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2. ACKNOWLEDGEMENTS Dr Nagendra Singh (Mentor) Kazuhiko Kasuya , Julie L. Boyer, Yadi Tan, and D.Olivier Alipui. Neil R. Hackett Ronald G. Crystal 3. TECHNIQUES USED IN THE ARTICLE Western blotting ELISA 4. INTRODUCTIONAnthrax, a disease caused by infection with Bacillus anthracis,manifests as cutaneous , gastrointestinal, or pulmonary disease.All can be fatal, but the inhalational form has the highest mortality,with a survival rate of only 55% in the recent U.S. bioterrorismincidents.Inhalational anthrax is acquired from aerosolized B. anthracisspores. In the lung, the spores are phagocytosed by alveolarmacrophages and transported to regional lymph nodes, wherethey germinate and the bacteria multiply.The virulence factors of B. anthracis are carried by two plasmids,one encoding genes for capsule components that limitphagocytosis and the other for three secreted exotoxin proteins,protective antigen (PA), lethal factor (LF), and edema factor (EF). 5. PA plays a central role in virulence in that it enablesLF and EF to function. Lethal toxin, comprising PA andLF, is responsible for the overwhelming systemic fatalpathophysiology associated with B. anthracisinfection.All available evidence supports the concept thatinduction of IgG antibodies against PA can beprotective against B. anthracis.An anthrax vaccine composed of an adenovirus(Ad) vector expressing PA requires only a singleadministration to be effective in protecting miceagainst the anthrax lethal toxin, but it requires 1week for anti-anthrax immunity to be effective. 6. METHODSAdenovirus vectors. AdaPAscAb is a serotype 5, Ad gene transfervector that expresses a murine/human single-chain antibodydirected against anthrax protective antigen.Binding of monoclonal antibodies to the carboxy-terminal domainof PA inhibits PA binding to cells.One of these antibodies (14B7) with specificity for PA and lethaltoxin neutralizing activity was engineered by Maynard et al. into asingle-chain antibody (scAb). 7. Using the anti-PA scAb construct, the expression cassette of AdaPAscAb contains (5V to 3V) the cytomegalovirus promoter/enhancer,an optimal Kozak sequence (GCCACCATG), the murine Ign-chainsecretion signal sequence, the anti-PA single-chain antibody cDNA(including the murine anti-PA heavy- and light-chain Fv sequencesand a sequence encoding a human constant n domain), tags to helpidentify the construct in preliminary studies (his tag + myc tag), andthe SV40 polyadenylation signal.Two control Ad vectors were used in the study. Ada CTXscAb isidentical to Ada PAscAb but, instead of the anti-PA single-chainantibody, the expression cassette encodes a single-chain antibodyreactive with a cobra toxin purified from Naja nigricollis venom.AdNull is a control vector with identical backbone to AdaPAscAb andAdaCTXscAb but it contains no transgene . 8. In vitro assessment of AdaPAscAbIn vitro assessment of Ad aPAscA. Expression of the anti-PAsinglechain antibody by Ada PAscAb was assessed in vitro byWestern analysis and by assessment of anti-neutralizing PAantibodies.In vivo assessment of anti-PA levelsFollowing administration of Ada PAscAb. Female BALB/c mice,4 to 6 weeks of age, were housed under pathogen-freeconditions. BALB/c mice were chosen for this study based ontheir sensitivity to lethal toxin. All administrations of Ad vectors diluted with PBS given intravenously. 9. Production and purification of lethal toxinLethal toxin was generated by combining PA and LF proteins producedand purified from bacteria. The PA or the LF gene was cloneddownstream of the T7 promoter in the prokaryotic expression plasmidfor expression as 6His fusion proteins. The resulting plasmids weretransformed into the BL21.DE3 strain of Escherichia coli.Bacteria were grown in Luria broth under ampicillin selection (50 Ag/ml)to an OD600 of 0.6 to 0.8 and expression of the PA.RESULTSIn Vitro Characterization of Ad-expressed Anti-PA Single-ChainAntibodyAda PAscAb is a serotype 5, Ad gene transfer vector containing acDNA encoding a murine /human single-chain antibody directedagainst anthrax protective antigen. 10. To examine the expression and secretion of the anti-PA single-chain antibody by Ada PAscAb, we infected A549 cells with AdaPAscAb and, as controls, we infected cells with Ada CTXscAbor AdNull. Supernatants collected from cells infected withAdaPAscAb at 48 h.We determined the specificity of the Ad-expressed PA-specificsingle-chain antibody for PA by Western analysis.The anti-PA single-chain antibody recognized PA, but not LF.Thus, AdaPAscAb expresses a single-chain antibody that hasspecificity for PA.The supernatants from AdaCTXscAb-infected and AdNull-infected cells had no neutralizing activity 11. In Vivo Expression by AdA PAscAbWe assessed the in vivo expression profile of Ada PAscAb inmouse serum 0 to 14 days after intra-venous administration byquantifying human Ign antibody levels.In contrast, we detected no single-chain antibody over time in micereceivingAdNull. 12. AdA PAscAb-Mediated Protection from in Vivo Challenge withLethal Toxin.Mice that had been injected with Ada PAscAb, Ada CTSscAb, orAdNull with lethal toxin at 72 h post administration, using naivemice as a negative control. 13. DISCUSSIONThe only vaccine approved for use in humans in the United States isused for military personnel and at-risk first-line responders; it is notavailable to the general civilian population.Using an adenovirus-delivered PA-specific single-chain antibody tomediate passive immunotherapy against B. anthracis lethal toxin, thepresent study presents a strategy to provide rapidly induced (24 h) andextended (14 days) protection against B. anthracis that can be used forat-risk individuals as well as those infected with B. anthracis.While effective, the half-life of the anti-PA single-chain antibody(identical to that encoded by the cDNA used in the present study) wasonly 10.4 min, a time far too short to be of use for protecting humans inthe context of ananthrax attack.