Title of Invention


Abstract The present inventor relates to a non-infections anklets and (RNA and DNA) contract consternated to expects a recombinant anybody or anybody frame-up in a most cell. The anybody nucleus confess protected to the host against a patent allergies or next. The host may be any animal deciding homes.
Full Text AMTIHOK ariHio »EirrtAfci3SHo VHSV (VXJIM. nucHoiuuaaxc SSSTICASHU
The present invention relates to a non-ir*f«ctioue nucleic acici (RJiA and DMA) construct constructed to express a rtcombinant antibody or Antibody fragment in a host cell. The antibody molecule confers protection to the host against a petncgen, allergen or toxin, Th* host may be any animal including a human.
immunization by injection of homo lego us or htterolcgous aerusn-artibociies is routinely ^sed in humans for inaaunaprophylaitis of people traveling to foreign rations ' involving risk of exposur* to exotic pathogens. In arxiaals a siriLlar atr-ategy may ae employed for protection of valuable apeciaseris , but. is centrally coc expansive for routine veterinary use. Pis*iva itnmuniaeticr. of aniroaia agaiiis: infectious diseases is thus mostly done en an experiment*! basis with the aim of studying the function of structures such as antibodies in vivo and relating the results to in vi~ro experiments.
During the recent decade, diverse technologies for th* in vitro production o£ antioodie* ay the use of jrecambinant DWA technology has beer, developed. Tha smallest functional recombir.ant antibody containing the actions of the heavy (K] and light U) polypeptide chains as in the native Bialecule hivs proved to be the single chain variafcli~f raiment construct (single chain fV} . The single chain FV conatruct ia cawpoaed of the variable parts of the H and'L chains connected by « flexible spaces region. Such avolecules have been ueed in varioua studies including 'virus neutralisation, cancer™ jLnsnuno therapy and recently also in the fcnr. of DMA vaccines where plasmios encoding anfci-idiotype single ..jgpbain rv

to induce an "antigen-specific
immune response. However, direct establishment of protective iWBunity to infectious diseases by prophylactic treatment with plasmid DKA carrying single chain FV genes encoding protective antibodies ha* not been described.
An object of the present invention is to provide 4 non-infectious nucleic acid construct which can product an antibody stoiecule in v*v$ thereby conferring iamtuaity to a
A further object o£ the present invention is to provide a method of establishing iramur.x^y tgfcinst & aachoger..
A yec further object of the pr«s«nc invention ia to provide a method of therapy for Animals which have 6 deficient iawune system.
An additional ofajacc of the preserve invention is to provide a method of cherapy for an anijnti suffering from ftn allargxc reaction or a mechod of prevent ing an allergic reaction.
For avoidance of doubt it should be noced that the word "animal" includes but is not restricted to usaitraais including humans .
According to an embodiment o£ cha present invention there ia provided a nucleic acid construct encoding a r»eemfeinanu antibody malaeule* isid construct being adapted for the ir> vivo escabliahnent of a protective immunity to an infectious disease in an animal.

According to & further embooimanc of the present invention there is pcovifiec a nucleic acici construct encoding a recombinant antibody molecule, said construct i* fcrsauleted for the in vivo prevention cf an allergic reaction to an in an animal.
According tc a yet further embodiment of the present invention there is provided & nucleic acid construct encoding a recojRbinant sntioody molecule, wherein laid construct is formulated for the in vivo prevention of a reaction caused by the presence of a toxic substance in an animal,
Che term reco-nabinant antibody molecule encompasses & full siz* mtibody, a single chair, variable fragment or any part of t&r mtibody which can recognia* an an;iger,. In this connection, :cnveni.«ntLy the intibody fragmen; aces not mve to be single ;hair. . However, i* some embodiments it La single chaif. ,
j* ties now b««n tour.ci tr.&t the intramuscular ^nrection of s nucleic field ccnsttuct, in the fora of a plasaid, encoding .a virus-r.eutcaLiair.'j single chain antibccy fragment cen neciate in vivo expression, of antibodies which protect an animal against a ooasibly lethal exposure to A virus. Thi» has been established tn an experimental model which involves a fish rhabdcvirus caliec viral h&emorrha According to a farther embodiment of the present Invention •.here is proviaeo « nucleic; acio construct, sue:, as a pltcniet*. lonwriai -".q an excreasioc, ve^tsi ana * gene sequar.ce for heavy ind/or light chair, variable domains nf an ar.tisooy.

Preferably tr.« heavy and light chain variable domains are linked by * iinkee sequence in order that they form whet is known in the art as a single chain variable-fragment.
It is thought that the antibody fragment as expressed in and secreted from a hcst cell carrying the vector wtil act with the same specificity as a natural antibody would in th* presence of a substance which it recognises. in this connection, for example, if the heavy and/or light chain variable domain were derived from a monoclonal anti-body raised against dengue virus then if dengue virus injected a host who hac received a nucleic construct expressing a single chain variable fragment produced Scon rh» heavy and light chain of the awnoclcm*! antibody, the fragment would recognise cells infacred with the dengue virus or the dengue virus particle itself and bind thereto thereby neutralising or inhibiting th* virus and/wr giving the host time to mour.t an ijsutiune response against zh» virus,
In pr»;«rreo eiraodi.Tsar.ts the expresaicr. vector is made for eujearyocic expression *rid/or is nor, infectious, for example, a bacterial plssniid, cr a smaller DMA fragment carrying t:ae variable fragment er.cibody gene within a eukacyotit fexpreasior. opetcn racludir.g regulatory elefter.ts such as an enhance::, promoter ana pclyadeny-ation signal caulct be uaeci, Alcernatively, stabilised messenger RNA including a positive atrand transcript of the variable-fragment antibody gene with translation signers may be employed.
The antibody -ragtnent genea can'be donee by any method known to these sXilied in the art., fo-c example from hvoridoma cell-s or directly from 3*lymphocytes from iHumurjiiai incividuaLs.. isic acid constructs «r.cocinq protective antieoey fragment-*

can oe prepared against any important pathogen7disease causing agent in animals including pathogens against which vaccines art not available or hiva proved insufficient. Furthermore, as a result of veterinary regulations, USB of live vaccines m*y not be allowed, In such cases an alcernacive prophylactic measure would have co be taker.. Such a aeaaure could be the administration of the nucleic acid construct of the present invention- A list of possible pathogens is given below,- this list is not intended to be exhaustive.
Viral haeraorrftacic septicaemia virus {fishi Infectious haematopoietic necrosis virus (fish) Infectious salman anemia virus (fisr.)
ra salmcniarum (fish) Pasracreiia (fish)
Ichci-.yopthtitijs ndiiiif iliis (fish; N«wCas:ls disease virus (fow;; Infectious cursai disease virus I fowl; Savin* respirarcry syncytial virus (cattle) Scvir.e v;rus diarrhoea virus icatcie) Parc;ne reproductive and respiratsry syndrome virus iaigs;
Equine herpes virus 1 (horses i
Ji.asmocyrosis virus (aink;
Rabies virus (dogs)
Feline leukemia virus (cats)
Fooc and mouth disease (cattle!
ftuoAn imrrune deficiency virus f human)
iepatitis A virus (human)
Jorreli* sp, (iiuiran)
m ap. inumanl

Rabies virus Epstain-Barr virus '(human)
In case of humans with either * congenital or acquired immunodeficiency, vaccines will generally be insufficient. in such casas, administration of a number of nucleic acid constructs according to Che present invention encoding antibodies against a broad spectrum of pathogens may b« considered.
For the purpose of prevention at allergic relations induced by luE response, administration cf nucleic acid constructs mediating expression of in allergen-specific recorcfainar.c snribody may be used to competitively inhibit binding of she allergen to the IgE molecules in the host. Alternatively ger* constructs encoding ar.ti-i?; antibodies may be used to -.nterfere with tne interaction. between Ij£ anc mast cells ir. che aliecgic individual.
ifls straticr. o£ Antibody ger-.e cor;scruc-wS encoding antiiseclies ta Eoxins or ver.o«s can be used fcr the prophylactic treatment cf individuals periodically being in high risk cf exposure co toxic organisms. Tha venoms could, for example, oe froft sr.&ices or spiders.
Conveniently the construct further comprises a gene encoding a signal sequence for the secretion of the product encoded bv the aer.e saquer.c*. Tn* sigr.jl sequence will allow the product ot tha gene sequence to be secreted trom a cell in which the gene has been expressed, int- ch'e blood so that the product of the gene sequence can circulate therein. For example, the genes fcr the signal sequence of either rainbow trout trans 'ermine, growch factor beta tTGr-be>ca;r or murine Ig

kappa-chain can be added to the :' end of a gene to be
administered tc the fish. Other secrttion signals/ preferably of homologous origin to the host species may te employed. Examples of gents which encode proteins which act as secretion signals include the ger.e for immunoglobulir. heavy and light chain, secretion signals or other glycoproteir. secretion signals> Preferably, the secretion signal should include a proteolytic cleavage site ensuring removal of the signal peptidt before secretion of the antibody fragment.
Preferably the construct further comprises a known gene sequence which encodes 8 short peptide sequence that can be usad to identify transferees cells. Such a gene sequence can b* attached to the 3' end of -he gene. Examples of such a sequence -include a human icapca light cftair; construe1: or sequence ftnccaing a six r.U';id.rie residue. In both cases, an a.-itioody specifically recogr.iaing the expressed peptide is commercially available.
The construct according ~.c ^he present invention may ba da'.iverad cy any suitaeia aiathed, such as ay i.-.:action la,g intramuscularly!, oy a spray on a nucosa surface ,'e.g incranasaliy! , by particle bcrrJaarcimenc on stcin/c.erai$ through use of 3 gane gut:, oy *i«ctroporation or by uptake by *n animal from an aqueous environment. In this connection, tne plasmid may be encased in a liposome for administraticn to an animal. The construct may be administered to the animal topically, through inhalation or orally. For oral administration the construct should be protected from degradation by proper encapsulation.
It is preferred ihat in * composition or formulation Jot adjnir.is~racion c-f the tor.st:u£Ls triere ars present genes

encoding the heavy and/or light chair, variable fragments against several different epitope* or an variable fragment antibody gens expression library against a given, pathogen. In this connection, the various fragments may be provided on one plasmid or they may b* provided on several different gene constcucts which are all present in the same formulation or other method of administration. in the alternative! each plasmid may have to be administered separately.
The invention alao provides for a method for treating an animal, for' example a mammal or a fish which comprises administering thereto a plasmid or ether nucleic acri construct encoding a prctective antibody fragment as previously described.
The invention thus provides far a method a? therapy for an animal which has a deficient i:wr.u.ie system.
7 he invention also provides far a therapeutic compos it 10?. comprising the piasmici as previously described ar.d a. pnarmaceutically acceptable diluent or carrier therefor. The composition may oe formula ceo such chat it is in the form of:, for example, a vaccine, dosage form, sreaa, ointment, liquid oc pa inc.
The i.-vtncion will now toe described Dy way off illustration only with reference to the following Example and Figures.
Figure 1 shows a schematic drawing of the pCDNA3 plasmid with a single chain antibody (ScAbi gene construct inserted downstream of a strong ./eukaryatic proinoter from cytsmegalovirus tCMV). PCDNA3 is a commercially available
en v»ccor

Figur* 2 shews a culture o! EPC cells (passaged fish cells! transfected with a pCDNA3-3Ul. BUI is a ScAfc cane construct encoding a recombinant antibody which is able to neutralise the fish pathogenic rhabdovirus, VHSV. 301 carries z part of the human kappa light chair, gene ss a residue or tag. Twelve days after the date of transaction the cells were fixed and stained iiwnunochwnically using horseradish peroxid*s«-conlucBtsc rebbit antibody to human kappa light chain, (HRP-Rabbit anti-kappa) for the detection of eella eor.ta.ining ScAb. These cells give a positive response and are darker -'nan the remaining cells; and
Figure 3 shows a histclogical sec'icn cf suisclB tissue samplec from a f;.s^ twelve days afzer intramuscular injection ct nCDNA3-BUi. The section was stained iwmunceheniicell/ using HRP-ranbit anri-kappa for t.^e detection s' ScAis. Several cells turr.ed out positix'e (arrow heads) aior.g the regenerating needi-j tracx fir]ecuicr. site) arrowed-
Gene Mae
The following gene nap is cha ON« sequence of the sonstruc: comosising a single chain antibody gene :BUr, inserted int;c E.csli pCDNA3 plasmid (rnvLtrogan) used in the Example described below.
1 cagtgtgcta acacgagggc uytgtgttLg atgcigaetr ccttattgat SI gctggaatat gtgtgccgga gtgaccaggt gcagcigcag gagtcaggac 101 ctogcrtcgt gaaacctcc- cagtctctgt ctctcacctg ctcrgtca;;: 151 ggctactcct tcaccagcgg ttatcactgg acctggetcc g^ca.gtrtfcc 2C1 agoaaacaaa ctggaAt^g* tgggctacat aagctscgac ggtaccaa-ca *il avtacaaesc a-^tcrcaca aatcgaatct ccatcactcg tgaeacatct 301 aaQaaecagc tr;crcc-=ea sttgaaatc? gtgactac:? aggac*c«t;c

351 tacatattac tgtgtaagag gg*-ctacta tggtaacgac tggtttgc:t
401 actggggcca agggaccacg gtcaccgtct cctcagaage, caaatcttcc
451 ggctctggct c-gaatctaa agtggatgac atcgagctca cccagtctcc
SOI tgectcccag tctgcatctc tgggagaaag tgtcaccatc acatgrctgg
551 caagtcagac cattggtaca cggctigcat ggtatcaaca gaaaccaggg
60. aaatctcc^c agctcctgat ttacgctgca accagttcgg cagatgggijc
651 ccceccaaigg ttcagtggta gcggatctgg cecaatAttt retttcaaga
70: -cngcagccc acaggctgaa gattrtgtaa gttactactg ccaacaacr.:
T51 -acageactc cgtacacgtt cggagggggg iccaigcccg agaccaaacg
801 qactgtggct gcaccatctg ccttcacctt cfcgccatct gatgagcagc
851 cgaaacct'gg aactgcctcc ytrgtgzgcc tgc"gaat*3 cttctatcerc
901 agagaggcca aagtacagtg gaaggtgger aacgrcccrcc aatcgggtaa
951 cccc;agg&g agcgtcacag agcaggacag caaggacagc acetacagcc
10C1 tcagcagcac cctgacgctg agcaaagca; actacgagaa acacaaagrc
1051 tacgcctgcg aagreaccca tcagggcctg agttcgcccg tcac*aag»g
llOi cttcaaecgc ggagagtc&r. aagccagac* tcci.t
The 3L1 insert (ScAis gene cor.S'ruci; is encoded by nuclaotides 10 u- 112.':. The =ca;ng r«gi=n .-.uclssTidts are 13 to 1122.
Tht aisov* icencifxed 4nqi;eac* can se found is tr.a S Che Accession Number is AF302252.
Sin.gi* chain antiisody genes wara preparad accorciing to proeaeuc* describ«d by McGregor «t al; Spontaneous Assembly of Divalent Sir.git Chain Antibody Fragrcanra in S-Coli; Mol. lanunal. February 31(3) pp 219 to 226; 1994. In short, the variable domains u£ the irmtianoglobulin H and L chain genes w«re clonac. frcn hybrictoma cell lines producing monoclonal antibodies co chs fish pathogen!:.- rhabdovitua viral haemorrhaeic sapcicaania virus iVKSV" . The H ana L chazn variael* acmaina were linked by a 3ent sequence encoding a 14

amir.s aeud Unk*r Co generate a single chain antibody (ScAb) gene. As a tag to allow specific detection, the human kappa light chain constant domain gene was included at the 3' end of the gene. In order to ensure accretion of the ScAb polypeptides in euicaryotic cells, the nucleotida sequence encoding the 20 amino acid signal peptide of rainbow trout trans forming growth factor beta (TGF-beta) was added at the 5' end of the gene.
The gen« construct was inserted by blunt-end ligation Lntc the enkaryctic expression vector pCCNA3 tlnvitrogen! in the EcoH I situ in the poly linker downstream oi a c/tomegaloviru* !CMV: promoter (see Ficruce 1). As a negative contra! in transfeetion experiments with cell cultures and imaunoprotecticn trials if. fish, the pCDNA2 elasmid without insert was used. Piasmid DNA was purified, from overnight cultures of £.co,i: by use of conurercial '/tits for aaian-•xchanga cr.comaccgraphy as reccumended by the supplier (Qiagen).
Other molecular viulcoy procedures used were as followed by Sambrcck st al in Molecular Cloning; A Lafioratcry Manual, Seca.-.ci Addition, Cold Spring Harbor -aooracory, USA, (1999i . The veriecle domair, ger.es from a hyiridorr.* ctll line secreting the VHSV-ntLtraiiair.g monacicnal ar.t^aody 3F1H10 ware used, cloning and sequencing of the variable domain genes has already been described. In the case o! antibody 3F1H10, twc am^no acids aubati-tutiona were made to the H-chair. (Asn35a tc Thr antt LyiS4 to Thr! . The ScAa carrying the variable domain.3 of ar.tibaay 3riH"»C w«s called BU1 .'
Passaged fish cells designated (E?C) wtre transfactad with ar anionis transfecti.cn reagent (Superftct, Qiageni . four to ai>

dayi lifter trans faction cell culture supernatant were harvested and analysed for antibody reactivity to VKSV. Af tar removal of the supernatant, the cells remaining attached to the cotton of the ctll culture wtils were fixed in 80% cold acetone and stair.ed by inwuno-peroxidase using horseradish. peroxidase-conjugated rabbit antibody to human Kappa ligtr: chain (KRP-Rabbit anti-kappa! (DAKO/ Denmark:) in order to detect cells expressing ScAb. The effect of transfecricn on the susceptibility of the cell cultures tc VHSV different dose* of live VHSV was examined by adding the different doses Co wells wir.H cult'jrif of transacted cells four days afteir ccenifection and the development cf cycopathagenic effacta (CPS! was recorded thersafcar.
disease *res raxr.bow t-ous fingerlingj, average weight 4.5c, rfere ana*sthesiaed with 3,001* be-zoica.:ne and given cwo 25^1 Injections of 20 ug plaamid DNA wen, ir. -.he epaxial muscles jelow the dersal firt. Tne 5i*h wers «fctsrw*ros iceoc in groups jf approximately 1 = 0 individuals ir. 120-liter can l AoaivMlJi — ggf g^arBga^OI — Q£
Twelve days after injection of plaaciia SNA, 10 Cish were sampled for each piasmiti construe1:. After termination of the fish a seccion cf muscle tissue was excised froa the sice cf injection. Th« iisaue w«s fixed in lOft phcschate buffered lin and aaaiyaed ty iiamunoniscschemistry. Horseradish

peroxidase-conjugated rabbit iamunoglobulin' (Ig) to human kappa light chain (HRp-rabbit anti kappa! (Dako, Denmark) was used for detection of expressed ScAb.
Saya 1,4,00 of jjplflsma {pom Fjph
Blood samples Mere collected 12 days After injection of plasmid DMA from fish not exposed to VHSV. Due to the small site of the fish, sampling wax performed with heperin-treated capillary tubes after cutting off the posterior fin of full/ anaasthetisad fish. The fish were terminated immediately afterwards. The blood samples were cantrifuged at 5000 xg ana plasma samples were collected and sacred at -80 *C until analysed.
$_g_yaij> The iTLISA assey *«s performed ir. 96-weil micrcticre pletss coacad Wi:r, purifiad VHSV. 3ou.*.d ScAb'a were cecsczei witr. HRF-Sabblt anT:i-k«pps. In orier :c aemonetrace thac th« virus-neutralising activity detecced in the Crout plsszta was due to the ScAijg produced by the fish and net by trout antibodies, z,wc variants of the 5C* PNT assay were also applied. One variant included parallel examination of the neutralising activity a^ainsc the virulent VHSV3592B and a neutralisation resistant variant of VMS 3592B (VHSV DK-3542B' selected by cultivating virus' in the presence of tne neutralising Mafc 3F1A2 which i,K'hiqh.'y similar to Mab 3F1H1C-The other variant involved pre-incabation of th» trout plasituj with rabsi: ar.ciboaies to human kappa iiaht chain cr with

rabbit antibodies to trout immunoglobulin before incubation with virus. The 50% PNT microplate assay was performed as described by Olesen «nd Jargensen in Detection of neutralising antibody to Egtved virus in rainbow trout by plaque neutralising with complement addition, J. Appl Ichthy61, Volume 2, pages 35 to 41.
ls Lr. Fi
Eleven days after injection if the plasraid, groups of fish were exposed to .'challenged with) the virulent VKSV DK-35S2B isolate by ' immersion in wacsr containing IOC DOC 30i tissue-suit ere inft-eci';* coses per mi. Challenge WAS perfcraed in 8-liter aquaria with 25-31 fish in each. Three replicate aquaria was includes fsr each plesrud construct. Dead fish were afterwards daily recorded and collected. Dead fish ttoti all tanks *ere analysed virologicaily Car the presence of VHSV. Maan water temperature was IS'C from ^he time cf irtjectior. z$ irrwiediately before challenge. Ar caaller.ge, the fish were adapted to « water tompecature of 12'C and this temperature waa ! f.f Sjtoreased SeA'c .r, cgll Culeur*
It wis founa that after iimunc-pscaxidase staining using the HFP-raabit ar-ti-human kappa, singls cells expressing Sc«J: could be aectcted in EPC eel. cultures transfected with the piasmid construct pCDNA3-5Ul (Fig. 2), whereas no positive cells were found in cultures cransfected with pCDNA3 without insert- Similarly, expression of ScAb could be demonstrated in muscle sections from injected fish (.Fig. 3t . No positive cells war* fou.-.cs ir. sis.-, injected wich pC3NA3 without insert.

q.C$ll Cultpr$
a: .5cRbs with. aroMgati art ef
Wh«rt mor.olayer* of epithelial cell iir.e of cap cei'_ cultures were inoculated with VHSv four days *f:er tr*nsfectio,*i. develcpnent of eytopathogRnir effect (CPE) as 4 result oi multiplication of VKSV was highly different in cultures ccanafected with pCDNA3 coaparec to call cultures transacted with pC5NA3-BUl. In the latter CAS« only ctrtain plequts of c«Ui oecaae ir.f»cted and died anc there was no further d«v»la.craent of C&E in •cr.a 8 -day observation period. In contrast, when cultures -rans£«cT:ed with pCDKA3 were inoculated, "'all cells becaa* infected and were d*s^roye4 within 3-5 day* as in a p.orrr.al prcpigation of VHSV in EFC jells '.Table 1) .


2uscB?tibiIi-y of cra

cell, culturea -:c

Construct ua«d for

Cytopathogtnie effect upon. inoculation with VESV*

(Table Remove) Concentrations of VHSV: 1G"-1CJ TClD-SO/iai cell cultura
When the plaswa froir. ift;,tct»ci fish w«s analyaec by SUSfi. fox ScAtos reeoqnisina VHSV, a strong reaction was found in plasma from fiah in^ecxtd with pCDNM-SUl. So reactivity was detected in plasma Sroni fist.' i'nj»c:t»d with pCDNA3 without . AJ i in is a tad " ^acie ^, the ii^izea aitioantj ct

plasma available made it necessary to perfonn' eh* analysis; on pools of fiv« individuals. The 501 PNT analysis was performed on individual plasma samples. All 10 individuals injected with pCCNA3-B'JI neutralised VHSV, whsreas no neutralising activity was detected in plasma from fish injected with che pCONA2 (Table 3). When plasma from fish injected with pCDNW-BCJ1 was _ preincjbated with Rabbit ar.ti-humar- kappa before sesrirtj in 50% PNT, the neutralising activity was eliminated, whereas r.s effect was observed upon pre-incubatian with norma; rabbit serum or wish rabbit serurr. to trout. Ig 'Table 4). Tne neutralising acti/ity of a positive trout serum cor.trel was ur.*ffec:*d by prs-incubation with normal rabbi- serum and wlch rabbit anti-nunian kapp*, but wes highly reduced upon pre-incubaticn with rabbic serum to trout Ig (Table 4). As WJLO. the parent nonoslor.al antibody 3F1H1Q, plasma saapies from fish in}*c=.ed with pCDKA3-lt;l could neutralise the virul«nt VHSV DK-3S9ZB isolate, Sut not a r.eucrelisation escape-.tit:taB= Cr.o* shown] . Table 2. Antibotiy reactivity ;n fish plasma: SLISA

(Table Remove) * The plasm* samples ware, analysed ir. pools of 5

(Table Remove). Antibcsy reac-ivity in fiir. plasma:' Neutralisation

Plasme samples were analysed individually. Titris represent the reciprocal v-alu* of plasma dilutions raducinc the number of plaques to approximately 50* compared cc A centre! well without:
4. Efiec: cf pteincubacion ot crout plasma with rabbit ar.tibodia* on PNT-tizres*

(Table Remove) In oraar so alinw dttectian of neucralLaing trout antibodies, trout complement was incluoea as described
When challenged with'VHSV DK-35923 11 days after injection o* plaamid DMA, most of the fish injected with pCCNA3-Bin survived whereas high morcaiiries were observed amor.g fish Injected with pCDNA3 (Table 5].
T*ble 5, Protection against VHSV

(Table Remove) To our knowledge, this is the first report demonstrating establishment cf protective ironur.ity ^o ar. infectious paihogen in higher vertebrates by adsinistration of ger.ea encciins pat.-.oqen specif^s single ch»ir. FV antibodies. The protective activity of the ?CDNA-3UI construct fully correlated wich the sccurranc* of neutralising anti-VHSV ScAbs in the plasma of1 injected fish, and a_though involvement o; non-specific necl**niajn$ cannot be completely exciudea, it appears likely :h»- the produced B'Jl scAb has been the ^1*3or cause of srotection fcllowiftg injection of the pCONA2-B01 piaimid DNA. Accordingly, in a later experiment: including cheliengt of the Jiah with a virus iaolate not recognised by the reoombinant: antibody fragnenL encoded by pCDNA-BDl, no protect lor. was obtained.
In contraat co DNA-vaccine*, iriciud.ing anti--diocype vaccines, the admir.istra~l3n of pi»smic,borne genes in u.iis instance dc
not involve specific activation of the immune system in the individual. The principle is simply thit iingle chain FV antibody polypeptides produced by the cells that take up th« administered piaswid will be systemically distributed by <:he body fluids and protect the individual if infection with tha pathogen occurs. this corresponds to mechanism of prophylaxis against infectious diseases in humans through administration antiserum or iflmunoglobulin from immunised donors ani-tials but without side effects such as risk roncoir.it4.it transfer induction lypersens-tivicy fallowing repeated administrations. arcler :o avoid variability overcoming immunity established by plasmid practical use may involve piasmids encoding genes single chain variable fragments several different epitopes fv antibody gene-expression library towards a giver painoge-i.> The principle of genetic imunoprcphylaxis according to the invention can be extended to mammals and to humans if. particular as it i« a valuable tori for transient protection of individuals such ss travelers agd.nsc exposure to pathogens or taxir.s where nc afficienc vaccines are available. Similarly, the invention may be used far induction of tile synthesis of antibodies of a desired specificity for use in imtnunodeficient individuals. Also the nucleic acid construct of the present invention could be used in individuals that produce an allergic response to certain allergens, such as pollen. In this connection, production or induction o£ antibody fragments to those 'allergens may be used for prevention of an allergic reactior.

sesicte the prophylactic aspects ot the invention, p la said constructs carrying genes encoding pathogen/disease antigen specific single chain F antibodies are of therapeutic use in certain diseases wherein the host immune system itself is unable to produce antibodies with the necessary activity.

1. A non-infectious nucleic acid construct encoding -a
recoweinant antibody molecule, said construct being adapted
for the in vivo establishment of a protective immunity to aft
infectious disease in an aninal,
2. A non-infectious nucleic acid construct encoding a
recombinanc antibody Kraiecule, said construct is formulate:;
lot the ifl vivo prevention of an allergic raaction, to an
allergen in'an animal.
3. ft non-infectious nucleic acid construct encoding &
ceccmbinant antibody molecule, ,wherein said construct is
foraulated for cne in vivo preventien at a reaction caused 6y
the presence of cosic subscaneii in s animal.
4. A construe- according to cliin 1 wherein -.he ar.ci'sody
molecule is derived froir. an ar.ti'socy saiaed against the"
pathogen causinr the disease.
5. A construct: according ta claim 2 wherein the antibcay
itolecule is derived from an antibody raised against the
5. A construct according to claia 2 wherein the antibody aolecule is derived frem an antioody raised against ZgE BOieeules.
r« K construct according ^o clain 3 wherein the antibody wlecuie is derived from antibodies raised against the toxic tubstance.

8. A construct, according tc claim 7 wherein the toxic
substance is a venom or toxin produced by a poisonous
9. A construct according to any preceding claim wherein the
antibody molecule comprises variable domains cf inmiunoglobuUn
Heavy and Light chain lir.fced together by a linker sequence.
10. ft construct according 10 any preceding claim further
comprising a genetic sequer.ee encoding secretion signal
11. A corvst~u.ee according to ar.y preceding claim formulated
for delivery by injection, spray or gent gun.
12. h construct according to any preceding ciain comprising
genes enctdi.-.g ar. antibody molecule to several different
epicopes on a given pathogen, allerge.*., or toxin.
13. A ccr.szruc- according to any preceding clain compriai-c:
genes erxcsaing in TV antibody gene-»xpressier, library co a
given patr.oger., allergen/ or coxin.
14. A cons-rues according :o any preceding claim including
a viral haemocrhagic aepticaemia virus VHSV-neutraliaiag
monoclonal ancibody 3T1H10 with two amino acida tufeatituencs.
in the H-chain gene respectively Aan 35a to Thr end. Lye 64 to
Thr and with the secretion tignal of rainbow crout
transforming growth factor (TGF-betaJ added to the 5* end of
the gene.
15. A twcnod of creating an nirr,ai coirpriaing administering
thereto a eonetrucT; »s ciaitved in any of ciaimj! 1 to 14.

iu. n pnarmaceuticti composition comprising' a construct as claimed in any on« of claims 1 to 14.










in-pct-2002-00412-del-description (complete).pdf











in-pct-2002-00412-del-pct-search report.pdf





IN-PCT-2002-412-DEL-Description (Complete)(18-12-2007).pdf





Patent Number 215831
Indian Patent Application Number IN/PCT/2002/00412/DEL
PG Journal Number 12/2008
Publication Date 21-Mar-2008
Grant Date 04-Mar-2008
Date of Filing 19-Apr-2002
Applicant Address HANGOVEJ 2, DK-8200 ARHUS N, DENMARK
# Inventor's Name Inventor's Address
PCT International Classification Number C12N 15/13
PCT International Application Number PCT/GB00/03605
PCT International Filing date 2000-09-20
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 PA 1999 01329 1999-09-20 Denmark