Production And Characterization Of A Cross Reactive Monoclonal Antibody To Lipopolysaccharide

Sepsis is the most common cause of death in many intensive care units.Despite extensive research on the pathophysiology of sepsis and the technical advances,the mortality associated with severe sepsis and sepsis shock are 25%to 30%and 40%to 70%,respectively.Sepsis is triggered by gram-negative and gram-positive bacteria,fungi,and other microorganisms in which gram-negative bacteria are responsible for approximately 40%of total episodes.Lipopolysaccharide(LPS),the major constituent of the outer membrane of gram-negative bacteria,is the predominant antigenic and toxic component,It thus has been qualified as a target for diagnostic and therapeutic treatment for infections associated with gram-negative bacteria.At present,there is no ideal antagonist against LPS and lipid A for clinical prophylaxis and therapy.But there were some evidences that the pre-existed of antibodies against LPS are protective and the amount of antibody against core glycolipid of LPS in vivo is correlated with the survival in sepsis patients.Monoclonal antibodies(mAbs) to the LPS have been evaluated in a variety of experimental models and used in therapeutic trials in human,and some were found to improve survival in patients with gram-negative sepsis.LPS is consisted of three distinct parts:the O-specific antigen,also called O-antigen or somatic antigen,which is a long chain polysaccharide;the oligosaccharide core,composed of approximately 10 monosaccharides;the lipid A. LPS is structurally complex,heterogeneous macromolecule,and belongs to typeⅠthymus-independent(TI) antigen.The respone to TI antigen is strikingly different from the response to most proteins.TI antigens generally fail to elicit a memory response and usually do not show affinity maturation,making it difficult to produce mAbs against LPS with high affinity and broadly cross-reactivity if using the traditional immunization schedual.A reasonable alternative may derive from the use of molecular mimicry technology,and the searching for effective formulations that mediate reactive immune responses to TI antigen has consequently focused on identifying peptides mimic of TI antigens.Peptides,which are intrinsical thymus-dependent(TD) antigen and mimic the epitope structure of LPS,have the potential to overcome the limitations of TI antigen and obtain high affinity, cross-reactive mAbs to LPS.Phage display is a simple methodology for screening and identifying protein-ligand interactions and is widely used in epitope mapping and infectious disease research. Actually,phage display has successfully identified peptides mimic of polysaccharides, for instance,peptides mimic of the capsular polysaccharide of three of the Neisseria meningitides serotypes A,B and C have been identified using phage display.The results of these studies are encouraging that the peptides identified in this way do elicit immune responses in murine models and are suitable for generating high affinity mAbs.Our objective is to screen and synthesize the peptide that mimic the conservative epitopes of LPS,conjugate the peptide with adequate carrier,then use the conjugation as immunogen to generate some high affinity cross reactive mAbs against lipopolysaccharide and investigate the potential protective activity of the mAbs against G~- bacteria infection in animal models.In our previous research,four peptides mimetic of the conservative structure of S.typhi-LPS were screened from phage display cyclic7-peptide library by using purified polyclonal anti- S.typhi LPS antibodies with cross protection against G~-bacteria infection.The antigenicity and immunogenicity of these peptides were identified by serials of experiment in vivo and in vitro,and one of the peptides mimicry to LPS epitope was synthesized and named as 13L.Our previous results suggested that the conjugation of 13L with Blue Carrier(BC) could be used as immunogen and the BALB/c mice immunized with peptide-BC conjugation were protected against G~- bacteria infection and endotoxic shock induced by injection of LPS.Based on these research,13L was chosen as the candidate to immunize the mice and generate high affinity cross reactive mAbs against lipopolysaccharide.This research can be divided into the following two parts.PartⅠGeneration and characterization of cross reactive mAbs To LipopolysaccharideThe peptide 13L was screened and synthesized as described previously.Briefly,the LPS mimotopes were screened from c7c phage display peptide library by using polyclonal antibody against E.coli LPS 2630(L2630) purified with affinity chromatograph.One of the peptides mimicry to LPS epitope was selected and named as L7.the origin sequence of L7 was added four additional amino acids on the two sides to stabilize the conformation and named as 13L,which was synthesized with 90%purity and stored at -20℃until use.2 mg 13L and 4 mg BC were added to 1 ml MES(0.1 M,pH4.5) followed by adding 1 mg EDC,subsequently,the mixed solution was agitated gently at 25℃for 2 hours to generate conjugation of 13L with BC.After that,the conjugation was dialysed against PBS to remove free EDC and labeled as 13L-BC,which is the immunogen for following immunization procedure.Six femal BALB/c mice,6-8 weeks old,each was immunized with 200μg 13L-BC emulsified in equal volum of complete freund's adjuvant(CFA) by footpad inoculation and subcutaneous(s.c.) injection.Subsequently,immunization were carried out with 100μg 13L-BC emulsified in equal volum of incomplete freund's adjuvant(IFA) by footpad inoculation and s.c.injection at two weeks intervals.After the 5th immunization,5×10~6 CFU of hot-killed E.coli O111:B4 and Salmonella typhimuria were injected intraperitoneally.Two weeks later,mice were bled from orbital vein and serum titers were screened for reactivity to L2630 and L7261,the LPS of E.coli O111:B4 add Salmonella typhimurium,using Enzyme-linked immunosorbent assay(ELISA).Mice were given the final booster of 10μg /body L2630 in 100μl phosphate-buffered saline(PBS,pH 7.4) by caudal vein injection. Three days after the final booster,mice were sacrificed and splenocytes were generated and fused with NS-1 myeloma cells which cutured in RPMI-1640 medium containing 20%fetal bovine serum in the presence of PEG4000 according to established protocols.Hybridomas were selected by using HAT medium,and positive wells were screened by indirect ELISAs coated with L2630 and L7261 separately.Sub-cloning was performed five times using the limiting dilution method. Monoclonal antibodys were harvested from ascites fluid of BABL/c mice in which hybridoma had been injected intraperitoneal(i.p.).Monoclonal antibodys were purified from ascites using HiTrap protein G HP column according to the manufacture's instructions.The concentration and isotypes of the mAbs produced were determined using BCA protein assay kit and a mouse monoclonal antibody isotyping kit respectively,according to the manufacture's instructions.After three fusions and cloning,one hybridoma cell line which secrete monoclonal antibodie against LPS was obtained and named as SMU-3A8,of which the isotype belongs to IgG2a.The binding characteristic of SMU-3A8 to different bacteria and LPSs was carried out by indirect ELISA,Dot-ELISA,western blotting,immunofluorescence, and flow cytometry,and the results indicated that SMU-3A8 can react with four commercial LPSs and seven G~- negative bacterial with high affinity.PartⅡThe characterizing protective activitys of SMU-3A8 in vitro and vivo1.Test for bactericidal activity.The bacteria were grown overnight in Luria-Bertani(LB) Medium,centrifuged for 10 mins at room temperature,8000rpm. The precipitation was washed three times,and resuspended to 10~4 CFU/ml in sterile PBS.SMU-3A8(10μg/ml) was serially diluted(2-fold dilutions) in PBS,mixed 1:1 (100μl/100μl) with bacteria,and placed inU-bottom 96-well plates.Fresh guinea pig complement was added to a 20%final concentration and the plates were incubated for 1h at 37℃.Complement alone was used as control.After the incubation,samples were diluted(20-fold dilutions in PBS) and plated on LB agar.The CFU were counted after overnight incubation at 37℃.The results suggested that SMU-3A8 has no batericidal activity against E.coli O111:B4,E.coli O128,S.syphimurium and dysentery bacilli in the presence of active complement.2.LPS-induced NO secretion.The murine macrophage cell line RAW264.7 were cultured in 0.5 ml of pyrogen-free medium in the presence of L2630 and in the presence or absence of increasing concentrations of SMU-3A8 or of an mAb belongs to IgG2a as control.Culture supernatants were collected after 24h for NO determination by using a Nitric Oxide Assay Kit.The data demenstrated that SMU-3A8 could inhibit the secretion of NO induced by LPS stimulation to the murine macrophage cell line RAW264.7 at high concentration.3.Effect of SMU-3A8 upon bacterial challenge and endotoxic shock.the G~-bacteria infection animal model was established by i.p injection of S.typhi to female BABL/c mice,which could lead to 100%mortality in 4-7 days.Endotoxin shock model was induced by i.v.injection of a 100%lethal dose of L7261 or of L2630 to female BALB/c mice within 12-36 h.SMU-3A8(range 10 to 1,000μg per mouse), diluted in pyrogen-free NaCl solution,was administered i.v.2 h prior to bacteria or LPS challenging.Survival rate was recorded.The results indicated that SMU-3A8 could not improve the survival rate of endotoxin shock mice induced by i.v.LPS injection,but could decrease the death rate of S.typhi infection mice from 100%to 75%.Conclusively,in this study,we successfully generated an mAb specific to LPS using a novel immunization procedure:peptide mimics of the epitope of LPS was screened by phage display and conjugated to protein carrier as the immunogen followed by boosting with bacteria ans LPS.The mAb can recognize seven different strains of gram-negative bacteria and four kinds of commercial LPSs with high affinity,and shows some protective activities against S.typhi infection in mouse model.It may provide a useful tool for research on LPS.Moreover,the immunization procedure may offer another consideration to generate mAbs for therapeutic treatment with sepsis,...