Patent Application: US-50641806-A

Abstract:
the invention relates to a novel non - radioactive method to detect cytolytic activity that provides a measure of the existence and magnitude of an immune response against a particular antigen or immunogen . provided is a method for detecting cytolytic activity of cells or a substance against a population of target cells , comprising the steps of providing target cells with a first nucleic acid sequence encoding a reporter molecule and a second nucleic acid sequence encoding an antigen of interest ; co - culturing the target cells with a sample containing cells or a substance suspected of having cytolytic activity ; and detecting the viability of target cells provided with the reporter molecule . also provided are a kit and a nucleic acid for use in a method according to the invention .

Description:
cytotoxicity is quantified by assessing the elimination of viable cells expressing an antigen of interest associated with a fluorescent reporter molecule . target cells can be generated by nucleofecting recombinant dna vectors encoding antigen - fluorescent fusion proteins into pbmc or cell lines ( fig1 , panel a ). from three to four hours later , expression of the antigen - reporter protein complex can be detected in sufficient cells to set up co - cultures with effector cell populations of interest . continuous expression of antigen - reporter molecule complexes in the target cells can be detected for several days , depending on the type of target cell and culture conditions . elimination of viable antigen - reporter molecule - expressing target cells ( t ) by cytotoxic effector cells ( e ) can be detected by any device or method that is designed to detect reporter gene expression , here gfp by flow cytometry ( fig1 , panel b ). if the total number of target cells does not significantly change during the co - culture period , specific target cell death can be derived from the change in the fraction dead cells ( to - pro - 3 +) among the cells expressing the reporter gene , using formula 1 of fig1 , panel b . if incubation periods are long and dying target cells disintegrate , specific target cell elimination can be expressed using formula 2 of fig1 , panel b . genes encoding viral proteins of hiv ( rev , tat , gag and nef ) and influenza a virus nucleoproteins np01 , np02 , np03 and matrix - proteinm1 were inserted in frame with gfp in the pegfp - n1 plasmid ( bd biosciences , erembodegem , belgium ) as depicted in fig2 . the sequences of the open reading frames ( orf ) are depicted in fig7 . direct cloning of influenza antigens nucleoprotein or matrix protein in pegfp - n1 was not possible , because the mcs of pegfp - n1 lacks a restriction site that would result in gfp expressed in frame with np / ma . the vector had to be adjusted and simultaneously a gfp construct expressing an env - epitope was created ( peryl - gfp ). for the insert dna , two primers were designed that code for the env - epitope erylkdqql followed by an ecorv restriction site necessary for cloning np / ma in frame with gfp . the primers were diluted to 100 pmol / ml and 2 ml of each primer was mixed , heated for five minutes at 95 ° c . and cooled down to room temperature . annealing of primers leads to double - strand dna with sticky ends complementary to the overhanging basepairs after digestion with xhoi and bamhi . after annealing , the sample was diluted to 400 μl with aqua bidest . after digestion of pegfp - n1 with xhoi x bamhi , the vector ( 4 . 7 kb ) was isolated from a 1 % agarose gel and used for ligation with the annealed primers as described above . correct cloning was confirmed by analysis on agarose gel after digestion with ecorvx noti ( bands 0 . 7 and 4 kb ) and sequencing . plasmid dna of peryl - gfp was digested with xhoi x ecorv . after dna precipitation , the vector was dephosphorylated for one hour at 37 ° c . with alkaline phosphatase ( roche ). the phosphatase was inactivated for ten minutes at 72 ° c . dna of the pb1 - np and pb1 - ma constructs [ 1 ] was digested with xhoi × hpai . bands were isolated ( np 1 . 5 kb , ma 0 . 8 kb , peryl - gfp 4 . 7 kb ) and used for cloning as described above . correct cloning was confirmed by analysis on agarose gel after digestion with xhoi × noti ( bands 1 . 5 / 2 . 2 kb and 3 . 9 kb ) and sequencing . nucleofection of cells of the ebv - transformed lymphoblastoid cell line ( blcl ) b157 with prev - gfp and ptat - gfp resulted in 50 to 60 % gfp + cells . antigen processing and presentation of antigen - gfp fusion protein was first assessed by co - culturing prev - gfp - transfected b157 cells with cells of the rev - specific ctl clone ( 709tcc108 ) at increasing effector - to - target cell ( e / t ) ratios . ptat - gfp - transfected b157 cells were used as negative control cells . after four hours of incubation , the percentages of dead target cells , i . e ., tp3 + gfp + cells , increased from 20 % to 84 % in an e / t ratio - dependent fashion . the proportion of non - viable control target cells did not increase ( fig3 , section a ). after correcting the values for spontaneous background dead cells , the antigen - specific cytolytic activity of the rev - specific ctl at e / t ratios of ˜ 0 . 3 , ˜ 1 and ˜ 3 were 18 %, 58 % and 80 %, respectively ( fig3 , section b , left panel ). next , we explored the use of fresh pbmc as target cells . nucleofection efficiency of un - stimulated pbmc , or cds + depleted pbmc was typically between 30 % and 70 % ( data not shown ), which proved to be sufficient for their use as target cells . mhc - class i matched pbmc , nucleofected with pnp01 - gfp , were lysed by ctl clone tcc - c10 . these data show that blcl cells as well as pbmc can be used as target cells in the fatt - ctl assay of the invention ( fig3 , section b , right panel ). a comparison between the performance of the fatt - ctl assay and the classical 51 cr - release assay the fatt - ctl assay was compared with a standard 51 cr - release assay using the same target cell and effector cell populations in both assays . again , 55 to 60 % viable gfp + events were detected among prev - gfp - and ptat - gfp - transfected b157 cells . assuming that ctl epitopes were generated in the gfp + cells only , this would be the maximum level of specific lysis that could be achieved in the 51 cr - release assay . indeed , 58 % specific lysis was observed at the highest e / t ratio of 10 ( fig4 ). using the fatt - ctl assay , more than 90 % of the gfp + cells were lysed by rev - specific ctl after four hours at the highest e / t ratio . specific lysis of ptat - gfp + cells was & lt ; 3 % for all e / t ratios tested in both assays ( data not shown ). overall , the fatt - ctl assay was capable of detecting cytotoxicity at significantly lower e / t ratios than the 51 cr - release assay ( fig4 ). these data show that the fatt - ctl assay detects the cytolytic activity of ctl and that it does so at lower e / t ratios than a classical 51 cr - release assay . to study the effects of epitope variation on the outcome of fatt - ctl assay , expression vectors encoding various influenza a virus nucleoprotein - and matrix - gfp fusion proteins were constructed . three vectors were generated using np - genes derived from distinct influenza virus strains : pnp01 -, pnp02 -, and pnp03 - gfp . these genes contained the same hla - a * 0101 epitope np44 - 52 sequence , but differed in the hla - b * 3501 epitope np418 - 426 ( fig5 ). pm1 - gfp encoded the hla - a * 0201 restricted epitope m158 - 66 . b3180 cells , which express hla - a * 0101 , - a * 0201 and - b * 3501 , were nucleofected with the different vectors and co - cultured the following day for three hours with or without cells of three different np - specific ctl clones , tcc1 . 7 , tcc - c10and tcc3180 , or the m1 - specific ctl clone m1 / a2 . between 60 % and 70 % specific lysis was detected among np01 -, np02 - and np03 - gfp + cells in cultures containing hla - a * 0101 - restricted tcc1 . 7 ctl , specific for the conserved np44 - 52 epitope ( fig5 ). the hla - b * 3501 - restricted tcc - c10 cells also specifically lysed np01 - gfp + cells ( 70 %), but not np02 - and np03 - gfp + cells , in concordance with previously determined ec50 values of the corresponding peptide variants , ˜ 0 . 8 , & gt ; 5000 and & gt ; 10000 nm , respectively [ 2 ]. npo1 - gfp + cells were lysed with similar efficiency by tcc3180 cells that recognized the np01 peptide variant with an ec50 value of 0 . 5 nm . the lower avidity of these cells for the np02 - variant peptide , ec50 = 26nm , was reflected by an approximately four - fold lower level of specific lysis of np02 - gfp + cells compared to np01 - gfp + cells ( fig5 ). cells expressing the np03 - variant , ec50 = l loonm , were not lysed by tcc3180 . the matrix - specific tcc - m1 / a2 ctl did not specifically lyse the np - gfp - expressing cells , but lysed 50 % of m1 - gfp + cells ( fig5 ). these data show that the fatt - ctl assay detects ctl - mediated lysis of target cells only if they express the correct epitope sequence , and that the assay detects subtle differences in the functional avidity of the ctl . it was also tested whether the fatt - ctl assay could be applied to detect antigen - specific cell - mediated cytotoxicity directly ex vivo . to this end , pbmc were obtained from four highly active antiretroviral therapy ( haart )- naïve hiv seropositive individuals and four seronegative individuals . part of the cells was used to generate target cells by nucleofection with pgag - gfp , pnef - gfp , or pegfp - n1 as a control . gag and nef were chosen as antigens because they are among the most frequently recognized . four hours later , nucleofected and autologous untreated pbmc were co - cultured at pbmc / gfp + cell ratios of ˜ 150 with or without ril - 2 . after overnight incubation , concentrations of viable gfp + events were used to calculate antigen - specific target cell elimination . specific elimination of gag - gfp - and / or nef - gfp -, compared to gfp - expressing cells , was observed in the absence ( individual rh1 - 021 ) or presence ( individuals rh1 - 022 , rh1 - 028 and rh1 - 029 ) of exogenous il - 2 ( fig6 ). for individuals rh1 - 022 , rh1 - 028 and rh1 - 029 , no significant cytotoxicity was observed in the absence of ril - 2 ( data not shown ). due to limiting cell numbers , we could not determine cytotoxicity in the presence of il - 2 for individual rh1 - 021 . no gag - or nef - specific cytotoxicity , compared to gfp alone , was observed for each of the four seronegative controls , irrespective of the presence of exogenous il - 2 ( data not shown ). these data illustrate the practical utility of the fatt - ctl assay to directly measure virus - specific ctl activity ex vivo . procedures for the generation and culturing of the cd8 + t - cell clones used have been previously described [ 2 , 4 , 5 ]: 709 tcc108 : specific for hiv rev67 - 75 epitope saepvplql ; tcc - c10 : influenza a , np418 - 426 epitope lpfekstvm , restricted via hla - b * 3501 ; tcc3180 : influenza a , np418 - 426 epitope lpfekstvm via hla - b * 3501 ; tcc1 . 7 : influenza a , np44 - 52 epitope ctelklsdy via hla - a * 0101 ; tccm1 / a2 : influenza a , m58 - 66 epitope gilgfvftl via hl - a * 0201 . the cells were cultured for at least seven days after stimulation with pha and feeder cells , before use as effector cells in ctl assays . the cloning strategy for the construction of vectors prev - gfp , ptat - gfp , pgag - gfp , pnef - gfp , pnpo1 - gfp , pnp02 - gfp , pnp03 - gfp and pm1 - gfp is depicted in fig2 . genes were cloned into the multiple cloning site of living colors ™ vectors pegfp - n1 , pdsredexpress - n1 and phcredl - n1 / 1 , in frame with the fluorescent protein ( fp ) orf using the indicated restriction enzymes . by omitting the stop - codon of the cloned genes , read - through of the fluorescent gene was achieved . hiv genes were codon - optimized consensus subtype b synthetic genes ( geneart , regensburg , germany ). influenza genes were derived from : np strain a / nl / 18 / 94 ( np01 ), np strain a / hk / 2 / 68 ( np02 ), np strain a / pr / 8 / 34 ( np03 ), m1 strain a / nl / 18 / 94 ( m1 ) [ 6 ]. inserts were sequenced to confirm that no errors had been introduced and that they were expressed in frame with the fluorescent protein orf . sequences ( see fig7 ) have been submitted to genebank . two ebv - transformed b - lymphoblastoid cell lines ( blcl ), b157 and b3180 , were used as source of autologous or hla - matched target cells for the ctl clones . antigen expression was achieved by transfecting blcl cells with plasmid dna vectors using the amaxa nucleofector ™ technology ( amaxa , cologne , germany ) according to the manufacturers &# 39 ; instructions . briefly , one to 2 × 10 6 cells in logarithmic growth phase were resuspended in 100 μl nucleofection buffer containing 2 to 4 μg dna , and subjected to one of the electroporation programs . subsequently , cells were cultured overnight in a final volume of 2 to 4 ml rpmi1640 supplemented with antibiotics and 10 % fetal calf serum ( riof ) at 37 ° c . 5 % co 2 . all buffers and programs of the cell line optimization nucleofector ™ kit ( amaxa ) were tested , and the combination of buffer v with program p - 16 resulted in the highest concentration of viable gfp - expressing cells , combined with high overall viability , i . e ., 50 % after 24 hours ( data not shown ). target cells for the ex vivo fatt - ctl assay were generated by nucleofecting freshly isolated pbmc using the optimized human t - cell nucleofector ™ kit ( amaxa ), as described below . target cells were washed and co - cultured with effector cells at increasing effector - to - target cell ( e / t ) ratios in 200 μl r10f , at 37 ° c . 5 % co 2 for three to four hours . cells were transferred to wells or tubes containing 5 μl edta ( 3 mm final concentration ) to reduce the number of cell to cell conjugates , and 5 μl to - pro - 3 iodide ( tp3 ; 25 nm final concentration , molecular probes , leiden , the netherlands ) to discriminate viable and non - viable cells [ 7 ]. in some experiments , edta / tp3 - treated cells were cooled on ice and stained with anti - cd8 - pe ( bd biosciences , erembodegem - aalst , belgium ) for 20 minutes prior to acquisition . the 5l cr - release assay was performed as described previously [ 4 ]. samples were acquired on a facs - calibur ( bd biosciences ) for a fixed period of 60 seconds per sample . the forward scatter ( fsc ) acquisition threshold was set to include non - viable events . debris was excluded by gating in fsc - tp3 dotplots during data analyses . the flow rate was plotted in a time - event histogram and generally proved to be constant in each of the samples per experiment . if not , we defined a region to select a shared period of constant flow rate . a region to exclude gfp events was defined in gfp - tp3 or gfp - fl3 dotplots of the data acquired from cultures containing blcl cells that had not been nucleofected . gfp + events derived from cultures containing nucleofected blcl cells were displayed in fsc - tp3 or gfp - tp3 dotplots to define viable gfp + ( vg ) events , i . e ., tp3 -, and non - viable or dead gfp + ( dg ) events , i . e ., tp3 + ( see fig2 , line a ). percentages of dead gfp + events (% dg ) were calculated by the formula : 100 * ( number of dg )/( number of vg + number of dg ). ctl - mediated target cell death was calculated with the formula 100 * (% dg + e −% dg − e )/( 100 −% dg − e ) where + e and − e denotes the presence or absence of effector cells in the cultures , respectively . pbmc were isolated by density centrifugation ( lymphoprep ™, nycomed , oslo , norway ) of heparin blood ( 28 to 30 ml ) obtained from four hiv - 1 seropositive individuals visiting the erasmusmc in rotterdam , the netherlands , who received no antiviral treatment , had cd4 counts of more than 300 cells / mm 3 and a viral load between 50 and 1 × 10 5 rna copies / ml . as controls , we isolated pbmc from buffy coats obtained from healthy blood donors . freshly isolated pbmc ( 2 × 10 6 cells / cuvette ) were nucleofected with plasmid dna vectors ( 2 μg ) using the human t - cell nucleofector ™ kit ( amaxa ) according to the manufacturer &# 39 ; s instructions , and incubated in 1 . 5 to 2 . 0 ml r10f medium at 37 ° c ., 5 % co 2 in a humidified incubator . four hours later , we determined the concentration of viable gfp + events in a 50 μl sample using trucount tubes ( bd biosciences ) and initiated co - cultures of ˜ 3000 gfp + events per well with untreated pbmc at a pbmc / gfp + cell ratio of 150 ( triplicates ) in 96 micro - well thermo - fast 96 detection plates ( abgene , surrey , uk ) in a total volume of 200 μl per well with or without ril - 2 ( 50 iu / ml ). after overnight incubation , the cultures were transferred to micronic tubes containing 5 μl edta ( 3 mm final concentration ) and 5 μl tp3 ( 25 nm final concentration ), incubated for 20 minutes at 37 ° c ., transferred to melting ice and acquired on a facs - calibur within two hours . to prevent event count rates exceeding 2000 total event / sec , we set an fl1 - threshold during acquisition to exclude the majority of gfp events , in addition to an fsc - threshold to exclude debris . because many killed gfp + cells can no longer be detected as tp3 + gfp + events after an overnight incubation period ( data not shown ), we used the difference between the number of viable gfp + ( vg ) events in cultures with ( vg + e ) and without ( vg − e ) effector pbmc to calculate the percentage of pbmc - mediated antigen - specific target cell death , i . e ., 100 * ( vg − e − vg + e )/ vg − e . 1 . voeten j . t ., g . f . rimmelzwaan , n . j . nieuwkoop , k . lovgren - bengtsson , and a . d . osterhaus . introduction of the haemagglutinin tran . smembrane region in the influenza virus matrix protein facilitates its incorporation into iscom and activation of specific cd8 (+) cytotoxic t - 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specific ctl response . j . immunol . 2004 ; 172 ( 7 ): 4435 - 43 . 6 . voeten j . t ., g . f . rimmelzwaan , n . j . nieuwkoop , r . a . fouchier , and a . d . osterhaus . antigen processing for mhc class i restricted presentation of exogenous influenza a virus nucleoprotein by b - lymphoblastoid cells . clin . exp . immunol . 2001 ; 125 ( 3 ): 423 - 31 . 7 . lee - macary a . e ., e . l . ross , d . davies , r . laylor , j . honeychurch , m . j . glennie , et al . development of a novel flow cytometric cell - mediated cytotoxicity assay using the fluorophores pkh - 26 and to - pro - 3 iodide . j . immunol . methods 2001 ; 252 ( 1 - 2 ): 83 - 92 . 8 . koksoy s ., a . j . phipps , k . a . hayes , and l . e . mathes . sv40 immortalization of feline fibroblasts as targets for mhc - restricted cytotoxic t - cell assays . vet . immunol . immunopathol . 2001 ; 79 ( 3 - 4 ): 285 - 95 .