Journal of Proteome Research ’ DISCUSSION Today with increasing eﬃciency, accuracy and speed we access completed genome sequences from thousands of infectious micro- organisms. However, systems- level studies to understand the complete network of antibody responses have not been widely performed. This work was motivated by the disturbing lack of a detailed scientiﬁc understanding of the antibody responses to infectious diseases. We have been taking a systems biology approach to account for all of the antibodies that develop after exposure to infectious microorganisms and to identify speciﬁc antibody signa- tures associated with each disease, in order to understand the molecular basis for antigen selection by the immune system and to predict serodiagnostic and vaccine antigens targets. This study is the ﬁrst full proteome-wide serological analysis of B. melitensis. Our previous study identiﬁed a set of serodiagnostic antigens from a randomly selected half of the B. melitensis proteome. However, an important diﬀerence between the present study and this previous report is that analysis of the complete proteome not only has allowed us to delineate additional serodiagnostic antigens but, more fundamentally, also provided the basis for a rigorous, comprehensive and quantitative determination of basic biologi- cal characteristics of the entire set of the serodominant antigens on a genomic level. The current standard serological screening assay, an aggluti- nation test that uses tinted, killed bacteria as antigen (Rose Bengal) is based primarily on identiﬁcation of antibodies to LPS in patient serum. In the present microarray study, we conﬁrmed LPS reactivity (with puriﬁed LPS spotted onto the microarray), and identiﬁed novel protein antigens. The signature antigens were validated using traditional Western blots. Although anti- body responses against all 33 protein antigens used together can predict disease with 92% accuracy, using the serological re- sponses against the top 3 individual antigens together improves accuracy to >98% for diagnosis of acute human brucellosis. One limitation is that the humans studied here only had acute or subacute brucellosis-fever but not focal or chronic disease. Future studies are needed to assess suspected cases that are agglutination test negative, for example chronic neurobrucellosis or focal disease such as orchitis or vertebral osteomyelitis. Eleven of the 33 serodiagnostic antigens identiﬁed in this study were also identiﬁed on the pilot array5 (Table S1, Support- ing Information). Our ﬁndings are in good agreement with published studies that identiﬁed well characterized antigens from other Brucella spp, including Bp26 (BMEI0536),20,25,31,56 HtrA/ DegP (BMEI1330),28 Omp16 (BMEI0340),29 the chaperonin GroEL protein (BMEII1048) and Omp 10 (BMEII0017).21,57 In addition to the well characterized antigens, we also identiﬁed 21 novel serodiagnostic antigens on the current array, including top antigen hypothetical protein BMEI0805. Two pyruvate dehy- drogenase complex molecules and the associated acetyl CoA hydrolase, which together form a large multimeric structure con- sisting of 60 subunits, are also diﬀerentially recognized; this large enzyme complex is found in most bacteria and is frequently a target of immune recognition for other infections.6,58 Protein microarrays enable enrichment analyses to identify proteomic features that are enriched in the immunodominant antigen set, and development of protein antigenicity prediction tools based on enriched features. The prediction tools then can be applied on a high-throughput scale to existing or new proteomes to identify key antigenic proteins that may have serodiagnostic or protective characteristics. Enrichment analysis identiﬁed 10 proteomic ARTICLE features that are enriched in the serodiagnostic signature anti- gens. The proteomic features fall into 3 categories: (i) COG annotations, (ii) computationally predicted proteomic features, and (iii) proteins with MS evidence of expression in viable organisms. No single proteomic feature or category of features is suﬃcient to identify all these signature antigens. Our data suggests that cloning of 37% of genome with these enriching features would reveal more than 90% of serodiagnostic antigens. We have classiﬁed the reactive immunodominant antigens for numerous agents and have consistently found these features predict antigenicity.6,7,10,59 These results describe the relationship between antigenicity and in vivo expression of individual proteins. In our study, we utilized the expression of proteins quantiﬁed by MS of a closely related strain, B. abortus during in vitro growth. We found that mass spec positive antigens were signiﬁcantly enriched among serodiagnostic antigens but not among the cross- reactive anti- gens. This validates the classiﬁcation of the serodiagnostic antigens being derived from actively replicating organisms, and validates the classiﬁcation of cross-reactive antigens being de- rived from previous exposure to nonbrucellosis infections. Although the number of peptides observed per protein has been applied to estimating protein abundance,60 62 we are aware that mass spectra of cultured organisms may not reﬂect the expression during infection in vivo. First, nutrients are not limiting in log phase growth in vitro, whereas in vivo, Brucella is likely to be within a nutrient-limited intracellular environment.63 Second, the MS method may not be particularly quantitative, especially for membrane proteins. Our protein array technology is also able to provide strong evidence of the comprehensive set of proteins expressed in vivo within a mammalian host by B. melitensis, by virtue of their exposure to the host immune system. The expression and abundance of proteins during in vivo growth merits further examination by other novel technologies, such as measure- ment of transcript abundance from RNA-seq (“deep sequencing”) technique.64 The B. melitensis immunoproteome comprises 1464 antigens that are signiﬁcantly reactive in at least one of the individuals in this study. Only 122 serodominant antigens are signiﬁcantly recognized by most of the individuals. Answers to questions of why and how the immune system focuses on 4% of the potential target antigens are not yet apparent from this work. One might expect that an immune response against a larger collection of antigens could result in a more eﬀective immune response attack against the infectious agent. But antibody responses against thousands of antigens from hundreds of clinical infections could accumulate during a lifetime, leading to cross reactivity against autologous antigens and autoimmune chaos. This could provide evolutionary selection pressure favoring a more focused response to infection. The observation that dozens of organism- speciﬁc antibody responses develop after B. melitensis infection is con- sistent with similar observations from other infectious agents, and has implications for subunit vaccine discovery and develop- ment. Mimicking the natural response to infection could be considered a viable strategy for vaccine development but most subunit vaccines aim to derive protection from immunization with only a single antigen. Attenuated or killed whole organism vaccines produce an antibody reactivity proﬁle against dozens of antigens more similar to natural infection.6 This systematic genome scale analysis of human antibody responses against B. melitensis proteins provides a top hit list of antigens worthy of assessing for improved diagnostics, and 4821 dx.doi.org/10.1021/pr200619r |J. Proteome Res. 2011, 10, 4813–4824