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Recombinant trimeric HA protein immunogenicity of H5N1 avian influenza viruses and their combined use with inactivated or adenovirus vaccines.

Lin SC, Huang MH, Tsou PC, Huang LM, Chong P, Wu SC - PLoS ONE (2011)

Bottom Line: The hemagglutinin (HA) envelope protein is the primary target for subunit vaccine development.We investigated trimeric rHA protein immunogenicity in mice via immunizations, and found that the highest levels of neutralizing antibodies resulted from coupling with a PELC/CpG adjuvant.We also found that the combined use of trimeric rHA proteins with (a) an inactivated H5N1 vaccine virus, or (b) a recombinant adenovirus encoding full-length HA sequences for prime-boost immunization, further improved antibody responses against homologous and heterologous H5N1 virus strains.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.

ABSTRACT

Background: The highly pathogenic avian influenza (HPAI) H5N1 virus continues to cause disease in poultry and humans. The hemagglutinin (HA) envelope protein is the primary target for subunit vaccine development.

Methodology/principal findings: We used baculovirus-insect cell expression to obtain trimeric recombinant HA (rHA) proteins from two HPAI H5N1 viruses. We investigated trimeric rHA protein immunogenicity in mice via immunizations, and found that the highest levels of neutralizing antibodies resulted from coupling with a PELC/CpG adjuvant. We also found that the combined use of trimeric rHA proteins with (a) an inactivated H5N1 vaccine virus, or (b) a recombinant adenovirus encoding full-length HA sequences for prime-boost immunization, further improved antibody responses against homologous and heterologous H5N1 virus strains. Data from cross-clade prime-boost immunization regimens indicate that sequential immunization with different clade HA antigens increased antibody responses in terms of total IgG level and neutralizing antibody titers.

Conclusion/significance: Our findings suggest that the use of trimeric rHA in prime-boost vaccine regimens represents an alternative strategy for recombinant H5N1 vaccine development.

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Related in: MedlinePlus

HA protein purification.Trimeric HA proteins were obtained via recombinant baculoviruses encoding H5N1 HA sequences from culture supernatant. Three truncated forms each of HA proteins from KAN-1 (A) and Anhui (B) strains were purified using metal affinity chromatography with Coomassie blue staining (left). Purified proteins were confirmed by western blotting (right) using anti-6xHis antibody. Purified HA proteins from KAN-1 (C) and Anhui (D) strains were digested with trypsin to confirm that the HA with a cleavage site mutation could be cleaved into HA1 (detected by polyclonal anti-H5HA antibodies, left) and HA2 (detected by polyclonal anti-6xHis antibodies, right).
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pone-0020052-g002: HA protein purification.Trimeric HA proteins were obtained via recombinant baculoviruses encoding H5N1 HA sequences from culture supernatant. Three truncated forms each of HA proteins from KAN-1 (A) and Anhui (B) strains were purified using metal affinity chromatography with Coomassie blue staining (left). Purified proteins were confirmed by western blotting (right) using anti-6xHis antibody. Purified HA proteins from KAN-1 (C) and Anhui (D) strains were digested with trypsin to confirm that the HA with a cleavage site mutation could be cleaved into HA1 (detected by polyclonal anti-H5HA antibodies, left) and HA2 (detected by polyclonal anti-6xHis antibodies, right).

Mentions: We used a baculovirus-insect cell expression system to generate three truncated forms of rHA (Tr1, Tr2, Tr3), with the transmembrane and cytoplasmic domains at the C terminus of full-length HA sequences replaced with the GCN4pII sequence KQIEDKIEEILSKIYHIENEIARIKKLIGEV and a His tag (Fig. 1). The polybasic cleavage site between HA1 and HA2 was changed from PQRRRKKRG to PQTRG to prevent unwanted cleavages in baculovirus-infected insect cells. We obtained rHA proteins from the culture supernatants of Sf9 cells infected with the recombinant baculoviruses. The three truncated rHA forms of the KAN-1 and Anhui strains were purified using Ni-NTA agarose chromatography (Fig. 2A–B). Hemagglutination of the three forms was tested for using turkey red blood cells; results indicate that the Tr1 proteins retained the highest HA titers compared to the Tr2 and Tr3 forms (Fig. 2C–D). We also treated the Tr1 rHA proteins of KAN-1 and Anhui strains with trypsin, revealing HA protein cleavage into HA1 and HA2 subunits (Fig. 3A–B). KAN-1 and Anhui rHA trimeric structures were evaluated by treatment with ethylene glycol-bis (EGS), a homobifunctional and cleavable cross-linking reagent previously used to analyze the trimeric form of HIV-1 gp120 [18]. According to our results, Tr1 monomers were cross-linked with a dimer, and then with a trimer, indicating that most of the rHA proteins of the KAN-1 and Anhui strains are trimers (Fig. 3C–D).


Recombinant trimeric HA protein immunogenicity of H5N1 avian influenza viruses and their combined use with inactivated or adenovirus vaccines.

Lin SC, Huang MH, Tsou PC, Huang LM, Chong P, Wu SC - PLoS ONE (2011)

HA protein purification.Trimeric HA proteins were obtained via recombinant baculoviruses encoding H5N1 HA sequences from culture supernatant. Three truncated forms each of HA proteins from KAN-1 (A) and Anhui (B) strains were purified using metal affinity chromatography with Coomassie blue staining (left). Purified proteins were confirmed by western blotting (right) using anti-6xHis antibody. Purified HA proteins from KAN-1 (C) and Anhui (D) strains were digested with trypsin to confirm that the HA with a cleavage site mutation could be cleaved into HA1 (detected by polyclonal anti-H5HA antibodies, left) and HA2 (detected by polyclonal anti-6xHis antibodies, right).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3104987&req=5

pone-0020052-g002: HA protein purification.Trimeric HA proteins were obtained via recombinant baculoviruses encoding H5N1 HA sequences from culture supernatant. Three truncated forms each of HA proteins from KAN-1 (A) and Anhui (B) strains were purified using metal affinity chromatography with Coomassie blue staining (left). Purified proteins were confirmed by western blotting (right) using anti-6xHis antibody. Purified HA proteins from KAN-1 (C) and Anhui (D) strains were digested with trypsin to confirm that the HA with a cleavage site mutation could be cleaved into HA1 (detected by polyclonal anti-H5HA antibodies, left) and HA2 (detected by polyclonal anti-6xHis antibodies, right).
Mentions: We used a baculovirus-insect cell expression system to generate three truncated forms of rHA (Tr1, Tr2, Tr3), with the transmembrane and cytoplasmic domains at the C terminus of full-length HA sequences replaced with the GCN4pII sequence KQIEDKIEEILSKIYHIENEIARIKKLIGEV and a His tag (Fig. 1). The polybasic cleavage site between HA1 and HA2 was changed from PQRRRKKRG to PQTRG to prevent unwanted cleavages in baculovirus-infected insect cells. We obtained rHA proteins from the culture supernatants of Sf9 cells infected with the recombinant baculoviruses. The three truncated rHA forms of the KAN-1 and Anhui strains were purified using Ni-NTA agarose chromatography (Fig. 2A–B). Hemagglutination of the three forms was tested for using turkey red blood cells; results indicate that the Tr1 proteins retained the highest HA titers compared to the Tr2 and Tr3 forms (Fig. 2C–D). We also treated the Tr1 rHA proteins of KAN-1 and Anhui strains with trypsin, revealing HA protein cleavage into HA1 and HA2 subunits (Fig. 3A–B). KAN-1 and Anhui rHA trimeric structures were evaluated by treatment with ethylene glycol-bis (EGS), a homobifunctional and cleavable cross-linking reagent previously used to analyze the trimeric form of HIV-1 gp120 [18]. According to our results, Tr1 monomers were cross-linked with a dimer, and then with a trimer, indicating that most of the rHA proteins of the KAN-1 and Anhui strains are trimers (Fig. 3C–D).

Bottom Line: The hemagglutinin (HA) envelope protein is the primary target for subunit vaccine development.We investigated trimeric rHA protein immunogenicity in mice via immunizations, and found that the highest levels of neutralizing antibodies resulted from coupling with a PELC/CpG adjuvant.We also found that the combined use of trimeric rHA proteins with (a) an inactivated H5N1 vaccine virus, or (b) a recombinant adenovirus encoding full-length HA sequences for prime-boost immunization, further improved antibody responses against homologous and heterologous H5N1 virus strains.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.

ABSTRACT

Background: The highly pathogenic avian influenza (HPAI) H5N1 virus continues to cause disease in poultry and humans. The hemagglutinin (HA) envelope protein is the primary target for subunit vaccine development.

Methodology/principal findings: We used baculovirus-insect cell expression to obtain trimeric recombinant HA (rHA) proteins from two HPAI H5N1 viruses. We investigated trimeric rHA protein immunogenicity in mice via immunizations, and found that the highest levels of neutralizing antibodies resulted from coupling with a PELC/CpG adjuvant. We also found that the combined use of trimeric rHA proteins with (a) an inactivated H5N1 vaccine virus, or (b) a recombinant adenovirus encoding full-length HA sequences for prime-boost immunization, further improved antibody responses against homologous and heterologous H5N1 virus strains. Data from cross-clade prime-boost immunization regimens indicate that sequential immunization with different clade HA antigens increased antibody responses in terms of total IgG level and neutralizing antibody titers.

Conclusion/significance: Our findings suggest that the use of trimeric rHA in prime-boost vaccine regimens represents an alternative strategy for recombinant H5N1 vaccine development.

Show MeSH
Related in: MedlinePlus