Serological Analysis of Herpes B Virus at Individual Epitope

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Serological Analysis of Herpes B Virus at Individual Epitope Resolution: From Two dimensional Peptide Arrays to Multiplex Bead Flow Assays Sven-Kevin Hotop, Ahmed Abd El Wahed, Ulrike Beutling, Florian Czerny, Claudia Sievers, Ulf Diederichsen, Ronald Frank, Christiane Stahl-Hennig, Mark Brönstrup, and Hans-Joachim Fritz Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01291 • Publication Date (Web): 31 Jul 2019 Downloaded from pubs.acs.org on August 3, 2019

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Analytical Chemistry

Serological Analysis of Herpes B Virus at Individual Epitope Resolution: From Two-dimensional Peptide Arrays to Multiplex Bead Flow Assays Sven-Kevin Hotop1, Ahmed Abd El Wahed2, Ulrike Beutling1, Florian Czerny3, Claudia Sievers4, Ulf Diederichsen3, Ronald Frank5, Christiane Stahl-Hennig6, Mark Brönstrup1*, Hans-Joachim Fritz7* 1 Department of Chemical Biology, Helmholtz Centre for Infection Research and German Centre for Infection research (DZIF), 38124 Braunschweig, Germany 2 Division of Microbiology and Animal Hygiene, Georg-August-University, 37077 Göttingen, Germany 3 Institute of Organic and Biomolecular Chemistry, Georg-August-University, 37077 Göttingen, Germany 4 Department for Epidemiology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany 5 AIMS Scientific Products GmbH, 13187 Berlin, Germany 6 Unit of Infection Models, German Primate Center, 37077 Göttingen, Germany 7 Akademie der Wissenschaften zu Göttingen, Theaterstr. 7, 37073 Göttingen, Germany

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. Step 1: Peptide microarray analysis of 42 monkey sera (30 of them tested BV-positive by ELISA) revealed 1635 responses against 469 different peptides. The latter could be grouped to 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 pre-analytical protocol that reduced unspecific binding of serum components to the cellulose-based matrix of the microarray. Step 2: Soluble peptides corresponding to eight ATRs of particularly high antigenicity were synthesized and coupled to fluorescently labelled 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 twodimensional high-resolution peptide arrays used for epitope discovery to a readily available bead array platform for serology applications is feasible.

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Introduction Viruses pose an omnipresent and dynamic threat to human and animal health, which necessitates accurate, robust and economical diagnostic methods. Diagnostics of virus infections can be grouped into two general classes: nucleic acid amplification (e.g. PCR) and serology (e.g. immunochemical detection of virus-specific antibodies by ELISA). The choice of one or the other method is governed by several considerations, most prominently the life cycle of the virus targeted. Infections by herpesviruses, for example, are characterized by phases of latency1 during which the virus may be undetectable by nucleic acid amplification. In particular, simplex viruses such as human HSV-1 and HSV-2 and their simian counterpart macacine herpesvirus 1 or herpes B virus (BV) persist in sensory ganglia for extended periods of time without shedding virions. This makes serology the option of choice. Colonies of monkeys kept in captivity must be monitored for BV infections, because of the small but finite risk of cross-species transmission and the associated high mortality rate in humans. For detection of anti-BV antibodies several ELISA systems are available that utilize inactivated HSV or lysate of cells infected by herpesvirus papio.2 Such tests, however, may fail due to sequence differences between homologous epitopes of different viruses.3 As an alternative, serological assays based on recombinant exo-domains of BV antigens exist4,5 but are prone to suffer from limited coverage of determinants due to protein truncations. This particularly applies to a highly antigenic epitope of BV which is located in the intracellular domain of glycoprotein D (gD) and was not included in the recombinant BV protein.6 Finally, assays based on whole BV virus lysate use the ideal antigen, but production and testing of large amounts of BV are challenging,7 and working with BV in the US and UK is limited to BSL-4 laboratories. In an earlier, preliminary study we tested the potential of short synthetic peptides as antibody baits for overcoming the shortcomings outlined above. In brief, we demonstrated that peptide microarrays of overlapping pentadekamers representing full sequences of just two BV glycoproteins (gB and gD) were sufficient to confirm 97% of positive results obtained with an anti-HSV ELISA.8 At the same time, however, this study demonstrated the need (i) to incorporate more than just two BV antigens in order to fill the remaining sensitivity gap and also (ii) to translate the microarray results to a simpler, more economical and more generally accessible assay format. Here we report on how both objectives were achieved.


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Experimental Methods Information on the origin of rhesus macaque and reference serum samples as well as on the chip layout (Supplementary Figure S1) is given under Supporting Information. Reduction of unspecific binding to conjugated cellulose in chip assays Small pieces of synthesis membrane (cellulose conjugated with N-acetyl-Ala-Alalinkers)9 were punched out as circles of 3 mm diameter and dissolved in 300 µl of a mixture of 4% trifluoromethanesulfonic acid; 88.5% trifluoroacetic acid; 5% H2O and 2.5% triisobutylsilane overnight. Dissolved cellulose conjugate was precipitated by adding 500 µl of ice-cold methyl tert-butyl ether (MTBE). After centrifugation at 3,000 x g at 4 °C, precipitates were washed three times with cold MTBE. After drying, the cellulose conjugate was dissolved in 500 μl DMSO. Then 100 µl DMSO stock solution was diluted with 500 µl coating buffer (100 mM sodium carbonate, pH 9.6). Thereafter, 300 µl of the so-obtained solution was used to coat each 1.5 ml Eppendorf tube overnight at 4 °C. Tubes were washed three times with Tris-buffered saline (TBS), followed by three times washing with absolute ethanol, and stored at -20 °C after drying. Serum samples were diluted 1:120 in blocking buffer (2% casein in TBS-T [TBS, 0.1 % Tween 20], final volume 120 μl each), and kept overnight at 4 °C in the precoated Eppendorf tubes. The protocol results from a method development that comprised the variation of the following parameters: Serum dilution; synthesis membrane (free cellulose vs. cellulose conjugated with N-acetyl-Ala-Ala-linkers and random peptides); dissolution method; buffer system, temperatures and volumes for precipitation; incubation times, incubation buffer and washing steps. Screening of serum samples by chip assays Serum samples, depleted as described above of unspecifically binding components, were transferred onto rehydrated, washed and blocked microarray slides, equipped for parallel screening of two individual samples with HybriWellTM dual well (100 µl capacity each) sealing chambers (Grace Bio-Labs, Bend, OR, USA). Slides were incubated at 4 °C overnight. After three times washing with TBS-T for 5 min each, bound antibodies were visualized using a macaque cross-reactive anti-human IgG, conjugated to Cy5 (Jackson ImmunoResearch, USA). The conjugate was diluted 240-fold in blocking buffer and allowed to react for 1.5 h at room temperature (RT). Subsequently, slides were washed three times with TBS-T and twice with dH2O, before drying in a stream of nitrogen. Finally, slides were scanned in an Agilent DNA microarray scanner. Positive responses were identified by processing raw scanner data with the spotcalling algorithm described earlier8. In brief, calculation of background was done per 3 ACS Paragon Plus Environment


half slide; thus, the background for each sample on each slide was calculated individually. We did not use a median intensity across all samples in order to avoid that weak signals over background on individual serum samples remained undetected due to strong signals and higher background in other samples. Signals were classified as positive if they have been detected over cut-off in 4/4 peptide spots (2 peptide spots as technical replicates per array and two positive calls in an independent experiment six weeks later with a second slide). Multiplex bead flow assay A general introduction to the LuminexTM technology of parallel, multiple flow assays using color-coded beads can be found in ref. 10. In brief, beads coded with distinct colors are coupled with a peptide antigen to measure antibodies in a serum sample recognizing the peptide. The wide range of color codes for beads allows a simultaneous analysis of multiple biomarkers (here: antibodies) using a flow cytometry approach. Here, eight selected ATRs of different length, plus five control peptides with random sequences, were used as capture antigens. The synthesis and analytical characterization of these N-terminally biotinylated peptides are described under Supporting Information. Bead-immobilized peptides were prepared as follows. NeutrAvidin (Thermo Scientific™) was coupled to carboxylated MagPlex™ microspheres (2.5 x 106/ml, LuminexTM, Luminex Corp., USA) with defined spectral properties using the AMG™ Activation Kit for Multiplex Microspheres according to the manufacturer´s protocol (Anteo Technologies, Cat # AI-LMPAKMM-01.02l.). Specifically, slurries of 13 different spectral types of activated magnetic beads (each ca. 200,000 beads in 100 l) were separately incubated with 10 g avidin for 1 h at room temperature (RT). Thereafter, beads were washed five times with blocking buffer (BB: phosphate-buffered saline (PBS), 1 mg/ml casein) using a magnetic separator (Dyna Mag™-2, Life technologies). Each slurry of avidin-conjugated beads was added to 1 ml of a 100 nM solution of one out of 13 N-terminally biotinylated peptides in BB, incubated at RT for 1 h, washed five times with BB, and finally resuspended in 200 µl of BB (final concentration 1x106 beads/ml). Aliquots of peptide-loaded beads were pooled and diluted to a final concentration of 2,000 beads per type in 50 µl BB, per serum sample to be measured. Two microliter of serum was diluted 100-fold in PVX buffer (PBS, 0.8% polyvinylpyrrolidone, 0.5% polyvinyl alcohol, 1 mg/ml Casein, 2.5% CBS-K100 (Millipore). 50 µl of diluted serum was incubated with the 50 µl of the bead pool solution for 1 h at RT in a 96 well plate. Beads were washed three times with 100 µl BB. All wash steps were performed using a hydroflex 96-Well washer with a magnetic plate (Tecan Austria GmbH). Bound serum-antibodies were visualized using 4 ACS Paragon Plus Environment

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R-phycoerythrin-conjugated goat anti-human IgG (Jackson ImmunoResearch, USA) in 1000-fold dilution of commercial stock solution. Finally, samples were washed three times with BB and resuspended in 100 µl of BB for measurement using a LuminexTM100 analyzer (xPONENTTM, Version 3.1 build 971, Luminex Corp., USA). The median fluorescence intensity (MFI) was determined from at least 100 beads per bead type. For each serum sample, two individual, intraday measurements were recorded. This procedure was repeated on a different day. The cut-off for positive signals was set to be at least 15-fold higher than the MFI of beads coated with random peptides. True positive signals were called if the MFI was 4 out of 4 times above cut-off.

Results Chip assay: Data Acquisition Cellulose conjugates of overlapping peptides (pentadecamers) covering complete sequences of BV immunogens gB, gD, gC, gG, gH and gL were synthesized and printed as ordered microarrays onto glass slides as described under Experimental Methods. Resulting chips were incubated with the samples of 42 different macaque sera characterized earlier,8 and bound serum antibodies were visualized by a macaque cross-reactive anti-human IgG secondary antibody conjugated to a Cy5 fluorescent dye. Individual array spots were classified as binding-positive or -negative by the automated SPOT calling procedure.8 Analysis of complex body fluids by immunosorbent assays (ISA) in general is often limited by low signal-to-noise ratios, and individual recipes for blocking unspecific binding and other options are part of many ISA’s. In our hands, enrichment of IgG by affinity-adsorption to proteins A/G before the SC2-chip analysis was only partially successful, because along with reduced background, signal intensity was also lost to a significant extent (data not shown). Since all peptides present on our SC2-chip share the structural feature of being bonded covalently to a cellulose/linker matrix, the latter is an obvious candidate for causing ubiquitous background. Therefore, serum samples were pre-incubated in Eppendorf tubes coated with conjugate that carried, instead of an oligopeptide, a -alanine dimer. This simple measure greatly enhanced signal-tonoise-ratios (typically from ca. 3:1 to ca. 20:1). Figure 1 illustrates the effect for serum #11, peptides D121 to D128. [Figure 1]

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This study addresses 42 well-characterized macaque sera. In previous experiments,8 the same set of sera had been characterized as follows: ELISA, based on inactivated HSV-1 antigen derived from infected simian kidney cells, classified 33 sera as BV-positive (subsets A, B and C, Table 1) and nine as negative (subsets D, E and F, Table 1). A peptide array chip assay carried out in parallel in that study confirmed all but one positive assignments (serum #33), while suggesting that the classification by ELISA of serum #42 as negative was erroneous.8 [Table 1] In the present study, serum #33 is now ruled positive by chip assay on the basis of three positive ATR-calls, in accord with the ELISA results. Also the serum #42 is clearly detected as positive due to 15 positive ATR-calls; arguments as to why the ELISA has probably yielded a false negative result are presented in the discussion section. Hence, the analysis of peptide reactions outlined below rests on a reference set of 34 positive sera (subsets A, B, C, and F, Table 1). With these, a total of 704 positively responding peptide spots were observed. A series of consecutive, positive array spot(s) represents the binding area of a single or multiple antibodies present in the serum. The peptide sequences behind locally clustered sets of positive spots were grouped to a single 'antibody target region' (ATR) applying previously published criteria,8 which led to 142 ATRs (Supplementary Table S2 and Supplementary Data Table). Newly included antigens gC, gG, gH and gL gave rise to 407 (58%) positively responding peptides. Antigens gB and gD contributed 297 (42%) positively responding peptides, 190 of which were not detected in the previous study.8 We attribute the strikingly increased sensitivity now observed to a combination of two independent but cooperating factors: improved signal-to-noise ratio achieved with the newly developed sample pre-incubation protocol and shorter shelf time spent by the new batch of slides between production and use. On the other hand, the new procedure missed seven out of 114 gB and gD signals seen earlier.8 The number of experimentally verified gB and gD ATRs increased from 18 to 50. The classification of serum #42 as BV positive (compare above) now rests on its clear chip-response to 15 different ATRs. The relevance of these responses is corroborated by 27 other positive serum samples producing signals with varying subsets of the same 15 ATRs. Significantly, the positive National B Virus Resource Center (NBVRC) reference sera (subset A, Table 1) displayed reactions with 11 of these.


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Altogether, these findings attest not only to improved sensitivity of the overall procedure but also to good batch-to-batch reproducibility of the chips – the latter in contrast to reports on other peptide microarray studies.11 [Figure 2]

Mapping of ATRs to known 3D-structures Knowledge of how ATRs are organized in the structural context of entire antigens helps to better understand B-cell responses. Wherever feasible,12,13,14 ATR sequences were projected onto known 3D-structures of homologous HSV antigens, as exemplified for ATR H108 and ATR H121 (Figure 2B). H108 is a typical ATR represented on the chip by a consecutive series of spots. Three spots (as in this case gH67 – 69) delineate an ATR length of nine amino acid residues which appear as a band on the surface of the protein molecule (dark blue in Figure 2). ATR H121 is unusual in that it is part of an alpha-helix, the middle of which is crossed by another stretch of polypeptide chain and one has to assume that an antibody giving rise to the observed spot response has to contact simultaneously both exposed ends. [Figure 3] A graphical overview of all signals observed with the 34 positive serum samples is given in Figure 3. The clustering of a large proportion of all positive responses in a comparably limited number of narrow regions (ATRs) is immediately obvious. As noted earlier8, ATR D18, located at the extreme C-terminus of gD, stands out as particularly immunodominant, closely followed by several others in various antigens. Glycoproteins gH and gL, however, are largely devoid of prominent ATRs. A ranking order of frequencies of occurrence for all ATRs lighting up with seven or more sera is depicted in Figure 4. [Figure 4]

Multiplex bead flow assay Next, the newly acquired insight into simian anti-BV B-cell responses was exploited to set up a convenient and economical high throughput assay based on the LuminexTM platform.15-17 To this end, soluble peptides corresponding to the top ten ATRs of the occurrence ranking list were synthesized (Figure 4, Supplementary Table S1). Eight 7 ACS Paragon Plus Environment


ATRs were successfully synthesized with lengths between 21 and 42 amino acids, while the synthesis of the ATRs H121 and B5 failed. Five additional peptides (15mers) were synthesized, whose sequences had been defined at random. The 13 peptides were coated to beads as described under Experimental Methods and used to re-screen 36 sera (31 positive, 5 negative according to chip data, sample subsets B, C, E, F). The six NBVRC reference sera could not be tested, because the amount of serum available was insufficient. [Figure 5] The median fluorescence intensity (MFI) in each sample was measured for ≥ 100 beads per bead type. The background was defined as the median MFI obtained by the five random peptides in a given sample, and the relative signal strength for a given ATR peptide was expressed as multiples of background level. To assess the status of each serum (BV positive/negative), an arbitrary threshold of 15-fold background level was set to determine a positive ATR-call (Figure 5). This protocol separated the 36 serum samples into 30 positives and 6 negatives. For the negative samples, the highest signal strength for an individual peptide was 11.5. In positive samples, the signal strength ranged from 15.5 to 349. As representative cases, results for sera #9 and #11 are displayed in Supplementary Figure S2. They match those obtained by the chip assay (see Supplementary Data Table) – with three exceptions: on the chip, serum #9 scored positive for ATR D12, which was missed in the bead assay. With serum #11, the same is true for ATRs B3b and D16. Nonetheless, with seven positive ATR calls left for serum #9 and three for serum #11, both sera are clearly identified as BV-positive, in accordance with the chip results. The classification of samples was in accordance with the chip data for 35 of 36 serum samples – but differed for sample #42. From the 15 ATRs detected by the chip assay for sample #42, only one (D12) was used in the bead assay, which however was not detected. In fact, D12 exhibited a particularly poor performance in the bead assay: while it led to positive responses against 18 sera on the chips, only a single response was observed in the bead format. Using the 31 confirmed BV positive samples, the overall frequency of positive ATRcalls was calculated for the chip and bead assay (Supplementary Figure S3). On average, a BV positive sample was detected with 4.2 signal peptides in the bead assay. For comparison, an average of 5.8 peptides corresponding to the eight ATR peptides was detected in the chip assay. Cumulatively, 186 positive responses were recorded for the eight peptides. 125 of 8 ACS Paragon Plus Environment

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these were shared between the two assay formats, 57 were only detected with chips and 4 only in the bead assay (Supplementary Table S3). Thus, signals observed in the chip assay could be recovered by bead-coupled ATRs in 69% of cases (Supplementary Table S3). Two concluding observations can be made: Firstly, as used here, chip assays are more sensitive than their bead counterparts. Secondly, since the two series of measurements rest on two different experimental formats and, in particular, the procedures of designating positive responses are entirely different, the fact that 97% of the positive ATR-calls by bead assay are mirrored on the chip side demonstrates the congruency of both series of measurements.

Discussion A characterization of antibody repertoires at the epitope- or even amino acid-level is achieved by high density peptide arrays, which have been applied to study a variety of disease conditions.18-21 Due to their complexity, however, manufacturing and use of peptide arrays is still confined to expert labs. A consequence of the small scale of production is that the costs per analysis are relatively high. We therefore asked the question whether it is possible to combine the analytical depth of peptide arrays with a more affordable and widespread technology for serology like multiplexed beads. Using BV diagnosis in macaque sera as a test case, we demonstrated that an epitope discovery based on an optimized protocol and comprehensive sets of peptides from six glycoproteins led to a set of 142 different ATRs. This represents a significant increase compared to 18 ATRs reported in a previous study with the same set of sera. An important enabler of this enhanced coverage was the modified pre-analytical sample preparation protocol that led to considerably improved signal-to-noise ratios. On the other hand, a few (6%) responses reported in the first screen8 could not be detected again. Possible reasons might be that the corresponding serum antibodies degraded over time during multiple freeze and thaw processes, or that they were binding to chemically modified cellulose fibers in the newly introduced pre-adsorption process. Indeed, higher absolute signal intensities were obtained when sera were processed directly without pre-adsorption, and it is not known which components of the serum samples are depleted by the cellulose carrier. However, the improvement of the signal-to-noise ratio from 3:1 in the previous method to 20:1 in this study clearly outweighs the loss in signal intensities, as reflected by the 27% increase of detected responses against gB and gD. The novel pre-analytical protocol is an important step to widen the scope of the peptide chip applications to translational research, as it facilitates the study of complex body fluids from primates. 9 ACS Paragon Plus Environment


Comparing the results from the current study with those obtained with a first generation chip,8 the high reproducibility of 94.7% of previous signals as well as the observation of 85% of additional signals are two noteworthy findings. Furthermore, 57% of signals came from peptide sequences of the four newly investigated antigens gC, gG, gH and gL. Whereas gC and gG have been described as antigenic before, this was not the case for gH and gL, that account now for 28.2% and 6.3% of all ATRs, respectively 5. Both glycoproteins are, like gB and gD, part of the herpes virus cell entry machinery and may therefore serve as targets for a vaccination approach. Indeed, our data suggest that some identified ATRs might induce neutralizing antibodies, as mapped targets on the 3D-structures matched known targets of neutralizing antibodies against HSV gB and gD or might block receptor binding sites for fusion and cell entry, like Nectin-1 on gD22-27. On the other hand, signals on gH were widely scattered over the antigen, and both gH and gL showed less relative antigenicity, when corrected for protein length. The fact that both proteins account for 34.5% of all ATRs also implies that they could be successfully applied together as full length, recombinant antigens. The conspicuous accumulation of responses in the C-terminal half of gG is in accord with findings by Perelygina et al., who detected diagnostic sensitivities of 80% with recombinant membrane-associated (mgG) segments of glycoprotein G (gG) in ELISA assays.5 The chip assay also disclosed the high antigenicity of the C-terminal region of gC. This domain, located inside the virus, has been cut off in previous cloning strategies and was not further considered.5 The importance of intracellular epitopes in BV is also underlined by the fact that the first linear epitope on gD is positioned behind the TM-region (i.e. inside the virus) at the end of its C-terminus.6 All coding sequences of BV virus proteins and miRNAs were found to be highly conserved, and differences were only observed for non-coding sequences. Thus, the peptides identified in this study do not correspond to highly variable regions in the BV glycoproteins, which renders them suitable as diagnostic targets.28 Beyond that, the peptide arrays highlight amino acid sequences of these glycoproteins preferentially addressed by the host’s immune system. The majority of targets lie within the so-called entry complex formed by gB/gD/gH and gL. Mainly gD in HSV and BV has been of interest for vaccine studies due to its binding of five different cell surface receptors.29 Also gB of HSV was studied in detail, as single amino-acid exchanges were found to reduce cell entry and pathogenesis in vivo.30 Further, it is known that gC binds C3b and therefore inhibits the complement system during infection of HSV. However, it is speculated that antibodies against gC may reduce virulence.31 In contrast, a secreted form of gG of HSV-2 was tested as prophylactic vaccine against genital infection, which lead to cellular immunity, but not to neutralizing antibody titers32 Thus, to test whether 10 ACS Paragon Plus Environment

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antibodies against major ATRs found in our studies may have neutralizing capacities would inform the design of a possible polypeptide-based BV vaccine in the future. A set of eight relevant ATRs from the chip array study was selected to develop a beadbased multiplex immunoassay on the well-established Luminex instrument platform. The method transfer was technically feasible and successful, but the recovery of signals observed in the chip assay was incomplete (i.e. 69%). One reason might be the use of a different secondary anti-human antibody, which might not have optimal binding capacity to macaque antibodies. In addition, the peptide length used in both assays was different: The sequences on the slides were composed of 15mers, whereas in the multiplex system whole ATRs (with up to 42 amino acids) were used as a target. This might have led to different readouts, as long peptides may bury the addressed epitopes due to inappropriate secondary structure formation or aggregation on the bead. On the other hand, other groups reported high values of accuracy, when testing surrogate markers of BV on recombinant gD alone or Herpes virus papio type 2 (HVP2) 15,16. Finally, the fact that peptides on the chip array were Cterminally bound to the surface, whereas the beads were loaded with N-terminally modified peptides, may have induced different binding properties. The analysis of the chip microarray data did not apply a fixed threshold value, but a dynamical cutoff was calculated for each individual serum from all peptide spots on the respective array.8 This provides higher analytical depth and allows detection of very weak signals. In contrast, a fixed threshold across all ATRs was applied in the bead assay, which led to discarding weak antibody detections. An improved setup with a larger number of negative reference sera would allow defining specific thresholds per ATR. We suggest that future work to further optimize the performance of the Luminex assay presentation should center on 1) altering the linker length and chemistry between peptides and beads, 2) attaching the peptides via the C-terminus to the beads, 3) replacing long ATRs by shorter peptides that closer resemble the 15mers used in the chip assay, and 4) increase the number of used ATRs in the bead assay to improve the coverage for each serum sample. The chip-based classification matched that of the ELISA assay with one exception: Serum #42 was ELISA-negative, but categorized as positive based on 15 independent ATRs. This clear result, and the observation that a further serum sample from this animal obtained two years later showed a positive reaction in the anti-HSV ELISA, suggest that serum #42 has been misclassified by the anti-HSV ELISA. This may be either due to a shortcoming of the assay, or because serum #42 was drawn at an early stage of seroconversion with not yet fully developed antibody responses. Serum #42 was also tested as negative in the bead assay. However, of the 15 ATRs binding to 11 ACS Paragon Plus Environment


serum sample #42 on the chip, only ATR D12 was represented in the bead assay. The 35 residue ATR D12 exhibited a particularly poor performance in all samples compared to the corresponding chip peptides. Factors discussed above may have led to a failure to detect D12 and to the false negative classification. The present study demonstrates that peptide-based assays need to be multiplexed, as more than one target sequence is required to avoid false negative results. This is especially important if no or low antibody titers are present against e.g. ATR18, which incorporates the described immunodominant linear epitope reported before.6 In the BV case study, the assay based on eight individual, carefully chosen ATRs delivered the same result as an ELISA based on HSV-1 antigens. However, the data imply that a higher accuracy than with ELISA is achievable by peptide arrays. This led us to define the chip method as the best available standard for classification that reaches perfect accuracy in this limited sample set. However, the bead based assay allows easier handling of samples and procedures and can be applied in routine settings. To reach the same high level of accuracy by a bead-based assay, a larger set of ATRrepresenting peptides may be required and this is feasible from a technical point of view. Thus, a validation of both methods and the proposed epitope set using an independent, larger set of sera is warranted. Conclusion High resolution methods for the analysis of B cell responses contribute significantly to a more informed diagnosis of infections by serology and to precision medicine in general. The present study suggests a biomarker discovery strategy that comprises an in-depth detection of ATRs using optimized peptide array analytics, followed by the definition of a multiplexed bead-based assay with the most relevant ATRs. The latter assay requires a low (2 l) amount of serum, has a simple pre-analytical protocol, a high throughput of 3 samples per minute and runs on an established, widely used instrument platform. With a classification accuracy of 97% (35 out of 36 samples) compared to the chip assay, we demonstrate that a switch from a chip-based epitope discovery format to a more practical bead format is indeed feasible and associated with a minor loss of diagnostic performance. We believe that its application to precisely diagnose BV infections, but also to characterize other infectious diseases, is an attractive subject of future studies. Keywords: Herpes B – multiplex immunoassays – peptide chips – serology – Luminex – SPOT method


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Author contributions: SKH and AW manufactured chips and performed chip experiments; UB developed serum pre-analytics; FC and UD designed and synthesized peptides; CSH characterized and provided the serum samples; CS performed bead immunoassay experiments; HJF, RF and MB conceived the study, analyzed the data and wrote the manuscript.

Supporting Information: The Supporting Information is available free of charge on the ACS Publications website at DOI: [XXX please insertXX] Origin of samples; chip layout; peptide synthesis; peptides used in the multiplex bead assays; Table with ATRs in Chip Assays; frequency of ATR detection; signal strengths; distribution of numbers of positive ATR calls (.pdf) All experimental values (.xlsx) Author Information: Corresponding Authors *Phone: +49 (0)531-6181-3400. Email: [email protected] and [email protected] ORCID: Mark Brönstrup: 0000-0002-8971-7045 Notes: The authors declare no competing ﬁnancial interest. Acknowledgements: S.K. Hotop was recipient of a fellowship from the GerhardHunsmann-Stiftung. We thank the HZI peptide facility (Dr. Dr. W. Tegge) for its peptide synthesis support.



Figures_and_Tables

Figure 1:

Figure 1. Primary chip data: Effect of pre-adsorption step on signal quality. Panels A and B display unprocessed data obtained with serum sample #11. White arrowheads in panels A and B (lower left corner) point out spot series (ATR D18: Spots D121 to D128) whose intensities are illustrated as corresponding bar graphs in panels C and D.


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Figure 2:

Figure 2. Microserology chip data. Panel A: The upper half of the chip was incubated with sample 24 (positive), the lower half with sample 37 (negative). Green spots in the unprocessed fluorescence pattern indicate location of biotin markers. Occasionally occurring spurious responses, as seen in lower half array field 'gG' (yellow spot in panel A, bar marked by asterisk in panel C), are recognized as artefactual by two criteria: a) Duplicate spot not responding. b) Comparable response strength in both red and green fluorescence channels. Panel B: Newly discovered ATR_H108 and ATR_H121, projected onto the surface of the 3D structure of gH (PDB: 3M1C). Color code: Brown: gH; grey: gL moiety of gH/gL heterodimer; blue: ATR_H108; magenta: ATR_H121. Panel C: Red fluorescence intensities of panel A) are represented as bar heights. The signals for ATR_H108 and ATR_H121 are marked by single and double hashtags, respectively.



Figure 3:

Figure 3. Distribution of positively reacting peptides across six BV antigens. Antigens are represented by boxes enclosing matrices with 34 rows each for 34 positive sera and as many columns as there are peptides on the chip covering the respective antigen (numbers in parentheses at bottom right corner of respective box; polypeptide chain length stated at upper right corner). Positively reacting peptides are indicated by a black dot at the appropriate matrix location. ATRs used in the bead assay are marked by asterisks.


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Figure 4:

Figure 4. ATRs ranked by frequency of detection in chip assay. The survey encompasses 34 verified BVpositive sera. ATRs reacting positive with seven or more sera are shown. None of the ATRs are recognized by BV-negative sera. Numbers in parentheses refer to amino acid residue positions in respective polypeptide chains. Asterisks indicate newly discovered epitopes. Arrows point to ATRs synthesized as soluble reporter peptides and employed in bead assays.



Figure 5:

Figure 5. Bead-based assay. A) Assay principle, showing beads that are coded by a characteristic fluorescence dye, coated with avidin and biotinylated peptides. The amount of analyte is quantified by measuring the MFI of a fluorescently labelled secondary antibody. B) Multiplex principle: The binding to different peptides, each one coated separately to beads of different coding dye can be detected in a single sample. In this example, only one peptide is recognized. C) Different signal strengths clearly discriminate positive sera (31 samples) from negative sera (5 samples) in the bead assay. ATRresponses were called positive if they exceeded an arbitrarily set threshold value of 15-fold background level. Left: Signal strength distribution for BV negative sera (subset E, in grey). Right: Signal strength distribution for BV positive sera (in black). With the exception of sample #42 all members of subsets B, C and F were represented by at least one positive ATR-call (compare Supplementary Figure S3). The values pointed out by arrows represent the following: 11.5: Signal strength of highest ATR-read seen with a verified BV-negative sample. 15.5: Signal strength of lowest ATR-read called positive. 27.1: Signal strength of lowest critical positive ATR-read, i.e. a read which if called negative would turn the classification of the entire sample from positive to negative (in this case sample #28).


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Table 1: Overview on serum samples, methods and test results

ELISA

Chip (2014)

Chip (this study)

Bead flow

Positive references (NBVRC)

pos.

pos.

pos.

n.t.

4-32

Part of test set (DPZ)

pos.

pos.

pos.

pos.

C

33


pos.

neg.

pos.

pos.

D

34-36

Negative references (NBVRC)

neg.

neg.

neg.

n.t.

E

37-41


neg.

neg.

neg.

neg.

F

42


neg.

pos.1

pos.2

neg.

Subset

Serum #

A

1-3

B

Description

1Tentative 2Confirmed.

n.t.: not tested. Erroneous assignments in bold



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