BACKGROUND
pertussis or whooping cough is an infectious respiratory disease caused by the bacterium bordetella pertussis.
despite being preventable by vaccination, pertussis remains one of the top ten causes of death worldwide in childhood, mainly in unvaccinated children  <cit> .
according to the world health organization , about  <dig>  million cases of pertussis occurred all over the world and about  <dig>  patients died of pertussis in  <dig>  <cit> .
most of deaths occurred in the developing countries.
immunization with whole cell pertussis vaccines  was started in the middle of 20th century and has significantly reduced the incidence of pertussis in many countries including china  <cit> .
however, these wpvs have drawbacks in causing side effects such as local pain, redness, swelling, fever, and fussiness  <cit> .
due to the fear of side effects and the need for booster immunizations in older age groups, acellular pertussis vaccines  were developed and they were first introduced in japan in  <dig>  <cit> .
typically current apvs are comprised of antigens directly purified from cultured b. pertussis bacteria.
they may include pertussis toxin , filamentous hemagglutin , prn, or fim <dig> and fim <dig>  clinical efficacy trials carried out in sweden and italy indicated that apvs containing two or three more components  were more effective than the pt alone and/or fha based vaccines  <cit> .
in china, two component apvs containing pt and fha have been developed and utilized since 1990s  <cit> .
prn, originally called 69-kda outer membrane protein, has been shown to play a role in invasion of eukaryotic cells by b. pertussis bacteria  <cit> .
it has also reported that prn elicits both humoral and cellular immune responses in mice and protects infant mice from respiratory challenge by b. pertussis  <cit> .
however, the low yield of prn from cells or the culture supernatant of b. pertussis has been a limiting factor in the production of prn-containing apvs  <cit> .
fimbriae , also known as pili and agglutinogen, belong to bacterial adhesins which are expressed on the b. pertussis surface.
fim <dig> and fim <dig> are closely related in molecular weight  but are serologically distinct  <cit> .
similar characteristics and molecular weight of fim <dig> and fim <dig> hampered the production of separate proteins from b. pertussis  <cit> .
so far there have been no separate purified fim <dig> and fim <dig> available.
in addition, antigenic divergence between vaccine strains and clinical isolates  <cit>  as well as the possible presence of other reactogenic contaminants  <cit> , should be considered during purification of those proteins.
to overcome these difficulties, attempts have been made to express the proteins in vitro by recombinant technology.
this technology has advantages regarding of higher yield and controlled production of recombinant proteins at a high homogeneity  <cit> .
if such a platform could be established, not only the cost for apv production could be reduced, but also the ability to deal with the antigenic shift could be enhanced.
in this report, we described a method that can be used to produce large amount of rprn, rfim <dig> and rfim <dig> proteins.
by using these proteins, we studied their immunogenicity and protective properties in mouse model.
RESULTS
expression and characterization of rprn, rfim <dig> and rfim3
to generate recombinant proteins rprn, rfim <dig> and rfim <dig> in escherichia coli, respective genes were amplified from a chinese vaccine strain cs and cloned into a protein expression vector.
three expression plasmids were constructed, and the dna sequences were confirmed by dna sequencing.
protein expression was induced by isopropyl-β-d-thiogalactopyranoside , and purification of the three recombinant proteins was achieved through nickel affinity chromatography with the histraptm hp column.
each purified protein migrated as a single band with the predicted size in sds-page, of which purity was more than 95% .
the specificity of the bands was confirmed by using specific antibodies generated against native forms of prn, fim <dig> or fim <dig>  respectively, in western blotting .
by using this approach, a large amount of proteins was obtained, at approximately  <dig> mg/l of rprn,  <dig> mg/l of rfim <dig>  and  <dig> mg/l of rfim <dig>
serum antibody responses to rprn, rfim <dig> and rfim3
in order to examine the antibody responses to rprn, rfim <dig> and rfim <dig>  sera of immunized mice were collected two weeks after the second immunization.
titres of serum igg antibodies were measured by elisa.
significant igg antibody responses were observed in the mice immunized with both high and low doses of rprn, rfim <dig> or rfim <dig> when compared to the control group  .
high levels of igg antibodies were induced in mice immunized with high doses of the three proteins.
however, the differences were not significant when compared to those in mice immunized with low doses .
when the same amount of rfim <dig> and rfim <dig> was used in immunization, igg responses appeared to be similar between the two groups .
serum cytokine responses to the rprn, rfim <dig> and rfim3
both th <dig>  and th <dig>  cytokine responses were determined after the second immunization with the recombinant proteins.
for il- <dig>  significant higher levels were induced in mice immunized with rprn, rfim <dig> or rfim <dig> when compared to the control mice .
for tnf-α, significant higher level was only observed in mice immunized with rprn , but not in those with rfim <dig> or rfim <dig>  the il- <dig> induction was not found in all groups of mice .
intranasal challenge with b. pertussis
seven days after the intranasal challenge with b. pertussis, the bacterial loads were significantly lower in the lungs of mice immunized with high or low doses of rprn, compared to those observed in the control mice .
for the mice immunized with rfim <dig> or rfim <dig>  no significant difference was observed in the bacterial loads in the lungs compared to the control mice .
intracerebral challenge with b. pertussis
two weeks after the intracerebral challenge with a lethal dose of b. pertussis, none of the mice in the control group survived .
in contrast, a dose-dependent protection was observed in mice immunized with different doses of the reference vaccine.
for the mice immunized with rprn, some protection against the lethal dose of intercerebral challenge was noticed when compared to the control mice .
the level of this protection provided from immunization with rprn was clearly higher than that from the immunization with  <dig>  iu of reference vaccine .
the result suggested that immunization with rprn alone can confer partial protection against a lethal intracerebral b. pertussis challenge.
such intracerebral challenge assays were also performed in the groups immunized with different doses of rfim <dig> and rfim <dig>  however, no significant protection was observed as none of mice were survived in the groups immunized with  <dig> μg dose of rfim <dig> and  <dig> μg dose of rfim <dig> and a few  survival mice in other dose groups.
discussion
because of its advantages in cost, yield and purity, vaccine based on recombinant components has been considered to be a valuable alternative for the vaccine production  <cit> , in particular for the developing countries.
in the present study, we showed that the recombinant prn, fim <dig> and fim <dig> proteins can be readily expressed and purified in large quantities from e. coli, and each recombinant protein solution is stable for up to twelve months when stored at below -20°c.
they were prepared in a large quantity and freeze-dried.
it was confirmed that the activity of freeze-dried preparation had no difference significantly compared with liquid preparation by elisa method and in some animal experiments .
the three recombinant proteins can elicit both humoral and cellular immune responses when they were investigated in mice.
furthermore, this recombinant technology makes it possible to avoid contaminations from the b. pertussis components that may cause side effects in vaccine preparations  <cit> .
availability of the purified fim <dig> and fim <dig> also provided an opportunity to assess their individual roles in the immunogenicity and protective efficacy.
as a virulence factor of b. pertussis, the ability of prn to function as adhesin has been investigated both in vitro and in vivo  <cit> .
it is reported that the prn-mediated protection may be afforded by blocking prn-mediated attachment of b. pertussis to the host cells  <cit> .
studies on the immunized children have also suggested that high level of circulating antibodies against prn are associated with protection  <cit> .
furthermore, evidence suggests that anti-prn antibodies may promote extracellular killing with complement or as opsonins, and mediate killing bacteria by phagocytes  <cit> .
however, although antibodies specific to b. pertussis antigens confer protection, many studies have indicated that humoral immunity alone is not sufficient to provide long-term protection against b. pertussis infection and that the protection against b. pertussis requires both t cell- and b cell-mediated immunity  <cit> .
our results showed that the antibody response increased significantly in mice immunized with rprn.
immunization of rprn also induced a th <dig> response that is characterized by the enhanced production of il-2- and tnf-α.
these results also indicated that the rprn shared a similar feature with the native form of prn, suggesting that b cell and t cell epitopes might be highly conserved in rprn.
murine intranasal and intracerebral challenge assays have been validated and used to demonstrate the protection of pertussis vaccine for many years  <cit> .
the results obtained from the intranasal and intracerebral challenge tests strongly suggest that rprn functions as a protective antigen.
these observations are consistent with previous reports that a higher th1-type response was associated with a stronger level of protection against b. pertussis  <cit> .
in this study, the bacterial loads were only evaluated on day  <dig> in lungs of the mince after the intranasal challenge.
however, a time course of infection would probably provide more information on the protective properties of the proteins studied.
so far, twelve, two and four different variants have been reported in prn, fim <dig> and fim <dig>  respectively  <cit> .
at present, the prevalent allele combinations of b. pertussis isolates are prn2/fim3b  <cit> .
the strains used in this study and the strains used for vaccine production are prn1/fim3a or prn6/fim3a.
as the difference occurred between b. pertussis vaccine strains and circulating isolates in many countries  <cit> , it has been proposed that the strain variation may have effect on the vaccine efficacy  <cit> .
in this case, engineering strategies will remedy antigenic shifts by performing genetic mutation on the antigen encoding genes, which is an advantage of using recombinant proteins compared with the ones purified from b. pertussis.
because of the similarity in the molecular weight, it is extremely difficult to purify separately fim <dig> and fim <dig> proteins from b. pertussis.
therefore, antibody responses against fim <dig> or fim <dig> were only measured in elisa using a mixture of fim <dig> and fim <dig> proteins as coating antigen in clinical vaccine trials  <cit> .
the exact role of fim <dig> and fim <dig> in protection against pertussis is not fully known.
in this study, recombinant fim <dig> and fim <dig> were expressed and purified separately.
for the first times, their functions in protection against pertussis were assessed separately in mice model.
the study demonstrated that higher antibody titres and cellular immune response characteristic of increased production of il- <dig> were induced in mice immunized with rfim <dig> and rfim <dig>  although monoclonal anti-fim <dig> and anti-fim <dig> antibodies were used in the study, it remains to be shown whether there is cross-reacting response between fim <dig> and fim <dig>
it is known that il- <dig>  tnf-α and ifn-γ are characteristic cytokines for th <dig> response, and il- <dig> and il- <dig> for th <dig> response  <cit> .
in this study, we have only measured serum concentrations of il- <dig>  tnf-α and il- <dig>  it is interesting to study concentrations of other cytokines such as ifn-γ in sera collected from mice after immunization and infection.
further, serum il- <dig> was not measurable in all mice tested in this study.
this was in contrast to the finding that il- <dig> and tnf-α were significantly induced.
the exact reason for the undetectable il- <dig> was unknown.
one explanation might be the nih mice used in this study.
it is known that nih mice predominate on cellular immunity.
another explanation might be timing of the serum sampling and possible posttranscriptional regulation of il- <dig>  no matter if il- <dig> was measurable or not, anti-pertussis antibodies were significantly induced in mice immunized with each of the three recombinant proteins.
previous vaccine efficacy trial in sweden indicated that inclusion of prn, fim <dig> and fim <dig> into acellular vaccine containing pt and fha provided higher protection against pertussis.
however, the contribution of individual components in the protection was not revealed  <cit> .
since fim of b. pertussis facilitates a variety of binding capabilities as adhesins  <cit> , some studies suggested that passive protection against b. pertussis infection might be conferred due to the existence of higher titres of anti-fim <dig> or anti-fim <dig> antibodies which might transmigrate into the lower respiratory tract in mice  <cit> .
in contrast, the results from intranasal and intracerebral challenges with b. pertussis indicated very limited role played by rfims in bacterial clearance, although higher titres of anti-fim antibodies have been observed in this study.
these data suggest that rfim <dig> or rfim <dig> alone may not be enough to provide the protection against b. pertussis and that they should be used in combination with other vaccine components such as pt, fha, and/or prn.
CONCLUSIONS
b. pertussis proteins prn, fim <dig>  and fim <dig> can be genetically manipulated and expressed in a large amount in vitro.
the three recombinant proteins can elicit both humoral and cellular immune responses.
immunization with rprn can confer certain protection in mouse infection models.
these recombinant proteins, especially rprn, have a potential for the development of a new generation of apvs in developing countries such as china.

 pertussis or whooping cough is an infectious respiratory disease caused by the bacterium bordetella pertussis. despite being preventable by vaccination, pertussis remains one of the top ten causes of death worldwide in childhood, mainly in unvaccinated children. according to the world health organization, about million cases of pertussis occurred all over the world and about patients died of pertussis in. most of deaths occurred in the developing countries. immunization with whole cell pertussis vaccines was started in the middle of 20th century and has significantly reduced the incidence of pertussis in many countries including china. however, these wpvs have drawbacks in causing side effects such as local pain, redness, swelling, fever, and fussiness. due to the fear of side effects and the need for booster immunizations in older age groups, acellular pertussis vaccines were developed and they were first introduced in japan in. typically current apvs are comprised of antigens directly purified from cultured b. pertussis bacteria. they may include pertussis toxin, filamentous hemagglutin, prn, or fim and fim clinical efficacy trials carried out in sweden and italy indicated that apvs containing two or three more components were more effective than the pt alone and/or fha based vaccines. in china, two component apvs containing pt and fha have been developed and utilized since 1990s. prn, originally called 69-kda outer membrane protein, has been shown to play a role in invasion of eukaryotic cells by b. pertussis bacteria. it has also reported that prn elicits both humoral and cellular immune responses in mice and protects infant mice from respiratory challenge by b. pertussis. however, the low yield of prn from cells or the culture supernatant of b. pertussis has been a limiting factor in the production of prn-containing apvs. fimbriae, also known as pili and agglutinogen, belong to bacterial adhesins which are expressed on the b. pertussis surface. fim and fim are closely related in molecular weight but are serologically distinct. similar characteristics and molecular weight of fim and fim hampered the production of separate proteins from b. pertussis. so far there have been no separate purified fim and fim available. in addition, antigenic divergence between vaccine strains and clinical isolates as well as the possible presence of other reactogenic contaminants, should be considered during purification of those proteins. to overcome these difficulties, attempts have been made to express the proteins in vitro by recombinant technology. this technology has advantages regarding of higher yield and controlled production of recombinant proteins at a high homogeneity. if such a platform could be established, not only the cost for apv production could be reduced, but also the ability to deal with the antigenic shift could be enhanced. in this report, we described a method that can be used to produce large amount of rprn, rfim and rfim proteins. by using these proteins, we studied their immunogenicity and protective properties in mouse model.  expression and characterization of rprn, rfim and rfim3 to generate recombinant proteins rprn, rfim and rfim in escherichia coli, respective genes were amplified from a chinese vaccine strain cs and cloned into a protein expression vector. three expression plasmids were constructed, and the dna sequences were confirmed by dna sequencing. protein expression was induced by isopropyl-β-d-thiogalactopyranoside, and purification of the three recombinant proteins was achieved through nickel affinity chromatography with the histraptm hp column. each purified protein migrated as a single band with the predicted size in sds-page, of which purity was more than 95%. the specificity of the bands was confirmed by using specific antibodies generated against native forms of prn, fim or fim respectively, in western blotting. by using this approach, a large amount of proteins was obtained, at approximately mg/l of rprn, mg/l of rfim and mg/l of rfim serum antibody responses to rprn, rfim and rfim3 in order to examine the antibody responses to rprn, rfim and rfim sera of immunized mice were collected two weeks after the second immunization. titres of serum igg antibodies were measured by elisa. significant igg antibody responses were observed in the mice immunized with both high and low doses of rprn, rfim or rfim when compared to the control group. high levels of igg antibodies were induced in mice immunized with high doses of the three proteins. however, the differences were not significant when compared to those in mice immunized with low doses. when the same amount of rfim and rfim was used in immunization, igg responses appeared to be similar between the two groups. serum cytokine responses to the rprn, rfim and rfim3 both th and th cytokine responses were determined after the second immunization with the recombinant proteins. for il- significant higher levels were induced in mice immunized with rprn, rfim or rfim when compared to the control mice. for tnf-α, significant higher level was only observed in mice immunized with rprn, but not in those with rfim or rfim the il- induction was not found in all groups of mice. intranasal challenge with b. pertussis seven days after the intranasal challenge with b. pertussis, the bacterial loads were significantly lower in the lungs of mice immunized with high or low doses of rprn, compared to those observed in the control mice. for the mice immunized with rfim or rfim no significant difference was observed in the bacterial loads in the lungs compared to the control mice. intracerebral challenge with b. pertussis two weeks after the intracerebral challenge with a lethal dose of b. pertussis, none of the mice in the control group survived. in contrast, a dose-dependent protection was observed in mice immunized with different doses of the reference vaccine. for the mice immunized with rprn, some protection against the lethal dose of intercerebral challenge was noticed when compared to the control mice. the level of this protection provided from immunization with rprn was clearly higher than that from the immunization with iu of reference vaccine. the result suggested that immunization with rprn alone can confer partial protection against a lethal intracerebral b. pertussis challenge. such intracerebral challenge assays were also performed in the groups immunized with different doses of rfim and rfim however, no significant protection was observed as none of mice were survived in the groups immunized with μg dose of rfim and μg dose of rfim and a few survival mice in other dose groups. discussion because of its advantages in cost, yield and purity, vaccine based on recombinant components has been considered to be a valuable alternative for the vaccine production, in particular for the developing countries. in the present study, we showed that the recombinant prn, fim and fim proteins can be readily expressed and purified in large quantities from e. coli, and each recombinant protein solution is stable for up to twelve months when stored at below -20°c. they were prepared in a large quantity and freeze-dried. it was confirmed that the activity of freeze-dried preparation had no difference significantly compared with liquid preparation by elisa method and in some animal experiments. the three recombinant proteins can elicit both humoral and cellular immune responses when they were investigated in mice. furthermore, this recombinant technology makes it possible to avoid contaminations from the b. pertussis components that may cause side effects in vaccine preparations. availability of the purified fim and fim also provided an opportunity to assess their individual roles in the immunogenicity and protective efficacy. as a virulence factor of b. pertussis, the ability of prn to function as adhesin has been investigated both in vitro and in vivo. it is reported that the prn-mediated protection may be afforded by blocking prn-mediated attachment of b. pertussis to the host cells. studies on the immunized children have also suggested that high level of circulating antibodies against prn are associated with protection. furthermore, evidence suggests that anti-prn antibodies may promote extracellular killing with complement or as opsonins, and mediate killing bacteria by phagocytes. however, although antibodies specific to b. pertussis antigens confer protection, many studies have indicated that humoral immunity alone is not sufficient to provide long-term protection against b. pertussis infection and that the protection against b. pertussis requires both t cell- and b cell-mediated immunity. our results showed that the antibody response increased significantly in mice immunized with rprn. immunization of rprn also induced a th response that is characterized by the enhanced production of il-2- and tnf-α. these results also indicated that the rprn shared a similar feature with the native form of prn, suggesting that b cell and t cell epitopes might be highly conserved in rprn. murine intranasal and intracerebral challenge assays have been validated and used to demonstrate the protection of pertussis vaccine for many years. the results obtained from the intranasal and intracerebral challenge tests strongly suggest that rprn functions as a protective antigen. these observations are consistent with previous reports that a higher th1-type response was associated with a stronger level of protection against b. pertussis. in this study, the bacterial loads were only evaluated on day in lungs of the mince after the intranasal challenge. however, a time course of infection would probably provide more information on the protective properties of the proteins studied. so far, twelve, two and four different variants have been reported in prn, fim and fim respectively. at present, the prevalent allele combinations of b. pertussis isolates are prn2/fim3b. the strains used in this study and the strains used for vaccine production are prn1/fim3a or prn6/fim3a. as the difference occurred between b. pertussis vaccine strains and circulating isolates in many countries, it has been proposed that the strain variation may have effect on the vaccine efficacy. in this case, engineering strategies will remedy antigenic shifts by performing genetic mutation on the antigen encoding genes, which is an advantage of using recombinant proteins compared with the ones purified from b. pertussis. because of the similarity in the molecular weight, it is extremely difficult to purify separately fim and fim proteins from b. pertussis. therefore, antibody responses against fim or fim were only measured in elisa using a mixture of fim and fim proteins as coating antigen in clinical vaccine trials. the exact role of fim and fim in protection against pertussis is not fully known. in this study, recombinant fim and fim were expressed and purified separately. for the first times, their functions in protection against pertussis were assessed separately in mice model. the study demonstrated that higher antibody titres and cellular immune response characteristic of increased production of il- were induced in mice immunized with rfim and rfim although monoclonal anti-fim and anti-fim antibodies were used in the study, it remains to be shown whether there is cross-reacting response between fim and fim it is known that il- tnf-α and ifn-γ are characteristic cytokines for th response, and il- and il- for th response. in this study, we have only measured serum concentrations of il- tnf-α and il- it is interesting to study concentrations of other cytokines such as ifn-γ in sera collected from mice after immunization and infection. further, serum il- was not measurable in all mice tested in this study. this was in contrast to the finding that il- and tnf-α were significantly induced. the exact reason for the undetectable il- was unknown. one explanation might be the nih mice used in this study. it is known that nih mice predominate on cellular immunity. another explanation might be timing of the serum sampling and possible posttranscriptional regulation of il- no matter if il- was measurable or not, anti-pertussis antibodies were significantly induced in mice immunized with each of the three recombinant proteins. previous vaccine efficacy trial in sweden indicated that inclusion of prn, fim and fim into acellular vaccine containing pt and fha provided higher protection against pertussis. however, the contribution of individual components in the protection was not revealed. since fim of b. pertussis facilitates a variety of binding capabilities as adhesins, some studies suggested that passive protection against b. pertussis infection might be conferred due to the existence of higher titres of anti-fim or anti-fim antibodies which might transmigrate into the lower respiratory tract in mice. in contrast, the results from intranasal and intracerebral challenges with b. pertussis indicated very limited role played by rfims in bacterial clearance, although higher titres of anti-fim antibodies have been observed in this study. these data suggest that rfim or rfim alone may not be enough to provide the protection against b. pertussis and that they should be used in combination with other vaccine components such as pt, fha, and/or prn.  b. pertussis proteins prn, fim and fim can be genetically manipulated and expressed in a large amount in vitro. the three recombinant proteins can elicit both humoral and cellular immune responses. immunization with rprn can confer certain protection in mouse infection models. these recombinant proteins, especially rprn, have a potential for the development of a new generation of apvs in developing countries such as china. 
the immunogenicity and protective efficacy of the three recombinant proteins were investigated in mouse model. three recombinant proteins with amount of to mg/l were produced. the three recombinant proteins induced both humoral and cellular immune responses in mice. our results indicated that the recombinant proteins still retain their immunological properties and highlighted the potential of the recombinant proteins for the future development of the b. pertussis vaccines. pertactin, fimbriae and fimbriae of b. pertussis are important virulence factors and immunogens which have been included in some acellular pertussis vaccines. immunization with rprn, but not with rfim or rfim significantly enhanced clearance of bacteria in the lungs of mice after intranasal challenge with b. pertussis. we have developed an efficient method to produce large amounts of rprn, rfim and rfim from b. pertussis. compared to the control mice only immunized with adjuvant, serum igg antibody responses were significantly induced in the mice immunized with rprn, rfim or rfim. immunization with rprn also conferred protection against pertussis in mouse infection models. when tested in a lethal intracerebral infection model, certain protection was observed in mice immunized with rprn. in this present study, we cloned, expressed and purified prn, fim and fim respectively.
 bordetella pertussis is a causative agent of pertussis or whooping cough in humans. pertactin, fimbriae and fimbriae of b. pertussis are important virulence factors and immunogens which have been included in some acellular pertussis vaccines. in this present study, we cloned, expressed and purified prn, fim and fim respectively. the immunogenicity and protective efficacy of the three recombinant proteins were investigated in mouse model.  three recombinant proteins with amount of to mg/l were produced. compared to the control mice only immunized with adjuvant, serum igg antibody responses were significantly induced in the mice immunized with rprn, rfim or rfim. furthermore, t cell responses characteristic of increased production of il- and tnf-α were elicited in the mice immunized with the three proteins. immunization with rprn, but not with rfim or rfim significantly enhanced clearance of bacteria in the lungs of mice after intranasal challenge with b. pertussis. when tested in a lethal intracerebral infection model, certain protection was observed in mice immunized with rprn.  we have developed an efficient method to produce large amounts of rprn, rfim and rfim from b. pertussis. the three recombinant proteins induced both humoral and cellular immune responses in mice. immunization with rprn also conferred protection against pertussis in mouse infection models. our results indicated that the recombinant proteins still retain their immunological properties and highlighted the potential of the recombinant proteins for the future development of the b. pertussis vaccines. 