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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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Prediction of potential discontinuous stalk epitopes.The spatial distribution of HA stalk-derived peptides and their relative orientations were assessed with a homologous model of the NC99 HA based on the available HA0 structure of the HA from PR8 (PDB#: 1RU7). The orientation of potential linear epitopes 66–68 (green), 86–89 (pink) and peptides 69 (blue), 73 (cyan) and 96 (yellow) that are part of potential discontinuous epitopes are given as sticks (A, left) or are highlighted in the respective colours in the protein structure (A, right). As peptide 69 is part of the potential linear epitope 66–69 and might also be part of the discontinuous epitope 69+73, it is indicated as separate epitope in the crystal structure. (B) Distances and angles between selected peptide pairs (circles) were calculated to evaluate whether their spatial distribution allows for the formation of a putative discontinuous epitope. Relative distances (abscissa) and relative orientation (ordinate) of HA stalk peptides were computed and peptide pairs as close as < 2 nm were considered suitable as single binding sites of a discontinuous epitope [24] (Black circles) peptide pairs formed by consecutive peptides; (red circles) non-linear peptide pairs. Distances exceeding 3.5 nm are not illustrated.
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pone.0153579.g002: Prediction of potential discontinuous stalk epitopes.The spatial distribution of HA stalk-derived peptides and their relative orientations were assessed with a homologous model of the NC99 HA based on the available HA0 structure of the HA from PR8 (PDB#: 1RU7). The orientation of potential linear epitopes 66–68 (green), 86–89 (pink) and peptides 69 (blue), 73 (cyan) and 96 (yellow) that are part of potential discontinuous epitopes are given as sticks (A, left) or are highlighted in the respective colours in the protein structure (A, right). As peptide 69 is part of the potential linear epitope 66–69 and might also be part of the discontinuous epitope 69+73, it is indicated as separate epitope in the crystal structure. (B) Distances and angles between selected peptide pairs (circles) were calculated to evaluate whether their spatial distribution allows for the formation of a putative discontinuous epitope. Relative distances (abscissa) and relative orientation (ordinate) of HA stalk peptides were computed and peptide pairs as close as < 2 nm were considered suitable as single binding sites of a discontinuous epitope [24] (Black circles) peptide pairs formed by consecutive peptides; (red circles) non-linear peptide pairs. Distances exceeding 3.5 nm are not illustrated.

Mentions: Besides linear epitopes we were also interested in putative discontinuous/conformational stalk epitopes. The calculation of angles and distances between epitopes served to identify peptides with sufficient proximity in the HA 3D structure to form a single binding footprint of a discontinuous epitope. We used a cut-off of 20 Å, as the footprint of an antibody paratope was shown to encompass a maximum area of approximately 20x30 Å2 [24]. Apart from mimotopes [25] linear peptides usually poorly mimic the conformation of discontinuous epitopes in the native protein. Nevertheless, discontinuous epitopes are composed of short linear stretches of 4–7 amino acid residues that form the binding site of an antibody [26]. We assumed that antibodies have only very low binding affinity to one of those single 'stretches' of a discontinuous epitope and would be poorly reactive in the epitope mapping assay. We have therefore also considered peptides with lower antigenicity in the binding assay (Fig 1) and included peptide pairs 69+73 and 73+96 in our experiments (indicated by arrows in Fig 2B). The crystallographic structure of an H1 HA from the closely-related influenza virus A/Puerto Rico/8/1934 (PR8) served to illustrate the native location of the chosen putative epitopes and revealed that peptide Nos. 73 and 96 are located closest to the virus envelope, peptide stretch 66–69 spans the HA1-HA2 intersubunit region including the fusion peptide, whereas peptide stretch 86–89 is located on the long alpha helix (LAH) (Fig 2A). The location of peptide stretch 106–109 could not be visualized as there was no structure available for this part of the HA. Bioinformatic analysis revealed that this stretch covers the HA transmembrane domain.


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)

Prediction of potential discontinuous stalk epitopes.The spatial distribution of HA stalk-derived peptides and their relative orientations were assessed with a homologous model of the NC99 HA based on the available HA0 structure of the HA from PR8 (PDB#: 1RU7). The orientation of potential linear epitopes 66–68 (green), 86–89 (pink) and peptides 69 (blue), 73 (cyan) and 96 (yellow) that are part of potential discontinuous epitopes are given as sticks (A, left) or are highlighted in the respective colours in the protein structure (A, right). As peptide 69 is part of the potential linear epitope 66–69 and might also be part of the discontinuous epitope 69+73, it is indicated as separate epitope in the crystal structure. (B) Distances and angles between selected peptide pairs (circles) were calculated to evaluate whether their spatial distribution allows for the formation of a putative discontinuous epitope. Relative distances (abscissa) and relative orientation (ordinate) of HA stalk peptides were computed and peptide pairs as close as < 2 nm were considered suitable as single binding sites of a discontinuous epitope [24] (Black circles) peptide pairs formed by consecutive peptides; (red circles) non-linear peptide pairs. Distances exceeding 3.5 nm are not illustrated.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153579.g002: Prediction of potential discontinuous stalk epitopes.The spatial distribution of HA stalk-derived peptides and their relative orientations were assessed with a homologous model of the NC99 HA based on the available HA0 structure of the HA from PR8 (PDB#: 1RU7). The orientation of potential linear epitopes 66–68 (green), 86–89 (pink) and peptides 69 (blue), 73 (cyan) and 96 (yellow) that are part of potential discontinuous epitopes are given as sticks (A, left) or are highlighted in the respective colours in the protein structure (A, right). As peptide 69 is part of the potential linear epitope 66–69 and might also be part of the discontinuous epitope 69+73, it is indicated as separate epitope in the crystal structure. (B) Distances and angles between selected peptide pairs (circles) were calculated to evaluate whether their spatial distribution allows for the formation of a putative discontinuous epitope. Relative distances (abscissa) and relative orientation (ordinate) of HA stalk peptides were computed and peptide pairs as close as < 2 nm were considered suitable as single binding sites of a discontinuous epitope [24] (Black circles) peptide pairs formed by consecutive peptides; (red circles) non-linear peptide pairs. Distances exceeding 3.5 nm are not illustrated.
Mentions: Besides linear epitopes we were also interested in putative discontinuous/conformational stalk epitopes. The calculation of angles and distances between epitopes served to identify peptides with sufficient proximity in the HA 3D structure to form a single binding footprint of a discontinuous epitope. We used a cut-off of 20 Å, as the footprint of an antibody paratope was shown to encompass a maximum area of approximately 20x30 Å2 [24]. Apart from mimotopes [25] linear peptides usually poorly mimic the conformation of discontinuous epitopes in the native protein. Nevertheless, discontinuous epitopes are composed of short linear stretches of 4–7 amino acid residues that form the binding site of an antibody [26]. We assumed that antibodies have only very low binding affinity to one of those single 'stretches' of a discontinuous epitope and would be poorly reactive in the epitope mapping assay. We have therefore also considered peptides with lower antigenicity in the binding assay (Fig 1) and included peptide pairs 69+73 and 73+96 in our experiments (indicated by arrows in Fig 2B). The crystallographic structure of an H1 HA from the closely-related influenza virus A/Puerto Rico/8/1934 (PR8) served to illustrate the native location of the chosen putative epitopes and revealed that peptide Nos. 73 and 96 are located closest to the virus envelope, peptide stretch 66–69 spans the HA1-HA2 intersubunit region including the fusion peptide, whereas peptide stretch 86–89 is located on the long alpha helix (LAH) (Fig 2A). The location of peptide stretch 106–109 could not be visualized as there was no structure available for this part of the HA. Bioinformatic analysis revealed that this stretch covers the HA transmembrane domain.

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