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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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SDS-PAGE and Western blot analysis of expressed proteins containing truncated HA1 fragments.Protein samples were either boiled at 95°C for 5 min, or not boiled, followed by addition of 6× SDS-PAGE sample buffer. SDS-PAGE with Coomassie Blue staining was then run (A), as well as Western blot (B) by using a HA1-specific mAb. The protein molecular weight marker (kDa) (Invitrogen) is indicated on the left.
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pone-0053568-g002: SDS-PAGE and Western blot analysis of expressed proteins containing truncated HA1 fragments.Protein samples were either boiled at 95°C for 5 min, or not boiled, followed by addition of 6× SDS-PAGE sample buffer. SDS-PAGE with Coomassie Blue staining was then run (A), as well as Western blot (B) by using a HA1-specific mAb. The protein molecular weight marker (kDa) (Invitrogen) is indicated on the left.

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)

SDS-PAGE and Western blot analysis of expressed proteins containing truncated HA1 fragments.Protein samples were either boiled at 95°C for 5 min, or not boiled, followed by addition of 6× SDS-PAGE sample buffer. SDS-PAGE with Coomassie Blue staining was then run (A), as well as Western blot (B) by using a HA1-specific mAb. 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-g002: SDS-PAGE and Western blot analysis of expressed proteins containing truncated HA1 fragments.Protein samples were either boiled at 95°C for 5 min, or not boiled, followed by addition of 6× SDS-PAGE sample buffer. SDS-PAGE with Coomassie Blue staining was then run (A), as well as Western blot (B) by using a HA1-specific mAb. The protein molecular weight marker (kDa) (Invitrogen) is indicated on the left.
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