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A critical HA1 neutralizing domain of H5N1 influenza in an optimal conformation induces strong cross-protection.

Du L, Zhao G, Sun S, Zhang X, Zhou X, Guo Y, Li Y, Zhou Y, Jiang S - PLoS ONE (2013)

Bottom Line: Consequently, the design of an effective and safe anti-H5N1 vaccine is essential.We then constructed three additional recombinants fused with Fd plus His tag (HA-13-263-Fd-His), Fc only (HA-13-263-Fc), and His tag only (HA-13-263-His), respectively.These results suggest that the oligomeric form of the CND containing the RBD can be further developed as an effective and safe vaccine for cross-protection against divergent strains of H5N1 viruses.

View Article: PubMed Central - PubMed

Affiliation: Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, United States of America.

ABSTRACT
The highly pathogenic avian influenza (HPAI) H5N1 viruses, especially the laboratory-generated H5N1 mutants, have demonstrated the potential to cross the species barrier and infect mammals and humans. Consequently, the design of an effective and safe anti-H5N1 vaccine is essential. We previously demonstrated that the full-length hemagglutinin 1 (HA1) could induce significant neutralizing antibody response and protection. Here, we intended to identify the critical neutralizing domain (CND) in an optimal conformation that can elicit strong cross-neutralizing antibodies and protection against divergent H5N1 strains. We thus constructed six recombinant proteins covering different regions of HA1 of A/Anhui/1/2005(H5N1), each of which was fused with foldon (Fd) and Fc of human IgG. We found that the critical fragment fused with Fd/Fc (HA-13-263-Fdc, H5 numbering) that could elicit the strongest neutralizing antibody response is located in the N-terminal region of HA1 (residues 13-263), which covers the receptor-binding domain (RBD, residues 112-263). We then constructed three additional recombinants fused with Fd plus His tag (HA-13-263-Fd-His), Fc only (HA-13-263-Fc), and His tag only (HA-13-263-His), respectively. We found that the HA-13-263-Fdc, which formed an oligomeric conformation, induced the strongest neutralizing antibody response and cross-protection against challenges of two tested H5N1 virus strains covering clade 1: A/VietNam/1194/2004 (VN/1194) or clade 2.3.4: A/Shenzhen/406H/06 (SZ/406H), while HA-13-263-Fc dimer and HA-13-263-Fd-His trimer elicited higher neutralizing antibody response and protection than HA-13-263-His monomer. These results suggest that the oligomeric form of the CND containing the RBD can be further developed as an effective and safe vaccine for cross-protection against divergent strains of H5N1 viruses.

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Detection of conformation of HA-13–263 proteins by native PAGE (N-PAGE) and cross-linker analysis.(A) N-PAGE analysis of expressed HA-13–263 proteins, followed by Coomassie Blue staining and Western blot (B), by using a HA1-specific mAb. The native protein molecular weight marker (kDa) (Invitrogen) is indicated on the left. (C) Cross-linker analysis followed by SDS-PAGE and Coomassie Blue staining of expressed HA-13–263 proteins (with cross-linker). The proteins without cross-linking (w/o cross-linker) were used as the controls. The protein molecular weight marker (kDa) (Invitrogen) is indicated on the left.
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pone-0053568-g005: Detection of conformation of HA-13–263 proteins by native PAGE (N-PAGE) and cross-linker analysis.(A) N-PAGE analysis of expressed HA-13–263 proteins, followed by Coomassie Blue staining and Western blot (B), by using a HA1-specific mAb. The native protein molecular weight marker (kDa) (Invitrogen) is indicated on the left. (C) Cross-linker analysis followed by SDS-PAGE and Coomassie Blue staining of expressed HA-13–263 proteins (with cross-linker). The proteins without cross-linking (w/o cross-linker) were used as the controls. The protein molecular weight marker (kDa) (Invitrogen) is indicated on the left.

Mentions: Since the molecular weight shown in reducing SDS-PAGE would not represent the actual size of the tested proteins, we next sought to detect the conformation of these proteins using non-reducing N-PAGE by removing the reducing agents in both sample and running buffers. As indicated in Fig. 5A, clear bands with high molecular weight were detected in HA-13–263-Fdc, HA-13–263-Fc and HA-13–263-Fd-His proteins, with HA-13–263-Fdc showing the highest molecular weight oligomer, while no clear band was shown in HA-13–263-His protein. Similarly, these bands were detected by a HA1-specific mAb in Western blot (Fig. 5B), indicating their high specificity to the HA1 protein. Comparison of the molecular weight from samples with and without cross-linker showed that the molecular weights of the cross-linker samples for HA-13–263 with Fd and/or Fc were about 1-, 2- or 3-fold higher than that of the corresponding without-cross-linker samples, but the molecular weight of the cross-linker HA-13–263-His was almost the same as that of the without-cross-linker HA-13–263-His (Fig. 5C). These results suggest that the identified neutralizing HA-13–263 fragment fused with Fd and/or Fc tags was able to form conformational structures of dimer, trimer, or oligomer.


A critical HA1 neutralizing domain of H5N1 influenza in an optimal conformation induces strong cross-protection.

Du L, Zhao G, Sun S, Zhang X, Zhou X, Guo Y, Li Y, Zhou Y, Jiang S - PLoS ONE (2013)

Detection of conformation of HA-13–263 proteins by native PAGE (N-PAGE) and cross-linker analysis.(A) N-PAGE analysis of expressed HA-13–263 proteins, followed by Coomassie Blue staining and Western blot (B), by using a HA1-specific mAb. The native protein molecular weight marker (kDa) (Invitrogen) is indicated on the left. (C) Cross-linker analysis followed by SDS-PAGE and Coomassie Blue staining of expressed HA-13–263 proteins (with cross-linker). The proteins without cross-linking (w/o cross-linker) were used as the controls. The protein molecular weight marker (kDa) (Invitrogen) is indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3539987&req=5

pone-0053568-g005: Detection of conformation of HA-13–263 proteins by native PAGE (N-PAGE) and cross-linker analysis.(A) N-PAGE analysis of expressed HA-13–263 proteins, followed by Coomassie Blue staining and Western blot (B), by using a HA1-specific mAb. The native protein molecular weight marker (kDa) (Invitrogen) is indicated on the left. (C) Cross-linker analysis followed by SDS-PAGE and Coomassie Blue staining of expressed HA-13–263 proteins (with cross-linker). The proteins without cross-linking (w/o cross-linker) were used as the controls. The protein molecular weight marker (kDa) (Invitrogen) is indicated on the left.
Mentions: Since the molecular weight shown in reducing SDS-PAGE would not represent the actual size of the tested proteins, we next sought to detect the conformation of these proteins using non-reducing N-PAGE by removing the reducing agents in both sample and running buffers. As indicated in Fig. 5A, clear bands with high molecular weight were detected in HA-13–263-Fdc, HA-13–263-Fc and HA-13–263-Fd-His proteins, with HA-13–263-Fdc showing the highest molecular weight oligomer, while no clear band was shown in HA-13–263-His protein. Similarly, these bands were detected by a HA1-specific mAb in Western blot (Fig. 5B), indicating their high specificity to the HA1 protein. Comparison of the molecular weight from samples with and without cross-linker showed that the molecular weights of the cross-linker samples for HA-13–263 with Fd and/or Fc were about 1-, 2- or 3-fold higher than that of the corresponding without-cross-linker samples, but the molecular weight of the cross-linker HA-13–263-His was almost the same as that of the without-cross-linker HA-13–263-His (Fig. 5C). These results suggest that the identified neutralizing HA-13–263 fragment fused with Fd and/or Fc tags was able to form conformational structures of dimer, trimer, or oligomer.

Bottom Line: Consequently, the design of an effective and safe anti-H5N1 vaccine is essential.We then constructed three additional recombinants fused with Fd plus His tag (HA-13-263-Fd-His), Fc only (HA-13-263-Fc), and His tag only (HA-13-263-His), respectively.These results suggest that the oligomeric form of the CND containing the RBD can be further developed as an effective and safe vaccine for cross-protection against divergent strains of H5N1 viruses.

View Article: PubMed Central - PubMed

Affiliation: Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, United States of America.

ABSTRACT
The highly pathogenic avian influenza (HPAI) H5N1 viruses, especially the laboratory-generated H5N1 mutants, have demonstrated the potential to cross the species barrier and infect mammals and humans. Consequently, the design of an effective and safe anti-H5N1 vaccine is essential. We previously demonstrated that the full-length hemagglutinin 1 (HA1) could induce significant neutralizing antibody response and protection. Here, we intended to identify the critical neutralizing domain (CND) in an optimal conformation that can elicit strong cross-neutralizing antibodies and protection against divergent H5N1 strains. We thus constructed six recombinant proteins covering different regions of HA1 of A/Anhui/1/2005(H5N1), each of which was fused with foldon (Fd) and Fc of human IgG. We found that the critical fragment fused with Fd/Fc (HA-13-263-Fdc, H5 numbering) that could elicit the strongest neutralizing antibody response is located in the N-terminal region of HA1 (residues 13-263), which covers the receptor-binding domain (RBD, residues 112-263). We then constructed three additional recombinants fused with Fd plus His tag (HA-13-263-Fd-His), Fc only (HA-13-263-Fc), and His tag only (HA-13-263-His), respectively. We found that the HA-13-263-Fdc, which formed an oligomeric conformation, induced the strongest neutralizing antibody response and cross-protection against challenges of two tested H5N1 virus strains covering clade 1: A/VietNam/1194/2004 (VN/1194) or clade 2.3.4: A/Shenzhen/406H/06 (SZ/406H), while HA-13-263-Fc dimer and HA-13-263-Fd-His trimer elicited higher neutralizing antibody response and protection than HA-13-263-His monomer. These results suggest that the oligomeric form of the CND containing the RBD can be further developed as an effective and safe vaccine for cross-protection against divergent strains of H5N1 viruses.

Show MeSH
Related in: MedlinePlus