitcovski et al . ( 19 ) reported the development and large - scale use of yeast - derived recombinant vp2 vaccine for the prevention of infectious bursal disease ( caused by another birnavirus ) of chickens . the west buxton ( wb ) strain of ipnv , obtained from american type culture collection ( atcc vr - 877 ), was used for this study . this virulent strain of ipnv is prevalent in maine and canada , where the major north american salmon aquaculture industry exists . the wb strain of ipnv was purified as previously described ( 26 ). the virus was propagated in chinook salmon embryo ( chse - 214 ) cell cultures ( atcc crl - 1681 ), maintained at 15 degrees celsius in eagle &# 39 ; s minimal essential medium ( emem ) and supplemented with 10 % fetal bovine serum ( fbs ), 100 units per milliliter penicillin , 100 micrograms per milliliter streptomycin and 1 microgram per milliliter fungizone . total viral rna was isolated from purified virus by digesting with proteinase k ( 200 micrograms per milliliter final concentration ) followed by phenol : chloroform extraction ( 23 ). the ipnv - vp2 and vp3 genes were amplified by reverse transcription - polymerase chain reaction ( rt - pcr ) and cloned into the pcr2 . 1 vector ( invitrogen , carlsbad , calif .) following previously published protocols ( 24 ). the primer pair used for vp2 cloning was wbabglf ( 5 ′- gagatctatgaacacaacaaaggcaaccgc - 3 ′; seq id no : 1 ), containing a 5 ′ bg / ii site , and wbavp2r ( 5 ′- aagcttaagcccatgtgtccatgac - 3 ′; seq id no : 2 ), containing a 5 ′ hindiii site . the primer pair used to clone the vp3 gene was wbavp3f ( 5 ′- ggatccatgtcagggatggacgaagaactg - 3 ; seq id no : 3 ) and fa3 ′ nchindr ( 5 ′- ataagcttgggggccccctggggggcc - 3 ′; seq id no : 4 ) with bambi or hindiii sites at the 5 ′ ends respectively . the integrity of the clones was verified by sequencing the plasmid dna in both directions using an automated dna sequencer ( applied biosystems ). to make a yeast expression vector containing the vp2 gene , the vp2 - containing plasmid was double digested with bg / ii and hindiii . the vp2 fragment was gel purified , blunt - ended with klenow enzyme , and inserted between the unique ecori and bg / ii sites of pesc - ura , which had been blunt - ended with klenow , behind the gal10 promoter ( fig1 a ). to make the vp3 yeast vector , the vp3 - containing plasmid was double digested with bamhi and hindiii enzymes . the vp3 fragment was gel purified and cloned between the unique bamhi and hindiii sites of pesc - ura behind the gal1 promoter ( fig1 b ). finally , to make the yeast vector that expressed both the vp2 and vp3 capsid protein genes , the vp2 gene was inserted into the unique ecori and bg / ii sites of pesc - ura behind the gal10 promoter in the vp3 - containing constructs ( fig1 c ). yeast ( saccharomyces cerivisiae strain yh501 ; stratagene , la jolla , calif .) were transformed using the ez yeast transformation kit ( zymed , sari francisco , calif . ): mutant colonies were selected for growth on autotrophic sg - ura medium containing galactose , yeast extract without amino acids , and amino acid dropout mixture ( all amino acids plus adenine , no uracil ). mutants were grown at 30 ° c . for 4 days , collected by centrifugation , then crude protein extracts prepared using y - per yeast breaking buffer ( pierce biotechnology , rockford , ill .). lysates were electrophoresed on 12 % sds - polyacrylamide gels ( biorad , richmond , calif .) and transferred to nitrocellulose by electroblotting . the blots were probed with sheep - anti - ipnv polyclonal antibody ( microtek international , inc , saanichton , b . c ., canada ) and detected with rabbit - anti - sheep polyclonal antibody conjugated to hrp ( bethyl laboratories , montgomery , tex .). detection was obtained using the colorimetric substrate tetramethyl benzidine ( tmb ) in a one step solution as described by the manufacturer ( pierce , rockford , ill .). svps were isolated from yeast cultures expressing recombinant vp2 according to a modified protoplasting protocol ( 18 ) to remove the yeast cell wall . the cells were lysed by three freeze thaw cycles then sonicated for five 60 - second cycles with 20 - second intervals . lipids were removed by performing two successive freon extractions . svps were then purified by passing them through a 26 % sucrose cushion at 82 , 705 × g ( average ) for 4 hours at 4 ° c . in a swinging bucket rotor ( beckman sw28 ), followed by cscl - gradient centrifugation overnight at 115 , 584 × g ( average ) at 4 ° c . in a swinging bucket rotor ( beckman sw41 ). the buoyant density of ipnv is 1 . 33 grams per cubic centimeter . bands were withdrawn with a syringe and dialyzed overnight at 4 ° c . in tn buffer ( 50 millimolar tris and 100 millimolar nacl , ph 8 . 0 ) to remove cscl . svp &# 39 ; s were prepared for negative staining transmission electron microscopy according to the previously published protocols ( 8 ). this method of producing svps is exemplary ; other methods can be used to produce svps . rainbow trout ( oncorhynchus mykiss ; approximately 25 grams ) originating from the clear springs food , inc . ( buhl , id .) and known to be free of ipnv were used for the immunization experiment . the vaccination and animal work was done at clear springs foods , inc . while the analytical work was performed at advanced bionutrition , inc . the fish were anesthetized and injected intraperitoneally ( ep ) with 100 microliters of vaccine ( 50 microliters of purified rvp2 - svps containing 100 micrograms antigen and 50 microliters of freund &# 39 ; s complete adjuvant ). there were three groups of fish : naive fish ( n = 9 ), fish injected with adjuvants only ( sham - injected treatment ; freund &# 39 ; s complete adjuvant , sigma , st . louis , mo . ; n = 8 ), and a treatment group that was injected with ipnv rvp2 - svps plus freud &# 39 ; s adjuvant ( n = 12 ). vaccinations were done at days 1 and 32 . for oral vaccination , recombinant yeast expressing rvp2 - svps ( without prior purification ) was mixed with feed . yeast were ground in liquid nitrogen then incorporated into a fish feed ( clear spring foods , inc ., proprietary blend ) that was first powdered using a coffee mill then supplemented with 10 % wheat gluten as binder . feed blends were mixed by hand with moisture added as required until a pliable dough was produced . this was then fed through a press to produce ribbons of feed that were chopped to approximately 0 . 5 cm in length . these were allowed to air dry at room temperature for several hours then spray coated with canola oil and frozen until use . the treatments for the oral vaccination include fish that were fed diet containing yeast expressing rvp2 - svps ( n = 13 ) or diet containing non - recombinant yeast ( control , n = 10 ). at day 60 , blood was withdrawn from caudal vessels of control and vaccinated fish and allowed to clot overnight at 4 degrees celsius . blood samples were centrifuged in a tabletop centrifuge at 12 , 568 × g ( average ) for 5 minutes , then serum was collected and stored at − 75 degrees celsius until analyzed . immuno breakapart microplates ( nunc , rochester , n . y .) were coated with purified ipnv rvp2 - svps at 150 micrograms per milliliter in a 50 millimolar carbonate coating buffer ( ph 9 . 6 ) at 4 ° c . for 16 hours . plates were washed 3 times in tbst ( ix tris buffered saline ( tbs )+ 0 . 05 % tween 20 ) for 5 minutes each wash . the plates were blocked with ix tbs containing 3 % bsa at room temperature . test sera were diluted 1 : 32 and 1 : 64 then 150 microliters was added per well and the plates were incubated for 1 hour at room temperature . following incubation with test sera , the microplates were washed again 3 times with tbst for 5 minutes per wash . the secondary antibody ( rabbit anti - rainbow trout igg ; jackson immunoresearch laboratories inc , west grove . pa .) was diluted 1 : 1000 and added to all wells ( 150 microliters / well ). the plates were incubated for 1 hour at room temperature and then washed 3 times in tbst , 5 minutes each wash . horseradish peroxidase - conjugated goat anti - rabbit igg ( biosource , camarillo , calif .) was added at a 1 : 1 . 000 dilution and detected by addition of the colorimetric substrate tetramethyl benzidine ( tmb , pierce , rockford , ill .). the absorbance was read at 450 nanometers using a spectrafluor ® plus fluorescent plate reader ( tecan , salzburg , austria ). negative controls consisted of wells that were coated as above , but a 3 % bsa solution was added instead of the fish serum at the capture step . three days after collecting the blood samples ( i . e ., at 63 days post - vaccination ), ipnv challenge was performed by injecting each fish with approximately 250 microliters of 10 7 tcid 50 / ml of ipnv ( buhl strain , lapatra unpublished ). naive fish injected with buffer served as negative control for the ipnv challenge . ten days post - injection , animals were sacrificed , spleen samples collected in tr1 reagent , then stored at − 75 degrees celsius until rna isolation was performed . total rna was isolated from spleen tissue of control and ipnv - injected rainbow trout using tri reagent following the manufacturer &# 39 ; s protocols ( molecular research center , cincinnati , ohio ). the rna samples were treated with dnase i ( ambion , enc , austin , tex .) then the rna quality assessed by running the samples on a 1 % formaldehyde agarose gel ( 23 ). the cdna synthesis was carried out in a 40 microliter reaction volume containing 1 microgram total rna , ix rt - pcr buffer . 1 millimolar dntps , 0 . 75 micromolar oligo dt , 4 units of rnase inhibitor , and 5 units of multiscribe reverse transcriptase ( applied biosystems , foster city , calif .) at 42 degrees celsius for 1 hour . the cdna was diluted 1 : 10 using dnase and rnase free molecular biology grade water and 2 microliters of the diluted cdna was taken for each reaction . the primers for the sybr green real - time rt - pcr were designed based on the nucleotide sequence of segment a of the ipnv genome that encodes the protease protein ( vp4 ) ( genbank accession no . nc — 001915 , forward primer 1916f : 5 ′ aggagatgac atgtgctacaccg 3 ′; seq id no : 5 , and reverse primer 1999r : 5 ′ ccagcgaata ttttctccacca 3 ′; seq id no : 6 ). the rainbow trout elongation factor 1 - α ( ef - 1 - α ) gene was used as an internal control for normalizing the viral load from sample to sample . the primers for rainbow trout elongation factor 1 - α ( ef - 1 - α ) were based on the published sequence of these genes ( genbank accession no af498320 , forward primer 136f : 5 ′ tgatctacaagtgcggaggca 3 ′; seq id no : 7 , and reverse primer 236r : 5 ′ cagcacccaggcatacttgaa 3 ′; seq id no : 8 ). the primers were designed using the primer express software version 1 . 0 ( perkin elmer - applied biosystem ). the real - time rt - pcr amplifications were performed in a biorad icycler iq ( biorad laboratories , inc ., richmond , calif .). the sybr green real - time rt - pcr mixture contained 12 . 5 microliters of 2 × sybr . green supermix ( iq sybr green supermix ), 300 nanomolar each of forward and reverse primers and 2 microliters of the 1 : 10 diluted cdna in a 25 microliters reaction volume . the amplifications were carried out in a 96 - well microplate with 3 replicates per sample . the thermal profile for sybr green real - time rt - pcr was 95 degrees celsius for 10 minutes , followed by 40 cycles of 95 degrees celsius for 10 seconds and 60 degrees celsius for 1 minute . after a sybr green pcr run , data acquisition and subsequent data analyses were performed using the icycler iq real - time pcr detection system ( biorad iq software version 1 . 3 ). the relative ipnv load in a sample was determined by subtracting the mean c t values for ef - 1α from the mean c t values of the ipnv amplicon . the differences in the c t value of the viral genes and the corresponding internal controls were expressed as δc t . the δc values were plotted using graphpad version 4 ( graphpad software , inc ., san diego , calif .). the difference in the δc t for one vaccine group compared to the δc t of the corresponding control was expressed as a δδc t and 2 represents the difference in viral load between the two treatments . the ipnv segment a has previously been cloned and expressed in hamster fibroblast cells , bhk - 21 , under the semliki forest virus promoter and in insect cells under the polyhedrin promoter ( polh ) and were shown to produce virus - like particles ( vlps ) that contain both vp2 and vp3 and are of similar size to the native virus but lack associated nucleic acid ( 15 ), ( 24 ). however , when we cloned the ipnv segment a in yeast , the polyprotein was expressed but no particles were observed under tem ( data not shown ). this might be due to the lack of post - translational processing of the polypeptide in yeast . therefore , we coexpressed vp2 and vp3 genes under different promoters into the pesc - ura vector so that the post - translational processing of the polyprotein would not be required . for clarity in the following discussion , the authors use the term virus - like particle ( vlp ) to describe viral - derived particles of similar size to the native virus that lack nucleic acid . for particles that are viral - derived and lack nucleic acid but do not have the same size or shape as the native virus the authors use the term sub - viral particle ( svp ) to differentiate the two sets of viral - derived particles . the predicted mature vp2 and vp3 genes were cloned separately behind gal10 and gal1 promoters in pesc - ura . recombinant yeast containing vp2 or both vp2 and vp3 genes were grown under galactose induction then analyzed by western blot analysis to determine if vp2 and vp3 were expressed ( fig2 ). two bands were observed that corresponded roughly to the molecular weights predicted for vp2 and vp3 in the co - expression system , 54 kda and 31 kda respectively ( fig2 , right panel ). the immune blots indicated the presence of both vp2 and vp3 in our yeast mutant designed to express both genes when grown under galactose induction . using the methods described above , vlp or svp preparations were prepared on the clones containing both vp2 & amp ; vp3 genes . several areas of high density were observed in the cscl gradients . the high molecular weight materials pelleted in the ultracentrifuge , and a band of moderate density was observed in the gradient . the moderate density band corresponded to a approximately 20 nm particle that contained only vp2 reacting materials ( fig3 ). however , 60 nm full sized ipnv virus - like particles , as seen previously in ipnv segment a expression in insect cells ( 24 ), were not observed . similar particles have been previously described for ipnv ( 10 ) and are thought to be due to an error in pvp2 processing . similar particles were also observed and characterized in ibdv ( 20 ). they are formed by 20 vp2 subunit trimers in a t = 1 fashion . vp3 is not involved in their formation . here , we saw the same thing whether vp2 was expressed in yeast simultaneously with the vp3 gene or alone in yeast . these particles are referred to herein as sub - viral particles ( svps ). similar methods can be used to produce svps for similar viruses ( e . g ., other viruses having capsid proteins from which svps can be formed , such as other birnaviridae family viruses and other viruses for which salmonids or other fish are a host ). the compositions described herein can be produced by a variety of methods available to skilled worker in this field , and svps made by any of these methods are expected to be useful in the methods described herein . rainbow trout that were free of ipnv were used for a vaccination experiment testing both intraperitoneal injection ( ip ) with adjuvant and by oral delivery in feed . the rvp2 - svps were delivered either as purified svps ( for ip injection ) or as crude yeast lysate incorporated into feeds ( for oral delivery ) to test the antigenicity of these ipnv subunit vaccines in particle form in rainbow trout . the experimental design is outlined in table 1 . to test the ability of rvp2 - svps to induce anti - ipnv antibody production , the most direct method is to use purified antigen and deliver by injection . purified rvp2 - svps were delivered by ip injection with freud &# 39 ; s adjuvant as described in tables 1 and 2 . a booster of the same composition was delivered after 32 days and fish bled at 63 days . all of the injected fish had significantly higher titers of anti - ipnv antibodies than either the naive or sham - injected controls ( fig4 a ). the naive fish and the sham - injected fish were not significantly different from each other at the 95 % confidence interval when compared using the student &# 39 ; s t - test . the purified rvp2 - svp injected fish showed 100 % seroconversion ( table 2 ; fig4 a ). student &# 39 ; s t - tests were run in statview version 5 . 01 ( sas institute , inc . ), testing for significant differences between antibody titers of vaccine injected or fed animals compared to both the naive fish and sham - injected fish ( negative controls ). at the 1 : 32 serum dilution , the rvp2 - svp injected fish had a significantly higher seroconversion rate when compared to the naive fish ( p = 0 . 013 ) and the sham - injected fish ( p = 0 . 001 ). the 1 : 64 serum dilution also demonstrated significant seroconversion differences between rvp2 - svp injected fish and negative controls ( p = 0 . 0003 , naive fish and p = 0 . 0007 , sham - injected fish ). oral vaccination would provide a number of advantages over injection such as ease of use , ability to vaccinate smaller fish , lower cost of vaccine , and easy ability to make multivalent vaccines ( through delivery of different clones in the feeds ). in order to test the ability of rvp2 - svps to induce an immune response , recombinant yeast expressing vlps were incorporated into fish feed and fed to one treatment group for seven days . at day 32 another seven day feeding of the recombinant yeast containing feed was done as a booster ( table 1 ). at 63 days the fish were bled and the anti - ipnv titers compared to that found in naive fish and fish fed a control feed supplemented with wild - type yeast in place of the recombinant yeast ( fig4 b ). it was apparent that the orally vaccinated fish had an immune response greater than that observed in either naive or yeast control fed fish ( p = 0 . 0002 for naive fish and p = 0 . 0053 for yeast control ). there appeared to be a higher anti - ipnv titer in the yeast control sera than in the naive fish , but the difference was not significant ( p = 0 . 1645 ) as determined by the student t - test . seroconversion of the orally vaccinated fish was slightly less than that observed in the ip injected animals with approximately 75 % conversion ( table 2 ). oral vaccination with rvp2 - svps provides an increase , albeit reduced relative to ip injection , in anti - ipnv titer . while these data do not demonstrate the effectiveness of these vaccination strategies on prevention of disease , they are an indication that oral vaccination could potentially provide an alternative to ip injection vaccination for the treatment of ipn . a challenge trial would provide definitive evidence that this approach could prevent disease . the results presented herein pertain to rainbow trout . however , the methods and compositions are not so limited in their applicability . one expects such compositions and methods to be effective in other types of fish as well , including not only salmonids . furthermore , the efficacy of the compositions described herein for enhancing immunity and preventing disease are not limited to the methods of administration that are explicitly described herein . other methods of administering immunogenic compositions to fish are expected to yield similar efficacy . there is no good challenge system for ipnv with mortality as the endpoint ( 1 ). using ipnv viral load , as determined by real - time rt pcr , could provide a convenient method to track the progress of the disease . in this study , the trout were vaccinated with either rvp2 - svps delivered in feed or by injection of purified rvp2 - svps derived from the west buxton strain of ipnv . after 63 days post - vaccination , fish were injected with the buhl strain of ipnv that had been isolated from rainbow trout in idaho ( la patra , unpublished data ). this was a different ipnv strain ( buhl ) than that from which the rvp2 - svps vaccine was derived ( west buxton strain ). therefore , the challenge was with a heterologous strain and may help evaluate the specificity of this approach . ip vaccinated rainbow trout had significantly less virus ( p = 0 . 0280 ) ( 22 fold ) than sham - injected control fish ( table 3 , fig5 ). when oral vaccinates were compared to the yeast only controls , a 12 - fold reduction in virus was found for ipnv vaccinated fish ( fig5 b ). this difference was visually apparent , but not significant at the 0 . 05 level ( p = 0 . 1179 ). these data indicate that rvp2 - svps produced in yeast could provide a novel means for amplification of a protective immune response in rainbow trout , and by extension to salmonid species like salmon , either by injection or by delivery in feeds . expression of a rvp2 - svp particle in yeast provides an interesting opportunity for its use as a vaccine for trout and salmon . the ability of these particles to induce the production of ipnv - specific antibodies was demonstrated by both oral and injection routes . the potential for use of the oral route as a vaccine needs further investigation to optimize the immune response and determine if the observed decrease in viral load directly correlates with prevention of ipn . this study sets the foundation for further studies to test in juvenile salmonids the utility of this approach to prevent early onset of ipn . the disclosure of every patent , patent application , and publication cited herein is hereby incorporated herein by reference in its entirety . while this subject matter has been disclosed with reference to specific embodiments , it is apparent that other embodiments and variations can be devised by others skilled in the art without departing from the true spirit and scope of the subject matter described herein . the appended claims include all such embodiments and equivalent variations . 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( 26 ) yao , k . and v . n . vakharia . 1998 . generation of infectious pancreatic necrosis virus from cloned cdna . j . virol . 72 ( 11 ): 8913 - 8920 . * naive fish were not injected and were fed normal fish feed , adjuvant control fish were ip injected with buffer and adjuvant , rvp2 - svps fish were injected with 100 μg of antigen plus adjuvant . control yeast fish were fed fish feed supplemented with wild - type yeast , and rvp2 - svps yeast fish were fed fish feeds containing the recombinant yeast . ** fish considered seropositive if a 450 was above the mean adjuvant control plus one standard error . * δct was first calculated for each fish using the ct values of ipnv for a fish minus the ct values of ef - i alpha gene for the same fish . then the average δct was calculated taking the ct value of all the fish in each treatment . ** δδ ct = average δct value of a treatment minus the average δct value of the corresponding control treatment