A complete description of the serological response following exposure of humans to complex pathogens is lacking and approaches suitable for accomplishing this are limited. Here we report, using malaria as a model, a method which elucidates the profile of antibodies that develop after natural or experimental infection or after vaccination with attenuated organisms, and which identifies immunoreactive antigens of interest for vaccine development or other applications. Expression vectors encoding 250 Plasmodium falciparum (Pf) proteins were generated by PCR/ recombination cloning; the proteins were individually expressed with >90% efficiency in E. coli cell-free in vitro transcription and translation reactions, and printed directly without purification onto microarray slides. The protein microarrays were probed with human sera from one of four groups which differed in immune status: sterile immunity or no immunity against experimental challenge following vaccination with radiation-attenuated Pf sporozoites, partial immunity acquired by natural exposure, and no previous exposure to Pf. Overall, 72 highly reactive Pf antigens were identified. Proteomic features associated with immunoreactivity were identified. AUTHOR CONTRIBUTIONSDLD and PLF conceived and designed the study, assisted in data analysis and interpretation, and wrote the manuscript. PLF and DHD contributed to the supervision and execution of the research. YM, BU, CV executed the research and assisted in data analysis and preparation of the manuscript figures. DM and XL provided the protein microarrays. SS, SH, AR and PB were responsible for the bioinformatic and statistical analysis. PLB and JCA assisted in the initial selected of open reading frames for analysis. DAF was responsible for the studies with irradiated sporozoite immunized volunteers that provided key specimens for analysis. JAO was responsible for the field studies with Kenyan volunteers that provided key specimens for analysis. COMPETING INTERESTSThe authors declare that no competing interests exist. NIH Public Access Author ManuscriptProteomics. Author manuscript; available in PMC 2011 January 16. Importantly, antibody profiles were distinct for each donor group. Information obtained from such analyses will facilitate identifying antigens for vaccine development, dissecting the molecular basis of immunity, monitoring the outcome of whole-organism vaccine trials, and identifying immune correlates of protection.

The eradication of smallpox by vaccination with vaccinia virus was probably one of the greatest achievements of vaccinology. However, the immunological basis of this protection is not fully understood. To this end, we have used protein microarrays of the vaccinia (Western Reserve, WR) proteome to profile antibody reactivities after primary infection or boosting with the licensed smallpox vaccine, Dryvax ® , and with archival convalescent smallpox sera. Some 25 antigens were consistently recognized by Dryvax ® sera, of which half were envelope proteins (notably, H3, A13, B5, and D8). The remainder consisted mainly of core proteins (e.g. A10, L4, and I1), proteins involved in intracellular morphogenesis (A11, D13), and the A-type inclusion protein, WR148. Convalescent smallpox sera also detected vaccinia antigens on the array, consistent with the notion that there is serological cross-reactivity between these two orthopox species that underlies protection. Moreover, the profiles of immunodominant antigens recognized by variola-infected individuals and Dryvax ® vaccinees were indistinguishable. This is the first description of antibody-specificity profiles induced after smallpox infection. The array data indicate that a significant component of the antibody response is not involved in virus neutralization, although these antigens should be considered alongside the envelope proteins as potential candidates for diagnostic and vaccine applications.

Stimulation of protective immune responses against intracellular pathogens is difficult to achieve using non-replicating vaccines. BALB/c mice immunized by intramuscular injection with killed Francisella tularensis (live vaccine strain) adjuvanted with preformed immune stimulating complexes admixed with CpG, were protected when systemically challenged with a highly virulent strain of F. tularensis (Schu S4). Serum from immunized mice was used to probe a whole proteome microarray in order to identify immunodominant antigens. Eleven out of the top 12 immunodominant antigens have been previously described as immunoreactive in F. tularensis. However, 31 previously unreported immunoreactive antigens were revealed using this approach. Twenty four (50%) of the ORFs on the immunodominant hit list belonged to the category of surface or membrane associated proteins compared to only 22% of the entire proteome. There were eight hypothetical protein hits and eight hits from proteins associated with different aspects of metabolism. The chip also allowed us to readily determine the IgG subclass bias, towards individual or multiple antigens, in protected and unprotected animals. These data give insight into the protective immune response and have potentially important implications for the rational design of non-living vaccines for tularemia and other intracellular pathogens.

Q fever is a widespread zoonosis caused by Coxiella burnetii. Diagnosis of Q fever is usually based on serological testing of patient serum. The diagnostic antigen of test kits is formalin-fixed phase I and phase II organisms of the Nine Mile reference strain. Deficiencies of this antigen include (i) potential for crossreactivity with other pathogens; (ii) an inability to distinguish between C. burnetii strains; and (iii) a need to propagate and purify C. burnetii, a difficult and potentially hazardous process. Consequently, there is a need for sensitive and specific serodiagnostic tests utilizing defined antigens, such as recombinant C. burnetii protein(s). Here we describe the use of a C. burnetii protein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of human C. burnetii infection or vaccination. Transcriptionally active PCR products corresponding to 1,988 C. burnetii open reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using an Escherichia coli-based in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50 C. burnetii proteins, including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated, and the immunoreactivity was confirmed by enzyme-linked immunosorbent assay. This sensitive and high-throughput method for identifying immunoreactive C. burnetii proteins will aid in the development of Q fever serodiagnostic tests based on recombinant antigen.Coxiella burnetii is a gram-negative obligate intracellular bacterium and the etiological agent of the zoonosis Q ("query") fever. Human populations most at risk for infection are those routinely exposed to infected animals and their products. The organism has a diverse animal reservoir that includes domestic livestock such as dairy cattle, goats, and sheep. Chronically infected dairy cattle shed C. burnetii in milk and other secretions, and the products of livestock parturition can deposit tremendous numbers of the organisms into the environment. The insidious nature of C. burnetii is further exacerbated by the organism's aerosol route of infection, low infectious dose, and pronounced extracellular stability. Q fever most commonly manifests as a self-limiting but debilitating influenza-like illness that includes signs and/or symptoms of prolonged high fever, headache, and malaise. Chronic infection can occur, normally in predisposed individuals, that typically presents as a life-threatening endocarditis (reviewed in reference 17).Two advancements that would aid in control of Q fever are (i) a specific and sensitive serodiagnostic test based on recombinant antigen and (ii) an efficacious and safe vaccine that d...