Monoclonal antibody targeting complement C9 binding domain of Trichinella spiralis paramyosin impairs the viability of Trichinella infective larvae in the presence of complement

Abstract

BackgroundTrichinella spiralis expresses paramyosin (Ts-Pmy) not only as a structural protein but also as an immunomodulator that inhibits host complement as a survival strategy. Previous studies demonstrated that Ts-Pmy bound to complement components C8 and C9 and inhibited the polymerization of C9 during the formation of the membrane attack complex (MAC). The C9 binding domain of Ts-Pmy was identified within 14 amino acid residues at the C-terminus of Ts-Pmy. The production of a monoclonal antibody that specifically targets the C9 binding site is necessary for further studies of Ts-Pmy function and may be used as a therapeutic agent for T. spiralis infection.MethodsIn this study, a monoclonal antibody against the complement C9 binding domain of Ts-Pmy (mAb 9G3) was produced using hybridoma technology. The binding activity of the mAb produced for recombinant or native Ts-Pmy and the blockade of Ts-Pmy binding to C9 by the mAb were assessed by Western blot analysis. The effect of the mAb on the viability of T. spiralis was observed by co-incubation of T. spiralis with mAb 9G3 in the presence of complement in vitro and by passive transfer of the mAb into naive mice following T. spiralis larval challenge.ResultsmAb 9G3 was successfully produced against the C9 binding domain of Ts-Pmy and bound specifically not only to recombinant Ts-Pmy but also to native Ts-Pmy expressed in different stages of T. spiralis, including adult worms, newborn larvae and muscle larvae. The binding of mAb 9G3 to Ts-Pmy efficiently blocked the binding of Ts-Pmy to human complement C9, resulting in a significant increase in the complement-mediated killing of newborn larvae in vitro and reduced infectivity of T. spiralis larvae in mice passively transferred with the mAb.ConclusionsmAb 9G3 is a specific antibody that binds to the C9 binding domain of Ts-Pmy and interferes with Ts-Pmy’s complement-binding activity. Therefore, this mAb is a protective antibody that has potential as a preventive and therapeutic agent for T. spiralis infection.

Protective immunity against Trichinella spiralis infection induced by TsNd vaccine in mice

“…An effective vaccine usually requires more than one immunization in the form of a prime-boost. In addition, the plasmid DNA is most commonly delivered by intramuscular inoculation or via the gene gun bombardment [ 7 ], but the later is practically inconvenient in terms of cost and simplicity. It was also reported that the intramuscular route was the best one among five delivery routes, including oral administration [ 34 ].…”

“…Trichinellosis is not only a public health hazard but also an economic problem in porcine animal production and food safety [ 6 ]. Due to the predominantly zoonotic importance of Trichinella infection, the development of vaccines capable of preventing swine from becoming infected is a promising measure for control of trichinellosis [ 7 - 9 ].…”

A Simple and Rapid Staining Technique for Sex Determination of Trichinella Larvae Parasites by Confocal Laser Scanning Microscopy

“…In studies of Trichinella genus, CLSM mainly was used to examine various execratory and secretory antigens (Li et al, 1999), immune response (Bai et al, 2012), or interaction between T. spiralis and muscle cells in vivo (Bai et al, 2011) and was used for various experiments where the pathological and/or therapeutic effects were evaluated (Li et al, 1999; Masetti et al, 2004; Hao et al, 2014). However, a structure investigation by Murata et al (1998) of Trichinella adults and larvae by CLSM showed stichocyte and their nuclear distribution and sizes.…”

Trichinella spiralis Paramyosin Binds Human Complement C1q and Inhibits Classical Complement Activation

“…Crude adult worm antigens were prepared from homogenized worm extracts based on a previously described protocol [ 18 ]. The anti- Ts -Pmy monoclonal antibody (mAb) 9G3 that specifically recognized Ts -Pmy was previously produced [ 19 ]. Recombinant T s-Pmy (r Ts -Pmy) with a His-tag at the C-terminus was expressed in baculovirus/insect cells (Invitrogen, USA) and purified by Ni-affinity chromatography (Qiagen, USA).…”

“…The C9 binding site on Ts -Pmy was determined to be located within the C-terminus of the protein (between 866 Val and 879 Met) [ 18 ]. A monoclonal antibody (mAb 9G3) targeting the binding site could block the binding of Ts -Pmy to human C9, resulting in a significant increase in the complement-mediated killing of newborn larvae of the parasite in vitro [ 18 , 19 ].…”