Patent Application: US-29749702-A

Abstract:
the invention relates to a composition comprising a t cell epitope or a mixture of t cell epitopes a polycationic peptide and a nucleic acid based on inosin and cytosin and its use as a vaccine .

Description:
the combined injection of polyinosinic - polycytidylic acid ( pic ) and poly - l - arginine ( pr ) synergistically enhances the immune response against ovalbumin - derived peptide . mice c57b1 / 6 ( harlan / olac ) peptide ova 257 – 264 - peptide ( siinfekl ), an mhc class i ( h - 2k b )- restricted epitope of chicken ovalbumin ( rotzschke et al ., 1991 ), was synthesized using standard solid phase f - moc synthesis , hplc purified and analyzed by mass spectroscopy for purity . dose : 300 μg / mouse poly - l - arginine ( pr ) poly - l - arginine with an average degree of polymerization of 60 arginine residues ; sigma chemicals dose : 100 μg / mouse polyinosinic - polycytidylic polyinosinic - polycytidylic acid acid ( pic ) ( sigma chemicals , p - 0913 , lot 125h4024 ) with the molecular weight between 220 , 000 to 460 , 000 dose : 100 μg / mouse on day 0 mice were injected into each hind footpad with a total volume of 100 μl ( 50 μl per footpad ) containing the above mentioned compounds . animals were sacrificed 4 days after injection and popliteal lymph nodes were harvested . single cell suspensions were prepared by mincing lymph nodes through a 70 μm cell strainer . thereafter , cells were washed twice with dmem medium ( life technologies ) containing 5 % fetal calf serum ( fcs , sigma chemicals ). cells were adjusted to 10 7 cells / ml in dmem / 5 % fcs . ifn - γ elispot assays were carried out in triplicates as described ( miyahira et al ., 1995 ). this method is a widely used procedure for the quantification of antigen - specific t cells . lymph node cells were re - stimulated in vitro either with ova - peptide , polyinosinic - polycytidylic acid ( pic ), poly - l - arginine ( pr ), concanavalin a ( cona ) or medium alone ( background ). each spot represents a single ifn - γ - producing t cell . spots were counted using a biosys reader ( biosys , germany ). number of background spots was subtracted from all samples . the results are expressed as the number of spots / 1 × 10 6 unseparated cells ± sd of triplicates . after the stimulation with cona we could detect many spots ( data not shown ) indicating a good condition of the used lymphocytes . as shown in fig1 , by injecting ova - derived peptide with a combination of pic and pr , we could induce almost 800 peptide - specific t cells among one million lymph node cells . in contrast , injections of peptide with either of the substances alone failed to induce peptide - specific t cells ( fig1 ). the combined injection of pic and pr enhances the immune response against ovalbumin - derived peptide in a concentration ( pic )- dependent manner . mice c57b1 / 6 ( harlan / olac ) peptide ova 257 – 264 - peptide ( siinfekl ), an mhc class i ( h - 2k b )- restricted epitope of chicken ovalbumin ( rotzschke et al ., 1991 ), was synthesized using standard solid phase f - moc synthesis , hplc purified and analyzed by mass spectroscopy for purity . dose : 300 μg / mouse poly - l - arginine ( pr ) poly - l - arginine with an average degree of polymerization of 60 arginine residues ; sigma chemicals dose : 100 μg / mouse polyinosinic - polycytidylic polyinosinic - polycytidylic acid acid ( pic ) ( sigma chemicals , p - 0913 , lot 125h4024 ) with the molecular weight ranging from 220 , 000 to 460 , 000 dose : 100 , 50 , 25 μg / mouse 1 . ova 257 - 264 - peptide + pic 100 μg + pr 2 . ova 257 - 264 - peptide + pic 50 μg + pr 3 . ova 257 - 264 - peptide + pic 25 μg + pr 4 . ova 257 - 264 - peptide + pic 100 μg 5 . ova 257 - 264 - peptide + pr on day 0 mice were injected into each hind footpad with a total volume of 100 μl ( 50 μl per footpad ) containing the above mentioned compounds . animals were sacrificed 4 days after injection and popliteal lymph nodes were harvested . lymph node cell suspensions were prepared and ifn - γ elispots were performed as described in example 1 . results are expressed as the number of spots / 1 × 10 6 cells ± sd of triplicates . even very low dose of pic ( 25 μg / mouse ) injected in a combination with pr plus peptide leads to the induction of antigen - specific t cells ( fig2 ). the combined injection of pic and pr enhances the induction of ovalbumin - peptide - specific t cells in a peptide concentration - dependent manner . mice c57b1 / 6 ( harlan / olac ) peptide ova 257 – 264 - peptide ( siinfekl ), an mhc class i ( h - 2k b )- restricted epitope of chicken ovalbumin ( rotzschke et al ., 1991 ), was synthesized using standard solid phase f - moc synthesis , hplc purified and analysed by mass spectroscopy for purity . dose : 300 , 100 , 50 μg / mouse poly - l - arginine ( pr ) poly - l - arginine with an average degree of polymerization of 60 arginine residues ; sigma chemicals dose : 100 μg / mouse polyinosinic - polycytidylic polyinosinic - polycytidylic acid acid ( pic ) ( sigma chemicals , p - 0913 , lot 125h4024 ) with the molecular weight ranging from 220 , 000 to 460 , 000 dose : 100 μg / mouse 1 . ova 257 - 264 - peptide 300 μg + pic + pr 2 . ova 257 - 264 - peptide 100 μg + pic + pr 3 . ova 257 - 264 - peptide 50 μg + pic + pr 4 . ova 257 - 264 - peptide 300 μg + pic 5 . ova 257 - 264 - peptide 300 μg + pr on day 0 mice were injected into each hind footpad with a total volume of 100 μl ( 50 μl per footpad ) containing the above mentioned compounds . animals were sacrificed 4 days after injection and popliteal lymph nodes were harvested . lymph node cell suspensions were prepared and ifn - γ elispots were performed as described in example 1 . results are expressed as the number of spots / 1 × 10 6 cells ± sd of triplicates . as shown in fig3 , comparably strong immune response can be induced even when lower peptide dose ( 100 μg instead of 300 μg / mouse ) is used for the vaccination . the combined injection of pic and pr synergistically enhances the immune response against a trp - 2 ( mouse tyrosinase - related protein - 2 )- derived peptide . mice c57b1 / 6 ( harlan / olac ) peptide trp - 2 - peptide ( vydffvwl ), an mhc class i ( h - 2k b )- restricted epitope of mouse tyrosinase - related protein - 2 ( bloom et al ., 1997 ) was synthesized by standard solid phase f - moc synthesis , hplc purified and analysed by mass spectroscopy for purity . dose : 100 μg / mouse poly - l - arginine ( pr ) poly - l - arginine with an average degree of polymerization of 60 arginine residues ; sigma chemicals dose : 100 μg / mouse polyinosinic - polycytidylic polyinosinic - polycytidylic acid acid ( pic ) ( sigma chemicals , p - 0913 , lot 125h4024 ) with the molecular weight ranging from 220 , 000 to 460 , 000 dose : 100 μg / mouse on day 0 mice were injected into each hind footpad with a total volume of 100 μl ( 50 μl per footpad ) containing the above mentioned compounds . animals were sacrificed 4 days after injection and popliteal lymph nodes were harvested . preparation of lymph nodes and ifn - γ elispots were carried out as described in example 1 . results are expressed as the number of spots / 1 × 10 6 cells ± sd of triplicates . our results show that pic and pr also synergistically act in inducing trp - 2 peptide - specific t cells ( fig4 ). the combined application of pic and pr strongly enhances the induction of t cells specific for a mastocytoma - derived peptide . mice dba / 2 ( harlan / olac ) peptide mouse mastocytoma p815 - derived peptide p1a ( lpylgwlvf ), restricted to mhc class i ( h2 - l d ) ( lethe et al ., 1992 ), synthesized by standard solid phase f - moc synthesis , hplc purified and analysed by mass spectroscopy for purity . dose : 300 μg / mouse poly - l - arginine ( pr ) poly - l - arginine with an average degree of polymerization of 60 arginine residues ; sigma chemicals dose : 100 μg / mouse polyinosinic - polycytidylic polyinosinic - polycytidylic acid acid ( pic ) ( sigma chemicals , p - 0913 , lot 125h4024 ) with the molecular weight ranging from 220 , 000 to 460 , 000 dose : 100 μg / mouse on day 0 mice were injected into each hind footpad . with a total volume of 100 μl ( 50 μl per footpad ) containing the above mentioned compounds . animals were sacrificed 4 days after injection and popliteal lymph nodes were harvested . preparation of lymph nodes and ifn - γ elispots were carried out as described in example 1 . results are expressed as the number of spots / 1 × 10 6 cells ± sd of triplicates . as shown in fig5 , the combined application of pic and pr induces strong antigen - specific response also in another mouse strain . the combined application of oligo - dic 26 and pr synergistically enhances the immune response against ovalbumin - derived peptide . mice c57b1 / 6 ( harlan / olac ) peptide ova 257 – 264 - peptide ( siinfekl ), an mhc class i ( h - 2k b )- restricted epitope of chicken ovalbumin ( rotzschke et al ., 1991 ), was synthesized using standard solid phase f - moc synthesis , hplc purified and analyzed by mass spectroscopy for purity . dose : 300 μg / mouse poly - l - arginine ( pr ) poly - l - arginine with an average degree of polymerization of 60 arginine residues ; sigma chemicals dose : 100 μg / mouse oligo - deoxy ic , 26 - mer oligo - dic was synthesized by standard ( oligo - dic 26 ) phosphoamidid chemistry on a 4 μmol scale and purified by hplc ( naps göttingen , germany ) dose : 5 nmol / mouse on day 0 mice were injected into each hind footpad with a total volume of 100 μl ( 50 μl per footpad ) containing the above mentioned compounds . animals were sacrificed 4 days after injection and popliteal lymph nodes were harvested . preparation of lymph nodes and ifn - γ elispots were carried out as described in example 1 . results are expressed as the number of spots / 1 × 10 6 cells ± sd of triplicates . as shown in fig6 , the combined application of oligo - dic 26 and pr induces strong peptide - specific t cell response . the antigen - specific immune response against ovalbumin - derived peptide induced by combined injection of poly - l - arginine ( pr ) and polyinosinic - polycytidylic acid ( pic ) is systemic . mice c57b1 / 6 ( harlan / olac ) peptide ova 257 – 264 - peptide ( siinfekl ), an mhc class i ( h - 2k b )- restricted epitope of chicken ovalbumin ( rotzschke et al ., 1991 ), was synthesized using standard solid phase f - moc synthesis , hplc purified and analyzed by mass spectroscopy for purity . dose : 300 μg / mouse poly - l - arginine ( pr ) poly - l - arginine with an average degree of polymerization of 60 arginine residues ; sigma chemicals , p - 4663 , lot 68h5903 ; with the average molecular weight ( mw ) 10 , 000 dose : 100 μg / mouse polyinosinic - polycytidylic polyinosinic - polycytidylic acid acid ( pic ) ( sigma chemicals , p - 0913 , lot 109h4037 ) with the molecular weight between 220 , 000 to 460 , 000 ( average length : 500 bp ) dose : 100 μg / mouse on day 0 , mice were injected either into hind footpads or into the flank with a total volume of 100 μl ( 50 μl per each footpad , 100 μl per flank ) containing the above listed compounds . four mice per group were sacrificed 4 days after injection and draining lymph nodes ( popliteal and inguinal lymph nodes for footpad and flank injection , respectively ) were harvested . preparation of lymph nodes and ifn - γ elispots were carried out as described in example 1 . as already shown in previous examples , by injecting ova - derived peptide in combination with pic and pr , strong peptide - specific t cell response could be induced on day 4 in draining lymph nodes . to investigate whether the immune response induced by one single injection of peptide with pic / pr is systemic , the rest of the animals ( four per group ) was bled from the tail vein at selected time points after injection , peripheral blood leukocytes ( pbls ) were isolated , re - stimulated either with the relevant peptide , pic , pr , cona ( positive control ) or medium alone ( background ) and the number of ifn - γ - secreting t lymphocytes was determined using an elispot assay . assays were carried out in duplicates . each spot represents a single ifn - γ - producing t cell . spots were counted using a biosys reader ( biosys , germany ). number of background spots was subtracted from all samples . results are expressed as the number of spots / 1 × 10 6 cells ± sd of duplicates . after the stimulation of pbls with cona , many spots demonstrating a good condition of the used lymphocytes could be detected . these results showed that injection of peptide with pic / pr indeed results in the systemic response as observed on day 7 in peripheral blood ( fig7 a , b ). in contrast , there was almost no or very weak peptide - specific immune response detectable on day 7 when mice were injected with peptide and pr or peptide and pic . the strong systemic response induced by single injection of peptide with combination of pr / pic declined rapidly within the next 30 days . to determine whether any component of the vaccine could have undesired effects for the host , e . g ., induce the systemic release of pro - inflammatory cytokines , animals were injected into hind footpads with combinations as mentioned before , sera from mice were collected one hour after injection and were screened for tnf - α and il - 6 by elisa . neither tnf - α nor il - 6 could be detected in the serum of any of the mice , whether injected with peptide / pr , peptide / pic or peptide and the combination of both substances . these results indicate that the response induced by injection of peptide antigen with a mixture composed of pic and pr is systemic . the combined injection of polyinosinic - polycytidylic acid ( pic ) and various polycationic compounds synergistically enhances the immune response against ovalbumin - derived peptide . mice c57b1 / 6 ( harlan / olac ) peptide ova 257 – 264 - peptide ( siinfekl ), an mhc class i ( h - 2k b )- restricted epitope of chicken ovalbumin ( rotzschke et al ., 1991 ), was synthesized using standard solid phase f - moc synthesis , hplc purified and analyzed by mass spectroscopy for purity . dose : 300 μg / mouse poly - l - arginine ( pr ) poly - l - arginine with an average degree of polymerization of 60 arginine residues ; sigma chemicals , p - 4663 , lot 68h5903 ; with the average molecular weight ( mw ) 10 , 000 dose : 100 μg / mouse poly - l - lysine ( pl ) poly - l - lysine , sigma chemicals , p - 6516 , lot 78h5910 ; with the average mw 9 , 500 dose : 100 μg / mouse poly - l - ornithine ( po ) poly - l - ornithine , sigma chemicals , p - 4538 , lot 57h5515 ; with the average mw 10 , 000 dose : 100 μg / mouse diethylaminoethyl - dextran deae - dextran , chloride form , prepared ( deae - dextran ) from dextran of the average molecular weight 500 , 000 ; sigma chemicals , d - 9885 , lot 39h1323 dose : 100 μg / mouse polyinosinic - polycytidylic polyinosinic - polycytidylic acid acid ( pic ) ( amersham pharmacia biotech , 27 – 4732 , lot 6034732012 ) dose : 50 μg / mouse 1 . ova 257 - 264 - peptide + pic + pr 2 . ova 257 - 264 - peptide + pic 3 . ova 257 - 264 - peptide + pr 4 . ova 257 - 264 - peptide 5 . ova 257 - 264 - peptide + pic + pl 6 . ova 257 - 264 - peptide + pl 7 . ova 257 - 264 - peptide + pic + po 8 . ova 257 - 264 - peptide + po 9 . ova 257 - 264 - peptide + pic + deae - dextran 10 . ova 257 - 264 - peptide + deae - dextran on day 0 , mice were injected into each hind footpad with a total volume of 100 μl ( 50 μl per footpad ) containing the above listed compounds . animals were sacrificed 4 days after injection and popliteal lymph nodes were harvested . lymph node cell suspensions were prepared and ifn - γ elispots were performed as described in example 1 . results are expressed as the number of spots / 1 × 10 6 cells ± sd of triplicates . as shown in fig8 , injection of peptide in combination with pic and any of the above listed polycationic compounds ( pr , pl , po , deae - dextran ) leads to the strong peptide - specific response . in contrast , injection of peptide with either of the substances alone did not induce peptide - specific t cells . interestingly , a 50 - fold higher amount of deae - dextran has to be used in combination with pic to induce the same numbers of peptide - specific ifn - γ - producing t cells as injection of pic with any of other used polycationic compounds ( pr , pl , po ). the combined injection of ovalbumin ( ova ) with poly - l - arginine ( pr ) and polyinosinic - polycytidylic acid ( plc ) synergistically enhances the ova - specific immune response . mice c57b1 / 6 ( harlan / olac ) ovalbumin ( ova ) ovalbumin from chicken egg , grade v , sigma chemicals , a - 5503 , lot 54h7070 dose : 50 μg / mouse peptides ova 257 – 264 - peptide ( siinfekl ), an mhc class i ( h - 2k b )- restricted dominant epitope of chicken ovalbumin ( rotzschke et al ., 1991 ), ova 265 – 280 - peptide ( tewtssnvmeerkikv ), an mhc class ii ( h - 2a b )- restricted epitope of chicken ovalbumin ( rotzschke et al ., 1991 ) were synthesized using standard solid phase f - moc synthesis , hplc purified and analyzed by mass spectro - scopy for purity . dose used for the re - stimulation of lymph node cells : 10 μg / ml . poly - l - arginine ( pr ) poly - l - arginine with an average degree of polymerization of 60 arginine residues ; sigma chemicals , p - 4663 , lot 68h5903 dose : 100 μg / mouse polyinosinic - polycytidylic polyinosinic - polycytidylic acid acid ( pic ) ( amersham pharmacia biotech , 27 – 4732 , lot 6034732012 ) dose : 50 μg / mouse on day 0 , mice were injected into each hind footpad with a total volume of 100 μl ( 50 μl per footpad ) containing the above listed compounds . animals were sacrificed 4 days after injection and popliteal lymph nodes were harvested . lymph node cell suspensions were prepared and ifn - γ elispots were performed as described in example 1 . results are expressed as the number of spots / 1 × 10 6 cells ± sd of triplicates . as shown in fig9 , injection of ovalbumin ( ova ) with either pic or pr leads to the antigen - specific immune response when compared with injection of ova alone . however , when ova is injected with a mixture of pic and pr , the synergizing effect of both substances can be observed , resulting in enhanced antigen - specific t cell response . importantly , the ifn - γ production was detected not only upon the re - stimulation with the whole ova protein but also with both , mhc class i ( ova 257 - 264 )- and ii ( ova 265 - 280 )- restricted ova - derived epitopes ( fig9 ). these data demonstrate that using the combination of pic and pr , not only peptides but also whole proteins can be used as an antigen for the vaccine composition . poly - l - arginine does not affect polyinosinic - polycytidylic acid ( pic )- induced in vitro maturation of dcs . lipopolysaccharide ( lps ) lipopolysaccharide from escherichia coli ; serotype 055 : b5 ( sigma chemicals ) dose : 1 μg / ml poly - l - arginine ( pr ) poly - l - arginine with an average degree of polymerization of 60 arginine residues ; sigma chemicals , p - 4663 , lot 68h5903 dose : 10 μg / ml polyinosinic - polycytidylic polyinosinic - polycytidylic acid acid ( pic ) ( amersham pharmacia biotech , 27 – 4732 , lot 6034732012 ) dose : 10 μg / ml 1 . medium 2 . lps 3 . pr 4 . pic 5 . pr + pic it has previously been described that pic , similar to influenza virus infection , triggers maturation of human dcs in vitro ( cella et al , 1999 ; verdijk et al ., 1999 ). human monocyte - derived dcs were used to investigate how poly - l - arginine affects this pic - induced dc maturation . human dcs were generated from monocytes . briefly , peripheral blood leukocytes ( pbls ) were isolated from buffy coats of healthy volunteers by ficoll gradient centrifugation . monocytes were isolated from pbls using cd14 - coated magnetic beads ( miltenyi biotec inc ., germany ) applied according to the manufacturer &# 39 ; s instructions . using this method , we obtained & gt ; 95 % cd14 + cells as determined by flow cytometry . these cd14 + monocytes were cultured in rpmi 1640 medium supplemented with 10 % fcs ( paa laboratories , linz , austria ), non - essential aminoacids , l - glutamin , gentamycin , sodium pyruvate , 100 ng / ml human gm - csf and 500 u / ml human il - 4 in 6 - well tissue plates for 6 – 7 days . to this end , the cultures contained & gt ; 80 % mhc class ii + / cd1a + cells (= dcs ). to determine phenotypic maturation , day 6 - cultured dcs were stimulated either with pic , pr or with a combination of both substances for 48 hours and were analyzed for the expression of several surface molecules by flow cytometry . for control purposes , dcs were also stimulated with lps or were left untreated . as shown in fig1 , pic induced an up - regulation of hla - dr , - dq and hla - i molecules , co - stimulatory molecules such as cd40 , cd54 , cd80 , de - novo expression of cd86 and the maturation marker cd83 as well as a down - regulation of cd1a molecules when compared to untreated dcs . the maturation effect of pic was in all cases comparable to that induced by lps . in addition , this analysis revealed a slight up - regulation of cd11a , cd11c , cd13 , cd25 , cd29 and cd50 antigens on dcs upon pic stimulation . none of the above described phenotypic changes could be observed when dcs were incubated with pr alone . the phenotype of dcs stimulated with a mixture of pic and pr was similar to that induced by pic alone ( fig1 ). therefore , pic has the capacity to induce maturation of human dcs in vitro , and pr does not affect this pic - induced differentiation process . the antigen - specific immune response induced by the combined application of antigen with oligo - deoxyic 26 - mer and poly - l - arginine ( pr ) is systemic . mice c57b1 / 6 ( harlan / olac ) peptide ova 257 – 264 - peptide ( siinfekl ), an mhc class i ( h - 2k b )- restricted epitope of chicken ovalbumin ( rotzschke et al ., 1991 ), was synthesized using standard solid phase f - moc synthesis , hplc purified and analyzed by mass spectroscopy for purity . dose : 300 μg / mouse poly - l - arginine ( pr ) poly - l - arginine with an average degree of polymerization of 60 arginine residues ; sigma chemicals dose : 100 μg / mouse oligo - deoxy ic , 26 - mer oligo - dic 26 - mer was synthesized by ( oligo - dic 26 - mer ) standard phosphoamidid chemistry on a 4 μmol scale and purified by hplc ( naps göttingen , germany ) dose : 5 nmol / mouse 1 . ova 257 - 264 - peptide + oligo - dic 26 - mer + pr 2 . ova 257 - 264 - peptide + oligo - dic 26 - mer 3 . ova 257 - 264 - peptide + pr 4 . ova 257 - 264 - peptide on day 0 , mice were injected into each hind footpad with a total volume of 100 μl ( 50 μl per footpad ) containing the above listed compounds . animals ( 4 mice / group ) were sacrificed 4 days after injection and popliteal lymph nodes were harvested . lymph node cell suspensions were prepared and ifn - γ elispots were performed as described in example 1 . as already shown previously , the combined application of peptide with oligo - dic 26 - mer and pr induced strong antigen - specific response against ova - derived peptide on day 4 in draining lymph node cells . to examine whether the immune response induced by one single injection of peptide with oligo - dic / pr is systemic , the rest of the mice ( 4 mice / group ) was investigated at selected time points after injection for the presence of peptide - specific ifn - γ - producing t cells in spleen or peripheral blood using an ifn - γ elispot assay . results are expressed as the number of spots / 1 × 10 6 cells ± sd of duplicates . fig1 shows that the response induced by the injection of ova - derived peptide with oligo - dic / pr is systemic and lasts at least until day 59 after one single injection ( the latest time point of investigation ). in contrast , the local or systemic response could not be observed when peptide was injected alone , with oligo - dic 26 - mer or pr . to determine whether any component of the vaccine could have undesired effects for the host , e . g ., induce the systemic release of pro - inflammatory cytokines , animals were injected into hind footpads with combinations as mentioned before , sera from mice were collected one hour after injection and were screened for tnf - α and il - 6 by elisa . neither tnf - α nor il - 6 could be detected in the serum of any of the mice , whether injected with peptide / pr , peptide / oligo - dic or peptide and the combination of both substances . these results indicate that the response induced by injection of peptide antigen with a mixture composed of oligo - dic and pr is systemic . the combined application of oligo - deoxyic 26 - mer and poly - l - arginine ( pr ) enhances the ovalbumin ( ova )- specific t cell response . mice c57b1 / 6 ( harlan / olac ) ovalbumin ( ova ) ovalbumin from chicken egg , grade v . sigma chemicals , a - 5503 , lot 54h7070 dose : 50 μg / mouse peptides ova 257 – 264 - peptide ( siinfekl ), an mhc class i ( h - 2k b )- restricted dominant epitope of chicken ovalbumin ( rotzschke et al ., 1991 ), ova 265 – 280 - peptide ( tewtssnvmeerkikv ), an mhc class ii ( h - 2a b )- restricted epitope of chicken ovalbumin ( rotzschke et al ., 1991 ) were synthesized using standard solid phase f - moc synthesis , hplc purified and analyzed by mass spectroscopy for purity . dose used for the re - stimulation of cells : 10 μg / ml poly - l - arginine ( pr ) poly - l - arginine with an average degree of polymerization of 60 arginine residues ; sigma chemicals , p - 4663 , lot 68h5903 dose : 100 μg / mouse oligo - deoxy ic , 26 - mer oligo - dic 26 - mer was synthesized by ( oligo - dic 26 - mer ) standard phosphoamidide chemistry on a 4 μmol scale and purified by hplc ( naps göttingen , germany ) dose : 5 nmol / mouse on day 0 , mice were injected into each hind footpad with a total volume of 100 μl ( 50 μl per footpad ) containing the above listed compounds . animals were sacrificed 4 days after injection and popliteal lymph nodes were harvested . lymph node cell suspensions were prepared and ifn - γ elispots were performed as described in example 1 . results are expressed as the number of spots / 1 × 10 6 cells ± sd of triplicates . as shown in fig1 a , injection of ovalbumin ( ova ) with a mixture of oligo - dic and pr results in enhanced antigen - specific t cell response . importantly , the ifn - γ production was detected not only upon the re - stimulation with the whole ova protein but also with both , mhc class i ( ova 257 - 264 )- and ii ( ova 265 - 280 )- restricted ova - derived epitopes ( fig1 a ). to further examine whether the immune response induced by one single injection of ova protein with oligo - dic / pr is systemic , the rest of the mice ( 4 mice / group ) was investigated at selected time points after injection for the presence of peptide - specific ifn - γ - producing t cells in peripheral blood using an ifn - γ elispot assay . results are expressed as the number of spots / 1 × 10 6 cells ± sd of duplicates . fig1 b shows that the response induced by the injection of ova with oligo - dic / pr is systemic and persisting at least until day 97 after injection ( the latest time point of investigation ). in contrast , the local or systemic response could not be observed when ova was injected alone , with oligo - dic 26 - mer or pr . these data demonstrate that using the combination of oligo - dic and pr , not only peptides but also whole proteins can be used as an antigen for the vaccine composition and that the response induced by injection of protein antigen with a mixture composed of oligo - dic and pr is systemic and longer - lasting . the combined application of oligo - deoxyic 26 - mer and poly - l - arginine ( pr ) enhances the ovalbumin ( ova )- specific humoral response . mice c57b1 / 6 ( harlan / olac ) ovalbumin ( ova ) ovalbumin from chicken egg , grade v , sigma chemicals , a - 5503 , lot 54h7070 dose : 50 μg / mouse poly - l - arginine ( pr ) poly - l - arginine with an average degree of polymerization of 60 arginine residues ; sigma chemicals , p - 4663 , lot 68h5903 dose : 100 μg / mouse oligo - deoxy ic , 26 - mer oligo - dic 26 - mer was synthesized by ( oligo - dic 26 - mer ) standard phosphoamidide chemistry on a 4 μmol scale and purified by hplc ( naps göttingen , germany ) dose : 5 nmol / mouse on day 0 , mice were injected into each hind footpad with a total volume of 100 μl ( 50 μl per footpad ) containing the above listed compounds . on day 24 after injection , serum was collected and screened by elisa for the presence of ova - specific antibodies . these results show that the injection of ova in combination with oligo - dic and pr enhanced the production of ova - specific igg antibodies when compared with injection of ova with each of the substances alone ( fig1 a , b ). interestingly , titers of both igg2a and igg1 were increased upon one single injection of ova with oligo - dic / pr , implying that both th1 and th2 cells were involved . however , after 115 days only the increased igg2a levels were still detectable in sera of mice injected with ova and oligo - dic / pr . these data demonstrate that the combined injection of ova with oligo - dic and pr enhances the ova - specific humoral response . this response is characterized by the production of both th1 - and th2 - induced antibody isotypes in the early phase , but later , mainly by th1 - induced antibodies . bloom , m . b ., perry - lalley , d ., robbins , p . f ., li , y ., el - gamil , m ., rosenberg , s . a ., and yang , j . c . 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