Patent Application: US-48479195-A

Abstract:
the l2 protein of papillomavirus has been cloned as a fusion protein with beta - galactosidase and gst ; both as the whole protein and as fragments . vaccination of calves is found to have both a prophylactic effect in tumour prevention and a therapeutic effect in tumour regression .

Description:
embodiments of the present invention will now be described by way of example only with reference to the following experimental protocol . fig1 shows the open reading frames for l2 protein of bpv - 2 ; fig2 and 4 show the results of vaccination experiments using l2 protein of bpv - 4 for control group , l2 vaccinated group , and l2 plus e7 vaccinated group respectively . fig1 referred to in the experimental protocol shows the l1 and l2 open reading frames ( orf &# 39 ; s ) of bpv2 and the restriction enzyme sites used for cloning . t = tata box ; a = polyadenylation site ; met = translation initiation codon ; taa = translational termination codon ; b = bamhi site ; hp = hpai site ; h = hindiii site . the dna fragment cloned in pur is indicated as l2 ( bamhi - bamhi ). the nucleotide numbering of potter and meinke ( 1985 ) is used . twenty one 12 - week old male friesian calves were obtained from a papilloma - free source . they were randomly assigned to three initial groups , which were housed in separate , clean , well ventilated pens in the isolation unit of the department of veterinary pathology , glasgow . all the calves were bled on arrival for haematological analysis and to obtain pre - inoculation serum samples . the experiment was started when the calves were 16 weeks old . the open reading frame ( orf ) encoding the l2 peptide was isolated by digesting the bpv - 2 genome cloned in pat 153 ( campo and coggins , 1982 ) with bam hi . this produced one fragment of 2030 bp ( nt 268 - 2298 ) numbered according to the nucleotide ( nt ) sequence of potter and meinke ( 1985 ), where nt 1 is the a of the atg codon of the l2 orf ( fig1 ); this fragment contains the majority of the l2 orf ( from aa 90 to aa 467 , l2 ), the l2 orf stop codon and the 5 ′ half of the l1 orf , which would not be expressed because of the termination codon . the fragment was cloned in the pur vector series ( ruther and muller - hill , 1983 ), giving rise to pl2 , and transfected into e . coli jm 109 . peptide for vaccination was prepared from mid - log phase cultures induced for 4 hours in l - broth supplemented with 100 ug / ml ampicillin and containing 1 mm iptg . bacterial pellets resuspended in lysozyme buffer ( 50 mm tris - hcl ph 8 . 0 , 10 mm mccl 2 , 50 mm glucose , 1 mg / ml lysozyme ) were left at 20 ° c . for 10 min , when edta was added to 50 mm . following cell lysis by the addition of triton x100 to 1 % ( v / v ), the fusion peptide was pelleted at 39000 g for 30 min and resuspended by boiling and sonication in 5 % sds , 50 mm b - mercaptoethanol , 50 mm tris - hcl , ph 8 . 0 purity of 90 - 95 % was achieved by preparative sds page , the final yields being up to 2 mg of product per gm wet weight of cells . the protein was stored at − 20 ° c . before use , but prolonged storage caused degradation . the vaccination experiments were designed as follows : in group a , six animals were vaccinated prophylactically with the gel - purified l2 ( one calf had to be withdrawn from the experiment because of pneumonia ); three of these animals were also vaccinated therapeutically with the gel - purified l2 nine weeks after callenge . in group b , eleven animals received no prophylactic vaccination ; after tumour formation three of these animals were therapeutically vaccinated with gel - purified l2 , while eight animals received no vaccine at all and were therefore used as controls . the calves receiving the l2 vaccine were given a 1 ml pbs suspension containing 650 ug of the l2 fusion protein plus 1 ml of freund &# 39 ; s incomplete adjuvant ( fia ) into the right quadriceps muscle . this was repeated fourteen days later as a boost , but with only 500 ug of protein . bpv - 2 was purified from a skin fibropapilloma ( campo et al , 1981 ) and the concentration of viral particles was estimated by the electron microscope assay ( jarrett et al , 1990a ). each calf was challenged at multiple sites with 10 12 virus particles as described by jarrett and other ( 1990a ) either four weeks after vaccination ( two weeks after the boost ) or nine weeks before vaccination . biopsies were performed as described by jarrett et al ( 1990a ). immunocytochemical studies were made by the peroxidase - anti - peroxidase ( hsu et al , 1981 ) or immunogold ( holgate et al , 1983 ) techniques using rabbit anti - bpv - 2 serum as described by jarrett et al ( 1984 ). the presence of neutralizing antibodies in serum samples was determined by the cell transformation inhibition assay described previously ( jarrett et al , 1990a ). this assay takes advantage of the ability of bpv - 2 to transform primary bovine fibroblasts in vitro ( jarrett , 1985b ), which is abrogated by pre - incubation of virus with immune serum . the size of the bpv - 2 b - gal - l2 fusion protein was estimated on page to be 180 kda well in agreement with the predicted size of 156 kda . the l2 fusion protein was characterized immunologically . it was injected into rabbits or calves and the antisera were tested against the fusion protein itself and against virion proteins in both ouchterlony and western blots assays . the antisera were reactive with both the engineered protein ( data not shown ) and its viral l2 ( 62 kda ) counterpart . in reciprocal experiments , rabbit or calf antisera raised against sds - disrupted virus were reactive with the fusion protein . although n - terminus truncated , the fusion protein therefore shares epitopes with virus and presents them effectively to the host immune system . five animals were vaccinated prophylactically ; three of these and three unvaccinated animals were vaccinated therapeutically nine weeks after challenge . as the same results were obtained with the two groups of calves , they will be considered together . all animals developed fibropapillomas four weeks after challenge ( table 1 ). six vaccinated animals were still bearing tumors at ten weeks . in the other two vaccinated calves the tumors were entering the rejection phase : the epithelium was virtually normal and the sub - epithelial tissue was mainly composed of hyalinised collagen . there was a drastic reduction in the number of fibroblasts and a massive infiltration of lymphocytes and macrophages in the sub - epithelial tissue . all vaccinated animals had reached that stage by week thirteen . by week sixteen the tumors had definitely regressed . there were small plaque - like lesions with hyperkeratosis , but virtually all the normal skin adnexal elements were present . some lymphocytes and macrophages were still present . the control animals were still bearing virus - producing tumors ( table 1 ). neutralizing antibodies appeared in the serum of the vaccinated calves at the same time and with the same titre as the control animals ( data not shown ). serum antibodies to l2 were however detected soon after vaccination and before challenge ( data not shown ). vaccination with the l2 fusion protein , whether delivered before or after challenge , induced early tumor regression . tumor regression was accompanied by infiltration of the lesion by macrophages and lymphocytes , a process consistently observed when natural regression takes place ( jarrett , 1985a ). thus it appears that the l2 protein encodes epitopes specific for the cellular effector arm of the immune system . zhou et al ( 1991 ) have recently shown that the l1 protein of hpv - 16 , when expressed in vaccinia virus , induces cytotoxic t - lymphocytes in infected mice , providing another example of t - cell activation by a structural protein . in field and experimental cases , rejection takes place approximately twelve months after infection and it generally follows ulceration of the lesion . this is consistent with the l2 being internal to the virion ( jin et al , 1989 ) and therefore not readily exposed to the host immune system ; ulceration of the tumor with associated bleeding would lead to the exposure of relatively large amounts of antigen to the immune cells . anti l2 antibodies were present in the serum of the vaccinated animals , but these had no activity in the neutralization assay . therefore , unless some neutralizing epitopes are present in the first n - terminus amino acids of l2 , which are missing in our fusion protein , it is unlikely that l2 plays a significant role in conferring prophylactic protection . l2 open reading frame ( orf ) of bpv - 4 was cloned following the general procedure of example 1 , except that plasmid pgex was employed which resulted in a l2 fusion protein with glutathione s - transferase ( gst ) as coprotein . the l2 orf was cloned as the whole orf ( encoding amino acids 8 to 525 ) and as the three fragments encoding amino acids 11 - 201 , 203 - 329 , and 330 - 525 . in the subsequent vaccination experiments a mixture of these four was used . expression was in e . coli and the proteins were purified by gel chromatography , as before . vaccination was carried out as in example 1 using freund &# 39 ; s incomplete adjuvant , except that doses of 1 mg total protein ( l2 and fragments ) was administered both as the dose ( day 0 ) and the booster ( day 28 ). 47 calves , of about 10 weeks of age at the start of the experiment , were housed in an isolation compound . they were divided into 2 groups of 15 and one of 17 ( controls ). all animals were examined and bled before day 0 . they were vaccinated on day 0 and day 28 . they were challenged with bpv - 4 virus on day 43 and examined for tumor formation 4 and 7 weeks later . the results are shown in fig2 ( controls ), fig3 ( l2 alone ) and fig3 ( l2 plus e7 ). the controls showed a good tumor response , 13 of the 17 animals being infected . in the l2 vaccinated group only one animal showed a response ( a small plaque ). in the group vaccinated with l2 plus e7 only one animal developed tumors . thus the l2 protein of bpv - 4 appears to be exerting a strong prophylactic effect in preventing tumor formation ( in contrast to bpv - 2 where a therapeutic effect was exhibited ). campo m s and coggins l w ( 1982 ) molecular cloning of bovine papillomavirus genomes and comparison of their sequence homologies by heteroduplex mapping . journal general virology , 63 , 255 - 264 . campo m s and jarrett w f h ( 1986 ) papillomavirus infection in cattle : viral and chemical cofactors in naturally occurring and experimentally induced tumors . ciba foundation symposium 120 ; 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( 1990 ) human t - cell responses to human papillomavirus type 16 l1 and e6 synthetic peptides : identification of t - cell determinants , hla - dr restriction and virus type specificity . journal general virology , 71 , 423 - 431 . tindle r w , smith j a , geysen h m , selvey l a and frazer i h ( 1990 ) identification of b epitopes in human papillomavirus type 16 e7 open reading frame protein . journal general virology , 71 , 1347 - 1354 . zhou j . mcindoe a , davies h , sun x - y and crawford l ( 1991 ) the induction of cytotoxic t - lymphocyte precursor cells by recombinantvaccinia virus expressing human papillomavirus type 16 l1 . virology , 181 , 203 - 210 . zur hausen h ( 1976 ) condyloma acuminata and human genital cancer . cancer research , 36 , 794 . atg agt gca cga aaa agg gtg aaa cgt gca aat gtc tat gac ctg tac 48 agg act tgc aag caa gcg ggc acc tgt cca cca gat gtg ata cct aag 96 arg thr cys lys gln ala gly thr cys pro pro asp val ile pro lys gta gaa ggt gac act ata gca gac aag att tta aaa tta gga ggc ctt 144 gca att tat ctg ggg ggc cta gct att gga aca tgg tct aca gga aga 192 gtg gct gca gga gga tca cct agg tat gta ccc tta aga acc tct gga 240 tcc act aca agc ctg gca tct gta gga tcc agg gct ggt gca gcc act 288 ggc act cgc agc agc atc aca gga atc ccc ctt gac acc cta gaa act 336 att ggg gct ctt cgt cct gga gct tat gaa gac act gtg ctc cca gag 384 ile gly ala leu arg pro gly ala tyr glu asp thr val leu pro glu gcc cct gct att gtc acc cct gat gct gta cct gcg gac aca ggg ata 432 gat ggc ctt tct ata ggc act gac tct tcc act gaa act tta atc aca 480 ttg tta gag cct gag ggt cct gaa gac gtg gca gtc tta gag ctg caa 528 cct cta gac cat gca aat tgg caa gtt agc aat gct gtt cat cag ggc 576 pro leu asp his ala asn trp gln val ser asn ala val his gln gly tct gca tac cac gcc cct ctg cag ctg cag tcc tcc att gca gaa aca 624 tct gga cta gaa aat att ttt gta gga ggg gct ggg tta ggg gat aca 672 ser gly leu glu asn ile phe val gly gly ala gly leu gly asp thr ggc gga gag aac ata gag ctc aca ttt ttt ggt tcc cca cgc aca agt 720 acc ccc cgt aac ctg cct caa act gca cgg ggc atc ttg aac tgg ttt 768 thr pro arg asn leu pro gln thr ala arg gly ile leu asn trp phe agc aaa aga tac tac aca caa ata ccc aca gaa gac cct gat gtc ttt 816 ser lys arg tyr tyr thr gln ile pro thr glu asp pro asp val phe tca tca cag aca ttt tca aac cca gtg tat gat cct gag cct gca gtg 864 ser ser gln thr phe ser asn pro val tyr asp pro glu pro ala val cta aaa ggt ccc agt ggc cgt gtg ggg cta agc caa gtg tat agg cct 912 gac tat att gaa aca cgg ggt ggg ggt cag gtg ggc cca cag ctg cat 960 asp tyr ile glu thr arg gly gly gly gln val gly pro gln leu his gtc agg tac tcc tta agc act atc aca gaa gat gtg gaa gcc ata cct 1008 val arg tyr ser leu ser thr ile thr glu asp val glu ala ile pro ata gca gtt gat gaa gac aca caa ggg cta gca ttt ctt cct tta cat 1056 ile ala val asp glu asp thr gln gly leu ala phe leu pro leu his gaa gaa cca ggg gac ttt gaa gaa att gag cta gat gat tta ggt gaa 1104 gag cac gcc ttg ctc ccc aag tca tct act gca cct att ggt agt gga 1152 gtt cgt agg gcg ctc att cca ggt caa ggc ttc agt gca aca cgg ccc 1200 val arg arg ala leu ile pro gly gln gly phe ser ala thr arg pro aca ggt gtg gta acc tat ggc tca cct gac atg tac cct gct agc cct 1248 gtt ggc cct gac tcg aca tcc cct agc cta gtt att gat gac aac aca 1296 aca aca cca ata atc att att gat ggc cac aca gtg gat ctg tat agc 1344 aat aac tat agc ttg cat ccc tcc ttg ttg agg aaa aga aaa aaa cgg 1392 aaa cat gcc taattttttt tgcag atg gcg ttg tgg caa caa ggc cag aag 1443 ctg tat ctc cct cca acc cct gta agc aag gtg cta tgc agt gaa acc 1491 tat gtg caa aga aaa agc ata ttt tat cat gca gaa acg gaa cgc ctg 1539 tyr val gln arg lys ser ile phe tyr his ala glu thr glu arg leu tta act gta gga cat cca tac tac caa gtc act gtg ggg gac aaa act 1587 gtt ccc aaa gtg tct gct aat caa ttt aga gtt ttt aaa ata cag ctc 1635 val pro lys val ser ala asn gln phe arg val phe lys ile gln leu ccc gat ccc aat cag ttt gca ttg cct gat agg act gtg cac aat cca 1683 pro asp pro asn gln phe ala leu pro asp arg thr val his asn pro agc aag gag cgc ctg gtg tgg gct gta ata ggg gtt caa gtg tct cgt 1731 ser lys glu arg leu val trp ala val ile gly val gln val ser arg ggc caa cca cta gga ggc aca gtt act ggg cac ccc act ttt aat gct 1779 ctg ctt gat gca gaa aat gtt aat aga aaa gtt act gca caa aca aca 1827 gat gac agg aag caa aca gga tta gat gct aag caa caa cag att ctg 1875 ttg ctg ggc tgt acc cct gca gaa ggg gaa tat tgg acc aca gcc cgt 1923 cca tgt gtt act gat aga ata gaa aat gtt gcg tgt cct cct tta gaa 1971 pro cys val thr asp arg ile glu asn val ala cys pro pro leu glu tta aag aac aaa cac ata gaa gat gga gac atg atg gaa ata ggg ttt 2019 ggt gct gct gac ttt aaa aca cta aat gcc agt aaa tca gat cta cct 2067 ttt gac att caa aat gaa ata tgc ctg tat cca gac tac ctc aaa atg 2115 phe asp ile gln asn glu ile cys leu tyr pro asp tyr leu lys met gct gaa gat gct gct gga aac agt atg ttc ttc ttt gca aga aaa gaa 2163 caa gtg tat gta agg cat ata tgg act cgg ggg ggc tct gaa aaa gaa 2211 gln val tyr val arg his ile trp thr arg gly gly ser glu lys glu gca ccc agt aaa gac ttc tac ctc aaa aat ggt aga ggt gaa gaa act 2259 ala pro ser lys asp phe tyr leu lys asn gly arg gly glu glu thr ctg aaa ata cct agt gtg cac ttt ggc agt ccc agt gga tcc ttg gtg 2307 tcc act gat aat caa ata ttt aac agg cct tat tgg cta ttc agg gct 2355 ser thr asp asn gln ile phe asn arg pro tyr trp leu phe arg ala cag ggc atg aac aat ggg att gca tgg gat aat tta tta ttt tta act 2403 gln gly met asn asn gly ile ala trp asp asn leu leu phe leu thr gta ggg gat aac aca cgg gga act aac ctt agt att agt gta gct gca 2451 gat gga aac gca ttg tca gag tat gat act ggc aaa ttt aac cta tac 2499 asp gly asn ala leu ser glu tyr asp thr gly lys phe asn leu tyr cat agg cat atg gaa gaa tat aag cta gca ttt ata ttg gag ctg gtc 2547 his arg his met glu glu tyr lys leu ala phe ile leu glu leu val tct gtt gag att act gca caa aca gtg tca cat ctg caa gga ctg atg 2595 ser val glu ile thr ala gln thr val ser his leu gln gly leu met ccc tct gtg cta caa aac tgg gaa atc ggg gtg caa cct cct gct tct 2643 pro ser val leu gln asn trp glu ile gly val gln pro pro ala ser tct att tta gaa gat acc tat agg tac ata gag tct cct gca act aaa 2691 ser ile leu glu asp thr tyr arg tyr ile glu ser pro ala thr lys tgt gca agt aat gtt ata cca ccc aaa gaa gac cct tat gca ggc ctt 2739 cys ala ser asn val ile pro pro lys glu asp pro tyr ala gly leu agg ttt tgg agc ata gac tta aaa gaa aag ctg tct ttg gac tta gac 2787 caa ttt ccc ttg gga aga aga ttc tta gct cag caa ggg gca gga tgt 2835 tca act gtg aga aag aga gct gtt gca acc aga aat tcc agt aag cct 2883 arg thr cys lys gln ala gly thr cys pro pro asp val ile pro lys ile gly ala leu arg pro gly ala tyr glu asp thr val leu pro glu pro leu asp his ala asn trp gln val ser asn ala val his gln gly ser gly leu glu asn ile phe val gly gly ala gly leu gly asp thr thr pro arg asn leu pro gln thr ala arg gly ile leu asn trp phe ser lys arg tyr tyr thr gln ile pro thr glu asp pro asp val phe ser ser gln thr phe ser asn pro val tyr asp pro glu pro ala val asp tyr ile glu thr arg gly gly gly gln val gly pro gln leu his val arg tyr ser leu ser thr ile thr glu asp val glu ala ile pro ile ala val asp glu asp thr gln gly leu ala phe leu pro leu his val arg arg ala leu ile pro gly gln gly phe ser ala thr arg pro pro thr pro val ser lys val leu cys ser glu thr tyr val gln arg lys ser ile phe tyr his ala glu thr glu arg leu leu thr val gly ser ala asn gln phe arg val phe lys ile gln leu pro asp pro asn gln phe ala leu pro asp arg thr val his asn pro ser lys glu arg asp arg ile glu asn val ala cys pro pro leu glu leu lys asn lys phe lys thr leu asn ala ser lys ser asp leu pro phe asp ile gln asn glu ile cys leu tyr pro asp tyr leu lys met ala glu asp ala ala gly asn ser met phe phe phe ala arg lys glu gln val tyr val arg his ile trp thr arg gly gly ser glu lys glu ala pro ser lys asp phe tyr leu lys asn gly arg gly glu glu thr leu lys ile pro gln ile phe asn arg pro tyr trp leu phe arg ala gln gly met asn leu ser glu tyr asp thr gly lys phe asn leu tyr his arg his met gln asn trp glu ile gly val gln pro pro ala ser ser ile leu glu asp thr tyr arg tyr ile glu ser pro ala thr lys cys ala ser asn val ile pro pro lys glu asp pro tyr ala gly leu arg phe trp ser