Screening Of Klebsiella Pneumoniae Immunity-Related Epitopes And Study On Experimental Immunization

Klebsiella pneumoniae is an important cause of respiratory infections and is responsible for significant morbidity and mortality in compromised individuals. Even though antibiotics are still the most effective treatment for K.pneumoniae infection, multiantibiotic resistance becomes a more pronounced problem.Thus, it is of great interest to find alternative ways to control the K.pneumoniae infection. Vaccination has become one of the most promising approaches against K.pneumoniae infection.Currently, most of vaccines against K.pneumoniae were reported to use native components from K.pneumoniae such as fimbriae and capsular polysaccharide and LPS.In general, these treatment are based on a natural form of the pathogen. In response to K. pneumoniae infection, the host triggers vigorous humoral and cellular immune response.Though K. pneumoniae specific antibodies have been detected at high titers both in inflamed gastricmucosa and in serum, the infection is still persistent and even for life long, suggesting that K. pneumoniae can evade both adaptive and innate immune responses and the immune responses triggered by K. pneumoniae in nature were not optimal to eliminate this pathogen.The immune responses evoked by natural infection or subunit vaccine immunization are not favorable. The immune responses should be improved and modified.Therefore, we assume that the modified immunotherapy is a feasible treatment which may be effective to combat against K. pneumoniae. This purpose can probably be achieved by artificially promoting qualitatively or quantitatively immunity different from natural infection.Epitope-based vaccines represent a modified immunotherapeutic approach which is based on the observation that in some instances. The potential advantages of the epitope-based approach include a specific immune response, increased safety, increased potency and breadth of rationally engineered epitopes and focusing on immune responses elicited by conserved epitopes.Accordingly, rational choices are made to isolate the components desired for the responses.The premise of these efforts is the identification of the appropriate epitopes. Phage display techniques have aroused interest as a new tool in the studies of mapping antigenic epitope since described by Smith.It was demonstrated that MrkD adhesin mediated K.pneumoniae to adhere human respiratory tissue.It has not been investigated that the MrkD adhesin stimulate a protective immune response in vivo. In this study, we have investigated the ability of MrkD-specific antibodies to protect against infection using the murine model of acute K.pneumoniae infection.The ability of immune sera to passively protect animals from infection was determined. Based on these observations, MrkD adhesion was selected as a potential target for treatment.Mapping the epitope of MrkD adhesion is one of the essential steps for the development of antibodies against K. pneumoniae.MrkD protein was expressed as a GST fusion protein in the E. coli BL21 to facilitate the purification.A pronounced band with the molecular weight of approximate 61 kDa was examined by SDS-PAGE in the supernatant of cell lysate after the induction,suggesting that the fusion protein was successfully expressed in the bacterial cells.MrkD—GST fusion protein was successfully purified by glutathione Sepharose 4B column chromatography and verified by SDS-PAGE.Three hybridomas, E01—E03, were established by cell fusion.All mAbs were IgG1 and showed high specificity to MrkD protein.The titer of culture supernatant was 1:1 000—1:4 000 and the titer of ascites was 1:128,000—1: 1:256,000.The antigen were used in ELISA assay to calculate the affinity of the monoclonal anti-MrkD antibody using the method given in'Materials and methods'section.MAb E01 displayed the highest affinity of about 0.3μg/mL. Competition studies using increasing concentrations of these three mAbs as inhibitors or competitors revealed that these three mAbs compete significantly with each other, indicating that recognition epitope of these three mAbs were the same or largely overlapped.In order to map the epitope of MrkD antigen recognized by the mAb E01, the Ph.D.-12 library was screened with the purified mAb E01.After three rounds of biopanning, the selected phages bound to mAb were well enriched indicated by the increased recovery.Then 36 clones of the phages were randomly selected to test for their immunoreactivity with mAb E01.Among 36 clones, 33clones were positively recognized by mAb E01 while BSA was used as negative control.The DNA sequences of 16 positive clones were determined. There were eight different peptides in these 16 phage clones and most clones were the same QKTLAKSTYMSA sequence. Comparison between the protein sequence of MrkD and the dodecapeptide sequence revealed that amino acid sequences T, L, A, Y, S were conserved in MrkD(residues 148—159aa) and most positive phage clones.This result suggested that T, L, A, Y, S was the core amino acid of MrkD to form an epitope recognized by mAb E01.This notion was further confirmed by competitive inhibition ELISA.Pre-incubating mAb E01 with the recombinant phage clone C1 inhibited its binding with MrkD protein while pre-incubation with control phage, for example, phage clone C6 did not affect mAb's binding with MrkD.To evaluate the immune responses of the phage clones which comprised of T L A Y S amino acids, phage clone C1 and clone C6, were chosen to immunize the BALB/c mice using the intraperitoneal administration. The serum of the mice immunized with phage clone C1 showed higher binding with pre-coated MrkD on ELISA plate while the serum of the mice immunized with phage clone C6 showed the similar low binding with MrkD as controls as M13 phage.The MrkD proteins recognized by the antisera (raised by phage clones and the controls) were verified further by Western blot analysis.The antisera of clone C1 reacted specifically with the MrkD protein (61 kDa), while the pre-immune serum, the antisera induced by C6 clone and wild type M13 phage did not.In order to study the immune response of BALB/c mice, the potential binding motifs for I-Ad and I-Ed in amino acid sequence of MrkD were scanned by the RANKPEP software. The I-Ad and I-Ed restricted epitopes were predicted, respectively. We selected five putative I-Ad restricted and one I-Ed restricted epitopes with higher scores. The selected peptides were synthesized and the purities of these peptides were all≥85% analyzed by high-pressure liquid chromatography.Having identified three CD4+ T cell epitopes in MrkD protein, we then evaluated whether lymphocytes primed by peptides could recognize naturally processec antigen. For this purpose, we immunized mice with these peptides emulsified in IFA, and assessed the responses of CD4+ T cells in vitro, as described above. Meanwhile, we evaluated the responses of CD4+ T cells isolated from mice vaccinated with PBS in IFA as negative controls. T cells from BALB/c mice immunized with peptides M221-235, M175-189, and M264-278 exhibited significant proliferation upon in vitro stimulation with respective immunizing peptide and rMrkD. Under the same conditions, T cells from the PBS injected control mice did not respond to any peptide and rMrkD (SI < 2). Thus, these peptides can evoke the CD4+ T cell response and the CD4+ T cell induced by MrkD derived peptides react to rMrkD. The FACS analyses were also performed to determine the relative percentage of CD4+ CD3+ T cells immunized with peptides. The percentage of CD4+ CD3+ T cells from mice immunized with peptides M221-235, M175-189, and M264-278 were higher than the control group treated with PBS. These results reconfirmed that the peptides can stimulate the CD4+ T cells.In conclusion, we have designed and constructed an epitope vaccine (KMepi) against K.pneumoniae. Although we have not assessed all the combination orders in the epitope vaccine, this study implies that epitope vaccine is a promising candidate for the development of K.pneumoniae treatment. Ongoing studies will evaluate the efficacies of other combination orders and compare the therapeutic effect of KMepi with rMrkD and K.pneumoniae lysates.We will also further evaluate the therapeutic effect of the epitope vaccine in mice. The data from mouse models will provide much information for the further development of therapeutic vaccines against K.pneumoniae and other pathogenic microorganism, and should lead to studies of the epitope-based vaccine for human use.