BACKGROUND
leptospira interrogans is the causative agent of zoonotic leptospirosis, which affects humans in both developing and developed countries.
it is a major public health problem in many areas, particularly after floods and heavy monsoons.
globally, it has is estimated that  <dig>  to  <dig> per  <dig>  people living in temperate climates are affected each year, with the numbers increasing to ten or more per  <dig>  people living in tropical climates .
in china, between  <dig> and  <dig> approximately  <dig> people were infected with l. interrogans of which  <dig> died  <cit> .
people are generally infected by contaminated water or soil sources through exposure of wounds in the skin or through exposure of the mucosal layers  <cit> .
the symptoms following infection can vary from a mild febrile illness to more severe icteric disease, which is characterized by potentially fetal renal and liver failure  <cit> .
despite the constant progress of antimicrobial therapeutics, vaccination is still expected to be the most effective method to protect people that come in close contact with infected animals or environments.
both inactivated and attenuated vaccines have been used in animals or even in humans.
however, these vaccines are associated with high rates of side-effects, such as aches and anaphylaxis, and they confer only short-term serovar-specific immunity  <cit> .
currently, studies on developing leptospirosis subunit vaccines are particularly focused on bacterial motility, lipopolysaccharides , lipoproteins, outer-membrane proteins  and potential virulence factors.
however, the protective efficacy of these candidates in experimental animals is low   <cit> .
thus far, the most promising subunit vaccine candidates are the lig proteins, which have been shown to confer high-level protection, approaching 100 % in mice and hamsters .
however, whether the lig proteins are able to elicit cross-protective immunity to a range of serovar remains to be determined, since the similarity of the amino acid sequence of this protein between different leptospira spp.
is 70–100 %  <cit> .
since the currently available vaccines are characterized by a short duration of immunity, side-effects or serovar specificity, it is important to develop a universal leptospirosis vaccine with high efficiency and low toxicity.
for this purpose, we screened different leptospira serotypes for conserved surface-exposed antigens.
bacterial outer membrane proteins  are a major target of the immune system in a variety of infectious diseases and they have been suggested as candidates for diagnosis and immunization  <cit> .
in leptospira, three classes of these omps have been identified: 1) lipoproteins, which are the most abundant class and include proteins such as lipl <dig>  lipl <dig> and lipl <dig> ; 2) trans-membrane proteins, which include ompl <dig>  <cit> ; and 3) peripheral membrane proteins, which include lipl <dig>  <cit> .
in our previously studies we have identified that ompl <dig>  lipl <dig> and lipl <dig> are expressed in all the pathogenic strains of  <dig> serovar/serogroup standard reference strains of leptospira in china .
furthermore, production of these proteins was down-regulated  in host-adapted leptospira  <cit> , suggesting that these proteins might not be involved in the pathogenesis of leptospira after it gains entry into the mammalian host, which indicates that these omps are safe vaccine candidates.
leptospira contains over  <dig> pathogenic serovars, which are divided over  <dig> serogroups.
the diversity of leptospira is also reflected in its omps, which are genetically diverse between different serogroups .
in addition, given the large size of omps, conserved epitopes might also be sterically blocked.
for these reasons, an omp vaccine might still provide only minimal protection against a heterologous leptospira infection.
alternatively, conserved functional omp epitopes could be used for the development of a peptide vaccine  <cit> .
however, it is difficult to develop an epitope-based peptide vaccine for human clinical trials, because of the technical limitations or economic reasons in synthesizing large quantities of highly purified peptides.
our strategy to overcome these difficulties was to develop a multi-epitope chimeric protein that is composed of the immunodominant epitopes from the omps ompl <dig>  lipl <dig> and lipl <dig>  which are separated by a tetraglycine linker.
the selected epitopes consisted of both b-cell epitopes, which are essential for protective antibody response, and t-cell epitopes, which serve to induce a cellular immune response.
RESULTS
expression, purification and immunogenicity of the chimeric r4r protein
based on the sequences of screened epitopes  <cit> , a gene fragment containing six selected epitope sequences was synthesized  and cloned as four consecutive repeats in an expression vector, thereby forming a single chimeric multi-epitope encoding gene.
the protein was successfully expressed in escherichia coli bl <dig>  and purified.
sds-page results showed that the full-length  <dig> kd protein was induced by iptg and present in the soluble fraction of lysed e. coli bl <dig>  cells .
furthermore, sds-page analysis of the purified protein under native conditions showed a single band at  <dig> kd .
the yield of the protein purification process was approximately  <dig>  mg of protein from  <dig> l culture.
the protein sample was passed through a detoxi-gel column, which contains a modified polymyxin prime b  that removes lipopolysaccharide  contaminations.
no residual lps was detected in the protein extract using a limulus test, which has a detection sensitivity of 10 pg .
the purified recombinant protein was named r4r.fig.
<dig> characterization of the expressed and purified chimeric r4r protein.
a sds-page analysis of the expression and purification steps of the r4r protein.
pellet  and supernatant  fractions of lysates from e. coli bl <dig>  cells containing the empty vector pet28a  or pet28a-4r  were electrophoresed in a 10 % sds-page gel.
m indicates the protein ladder.
lane  <dig> contains the purified recombinant 4r protein.
b anti-leptospira western blot analysis.
the purified chimeric r4r protein was run on a sds-page gel and transferred to a pvdf membrane.
serum from heat-killed l. interrogans strain lai immunized rabbits was used as a primary antibody to detect r4r.
serum from pbs injected rabbits was used as control.
c anti-r4r western analysis.
the purified chimeric r4r protein was run on sds-page gel and transferred to a pvdf membrane.
serum from guinea pigsimmunized with pbs  or chimeric protein, were used as primary antibody
to evaluate reactivity of r4r with antibodies raised in rabbits against heat-killed l. interrogans strain lai, western blot analysis was performed.
antibodies raised in rabbits against heat-killed l. interrogans were showing a strong interaction with r4r , indicating that the epitopes of r4r are present on the surface of l. interrogans.
to evaluate the immunogenicity of r4r, guinea pigs were immunized with r4r and the collected serum analyzed.
western analysis showed that the chimeric multi-epitope r4r protein was able to induce an antigen-specific humoral immune response .
the immunodiffusion titer of the antisera against the chimeric r4r protein was 1: <dig>
to evaluate the polarity of the immune response against r4r, antigen-specific igg subtype  responses in the sera of vaccinated and control guinea pigs were measured by elisa.
the results showed that although the levels of both igg <dig> and igg2a were increased in the sera of animals immunized with the recombinant protein compared with the control sera from pbs treated animals, the level of igg2a was higher than that of igg <dig> .
the elevated igg2a/igg <dig> ratio points to a th <dig> polarized immune response in the r4r-immunized guinea pigs.
to determine the cross-reactivity of serum obtained from r4r-immunized guinea pigs, mat analysis was performed using the most commonly encountered serogroups of l. interrogans in china, namely icterohaemorrhagiae, pomona, canicola, hebdomadis, autumnalis, grippotyphosa and australis.
serum from the r4r-immunized guinea pigs was able to cross-agglutinate each of the l. interrogans serogroup-specific standard reference strains with titers ranging from 1: <dig> to 1: <dig> .
these results indicate that r4r is an immunological cross-reactive protein, which is important for the coverage of a leptospiral vaccine.fig.
<dig> response of igg subclasses on the chimeric protein.
igg subclasses induced by the chimeric protein in guinea pigs were detected by elisa
lymphocyte proliferation and cytokine production in response to chimeric r4r protein
lymophocytes from r4r-immunized guinea pigs were stimulated ex vivo with the r4r protein and analyzed for proliferation and cytokine production.
lymphocytes stimulated with r4r showed a significantly higher proliferation response compared with the controls .
the supernatant of the r4r stimulated lymphocytes was analyzed for ifn-γ and il- <dig> cytokine levels by elisa.
ifn-γ production was 20-fold higher in lymphocytes that were isolated from animals immunized with the chimeric r4r protein and subsequently stimulated with r4r compared with the controls .
no significant differences in il- <dig> cytokine production were observed , indicating again that r4r specifically stimulates a th1-biased immune response.fig.
<dig> proliferative and immune responses of splenocytes immunized with chimeric protein.
guinea pigs were immunized with the chimeric r4r protein.
ten days after the last immunization, the splenocytes were isolated, stimulated with the r4r protein and cultured for 72 h. a splenocyte proliferation analysis of pbs, cona and r4r stimulated splenocytes.
b elisa measurements of il- <dig> and inf-γ secreted by pooled control, cona and r4r stimulated splenocytes and lymphocytes in the culture supernatants
protective efficacy of the chimeric r4r protein vaccine
to investigate whether the chimeric r4r protein is able to induce protective immunity against a lethal l. interrogans infection, guinea pigs were immunized and challenged with l. interrogans strain lai, for which the ld <dig> was determined at 1 ×  <dig>  pbs-immunized guinea pigs challenged with a 2 × ld <dig> died within 8– <dig> post-infection, while 80 %  of the r4r-immunized guinea pigs survived challenges with this dose.
liver, kidney and lung tissues of the challenged pbs-immunized guinea pigs showed severe leptospirosis symptoms, including necrosis .
in the kidney and liver, severe lesions were observed, including hemorrhages, while histopathological lesions in the kidney showed renal tubular necrosis with occasional polymorphonuclear cell infiltration and loss of normal liver architecture.
in contrast, liver, kidney and lung tissues of r4r-immunized animals showed only mild edema symptoms , and there was little or no evidence of lesion formation.
furthermore, in fontana silver-stained kidney samples, no leptospira were detected in the r4r-immunized animals, while many leptospira were detected in the samples from pbs-immunized animals .
in addition, after incubation of the lysed kidney samples in emjh medium at 30 °c for 28 days, floccules were observed in the emjh medium by tungsten lamp analysis of the pbs-immunized guinea pig samples, while none were detected in the samples from r4r-immunized animals .
finally, no leptospires were detected in the urine samples of r4r-immunized guinea pigs, while some were encountered in the urine samples of pbs-immunized animals .
together, these results indicate that immunization of guinea pigs with the chimeric multi-antigen r4r protein provides protection of the animals against lethal challenges of leptospira and prevents severe tissue damage.fig.
<dig> histopathological effects of leptospira challenges on immunized guinea pig tissues.
representative images of liver, kidney and lung tissues of healthy guinea pigs  or guinea pigs immunized with the chimeric r4r protein  or pbs  and challenged with l. interrogans strain lai.
tissues were stained with hematoxylin and eosin
fig.
<dig> colonization of l. interrogans in chimeric r4r immunized guinea pigs.
representative images of kidney lysates  or urine samples  that were collected from healthy control guinea pigs  of guinea pigs immunized with r4r or pbs and subsequently challenged with l. interrogans strain lai.
all samples were strained with the fontana silver staining method and observed using an optical microscope
discussion
l. interrogans is a bacterial pathogen that causes widespread disease in both animals and humans.
leptospirosis poses a significant public health problem since it is potentially fatal due to the damage it can cause to multiple organs, including the liver, lungs, kidney and brain  <cit> .
leptospirosis is prevalent throughout china, except for some of the western provinces .
particularly hard hit are the regions in the yangtze river basin, such as the provinces sichuan, hubei and hunan and the area south of the five ridges.
the most commonly encountered l. interrogans serogroups in china are icterohaemorrhagiae, pomona, canicola, hebdomadis, autumnalis, grippotyphosa and australis  <cit> .
the currently available and widely used vaccine against leptospirosis is a multivalent inactivated whole-cell vaccine.
however, this vaccine shows high side-effects and elicits little cross-protection against strains from different serovars.
therefore, a new effective and safe vaccine that is able to provide cross-protection of at risk humans or animals is urgently needed.
currently, vaccine research against leptospira is focused on the discovery of cross-reactive conserved antigens that are able to provide long-term protection against a broad range of leptospira spp.
<cit> .
it is well established that omps show a high degree of conservation in leptospira spp.. furthermore, they are immunodominant antigens in both humans and animals and contain multiple b- and t-cell epitopes that are able to elicit both neutralizing antibody response and t-cell mediated immunity  <cit> .
therefore, omps are likely the most successful candidate antigens for potential subunit vaccines.
an ideal peptide-based vaccine should also contain both b- and t-cell epitopes.
in our previous research, we identified six b- and t-cell combined immunodominant epitopes in the omps ompl <dig>  lipl <dig> and lipl <dig> from leptospira, and tested whether they were able to induce specific immune responses  <cit> .
considering the diversity among leptospira serovars, these epitopes fragments are much conserved and show a high degree of homology among  <dig> serovar/serogroup standard reference strains of leptospira in china.
these six selected epitopes are only about  <dig> amino acids in length, which complicates mass production and makes them susceptible to host hydrolysis mechanisms.
therefore, in the current study, a gene fragment was synthesized that encoded all six epitopes separated by a four glycine spacer and which was codon optimized for expression in e. coli.
this gene fragment was cloned as four consecutive copies in an expression vector and produced a  <dig> kd fusion protein.
this recombinant protein was expressed in the soluble fraction, which is important to maintain its antigenicity and immunogenicity.
antibodies are a major contributor to the phagocytosis and destruction of pathogenic leptospira by macrophages  <cit> .
our r4r protein showed a strong immunogenicity in guinea pigs, which resulted in the production of high levels of specific antibodies.
this is essential for the killing of leptospira through complement-mediated lysis or through opsonin-dependent phagocytosis.
to confirm that antibodies were reactive with a variety of leptospiral serovars, the mat test was used, which is considered as the “golden standard” in the serodiagnosis of leptospirosis and the serological classification of leptospira species.
our results revealed that the antisera obtained after immunization with r4r were able to agglutinate all standard reference strains of leptospira in china, with mat titers up to 1: <dig>  therefore, we expect that antibodies raised against our r4r protein are able to provide cross-protection against a wide variety of leptospira serovars, although this still requires further confirmation using animal experiments.
protective immunity to leptospira is highly serovar-specific, which points to a dominant role for humoral immunity in the protection against leptospira infection.
however, recently it was shown that during an infection with leptospira, cellular immunity still plays an important role in the control of disease  <cit> .
immunization with our r4r multi-epitope chimeric protein resulted in a dominant th <dig> response, which was reflected by the yield of antigen-specific igg subtypes .
a th <dig> response generally induces igg2a expression, while a th <dig> response results predominantly in igg <dig> production  <cit> .
besides mediating intracellular killing of a variety of pathogenic microorganisms, th <dig> cells also stimulate b cells to produce antibodies that are important for mucosal immunity, such as igm, igg2a and iga  <cit> , which is regulated via secretion of ifn-γ and il- <dig> and via expression of cd40l  <cit> .
cytokine measurements in the culture supernatant of splenocytes from immunized guinea pigs, after stimulation with our r4r protein, showed high induction of ifn-γ compared with il- <dig>  which supports that the r4r multi-epitope chimeric protein vaccine is biased towards a th1-type response.
severe leptospirosis is typically associated with an overactive host response that damages its own tissues, such as the liver, kidney and lung, which can furthermore lead to hemorrhages.
guinea pigs are susceptible to leptospira infection and frequently present these symptoms.
in order to determine the ability of our r4r protein to induce a protective immune response against a leptospira infection, guinea pigs were used as an infection model.
compared with unvaccinated guinea pigs, immunized animals showed increased survival, reduced tissue damage, decreased colonization of the kidneys and reduced shedding of leptospira in the urine.
however, immunized guinea pigs also showed some inflammatory reactions, such as edema symptoms.
this may be the result of increased ifn-γ production after immunization, which can cause inflammation or a delayed type hypersensitivity  <cit> .
CONCLUSIONS
this study showed that the chimeric multi-epitope protein r4r is a promising vaccine candidate for the development of a universal vaccine against leptospira infections.
immunized guinea pigs showed a strong antigen-specific humoral and cellular immune response, a reduction in tissue damage and colonization and protection against a lethal leptospira infection.
thus, the multi-epitope chimeric r4r vaccine, containing both t- and b-cell epitopes, may represent an effective antigen to increase the efficacy of vaccines against leptospirosis.

 leptospira interrogans is the causative agent of zoonotic leptospirosis, which affects humans in both developing and developed countries. it is a major public health problem in many areas, particularly after floods and heavy monsoons. globally, it has is estimated that to per people living in temperate climates are affected each year, with the numbers increasing to ten or more per people living in tropical climates. in china, between and approximately people were infected with l. interrogans of which died. people are generally infected by contaminated water or soil sources through exposure of wounds in the skin or through exposure of the mucosal layers. the symptoms following infection can vary from a mild febrile illness to more severe icteric disease, which is characterized by potentially fetal renal and liver failure. despite the constant progress of antimicrobial therapeutics, vaccination is still expected to be the most effective method to protect people that come in close contact with infected animals or environments. both inactivated and attenuated vaccines have been used in animals or even in humans. however, these vaccines are associated with high rates of side-effects, such as aches and anaphylaxis, and they confer only short-term serovar-specific immunity. currently, studies on developing leptospirosis subunit vaccines are particularly focused on bacterial motility, lipopolysaccharides, lipoproteins, outer-membrane proteins and potential virulence factors. however, the protective efficacy of these candidates in experimental animals is low. thus far, the most promising subunit vaccine candidates are the lig proteins, which have been shown to confer high-level protection, approaching 100 % in mice and hamsters. however, whether the lig proteins are able to elicit cross-protective immunity to a range of serovar remains to be determined, since the similarity of the amino acid sequence of this protein between different leptospira spp. is 70–100 %. since the currently available vaccines are characterized by a short duration of immunity, side-effects or serovar specificity, it is important to develop a universal leptospirosis vaccine with high efficiency and low toxicity. for this purpose, we screened different leptospira serotypes for conserved surface-exposed antigens. bacterial outer membrane proteins are a major target of the immune system in a variety of infectious diseases and they have been suggested as candidates for diagnosis and immunization. in leptospira, three classes of these omps have been identified: 1) lipoproteins, which are the most abundant class and include proteins such as lipl lipl and lipl; 2) trans-membrane proteins, which include ompl; and 3) peripheral membrane proteins, which include lipl. in our previously studies we have identified that ompl lipl and lipl are expressed in all the pathogenic strains of serovar/serogroup standard reference strains of leptospira in china. furthermore, production of these proteins was down-regulated in host-adapted leptospira, suggesting that these proteins might not be involved in the pathogenesis of leptospira after it gains entry into the mammalian host, which indicates that these omps are safe vaccine candidates. leptospira contains over pathogenic serovars, which are divided over serogroups. the diversity of leptospira is also reflected in its omps, which are genetically diverse between different serogroups. in addition, given the large size of omps, conserved epitopes might also be sterically blocked. for these reasons, an omp vaccine might still provide only minimal protection against a heterologous leptospira infection. alternatively, conserved functional omp epitopes could be used for the development of a peptide vaccine. however, it is difficult to develop an epitope-based peptide vaccine for human clinical trials, because of the technical limitations or economic reasons in synthesizing large quantities of highly purified peptides. our strategy to overcome these difficulties was to develop a multi-epitope chimeric protein that is composed of the immunodominant epitopes from the omps ompl lipl and lipl which are separated by a tetraglycine linker. the selected epitopes consisted of both b-cell epitopes, which are essential for protective antibody response, and t-cell epitopes, which serve to induce a cellular immune response.  expression, purification and immunogenicity of the chimeric r4r protein based on the sequences of screened epitopes, a gene fragment containing six selected epitope sequences was synthesized and cloned as four consecutive repeats in an expression vector, thereby forming a single chimeric multi-epitope encoding gene. the protein was successfully expressed in escherichia coli bl and purified. sds-page results showed that the full-length kd protein was induced by iptg and present in the soluble fraction of lysed e. coli bl cells. furthermore, sds-page analysis of the purified protein under native conditions showed a single band at kd. the yield of the protein purification process was approximately  mg of protein from l culture. the protein sample was passed through a detoxi-gel column, which contains a modified polymyxin prime b that removes lipopolysaccharide contaminations. no residual lps was detected in the protein extract using a limulus test, which has a detection sensitivity of 10 pg. the purified recombinant protein was named r4r.fig. characterization of the expressed and purified chimeric r4r protein. a sds-page analysis of the expression and purification steps of the r4r protein. pellet and supernatant fractions of lysates from e. coli bl cells containing the empty vector pet28a or pet28a-4r were electrophoresed in a 10 % sds-page gel. m indicates the protein ladder. lane contains the purified recombinant 4r protein. b anti-leptospira western blot analysis. the purified chimeric r4r protein was run on a sds-page gel and transferred to a pvdf membrane. serum from heat-killed l. interrogans strain lai immunized rabbits was used as a primary antibody to detect r4r. serum from pbs injected rabbits was used as control. c anti-r4r western analysis. the purified chimeric r4r protein was run on sds-page gel and transferred to a pvdf membrane. serum from guinea pigsimmunized with pbs or chimeric protein, were used as primary antibody to evaluate reactivity of r4r with antibodies raised in rabbits against heat-killed l. interrogans strain lai, western blot analysis was performed. antibodies raised in rabbits against heat-killed l. interrogans were showing a strong interaction with r4r, indicating that the epitopes of r4r are present on the surface of l. interrogans. to evaluate the immunogenicity of r4r, guinea pigs were immunized with r4r and the collected serum analyzed. western analysis showed that the chimeric multi-epitope r4r protein was able to induce an antigen-specific humoral immune response. the immunodiffusion titer of the antisera against the chimeric r4r protein was 1: to evaluate the polarity of the immune response against r4r, antigen-specific igg subtype responses in the sera of vaccinated and control guinea pigs were measured by elisa. the results showed that although the levels of both igg and igg2a were increased in the sera of animals immunized with the recombinant protein compared with the control sera from pbs treated animals, the level of igg2a was higher than that of igg. the elevated igg2a/igg ratio points to a th polarized immune response in the r4r-immunized guinea pigs. to determine the cross-reactivity of serum obtained from r4r-immunized guinea pigs, mat analysis was performed using the most commonly encountered serogroups of l. interrogans in china, namely icterohaemorrhagiae, pomona, canicola, hebdomadis, autumnalis, grippotyphosa and australis. serum from the r4r-immunized guinea pigs was able to cross-agglutinate each of the l. interrogans serogroup-specific standard reference strains with titers ranging from 1: to 1:. these results indicate that r4r is an immunological cross-reactive protein, which is important for the coverage of a leptospiral vaccine.fig. response of igg subclasses on the chimeric protein. igg subclasses induced by the chimeric protein in guinea pigs were detected by elisa lymphocyte proliferation and cytokine production in response to chimeric r4r protein lymophocytes from r4r-immunized guinea pigs were stimulated ex vivo with the r4r protein and analyzed for proliferation and cytokine production. lymphocytes stimulated with r4r showed a significantly higher proliferation response compared with the controls. the supernatant of the r4r stimulated lymphocytes was analyzed for ifn-γ and il- cytokine levels by elisa. ifn-γ production was 20-fold higher in lymphocytes that were isolated from animals immunized with the chimeric r4r protein and subsequently stimulated with r4r compared with the controls. no significant differences in il- cytokine production were observed, indicating again that r4r specifically stimulates a th1-biased immune response.fig. proliferative and immune responses of splenocytes immunized with chimeric protein. guinea pigs were immunized with the chimeric r4r protein. ten days after the last immunization, the splenocytes were isolated, stimulated with the r4r protein and cultured for 72 h. a splenocyte proliferation analysis of pbs, cona and r4r stimulated splenocytes. b elisa measurements of il- and inf-γ secreted by pooled control, cona and r4r stimulated splenocytes and lymphocytes in the culture supernatants protective efficacy of the chimeric r4r protein vaccine to investigate whether the chimeric r4r protein is able to induce protective immunity against a lethal l. interrogans infection, guinea pigs were immunized and challenged with l. interrogans strain lai, for which the ld was determined at 1 ×  pbs-immunized guinea pigs challenged with a 2 × ld died within 8– post-infection, while 80 % of the r4r-immunized guinea pigs survived challenges with this dose. liver, kidney and lung tissues of the challenged pbs-immunized guinea pigs showed severe leptospirosis symptoms, including necrosis. in the kidney and liver, severe lesions were observed, including hemorrhages, while histopathological lesions in the kidney showed renal tubular necrosis with occasional polymorphonuclear cell infiltration and loss of normal liver architecture. in contrast, liver, kidney and lung tissues of r4r-immunized animals showed only mild edema symptoms, and there was little or no evidence of lesion formation. furthermore, in fontana silver-stained kidney samples, no leptospira were detected in the r4r-immunized animals, while many leptospira were detected in the samples from pbs-immunized animals. in addition, after incubation of the lysed kidney samples in emjh medium at 30 °c for 28 days, floccules were observed in the emjh medium by tungsten lamp analysis of the pbs-immunized guinea pig samples, while none were detected in the samples from r4r-immunized animals. finally, no leptospires were detected in the urine samples of r4r-immunized guinea pigs, while some were encountered in the urine samples of pbs-immunized animals. together, these results indicate that immunization of guinea pigs with the chimeric multi-antigen r4r protein provides protection of the animals against lethal challenges of leptospira and prevents severe tissue damage.fig. histopathological effects of leptospira challenges on immunized guinea pig tissues. representative images of liver, kidney and lung tissues of healthy guinea pigs or guinea pigs immunized with the chimeric r4r protein or pbs and challenged with l. interrogans strain lai. tissues were stained with hematoxylin and eosin fig. colonization of l. interrogans in chimeric r4r immunized guinea pigs. representative images of kidney lysates or urine samples that were collected from healthy control guinea pigs of guinea pigs immunized with r4r or pbs and subsequently challenged with l. interrogans strain lai. all samples were strained with the fontana silver staining method and observed using an optical microscope discussion l. interrogans is a bacterial pathogen that causes widespread disease in both animals and humans. leptospirosis poses a significant public health problem since it is potentially fatal due to the damage it can cause to multiple organs, including the liver, lungs, kidney and brain. leptospirosis is prevalent throughout china, except for some of the western provinces. particularly hard hit are the regions in the yangtze river basin, such as the provinces sichuan, hubei and hunan and the area south of the five ridges. the most commonly encountered l. interrogans serogroups in china are icterohaemorrhagiae, pomona, canicola, hebdomadis, autumnalis, grippotyphosa and australis. the currently available and widely used vaccine against leptospirosis is a multivalent inactivated whole-cell vaccine. however, this vaccine shows high side-effects and elicits little cross-protection against strains from different serovars. therefore, a new effective and safe vaccine that is able to provide cross-protection of at risk humans or animals is urgently needed. currently, vaccine research against leptospira is focused on the discovery of cross-reactive conserved antigens that are able to provide long-term protection against a broad range of leptospira spp.. it is well established that omps show a high degree of conservation in leptospira spp.. furthermore, they are immunodominant antigens in both humans and animals and contain multiple b- and t-cell epitopes that are able to elicit both neutralizing antibody response and t-cell mediated immunity. therefore, omps are likely the most successful candidate antigens for potential subunit vaccines. an ideal peptide-based vaccine should also contain both b- and t-cell epitopes. in our previous research, we identified six b- and t-cell combined immunodominant epitopes in the omps ompl lipl and lipl from leptospira, and tested whether they were able to induce specific immune responses. considering the diversity among leptospira serovars, these epitopes fragments are much conserved and show a high degree of homology among serovar/serogroup standard reference strains of leptospira in china. these six selected epitopes are only about amino acids in length, which complicates mass production and makes them susceptible to host hydrolysis mechanisms. therefore, in the current study, a gene fragment was synthesized that encoded all six epitopes separated by a four glycine spacer and which was codon optimized for expression in e. coli. this gene fragment was cloned as four consecutive copies in an expression vector and produced a kd fusion protein. this recombinant protein was expressed in the soluble fraction, which is important to maintain its antigenicity and immunogenicity. antibodies are a major contributor to the phagocytosis and destruction of pathogenic leptospira by macrophages. our r4r protein showed a strong immunogenicity in guinea pigs, which resulted in the production of high levels of specific antibodies. this is essential for the killing of leptospira through complement-mediated lysis or through opsonin-dependent phagocytosis. to confirm that antibodies were reactive with a variety of leptospiral serovars, the mat test was used, which is considered as the “golden standard” in the serodiagnosis of leptospirosis and the serological classification of leptospira species. our results revealed that the antisera obtained after immunization with r4r were able to agglutinate all standard reference strains of leptospira in china, with mat titers up to 1: therefore, we expect that antibodies raised against our r4r protein are able to provide cross-protection against a wide variety of leptospira serovars, although this still requires further confirmation using animal experiments. protective immunity to leptospira is highly serovar-specific, which points to a dominant role for humoral immunity in the protection against leptospira infection. however, recently it was shown that during an infection with leptospira, cellular immunity still plays an important role in the control of disease. immunization with our r4r multi-epitope chimeric protein resulted in a dominant th response, which was reflected by the yield of antigen-specific igg subtypes. a th response generally induces igg2a expression, while a th response results predominantly in igg production. besides mediating intracellular killing of a variety of pathogenic microorganisms, th cells also stimulate b cells to produce antibodies that are important for mucosal immunity, such as igm, igg2a and iga, which is regulated via secretion of ifn-γ and il- and via expression of cd40l. cytokine measurements in the culture supernatant of splenocytes from immunized guinea pigs, after stimulation with our r4r protein, showed high induction of ifn-γ compared with il- which supports that the r4r multi-epitope chimeric protein vaccine is biased towards a th1-type response. severe leptospirosis is typically associated with an overactive host response that damages its own tissues, such as the liver, kidney and lung, which can furthermore lead to hemorrhages. guinea pigs are susceptible to leptospira infection and frequently present these symptoms. in order to determine the ability of our r4r protein to induce a protective immune response against a leptospira infection, guinea pigs were used as an infection model. compared with unvaccinated guinea pigs, immunized animals showed increased survival, reduced tissue damage, decreased colonization of the kidneys and reduced shedding of leptospira in the urine. however, immunized guinea pigs also showed some inflammatory reactions, such as edema symptoms. this may be the result of increased ifn-γ production after immunization, which can cause inflammation or a delayed type hypersensitivity.  this study showed that the chimeric multi-epitope protein r4r is a promising vaccine candidate for the development of a universal vaccine against leptospira infections. immunized guinea pigs showed a strong antigen-specific humoral and cellular immune response, a reduction in tissue damage and colonization and protection against a lethal leptospira infection. thus, the multi-epitope chimeric r4r vaccine, containing both t- and b-cell epitopes, may represent an effective antigen to increase the efficacy of vaccines against leptospirosis. 
microscopic agglutination tests showed that r4r antisera was immunological cross-reactive with a range of chinese standard reference strains of leptospira belonging to different serogroups. finally, r4r-immunized guinea pigs showed increased survival of lethal leptospira challenges compared with pbs-immunized animals and tissue damage and leptospiral colonization of the kidney were reduced. in this study, a chimeric multi-epitope protein-based vaccine was designed and tested for its potency to induce a specific immune response and provide protection against l. interrogans infection. in guinea pigs, the r4r vaccine induced a th1-biased immune response, as reflected by the igg2a/igg ratio and cytokine production of stimulated splenocytes derived from immunized animals. western blot analysis showed that the recombinant protein mainly expressed in a soluble pattern, and reacted with antibodies raised in rabbit against heat-killed leptospira and in guinea pigs against the r4r vaccine. development of a universal vaccine is one of the major strategic goals to overcome the disease burden of leptospirosis.
 leptospirosis is an important reemerging zoonosis, with more than half a million cases reported annually, and is caused by pathogenic leptospira species. development of a universal vaccine is one of the major strategic goals to overcome the disease burden of leptospirosis. in this study, a chimeric multi-epitope protein-based vaccine was designed and tested for its potency to induce a specific immune response and provide protection against l. interrogans infection.  the protein, containing four repeats of six t- and b-cell combined epitopes from the leptospiral outer membrane proteins, ompl lipl and lipl was expressed and purified. western blot analysis showed that the recombinant protein mainly expressed in a soluble pattern, and reacted with antibodies raised in rabbit against heat-killed leptospira and in guinea pigs against the r4r vaccine. microscopic agglutination tests showed that r4r antisera was immunological cross-reactive with a range of chinese standard reference strains of leptospira belonging to different serogroups. in guinea pigs, the r4r vaccine induced a th1-biased immune response, as reflected by the igg2a/igg ratio and cytokine production of stimulated splenocytes derived from immunized animals. finally, r4r-immunized guinea pigs showed increased survival of lethal leptospira challenges compared with pbs-immunized animals and tissue damage and leptospiral colonization of the kidney were reduced.  the multi-epitope chimeric r4r protein is a promising antigen for the development of a universal cross-reactive vaccine against leptospirosis. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords leptospiraouter membrane proteinmulti-epitopevaccinecross protectionnational natural science foundation of china 81271781issue-copyright-statement© the author 2016 