B. anthracis IVIAT Source/Reference [50,51] ATCC [53] [54] ATCC #14579 ATCC #23857 US Army Medical Research Institute of Infectious Diseases, Frederick, MD Relevant Characteristics Sterne strain; pXO1+, pXO2- Pasteur strain; pXO1-, pXO2+ Sterne strain 7702 derivative cured of pXO1; pXO1-, pXO2- [52] 9131(pXO2) 9131 containing pXO2 from 6602; pXO1-, pXO2+ Sterne strain 7702 derivative; pXO1+ (Dlef, Dcya), pXO2- Virulent wild type; pXO1+, pXO2+ Wild type strain Wild type strain Table 3. Bacillus strains used in this study. Species Bacillus anthracis Strain 7702 6602 9131 RP42 Ames 14579 168 [EMG 51] Bacillus cereus Bacillus subtilis doi:10.1371/journal.pone.0001824.t003 to the study start. We obtained convalescent sera from nine macaques that had been immunized with three doses (week 0, 4, 26) of a 1:20 or 1:40 dilution of anthrax vaccine adsorbed (AVA), and survived aerosol challenge with 20–422 LD50 equivalents (1.16106–23.26106 CFUs) of B. anthracis Ames strain at week 52. Sera were harvested on days 14 and 30 post-challenge. B. anthracis Ames infection of macaques was confirmed by seroconversion to anti-PA IgG following aerosol exposure. We pooled sera containing the highest anti-PA antibody levels from each surviving macaque. We removed antibodies binding in vitro expressed proteins by six successive direct adsorptions with concentrated whole B. anthracis cells (1011 CFUs/ml RP42 and 9131 [pXO2] mixed in equal volumes) grown in vitro in BHI broth in air at 37uC until mid/late log phase [3,6]. We further adsorbed pooled sera with both native and heat-denatured cell lysates of mid/late- log phase cultures of B. anthracis RP42 and B. anthracis 9131(pXO2) (1:1) and E. coli BL21 (DE3), as previously described [3]. Primary screening of the protein expression library, identification of inserts, and prediction of function of antigens identified by IVIAT For primary screening, we followed a modification of the method of Hang et al [6]. Briefly, we plated aliquots of the protein expression library on LB agar-kanamycin plates and incubated plates for approximately 10 hours at 37uC, and then used nitrocellulose disks to replica plate colonies onto LB agar- kanamycin plates containing 1 mM isopropyl-beta-D-thiogalacto- side (IPTG), and incubated plates at 37uC for 3–4 hours. To expose induced antigens, we lysed bacterial colonies by saturating disks with chloroform for ten seconds and air-drying. Following washing and blocking of disks, we incubated membranes with adsorbed sera at a 1:6,000 dilution in PBS-Tween at 4uC overnight with mild agitation, and detected immuno-reactive clones using horse-radish peroxidase-conjugated goat anti-monkey IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD) at a 1:20,000 dilution in PBS-Tween and an ECL chemiluminesence kit with Hyperfilm ECL (Amersham, Pascataway, NJ). We recovered plasmid DNA from positive clones and sequenced B. anthracis DNA inserts. We surveyed insert DNA for open reading frames, regulatory sequences, and signal sequences. Functional classification was based, when available, on published studies of identified proteins in B. anthracis. When no published functional studies of the B. anthracis genes were available, protein functional classification was based on annotation from the B. anthracis Ames chromosome (NCBI accession # NC_003997), pXO1 (NCBI accession # NC_001496), pXO2 (NCBI accession # AF188935), and predictive models using BLASTP protein alignment analysis and a Conserved Domain Database (CDD) domain search (http://www.ncbi.nlm.nih.gov/BLAST/). Cellular localization of antigens identified by IVIAT was predicted using PSORTb version 2.0.4. [26]; http://www.psort.org/psortb/). Immuno-screening of individual gene products, paralogs, and similar proteins We used PCR to generate the entire predicted B. anthracis ORF contained within each insert and cloned these into pET30abc; we confirmed reactive ORF products with serological immuno- screening, as above. We also evaluated serological reactivity of bioinformatically selected paralog and similar protein ORF clones. Specifically, for ORF pXO2-08, we evaluated paralogs BA1952, BA2849, and BA5427; and for ORF BA4073, we evaluated structurally or functionally related amidases including BA0485, BA0872, BA2446, BA2805, BA3737, BA3767, and pXO2-42 (amiA). Assessment of in vivo expression of IVIAT-identified, paralog and similar B. anthracis genes To assess in vivo expression of B. anthracis genes identified by IVIAT, we inoculated tail veins of BALB/c mice (Harlan Laboratories, Indianapolis, IN) with 200-LD50 of B. anthracis Ames vegetative cells growing in log phase under conditions associated with expression of virulence factors and capsule [43]; we inoculated control mice with PBS. We collected blood, lungs, and spleens of mice sacrificed 6, 12, or 18 hours following inoculation, immediately homogenizing tissues and adding RNAwiz reagent (Ambion; Austin, TX) to recovered samples. We extracted RNA using an Ambion RiboPure-Bacteria kit (Austin, TX), following manufacturer’s specifications. To measure B. anthracis gene expression in vitro, we similarly recovered RNA from Ames strain B. anthracis vegetative cells grown in BHI and air to mid/late log phase. We quantified RNA preparations spectro- photometerically, removed residual DNA using an Ambion DNAfree kit, and generated cDNAs using an Ambion Retroscript kit. We designed primers (Table 4) using Beacon Design (Premier Biosoft; Palo Alto, CA), www.primer3.com, and Blast software. We performed RT-PCR analysis on collected mouse and in vitro grown samples using iQ SYBR Green Supermix reagent (Bio-Rad; Hercules, CA) and a MJ Research Chromo4 thermocycler and MJ Opticon Monitor software version 3.1 (Bio-Rad; Hercules, CA). PLoS ONE | www.plosone.org 7 March 2008 | Volume 3 | Issue 3 | e1824