First contribution to water buffalo (Bubalus bubalis) type-I/III interferons – induced Mx1 detection and anti-viral activity
Myxovirus resistance protein 1 (Mx1) is a type I/III interferons-induced protein conferring the hosts an antiviral property against several viruses. The protein is also found to be a potential biomarker for early detection of viral infections in many vertebrates. In humans, MxA and MxB proteins have been studied intensively for decades and it has been found that the normal concentration of MxA protein in peripheral blood of healthy people is less than 50 ng/mL, that it is induced very shortly (1-2 hours) after exposure to viral infections and that it displays a half-life of 2-3 days. Human MxA protein in peripheral blood of patients has been shown to be clinically sensitive and specific biomarker for viral infections. The blood MxA concentration is increased specifically by infections by Rhinoviruses, respiratory syncytial viruses, parainfluenza viruses, influenza viruses, coronaviruses, and human metapneumoviruses. In human medicine, MxA protein has been used successfully as a specific biomarker for distinguishing viral to bacterial infections, providing good guidance for diagnostic and prescription of antibiotics. Several studies have been conducted on Mx1 protein of many livestock including pig, dog, horse and cow. However, there is no research conducted on the proteins Mx of water buffalo, even if this valuable ruminant, which is vulnerable to several dangerous viral diseases, has been increasingly economically important in Asia.
In an attempt to develop a new, effective, cheap and rapid diagnostic tool, which can be used effectively for the control of viral diseases by screening for elimination of domestic water buffalo carrying viruses including asymptomatic individuals, in the present study, we initially succeeded in production and characterization of recombinant buffalo (Bubalus bubalis) Mx1 protein (bbMx1) and a series of monoclonal antibodies specific to the bbMx1 protein.
In order to produce recombinant bbMx1 protein, two vectors were synthesized for expressing a reference buffalo Mx1 either in Escherichia coli or in mammalian cells. In both cases, the recombinant water buffalo Mx1 was intendedly fused to a N-term functional group with either a poly-histidine tract or a V5 epitope. In the latter, the V5-bbMx1 protein was further fused to a N-term bipartite group consisting of the fluorescent marker protein RFP followed by the spontaneously cleavable peptide 2A. The recombinant bbMx1 was expressed successfully in both E. coli and mammalian cells by transient transfection using Lipofectamin 3,000 reagent. SDS-PAGE analysis of cells lysate from transfected cell population and poly (I:C)-incubated buffalo cells illustrated of bbMx1 protein with molecular weight of almost 75kDa.
The hybridomas expressing antibodies specific to bbMx1 were established by fusion of SP2/0myeloma cells with spleenocytes derived from female BALB/c mice, which had been hyper-immunized with the recombinant V5-bbMx1 protein. A set of 10 mouse hybridomas producing mAbs to bbMx1 was obtained. The 10 corresponding mAbs were further characterized using indirect ELISA, Western blot analysis and immunocytofluorescent staining.
Eight mAbs, designated 11C7, RD5, NF3, 9D1, FD4, PE6, 11A7 and 10F5, displayed binding abilities and specificity to recombinant bbMx1 in the three formats. Moreover, combining NF3 (for capture) with RD5 (for detection), 9D1 with RD5, FD4 with 11C7 or PE6 with 11C7 generated a strong signal in a prototype sandwich-ELISA. In addtion, mAbs RD5 and 11A7 also illustrated to bind specifically to wildtype bbMx1 protein, which was expressed by bubaline poly-I/C-stimulated mesenchymal stem cells in immunoblotting format. Interestingly, it was also observed that there were cross reactions between mAbs and recombinant proteins among species. NF3, which was a specifically designed mAb targeting bovine Mx1 protein, bound specifically to buffalo Mx1 protein too. Meanwhile, 10B4, 11A7, 11C7 and 9D1, the mAbs specific to buffalo Mx1 protein also showed binding abilities to recombinant porcine Mx1 protein in western blot tests. The results suggest that the mAbs developed and characterized here provide an excellent starting point for developing diagnostic tools aimed at detecting viral infections in the water buffalo, whatever using immunoblotting, immunocytostaining, flow cytometry or sandwich-ELISA.
Furthermore, type I/III interferons provide powerful and universal innate intracellular defense mechanisms against viruses. Among the antiviral effectors induced, Mx proteins of some species appear as key components of defense against influenza A viruses. It is expected that such an antiviral protein must display a platform dedicated to the recognition of said viruses. In my thesis, the anti-influenza activity of bbMx1 and other four distinct mammalian Mx1 proteins was measured by comparing the number of viral nucleoprotein-positive cells 7 hours after infection in a sample of 100,000 cells expected to contain both Mx1-positive and Mx1-negative cell subpopulations. The systematic depletion (p < 0.001) of virus nucleoprotein-positive cells among equine, bubaline, porcine and bovine Mx1-expressing cell populations compared to Mx-negative cells suggests a strong anti-influenza A activity. Looking for common anti-influenza signature elements in the sequence of these Mx proteins, we found that an aromatic residue at positions 561 or 562 in the L4 loop seems critical for the anti-influenza function and/or specificity of mammalian Mx1.
Từ khóa: Bubalus bubalis, myxovirus resistance protein_1, water bufalo, monocnonal antibodies, water bufalo
PHAI DAM VAN
Vietnam National University of Agriculture