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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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Construction of recombinant truncated HA1 protein fragments fused with Fd and Fc.(A) Schematic structure of HA protein of A/Anhui/1/2005 (H5N1 HA). SP, signal peptide. FP, fusion peptide. HA1-Fdc protein was previously expressed covering HA residues +3–322 [15], corresponding to residues 13–325 of H5 numbering as described in the published literature [9], [20], [21]. (B) Truncated HA fragments covering various lengths of HA1 were constructed by fusing HA1 fragments with Fd and Fc. Each fragment contains IL2ss signal peptide at the 5′ terminus for the purpose of leading expressed proteins to the culture supernatant. The predicted RBD region was indicated in black.
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pone-0053568-g001: Construction of recombinant truncated HA1 protein fragments fused with Fd and Fc.(A) Schematic structure of HA protein of A/Anhui/1/2005 (H5N1 HA). SP, signal peptide. FP, fusion peptide. HA1-Fdc protein was previously expressed covering HA residues +3–322 [15], corresponding to residues 13–325 of H5 numbering as described in the published literature [9], [20], [21]. (B) Truncated HA fragments covering various lengths of HA1 were constructed by fusing HA1 fragments with Fd and Fc. Each fragment contains IL2ss signal peptide at the 5′ terminus for the purpose of leading expressed proteins to the culture supernatant. The predicted RBD region was indicated in black.

Mentions: In this study, we constructed six recombinant proteins covering different lengths of HA1 of H5N1 virus, including full-length RBD (HA-112–263, HA-38–263, and HA-13–263), or partial-length RBD (HA-13–158 and HA-13–218) or no RBD (HA-13–90), and containing Fd and Fc at the C-termini (Fig. 1). All six constructs were able to express soluble proteins in the culture supernatant of transfected 293T cells, maintaining high expression with strong purity, as shown in SDS-PAGE (Fig. 2A). The expressed proteins were shown as monomers in the boiled sample lines, but the molecular weight shown in the non-boiled proteins is around 2-fold higher than that of the boiled samples after addition of the sample buffer containing 2% SDS and 1% 2-mercaptoethanol reducing agent and migration of the gels in the presence of 0.1% SDS, indicating that all expressed proteins fused with Fd and Fc formed suitable conformational structures similar to dimers in the reducing SDS-PAGE (Fig. 2A). In addition, Western blot results revealed strong bands corresponding to the relative molecular weight of the recombinant proteins, demonstrating that all proteins were recognized by a HA1-specific mAb (Fig. 2B).


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)

Construction of recombinant truncated HA1 protein fragments fused with Fd and Fc.(A) Schematic structure of HA protein of A/Anhui/1/2005 (H5N1 HA). SP, signal peptide. FP, fusion peptide. HA1-Fdc protein was previously expressed covering HA residues +3–322 [15], corresponding to residues 13–325 of H5 numbering as described in the published literature [9], [20], [21]. (B) Truncated HA fragments covering various lengths of HA1 were constructed by fusing HA1 fragments with Fd and Fc. Each fragment contains IL2ss signal peptide at the 5′ terminus for the purpose of leading expressed proteins to the culture supernatant. The predicted RBD region was indicated in black.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0053568-g001: Construction of recombinant truncated HA1 protein fragments fused with Fd and Fc.(A) Schematic structure of HA protein of A/Anhui/1/2005 (H5N1 HA). SP, signal peptide. FP, fusion peptide. HA1-Fdc protein was previously expressed covering HA residues +3–322 [15], corresponding to residues 13–325 of H5 numbering as described in the published literature [9], [20], [21]. (B) Truncated HA fragments covering various lengths of HA1 were constructed by fusing HA1 fragments with Fd and Fc. Each fragment contains IL2ss signal peptide at the 5′ terminus for the purpose of leading expressed proteins to the culture supernatant. The predicted RBD region was indicated in black.
Mentions: In this study, we constructed six recombinant proteins covering different lengths of HA1 of H5N1 virus, including full-length RBD (HA-112–263, HA-38–263, and HA-13–263), or partial-length RBD (HA-13–158 and HA-13–218) or no RBD (HA-13–90), and containing Fd and Fc at the C-termini (Fig. 1). All six constructs were able to express soluble proteins in the culture supernatant of transfected 293T cells, maintaining high expression with strong purity, as shown in SDS-PAGE (Fig. 2A). The expressed proteins were shown as monomers in the boiled sample lines, but the molecular weight shown in the non-boiled proteins is around 2-fold higher than that of the boiled samples after addition of the sample buffer containing 2% SDS and 1% 2-mercaptoethanol reducing agent and migration of the gels in the presence of 0.1% SDS, indicating that all expressed proteins fused with Fd and Fc formed suitable conformational structures similar to dimers in the reducing SDS-PAGE (Fig. 2A). In addition, Western blot results revealed strong bands corresponding to the relative molecular weight of the recombinant proteins, demonstrating that all proteins were recognized by a HA1-specific mAb (Fig. 2B).

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