Patent Application: US-7396293-A

Abstract:
what is described is a recombinant poxvirus , such as vaccinia virus or canarypox virus , containing foreign dna from morbillivirus . in one embodiment , the foreign dna is expressed in a host by the production of a measles virus glycoprotein . in another embodiment , the foreign dna is expressed in a host by the production of at least two measles virus glycoproteins . what is also described is a vaccine containing the recombinant poxvirus for inducing an immunological response in a host animal inoculated with the vaccine . by the present invention , cross - protection of dogs against canine distemper is obtained by inoculating the dog with the recombinant poxvirus .

Description:
a better understanding of the present invention and of its many advantages will be had from the following examples , given by way of illustration . the rescuing virus used in the production of both recombinants was the copenhagen strain of vaccinia virus from which the thymidine kinase gene had been deleted . all viruses were grown and titered on vero cell monolayers . the early / late vaccinia virus h6 promoter ( rosel et al ., 1986 ; taylor et al ., 1988a , b ) was constructed by annealing four overlapping oligonucleotides , h6syn a - d . the resultant h6 sequence is as follows : referring now to fig1 the annealed h6syn oligonucleotides were ligated into pmp2lvc digested with xhoi / hindiii to yield plasmid psp131 . the plasmid pmp2lvc contains the leftmost 0 . 4 kbp of the vaccinia virus ( copenhagen strain ) hindiii k region within puc18 . the construction of pmp2lvc was performed as follows : a 0 . 4 kbp hindiii / sali fragment from the hindiii k region was isolated and blunt - ended with the klenow fragment of the e . coli dna polymerase in the presence of 2 mm dntps . this fragment was inserted into puc18 which had been digested with pvuii . the resulting plasmid was designated pmp2vc . the plasmid pmp2vc was linearized with sspi . synthetic oligonucleotides mpsyn52 ( seq id no : 3 ) ( 5 &# 39 ;- attatttttataagcttggatccctcgagggtacccccggggagctcgaattct - 3 &# 39 ;) and mpsyn53 ( seq id no : 4 ) ( 5 &# 39 ;- agaattcgagctccccgggggtaccctcgagggatccaagcttataaaaataat - 3 &# 39 ;) were annealed and inserted into the leftmost of the two sspi sites located within the vaccinia virus sequences . the resultant plasmid pmp2lvc contains a multiple cloning region in the intergenic region between the k1l and k2l open reading frames . annealed oligonucleotides 3p1 ( seq id no : 5 ) ( 5 &# 39 ;- gggaagatggaaccaatcgcagatag - 3 &# 39 ;) and 3p2 ( seq id no : 6 ) ( 5 &# 39 ;- aattctatctgcgattggggttccatcttccc - 3 &# 39 ;) containing the extreme 3 &# 39 ; sequences of the ha gene and a sticky ecori end were ligated to a 1 . 8 kbp xhoi / smai fragment from pmh22 containing the remainder of the ha gene and psp131 digested with xhoi and ecori . the resultant plasmid was designated pspmha11 . the plasmid pmh22 was derived from a full length cdna clone of the measles ha gene by creating a xhoi site at the atg initiation codon ( alkhatib et al ., 1986 ). a 1 . 9 kbp hindiii / ecori fragment from pspmha11 , containing the measles ha gene , was isolated and blunt - ended with the klenow fragment of the e . coli dna polymerase in the presence of 2 mm dntps . the isolated fragment was inserted into pmp409dvc ( guo et al ., 1989 ) digested with bgliii and blunt - ended by treatment with mung bean nuclease . insertion into this vector yielded plasmid pspmha41 . the xhoi site between the h6 promoter and the initiation codon of the ha gene was removed by oligonucleotide directed double strand break mutagenesis ( mandecki , 1982 ) using oligonucleotide haxhod ( seq id no : 7 ) ( 5 &# 39 ;- atatccgttaagtttgtatcgtaatgtcaccacaacgagaccggat - 3 &# 39 ;). plasmid pspm2lhavc was generated by this procedure . insertion plasmid pspm2lhavc was used in in vitro recombination experiments with vaccinia virus vp458 as the rescue virus to generate recombinant vp557 . vp458 contains the e . coli lac z gene in the m2l insertion site of vp410 . this vaccinia virus recombinant contains the measles ha gene in the m2l locus of the genome , replacing the lac z gene . referring now to fig2 annealed oligonucleotides 3pa ( seq id no : 8 ) ( 5 &# 39 ;- cctaaagcctgatcttacgggaacatcaaaatcctatgtaaggtcgctctgatttttatcggccga - 3 &# 39 ;) and 3pb ( seq id no : 9 ) ( 5 &# 39 ;- agcttcggccgataaaaatcagagcgaccttacataggattttgatgttcccgtaagatcaggctttagg - 3 &# 39 ;) containing the 3 &# 39 ; end of the measles fusion gene , a vaccinia virus early transcription termination signal ( yuen et al ., 1987 ) and eagi and hindiii ends were ligated to a lkbp sali / haeiii fragment from pcrf2 ( obtained from c . richardson , national research council of canada ( biotechnology institute ), montreal , canada h3a 1a1 ) and puc8 digested with sali and hindiii . the resulting plasmid pmf3pr14 contains the 3 &# 39 ; end of the 1 kbp fragment of the measles fusion gene . annealed oligonucleotides 5pa ( seq id no : 10 ) ( 5 &# 39 ;- gggatgggtctcaaggtgaacgtctctgccatattc - 3 &# 39 ;) and 5pb ( seq id no : 11 ) ( 5 &# 39 ;- atggcagagacgttcaccttgagacccatccc - 3 &# 39 ;), containing a 5 &# 39 ; smai site and a 3 &# 39 ; bstxi site , were ligated to a 820 bp bstxi / sali fragment from pcrf2 and puc8 digested with smai and sali . the resultant plasmid pspmf5p16 contains the 5 &# 39 ; portion of the measles fusion gene . the 820 bp smai / sali fragment from pspmf5p16 and the 1 kbp sali / eagi fragment from pmf3pr14 were ligated into ptp15 digested with smai and eagi . the plasmid ptp15 ( guo et al ., 1989 ) contains the vaccinia virus early / late h6 promoter flanked by sequences from the ha locus of the vaccinia virus ( copenhagen strain ) genome . the resultant plasmid containing the measles fusion gene juxtaposed 3 &# 39 ; to the h6 promoter within the ha insertion plasmid was designated psphmf7 . oligonucleotide directed mutagenesis was performed on psphmf7 . initially an in vitro mutagenesis reaction ( mandecki , 1982 ) was performed to create a precise atg : atg linkage of the h6 promoter with the measles fusion gene by removing the smai site using the oligonucleotide spmad ( seq id no : 12 ) ( 5 &# 39 ;- tatccgttaagtttgtatggtaatgggtctcaaggtgaacgtct - 3 &# 39 ;). this resulted in the generation of pspmf75m20 . subsequently , the bglii site at the 5 &# 39 ; end of the h6 promoter was removed using oligonucleotide spbgld ( seq id no : 13 ) ( 5 &# 39 ;- aataaatcactttttatactaattctttattctatacttaaaaagt - 3 &# 39 ;) according to a known procedure ( mandecki , 1982 ). the resultant plasmid was designated pspmfvc . this plasmid was used in in vitro recombination experiments with vaccinia virus vp410 as rescue virus to generate vp455 . in order to determine that recombinants vp455 and vp557 expressed authentic proteins , immunoprecipitation experiments were performed essentially as described ( taylor et al ., 1990 ). briefly , vero cell monolayers were infected at 10 pfu per cell with either parental or recombinant viruses in the presence of 35 s - methionine . the fusion protein was specifically precipitated from the infected cell lysate using a rabbit antiserum directed against a carboxy terminal fusion peptide . the hemagglutinin protein was specifically precipitated from the infected cell lysate using a polyclonal monospecific anti - hemagglutinin serum . with respect to immunoprecipitation using a fusion specific serum , no radiolabelled products were detected in uninfected vero cells , parentally infected vero cells , or cells infected with the ha recombinant vp557 . in cells infected with the fusion recombinant vp455 , the fusion precursor f 0 with a molecular weight of approximately 60 kd and the two cleavage products f 1 and f 2 with molecular weights of 44 kd and 23 kd were detected . similarly , with respect to immunoprecipitation of the glycosylated form of the ha protein with a molecular weight of approximately 75 - 77 kd , no products were detected in uninfected vero cells , parental infected cells , or vero cells infected with vp455 . in addition , immunofluorescence studies indicated that both proteins were expressed on the infected cell surface . a characteristic of morbillivirus cytopathogenicity is the formation of syncytia which arise by fusion of infected cells with surrounding uninfected cells followed by migration of the nuclei toward the center of the syncytium ( norrby et al ., 1982 ). this has been shown to be an important method of viral spread , which for paramyxoviruses can occur in the presence of hemagglutinin specific antibody ( merz et al ., 1980 ). this ability has been assigned by analogy with other paramyxoviruses to the amino terminus of the f1 peptide ( choppin et al ., 1981 ; novick et al ., 1988 ; paterson et al ., 1987 ). in order to determine that the measles proteins expressed in vaccinia virus were functionally active , vero cell monolayers were inoculated with parental or recombinant viruses vp455 and vp557 , respectively , at 1 pfu per cell . after 1 h absorption at 37 ° c . the inoculum was removed , the overlay medium replaced , and the dishes incubated overnight at 37 ° c . at 18 h post - infection , plates were examined with a microscope and photographed . no cell fusing activity was evident in vero cells inoculated with parental virus , vp455 or vp557 . however , when vp455 and vp557 were co - inoculated , efficient cell fusing activity was observed . this result has recently been confirmed by wild et al . ( 1991 ) who determined that syncytium formation in a variety of cell lines infected with measles / vaccinia virus recombinants required expression of both fusion and hemagglutinin genes . the result , however , is in contrast to a previous report ( alkhatib , 1990 ) which described cell fusion in 293 cells infected with high multiplicities of an adenovirus recombinant expressing the measles fusion protein . similarly , it has been reported ( vialard et al ., 1990 ) that cell fusion was observed in insect cells infected with a baculovirus recombinant expressing the measles fusion protein but only when incubated at ph 5 . 8 . in neither case was the fusion activity enhanced by co - infection with the appropriate recombinant expressing the measles hemagglutinin protein . variables which may be involved in the fusion process are cell type ( giraudon et al ., 1984 ), ph of medium ( vialard et al ., 1990 ) and level of expression of the fusion protein ( norrby et al ., 1982 ). the technique for virus neutralizing ( vn ) antibody testing was previously described in detail ( appel et al ., 1973 ). testing for cdv - vn antibody titers was made in vero cells with the adapted onderstepoort strain of cdv . testing for mv - vn antibody titers was made in vero cells with the adapted edmonston strain of mv . the results of the serological tests are shown in table 1 . dogs immunized as described in example 6 with either the vaccinia parental virus or vp455 expressing the measles fusion protein did not develop neutralizing antibody to mv . dogs immunized with either vp557 expressing the ha protein or co - inoculated with both recombinants vp455 and vp557 did develop neutralizing antibodies after one inoculation . levels of antibody were equivalent to those induced by inoculation with the attenuated edmonston strain of mv . table 1______________________________________measles virus neutralizing antibody titersin response to vaccination days past vaccinationimmunization dog no . 0 . sup . a 7 14 21 . sup . b 28 35 . sup . c______________________________________vacc . 4 / 1 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 4 / 2 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0vp455 4 / 3 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 4 / 4 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0vp557 4 / 5 & lt ; 1 . 0 2 . 2 . sup . d 2 . 9 2 . 9 3 . 4 3 . 4 4 / 6 & lt ; 1 . 0 2 . 7 2 . 9 2 . 9 3 . 9 3 . 4vp455 & amp ; vp557 4 / 7 & lt ; 1 . 0 2 . 7 3 . 4 3 . 2 3 . 4 3 . 4 4 / 8 & lt ; 1 . 0 2 . 5 2 . 9 2 . 9 3 . 6 2 . 9mv 4 / 14 & lt ; 1 . 0 2 . 9 4 / 15 & lt ; 1 . 0 3 . 2______________________________________ . sup . a time of first immunization . sup . b time of second immunization ( first immunization with mv ) . sup . c time of challenge . sup . d titer expressed as log . sub . 10 of last antibody dilution showing complete neutralization of infectivity in a microtiter neutralization tes as described by appel et al . ( 1973 ). in order to determine whether expression of the measles virus proteins in dogs inoculated with the recombinants was sufficient to induce a protective immune response against cdv challenge , fourteen 10 week old specific pathogen free beagle dogs were studied . blood samples were collected at the initiation of the experiment and repeatedly thereafter . four groups with two dogs in each group were immunized with two injections three weeks apart . the first group received vaccinia virus only . the second group received vaccinia virus with an insert for the f protein of measles virus ( vp455 ). the third group received vaccinia virus with an insert for the ha antigen of mv ( vp557 ), and the fourth group received a combination of 2 and 3 . each dog was inoculated with approximately 4 × 10 8 pfu of vaccinia virus in 1 ml amounts ( 0 . 6 ml subcutaneously and 0 . 4 ml intramuscularly ). two control dogs received 10 5 50 % tissue culture infectious doses ( tcid 50 ) of the attenuated edmonston strain of mv intramuscularly ( 1 ml amount ) and two control dogs received 10 4 tcid 50 of the attenuated rockborn strain of cdv subcutaneously two weeks before challenge with virulent cdv . two control dogs remained uninoculated before challenge . all dogs were challenged by intranasal inoculation of 1 ml of tissue culture fluid containing 10 4 tcid 50 of the snyder hill strain of virulent cdv two weeks after the last inoculation . the clinical reactions of the dogs were monitored by daily observations and recording of body temperature and by biweekly recording of weight gain or losses . circulating blood lymphocytes were counted before challenge and on days post challenge ( dpc ) 3 , 5 , 7 and 10 . virus isolation from buffy coat cells by co - cultivation with dog lung macrophages ( appel et al ., 1967 ) was attempted on dpc 3 , 5 , 7 and 10 . blood samples for serological tests were collected before vaccination and in weekly intervals until time of challenge , and on dpc 7 , 10 and 20 . table 2__________________________________________________________________________effects of immunization on clinical signs after exposure of dogs tovirulent cdv no . of days after inoculation with virulent cdv dog weight elevated lympho - virusimmunization number depression loss body temp .. sup . a penia . sup . b isolation . sup . c death__________________________________________________________________________vacc . 4 / 1 4 - 10 . sup . d 3 - 10 4 , 5 , 7 , 10 7 - 10 3 - 7 10 4 / 2 4 - 10 . sup . d 3 - 10 4 , 5 , 8 - 10 3 - 10 3 - 7 10vp455 4 / 3 4 - 8 7 - 10 4 , 5 , 7 - 10 5 , 7 5 - 7 -- 4 / 4 4 - 6 7 - 10 4 - 6 7 5 - 7 -- vp557 4 / 5 nd . sup . e nd 6 , 7 10 7 -- 4 / 6 nd nd nd nd nd -- vp455 & amp ; vp557 4 / 7 nd nd 7 7 7 -- 4 / 8 nd 7 6 nd 7 -- mv 4 / 14 nd nd nd 7 nd -- 4 / 15 nd 7 5 5 nd -- cdv - ro 4 / 16 nd nd nd nd nd -- 4 / 17 nd nd nd nd nd -- none 4 / 18 6 , 14 - 17 . sup . d 7 - 17 5 , 7 13 - 17 10 17 4 / 19 4 - 10 . sup . d 3 - 10 4 , 5 , 7 3 , 7 , 10 3 - 10 10__________________________________________________________________________ . sup . a above 39 . 5 ° c . . sup . b less than 2 × 10 . sup . 3 lymphocytes per . sup . c isolated from buffy coat cells cocultivated with dog lung macrophages . sup . d dog became dehydrated and was euthanized . sup . e none detected non - immunized control dogs and dogs vaccinated with parental vaccinia virus developed clinical signs of severe disease and were euthanized when dehydration was evident . both dogs immunized with vp455 showed some signs of infection with cdv including weight loss , elevated body temperature , and lymphopenia although these symptoms were of shorter duration than were seen in control dogs . nonetheless , both dogs survived lethal challenge with cdv . dogs inoculated with vp557 or co - inoculated with both recombinants showed minimal signs of infection and survived challenge . dogs inoculated with either attenuated edmonston strain of mv or the attenuated rockborn strain of cdv also survived challenge with minimal signs of disease . referring now to fig3 a second vaccinia virus recombinant containing the measles ha gene within the tk locus was generated ( vp756 ) using insertion plasmid prw843 . prw843 was constructed in the following manner . a 1 . 8 kbp ecorv / smai fragment containing the 3 &# 39 ;- most 24 bp of the h6 promoter fused in a precise atg : atg configuration with the ha gene lacking the 3 &# 39 ;- most 26 bp was isolated from pspm2lhavc . this fragment was used to replace the 1 . 8 kbp ecorv / smai fragment of pspmha11 to generate prw803 . plasmid prw803 contains the entire h6 promoter linked precisely to the entire measles ha gene . in the confirmation of previous constructs with the measles ha gene it was noted that the sequence for codon 18 ( ccc ) was deleted as compared to the published sequence ( alkhatib et al ., 1986 ). the ccc sequence was replaced by oligonucleotide mutagenesis via the kunkel method ( kunkel , 1985 ) using oligonucleotide rw117 ( seq id no : 14 ) ( 5 &# 39 ;- gactatcctacttcccttgggatgggggttatctttgta - 3 &# 39 ;). single stranded template was derived from plasmid prw819 which contains the h6 / ha cassette from prw803 in pibi25 ( ibi , new haven , conn .). the mutagenized plasmid containing the inserted ( ccc ) to encode for a proline residue at codon 18 was designated prw820 . the sequence between the hindiii and xbai sites of prw820 was confirmed by nucleotide sequence analysis . the hindiii site is situated at the 5 &# 39 ; border of the h6 promoter while the xbai site is located 230 bp downstream from the initiation codon of the ha gene . a 1 . 6 kbp xbai / ecori fragment from prw803 , containing the ha coding sequences downstream from the xbai and including the termination codon , was used to replace the equivalent fragment of prw820 resulting in the generation of prw837 . the mutagenized expression cassette contained within prw837 was derived by digestion with hindiii and ecori , blunt - ended using the klenow fragment of e . coli dna polymerase in the presence of 2 mm dntps , and inserted into the smai site of psd573vcvq to yield prw843 . the plasmid prw843 was used in in vitro recombination experiments with vp618 as the rescue virus to yield vp756 . parental virus vp618 is a copenhagen strain virus from which the thymidine kinass , hemorrhagic and a - type inclusion genes have been deleted . recombinant vp756 has been shown by immunoprecipitation analysis to correctly express a hemagglutinin glycoprotein of approximately 75 kd . referring now to fig4 a second vaccinia virus recombinant ( vp800 ) harboring the measles fusion gene in the ati locus of the genome was generated using insertion plasmid prw850 . to construct prw850 , the following manipulations were performed . the plasmid pspmf75m20 containing the measles fusion gene linked in a precise atg : atg configuration with the h6 promoter was digested with nrui and eagi . the 1 . 7 kbp blunt ended fragment containing the 3 &# 39 ;- most 28 bp of the h6 promoter and the entire fusion gene was isolated and inserted into prw823 digested with nrui and xbai and blunt - ended . the resultant plasmid prw841 contains the h6 promoter linked to the measles fusion gene in the pibi25 plasmid vector ( ibi , new haven , conn .). the h6 / measles fusion expression cassette was derived from prw841 by digestion with smai and the resulting 1 . 8 kbp fragment was inserted into psd494vc digested with smai to yield prw850 . the plasmid prw850 was used in in vitro recombination experiments with vp618 as the rescue virus to yield vp800 . recombinant vp800 has been shown by immunoprecipitation analysis to express an authentically processed fusion glycoprotein . assessment of measles neutralizing antibody in guinea pigs and rabbits inoculated with vp455 two rabbits were inoculated intradermally at 5 sites with a total of 1 × 10 8 pfu of recombinant vp455 expressing the measles fusion protein . both rabbits were boosted with an identical inoculation at week 12 . serial bleeds were collected , and at week 14 , two weeks after the boost , the rabbits were tested for the presence of serum neutralizing antibodies . four guinea pigs were inoculated subcutaneously with 1 × 10 8 pfu each of recombinant vp455 . an identical booster inoculation was given at 21 days . serial bleeds were collected . the presence of measles virus serum neutralizing antibody was assessed using a microtiter test ( appel et al ., 1973 ) using 10 tcid 50 of virus per microtiter well . the results are shown in table 3 . table 3__________________________________________________________________________results of measles virus serum neutralizing antibodies in guinea pigs andrabbitsinoculated with vp455week post - inoculationanimal 0 2 3 4 5 7 14__________________________________________________________________________guinea pig1 n . d .. sup . a n . d . n . d .-. 8 . sup . b 1 . 3 -- 1 . 3 1 . 3 - 1 . 5 1 . 3 n . t . sup . c2 n . d . n . d . . 8 - 1 . 0 . 8 - 1 . 3 1 . 3 - 1 . 5 n . d . n . t . 3 n . d . n . d . n . d .-. 8 . 8 - 1 . 5 1 . 0 - 1 . 3 1 . 0 n . t . 6 n . d . n . d . . 8 --. 8 . 8 --. 8 1 . 0 - 1 . 3 1 . 0 n . t . rabbitw44 n . d . n . t . n . t . n . t . n . t . n . t . 1 . 5w86 n . d . n . t . n . t . n . t . n . t . n . t . 1 . 5__________________________________________________________________________ . sup . a not detectable . sup . b results of two assays . sup . c not tested measles / canarypox virus recombinants were developed using a similar strategy to that previously described for fowlpox virus ( taylor et al ., 1988a , b ). plasmids for insertion of the measles f and ha genes into canarypox virus were generated as follows . referring now to fig5 the 1 . 8 kbp blunt - ended bglii / eagi fragment from pspmf75m20 containing the h6 promoted measles f gene was inserted into the blunt - ended ecori site of prw764 . 2 . plasmid prw764 . 2 contains a 3 . 4 kbp pvuii fragment from the canarypox genome having a unique ecori site which has been determined to be non - essential for viral replication . the resultant plasmid containing the measles f gene was designated prw800 and was used in recombination experiments with canarypox as the rescuing virus to generate vcp40 . referring now to fig6 the 1 . 8 kbp ecorv / smai fragment from pspm2lha containing the 3 &# 39 ;- most 28 bp of the h6 promoter fused in a precise atg : atg configuration with ha was inserted between the ecorv and smai sites of pspmha11 . the resultant plasmid was designated prw803 . a 2 kbp hindiii / ecori fragment of prw803 containing the h6 promoted measles ha gene was blunt - ended and inserted into the blunt - ended bglii site of plasmid prw764 . 5 . plasmid prw764 . 5 contains an 800 bp pvuii fragment of the canarypox genome having a unique bglii site which has previously been determined to be non - essential for viral growth . this insertion created plasmid prw810 which was used in recombination tests to generate vcp50 . insertion of the measles f and ha sequences individually led to the development of recombinants vcp40 and vcp50 , respectively . in order to create a double recombinant , the single f recombinant vcp40 was used as a rescue virus for insertion of the ha gene contained in prw810 . this led to the development of double recombinant vcp57 . in order to confirm that recombinants vcp40 , vcp50 and vcp57 expressed authentic proteins , immunoprecipitation analysis was performed using mono - specific sera directed against either the ha or f proteins . a correctly processed fusion polypeptide was specifically precipitated from lysates of cells infected with vcp40 and vcp57 . the fusion precursor f 0 with a molecular weight of approximately 60 kd and the two cleavage products f 1 and f 2 with molecular weights of approximately 44 and 23 kd , respectively , were detected . no fusion specific products were detectable in uninfected cef cells , parentally infected cef cells or cef cells infected with the ha recombinant vcp50 . similarly , a glycoprotein of approximately 75 kd was specifically precipitated from cef cells infected with the single ha recombinant vcp50 and double recombinant vcp57 . no ha specific products were detected in uninfected cells , parentally infected cells or cells infected with fusion recombinant vcp40 . in order to determine that the measles virus recombinants were functionally active , cell fusion assays were performed . vero cell monolayers were infected with 1 pfu per cell of cp parental or recombinant viruses and examined for cytopathic effects at 18 hours post infection . no cell fusing activity was evident in vero cells inoculated with parental , vcp40 or vcp50 viruses . however , when vero cells were inoculated with the double recombinant vcp57 or when cells are co - infected with both vcp40 and vcp50 , efficient cell fusing activity is evident . dogs inoculated as described in example 13 with the canarypox / ha recombinant vcp50 , vaccinia / ha recombinant vp557 , the canarypox / ha / f double recombinant vcp57 or co - inoculated with vp455 and vp557 developed significant serum neutralizing antibody to measles virus after one inoculation . neither of the two dogs inoculated with the canarypox / f recombinant vcp40 developed neutralizing antibody after one or two inoculations . the results of the serological tests are shown in table 4 . in addition , guinea pigs inoculated with the vcp40 recombinant did develop low but reproducible levels of serum neutralizing antibody . table 4______________________________________measles virus neutralizing antibody titers ( in log . sup . 10 ) dog days post vaccinationimmunization no . 0 . sup . a 7 14 21 . sup . b 28 35 . sup . c______________________________________canary pox virus 9 / 1 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 ( cpv ) 9 / 2 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0vcp50 9 / 3 & lt ; 1 . 0 2 . 7 . sup . d 2 . 9 3 . 2 4 . 4 4 . 1 9 / 4 & lt ; 1 . 0 1 . 7 2 . 7 2 . 7 3 . 9 3 . 9vcp40 9 / 5 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 9 / 6 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0vcp57 9 / 7 & lt ; 1 . 0 2 . 0 2 . 7 2 . 5 3 . 9 3 . 6 9 / 8 & lt ; 1 . 0 1 . 0 2 . 2 2 . 0 3 . 6 3 . 4vp455 9 / 9 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0 1 . 0 1 . 0 1 . 0vp557 9 / 10 & lt ; 1 . 0 2 . 9 2 . 5 3 . 2 3 . 4 3 . 4vp455 & amp ; vp557 9 / 11 & lt ; 1 . 0 1 . 3 2 . 9 2 . 9 2 . 9 2 . 9mv 9 / 12 & lt ; 1 . 0 2 . 5 2 . 5control 9 / 13 & lt ; 1 . 0 9 / 14 & lt ; 1 . 0cdv - ro 9 / 15 & lt ; 1 . 0 & lt ; 1 . 0 & lt ; 1 . 0______________________________________ . sup . a time of first immunization . . sup . b time of second immunization . ( first immunization with mv and cdvro ). . sup . c time of challenge . . sup . d assessment of serum neutralization titers assessed in known manner ( appel et al ., 1973 ). in order to determine whether non - replicating canarypox vectors expressing measles virus proteins would induce a protective immune response against cdv challenge , ten week old specific pathogen free beagle dogs were inoculated with canarypox parental and recombinant viruses . two dogs were inoculated simultaneously with two subcutaneous injections of 1 × 10 8 pfu of each recombinant at three week intervals . for comparison , one dog was inoculated in the same regimen with each of the single vaccinia virus recombinants vp455 and vp557 and a combination of both . one dog was also inoculated intramuscularly with one dose of 10 5 tcid 50 of the attenuated edmonston strain of mv . one dog was inoculated subcutaneously with one dose of 10 4 tcid 50 of the attenuated rockborn strain of cdv . dogs were challenged two weeks after the final inoculation via intranasal inoculation with a lethal dose of 10 4 tcid 50 of the virulent snyder hill strain of cdv . clinical reactions of dogs were monitored daily . the results are shown in table 5 . table 5__________________________________________________________________________effects of immunization on clinical signs after exposure of dogs tovirulent cdv no . of days after inoculation with virulent cdv elevated lympho virusimmunization dohg no . depression loss body temp .. sup . a penia . sup . b isolation . sup . c__________________________________________________________________________canary pox 9 / 1 4 - 10 . sup . d 3 - 10 4 , 5 , 7 , 8 5 - 10 3 - 10virus ( cpv ) 9 / 2 4 - 10 . sup . d 7 - 10 4 , 5 , 7 , 8 3 - 10 3 - 10vcp50 9 / 3 nd . sup . e 7 - 10 5 - 7 5 - 7 7 9 / 4 nd 7 6 , 7 7 ndvcp40 9 / 5 4 - 10 3 - 10 4 5 - 10 5 - 7 9 / 6 4 - 6 3 - 10 4 - 6 5 5 - 7vcp57 9 / 7 nd 7 - 10 5 , 6 5 nd 9 / 8 nd 7 - 10 4 - 7 , 10 7 - 10 5 - 7vp455 9 / 9 6 - 8 7 - 10 4 , 5 , 8 , 9 5 - 10 3 - 10vp557 9 / 10 nd 3 - 10 nd nd ndvp455 & amp ; 9 / 11 nd 3 - 10 nd 5 - 7 ndvp557mv 9 / 12 nd 7 - 10 nd 5 5none 9 / 13 4 - 10 . sup . d 3 - 10 4 - 6 5 - 10 5 - 10 9 / 14 4 - 10 . sup . d 3 - 10 4 - 5 3 - 10 5 - 7cdv - ro 9 / 15 nd nd nd nd nd__________________________________________________________________________ . sup . a above 39 . 5 ° c . . sup . b less than 2 × 10 . sup . 3 lymphocytes per mm . sup . 3 . . sup . c isolated from buffy coat cells cocultivated with dog lung macrophages in known manner ( appel et al ., 1967 ). . sup . d dog became dehydrated and was euthanized . . sup . e none detected . no adverse reactions to vaccination were noticed in any of the dogs during the course of the experiment . the two dogs immunized with parental canarypox virus and two non - immunized control dogs showed severe disease after challenge with virulent cdv . all four dogs became depressed , showed elevated body temperature , weight loss , lymphopenia and severe dehydration . dogs immunized with cdv - rockborn developed serum neutralizing antibodies against cdv but not against mv prior to challenge and survived challenge , symptom free . dogs immunized with attenuated mv developed serum neutralizing antibodies to mv but not cdv prior to challenge , and survived challenge with mild signs of infection . dogs inoculated with vcp50 , vcp57 , vp557 or co - inoculated with vp455 and vp557 developed significant serum neutralizing antibody to mv after one inoculation and survived challenge with only minor signs of infection . referring now to fig7 to generate a canarypox virus recombinant expressing the mv ha gene the following insertion plasmids were created . a 1 . 8 kbp ecorv / ecori fragment from prw837 containing the 3 &# 39 ;- most 26 bp of the h6 promoter linked precisely to the measles ha , was ligated to a 3 . 2 kbp ecorv / ecori fragment from prw838 . the prw838 derived fragment includes the 5 &# 39 ; portion of the h6 promoter and c5 locus flanking arms . plasmids prw838 and prw831 ( see below ) were derived as follows . an 880 bp pvuii canarypox genomic fragment was inserted between the pvuii sites of puc9 . the resultant plasmid was designated prw764 . 5 . the nucleotide sequence of the 880 bp canarypox fragment was determined using the modified t7 enzyme sequenase ™ kit ( united states biochemical , cleveland , ohio ) according to manufacturer &# 39 ; s specifications . sequence reactions utilized custom synthesized primers ( 17 - 18 mere ) prepared with the biosearch 8700 ( san rafael , calif .) or applied biosystems 3800 ( foster city , calif .). this enabled the definition of the c5 open reading frame . to specifically delete the c5 open reading frame , prw764 . 5 was partially cut with rsai and the linear product was isolated . the rsai linear fragment was recut with bglii and the prw764 . 5 fragment with a rsai - bglii deletion from position 156 to position 462 was isolated and used as a vector for the following synthetic oligonucleotides : rw145 ( seq id no : 15 ): ( 5 &# 39 ;- actctcaaaagcttcccgggaattctagctagctagtttttataaa - 3 &# 39 ;) rw146 ( seq id no : 16 ): ( 5 &# 39 ;- gatctttataaaaactagctagctagaattcccgggaagcttttgagagt - 3 &# 39 ;) oligonucleotides rw145 and rw146 were annealed and inserted into the prw764 . 5 rsai - bglii vector described above . the resulting plasmid is prw831 . this c5 deletion plasmid was constructed without interruption of other canarypox virus open reading frames . the c5 coding sequence was replaced with the above annealed oligonucleotides ( rw145 and rw146 ) which include the restriction sites for hindiii , smai , and ecori . the plasmid prw838 , was derived from prw831 by the insertion of a smai fragment containing the rabies g gene ( taylor et al ., 1988b ) juxtaposed 3 &# 39 ; to the vaccinia virus h6 promoter . ligation of the 1 . 8 kbp ecorv / ecori fragment from prw837 with the 3 . 2 kbp ecorv / ecori fragment from prw838 led to the construction of plasmid prw852 . plasmid prw852 was used in recombination experiments with a canarypox isolate designated alvac to yield vcp85 . alvac is a plaque cloned isolate of canarypox virus ( cpv ) derived from the rentschler strain , a highly attenuated strain of cpv used for vaccination of canaries . replication of alvac and derived recombinants is restricted to avian species . immunoprecipitation analysis has confirmed that a protein of approximately 75 kd recognized by a rabbit anti - ha serum is expressed in cef cells infected with recombinant vcp85 . referring now to fig8 to generate a canarypox virus recombinant harboring both the mv ha and f genes the following constructs were engineered . smai restriction sites were added to the ends of the h6 promoted measles fusion gene . to accomplish this , prw823 , which is pibi25 containing the vaccinia virus h6 promoter , was digested downstream of the promoter sequence at the xbai site . the ends were blunted with the klenow fragment of the e . coli dna polymerase in the presence of 2 mm dntps . the blunt - ended dna was subsequently digested with nrui to liberate a 3 . 0 kbp fragment containing the 5 &# 39 ;- most 100 bp of the h6 promoter . this fragment was isolated and ligated to a 1 . 7 kbp blunt - ended eagi / nrui fragment from pspmf75 . the resultant plasmid was designated as prw841 . the 1 . 8 kbp smai fragment derived by digestion of prw841 was inserted into the c5 deletion vector , prw831 . the plasmid prw851 was linearized at the ecori site situated 3 &# 39 ; to the fusion gene and was blunt - ended with the klenow fragment of the e . coli dna polymerase in the presence of 2 mm dntps . the plasmid prw837 , containing the measles ha gene juxtaposed 3 &# 39 ; to the h6 promoter sequences , was digested with hindiii and ecori and blunt - ended with the klenow fragment . the resultant 1 . 8 kbp fragment was isolated and inserted into prw851 that had been linearized with ecori and blunt - ended . the resultant plasmid , which contains both genes in a tail to tail configuration , was designated prw853a and was utilized in in vitro recombination experiments with canarypox ( alvac ) as the rescue virus to generate vcp82 also designated alvac - mv . expression analysis using immunoprecipitation and immunofluorescence confirmed that in cells infected with recombinant vcp82 authentically processed ha and f proteins were expressed . the recombinant was also functional for cell fusing activity . results of serological analysis of sera of rabbits and guinea pigs inoculated with alvac - mv ( vcp82 ) four guinea pigs were inoculated by the subcutaneous route with alvac - mv ( vcp82 ). two animals ( 026 and 027 ) each received 1 × 10 8 pfu and two animals ( 028 and 029 ) each received 1 × 10 7 pfu . at 28 days , animals were re - inoculated with an identical dose . two rabbits were inoculated with 1 × 10 8 pfu of alvac - mv ( vcp82 ) by the subcutaneous route . at 28 days , animals were re - inoculated with an identical dose . serial bleeds of these animals were analyzed for measles virus neutralizing activity using either a microtiter neutralization test described by appel and robson ( 1973 ) or a plaque reduction neutralization test described by albrecht et al . ( 1981 ). in addition , sera were analyzed for the presence of antibody capable of blocking measles virus induced cell - cell fusion in an anti - fusion assay performed as described in merz et al . ( 1980 ). the results of analysis for the presence of measles virus serum neutralizing antibody are shown in tables 6 and 7 . both guinea pigs ( 026 and 027 ) receiving 1 × 10 8 pfu of alvac - mv sero - converted after a single inoculation and sera showed an antibody rise after the booster inoculation . one animal ( 029 ) receiving 1 × 10 7 pfu also sero - converted after one inoculation . the fourth animal ( 028 ) did not show a detectable response after one inoculation but did achieve equivalent titers after the second inoculation . rabbit sera were also analyzed using a plaque reduction neutralization method . the results are shown in table 7 . both animals sero - converted after one inoculation . sera of rabbit 063 was tested by both the micro - titer neutralization test and the plaque reduction neutralization test . titers achieved were similar using both methods . it has been reported that a minimal serum neutralizing titer of 1 . 2 to 1 . 9 in vaccinated children is required for protection from disease ( lennon and black , 1986 ; black et al ., 1984 ). using this criteria , all animals , except the one guinea - pig which did not sero - convert until the second inoculation showed a protective level of antibody after one inoculation . table 6______________________________________serological analysis of sera of guinea pigs inoculated withalvac - mv ( vcp82 ): analysis performed by microtiter serumneutralization assay . animal days post - inoculationguinea pigs 0 14 21 28 . sup . c 42 48 56______________________________________026 . sup . d -- n . t .. sup . a 1 . 25 . sup . b 1 . 49 2 . 45 2 . 68 2 . 92027 -- n . t . 1 . 97 1 . 49 2 . 68 2 . 45 2 . 21028 . sup . e -- n . t . -- -- 1 . 73 2 . 45 1 . 97029 -- n . t . 0 . 8 1 . 49 2 . 45 2 . 45 2 . 45______________________________________ . sup . a not tested . . sup . b titer expressed as log . sub . 10 of reciprocal of last dilution showing complete neutralization of cytopathic effect . . sup . c animals boosted at 28 days postinoculation . . sup . d animals 026 and 027 received 1 × 10 . sup . 8 pfu . . sup . e animals 028 and 029 received 1 × 10 . sup . 7 pfu . table 7______________________________________serological analysis of sera of rabbits inoculated withalvac - mv ( vcp82 ) days post - inoculationanimal 0 14 21 28 . sup . b 42 56______________________________________plaque reduction method063 -- 1 . 9 . sup . a 2 . 8 1 . 6 2 . 2 2 . 2064 -- 2 . 2 2 . 5 2 . 8 3 . 1 2 . 8microtiter neutralization method063 -- 1 . 5 . sup . c 1 . 7 1 . 5 1 . 7______________________________________ . sup . a titer expressed as log . sub . 10 of reciprocal of last dilution showing a 50 % reduction in plaque number as compared to preinoculation serum . . sup . b animals boosted at 28 days postinoculation . . sup . c titer expressed as log . sub . 10 of reciprocal of last dilution showing complete neutralization of cytophatic effect . previous studies have shown that an inactivated vaccine was associated with poor protective efficacy and an enhanced measles disease on re - exposure to the virus . recipients of the inactivated vaccine demonstrated an absence of antibody to the fusion protein and it was proposed that the inactivation process had rendered the protein non - immunogenic ( norrby and gollmar , 1975 ; norrby et al ., 1975 ). in addition , it has been shown for other paramyxoviruses that antibody to the f protein is able to inhibit cell to cell spread of virus in tissue culture while antibody to the hemagglutinin component is not ( merz et al ., 1980 ). it was therefore significant to demonstrate that animals inoculated with alvac - mv ( vcp82 ) were able to induce antibody to the f component which was capable of blocking cell to cell transmission of measles virus . the results of this antifusion assay are shown in table 8 . anti - fusion activity was evident in sera of both guinea - pigs and rabbits inoculated with alvac - mv ( vcp82 ). the sera analyzed was taken two or three weeks after the boost inoculation . no anti - fusion activity could be detected in sera of rabbits inoculated with alvac parental virus . table 8______________________________________analysis of sera of guinea pigs and rabbits inoculated withalvac - mv for anti - fusion activity anti - fusion titeranimal designation immunogen pre - inoc . post - vacc . ______________________________________guinea - pig 026 alvac - mv -- 2 . 4 . sup . a , b 027 alvac - mv -- 1 . 2rabbit 063 alvac - mv -- 1 . 8 . sup . c 064 alvac - mv -- 1 . 8rabbit w121 alvac -- -- w123 alvac -- -- ______________________________________ . sup . a guinea pig sera tested at 7 weeks postvaccination . . sup . b titer expressed as log . sub . 10 of reciprocal of highest dilution showing complete inhibition of measles virus induced cell fusing activity . sup . c rabbit sera test at 6 weeks postvaccination . in further tests to demonstrate the presence of antibody to both the mv hemagglutinin and mv fusion proteins in sera of animals inoculated with alvac - mv , immunoprecipitation experiments were performed . sera of rabbits inoculated with alvac - mv was shown to specifically precipitate both the hemagglutinin and fusion proteins from radiolabelled lysates of vero cells infected with edmonston strain mv . in a similar study , groups of guinea pigs , rabbits and mice were inoculated by the intra muscular route with alvac - mv , and their serological response to measles virus monitored using the hemagglutination - inhibition ( hi ) test . the serological response to canarypox virus was monitored by elisa assay . in this study , five guinea pigs were inoculated with 5 . 5 log 10 tcid 50 , thirty mice were inoculated with 4 . 8 log 10 tcid 50 , and five rabbits were inoculated with 5 . 8 log 10 tcid 50 . all animals were re - inoculated at 28 days with an equivalent dose . animals were bled at regular intervals and their response to measles virus assessed in an hi assay . the limit of detection in the hi assay corresponds to a log 10 titer of 1 and it is considered that sero - positive ( protected ) children have a serum titer in the range of 1 . 6 to 2 . 8 . the results of analysis are shown in tables 9 , 10 and 11 . sera of mice were analyzed in groups of 5 animals ( table 9 ). all animals showed a primary response to canarypox virus which was boosted after the second inoculation . the mice did not show a response to mv after one inoculation . three of the six groups showed titers within the protective range at 8 weeks post - inoculation . similarly , all guinea - pigs ( table 10 ) showed a response to canarypox virus after one inoculation which was boosted after the second inoculation . four of five animals developed anti - hi titers after one inoculation , one of these being in the protective range . one week after the second inoculation , the titers of all animals were in the protective range . these titers were maintained through 8 weeks post - inoculation when the experiment was concluded . all rabbits ( table 11 ) inoculated with alvac - mv ( vcp82 ) responded serologically to canarypox inoculation . four of five animals sero - converted to measles virus after one inoculation ( one in the protective range ). serum titers of all animals were in the protective range one week after the second inoculation . table 9______________________________________serological response of mice to inoculation with alvac - mv ( vcp82 ) week post - inoculationmouse group 0 2 4 5 6 8______________________________________anti - canarypox responseelisa titer1 . sup . a - 0 . 009 . sup . b 0 . 364 0 . 193 1 . 821 1 . 616 1 . 1232 - 0 . 026 0 . 047 0 . 240 1 . 739 1 . 963 1 . 9863 - 0 . 006 0 . 148 0 . 641 1 . 860 1 . 861 1 . 9474 - 0 . 005 0 . 130 0 . 451 1 . 506 1 . 937 1 . 1245 0 . 687 0 . 542mean - 0 . 012 0 . 275 0 . 413 1 . 732 1 . 844 1 . 395anti - measles responsehi titer1 & lt ; 1 . sup . c & lt ; 1 & lt ; 1 & lt ; 1 1 12 & lt ; 1 & lt ; 1 & lt ; 1 1 1 . 6 1 . 63 & lt ; 1 & lt ; 1 1 1 2 . 2 2 . 24 & lt ; 1 & lt ; 1 & lt ; 1 1 . 6 1 1 . 85 & lt ; 1 & lt ; 1 & lt ; 1 1 . 3 1 . 8 1 . 2mean -- -- 1 1 . 2 1 . 5 1 . 5______________________________________ . sup . a groups of five mice were exsanguinated and sera pooled . . sup . b optical density in an elisa assay on sera at dilution of 1 : 800 . sup . c limit of detection in hi test corresponds to a log . sub . 10 titer of 1 i . e . 1 : 10 dilution . titer expressed as log . sub . 10 of reciprocal of highest dilution showing inhibition of hemagglutination . table 10______________________________________serological response of guinea - pigs to inoculation withalvac - mv ( vcp82 ) week post - inoculationguinea - pig 0 2 4 5 6 8______________________________________anti - canarypox responseelisa titer1 0 . 038 . sup . a 0 . 045 0 . 111 1 . 771 1 . 970 1 . 8562 0 . 010 0 . 072 0 . 234 1 . 768 1 . 786 1 . 7853 - 0 . 011 0 . 426 0 . 529 1 . 567 1 . 586 1 . 7004 0 . 016 0 . 045 0 . 076 1 . 583 1 . 696 1 . 6355 - 0 . 020 0 . 012 0 . 050 1 . 583 1 . 859 1 . 847anti - measles responsehi titer1 & lt ; 1 . sup . b 1 . 18 1 . 90 3 . 11 3 . 41 3 . 112 & lt ; 1 & lt ; 1 1 . 00 2 . 20 2 . 20 2 . 083 & lt ; 1 & lt ; 1 1 . 18 2 . 51 2 . 68 2 . 984 & lt ; 1 & lt ; 1 & lt ; 1 1 . 60 1 . 90 1 . 905 & lt ; 1 & lt ; 1 1 . 30 1 . 90 2 . 20 2 . 20______________________________________ . sup . a optical density in an elisa assay on serum at a 1 : 3200 dilution . . sup . b limit of detection in hi test corresponds to a log . sub . 10 titer of 1 i . e . 1 : 10 dilution . titer expressed as in legend to table 9 . table 11______________________________________serological response of rabbits to inoculation with alvac - mv ( vcp82 ) week post - inoculationrabbit 0 2 4 5 6 8______________________________________anti - canarypox responseelisa titer1 . sup . a - 0 . 009 . sup . a 0 . 085 0 . 113 1 . 953 1 . 754 1 . 2492 - 0 . 002 0 . 065 0 . 068 0 . 717 0 . 567 0 . 3533 - 0 . 003 0 . 090 0 . 079 0 . 921 0 . 692 0 . 4814 - 0 . 005 0 . 034 0 . 068 1 . 558 1 . 324 1 . 0765 - 0 . 003 0 . 072 0 . 092 1 . 785 1 . 226 0 . 710anti - measles responsehi titer1 & lt ; 1 . sup . b & lt ; 1 1 . 00 2 . 81 2 . 51 2 . 202 & lt ; 1 & lt ; 1 & lt ; 1 2 . 20 1 . 90 1 . 603 & lt ; 1 & lt ; 1 1 . 30 2 . 81 2 . 51 2 . 384 & lt ; 1 1 . 30 1 . 60 3 . 11 3 . 11 2 . 515 & lt ; 1 1 . 00 1 . 30 2 . 68 2 . 38 1 . 90______________________________________ . sup . a optical density in an elisa assay on sera at a dilution of 1 : 1600 . . sup . b limit of detection in hi test corresponds to a log . sub . 10 titer of 1 i . e . 1 : 10 dilution . titer expressed as in legend to table 9 . results of serological analysis of sera of squirrel monkeys inoculated with alvac - mv ( vcp82 ): influence of prior exposure to poxvirus on induction of a measles virus specific immune response nine squirrel monkeys ( saimiri sciureus ) were inoculated with alvac - mv ( vcp82 ). all monkeys were naive to measles virus . seven of the monkeys had prior exposure to vaccinia virus and / or canarypox virus . the previous immunization history is shown in table 12 . all monkeys were inoculated with one dose of 5 . 8 log 10 pfu by the subcutaneous route . four of the animals (# 39 , 42 , 53 and 58 ) were re - inoculated with an equivalent dose fifteen weeks after the primary inoculation . anti - measles antibody was measured in the hi test . the results are shown in table 12 . after the first inoculation , two of the nine monkeys showed a low response to inoculation with alvac - mv . after the second inoculation , the four monkeys re - inoculated all sero - converted with significant antibody titers in the range required for protective immunity . the titers achieved were equivalent whether the monkey had prior exposure to vaccinia virus and alvac or no prior poxvirus exposure . table 12______________________________________inoculation of squirrel monkeys with alvac - mv ( vcp82 ): immune response in the face of preexisting alvac immunity . anti - himonkey previous immunity measles response # to poxviruses primary . sup . a boost . sup . b______________________________________36 vv , alvac & lt ; 1 n . b . 37 vv , alvac - rg & lt ; 1 n . b . 39 vv , alvac - rg , cp - felv 1 2 . 2 . 40 vv , cp - felv & lt ; 1 n . b . 42 none & lt ; 1 2 . 2 . 52 alvac & lt ; 1 n . b . 53 alvac - rg , alvac - rg & lt ; 1 1 . 6 . 56 cp - felv & lt ; 1 n . b . 58 none 1 2 . 2 . ______________________________________ vv : vaccinia virus , copenhagen strain alvacrg : alvac recombinant expressing rabies g gene cpfelv : canarypox recombinant expressing felv env gene nb : not boosted . sup . a animals received 5 . 8 log . sub . 10 pfu by s . c . route . . sup . b animals 39 , 42 , 52 and 53 were boosted with an identical dose 15 weeks after the first inoculation . to develop a new vaccinia vaccine strain , the copenhagen vaccine strain of vaccinia virus was modified by the deletion of six nonessential regions of the genome encoding known or potential virulence factors . the sequential deletions are detailed below . all designations of vaccinia restriction fragments , open reading frames and nucleotide positions are based on the terminology reported in goebel et al . ( 1990a , b ). the deletion loci were also engineered as recipient loci for the insertion of foreign genes . the regions sequentially deleted in nyvac are listed below . also listed are the abbreviations and open reading frame designations for the deleted regions ( goebel et al ., 1990a , b ) and the designation of the vaccinia recombinant ( vp ) containing all deletions through the deletion specified : plasmids were constructed , screened and grown by standard procedures ( maniatis et al ., 1986 ; perkus et al ., 1985 ; piccini et al ., 1987 ). restriction endonucleases were obtained from gibco / brl , gaithersburg , md ., new england biolabs , beverly , mass . ; and boehringer mannheim biochemicals , indianapolis , ind . klenow fragment of e . coli polymerase was obtained from boehringer mannheim biochemicals . bal - 31 exonuclease and phage t4 dna ligase were obtained from new england biolabs . the reagents were used as specified by the various suppliers . synthetic oligodeoxyribonucleotides were prepared on a biosearch 8750 or applied biosystems 380b dna synthesizer as previously described ( perkus et al ., 1989 ). dna sequencing was performed by the dideoxy - chain termination method ( sanger et al ., 1977 ) using sequenase ( tabor et al ., 1987 ) as previously described ( guo et al ., 1989 ). dna amplification by polymerase chain reaction ( pcr ) for sequence verification ( engelke et al ., 1988 ) was performed using custom synthesized oligonucleotide primers and geneamp dna amplification reagent kit ( perkin elmer cetus , norwalk , conn .) in an automated perkin elmer cetus dna thermal cycler . excess dna sequences were deleted from plasmids by restriction endonuclease digestion followed by limited digestion by bal - 31 exonuclease and mutagenesis ( mandecki , 1986 ) using synthetic oligonucleotides . the origins and conditions of cultivation of the copenhagen strain of vaccinia virus has been previously described ( guo et al ., 1989 ). generation of recombinant virus by recombination , in situ hybridization of nitrocellulose filters and screening for beta - galactosidase activity are as previously described ( panicali et al ., 1982 ; perkus et al ., 1989 ). referring now to fig9 plasmid psd406 contains vaccinia hindiii j ( pos . 83359 - 88377 ) cloned into puc8 . psd406 was cut with hindiii and pvuii , and the 1 . 7 kb fragment from the left side of hindiii j cloned into puc8 cut with hindiii / smai , forming psd447 . psd447 contains the entire gene for j2r ( pos . 83855 - 84385 ). the initiation codon is contained within an nlaiii site and the termination codon is contained within an sspi site . direction of transcription is indicated by an arrow in fig9 . to obtain a left flanking arm , a 0 . 8 kb hindiii / ecori fragment was isolated from psd447 , then digested with nlaiii and a 0 . 5 kb hindiii / nlaiii fragment isolated . annealed synthetic oligonucleotides mpsyn43 / mpsyn44 ( seq id no : 17 / seq id no : 18 ) ## str2 ## were ligated with the 0 . 5 kb hindiii / nlaiii fragment into puc18 vector plasmid cut with hindiii / ecori , generating plasmid psd449 . to obtain a restriction fragment containing a vaccinia right flanking arm and puc vector sequences , psd447 was cut with sspi ( partial ) within vaccinia sequences and hindiii at the puc / vaccinia junction , and a 2 . 9 kb vector fragment isolated . this vector fragment was ligated with annealed synthetic oligonucleotides mpsyn45 / mpsyn46 ( seq id no : 19 / seq id no : 20 ) ## str3 ## generating psd459 . to combine the left and right flanking arms into one plasmid , a 0 . 5 kb hindiii / smai fragment was isolated from psd449 and ligated with psd459 vector plasmid cut with hindiii / smai , generating plasmid psd460 . psd460 was used as donor plasmid for recombination with wild type parental vaccinia virus copenhagen strain vc - 2 . 32 p labeled probe was synthesized by primer extension using mpsyn45 ( seq id no : 19 ) as template and the complementary 20 mer oligonucleotide mpsyn47 ( seq id no : 21 ) ( 5 &# 39 ;- ttagttaattaggcggccgc - 3 &# 39 ;) as primer . recombinant virus vp410 was identified by plaque hybridization . referring now to fig1 , plasmid psd419 contains vaccinia sali g ( pos . 160 , 744 - 173 , 351 ) cloned into puc8 . psd422 contains the contiguous vaccinia sali fragment to the right , sali j ( pos . 173 , 351 - 182 , 746 ) cloned into puc8 . to construct a plasmid deleted for the hemorrhagic region , u , b13r - b14r ( pos . 172 , 549 - 173 , 552 ), psd419 was used as the source for the left flanking arm and psd422 was used as the source of the right flanking arm . the direction of transcription for the u region is indicated by an arrow in fig1 . to remove unwanted sequences from psd419 , sequences to the left of the ncoi site ( pos . 172 , 253 ) were removed by digestion of psd419 with ncoi / smai followed by blunt ending with klenow fragment of e . coli polymerase and ligation generating plasmid psd476 . a vaccinia right flanking arm was obtained by digestion of psd422 with hpai at the termination codon of b14r and by digestion with nrui 0 . 3 kb to the right . this 0 . 3 kb fragment was isolated and ligated with a 3 . 4 kb hincii vector fragment isolated from psd476 , generating plasmid psd477 . the location of the partial deletion of the vaccinia u region in psd477 is indicated by a triangle . the remaining b13r coding sequences in psd477 were removed by digestion with clai / hpai , and the resulting vector fragment was ligated with annealed synthetic oligonucleotides sd22mer / sd20mer ( seq id no : 22 / seq id no : 23 ) ## str4 ## generating psd479 . psd479 contains an initiation codon ( underlined ) followed by a bamhi site . to place e . coli beta - galactosidase in the b13 - b14 ( u ) deletion locus under the control of the u promoter , a 3 . 2 kb bamhi fragment containing the beta - galactosidase gene ( shapira et al ., 1983 ) was inserted into the bamhi site of psd479 , generating psd479bg . psd479bg was used as donor plasmid for recombination with vaccinia virus vp410 . recombinant vaccinia virus vp533 was isolated as a blue plaque in the presence of chromogenic substrate x - gal . in vp533 the b13rb14r region is deleted and is replaced by beta - galactosidase . to remove beta - galactosidase sequences from vp533 , plasmid psd486 , a derivative of psd477 containing a polylinker region but no initiation codon at the u deletion junction , was utilized . first the clai / hpai vector fragment from psd477 referred to above was ligated with annealed synthetic oligonucleotides sd42mer / sd40mer ( seq id no : 24 / seq id no : 25 ) ## str5 ## generating plasmid psd478 . next the ecori site at the puc / vaccinia junction was destroyed by digestion of psd478 with ecori followed by blunt ending with klenow fragment of e . coli polymerase and ligation , generating plasmid psd478e - . psd478e - was digested with bamhi and hpai and ligated with annealed synthetic oligonucleotides hem5 / hem6 ( seq id no : 26 / seq id no : 27 ) ## str6 ## generating plasmid psd486 . psd486 was used as donor plasmid for recombination with recombinant vaccinia virus vp533 , generating vp553 , which was isolated as a clear plaque in the presence of x - gal . referring now to fig1 , psd414 contains sali b cloned into puc8 . to remove unwanted dna sequences to the left of the a26l region , psd414 was cut with xbai within vaccinia sequences ( pos . 137 , 079 ) and with hindiii at the puc / vaccinia junction , then blunt ended with klenow fragment of e . coli polymerase and ligated , resulting in plasmid psd483 . to remove unwanted vaccinia dna sequences to the right of the a26l region , psd483 was cut with ecori ( pos . 140 , 665 and at the puc / vaccinia junction ) and ligated , forming plasmid psd484 . to remove the a26l coding region , psd484 was cut with ndei ( partial ) slightly upstream from the a26l orf ( pos . 139 , 004 ) and with hpai ( pos . 137 , 889 ) slightly downstream from the a26l orf . the 5 . 2 kb vector fragment was isolated and ligated with annealed synthetic oligonucleotides ati3 / ati4 ( seq id no : 28 / seq id no : 29 ) ## str7 ## reconstructing the region upstream from a26l and replacing the a26l orf with a short polylinker region containing the restriction sites bglii , ecori and hpai , as indicated above . the resulting plasmid was designated psd485 . since the bglii and ecori sites in the polylinker region of psd485 are not unique , unwanted bglii and ecori sites were removed from plasmid psd483 ( described above ) by digestion with bglii ( pos . 140 , 136 ) and with ecori at the puc / vaccinia junction , followed by blunt ending with klenow fragment of e . coli polymerase and ligation . the resulting plasmid was designated psd489 . the 1 . 8 kb clai ( pos . 137 , 198 )/ ecorv ( pos . 139 , 048 ) fragment from psd489 containing the a26l orf was replaced with the corresponding 0 . 7 kb polylinker - containing clai / ecorv fragment from psd485 , generating psd492 . the bglii and ecori sites in the polylinker region of psd492 are unique . a 3 . 3 kb bglii cassette containing the e . coli beta - galactosidase gene ( shapira et al ., 1983 ) under the control of the vaccinia 11 kda promoter ( bertholet et al ., 1985 ; perkus et al ., 1990 ) was inserted into the bglii site of psd492 , forming psd493kbg . plasmid psd493kbg was used in recombination with rescuing virus vp553 . recombinant vaccinia virus , vp581 , containing beta - galactosidase in the a26l deletion region , was isolated as a blue plaque in the presence of x - gal . to generate a plasmid for the removal of beta - galactosidase sequences from vaccinia recombinant virus vp581 , the polylinker region of plasmid psd492 was deleted by mutagenesis ( mandecki , 1986 ) using synthetic oligonucleotide mpsyn177 ( seq id no : 30 ) ( 5 &# 39 ;- aaaatgggcgtggattgttaactttatataacttattttttgaatatac - 3 &# 39 ;). in the resulting plasmid , pmp494δ , vaccinia dna encompassing positions 137 , 889 - 138 , 937 !, including the entire a26l orf is deleted . recombination between the pmp494δ and the beta - galactosidase containing vaccinia recombinant , vp581 , resulted in vaccinia deletion mutant vp618 , which was isolated as a clear plaque in the presence of x - gal . referring now to fig1 , vaccinia sali g restriction fragment ( pos . 160 , 744 - 173 , 351 ) crosses the hindiii a / b junction ( pos . 162 , 539 ). psd419 contains vaccinia sali g cloned into puc8 . the direction of transcription for the hemagglutinin ( ha ) gene is indicated by an arrow in fig1 . vaccinia sequences derived from hindiii b were removed by digestion of psd419 with hindiii within vaccinia sequences and at the puc / vaccinia junction followed by ligation . the resulting plasmid , psd456 , contains the ha gene , a56r , flanked by 0 . 4 kb of vaccinia sequences to the left and 0 . 4 kb of vaccinia sequences to the right . a56r coding sequences were removed by cutting psd456 with rsai ( partial ; pos . 161 , 090 ) upstream from a56r coding sequences , and with eagi ( pos . 162 , 054 ) near the end of the gene . the 3 . 6 kb rsai / eagi vector fragment from psd456 was isolated and ligated with annealed synthetic oligonucleotides mpsyn59 ( seq id no : 31 ), mpsy62 ( seq id no : 32 ), mpsyn60 ( seq id no : 33 ), and mpsyn 61 ( seq id no : 34 ) ## str8 ## reconstructing the dna sequences upstream from the a56r orf and replacing the a56r orf with a polylinker region as indicated above . the resulting plasmid is psd466 . the vaccinia deletion in psd466 encompasses positions 161 , 185 - 162 , 053 !. the site of the deletion in psd466 is indicated by a triangle in fig1 . a 3 . 2 kb bglii / bamhi ( partial ) cassette containing the e . coli beta - galactosidase gene ( shapira et al ., 1983 ) under the control of the vaccinia 11 kda promoter ( bertholet et al ., 1985 ; guo et al ., 1989 ) was inserted into the bglii site of psd466 , forming psd466kbg . plasmid psd466kbg was used in recombination with rescuing virus vp618 . recombinant vaccinia virus , vp708 , containing beta - galactosidase in the a56r deletion , was isolated as a blue plaque in the presence of x - gal . beta - galactosidase sequences were deleted from vp708 using donor plasmid psd467 . psd467 is identical to psd466 , except that ecori , smai and bamhi sites were removed from the puc / vaccinia junction by digestion of psd466 with ecori / bamhi followed by blunt ending with klenow fragment of e . coli polymerase and ligation . recombination between vp708 and psd467 resulted in recombinant vaccinia deletion mutant , vp723 , which was isolated as a clear plaque in the presence of x - gal . construction of plasmid pmpcsk1δ for deletion of open reading frames c7l - k1l ! referring now to fig1 , the following vaccinia clones were utilized in the construction of pmpcsk1δ . psd420 is sali h cloned into puc8 . psd435 is kpni f cloned into puc18 . psd435 was cut with sphi and religated , forming psd451 . in psd451 , dna sequences to the left of the sphi site ( pos . 27 , 416 ) in hindiii m are removed ( perkus et al ., 1990 ). psd409 is hindiii m cloned into puc8 . to provide a substrate for the deletion of the c7l - k1l ! gene cluster from vaccinia , e . coli beta - galactosidase was first inserted into the vaccinia m2l deletion locus ( guo et al ., 1990 ) as follows . to eliminate the bglii site in psd409 , the plasmid was cut with bglii in vaccinia sequences ( pos . 28 , 212 ) and with bamhi at the puc / vaccinia junction , then ligated to form plasmid pmp409b . pmp409b was cut at the unique sphi site ( pos . 27 , 416 ). m2l coding sequences were removed by mutagenesis ( guo et al ., 1990 ; mandecki , 1986 ) using synthetic oligonucleotide ## str9 ## the resulting plasmid , pmp409d , contains a unique bglii site inserted into the m2l deletion locus as indicated above . a 3 . 2 kb bamhi ( partial )/ bglii cassette containing the e . coli beta - galactosidase gene ( shapira et al ., 1983 ) under the control of the 11 kda promoter ( bertholet et al ., 1985 ) was inserted into pmp409d cut with bglii . the resulting plasmid , pmp409dbg ( guo et al ., 1990 ), was used as donor plasmid for recombination with rescuing vaccinia virus vp723 . recombinant vaccinia virus , vp784 , containing beta - galactosidase inserted into the m2l deletion locus , was isolated as a blue plaque in the presence of x - gal . a plasmid deleted for vaccinia genes c7l - k1l ! was assembled in puc8 cut with smai , hindiii and blunt ended with klenow fragment of e . coli polymerase . the left flanking arm consisting of vaccinia hindiii c sequences was obtained by digestion of psd420 with xbai ( pos . 18 , 628 ) followed by blunt ending with klenow fragment of e . coli polymerase and digestion with bglii ( pos . 19 , 706 ). the right flanking arm consisting of vaccinia hindiii k sequences was obtained by digestion of psd451 with bglii ( pos . 29 , 062 ) and ecorv ( pos . 29 , 778 ). the resulting plasmid , pmp581ck is deleted for vaccinia sequences between the bglii site ( pos . 19 , 706 ) in hindiii c and the bglii site ( pos . 29 , 062 ) in hindiii k . the site of the deletion of vaccinia sequences in plasmid pmp581ck is indicated by a triangle in fig1 . to remove excess dna at the vaccinia deletion junction , plasmid pmp581ck , was cut at the ncoi sites within vaccinia sequences ( pos . 18 , 811 ; 19 , 655 ), treated with bal - 31 exonuclease and subjected to mutagenesis ( mandecki , 1986 ) using synthetic oligonucleotide mpsyn233 ( seq id no : 36 ) 5 &# 39 ;- tgtcatttaacactatactcatattaataaaaataatatttatt - 3 &# 39 ;. the resulting plasmid , pmpcsk1δ , is deleted for vaccinia sequences positions 18 , 805 - 29 , 108 , encompassing 12 vaccinia open reading frames c7l - k1l !. recombination between pmpcsk1δ and the beta - galactosidase containing vaccinia recombinant , vp784 , resulted in vaccinia deletion mutant , vp804 , which was isolated as a clear plaque in the presence of x - gal . construction of plasmid psd548 for deletion of large subunit , ribonucleotide reductase ( i4l ) referring now to fig1 , plasmid psd405 contains vaccinia hindiii i ( pos . 63 , 875 - 70 , 367 ) cloned in puc8 . psd405 was digested with ecorv within vaccinia sequences ( pos . 67 , 933 ) and with smai at the puc / vaccinia junction , and ligated , forming plasmid psd518 . psd518 was used as the source of all the vaccinia restriction fragments used in the construction of psd548 . the vaccinia i4l gene extends from position 67 , 371 - 65 , 059 . direction of transcription for i4l is indicated by an arrow in fig1 . to obtain a vector plasmid fragment deleted for a portion of the i4l coding sequences , psd518 was digested with bamhi ( pos . 65 , 381 ) and hpai ( pos . 67 , 001 ) and blunt ended using klenow fragment of e . coli polymerase . this 4 . 8 kb vector fragment was ligated with a 3 . 2 kb smai cassette containing the e . coli beta - galactosidase gene ( shapira et al ., 1983 ) under the control of the vaccinia 11 kda promoter ( bertholet et al ., 1985 ; perkus et al ., 1990 ), resulting in plasmid psd524kbg . psd524kbg was used as donor plasmid for recombination with vaccinia virus vp804 . recombinant vaccinia virus , vp855 , containing beta - galactosidase in a partial deletion of the i4l gene , was isolated as a blue plaque in the presence of x - gal . to delete beta - galactosidase and the remainder of the i4l orf from vp855 , deletion plasmid psd548 was constructed . the left and right vaccinia flanking arms were assembled separately in puc8 as detailed below and presented schematically in fig1 . to construct a vector plasmid to accept the left vaccinia flanking arm , puc8 was cut with bamhi / ecori and ligated with annealed synthetic oligonucleotides 518a1 / 518a2 ( seq id no : 37 / seq id n0 : 38 ) ## str10 ## forming plasmid psd531 . psd531 was cut with rsai ( partial ) and bamhi and a 2 . 7 kb vector fragment isolated . psd518 was cut with bglii ( pos . 64 , 459 )/ rsai ( pos . 64 , 994 ) and a 0 . 5 kb fragment isolated . the two fragments were ligated together , forming psd537 , which contains the complete vaccinia flanking arm left of the i4l coding sequences . to construct a vector plasmid to accept the right vaccinia flanking arm , puc8 was cut with bamhi / ecori and ligated with annealed synthetic oligonucleotides 518b1 / 518b2 ( seq id no : 39 / seq id no : 40 ) ## str11 ## forming plasmid psd532 . psd532 was cut with rsai ( partial )/ ecori and a 2 . 7 kb vector fragment isolated . psd518 was cut with rsai within vaccinia sequences ( pos . 67 , 436 ) and ecori at the vaccinia / puc junction , and a 0 . 6 kb fragment isolated . the two fragments were ligated together , forming psd538 , which contains the complete vaccinia flanking arm to the right of i4l coding sequences . the right vaccinia flanking arm was isolated as a 0 . 6 kb ecori / bglii fragment from psd538 and ligated into psd537 vector plasmid cut with ecori / bglii . in the resulting plasmid , psd539 , the i4l orf ( pos . 65 , 047 - 67 , 386 ) is replaced by a polylinker region , which is flanked by 0 . 6 kb vaccinia dna to the left and 0 . 6 kb vaccinia dna to the right , all in a puc background . the site of deletion within vaccinia sequences is indicated by a triangle in fig1 . to avoid possible recombination of beta - galactosidase sequences in the puc - derived portion of psd539 with beta - galactosidase sequences in recombinant vaccinia virus vp855 , the vaccinia i4l deletion cassette was moved from psd539 into prc11 , a puc derivative from which all beta - galactosidase sequences have been removed and replaced with a polylinker region ( colinas et al ., 1990 ). psd539 was cut with ecori / psti and the 1 . 2 kb fragment isolated . this fragment was ligated into prc11 cut with ecori / psti ( 2 . 35 kb ), forming psd548 . recombination between psd548 and the beta - galactosidase containing vaccinia recombinant , vp855 , resulted in vaccinia deletion mutant vp866 , which was isolated as a clear plaque in the presence of x - gal . dna from recombinant vaccinia virus vp866 was analyzed by restriction digests followed by electrophoresis on an agarose gel . the restriction patterns were as expected . polymerase chain reactions ( pcr ) ( engelke et al ., 1988 ) using vp866 as template and primers flanking the six deletion loci detailed above produced dna fragments of the expected sizes . sequence analysis of the pcr generated fragments around the areas of the deletion junctions confirmed that the junctions were as expected . recombinant vaccinia virus vp866 , containing the six engineered deletions as described above , was designated vaccinia vaccine strain &# 34 ; nyvac .&# 34 ; cdna copies of the sequences encoding the ha and f proteins of measles virus mv ( edmonston strain ) were inserted into nyvac to create a double recombinant designated nyvac - mv ( vp913 ). the recombinant authentically expressed both measles glycoproteins on the surface of infected cells . immunoprecipitation analysis demonstrated correct processing of both f and ha glycoproteins . the recombinant was also shown to induce syncytia formation . the rescuing virus used in the production of nyvac - mv was the modified copenhagen strain of vaccinia virus designated nyvac . all viruses were grown and titered on vero cell monolayers . referring now to fig1 and taylor et al . ( 1991 ), plasmid pspm2lha contains the entire measles ha gene linked in a precise atg to atg configuration with the vaccinia virus h6 promoter which has been previously described ( taylor et al ., 1988a , b ; guo et al ., 1989 ; perkus et al ., 1989 ). a 1 . 8 kpb ecorv / smai fragment containing the 3 &# 39 ; most 24 bp of the h6 promoter fused in a precise atg : atg configuration with the ha gene lacking the 3 &# 39 ; most 26 bp was isolated from pspm2lha . this fragment was used to replace the 1 . 8 kbp ecorv / smai fragment of pspmha11 ( taylor et al ., 1991 ) to generate prw803 . plasmid prw803 contains the entire h6 promoter linked precisely to the entire measles ha gene . plasmid psd513vcvq was derived from plasmid psd460 by the addition of polylinker sequences . plasmid psd460 was derived to enable deletion of the thymidine kinase gene from vaccinia virus ( fig9 ). to insert the measles virus f gene into the ha insertion plasmid , manipulations were performed on psphmf7 . plasmid psphmf7 ( taylor et al ., 1991 ) contains the measles f gene juxtaposed 3 &# 39 ; to the previously described vaccinia virus h6 promoter . in order to attain a perfect atg for atg configuration and remove intervening sequences between the 3 &# 39 ; end of the promoter and the atg of the measles f gene oligonucleotide directed mutagenesis was performed using oligonucleotide spmad ( seq id no : 41 ). ## str12 ## the resultant plasmid was designated pspmf75m20 . the plasmid pspmf75m20 which contains the measles f gene now linked in a precise atg for atg configuration with the h6 promoter was digested with nrui and eagi . the resulting 1 . 7 kbp blunt ended fragment containing the 3 &# 39 ; most 27 bp of the h6 promoter and the entire fusion gene was isolated and inserted into an intermediate plasmid prw823 which had been digested with nrui and xbai and blunt ended . the resultant plasmid prw841 contains the h6 promoter linked to the measles f gene in the pibi25 plasmid vector ( ibi , new haven , conn .). the h6 / measles f cassette was excised from prw841 by digestion with smai and the resulting 1 . 8 kb fragment was inserted into prw843 ( containing the measles ha gene ). plasmid prw843 was first digested with noti and blunt - ended with klenow fragment of e . coli dna polymerase in the presence of 2 mm dntps . the resulting plasmid , prw857 , therefore contains the measles virus f and ha genes linked in a tail to tail configuration . both genes are linked to the vaccinia virus h6 promoter . plasmid prw857 was transfected into nyvac ( vp866 ) infected vero cells by using the calcium phosphate precipitation method previously described ( panicall et al ., 1982 ; piccini et al ., 1987 ). positive plaques were selected on the basis of in situ plaque hybridization to specific mv f and ha radiolabeled probes and subjected to 6 sequential rounds of plaque purification until a pure population was achieved . one representative plaque was then amplified and the resulting recombinant was designated nyvac - mv ( vp913 ). indirect immunofluorescence was performed as previously described ( taylor et al ., 1990 ). mono - specific reagents used were sera generated by inoculation of rabbits with canarypox recombinants expressing either the measles f or ha genes . immunoprecipitation reactions were performed as previously described ( taylor et al ., 1990 ) using a guinea - pig anti measles serum ( whittaker m . a . bioproducts , walkersville , md .). vero cell monolayers in 60 mm dishes were inoculated at a multiplicity of 1 pfu per cell with parental or recombinant viruses . after 1 h absorption at 37 ° c . the inoculum was removed , the overlay medium replaced and the dishes inoculated overnight at 37 ° c . at 20 h post - infection , dishes were examined . in order to determine that the expression products of both measles virus f and ha genes were presented on the infected cell surface , indirect immunofluorescence analysis was performed using mono - specific sera generated in rabbits against canarypox recombinants expressing either the measles f or ha genes . the results indicated that both f and ha gene products were expressed on the infected cell surface , as demonstrated by strong surface fluorescence with both mono - specific sera . no background staining was evident with either sera on cells inoculated with the parental nyvac strain , nor was there cross - reactive staining when mono - specific sera were tested against vaccinia single recombinants expressing either the ha or f gene . in order to demonstrate that the proteins expressed by nyvac - mv were immunoreactive with measles virus specific sera and were authentically processed in the infected cell , immunoprecipitation analysis was performed . vero cell monolayers were inoculated at a multiplicity of 10 pfu / cell of parental or recombinant viruses in the presence of 35 s - methionine . immunoprecipitation analysis revealed a ha glycoprotein of approximately 76 kda and the cleaved fusion products f 1 and f 2 with molecular weights of 44 kda and 23 kda , respectively . no measles specific products were detected in uninfected vero cells or vero cells infected with the parental nyvac virus . a characteristic of mv cytopathology is the formation of syncytia which arise by fusion of infected cells with surrounding infected or uninfected cells followed by migration of the nuclei toward the center of the syncytium ( norrby et al ., 1982 ). this has been shown to be an important method of viral spread , which for paramyxoviruses , can occur in the presence of ha - specific virus neutralizing antibody ( merz et al ., 1980 ). in order to determine that the mv proteins expressed in vaccinia virus were functionally active , vero cell monolayers were inoculated with nyvac and nyvac - mv and observed for cytopathic effects . strong cell fusing activity was evident in nyvac - mv infected vero cells at approximately 18 hours post infection . no cell fusing activity was evident in cells infected with parental nyvac . in this study , two rabbits were inoculated with 1 × 10 8 pfu of nyvac - mv ( vp913 ) by the subcutaneous route . at 28 days , animals were boosted with an equivalent dose . serial bleeds were analyzed for mv neutralizing activity using the plaque reduction method . the results are shown in table 13 . the results indicate that neither rabbit responded to the initial inoculation of nyvac - mv . however , the sharply rising response after the second inoculation indicates that the animals were primed . both animals achieved neutralizing antibody titers in the protective range . the in vivo analysis of immunogenicity of alvac - 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nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 2 : tcgagtacgatacaaacttaacggatatcgcgataatgaaataatttatgattatttctc 60gctttcaatttaacacaaccctcaagaacctttgtatttattttcactttttaagtatag120aataaaga128 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 54 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 3 : attatttttataagcttggatccctcgagggtacccccggggagctcgaattct54 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 54 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 4 : agaattcgagctccccgggggtaccctcgagggatccaagcttataaaaataat54 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 26 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 5 : gggaagatggaaccaatcgcagatag26 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 32 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 6 : aattctatctgcgattggggttccatcttccc32 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 46 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 7 : atatccgttaagtttgtatcgtaatgtcaccacaacgagaccggat46 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 66 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 8 : cctaaagcctgatcttacgggaacatcaaaatcctatgtaaggtcgctctgatttttatc60ggccga 66 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 70 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 9 : agcttcggccgataaaaatcagagcgaccttacataggattttgatgttcccgtaagatc60aggctttagg70 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 36 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 10 : g ggatgggtctcaaggtgaacgtctctgccatattc36 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 32 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 11 : atggca gagacgttcaccttgagacccatccc32 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 44 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 12 : tatccgttaa gtttgtatggtaatgggtctcaaggtgaacgtct44 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 46 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 13 : aataaatcacttttt atactaattctttattctatacttaaaaagt46 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 39 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 14 : gactatcctacttcccttgg gatgggggttatctttgta39 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 46 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 15 : actctcaaaagcttcccgggaatt ctagctagctagtttttataaa46 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 16 : gatctttataaaaactagctagctagaat tcccgggaagcttttgagagt50 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 17 : taattaactagctacccggg 20 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 28 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 18 : aattcccgggtagctagttaattacatg 28 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 73 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 19 : agcttcccgggtaagtaatacgtcaaggagaaaacgaaacga tctgtagttagcggccgc60ctaattaactaat73 ( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 69 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 20 : attagttaattaggcggccgctaactacagatcgtttcgttttctccttgacgtattact60tacccggga69 ( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 21 : ttagttaattaggcggccgc20 ( 2 ) information for seq id no : 22 :( i ) sequence characteristics : ( a ) length : 22 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 22 : cgattactatgaaggatccgtt22 ( 2 ) information for seq id no : 23 :( i ) sequence characteristics : ( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 23 : aacggatccttcatagtaat20 ( 2 ) information for seq id no : 24 :( i ) sequence characteristics :( a ) length : 41 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 24 : cgattactagatctgagctccccgggctcgagggatccgtt41 ( 2 ) information for seq id no : 25 :( i ) sequence characteristics :( a ) length : 39 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 25 : aacggatccctcgagcccggggagctcagatctagtaat39 ( 2 ) information for seq id no : 26 :( i ) sequence characteristics :( a ) length : 16 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 26 : gatccgaattctagct16 ( 2 ) information for seq id no : 27 :( i ) sequence characteristics :( a ) length : 12 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 27 : agctagaattcg12 ( 2 ) information for seq id no : 28 :( i ) sequence characteristics :( a ) length : 75 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 28 : tatgagtaacttaactcttttgttaattaaaagtatattcaaaaaataagttatataaat60agatctgaattcgtt 75 ( 2 ) information for seq id no : 29 :( i ) sequence characteristics :( a ) length : 73 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 29 : aacgaattcagatctatttatataacttattttttgaatatacttttaattaacaaaaga 60gttaagttactca73 ( 2 ) information for seq id no : 30 :( i ) sequence characteristics :( a ) length : 49 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 30 : aaaatgggcgtggattgttaactttatataacttattttttgaatatac49 ( 2 ) information for seq id no : 31 :( i ) sequence characteristics :( a ) length : 67 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 31 : ac acgaatgattttctaaagtatttggaaagttttataggtagttgatagaacaaaatac60ataattt67 ( 2 ) information for seq id no : 32 :( i ) sequence characteristics :( a ) length : 51 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 32 : tctatcaactacctataaaactttccaaatactttagaaaatcattcgtgt51 ( 2 ) information for seq id no : 33 :( i ) sequence characteristics :( a ) length : 46 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 33 : tgtaaaaataaatcactttttatactaagatctcccgggctgcagc46 ( 2 ) information for seq id no : 34 :( i ) sequence characteristics :( a ) length : 66 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 34 : ggccgctgcagcccgggagatcttagtataaaaagtgatttatttttacaaaattatgta60ttttgt 66 ( 2 ) information for seq id no : 35 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 35 : tttctgtatatttgcaccaatttagatcttactcaaaatatgtaacaata 50 ( 2 ) information for seq id no : 36 :( i ) sequence characteristics :( a ) length : 44 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 36 : tgtcatttaacactatactcatattaataaaaataatatttatt44 ( 2 ) information for seq id no : 37 :( i ) sequence characteristics :( a ) length : 72 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 37 : gatcctgagtactttgtaatataatgatatatattttcactttatctcatttgagaataa60aaag atcttagg72 ( 2 ) information for seq id no : 38 :( i ) sequence characteristics :( a ) length : 72 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 38 : aattcctaa gatctttttattctcaaatgagataaagtgaaaatatatatcattatatta60caaagtactcag72 ( 2 ) information for seq id no : 39 :( i ) sequence characteristics :( a ) length : 72 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 39 : gatccagatctcccgggaaaaaaattatttaacttttcattaatagggatttgacgtatg60tagcgtactagg 72 ( 2 ) information for seq id no : 40 :( i ) sequence characteristics :( a ) length : 72 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 40 : aattcctagtacgcatcatacgtcaaatccctattaatgaaaagttaaataatttttttc 60ccgggagatctg72 ( 2 ) information for seq id no : 41 :( i ) sequence characteristics :( a ) length : 44 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 41 : tatccgttaagtttgtatcgtaatgggtctcaaggtgaacgtct44