acid pretreatment of gram - positive bacteria enhances binding of acma protein anchor fusions bacterial strains and growth conditions . lactococcus lactis strain mg1363 ( gasson 1983 ) or derivatives thereof , such as mg1363δacma ( buist et al . 1995 ) or nz9000δacma , were used as recipients for binding of reporter fusion protein . nz9000 ( kuipers et al . 1997 ), which carries one of the reporter plasmids , was used as a production strain . l . lactis strains were grown in m17 broth ( oxoid ) supplemented with 0 . 5 % glucose in standing cultures at 30 ° c . chloramphenicol was added to the m17 medium to an end - concentration of 5 μg / ml when appropriate . for expression , mid - log phase cultures were induced for 2 hours with the culture supernatant of the nisin producing l . lactis strain nz9700 as described by kuipers et al . ( 1997 ). lactobacillus casei , atcc393 , was grown in mrs broth ( oxoid ) in standing cultures at 30 ° c . mycobacterium smegmatis , atcc700084 , was grown in middlebrook medium ( oxoid ) at 37 ° c . in aerated cultures . bacillus subtilis , 168 , was grown in ty broth ( per liter : 10 g tryptone , 5 g yeast extract , 5 g nacl ph 7 . 4 ) at 37 ° c . in aerated cultures . construction of reporter plasmids . the merozoite surface antigen 2 ( msa2 ) of plasmodium falciparum strain 3d7 ( ramasamy et al . 1999 ) fused to the three repeats of acma ( msa2 :: ca ) was used as the reporter anchor protein . the reporter anchor protein is encoded by plasmid png3041 based on the nisin inducible expression vector pnz8048 ( kuipers et al . 1997 ) and contains a modified multiple cloning site in which the hybrid reporter gene was cloned . an in - frame fusion of the reporter was made at the 5 ′ end , the lactococcal prtp signal - and prosequence , and at the 3 ′ end , the acma protein anchor sequence . the sequence of the msa2 gene that was included in the construct corresponds to nucleotides ( nt ) 61 to 708 in genbank accession number a06129 . primers used for the amplification of the msa2 gene were msa2 . 1 ( 5 ′- accatggcaaaaaatgaaagtaaatatagc ( seq id no : 2 )) and msa2 . 4 ( 5 ′- cggtctctagcttataagcttagaattcgggatgttgctgctcc acag ( seq id no : 3 )). the primers contain tags with restriction endonuclease recognition sites that were used for cloning . for cloning of the prtp signal and prosequence ( nt 1206 to 1766 in kok et al . 1988 ), the primers prtp . sspro . fw ( 5 ′- ccgtctcccatgcaaaggaaaaaaga aagggc ( seq id no : 4 )) and prtp . sspro . rev ( aaaaaaagcttgaattcccat ggcagtcggataataaactttcgcc ( seq id no : 5 )) were used . the primers include restriction sites that were used for cloning . the acma protein anchor gene fragment ( nt 833 to 1875 ) was obtained by subcloning a pvuii - hindiii fragment from plasmid pal01 ( buist et al . 1995 ). restriction endonuclease enzymes and expand high fidelity pcr polymerase were used in accordance with the instructions of the supplier ( roche ). the final expression vector was designated png3041 ( fig1 ). a construct including a stop codon introduced after the msa2 sequence in png3041 was designated png304 . the protein secreted using this construct is substantially the same as the protein expressed from the png3041 plasmid except that the protein produced from png304 does not contain the acma protein anchor . the protein produced from png304 is used as a negative control in the binding assays . a vector was also made in which the acma protein anchor was exchanged for a protein anchor . the putative cell - wall binding domain of l . lactis acmd ( bolotin et al . 2001 ) was cloned ( nt 1796 to 2371 in genbank accession number ae006288 ) using primers pacmb2 ( 5 ′- cgcaagcttctgcagagctcttagattctaatt gtttgtcctgg ( seq id no : 6 )) and pacmb3 ( 5 ′- cggaattcaaggaggagaaata tcaggagg ( seq id no : 7 )) to produce the plasmid png3042 . png3042 contains an in - frame fusion between msa2 and the protein anchor of acmd ( msa2 :: cd ) and differs from plasmid png3041 only in the gene fragment encoding the protein anchor . cell pretreatment and binding conditions . chemical pretreatment of l . lactis nz9000äacma was done with 10 % tca ( 0 . 6 m ) in the following manner . cells of 0 . 5 ml stationary phase cultures were sedimented by centrifugation and washed once with 2 volumes of demineralized water . cells were resuspended in 1 volume of a 10 % tca solution and incubated by placing the reaction tube in boiling water for 15 minutes . subsequently , cells were washed once with 2 volumes pbs ( 58 mm na 2 hpo 4 . 2h 2 o , 17 mm nah 2 po 4 . h 2 o , 68 mm nacl ; ph 7 . 2 ) and three times with 2 volumes demineralized water . the cells were used directly for binding experiments or stored ( as described herein ) until further use . the following chemicals and conditions were used to examine the effect of different chemicals on the binding capacity of l . lactis cells for acma - type protein anchor fusions : acetic acid ( hac ), hydrochloric acid ( hcl ), sulfuric acid ( h 2 so 4 ), tca , trifluoroacetic acid ( tfa ), and monochloro acetic acid ( mca ). the acids were used at a final concentration of 0 . 6 m and incubated for 15 minutes in boiling water . sds , dimethyl formamide ( dmf ) and dimethyl sulfoxide ( dmso ) were used at a concentration of 10 %. the sds pretreatment was incubated for 15 minutes in boiling water and dmf and dmso treatments were incubated at room temperature for 15 minutes . cells were also pretreated with phenol ( tris buffer saturated ) and incubated for 15 minutes at 55 ° c . other chemicals pretreated at the 55 ° c . incubation temperature were : 4 m guanidine hydrochloride ( gnhcl ), 37 % formaldehyde , chloroform : methanol ( chcl 3 : ch 3 oh ( 2 : 1 )) and 0 . 1 % sodium hypochlorite ( naocl ). in addition , incubation with 25 mm dithiothrietol ( dtt ) for 30 minutes at 37 ° c . and a pretreatment with hexane ( 100 %) were analyzed . the effect of enzymatic pretreatment of cells with lysozyme was also tested . for lysozyme pretreatment , the cells were resuspended in buffer ( 20 % sucrose , 10 mm tris ph 8 . 1 , 10 mm edta , 50 mm nacl ) with lysozyme ( 2 mg / ml ) and incubated at 55 ° c . for 15 minutes . after the chemical and enzymatic pretreatments , the washing steps were the same as the washing steps used for the tca - treated cells . tca pretreatment of bacillus subtilis , lactobacillus casei and mycobacterium smegmatis was done as described herein for l . lactis . cell - free culture supernatants containing msa2 :: ca , msa2 :: cd or msa2 without anchor were incubated in four - fold excess for 10 minutes at room temperature with pretreated cells ( e . g ., cells from 0 . 5 ml culture were incubated with 2 . 0 ml culture supernatant ). after binding , cells were sedimented by centrifugation , washed twice in 2 volumes of demineralized water , resuspended in sds - denaturation buffer , heated for 5 minutes at 98 ° c ., subjected to sds - page , and analyzed by western blot analysis . storage conditions . cell - free supernatants containing msa2 :: ca , msa2 :: cd or msa2 were stored at − 20 ° c . with or without 10 % glycerol prior to binding . tca - pretreated l . lactis cells were stored at − 80 ° c . in 10 % glycerol prior to binding . tca pretreated l . lactis cells with bound msa2 :: ca were stored at + 4 ° c . or − 80 ° c . with or without 10 % glycerol . cells stored in 10 % glycerol were washed once with 1 volume of demineralized water prior to binding . cell pellets ( in demineralized water ) of tca - pretreated l . lactis cells with or without bound msa2 :: ca were frozen by contacting the vials with liquid nitrogen and removing the water with lyophilization . alternatively , non - frozen cell pellets were dried under vacuum at 30 ° c . for 2 hours prior to binding . western blotting . for detection of msa2 proteins , cell pellets corresponding to 500 μl culture were resuspended in 50 μl sds - denaturation buffer . cell - free culture supernatants ( 1 ml ) were concentrated by phenol - ether precipitation ( sauvé et al . 1995 ), vacuum dried and resuspended in 50 μl sds - denaturation buffer . proteins were separated with standard sds - page techniques . after separation , proteins were electroblotted onto pvdf membranes ( roche ). in immunoblots , msa2 proteins were detected with 1 : 10 , 000 diluted rabbit msa2 - specific antiserum ( ramasamy et al . 1999 ) and 1 : 5 , 000 diluted anti - rabbit igg - conjugated alkaline phosphatase ( roche ) using known procedures . fluorescence microscopy . 100 μl cell suspensions incubated with msa2 :: ca , msa2 :: cd or msa2 fusion proteins were washed twice with demineralized water and resuspended in an equal volume of pbs containing 1 % bsa and msa2 - specific rabbit antiserum diluted to 1 : 200 . after incubation for 20 minutes at room temperature , the cells were washed three times with 2 volumes pbs . subsequently , the cells were incubated for 20 minutes in 1 volume pbs with 1 % bsa and 1 : 100 diluted oregon green labeled goat anti - rabbit immunoglobulin g ( molecular probes ). after washing once with 2 volumes pbs and twice with 2 volumes demineralized water , the cells were resuspended in 100 μl demineralized water . a 10 μl aliquot of the resuspended cells was spread onto a polysin microslide ( menzel - gläser ), air dried , and examined under a fluorescence microscope ( zeiss ). electron microscopy . tca - pretreated l . lactis cells incubated with msa2 :: ca , msa2 :: cd or msa2 were collected and washed as described herein . immunogold labeling was performed on whole mount preparations of glutaraldehyde fixed cells on formvar - carbon coated nickel grids using auroprobe 15 nm goat anti - rabbit igg gold marker ( amersham ). primary antibodies against msa2 were diluted 1 : 1000 in pbs - glycine buffer . the labeled samples were stained with 0 . 1 % uranyl acetate ( w / v in water ) and examined in a philips cm10 transmission electron microscope at 100 kv . pretreatment of l . lactis cells with different chemicals . the ca protein anchor of l . lactis acma can be used to bind fusion proteins to a wide variety of gram - positive bacteria . however , the amount of fusion protein that binds varies greatly among this group of bacteria . binding of msa2 :: ca that covers the entire cell surface of some lactobacilli was observed , whereas other bacteria such as l . lactis showed only limited localized binding ( fig2 a ). this phenomenon may be due to the fact that the cell walls of some bacterial species contain components that interfere with ca anchor binding . since chemicals like sds , tca , chloroform / methanol and others may be used to remove components from isolated bacterial cell walls ( morata de ambrosini et al . 1998 ), the effect of the removal of cell - wall components from l . lactis whole cells on the binding of the reporter fusion protein msa2 :: ca was investigated . l . lactis cells were pretreated as described herein with various chemicals or with lysozyme . fig3 shows typical western blots of pretreated whole cells to which msa2 :: ca was bound . mature msa2 :: ca migrates at a position of a 75 kda protein ( indicated by an asterisk ). the arrow represents msa2 :: ca that contains the prtp prosequence . the double asterisks represent msa2 :: ca from which one or two of the repeats have been removed . a cell membrane anchored protease htra has been shown to be involved in processing proproteins and in removing repeats from acma ( poquet et al . 2000 ). from the results of fig3 , it may be concluded that pretreatment with tca ( lanes 8 and 16 contain the same samples , the difference in signal intensity is due to differences in stain developing time ), hcl , h 2 so 4 and hac substantially improves the subsequent binding of msa2 :: ca ( compare with the negative control in lane 15 ). other tested acids , tfa and mca , had similar effects ( not shown ). phenol , gnhcl , formamide and chloroform / methanol pretreatments showed a moderate improvement of binding ( lanes 4 , 5 , 6 , 7 , respectively ). minor binding improvements were observed after pretreatment with sds , dmf , dmso and dtt . the results are summarized in table 1 . based on the results , it appears that pretreatment of l . lactis cells with the acids tca , tfa , mca , hcl , h 2 so 4 and hac are the most effective agents for improving binding of ca anchor fusion proteins to lactococcal cells . acids such as tca are known to remove lipoteichoic acids from cell walls . whether proteins are removed from the cell walls by these acid treatments was also analyzed . fig4 shows a coomassie stained gel of lysed pretreated cells . most of the acid treatments , except for hac , removed a substantial amount of proteins from the lactococcal cells . since hac removed only a trace amount of proteins ( compare lane 1 and 4 ) and sds pretreatment ( which is known to remove proteins from the cell walls ) showed only a minor improvement of msa2 :: ca binding ( fig3 , lane 1 ), it may be concluded that removal of proteins from the cell wall is not critical for improving the binding of ca anchor fusions . this conclusion may be due to the fact that lipoteichoic acids or carbohydrates occupy sites in the cell walls of l . lactis that interfere with efficient binding . alternatively , acid pretreatment may result in altering the compactness of peptidoglycan strands that make ca binding sites more available . tca pretreatment was also used in all other experiments . the optimal tca concentration in the boiling procedure was determined . tca percentages of 1 , 5 , 10 and 20 % were tested . although 1 % tca pretreatment already showed a significant improvement in binding of msa2 :: ca and 5 % tca pretreatment showed a further increase , no further improvement was observed at concentrations higher than 10 % tca ( fig5 ). therefore , the boiling procedure with 10 % tca was selected as the standard procedure for the experiments . the binding characteristics of the lactococcal ca homolog cd in an msa2 fusion were analyzed using the standard tca pretreatment procedure . two of the three acmd repeats are highly homologous to those of acma . an alignment is shown in fig6 . secreted msa2 without an anchoring domain was included in these experiments as a negative control . in western blots , the effect of tca pretreatment on the binding of msa2 :: ca was evident ( fig7 , compare lanes 1 and 4 ). the effect of tca pretreatment was also studied using fluorescence microscopy ( fig2 , compare l . lactis in a and b ; fig8 ) and electron microscopy ( fig9 , compare a and b ). independent of the technique used , the effect of tca pretreatment on the binding of msa2 :: ca can be detected . the binding of msa2 :: cd to non - tca - pretreated l . lactis cells was low as detected in western blots ( fig7 , lane 2 ) and was undetectable in fluorescence microscopy and electron microscopy ( fig9 a ). tca pretreatment only had minor effects on the intensity of the msa2 :: cd signal in western blots ( fig7 , lane 5 ). at the same time , no msa2 :: cd specific signal associated with the pretreated cells could be observed in fluorescence microscopy ( fig8 ) and only low levels of labeling were observed in electron microscopy ( fig9 c ). some cell - associated signal was observed for msa2 without anchoring domain for both non - tca pretreated and tca - pretreated l . lactis cells ( fig7 , lanes 3 and 6 , respectively ). however , for msa2 :: cd , this was not observed in fluorescence microscopy ( not shown ) and only minor labeling signals were found in electron microscopy ( fig9 d ). taken together , it may be concluded that : ( i ) the reporter protein msa2 does have some low degree of affinity for bacterial cell walls that can be detected in western blots ; ( ii ) the ca anchor domain specifically stimulates the binding of the reporter fusion to non - pretreated cells ; ( iii ) chemical pretreatment , especially with acids , enhances this binding ; and ( iv ) the cd anchor domain does not promote binding of fusion proteins under the conditions applied . the fluorescence microscopic images and electron microscopic images of tca pretreated lactococcal cells ( fig2 , 8 and 9 ) showed that pretreatment leaves the integrity of the cell intact . however , cells are no longer viable ( plating efficiency 0 ) and therefore may be considered as inert spherical peptidoglycan microparticles with a diameter of approximately 1 μm , “ ghost cells .” binding to other gram - positives . the binding of msa2 :: ca , msa2 :: cd and msa2 without anchor domain to the gram - positive bacteria b . subtilis , lb . casei and m . smegmatis was also analyzed . fig1 shows a western blot summarizing binding of msa2 :: ca , msa2 :: cd and msa2 to non - pretreated and tca - pretreated b . subtilis cells . as for l . lactis , an increase in binding is observed for msa2 :: ca . an msa :: ca specific signal could also be visualized in fluorescence microscopy of non - pretreated b . subtilis cells , but with a highly improved signal for the tca - pretreated cells ( not shown ). binding of msa2 :: cd and msa2 to non - pretreated or tca - pretreated cells could not be demonstrated in fluorescence microscopy ( not shown ). similar results were obtained for lb . casei and m . smegmatis . the improved binding of msa2 :: ca to tca - pretreated lb . casei cells is shown in fig1 . for msa2 :: cd and msa2 , no fluorescence signals were detected ( not shown ). the tca pretreatment of m . smegmatis also had a positive effect on the binding of msa2 :: ca , whereas no binding was observed for msa2 :: cd or msa2 ( fig1 ). taken together , it may be concluded that acid pretreatment , such as with tca , improves the binding of ca protein anchor fusions to the cell surface of gram - positive bacteria . binding strength and storage conditions . the strength of the msa2 :: ca binding to tca - pretreated l . lactis cells was analyzed with a treatment of licl after the binding . licl is commonly used to remove proteins from bacterial cell walls . from the western blot of fig1 , it may be concluded that 8 m licl partially removes msa2 :: ca from the l . lactis cells ( compare lanes 4 and 5 ). therefore , although msa2 :: ca binds non - covalently to cell walls , the binding interactions are most likely very strong . cell - free culture supernatants with msa2 :: ca were stored with or without 10 % glycerol at − 20 ° c . msa2 :: ca stored in this manner for several weeks had the same capacity to bind to tca - pretreated l . lactis cells ( not shown ). tca - pretreated l . lactis cells with bound msa2 :: ca were stored for 3 weeks at + 4 ° c . in demineralized water or at − 80 ° c . in demineralized water with or without 10 % glycerol . the samples were analyzed in western blots . storing pretreated cells with bound msa2 :: ca for 3 weeks in water at + 4 ° c . only resulted in a loss of signal of about 50 % ( fig1 , compare lanes 4 and 6 ). whether this loss of signal was due to degradation or due to release of the protein into the water was not determined . storage at − 80 ° c . with or without 10 % glycerol had no effect on the binding ( fig1 , compare lanes 4 , 7 and 8 ). in addition , the effects of drying and lyophilization on the binding of msa2 :: ca to tca - pretreated l . lactis cells were studied . drying of pretreated cells had no observable negative effect on binding of msa2 :: ca afterwards . dried pretreated cells with bound msa2 :: ca could be resuspended in water without losing bound fusion protein . this was also observed for lyophilized cells with bound msa2 :: ca . lyophilization of tca - pretreated cells prior to binding resulted in loss of the binding capacity for msa2 :: ca ( results not shown ). from these data , it may be concluded that : ( i ) in spite of the non - covalent character of ca anchor binding to cell walls , the binding is very strong ; ( ii ) cell - free culture supernatants can be stored safely at − 20 ° c . ; and ( iii ) drying of tca - pretreated cells provides an efficient and simple method for storage of such cells either with or without bound ca - anchor fusions . oral immunizations of rabbits with non - recombinant lactococcus lactis preloaded with the plasmodium falciparum malaria antigen msa2 fused to the lactococcal acma protein anchor in example 1 , a technology is described that efficiently binds protein hybrids when externally added to the cell surface of non - recombinant gram - positive bacteria by means of an acma - type protein anchor . this technology provides the possibility to provide bacteria or bacterial cell walls with new traits without introducing recombinant dna into them . the immunogenicity in rabbits of the plasmodium falciparum merozoite surface protein , msa2 of strain 3d7 ( ramasamy et al . 1999 ), presented on the cell surface of non - recombinant non - living l . lactis cells as an acma anchor fusion protein was investigated . bacterial strains and growth conditions . the l . lactis strain which produces msa2 :: ca , the strain &# 39 ; s growth conditions , the induction for expression , the tca pretreatment of the l . lactis recipient cells and the binding of msa2 :: ca to the cells was described in example 1 with the following modification : a ratio of 1 ( tca - pretreated cells ) to 5 ( cell - free culture supernatant with msa2 :: ca ) was used for binding . an l . lactis nz9000 strain carrying plasmid png3043 was used as a positive control in the immunization experiments ( was positive in a previous , unpublished experiment ). plasmid png3043 encodes an msa2 hybrid protein that contains the lactococcal prtp cell - wall anchoring domain at its c - terminus ( msa2 :: cp ) instead of the acma protein anchor . the prtp cell - wall anchoring domain contains the lpxtg ( seq id no : 1 ) motif that enables a membrane - linked sortase to covalently couple the protein to the cell wall ( navarre and schneewind 1994 ). the cp domain used in construct png3043 corresponds to nt 6539 to 6914 in kok et al . ( 1988 ). primers used for the amplification of this fragment were prtp . cwa . fw3 ( 5 ′- atataaagcttgcaaagtctgaaaacgaagg ( seq id no : 8 )) and prtp . cwa . rev ( 5 ′- ccgtctcaagctcactattcttcacgttgtttccg ( seq id no : 9 )). the primers include restriction endonuclease recognition sites for cloning . plasmid png3043 differs from plasmid png3041 in the cell - wall binding domain . growth conditions and induction of expression of strain nz9000δacma ( png3043 ) were the same as for strain nz9000δacma ( png3041 ). rabbit immunizations . ten barrier - reared , new zealand white rabbits obtained from harlan laboratories , the netherlands , were used in groups of 2 for experimental immunizations . the care and use of animals were according to who guidelines ( who / lab / 88 . 1 ). the rabbits were ear bled prior to immunization to obtain preimmune sera . details of the rabbits and immunogens are as follows : rabbits a1 and a2 were subcutaneously immunized with nz9000δacma ( png3041 ) cells ( recombinant , msa2 :: ca partly surface anchored ). rabbits b1 and b2 were subcutaneously immunized with nz9000δacma ( negative control ). rabbits c1 and c2 were orally immunized with nz9000δacma ( png3043 ) cells ( recombinant , msa2 :: cp surface anchored ). rabbits d1 and d2 were orally immunized with nz9000δacma ( png3041 ) cells ( recombinant , msa2 :: ca surface anchored ). rabbits e1 and e2 were orally immunized with tca treated nz9000δacma to which msa2 :: ca had been bound from nz9000δacma ( png3041 ) culture supernatant ( non - recombinant , msa2 :: ca surface anchored ). stocks of nz9000δacma ( png3043 ) with msa2 :: cp expressed at its surface were stored in aliquots of 10 11 cells in growth medium containing 10 % glycerol at − 80 ° c . the cells remain viable under these conditions and retain msa2 on the surface as demonstrated by immunofluorescence ( not shown ). the first immunization was carried out with freshly grown bacteria . for subsequent immunizations , stocks of bacteria were freshly thawed , washed and resuspended in buffer at the appropriate concentration for immunizations . on the other hand , the non - pretreated nz9000δacma ( negative control ), the non - pretreated nz9000δacma ( png3041 ) and the tca - pretreated nz9000δacma with the externally bound msa2 :: ca were prepared daily from fresh cultures . subcutaneous injections were performed with a total of 5 × 10 9 cells in 100 μl pbs without any adjuvant into two sides on either side of the spine . the subcutaneous injections were repeated two more times at three - week intervals . prior to oral immunization , the rabbits were deprived of water and food for two to four hours . the rabbits were then fed 5 × 10 10 cells resuspended in 1 ml of 0 . 5 % sucrose . each dose was repeated for three successive days to obtain reproducible oral immunization . altogether , three series of oral immunizations were given at three - week intervals . adverse effects consequent to the immunizations , including granulomas at the sites of subcutaneous injections , were not observed , indicating that l . lactis was well tolerated by the animals . serum antibody responses . rabbits were ear bled two weeks after each immunization to obtain sera for antibody assays . the sera were stored at − 20 ° c . until use . ten - fold serial dilutions of the antisera in 2 % bsa in pbs were used in immunofluorescence assays ( ifa ) to determine the titer of the antibodies against msa2 on the surface of 3d7 p . falciparum merozoites . ifa was performed on acetone - methanol fixed late stage 3d7 p . falciparum parasites as previously described ( ramasamy 1987 ). for detection of antibody isotypes , oregon green conjugated goat anti - rabbit ig ( molecular probes ) was used as the second antibody . for detection of igg antibodies , a fluorescein conjugated , affinity purified , mouse monoclonal with specificity against rabbit igg chains ( rockland ) was used . surface expression of msa2 in different l . lactis strains . coomassie staining of sds - page gels and fluorescence microscopy were used to determine , in a semi - quantitative way , the number of msa2 molecules expressed and surface exposed by the recombinant lactococcal strains carrying plasmid png3041 or png3043 that produce msa2 :: ca or msa2 :: cp , respectively , and by the non - recombinant tca - pretreated l . lactis cells to which msa2 :: ca had been bound from the outside . the recombinant strains were estimated to produce approximately 1 . 4 × 10 5 molecules of msa2 :: ca or msa2 :: cp . the surface exposure of msa2 :: ca and msa2 :: cp differed considerably as shown by fluorescence microscopy in fig1 . the non - recombinant tca - pretreated l . lactis cells with bound msa2 :: ca showed a uniform staining of the entire cell surface . however , the semiquantitative sds - page analysis indicated that about 1 × 10 4 molecules of msa2 :: ca per cell were represented . accordingly , it may be concluded that the number of surface - exposed msa2 :: ca and msa2 :: cp on the recombinant lactococcal strains is less than 10 % of the total number of molecules produced by these strains . the other molecules are most likely trapped in the membrane or the cell wall . similar observations were made by norton et al . ( 1996 ) for the expression of ttfc fused to the cp cell - wall anchoring domain . in that study , only membrane - associated or cell - wall - associated ttfc could be demonstrated and no surface - exposed ttfc :: cp was demonstrated . thus , it appears that binding from the outside to tca - pretreated cells is a more efficient method to surface - exposed proteins on l . lactis cells . anti - msa2 antibody responses in orally immunized rabbits . characteristics of the anti - msa2 antibody response to the immunizations are summarized in table 2 . the oral immunizations with the recombinant l . lactis that produces msa2 :: cp ( rabbits c1 and c2 ) were done before ( unpublished results ) and used as a positive control . in the previous experiment , a similar antibody response was found . the present experiment showed that specific antibodies against near native msa2 were detectable after two immunizations for group a , d and e rabbits , and that antibody titers increased in all instances after a third immunization . igg antibodies were predominant after three immunizations in either the subcutaneous or oral route . a comparatively weak anti - msa2 surface ifa , attributable to the generation of cross - reactive antibodies ( as described herein ), was also observed after three control subcutaneous immunizations with l . lactis cells alone . taken together , the results indicate that : ( i ) msa2 produced by lactococcal cells elicits serum antibodies that recognize native p . falciparum parasite msa2 ; ( ii ) msa2 - specific t h cells are activated through mucosal immunization due to the presence of systemic igg antibodies ( table 2 ) that can be boosted ( unpublished results ); and ( iii ) oral immunizations with msa2 :: ca bound to non - recombinant non - living tca - pretreated l . lactis cells are as efficient in evoking specific serum antibody responses as the live recombinant strain producing msa2 :: ca that was administered subcutaneously or orally , or as efficient as the live recombinant strain producing msa2 :: cp that binds msa2 covalently to its cell wall delivered orally . anti - lactococcal antibody responses . western blots ( fig1 ) demonstrated significant antibody responses against l . lactis antigens after two and three immunizations of the rabbits . the responses were notably greater after subcutaneous ( group a and b rabbits ) than oral immunization with l . lactis ( group c rabbits ). oral immunization with the tca - pretreated lactococcal cells ( group e rabbits ) elicited antibodies that reacted at a lower intensity with fewer l . lactis antigens than oral immunization with viable l . lactis cells . this is most likely due to the fact that proteins are removed from the lactococcal cells by the tca pretreatment ( see , example 1 ). the lower anti - carrier response observed for the tca - pretreated ( non - recombinant ) cells renders this type of delivery vehicle more suitable for repeated immunization strategies than its untreated ( recombinant ) counterpart . the cell - wall binding domain or anchor of the lactococcal cell - wall hydrolase acma includes three repeats of 45 amino acids that show a high degree of homology ( buist et al . 1995 ). these three repeats belong to a family of domains that meet the consensus criteria as defined in pct publication wo 99 / 25836 and can be found in various surface - located proteins in a wide variety of organisms . another feature that most of these domains have in common is that their calculated pi values are high , approximately 8 or higher ( table 3 ). the ph used in previous binding experiments with msa2 :: ca ( i . e ., examples 1 and 2 ) was approximately 6 , indicating that the binding domain was positively charged . the acma protein anchor homolog of the lactococcal cell - wall hydrolase acmd ( cd ) ( bolotin et al . 2001 ) also includes three repeats ( fig1 ) with a calculated pi that is lower ( approximately pi 3 . 8 ) than that of the ca domain ( table 4 ). consequently , the cd anchor was negatively charged at the binding conditions used in example 1 . no binding of the msa2 :: cd reporter protein occurred under these conditions as demonstrated herein . therefore , the influence of the ph during binding of a cd fusion protein ( msa2 :: cd ) was investigated . furthermore , a hybrid protein anchor including the three cd repeats and one ca repeat that has a calculated pi value that is higher than that of the cd repeats alone was constructed . the hybrid protein anchor showed better binding ph values above the pi of the cd repeats alone , indicating that the ph binding range of acma - type protein anchors can be manipulated by using the pi values of the individual repeats in hybrids . bacterial strains , growth and induction conditions , tca pretreatment of l . lactis cells , incubation of the msa2 protein anchor fusion proteins to tca - pretreated cells , washing conditions , protein gel electrophoresis , western blotting and immunodetection were the same as described herein with reference to example 1 . the cell - free culture supernatants with msa2 :: ca , msa2 :: cd or a3d1d2d3 have a ph of approximately 6 . 2 . the influence of ph was examined by adjusting the ph of the cultures by the addition of hcl or naoh to obtain the required ph . plasmid constructions . the plasmid that expresses the msa2 :: cd fusion was described herein with reference to example 1 . plasmid ppa43 is based on the same expression plasmid and contains an in - frame fusion of the lactococcal signal sequence of usp45 ( ssusp ; van asseldonk et al . 1990 . gene 95 : 155 - 160 ), the c - myc epitope for detection purposes , the a3 ca repeat and repeats d1 , d2 and d3 of cd . primers used for cloning a3 were carepeat3 . fw ( ccg tct cca att caa tct gct gct gct tca aat cc ( seq id no : 10 )) and ca repeat3 . rev ( taa taa gct taa agg tct cca att cct ttt att cgt aga tac tga cca att aaa ata g ( seq id no : 11 )) ( the primers include the a3 specific sequences ). the primers used for cloning the three cd repeats were cdrepeat1 . fw ( ccgtctccaatttcaggaggaactgctgttacaactag ) ( seq id no : 12 ) and cdrepeat3 . rev ( taataagcttaaaggtctccaattccagcaacttgcaaaacttctcct ac ) ( seq id no : 13 ) ( the primers include the cd specific sequences ). binding of msa2 :: cd at low ph . since binding of msa2 :: cd was not observed at a ph ( the ph of the culture medium after growth and induction is about 6 . 2 ) higher than the calculated pi for the cd domain ( i . e ., pi 3 . 85 ), binding was studied when the ph of the medium was adjusted to ph 3 . 2 . tca - pretreated l . lactis cells were used as the binding substrate and the relative amounts of bound msa2 :: cd were analyzed in western blots . the amounts of unbound reporter protein remaining in the culture supernatant after binding were also analyzed . fig1 shows a clear increase in bound msa2 :: cd when binding is performed at ph 3 . 2 ( compare lanes 1 and 3 ). at the same time , less unbound reporter protein remained in the supernatant ( compare lanes 2 and 4 ). this result indicates that positive charges are important for binding of ca - type anchoring domains . binding of cacd hybrid anchors . analysis of the pi values of the ca homologs in table 3 indicates that two classes of repeats can be distinguished : a majority ( 99 out of 148 ) of homologs that have a high pi value (& gt ; 8 ) and a smaller group ( 33 out 148 ), of which cd is a representative , that has pi values lower than 6 . based on the experimental results , it is shown that these types of anchoring domains only bind to bacterial cell walls at a ph that is lower than the anchoring domains pi . notably , most cell - wall binding domain homologs include repeats with a pi that are representatives of one of the two groups , i . e ., only repeats with a high or low pi . some proteins with cell - wall binding domains , e . g ., those of dnir of trepanoma pallidum and an amidase of borrelia burgdorferi , include repeats with high and low pi . since the binding ph of such “ natural hybrid ” cell - wall binding domains is below the intermediate pi value of the total number of repeats present in the domain , a hybrid cell - wall protein anchor was constructed using the ca and cd repeats with an intermediate pi value . table 5 lists the native acma and acmd anchors and a number of examples of ca / cd hybrids . the constructed hybrid protein anchor ( a3 d1d2d3 ) has a calculated pi value of approximately 5 . 1 . a protein anchor including only d1d2d3 shows little binding at a ph above its calculated pi ( as described herein ). the a3 ( pi 10 ) domain shows similar binding at ph 5 and ph 7 . the binding of the hybrid anchor a3d1d2d3 was tested at ph 3 , ph 5 and ph 7 . at ph 3 , most protein had been bound to the ghost cells ( fig1 ). at ph 5 , there was considerable binding (+/− 40 %), whereas there was only minimal binding at ph 7 (+/− 20 %). this result indicates the ph range of binding for cd repeats was shifted to higher ph values by the addition of one ca repeat ( a3 ) that caused a shift in calculated pi values of 3 . 8 to 5 . 1 . the increase of binding at ph 5 for the a3d1d2d3 hybrid cannot be attributed to binding of the a3 repeat alone . if this was the case , then the same level of binding should occur at ph 7 since the a repeats show the same binding at these ph values . in addition , the increased binding at ph 5 is not an additive effect in the sense that an extra binding domain results in increased binding . it has previously been shown that addition of one repeat to the ca anchor did not result in increased binding . the binding at the higher ph values of the a3d1d2d3 repeats , as compared to the d1d2d3 repeats alone , thus may be attributed to the increase in the calculated pi value of the hybrid ca / cd anchor . this demonstrates that ph binding properties of these types of protein anchors may be manipulated on the basis of the pi values of individual repeats present in the hybrid anchor . induction of cellular immune responses in mice after oral immunizations with lactococcal ghosts displaying the malaria plasmodium falciparum antigen msa2 fused to the lactococcal acma protein anchor non - genetically modified non - living lactococcus lactis cells ( ghosts ) preloaded with the plasmodium falciparum msa2 antigen fused to the acma protein anchor ( msa2 :: ca ) were used to orally immunize mice in a similar way as described herein with reference to example 2 . in this experiment , the question of whether immunizations through the oral route with the non - recombinant non - living ghosts carrying msa2 :: ca on their surface ( ghosts - msa2 :: ca ) can elicit typical th1 - type immune responses , such as igg2 antibodies and gamma - interferon ( γifn )- producing t cells in the spleen is addressed . these responses are particularly relevant to obtain immunity for pathogens , such as malaria , that undergo stages in their life cycle where they are not in the blood but hide in cells . groups of five mice of different strains were used for immunization . the strains of mice used were balb / c ( with the major histocompatibility locus allotype of h2d ), c57 black ( h2b ), c3h ( h2k ) and icr ( outbred , i . e ., of varying h2 types ). oral immunizations were performed at three weekly intervals . immunizations were performed with msa2 :: ca absorbed onto the surfaces of tca - treated lactococcus lactis cells ( ghosts - msa2 :: ca ) or with recombinant l . lactis that displayed msa2 on the surface through the use of a covalently linked cell - wall anchor ( l . lactis ( msa2 :: cp )) as described herein with reference to example 2 . the mice were tail bled to obtain serum samples two weeks after the second , third and fourth immunizations . fecal pellets were collected and extracted to examine intestinal iga antibody production . the mice were sacrificed at the end of each experiment and the spleens were removed for examining t - cell responses by elispot . msa2 - his tag produced in e . coli was used as antigen in the elisa and elispot assay . the growth of bacterial strains and the preparation of ghost cells were as described herein with reference to example 2 . kinetics and isotypes of the serum igg antibodies generated oral immunizations . differences in the kinetics of the antibody response and the isotype distribution were observed between different murine strains . the antibody response was also different when living recombinant l . lactis ( msa2 :: cp ) or ghosts - msa2 :: ca were used as immunogens . with ghosts - msa2 :: ca , high serum antibody levels were detectable in the c3h mice after two immunizations . igg antibodies were detectable in all four murine strains after three and four immunizations . antibody titers were highest in c3h mice . igg antibodies that reacted with native msa2 on parasites were detected in the sera of immune mice by fluorescence microscopy ( ifa ) confirming that the immunizing form of the protein elicits biologically relevant antibodies . control immunizations were performed with ghosts alone where no msa2 - specific antibodies were elicited . in parallel experiments using msa2cp as the immunogen , high serum igg antibody levels were only seen with balb / c mice after two immunizations . after three and four immunizations , good antibody responses developed in c3h mice . antibody titers were highest in balb / c mice . significant differences existed between the strains in the isotypes of the elicited serum igg antibodies in response to immunization with ghosts - msa2 :: ca . balb / c mice showed higher levels of igg2a and igg2b antibodies , some igg3 antibodies and negligible igg1 , which demonstrates a possible th1 bias . on the other hand , c57 black and c3h mice had high igg1 , igg2a and igg2b , and lower igg3 antibodies to msa2 , which is more characteristic of a mixed th1 and th2 response . icr mice , as expected , showed a range of responses . some icr mice had the balb / c and others the c3h / c57 black pattern of igg isotypes . formation of mucosal antibodies . iga antibodies were detected by elisa in the fecal pellets of the icr and balb / c mice , but were not detected in c3h or c57 black mice when immunization was performed with living recombinant l . lactis ( msa2 :: cp ) or ghost - msa2 :: ca . t - cell responses . the increase of the intensity of the igg elisa reactions seen in mice immunized with ghosts - msa2 :: ca with each immunization demonstrates that boosting takes place and that a th - dependent antibody response exists in these animals . the igg isotype distribution further confirms this conclusion . therefore , th cells are generated in icr , balb / c , c57 black and c3h mice . the elispot assay for detecting gamma - interferon ( γifn ) producing cells detects mainly cd8 + tc cells , which are an important component of the immune response to many pathogens , including malaria parasites . his - tagged msa2 produced in e . coli was used as antigen in the assay . msa2 - specific γifn producing cells could be detected in the spleens of balb / c , c57 black and c3h mice that were immunized with ghosts - msa2 :: ca . msa2 - specific γifn producing cells were not observed in the spleens of control mice immunized with ghosts alone or with the living recombinant l . lactis ( msa2 - cp ). the latter group showed a high level of non - specific γifn producing cells . the high background observed may be due to ongoing inflammation . the sensitization of msa2 - specific tc cells in the spleen after immunization with the non - recombinant non - living l . lactis ghost system carrying a foreign protein is a novel finding which is applicable to malaria since protection against sporozoite - infection is associated with γifn producing cells being produced in the spleen . the non - recombinant non - living ghost system can be used in oral immunizations to elicit typical th1 - type immune responses . these types of responses are particularly relevant to obtain immunity for pathogens that undergo stages in their life cycle where the pathogens are not in the blood but rather hide in cells . the responses are more pronounced and more specific for the ghost system than for the living recombinant system . the ghost system has the additional advantage of eliminating the risk of spreading recombinant dna into the environment . protection of mice for lethal streptococcus pneumoniae challenge after oral immunizations with lactococcal ghosts preloaded with ppma antigen fused to the lactococcal acma protein anchor streptococcus pneumoniae is the leading etiological agent of severe infections including septicemia , meningitis , pneumonia , and otitis media . recent studies on the molecular epidemiology and pathogenesis of s . pneumoniae have identified pneumococcal proteins with vaccine potential . one of these proteins , the protease maturation protein ppma , has been shown to elicit immune protective potential in a mouse pneumonia model . the non - genetically modified lactococcal ghosts have been shown to be an efficient carrier for use in oral immunizations of rabbits and mice in order to elicit strong anti - malaria immune responses . the construction of lactococcal ghosts that display the s . pneumoniae ppma fused to the lactococcal acma cell - wall binding domain on their surface is described herein . the ability of these ghosts to protect orally immunized mice from a lethal nasal dose of s . pneumoniae was investigated . bacterial strains and growth conditions . l . lactis was grown and ghost cells were prepared as described herein with reference to example 1 . s . pneumoniae was grown as described before ( gingles et al . 2001 , infect . immun . 69 : 426 - 434 ). construction of ppma protein anchor fusion expression plasmid . the expression plasmid for ppma protein anchor fusion ( ppma :: ca ) was substantially similar to the expression plasmid for the msa2 protein anchor fusion as described herein with reference to example 2 . for the secretion of ppma :: ca , the secretion signal sequence of the usp45 protein ( ssusp ) of l . lactis ( van asseldonk et al . 1990 . gene 95 : 155 - 160 ) was used . the ppma gene was cloned by pcr using primers ppma . 1 ( cggtctcacatgtcgaaagggtcagaaggtg cagacc ) ( seq id no : 14 ) and ppma . 2 ( cggtctcgaattgcttcgtttgatgtactactg cttgag ) ( seq id no : 15 ) resulting in plasmid ppa32 which contains ppma as an in - frame fusion with ssusp45 and the protein anchor ( ssusp :: ppma :: ca ). expression of the fusion gene results in the secreted product ppma :: ca . the primers include an eco31i restriction enzyme recognition site that was used for digestion of the pcr fragment . this restriction digest produced ncoi and ecori sticky ends which were used for cloning . the primers also included the ppma sequences . chromosomal dna of s . pneumoniae strain d39 was used as a template for the pcr reactions . preparation of the vaccine . three liters of m17 medium with ppma :: ca , obtained after growth and used to induce producer cells for expression of l . lactis ( ppa32 ) was centrifuged and filter sterilized ( 0 . 2 μm ) to remove the producer cells . ghost cells were prepared from 0 . 5 liter of l . lactis nz9000 ( δacma ). after binding , the ghost cells with ppma :: ca ( ghosts - ppma :: ca ) were isolated by centrifugation and washed with pbs . the ghost cells were stored in pbs in aliquots of 2 . 5 × 10 10 ghosts / ml at − 80 ° c . two control groups included : ( i ) ghosts without bound ppma :: ca ; for the sample preparation , the same amounts of ghost cells were used and the same centrifugation and washing steps were performed , but the binding step was omitted ; and ( ii ) soluble ppma was isolated as a his - tagged fusion . mice immunizations . groups of 10 mice ( cd - 1 ) were used in the immunizations . oral doses included 5 × 10 9 ghosts with or without ppma :: ca ( 50 μg ) or 50 μg soluble ppma in pbs . nasal doses included 5 × 10 8 ghosts with or without ppma :: ca ( 5 μg ) or 5 μg soluble ppma . 10 8 ghosts - ppma :: ca ( 1 μg ) were subcutaneously injected . for intranasal immunizations , the mice were slightly anesthetized with isofluorane . intranasal challenge . the groups of orally immunized mice were intranasally challenged 14 days after the last booster immunization with a dose of 10 6 colony forming units ( cfu ) s . pneumoniae d39 as described ( kadioglu et al . 2000 , infect . immun . 68 : 492 - 501 ). mice were monitored after the challenge for visible clinical symptoms for 7 days , at which point the experiment was ended . mice that were alive after 7 days were considered to have survived the pneumococcal challenge and mice that became moribund during the 7 - day period were judged to have reached the endpoint of the assay . the time the animal became moribund was recorded , and the animal was sacrificed by cervical dislocation . elisa analysis . serum samples were taken from each mouse before the intranasal challenge and stored at − 20 ° c . before use . microtiter plates were coated with 100 μg ppma / ml in 0 . 05 carbonate buffer . serial 10 - fold dilutions of pooled serum of each group were incubated on the plates as described ( gingles et al . 2001 , infect . immun . 69 : 426 - 434 ). anti - mouse immunoglobulin - horseradish peroxidase conjugate was used for detection and the absorbance was measured at 492 nm . serum antibody response . mice were immunized orally , nasally and subcutaneously according to the scheme shown in fig1 . anti - ppma antibody titers in the blood serum were determined for each group by elisa assays . the results are given in fig2 . as expected , ghosts alone administered orally or nasally , ov ghosts or in ghosts , respectively , did not induce anti - ppma antibodies . soluble ppma given by the nasal route resulted in only a low anti - ppma antibody titer , which agrees with the general findings that soluble antigens are not very immunogenic when given by the mucosal routes . ghosts - ppma :: ca provided by the oral route ( ov ppma + ghost ) induced only a low level of anti - ppma serum antibodies . this contrasts the results for the oral immunization experiments described herein with reference to examples 2 and 4 with msa2 :: ca . however , the contrast may be antigen - type related . intranasal administration of ghosts - ppma :: ca resulted in a high titer of anti - ppma antibodies ( in ppma + ghosts ). a high titer was also obtained by subcutaneous administration of ghosts - ppma :: ca . these titers were lower by a factor of 5 to 10 when compared to soluble ppma that was subcutaneously administered and formulated with the strong freund &# 39 ; s complete adjuvant ( peter adrian , erasmus university rotterdam , the netherlands , unpublished results ). in addition , the freund &# 39 ; s ppma vaccine contained 50 μg ppma per dose , whereas the intranasally administered ghosts - ppma : ca contains only 5 μg / dose and the subcutaneous ghost - ppma :: ca vaccine contains only 1 μg ppma / dose . this result demonstrates the adjuvant effect of the ghost cells . side effects of the orally , nasally or subcutaneously administered ghosts were not observed , which is in contrast to the severe side effects that are usually seen with the use of freund &# 39 ; s adjuvants . the results demonstrate that high titer serum antibodies can be obtained by the mucosal route of administration . these data also show that ghost cells may be safely used in traditionally injected vaccines without side effects in order to induce high titer serum antibodies . protection against challenge . the mice orally immunized with soluble ppma , ghosts alone or ghosts - ppma :: ca were challenged 14 days post - immunization with a lethal intranasal dose of s . pneumoniae . the mice immunized with soluble ppma or ghosts alone died within 72 hours after challenge . the group immunized with ghosts - ppma :: ca showed a survival rate of 40 % ( fig2 ). these results show that mucosal immunization of mice with ghosts - ppma induces protective immunity against a lethal s . pneumoniae challenge . in conclusion , the non - recombinant non - living ghost system may be used to elicit high titer serum antibodies and the mucosal route of administration may be used to obtain protective immunity against a mucosally acquired pathogen . titers are expressed as the negative logarithms of the lowest ten - fold dilution of sera giving a detectable reaction on the merozoite surface . the last column represents the igg fraction of the antibody response . 0 : indicates no detectable reaction at a 1 : 10 dilution of the serum .