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Cross-clade protective immune responses to influenza viruses with H5N1 HA and NA elicited by an influenza virus-like particle.

Bright RA, Carter DM, Crevar CJ, Toapanta FR, Steckbeck JD, Cole KS, Kumar NM, Pushko P, Smith G, Tumpey TM, Ross TM - PLoS ONE (2008)

Bottom Line: However, an apparent association rate of antibody binding to HA correlated with protection and was enhanced using VLPs, particularly when delivered intranasally, compared to rHA vaccines.This is the first report describing the use of an H5N1 VLP vaccine created from a clade 2 isolate.The results show that a non-replicating virus-like particle is effective at eliciting a broadened, cross-clade protective immune response to proteins from emerging H5N1 influenza isolates giving rise to a potential pandemic influenza vaccine candidate for humans that can be stockpiled for use in the event of an outbreak of H5N1 influenza.

View Article: PubMed Central - PubMed

Affiliation: Novavax, Inc., Rockville, Maryland, USA. rbright@novavax.com

ABSTRACT

Background: Vaccination is a cost-effective counter-measure to the threat of seasonal or pandemic outbreaks of influenza. To address the need for improved influenza vaccines and alternatives to egg-based manufacturing, we have engineered an influenza virus-like particle (VLP) as a new generation of non-egg or non-mammalian cell culture-based candidate vaccine.

Methodology/principal findings: We generated from a baculovirus expression system using insect cells, a non-infectious recombinant VLP vaccine from both influenza A H5N1 clade 1 and clade 2 isolates with pandemic potential. VLPs were administered to mice in either a one-dose or two-dose regimen and the immune responses were compared to those induced by recombinant hemagglutinin (rHA). Both humoral and cellular responses were analyzed. Mice vaccinated with VLPs were protected against challenge with lethal reassortant viruses expressing the H5N1 HA and NA, regardless if the H5N1 clade was homologous or heterologous to the vaccine. However, rHA-vaccinated mice showed considerable weight loss and death following challenge with the heterovariant clade virus. Protection against death induced by VLPs was independent of the pre-challenge HAI titer or cell-mediated responses to HA or M1 since vaccinated mice, with low to undetectable cross-clade HAI antibodies or cellular responses to influenza antigens, were still protected from a lethal viral challenge. However, an apparent association rate of antibody binding to HA correlated with protection and was enhanced using VLPs, particularly when delivered intranasally, compared to rHA vaccines.

Conclusion/significance: This is the first report describing the use of an H5N1 VLP vaccine created from a clade 2 isolate. The results show that a non-replicating virus-like particle is effective at eliciting a broadened, cross-clade protective immune response to proteins from emerging H5N1 influenza isolates giving rise to a potential pandemic influenza vaccine candidate for humans that can be stockpiled for use in the event of an outbreak of H5N1 influenza.

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Expression of H5N1 virus-like particles.A) Baculovirus construct for expression of influenza A/Indonesia/05/2005 (H5N1) VLPs. Indicated are the polyhedrin promoters (PolH), polyadenylation signals, Tn7 regions, gentamicin resistance gene (Gm), and influenza genes (HA, hemagglutinin, M1, matrix 1 protein, NA, neuraminidase); B). Scanning densitometry analysis of purified Indo/05 VLPs. A sample of purified VLPs (4 µg) was electrophoresed on 4–12% polyacrylamide gel and stained with Coomassie blue (right panel, lane 3). A scanned image (left panel, lane 3) was used to determine the relative optical density (OD) of HA, NA, and M1. Purity is = OD HA+NA+M1/OD Total in the lane. Purity of this lot of Indo/05 VLPs was 96%. The location of HA, NA, and M1 structural proteins are marked. C). Immunogold electron microsopy of purified Indo/05 VLPs. Left Panel. Primary antibody: Influenza A H5N1 Anti-HA antibody (Biodesign). Secondary antibody: Goat anti-rabbit conjugated to 10 nm gold beads. Right Panel. Control antibody and goat anti-rabbit secondary antibody conjugated to 10 nm gold beads. Bar represents 100 nm scale.
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pone-0001501-g001: Expression of H5N1 virus-like particles.A) Baculovirus construct for expression of influenza A/Indonesia/05/2005 (H5N1) VLPs. Indicated are the polyhedrin promoters (PolH), polyadenylation signals, Tn7 regions, gentamicin resistance gene (Gm), and influenza genes (HA, hemagglutinin, M1, matrix 1 protein, NA, neuraminidase); B). Scanning densitometry analysis of purified Indo/05 VLPs. A sample of purified VLPs (4 µg) was electrophoresed on 4–12% polyacrylamide gel and stained with Coomassie blue (right panel, lane 3). A scanned image (left panel, lane 3) was used to determine the relative optical density (OD) of HA, NA, and M1. Purity is = OD HA+NA+M1/OD Total in the lane. Purity of this lot of Indo/05 VLPs was 96%. The location of HA, NA, and M1 structural proteins are marked. C). Immunogold electron microsopy of purified Indo/05 VLPs. Left Panel. Primary antibody: Influenza A H5N1 Anti-HA antibody (Biodesign). Secondary antibody: Goat anti-rabbit conjugated to 10 nm gold beads. Right Panel. Control antibody and goat anti-rabbit secondary antibody conjugated to 10 nm gold beads. Bar represents 100 nm scale.

Mentions: The HA, NA, and M1 genes for the H5N1 VLP vaccine were synthesized by GeneArt (Germany) based upon sequences ISDN125873, ISDN125875, ISDN125876 [17] and followed by cloning into E. coli bacmids (Fig. 1A), plaque-purification of recombinant baculoviruses with HA, NA, and M1 genes in a single vector and expression in Spodoptera frugiperda Sf9 insect cells (ATCC CRL-1711) as previously described [14]. At 72 h post-transfection, cells were harvested for VLP production and recovery of recombinant baculoviruses in the culture medium. Particle expression was analyzed by sucrose gradient ultracentrifugation and SDS-PAGE followed by Western blot and purification of VLPs were essentially as previously described [14]. Indo/05 rHA (Lot # 31-10-06) was purified from the supernatants of Sf9 insect cells in-house and VN/04 rHA (Lot # 15-06-06) was purified from the supernatants of Sf9 insect cells acquired from Protein Sciences Corp., Meriden, CT, USA. Vaccines and protein were stored at 4°C prior to use.


Cross-clade protective immune responses to influenza viruses with H5N1 HA and NA elicited by an influenza virus-like particle.

Bright RA, Carter DM, Crevar CJ, Toapanta FR, Steckbeck JD, Cole KS, Kumar NM, Pushko P, Smith G, Tumpey TM, Ross TM - PLoS ONE (2008)

Expression of H5N1 virus-like particles.A) Baculovirus construct for expression of influenza A/Indonesia/05/2005 (H5N1) VLPs. Indicated are the polyhedrin promoters (PolH), polyadenylation signals, Tn7 regions, gentamicin resistance gene (Gm), and influenza genes (HA, hemagglutinin, M1, matrix 1 protein, NA, neuraminidase); B). Scanning densitometry analysis of purified Indo/05 VLPs. A sample of purified VLPs (4 µg) was electrophoresed on 4–12% polyacrylamide gel and stained with Coomassie blue (right panel, lane 3). A scanned image (left panel, lane 3) was used to determine the relative optical density (OD) of HA, NA, and M1. Purity is = OD HA+NA+M1/OD Total in the lane. Purity of this lot of Indo/05 VLPs was 96%. The location of HA, NA, and M1 structural proteins are marked. C). Immunogold electron microsopy of purified Indo/05 VLPs. Left Panel. Primary antibody: Influenza A H5N1 Anti-HA antibody (Biodesign). Secondary antibody: Goat anti-rabbit conjugated to 10 nm gold beads. Right Panel. Control antibody and goat anti-rabbit secondary antibody conjugated to 10 nm gold beads. Bar represents 100 nm scale.
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Related In: Results  -  Collection

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pone-0001501-g001: Expression of H5N1 virus-like particles.A) Baculovirus construct for expression of influenza A/Indonesia/05/2005 (H5N1) VLPs. Indicated are the polyhedrin promoters (PolH), polyadenylation signals, Tn7 regions, gentamicin resistance gene (Gm), and influenza genes (HA, hemagglutinin, M1, matrix 1 protein, NA, neuraminidase); B). Scanning densitometry analysis of purified Indo/05 VLPs. A sample of purified VLPs (4 µg) was electrophoresed on 4–12% polyacrylamide gel and stained with Coomassie blue (right panel, lane 3). A scanned image (left panel, lane 3) was used to determine the relative optical density (OD) of HA, NA, and M1. Purity is = OD HA+NA+M1/OD Total in the lane. Purity of this lot of Indo/05 VLPs was 96%. The location of HA, NA, and M1 structural proteins are marked. C). Immunogold electron microsopy of purified Indo/05 VLPs. Left Panel. Primary antibody: Influenza A H5N1 Anti-HA antibody (Biodesign). Secondary antibody: Goat anti-rabbit conjugated to 10 nm gold beads. Right Panel. Control antibody and goat anti-rabbit secondary antibody conjugated to 10 nm gold beads. Bar represents 100 nm scale.
Mentions: The HA, NA, and M1 genes for the H5N1 VLP vaccine were synthesized by GeneArt (Germany) based upon sequences ISDN125873, ISDN125875, ISDN125876 [17] and followed by cloning into E. coli bacmids (Fig. 1A), plaque-purification of recombinant baculoviruses with HA, NA, and M1 genes in a single vector and expression in Spodoptera frugiperda Sf9 insect cells (ATCC CRL-1711) as previously described [14]. At 72 h post-transfection, cells were harvested for VLP production and recovery of recombinant baculoviruses in the culture medium. Particle expression was analyzed by sucrose gradient ultracentrifugation and SDS-PAGE followed by Western blot and purification of VLPs were essentially as previously described [14]. Indo/05 rHA (Lot # 31-10-06) was purified from the supernatants of Sf9 insect cells in-house and VN/04 rHA (Lot # 15-06-06) was purified from the supernatants of Sf9 insect cells acquired from Protein Sciences Corp., Meriden, CT, USA. Vaccines and protein were stored at 4°C prior to use.

Bottom Line: However, an apparent association rate of antibody binding to HA correlated with protection and was enhanced using VLPs, particularly when delivered intranasally, compared to rHA vaccines.This is the first report describing the use of an H5N1 VLP vaccine created from a clade 2 isolate.The results show that a non-replicating virus-like particle is effective at eliciting a broadened, cross-clade protective immune response to proteins from emerging H5N1 influenza isolates giving rise to a potential pandemic influenza vaccine candidate for humans that can be stockpiled for use in the event of an outbreak of H5N1 influenza.

View Article: PubMed Central - PubMed

Affiliation: Novavax, Inc., Rockville, Maryland, USA. rbright@novavax.com

ABSTRACT

Background: Vaccination is a cost-effective counter-measure to the threat of seasonal or pandemic outbreaks of influenza. To address the need for improved influenza vaccines and alternatives to egg-based manufacturing, we have engineered an influenza virus-like particle (VLP) as a new generation of non-egg or non-mammalian cell culture-based candidate vaccine.

Methodology/principal findings: We generated from a baculovirus expression system using insect cells, a non-infectious recombinant VLP vaccine from both influenza A H5N1 clade 1 and clade 2 isolates with pandemic potential. VLPs were administered to mice in either a one-dose or two-dose regimen and the immune responses were compared to those induced by recombinant hemagglutinin (rHA). Both humoral and cellular responses were analyzed. Mice vaccinated with VLPs were protected against challenge with lethal reassortant viruses expressing the H5N1 HA and NA, regardless if the H5N1 clade was homologous or heterologous to the vaccine. However, rHA-vaccinated mice showed considerable weight loss and death following challenge with the heterovariant clade virus. Protection against death induced by VLPs was independent of the pre-challenge HAI titer or cell-mediated responses to HA or M1 since vaccinated mice, with low to undetectable cross-clade HAI antibodies or cellular responses to influenza antigens, were still protected from a lethal viral challenge. However, an apparent association rate of antibody binding to HA correlated with protection and was enhanced using VLPs, particularly when delivered intranasally, compared to rHA vaccines.

Conclusion/significance: This is the first report describing the use of an H5N1 VLP vaccine created from a clade 2 isolate. The results show that a non-replicating virus-like particle is effective at eliciting a broadened, cross-clade protective immune response to proteins from emerging H5N1 influenza isolates giving rise to a potential pandemic influenza vaccine candidate for humans that can be stockpiled for use in the event of an outbreak of H5N1 influenza.

Show MeSH
Related in: MedlinePlus