| Staphylococcus aureus(S.aureus),one of the most common Gram-positive,commensal bacterium that colonizes its human host,can cause a broad spectrum of diseases including skin and soft tissue infections,bacteremia,pnemonia and septic shock.The emergence of some antibiotics-resistant strains,such as methicillin resistant S.aureus(MRSA)makes the treatment of the infection toughter and leads to higher healthcare costs and higher mortality rate.Given this fact,there is a need to develop novel immunoprophylaxis and immunotherapy startegies against this pathogen.Lipoteichoic acid(LTA)is an essential component of cell wall that is expressed steadily on the surface of S.aureus.The structure of LTA varies and has been grouped into five different types(type I to V).The basic structure of type I LTA,found in a large range of Gram-positive bacteria such as S.aureus,is formed by a conserved,unbranched,repeating units of 1,3-glycerolphosphate that is linked to the cytoplasmic membrane via a glycolipid anchor.Recent works have demonstrated that LTA is essential for the pathogenicity and survival of S.aureus under low-osmolarity.However,as a thymus-independent antigen(TI antigen)with weak immunogenicity,LTA is not considered as a rational vaccine candidate.Phage-displayed peptide library is a useful tool for identifying peptide sequences that mimic epitopes of carbohydrates.In our previous work,we screened a phage displayed peptide library and obtained a series of peptide that can mimic the epitope of LTA.In light of one of positive sequence,GHKEDRQWCQHS,a tetra-branched multiple antigenic peptide(named MAP2-3)was synthesized.Immunization with MAP2-3 significantly prolonged the survival time of mice in S.aureus septic shock model and decreased the bacterial burden in organs of mice in bacteremia model.In this study,we further investigate the protective properites of the anti-MAP2-3 sera in different mouse models infected with S.aureus and study whether vaccination with MAP2-3 elicts speicifc immune response and immune memory.This work including the following parts:Part I Immunization with MAP2-3 elicited specific antibodies.1.The hydrophyilicity of three positive sequences were analyzed by bioinformatics softwares ProtParam(http://web.expasv.org/cgi-bin/protparam/otparam)and DNAstar.The result showed that sequence GHKEDRQWCQHS has the lowest grand average of hydropathicity(GRAVY),the highest surface probability and the highest antigenic index score.Based on this sequence,we synthesized a tetra-branched multiple antigenic peptide(named MAP2-3).ELISA result showed that MAP2-3 could react with two different anti-LTA mAbs,respectively.2.The BALB/c mice were subcutaneously(s.c.)immunized with MAP2-3(100?g/mice)or MAPctrl(the lysine-based,tetra-branched scaffold for MAP,100?g/mice)emulsified with Freund's adjuvant,respectively,for four times at 2-week intervals.The third group of mice,without any peptide immunization,was breed at the same time and used as a blank control.Serum samples were collected through retro-orbital bleeding 7 days after the last immunization.3.The serum samples from MAP2-3-immunized mice reacted with MAP2-3 as well as with LTA from S.aureus.In contrast,sera from either the MAPctrl-immunized mice or blank control mice failed to react with these two antigens.4.Ig class/subclass assay showed that MAP2-3 immunization elicited the production of LTA-specific IgG.Part ? MAP2-3 immunization protected the mice from S.aureus infection by elicitating functional antibodies.1.The rabbit anti-MAP2-3 sera significantly reacted with MAP2-3,LTA from S.aureus or the ultrasonic products of S.aureus compared with the normal sera(NRS).2.Opsonophagocytosis assay showed that antisera from MAP2-3 immunized mice enhanced mouse neutrophils to kill S.aureus.In the presence or absence of complement,the rabbit anti-MAP2-3 serum mediated the mouse neutrophils to kill S.aureus.3.Passive immunization with rabbit antisera protected BALB/c mice in three different S.aureus infection models:(1)In bacteremia model,the bacterial load in blood and organs(lung and kidney)from the mice that received anti-MAP2-3 sera was significantly lower than that from the mice that received normal rabbit sera(NRS)or PBS.(2)In pneumonia model,the bacterial load in lung from mice received MAP2-3 antisera was significantly lower than that from the mice received NRS or PBS.Moreover,administration of anti-MAP2-3 sera significantly reduced acute lung injury and inflammatory cells infiltrated in lung.(3)In skin infection,passive immunization with anti-MAP2-3 sera significantly reduced the abscess volume and dermonecrosis area compared to those received PBS or NRS within 14 days after infection.In addition,passive immunization with anti-MAP2-3 sera also reduced the bacterial counts in skins from mice 5 days after the infection.Part III Immune response and immunological memory was induced after MAP2-3 immunization.Since MAP2-3 immunization can elicit IgG class specific antibodies,memory B cells would be activated in vivo and developed into plasma cells.In addition,it is speculated that specific T cell response might contribute to the IgG formation.To test this hypothesis,all mice were injected with heat-killed S.aureus one month after the final immunizaation and the cells of spleen,lymphod,bone marrow were isolated.1.ELISPOT results showed that the number of LTA-specific IgG antibody secreted cells(ASCs)and S.aureus-specific IgG ASCs in splenocytes from MAP2-3 mice were significantly higher than that from MAPctrl mice or blank control.2.Flow cytometory assay was performed.Compared with MAPctrl mice or blank control mice:(1)The frequency of plasma cells(B220-CD138+)in splenocytes and in bone marrow from MAP2-3 immunization mice was higher.(2)The percentage of GC-B cells(CD19+ CD95+ GL7+)was significantly increased in the MAP2-3 immunized mice.(3)The frequency of TFH cells(CD4+ CXCR5+ PD-1+)that could trigger the formation of GC reaction and maintance of immune memory were significantly increased in the MAP2-3 immunized mice.(4)The percentage of memory B cells(B220+ CD38+ IgD-GL7-IgG+)in spleen/bone marrow from MAP2-3 immunized mice was significantly increased.(5)The expression of CD80 on memory B cells in splenocytes from MAP2-3 immunized mice was higher than that from MAPctrl or blank mice.(6)The percentage of effector T cells(TEM,CD4+ CD44+ CD62L-)in spleen/lymph node from MAP2-3 immunized mice was significantly increased.3.In vitro LTA treatment stimulated the splenocytes from MAP2-3-immunized mice to produce IL-6.Moreover,splenocytes from MAP2-3-immunized mice specifically recognized heat-killed S.aureus and produced IL-2,IFN-y and IL-6.In summary,as a surrogate of LTA,MAP2-3 is efficient at eliciting immune response and immune memory to protect BALB/c mice from S.aureus infection.This study provides a new option to design vaccines against S.aureus. |
PDF Full Text Request