Peptide Mimics To Peptidoglycan Are Vaccine Candidates And Protect Mice From Infection With Staphylococcus Aureus

Staphylococcus aureus, a gram-positive bacterium, is a leading cause of community-associated and nosocomial infections which colonizatied in human skin and mucosa, particularly the anterior nares. However, it often causes bacteraemia with high morbidity and mortality in immunocompromised hosts, surgical patients and in those with indwelling medical devices in spite of adequate antibiotic therapy. Since appearance of multidrug resistant strains especially methicillin-resistant S.aureus (MRSA) in past 50 years, which indicates S.aureus cannot always be controlled by commonly used antibiotics, additional control strategies are greatly needed. Therefore, the immunotherapy and prevention have been explored and emphasised since the 1960s, especially, the development with vaccine has been expected and been made great efforts. However, about ten vaccine candidates with different components of S.aureus including StaphVAX (the polysaccharide-carrier conjugates, failed in Trial III), PentaStaph (containing CP8/5, a-toxoid and PVL) a-hemolysin, Panton-Valentine leukocidin (PVL), Toxic shock syndrome toxin-1 (TSST-1), S. aureus attenuated toxin strain and Poly-N-acetyl-glucosamine (PNAG), poly-N-succinyl beta-1-6 glucosamine (PNSG) protein or capsule polysaccharide-protein conjugates, surface protein vaccines (IsdA, IsdB, SdrD and SdrE), and a DNA vaccine prepared from FnBPA-CIfA, have not been approved to be used in clinical.PGN is a vital component on cell wall of many kinds of bacteria, especially in Gram positive bacteria such as S.aureus. Recently, PGN was considered as 'the bacterial Achille' sheel, providing the weak point for targeting. However, PGN has never been used as vaccine candidate because PGN as nonprotein component, belongs to thymus independent antigen (TI-Ag), and neither induces the production of memory B cells nor effective secondary antibody response. Our strategy is to convert the PGN, a TI-Ag, to becoming peptide mimics to PGN as thymus dependent antigen (TD-Ag) that can provoke secondary immune responese.The work on peptide mimics to PGN has been not reported before, however, this strategy and technique have been widely used in research of non-protein or protein vaccines, as well as single conformation-dependant antigenic determinant and antigenic epitopes mapping, These reports include the peptide mimics to provoke the production of protective antibodies against Cryptococcus neoformans, group B meningococcal, Haemophilus influenzae, Streptococcus pneumoniae, Vibrio cholerae, Brucella, Entamoeba histolytica, as well as the production of cytotoxic antibody against tumor cells.In recent years, multiple antigen peptide (MAP) has been considered and used as a valuable approach to amplify peptide immunogens. The advantages of MAP are as follows:MAP is consisted of nearly pure antigenic sequences without protein carrier to make conjugate; and MAP can be used an immunogen without adjuvant; additionally, MAP can provoke T cell mediated immune response.We screened the peptide mimics to PGN from a random phage display peptide library using an anti-PGN monoclonal antibody (McAb) and synthesised the four-branch multiple antigenic peptides that mimics epitopes on PGN. These MAPs were used as vaccine candidates to immunize the mice, and antigenicity and immunogenicity were identified by serial of experiments in vivo and in vitro. We also studied whether these peptide mimics can induce an effective protection immune against S.aureus or not.This research can be divided into the following four parts. PartⅠ. Screening of the peptide mimics to PGN from phage displayed peptide librariesThe PGN mimotopes were screened from 12-mers linear phage display peptide library by using monoclonal antibody against PGN and the antigenicity of selected clones was identified by ELISA. The screening of phage clones showed an enrichment process from first round to third round, and 14 of 49 phage clones were defined as positive clones that bound with anti-PGN McAb identified by indirect ELISA. The amino acid sequences deduced from DNA sequences were analysed and were shown that three phage clones as:No.31, No.28 and No.43, had the consensus sequence ATWxHxLxSAGL, and showed higher reactivity with anti-PGN McAb than other clones. Phage clones No.39, No.27 and No.1 shared the conserved sequence xHx or Hx found in clone No.31, these phages may display the mimotopes of PGN.PartⅡ. The design, synthesis and identification to peptide mimics of PGN in vitroIn this part, three peptide mimics sequences'SP31, SP27, and SP39'were redesigned and synthesized by adding S (serine) A (alanine) and G G (glycine) on the two sides of origin sequence for stability of the conformation as well as adding biotin on the N- terminal as a tag. Four branch mutilgen peptides (MAP) were also synthesized in order to enhance antigenicity and immunogenicity of the peptides SP31 and SP27. Finally, the antigenicity of peptide mimics or MAPs was identified.In the design of peptide mimic for systhesis, we estimated or predicted the antigenic epitopes, T cell epitopes and scores binding to MHC of these peptides by using DNASTAR and online softwares (http://bio.dfci.harvard.edu/Tools/antigenic.pl, www.syfpeithi.de, http://www.darrenflower.info/mhcpred), indicating that three peptides contain sites bound both mice and human MHC. The Sequences-TWxHxLx-' and 'WxHxVxW-' may be antigenic epitope as SP31 and SP39 respectively, which contains a T cell epitope by adding 'S' 'A' and 'G' 'G' on the two sides. However, SP27 does not contain a T cell epitope even if was added different assemblies of amino acid residue 'S A G G' and the sequence '-SPHxH000RS-'as an antigenic epitope.According to above design, the sequence of clone No.31,27 and No.39 were synthesized and termed as SP31, SP27 and SP39, respectively. To enhance the immunogenicity, a four-branch MAP based on the sequence SP31 and SP27 were synthesized and termed as MAP-P31 and MAP-P27, respectively, Then the antigenicity of peptide mimics or MAPs was identified by indirect and competitive ELISA. Our results show that both SP31 and MAP-P31 could bind to both anti-PGN McAb and a polyclonal antibody against S.aureus in a dose-dependent manner (FSP31 binding to anti-PGN McAb=367.090, P SP31 binding to anti-PGN McAb=0.000; FSP31 binding to anti-S.aureus PcAb=367.090,PSP31 binding to anti-S.aureus PcAb=0.000; FMAP-P31 to anti-PGN McAb=2021.727, PMAP-P31 to anti-PGN McAb=0.000; FMAP-P31 to binding to anti-S.aureus PcAb=178.344,PMAP-P31 to binding to anti-S.aureus PcAb=0.000, respectively). Moreover, both SP31 and MAP-P31 could inhibit the binding of PGN to anti-PGN McAb in a dose-dependent manner(F SP31=12.286, P SP31=0.039; FMAP-P31=17.532, PMAP-P31=0.053; respectively); conversely, PGN could inhibit the binding of SP31 and MAP-P31 to the anti-PGN McAb. However, MAP-P27 could not bind to McAb against PGN even if SP27 better bound to both PcAb and McAb against PGN. Additionally, SP39 could bind to antibodies against PGN or S.aureus but not in a dose-dependent manner. These data indicate that both SP31 and MAP-P31 mimic to epitopes on PGN.Since PGN can stimulate murine peritoneal macrophage (pM0) to produce some proinflammatory cytokines such as TNF-αand IL-6, we have tested whether the peptide mimics to PGN likde MAP-P31 could stimulate the production of proinflammatory cytokines or not. Polymyxin B was used to exclude the influence of lipopolysaccharide (LPS) if any LPS presented in the supernatant. The results show that TNF-a and IL-6 were produced by pMΦstimulated with MAP-P31 in dose-dependent manner(FTNF-α=1265.415,PTNF-α=0.000, FIL-6=248.353, PIL-6=0.000, respectively), suggesting that MAP-P31 can also mimic the biological activity of PGN despite the weaker activity of MAP-P31 to stimulate cytokine production than that of PGN.PartⅢ. The characteristization of active immunity in mice immunized with peptide mimics to PGNIn order to characterize MAP-P31 mimics to PGN as candidate vaccine, six-eight week-old Balb/C mice were randomly divided into five groups to be immunised as follows:MAP-P31 emulsified with FCA for the first immunisation, followed by boost with Freund incomplete adjuvant; FCA as basic immunization for one week prior to MAP-P31; MAP-P31 alone; and unrelated MAP emulsified with FCA and PBS were used as controls. The antisera were collected for identification of antibodies to MAP-P31, PGN and S.aureus, S.epidermidis, E.coli and P.aeruginosa by indirect ELISA. The results show that the better antibody response was provoked in mice immunised with MAP-P31 emulsified with Freund adjuvant(P=0.001 vs FCA prior to MAP-P31; P=0.005 vs MAP-P31), and the antiserum can bind with PGN well(FPGN=70.122, PPGN=0.000), but bind with LTA and LPS weakly. Consistent with our expectations, the antiserum can also bind to ultrasonic cell lysates of S.aureus, S.epidermidis, E.coli, and P.aeruginosus (P<0.05 vs anti-unrelated MAP). In addition, the serum IgG type antibodies against PGN and S.aureus endured in mice, which demonstrates that the MAP-P31 as peptide mimics to epitopes on PGN, provokes an effective secondary or recall antibody response that can only be induced by a TD-Ag and protects against infection with S.aureus.We estimated the bactericidal/acteriostatic activity as well as opsonophagocytosis activity of the antiserum. The results show that anti-MAP-P31 serum possess a more potent bactericidal/bacteriostatic activity than that of anti-unrelated MAP serum or normal serum in a dose-dependent manner with or without complements, and enchance phagocytosis of murine peritoneal macrophages (pMΦ)to FITC-labeled s.aureus (ATCC 25923)(F=981.862, P=0.000). In addition, anti-MAP-P31 serum also has complement-independent bactericidal activity against F.enterococcus, S.hominis, P.aeruginosa and E.coli but not against clinic isolated and standard methicillin resistant S.aureus (MRSA).Since the most important evaluation for a vaccine should be protection of mice from lethal bacterial attack, the bacterial clearance and protection against infection with lethal live S.aureus were observed in 6 to 8 week-old Balb/c mice immunised with MAP-P31 or an unrelated MAP and PBS as controls. The results show that bacterial clearance in spleen, liver and kidney tissues was enhanced significantly in mice immunised with MAP-P31 compared to that in mice immunised with an unrelated MAP(Pliver=0.018 vs unrelated MAP,Pspieen=0.015 vs unrelated MAP and Pkindey=0.020 vs unrelated MAP). Moreover, mice were challenged with 6×108 live S.aureus by intraperitoneal injection (ip) at two weeks after the fifth immunisation, the survival of the mice immunised with MAP-P31 was notably longer than that of mice immunised with an unrelated MAP or PBS control, and mice never died after one week of attack. Consequently, we conclude that MAP-P31 can provoke protection against a lethal infection with S.aureus (MSSA), which may be associated with enhancing the bacterial clearance in vivo. However, MAP-P31 could not protect mice infected with MRSA, which would be a serious problem and challenge for development of vaccine.Part IV. The estimation of passive immunity by antiserum to modified MAP-P31In order to make PGN peptide mimics induce a protective immunity to MRSA, we have modified MAP-P31 by changing a few of amino acide to form a Th epitope, the modified MAP-P31 was named as MAP-P31.1. All of MAP-P31.1, MAP-P31 and MRSA with Freund ajuvant were used to immunize 6-8 week-old Balb/C or rabbits.The ten folds increasing antibody titer was found in mice immunized with MAP-P31.1 and followed by boost with heat-killed S.aureus, which suggests that natural infection with S.aureus could become a vaccine boost to enhance the specific immunity to infection.The estimation in vitro for bactericidal/bacteriostatic activity show that anti-MAP-P31.1 antisera had a more potent bactericidal/bacteriostatic activity against MSSA (ATCC 25923) than that of anti-MAP-P31 serum or anti-S.aureus serum with or without complements(FMSSA(inactive complements)16.490,PMSSA(inactive complements)=0.001); importantly, anti-MAP-P31.1 antisera also show a bactericidal/bacteriostatic activit on MRSA such as standard (ATCC43300) or clinic isolation MRSA strains (317177, identifying by clinical labs) (FMRSA(inactive complements)=13.213, PMRSA(inactive complements)=O.006; FMRSA(complements)=14.150 and PMRSA(complements)=0.005), whereas anti-MAP-P31 antisera could not. The antisera to S.aureus were not shown the bactericidal or bacteriostatic activity against MSSA and MRSA without complements, although it showed a better binding to PGN and S.aureus than antisera against MAP-P31 and MAP-P31.1. These results suggest that MAP31.1 can provoke an antibody response with bactericidal or bacteriostatic activity against both MSSA and MRSA in vitro.For the evaluation of passive immunity by injecting anti-MAP-P31.1 rabbit IgG antisera into mice with S.aureus infection, we found that anti-MAP-P31.1 purified IgG notably prolonged living time and survival of mice infected with MSSA but not MRSA. Additionally, bacterial clearance in spleen, liver and kidney tissues was enhanced in mice challenging with MSSA after administering anti-MAP-P31.1, compared to that of administering normal rabbit IgG(Pliver=0.006 vs NRS, Pspleen=0.013 vs NRS,Pkidey=0.010 vs NRS); and the inhibition of renal abscess causing by MSSA (ATCC25923) but not MRSA (ATCC43300) were also observed.In this work, the modified peptide mimics MAP-P31.1 could induce an improved antibody response to MRSA in vitro, compare to MAP-P31, but it still could not provoke an effective protection against infecton with MRSA.