Patent Abstract:
the present invention relates to combined vaccines against measles and human papilloma virus . in particular , the invention relates to recombinant measles virus vectors containing heterologous nucleic acid encoding single or several antigens derived from hpv , preferably , the major capside antigen l1 , the minor capside antigen l2 , the early gene e6 and the early gene e7 oncoproteins of hpv type 16 , and optionally of types 18 , 6 and 11 . in a first embodiment , prophylactic vaccines are generated expressing hpv antigens , preferably l1 and / or l2 such that they induce a potent long - lasting immune response in mammals , preferably humans , to protect against hpv and mv infection . in another embodiment , therapeutic vaccines are generated expressing e6 and e7 proteins , and optionally l1 and l2 , such that they induced strong immune responses will resolve persistent hpv infections at early or late stages , including hpv - induced cervical carcinoma . in a preferred embodiment , the combined vaccines are easy to produce on a large scale and can be distributed at low cost .

Detailed Description:
the object of the invention is the production of combined measles - hpv vaccines from a recombinant measles vector capable of containing stably integrated nucleotide sequences which code for l1 , l2 , e6 and / or e7 protein from different hpv types . the invention includes the rescue of recombinant mv - hpv viruses which are capable of infection , replication and expression of l1 , l2 , e6 or e7 protein in susceptible transgenic mice , monkeys and human host . furthermore , the invention includes the construction of multivalent recombinant measles - hpv vectors , in which two different antigens are simultaneously cloned and expressed in the same vector , conferring immunity against both of them . moreover , the invention relates to the combination of different recombinant measles - hpv viruses , each carrying and expressing a gene from a different hpv type , in order to elicit immune response in the host , directed against the different hpv types . furthermore , the invention comprises a method to produce a vaccine containing such recombinant viruses . moreover the invention also relates to the use of interleukin or interleukin - 2 as adjuvent in order to increase the response of combined vaccine . the invention finally relates to a vaccine capable to induce a potent and lifelong immune response against hpv and measles virus in human and to prevent from infection and / or treat diseases associated with infection . fig1 . schematic representation of the antigenomic p (+) mv of measles virus . p (+) mv - ez is a plasmid derived from pbluescript containing the complete sequence of the measles virus ( edmoston zagreb ), under the control of the t7 rna polymerase promoter ( t7 ), containing three atu respectively in position 1 ( before the n gene of the measles virus ), 2 ( between the p and the m genes of the measles virus ) and 3 ( between the h and the l genes of the measles virus ), and exactly terminated by the hepatitis delta ribozyme and t7 rna polymerase terminator (□ t7t ). the size of the plasmid is 18941 bp . fig2 . schematic representation of the recombinant measles - papilloma plasmid , p (+) mv 2 - ez - l1 . it is a plasmid derived from p (+) mv - ez containing hpv - l1 gene ( type 16 ), 1518 bp , cloned in position two of the measles genome by bsshii - aatii digestion . the size of the recombinant plasmid is 20561 bp . fig3 . schematic representation of the recombinant measles - papilloma plasmid , p (+) mv 3 - ez - hpv - l1 it is a plasmid derived from p (+) mv - ez containing the hpv - l1 gene ( type 16 ), 1518 bp , cloned in position three of the measles genome by bsshii - aatii digestion . the recombinant plasmid p (+) mv 3 - ez - hpv - l1 is 20561 bp . fig4 a . complete nucleotide sequence of p (+) mv 2 ez - gfp ( 19774 bp ). the sequence can be described as follows with reference to the position of the nucleotides : fig4 b . complete nucleotide sequence of p (+) mv 3 ez - gfp ( 19774 bp ). the sequence can be described as follows with reference to the position of the nucleotides : fig5 a . anl1te : this is the hpv16 - l1 sequence orf ( 1518 bp ) cloned by the inventors . the sequence can be described as follows with reference to the position of the nucleotides : fig5 b . hpv16 - l2 sequence orf . the sequence can be described as follows with reference to the position of the nucleotides : fig5 c . hpv 16 - e6 sequence orf . the sequence can be described as follows with reference to the position of the nucleotides : fig5 d . hpv 16 - e7 sequence orf . the sequence can be described as follows with reference to the position of the nucleotides : fig5 e . hpv18 - l1 sequence orf . the sequence can be described as follows with reference to the position of the nucleotides : fig5 f . hpv 18 - l2 sequence orf . the sequence can be described as follows with reference to the position of the nucleotides : fig5 g . hpv18 - e6 sequence orf . the sequence can be described as follows with reference to the position of the nucleotides : fig5 h . hpv18 - e7 sequence orf . the sequence can be described as follows with reference to the position of the nucleotides : fig5 i . hpv6 - l1 sequence orf . the sequence can be described as follows with reference to the position of the nucleotides : fig5 j . hpv6 - l2 sequence orf . the sequence can be described as follows with reference to the position of the nucleotides : fig5 k . hpv6 - e6 sequence orf . the sequence can be described as follows with reference to the position of the nucleotides : fig5 l . hpv6 - e7 sequence orf . the sequence can be described as follows with reference to the position of the nucleotides : fig6 . rescue of recombinant virus mv2ez - l1 on 293 - 3 - 46 helper cells . cells were co - transfected with 25 ng pemcla and 5 □ g of the recombinant p (+) mv2ez - l1 plasmid . fig7 . syncytium formation in vero cells transfected with mv2ez - l1 . at 24 h p . i ., cells were fixed with ethanol and stained with crystal violet . fig8 . growth curves of cell - associated ( ca ) and cell - free ( cf ) virus in mrc5cells . monolayers were infected at an moi of 0 . 05 with mv2ez - l1 ed mvez virus . fig9 . expression of hpv - l1 from recombinant mvs by immunofluorescence analysis . vero cells were infected with either mv2ez - l1 ( a ) or mvez ( b ) at moi 0 . 05 for 48 h and processed for indirect immunofluorescence . in upper panels specific hpv - l1 signal were detected with mouse monoclonal anti - hpv - l1 antibody , followed by goat anti - mouse antibody — fitc conjugate , in lower panels the same slides were stained with dapi ( 4 ′, 6 - diamidine - 2 - phenilindol chloridrate ) and observed respectively with fluorescent or natural light . fig1 . expression of hpv - l1 from recombinant mvs by western blot analysis . vero cells were infected with either mvez ( 1 ) or mv2ez - l1 ( 2 ) at moi 0 . 05 for 48 h . expression of hpv - l1 in supernatant ( a ) and cell lysate ( b ) after infection with mvez ( line 1 ) or mv2ez - l1 ( line 2 ). fig1 . western blot analysis of hpv - l1 in the different cscl gradient fractions . all cloning procedures were basically as described in sambrook et al . ( 1989 ). all the restriction enzymes were from new england biolabs ; the oligonucleotides pcr primers were from invitrogen . the l1 sequence has been amplified by pcr , and direct cloned into the definitive mv vectors , obtaining two recombinant mv - hpv16 - l1 plasmids : p (+) mv2ez - hpv - l1 and p (+) mv3ez - hpv - l1 . pcr amplification was carried out using the proof - reading pfu dna polymerase ( stratagene ). dna sequences of the synthetic oligonucleotides primers are given in upper case for the mv nucleotides and in lower case for non - mv nucleotides ; sequences of relevant restriction endonucleases recognition sites are underlined . the following primers have been used : for - l1 5 ′- ttg gcgcgc catgagcctgtggctgccc - 3 ′; rev - l1 5 ′- at gacgtc tcacagcttcctcttcttcctc - 3 ′. for - l1 contains an overhang ( in lower case ) with bsshii restriction site ( gcgcgc ), after 3 - bp long - protection site ( ttg ). rev - l1 contains an overhang ( in lower case ) with aatii restriction site ( gacgtc ). the obtained pcr - hpv16 - l1 ( 1536 bp ) has been cloned in the p (+) mvez vector ( fig1 ) between genes for p and m protein ( position 2 , fig2 ) or between h and l ( position 3 , fig3 ), after digestion with bsshii + aatii . ligations were performed overnight at 16 ° c . in an equimolar ratio respect to the pre - digested mev 2 ez and mev 3 ez vectors bsshii + aatii ( 19 kb in length ), using one unit of t4 dna ligase , obtaining , respectively , p (+) mv 2 ez - l1 ( 20561 bp ) and p (+) mv 3 ez - l1 ( 20561 bp ). in fig4 and 5 are listed , respectively , the sequences of the vectors p (+) mv 2 ez - gfp and p (+) mv 3 ez - gfp . xl10 gold chemical competent cell were then transformed with all ligation volume , following a standard transformation protocol ( sambrook et al . 1989 ), plated and selected on lb - agar plates for ampicillin resistance . colonies were screened by dna plasmid preparation ( qiagen , mini - midi and maxi kit ) and restriction enzymes digestion . the right clones were sent to mwg for sequencing : the sequences , aligned with the assumed ones using a dna strider software , showed 100 % identity . nucleotide sequence encoding for hpv16 - l1 is presented in fig5 a . all the other antigens l2 , e6 , and e7 from hpv16 , and l1 , l2 , e6 , and e7 from hpv 18 and 6 types , have been cloned as above detailed . the orfs sequences are listed in fig5 ( 5 b to 5 l ). list of the recombinant mv - hpv plasmids from hpv 16 , 18 , and 6 types : p (+) mv2ez - hpv16 - l1 plasmid p (+) mv3ez - hpv16 - l1 plasmid p (+) mv2ez - hpv16 - l2 plasmid p (+) mv3ez - hpv16 - l2 plasmid p (+) mv2ez - hpv16 - e6 plasmid p (+) mv3ez - hpv16 - e6 plasmid p (+) mv2ez - hpv16 - e7 plasmid p (+) mv3ez - hpv16 - e7 plasmid p (+) mv2ez - hpv18 - l1 plasmid p (+) mv3ez - hpv18 - l1 plasmid p (+) mv2ez - hpv18 - l2 plasmid p (+) mv3ez - hpv18 - l2 plasmid p (+) mv2ez - hpv18 - e6 plasmid p (+) mv3ez - hpv18 - e6 plasmid p (+) mv2ez - hpv18 - e7 plasmid p (+) mv3ez - hpv6 - e7 plasmid p (+) mv2ez - hpv6 - l1 plasmid p (+) mv3ez - hpv6 - l1 plasmid p (+) mv2ez - hpv6 - l2 plasmid p (+) mv3ez - hpv6 - l2 plasmid p (+) mv2ez - hpv6 - e6 plasmid p (+) mv3ez - hpv6 - e6 plasmid p (+) mv2ez - hpv6 - e7 plasmid p (+) mv3ez - hpv6 - e7 plasmid cells were maintained as monolayers in dulbecco &# 39 ; s modified eagles medium ( dmem ), supplemented with 5 % foetal calf serum ( fcs ) for vero cells ( african green monkey kidney ) and with 10 % fcs and 1 % penicillin / streptomycin for 293t cells ( human embryonic kidney ); dmem supplemented with glutamax ( f12 ) and 10 % fcs for mrc - 5 ( human foetal fibroblast ); dmem supplemented with 10 % fcs and 1 . 2 mg / ml of g 418 for 293 - 3 - 46 . to grow mv virus stocks reaching titers of about 10 7 pfu / ml , recombinant viruses and the vaccine strain edmoston zagreb were propagated in mrc - 5 cells : plaque purification was carried out by transferring a syncythium to 35 mm mrc - 5 cell culture which was expanded first to a 10 cm dish , and afterwards to a 175 cm flask . virus stocks were made from 175 cm 2 cultures when syncythia formation was about 90 % pronounced . medium corresponding to the so - called “ free - cell virus fraction ” was collected , freeze and thaw three times and spin down to avoid cell debris . the medium was then stored at − 80 ° c . cells , which correspond to the so - called “ cell - associated virus fraction ”, were scraped into 3 ml of optimem ( gibco brl ) followed by three rounds freezing and thawing , spin down and the cleared surnatant stored at − 80 ° c . recombinant measles - hpv vaccine viruses have been obtained using the 293 - 3 - 46 helper cell ( human embryonic kidney cells ), stably expressing the measles n and p proteins as well as the t7 rna polymerase . the viral rna polymerase ( large protein , l ) was expressed by co - transfecting the cells with 15 ng of the plasmid pemcla . calcium - phosphate method was used for transfection . 293t - 3 - 46 cells were seeded into a 35 mm well to reach ˜ 50 - 70 % confluence when being transfected . 4 h before transfection , the medium was replaced with 3 ml dmem containing 10 % fcs . all recombinant plasmids were prepared according to the qiagen plasmid preparation kit . the kit for the ca2 + phosphate coprecipitation of dna was from invitrogen . cells were co - transfected with the plasmids in the follows final concentration : pemcla 25 ng and the recombinant p (+) mv2ez - l1 plasmid 5 μg . all plasmids , diluted in h2o , were added in a eppendorf tube containing 2m cacl2 , the mix was added to another eppendorf tube containing hepes buffer under shaking conditions , and was incubated 30 min at room temperature ( rt ). thus , the co - precipitates were added dropwise to the culture and the transfection was carried out at 37 ° c . and 5 % co2 for about 18 h . then , the transfection medium was replaced with 3 ml of dmem containing 10 % fcs . first syncytia appeared 3 - 4 days after transfection when the cells were still subconfluent as shown in fig6 . to allow syncytia formation to progress more easily , almost confluent cell monolayers of each 35 mm well were then transferred to a 10 cm dish . each syncytium was taken up in 300 μl of transfection medium and put in a sterile eppendorf tube containing 700 μl of optimem , freeze and thaw for three rounds , and stored at − 80 ° c . serial 10 - times dilutions of virus preparations were carried out using optimem to a final volume of 0 . 5 ml . each dilution was added on 35 mm vero cell cultures . after 1 h of virus adsorption , the inoculum was removed and the infected cells were overlaid with 2 ml of dmem containing 5 % fcs and 1 % low melting point agarose ( lmp agarose ). after 5 days of incubation at 37 ° c . and 5 % co2 , cultures were fixed with 1 ml of 10 % tca for 1 h , then uv cross - linked for 30 min . after removal of the agarose overlay , cell monolayers were stained with crystal violet dissolved in 4 % ethanol , washed with water and the plaques were counted under the inverted microscope ( fig7 ). rescued viruses were serially passaged 10 - times on mrc5 cells , seeded into 10 cm diameter plates , that were infected with the standard and the recombinant mv viruses at moi of 0 . 01 pfu / cells . after monolayer was full infected , 1 % surnatant of each culture was used to infect the subsequent mrcs cells monolayer . to test transgene expression and stability , viruses from passage 1 , 5 , and 10 were used for further characterisation of expression by western blot and immunofluorescence . mrc5 cells seeded on 35 mm dish ( 1 - 5 × 105 ) were monitored for 90 % confluence and infected with cleared virus suspension from cell - associated virus fraction at 0 . 05 moi , including mvez as control . samples , corresponding to the so - called “ free - cell virus fraction ” and to the so - called “ cell - associated virus fraction ”, were collected daily for one week and titrated . from growth curve comparison of it is interesting that the replication of mv2ez - l1 was only slightly impaired : the recombinant virus reached peak titers of 6 . 12 × 106 tcid50s / ml 48 hpi , whereas mvez gave final titers of 6 . 8 × 106 tcid50s / ml 36 hpi ( fig8 ). the slightly slower progression of replication was also reflected in somewhat reduced plaque sizes . mv2ez - l1 produced plaques with an average diameter of 0 . 83 mm , while mvez produced plaques with an average diameter of 0 . 91 mm . to analyse the expression either mv and hpv antigen , immunofluorescence , western blot and isolation of vlp were carried out . vero cells were seeded on 24 mm × 24 mm glass cover slips in 35 mm wells , cultured overnight and infected with rescued recombinant virus mv2ez - l1 or with negative control virus mvez at 0 . 05b m . o . i . 48 hours after infection cells on coverslips were fixed with 4 % paraformaldehyde in pbs , and permeabilized with 0 . 1 % tx - 100 , washed with blocking solution ( pbs containing 1 % bsa ) for 1 h , and stained with the specific hpv - l1 mouse monoclonal antibody ( biogenesis ) and by fitch conjugated goat anti - mouse secondary antibody . all syncytia of rescued mv2ez - l1 showed positive signals ( fig9 upper panel , left images ), whereas the syncytia of rescued mvez ( fig9 , upper panel right images ) showed no fluorescence , that indicates that all syncytia induced by mv2ez - l1 expressed l1 antigen . for western blot , vero cells seeded on 35 mm dish ( 1 - 5 × 105 ) were monitored the next day for 90 % confluence and infected with cleared virus suspension from cell - associated virus fraction , using 0 . 05 moi ( multiplicity of infection ), including mvez as control . when about 80 % syncythia formation was observed , proteins from medium and from cells were analysed . cells were first washed with pbs and then scraped in 1 ml pbs and collected in an eppendorf tube , and centrifuge at 2000 rpm / 4 min . cells were then lysed 5 min / rt with 70 μl of lysis buffer ( 1 % np - 40 , 50 mm tris ph 8 , 150 mm nacl ) supplemented with protease inhibitor cocktail ( complete mini , roche , 1 836 153 ). surnatants were cleared by centrifuge at 13000 rpm / 5 min , and transferred into a new tube : 30 □ l of 4 × loading buffer ( invitrogen ) were added ; samples were mixed and boiled at 95 ° c ./ 2 min , spun down and stored at − 20 ° c . an sds - page migration was performed , running a nupage 12 % bis - acrylamide gel in reducing conditions , using 1 × running buffer , for 50 min at 200v ( start 100 - 125 ma , end 60 - 80 ma ). then , semi - dry method was used to transfer separated cell - proteins to nitrocellulose membrane , at 14v / 1h30 . mouse monoclonal antibody against hpv - l1 ( biogenesis ) was used as first antibody . the second antibody was a goat anti - mouse antibody coupled to horse - radish peroxidase allowing the visualization of the bands by the enhanced chemiluminescence kit ( ecltm , amersham lifescience ). the anti - hpvl1 antibody reacted with a protein of approximately 55 kda released in the culture medium of mv2ez - l1 - infected cells , whereas no such protein was detected in culture medium of mvez - infected cells ( fig1 , panel a ) as well as with a protein of approximately 55 kda synthesized in the mv2ez - l1 - infected cells , whereas no such protein was detected in mvez - infected cell lysates ( fig1 , panel b ). this result indicates that mv2ez - l1 - infected cells express and secrete the l1 protein that is similar in size to the authentic l1 protein from hpv . monolayer vero cells grown were infected at 0 , 1 moi with recombinant virus mv2ez - l1 or negative control virus mvez and incubated at 37 °. 1 hour after viral adsorption medium was substituted by dmem containing 5 % fcs and incubated at 37 ° for 48 hours to obtain 90 % syncytia . medium from infected cells has been collected , centrifugated and submitted to centrifugation on a 40 % ( w / v ) saccharose layer to separate proteins from particles at 110 , 00 × g for 2 . 5 h at 4 °. pellet was successively solubilised in cesium chloride 27 % ( w / w ) in pbs and analysed on density gradient centrifugation in cesium chloride 27 % ( w / w ) in pbs for 20 h at 141 , 000 g at 4 °. gradient fractions were analyzed for the presence of hpv - l1 by sds - page electrophoresis and western blot ( fig1 ). the immunogenic power of the rescued recombinant mv - hpv viruses described was proved by immunisation tests performed on transgenic mice cd46 , susceptible for mv infections . the animals were kept under optimal hygienic conditions and were immunized at 6 - 8 weeks of age . immunisation was performed intra - peritoneal using 10 5 pfu of each recombinant mv - hpv in two injections , at 0 and 4 weeks . non - infected mice as well as mice immunized with pbs served as control . uv inactivated mv was used as a control to determine the effect of virus replication on activation of immune responses . the presence of mv - specific antibodies in the sera from the immunised cd46 mice ( at least 6 per group ) was deteimined by elisa using 96 - microwell plates , coated with measles virus eia bulk ( atcc vr - 24 ), for igg antibody detection . protein was diluted 0 . 6 μg / ml with 0 . 05 m carbonate buffer ( ph 9 . 4 ), and 100 μl per well was added to 96 - well - microtiter plates . the plates were incubated overnight at 4 ° c ., washed with pbs / 0 . 05 % tween 20 ( pt ) ( ph 7 . 4 ), incubated with pt ( 0 . 1 ml / well )- 10 % bsa for 60 min at 37 ° c ., and washed again with pt . serial 2 - folds dilutions of the tested sera were added ( 100 μl / well ), and the plates were incubated for 60 min at 37 ° c . the plates were washed with pt and were incubated with 100 μl of goat anti - mouse igg hrp diluted 1 : 2000 in pbs - 0 . 05 % tween 20 for 30 min at 37 ° c . the plates were washed with pt and incubated with 100 μl opd ( o - phenylendiamin , fluka 78411 ). the reaction was stopped after 3 - 4 min . plates were read on a microelisa reader at a wave length of 490 nm . readings higher than three - folds negative controls were scored as positive reaction . the presence hpvl1 - specific antibodies in the sera of immunised cd46 mice was determined by elisa assay and by neutralization assay . briefly , for the elisa assay , 96 - microwell plates were coated with hpvl1 antigen , diluted with carbonate buffer ph 9 . 4 at a concentration of 2 - 50 ng / well . the plates were incubated overnight at 4 ° c ., washed with pbs / 0 . 05 % tween 20 ( pt ). subsequently , unspecific interaction were blocked with 10 % defatted milk dissolved in pt for 1 hour at 37 ° c . and wells were washed again with pt . the plates were consecutively incubated with various dilutions of mouse sera ( starting at 1 : 200 , followed by serial two - fold dilutions ), peroxidase - conjugate goat anti - mouse igg and with opd substrate . optical density values were measured at 490 nm . values above the cut - off background level ( mean value of sera from mv immunised mice multiplied by a factor of 2 . 1 ) were considered positive . titres were depicted as reciprocal end - dilutions . to assay for neutralizing antibodies twofold serum dilutions were incubated with 50 pfu of recombinant for 1 h at 37 ° c . and plated in duplicate onto 104 vero cells / well ( 96 - well plate ). five days later titers were determined microscopically . the endpoint titer was calculated as the highest serum dilution tested that reduced the number of pfu by at least 50 % the specific immune responses to hpv - l1 is shown in table i . the titers in both the elisa and neutralization assays are similar to those observed in women or mice after three injections of vlps . moreover , the very similar titers observed in the two assays indicate that most of the l1 expressed is conformationally correct . purification of recombinant measles virus expressing malaria antigens from defecting interfering particles ( dis ) by plaque purification it is known from literature that after a certain number of passages with paramyxoviruses , and in particular with measles virus , an accumulation of defective interfering particles ( dis ) will occur ( 23 , 24 ). it has been described that these dis develop various defects : negative impact on vaccine safety , negative influence on virus yields in production , genome instability and suppression of immune reaction after vaccination . in order to avoid such dis with our new recombinant viruses , we have applicated the method of plaque purification as described in example 7 with the exception that we use mrc5 cell instead of vero cell . after the formation of clear , well defined syncytia we aspirated under the microscope with a micropipette such material for further passaging in a fresh mrcs tissue colture . purification of recombinant measles virus expressing malaria antigens from defecting interfering particles ( dis ) by end point dilution the end point dilution technique was applicated in microplates : in all wells a fresh monolayer with mrc5 cells had just developed . the virus suspension containing recombinant measles - malaria viruses was prepared in two fold dilutions . from the well of the latest monolayer where a syncytia was detected the supernatant was aspirated with a pipette . the supernatant was mixed with a suspension containing mrc5 cells . this mixture was incubated at 4 ° c . for 1 hour . finally , it was transferred in a small costar flask and incubated at 35 ° c .+ 5 % co2 and harvested for purify recombinant measles - malaria virus after ten days . the working seed of the described recombinant measles - malaria virus has been incubated on mrc5 cell monolayer in 1750 cm2 roller bottles at 35 ° c . for ten days . the cells have been monitored every day for status of health and confluence . on day ten at highest level of syncytia formation , the supernatant was pumped in a steel cylinder for storage in liquid nitrogen . the same procedure was repeated two days later . after performing of all the tests ( virus titer , genome stability , virus safety , cell safety , chemical analysis , sterility and others ), the harvests have been thawed up and mixed with stabilizer containing gelatine , sorbitol , amminoacids and other sugars to final dilution of 105 . with a automated filling machine small lyo bottles ( f3 ) have been inoculated with 0 . 5 ml each . a specially calculated lyophilisation program is used to guarantee maximal survival of the product during the freeze - 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