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Iterative structure-based improvement of a respiratory syncytial virus fusion glycoprotein vaccine

View Article: PubMed Central - PubMed

ABSTRACT

Structure-based design of vaccines has been a long-sought goal, especially the iterative optimization used so successfully with structure-based design of drugs. We previously developed a 1st-generation vaccine antigen called DS-Cav1, comprising a pre-fusion-stabilized form of the fusion (F) glycoprotein, which elicited high titers of protective responses against respiratory syncytial virus (RSV) in mice and macaques. Here we report the improvement of DS-Cav1 through iterative cycles of structure-based design that significantly increased the titer of RSV-protective responses. The resultant 2nd-generation “DS2”-stabilized immunogens have F subunits genetically linked, fusion peptide deleted, and interprotomer movements stabilized by an additional disulfide bond. These DS2-immunogens are promising vaccine candidates with superior attributes, such as the absence of a requirement for furin cleavage and increased antigenic stability to heat inactivation. The iterative structure-based improvement described here may have utility in the optimization of other vaccine antigens.

No MeSH data available.


Design, structure, immunogenicity, and informatics of single-chain RSV F glycoproteins with interprotomer disulfides (DS2) from design cycle 3. (a) Crystal structure of RSV sc9-10 DS-Cav1 trimer in ribbon. Insets show close-ups of Cycle 3 designs for residues mutated to Cys to form interprotomer disulfides (mutations labeled and in stick representation). (b) Engineered single-chain RSV F glycoproteins with interprotomer disulfides by SDS-PAGE (uncropped form is shown in Supplementary Fig. 1c). (c) Negative-stain EM class-averaged images of RSV F DS2 variants. (d) Structure of single-chain RSV F glycoproteins sc9-10 with interprotomer disulfide A149C Y458C shown in close-up with electron density. (e) Serum neutralization titers of 10 mice immunized with sc9-10 DS-Cav1 A149C Y458C and 15 mice immunized with sc9-10 DS-Cav1 N183GC N428C. Titers from each mouse are shown as individual purple symbols, with geometric means indicated by horizontal red lines (values shown at the bottom). Sera titers from 20 mice immunized with sc9-10 DS-Cav1 (blue symbols) and 39 mice immunized with RSV F DS-Cav1 (gray symbols) are shown for reference. P values <0.0001 (****) or <0.001 (***) were determined by two-tailed Mann-Whitney tests. (f)Statistical analysis of design cycles 1–3. Left: spearman correlation of each physical property with neutralization titers for design cycle 1, 2, and 3 variants (including DS-Cav1). Correlations with adjusted P value (Bonferroni correction) of less than 0.05 were marked with “*”. Right: correlation of quaternary antibody antigenicity and physical stability with neutralization titer. Constructs containing a mutation at residue 183 were not included in the correlation for quaternary antibody antigenicity as N183 is part of the AM14 epitope. “10,000” was used for antigenicity with “N.B.” values.
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Figure 3: Design, structure, immunogenicity, and informatics of single-chain RSV F glycoproteins with interprotomer disulfides (DS2) from design cycle 3. (a) Crystal structure of RSV sc9-10 DS-Cav1 trimer in ribbon. Insets show close-ups of Cycle 3 designs for residues mutated to Cys to form interprotomer disulfides (mutations labeled and in stick representation). (b) Engineered single-chain RSV F glycoproteins with interprotomer disulfides by SDS-PAGE (uncropped form is shown in Supplementary Fig. 1c). (c) Negative-stain EM class-averaged images of RSV F DS2 variants. (d) Structure of single-chain RSV F glycoproteins sc9-10 with interprotomer disulfide A149C Y458C shown in close-up with electron density. (e) Serum neutralization titers of 10 mice immunized with sc9-10 DS-Cav1 A149C Y458C and 15 mice immunized with sc9-10 DS-Cav1 N183GC N428C. Titers from each mouse are shown as individual purple symbols, with geometric means indicated by horizontal red lines (values shown at the bottom). Sera titers from 20 mice immunized with sc9-10 DS-Cav1 (blue symbols) and 39 mice immunized with RSV F DS-Cav1 (gray symbols) are shown for reference. P values <0.0001 (****) or <0.001 (***) were determined by two-tailed Mann-Whitney tests. (f)Statistical analysis of design cycles 1–3. Left: spearman correlation of each physical property with neutralization titers for design cycle 1, 2, and 3 variants (including DS-Cav1). Correlations with adjusted P value (Bonferroni correction) of less than 0.05 were marked with “*”. Right: correlation of quaternary antibody antigenicity and physical stability with neutralization titer. Constructs containing a mutation at residue 183 were not included in the correlation for quaternary antibody antigenicity as N183 is part of the AM14 epitope. “10,000” was used for antigenicity with “N.B.” values.

Mentions: We analyzed the structure of sc9-10 DS-Cav1 for interprotomer positions where disulfide linkages might be introduced (Fig. 3a). We synthesized 17 variants of sc9-10 with introduced interprotomer disulfides (Supplementary Table 1) and tested the expression of these in the 96-well microplate-formatted transient transfection format. Four of these constructs expressed at levels which allowed characterization by reducing and non-reducing SDS-PAGE (Fig. 3b); in the absence of reducing agent, all of these constructs ran as disulfide-linked oligomers of over 150 kDa without detectable dimer or monomer populations. In the presence of reducing agent, all four ran as monomers of ~60 kDa.


Iterative structure-based improvement of a respiratory syncytial virus fusion glycoprotein vaccine
Design, structure, immunogenicity, and informatics of single-chain RSV F glycoproteins with interprotomer disulfides (DS2) from design cycle 3. (a) Crystal structure of RSV sc9-10 DS-Cav1 trimer in ribbon. Insets show close-ups of Cycle 3 designs for residues mutated to Cys to form interprotomer disulfides (mutations labeled and in stick representation). (b) Engineered single-chain RSV F glycoproteins with interprotomer disulfides by SDS-PAGE (uncropped form is shown in Supplementary Fig. 1c). (c) Negative-stain EM class-averaged images of RSV F DS2 variants. (d) Structure of single-chain RSV F glycoproteins sc9-10 with interprotomer disulfide A149C Y458C shown in close-up with electron density. (e) Serum neutralization titers of 10 mice immunized with sc9-10 DS-Cav1 A149C Y458C and 15 mice immunized with sc9-10 DS-Cav1 N183GC N428C. Titers from each mouse are shown as individual purple symbols, with geometric means indicated by horizontal red lines (values shown at the bottom). Sera titers from 20 mice immunized with sc9-10 DS-Cav1 (blue symbols) and 39 mice immunized with RSV F DS-Cav1 (gray symbols) are shown for reference. P values <0.0001 (****) or <0.001 (***) were determined by two-tailed Mann-Whitney tests. (f)Statistical analysis of design cycles 1–3. Left: spearman correlation of each physical property with neutralization titers for design cycle 1, 2, and 3 variants (including DS-Cav1). Correlations with adjusted P value (Bonferroni correction) of less than 0.05 were marked with “*”. Right: correlation of quaternary antibody antigenicity and physical stability with neutralization titer. Constructs containing a mutation at residue 183 were not included in the correlation for quaternary antibody antigenicity as N183 is part of the AM14 epitope. “10,000” was used for antigenicity with “N.B.” values.
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Related In: Results  -  Collection

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Figure 3: Design, structure, immunogenicity, and informatics of single-chain RSV F glycoproteins with interprotomer disulfides (DS2) from design cycle 3. (a) Crystal structure of RSV sc9-10 DS-Cav1 trimer in ribbon. Insets show close-ups of Cycle 3 designs for residues mutated to Cys to form interprotomer disulfides (mutations labeled and in stick representation). (b) Engineered single-chain RSV F glycoproteins with interprotomer disulfides by SDS-PAGE (uncropped form is shown in Supplementary Fig. 1c). (c) Negative-stain EM class-averaged images of RSV F DS2 variants. (d) Structure of single-chain RSV F glycoproteins sc9-10 with interprotomer disulfide A149C Y458C shown in close-up with electron density. (e) Serum neutralization titers of 10 mice immunized with sc9-10 DS-Cav1 A149C Y458C and 15 mice immunized with sc9-10 DS-Cav1 N183GC N428C. Titers from each mouse are shown as individual purple symbols, with geometric means indicated by horizontal red lines (values shown at the bottom). Sera titers from 20 mice immunized with sc9-10 DS-Cav1 (blue symbols) and 39 mice immunized with RSV F DS-Cav1 (gray symbols) are shown for reference. P values <0.0001 (****) or <0.001 (***) were determined by two-tailed Mann-Whitney tests. (f)Statistical analysis of design cycles 1–3. Left: spearman correlation of each physical property with neutralization titers for design cycle 1, 2, and 3 variants (including DS-Cav1). Correlations with adjusted P value (Bonferroni correction) of less than 0.05 were marked with “*”. Right: correlation of quaternary antibody antigenicity and physical stability with neutralization titer. Constructs containing a mutation at residue 183 were not included in the correlation for quaternary antibody antigenicity as N183 is part of the AM14 epitope. “10,000” was used for antigenicity with “N.B.” values.
Mentions: We analyzed the structure of sc9-10 DS-Cav1 for interprotomer positions where disulfide linkages might be introduced (Fig. 3a). We synthesized 17 variants of sc9-10 with introduced interprotomer disulfides (Supplementary Table 1) and tested the expression of these in the 96-well microplate-formatted transient transfection format. Four of these constructs expressed at levels which allowed characterization by reducing and non-reducing SDS-PAGE (Fig. 3b); in the absence of reducing agent, all of these constructs ran as disulfide-linked oligomers of over 150 kDa without detectable dimer or monomer populations. In the presence of reducing agent, all four ran as monomers of ~60 kDa.

View Article: PubMed Central - PubMed

ABSTRACT

Structure-based design of vaccines has been a long-sought goal, especially the iterative optimization used so successfully with structure-based design of drugs. We previously developed a 1st-generation vaccine antigen called DS-Cav1, comprising a pre-fusion-stabilized form of the fusion (F) glycoprotein, which elicited high titers of protective responses against respiratory syncytial virus (RSV) in mice and macaques. Here we report the improvement of DS-Cav1 through iterative cycles of structure-based design that significantly increased the titer of RSV-protective responses. The resultant 2nd-generation &ldquo;DS2&rdquo;-stabilized immunogens have F subunits genetically linked, fusion peptide deleted, and interprotomer movements stabilized by an additional disulfide bond. These DS2-immunogens are promising vaccine candidates with superior attributes, such as the absence of a requirement for furin cleavage and increased antigenic stability to heat inactivation. The iterative structure-based improvement described here may have utility in the optimization of other vaccine antigens.

No MeSH data available.