Identification And Immunogenicity Analysis Of Surface-exposed Proteins And T Cell Epitopes Of Coxiella Burnetii

The obligate intracellular Gram-negative bacterium Coxiella burnetii is the pathogen responsible for Q fever,a worldwide zoonosis.Human infection with C.burnetii is characterized by pneumonia(acute Q fever),hepatitis,endocarditis(chronic Q fever)and other serious diseases.Livestock infected with C.burnetii is the main source of Q fever infection crowd and persistence.Acute Q fever is treated effectively using antibiotics,and however,acute Q fever may progress to chronic disease which is sometimes incurable.Currently,vaccination is an effective measure to prevent C.burnetii infection.Although the inoculation of inactivated whole cell vaccine can induce the body to effectively prevent the C.burnetii infection,serious side effects of the body makes it difficult to promote it.At present,researchers try to use advanced molecular biology techniques to study antigens of C.burnetii in order to discover protective antigens so as to develop safe and effective molecular vaccines of Q fever.Recent studies of Q fever vaccine is only limited to a few outer membrane proteins of C.burnetii whose protective effect is limited,and numerous studies indicate that the protective effect of a single recombinant antigen or epitope is significantly weaker than the whole-cell antigen(WCA).In our study,surface-exposed proteins(SEPs)of C.burnetii were isolated with biotin-streptavidin affinity purification from the lysates coupled with two-dimensional electrophoresis,and then thirty-seven SEPs were identified by using electrospray ionization tandem mass spectrometry.Bioinformatics analysis found that the 37 SEPs were classified into 11 categories,and 8 SEPs were predicted to contain a signal peptide cleavage site which strongly suggests that they are membrane-associated or secreted proteins.Ten proteins may be located in the outer membrane,4 proteins may be located in non-cytoplasmic(outer membrane,inner membrane,or periplasmic),and the subcellular localization of 6 proteins was unknown.Thirty recombinant proteins(rSEPs)were successfully expressed in Escherichia coli using molecular cloning technique and purified using Ni-NTA affinity purification,and then the purified proteins were obtained to fabricate an epoxy protein microarray.The protein microarray was probed with sera from C.burnetii-infected mice or Q fever patients.As a result,23 rSEPs reacted positively with more than half of the sera obtained from mice infected with C.burnetii on days 28 post infection,of whom 15 rSEPs could be recognized by more than half of the sera from Q fever patients but could not react with sera from patients with brucellosis or mycoplasma pneumonia and showed a good serological specificity which suggested that they could serve as serodiagnostic antigens or as subunit vaccine antigens against C.burnetii infection.Seven SEPs were scanned for 15-mer epitope peptides predicted to have a high-affinity binding capacity for the major histocompatibility complex(MHC)class II molecule H2 I-Ab using bioinformatics predictions.As a result,121 epitop peptides were selected and synthesized chemically.The 7 rSEPs were used to immunize C57BL/6 mice,and then CD4~+ T cells were isolated from the spleen of immunized mice.Each of 121 CD4~+ T cell epitope peptides was incubated with the CD4~+ T cells to test for antigenicity by IFN-?-ELISPOT assays and 34 out of these candidate peptides elicited positive responses.The epitope peptides with highly similar amino acid sequence were excluded and 22 of them were selected as the effective CD4~+ T cell epitopes.The 22 epitope peptides were tested for recognition by CD4~+ T cells isolated from C57BL/6 mice immunized with C.burnetii WCA and 7 distinct epitope peptides identified in 7 different C.burnetii SEPs were recognized as more effective CD4~+ T cell epitopes that could stimulate significantly increased levels of IFN-? secretion.Then the 7 epitope peptides were used individually or pooled to stimulate CD4~+ T cells from C.burnetii WCA-immunized C57BL/6 mice,and the levels of IFN-? and TNF-? expressing in CD4~+ T cells were analyzed by flow cytometry.Compared with mock stimulation,each of the 7 epitope peptides could induce significantly higher levels of IFN-? expression,and three could stimulate significantly higher levels of both IFN-? and TNF-? expression.Furthermore,treatment with the pooled 7 epitope peptides resulted in IFN-? and TNF-? expression of CD4~+ T cells that were increased significantly compared with any individual epitope.These results suggested that pooled epitope peptides have a better immunoprotective effect compared to a single epitope peptide.For further study,C57BL/6 mice were immunized with the 7 epitope peptides individually or pooled,and then infected with C.burnetii.Both the bacterial loads and spleen weights of mice immunized with the 7 epitope peptides pooled were significantly lower than these with a single epitope peptide.The results showed that the immunoprotectivity of the 7 epitope peptides pooled is better than a single epitope peptide.While twenty-four Type IV secretion system(T4SS)effector proteins and 6 previously identified C.burnetii immunodominant proteins were scanned for 9-mer epitope peptides based on bioinformatics predictions of binding to murine MHC class I molecule H2 I-Kb or Db.As a result,157 epitope peptides were selected and synthesized chemically.Each of these 157 CD8~+ T cell epitope peptides was used to stimulate in vitro CD8~+ T cells of C57BL/6 mice infected with C.burnetii to test their antigenicity by IFN-?-ELISPOT assays.Twenty-nine of these candidate peptides elicited positive responses in IFN-?-ELISPOT assays.Interestingly,22 of the 29 peptides are derived from 17 T4 SS related proteins,none of which was identified as immunodominant antigen before.The 29 peptides were used individually to stimulate CD8~+ T cells from C.burnetii-infected C57BL/6 mice,and then the frequency of epitope specific IFN-? producing CD8~+ T cells were quantified by flow cytometry.As a result,all peptides induced detectable IFN-? responses,the frequency of responding cells ranged from 0.44% to 3.78% of total CD8~+ T cells.The genes encoding the 29 CD8~+ T cell epitopes linked in series were ligated into prokaryotic expression plasmid,and then the recombinant plasmid was electrotransformed into Listeria monocytogenes vaccine vector strain(LM-Cb).C57BL/6 mice were immunized with LM-Cb,and then infected with C.burnetii to evaluate its immunoprotective effect.Both the bacterial loads and spleen weights of mice immunized with LM-Cb were significantly lower than these with LM or negative control.The results suggested that these CD8~+ T cell epitope peptides could induce an effective immune response against C.burnetii infection.In summary,we isolated and identified 37 SEPs and completed its bioinformatics analysis;30 rSEPs were successfully expressed and then were used to fabricate a protein microarray.The protein microarray was probed with C.burnetii-infected sera,and 15 of them were considered as major seroreactive rSEPs and showed a good serological specificity.One hundred and twenty-one CD4~+ T cell epitope peptides and 157 CD8~+ T cell epitope peptides were predicted and synthesized,of which 7 CD4~+ T cell epitope peptides and 29 CD8~+ T cell epitope peptides were screened as positive peptides by stimulation assay of CD4~+ T cells or CD8~+ T cells.Further study suggested that the activation levels of CD4~+ or CD8~+ T cells stimulated by the 7 CD4~+ T cell epitope peptides pooled or the 29 CD8~+ T cell epitope peptides ligated were significantly higher than those by a single epitope.Mice immunized with the 7 CD4~+ T cell epitope peptides pooled or the 29 CD8~+ T cell epitopes expressed in Listeria monocytogenes(LM-Cb)were efficiently protected from C.burnetii infection.These results demonstrate that a combination of CD4~+ T cell epitope peptides or CD8~+ T cell epitope peptides can significantly enhance specific cellular immune responses against C.burnetii infectionOur study has laid a good theoretical and working basis for the development of serological diagnostic reagents and molecular vaccines of Q fever.