Rapid Response to Pandemic Threats: Immunogenic Epitope Detection of Pandemic Pathogens for Diagnostics and Vaccine Development Using Peptide Microarrays

Abstract

Emergence and re-emergence of pathogens bearing the risk of becoming a pandemic threat are on the rise. Increased travel and trade, growing population density, changes in urbanization, and climate have a critical impact on infectious disease spread. Currently, the world is confronted with the emergence of a novel coronavirus SARS-CoV-2, responsible for yet more than 800 000 deaths globally. Outbreaks caused by viruses, such as SARS-CoV-2, HIV, Ebola, influenza, and Zika, have increased over the past decade, underlining the need for a rapid development of diagnostics and vaccines. Hence, the rational identification of biomarkers for diagnostic measures on the one hand, and antigenic targets for vaccine development on the other, are of utmost importance. Peptide microarrays can display large numbers of putative target proteins translated into overlapping linear (and cyclic) peptides for a multiplexed, high-throughput antibody analysis. This enabled for example the identification of discriminant/diagnostic epitopes in Zika or influenza and mapping epitope evolution in natural infections versus vaccinations. In this review, we highlight synthesis platforms that facilitate fast and flexible generation of high-density peptide microarrays. We further outline the multifaceted applications of these peptide array platforms for the development of serological tests and vaccines to quickly encounter pandemic threats.

Keywords: array synthesis technologies; epitope mapping; infectious diseases; microarrays.

Figure 1

Typical workflow of a peptide microarray experiment. The pathogens of interest are selected; their protein sequences are cut into overlapping peptides, and these peptides are then synthesized on peptide microarrays. Patient samples are incubated on the arrays and serum antibodies bind to distinct epitopes. This information is the basis for many different applications.

Figure 2

SPOT synthesis technology. (1) Syringe or pipet tip is used to dispense solvents with solubilized amino acid building blocks onto a surface. (2) Coupling reaction proceeds directly upon contact with the surface-bound free reactive groups. (3) Excess and nonreacted building blocks are removed by washing and (4) the fluorenylmethyloxycarbonyl (Fmoc) protecting group is removed in a subsequent chemical washing step. SPOT synthesis is the gold standard in the field, offering reliable access to peptides, but with a limited number of peptides.

Figure 3

Selected high-throughput technologies for the synthesis of peptide microarrays. Lithographic methods (a) use a digital micromirror device to cleave photolabile protecting groups from amino acid building blocks via illumination, offering much higher spot densities. Solid material-based synthesis methods, such as the laser printer technology (b) or the combinatorial laser-induced forward transfer approach (c), offer a highly parallelized peptide array synthesis. Both rely on the deposition of at room temperature solid polymer, which embeds the amino acid building blocks. Only after several minutes of heating in an oven, the coupling reaction begins. Since this allows for the separation of patterning and coupling steps, these approaches can yield shorter process times for a rapid production of arrays.

Figure 4

Immunogenic epitopes of the EBOV GP (676 AA), identified with peptide microarrays (figure derived from Heidepriem et al.; reprinted with permission). (A) Comparison of the immunogenic IgG epitopes from Heidepriem et al. (S, survivor; V, vaccinee) in the EBOV GP with the published epitopes from other human response studies (Becquart et al., Rijal et al.).^, (B–D) 3D view of the EBOV GP trimer structure with the in Heidepriem et al. identified IgG epitopes highlighted in cyan.

Figure 5

In an initial prescreen, up to 10⁹ random peptides displayed on phage were screened for their binding to serum antibodies, immobilized on beads. Next, the identified epitope peptides were validated with solid material-based peptide microarray technology. Finally, the validated epitopes were fine mapped by comprehensive substitution analysis. The resulting “binding fingerprints” enable the identification of those proteins that match the antibody specificity, and, eventually, the correlation to disease causing agents. Reprinted with permission from ref (146). Copyright 2017 Elsevier B.V.