Limits...
Properly folded bacterially expressed H1N1 hemagglutinin globular head and ectodomain vaccines protect ferrets against H1N1 pandemic influenza virus.

Khurana S, Verma S, Verma N, Crevar CJ, Carter DM, Manischewitz J, King LR, Ross TM, Golding H - PLoS ONE (2010)

Bottom Line: Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes.However, the HA1 (1-330) protein that had higher content of multimeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1-480).Protein yield for the HA1 (1-330) ranged around 40 mg/Liter, while the HA (1-480) yield was 0.4-0.8 mg/Liter.

View Article: PubMed Central - PubMed

Affiliation: Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America.

ABSTRACT

Background: In the face of impending influenza pandemic, a rapid vaccine production and mass vaccination is the most effective approach to prevent the large scale mortality and morbidity that was associated with the 1918 "Spanish Flu". The traditional process of influenza vaccine production in eggs is time consuming and may not meet the demands of rapid global vaccination required to curtail influenza pandemic.

Methodology/principal findings: Recombinant technology can be used to express the hemagglutinin (HA) of the emerging new influenza strain in a variety of systems including mammalian, insect, and bacterial cells. In this study, two forms of HA proteins derived from the currently circulating novel H1N1 A/California/07/2009 virus, HA1 (1-330) and HA (1-480), were expressed and purified from E. coli under controlled redox refolding conditions that favoured proper protein folding. However, only the recombinant HA1 (1-330) protein formed oligomers, including functional trimers that bound receptor and caused agglutination of human red blood cells. These proteins were used to vaccinate ferrets prior to challenge with the A/California/07/2009 virus. Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes. However, the HA1 (1-330) protein that had higher content of multimeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1-480). Protein yield for the HA1 (1-330) ranged around 40 mg/Liter, while the HA (1-480) yield was 0.4-0.8 mg/Liter.

Conclusions/significance: This is the first study that describes production in bacterial system of properly folded functional globular HA1 domain trimers, lacking the HA2 transmembrane protein, that elicit potent neutralizing antibody responses following vaccination and protect ferrets from in vivo challenge. The combination of bacterial expression system with established quality control methods could provide a mechanism for rapid large scale production of influenza vaccines in the face of influenza pandemic threat.

Show MeSH

Related in: MedlinePlus

Development of neutralizing and anti-HA binding antibodies following wt H1N1 (A/California/7/2009) infection in ferrets & post-H1N1 vaccination (inactivated vaccine) in humans.(A) Microneutralization of H1N1 A/California/2009 virus with post-H1N1-infected ferret samples. End-point titers (mean of three replicates) using post-infection sera from multiple ferrets at each time point in a microneutralization assay performed with A/California/07/2009 (X-179A). For day 21, sera of ten animals were pooled. Each dot in other time-points represents an individual H1N1 infected ferret. (B–D) Antibody kinetics following H1N1 challenge in ferrets. Steady-state equilibrium analysis of post-H1N1 infected ferret sera or pre- & post-H1N1 vaccinated human sera to mammalian H1N1 HA0 (Immune Technologies, NY) and properly folded bacterially expressed H1N1 HA1 (1–330) or H1N1 HA (1–480) fragment were measured using SPR. Ten-fold diluted individual post-infection sera from each time point, were injected simultaneously onto recombinant mammalian H1N1 HA0 in (B) and properly folded bacterially expressed H1N1 HA1 (1–330) in (C) or H1N1 HA (1–480) in (D), immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. SPR binding of pre-vaccine and post-H1N1 vaccination sera from two individuals with different neutralizing antibody titers (in parenthesis) is shown with recombinant mammalian H1N1 HA0 in (E) and properly folded bacterially expressed H1N1 HA1 (1–330) in (F) or H1N1 HA (1–480) in (G). Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2902520&req=5

pone-0011548-g002: Development of neutralizing and anti-HA binding antibodies following wt H1N1 (A/California/7/2009) infection in ferrets & post-H1N1 vaccination (inactivated vaccine) in humans.(A) Microneutralization of H1N1 A/California/2009 virus with post-H1N1-infected ferret samples. End-point titers (mean of three replicates) using post-infection sera from multiple ferrets at each time point in a microneutralization assay performed with A/California/07/2009 (X-179A). For day 21, sera of ten animals were pooled. Each dot in other time-points represents an individual H1N1 infected ferret. (B–D) Antibody kinetics following H1N1 challenge in ferrets. Steady-state equilibrium analysis of post-H1N1 infected ferret sera or pre- & post-H1N1 vaccinated human sera to mammalian H1N1 HA0 (Immune Technologies, NY) and properly folded bacterially expressed H1N1 HA1 (1–330) or H1N1 HA (1–480) fragment were measured using SPR. Ten-fold diluted individual post-infection sera from each time point, were injected simultaneously onto recombinant mammalian H1N1 HA0 in (B) and properly folded bacterially expressed H1N1 HA1 (1–330) in (C) or H1N1 HA (1–480) in (D), immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. SPR binding of pre-vaccine and post-H1N1 vaccination sera from two individuals with different neutralizing antibody titers (in parenthesis) is shown with recombinant mammalian H1N1 HA0 in (E) and properly folded bacterially expressed H1N1 HA1 (1–330) in (F) or H1N1 HA (1–480) in (G). Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA).

Mentions: The differences in the functional properties of HA1 (1–330) compared with the larger proteins containing the HA1+HA2 ectodomain needed further investigation. It was important to confirm that all three proteins expressed conformational “native” antigenic epitopes, recognized by antibodies elicited by H1N1 infection or by immunization with traditional (inactivated) vaccine. Ferrets are a good animal model for influenza virus pathogenesis. Following H1N1 infection, ferrets undergo transient loss of body weight, elevation in body temperature, and extensive viral replication in the upper and lower respiratory track on days 1–5, followed by viral clearance and recovery between Days 7–14 [25]. In the current study, consecutive post-H1N1 infection sera were evaluated for virus neutralizing antibody titers in a microneutralization assay (MN) (Fig. 2A) and for binding to recombinant H1N1 HA proteins by surface plasmon resonance (SPR), using either mammalian cell expressed HA0 or the bacterially expressed H1N1 HA1 (1–330) and HA (1–480) proteins (Fig. 2B–D). MN titers in the ferret sera were <20 during the first 5 days, followed by a rapid rise on days 7 and 14, and started to decline there after (Fig. 2A). On the other hand, using SPR, HA binding antibodies were measured as early as day 5 post infection and peaked on day 14. Importantly, binding of post-H1N1 infection ferret sera to HA0 from mammalian cells and to the bacterially expressed HA1 (1–330) and HA (1–480) proteins demonstrated similar kinetics and binding avidity profiles (Fig. 2 B-C-D), confirming that the bacterially expressed proteins were antigenically similar to the mammalian cell derived HA. The significant increase in binding to H1N1-HA proteins on days 7 and 14 correlated with the appearance of neutralizing antibodies against A/California/07/2009 (Fig. 2A). We also evaluated the binding of pre-and post-vaccination sera from two individuals that were immunized with a licensed inactivated subunit A/California/7/2009 vaccine (Fig. 2 E-F-G). Post-vaccination sera bound to mammalian-expressed HA0 (Fig. 2E) and to bacterially-expressed HA1 (1–330) (Fig. 2F) & HA (1–480) (Fig. 2G) proteins. The SPR results confirmed that all three proteins were properly folded and expressed native conformational epitopes. Interestingly, in both ferret and human studies, antibody binding to the HA1 (1–330) (containing trimers) was superior to that observed with the mammalian cell derived HA0 and the bacterially expressed HA ectodomain proteins (Fig. 2C vs. 2B and 2D, and Fig. 2F vs. 2E and 2G).


Properly folded bacterially expressed H1N1 hemagglutinin globular head and ectodomain vaccines protect ferrets against H1N1 pandemic influenza virus.

Khurana S, Verma S, Verma N, Crevar CJ, Carter DM, Manischewitz J, King LR, Ross TM, Golding H - PLoS ONE (2010)

Development of neutralizing and anti-HA binding antibodies following wt H1N1 (A/California/7/2009) infection in ferrets & post-H1N1 vaccination (inactivated vaccine) in humans.(A) Microneutralization of H1N1 A/California/2009 virus with post-H1N1-infected ferret samples. End-point titers (mean of three replicates) using post-infection sera from multiple ferrets at each time point in a microneutralization assay performed with A/California/07/2009 (X-179A). For day 21, sera of ten animals were pooled. Each dot in other time-points represents an individual H1N1 infected ferret. (B–D) Antibody kinetics following H1N1 challenge in ferrets. Steady-state equilibrium analysis of post-H1N1 infected ferret sera or pre- & post-H1N1 vaccinated human sera to mammalian H1N1 HA0 (Immune Technologies, NY) and properly folded bacterially expressed H1N1 HA1 (1–330) or H1N1 HA (1–480) fragment were measured using SPR. Ten-fold diluted individual post-infection sera from each time point, were injected simultaneously onto recombinant mammalian H1N1 HA0 in (B) and properly folded bacterially expressed H1N1 HA1 (1–330) in (C) or H1N1 HA (1–480) in (D), immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. SPR binding of pre-vaccine and post-H1N1 vaccination sera from two individuals with different neutralizing antibody titers (in parenthesis) is shown with recombinant mammalian H1N1 HA0 in (E) and properly folded bacterially expressed H1N1 HA1 (1–330) in (F) or H1N1 HA (1–480) in (G). Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2902520&req=5

pone-0011548-g002: Development of neutralizing and anti-HA binding antibodies following wt H1N1 (A/California/7/2009) infection in ferrets & post-H1N1 vaccination (inactivated vaccine) in humans.(A) Microneutralization of H1N1 A/California/2009 virus with post-H1N1-infected ferret samples. End-point titers (mean of three replicates) using post-infection sera from multiple ferrets at each time point in a microneutralization assay performed with A/California/07/2009 (X-179A). For day 21, sera of ten animals were pooled. Each dot in other time-points represents an individual H1N1 infected ferret. (B–D) Antibody kinetics following H1N1 challenge in ferrets. Steady-state equilibrium analysis of post-H1N1 infected ferret sera or pre- & post-H1N1 vaccinated human sera to mammalian H1N1 HA0 (Immune Technologies, NY) and properly folded bacterially expressed H1N1 HA1 (1–330) or H1N1 HA (1–480) fragment were measured using SPR. Ten-fold diluted individual post-infection sera from each time point, were injected simultaneously onto recombinant mammalian H1N1 HA0 in (B) and properly folded bacterially expressed H1N1 HA1 (1–330) in (C) or H1N1 HA (1–480) in (D), immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. SPR binding of pre-vaccine and post-H1N1 vaccination sera from two individuals with different neutralizing antibody titers (in parenthesis) is shown with recombinant mammalian H1N1 HA0 in (E) and properly folded bacterially expressed H1N1 HA1 (1–330) in (F) or H1N1 HA (1–480) in (G). Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA).
Mentions: The differences in the functional properties of HA1 (1–330) compared with the larger proteins containing the HA1+HA2 ectodomain needed further investigation. It was important to confirm that all three proteins expressed conformational “native” antigenic epitopes, recognized by antibodies elicited by H1N1 infection or by immunization with traditional (inactivated) vaccine. Ferrets are a good animal model for influenza virus pathogenesis. Following H1N1 infection, ferrets undergo transient loss of body weight, elevation in body temperature, and extensive viral replication in the upper and lower respiratory track on days 1–5, followed by viral clearance and recovery between Days 7–14 [25]. In the current study, consecutive post-H1N1 infection sera were evaluated for virus neutralizing antibody titers in a microneutralization assay (MN) (Fig. 2A) and for binding to recombinant H1N1 HA proteins by surface plasmon resonance (SPR), using either mammalian cell expressed HA0 or the bacterially expressed H1N1 HA1 (1–330) and HA (1–480) proteins (Fig. 2B–D). MN titers in the ferret sera were <20 during the first 5 days, followed by a rapid rise on days 7 and 14, and started to decline there after (Fig. 2A). On the other hand, using SPR, HA binding antibodies were measured as early as day 5 post infection and peaked on day 14. Importantly, binding of post-H1N1 infection ferret sera to HA0 from mammalian cells and to the bacterially expressed HA1 (1–330) and HA (1–480) proteins demonstrated similar kinetics and binding avidity profiles (Fig. 2 B-C-D), confirming that the bacterially expressed proteins were antigenically similar to the mammalian cell derived HA. The significant increase in binding to H1N1-HA proteins on days 7 and 14 correlated with the appearance of neutralizing antibodies against A/California/07/2009 (Fig. 2A). We also evaluated the binding of pre-and post-vaccination sera from two individuals that were immunized with a licensed inactivated subunit A/California/7/2009 vaccine (Fig. 2 E-F-G). Post-vaccination sera bound to mammalian-expressed HA0 (Fig. 2E) and to bacterially-expressed HA1 (1–330) (Fig. 2F) & HA (1–480) (Fig. 2G) proteins. The SPR results confirmed that all three proteins were properly folded and expressed native conformational epitopes. Interestingly, in both ferret and human studies, antibody binding to the HA1 (1–330) (containing trimers) was superior to that observed with the mammalian cell derived HA0 and the bacterially expressed HA ectodomain proteins (Fig. 2C vs. 2B and 2D, and Fig. 2F vs. 2E and 2G).

Bottom Line: Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes.However, the HA1 (1-330) protein that had higher content of multimeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1-480).Protein yield for the HA1 (1-330) ranged around 40 mg/Liter, while the HA (1-480) yield was 0.4-0.8 mg/Liter.

View Article: PubMed Central - PubMed

Affiliation: Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America.

ABSTRACT

Background: In the face of impending influenza pandemic, a rapid vaccine production and mass vaccination is the most effective approach to prevent the large scale mortality and morbidity that was associated with the 1918 "Spanish Flu". The traditional process of influenza vaccine production in eggs is time consuming and may not meet the demands of rapid global vaccination required to curtail influenza pandemic.

Methodology/principal findings: Recombinant technology can be used to express the hemagglutinin (HA) of the emerging new influenza strain in a variety of systems including mammalian, insect, and bacterial cells. In this study, two forms of HA proteins derived from the currently circulating novel H1N1 A/California/07/2009 virus, HA1 (1-330) and HA (1-480), were expressed and purified from E. coli under controlled redox refolding conditions that favoured proper protein folding. However, only the recombinant HA1 (1-330) protein formed oligomers, including functional trimers that bound receptor and caused agglutination of human red blood cells. These proteins were used to vaccinate ferrets prior to challenge with the A/California/07/2009 virus. Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes. However, the HA1 (1-330) protein that had higher content of multimeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1-480). Protein yield for the HA1 (1-330) ranged around 40 mg/Liter, while the HA (1-480) yield was 0.4-0.8 mg/Liter.

Conclusions/significance: This is the first study that describes production in bacterial system of properly folded functional globular HA1 domain trimers, lacking the HA2 transmembrane protein, that elicit potent neutralizing antibody responses following vaccination and protect ferrets from in vivo challenge. The combination of bacterial expression system with established quality control methods could provide a mechanism for rapid large scale production of influenza vaccines in the face of influenza pandemic threat.

Show MeSH
Related in: MedlinePlus