Antibody recognition of the dengue virus proteome and implications for development of vaccines.

Dengue is a mosquito-borne infection caused by four distinct serotypes of dengue virus, each appearing cyclically in the tropics and subtropics along the equator. Although vaccines are currently under development, none are available to the general population. One of the main impediments to the successful advancement of these vaccines is the lack of well-defined immune correlates of protection. Here, we describe a protein microarray approach for measuring antibody responses to the complete viral proteome comprised of the structural (capsid, membrane, and envelope) and nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) components of all four dengue virus serotypes (1 to 4). We examined rhesus macaques vaccinated with tetravalent vaccines consisting of live-attenuated virus (LAV) or purified inactivated virus (PIV), followed by boosting with LAV and challenging with wild-type dengue virus. We detected temporal increases in antibodies against envelope proteins in response to either vaccine, while only the PIV/LAV vaccination strategy resulted in anticapsid antibodies. In contrast to results from vaccination, naïve macaques challenged with wild-type viruses of each serotype demonstrated a balanced response to nonstructural and structural components, including responses against the membrane protein. Our results demonstrate discriminating details concerning the nature of antibody responses to dengue virus at the proteomic level and suggest the usefulness of this information for vaccine development.

Immune response biomarker profiling application on ProtoArray protein microarrays.

The development of autoantibodies is observed in autoimmune disorders and numerous cancers. Consequently, autoantibodies form the basis of potential diagnostic and prognostic assays, as well as approaches for monitoring disease progression and treatment response. The effective use of autoantigen biomarkers for these applications, however, is contingent upon the identification of not one but multiple biomarkers. This is a consequence of the observation that the development of autoantibodies to any given protein is typically seen only in a fraction of patients. We have previously demonstrated the utility of functional protein microarrays containing thousands of different human proteins (ProtoArrays) for discovering novel autoimmune biomarkers in serum and plasma. Here, we describe a protocol for detecting autoantibodies in urine.

Proteomic basis of the antibody response to monkeypox virus infection examined in cynomolgus macaques and a comparison to human smallpox vaccination.

Monkeypox is a zoonotic viral disease that occurs primarily in Central and West Africa. A recent outbreak in the United States heightened public health concerns for susceptible human populations. Vaccinating with vaccinia virus to prevent smallpox is also effective for monkeypox due to a high degree of sequence conservation. Yet, the identity of antigens within the monkeypox virus proteome contributing to immune responses has not been described in detail. We compared antibody responses to monkeypox virus infection and human smallpox vaccination by using a protein microarray covering 92-95% (166-192 proteins) of representative proteomes from monkeypox viral clades of Central and West Africa, including 92% coverage (250 proteins) of the vaccinia virus proteome as a reference orthopox vaccine. All viral gene clones were verified by sequencing and purified recombinant proteins were used to construct the microarray. Serum IgG of cynomolgus macaques that recovered from monkeypox recognized at least 23 separate proteins within the orthopox proteome, while only 14 of these proteins were recognized by IgG from vaccinated humans. There were 12 of 14 antigens detected by sera of human vaccinees that were also recognized by IgG from convalescent macaques. The greatest level of IgG binding for macaques occurred with the structural proteins F13L and A33R, and the membrane scaffold protein D13L. Significant IgM responses directed towards A44R, F13L and A33R of monkeypox virus were detected before onset of clinical symptoms in macaques. Thus, antibodies from vaccination recognized a small number of proteins shared with pathogenic virus strains, while recovery from infection also involved humoral responses to antigens uniquely recognized within the monkeypox virus proteome.

Robust-linear-model normalization to reduce technical variability in functional protein microarrays.

Protein microarrays are similar to DNA microarrays; both enabling the parallel interrogation of thousands of probes immobilized on a surface. Consequently, they have benefited from technologies previously developed for DNA microarrays. However, assumptions for the analysis of DNA microarrays do not always translate to protein arrays, especially in the case of normalization. Hence, we have developed an experimental and computational framework to assess normalization procedures for protein microarrays. Specifically, we profiled two sera with markedly different autoantibody compositions. To analyze intra- and interarray variability, we compared a set of control proteins across subarrays and the corresponding spots across multiple arrays, respectively. To estimate the degree to which the normalization could help reveal true biological separability, we tested the difference in the signal between the sera relative to the variability within replicates. Next, by mixing the sera in different proportions (titrations), we correlated the reactivity of proteins with serum concentration. Finally, we analyzed the effect of normalization procedures on the list of reactive proteins. We compared global and quantile normalization, techniques that have traditionally been employed for DNA microarrays, with a novel normalization approach based on a robust linear model (RLM) making explicit use of control proteins. We show that RLM normalization is able to reduce both intra- and interarray technical variability while maintaining biological differences. Moreover, in titration experiments, RLM normalization enhances the correlation of protein signals with serum concentration. Conversely, while quantile and global normalization can reduce interarray technical variability, neither is as effective as RLM normalization in maintaining biological differences. Most importantly, both introduce artifacts that distort the signals and affect the correct identification of reactive proteins, impairing their use for biomarker discovery. Hence, we show RLM normalization is better suited to protein arrays than approaches used for DNA microarrays.

Profiling protein interaction networks with functional protein microarrays.

The word protein is derived from the Greek "prota" meaning "of primary importance", a designation which appropriately acknowledges the central role proteins play in biological systems. Following translation and folding into a remarkable array of three-dimensional structures, individual proteins achieve added complexity and functionality through the addition of modifications including glycosylation, acetylation, methylation, and phosphorylation. This complexity is further expanded through the non-covalent interactions that occur between proteins, and it is these interactions that form the foundation for many of the exquisitely regulated cellular processes essential to life. As a result, protein-protein interactions comprise an important class of targets for drug discovery, and modulation of protein-protein binding represents an emerging therapeutic paradigm. Protein microarrays are an important tool to identify and characterize protein interactions, providing the ability to rapidly develop binding profiles between thousands of proteins in a simple multiplex assay. These assays are highly reproducible, sensitive, and scalable and provide an enabling technology for proteomic research within the rubric of systems biology.

Antibody specificity profiling on functional protein microarrays.

Antibodies represent the end product of an exquisitely complex biological process including recombination, somatic hypermutation, affinity maturation, and self-tolerance, culminating in binding reagents directed against a vast repertoire of antigens. The resultant high affinity and diversity of specificity of these biomolecules has been exploited through the development of immunoassays and biotherapeutics that inaugurated a new era in experimental molecular biology and pharmaceutical drug development. Despite the utility of antibodies for research applications and in disease treatment, they must be employed in the context of an accurate understanding of their binding profile. High-content microarrays comprised of thousands of native, full length human proteins are an important tool in the assessment of antibody specificity.

Extensive antibody cross-reactivity among infectious gram-negative bacteria revealed by proteome microarray analysis.

Antibodies provide a sensitive indicator of proteins displayed by bacteria during sepsis. Because signals produced by infection are naturally amplified during the antibody response, host immunity can be used to identify biomarkers for proteins that are present at levels currently below detectable limits. We developed a microarray comprising approximately 70% of the 4066 proteins contained within the Yersinia pestis proteome to identify antibody biomarkers distinguishing plague from infections caused by other bacterial pathogens that may initially present similar clinical symptoms. We first examined rabbit antibodies produced against proteomes extracted from Y. pestis, Burkholderia mallei, Burkholderia cepecia, Burkholderia pseudomallei, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri, and Escherichia coli, all pathogenic Gram-negative bacteria. These antibodies enabled detection of shared cross-reactive proteins, fingerprint proteins common for two or more bacteria, and signature proteins specific to each pathogen. Recognition by rabbit and non-human primate antibodies involved less than 100 of the thousands of proteins present within the Y. pestis proteome. Further antigen binding patterns were revealed that could distinguish plague from anthrax, caused by the Gram-positive bacterium Bacillus anthracis, using sera from acutely infected or convalescent primates. Thus, our results demonstrate potential biomarkers that are either specific to one strain or common to several species of pathogenic bacteria.

Protein kinase substrate identification on functional protein arrays.

BACKGROUND: Over the last decade, kinases have emerged as attractive therapeutic targets for a number of different diseases, and numerous high throughput screening efforts in the pharmaceutical community are directed towards discovery of compounds that regulate kinase function. The emerging utility of systems biology approaches has necessitated the development of multiplex tools suitable for proteomic-scale experiments to replace lower throughput technologies such as mass spectroscopy for the study of protein phosphorylation. Recently, a new approach for identifying substrates of protein kinases has applied the miniaturized format of functional protein arrays to characterize phosphorylation for thousands of candidate protein substrates in a single experiment. This method involves the addition of protein kinases in solution to arrays of immobilized proteins to identify substrates using highly sensitive radioactive detection and hit identification algorithms. RESULTS: To date, the factors required for optimal performance of protein array-based kinase substrate identification have not been described. In the current study, we have carried out a detailed characterization of the protein array-based method for kinase substrate identification, including an examination of the effects of time, buffer compositions, and protein concentration on the results. The protein array approach was compared to standard solution-based assays for assessing substrate phosphorylation, and a correlation of greater than 80% was observed. The results presented here demonstrate how novel substrates for protein kinases can be quickly identified from arrays containing thousands of human proteins to provide new clues to protein kinase function. In addition, a pooling-deconvolution strategy was developed and applied that enhances characterization of specific kinase-substrate relationships and decreases reagent consumption. CONCLUSION: Functional protein microarrays are an important new tool that enables multiplex analysis of protein phosphorylation, and thus can be utilized to identify novel kinase substrates. Integrating this technology with a systems biology approach to cell signalling will help uncover new layers in our understanding of this essential class of enzymes.

Analysis of the human immune response to vaccinia by use of a novel protein microarray suggests that antibodies recognize less than 10% of the total viral proteome.

Control of smallpox by mass vaccination was one of the most effective public health measures ever employed for eradicating a devastating infectious disease. However, new methods are needed for monitoring smallpox immunity within current vulnerable populations, and for the development of replacement vaccines for use by immunocompromized or low-responding individuals. As a measure for achieving this goal, we developed a protein microarray of the vaccinia virus proteome by using high-throughput baculovirus expression and purification of individual elements. The array was validated with therapeutic-grade, human hyperimmune sera, and these data were compared to results obtained from individuals vaccinated against smallpox using Dryvax. A high level of reproducibility with a very low background were apparent in repetitive assays that confirmed previously reported antigens and identified new proteins that may be important for neutralizing viral infection. Our results suggest that proteins recognized by antibodies from all vaccinees constituted <10% of the total vaccinia proteome.

A critical role for cortactin phosphorylation by Abl-family kinases in PDGF-induced dorsal-wave formation.

Proper regulation of cell morphogenesis and migration by adhesion and growth-factor receptors requires Abl-family tyrosine kinases [1-3]. Several substrates of Abl-family kinase have been identified, but they are unlikely to mediate all of the downstream actions of these kinases on cytoskeletal structure. We used a human protein microarray to identify the actin-regulatory protein cortactin as a novel substrate of the Abl and Abl-related gene (Arg) nonreceptor tyrosine kinases. Cortactin stimulates cell motility [4-6], and its upregulation in several cancers correlates with poor prognosis [7]. Even though cortactin can be tyrosine phosphorylated by Src-family kinases in vitro [8], we show that Abl and Arg are more adept at binding and phosphorylating cortactin. Importantly, we demonstrate that platelet-derived growth-factor (PDGF)-induced cortactin phosphorylation on three tyrosine residues requires Abl or Arg. Cortactin triggers F-actin-dependent dorsal waves in fibroblasts after PDGF treatment and thus results in actin reorganization and lamellipodial protrusion [9]. We provide evidence that Abl/Arg-mediated phosphorylation of cortactin is required for this PDGF-induced dorsal-wave response. Our results reveal that Abl-family kinases target cortactin as an effector of cytoskeletal rearrangements in response to PDGF.

Identification of small molecule targets on functional protein microarrays.

Small molecules, such as metabolites and hormones, interact with proteins to regulate numerous biological pathways, which are often aberrant in disease. Small molecule drugs have been successfully exploited to specifically perturb such processes and thereby, decrease and even eliminate disease progression. Although there are compelling reasons to fully characterize interactions of small molecules with all proteins from an organism for which an intended drug regimen is planned, currently available technologies are not yet up to this task. High-content functional protein microarrays, containing hundreds to thousands of proteins, are new tools that show great potential for meeting this need. In this chapter, we review examples and methods for profiling small molecules on high-content functional protein arrays and discuss considerations for troubleshooting.

Functional protein arrays to facilitate drug discovery and development.

Protein microarrays are miniaturized formats for studying proteins. This technology is empowering investigators with the ability to profile numerous types of interactions to progress basic science research and to advance drug discovery and development. Protein microarrays are poised to make significant contributions to our understanding of biology and disease because: (i) both covalent and non-covalent interactions can be reconstituted on solid-state supports; and (ii) a wealth of knowledge can be generated rapidly from such simple experiments. This feature focuses on applications of protein microarrays that have tremendous potential for addressing bottlenecks in disease-focused discovery efforts.

Global analysis of protein phosphorylation in yeast.

Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology, the in vitro substrates recognized by most yeast protein kinases: we identified over 4,000 phosphorylation events involving 1,325 different proteins. These substrates represent a broad spectrum of different biochemical functions and cellular roles. Distinct sets of substrates were recognized by each protein kinase, including closely related kinases of the protein kinase A family and four cyclin-dependent kinases that vary only in their cyclin subunits. Although many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, indicating possible new roles for several kinases. Furthermore, integration of the phosphorylation results with protein-protein interaction and transcription factor binding data revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast. Because many yeast proteins and pathways are conserved, these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.

Biomarker discovery using protein microarray technology platforms: antibody-antigen complex profiling.

Protein microarrays represent an important new tool in proteomic systems biology. This review focuses on the contributions of protein microarrays to the discovery of novel disease biomarkers through antibody-based assays. Of particular interest is the use of protein microarrays for immune response profiling, through which a disease-specific antibody repertoire may be defined. The antigens and antibodies revealed by these studies are useful for clinical assay development, with enormous potential to aid in diagnosis, prognosis, disease staging and treatment selection. The discovery and characterization of novel biomarkers specifically tailored to disease type and stage are expected to enable personalized medicine by facilitating preventative medicine, predictive diagnostics and individualized curative therapies.

Protein microarray-based screening of antibody specificity.

The increased use of antibodies as therapeutics, as well as the growing demand for large numbers of antibodies for high-throughput protein analyses, has been accompanied by a need for more specific antibodies. An array containing every protein for the relevant organism represents the ideal format for an assay to test antibody specificity since it allows the simultaneous screening of thousands of proteins in relatively normalized quantities. Indeed, the use of a yeast proleome array to profile the specificity of several antibodies directed against yeast proteins has recently been described. In this chapter, we present a detailed description of the methods used to probe protein arrays with antibodies as well as the technical issues to consider when carrying out such experiments.

Functional protein microarrays: just how functional are they?

Arrays of immobilized proteins have been developed for the discovery and characterization of protein functions ranging from molecular recognition to enzymatic activity. The success of these applications is highly dependent upon the maintenance of protein structure and function while in an immobilized state - a largely untested hypothesis. However, the immobilization of functional proteins is not without precedent. Active enzymes have been successfully immobilized for industrial applications for several decades. Furthermore, a survey of recent protein microarray literature reveals that an even wider range of proteins can maintain 'proper' function while immobilized. These reports help to validate the functionality of so-called functional protein microarrays.

Protein microarrays: a new tool for profiling antibody cross-reactivity.

Antibody cross-reactivity can compromise interpretation of experiments and derail therapeutic antibody development. Standard techniques such as immunohistochemistry or Western analysis provide important but often inadequate approaches to assess antibody specificity. Protein microarrays are providing a new approach to rapidly characterize antibody cross-reactivity against 1,000s of proteins simultaneously. This review will focus on reported examples of antibody cross-reactivity, methods used to characterize them, and the recent development and use of protein microarrays for assessing antibody specificity.

Human ribonuclease A superfamily members, eosinophil-derived neurotoxin and pancreatic ribonuclease, induce dendritic cell maturation and activation.

A number of mammalian antimicrobial proteins produced by neutrophils and cells of epithelial origin have chemotactic and activating effects on host cells, including cells of the immune system. Eosinophil granules contain an antimicrobial protein known as eosinophil-derived neurotoxin (EDN), which belongs to the RNase A superfamily. EDN has antiviral and chemotactic activities in vitro. In this study, we show that EDN, and to a lesser extent human pancreatic RNase (hPR), another RNase A superfamily member, activates human dendritic cells (DCs), leading to the production of a variety of inflammatory cytokines, chemokines, growth factors, and soluble receptors. Human angiogenin, a RNase evolutionarily more distant to EDN and hPR, did not display such activating effects. Additionally, EDN and hPR also induced phenotypic and functional maturation DCs. These RNases were as efficacious as TNF-alpha, but induced a different set of cytokine mediators. Furthermore, EDN production by human macrophages could be induced by proinflammatory stimuli. The results reveal the DC-activating activity of EDN and hPR and suggest that they are likely participants of inflammatory and immune responses. A number of endogenous mediators in addition to EDN have been reported to have both chemotactic and activating effects on APCs, and can thus amplify innate and Ag-specific immune responses to danger signals. We therefore propose these mediators be considered as endogenous multifunctional immune alarmins.