No abstract

Transmission blocking monoclonal antibodies, 4B7 and 1245, targeting Pfs25, have been demonstrated to block the developmental stages of the malarial parasite inside the mosquitoes. In this work, anti-plasmodium antibody fragments of 4B7 and 1245, designated as diabodies, were expressed in bacteria and refolded using a standardized method, with a yield of 0.1 and 0.4 mg per litre of culture, respectively. Dimeric species was detected for 4B7-Db, but not for 1245-Db, using glutaraldehyde cross-linking experiment. The quality of the purified refolded fraction was assessed with differential scanning fluorimetry (DSF). Refolded 4B7-Db, with a melting temperature of 59 degrees C, recognized Pfs25 expressed on the surface of ookinete and zygote stages of Plasmodium in an immunofluorescence-based assay, whereas, 1245-Db, exhibiting a skewed melt profile, showed weak recognition. Further, refolded 4B7-Db recognized the linear epitope, on purified Pfs25 protein, both in the denatured and native state. Differential scanning fluorimetry can be potentially employed as a qualitative measure of functionality, to evaluate refolded proteins, with applicability for antibody engineering in passive immunization.

No abstract

Kyasanur forest disease is a neglected zoonotic disease caused by a single stranded RNA based flavivirus, the incidence of which, was first recorded in 1957, in the Southern part of India. Kyasanur forest disease virus is transmitted to monkeys and humans, through the infected tick bite of Haemophysalis spinigera. Kyasanur forest disease is a febrile illness, which in severe cases, results in neurological manifestations leading to mortality. The current treatment regimens are symptomatic and supportive in nature. There are no targeted therapies available for this disease. In this study, we evaluated the ability of the FDA approved drug, Sofosbuvir to inhibit the RNA dependent RNA polymerase activity of NS5 protein from Kyasanur forest disease virus. NS5 protein containing the N-terminal methyl transferase domain and C-terminal RNA dependent RNA polymerase domain was expressed in Escherichia coli and RNA dependent RNA polymerase activity, demonstrated with the purified protein. The RNA dependent RNA polymerase assay conditions were optimized, followed by determination of apparent Km,ATP. IC50 of Sofosbuvir against RNA dependent RNA polymerase activity was determined under the optimized conditions with the purified NS5 protein. Drug repurposing strategies can accelerate the development of targeted therapies for Kyasanur forest disease. This is the first demonstration of inhibition of RNA dependent RNA polymerase activity of Kyasanur forest disease virus with a small molecule inhibitor.