Abstract
Botulinum neurotoxins (BoNTs) represent a family of bacterial toxins responsible for neuroparalytic disease ‘botulism’ in human and animals. Their potential use as biological weapon led to their classification in category ‘A’ biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. In present study, gene encoding full length catalytic domain of BoNT/E-LC was cloned, expressed and protein was purified using Ni–NTA chromatography. Humoral immune response was confirmed by Ig isotyping and cell-mediated immunity by cytokine profiling and intracellular staining for enumeration of IFN-γ secreting CD4+ and CD8+ T cells. Increased antibody titer with the predominance of IgG subtype was observed. An interaction between antibodies produced against rBoNT/E-LC was established that showed the specificity against BoNT/E in SPR assay. Animal protection with rBoNT/E-LC was conferred through both humoral and cellular immune responses. These findings were supported by cytokine profiling and flow cytometric analysis. Splenocytes stimulated with rBoNT/E-LC showed a 3.27 and 2.8 times increase in the IFN-γ secreting CD4+ and CD8+ T cells, respectively; in immunized group (P < 0.05). Protection against BoNT/E challenge tended to relate with increase in the percentage of rBoNT/E-LC specific IL-2 in the splenocytes supernatant (P = 0.034) and with IFN-γ-producing CD4+ T cell responses (P = 0.045). We have immunologically evaluated catalytically active rBoNT/E-LC. Our results provide valuable investigational report for immunoprophylactic role of catalytic domain of BoNT/E.
Highlights
Botulinum neurotoxins (BoNTs) represent a family of bacterial toxins responsible for neuroparalytic disease ‘botulism’ in human and animals
Purified inclusion bodies were solubilized in 6 M GuHCl and after binding to Ni– NTA the effort was proceeded for on-column renaturation by washing with reducing concentration of GuHCl
This study is the first report of complete immunological profiling induced by light chain (LC) of BoNT/E
Summary
Botulinum neurotoxins (BoNTs) represent a family of bacterial toxins responsible for neuroparalytic disease ‘botulism’ in human and animals Their potential use as biological weapon led to their classification in category ‘A’ biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Human-derived immune globulins, BabyBIG is being supplied by the California Department of Public Health (CDPH)[16,17] These post-exposure antitoxin therapies have several drawbacks owing to their side effects due to hypersensitivity and are effective towards the extracellular toxin but are unable to neutralize internalized toxin[16]. Prevention can be achieved by vaccination that generates neutralizing antibodies against botulinum neurotoxin These toxoids based vaccine made from purified BoNTs. mass application necessitates the large-scale production of BoNTs, that require high containment facility and direct handling of toxins which imposes risk for workers. To overcome all these problems, continuous efforts being made toward the development of vaccines based on DNA and recombinant proteins that exhibit lesser side effects[25]
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