Patent Application: US-38354509-A

Abstract:
the invention relates to polypeptide carrier proteins that comprise at least five cd4 + t cell epitopes , for conjugation to capsular polysaccharides . the carrier proteins are useful as components of vaccines that can elicit a t - cell dependent immune response . these vaccines are particularly useful to confer protection against infection from encapsulated bacteria in infants between the ages of 3 months and about 2 years .

Description:
the practice of the present invention will employ , unless otherwise indicated , conventional techniques of molecular biology , microbiology , recombinant dna , and immunology , which are within the skill of the art . such techniques are explained fully in the literature eg . sambrook molecular cloning ; a laboratory manual , second edition ( 1989 ); dna cloning , volumes i and ii ( d . n glover ed . 1985 ); oligonucleotide synthesis ( m . j . gait ed , 1984 ); nucleic acid hybridization ( b . d . hames & amp ; s . j . higgins eds . 1984 ); transcription and translation ( b . d . hames & amp ; s . j . higgins eds . 1984 ); animal cell culture ( r . i . freshney ed . 1986 ); immobilised cells and enzymes ( irl press , 1986 ); b . perbal , a practical guide to molecular cloning ( 1984 ); the methods in enzymology series ( academic press , inc . ), especially volumes 154 & amp ; 155 ; gene transfer vectors for mammalian cells ( j . h . miller and m . p . calos eds . 1987 , cold spring harbor laboratory ); mayer and walker , eds . ( 1987 ), immunochemical methods in cell and molecular biology ( academic press , london ); scopes , ( 1987 ) protein purification : principles and practice , second edition ( springer - verlag , n . y . ), and handbook of experimental immunology , volumes i - iv ( d . m . weir and c . c . blackwell eds 1986 ). pemblex2 plasmid was derived from pembl8m ( dente l . and cortese r ., meth . enzymol . ( 1987 ), 155 : 111 - 9 ) by inserting a λp l promoter and a polylinker into the ecori and hindiii sites . the commercial vectors ptrc - his and pqe30 were purchased from invitrogen and qiagen respectively . e . coli strains used as recipients of the above plasmids were : k12δh1δtrp for pemblex2 , top10 for ptrc - his and tg1 for pqe30 . human t cell clones ksmik 140 and gg - 22 specific for p2tt and p30tt respectively were kindly provided by dr . a . lanzavecchia ( basel , switzerland ). construction of recombinant plasmids that express the n6 polyepitope carrier protein . complementary oligodeoxyribonucleotide pairs coding for p2tt , p21tt , p23tt , p30tt1 , p32tt and pft3 t cell epitopes ( table 1 ) and for a flag peptide were synthesised using the dna synthesiser abi394 ( perkin elmer ) and the reagents from cruachem ( glasgow , scotland ). the oligo pairs were separately annealed in t4 dna ligase buffer ( boehringer mannheim ) and equimolar amounts of each annealing reaction were mixed and ligated at room temperature for 3 hours using t4 dna ligase ( boehringer manheim ). the ligase reaction was then loaded onto a 1 % agarose gel and subjected to electrophoresis . the bands corresponding to the dna fragments of expected size were isolated , purified and cloned into the pemblex2 expression vector using standard protocols ( sambrook et al ., 1989 ). after transformation , a rabbit antiserum specific for the flag peptide was used to perform colony - screenings ( sambrook et al ., 1989 ) in order to identify recombinant protein producing clones . protein extracts from positive clones were analysed using sds - page to further select for clones on the basis of recombinant protein size . cd4 + t cell epitopes inserted in the recombinant polyepitope carrier proteins . after nucleotide sequencing of the selected clones , a clone named pembln6 was shown to contain six different t cell epitopes with no repetitive sequences . the n6 insert was then pcr - amplified and transferred to ptrc - his expression vector ( invitrogen ) using standard techniques ( sambrook et al ., 1989 ). the generation of the n6 expressing plasmids is summarised in fig1 . construction of recombinant plasmids that express the n10 polyepitope carrier protein using synthetic oligodeoxyribonucleotides and standard cloning techniques ( sambrook et al ., 1989 ), four additional cd4 + t cell epitopes were added to the n6 protein : hbvnc , ha , hbsag , and mt ( table i ). hbvnc and ha were sequentially introduced into ptrc - n6 by means of two consecutive cloning steps ; to the resulting plasmid the hbsag and mt epitopes were added in a single cloning step . after dna sequencing , a correct construct ( ptrc - n10 ) coding for the expected ten epitope polyepitope protein was identified . the n10 coding insert was then transferred from ptrc - n10 to pqe30 ( qiagen ) by means of pcr . the sequence of the resulting pqe - n10 construct was then confirmed by dna sequencing . two complementary oligodeoxyribonucleotides were synthesised and annealed to obtain a dna linker coding for the hsp70 cd4 + t cell epitope ( table i ). the linker was inserted in ptrc - n10 plasmid downstream from n10 coding region and in frame with it . after transformation in top10 e . coli strain , the transformants were selected using protein expression and dna sequencing analyses . glycerol batches of a selected clone ( top 10 / ptrc - n11 ) having the correct coding sequence and expressing a protein of the expected molecular weight were stored to − 80 ° c . construction of recombinant plasmids that express the n19 polyepitope carrier protein . the dna fragment encompassing the coding region from p23tt to hbsag was pcr amplified using the plasmid ptrc - n10 as template and two oligonucleotide primers which allow the insertion of bglii and psti restriction sites respectively at the 5 ′ and 3 ′ ends of the pcr product . the plasmid ptrc - n10 was digested with bamhi and psti restriction enzymes and ligated to the pcr product digested with bglii and psti . after transformation in top10 cells and selection of the transformants using protein expression and dna sequencing analyses , glycerol batches of a selected clone ( top10 / ptrc - n19 ) having the correct coding sequence and expressing a protein of the expected molecular weight were stored to − 80 ° c . the ptrc - n19 plasmid was digested with ecorv and psti and the insert was cloned in pqe - n10 digested with the same enzymes . after transformation in tg1 cells and selection of the transformants using protein expression and dna sequencing analyses , glycerol batches of a selected clone ( tg1 / pqe - n19 ) having the correct coding sequence and expressing a protein of the expected molecular weight were stored to − 80 ° c . all the recombinant polyepitope carrier proteins were purified using a similar strategy . briefly , e . coli cultures were grown in 500 ml lb medium containing 100 μg / ml ampicillin , at 37 ° c . at 0 . 3 - 0 . 5 od 600 , the expression of the polyepitope proteins was induced for 3 - 5 hours by adding 0 . 1 - 1 mm iptg . cells were disrupted by sonication or french press , the insoluble fraction was collected by centrifugation , dissolved with buffer a ( 6 m guanidiniwn - hcl , 100 mm nah 2 po 4 , 10 mm tris base , ph 8 ) and adsorbed with 2 ml of ni 2 + nta resin ( qiagen ). then , the resin was packed in a column and washed with buffer a . guanidinium - hcl was removed from the column by washing with buffer b ( 8 m urea , 100 mm nah 2 po 4 , 10 mm tris base ) ph 8 . after a wash with buffer b ph 6 . 5 , recombinant proteins were eluted with a 20 ml buffer b gradient from ph 6 . 5 to ph 4 . the factions containing the purified recombinant proteins were pooled and dialysed against pbs , ph 7 . 2 . proteins were analysed by sds_page and protein content was determined using the bradford method . alternatively , cell pellets obtained from e coli cultures were solubilized by heating at 37 ° c . in buffer a , the lysates were centrifuged to 15 , 000 g for 20 min . the supernatants were subjected to column chromatography on nickel activated chelating sepharose fast flow ( pharmacia ). after a wash with buffer a and a wash with buffer b , ph 7 , the proteins were separated by collecting fractions from a 0 - 200 mm gradient of imidazole in buffer b , ph 7 . the fractions containing the purified recombinant proteins ( as judged by sds - page and coomassie staining ) were pooled and dialysed against pbs , ph 7 . 2 . the hib capsular polysaccharide can be prepared according to the protocol described in gotschlich et al . ( 1981 ) j . biol . chem . 256 : 8915 - 8921 . 1 . 99 l of a 10 mg / ml solution of hib polysaccharide was hydrolysed in 0 . 01m acetic acid at 76 ° c . for 5 hours . after chilling , neutralization and 0 . 2 μm filtration , the resulting oligosaccharide population had an average degree of polymerisation ( avdp ) of 8 as measured by the chemical ratio between ribose and reducing groups . nacl was then added to the hydrolysate until a concentration of 0 . 16 m was attained , then diluted 1 : 1 with 0 . 16m nacl / 10 mm acetate ph 6 and submitted to tangential flow ultrafiltration on a 10 kda membrane in order to remove high molecular weight species . ultrafiltration comprised approximately 11 - fold concentration followed by 15 cycles of diafiltration against 0 . 16 m nacl / 10 mm acetate , ph 6 . the retentate was discarded . the permeate was diluted : 1 with water and 0 . 22 μm filtered . chemical analysis revealed an avdp of 8 . 1 . the permeate obtained from 10 kda uf was loaded , at a linear flow rate of 150 cm / h , onto a q - sepharose fast flow column [ 10 cm ( id ); 5 . 5 cm ( h )] equilibrated with 0 . 08 m nacl / 0 . 05 m sodium acetate ph 6 . after adsorption , low molecular weight fragments ( up to 5 repeats ) were removed by washing the column with 10 column volumes of equilibrating buffer and then eluted with 3 column volumes of 0 . 5 m nacl / 0 . 005m sodium acetate ph 6 . the eluate was 0 . 2 μm filtered and then analysed for avdp and ion exchange analytical chromatography . avdp resulted at 17 . 3 , ion exchange analytical chromatography on mono q hr 5 / 5 revealed the absence of any small fragments until dp 5 . to introduce a terminal amino group , reductive amination was then performed ; to the fractionated hib oligosaccharide obtained from q - sepharose chromatography , ammonium chloride 35 mg / ml and sodium cyanoboroidride 12 mg / ml final concentrations were added . after stirring , the solution was 0 . 2 μm filtered and incubated at 37 ° c . for 120 hours . the amino oligosaccharide was then purified from excess of reagents by precipitation with 95 ° etoh ( 81 ° final concentration ) in the cold for 15 - 20 hours . the precipitated oligosaccharide was then recovered by centrifugation , solubilized in nacl 0 . 4m using approximately ¼ of the starting volume and precipitated again at 81 ° etoh in the cold for 15 - 20 hours . the amino - oligosaccharide was again recovered by centrifugation and solubilized in about 300 ml of 0 . 02 m nacl . after having taken a sample for analysis , the resulting solution was then dried using a rotary evaporator . colorimetric amino group analysis confirmed the introduction of a primary amino group into the oligosaccharide . derivatisation to active ester was then performed as follows . the amino - oligosaccharide was solubilised in distilled water at a concentration of 40 μmol of amino groups per ml . the solution was then diluted 10 - fold with dmso . triethylamine was added in molar ratio to the amino groups of 2 : 1 . n - hydroxysuccinimido diester of adipic acid was then added in a molar ratio to the amino groups of 12 : 1 . the reaction mixture was kept under gentle stirring for 2 hours at rt . the activated oligosaccharide was then purified from the excess of reagents by precipitation into 10 volumes of 1 - 4 dioxane under stirring . after 30 minutes in the cold the precipitate was collected onto a syntered glass filter , washed onto the filter with dioxane and then dried under vacuum . the dried activated oligosaccharide was analysed for its content of active ester groups by a colorimetric method ; this test showed the presence of 62 . 1 μmol of active ester per mg of dried oligosaccharide . conjugation of the polyepitope carrier protein with hib capsular oligosaccharides and purification of the conjugates . 33 . 4 nmoles of recombinant carrier protein and 669 nmoles of activated hib oligosaccharide in a final volume of 0 . 5 ml 10 mm phosphate buffer , ph 7 , were gently stirred overnight at rt and brought up to 5 ml 1m ( nh 4 ) 2 so 4 , 10 mm phosphate ph7 . the sample was subjected to fplc on a 1 ml phenyl sepharose 5 / 5 hr column ( pharmacia ). 1 ml fractions were collected both during washing ( 1m ( nh 4 ) 2 so 4 . 10 mm phosphate , ph 7 ) and elution ( 10 mm phosphate , ph 7 ). two peaks corresponding to the non - adsorbed material and to the eluted material were obtained . the pooled fractions corresponding to the non - adsorbed material and the pooled fractions corresponding to the elution peak were subjected to protein and ribose content determination and to sds - page and western blot analysis . a protocol to conjugate recombinant proteins to oligosaccharides directly on ni 2 + - nta resin was also developed . recombinant proteins were purified as described above , but the final dialysis step was omitted . the protein content of the 8m urea fraction pool was measured with the bradford assay . the ph of the eluted proteins was adjusted to ph8 and adsorption on 1 ml pre - equilibrated ni 2 + - nta resin was again performed in a batch mode . urea was removed by washing with 4 × 25 ml 100 mm phosphate buffer ph 7 . 5 . the resin was suspended in 1 ml 100 mm phosphate buffer ph 7 . 5 and a 20 - fold molar excess of activated hib oligosaccharide ( as compared to the protein that was adsorbed on the resin ) was added to the suspension . the mixture was gently stirred overnight at rt packed in a column , and washed with 50 ml 100 mm phosphate buffer ph 7 . 5 to remove unconjugated oligosaccharide . elution of the conjugate was performed with 100 mm phosphate buffer ph 4 . peak fractions were pooled and dialysed against pbs , ph 7 . 2 . the conjugate was analysed by coomassie staining of sds - page gels and western immunoblot using an anti - flag rabbit antibody . the protein / carbohydrate ratio of the glycoconjugate was determined upon bradford assay and ribose content determination . culture medium for pbmcs was rpmi 1640 ( gibco laboratories , paisley , scotland ) supplemented with 2 mm l - glutamine , 1 % nonessential amino acids , 1 mm sodium pyruvate , gentamycin ( 50 μg / ml ), and 5 % human serum ( rpmi - hs ) or 10 % foetal calf serum ( rpmi - fcs ). for the growth of t - cell lines and clones , rpmi - hs was supplemented with 50 u of recombinant interleukin - 2 ( ril - 2 : hoffmann la roche , nutley , n . j .) per ml . frozen pbmc ( 10 5 ) from healthy adults immune to tetanus toxoid were thawed and cultured in duplicate wells of 96 - well flat - bottomed microplates , in 0 . 2 ml of rpmi - hs ( di tommaso et al , 1997 ). the recombinant proteins and tetanus toxoids ( chiron , siena ) were added to wells at the final concentration of 10 μg / ml . after 5 days of culture . 1 μci of [ 3 h ] thymidine ( specific activity : 5 ci / mmol , amersham ) was added to each well and dna - incorporated radioactivity was measured after an additional 16 hrs by liquid scintillation counting . two human t cell clones , ksmik 140 , and gg - 22 , specific for p2tt and p30tt respectively , and the respective peptides were kindly provided by dr . a . lanzavecchia ( basel , switzerland ). t cells ( 2 × 10 4 ) were cultured with autologous irradiated epstein barr virus - transformed b lymphocytes ( 3 × 10 4 ) in 0 . 2 ml of rpmi - fcs in 96 - well flat - bottomed microplates in duplicate wells . synthetic peptides and conjugated or unconjugated recombinant proteins were added to cultures at a final concentration of 10 μg / ml . after 2 days , 1 μci of [ 3 h ] thymidine was added and the radioactivity incorporated was measured by liquid scintillation counting after an additional 16 hours . in some experiments , carrier proteins and their conjugates were pre - incubated with apcs for 2 - 4 hours , then apcs were washed and cultured with t cell clones . this procedure was used to limit possible proteolytic degradation by serum proteases and to be more confident that epitope presentation would be due to intracellularly - processed epitopes . in a first experiment , equal doses of the glycoconjugates and of the polysaccharide ( 2 . 5 μg as polysaccharide ) in presence of 0 . 5 mg of aluminium hydroxide as adjuvant were injected subcutaneously into groups of eight balb / c and c57bl / 6 mice ( female , 7 - week - old ) on days 0 , 21 and 35 . mice were bled on day − 1 ( pre - immune ), 20 ( pre - 2 ), 34 ( pre - 3 ) and 45 ( post - 3 ) and individual sera collected and stored at − 80 ° c . before elisa assay . in a second experiment , equal doses of the glycoconjugates and of the polysaccharide ( 2 . 5 μg as polysaccharide ) in the presence of 0 . 5 mg of aluminium hydroxide as adjuvant were injected subcutaneously into groups of eight swiss (&# 39 ; d1 and balb / c mice ( female , 7 - week - old ) on days 0 , 10 and 20 . a boost of 2 . 5 μg of purified hib polysaccharide ( hibcps ) in presence of 0 . 5 mg of aluminium hydroxide was then given to each mouse at day 70 . mice were bled on day − 1 ( pre - immune ). 35 ( post - vaccination ), 68 ( pre - boost ) and 85 ( post - boost ) and individual sera collected and stored at − 80 ° c . before elisa assay . in a third experiment , equal doses of crm - hib , n10 - hib , and n19 - hib ( 2 . 5 μg as polysaccharide ) in presence of 0 . 5 mg of aluminium hydroxide as adjuvant were injected subcutaneously in groups of 6 swiss cd1 mice ( female , 7 - week - old ) on days 0 , 15 , and 28 in order to compare the carrier effects . different groups of mice were also subjected to the same schedule but were previously primed with unconjugated carriers in order to check for potential immunosuppression phenomena . in the latter groups equal doses of carrier proteins ( 50 μg ) in 0 . 5 mg alum were injected on day − 30 . all mice were bled on day − 32 ( pre - priming ), − 2 ( pre - immune ), 14 ( post - 1 ), 27 ( post - 2 ), and 45 ( post - 3 ) and the sera were collected and stored to − 80 ° c . before elisa assay . nunc maxisorp 96 - well flat - bottomed plates were coated by overnight incubation at 4 ° c . with 1 μg / ml ( as polysaccharide ) of a human serum albumin ( hsa ) and h . influenzae type b polysaccharide conjugate ( hsa - hib ). after washing , wells were over - coated using 1 % ( w / v ) gelatin in pbs , ph 7 . 2 for 3 additional hours at 37 ° c . serum samples were diluted 1 : 50 in 5 mm phosphate buffer , ph 7 . 2 containing 75 mm nacl 1 % ( w / v ) bsa and 0 . 05 % ( w / v ) tween - 20 and dispensed in duplicate into the wells . sera from untreated mice were pooled and diluted 1 : 50 as above and dispensed into 8 wells . after overnight incubation at 4 ° c ., plates were washed three times with 5 mm phosphate buffer , ph 7 . 2 containing 75 mm nacl and 0 . 05 % ( w / v ) tween - 20 . then , alkaline phosphate - conjugated goat igg anti - mouse igg diluted 1 : 1000 and 5 mm phosphate buffer , ph 7 . 2 containing 75 mm nacl . 1 % ( w / v ) bsa and 0 . 05 % ( w / v ) tween - 20 were added to each well , and incubated 3 hours at 37 ° c . after repeated washing , 100 μl of a chromogen - substrate , p - nitrophenylphosphate , in a diethylenamine solution , were added to each well . reaction was stopped after 20 min by adding a 4n naoh solution . then , the plate was read at 405 mm with a reference wavelength of 595 mm . titres were expressed as absorbencies at 405 mm ( a 405mm ). mice were considered responders when the average a 405mm was found equal to or higher than four times the average of absorbencies of the eight wells with the sera from untreated animals . according to the european pharmacopoeia [ pa / ph / exp15 / t ( 93 ) 3anp ] four out of eight mice should be responders . in the second experiment , mice were considered responders when the average a 405mm was found four times the average of the absorbencies of eight pre - immune sera of the same group of treatment . the anti - carrier response was assayed as above described for anti - hib response using plates coated with n10 or n6 ( coating concentration = 2 μg / ml ). using the approaches described in materials and methods , we created several e . coli clones expressing different carrier proteins . the following table lists only the six clones we utilised to purify the recombinant polyepitope carrier proteins : the clone expressing n6 protein comprised the plasmid ptrc - n6 transformed in the top 10 e . coli strain . as deduced from plasmid dna sequencing , this plasmid code for a protein having an hexahistidine amino terminal tail followed in sequence by a flag peptide , a fxa site , and the following t cell epitopes : p23tt , p32tt , p21tt , pft3 , p30tt , and p2tt . all the epitopes were spaced by a kg amino acid sequence ( fig2 ). the two clones that produced n10 protein were the top 10 e . coli strain containing the plasmid ptrc - n10 , and the tg1 e . coli strain containing the plasmid pqe - n10 . both these clones contained the n6 coding sequence fused to a carboxy terminal sequence coding for four additional t cell epitopes which were in the order : hbvnc , ha , hbsag , and mt ( fig2 ). the clone that produced n11 protein comprised the plasmid ptrc - n10 transformed in the top10 e . coli strain . as deduced from plasmid dna sequencing , this plasmid code for a protein consisting in the n10 sequence fused to a carboxy terminal sequence coding for the hsp70 t cell epitope ( fig7 ). the two clones that produced n19 protein were the top10 e . coli strain containing the plasmid ptrc - n19 , and the tg1 e . coli strain containing the plasmid pqe - n19 . both these clones contained the n10 coding sequence fused to a carboxy terminal sequence coding for nine additional t cell epitopes which were in the order : p23tt , p32tt , p21tt , pft3 , p30tt , p2tt , hbvnc , ha , and hbsag ( fig8 ). fig3 and 4 depict protein expression of the three synthetic proteins . the addition of four new epitopes ( hbvnc , ha , hbsag , and mt ) to n6 in ptrc - his ( lane d ) to obtain n10 protein ( lane c ) resulted in a remarkable reduction of protein expression . an attempt to increase the expression level of n10 simply involved changing the expression vector ( from ptrc )- his to pqe30 ) and the e . coli strain ( from top10 to tg1 ). as seen in fig3 and 4 , the amount of n10 expressed by pqe30 - n10 in tg1 ( lane b ) was notably higher than the same protein expressed by ptrc - n10 ( lane c ). this is thought possibly to be due to the fact that whereas n6 protein was effectively assembled by the e . coli strain in the order of epitopes most suited to the organism , whereas the addition of four further epitopes was effectively forced and thus was less natural . however , the fact that the level of n10 expression was notably increased by simply changing expression vector ( from ptrc - his to pqe30 ) and e . coli strain ( from top - 10 to tg1 ) suggests that additional factors , other than epitope combination , play a role in protein expression . fig9 shows protein expression and purification of the n11 protein ( sds - page and coomassie staining ). total extract coming from an induced culture ( lane b ) shows an induced band , corresponding roughly to the expected molecular weight of n11 protein , that is not present in uninduced extract ( lane a ). the identity of the induced band was established also by western blot using an anti - flag antibody , and was also deduced from plasmid dna sequencing ( fig7 ). n11 purification ( fig9 , lane c ) was done by solubilising whole cell pellets in guanidinium and by subjecting the whole extract to imac chromatography , with this procedure we obtained 14 mg of recombinant n11 protein from one litre of top10 - trc - n11 flask culture . the addition of hsp70 t cell epitope to the carboxy terminus of n10 resulted in a construct ( ptrc - n11 ) that was able to notably improve the expression of the polyepitope protein as compared to the expression obtained from ptrc - n10 . as it was for the n10 protein , also the expression of n19 protein was improved by charging the expression vector ( from ptrc - his to pqe30 ) and the host strain ( from top10 to tg1 ). tg1 ( qe - n19 ) was used to purify n19 polyepitope protein . by subjecting solubilised inclusion bodies to imac chromatography , we purified ( see fig1 a ) 5 . 42 mg of n19 protein from one litre of flask culture . the identity of n19 was identified in sds - page as an induced band having the expected molecular weight , in immuno western blot using an anti - flag antibody , and was also deduced after plasmid dna sequencing ( fig8 ). all clones expressing recombinant polyepitope proteins produced them mainly in the form of inclusion bodies . purification of n6 and n10 proteins from inclusion bodies solubilised with 8m urea using an immobilised metal affinity chromatography ( imac ) procedure in the presence of 8m urea resulted in the loss of a high percentage of protein which was elutable with a 6 . 5 - 4 ph gradient ( data not shown ). on the contrary , almost all of the histidine - tagged protein was eluted with the 6 . 5 - 4 ph gradient when starting inclusion bodies were solubilised with 6m guanidine hydrochloride ( fig5 and 6 ). using this protocol 7 . 8 mg of n6 was purified from a litre of culture . the n10 protein that was employed in immunisation and t cell proliferation experiments was purified from ptrc - n10 clone . given the lower expression of recombinant protein shown by this clone we decided to purify n10 protein by solubilising whole cells with guanidinium in such a way as to exploit soluble and insoluble ( inclusion bodies ) proteins for imac purification . with this procedure 1 . 5 mg of purified n10 protein was obtained from a litre of culture . the higher success of solubilisation using 6m guanidium is thought to be due to the ability of this compound to solubilise the carrier proteins in monomeric form . using the phenyl sepharose fplc protocol we obtained a purified n6 - hib conjugate having a protein content of 79 . 4 μg / ml , and an oligosaccharide content of 42 . 7 μg / ml . we observed that 30 % of conjugated protein was unable to bind to phenyl sepharose in the presence of 1m ( nh 4 ) 2 so 4 . in addition , 30 - 40 % of carrier protein was previously lost during a dialysis step to remove urea before the conjugation reaction . to overcome these problems it was checked if it was possible to perform the conjugation reactions when the protein was adsorbed on the ni 2 + - nta resin . we observed that the hib oligosaccharide was unable to bind ni 2 + - nta resin at any ph , suggesting the feasibility of this approach and predicting that no interference due to the oligosaccharide could influence the elution of the protein once conjugation had taken place . a reaction was thus set up involving protein adsorption on ni 2 + - nta resin in the presence of 8m urea , urea removal , conjugation with oligosaccharide , washing , and conjugate elution . no aggregation phenomena were observed for the eluted conjugate . using this procedure we obtained a purified n6 - hib conjugate having a protein content of 320 μg / ml and an oligosaccharide content of 370 μg / ml . and a purified n10 - hib having a protein content of 113 μg / ml and an oligosaccharide content of 114 μg / ml . by using a 1 : 10 protein to carbohydrate molar ratio to conjugate oligosaccharide to recombinant carriers , we observed that a fraction of protein remained unconjugated ( as judged by coomassie staining of sds - page gel and western immunoblot ; data not shown ). when a 1 : 20 protein to carbohydrate stoichiometric ratio was used , all the purified recombinant proteins were found to be completely conjugated , in fact , by analysing coomassie - stained gels and western immunoblots using an anti - flag antibody . we observed that after conjugation of n6 and n10 with hib oligosaccharides these molecules increased their molecular weight , appearing as a high molecular weight smear , and proteins were no longer visible at the expected molecular weight for n6 and n10 monomers . this suggested that the synthetic proteins were completely conjugated to hib oligosaccharides ( data not shown ). the conjugation of activated hib oligosaccharide to n19 protein resulted in a protein content of 173 μg / ml and in an oligosaccharide content of 127 μg / ml . fig1 b depicts an sds - page and coomassie staining analysis of the fractions obtained from imac chromatography of the n19 conjugated to hib polysaccharide . all n19 protein resulted to be conjugated , as judged by the high molecular weight of the conjugate and by the absence of 43 , 000 kda unconjugated n19 protein . fig1 c shows the corresponding western immuno - blot using an anti - flag antibody . also here it can be appreciated that all n19 protein migrated as a very high molecular weight after conjugation to hib polysaccharide , and that there is not unconjugated n19 protein migrating at 43 , 000 kda . recognition of carrier proteins and their conjugates by human t lymphocytes . to investigate whether t cell epitopes contained in the polypeptides were recognised by human t cells we used t cell clones specific for the tt universal epitopes p2tt and p30tt ( demotz et al . 1993 ). fig1 shows that n6 is recognised by both clones not only as a simple polypeptide but also after it has been conjugated with polysaccharide . remarkably , n6 - hib is recognised even better than unconjugated n6 by the t cell clone specific for p2tt . n10 - hib is recognised by the clone specific for p2tt but is poorly recognised by the clone specific for p30tt . in both cases n10 - hib exerts the same stimulatory activity as the synthetic peptide . the n10 clone was not tested in these experiments . once assessed that the t cell epitopes contained in the carrier proteins are correctly presented to t lymphocytes , we asked whether these carriers maintain their stimulatory capacity when presented to a heterogeneous population of lymphocytes such as pbmc . this could be predictive of whether our carriers might function as such once injected into subjects immune to antigens whose epitopes are included in the carriers themselves . for this purpose we used pbmc from donors immune to tt ( a . di tommaso et al . 1997 ), since tt epitopes are the most represented in our polypeptides . fig1 shows that all the formulations were able to stimulate pbmc proliferation . however , the incubation of pbmc with a synthetic peptide representing one of the epitopes included in both n6 and n10 constructs failed to exert a stimulatory effect . as a positive control , the pbmc were also incubated with 10 μg / ml of tt , that in all cases induced a proliferative response . interestingly , the n6 polyepitope protein turned out to be the most potent pbmc stimulator among those tested in two out of three volunteers . the carrier effect of the proteins n10 and n6 in comparison with crm197 was assayed in mice in several glycoconjugate vaccines . once coupled to hib oligosaccharides the carrier proteins were injected in different mouse strains to verify the potential of their carrier effect . in balb / c mice , an equivalent anti - hib response was found when crm197 and n10 were used as carrier proteins , whilst a lower response was found when n6 was used as carrier protein . this result was evident when the results were expressed using titres , while responder percentages failed to evidence the lower anti - hib response obtained with the n6 protein carrier . in c57bl / 6 mice , the n6 protein gave a negative result , while positive results were obtained with crm197 and n10 , even if to a lower extent . these results were evident both using titres or responder percentages to express the results . when the results were expressed as a responder percentage , the high carrier effect of crm197 and n10 was well evidenced with respect to n6 , whose results were lower than 50 % at day − 34 and day − 45 bleedings , after a comparable primary response ( pre - 2 bleeding , day 20 ). table ii reports the results of the experiments in balb / c and c57bl / 6 mice . in swiss cdi mice , the titres obtained with the n10 carrier protein were equivalent to those obtained with crm197 . the anti - hib titres increased after immunisation up to the 70th day , when a polysaccharide boost was given to assay whether or not an immunological memory was induced in the treated mice . no boost effect was observed with any carrier , although when crm197 or n10 were used as carrier protein the titre did not decrease . in this mouse strain the immunisation with n6 - hib glycoconjugate give results very similar to the controls ( polysaccharide and alum ). the boost effect was not evidenced even in balb / c mice that evoke a lower response with respect to swiss cd1 mice . immunisation of different mice strains with hib oligosaccharides conjugated to the artificial carrier proteins resulted in a good carrier effect exerted by n10 , whilst n6 gave unsatisfactory results . this suggests that the size of the protein or the number of t cell epitopes has a high influence in providing t cell help to the oligosaccharides . we used outbred cd1 mice to perform an immunogenicity experiment in which the carrier effect of n19 protein was compared to the carrier effects of n10 and crm197 . in addition , in order to explore potential carrier - induced immunosuppression phenomena , the three doses of n10 - hib , n19 - hib and crm - hib were given to groups of mice that did not received carrier priming and to groups of mice that one month before were primed with 50 μg of the respective unconjugated carrier ( see materials and methods ). * for priming we used a chemically detoxified diphtheria toxin ( dt : diphtheria toxoid ) instead of the non toxic mutant ( crm - 197 ) of diphtheria toxin . the results are depicted in fig1 . in unprimed mice the best anti - hib titres were obtained using n19 - hib , whilst crm - hib and n10 - hib gave lower titres . according to the known direct proportion between the size of the carrier molecules and the exerted carrier effect , n19 - hib elicited a clearly improved anti - hib response as compared to n10 - hib . in addition n19 - hib seems slightly superior also when compared to crm - hib suggesting the feasibility to substitute “ classical ” carrier proteins with the recombinant cd4 + polyepitope proteins . in contrast to the previous immunogenicity test performed on cd1 mice , were the carrier effects of n10 and crm - 197 were similar , in this new test the mean anti - hib titre elicited by n10 - hib was notably lower than the one obtained with crm - hib . in primed mice the best results were obtained with n19 - hib , which elicited a better response also when compared to the response obtained in unprimed mice , suggesting a potentiation due to the priming with n19 protein . a slight potentiation was also obtained after priming with n10 . conversely , anti - hib response obtained with crm - hib in primed mice was notably lower of the response obtained in unprimed mice , confirming the carrier induced immunosuppression often observed with the carriers in current use . since n10 and n19 contains five and ten tetanus toxoid t cell epitopes respectively , we subjected n10 - hib and n19 - hib to an immunogenicity test in cd1 mice primed with tetanus toxoid . the goal of this experiment was to check whether in primed mice the anti - hib titers were improved in comparison to non - primed mice . surprisingly , tetanus toxoid priming potentiated the immunoresponse to hib in mice immunised with n10 - 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