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Globular Head-Displayed Conserved Influenza H1 Hemagglutinin Stalk Epitopes Confer Protection against Heterologous H1N1 Virus.

Klausberger M, Tscheliessnig R, Neff S, Nachbagauer R, Wohlbold TJ, Wilde M, Palmberger D, Krammer F, Jungbauer A, Grabherr R - PLoS ONE (2016)

Bottom Line: Peptide-based vaccines have gained much interest as they allow the immune system to focus on relevant but less immunogenic epitopes.Flow cytometry and competition assays suggest that the identified stalk sequences do not recapitulate the epitopes of already described broadly neutralizing stalk antibodies.Vaccine constructs displaying 25-mer stalk sequences provided up to 75% protection from lethal heterologous virus challenge in BALB/c mice and induced antibody responses against the H1 hemagglutinin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria.

ABSTRACT
Significant genetic variability in the head region of the influenza A hemagglutinin, the main target of current vaccines, makes it challenging to develop a long-lived seasonal influenza prophylaxis. Vaccines based on the conserved hemagglutinin stalk domain might provide broader cross-reactive immunity. However, this region of the hemagglutinin is immunosubdominant to the head region. Peptide-based vaccines have gained much interest as they allow the immune system to focus on relevant but less immunogenic epitopes. We developed a novel influenza A hemagglutinin-based display platform for H1 hemagglutinin stalk peptides that we identified in an epitope mapping assay using human immune sera and synthetic HA peptides. Flow cytometry and competition assays suggest that the identified stalk sequences do not recapitulate the epitopes of already described broadly neutralizing stalk antibodies. Vaccine constructs displaying 25-mer stalk sequences provided up to 75% protection from lethal heterologous virus challenge in BALB/c mice and induced antibody responses against the H1 hemagglutinin. The developed platform based on a vaccine antigen has the potential to be either used as stand-alone or as prime-vaccine in combination with conventional seasonal or pandemic vaccines for the amplification of stalk-based cross-reactive immunity in humans or as platform to evaluate the relevance of viral peptides/epitopes for protection against influenza virus infection.

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Human immune serum IgGs bind to synthetic HA stalk peptides.Human pre-immune and immune sera from individuals receiving a monovalent live-attenuated A (H1N1) virus vaccine based on NC99 (A) and placebo controls (B) were used in an epitope mapping experiment. Individual sera were evaluated for binding to 112 decapeptides (having overlaps of 5 aa) representing the entire NC99 HA sequence in a chemiluminescence binding assay. Image spots were normalized and negative (deca-alanin) and positive control (Strep-tag) peptide spots were defined as 0% and 100% intensity respectively. Signal intensities (%) of serum IgGs binding to the individual hemagglutinin peptide spots were calculated. Bars represent mean differences in peptide-specific chemiluminescence signal intensities between immune and pre-immune sera from twelve vaccinated individuals (Fig 1A) and four placebo controls (Fig 1B). Arrows indicate interesting antigenic peptide stretches and individual peptides of the stalk domain and include peptide Nos. 64–69 (green), 73 (cyan), 87–89 (pink) and 106–109 (black). HA stalk peptides are highlighted in orange. The two HA domains are roughly demarcated by peptides no. 11 and 58, as they harbour the two conserved cystine residues that act as boundary between them [20].
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pone.0153579.g001: Human immune serum IgGs bind to synthetic HA stalk peptides.Human pre-immune and immune sera from individuals receiving a monovalent live-attenuated A (H1N1) virus vaccine based on NC99 (A) and placebo controls (B) were used in an epitope mapping experiment. Individual sera were evaluated for binding to 112 decapeptides (having overlaps of 5 aa) representing the entire NC99 HA sequence in a chemiluminescence binding assay. Image spots were normalized and negative (deca-alanin) and positive control (Strep-tag) peptide spots were defined as 0% and 100% intensity respectively. Signal intensities (%) of serum IgGs binding to the individual hemagglutinin peptide spots were calculated. Bars represent mean differences in peptide-specific chemiluminescence signal intensities between immune and pre-immune sera from twelve vaccinated individuals (Fig 1A) and four placebo controls (Fig 1B). Arrows indicate interesting antigenic peptide stretches and individual peptides of the stalk domain and include peptide Nos. 64–69 (green), 73 (cyan), 87–89 (pink) and 106–109 (black). HA stalk peptides are highlighted in orange. The two HA domains are roughly demarcated by peptides no. 11 and 58, as they harbour the two conserved cystine residues that act as boundary between them [20].

Mentions: To identify antigenic regions in the influenza HA protein, we performed a chemiluminescence binding assay with purified biotinylated human sera from donors participating in a clinical trial (Clinical.Trials.gov Identifier: NCT00724997) and 112 immobilized decapeptides spanning the entire HA sequence of the monovalent seasonal NC99 (sH1N1) vaccine strain [3]. Calculation of mean differences in peptide-specific chemiluminescence signal intensities between pre- and post-vaccination sera from twelve vaccinated individuals suggest that vaccination with the LAIV induced antibodies that are able to bind to several synthetic peptides located in the HA head and stalk region (Fig 1A). The greatest mean increase in peptide-specific serum-reactivity (Fig 1A) and greatest diversity among individual sera (S1A Fig) was observed for HA stalk peptide 9 (mean: +111%; range: (-58)–564) and HA head peptide 55 (mean: +135%; range: (-39)–704) (see S1 Table for peptide sequences). For several peptides we also observed considerable sero-reactivity (Fig 1B) and highly diverse individual serological profiles with control sera (S1B Fig). The fluctuation in mean responses with control sera, however, seemed to be largely driven by a single donor (S1B Fig). To investigate whether the magnitude of head-specific HI response post vaccination correlates with the binding profile to lead head and stalk peptides, we clustered the study group into non, medium or high HI-responders (no, 2-4-fold or 16-fold-increase of HI activity in post-vaccination sera respectively). Non and high-HI responders showed rather consistent weak peptide-specific responses with all peptides, apart from stalk peptide 9. Medium-responders were shown to be largely responsible for the wide-ranging binding spectrum and for highest responses with head and stalk peptides (S2 Fig). As our focus were antigenic peptides from the HA stalk, we graphically identified and selected stalk peptides with highest mean increases in sero-reactivity and little sero-reactivity with control sera. As stalk peptide 9 showed the highest mean increase in sero-reactivity with control sera (+70%, Fig 1B) it was omitted from further experiments. Three separate stretches of two to five contiguous decapeptides in the HA stalk (peptide Nos. 65–69, 87–89 and 106–109, excluding peptide No. 107) exhibited considerable increases in mean serum-reactivity (up to +90%) after vaccination as did peptide No. 73, while control serum reactivity to these peptides was low (Fig 1). Therefore, these peptides were chosen to be further investigated. To prove that serum-reactivity of selected peptides is method-independent binding specificity was re-confirmed using Surface Plasmon Resonance (data not shown).


Globular Head-Displayed Conserved Influenza H1 Hemagglutinin Stalk Epitopes Confer Protection against Heterologous H1N1 Virus.

Klausberger M, Tscheliessnig R, Neff S, Nachbagauer R, Wohlbold TJ, Wilde M, Palmberger D, Krammer F, Jungbauer A, Grabherr R - PLoS ONE (2016)

Human immune serum IgGs bind to synthetic HA stalk peptides.Human pre-immune and immune sera from individuals receiving a monovalent live-attenuated A (H1N1) virus vaccine based on NC99 (A) and placebo controls (B) were used in an epitope mapping experiment. Individual sera were evaluated for binding to 112 decapeptides (having overlaps of 5 aa) representing the entire NC99 HA sequence in a chemiluminescence binding assay. Image spots were normalized and negative (deca-alanin) and positive control (Strep-tag) peptide spots were defined as 0% and 100% intensity respectively. Signal intensities (%) of serum IgGs binding to the individual hemagglutinin peptide spots were calculated. Bars represent mean differences in peptide-specific chemiluminescence signal intensities between immune and pre-immune sera from twelve vaccinated individuals (Fig 1A) and four placebo controls (Fig 1B). Arrows indicate interesting antigenic peptide stretches and individual peptides of the stalk domain and include peptide Nos. 64–69 (green), 73 (cyan), 87–89 (pink) and 106–109 (black). HA stalk peptides are highlighted in orange. The two HA domains are roughly demarcated by peptides no. 11 and 58, as they harbour the two conserved cystine residues that act as boundary between them [20].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835069&req=5

pone.0153579.g001: Human immune serum IgGs bind to synthetic HA stalk peptides.Human pre-immune and immune sera from individuals receiving a monovalent live-attenuated A (H1N1) virus vaccine based on NC99 (A) and placebo controls (B) were used in an epitope mapping experiment. Individual sera were evaluated for binding to 112 decapeptides (having overlaps of 5 aa) representing the entire NC99 HA sequence in a chemiluminescence binding assay. Image spots were normalized and negative (deca-alanin) and positive control (Strep-tag) peptide spots were defined as 0% and 100% intensity respectively. Signal intensities (%) of serum IgGs binding to the individual hemagglutinin peptide spots were calculated. Bars represent mean differences in peptide-specific chemiluminescence signal intensities between immune and pre-immune sera from twelve vaccinated individuals (Fig 1A) and four placebo controls (Fig 1B). Arrows indicate interesting antigenic peptide stretches and individual peptides of the stalk domain and include peptide Nos. 64–69 (green), 73 (cyan), 87–89 (pink) and 106–109 (black). HA stalk peptides are highlighted in orange. The two HA domains are roughly demarcated by peptides no. 11 and 58, as they harbour the two conserved cystine residues that act as boundary between them [20].
Mentions: To identify antigenic regions in the influenza HA protein, we performed a chemiluminescence binding assay with purified biotinylated human sera from donors participating in a clinical trial (Clinical.Trials.gov Identifier: NCT00724997) and 112 immobilized decapeptides spanning the entire HA sequence of the monovalent seasonal NC99 (sH1N1) vaccine strain [3]. Calculation of mean differences in peptide-specific chemiluminescence signal intensities between pre- and post-vaccination sera from twelve vaccinated individuals suggest that vaccination with the LAIV induced antibodies that are able to bind to several synthetic peptides located in the HA head and stalk region (Fig 1A). The greatest mean increase in peptide-specific serum-reactivity (Fig 1A) and greatest diversity among individual sera (S1A Fig) was observed for HA stalk peptide 9 (mean: +111%; range: (-58)–564) and HA head peptide 55 (mean: +135%; range: (-39)–704) (see S1 Table for peptide sequences). For several peptides we also observed considerable sero-reactivity (Fig 1B) and highly diverse individual serological profiles with control sera (S1B Fig). The fluctuation in mean responses with control sera, however, seemed to be largely driven by a single donor (S1B Fig). To investigate whether the magnitude of head-specific HI response post vaccination correlates with the binding profile to lead head and stalk peptides, we clustered the study group into non, medium or high HI-responders (no, 2-4-fold or 16-fold-increase of HI activity in post-vaccination sera respectively). Non and high-HI responders showed rather consistent weak peptide-specific responses with all peptides, apart from stalk peptide 9. Medium-responders were shown to be largely responsible for the wide-ranging binding spectrum and for highest responses with head and stalk peptides (S2 Fig). As our focus were antigenic peptides from the HA stalk, we graphically identified and selected stalk peptides with highest mean increases in sero-reactivity and little sero-reactivity with control sera. As stalk peptide 9 showed the highest mean increase in sero-reactivity with control sera (+70%, Fig 1B) it was omitted from further experiments. Three separate stretches of two to five contiguous decapeptides in the HA stalk (peptide Nos. 65–69, 87–89 and 106–109, excluding peptide No. 107) exhibited considerable increases in mean serum-reactivity (up to +90%) after vaccination as did peptide No. 73, while control serum reactivity to these peptides was low (Fig 1). Therefore, these peptides were chosen to be further investigated. To prove that serum-reactivity of selected peptides is method-independent binding specificity was re-confirmed using Surface Plasmon Resonance (data not shown).

Bottom Line: Peptide-based vaccines have gained much interest as they allow the immune system to focus on relevant but less immunogenic epitopes.Flow cytometry and competition assays suggest that the identified stalk sequences do not recapitulate the epitopes of already described broadly neutralizing stalk antibodies.Vaccine constructs displaying 25-mer stalk sequences provided up to 75% protection from lethal heterologous virus challenge in BALB/c mice and induced antibody responses against the H1 hemagglutinin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria.

ABSTRACT
Significant genetic variability in the head region of the influenza A hemagglutinin, the main target of current vaccines, makes it challenging to develop a long-lived seasonal influenza prophylaxis. Vaccines based on the conserved hemagglutinin stalk domain might provide broader cross-reactive immunity. However, this region of the hemagglutinin is immunosubdominant to the head region. Peptide-based vaccines have gained much interest as they allow the immune system to focus on relevant but less immunogenic epitopes. We developed a novel influenza A hemagglutinin-based display platform for H1 hemagglutinin stalk peptides that we identified in an epitope mapping assay using human immune sera and synthetic HA peptides. Flow cytometry and competition assays suggest that the identified stalk sequences do not recapitulate the epitopes of already described broadly neutralizing stalk antibodies. Vaccine constructs displaying 25-mer stalk sequences provided up to 75% protection from lethal heterologous virus challenge in BALB/c mice and induced antibody responses against the H1 hemagglutinin. The developed platform based on a vaccine antigen has the potential to be either used as stand-alone or as prime-vaccine in combination with conventional seasonal or pandemic vaccines for the amplification of stalk-based cross-reactive immunity in humans or as platform to evaluate the relevance of viral peptides/epitopes for protection against influenza virus infection.

Show MeSH
Related in: MedlinePlus