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A high-affinity CDR-grafted antibody against influenza A H5N1 viruses recognizes a conserved epitope of H5 hemagglutinin.

Xiong F, Xia L, Wang J, Wu B, Wang D, Yuan L, Cheng Y, Zhu H, Che X, Zhang Q, Zhao G, Wang Y - PLoS ONE (2014)

Bottom Line: While vaccines and antiviral drugs are currently under development, neutralizing antibodies could offer an alternative strategy to prevent and treat H5N1 virus infection.Our results for the first time reported a humanized antibody against H5N1 viruses by CDR grafting method.With the expected lower immunogenicity, this humanized antibody was expected to be more efficacious than murine or human-mouse chimeric antibodies for future application in humans.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Technology, Tongji University, Shanghai, China ; Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, China.

ABSTRACT
Highly pathogenic avian influenza (HPAI) H5N1 virus infection is still a potential threat to public health worldwide. While vaccines and antiviral drugs are currently under development, neutralizing antibodies could offer an alternative strategy to prevent and treat H5N1 virus infection. In the present study, we had developed a humanized antibody against H5N1 viruses from mouse-derived hybridoma in order to minimize its immunogenicity for potential clinical application. The humanized antibody hH5M9 was generated by transferring the mouse complementarity determining region (CDR) residues together with four key framework region (FR) residues onto the FR of the human antibody. This humanized antibody exhibited high affinity and specificity comparable to the parental mouse or chimeric counterpart with broad and strong neutralization activity against all H5N1 clades and subclades except for Egypt clades investigated. Furthermore, through epitope mapping we identified a linear epitope on the top region of hemagglutinin (HA) that was H5N1 specific and conserved. Our results for the first time reported a humanized antibody against H5N1 viruses by CDR grafting method. With the expected lower immunogenicity, this humanized antibody was expected to be more efficacious than murine or human-mouse chimeric antibodies for future application in humans.

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Epitope mapping of hH5M9.(A) Western blotting analysis of hH5M9 with HA. Purified A/Anhui/1/05 HA was applied to SDS-PAGE under reducing conditions. The hH5M9 was used as primary antibody. A rabbit polyclonal antibody against A/Anhui/1/05 was used as positive control (PC). (B) Binding analysis of synthesized epitope “KPNDAINF” with hH5M9 was measured by an indirect ELISA. (C) Schematic representation of the epitope recognized by hH5M9. The linear epitope (KPNDAINF, amino acids 234–241) on the three dimensional structure of the H5 HA (VN1194) mono-structure (PDB No. 2IBX) was colored in green while the receptor binding domain (RBD) was highlighted in yellow. (D) Cartoon illustration of the three dimensional structure of the linear epitope for hH5M9.
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pone-0088777-g003: Epitope mapping of hH5M9.(A) Western blotting analysis of hH5M9 with HA. Purified A/Anhui/1/05 HA was applied to SDS-PAGE under reducing conditions. The hH5M9 was used as primary antibody. A rabbit polyclonal antibody against A/Anhui/1/05 was used as positive control (PC). (B) Binding analysis of synthesized epitope “KPNDAINF” with hH5M9 was measured by an indirect ELISA. (C) Schematic representation of the epitope recognized by hH5M9. The linear epitope (KPNDAINF, amino acids 234–241) on the three dimensional structure of the H5 HA (VN1194) mono-structure (PDB No. 2IBX) was colored in green while the receptor binding domain (RBD) was highlighted in yellow. (D) Cartoon illustration of the three dimensional structure of the linear epitope for hH5M9.

Mentions: To anchor the epitope recognized by hH5M9, two fragments of HA, HA1 and HA2, were first prepared and adapted for epitope mapping. Through western blotting analysis, it was demonstrated that hH5M9 antibody reacted with the viral HA1 protein, rather than HA2. Considering the denaturation of HA1 during the western blotting analysis, the epitope targeted by hH5M9 appeared to be linear rather than conformational (Figure 3A). According to our above results that hH5M9 displayed broad cross-reactivity to H5N1 HAs but not A/Egypt/N05056/09 (Table 2), we first aligned HA1 amino acid sequences of these nine H5N1 isolates used in the binding assay to find out the unique amino acid residues of HA1 of A/Egypt/N05056/09 (Table 3). We found that Arg22, Asn120, Thr151, Gln152, Asp154, Ile210 and Ser235 were unique to A/Egypt/N05056/09, which inferred these seven amino acids were the candidates for antigenic epitope according to the previous study [14].


A high-affinity CDR-grafted antibody against influenza A H5N1 viruses recognizes a conserved epitope of H5 hemagglutinin.

Xiong F, Xia L, Wang J, Wu B, Wang D, Yuan L, Cheng Y, Zhu H, Che X, Zhang Q, Zhao G, Wang Y - PLoS ONE (2014)

Epitope mapping of hH5M9.(A) Western blotting analysis of hH5M9 with HA. Purified A/Anhui/1/05 HA was applied to SDS-PAGE under reducing conditions. The hH5M9 was used as primary antibody. A rabbit polyclonal antibody against A/Anhui/1/05 was used as positive control (PC). (B) Binding analysis of synthesized epitope “KPNDAINF” with hH5M9 was measured by an indirect ELISA. (C) Schematic representation of the epitope recognized by hH5M9. The linear epitope (KPNDAINF, amino acids 234–241) on the three dimensional structure of the H5 HA (VN1194) mono-structure (PDB No. 2IBX) was colored in green while the receptor binding domain (RBD) was highlighted in yellow. (D) Cartoon illustration of the three dimensional structure of the linear epitope for hH5M9.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928294&req=5

pone-0088777-g003: Epitope mapping of hH5M9.(A) Western blotting analysis of hH5M9 with HA. Purified A/Anhui/1/05 HA was applied to SDS-PAGE under reducing conditions. The hH5M9 was used as primary antibody. A rabbit polyclonal antibody against A/Anhui/1/05 was used as positive control (PC). (B) Binding analysis of synthesized epitope “KPNDAINF” with hH5M9 was measured by an indirect ELISA. (C) Schematic representation of the epitope recognized by hH5M9. The linear epitope (KPNDAINF, amino acids 234–241) on the three dimensional structure of the H5 HA (VN1194) mono-structure (PDB No. 2IBX) was colored in green while the receptor binding domain (RBD) was highlighted in yellow. (D) Cartoon illustration of the three dimensional structure of the linear epitope for hH5M9.
Mentions: To anchor the epitope recognized by hH5M9, two fragments of HA, HA1 and HA2, were first prepared and adapted for epitope mapping. Through western blotting analysis, it was demonstrated that hH5M9 antibody reacted with the viral HA1 protein, rather than HA2. Considering the denaturation of HA1 during the western blotting analysis, the epitope targeted by hH5M9 appeared to be linear rather than conformational (Figure 3A). According to our above results that hH5M9 displayed broad cross-reactivity to H5N1 HAs but not A/Egypt/N05056/09 (Table 2), we first aligned HA1 amino acid sequences of these nine H5N1 isolates used in the binding assay to find out the unique amino acid residues of HA1 of A/Egypt/N05056/09 (Table 3). We found that Arg22, Asn120, Thr151, Gln152, Asp154, Ile210 and Ser235 were unique to A/Egypt/N05056/09, which inferred these seven amino acids were the candidates for antigenic epitope according to the previous study [14].

Bottom Line: While vaccines and antiviral drugs are currently under development, neutralizing antibodies could offer an alternative strategy to prevent and treat H5N1 virus infection.Our results for the first time reported a humanized antibody against H5N1 viruses by CDR grafting method.With the expected lower immunogenicity, this humanized antibody was expected to be more efficacious than murine or human-mouse chimeric antibodies for future application in humans.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Technology, Tongji University, Shanghai, China ; Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, China.

ABSTRACT
Highly pathogenic avian influenza (HPAI) H5N1 virus infection is still a potential threat to public health worldwide. While vaccines and antiviral drugs are currently under development, neutralizing antibodies could offer an alternative strategy to prevent and treat H5N1 virus infection. In the present study, we had developed a humanized antibody against H5N1 viruses from mouse-derived hybridoma in order to minimize its immunogenicity for potential clinical application. The humanized antibody hH5M9 was generated by transferring the mouse complementarity determining region (CDR) residues together with four key framework region (FR) residues onto the FR of the human antibody. This humanized antibody exhibited high affinity and specificity comparable to the parental mouse or chimeric counterpart with broad and strong neutralization activity against all H5N1 clades and subclades except for Egypt clades investigated. Furthermore, through epitope mapping we identified a linear epitope on the top region of hemagglutinin (HA) that was H5N1 specific and conserved. Our results for the first time reported a humanized antibody against H5N1 viruses by CDR grafting method. With the expected lower immunogenicity, this humanized antibody was expected to be more efficacious than murine or human-mouse chimeric antibodies for future application in humans.

Show MeSH
Related in: MedlinePlus