High-density Peptide Microarrays Research Articles

Identification of highly conserved Trypanosoma cruzi antigens for the development of a universal serological diagnostic assay

ABSTRACT Chagas Disease is an important neglected tropical disease caused by Trypanosoma cruzi. There is no gold standard for diagnosis and commercial serological tests perform poorly in certain locations. By aligning T. cruzi genomes covering parasite genetic and geographic diversity, we identified highly conserved proteins that could serve as universal antigens for improved diagnosis. Their antigenicity was tested in high-density peptide microarrays using well-characterized plasma samples, including samples presenting true infections but discordant serology. Individual and combination of epitopes were also evaluated in peptide-ELISAs. We identified >1400 highly conserved T. cruzi proteins evaluated in microarrays. Remarkably, T. cruzi positive controls had a different epitope recognition profile compared to serologically discordant samples. In particular, multiple T. cruzi antigens used in current tests and their strain-variants, and novel epitopes thought to be broadly antigenic failed to be recognized by discordant samples. Nonetheless, >2000 epitopes specifically recognized by IgGs from both positive controls and discordant samples were identified. Evaluation of selected peptides in ELISA further illustrated the extensive variation in antibody profiles among subjects and a peptide combination could outperform a commercial ELISA, increasing assay sensitivity from 52.3% to 72.7%. Individual variation in antibody profiles rather than T. cruzi diversity appears to be the main factor driving differences in serological diagnostic performance according to geography, which will be important to further elucidate. ELISA with a combination of peptides recognized by a greater number of individuals could better capture infections, and further development may lead to an optimal antigen mixture for a universal diagnostic assay.

ASFV epitope mapping by high density peptides microarrays

African swine fever (ASF) is an acute, highly contagious and deadly infectious disease. It is a threat to animal health with major potential economic and societal impact. Despite decades of ASF vaccine research, still some gaps in knowledge are hindering the development of a functional vaccine. Worth mentioning are gaps in understanding the mechanism of ASF infection and immunity, as well as the fact that - in case of this disease - virus proteins, so-called protective antigens, responsible for inducing protective immune responses in pigs are not identified yet. In this paper we elaborate on a methodology to identify protective antigens based on epitope mapping by microarray technology. High density peptide microarrays, combined with fluorescence scanning, have been used to analyze the interaction of peptide sequences of African swine fever virus (ASFV) proteins with antibodies present in inactivated serum from infected and healthy animals. The study evidenced ASFV proteins already under the radar for vaccine development, such as p54, and identified specific sequences in those proteins that may become the focus for future vaccine candidates. Such methodology is amenable to automation and high-throughput and may help developing better targeting for next generation vaccines.

Genome-wide epitope mapping across multiple host species reveals significant diversity in antibody responses to Coxiella burnetii vaccination and infection.

Coxiella burnetii is an important zoonotic bacterial pathogen of global importance, causing the disease Q fever in a wide range of animal hosts. Ruminant livestock, in particular sheep and goats, are considered the main reservoir of human infection. Vaccination is a key control measure, and two commercial vaccines based on formalin-inactivated C. burnetii bacterins are currently available for use in livestock and humans. However, their deployment is limited due to significant reactogenicity in individuals previously sensitized to C. burnetii antigens. Furthermore, these vaccines interfere with available serodiagnostic tests which are also based on C. burnetii bacterin antigens. Defined subunit antigen vaccines offer significant advantages, as they can be engineered to reduce reactogenicity and co-designed with serodiagnostic tests to allow discrimination between vaccinated and infected individuals. This study aimed to investigate the diversity of antibody responses to C. burnetii vaccination and/or infection in cattle, goats, humans, and sheep through genome-wide linear epitope mapping to identify candidate vaccine and diagnostic antigens within the predicted bacterial proteome. Using high-density peptide microarrays, we analyzed the seroreactivity in 156 serum samples from vaccinated and infected individuals to peptides derived from 2,092 open-reading frames in the C. burnetii genome. We found significant diversity in the antibody responses within and between species and across different types of C. burnetii exposure. Through the implementation of three different vaccine candidate selection methods, we identified 493 candidate protein antigens for protein subunit vaccine design or serodiagnostic evaluation, of which 65 have been previously described. This is the first study to investigate multi-species seroreactivity against the entire C. burnetii proteome presented as overlapping linear peptides and provides the basis for the selection of antigen targets for next-generation Q fever vaccines and diagnostic tests.

Fast Preparation of Peptide Tools to address new emerging Viruses: Case example SARS-CoV-2

Abstract The emergence of new virus species or variants poses a major challenge for healthcare systems. Countermeasures, including vaccine development, need to be developed without delay to ensure success. Vaccine and antibody development depend on highly efficient tools for antigen discovery, epitope mapping, diagnostics and (clinical) immune monitoring. Here we present a workflow for peptide tool development that can be rapidly adapted to emerging viruses. It combines bioinformatics algorithms and an ultra-high throughput peptide synthesis method alongside high-content assay formats. Data from different SARS-CoV-2 studies are presented addressing elucidation of both cellular and humoral immune responses. Robust spike-specific cellular immune responses were measured using SARS-CoV-2 Spike PepMix ™peptide pools during infection and/or vaccine response monitoring [ 1]. Using epitope mapping (matrix) pools an immunodominant T-cell epitope was identified that may contribute to the optimization of existing vaccines [ 2]. In addition, the immune response of Covid-19 convalescents against all major SARS-CoV-2 proteins was analyzed [ 2]. Humoral immune response analysis by high-density peptide microarrays identified immunodominant B cell epitopes as starting points for the development of alternative vaccines and diagnostic tests [ 3]. In conclusion, we show that readily available and tailored peptide formats, as exemplified for SARS-CoV-2, are important tools that contribute to the fast development and optimization of vaccines and diagnostic tests for emerging viruses of concern.

Immunoglobulin profiling with large high-density peptide microarrays as screening method to detect candidate proteins for future biomarker detection in dogs with steroid-responsive meningitis-arteritis.

Large high-density peptide microarrays are a suitable screening method to detect possible autoantigens which might be involved in the pathogenesis of SRMA. The IgA and IgG profile of pooled serum samples of 5 dogs with SRMA and 5 dogs with neck pain due to intervertebral disc herniation (IVDH) without ataxia or paresis were compared via commercially available high-density peptide microarrays (Discovery Microarray) containing 29,240 random linear peptides. Canine distemper virus nucleoprotein (CDVN) served as positive control as all dogs were vaccinated. Common motifs were compared to amino acid sequences of known proteins via databank search. One suitable protein was manually selected for further analysis with a smaller customized high-density peptide microarray. Pooled serum of dogs with SRMA and IVDH showed different IgA and IgG responses on Discovery Microarray. Only top IgG responses of dogs with SRMA showed a common motif not related to the control protein CDVN. This common motif is part of the interleukin 1 receptor antagonist protein (IL1Ra). On IL1Ra, dogs with SRMA displayed IgA binding to an additional epitope, which dogs with IVDH did not show. IL1Ra is an anti-inflammatory acute phase protein. Different immunoglobulin binding patterns on IL1Ra could be involved in the pathogenesis of SRMA and IL1Ra might be developed as future biomarker for SRMA.

Current situation of snakebites envenomation in the Neotropics: Biotechnology, a versatile tool in the production of antivenoms.

Snakebite envenomation is a neglected tropical disease that affects millions of people around the world with a great impact on health and the economy. Unfortunately, public health programs do not include this kind of disease as a priority in their social programs. Cases of snakebite envenomations in the Neotropics are inaccurate due to inadequate disease management from medical records to the choice of treatments. Victims of snakebite envenomation are primarily found in impoverished agricultural areas where remote conditions limit the availability of antivenom. Antivenom serum is the only Food and Drug Administration-approved treatment used up to date. However, it has several disadvantages in terms of safety and effectiveness. This review provides a comprehensive insight dealing with the current epidemiological status of snakebites in the Neotropics and technologies employed in antivenom production. Also, modern biotechnological tools such as transcriptomic, proteomic, immunogenic, high-density peptide microarray and epitope mapping are highlighted for producing new-generation antivenom sera. These results allow us to propose strategic solutions in the Public Health Sector for managing this disease. Keywords: antivenom, biotechnology, neglected tropical disease, omics, recombinant antibody.

Manufacturing of Peptide Microarrays Based on Catalyst-Free Click Chemistry.

Immobilization of peptides to a solid surface is frequently an important first step before they can be probed with a variety of biological samples in a heterogeneous assay format for research and clinical diagnostic purposes. Peptides can be derivatized in many ways to subsequently covalently attach them to an activated solid surface such as, for instance, epoxy-functionalized glass slides. Here, we describe a clean, efficient, and reproducible fabrication process based on catalyst-free click chemistry compatible with the construction of low- to high-density peptide microarrays.

High-Throughput Screening Identifies Synthetic Peptides with Antibacterial Activity against Mycobacterium abscessus and Serum Stability.

The rise in antibiotic resistance in bacteria has spawned new technological approaches for identifying novel antimicrobials with narrow specificity. Current antibiotic treatment regimens and antituberculosis drugs are not effective in treating Mycobacterium abscessus. Meanwhile, antimicrobial peptides are gaining prominence as alternative antimicrobials due to their specificity toward anionic bacterial membranes, rapid action, and limited development of resistance. To rapidly identify antimicrobial peptide candidates, our group has developed a high-density peptide microarray consisting of 125,000 random synthetic peptides screened for interaction with the mycobacterial cell surface of M. abscessus morphotypes. From the array screening, peptides positive for interaction were synthesized and their antimicrobial activity was validated. Overall, six peptides inhibited the M. abscessus smooth morphotype (IC50 = 1.7 μM for all peptides) and had reduced activity against the M. abscessus rough morphotype (IC50 range: 13–82 μM). Peptides ASU2056 and ASU2060 had minimum inhibitory concentration values of 32 and 8 μM, respectively, against the M. abscessus smooth morphotype. Additionally, ASU2060 (8 μM) was active against Escherichia coli, including multidrug-resistant E. coli clinical isolates, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. ASU2056 and ASU2060 exhibited no significant hemolytic activity at biologically relevant concentrations, further supporting these peptides as promising therapeutic candidates. Moreover, ASU2060 retained antibacterial activity after preincubation in human serum for 24 h. With antimicrobial resistance on the rise, methods such as those presented here will streamline the peptide discovery process for targeted antimicrobial peptides.

High-resolution analysis of long-term serum antibodies in humans following convalescence of SARS-CoV-2 infection

Long-term antibody responses to SARS-CoV-2 have focused on responses to full-length spike protein, specific domains within spike, or nucleoprotein. In this study, we used high-density peptide microarrays representing the complete proteome of SARS-CoV-2 to identify binding sites (epitopes) targeted by antibodies present in the blood of COVID-19 resolved cases at 5 months post-diagnosis. Compared to previous studies that evaluated epitope-specific responses early post-diagnosis (< 60 days), we found that epitope-specific responses to nucleoprotein and spike protein have contracted, and that responses to membrane protein have expanded. Although antibody titers to full-length spike and nucleoprotein remain steady over months, taken together our data suggest that the population of epitope-specific antibodies that contribute to this reactivity is dynamic and evolves over time. Further, the spike epitopes bound by polyclonal antibodies in COVID-19 convalescent serum samples aligned with known target sites that can neutralize viral activity suggesting that the maintenance of these antibodies might provide rapid serological immunity. Finally, the most dominant epitopes for membrane protein and spike showed high diagnostic accuracy providing novel biomarkers to refine blood-based antibody tests. This study provides new insights into the specific regions of SARS-CoV-2 targeted by serum antibodies long after infection.

EPIphany-A Platform for Analysis and Visualization of Peptide Immunoarray Data.

Antibodies are critical effector molecules of the humoral immune system. Upon infection or vaccination, populations of antibodies are generated which bind to various regions of the invading pathogen or exogenous agent. Defining the reactivity and breadth of this antibody response provides an understanding of the antigenic determinants and enables the rational development and assessment of vaccine candidates. High-resolution analysis of these populations typically requires advanced techniques such as B cell receptor repertoire sequencing, mass spectrometry of isolated immunoglobulins, or phage display libraries that are dependent upon equipment and expertise which are prohibitive for many labs. High-density peptide microarrays representing diverse populations of putative linear epitopes (immunoarrays) are an effective alternative for high-throughput examination of antibody reactivity and diversity. While a promising technology, widespread adoption of immunoarrays has been limited by the need for, and relative absence of, user-friendly tools for consideration and visualization of the emerging data. To address this limitation, we developed EPIphany, a software platform with a simple web-based user interface, aimed at biological users, that provides access to important analysis parameters, data normalization options, and a variety of unique data visualization options. This platform provides researchers the greatest opportunity to extract biologically meaningful information from the immunoarray data, thereby facilitating the discovery and development of novel immuno-therapeutics.

Dynamics of the Magnitude, Breadth and Depth of the Antibody Response at Epitope Level Following Dengue Infection.

Dengue is a major public health problem in tropical and sub-tropical regions worldwide. Since the Zika epidemic and the increased co-circulation of other arboviruses, the serology-based diagnosis of dengue has become more problematic due to the high antigenic resemblance, especially among the flavivirus family. Therefore, a more comprehensive understanding of the diversity, specificity and temporal evolution of the antibody response following dengue infection is needed. In order to close this knowledge gap, we used a high-density peptide microarray of 9,072 linear peptides covering the entire proteome diversity of dengue, Zika, yellow fever and chikungunya viruses. The IgM and IgG antibody responses were measured against the designed microarray in symptomatic dengue infected individuals from an arbovirus endemic area in Peru and in overseas travelers returning to Belgium, as representatives of multiple-exposed and primary infections, respectively. Serum samples were collected longitudinally across four time points over the period of six months in Peru and over two time points in travelers. We show that epitopes eliciting the strongest flavivirus cross-reactive antibodies, in both primary and secondary infections were concentrated in the capsid, E, NS1, NS3 and NS5 proteins. The IgG antibody responses against NS1 and NS3 followed a rise-and-fall pattern, with peak titers between two to four weeks after onset of illness. The response to the E and NS5 proteins increased rapidly in the acute phase and was maintained at stable levels until at least 6 months after illness. A more scattered IgM antibody reactivity across the viral proteome was observed in the acute phase of the disease and that persisted through the 6-month window. The magnitude, breadth (i.e. number of unique epitopes targeted) and depth (i.e. number of epitope variants recognized) of the IgG response was higher in secondary infections compared to primary infections. For IgM antibodies, the magnitude of the response was higher in primary infected individuals whereas the breadth and depth of the response was lower in this group compared with the endemic subjects. Finally, through this arboviral proteome-wide epitope mapping, we were able to identify IgM and IgG dengue-specific epitopes which can be useful serological markers for dengue diagnosis and serostatus determination.

Identification of antigenic linear peptides in the soil-transmitted helminth and Schistosoma mansoni proteome.

The scientific community identified non stool-based biomarkers as the way forward to support soil-transmitted helminth (STH; Ascaris lumbricoides, Trichuris trichiura and the hookworms Ancylostoma duodenale and Necator americanus) and schistosome (S. mansoni and S. haematobium) deworming programs. This support is needed in making the decision of whether or not to stop preventive chemotherapy intervention efforts and to ultimately transition towards a post-intervention surveillance phase. We applied a two-step micro-array approach to identify antigenic linear epitopes in the STH and S. mansoni proteomes. In a first experiment, we identified antigenic peptides by applying sera from 24 STH and/or S. mansoni infected Ethiopian children on a high-density peptide microarray containing 3.3 million peptides derived from the complete STH and S. mansoni proteomes. A second array experiment with 170,185 peptides that were recognized in the first array was designed to identify non-specific antibody reactivity by applying sera from 24 healthy individuals from Belgium (a non-endemic country). From this array testing cascade, several peptides were identified for STH but none of them appeared to be unique for one species. We therefore concluded that for STH, none of the peptides revealed to be sufficiently sensitive or species specific. For S. mansoni, some promising peptides were identified prompting future investigation. Based on these results, it is unlikely that linear epitopes would be highly useful in detecting species-specific antibody responses to STH in endemic communities. For S. mansoni, one particular peptide of the micro-exon gene 12 (MEG-12) protein deserves further research. In addition, this study emphasizes the need of well-characterized biobanks for biomarker discovery, particularly when the integration of multiple disease programs is envisioned.

Immunoreactive peptide maps of SARS-CoV-2

Serodiagnosis of SARS-CoV-2 infection is impeded by immunological cross-reactivity among the human coronaviruses (HCoVs): SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43, 229E, HKU1, and NL63. Here we report the identification of humoral immune responses to SARS-CoV-2 peptides that may enable discrimination between exposure to SARS-CoV-2 and other HCoVs. We used a high-density peptide microarray and plasma samples collected at two time points from 50 subjects with SARS-CoV-2 infection confirmed by qPCR, samples collected in 2004–2005 from 11 subjects with IgG antibodies to SARS-CoV-1, 11 subjects with IgG antibodies to other seasonal human coronaviruses (HCoV), and 10 healthy human subjects. Through statistical modeling with linear regression and multidimensional scaling we identified specific peptides that were reassembled to identify 29 linear SARS-CoV-2 epitopes that were immunoreactive with plasma from individuals who had asymptomatic, mild or severe SARS-CoV-2 infections. Larger studies will be required to determine whether these peptides may be useful in serodiagnostics.

Major antigen and paramyosin proteins as candidate biomarkers for serodiagnosis of canine infection by zoonotic Onchocerca lupi.

Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dog is mandatory. In this study, an immunoproteomic analyses was performed using a combination of immunoblotting and mass spectrometry techniques. Onchocerca lupi major antigen (Ol-MJA) and paramyosin (Ol-PARA) proteins were identified as potential biomarkers for serodiagnosis. Linear epitopes were herein scanned for both proteins using high-density peptide microarray. Sera collected from dog infected with O. lupi and healthy animal controls led to the identification of 11 immunodominant antigenic peptides (n = 7 for Ol-MJA; n = 4 for Ol-PARA). These peptides were validated using sera of dogs uniquely infected with the most important filarioids infesting dogs either zoonotic (Dirofilaria repens, Dirofilaria immitis) or not (Acanthocheilonema reconditum and Cercopithifilaria bainae). Overall, six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential antigens for the serological detection of canine O. lupi infection. The molecular and proteomic dataset herein reported should provide a useful resource for studies on O. lupi toward supporting the development of new interventions (drugs, vaccines and diagnostics) against canine onchocercosis.

Substitutional landscape of a split fluorescent protein fragment using high-density peptide microarrays.

Split fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in super-resolution microscopy. We have here established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover. We have screened more than 12,000 variants of the beta-strand split fragment using high-density peptide microarrays for binding and functional complementation in Green Fluorescent Protein. We studied the effect of peptide length and the effect of different linkers to the solid support. We further mapped the effect of all possible amino acid substitutions on each position as well as in the context of some single and double amino acid substitutions. As all peptides were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing for confirmation of the robustness and precision of the method. Based on experiments in solution, we conclude that under the given conditions, the signal intensity on the peptide microarray faithfully reflects the binding affinity between the split fragments. With this, we are able to identify a peptide with 9-fold higher affinity than the starting peptide.

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

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.

Unbiased Characterization of Peptide-HLA Class II Interactions Based on Large-Scale Peptide Microarrays; Assessment of the Impact on HLA Class II Ligand and Epitope Prediction.

Human Leukocyte Antigen class II (HLA-II) molecules present peptides to T lymphocytes and play an important role in adaptive immune responses. Characterizing the binding specificity of single HLA-II molecules has profound impacts for understanding cellular immunity, identifying the cause of autoimmune diseases, for immunotherapeutics, and vaccine development. Here, novel high-density peptide microarray technology combined with machine learning techniques were used to address this task at an unprecedented level of high-throughput. Microarrays with over 200,000 defined peptides were assayed with four exemplary HLA-II molecules. Machine learning was applied to mine the signals. The comparison of identified binding motifs, and power for predicting eluted ligands and CD4+ epitope datasets to that obtained using NetMHCIIpan-3.2, confirmed a high quality of the chip readout. These results suggest that the proposed microarray technology offers a novel and unique platform for large-scale unbiased interrogation of peptide binding preferences of HLA-II molecules.

HLA Class II Specificity Assessed by High-Density Peptide Microarray Interactions.

The ability to predict and/or identify MHC binding peptides is an essential component of T cell epitope discovery, something that ultimately should benefit the development of vaccines and immunotherapies. In particular, MHC class I prediction tools have matured to a point where accurate selection of optimal peptide epitopes is possible for virtually all MHC class I allotypes; in comparison, current MHC class II (MHC-II) predictors are less mature. Because MHC-II restricted CD4+ T cells control and orchestrated most immune responses, this shortcoming severely hampers the development of effective immunotherapies. The ability to generate large panels of peptides and subsequently large bodies of peptide-MHC-II interaction data are key to the solution of this problem, a solution that also will support the improvement of bioinformatics predictors, which critically relies on the availability of large amounts of accurate, diverse, and representative data. In this study, we have used rHLA-DRB1*01:01 and HLA-DRB1*03:01 molecules to interrogate high-density peptide arrays, in casu containing 70,000 random peptides in triplicates. We demonstrate that the binding data acquired contains systematic and interpretable information reflecting the specificity of the HLA-DR molecules investigated, suitable of training predictors able to predict T cell epitopes and peptides eluted from human EBV-transformed B cells. Collectively, with a cost per peptide reduced to a few cents, combined with the flexibility of rHLA technology, this poses an attractive strategy to generate vast bodies of MHC-II binding data at an unprecedented speed and for the benefit of generating peptide-MHC-II binding data as well as improving MHC-II prediction tools.

An interactive database for the investigation of high-density peptide microarray guided interaction patterns and antivenom cross-reactivity.

Snakebite envenoming is a major neglected tropical disease that affects millions of people every year. The only effective treatment against snakebite envenoming consists of unspecified cocktails of polyclonal antibodies purified from the plasma of immunized production animals. Currently, little data exists on the molecular interactions between venom-toxin epitopes and antivenom-antibody paratopes. To address this issue, high-density peptide microarray (hdpm) technology has recently been adapted to the field of toxinology. However, analysis of such valuable datasets requires expert understanding and, thus, complicates its broad application within the field. In the present study, we developed a user-friendly, and high-throughput web application named “Snake Toxin and Antivenom Binding Profiles” (STAB Profiles), to allow straight-forward analysis of hdpm datasets. To test our tool and evaluate its performance with a large dataset, we conducted hdpm assays using all African snake toxin protein sequences available in the UniProt database at the time of study design, together with eight commercial antivenoms in clinical use in Africa, thus representing the largest venom-antivenom dataset to date. Furthermore, we introduced a novel method for evaluating raw signals from a peptide microarray experiment and a data normalization protocol enabling intra-microarray and even inter-microarray chip comparisons. Finally, these data, alongside all the data from previous similar studies by Engmark et al., were preprocessed according to our newly developed protocol and made publicly available for download through the STAB Profiles web application (http://tropicalpharmacology.com/tools/stab-profiles/). With these data and our tool, we were able to gain key insights into toxin-antivenom interactions and were able to differentiate the ability of different antivenoms to interact with certain toxins of interest.The data, as well as the web application, we present in this article should be of significant value to the venom-antivenom research community. Knowledge gained from our current and future analyses of this dataset carry the potential to guide the improvement and optimization of current antivenoms for maximum patient benefit, as well as aid the development of next-generation antivenoms.

Antibody responses to Zika virus proteins in pregnant and non-pregnant macaques.

The specificity of the antibody response against Zika virus (ZIKV) is not well-characterized. This is due, in part, to the antigenic similarity between ZIKV and closely related dengue virus (DENV) serotypes. Since these and other similar viruses co-circulate, are spread by the same mosquito species, and can cause similar acute clinical syndromes, it is difficult to disentangle ZIKV-specific antibody responses from responses to closely-related arboviruses in humans. Here we use high-density peptide microarrays to profile anti-ZIKV antibody reactivity in pregnant and non-pregnant macaque monkeys with known exposure histories and compare these results to reactivity following DENV infection. We also compare cross-reactive binding of ZIKV-immune sera to the full proteomes of 28 arboviruses. We independently confirm a purported ZIKV-specific IgG antibody response targeting ZIKV nonstructural protein 2B (NS2B) that was recently reported in ZIKV-infected people and we show that antibody reactivity in pregnant animals can be detected as late as 127 days post-infection (dpi). However, we also show that these responses wane over time, sometimes rapidly, and in one case the response was elicited following DENV infection in a previously ZIKV-exposed animal. These results suggest epidemiologic studies assessing seroprevalence of ZIKV immunity using linear epitope-based strategies will remain challenging to interpret due to susceptibility to false positive results. However, the method used here demonstrates the potential for rapid profiling of proteome-wide antibody responses to a myriad of neglected diseases simultaneously and may be especially useful for distinguishing antibody reactivity among closely related pathogens.