Mechanism Of Protection Against S.Aureus Systemic Infection By Immunization With Peptidoglycan-mimicking Peptide In Murine Model And Screening The Mimotopes Of Lipoteichoic Acid

Staphylococcus aureus, one of the most common Gram-positive bacterium, causes not only skin and soft tissue infections, but also a series of life-threatening diseases, such as bacteraemia, pneumonia, osteomyelitis, meningitis,and sepsis. The treatment of S.aureus infection has become difficulty responsible for the emergence of the methicillin-resistant S.aureus (MRSA). Consequently, prevention and immunotherapy has been explored. Nevertheless, attempts to develop an efficacious vaccine which can be propHylactic against S.aureus infection have so far been unsuccessful. This failure has been attributed partly to the ability of numerous virulence factors engendered by this organism whose expression pattern can change widely among individual clinical isolates and display on diverse infection state. Thus, it is imperative to select a conserved antigen that express on bacterium persistently and steadily as vaccine candidate from the variety molecules in S.aureus. The second obstacle for development of S.aureus vaccine is that, the relationship between the bacterium and the immune system remains ambiguous. Opsonization was initially considered as an efficacious approach. However, the effect of antibody-mediated opsonization is controversial. Recently, more and more reports indicated that T cells-mediated immune responses, especially stimulating and secreting IL-17 or IFN-y, might play an important role in prevention of S.aureus infection.Both Peptidoglycan (PGN) and lipoteichoic acid (LTA), are the crucial and conserved molecule in S.aureus. However, as thymus-independent antigen (TI antigen) with weak immunogenicity, PGN and LTA are not considered as rational vaccine candidates. Mimetic peptides have been represented a promising tool to overcome this handicap of TI antigen. Immuniziation with peptide as a surrogate, which mimic to carbohydrate antigen of tumor cells or polysaccharid in bacterium, induced protective antibodies response as well as augment tumor-specific cellular response.In our previous work, we successfully obtained a series of PGN peptide mimic sequences from pHage peptide library by using an anti-PGN mAb as the target. Immunization with one of the peptide mimic induce effective antibdoy response and protects mice against S.aureus infection, but not stimulate T cell response. In this study, we synthesized a multiple antigenic peptide (MAP), named MAP27, carrying four copies of an identical peptide derived from another mimic sequence (SASPHxHSRLRSESGG). The mechanism of protection against S.aureus systemic infection by immunization with MAP27 was investigated. Meanwhile, the peptide mimic to epitope of LTA was screened and identification.Part Ⅰ A multiple antigenic peptide mimicking-peptidoglycan elicted T cells response protects mice against S.aureus systemic infection.1. Both linear-peptide SP27 and tetra-branched, multiple antigenic peptide MAP27 bind to antibody specifically.Linear-peptide, SP27 (Biotin-SASPHxHSRLRSESGG) and a multiple antigenic peptide, named MAP27 that contains four copies of SP27 was synthesized by Shenzhen Hanyu Technology Co. Ltd. The antigenicity of SP27 and MAP27 was identified by ELISA. The results showed that SP27 bound to anti-PGN mAb in a dose-dependent manner, but didn’t react with anti-LTA mAb. MAP27 also bound to anti-PGN McAb and polyclonal antibodies against S.aureus (PSP27 binding to anti-PGN McAb=0.02;PSP27 binding to anti-s.aureu PcAb=0.002). These data suggested that both SP27 and MAP27 mimic epitopes of PGN. MAP27 stimulated production of IL-6 and IL-1β from murine peritoneal macrophage (MAP27 vs MAP(ctrl),PIL-6 =0.045;MAP27 vs blank,PIL-6=0.018)、IL-1β(MAP27 vs MAP(ctrl),P IL-1β=0.002;MAP27 vs blank, PIL-1β=0.000). These results suggested that MAP27 mimic the biological activity of PGN partly.2. Last boost with heat-killed bacterium produced low titer antisera against S.aureus and PGNFour to six week-old BALB/c mice were randomly divided into three groups, then immunized with MAP27 or MAP(ctrl) emulsified with Fruend’s adjuvant for five times. The third group of mice without any MAP immunization were breed at the same time and used as a blank control. All of the three groups of mice were administrated with heat-killed bacterium via intrapertioneal injection one time. The antisera were detected during immunization. The titer of antibodies against MAP27 in MAP27-immunized mice reached to 1/104 after the third time of vaccination. After boosting with heat-killed bacterium one time, anti-S. aureus antibodies and anti-PGN antibodies could be detected with the titer 1/200 (MAP27 vs MAP(ctrl),P anti-S.aureus antibodies=0.000,MAP27 vs blank,P anti-S.aureus antibodies=0.000;MAP27 vs MAP(ctrl),P anti-PGN antibodies=0.026,MAP27 vs blank,P anti-PGN antibodies=0.015).3.Vaccination with MAP27 protected mice against S.aureus lethal-challenge and decrease the bacterial burden in organs.All of mice were challenged with S.aureus with lethal dose on the fifth days post the last boost of heat-killed bacterium. The survival percent was then monitered. After seven days of lethal challenge,50% of the MAP27-immunized mice were still alive, whereas, all of the naive mice and MAP(ctrl)-immunized mice died on day two and day five, respectively (MAP27 vs MAP(ctrl),P=0.000;MAP27 vs blank, P=0.000).To further determine the effect of MAP27 immunization on bacterial growth in animal organs, all of the mice were sacrificed after S.aureus infection three days. The number of bacterium in kidney and lung of MAP27-immunized mice is significantly fewer than that of MAP(ctrl)-immunized mice and naive mice (MAP27 vs MAP(ctrl),Pkidney =0.029,MAP27 vs blank, Pkidney=0.029; MAP27 vs MAP(ctrl),Plung=0.016, MAP27 vs blank,Plung=0.008).4.Levels of IFN-γ, IL-17A/F and CCL3 are increased in organs of MAP27-immunized mice after S.aureus systemic infection.In light of immunization with MAP27 protects the mice against S.aureus systemic infection accompanied with low titer antiserum against S.aureus and PGN, we speculated that the protection effect of MAP27 immunization related with T cell-mediate response in vivo. To examine this hypothesis, the profiles of cytokine in the homogenate of spleen and lung from the three groups mice were detected by ELISA after infection three days. As expected, the level of IFN-γ and IL-17A/F that are cytokines mainly secreted by T cell were remarkably higher in MAP27-immunized mice than MAP(ctrl)-immunized mice and naive mice (IFN-γ:MAP27 vs MAP(ctrl), Pspleen=0.001, MAP27 vs blank, Pspieen=0.000; MAP27 vs MAP(ctrl),Plung =0.000,MAP27 vs blank,Plung=0.000;IL-17A/F:MAP27 vs MAP(ctrl),Pspleen=0.01, MAP27 vs blank,Pspieen=0.014; MAP27 vs MAP(ctrl),Piung=0.000, MAP27 vs blank,Plung=0.000). Concentration of CCL3 was also increased in MAP27-immunized mice(MAP27 vs MAP(ctrl),Pspieen=0.022,MAP27 vs blank, Pspleen=0.000; MAP27 vs MAP(ctrl), Plung=0.033,MAP27 vs blank,Plung=0.016).5.The IFN-γ+CD3+ and IL-17A+CD4+ T cells were increased in spleen of MAP27-immunized mice post S.aureus systemic infection.To further characterize the cytokine-expression profiles of infiltration T cell population over the course of infection, intracellular cytokine staining was performed by flow cytometry. The percent of IFN-γ+CD3+CD4+ T cells from MAP27-immunized mice was significantly higher than from MAP(ctrl)-immunized mice or naive mice(MAP27 vs MAP (ctrl),P=0.021;MAP27 vs blank,P=0.03). Similar results were also observed in IFN-y+CD3+CD4- T cells (MAP27 vs MAP (ctrl), P=0.029;MAP27vs blank,P=0.021). we also found the percent of IL-17+CD3+CD4+T cells from MAP27-immunized mice was higher than from MAP(ctrl)-immunized mice and naive mice(MAP27 vs MAP (ctrl),P=0.021;MAP27 vs blank,P=0.012). Taken together, not only IFN-y-but also IL-17-producing T cells of MAP27-immunized mice were stimulated effectively upon S.aureus infection.6.In vitro, MAP27 treatment stimulated the splenocytes from MAP27-immunized mice to produce IFN-y.To further assess whether immunization with MAP27 is able to induce IFN-y and IL-17 in vitro, the splenocytes were harvested after the last boost with heat-inactivated bacteria five days, and incubated with 100μg/ml MAP27 for 24h. The supernatant was then collected and cytokine induction was analyzed by ELISA. The splenocytes from MAP27-immunized mice produced significantly more IL-2, IFN-y than cells from control mice upon stimulation by MAP27(IL-2:MAP27 vs MAP (ctrl),P=0.000, MAP27 vs blank, P=0.000;IFN-y:MAP27 vs MAP (ctrl),P=0.000,MAP27 vs blank,P=0.000). In contrast, IL-4 that is mainly produced by Th2 cells could be detected very weakly in all three groups (P>0.05). In ELISPOT assay, the number of IFN-y-producing cells from MAP27-vaccinated mice significantly increased than from MAP(ctrl)-immunized mice and naive mice (MAP27 vs MAP (ctrl),P=0.001;MAP27 vs blank,P=0.001). In light of this, we speculate that immunization with MAP27 might predominantly induce Thl cell response.7.Splenocytes from MAP27-immunized mice specifically recognized S.aureus and produced IFN-y and IL-17.Splenocytes isolated from three groups of mice were incubated with heat-inactivated S.aureus for 72h, and cytokines in the culture supernatant were analyzed by ELISA. Two classical cytokines produced by T cells, IL-2 and IFN-y, were significantly produced in splenocytes from MAP27-immunized mice than from MAP(ctrl)-immunized mice and naive mice (IL-2:MAP27 vs MAP (ctrl),P=0.041, MAP27 vs blank,P=0.024;IFN-γ:MAP27 vs MAP (ctrl),P=0.028,MAP27 vs blank,P =0.032). Interestingly, IL-17A/F, cytokine secreted mainly by Th17 cells, is also significantly induced by splenocytes from MAP27-immunized mice than from control mice (MAP27 vs MAP (ctrl),P=0.039,MAP27 vs blank,P=0.043). In ELISPOT assay, the number of IFN-γ-or IL-17A-producing cells from MAP27-immunized mice was significantly higher than from control groups (IFN-γ:MAP27 vs MAP (ctrl), P=0.0126,MAP27 vs blank,P=0.0041;IL-17A:MAP27 vs MAP (ctrl), P=0.0034, MAP27 vs blank,P=0.0039). Collectively, these results indicate that T cells, especially IFN-γ- and IL-17-producing T cells in MAP27-immunized mice recognized effectively the nature antigen S.aureus in vitro.Part Ⅱ Screening and Identification of Peptide mimics to lipoteichoic acid1.Screening of the LTA mimotopes from phage displayed peptide libraryThe LTA mimotopes were screened from 12-mers linear phage display peptide library. After 3 rounds of selection,65 phage clones were selected by direct biopanning and 45 phage clones were selected by solution-phase panning.The positive phage clones were identified by ELISA. The results showed 18 of 65 clones from direct biopanning as well as 4 clones from solution-phase panning were identified as positive clones. The amino acid sequences deduced from DNA sequences were analysed and were shown that VHxDFRQxxQPS and GHxDFRQxxQPS are the two conserved sequences.2. Synthesis peptide mimics and IdentificaionAccording to the conserved seuqences, two linear-peptides, name L3-1 (Biotin-HSVHxDFRQxxQPSGGG and L2 (Biotin-HSGHxDFRQxxQPSGGGS) were synthesized. As shown in ELISA, both L3-1 and L2 bind to anti-LTA mAb in a dose-dependent manner. In light of the sequence of L3-1,a multiple antigenic peptide, named MAP3 was synthesized by using standard Fmoc chemistry. MAP3 contains four copies of L3-1, with each peptide linked with the non-immunogenic lysine-based dendritic scaffold. The result of ELISA showed that MAP3 binds to anti-LTA mAb specifically.