Despite the increasing availability of genome sequences from many human pathogens, the production of complete proteomes remains at a bottleneck. To address this need, a high-throughput PCR recombination cloning and expression platform has been developed that allows hundreds of genes to be batch-processed by using ordinary laboratory procedures without robotics. The method relies on high-throughput amplification of each predicted ORF by using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription͞translation system, and the crude reactions containing expressed proteins are printed directly onto nitrocellulose microarrays without purification. The protein microarrays are useful for determining the complete antigen-specific humoral immuneresponse profile from vaccinated or infected humans and animals. The system was verified by cloning, expressing, and printing a vaccinia virus proteome consisting of 185 individual viral proteins. The chips were used to determine Ab profiles in serum from vaccinia virus-immunized humans, primates, and mice. Human serum has high titers of anti-E. coli Abs that require blocking to unmask vaccinia-specific responses. Naïve humans exhibit reactivity against a subset of 13 antigens that were not associated with vaccinia immunization. Naïve mice and primates lacked this background reactivity. The specific profiles between the three species differed, although a common subset of antigens was reactive after vaccinia immunization. These results verify this platform as a rapid way to comprehensively scan humoral immunity from vaccinated or infected humans and animals.pox virus ͉ proteomics ͉ vaccine ͉ Francisella tularensis

Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, we monitored repair of DNA ends with long 3′ single-stranded overhangs. End joining events retaining much of the overhang were dependent on POLQ, and independent of Ku70. To analyze the repair function in more detail, we examined immunoglobulin class switch joining between DNA segments in antibody genes. POLQ participates in end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers. DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. We found a marked increase in Myc/IgH translocations in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone.

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 smallpox vaccine is the prototypic vaccine, yet the viral targets critical for vaccine-mediated protection remain unclear in humans. We have produced protein microarrays of a near-complete vaccinia proteome and used them to determine the major antigen specificities of the human humoral immune response to the smallpox vaccine (Dryvax). H3L, an intracellular mature virion envelope protein, was consistently recognized by hightiter antibodies in the majority of human donors, particularly after secondary immunization. We then focused on examining H3L as a valuable human antibody target. Purified human anti-H3L antibodies exhibited substantial vaccinia virus-neutralizing activity in vitro (50% plaque reduction neutralization test [PRNT 50 ] ‫؍‬ 44 g/ml). Mice also make an immunodominant antibody response to H3L after vaccination with vaccinia virus, as determined by vaccinia virus protein microarray. Mice were immunized with recombinant H3L protein to examine H3L-specific antibody responses in greater detail. H3L-immunized mice developed hightiter vaccinia virus-neutralizing antibodies (mean PRNT 50 ‫؍‬ 1:3,760). Importantly, H3L-immunized mice were subsequently protected against lethal intranasal challenges with 1 or 5 50% lethal doses (LD 50 ) of pathogenic vaccinia virus strain WR, demonstrating the in vivo value of an anti-H3L response. To formally demonstrate that neutralizing anti-H3L antibodies are protective in vivo, we performed anti-H3L serum passive-transfer experiments. Mice receiving H3L-neutralizing antiserum were protected from a lethal challenge with 3 LD 50 of vaccinia virus strain WR (5/10 versus 0/10; P < 0.02). Together, these data show that H3L is a major target of the human anti-poxvirus antibody response and is likely to be a key contributor to protection against poxvirus infection and disease.