Serological Analysis of Herpes B Virus at Individual Epitope Resolution: From Two-Dimensional Peptide Arrays to Multiplex Bead Flow Assays

Abstract

Macacine herpesvirus or B Virus (BV) is a zoonotic agent that leads to high mortality rates in humans if transmitted and untreated. Here, BV is used as a test case to establish a two-step procedure for developing high throughput serological assays based on synthetic peptides. In step 1, peptide microarray analysis of 42 monkey sera (30 of them tested BV positive by ELISA) revealed 1148 responses against 369 different peptides. The latter could be grouped into 142 different antibody target regions (ATRs) in six different glycoproteins (gB, gC, gD, gG, gH, and gL) of BV. The high number of newly detected ATRs was made possible inter alia by a new preanalytical protocol that reduced unspecific binding of serum components to the cellulose-based matrix of the microarray. In step 2, soluble peptides corresponding to eight ATRs of particularly high antigenicity were synthesized and coupled to fluorescently labeled beads, which were subsequently employed in immunochemical bead flow assays. Their outcome mirrored the ELISA results used as reference. Hence, convenient, fast, and economical screening of arbitrarily large macaque colonies for BV infection is now possible. The study demonstrates that a technology platform switch from two-dimensional high-resolution peptide arrays used for epitope discovery to a readily available bead array platform for serology applications is feasible.

Peptide microarrays coupled to machine learning reveal individual epitopes from human antibody responses with neutralizing capabilities against SARS-CoV-2

“…Also, the intraparticular C-terminus of M was frequently targeted. This underlines the importance of intraparticular epitopes for immunogenicity, which was highlighted in previous studies [ 3 , 23 ].…”

“…On the other hand, strong responses to the N-terminal region of the M protein were found in both studies. Overall the high complexity of peptide array manufacturing and testing does not render this approach competitive for a simple, binary diagnostic application, in particular when compared to optimized multiplexed immunoassays [ 30 , 31 ], however, we note that a transfer of findings from discovery microarrays to more common platforms has been shown to be feasible by us [ 3 ]. Instead, the ability to reveal functionally relevant epitopes is a strength of the microarray technology.…”

“…Peptides were synthesized via Fmoc solid-phase peptide synthesis in an Intavis peptide synthesizer (Tübingen, Germany) on cellulose membranes by the SPOT method as described earlier [ 3 , 17 , 18 ]. In total, 648 peptides with a length of 15 amino acids and an offset of three amino acids to the following peptide of the protein sequences S, N, M and E derived from Ref.…”

“…To reduce unspecific binding, slides were blocked overnight in a blocking buffer that consisted of 2% casein in TTBS (1% Tween 20 (w/v) in TBS). Due to the sufficient signal–noise ratios, a pre-incubation of samples with cellulose/linker matrix as reported for other sample matrices [ 3 ], was not necessary and therefore not performed. Antibody-containing crude serum samples were first heat inactivated by 56°C for 30 min in order to eliminate their infectivity [ 19 ].…”

“…Such privileged, immunogenic epitopes would be prime candidates for precision diagnostics, vaccination approaches, or therapeutic antibodies, in particular, if their recognition is associated with neutralization. To characterize antibodies at the epitope level, microarrays of short synthetic peptides as antibody baits have been successfully applied for a variety of disease conditions, including viral infections [ 3–5 ]. Therefore, microarrays have been quickly adapted to investigate SARS-CoV-2 infections.…”