Patent Application: US-201113576672-A

Abstract:
the present invention provides attenuated viruses for use as vaccines and for the treatment and / or prevention of viral diseases and / or infections .

Description:
the present invention will now be described in detail with reference to the following figures which show : fig1 : construction of the chimeric s - m genomic segment of rvfv strain mp12 . the coding strategy of the s segment is shown at the top in the viral - complementary sense . excision pcr mutagenesis was used to remove the nss orf and to introduce unique pmli and spei restriction enzyme sites . the coding sequence for gn and gc was amplified from ptvt7 - gm by pcr using primers that introduced pmli and spei restriction enzyme sites , and allowed insertion of the gn / gc coding sequence into the modified s segment . utr , untranslated region ; igr , intergenic region . fig2 : analysis of s segment rna from three segment ( mp12 ) or two segment ( r2segmp12 ) recombinant viruses . ( a ) schematic depiction of the parental s segment and the chimeric s - m segment , and the sites at which oligonucleotides ( α , β , ψ ) anneal . ( b ) reverse transcription - pcr analysis was used to confirm that the two - segmented virus had an s segment that encoded the viral glycoproteins . bhk cells were infected with mp12 or the two segmented r2segmp12 viruses at a m . o . i of 1 p . f . u . per cell . total cellular rna was extracted from the infected cells 24 h . p . i and s segment rt - pcr was performed with the pairs of primers as indicated . no product was obtained using primer pairs α and β with mp12 s rna as the template , as no glycoprotein encoding sequences were present for primer a to anneal . all other amplified product were the correct sizes . fig3 : northern blot analysis of viral rnas in infected bhk - 21 . cells were infected at 1 pfu / cell with either 3 - segment mp12 or 2 segment r2segmp12 virus as indicated , and total infected cell rna was extracted at 24 hr post infection . northern blotting was performed using dig - labelled probes complementary to n , nss , gn , & amp ; l coding sequences in the viral genomic rna (−) ( a , b , d , & amp ; f ) and gn & amp ; n coding sequences in the viral anti - genomic rna (+) ( c & amp ; e ). the sizes of the rna species are indicated on the left and the sizes of the detected products were as expected , fig4 : characterisation of protein expression from the recombinant two - segmented virus . ( a ) western blot analysis of bhk - 21 cells infected with rmp12 or r2segmp12 at a m . o . i . of 1 . cell extracts were harvested 48 h p . i and loaded into 4 - 12 % nupage gel ( invitrogen ) and the blot was probed with anti - n and anti - nss antibodies . no nss band was detected in r2segmp12 - infected cells . ( b ) inhibition of host cell protein synthesis by rmp12 or the recombinant r2segmp12 virus . bhk - 21 cells were infected at a m . o . i of 5 , and harvested at 24 h p . i . the cells were labelled with 50 μci [ 35 s ] methionine for 2 . 5 h and cell lysates analysed by sds - page . the positions of the viral proteins n and nss are indicated on the right . fig5 : growth kinetics , viral protein synthesis , plaque phenotype and serial passage of rmp12 and the recombinant r2segmp12 viruses . viral growth curves in ( a ) bhk - 21 , ( b ) vero e6 and ( c ) c6 / 36 cells . cells were infected with either rmp12 or r2segmp12 at a m . o . i of 1 . viruses were harvested at the time points indicated and titrated by plaque assay . ( d ) comparison of plaque sizes on bhk - 21 cells . cell monolayers were fixed 96 h p . i . with 4 % paraformaldehyde and stained with giemsa solution . ( e ) western blot of s segment encoded proteins from rmp12 and r2segmp12 in infected cells . in tandem with the viral titres , infected cell extracts were harvested at the time points indicated , loaded into 4 - 12 % nupage gel ( invitrogen ) and blots were probed with anti - n , anti - nss and anti - tubulin antibodies . ( f ) serial passaging of rmp12 and r2segmp12 in bhk - 21 cells . western blots probed with anti - n , anti - nss and anti - tubulin antibodies . fig6 : construction of a recombinant rvfv virus expressing v5 epitope - tagged l protein . ( a ) the 14 amino acid v5 epitope tag was inserted into the coding region of the l protein by pcr mutagenesis with both ptm1 - rvfv l and ptvt7 - gl dna as template . insertion sites are indicated by triangles at the indicated nucleotide positions and were designated l1v5 - l3v5 . ( b ) activity of v5 - tagged l proteins in a minireplicon assay . bsr - t7 cells were transfected with ptm1 - rvfv n , ptvt7 - gs : hren ( encoding an mp12 s segment in which the coding sequence for the non - structural protein nss has been replaced with that of humanised renilla luciferase ( hren )) and either parental ptm1 - rvfv l or v5 - tagged l mutants . renilla luciferase activity in cell lysates was measured at 24 h post - transfection and is shown in arbitrary light units . ( c ) generation of a lv5 - tagged rvfv . construct ptvt7 - gl3v5 was used in place of parental rvfv l plasmid and a recombinant virus expressing the v5 tag recovered by reverse genetics . plaque phenotype , growth kinetics and viral protein synthesis of the recombinant virus rmp12l3v5 are shown compared to mp12 virus . vero cells were grown in dulbecco &# 39 ; s modified minimal essential medium ( dmem ) supplemented with 10 % fetal bovine serum ( fbs ). bhk - 21 cells were grown in glasgow modified mem ( gmem ) supplemented with 10 % heat - inactivated fbs , and bsr - t7 / 5 cells , which stably express t7 rna polymerase ( buchholz et al ., 1999 ) were grown in gmem supplemented with 10 % fbs and 1 mg / ml g418 . all mammalian cell lines were grown at 37 ° c . with 5 % co 2 unless otherwise stated . the aedes albopictus derived cell line c6 / 36 was maintained in leibovitz &# 39 ; s l - 15 medium supplemented with 10 % fcs and 8 % tpb . these cells were incubated at 28 ° c . in the absence of co 2 . recombinant rift valley fever viruses created by reverse genetics were grown under bsl3 conditions by infecting bhk - 21 cells at a multiplicity of infection ( moi ) of 0 . 01 pfu / cell and harvesting the culture medium after incubation for 72 h . for serial blind passage of viruses at low multiplicity , infected bhk - 21 cells were grown at 33 ° c . in medium supplemented with 2 % ncs until marked cytopathic effect ( cpe ) was observed ; the supernatant was clarified and an aliquot used to inoculate fresh bhk - 21 cell monolayers . plasmids which contain full - length cdna copies of the rvfv mp12 genome segments or which express rvfv mp12 proteins have been described previously ( billecocq et al ., 2008 ). to delete the nss gene in the s segment , plasmid ptvt7 - gs was used as template in an excision pcr reaction ( shi and elliott , 2002 ), using outward - facing oligonucleotides 5 ′- acaggaaagtggtacctgatacacgtgataagcactag - 3 ′ and 5 ′- gaggaggagaggtaccatgatggactagttgaggttgattag - 3 ′. the pcr product generated lacked nt 19 to 819 of the rvfv s segment , and possessed at its 3 ′ end spei and kpni restriction sites and at its 5 ′ end kpni and pmii sites . the pcr product was digested with kpni and religated to form plasmid ptvt7 - gsδnss - kpni . digestion of ptvt7 - gsδnss - kpni with pmli and spei allowed directional insertion of foreign genes into the s segment in an ambisense orientation . the coding sequences for enhanced gfp , humanized renilla luciferase , and rvfv gn - gc sequences which were amplified by pcr from pegfp - n1 ( clontech ), phrl - cmv ( promega ) or ptvt7 - gm ( billecocq et al ., 2008 ) respectively . the sequences were then verified to ensure no mutations had occurred during the cloning process . all sequences of the specific oligonucleotides used for the different constructs are available on request . to insert the v5 epitope tag into the l protein or the genomic l segment , the plasmids ptvt7 - gl and ptm1 - l were used as templates in pcr mutagenesis reactions ( shi and elliott , 2009 ), using outward facing oligonucleotides with an additional 5 ′ phosphate group . the pcr products generated contained one half of the v5 tag at their 5 ′ ends and the other at their 3 ′ ends . the resulting pcr products were ligated overnight with t4 - ligase ( promega ). the sequences were then verified to ensure no mutations had occurred during the cloning process . all sequences of the specific oligonucleotides used for the different constructs are available on request . the reverse genetics protocol essentially followed that described by lowen et al ( lowen et al ., 2004 ). bsr - t7 / 5 cells ( 7 × 10 5 cells in a t - 25 flask ) were transfected with the expression plasmids ptm1 - l ( 0 . 5 μg ) and ptm1 - n ( 0 . 5 μg ) together with 1 μg each of the transcription plasmids ptvt7 - gl , ptvt7 - gm and ptvt7 - gs ( to generate rvfv strain mp12 ) or ptvt7 - gl and one of the modified s segment cdna constructs , using 2 μl lipofectamine 2000 ( invitrogen ) per μg of plasmid dna . in the case of the v5 epitope - tagged l protein - containing virus , bsr - t7 / 5 cells ( 7 × 10 5 cells in a t - 25 flask ) were transfected with the expression plasmids ptm1 - l ( 0 . 5 μg ) and ptm1 - n ( 0 . 5 μg ) together with 1 μg each of the transcription plasmids ptvt7 - gl3v5 , ptvt7 - gm and ptvt7 - gs , to generate rvfv strain mp12lv5 . after 5 to 7 days , when extensive cytopathic effect was observed , the culture media were collected and stored at − 80 ° c . bhk cells were infected with serial dilutions of virus and incubated under an overlay consisting of dmem , 2 % fcs and 1 % agarose at 37 ° c . for 7 days . cell monolayers were fixed with 4 % formaldehye , and plaques were visualized by staining with giemsa . bhk - 21 cells in t - 25 flasks were infected at a moi of 1 . 0 pfu / cell with the different viruses . one hour post - infection , the inoculum was removed and cells were washed with phosphate - buffered saline to remove unattached viruses . at the time point indicated , viruses in the supernatant fluid were titrated by plaque assay on bhk - 21 cells . bhk - 21 cells in t - 25 flasks were infected at a moi of 1 . 0 pfu / cell with the different viruses . twenty - four hours post - infection , cells were labeled with 50 μci per flask of [ 35 s ] methionine for 2 . 5 h . cell lysates were prepared and analyzed by sodium dodecyl sulfate ( sds )- polyacrylamide gel electrophoresis ( page ) as described previously ( watret et al ., 1985 ). bhk - 21 cells in t - 25 flasks were infected at a moi of 1 . 0 pfu / cell with the different viruses . twenty - four hour post - infection cell lysates were prepared by the addition of 1 ml cell lysis buffer ( 100 mm tris ; 4 % ( wt / vol ) sds ; 20 % ( wt / vol ) glycerol ; 200 mm dtt , 0 . 2 % ( wt / vol ) bromophenol blue and 25 u benzonase ( novagen )). equal amounts of cell extract were separated by sds - 4 - 12 % page ( invitrogen ) and transferred to a hybond - c extra membrane ( amersham ), followed by 2 h incubation in saturation buffer ( phosphate - buffered saline containing 5 % dry milk and 0 . 1 % tween 20 ). the membrane was incubated overnight in saturation buffer containing an anti - n antibody at a dilution of 1 : 20 , 000 , anti - nss antibody at a dilution of 1 : 20 , 000 , anti - tubulin antibody at a dilution of 1 : 5 , 000 ( sigma ) or anti - v5 antibody ( serotec ) at a dilution of 1 : 1000 , followed by incubation with horse - radish peroxidase ( hrp )- labeled anti - rabbit antibody ( cell signaling technology ) diluted 1 : 1000 in saturation buffer for 1 h . after three washes in phosphate - buffered saline - 0 . 1 % tween 20 , the membrane was rinsed in phosphate - buffered saline , and detection was performed using supersignal westpico chemiluminescent substrate ( pierce ). analysis of rna by northern blot was as described previously ( lowen et al ., 2005 ). briefly , bhk - 21 cells in t - 75 flasks were infected at moi of 1 . 0 pfu / cell with the different viruses . forty - eight hours post infection total cellular rna was extracted using trizol reagent ( invitrogen ), and 4 μg of rna was electrophoresed through a 1 . 2 % agarose gel . the rna was then transferred to a positively charged nylon membrane ( roche ), and hybridized with digoxigenin - labelled segment - and strand - specific rna probes ( 150 ng of each probe ), followed by detection with the dig northern starter kit ( roche ). for reverse transcription - pcr analysis , 1 μg of total cellular rna was mixed with 100 pmole of a segment specific oligonucleotide , in buffer containing 0 . 5 mm each dntp , 40 u rrnasin ( promega ) and 200 u m - mlv reverse transcriptase , and incubated at 42 ° c . for 3 hours . the resulting cdna was the used in pcr reactions with the primer sets ψ ( 5 ′- acacaaagaccccctagtgcttatc - 3 ′) and β ( 5 ′- gaatagcaactttataagccatg - 3 ′) and α ( 5 ′- gtatgagctcactgaagactgcaac - 3 ′) and β . the first pair of primers determined the size of the inserted coding sequence , and the second confirmed the insertion of the gn / gc coding sequence ( fig2 ). the amplified dna products were characterized by agarose gel electrophoresis and uv illumination . previous studies have shown that the rvfv nss protein , that is encoded in an ambisense strategy in the genomic s rna segment ( giorgi et al ., 1991 ), is not essential for growth in tissue culture or in animals , and acts a major virulence factor ( vialat et al ., 2000 , bouloy et al ., 2001 , billecocq et al ., 2004 , ikegami et al ., 2006 ). the nsm coding region at the n - terminus of the glycoprotein precursor encoded on the viral m segment is similarly not essential for viral replication , and rift valley fever viruses lacking both the nss and nsm genes were highly attenuated in mammalian cell culture systems and small animal models ( won et al ., 2006 , bird et al ., 2007b , gerrard et al ., 2007b ). such recombinant viruses have potential as live attenuated vaccines ( bird et al ., 2008 ). however , the possibility exists that reverse to virulence could occur via genome segment reassortment with a wild type strain in the field situation ( sall et al ., 1999 , maclachlan and hirsch , 2004 ). in order to reduce this possibility , as well as creating a recombinant virus with a clearly distinguishable genome structure , we investigated whether we could express the viral glycoproteins in an ambisense orientation in a modified s segment in place of the nss protein . this would obviate the need for the m genome segment , and convert rvfv into a two - segmented virus . to this end , we inserted the gn / gc coding sequence in place of the nss orf ( fig1 ), and using reverse genetics we attempted to recover infectious virus . recovery of rvfv using t7 promoter - based plasmids into t7 polymerase expressing cells has been reported ( ikegami , won et al . 2006 ; bird , albarino et al . 2007 ; billecocq , gauliard et al . 2008 ; habjan , penski et al . 2008 , using essentially the procedures first developed for bunyamwera virus ( lowen , noonan et al . 2004 . in this system “ transcription ” plasmids generate full - length rnas corresponding to antigenome - sense rnas and “ support ” plasmids express viral proteins . therefore , bsr - t7 / 5 cells were transfected with transcription plasmids ( based on ptvt7 ) containing parental mp12 l cdna and the chimeric s segment , along with support plasmids ( based on ptm1 ) encoding the viral n and l proteins . as control , the three parental mp12 cdna transcription plasmids were used in a parallel transfection . both transfections yielded virus at the first attempt ; parental mp12 gave 2 × 10 6 pfu / ml whereas as the transfection using the modified s segment gave 3 × 10 5 pfu / ml . independent isolates were obtained by plaque purification on bhk - 21 cells , and after amplification in bhk - 21 cells , the resulting viral stocks were characterised . the virus obtained using the modified s segment was designated r2segmp12 . confirmation of a two segmented rvf virus lacking nss and nsm the s segment rna of each recovered virus was analyzed by reverse transcription - pcr using specific primers as shown in fig2 . reverse transcription was primed with an oligonucleotide specific for the 3 ′ end of the virion - sense s segment , and the cdna used in pcr reactions with primers designed to anneal to the glycoprotein orf ( a ), the 3 ′ end of n orf ( b ) or the s segment 3 ′ utr ( c ). a pcr product of 1041 nt was produced from mp12 derived cdna when using primers b and c that corresponds to the viral 3 ′ utr , nss orf and intergenic region . however , as expected when primer set a and b was used , no pcr product was seen . when the r2segmp12 - derived cdna was used with primer set a and b , a product of 2983 nt was obtained , indictaing that the s rna segment indeed contained sequences encoding the viral glycoproteins . when primer set b and c was used , a product of 3446 nt was obtained , corresponding to the predicted size of the chimeric segment . nuceotide sequence analysis of the amplified dna products confirmed that the recombinant virus s segments had sequences corresponding to the palsmids used for their rescue ( data not shown ). the genome of r2segm12 would comprise an l segment of 6404 nt , and a chimeric s segment of 4105 nt , whereas mp12 virus has genome segment lengths of 6404 nt ( l ), 3885 nt ( m ) and 1690 nt ( s ). northern blot analysis of total rna from infected bhk - 21 cells using segment and strand - specific probes provided further evidence that the recombinant mp12 and r2segmp12 viruses had the predicted genome structures . sizes of rna species detected by the different probes were estimated by reference to an rna size marker ladder on the same gel that was stained with ethidium bromide . the s - probe ( that hybrised to n orf sequences ) detected an rna in mp12 virus - infected cells of approximately 1700 nt , the size expected of the native s genome segment . however , the same s − probe detected an rna species of approximately 4100 nt in r2segmp12 infected cells , as expected for the chimeric s segment . the l − probe detected l segment rna with the expected size in both the mp12 and r2segmp12 infected cells . two m segment derived probes were used , one ( m −) to detect virion - sense rna and the other ( m +) to detect virion - complementary rna . the m − probe detected an rna species of the expected size of the m segment in mp12 - infected cells , and an equivalent sized rna to that detected by the s − probe in the r2segmp12 intracellular rna . when the m + probe was used , a single band was observed in mp12 infected cells , corresponding to m segment antigenome and mrna that would comigrate on the gel . however , two rnas were detected with this probe in r2segmp12 infected cells , the larger corresponding the full - length chimeric s - m segment , and a smaller band corresponding to the subgenomic mrna encoding the gn - gc precursor . as the glycoprotein coding sequence is in the ambisense orientation in the chimeric s segment its messenger rna would be detected by the m + probe . western blot analysis was performed on extracts of cells infected with mp12 and r2segmp12 viruses using monospecific antibodies raised against the rvfv n and nss proteins produced in e . coli . as seen in fig4 a , whereas n protein was clearly detected in lysates from cells infected with either virus , nss was only detected in mp12 virus infected cells . metabolic labeling of infected cells with 35s - methionine produced a similar pattern . compared to mock infected cells , a band of the appropriate size ( approx 25 kda ) was seen in both mp12 and r2segmp12 virus infected cells , but a slightly slower migrating band corresponding to nss ( approx 28 kda ) was only seen in mp12 virus infected cells . also noteworthy is that there little shut - off of host protein synthesis in r2segmp12 - infected cells compared to mp12 infected cells , which correlates with the known effects of nss in inhibiting host cell transcription . finally , immunofluorescent staining of infected cells with monospecific antibodies confirmed that whereas n protein was detected in the cytoplasm of cells infected with both viruses ( fig4 c ), nss was only detected ( in the nucleus , as expected ) in cells infected with mp12 . the growth of r2segmp12 was compared to that of mp12 virus in bhk cells at two multiplicities of infection , 0 . 001 and 1 . 0 pfu / cell ( fig5 a ). at the lower multiplicity , growth of rmp12 was retarded compared to mp12 , though both viruses reached their peak titres by 48 hr p . i . the titre of the two - segment virus was about 75 - fold lower than that of the parental mp12 virus ( 7 × 10 5 pfu / ml vs . 7 × 10 7 pfu / ml ). growth kinetics were similar at the higher multiplicity , and the difference in peak titre was less marked ( 4 . 5 × 10 7 pfu / ml vs . 3 . 5 × 10 8 pfu / ml ). cell extracts taken from the same cultures as above showed that the synthesis of viral n and nss ( where synthesized ) as monitored by western blot analysis with monospecific antibodies reflected the growth curves fig5 b ). the plaque phenotypes of the two - segmented r2segmp12 and the parental mp12 virus were examined in bhk cells ( fig5 c ). the r2segmp12 viral plaques were consistently smaller than mp12 , consistent with the growth kinetics observed in the time course experiments . bunyavirus l proteins are difficult to detect in infected cells , but recently we described the generation of a recombinant bunv expressing l protein tagged with the v5 - epitope ( shi and elliott , 2009 ); the epitope was inserted towards the c terminus of the protein . we sought to create the analogous insertion of the epitope into the rvfv l protein , and based on comparison of available phlebovirus l protein sequences ( data not shown ) we selected 3 positions to insert the v5 epitope ( fig6 a ). the effect of inserting the 14 - residue sequence on l protein function was first assessed in the rvfv minireplicon system ( gauliard et al ., 2006 ). no minireplicon activity was observed when the epitope was inserted at positions l1 and l2 , but a modest activity , approx 17 % of unmodified l protein , was observed when the epitope was inserted in the most c - terminal position , l3 ( fig6 b ). therefore , virus rescue was attempted with the modified l construct , and a recombinant virus was recovered and designated rmp12 - lv5 . this virus gave a smaller plaque size , and grew to slightly lower titres , than parental mp12 fig6 c ). analysis of viral proteins by western blotting revealed that the anti - v5 antibody clearly detected the tagged l protein by 6 hr pi . 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