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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

Neutralization against H5 pseudotyped particles in HA-immunized mice.Neutralization antibody titers were measured as reduction in luciferase activity of the H5HA-pseudotyped particle (H5pp) following the incubation of sera with H5 pseudotyped particles. p24 of H5pp (10 ng) was incubated with four-fold serial dilutions of serum for 1 h at 37°C and then transferred to MDCK cells. Luciferase assays were performed 48 h later. Dose-dependent neutralization curves were plotted against homologous KAN-1 (A) and Anhui (B) strains. Neutralization titers against homologous KAN-1 (C) and Anhui (D) strains and standard deviations were calculated using the ID50 program developed by John Spouge of the National Center for Biotechnology Information, National Library of Medicine, US National Institutes of Health. PELC/CpG elicited the highest level of neutralization titers, and Alum the lowest. Asterisk (*) indicates a statistically significant difference compared to the PELC/CpG group (p<0.05, Student t test).
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pone-0020052-g005: Neutralization against H5 pseudotyped particles in HA-immunized mice.Neutralization antibody titers were measured as reduction in luciferase activity of the H5HA-pseudotyped particle (H5pp) following the incubation of sera with H5 pseudotyped particles. p24 of H5pp (10 ng) was incubated with four-fold serial dilutions of serum for 1 h at 37°C and then transferred to MDCK cells. Luciferase assays were performed 48 h later. Dose-dependent neutralization curves were plotted against homologous KAN-1 (A) and Anhui (B) strains. Neutralization titers against homologous KAN-1 (C) and Anhui (D) strains and standard deviations were calculated using the ID50 program developed by John Spouge of the National Center for Biotechnology Information, National Library of Medicine, US National Institutes of Health. PELC/CpG elicited the highest level of neutralization titers, and Alum the lowest. Asterisk (*) indicates a statistically significant difference compared to the PELC/CpG group (p<0.05, Student t test).

Mentions: To determine the immunogenicity of KAN-1 and Anhui trimeric rHA proteins, we intramuscularly immunized a group of BALB/c mice with 15 µg rHA per mouse, coupled with an adjuvant of Alum (300 µg/dose), CpG oligodeoxynucleotides (10 µg/dose), Alum/CpG, PELC (10%), or PELC/CpG. Two immunizations were given over a 3-week period. Anti-sera were collected 2 weeks after the second immunization, and used to evaluate elicited antibody responses. According to results from an ELISA coated with the trimeric rHA proteins of KAN-1 (Fig. 4A) or Anhui (Fig. 4B), mice immunized with either rHA protein plus the PELC/CpG adjuvant produced the highest levels of HA-specific total IgG titers. Immunization with trimeric rHA proteins plus PELC or PELC/CpG adjuvants also induced higher IgG1 and IgG2a subtype titers compared to trimeric rHA proteins plus Alum, CpG, or Alum/CpG (Fig. 4C–D). Neutralization curves against KAN-1 or Anhui H5pp indicate that immunization with the trimeric rHA proteins plus any of the adjuvant formulations that were investigated in this study induced neutralizing antibody responses in a dose-dependent manner (Fig. 5A–B). Corresponding log (ID-50) values against KAN-1 H5pp and Anhui H5pp are shown in Figures 5C–D. According to these results, trimeric Anhui rHA proteins were more immunogenic than those of the KAN-1 strain for all of the investigated adjuvant formulations. The PELC/CpG adjuvant was the most effective of the four adjuvant formulations in terms of enhancing antibody responses in mice immunized with trimeric rHA proteins.


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)

Neutralization against H5 pseudotyped particles in HA-immunized mice.Neutralization antibody titers were measured as reduction in luciferase activity of the H5HA-pseudotyped particle (H5pp) following the incubation of sera with H5 pseudotyped particles. p24 of H5pp (10 ng) was incubated with four-fold serial dilutions of serum for 1 h at 37°C and then transferred to MDCK cells. Luciferase assays were performed 48 h later. Dose-dependent neutralization curves were plotted against homologous KAN-1 (A) and Anhui (B) strains. Neutralization titers against homologous KAN-1 (C) and Anhui (D) strains and standard deviations were calculated using the ID50 program developed by John Spouge of the National Center for Biotechnology Information, National Library of Medicine, US National Institutes of Health. PELC/CpG elicited the highest level of neutralization titers, and Alum the lowest. Asterisk (*) indicates a statistically significant difference compared to the PELC/CpG group (p<0.05, Student t test).
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Related In: Results  -  Collection

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pone-0020052-g005: Neutralization against H5 pseudotyped particles in HA-immunized mice.Neutralization antibody titers were measured as reduction in luciferase activity of the H5HA-pseudotyped particle (H5pp) following the incubation of sera with H5 pseudotyped particles. p24 of H5pp (10 ng) was incubated with four-fold serial dilutions of serum for 1 h at 37°C and then transferred to MDCK cells. Luciferase assays were performed 48 h later. Dose-dependent neutralization curves were plotted against homologous KAN-1 (A) and Anhui (B) strains. Neutralization titers against homologous KAN-1 (C) and Anhui (D) strains and standard deviations were calculated using the ID50 program developed by John Spouge of the National Center for Biotechnology Information, National Library of Medicine, US National Institutes of Health. PELC/CpG elicited the highest level of neutralization titers, and Alum the lowest. Asterisk (*) indicates a statistically significant difference compared to the PELC/CpG group (p<0.05, Student t test).
Mentions: To determine the immunogenicity of KAN-1 and Anhui trimeric rHA proteins, we intramuscularly immunized a group of BALB/c mice with 15 µg rHA per mouse, coupled with an adjuvant of Alum (300 µg/dose), CpG oligodeoxynucleotides (10 µg/dose), Alum/CpG, PELC (10%), or PELC/CpG. Two immunizations were given over a 3-week period. Anti-sera were collected 2 weeks after the second immunization, and used to evaluate elicited antibody responses. According to results from an ELISA coated with the trimeric rHA proteins of KAN-1 (Fig. 4A) or Anhui (Fig. 4B), mice immunized with either rHA protein plus the PELC/CpG adjuvant produced the highest levels of HA-specific total IgG titers. Immunization with trimeric rHA proteins plus PELC or PELC/CpG adjuvants also induced higher IgG1 and IgG2a subtype titers compared to trimeric rHA proteins plus Alum, CpG, or Alum/CpG (Fig. 4C–D). Neutralization curves against KAN-1 or Anhui H5pp indicate that immunization with the trimeric rHA proteins plus any of the adjuvant formulations that were investigated in this study induced neutralizing antibody responses in a dose-dependent manner (Fig. 5A–B). Corresponding log (ID-50) values against KAN-1 H5pp and Anhui H5pp are shown in Figures 5C–D. According to these results, trimeric Anhui rHA proteins were more immunogenic than those of the KAN-1 strain for all of the investigated adjuvant formulations. The PELC/CpG adjuvant was the most effective of the four adjuvant formulations in terms of enhancing antibody responses in mice immunized with trimeric rHA proteins.

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