Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth

Abstract

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite β-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or “camelization” of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs.

Treatment of Microsporidium Nosema bombycis Spores with the New Antiseptic M250 Helps to Avoid Bacterial and Fungal Contamination of Infected Cultures without Affecting Parasite Polar Tube Extrusion

“…Thus, the novel antiseptic M250 at a concentration not exceeding 0.1% can be successfully applied for the treatment of microsporidian spores non-sterilely isolated from infected host animals to establish long-term cell cultures infected with these parasites. In our recent studies, we used 0.1% M250 to sterilize Vairimorpha (Nosema) ceranae and N. bombycis spores obtained from artificially infected insects, and these treatments did not affect the intracellular growth of either parasite [ 52 , 53 , 54 ].…”

A monoclonal antibody targeting spore wall protein 1 inhibits the proliferation of Nosema bombycis in Bombyx mori

“…Our previous study on the scFvG4 showed potent inhibitory effects against microsporidia in the cellular level ( 38 , 39 ). Recently, the scFv antibodies against the outer loops of N. bombycis ATP/ADP-transporters could efficiently inhibit the parasite growth in Sf9 cell line ( 40 ). In the current study, we used the spore germination fluid proteins of N. bombycis as immunogens to prepare monoclonal antibody.…”

MicrosporidianNosema bombycissecretes serine protease inhibitor to suppress host cell apoptosisviacaspase BmICE

“…The copyright holder for this preprint this version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600942 doi: bioRxiv preprint silkworms from infection with N. bombycis. Previous studies have shown that the transgenic expression of monoclonal antibody single-chain variable fragments (scFvs) targeting N. bombycis protein in silkworm or host cell lines reduced N. bombycis infection and proliferation [56][57][58]. As host cell apoptosis promotes the clearing of infected cells and reduces the load of N. bombycis, we can express monoclonal antibody scFv against NbSPN14 in silkworm to block the N. bombycis serpin inhibiting host cell apoptosis; as a result, the resistance of the host silkworm to N. bombycis will enhance.…”