Patent Application: US-201414486578-A

Abstract:
a combined measles - malaria vaccine containing different attenuated recombinant measles - malaria vectors comprising a heterologous nucleic acid encoding several plasmodium falciparum antigens is described . preferably , it relates to viral vectors that comprise nucleic acids encoding the circumsporozoite protein of p . falciparum , the merozoite surface protein 1 of p . falciparum , and its derivatives in its glycosylated and secreted forms , and apical membrane antigen1 of p . falciparum , in its anchored or secreted form . the viral vector stems from an attenuated measles virus , based on a strain that is used as a vaccine and is efficient in delivering the gene of interest and that binds to and infects the relevant immune cells efficiently .

Description:
the object of the invention is the production of a combined measles - malaria vaccine from a recombinant measles vectors capable of containing stably integrated dna sequences which code for cs , msp - 1 or partial sections of it and ama - 1 or partial sections , in the secreted or surface anchored forms , of p . falciparum . the invention shall also include the rescue of recombinant mv - malaria viruses which are capable of infection , replication and expression of pfcs , pfmsp - 1 and pfama - 1 antigens in susceptible transgenic mice , monkeys and human host . furthermore , the invention intends to include the construction of multivalent recombinant measles - malaria 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 three different recombinant measles - malaria viruses , each carrying a different gene and expressing different antigens , in a manner to elicit immuno response in the host , directed against the different stages of the parasite &# 39 ; s life - cycle . in addition , the invention includes a process to produce recombinant measles - malaria viruses which are avoided of defective interfering particles ( dis ). the dis are known to significantly inhibit the growth of virus in any production system and to successfully suppress immune response in human individuals . furthermore , the invention comprises a method to produce a vaccine containing such recombinant viruses . the examples below describe the preferred mode of carrying out the invention . it should be understood that these examples are provided for illustration and should not be construed as limiting the scope of the invention in any way . all cloning procedures were done as per the techniques described in sambrook et al . ( 1989 ). all the restriction enzymes were from new england biolabs ; the oligonucleotides pcr primers and dna polylinkers were from invitrogen . pfmsp1 and its fragments ( d - 83 - 30 - 38 and d - 42 ) either in the secreted and anchored form , have been chemically synthesized and human codon optimised . they have been cloned into the pze21mv intermediate vector and have been slightly modified by adding sgrai cloning site at the 5 ′ end followed by an optimised kozak sequence ( tcatca ). these modifications have been checked by sequencing at mwg biotech . list of the recombinant mv - pfmsp - 1 plasmids , gpi - anchored and secreted (*) forms , from 3d7 strain , which belongs to the mad20 prototype , and from fcb1 strain , which belongs to the k1 prototype : 1a ) construction of p (+) mv 2 ez - d - 190 - sgrai ( 3d7 , 24323 bp ) and p (+) mv 3 ez - d - 190 - sgrai ( 3d7 , 24323 bp ). 1 μg of mv plasmid dna containing the green fluorescent protein ( gfp ) ( p (+) mv 2 - 3 ez - gfp berna strain , 19774 bp : fig2 and 25 ) was digested with one unit of both sgrai and bsshii restriction enzymes , for two hours at their optimal temperature , in 50 μl final volume . all the digested dna was loaded onto a 1 % agarose gel , run at 80 volt for about 2 hours . then , the proper band ( 19048 bp ) was excised from the gel , purified by qiaex gel purification and the dna concentration was calculated by absorbance at 260 nm and adjusted to 1 μg / ml . 1 μg of d - 190 gene , inserted into an intermediate plasmid ( pze21mv - d - 190 sgrai , 7564 bp ,) was taken out by sgrai - bsshii digestion ( one unit of each enzyme ), for two hours at their optimal temperature , in 50 μl final volume . all the digested dna was loaded onto a 1 % agarose gel , run at 80 volt for about 2 hours . then , the proper band ( 5275 bp ) was excised from the gel , purified by qiaex gel purification kit and the dna concentration was calculated by absorbance at 260 nm and adjusted to 1 μg / ml . thus , the vector ( mv dna : fig1 ) and the insert ( d - 190 dna : fig2 ), were ligated in an equimolar ratio overnight at 16 ° c ., using one unit of t4 dna ligase and its own reaction buffer in 10 μl final volume . 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 . the d - 190 - 3d7 gene , inserted into position 2 of the mv vector ( sgrai , pos . 4060 , and bsshii , pos . 9335 ) is represented in fig4 and its open reading frame ( orf ) is listed in fig2 . the d - 190 - 3d7 gene , inserted into position 3 of the mv vector ( sgrai , pos . 9862 , and bsshii , pos . 15137 ) is represented in fig6 . the genome &# 39 ; s length ( starting at acc , pos . 609 , to ggt , pos . 21884 ) of the recombinant measles - malaria plasmids was a multiple of six , allowing the rescue of the recombinant mv 2 - 3 - d - 190 - 3d7 viruses . 1b ) construction of p (+) mv 2 ez - d - 83 - 30 - 38 - sgrai ( 3d7 , 23195 bp ) and p (+) mv 3 ez - d - 83 - 30 - 38 - sgrai ( 3d7 , 23195 bp ). the measles vectors were prepared as detailed described in example 3a . the pze21mv - d - 190 sgrai was digested bsteii - acli to cut out the d - 42 fragment ; a polylinker , with cohesive bsteii and acli ends , had been ligated to obtain the intermediate plasmid pze21mv - d - 83 - 30 - 38 - sgrai ( 6436 bp ). 1 μg of pze21mv - d - 83 - 30 - 38 sgrai was digested sgrai - bsshii ( one unit of each enzyme ), for two hours at their optimal temperature , in 50 μl final volume . all the digested dna was loaded onto a 1 % agarose gel , run at 80 volt for about 2 hours . then , the proper band ( 4147 bp ) was excised from the gel , purified by qiaex gel purification kit and the dna concentration was calculated by absorbance at 260 nm and adjusted to 1 μg / ml . thus , the vector ( mv dna : fig1 ) and the insert ( d - 83 - 30 - 38 dna : fig2 ), were ligated in an equimolar ratio overnight at 16 ° c ., using one unit of t4 dna ligase and its own reaction buffer in 10 μl final volume . 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 were then aligned with the assumed ones using a dna strider software . the right clones were sent to mwg for sequencing : the sequences , aligned with the assumed ones using a dna strider software , showed 100 % identity . the d - 83 - 30 - 38 - 3d7 gene , inserted into position 2 of the mv vector ( sgrai , pos . 4060 , and bsshii , pos . 8207 ) is represented in fig7 and its open reading frame ( orf ) is listed in fig2 . the d - 83 - 30 - 38 - 3d7 gene , inserted into position 3 of the mv vector ( sgrai , pos . 9862 , and bsshii , pos . 14006 ) is represented in fig9 . the genome &# 39 ; s length ( starting at acc , pos . 609 , to ggt , pos . 20756 ) of the recombinant measles - malaria plasmids was a multiple of six , allowing the rescue of the recombinant mv 2 - 3 - d - 83 - 30 - 38 - 3d7 viruses . 1c ) construction of p (+) mv 2 ez - d - 42 - sgrai ( 3d7 , 20417 bp ) and p (+) mv 3 ez - d - 42 - sgrai ( 3d7 , 20417 bp ). the measles vectors were prepared as detailed described in example 3a . 1 μg of d - 42gene , inserted into an intermediate plasmid ( pze21mv - d - 42 sgrai , 3658 bp ) was taken out by sgrai - bsshii digestion ( one unit of each enzyme ), for two hours at their optimal temperature , in 50 μl final volume . all the digested dna was loaded onto a 1 % agarose gel , run at 80 volt for about 2 hours . then , the proper band ( 1369 bp ) was excised from the gel , purified by qiaex gel purification kit and the dna concentration was calculated by absorbance at 260 nm and adjusted to 1 μg / ml . thus , the vector ( mv dna : fig1 ) and the insert ( d - 42 dna : fig2 ), were ligated in an equimolar ratio overnight at 16 ° c ., using one unit of t4 dna ligase and its own reaction buffer in 10 μl final volume . 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 . the d - 42 - 3d7 gene , inserted into position 2 of the mv vector ( sgrai , pos . 4060 , and bsshii , pos . 5429 ) is represented in fig1 and its open reading frame ( orf ) is listed in fig3 . the d - 42 - 3d7 gene , inserted into position 3 of the mv vector ( sgrai , pos . 9862 , and bsshii , pos . 11231 ) is represented in fig1 . the genome &# 39 ; s length ( starting at acc , pos . 609 , to ggt , pos . 17978 ) of the recombinant measles - malaria plasmids was a multiple of six , allowing the rescue of the recombinant mv 2 - 3 - d - 42 - 3d7 viruses . the recombinant measles - p - 42 malaria viruses and mv vaccine induced similar cytopathic effect ( fig3 ). the transgene is rather stably expressed : its expression was completely maintained in all analysed progeny clones derived from single original rescued clones after ten serial virus passages in human diploid cell mrc5 ( fig3 - 38 ). the growth curves of recombinant mv - malaria virus and mv vaccine showed the same kinetics ( fig3 ). 1d ) construction of p (+) mv 2 ez - d - 190 *- sgrai ( 3d7 , 24227 bp ) and p (+) mv 3 ez - d - 190 *- sgrai ( 3d7 , 24227 bp ). the measles vectors were prepared as detailed described in example 3a . using the intermediate vector pze21mvd - 190 - sgrai as template , a pcr reaction has been performed to delete the gpi anchor region , which is located between acli ( pos . 5434 ) and clai ( pos . 5536 ) sites . pcr amplifications were carried out using the proofreading pfu dna polymerase ( stratagene ). dna sequences of the synthetic oligonucleotides primers are given in lower case for the mv nucleotides and in upper case for non mv nucleotides ; sequences of relevant restriction endonucleases recognition sites are underlined . the following oligonucleotides primers have been used : for - clai , 5 ′- ccaata aacgtt taat ag atcgat tac gcgcgc tctagc - 3 ′, and rev - avrii , 5 ′- gcctttgagtgagctgatacc - 3 ′. for - clai is homologous to the template at the level of the clai and bsshii sites and contains an overhang ( in upper case ) with two stop codons ( taatag ), the acli site ( aacgtt ), and a 6 bp long - protection site for acli ( ccaata ). in the so - called pcr - gpi and in the final construct d - 190 *, acli will become close to clai . pcr product was 207 bp - long : its digestion with acli + avrii and ligation with the pre - digested acli + avrii intermediate vector pze21mvd - 190 - sgrai has produced pze21 mvd - 190 *- sgrai . in detail , the digestion of the vector with acli + avrii has produced two bands of 7318 bp and 246 bp ( containing the gpi region to delete ): the 7 . 3 kb - fragment was purified from agarose gel by using qiaex ii purification kit ( qiagen ) and was ligated to the digested acli - avrii pcr ( insert ) to obtain pze21mvd - 190 *- sgrai . to screen for positive clones , ncoi digestion has be done , producing a single band of 7 kb from the d - 190 * intermediate vector , and two bands of 1 . 3 and 5 . 7 kb from the original gpi - anchor construct . to construct the definitive recombinant p (+) mev 2 ez - d190 * and p (+) mev 3 ez - d190 * ( fig5 and fig6 ), according to the “ rule of six ”, mev vectors and intermediate plasmid were digested with sgrai + bsshii and afterwards ligated each other . in detail , pze21mvd - 190 *- sgrai digested sgrai + bsshii has produced three bands , 5 . 2 kb + 1 . 3 kb + 900 bp . d - 190 * sequence was contained in the 5 . 2 kb fragment , that has been cut , purified and ligated with mev 2 ez and mev 3 ez vectors sgrai + bsshii digested ( 19 kb in length ), in an equimolar ratio overnight at 16 ° c ., using one unit of t4 dna ligase . 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 . the d - 190 *- 3d7 gene , inserted into position 2 of the mv vector ( sgrai , pos . 4060 , and bsshii , pos . 9239 ) is represented in fig5 and its open reading frame ( orf ) is listed in fig2 . the d - 190 *- 3d7 gene , inserted into position 3 of the mv vector ( sgrai , pos . 9862 , and bsshii , pos . 15041 ) is represented in fig6 . the genome &# 39 ; s length ( starting at acc , pos . 609 , to ggt , pos . 21788 ) of the recombinant measles - malaria plasmids was a multiple of six , allowing the rescue of the recombinant mv 2 - 3 - d - 90 *- 3 d7 viruses . 1e ) construction of p (+) mv 2 ez - d - 83 - 30 - 38 *- sgrai ( 3d7 , 23105 bp ) and p (+) mv 3 ez - d - 83 - 30 - 38 *- sgrai ( 3d7 , 23105 bp ). the measles vectors were prepared as detailed described in example 3a . the intermediate vector pze21mvd - 190 - sgrai was digested bsteii - clai to cut out the d - 42 fragment and the gpi region , which is located between acli ( pos . 5434 ) and clai ( pos . 5536 ) sites ; a polylinker , with cohesive bsteii and clai ends , had been ligated to obtain the intermediate plasmid pze21m v - d - 83 - 30 - 38 *- sgrai ( 6346 bp ). the sequence of the polylinker was : 5 ′- gtcacc ggggaataatagcgc at - 3 ′. dna sequence of the synthetic oligonucleotide polylinker is given in upper case for non mv nucleotides ; sequences of relevant restriction endonucleases recognition sites are underlined . polylinker contains the bsteii ( gtcacc ) and clai ( at ) sticky ends , two stop codons ( taatag ), and a triplet ( gcg ) to keep the rule of six . 1 μg of pze21mv - d - 83 - 30 - 38 * sgrai was digested sgrai - bsshii ( one unit of each enzyme ), for two hours at their optimal temperature , in 50 μl final volume . all the digested dna was loaded onto a 1 % agarose gel , run at 80 volt for about 2 hours . then , the proper band ( 4057 bp ) was excised from the gel , purified by qiaex gel purification kit and the dna concentration was calculated by absorbance at 260 nm and adjusted to 1 μg / ml . thus , the vector ( mv dna : fig1 ) and the insert ( d - 83 - 30 - 38 * dna : fig2 ), were ligated in an equimolar ratio overnight at 16 ° c ., using one unit of t4 dna ligase and its own reaction buffer in 10 μl final volume . 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 were then aligned with the assumed ones using a dna strider software . the right clones were sent to mwg for sequencing : the sequences , aligned with the assumed ones using a dna strider software , showed 100 % identity . the d - 83 - 30 - 38 *- 3d7 gene , inserted into position 2 of the mv vector ( sgrai , pos . 4060 , and bsshii , pos . 8117 ) is represented in fig8 and its open reading frame ( orf ) is listed in fig2 . the d - 83 - 30 - 38 *- 3d7 gene , inserted into position 3 of the mv vector ( sgrai , pos . 9862 , and bsshii , pos . 13919 ) is represented in fig9 . the genome &# 39 ; s length ( starting at acc , pos . 609 , to ggt , pos . 20666 ) of the recombinant measles - malaria plasmids was a multiple of six , allowing the rescue of the recombinant mv 2 - 3 - d - 83 - 30 - 38 *- 3d7 viruses . 1f ) construction of p (+) mv 2 ez - d - 42 *- sgrai ( 3d7 , 20345 bp ) and p (+) mv 3 ez - d - 42 *- sgrai ( 3d7 , 20345 bp ). the measles vectors were prepared as detailed described in example 3a . using the intermediate vector pze21mvd - 42 - sgrai ( 3658 bp ) as template , a pcr reaction has been performed to delete the gpi anchor region , which is located between acli ( pos . 1528 ) and clai ( pos . 1630 ) sites . pcr amplifications were carried out using the proofreading pfu dna polymerase ( stratagene ). dna sequences of the synthetic oligonucleotides primers are given in lower case for the mv nucleotides and in upper case for non mv nucleotides ; sequences of relevant restriction endonucleases recognition sites are underlined . the following oligonucleotides primers have been used : for - clai , 5 ′- ccaata aacgtt taat ag atcgat tac gcgcgc tctagc - 3 ′, and rev - avrii , 5 ′- gcctttgagtgagctgatacc - 3 ′. for - clai is homologous to the template at the level of the clai ( pos . 1630 ) and bsshii ( pos . 1639 ) sites and contains an overhang ( in upper case ) with two stop codons ( taatag ), the acli site ( aacgtt ), and a 6 bp long - protection site for acli ( ccaata ). in the so - called pcr - gpi and in the final construct d - 42 *, acli will become close to clai . pcr product was 207 bp - long : its digestion with acli + avrii and ligation with the pre - digested acli + avrii intermediate vector pze21mvd - 42 - sgrai has produced pze21 mvd - 42 *- sgrai . in detail , the digestion of the vector with acli + avrii has produced two bands of 3412 bp and 246 bp ( containing the gpi region to delete ): the 3 . 4 kb - fragment was purified from agarose gel by using qiaex ii purification kit ( qiagen ) and was ligated to the digested acli - avrii pcr ( insert ) to obtain pze21mvd - 42 *- sgrai . to screen for positive clones , ncoi digestion has be done , producing a single band of 3 . 4 kb from the d - 42 * intermediate vector , and two bands of 1 . 3 and 2 . 3 kb from the original gpi - anchor construct . to construct the definitive recombinant p (+) mev 2 ez - d42 * and p (+) mev 3 ez - d42 *, according to the “ rule of six ”, mev vectors and intermediate plasmid were digested with sgrai + bsshii and afterwards ligated each other . in detail , pze21mvd - 42 *- sgrai digested sgrai + bsshii + spei has produced four bands , 1 . 3 kb + 936 bp + 800 bp + 400 bp . d - 42 * sequence was contained in the 1 . 3 kb fragment , that has been cut , purified and ligated with mev 2 ez and mev 3 ez vectors sgrai + bsshii digested ( 19 kb in length ), in an equimolar ratio overnight at 16 ° c ., using one unit of t4 dna ligase . 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 . the d - 42 *- 3d7 gene , inserted into position 2 of the mv vector ( sgrai , pos . 4060 , and bsshii , pos . 5357 ) is represented in fig1 and its open reading frame ( orf ) is listed in fig3 . the d - 42 *- 3d7 gene , inserted into position 3 of the mv vector ( sgrai , pos . 9862 , and bsshii , pos . 11159 ) is represented in fig1 . the genome &# 39 ; s length ( starting at acc , pos . 609 , to ggt , pos . 17906 ) of the recombinant measles - malaria plasmids was a multiple of six , allowing the rescue of the recombinant mv 2 - 3 - d - 42 *- 3d7 viruses . 1g ) construction of p (+) mv 2 ez - d - 190 - sgrai ( fcb1 , 24083 bp ) and p (+) mv 3 ez - d - 190 - sgrai ( fcb1 , 24083 bp ). first of all , the cloning of the synthetic gene for msp - 1 of the fcb1 strain into the intermediate plasmid pze21mv - sgrai has been performed , keeping the signal peptide and the gpi - anchor region from msp - 1 of 3d7 strain . d - 190 gene ( fcb1 ) was obtained stepwise from an intermediate vector , called pze23f - gx - 190h , as follow : i ). 1 μg of the plasmid pze21mv - d - 190 - sgrai ( 3d7 ) was digested with hindiii + acli restriction enzymes , for two hours at their optimal temperature , in 501 final volume . all the digested dna was loaded onto a 1 % agarose gel , run at 80 volt for about 2 hours . then , the proper band ( 2558 bp ), corresponding to the vector , was excised from the gel , purified by qiaex gel purification and the dna concentration was calculated by absorbance at 260 nm . ii ). a pcr reaction was performed , using the pze23f - gx - 190h as template , in order to amplify and recover the d - 42 portion of the msp - 1 / fcb1 . pcr amplification was carried out using the proofreading pfu dna polymerase ( stratagene ). dna sequences of the synthetic oligonucleotides primers are given in lower case for the mv nucleotides and in upper case for non mv nucleotides ; sequences of relevant restriction endonucleases recognition sites are underlined . the following oligonucleotides primers have been used , designed on the pze23f - gx - 190h sequence : for - 1 fcb1 , 5 ′- ccc aagctt ccaggt ggtcacc ggagagctgtcactcc - 3 ′, and rev - 1 fcb1 , 5 ′- gcctgc aacgtt gctagagctggagcagaagatcccgtcg - 3 ′. for - 1 fcb1 is homologous to the template from pos . 4509 to pos . 4538 , comprising the bsteii site ( ggtcacc ). the a ( in upper case ) was a t in the template , and it has been modified to eliminate a sgrai site . it contains an overhang ( in upper case ) with the hindiii site ( aagctt ), after its 3 bp long - protection site ( ccc ). rev - 1 fcb1 contains an acli site ( aacgtt ), preceded by a 6 - bp protection site ( gcctgc ). it was introduced a triplet gct , coding for a serine , to keep the rule of six ; two a have been modified in g to avoid a poly ( a ) site . the obtained pcr - hindiii - acli ( 1 . 1 kb ) has been digested hindiii + acli and ligated , overnight at 16 ° c . in an equimolar ratio , to the pre - digested pze21mv - d - 190 - sgrai with hindiii + acli ( step i ), obtaining the pze21mv - d - 42 - sgrai - fcb1 ( 3657 bp ). 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 by restriction enzymes digestion with hindiii + acli ( expected fragments 2558 bp + 1099 bp ). iii ). the pze21mv - d - 42 - sgrai - fcb1 , obtained as described in step ii , has been digested hindiii + bsteii ( hindiii , pos . 428 , and bsteii , pos . 440 ), and the proper band ( 3645 bp ), corresponding to the opened vector , was loaded on a 1 % agarose gel , excised from the gel , purified by qiaex gel purification and the dna concentration was calculated by absorbance at 260 nm . iv ). the pze23f - gx - 190h was digested hindiii + bsteii and the proper band of 3679 bp ( insert ), corresponding to the d - 83 - 30 - 38 / fcb1 fragment , was purified from the gel , as previously described . v ). the hindiii + bsteii digested fragment of 3657 bp ( vector ), obtained from pze21mv - d - 42 - sgrai - fcb1 , has been ligated to the hindiii + bsteii fragment of 3679 bp ( insert ), containing the d - 83 - 30 - 38 / fcb1 and obtained by digestion from pze23f - gx - 190h . ligation was done in an equimolar ratio overnight at 16 ° c ., using one unit of t4 dna ligase , obtaining the pze21mv - d - 190 - sgrai - fcb1 ( 7324 bp ). afterwards , 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 . to construct the p (+) mv 2 ez - d - 190 - sgrai - fcb1 and p (+) mv 3 ez - d - 190 - sgrai - fcb1 , the measles vectors were prepared as detailed described in example 3a . 1 μg of d - 190 / fcb1 gene , inserted into an intermediate plasmid ( pze21mv - d - 190 sgrai - fcb1 , 7324 bp ), was taken out by sgrai - bsshii digestion ( one unit of each enzyme ), for two hours at their optimal temperature , in 50 μl final volume . all the digested dna was loaded onto a 1 % agarose gel , run at 80 volt for about 2 hours . then , the proper band ( 5035 bp ) was excised from the gel , purified by qiaex gel purification kit and the dna concentration was calculated by absorbance at 260 nm and adjusted to 1 μg / ml . thus , the vector ( mv dna : fig1 ) and the insert ( d - 190 / fcb1 dna : fig3 ), were ligated in an equimolar ratio overnight at 16 ° c ., using one unit of t4 dna ligase and its own reaction buffer in 10 μl final volume . 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 . the d - 190 - fcb1 gene , inserted into position 2 of the mv vector ( sgrai , pos . 4060 , and bsshii , pos . 9095 ) is represented in fig1 and its open reading frame ( orf ) is listed in fig3 . the d - 190 - fcb1 gene , inserted into position 3 of the mv vector ( sgrai , pos . 9862 , and bsshii , pos . 14897 ) is represented in fig1 . the genome &# 39 ; s length ( starting at acc , pos . 609 , to ggt , pos . 21884 ) of the recombinant measles - malaria plasmids was a multiple of six , allowing the rescue of the recombinant mv 2 - 3 - d - 190 - fcb1 viruses . the transgene is rather stably expressed : its expression was completely maintained in all analysed progeny clones derived from single original rescued clones after ten serial virus passages in human diploid cell mrc5 ( fig4 ). the growth curves of recombinant mv - malaria virus and mv vaccine showed the same kinetics ( fig4 ). starting from the aminoacidic dico1 sequence ( ecto , trans and cytoplasmic domains : aa 97 - 622 ) and using the dna strider software , a correspondent nucleic acid sequence has been designed comparing the dico1 dna degenerate sequence to a selected pfama1 gene ( accession number aag141 . 1 ), which represents the most similar sequence to the dico1 after blast alignment . at the 5 ′ end suitable unique restriction sites has been added ( mlui and sgrai ) as cloning sites , followed by an optimal kozac sequence and a human optimised signal peptide ( sp ). at the 3 ′ end , two stop codons and a bsshii cloning site have been added . following this scheme , we designed two nucleotides sequences ( respecting the “ rule of six ” for the further expression into the measles vector ), encoding the anchored and the secreted forms of the dico1 protein : the first gene comprises the ectoplasmic , the transmembrane and cytoplasmic domains ( fig1 ), while the second one corresponds to the ectodomain alone ( fig1 ). the two sequences has been human codon optimised by geneart , to reduce at % content , to avoid poly ( a ) sequence and rna instability motif . dico1 complete orf and dico1 ectodomain orf are listed respectively in fig3 and 35 . all cloning procedures were done as per techniques described in sambrook et al . ( 1989 ). pfama1 , and in particular diversity covering sequences 1 ( dico1 ) either in the secreted and anchored form , have been chemically synthesized and human codon optimised . the codon optimised dico1 secreted and anchored forms were digested sgrai + bsshii and ligated , overnight at 16 ° c . in an equimolar ratio , to the pre - digested mev 2 ez and mev 3 ez vectors ( 19 kb in length ), using one unit of t4 dna ligase , obtaining the following recombinant mv - pfama - 1 plasmids : p (+) mv 2 ez - dico1 - complete ( fig2 ), p (+) mv 3 ez - dico1 - complete ( fig2 ), p (+) mv 2 ez - dico1 - ecto ( fig2 ), and p (+) mv 3 ez - dico1 - ecto ( fig2 ). all cloning procedures were basically as described in sambrook et al . ( 1989 ). pfcs1 , cloned into an intermediate vector padapt35bsu . cs . pfalc . aa - sub . gcc , has been amplified by pcr , and directly cloned into the definitive mv vectors , obtaining two recombinant mv - pfcs plasmids : p (+) mv 2 ez - cs and p (+) mv 3 ez - cs . in detail , a pcr reaction was performed , using the padapt35bsu . cs . pfalc . aa - sub . gcc as template , in order to amplify and recover the cs gene ( fig1 ). pcr amplification was carried out using the proofreading pfu dna polymerase ( stratagene ). dna sequences of the synthetic oligonucleotides primers are given in lower case for the mv nucleotides and in upper case for non mv nucleotides ; sequences of relevant restriction endonucleases recognition sites are underlined . the following oligonucleotides primers have been used , designed on the padapt35bsu . cs . pfalc . aa - sub . gcc sequence : for - sgrai , 5 ′- actttct caccggtg tgg aagctt gccac catgat - 3 ′, and rev - bsshii - cs 5 ′- ta gcgcgctctagagga tccttatcagc - 3 ′. for - sgrai is homologous to the template from pos . 1356 to pos . 1375 , comprising the hindiii site ( aagctt ). it contains an overhang ( in upper case ) with sgrai restriction site ( caccggtg ), after 6 - bp long - protection site ( acttct ). rev - bsshii - cs contains an overhang ( in upper case ) with bsshii restriction site ( gcgcgc ), which will be close to xbai ( tctaga ) in the pcr - cs ( 1187 bp ). the obtained pcr - cs has been digested sgrai + bsshii and ligated , overnight at 16 ° c . in an equimolar ratio , to the pre - digested mev 2 ez and mev 3 ez vectors sgrai + bsshii ( 19 kb in length ), using one unit of t4 dna ligase , obtaining , respectively , p (+) mv 2 ez - cs - sgrai ( 20219 bp , fig1 ) and p (+) mv 3 ez - cs - sgrai ( 20219 bp , fig1 ). the cs orf is listed in fig3 . 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 . 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 ( p / s ) 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 thawed three times and spun 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 , spun down and the cleared surnatant stored at − 80 ° c . 293t 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 ca 2 + phosphate coprecipitation of dna was from invitrogen . cells were co - transfected with the plasmids in the follows final concentration : pca - l 0 . 5 μg , pca - n 0 . 5 μg , pca - p 0 . 1 μg , pca t7 1 μg and the recombinant measles - malaria plasmid 4 μg . all five plasmids , diluted in h 2 o , were added in a eppendorf tube containing 2m cacl 2 , 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 % co 2 for about 18 h . then , the transfection medium was replaced with 3 ml of dmem containing 10 % fcs . another way to obtain recombinant measles - malaria vaccine viruses is described hereafter , 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 . to improve transfection efficiency 300 ng of psc6 - t7 neo were added . calcium - phosphate method was used for transfection . first syncytia appeared 3 - 4 days after transfection when the cells were still subconfluent . to allow syncytia formation to progress more easily , almost confluent cell monolayer 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 % co 2 , 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 . 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 mrc5 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 . to analyse the expression either mv and malaria , western blot and immunofluorescence were carried out . for western blot , vero cells seeded on 35 mm dish ( 1 - 5 × 10 5 ) were monitored the next day for 90 % confluence and infected with cleared virus suspension from cell - associated virus fraction , using 0 . 1 moi ( multiplicity of infection ), including mvez as control . when about 80 % syncythia formation was observed , 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 lysated 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 μl 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 / 1 h30 . as first antibodies , rabbit polyclonal against msp1 - p - 83 , diluted in pbst at least 1 : 30000 , and against msp1 - p - 42 , diluted at least 1 : 50000 , were used . the second antibody was a swine anti - rabbit antibody coupled to horse - radish peroxidase allowing the visualization of the bands by the enhanced chemiluminescence kit ( ecl ™, amersham lifescience ). for immunofluorescence , vero cells were seeded on a 24 mm × 24 mm glass cover slips in 35 mm wells , cultured overnight and infected with rescued recombinant virus . 3 days after infection cells on coverslips were fixed with 3 . 7 % 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 antibodies . mouse hybridoma supernatant mab 5 . 2 , which recognises a egf - like domain in the p - 19 portion of p - 42 , was used in a dilution 1 : 100 followed by fitch conjugated goat anti - mouse serum , diluted 1 : 250 . mrc5 cells seeded on 35 mm dish ( 1 - 5 × 10 5 ) were monitored for 90 % confluence and infected with cleared virus suspension from cell - associated virus fraction , using 0 . 1 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 . the immunogenic power of the rescued recombinant mv - malaria viruses described was proven by immunisation tests performed on transgenic mice ifnar / cd46 , susceptible to mv infections . the animals were kept under optimal hygienic conditions and were immunized at 6 - 8 weeks of age . below is provided an example of mice immunization with two recombinant measles - malaria virus : the mev2ez - d - p42 - sgrai ( the gpi anchored form ) and the mev2ez - d - p42 * ( the secreted form ). immunisation was performed intra - muscularly using 10 5 pfu of each recombinant mv - malaria in three injections at 0 , 4 and 8 weeks . mice immunized with recombinant - empty measles ( rmvez13 — empty cloned ) served as negative control . uv inactivated rmv was used as a control to determine the effect of virus replication on activation of immune responses . the immune response of the mv vectored antigen was tested compared to the purified d - 42 protein ( 0 . 5 mg / ml ): mice were immunized sub cutaneously with 20 μg of protein in incomplete freund &# 39 ; s adjuvant . the presence of mv - specific antibodies in the sera from the immunised ifnar / cd46 mice ( 6 per test group and 3 for control group ) was determined 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 j / 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 pt 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 of mv - malaria - specific antibodies in the sera of immunised cd46 mice ( at least 10 per test group ) was determined by elisa assay . briefly , 96 - microwell plates were coated 50 ng / well msp - 1 - d42 3d7 strains , diluted with carbonate buffer ph 9 . 4 . 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 . the humoral immune responses against measles are shown in fig4 . the humoral immune responses against malaria p42 are shown in fig4 . 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 applied the method of plaque purification as described in example 6 with the exception that we use mrc5 cell instead of 293t cells . after the formation of clear , well defined syncytia we aspirated under the microscope with a micropipette such material for further passaging in a fresh mrc5 tissue culture . purification of recombinant measles virus expressing malaria antigens from defecting interfering particles ( dis ) by end point dilution the end point dilution technique was applied in microplates : in all wells a fresh monolayer of 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 % co 2 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 cm 2 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 10 5 . with a automated filling machine small lyo bottles ( f3 ) have been inoculated with 0 . 5 ml each . a specially calculated lyophilisation program was used to guarantee maximal survival of the product during the freeze - 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