Cloning antifungal single chain fragment variable antibodies by phage display and competitive panning elution

J of Molecular Recognition

et al. 2011

Self Cite

Existing antifungal drugs are notable for their inability to act rapidly, as well as their toxicity and limited spectrum. The identification of fungal-specific genes and virulence factors would provide targets for new and influential drugs. The display of repertories of antibody fragments on the surface of filamentous phage offers a new way to produce immunoreagents as defined specificities. Here we report the selection of Cryptococcus-specific targets by using phage-display panning from a cDNA library, where bactericidal antibodies have been developed against conserved surface-exposed antigens. A single-chain variable fragment (scFv) phage library was constructed from splenocyte of an immunized mouse by idiotypic vaccination with HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) that was used for selection against Cryptococcus neoformans membrane fraction (CnMF). Key elements were the selection against antigen (nmAb-KT and CnMF) and the release of bound phages using competitive panning elution with CnMF at neutral pH condition. Isolated scFvs react specifically with C. neoformans and some other pathogenic and non-pathogenic fungal strain's cell wall receptors by exerting strong antifungal activity in vitro. A high affinity clone, designated M1 was selected for detailed characterization and tested anti-cryptococcal activity with IC(50) values at 5.33 × 10(-7) to 5.56 × 10(-7) M against C. neoformans. The method described here is a new technique for the isolation of cell membrane specific immunoreactive phages in the form of scFv using CnMF that contained cell membrane associated proteins.

Section: Discussionmentioning

confidence: 63%

Section: Sequence Analysis Of Scfv Clonesmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

New avenues for phage‐display library to produce a Cryptococcus‐specific anti‐idiotypic antibody of HM‐1 killer toxin

Rahman

J of Molecular Recognition

et al. 2011

Self Cite

“…To overcome the intrinsic toxicity and chemical instability of HM‐1 and simultaneously take advantage of its immunogenicity, nmAb‐KT has been produced . With the aim of producing a new anti‐fungal drug, we used nmAb‐KT as an immunogen in mice to produce an antigen‐specific anti‐idiotypic antibody that shares structural and/or functional similarities with the active site of HM‐1 .…”

mentioning

confidence: 99%

Antigen itself antibody: an alternative one‐step immunoassay for measuring the anti‐idiotypic antibody titer

Microbiology and Immunology

Selvakumar

et al. 2014

Self Cite

Immunoassay designs rely on the great specificity of antibodies and a suitable marker that facilitates generation of a quantitative signal. Currently, there is no reliable method for measuring the titers of an anti-idiotypic antibody. Our initial attempt to measure titers of mouse anti-idiotypic antibody after idiotypic vaccination with HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT) failed. Because the injected antigen, nmAb-KT, is a mouse IgG, using a commercial antibody to measure the antibody titer always gave a false positive signal against control mouse serum antibody in parallel with the antigen-treated immunized serum antibodies. To get a reliable and clearly differentiable signal by ELISA, idiotypic antigen was labeled with HRP and HRP-conjugated-nmAb-KT used to measure the antibody titers in the antigen-treated mice. Compared with control mice, signals were found in high anti-nmAb-KT IgG responses in test mice; however, untreated control mice had a significant amount of purified non-specific IgG. This method is amenable to long read lengths and will likely enable anti-idiotypic antibody titer measurement in a more specific and cost effective way without requiring commercial antibody.Key words anti-idiotypic antibody, killer toxin neutralizing monoclonal antibody, one-step immunoassay, titer measurement.Killer toxins are protein molecules that disrupt cell functions in a number of ways (1, 2). Among the yeast killer toxins, HM-1 is highly stable against heat treatment and a wide range of pH values (pH 2-11); it also exhibits a wide spectrum of anti-fungal activity (3, 4). These unique properties have made this toxin attractive for therapeutic applications, but not for clinical use both because of its instability in host physiological conditions and its antigenicity (2, 4, 5). To overcome the intrinsic toxicity and chemical instability of HM-1 and simultaneously take advantage of its immunogenicity, nmAb-KT has been produced (6). With the aim of producing a new anti-fungal drug, we used nmAb-KT as an immunogen in mice to produce an antigenspecific anti-idiotypic antibody that shares structural and/or functional similarities with the active site of HM-1 (7-15).Antibodies are highly specific, naturally evolved molecules that recognize specific antigens and can be classified according to their mode of action as they react to and set about defending the body against foreign invaders (16). Anti-idiotypic antibodies are a special set

Peptide derived from anti-idiotypic single-chain antibody is a potent antifungal agent compared to its parent fungicide HM-1 killer toxin peptide

Karim

Appl Microbiol Biotechnol

et al. 2011

Based on anti-idiotypic network theory in light of the need for new antifungal drugs, we attempted to identify biologically active fragments from HM-1 yeast killer toxin and its anti-idiotypic antibody and to compare their potency as an antifungal agent. Thirteen overlapping peptides from HM-1 killer toxin and six peptides from its anti-idiotypic single-chain variable fragment (scFv) antibodies representing the complementarity determining regions were synthesized. The binding affinities of these peptides were investigated and measured by Dot blot and surface plasmon resonance analysis and finally their antifungal activities were investigated by inhibition of growth, colony forming unit assay. Peptide P6, containing the potential active site of HM-1 was highly capable of inhibiting the growth of Saccharomyces cerevisiae but was less effective on pathogenic fungi. However, peptide fragments derived from scFv antibody exerted remarkable inhibitory effect on the growth of pathogenic strains of Candida and Cryptococcus species in vitro. One scFv-derived decapeptide (SP6) was selected as the strongest killer peptide for its high binding affinity and antifungal abilities on both Candida and Cryptococcus species with IC(50) values from 2.33 × 10(-7) M to 36.0 × 10(-7) M. SP6 peptide activity was neutralized by laminarin, a β-1,3-glucan molecule, indicating this peptide derived from scFv anti-idiotypic antibody retains antifungal activity through interaction with cell wall β-glucan of their target fungal cells. Experimental evidence strongly suggested the possibility of development of anti-idiotypic scFv peptide-based antifungal agents which may lead to improve therapeutics for the management of varieties of fungal infections.