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An influenza A/H1N1/2009 hemagglutinin vaccine produced in Escherichia coli.

Aguilar-Yáñez JM, Portillo-Lara R, Mendoza-Ochoa GI, García-Echauri SA, López-Pacheco F, Bulnes-Abundis D, Salgado-Gallegos J, Lara-Mayorga IM, Webb-Vargas Y, León-Angel FO, Rivero-Aranda RE, Oropeza-Almazán Y, Ruiz-Palacios GM, Zertuche-Guerra MI, DuBois RM, White SW, Schultz-Cherry S, Russell CJ, Alvarez MM - PLoS ONE (2010)

Bottom Line: It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture.Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnología-FEMSA, Tecnológico de Monterrey at Monterrey, Monterrey, México.

ABSTRACT

Background: The A/H1N1/2009 influenza pandemic made evident the need for faster and higher-yield methods for the production of influenza vaccines. Platforms based on virus culture in mammalian or insect cells are currently under investigation. Alternatively, expression of fragments of the hemagglutinin (HA) protein in prokaryotic systems can potentially be the most efficacious strategy for the manufacture of large quantities of influenza vaccine in a short period of time. Despite experimental evidence on the immunogenic potential of HA protein constructs expressed in bacteria, it is still generally accepted that glycosylation should be a requirement for vaccine efficacy.

Methodology/principal findings: We expressed the globular HA receptor binding domain, referred to here as HA(63-286)-RBD, of the influenza A/H1N1/2009 virus in Escherichia coli using a simple, robust and scalable process. The recombinant protein was refolded and purified from the insoluble fraction of the cellular lysate as a single species. Recombinant HA(63-286)-RBD appears to be properly folded, as shown by analytical ultracentrifugation and bio-recognition assays. It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.

Conclusions/significance: Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture. Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy.

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Serum from patients infected with Influenza A H1N1/2009 specifically recognize HA63–286-RBD.(A) Bars 1–8, in gray tones, correspond to absorbance signals from non-exposed subjects (samples taken from March to May 2008). Bar 9, in black, shows the average absorbance value of samples 1 to 8. Bars 10 to 14, shown in colour, correspond to absorbance signals from Influenza A/H1N1 negative subjects. Bars 15–23, shown in colour, correspond to absorbance signals from samples of Influenza A H1N1 positive subjects (diagnosed two or three weeks previously by RT-PCR). Error bars represent one standard deviation (B) Proper refolding (biorecognition of antibodies from a positive patient), was evaluated for 4 different production batches of HA63–286-RBD. Batch 5 is a reference batch where HA63–286-RBD was expressed in its soluble form using a signal peptide for periplasmic expression.
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pone-0011694-g005: Serum from patients infected with Influenza A H1N1/2009 specifically recognize HA63–286-RBD.(A) Bars 1–8, in gray tones, correspond to absorbance signals from non-exposed subjects (samples taken from March to May 2008). Bar 9, in black, shows the average absorbance value of samples 1 to 8. Bars 10 to 14, shown in colour, correspond to absorbance signals from Influenza A/H1N1 negative subjects. Bars 15–23, shown in colour, correspond to absorbance signals from samples of Influenza A H1N1 positive subjects (diagnosed two or three weeks previously by RT-PCR). Error bars represent one standard deviation (B) Proper refolding (biorecognition of antibodies from a positive patient), was evaluated for 4 different production batches of HA63–286-RBD. Batch 5 is a reference batch where HA63–286-RBD was expressed in its soluble form using a signal peptide for periplasmic expression.

Mentions: The resulting HA63–286-RBD protein is specifically recognized by antibodies in serum samples from patients positive for the 2009 H1N1 virus (Figure 5). In comparative experiments, serum from positive patients diagnosed by the RT-PCR protocols established by the CDC and recommended by the WHO [27], or serum from subjects negative for influenza A H1N1, were measured in an HA63–286-RBD-specific ELISA as described by Alvarez et al. [28]. At 1∶50 dilution, the absorbance signal observed in samples from positive patients was between 2 to 4-fold higher when compared to signal from samples from negative subjects (Figure 5a).


An influenza A/H1N1/2009 hemagglutinin vaccine produced in Escherichia coli.

Aguilar-Yáñez JM, Portillo-Lara R, Mendoza-Ochoa GI, García-Echauri SA, López-Pacheco F, Bulnes-Abundis D, Salgado-Gallegos J, Lara-Mayorga IM, Webb-Vargas Y, León-Angel FO, Rivero-Aranda RE, Oropeza-Almazán Y, Ruiz-Palacios GM, Zertuche-Guerra MI, DuBois RM, White SW, Schultz-Cherry S, Russell CJ, Alvarez MM - PLoS ONE (2010)

Serum from patients infected with Influenza A H1N1/2009 specifically recognize HA63–286-RBD.(A) Bars 1–8, in gray tones, correspond to absorbance signals from non-exposed subjects (samples taken from March to May 2008). Bar 9, in black, shows the average absorbance value of samples 1 to 8. Bars 10 to 14, shown in colour, correspond to absorbance signals from Influenza A/H1N1 negative subjects. Bars 15–23, shown in colour, correspond to absorbance signals from samples of Influenza A H1N1 positive subjects (diagnosed two or three weeks previously by RT-PCR). Error bars represent one standard deviation (B) Proper refolding (biorecognition of antibodies from a positive patient), was evaluated for 4 different production batches of HA63–286-RBD. Batch 5 is a reference batch where HA63–286-RBD was expressed in its soluble form using a signal peptide for periplasmic expression.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908544&req=5

pone-0011694-g005: Serum from patients infected with Influenza A H1N1/2009 specifically recognize HA63–286-RBD.(A) Bars 1–8, in gray tones, correspond to absorbance signals from non-exposed subjects (samples taken from March to May 2008). Bar 9, in black, shows the average absorbance value of samples 1 to 8. Bars 10 to 14, shown in colour, correspond to absorbance signals from Influenza A/H1N1 negative subjects. Bars 15–23, shown in colour, correspond to absorbance signals from samples of Influenza A H1N1 positive subjects (diagnosed two or three weeks previously by RT-PCR). Error bars represent one standard deviation (B) Proper refolding (biorecognition of antibodies from a positive patient), was evaluated for 4 different production batches of HA63–286-RBD. Batch 5 is a reference batch where HA63–286-RBD was expressed in its soluble form using a signal peptide for periplasmic expression.
Mentions: The resulting HA63–286-RBD protein is specifically recognized by antibodies in serum samples from patients positive for the 2009 H1N1 virus (Figure 5). In comparative experiments, serum from positive patients diagnosed by the RT-PCR protocols established by the CDC and recommended by the WHO [27], or serum from subjects negative for influenza A H1N1, were measured in an HA63–286-RBD-specific ELISA as described by Alvarez et al. [28]. At 1∶50 dilution, the absorbance signal observed in samples from positive patients was between 2 to 4-fold higher when compared to signal from samples from negative subjects (Figure 5a).

Bottom Line: It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture.Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnología-FEMSA, Tecnológico de Monterrey at Monterrey, Monterrey, México.

ABSTRACT

Background: The A/H1N1/2009 influenza pandemic made evident the need for faster and higher-yield methods for the production of influenza vaccines. Platforms based on virus culture in mammalian or insect cells are currently under investigation. Alternatively, expression of fragments of the hemagglutinin (HA) protein in prokaryotic systems can potentially be the most efficacious strategy for the manufacture of large quantities of influenza vaccine in a short period of time. Despite experimental evidence on the immunogenic potential of HA protein constructs expressed in bacteria, it is still generally accepted that glycosylation should be a requirement for vaccine efficacy.

Methodology/principal findings: We expressed the globular HA receptor binding domain, referred to here as HA(63-286)-RBD, of the influenza A/H1N1/2009 virus in Escherichia coli using a simple, robust and scalable process. The recombinant protein was refolded and purified from the insoluble fraction of the cellular lysate as a single species. Recombinant HA(63-286)-RBD appears to be properly folded, as shown by analytical ultracentrifugation and bio-recognition assays. It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.

Conclusions/significance: Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture. Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy.

Show MeSH
Related in: MedlinePlus