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Hemagglutinin Sequence Conservation Guided Stem Immunogen Design from Influenza A H3 Subtype.

Mallajosyula VV, Citron M, Ferrara F, Temperton NJ, Liang X, Flynn JA, Varadarajan R - Front Immunol (2015)

Bottom Line: We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis.The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, "isoleucine-zipper", or "foldon".Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science , Bangalore , India.

ABSTRACT
Seasonal epidemics caused by influenza A (H1 and H3 subtypes) and B viruses are a major global health threat. The traditional, trivalent influenza vaccines have limited efficacy because of rapid antigenic evolution of the circulating viruses. This antigenic variability mediates viral escape from the host immune responses, necessitating annual vaccine updates. Influenza vaccines elicit a protective antibody response, primarily targeting the viral surface glycoprotein hemagglutinin (HA). However, the predominant humoral response is against the hypervariable head domain of HA, thereby restricting the breadth of protection. In contrast, the conserved, subdominant stem domain of HA is a potential "universal" vaccine candidate. We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis. The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, "isoleucine-zipper", or "foldon". These immunogens elicited cross-reactive, antiviral antibodies and conferred partial protection against a lethal, homologous HK68 virus challenge in vivo. Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up.

No MeSH data available.


Related in: MedlinePlus

HA sequence conservation guided immunogen design. (A) The residue conservation among H3 HA human isolates is mapped onto a surface representation of H3N2 A/Hong Kong/1/68 HA trimer [PDB ID: 1HGD (38)]. The quality score (Q-score) at every position in the protein sequence, which is a measure of residue conservation, was obtained from a multiple sequence alignment of H3 HA sequences (n = 3169) and binned. The HA stem is well conserved within a subtype. One monomer is colored according to the Q-score scale. Rest of HA (gray). (B) Conserved HA stem-fragments are “stitched” together in H3HA10 (cartoon). One monomer is colored according to the Q-score scale [indicated in (A)] to highlight the residue conservation in the designed immunogen. Stable structural breakpoints with optimal Cα–Cα distances (shown in another monomer) were determined by analysis of the H3 HA distance matrix. Soluble, flexible linker as indicated (black, dashed curve) was used to connect the HA1-subunit fragment 191-461 (pale yellow) to 2901-3211 (brown). A 6-residue linker (GSAGSA) connects the HA1 (brown) and HA2 (magenta) subunits in H3HA10. The third monomer is in gray. (C) Derivative(s) of H3HA10 were made with C-terminal trimerization motif(s), IZ, and foldon (34, 35). The figures (A,B) were rendered using PyMOL.
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Figure 1: HA sequence conservation guided immunogen design. (A) The residue conservation among H3 HA human isolates is mapped onto a surface representation of H3N2 A/Hong Kong/1/68 HA trimer [PDB ID: 1HGD (38)]. The quality score (Q-score) at every position in the protein sequence, which is a measure of residue conservation, was obtained from a multiple sequence alignment of H3 HA sequences (n = 3169) and binned. The HA stem is well conserved within a subtype. One monomer is colored according to the Q-score scale. Rest of HA (gray). (B) Conserved HA stem-fragments are “stitched” together in H3HA10 (cartoon). One monomer is colored according to the Q-score scale [indicated in (A)] to highlight the residue conservation in the designed immunogen. Stable structural breakpoints with optimal Cα–Cα distances (shown in another monomer) were determined by analysis of the H3 HA distance matrix. Soluble, flexible linker as indicated (black, dashed curve) was used to connect the HA1-subunit fragment 191-461 (pale yellow) to 2901-3211 (brown). A 6-residue linker (GSAGSA) connects the HA1 (brown) and HA2 (magenta) subunits in H3HA10. The third monomer is in gray. (C) Derivative(s) of H3HA10 were made with C-terminal trimerization motif(s), IZ, and foldon (34, 35). The figures (A,B) were rendered using PyMOL.

Mentions: Current influenza vaccines elicit a robust strain-specific antibody response which neutralizes the virus and confers protection (1). The primary response is against the immunodominant antigenic sites in the head domain of HA (43). However, the virus has evolved a mechanism of “antigenic drift” wherein mutations accumulated in these antigenic sites facilitate escape from the host immune pressure. Immune selection pressures coerce influenza virus into presenting a continually “moving target”. Therefore, we analyzed a large dataset of full-length H3 HA sequences (3169 sequences) reported from human hosts to identify conserved regions on HA. In agreement with previous results (1, 21, 22), the HA stem is on average more conserved than the variable head domain within a subtype (Figure 1A). However, there are pockets of high conservation even within the head domain of HA and monoclonal antibodies binding within the RBS have been isolated that neutralize drift variants of a HA subtype (6). The conservation within the HA stem has been ascribed to the less-than-optimal accessibility on the virion surface and the functional restraint imposed by conformational changes in the stem domain that are essential for infection. However, recent cryoEM studies of H1N1 A/California/7/2009 virus pre-incubated with the stem-directed bnAb C179 demonstrated that ~75% of HA trimers on the virion surface were in complex with the antibody (44). The study demonstrated that antibody binding to the HA stem on the virion is not severely impeded by accessibility.


Hemagglutinin Sequence Conservation Guided Stem Immunogen Design from Influenza A H3 Subtype.

Mallajosyula VV, Citron M, Ferrara F, Temperton NJ, Liang X, Flynn JA, Varadarajan R - Front Immunol (2015)

HA sequence conservation guided immunogen design. (A) The residue conservation among H3 HA human isolates is mapped onto a surface representation of H3N2 A/Hong Kong/1/68 HA trimer [PDB ID: 1HGD (38)]. The quality score (Q-score) at every position in the protein sequence, which is a measure of residue conservation, was obtained from a multiple sequence alignment of H3 HA sequences (n = 3169) and binned. The HA stem is well conserved within a subtype. One monomer is colored according to the Q-score scale. Rest of HA (gray). (B) Conserved HA stem-fragments are “stitched” together in H3HA10 (cartoon). One monomer is colored according to the Q-score scale [indicated in (A)] to highlight the residue conservation in the designed immunogen. Stable structural breakpoints with optimal Cα–Cα distances (shown in another monomer) were determined by analysis of the H3 HA distance matrix. Soluble, flexible linker as indicated (black, dashed curve) was used to connect the HA1-subunit fragment 191-461 (pale yellow) to 2901-3211 (brown). A 6-residue linker (GSAGSA) connects the HA1 (brown) and HA2 (magenta) subunits in H3HA10. The third monomer is in gray. (C) Derivative(s) of H3HA10 were made with C-terminal trimerization motif(s), IZ, and foldon (34, 35). The figures (A,B) were rendered using PyMOL.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481277&req=5

Figure 1: HA sequence conservation guided immunogen design. (A) The residue conservation among H3 HA human isolates is mapped onto a surface representation of H3N2 A/Hong Kong/1/68 HA trimer [PDB ID: 1HGD (38)]. The quality score (Q-score) at every position in the protein sequence, which is a measure of residue conservation, was obtained from a multiple sequence alignment of H3 HA sequences (n = 3169) and binned. The HA stem is well conserved within a subtype. One monomer is colored according to the Q-score scale. Rest of HA (gray). (B) Conserved HA stem-fragments are “stitched” together in H3HA10 (cartoon). One monomer is colored according to the Q-score scale [indicated in (A)] to highlight the residue conservation in the designed immunogen. Stable structural breakpoints with optimal Cα–Cα distances (shown in another monomer) were determined by analysis of the H3 HA distance matrix. Soluble, flexible linker as indicated (black, dashed curve) was used to connect the HA1-subunit fragment 191-461 (pale yellow) to 2901-3211 (brown). A 6-residue linker (GSAGSA) connects the HA1 (brown) and HA2 (magenta) subunits in H3HA10. The third monomer is in gray. (C) Derivative(s) of H3HA10 were made with C-terminal trimerization motif(s), IZ, and foldon (34, 35). The figures (A,B) were rendered using PyMOL.
Mentions: Current influenza vaccines elicit a robust strain-specific antibody response which neutralizes the virus and confers protection (1). The primary response is against the immunodominant antigenic sites in the head domain of HA (43). However, the virus has evolved a mechanism of “antigenic drift” wherein mutations accumulated in these antigenic sites facilitate escape from the host immune pressure. Immune selection pressures coerce influenza virus into presenting a continually “moving target”. Therefore, we analyzed a large dataset of full-length H3 HA sequences (3169 sequences) reported from human hosts to identify conserved regions on HA. In agreement with previous results (1, 21, 22), the HA stem is on average more conserved than the variable head domain within a subtype (Figure 1A). However, there are pockets of high conservation even within the head domain of HA and monoclonal antibodies binding within the RBS have been isolated that neutralize drift variants of a HA subtype (6). The conservation within the HA stem has been ascribed to the less-than-optimal accessibility on the virion surface and the functional restraint imposed by conformational changes in the stem domain that are essential for infection. However, recent cryoEM studies of H1N1 A/California/7/2009 virus pre-incubated with the stem-directed bnAb C179 demonstrated that ~75% of HA trimers on the virion surface were in complex with the antibody (44). The study demonstrated that antibody binding to the HA stem on the virion is not severely impeded by accessibility.

Bottom Line: We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis.The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, "isoleucine-zipper", or "foldon".Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science , Bangalore , India.

ABSTRACT
Seasonal epidemics caused by influenza A (H1 and H3 subtypes) and B viruses are a major global health threat. The traditional, trivalent influenza vaccines have limited efficacy because of rapid antigenic evolution of the circulating viruses. This antigenic variability mediates viral escape from the host immune responses, necessitating annual vaccine updates. Influenza vaccines elicit a protective antibody response, primarily targeting the viral surface glycoprotein hemagglutinin (HA). However, the predominant humoral response is against the hypervariable head domain of HA, thereby restricting the breadth of protection. In contrast, the conserved, subdominant stem domain of HA is a potential "universal" vaccine candidate. We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis. The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, "isoleucine-zipper", or "foldon". These immunogens elicited cross-reactive, antiviral antibodies and conferred partial protection against a lethal, homologous HK68 virus challenge in vivo. Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up.

No MeSH data available.


Related in: MedlinePlus