title: Generation and characterization of recombinant bivalent fusion protein r-Cpib for immunotherapy against Clostridium perfringens beta and iota toxemia.
abstract: Clostridium perfringens beta (CPB) and iota (CPI) toxaemias result in some of the most lethal forms of haemorrhagic and necrotic enteritis and sudden death syndrome affecting especially neonates. While CPB enterotoxemia is one of the most common forms of clostridial enterotoxemia, CPI enterotoxemia though putatively considered to be rare is an emerging cause of concern. The similarities in clinical manifestation, gross and histopathology findings of both types of toxaemias coupled to the infrequency of CPI toxaemia might lead to symptomatic misidentification with Type C resulting in therapeutic failure due to habitual administration of CPB anti-toxin which is ineffective against CPI. Therefore in the present study, to generate a composite anti-toxin capable of neutralizing both toxaemias, a novel bivalent chimera r-Cpib was constructed by splicing the non-toxic C terminal binding regions of CPB and CPI, via a flexible glycine linker (G4S) by overlap-extension PCR. The fusion protein was characterized for its therapeutic abilities toward CPI and CPB toxin neutralizations. The r-Cpib was found to be non-toxic and could competitively inhibit binding of CPB to host cell receptors thereby reducing its cytotoxicity. Immunization of mice with r-Cpib generated specific antibodies capable of neutralizing the above toxaemias both in vitro and in vivo. Caco-2 cells exposed to a mixture of anti-r-Cpib sera and native CPI or CPB, displayed significantly superior protection against the respective toxins while passive challenge of mice with a similar mixture resulted in 83 and 91% protection against CPI and CPB respectively. Alternatively, mice exposed to a mixture of sham sera and native toxins died within 2-3 days. This work thus demonstrates r-Cpib as a novel bivalent fusion protein capable of efficient immunotherapy against C. perfringens CPI and CPB toxaemia.
authors.affiliation: Division of Microbiology, Defence Food Research Laboratory, Siddartha Nagar, Mysore 570011, India.
authors.forename: Shreya
authors.initials: S
authors.lastname: Das
authors.affiliation: Division of Microbiology, Defence Food Research Laboratory, Siddartha Nagar, Mysore 570011, India.
authors.forename: Saugata
authors.initials: S
authors.lastname: Majumder
authors.affiliation: Division of Microbiology, Defence Food Research Laboratory, Siddartha Nagar, Mysore 570011, India. Electronic address: joseph@dfrl.drdo.in.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Kingston
authors.affiliation: Division of Microbiology, Defence Food Research Laboratory, Siddartha Nagar, Mysore 570011, India.
authors.forename: Harsh V
authors.initials: HV
authors.lastname: Batra
mesh_terms: D036002:ADP Ribose Transferases / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000276:immunology*; D015153:Blotting, Western; D018938:Caco-2 Cells; D003015:Clostridium Infections / Q000276:immunology*; D003016:Clostridium perfringens; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000276:immunology*; D014115:Toxemia / Q000276:immunology*
chemical_list: D001427:Bacterial Toxins; C031884:CPB protein, Clostridium perfringens; D011993:Recombinant Fusion Proteins; C050737:iota toxin, Clostridium perfringens; D036002:ADP Ribose Transferases
keywords: Bivalent fusion protein r-Cpib; Clostridium perfringens beta toxin; Immuno therapy; Iota toxin; Toxin neutralization