Patent Application: US-15588893-A

Abstract:
a first embodiment is a specific plasmid vector , pdip / pycsp . 1 , into which nucleotides encoding the targets of specific immune responses are inserted . these targets include , but are not limited to proteins and peptides . these plasmid constructs are incorporated in a composition comprising a suitable and acceptable art recognized pharmaceutical reagent that is benign to the plasmid construct . the plasmid construct provides protective immune responses to the disease associated with the selected targets . a second embodiment is a construct having , at a minimum , the nucleotide sequences encoding one or more plasmodium species proteins in a pharmaceutically acceptable vector . a third embodiment is a method of controlling malaria in mammals comprising injecting a polynucleotide delivery vector into a mammal .

Description:
in the first embodiment , a plasmid vector was constructed for use in the delivery in vivo of polynucleotides ( cdna , dna , rna ,) the sequences of which encode the synthesis of molecules that provide beneficial therapeutic or immunological ( protective ) responses in mammalian subjects . in research results published by cullen 27 , the plasmid vector pbc12 / cmv / il - 2 when transfected in vitro into mammalian tissue culture cells effectively utilized the cytomegalovirus ( cmv ) promoter of transcription to drive expression of human interleukin 2 ( il - 2 ). these results suggested a rationale for a plasmid based on cullen &# 39 ; s plasmid vector that could be used in vivo ( contrary to cullen &# 39 ; s vector that included the intact il2 ) to express any polynucleotide sequence from inside mammalian cells following delivery of the plasmid by injection ( intramuscular , subcutaneous , intradermal , intravenous ), inhalation , topical application , or ingestion into the body of a mammalian subject . this vector leads to the expression of polynucleotides encoding the first six amino acids of the rat preproinsulin signal peptide , the first 82 amino acids of the human il - 2 protein , and the targets in parasites , viruses , bacteria , fungi , toxins , and other molecules of mammalian protective immune responses . depicted in fig3 pdip / x is an illustration of a derived construct of pdip / pycsp . 1 in which any polynucleotide x ( cdna , dna , rna ) is inserted downstream of the cmv promoter using for example one or more combinations of the following restriction endonucleases to modify the parent pdip / pycsp . 1 plasmid for other uses : hindiii , nhei , xbai , bamhi , hincii , kpni , smai , ecori , or any endonucleases that recognize sites within the pycsp gene sequence ( not shown ). the resultant plasmid from any modification of pdip / pycsp . 1 in which polynucleotide sequences are inserted downstream of the cmv promoter will be capable of being expressed in mammalian cells and therefore the plasmid used as a vaccine , as we have shown in malaria studies with mice , or in any case where this plasmid vector is used to deliver polynucleotide sequences for expression in mammalian subjects that will have beneficial therapeutic or immunological effects . in the second embodiment , we have found that intramuscular injection with a plasmid containing polynucleotides encoding a specific protein of the plasmodium species induces antibodies and ctl against the specific protein , and protection against challenge with the malaria parasite . nucleotides encoding any plasmodium sp . proteins capable of inducing protection against malaria usable in this invention can be included . specific proteins include the known p . falciparum , p . vivax , p . malariae , and p . ovale csp ; ssp2 ( trap ); pfs16 ( sheba ); lsa - 1 ; lsa - 2 ; lsa - 3 ; msa - 1 ( pmmsa , psa , p185 , p190 ); msa - 2 ( gymmnsa , gp56 , 38 - 45 kda antigen ); resa ( pf155 ); eba - 175 ; ama - 1 ( pf83 ); sera ( p113 , p126 , serp , pf140 ); rap - 1 ; rap - 2 ; rhoph3 ; pfhrp - ii ; pf55 ; pf35 ; gbp ( 96 - r ); abra ( p101 ); exp - 1 ( cra , ag5 . 1 ); aldolase ; duffy binding protein of p . vivax ; reticulocyte binding proteins ; hsp70 - 1 ( p75 ); pfg25 ; pfg28 ; pfg48 / 45 ; and pfg230 , and the nucleotides encoding the analogues of these proteins in the other species when not yet defined . specifically we have found that im injection of balb / c mice with pycsp dna induces antibodies and ctl against the protein , and protects against challenge with the protozoan parasite , p . yoelii . polynucleotide sequences encoding one or any of the above proteins is ligated into the pdip / pycsp . 1 plasmid or any other vector as depicted in fig1 . it is believed that protection can be improved by altering immunization regimens , or immunizing with several genes or short portions of genes encoding protective b and t cell epitopes ; to determine the mechanisms by which dna immunization induces protective antibody and t cell responses ; to establish the safety of this method of immunization ; and to establish similar immunogenicity in non - human primates . having described the invention , the following examples are given to illustrate specific applications of the invention including the best mode now known to perform the invention . these specific examples are not intended to limit the scope of the invention described in this application . our initial attempts to induce antibodies , ctl , or protective immunity by immunizing balb / c mice by the intravenous ( iv ) route with a plasmid containing the entire coding region of py sporozoite surface protein 2 ( pyssp2 ) 28 , 29 in expression vector pcexv - 3 30 were unsuccessful . because of the success of dna immunization with vectors containing cytomegalovirus ( cmv ) promoters 26 , we cloned the entire coding region of pycsp 31 into a cmv promoter vector 27 that we modified to accommodate the pycsp gene to form pdip / pycsp . 1 ( fig2 ). the pycsp gene was obtained from plasmid b155 32 as a 1468 bp dra1 / ecorv fragment . this fragment was ligated into puc18 , which was previously digested with sma1 followed by calf intestinal alkaline phosphatase , to form puc18 / pycsp . a 1486bp xbal / kpl fragment encompassing the pycsp gene was excised from puc18 / pycsp and used to replace the 666 bp xbal / kpnl fragment of the expression vector pbc12 / cmv / il - 2 27 to form pdip / pycsp . 1 . dna sequencing of the xbal junction of pdip / pycsp . 1 predicted that the entire csp coding region was fused in - frame with the sequence encoding the first 82 amino acids of il - 2 . the sequence of pdip / pycsp . 1 plasmid is shown in sequence id no . 1 . sequence id no . 2 is the translated peptides . plasmid dna for injections was purified by cesium chloride gradient centrifugation , sterilized by ethanol precipitation , and dissolved in sterile pbs . mice were injected im in each thigh with 100 mcg of plasmid dna dissolved in pbs with or without lipofectin ® reagent ( brl ). initial experiments indicated that delivery with lipofectin ® reagent did not augment antibody responses as measured in an indirect fluorescent antibody test ( ifat ) against sporozoites ( data not shown ). therefore , in subsequent experiments , plasmid dna was delivered im in pbs alone . negative control mice were injected with unmodified plasmid dna lacking the pycsp gene . in initial experiments antibody responses were inconsistent . after 3 doses only 9 of 13 mice had antibodies to sporozoites , and 7 of the 9 had low levels of antibodies . however , after the fourth immunization 12 of the 13 mice had moderate to high antibody titers against sporozoites ( data not shown ). after experience was gained with injections , and the caliber of the needle reduced from 26 gauge to 30 gauge , the frequency of antibody response improved ( table 1 ). table 1______________________________________antibodies against sporozoites . mice were immunized withpdip / pycsp . 1 at 0 and 8 weeks , or at 0 , 5 and 8 weeks . sera weretested for antibodies to air - dried sporozoites by ifat ( 7 ) 5 , 8and 10 weeks after the first immunization . control mice received thepbc12 / cmv / il - 2 plasmid ( 16 , 17 ) without the pycsp insert . pooled sera taken 2 weeks after the third immunization withp . yoelii irrspz ( 7 ), and tested at the same time had anifat titer of 1280 . ifat titers weeks after first immunizationmouse immunizations 5 8 10______________________________________1a 0 , 8 weeks 40 40 20 , 4802a 0 , 8 weeks 320 160 20 , 4803a 0 , 8 weeks 320 160 20 , 4807a 0 , 8 weeks 160 320 20 , 4805a 0 , 8 weeks & lt ; 10 & lt ; 10 10 , 2406a 0 , 8 weeks 320 320 2 , 5604a 0 , 8 weeks 80 80 2 , 5603b 0 , 5 , 8 weeks 640 20 , 480 20 , 4804b 0 , 5 , 8 weeks 640 10 , 240 20 , 4805b 0 , 5 , 8 weeks 160 2 , 560 20 , 4801b 0 , 5 , 8 weeks 320 5 , 120 5 , 1202b 0 , 5 , 8 weeks 160 2 , 560 2 , 5606b 0 , 5 , 8 weeks 160 2 , 560 2 , 560controls ( n = 6 ) 0 , 8 weeks & lt ; 10 & lt ; 10 & lt ; 10controls ( n = 6 ) 0 , 5 , 8 weeks & lt ; 10 & lt ; 10 & lt ; 10______________________________________ data also indicated that delay in delivery of the second dose improved the response to 2 doses ( table 1 ). antibodies in sera from mice immunized with pdip / pycsp . 1 recognized purified recombinant p . yoelii csp ( fig4 ). the results shown in fig4 were obtained from pooled sera taken from 3 mice 2 weeks after the third dose where the mice were respectively immunized with the pdip / pycsp . 1 vaccine , irradiated p . yoelii sporozoites ( irrspz ), and plasmid control . the sera of each was assessed by elisa as described 14 for antibodies to a recombinant fusion protein , py cs . 1 , including amino acids 64 to 321 of the pycsp . the serum dilution at which the absorbance ( 405 nm ) was 1 . 0 by elisa was 7 . 4 times higher in mice immunized with pdip / pycsp . 1 as compared to those immunized with irradiated sporozoites ( fig4 ). fig5 is a graph of induction of mhc restricted , cd8 + t cell - dependent ctl against pycsp by immunization with pdip / pycsp . 1 . two weeks after the second dose of pdip / pycsp . 1 , mice were euthanized and spleen cells isolated , stimulated in vitro for 5 days with peptide pycsp ( 281 - 296 ; syvpsaeqilefvkqi ), and then assessed for cytolytic activity as previously described 21 . at an effector to target ratio of 60 : 1 t cells lysed mhc matched p815 cells ( h - 2d ) pulsed with the same pycsp ( 281 - 296 ) peptide , but did not lyse peptide - pulsed el - 4 cells ( h - 2b ). this cytolytic activity was eliminated by depletion of cd8 + t cells , but unaffected by depletion of cd4 + t cells . these sera also inhibited sporozoite invasion and development within primary mouse hepatocytes in vitro 33 . hepatocytes isolated from balb / c mice were seeded in 8 - chamber lab - tek plastic slides at 1 × 10 5 cells / chamber . after 24 h of incubation at 37 ° c . in an atmosphere of 5 % co 2 in air , medium was removed and 5 × 10 4 salivary gland dissected sporozoites suspended in 25 μl of medium , and 25 μl of diluted sera from immunized or control mice added . after 3 hours incubation cultures were washed to remove unattached sporozoites and fresh medium was added . at 24 hours medium was changed and at 48 hours cultures were fixed and incubated with a mab directed against liver stage parasites of p . yoelii ( nylsi ) before incubating with fitc - labelled goat anti - mouse ig . the number of liver - stage schizonts in triplicate cultures was counted using an olympus fluorescence microscope . a 1 : 10 dilution of serum from a mouse immunized with 3 doses of vaccine , serum with an ifat titer against sporozoites of 20 , 480 , inhibited sporozoite invasion and development by 80 ± 5 % as compared to serum from a mouse immunized with plasmid control ( 9 . 3 ± 2 . 5 schizonts per well vs 46 . 0 ± 3 . 6 schizonts / well , p = 0 . 001 , student &# 39 ; s t test , 2 - tailed ). sera from mice immunized with irradiated p . yoelii sporozoites do not inhibit sporozoite invasion and development in this assay 33 . since cd8 + ctl against the pycsp have been shown to adoptively transfer protection 16 , and cd8 + t cells are required for the protection against p . yoelii induced by immunization with irradiated sporozoites 34 , recombinant p815 mastocytoma cells expressing pycsp 30 , or recombinant viruses expressing pycsp 22 , we wanted to determine if immunization with the pycsp plasmid , pdip / pycsp . 1 , induced ctl . the results indicate that immunization with pdip / pycsp . 1 induces mhc restricted , cd8 + t cell - dependent cytolytic activity ( fig5 ). furthermore , the cytolytic activity is significantly greater than that found after immunization with irradiated sporozoites ( fig6 a , b ). before achieving consistency of induction of antibodies by modifying injection techniques , we tested 2 immunized mice without antibodies to sporozoites , and 4 immunized mice that had antibodies to sporozoites for ctl . all 4 of the mice with antibodies and none of the mice without antibodies had demonstrable ctl ( data not shown ). this suggests that when this vaccine induces immune responses , it induces antibodies and ctl . the data shown in fig6 a & amp ; b were taken from a ctl assay performed 2 weeks after the last immunization as described for fig5 . significantly more cytolytic activity was demonstrated at all effector to target ratios against p815 cells pulsed with the pycsp ( 281 - 296 ) peptide using cells from mice immunized with pdip / pycsp . 1 . these effectors did not lyse targets pulsed with a peptide from the p . falciparum csp , pfcsp ( 368 - 390 ) that includes a ctl epitope and did not lyse p815 cells that had not been exposed to peptide ( fig6 a & amp ; 6b ). spleen cells from naive mice and mice immunized with the plasmid control , and stimulated in vitro with peptide pycsp ( 281 - 296 ), had no greater activity against p815 cells targets pulsed with pycsp ( 281 - 296 ) peptide than against targets pulsed with the pfcsp ( 368 - 390 ) control peptide or targets not exposed to peptide ( data not shown ). having established that immunization with pdip / pycsp . 1 induced biologically active antibodies and ctl against the pycsp , we asked if immunization with the vaccine would reduce liver stage infections . groups of mice were immunized with 3 doses of pdip / pycsp . 1 or control plasmid at 3 week intervals . two weeks after the third dose 3 immunized mice with highest ifat titers ( 20 , 480 ), and 3 controls were selected for iv challenge with 5 × 10 5 p . yoelii sporozoites . since the id 50 for p . yoelii sporozoites is often less than 2 sporozoites 30 , this is an enormous challenge representing greater than 10 5 id 50 s . forty - two hours after iv inoculation of 5 × 10 5 sporozoites into immune or control mice , mice were euthanized . livers were removed and used to prepare single cell suspensions of hepatocytes in medium . after cells were counted , 2 × 10 5 hepatocytes were distributed into 10 wells of a multi - well slide . slides were dried and frozen at - 70 ° c . until studied . to count the number of schizonts , slides were dried and incubated with nyls1 before incubating with fitc - labelled goat anti - mouse ig . the numbers of liver - stage schizonts in each well of coded slides were counted blindly by fluorescence microscopy . 70 wells containing a total of 1 . 4 × 10 6 hepatocytes were read per liver . there was an 85 . 6 ± 4 . 0 % reduction in the number of liver stage schizonts in the group that received pdip / pycsp . 1 as compared to the mice that received the plasmid without the pycsp insert ( 12 . 7 ± 3 . 5 schizonts / 1 . 4 × 10 6 hepatocytes vs 88 . 0 ± 17 . 8 schizonts / 1 . 4 × 10 6 hepatocytes , p = 0 . 002 , student &# 39 ; s t test , 2 - tailed ). having demonstrated that immunization with this vaccine significantly reduced the numbers of infected hepatocytes , we asked if it could protect against blood stage infection . the results of an initial experiment clearly demonstrate that it can ( table 2 ), and indicate that the protection is comparable to that induced by immunization with recombinant p815 cells expressing pycsp 30 . table 2______________________________________protection against sporozoite challenge . mice were immunized withpdip / pycsp . 1 at 0 and 8 weeks , and challenged 2 weeks later byiv injection of 100 p . yoelii sporozoites . plasmid controlsreceived the pbc12 / cmv / il - 2 plasmid without the pycsp insert , and naive controls were not immunized . protection was definedas absence of p . yoelii parasites on blood smears obtainedon days 4 , 7 , 8 , 9 , 11 and 14 after infection . mouse ifat titer at challenge protection______________________________________1a 20 , 480 yes2a 20 , 480 yes5a 10 , 240 noplasmid controls ( n = 3 ) & lt ; 10 nonaive controls ( n = 7 ) & lt ; 10 no______________________________________ pdip / pycsp . 1 is modified to remove the gene encoding pycsp and dna encoding influenza a nucleoprotein is inserted to form , pdip / infnp . 1 . after appropriate production and purification , mice are injected intramuscularly with 3 doses of 200 mcg of this plasmid dna at 4 week intervals . four weeks after the third dose , specimens from the mice are assessed for specific antibody and cellular immune responses against influenza a nucleoprotein , and the mice are challenged by intranasal administration of 10 25 tcid 50 ( mean tissue culture infectious dose ) of influenza a virus . after establishing the efficacy in mice , large numbers of humans are immunized in a double blind placebo controlled field trial . pdip / pycsp . 1 is modified to remove nucleotides 1383 to 2155 of the pycsp and the gene encoding influenza a nucleoprotein is inserted to form , pdip / cs - infnp . 1 . after appropriate production and purification , mice are injected intramuscularly with 3 doses of 200 mcg of this plasmid dna at 4 week intervals . four weeks after the third dose , specimens from the mice are assessed for specific antibody and cellular immune responses against influenza nucleoprotein , and the mice are challenged by intranasal administration of 10 25 tcid 50 ( mean tissue culture infectious dose ) of influenza a virus . after establishing the efficacy in mice , large numbers of humans are immunized in a double blind placebo controlled field trial . pdip / pycsp . 1 is modified to remove the pycsp gene and the gene encoding outer surface protein ( osp - a ) of borrelia burgdorferi ( a candidate lyme disease vaccine ) is inserted to form , pdip / bbospa . 1 . after appropriate production and purification , mice are injected intramuscularly with 3 doses of 200 mcg of this plasmid dna at 4 week intervals . four weeks after the third dose , specimens from the mice are assessed for specific antibody and cellular immune responses against recombinant purified osp - a . after establishing the safety and immunogenicity of this vaccine in mice , human volunteers chosen from a population of individuals at high risk of contracting lyme disease are immunized in a double blind placebo controlled study . pdip / pycsp . 1 is modified to remove the nucleotides 1383 to 2155 of the pycsp and the gene encoding the outer surface protein ( osp - a ) of borrelia burgdorferi ( a candidate lyme disease vaccine ) is inserted to form , pdip / cs - bbospa . 1 . after appropriate production and purification , mice are injected intramuscularly with 3 doses of 200 mcg of this plasmid dna at 4 week intervals . four weeks after the third dose , specimens from the mice are assessed for specific antibody and cellular immune responses against purified recombinant osp - a . after establishing the safety and immunogenicity of this vaccine in mice , human volunteers chosen from a population of individuals at high risk of contracting lyme disease are immunized in a double blind placebo controlled study . pdip / pycsp . 1 is modified to remove the pycsp gene and the gene encoding his - 62 ( a protective antigen ) from histoplasma capsulatum is inserted to form , pdip / his62 . 1 . after appropriate production and purification , mice are injected intramuscularly with 3 doses of 200 mcg of this plasmid dna at 4 week intervals . four weeks after the third dose , specimens from the mice are assessed for specific antibody and cellular immune responses against purified recombinant his - 62 . after establishing the safety and immunogenicity of this vaccine in mice , human volunteers chosen from a population of individuals at high risk of contracting histoplasmosis are immunized in a double blind placebo controlled study . pdip / pycsp . 1 is modified to remove nucleotides 1383 to 2155 of the pycsp and the gene encoding his - 62 ( a protective antigen ) from histoplasma capsulatum is inserted to form , pdip / cs - his62 . 1 . after appropriate production and purification , mice are injected intramuscularly with 3 doses of 200 mcg of this plasmid dna at 4 week intervals . four weeks after the third dose , specimens from the mice are assessed for specific antibody and cellular immune responses against his - 62 . after establishing the safety and immunogenicity of this vaccine in mice , human volunteers chosen from a population of individuals at high risk of contracting histoplasmosis are immunized are immunized in a double blind placebo controlled trial . pdip / pycsp . 1 is modified to remove the pycsp gene and dna encoding tetanus toxin is inserted to form , pdip / tet . 1 . after appropriate production and purification , mice are injected intramuscularly with 3 doses of 200 mcg of this plasmid dna at 4 week intervals . four weeks after the third dose , specimens from the mice are assessed for specific antibody and cellular immune responses against tetanus toxin . after establishing the safety and immunogenicity of this vaccine in mice , human volunteers are immunized in a double blind placebo controlled trial comparing pdip / tet . 1 to standard tetanus toxoid vaccine for their capacities to induce antibodies and t cell proliferative responses against tetanus toxin . pdip / pycsp . 1 is modified to remove nucleotides 1383 to 2155 of the pycsp and the gene encoding tetanus toxin is inserted to form , pdip / cs - tet . 1 . after appropriate production and purification , mice are injected intramuscularly with 3 doses of 200 mcg of this plasmid dna at 4 week intervals . four weeks after the third dose , specimens from the mice are assessed for specific antibody and cellular immune responses against tetanus toxin . after establishing the safety and immunogenicity of this vaccine in mice , human volunteers are immunized in a double blind placebo controlled trial comparing pdip / cs - tet . 1 to standard tetanus toxoid vaccine for their capacities to induce antibodies and t cell proliferative responses against tetanus toxin . pdip / pycsp . 1 is modified to remove the pycsp gene and the gene encoding the p . falciparum csp inserted to form , pdip / pfcsp . 1 . after appropriate production and purification , mice are injected intramuscularly with 3 doses of 200 mcg of this plasmid dna at 4 week intervals . four weeks after the third dose , specimens from the mice are assessed for antibodies to the pfcsp , t cell proliferative responses to pfcsp , and ctl against pfcsp . after establishing the safety and immunogenicity of this vaccine in mice , human volunteers are immunized with pdip / pfcsp . 1 in a double blind placebo controlled trial . pdip / pycsp . 1 is modified to remove nucleotides 1383 to 2155 of the pycsp and the gene encoding the p . falciparum csp inserted to form , pdip / cs - pfcsp . 1 . after appropriate production and purification , mice are injected intramuscularly with 3 doses of 200 mcg of this plasmid dna at 4 week intervals . four weeks after the third dose , specimens from the mice are assessed for antibodies to the pfcsp , t cell proliferative responses to pfcsp , and ctl against pfcsp . after establishing the safety and immunogenicity of this vaccine in mice , human volunteers are immunized with pdip / cs - pfcsp . 1 in a double blind placebo controlled trial . any polynucleotide delivery vector ( pdv ) designed for in vivo use ( injection , inhalation , topical application , ingestion ) is modified and the gene encoding the p . falciparum csp inserted to form , ppdv / pfcsp . 1 ( a double stranded dna plasmid in this example but not limited to plasmid vectors only ). after appropriate production and purification , mice are injected intramuscularly with 3 doses of 200 mcg of this plasmid dna at 4 week intervals . four weeks after the third dose , specimens from the mice are assessed for antibodies to the pfcsp , t cell proliferative responses to pfcsp , and ctl against pfcsp . after establishing the safety and immunogenicity of this vaccine in mice , human volunteers are immunized with ppdv / pfcsp . 1 in a double blind placebo controlled trial . polynucleotide vaccines provide an entirely new approach to developing multi - component vaccines against the microorganisms that cause malaria without the often difficult , time - consuming , and expensive requirement for production , purification , and mixing of synthetic peptides , purified recombinant proteins , recombinant live vectors , and adjuvants ; processes that have greatly impeded the development of truly effective malaria vaccines as described in the institute of medicine report ( page 3 , line 10 to page 8 , line 20 , quoted above ). obviously , many modifications and variations of the present invention are possible in light of the above teachings . it is therefore to be understood that , within the scope of the appended claims , the invention may be practiced otherwise than as specifically described . 1 . institute of medicine . malaria : obstacles and opportunities . s . c . oaks , v . s . mitchell , g . w . pearson and c . carpenter , eds . national academy press , washington d . c . 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( 1992 ) ( in press ). 11 . r . edelman et al .. j . lnfect . dis . 168 : 1066 ( 1993 ). 12 . n . h . fairley . trans . roy . soc . trop . med . hyg 40 : 621 ( 1947 ). 13 . s . l . hoffman , v . nussenzweig , j . c . sadoff and r . nussenzweig . science 252 : 520 ( 1991 ). 16 . w . r . weiss et al ., j . immunol . 149 : 2103 ( 1992 ). 18 . a . a . lal et al ., proc . natl . acad . sci . u . s . a . 84 : 8647 ( 1987 ). 19 . m . sedegah et al . in technological advances in vaccine development , l . lasky , ed . ( alan r . liss inc ., new york , 1988 ), pp . 295 - 309 . 20 . m . sedegah et al ., bull . world health organ . 68 ( suppl .) 109 ( 1990 ). 22 . s . li et al ., proc . natl . acad . sci . u . s . a . 90 : 5214 ( 1993 ). 23 . d . tang , m . devit and s . a . johnston . nature 356 : 152 ( 1992 ). 24 . b . wang et al ., proc . natl . acad . sci . u . s . a . 90 : 4156 ( 1993 ). 25 . g . j . m . cox et al ., j . of virology 67 : 5664 ( 1993 ). 28 . r . c . hedstrom et al ., bull . world health organ . 68 ( suppl .) 152 ( 1990 ). 29 . w . o . rogers , m . d . rogers , r . c . hedstrom and s . l . hoffman . mol . biochem . parasitol . 53 : 45 ( 1992 ). 31 . a . a . lal , v . f . de la cruz and j . a . welsh . j . biol . chem . 262 : 2937 ( 1987 ). 33 . s . mellouk et al ., bull . world health organ . 68 ( suppl .) 52 ( 1990 ). 34 . w . r . weiss et al ., proc . natl . acad . sci u . s . a . 85 : 573 ( 1988 ). __________________________________________________________________________ # sequence listing - ( 1 ) general information :- ( iii ) number of sequences : 2 - ( 2 ) information for seq id no : 1 :- ( i ) sequence characteristics :# pairs ( a ) length : 5552 base ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : circular - ( iii ) hypothetical : no - ( iv ) anti - sense : no - ( ix ) feature : ( a ) name / key : promoter ( b ) location : 1 .. 755 ( c ) identification method : - # experimental #/ function = &# 34 ; promoter &# 34 ; formation : / evidence =- # experimental # cmv - ie / label =#&# 34 ; this feature acts as a promoter for any # dna sequence .&# 34 ; downstream / citation =- # ([ 2 ])- ( ix ) feature : ( a ) name / key : cds ( b ) location : 933 .. 2367 ( c ) identification method : - # experimental #/ codon . sub .-- start = 933mation : / function =- # &# 34 ; protein protective against malaria &# 34 ; / product =- # &# 34 ; protein &# 34 ; / evidence =- # experimental # 1 / number =# il2 - csp / label = / citation =- # ([ 1 ])- ( x ) publication information : ( a ) authors : sedegah , m - # artha # richard c . hedstrom ,# stephen l . hoffman ,# with plasmodium yoelii cs protein plasmid d - # na protects against malaria ( c ) journal : science - ( x ) publication information : ( a ) authors : cullen , br - # yan r . ( b ) title : trans - activatio - # n of human immunodeficiency virus occ - # urs via a bimodal mechanism ( c ) journal : cell ( d ) volume : 46 ( f ) pages : 973 - 982 ( g ) date : 26 sep - 1986 ( k ) relevant residues i - # n seq id no : 1 : from 1 to 4732 - ( xi ) sequence description : seq id no : 1 :- agcttcccat tgcatacgtt gtatccatat cataatatgt acatttatat tg - # gctcatgt 60 - ccaacattac cgccatgttg acatggatta ttgactagtt attaatagta at - # caattacg 120 - gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac gg - # taaatggc 180 - ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gt - # atgttccc 240 - atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt ac - # ggtaaact 300 - gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tg - # acgtcaat 360 - gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ct - # ttcctact 420 - tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt tt - # ggcagtac 480 - atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca cc - # ccattgac 540 - gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tc - # gtaacaac 600 - tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta ta - # taagcaga 660 - gctcgtttag tgaaccgcca gatcgcctgg agacgccatc cacgctgttt tg - # acctccat 720 - agaagacacc gggaccgatc cagcctcccc tcgaagcttg gtaagtgacc ag - # ctacagtc 780 - ggaaaccatc agcaagcagg tatgtactct ccagggtggg cctggcttcc cc - # agtcaaga 840 - ctccagggat ttgagggacg ctgtgggctc ttctcttaca tgtacctttt gc - # tagcctca 900 - accctgacta tcttccaggt cattgttcca ac atg gcc ctg tgg - # atc gac agg 953 # met ala leu trp ile asp arg # 1 5 - atg caa ctc ctg tct tgc att gca cta agt ct - # t gca ctt gtc aca aac1001met gln leu leu ser cys ile ala leu ser le - # u ala leu val thr asn # 20 - agt gca cct act tca agt tct aca aag aaa ac - # a cag cta caa ctg gag1049ser ala pro thr ser ser ser thr lys lys th - # r gln leu gln leu glu # 35 - cat tta ctg ctg gat tta cag atg att ttg aa - # t gga att aat aat tac1097his leu leu leu asp leu gln met ile leu as - # n gly ile asn asn tyr # 55 - aag aat ccc aaa ctc acc agg atg ctc aca tt - # t aag ttt tac atg ccc1145lys asn pro lys leu thr arg met leu thr ph - # e lys phe tyr met pro # 70 - aag aag gcc aca gaa ctg aaa cat ctt cag tg - # t cta gag gat ccc aaa1193lys lys ala thr glu leu lys his leu gln cy - # s leu glu asp pro lys # 85 - atg aag aag tgt acc att tta gtt gta gcg tc - # a ctt tta tta gtt gat1241met lys lys cys thr ile leu val val ala se - # r leu leu leu val asp # 100 - tct cta ctt cca gga tat gga caa aat aaa ag - # t gtc caa gcc caa aga1289ser leu leu pro gly tyr gly gln asn lys se - # r val gln ala gln arg # 115 - aac tta aac gag cta tgt tac aat gaa gaa aa - # t gat aat aaa ttg tat1337asn leu asn glu leu cys tyr asn glu glu as - # n asp asn lys leu tyr120 1 - # 25 1 - # 30 1 -# 35 - cac gtc ctt aac tcg aag aat gga aaa ata ta - # c aat cga aat ata gtc1385his val leu asn ser lys asn gly lys ile ty - # r asn arg asn ile val # 150 - aac aga tta ctt ggc gat gct ctc aac gga aa - # a cca gaa gaa aaa aaa1433asn arg leu leu gly asp ala leu asn gly ly - # s pro glu glu lys lys # 165 - gat gat ccc cca aaa gat ggc aac aaa gat ga - # t ctt cca aaa gaa gaa1481asp asp pro pro lys asp gly asn lys asp as - # p leu pro lys glu glu # 180 - aaa aaa gat gat ctt cca aaa gaa gaa aaa aa - # a gat gat ccc cca aaa1529lys lys asp asp leu pro lys glu glu lys ly - # s asp asp pro pro lys # 195 - gat cct aaa aaa gat gat cca cca aaa gag gc - # t caa aat aaa ttg aat1577asp pro lys lys asp asp pro pro lys glu al - # a gln asn lys leu asn200 2 - # 05 2 - # 10 2 -# 15 - caa cca gta gtg gca gat gaa aat gta gat ca - # a ggg cca gga gca cca1625gln pro val val ala asp glu asn val asp gl - # n gly pro gly ala pro # 230 - caa ggg cca gga gca cca caa ggg cca gga gc - # a cca cag ggg cca gga1673gln gly pro gly ala pro gln gly pro gly al - # a pro gln gly pro gly # 245 - gca cca cag ggg cca gga gca cca caa ggg cc - # a gga gca cca caa gga1721ala pro gln gly pro gly ala pro gln gly pr - # o gly ala pro gln gly # 260 - cca gga gca cca caa ggg cca gga gca cca ca - # a ggg cca gga gca cca1769pro gly ala pro gln gly pro gly ala pro gl - # n gly pro gly ala pro # 275 - caa ggg cca gga gca cca cag ggg cca gga gc - # a cca caa ggg cca gga1817gln gly pro gly ala pro gln gly pro gly al - # a pro gln gly pro gly280 2 - # 85 2 - # 90 2 -# 95 - gca cca caa gga cca gga gca cca cag ggt cc - # a gga gca cca caa gga1865ala pro gln gly pro gly ala pro gln gly pr - # o gly ala pro gln gly # 310 - cca gga gca cca caa gga cca gga gca cca ca - # a ggt cca gga gca cca1913pro gly ala pro gln gly pro gly ala pro gl - # n gly pro gly ala pro # 325 - cag ggg cca gga gca cca caa ggg cca gga gc - # a cca caa gaa cca ccc1961gln gly pro gly ala pro gln gly pro gly al - # a pro gln glu pro pro # 340 - caa caa cca ccc caa caa cca cca caa cag cc - # a cca caa cag cca cca2009gln gln pro pro gln gln pro pro gln gln pr - # o pro gln gln pro pro # 355 - caa cag cca cca caa cag cca cca caa caa cc - # a cgc cca cag cca gat2057gln gln pro pro gln gln pro pro gln gln pr - # o arg pro gln pro asp360 3 - # 65 3 - # 70 3 -# 75 - ggt aat aac aac aat aac aat aat aat ggt aa - # t aat aat gaa gat tct2105gly asn asn asn asn asn asn asn asn gly as - # n asn asn glu asp ser # 390 - tat gtc cca agc gcg gaa caa ata cta gaa tt - # t gtt aaa cag ata agt2153tyr val pro ser ala glu gln ile leu glu ph - # e val lys gln ile ser # 405 - agt caa ctc aca gag gaa tgg tct caa tgt ag - # t gta acc tgt ggt tct2201ser gln leu thr glu glu trp ser gln cys se - # r val thr cys gly ser # 420 - ggt gta aga gtt aga aaa cga aaa aat gta aa - # c aag caa cca gaa aat2249gly val arg val arg lys arg lys asn val as - # n lys gln pro glu asn # 435 - ttg acc tta gag gat att gat act gaa att tg - # t aaa atg gat aaa tgt2297leu thr leu glu asp ile asp thr glu ile cy - # s lys met asp lys cys440 4 - # 45 4 - # 50 4 -# 55 - tca agt ata ttt aat att gta agc aat tca tt - # a gga ttt gta ata tta2345ser ser ile phe asn ile val ser asn ser le - # u gly phe val ile leu # 470 - tta gta tta gta ttc ttt aat t aaataaacat taca - # cattat tataaatatt2397leu val leu val phe phe asn 475 - tatatattat ataaatattt tatatacata taatgtgtgt agactttatt tt - # ttgtattg2457 - tgaactttcc tcatttatta cgattatttt tatatatata catatttaat at - # gtaaatta2517 - aaagaaaaaa gaaataatag aaatcttatt atatttatga tataaattaa aa - # aaataaaa2577 - tatatataca ttacaaaatt tacttttttt agtttatttt tttcgtgttt at - # tatatatg2637 - taattaactt gttatgacga tgggtaccca tttggggacc ccatagagca cc - # gcaccgac2697 - cgagggatgg taacaggatg tgtaggtttt ggaggcccat atgtccattc at - # gaccagtg2757 - acttgtctca cagccatgca acccttgcct cctgtgctga cttagcaggg ga - # taaagtga2817 - gagaaagcct gggctaatcg gggggtcgct cggctcctcc taactggatt gt - # cctatgtg2877 - tctttgcttc tgtgctgctg atgctctgcc ctgtgctgac atgacctccc tg - # gcagtggc2937 - acaactggag ctgggtggag gcccgggggc cggtgacctt cagaccttgg ca - # ctggaggt2997 - ggcccggcag aagcgcggca tcgtggatca gtgctgcacc agcatctgct ct - # ctctacca3057 - actggagaac tactgcaact aggcccacca ctaccctgtc cacccctctg ca - # atgaataa3117 - aacctttgaa agagcactac aagttgtgtg tacatgcgtg cgtgtgcata tg - # tggtgcgg3177 - ggggaacatg agtggggtcg gctggagtgg tcgcggctta atctatctgg cg - # atgataag3237 - ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt gatacgccta tt - # tttatagg3297 - ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg gg - # aaatgtgc3357 - gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ct - # catgagac3417 - aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt at - # tcaacatt3477 - tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gc - # tcacccag3537 - aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg gg - # ttacatcg3597 - aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cg - # ttttccaa3657 - tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt ga - # cgccgggc3717 - aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag ta - # ctcaccag3777 - tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gc - # tgccataa3837 - ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga cc - # gaaggagc3897 - taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tg - # ggaaccgg3957 - agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gc - # aatggcaa4017 - caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg ca - # acaattaa4077 - tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc ct - # tccggctg4137 - gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt at - # cattgcag4197 - cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gg - # gagtcagg4257 - caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg at - # taagcatt4317 - ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa ct - # tcattttt4377 - aatttaaaag gatctaggtg aagatccgta atctgctgct tgcaaacaaa aa - # aaccaccg4437 - ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc ga - # aggtaact4497 - ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gt - # taggccac4557 - cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gt - # taccagtg4617 - gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg at - # agttaccg4677 - gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag ct - # tggagcga4737 - acgacctaca ccgaactgag atacctacag cgtgagcatt gagaaagcgc ca - # cgcttccc4797 - gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg ag - # agcgcacg4857 - agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tc - # gccacctc4917 - tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg ga - # aaaacgcc4977 - agcaacgtcg ggatgcgccg cgtgcggctg ctggagatgg cggacgcgat gg - # atatgttc5037 - tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc tc - # caattctt5097 - ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag gt - # ggcccggc5157 - tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg cc - # tacaatcc5217 - atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg at - # cagcggtc5277 - cagtgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt cc - # ctgatggt5337 - cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg cc - # gccggaag5397 - cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgagcttttt gc - # aaaagcct5457 - aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga gg - # cggcctcg5517 # 5552 aaaa aattagtcag ccatg - ( 2 ) information for seq id no : 2 :- ( i ) sequence characteristics :# acids ( a ) length : 478 amino ( b ) type : amino acid ( d ) topology : linear - ( ii ) molecule type : protein - ( xi ) sequence description : seq id no : 2 :- met ala leu trp ile asp arg met gln leu le - # u ser cys ile ala leu # 15 - ser leu ala leu val thr asn ser ala pro th - # r ser ser ser thr lys # 30 - lys thr gln leu gln leu glu his leu leu le - # u asp leu gln met ile # 45 - leu asn gly ile asn asn tyr lys asn pro ly - # s leu thr arg met leu # 60 - thr phe lys phe tyr met pro lys lys ala th - # r glu leu lys his leu # 80 - gln cys leu glu asp pro lys met lys lys cy - # s thr ile leu val val # 95 - ala ser leu leu leu val asp ser leu leu pr - # o gly tyr gly gln asn # 110 - lys ser val gln ala gln arg asn leu asn gl - # u leu cys tyr asn glu # 125 - glu asn asp asn lys leu tyr his val leu as - # n ser lys asn gly lys # 140 - ile tyr asn arg asn ile val asn arg leu le - # u gly asp ala leu asn145 1 - # 50 1 - # 55 1 -# 60 - gly lys pro glu glu lys lys asp asp pro pr - # o lys asp gly asn lys # 175 - asp asp leu pro lys glu glu lys lys asp as - # p leu pro lys glu glu # 190 - lys lys asp asp pro pro lys asp pro lys ly - # s asp asp pro pro lys # 205 - glu ala gln asn lys leu asn gln pro val va - # l ala asp glu asn val # 220 - asp gln gly pro gly ala pro gln gly pro gl - # y ala pro gln gly pro225 2 - # 30 2 - # 35 2 -# 40 - gly ala pro gln gly pro gly ala pro gln gl - # y pro gly ala pro gln # 255 - gly pro gly ala pro gln gly pro gly ala pr - # o gln gly pro gly ala # 270 - pro gln gly pro gly ala pro gln gly pro gl - # y ala pro gln gly pro # 285 - gly ala pro gln gly pro gly ala pro gln gl - # y pro gly ala pro gln # 300 - gly pro gly ala pro gln gly pro gly ala pr - # o gln gly pro gly ala305 3 - # 10 3 - # 15 3 -# 20 - pro gln gly pro gly ala pro gln gly pro gl - # y ala pro gln gly pro # 335 - gly ala pro gln glu pro pro gln gln pro pr - # o gln gln pro pro gln # 350 - gln pro pro gln gln pro pro gln gln pro pr - # o gln gln pro pro gln # 365 - gln pro arg pro gln pro asp gly asn asn as - # n asn asn asn asn asn # 380 - gly asn asn asn glu asp ser tyr val pro se - # r ala glu gln ile leu385 3 - # 90 3 - # 95 4 -# 00 - glu phe val lys gln ile ser ser gln leu th - # r glu glu trp ser gln # 415 - cys ser val thr cys gly ser gly val arg va - # l arg lys arg lys asn # 430 - val asn lys gln pro glu asn leu thr leu gl - # u asp ile asp thr glu # 445 - ile cys lys met asp lys cys ser ser ile ph - # e asn ile val ser asn # 460 - ser leu gly phe val ile leu leu val leu va - # l phe phe asn465 4 - # 70 4 - # 75__________________________________________________________________________