Patent Application: US-201213624304-A

Abstract:
the invention relates to recombinant mva which is capable of expressing structural hcv antigens , functional parts of said structural antigens or epitopes of said structural antigens . the invention further relates to a pharmaceutical composition , especially in the form of a vaccine and containing the recombinant mva according to the invention , to eukaryotic cells that contain the inventive recombinant mva and to various uses of the recombinant mva , for example for producing recombinant structural proteins , for producing a pharmaceutical preparation that is suitable for the therapy and prophylaxis of hcv infections and diseases thereby caused . the invention further relates to methods for producing recombinant mva and recombinant structural hcv polypeptides encoded by said recombinant mva , and to dna or rna of said recombinant mva .

Description:
primary chicken embryo fibroblasts ( cef ), baby hamster kidney bhk - 21 ( atcc ccl - 10 ), rabbit kidney - rk - 13 ( atcc ccl - 37 ) and mouse p815 ( h - 2d , atcc tib64 ) cells were grown in minimal essential medium ( mem ) or rpmi 1640 medium supplemented with 10 % fetal calf serum ( fcs ). the cells were maintained in a humid air 5 % co 2 atmosphere at 37 ° c . vaccinia virus mva ( cloned isolate f6 , 45 , 60 ) was routinely propagated and its titer was detected by a vaccinia virus specific immune staining in cef to determine the infectious units ( iu )/ ml . virus from the 582 nd cef run were used for this study . recombinant virus mva - lz encoding the e . coli lacz reporter gene ( 60 ) was used in the plaque reduction assay to determine vaccinia virus mva specific neutralizing antibodies . for the construction of mva vector plasmids containing structural hcv genes , dna fragments from cloned hcv - cdna were prepared which originally were obtained from y . wang ( university of beijing , genbank ® accession number d10934 , 63 , 38 ). a dna segment containing the coding sequence for the hcv amino acids ( aa ) 1 to 742 was obtained by ecori and hindiii cleavage of puc18 / ce1e2 , treated with klenow polymerase , for the formation of blunt ends and cloned into the single pmei - site of piiidhr - p7 . 5 , to obtain piiidhr - p7 . 5 - hcv ( 1 - 742 ). to obtain mva vector plasmids piiidhr - p7 . 5 - hcv ( 1 - 661 ) and piiidhr - p7 . 5 - hcv ( 151 - 661 ), dna fragments encoding hcv - aa 1 - 661 or aa 151 - 651 were amplified by means of a polymerase chain reaction ( pcr ) from the same hcv - cdna matrix , wherein the following oligonucleotides were used : hcv - 5 ′- 1 5 ′- cat gg g aat tc c cat gag - 3 ′, hcv - 5 ′- 151 5 ′- ggc gct gc g aat tc c atg gcg cat ggc gtc cgg - 3 ′ and hcv - 3 ′- 661 5 ′- ggg ggg gaa ttc tca ctc tga tct atc cct gtc - 3 ′ ( eco ri - sites are underlined ). the pcr products were cleaved with ecori , blunt ended with klenow and cloned into the pmei site of piiidhr - p7 . 5 . monolayers of nearly confluent bhk - 21 cells in tissue culture plates with six wells ( corning , corning , n . y ., usa ) were infected with mva at a multiplicity of 0 . 01 infectious units per cell and transfected 90 min after infection with 1 . 5 μg plasmid dna per well by means of fugene 6 - transfection reagent ( roche molecular biochemicals mannheim , germany ) according to the manufacturer &# 39 ; s specifications . 24 h after infection the cells were harvested and processed as described for the isolation of rmva by selection for transient extension of the host cell spectrum ( 58 ). recombinant viruses were seen after the formation of typical infection foci in rk - 13 cell monolayers . the rk - 13 cells did not support the productive growth of parenteral mva . after 3 propagation cycles of plaque purification in rk - 13 cells , the rmva were sent through bhk - 21 cells to remove the selectable marker gene k1 l . virus starting materials were prepared in bhk - 21 cells , purified via a two - step ultra centrifugation through a sucrose pad , the titre was determined on cef - cells by means of vaccinia virus specific immune staining , separated into aliquots and stored at − 80 ° c . monolayers of bhk - 21 cells were infected with 10 infectious rmva units per cells . after 24 h the infected cells were harvested , collected by centrifugation and lysed in sds gel loading buffer ( 50 mm tris - hcl , ph 6 . 8 , 2 % sds , 0 . 04 % bromophenolblue , 84 mm β - mercaptoethanol , 20 % glycerol ). total cell proteins were electrophoresed on a sds polyacrylamide gel and electroblotted onto nitrocellulose . the blot membranes were blocked for 1 h in phosphate buffered saline buffer ( pbs ) with 2 % ( w / v ) bovine serum albumin ( bsa ) and 0 . 05 % ( v / v ) tween 20 , then incubated for 4 h with polyclonal rabbit anti - hcv - e2 antibodies ( serum re2116 , 1 : 5000 ), polyclonal rabbit anti - hcv - antibodies ( antigenix , amerika inc ./ biotrade , vienna , austria , 1 : 10000 ) or monoclonal mouse anti - hcv - core - antibodies c1 ( 1 : 5000 , kindly offered by ramsey c . cheung , stanford university school of medicine , stanford , calif ., usa ), diluted in the same pbs buffer . after washing with 0 . 05 % tween 20 in pbs , the blots were incubated for 1 h with anti - rabbit or anti - mouse igg - antibody , conjugated to horseradish peroxidase ( dianova , hamburg , germany ) and diluted 10000 fold in blocking buffer , incubated , again washed and developed by means of lumi - light western blotting substrate ( roche molecular biochemicals , mannheim , germany ). for the control of secreted recombinant hcv proteins cell free supernatants from mva infected cultures which were grown in serum free opti - mem ® ( gibco brl ), were collected 24 hours after infection . proteins from the supernatants were precipitated with an equal volume of ice - cold ethanol , obtained by an 1 h centrifugation at 10000 × g / 4 ° c ., resuspended in pbs and subjected to a western blot analysis . the endoglycosidases endoh and pngasef ( new england biolabs , frankfurt am main , germany ) were used for the deglycosylation of recombinant proteins . the total proteins precipitated from infected cells or from culture supernatants were denatured for 10 min at 100 ° c ., 2 h at 37 ° c . in 0 . 5 % sds , 1 % β - mercaptoethanol , with endoh or pngase f , as described by the manufacturer &# 39 ; s instructions , cleaved and analyzed by western blotting . groups of five balb / c mice ( 6 to 8 weeks old , obtained by charles river , sulzfeld , germany ) were intraperitoneally immunized with 1 × 10 8 infectious mva or rmva units ( in 0 . 5 ml sterile pbs ) per animal at the days 0 , 38 und 81 . the mice were bled through the retroorbital - plexus at days − 7 , 21 , 48 , 103 and 158 . the blood was collected in a microcentrifuge tube , incubated for 4 h at room temperature and centrifuged for 10 min at 2700 × g / 4 ° c . the sera obtained were stored at − 20 ° c . enzyme immunosorbent assays ( elisa ) were used for the determination of the presence of antibodies against hcv nucleus und e2 - antigens in serum probes . the antigens used for coating the 96 well flat bottom plates ( maxisorp surface , nunc , wiesbaden , germany ) in a concentration of 1 μg / ml were hcv nucleus protein ( aa 2 to 192 , bio trade , vienna , austria ) or hcv e2 produced in e . coli ( aa 450 to 565 ). the antigens were suspended in pbs with 0 . 02 % sodium azide , plated with 50 μl / well and incubated over night at 4 ° c . subsequently , the content of each well was removed and washed three times with pbs plus 0 . 05 % tween 20 ( pbs - t ). blocking buffer ( 1 % bsa in pbs - t ) was added at 200 μl / well , and the plates were incubated for 1 h at 37 ° c . the plates were washed with pbs - t , and twofold serial dilutions of the serum samples in blocking buffer were added in a volume of 100 μl / well and incubated for 3 h at 37 ° c . after three washing steps with pbs - t , alkaline phosphatase conjugated goat anti mouse immunoglobulin g ( dianova , hamburg , deutschland , 1000 × diluted in blocking buffer ) was added and incubated for 1 h at 37 ° c . after 8 times washing of the plates with pbs - t , the wells were developed with para - nitrophenylphosphate substrate ( sigma , deisenhofen , germany ). after 30 min incubation at room temperature the reaction was stopped by the addition of 0 . 5 m naoh , and the extinction was measured at 405 nm with a microplate reading device ( model 550 , bio rad laboratories , munich , germany ). the antibody titers were calculated as a twofold serial dilution resulting in an optical density ( od ), which was twice as high as the cutoff . the cutoff value was estimated as the average od of sera from control mice , which were vaccinates with non - recombinant mva . vaccinia virus mva specific antibodies were analyzed by a plaque reduction test by means of recombinant mva - lz . twofold serial dilutions of sera from immunized mice were reacted with 200 infectious mva - lz units in a total volume of 200 μl of pbs and incubated for 3 h at 37 ° c . thereafter , the confluent bhk - 21 monolayer were incubated in a double approach , and foci of virus infected cells were visualized 48 h after vaccination by staining with 5 - bromo - 4 - chloro - 3 - indolyl - β - galactopyranoside substrate ( x - gal , roche molecular biochemicals , mannheim , germany ) as described above ( 16 ). the blue colored foci were counted , and the number obtained with each serum was compared to controls with preimmune sera or without mouse serum . the antibodies were calculated as twofold serial dilution , which resulted in a 50 % reduction of the foci number . the methods were done essentially as described before ( 5 ). splenocytes , obtained 12 weeks after the last virus vaccination of the mice , were used as in vitro responder cells . 5 × 10 6 cells were grown in the presence of 5 μg / ml of e1 peptide ( hcv aa 312 - 326 ) in rpmi1640 medium supplemented with 10 % fcs , 25 μm 2 - mercaptoethanol , 1 mm sodium pyruvate and 2 mm l - glutamine . on the second day , 10 % tcgf was added to the cultures . after 7 days the effector cells were harvested and tested for specific cytotoxicity , wherein a 51 cr release test was used . 1 × 10 6 p815 or t2 target cells were incubated with 75 μci na 51 cro 4 in 200 μl rpmi / 10 % fcs 1 h at 37 ° c . the cells were washed three times with rpmi / 10 % fcs and incubated three times with 5 μg / ml e1 peptide ( hcv aa 312 - 326 ) or 1 μg / ml a / pr / 8 / 34 - influenza virus matrix protein m1 peptide for 30 min at 37 ° c . responder and target cells were co - cultivated in different ratios in plates with 96 wells for 5 h at 37 ° c . 30 μl supernatant were harvested to determine the amount of released 51 cr . all samples were counted in a topcount nxt ( packard , downers grove ill . ), and the percentage of specific release was calculated as : [( experimental release − spontaneous release )/( total release − spontaneous release )]× 100 . the release was measured in counts per min . the tests were carried out in a double approach . for restimulation , 5 × 10 6 irradiated splenocytes ( 3000 rad ) which were derived from non - immunized balb / c mice and which were incubated for 2 h with 10 μg / ml e1 peptide ( hcv aa 312 - 326 ) were added to 1 × 10 6 effector cells and grown for 7 days in rpmi medium supplemented with 10 % fcs , 25 μm 2 - mercaptoethanol , 1 mm sodium pyruvate , 2 mm l - glutamine and 10 % tcgf . prior investigations showed a suitable formation and isolation of rmva by means of vector plasmids which enables a host range selection on the basis of a transient expression of the vaccinia virus k1 l gene ( 58 ). for the use of said novel technique the hcv target gene sequences were cloned into the mva vector plasmid piiidhr - p7 . 5 . the resulting plasmids piiidhr - hcv / 1 - 742 , piiidhr - hcv / 1 - 661 and piiidhr - hcv / 151 - 661 contained hcv - cdna encoding amino acids 1 to 742 , 1 to 661 or 151 to 651 of the polyprotein . rmva was formed in bhk - 21 cells which have been infected with mva , and which were transfected with piiidhr - hcv / 1 - 742 , piiidhr - hcv / 1 - 661 or piiidhr - hcv / 151 - 661 . dilution series of the infected cell lysates were plated to rk - 13 cells which enabled a selective growth of recombinant viruses at transient expression of the selectable marker gene k1 l . after 3 cloning passages on rk - 13 cells only rmva was detectable , which was confirmed by pcr analysis of viral dna ( data not shown ), and the virus isolates were further plaque purified in bhk 21 cells . the growth of rmva in said cells was independent of k1 l gene expression and resulted to the loss of the marker gene by homologous recombination between repetitive dna sequences which flanked the k1l gene within the viral genomes ( fig1 a ). after several passages through bhk - 21 cells , the pcr analysis of viral dna revealed that the viral genome contained stably integrated hcv target gene sequences but no k1l genes any longer ( fig1 b ). amplification and purification of the obtained viruses mva - hcv / 1 - 742 , mva - hcv / 1 - 661 and mva - hcv / 151 - 661 resulted in virus preparation with high titers which were used for further analysis . since the formed rmva should be tested as a vector vaccine candidate for the delivery and immunological characterization of hcv antigens , the production of hcv proteins in tissue culture infection should be thoroughly determined . all rmva containing different coding sequences of the core , e1 and e2 proteins controlled the synthesis of the structural hcv proteins , as has been shown by sds page analysis and immunoblotting of bhk 21 cell lysates which were harvested 24 h after infection ( fig2 a , b ). monoclonal antibodies raised against the hcv core protein , resulted in specifically equal amounts of an about 21 kda core protein , prepared in mva - hcv / 1 - 742 or mva - hcv / 1 - 661 infected cells , whereas no such protein band could be detected after infection with mva - hcv / 151 - 661 or non - recombinant mva ( fig2 a ). at later times and with increased amounts of core protein a second 23 kda protein band could be detected . the synthesis of hcv envelope proteins was controlled with an e2 specific polyclonal rabbit antiserum . according to fig2 b , all three recombinant viruses formed e2 polypeptides which had been visualized as proteins with molecular weights of about 60 kda for mva - hcv / 1 - 742 and about 50 kda for mva - hcv / 1 - 661 and mva - hcv / 151 - 661 . the latter viruses both contain expression cassettes for hcv polyproteins having e2 sequences truncated at the c - terminus . several other smaller protein bands were also stained by the polyclonal serum and possibly represent proteins which are not hcv encoded , since they have been also detected in control lysates from cells infected with wild type mva . further , the kinetics of the hcv protein synthesis in rmva infection should be controlled . we synchronously infected bhk - 21 cell monolayers with 10 infectious units / cell of mva - hcv / 1 - 742 and prepared cell lysates for western blot analysis at several times during a period of 2 days after infection ( fig2 c , d ). already 4 h after infection the synthesis of hcv core protein could be detected fig2 c ), whereas the first recombinant e2 protein ( fig2 d ) clearly occurred 8 h after infection . the amounts of both recombinant proteins clearly increased during a time period of 34 h after infection . lysates of cells which had been infected for 24 h with wild type mva served as negative controls . analysis of cell associated and secreted forms of e2 antigens , prepared by rmva posttranslational modifications of the delivered antigens may substantially influence the immunogenicity and the protection capacity . the hcv envelope proteins e1 and e2 are highly modified by glycosylation and are possibly type i transmembrane glycoproteins with a carboxyterminal hydrophobic anchor domain . the removal of the e2 transmembrane anchor results in the secretion of the e2 ectodomain ( overview c . f . 17 ). upon infection of mammalian cells the eukaryotic mva expression system should result in the occurrence of authentic posttranslational modifications of recombinant proteins . for the control of said processes the e2 proteins were biochemically characterized , which are synthesized during infection with rmvas . bhk - 21 cells were infected with mva - hcv / 1 - 742 , mva - hcv / 1 - 661 and mva - hcv / 151 - 661 . total cell lysates ( fig3 a ) or precipitates of supernatants from infected cells ( fig3 b ) were treated with pngase f and analyzed by western blotting by means of polyclonal anti - e2 - antiserum . expressed e2 proteins with ˜ 60 kda ( mva - hcv / 1 - 742 ) or ˜ 50 kda ( mva - hcv / 1 - 661 and mva - hcv / 151 - 661 ) in cell lysates were reduced to a size of about 33 kda ( full length e2 ) or 31 kda ( truncated e2 ), respectively , corresponding to the unglycosylated peptide backbone of e2 ( fig3 a ). secreted e2 could only be detected after infection with the recombinant viruses mva - hcv / 1 - 661 and mva - hcv / 151 - 661 , which produce a truncated e2 in the transmembrane domain . secreted e2 is more strongly modified than cell associated e2 with an apparent molecular weight of ˜ 75 kda . upon treatment with pngase f said proteins will also be reduced to a size of about 31 kda , corresponding to a peptide backbone of the truncated e2 . in the next step the glycosylation type in said secreted e2 proteins was further characterized . cells were infected with mva - hcv / 1 - 661 and mva - hcv / 151 - 661 , cell lysates or supernatants of infected cells were treated with endoglycosidase h ( fig4 ). however , intracellular e2 is sensitive against endoh , as has been detected by a reduction of mw of ˜ 50 kda to ˜ 32 kda ( fig4 a ), but secreted e2 is not affected as far as possible ( fig4 b ). the e2 bands of the pngase f cleavage had a lower mw than the corresponding bands of the endo h cleavage , since the latter enzyme leaves a glcnac residue at the side chain of asn after the cleavage . this observation corresponds to a more complex glycosylation of c - terminally truncated e2 during the passage through the golgi apparatus . vaccination of mice with rmva causes hcv and vaccinia virus specific immune responses now , the immunogenicity of different rmva vaccines was tested in mice , since it was known , that the production of the hcv core protein by vaccinia virus recombinants mediates an immunosuppression in mice ( 36 ). additionally , the possible modulation of the immune responses by full length hcv core protein , raised by 2 of our rmva vaccines , should be determined . thus , hcv specific humoral or cellular responses were analyzed , which were directed against hcv - e1 and - e2 antigens . after three times of intraperitoneal immunization of balb / c mice with recombinant or wild type mva hcv - e1 , specific ctl responses with an e1 ( 312 - 326 )- balb / c - epitope were investigated . splenocytes which were derived from animals immunized with mva / hcv / 151 - 661 or mva - hcv / 1 - 661 , showed e1 specific lysis of 75 % with an e : t ratio of 9 : 1 , splenocytes of animals which were immunized with mva - hcv / 1 - 742 resulted in a specific lysis of 47 %, when they were stimulated with the e1 peptide ( fig5 ). a higher e1 specific lysis in cultures with splenocytes of mice was observed which were immunized with rmva expressing c - terminally truncated e2 proteins . serum samples of the same balb / c mice were furthermore investigated for antibody responses against the hcv e2 protein by elisa ( fig6 ). e2 specific antibodies with an average of the titers of 1 : 2512 ( mva - hcv / 151 - 661 ), 1 : 933 ( mva - hcv / 1 - 661 ) and 1 : 176 ( mva - hcv / 1 - 742 ) were detected . higher amounts of anti - e2 antibodies were found in animals which had been immunized with mva - hcv / 151 - 661 , although the differences beyond the groups were statistically not significant . the hcv core protein may exhibit as already mentioned an immunomodulatory function . now , it was investigated whether the higher anti e2 antibody response of mva - hcv / 151 - 661 - injected mice could be addressed to the large truncation of the core protein in said virus . it was expected , that the hcv nucleus also modulates the induction of the vaccinia virus mva specific immune responses . thus , the amounts of mva neutralizing antibodies in the same mouse sera as were used for elisa were measured . according to table 1 , no difference of the anti mva antibody response beyond the groups immunized with recombinant or wild type mva expressing full length or truncated core protein was detected . prior experiments established data for the immunogenicity and protection effect of recombinant mva vaccines delivering viral antigens in a series of animal model systems ( 61 , 29 , 27 , 64 , 65 , 44 , 59 ). however , there was still an urgent need for a vaccine against hcv infection and a vaccine candidate had to be identified which has a suitable immunogenic and possibly protective capacity . for the investigation of the suitability of mva for the production of hcv antigens a series of rmva with inserted expression cassettes of hcv gene sequences were constructed , which code for the structural proteins c - e1 - e2 . we wanted to determine the characteristics of recombinant mva , which was designed to produce hcv - e1 - e2 envelope proteins or secreted e2 antigen with or without full length hcv capsid / core protein . the formation and isolation of rmva resided in the transient host range gene selection ( 58 ) and showed that this technique is particularly suited , when several mva vector viruses have to be prepared for a comparative test . recombinant mva which expresses the different hcv encoding sequences under the control of the vaccinia virus early late promoter p7 . 5 , could easily be obtained and initiating experiments showed , that all constructs produced similar amounts of structural hcv proteins in a corresponding manner . the hcv core sequence is one of the most highly conserved regions in the viral genome reaching up to the viruses from different hcv genotypes and which renders the core protein interesting as an antigen for immunization ( 6 , 57 , 7 ). however , the inclusion of core into vaccine candidates is highly questionable , since its multifunctional characteristics are possibly involved in the regulation of the host cell apoptosis , transcription , transformation and immune presentation ( 43 , 4 ). the expression of hcv sequences encoding polyprotein aa 1 - 742 and 1 - 661 by recombinant mva primarily led to the synthesis of the 21 - kda - core proteins ( fig2 a ). only at later times of infection with increasing amounts of produced recombinant protein a second core - specific protein band at 23 kda size was visible . the 23 kda protein possibly is the non - mature core protein with 191 amino acids derived from an initial polyprotein cleavage , whereas the 21 - kda protein possibly is the mature core protein present in virus particles ( 28 , 26 , 51 , 55 , 66 ). said data show an efficient early synthesis and processing of the core polypeptide in mva - hcv vector infected cells . after the infection with mva - hcv / 1 - 742 the synthesis of an approx . 60 kda e2 protein was verified which strictly remained cell associated ( fig2 b ). the amplification of a 75 kda c - terminally truncated e2 protein in cell culture supernatants suggested for the functionality of the secretory pathway in hcv infected cells . the comparison of cell - associated and secreted truncated e2 , produced by mva - hcv / 1 - 661 and mva - hcv / 151 - 661 , by sds - page ( fig3 ab ) showed , that the secreted form migrated more slowly . this occurred due to a different glycosylation . accordingly both e2 proteins migrate after deglycosylation with pgnase f on the same level . furthermore , the secreted e2 was insensitive against a treatment with endoglycosidase h , which clearly shows the uptake of complex sugars during transport by the golgi apparatus . however , the expression of the hcv - e1 protein was not analyzed formally , but the e2 protein forms a downstream portion of the hcv polyprotein and the demonstration of the e2 synthesis suggests for the production of the e1 protein sequences . the correctness of this thought was verified by the detection of e1 specific t cell responses after vaccination with recombinant mva - hcv viruses . for the comparative test of the immunogenicity of the vaccines the balb / c mice were vaccinated and investigated for the induction of immune responses , which were directly directed against hcv envelope proteins produced by all vector viruses . for the envelope antigens e1 and e2 specific ctl are present in the liver and in peripheral blood of hcv infected humans and chimpanzees ( walker 1996 , sem . virol .). more recent data of the investigation of immune responses in hcv infected chimpanzees suggest that cd8 + ctl are accompanied by an acute cessation of the infection ( 12 ). the analysis of the fine specificity of long durable ctl revealed 9 different peptide epitopes of the hcv proteins e1 , e2 , p7 , ns2 , ns3 , ns5a , wherein 4 of said epitopes are present in e1 / e2 sequences . the e1 epitope 312 - 326 was successfully used for in - vitro restimulation of splenocytes from vaccinated balb / c mice , and epitope - specific ctl were detected which were induced by all 3 rmva vaccines . interestingly , the detection of e1 specific ctl was also possible more than 12 weeks after the last immunization . this suggests that immunizations over a long period could induce long lasting ( memory phase ) e1 specific t cell responses . the co - synthesis of the hcv core protein did not affect the induction of e1 specific ctl responses , however , an immunization with vector viruses producing c - terminally truncated secreted e2 proteins , seemed to elicit higher e1 specific cytotoxic activities . the glycoproteins e1 and e2 form complexes , which in endoplasmic reticulum ( er ) are retained at the c - termini of the proteins due to the interaction of hydrophobic transmembrane sequences ( 10 ). the removal or mutation of the e2 transmembrane region inhibits the formation of native e1 - e2 complexes and prevents the correct folding of the e1 glycoprotein ( 11 , 48 ). for a presentation by mhc - 1 - molecules the e1 antigen seems to be derived from the endoplasmic reticulum , but requires a cytoplasmic processing ( 54 ) and resides probably on a secondary export pathway for defect transmembrane glycoproteins into the cytosol as has been suggested for the influenza nucleoprotein or the cellular glycoprotein tyrosinase ( 2 , 47 ). due to this finding of the higher ctl activity after immunization with vector viruses producing secreted e2 one might speculate that the mobilization of truncated e2 for the secretory pathway makes available e1 preferably for the above processing pathway and enables an enhanced mhc - i restricted presentation of e1 antigens . the protection achieved after the prophylactic vaccination with recombinant hcv - envelope antigens in the chimpanzee model was correlated with e2 specific antibodies ( 9 ). accordingly , great attention was paid to the e2 protein as a vaccination antigen candidate for the induction of hcv neutralizing antibodies ( 68 , 56 ). during the investigation for e2 - specific humoral immune responses in the experiments specific antibodies were found which were formed after immunization in all 3 rmva vaccines . higher antibody titers were obtained in sera from animals vaccinates with vectors which synthesize secreted e2 proteins with truncated transmembrane domain . naturally , the secretion of e2 could elicit better antibody responses , whereas the interaction of full length e1 and - e2 proteins for the formation of intracellular retained complexes could mask important epitopes and prevent an optimal immune presentation . the hcv core protein , produced by replication competent vaccinia virus , seems to suppress host immune responses , in particular to suppress the formation of vaccinia virus specific immune responses ( large et al ., 1999 , j . immunol 162 : 931 - 938 ). in the vaccination trials shown herein , corresponding to these data the best e2 specific antibody responses seemed to be induced by the mva - hcv / 151 - 661 vaccine , which produced truncated e2 in the absence of core antigen . the accompanying analysis of the vaccinia virus specific neutralizing antibody responses showed however , that nearly equal amounts of vaccinia virus specific immunity was elicited by all rmva - hcv vaccines . however , the evaluation of the results of all immunization trials led to the conclusion that the hcv core antigen ( at least in the present experiments ) with replication deficient vaccinia virus mva as expression vector had no detectable effect . in summary , this investigation proves that rmva may be used for an efficient production of mature core and envelope proteins . moreover , the vaccination of balb / c mice with rmva vaccines induced comparable amounts of e2 specific antibodies and long lasting e1 specific cytotoxic t cell responses and evidences the usability of said viral vector for the further evaluation as a promising vaccine against hcv infection . thus according to the present invention rmva was investigated as a potential vaccine candidate against a hcv infection . in the first step mva vector vaccines were constructed which express all sequences encoding the main hcv virion components , to characterize the synthesis of recombinant hcv proteins upon mva infection and to evaluate their immunogenicity in the use as vaccines . in this investigation , a strong synthesis and an efficient posttranslational maturation of full length hcv structural proteins was detected , which were prepared upon in - vitro infection with rmva . an induction of hcv - e1 and e2 - antigen specific humoral or cellular immune responses in balb / c mice for rmva immunization could be shown . all vaccines caused balanced amounts of vaccinia virus specific circulating antibodies . the present data suggest for a high immunogenicity of rmva expressed hcv structural antigens , including the complete hcv core antigen . 2 . bacik , i ., h . l . snyder , l . c . anton , g . russ , w . chen , j . r . bennink , l . urge , l . otvos , b . dudkowska , l . eisenlohr , and j . w . yewdell 1997 . introduction of a glycosylation site into a secreted protein provides evidence for an alternative antigen processing pathway : transport of precursors of major histocompatibility complex class i - restricted peptides from the endoplasmic reticulum to the cytosol j exp med . 186 : 479 - 87 . 3 . belyakov , i . m ., l . s . wyatt , j . d . ahlers , p . earl , c . d . pendleton , b . l . kelsall , w . strober , b . moss , and j . a . berzofsky 1998 . induction of a mucosal cytotoxic t - 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