Biomimetic systems responsive to environmental stimuli are of growing interest due to their useful, intriguing, and sometimes unexpected properties. Hierarchical self-assembly of biological building blocks has emerged as a powerful means of creating biomaterials with collective properties. Here, we show P22 virus-like particles (VLPs) functionalized with a spider silk protein derivative on the capsid exterior assemble into a hierarchical structure due to spider silk/spider silk interactions at low pH and reversibly dissemble upon raising the pH. We also show that the capsid arrays can be assembled through electrostatic interaction between the negatively charged capsids and a positively supercharged GFP mutant and can be reversed by raising the ionic strength. Most notably, we found that the supercharged GFP could bind to the hierarchically assembled material under high salt conditions but not to the individual capsids under the same high salt conditions. The binding of the charged macromolecule under high salt conditions demonstrates a collective behavior of the hierarchically assembled system that is not present with the unassembled, individual components of the system.

Antibody-antigen interaction–at antigenic local environments called B-cell epitopes–is a prominent mechanism for neutralization of infection. Effective mimicry, and display, of B-cell epitopes is key to vaccine design. Here, a physical approach is evaluated for the discovery of epitopes which evolve slowly over closely related pathogens (conserved epitopes). The approach is 1) protein flexibility-based and 2) demonstrated with clinically relevant enveloped viruses, simulated via molecular dynamics. The approach is validated against 1) seven structurally characterized enveloped virus epitopes which evolved the least (out of thirty-nine enveloped virus-antibody structures), 2) two structurally characterized non-enveloped virus epitopes which evolved slowly (out of eight non-enveloped virus-antibody structures), and 3) eight preexisting epitope and peptide discovery algorithms. Rationale for a new benchmarking scheme is presented. A data-driven epitope clustering algorithm is introduced. The prediction of five Zika virus epitopes (for future exploration on recombinant vaccine technologies) is demonstrated. For the first time, protein flexibility is shown to outperform solvent accessible surface area as an epitope discovery metric.

Conjugate-vaccine immunogens require three components: a carrier protein, an antigen, and a crosslinker, capable of coupling antigen to carrier protein, while preserving both T-cell responses from carrier protein and B-cell responses from antigen. We previously showed that the N-terminal eight residues of the HIV-1 fusion peptide (FP8) as an antigen could prime for broad cross-clade neutralizing responses, that recombinant heavy chain of tetanus toxin (rTTHC) as a carrier protein provided optimal responses, and that choice of crosslinker could impact both antigenicity and immunogenicity. Here, we delve more deeply into the impact of varying the linker between FP8 and rTTHC. In specific, we assessed the physical properties, the antigenicity, and the immunogenicity of conjugates for crosslinkers ranging in spacer-arm length from 1.5 to 95.2 Å, with varying hydrophobicity and crosslinking-functional groups. Conjugates coupled with different degrees of multimerization and peptide-to-rTTHC stoichiometry, but all were well recognized by HIV-fusion-peptide-directed antibodies VRC34.01, VRC34.05, PGT151, and ACS202 except for the conjugate with the longest linker (24-PEGylated SMCC; SM(PEG)24), which had lower affinity for ACS202, as did the conjugate with the shortest linker (succinimidyl iodoacetate; SIA), which also had the lowest peptide-to-rTTHC stoichiometry. Murine immunizations testing seven FP8-rTTHC conjugates elicited fusion-peptide-directed antibody responses, with SIA- and SM(PEG)24-linked conjugates eliciting lower responses than the other five conjugates. After boosting with prefusion-closed envelope trimers from strains BG505 clade A and consensus clade C, trimer-directed antibody-binding responses were lower for the SIA-linked conjugate; elicited neutralizing responses were similar, however, though statistically lower for the SM(PEG)24-linked conjugate, when tested against a strain especially sensitive to fusion-peptide-directed responses. Overall, correlation analyses revealed the immunogenicity of FP8-rTTHC conjugates to be negatively impacted by hydrophilicity and extremes of length or low peptide-carrier stoichiometry, but robust to other linker parameters, with several commonly used crosslinkers yielding statistically indistinguishable serological results.

<p>Antibody-antigen interaction – at antigenic local environments called B-cell epitopes – is a prominent mechanism for neutralization of infection. Effective mimicry, and display, of B-cell epitopes is key to vaccine design. Here, a physical approach is evaluated for the discovery of epitopes which evolve slowly over closely related pathogens (conserved epitopes). The approach is 1) protein flexibility-based and 2) demonstrated with the Zika virus, simulated via molecular dynamics. The approach is validated against 1) the seven structurally characterized flavivirus epitopes which have evolved the least (out of thirty-eight flavivirus-antibody structures) and 2) eight preexisting epitope and peptide discovery algorithms. For the first time, protein flexibility outperforms solvent accessible surface area as an epitope discovery metric.</p>

<p></p><p>Antibody-antigen interaction – at antigenic local environments called B-cell epitopes – is a prominent mechanism for neutralization of infection. Effective mimicry, and display, of B-cell epitopes is key to vaccine design. Here, a physical approach is evaluated for the discovery of epitopes which evolve slowly over closely related pathogens (conserved epitopes). The approach is 1) protein flexibility-based and 2) demonstrated with clinically relevant enveloped viruses, simulated via molecular dynamics. The approach is validated against 1) seven structurally characterized enveloped virus epitopes which evolved the least (out of thirty-eight enveloped virus-antibody structures) and 2) eight preexisting epitope and peptide discovery algorithms. Rationale for a new benchmarking scheme is presented. A data-driven epitope clustering algorithm is introduced. The prediction of eleven Zika virus epitopes (for future exploration on recombinant vaccine technologies) is demonstrated. For the first time, protein flexibility is shown to outperform solvent accessible surface area as an epitope discovery metric.</p><p></p>