Development And Identification Of Human Anti-H5N1Avian Influenza Virus ScFv Antibody Having Broad Neutralization And Mapping Of The Conserved Neutralizing Epitope

Avian influenza is a highly contagious disease of birds caused by A influenza viruses. The circulation in humans by the highly pathogenic H5N1avian flu in the past few years, with either high mortality or morbidity, have caused most pandemics and have heightened fear that the next influenza pandemic is due. Lacking of the antibodies against H5N1in human, there is a higher mortality and now still poses threats to public health. Vaccination undoubtedly is the principal strategy for prevention and control of influenza. However, the current annual vaccine against seasonal influenza has limitations, in that the vaccine only protects those at risk from the viruses circulating at the time; and prediction of the next potential pandemic virus subtype is guesswork. Chemical-based therapeutic agents with broad antiviral spectrum have been clinically used, and they exert their activity by either blocking the M2ion channel, interfering with viral transcription and genome replication, or by inhibiting the NA function. However, the excess use has been associated with the generation of resistant viruses, and its application is now restricted. Another possible effective countermeasure against influenza is a resurgence of antibody-based therapy. This has been fuelled by the reports that people survived severe influenza infection by transfusion of convalescent plasma. Humanized or human antibodies could be used as an efficient anti-virus agent in clinical therapy.A human immune single chain Fv fragment(scFv) phage-display library was constructed and scFv antibodies that have broad neutralizing effect were selected from the library. The scFv antibodies could be used in therapies to counter the H5N1virus, and the conserved epitope could be the key point for the design and implementation of vaccines with H5N1viruses.Methods1. Construction of specific phage antibody library Lymphocytes were isolated from normal healthy H5N1vaccinated individuals peripheral blood cells. RT-PCR amplified Vh and VL genes and scFv was spliced by overlap-PCR and were ligated with phagemid vector pComb3XSS. A scFv phage display library was constructed after the ligations were introduced into competent E. coli XL1-Blue cells by electroporation. The library was rescued and amplified, using VCSM13helper phages.2. Expression of H5N1recombinant HA1(rHAl) protein in Bac-to-Bac baculovirus expression systemThe total RNA was isolated and HA1gene amplified by RT-PCR. The amplified products were subcloned into plasmid pFastBac and transformed into E.Coli DH10Bac competent cells. The positive recombinant bacmid was isolated and was transfected into Sf9cells. Proteins were detected by Western blot. The rHA1were purified by an affinity chromatography column and identified by SDS-PAGE and tandem mass spectrography.3. Selection of rHA1specific clones from the scFv libraryFor phage bio-panning, the recombinant HA1coated onto a microtiter ELISA plate. Amplified phage mixtures were then incubated with the coated wells. Following three rounds of panning, bacterial clones were selected from the overnight plates. The positive scFv sequence were expressed and purified with HisTrap FF crude column.4. Microneutralization assay and Epitope mapping of HAScFv antibodies that have neutralizing effect were selected using microneutralization assays based on MDCK, western blot, immunofluorescence assays and hemagglutination-inhibition assay. HA epitopes were identified by the phage peptide display approach, using the Ph.D.-12library, three dimensional (3D) models and amino acid mutant assay.5. Prophylactic and therapeutic of H5N1viruses in embryonated chicken eggsGroups of embryonated chicken eggs were used in all experiments. For the prophylactic groups, the eggs were given several treatment doses (25,50,75,100and200μg/kg) of the scFv antibodies,0.5h prior to the viral challenge (10LD50). For therapeutic treatment, the scFv antibodies were employed after the eggs were infected by viruses (10LD50)1h later, and several doses of the antibody (100,150,200,250and500μg/kg) were included for the therapeutic experiment.The survival rates of the embryos were recorded until post-inoculation day8.Results1. After RNA extraction, RT-PCR, and gene splicing by overlap PCR, about750bp single chain Fv fragment(scFv) gene fragments were generated and ligated with the phagemid and were transformed into competent cells. A phage display library containing6.0×108clones was constructed successfully.2. The full-length gene sequence encoding HA1of the A/Jiangsu/1/2007(H5N1) was amplified and successfully subcloned to the pFastBac vector. The recombinant plasmids were shown two bands of pFastBac about4.7kb and HA1about1.0kb by restriction enzyme digestion. The Sf9cells were infected with the recombinant bacmid DNA virus at10MOI. The western blotting showed that the recombinant protein was presented both in the cell culture supernatant and in the cell pellets. The purified protein was analyzed by SDS-PAGE showed a43kD band. The best matching protein was HA A/Jiangsu/1/2007(H5N1) by searching sequence databases using mass spectrometry, this can confirm the recombinant protein required.3. The purified HA1was used as the antigen for the selection of phage clones displaying HA1-bound scFv. Following three rounds of the phage panning,25clones were high positive against recombinant HA1by phage-ELISA. The9unique scFv sequences were expressed and purified with HisTrap FF crude column.4. In the neutralization assay, all9purified scFv antibodies were tested for their neutralizing efficacy. Four of the antibodies (3D1,3F5,4F5and6F9) were able to broad neutralize all of the6viruses and the IC50was from0.39μg/ml to45μg/ml.When detected with scFv antibodies, all6viruses showed70kD and43kD protein band, which is the predicted the full length HA protein and HA1protein. This confirms that the binding site of the scFv antibodies were located at the HA1domain. The nucleuses of MDCK cells infected by6viruses were colored purple, and red around the nucleus. All the4scFv antibodies showed HI activity to the6viruses, the HI has a positive correlation with neutralization concentration IC50, which also further confirms that the binding site of the scFv antibodies were located at the HA1domain.The4scFv antibodies have the same HCDRs and the competitive ELISA results indicted that the4scFv antibodies were binding to the same unique site of the HA.5. Dodecamer mimotope peptides recognized by these4antibodies were screened from Ph.D-12libraries. Twenty clones were randomly isolated and18sequences were shown to share a common sequence, WLLP, homologous to WLLGNP in the amino acid sequence of HA. The putative binding site was predicted using3D structure and the target binding site was localized in the E epitope region. When the mutant HA1protein was probed with the scFv antibody, there was no specific band, This further verified that the peptide WLLGNP was the binding site for the scFv antibodies.6. The antibody had a complete100%protection effect on the H5N1viruses in avian host in the prophylactic and therapeutic groups. The100%preventive protection effect could be reached when challenged with H5N1viruses in human host in the prophylactic groups, and there is also a62.5%protection effect with H5N1viruses in human host in the therapeutic groups. Conclusion1. A human immune scFv phage-display library containing6.0×10clones was constructed successfully.2. Four scFv antibodies that have broad neutralizing effect on both clade2and clade9H5N1viruses were selected from the library. The conserved peptide (76)WLLGNP(81) could be the key point for the design and implementation of vaccines with H5N1viruses.3.The scFv antibody had a satisfied protection effect on the H5N1viruses in the prophylactic and therapeutic groups on H5N1influenza A inviruses infection in an embryonated chicken eggs model.Thereby, the study suggests that the scFv antibodies in our study could be used in therapies to counter the H5N1virus.