The coding capacity of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic1. To understand the pathogenicity and antigenic potential of SARS-CoV-2 and to develop therapeutic tools, it is essential to profile the full repertoire of its expressed proteins. The current map of SARS-CoV-2 coding capacity is based on computational predictions and relies on homology with other coronaviruses. As the protein complement varies among coronaviruses, especially in regard to the variety of accessory proteins, it is crucial to characterize the specific range of SARS-CoV-2 proteins in an unbiased and open-ended manner. Here, using a suite of ribosome-profiling techniques2-4, we present a high-resolution map of coding regions in the SARS-CoV-2 genome, which enables us to accurately quantify the expression of canonical viral open reading frames (ORFs) and to identify 23 unannotated viral ORFs. These ORFs include upstream ORFs that are likely to have a regulatory role, several in-frame internal ORFs within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. We further show that viral mRNAs are not translated more efficiently than host mRNAs; instead, virus translation dominates host translation because of the high levels of viral transcripts. Our work provides a resource that will form the basis of future functional studies.

rVSV-ΔG-SARS-CoV-2-S vaccine: repeated intramuscular (IM) toxicity, local tolerance, immunogenicity and biodistribution study in NZW rabbits.

BriLife®, a vector-based vaccine that utilizes the recombinant vesicular stomatitis virus (VSV) platform to express and present the spike antigen of SARS-CoV-2, is undergoing testing in a phase 2 clinical trial in Israel. A nonclinical repeated-dose (GLP) toxicity study in New Zealand white rabbits was performed to evaluate the potential toxicity, local tolerance, immunogenicity and biodistribution of the vaccine. rVSV-ΔG-SARS-CoV-2-S (or vehicle) was administered intramuscularly to two groups of animals (106, 107 PFU/animal, n = 10/sex/group) on three occasions, at 2-week intervals, followed by a 3-week recovery period. Systemic clinical signs, local reactions, body weight, body temperature, food consumption, ophthalmology, urinalysis, clinical pathology, C-reactive protein, viremia and antibody levels were monitored. Gross pathology was performed, followed by organs/tissues collection for biodistribution and histopathological evaluation. Treatment-related changes were restricted to multifocal minimal myofiber necrosis at the injection sites, and increased lymphocytic cellularity in the iliac and mesenteric lymph nodes and in the spleen. These changes were considered related to the inflammatory reaction elicited, and correlated with a trend for recovery. Detection of rVSV-ΔG-SARS-CoV-2-S vaccine RNA was noted in the regional iliac lymph node in animals assigned to the high-dose group, at both termination time points. A significant increase in binding and neutralizing antibody titers was observed following vaccination at both vaccine doses. In view of the findings, it was concluded that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe. These results supported the initiation of clinical trials.

Early, Real-Time Medical Diagnosis of Botulism by Endopeptidase-Mass Spectrometry.

Botulinum toxin was detected in patient serum using Endopeptidase-mass-spectrometry assay, although all conventional tests provided negative results. Antitoxin was administered, resulting in patient improvement. Implementing this highly sensitive and rapid assay will improve preparedness for foodborne botulism and deliberate exposure.

Active vaccination with vaccinia virus A33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response.

Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104-120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox.

Medicaid Costs and Utilization of Collaborative Versus Colocation Care for Patients With Depression.

OBJECTIVE: The authors examined cost and utilization metrics for racially diverse Medicaid primary care patients with depression receiving care through either a collaborative care model (CoCM) of integration or the standard colocation model. METHODS: Data from a retrospective cohort of Medicaid patients screening positive for clinically significant depression during January 2016-December 2017 were analyzed to assess health care costs and selected utilization measures. Seven primary care clinics providing CoCM were compared with 16 clinics providing colocated behavioral health care. Data for the first year and second year after a patient received an initial Patient Health Questionnaire-9 score ≥10 were analyzed. RESULTS: In the first year, compared with patients receiving colocated care (N=3,061), CoCM patients (N=4,315) had significantly lower odds of emergency department (ED) visits (OR=0.95) and medical specialty office visits (OR=0.92), with slightly higher odds of primary care provider (PCP) visits (OR=1.03) and behavioral health office visits (OR=1.03). In year 2, CoCM patients (N=2,623) had significantly lower odds of inpatient medical admissions (OR=0.87), ED visits (OR=0.84), medical specialty office visits (OR=0.89), and PCP visits (OR=0.94) than the colocated care patients (N=1,838). The two groups did not significantly differ in total cost in both years. CONCLUSIONS: Access to CoCM treatment in primary care for racially diverse Medicaid patients with depression was associated with more positive health care utilization outcomes than for those accessing colocated treatment. As organizations continue to seek opportunities to integrate behavioral health care into primary care, consideration of health care costs and utilization may be helpful in the selection and implementation of integration models.

Collaborative Care for Low-Income Patients From Racial-Ethnic Minority Groups in Primary Care: Engagement and Clinical Outcomes.

OBJECTIVE: To assess model impact and opportunities for improvement, this study examined collaborative care model (CoCM) engagement and clinical outcomes among low-income patients from racial-ethnic minority groups with depression and anxiety. METHODS: Starting in 2015, the CoCM was implemented in seven primary care practices of an urban academic medical center serving patients from racial-ethnic minority backgrounds, predominantly Medicaid beneficiaries. Eligible individuals scored positive for depressive or anxiety symptoms (or both) on the Patient Health Questionnaire-2 (PHQ-2) and PHQ-9 and the Generalized Anxiety Disorder Scale-2 (GAD-2) and GAD-7 during systematic screening in primary care settings. Screening rates and yield, patient characteristics, and CoCM engagement and outcomes were examined. Clinical improvement was measured by the difference in PHQ-9 and GAD-7 scores at baseline and at 10-to-14-week follow-up. RESULTS: High rates of screening (87%, N=88,236 of 101,091) and identification of individuals with depression or anxiety (13%, N=11,886) were observed, and 58% of 3,957 patients who engaged in minimally adequate CoCM treatment had significant clinical improvement. Nevertheless, only 56% of eligible patients engaged in the model, and 25% of those individuals did not return for at least one follow-up appointment. Being female with clinically significant comorbid anxiety and depressive symptoms and having Medicaid or commercial insurance increased the likelihood of CoCM engagement. CONCLUSIONS: CoCM can help engage vulnerable patients in behavioral health care and improve clinical symptoms. However, significant opportunity exists to advance the model's impact in treating depressive and anxiety disorders and decreasing health disparities by addressing engagement barriers.

Mutation Profile of SARS-CoV-2 Genome Sequences Originating from Eight Israeli Patient Isolates.

We report the genome sequences and the identification of genetic variations in eight clinical samples of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Samples were collected from nasopharyngeal swabs of symptomatic and asymptomatic individuals from five care homes for elderly and infirm persons in Israel. The sequences obtained are valuable, as they carry a newly reported nonsynonymous substitution located within the nucleoprotein open reading frame.

Mice with induced pulmonary morbidities display severe lung inflammation and mortality following exposure to SARS-CoV-2.

Mice are normally unaffected by SARS coronavirus 2 (SARS-CoV-2) infection since the virus does not bind effectively to the murine version of the angiotensin-converting enzyme 2 (ACE2) receptor molecule. Here, we report that induced mild pulmonary morbidities rendered SARS-CoV-2-refractive CD-1 mice susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low doses of the acute lung injury stimulants bleomycin or ricin caused severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates greater than 50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart, and serum of low-dose ricin-pretreated mice compared with non-pretreated mice. Furthermore, lung extracts prepared 2-3 days after viral infection contained subgenomic mRNA and virus particles capable of replication only when derived from the pretreated mice. The deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against the SARS-CoV-2 receptor binding domain (RBD). Thus, viral cell entry in the sensitized mice seems to depend on viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. This unique mode of viral entry, observed over a mildly injured tissue background, may contribute to the exacerbation of coronavirus disease 2019 (COVID-19) pathologies in patients with preexisting morbidities.

Accommodation of physostigmine and its analogues by acetylcholinesterase is dominated by hydrophobic interactions.

The role of the functional architecture of the HuAChE (human acetylcholinesterase) in reactivity toward the carbamates pyridostigmine, rivastigmine and several analogues of physostigmine, that are currently used or considered for use as drugs for Alzheimer's disease, was analysed using over 20 mutants of residues that constitute the interaction subsites in the active centre. Both steps of the HuAChE carbamylation reaction, formation of the Michaelis complex as well as the nucleophilic process, are sensitive to accommodation of the ligand by the enzyme. For certain carbamate/HuAChE combinations, the mode of inhibition shifted from a covalent to a noncovalent type, according to the balance between dissociation and covalent reaction rates. Whereas the charged moieties of pyridostigmine and rivastigmine contribute significantly to the stability of the corresponding HuAChE complexes, no such effect was observed for physostigmine and its analogues, phenserine and cymserine. Moreover, physostigmine-like ligands carrying oxygen instead of nitrogen at position -1 of the tricyclic moiety (physovenine and tetrahydrofurobenzofuran analogues) displayed comparable structure-function characteristics toward the various HuAChE enzymes. The essential role of the HuAChE hydrophobic pocket, comprising mostly residues Trp(86) and Tyr(337), in accommodating (-)-physostigmine and in conferring approximately 300-fold stereoselectivity toward physostigmines, was elucidated through examination of the reactivity of selected HuAChE mutations toward enantiomeric pairs of different physostigmine analogues. The present study demonstrates that certain charged and uncharged ligands, like analogues of physostigmine and physovenine, seem to be accommodated by the enzyme mostly through hydrophobic interactions.

Enhancing the scalability of the collaborative care model for depression using mobile technology.

The collaborative care model (CoCM) has substantial support for improving behavioral health care in primary care. However, large-scale CoCM adoption relies on addressing operational and financial implementation challenges across health care settings with varying resources. An academic medical center serving socioeconomically and racially diverse patients implemented the CoCM in seven practices. A smartphone application was introduced to facilitate CoCM care management during depression treatment (app-augmented CoCM). App features included secure texting, goal/appointment reminders, symptom monitoring, and health education material. A nonrandomized convenience patient sample (N = 807) was enrolled in app-augmented CoCM and compared with patients in standard CoCM (N = 3,975). Data were collected on clinical contact frequency, engagement, and clinical outcomes. App-augmented CoCM patients received more health care team contacts (7.9 vs. 4.9, p < .001) and shorter time to follow up compared with the standard CoCM sample (mean = 11 vs. 19 days, p < .001). App-augmented CoCM patients had clinical outcomes similar to the standard CoCM group (47% vs. 46% with ≥50% depression improvement or score <10), despite app-augmented patients having more prior depression treatment episodes. Further, the app-augmented group with greater app engagement demonstrated increased behavioral health appointment compliance, including more completed appointments and fewer no shows, and greater depression symptom improvement than those with less app engagement. App-augmented CoCM may improve patient engagement in treatment and provide opportunities to implement key CoCM elements without overburdening practice resources. CoCM sustainability and scalability in primary care may be enhanced by using this technology.

Evaluating the efficacy of RT-qPCR SARS-CoV-2 direct approaches in comparison to RNA extraction.

The genetic identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is based on viral RNA extraction prior to RT-qPCR assay. However, recent studies have supported the elimination of the extraction step. This study was performed to assess the necessity for the RNA extraction, by comparing the efficacy of RT-qPCR in several direct approaches versus the gold standard RNA extraction, in the detection of SARS-CoV-2 in laboratory samples, as well as in clinical oro-nasopharyngeal SARS-CoV-2 swabs. The findings showed an advantage for the extraction procedure; however a direct no-buffer approach might be an alternative, since it identified more than 60% of positive clinical specimens.

Coding-Complete Genome Sequences of Two SARS-CoV-2 Isolates from Early Manifestations of COVID-19 in Israel.

We announce the genome sequences of two strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated in Israel, one imported by a traveler who returned from Japan and the second strain collected from a patient infected by a traveler returning from Italy. The sequences obtained are valuable as early manifestations for future follow-up of the local spread of the virus in Israel.

A single dose of recombinant VSV-∆G-spike vaccine provides protection against SARS-CoV-2 challenge.

The COVID-19 pandemic caused by SARS-CoV-2 imposes an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we show the development of a replication competent recombinant VSV-∆G-spike vaccine, in which the glycoprotein of VSV is replaced by the spike protein of SARS-CoV-2. In-vitro characterization of this vaccine indicates the expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in-vivo model for COVID-19 is implemented. We show that a single-dose vaccination results in a rapid and potent induction of SARS-CoV-2 neutralizing antibodies. Importantly, vaccination protects hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss, and  alleviation of the extensive tissue damage and viral loads in lungs and nasal turbinates. Taken together, we suggest the recombinant VSV-∆G-spike as a safe, efficacious and protective vaccine against SARS-CoV-2.

Identification and genetic characterization of a novel Orthobunyavirus species by a straightforward high-throughput sequencing-based approach.

Identification and characterization of novel unknown viruses is of great importance. The introduction of high-throughput sequencing (HTS)-based methods has paved the way for genomics-based detection of pathogens without any prior assumptions about the characteristics of the organisms. However, the use of HTS for the characterization of viral pathogens from clinical samples remains limited. Here, we report the identification of a novel Orthobunyavirus species isolated from horse plasma. The identification was based on a straightforward HTS approach. Following enrichment in cell culture, RNA was extracted from the growth medium and rapid library preparation, HTS and primary bioinformatic analyses were performed in less than 12 hours. Taxonomical profiling of the sequencing reads did not reveal sequence similarities to any known virus. Subsequent application of de novo assembly tools to the sequencing reads produced contigs, of which three showed some similarity to the L, M, and S segments of viruses belonging to the Orthobunyavirus genus. Further refinement of these contigs resulted in high-quality, full-length genomic sequences of the three genomic segments (L, M and S) of a novel Orthobunyavirus. Characterization of the genomic sequence, including the prediction of open reading frames and the inspection of consensus genomic termini and phylogenetic analysis, further confirmed that the novel virus is indeed a new species, which we named Ness Ziona virus.

Aging-resistant organophosphate bioscavenger based on polyethylene glycol-conjugated F338A human acetylcholinesterase.

The high reactivity of cholinesterases (ChEs) toward organophosphorus (OP) compounds has led to propose recombinant ChEs as bioscavengers of nerve agents. The bioscavenging potential of recombinant ChEs can be enhanced by conjugation of polyethylene glycol (PEG) moieties, to extend their circulatory residence. However, the ability of exogenously administered ChEs to confer long-term protection against repeated exposures to nerve agents is still limited due to the aging process, whereby organophosphate-ChE adducts undergo irreversible dealkylation, which precludes oxime-mediated reactivation of the enzyme. To generate an optimal acetylcholinesterase (AChE)-based OP bioscavenger, the F338A mutation, known to decelerate the rate of aging of AChE-OP conjugates, was incorporated into polyethylene glycol-conjugated (PEGylated) human AChE. The PEGylated F338A-AChE displayed unaltered rates of hydrolysis, inhibition, phosphylation, and reactivation and could effectively protect mice against exposure to soman (pinacolylmethyl phosphonofluoridate), sarin (O-isopropyl methylphosphonofluoridate), or O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothioate (VX). Unlike PEGylated wild-type (WT)-AChE, the PEGylated F338A-AChE exhibits significantly reduced aging rates after soman inhibition and can be efficiently reactivated by the 1-[[[4(aminocarbonyl)-pyridinio]methoxy]methyl]-2(hydroxyimino)methyl]pyridinium dichloride (HI-6) oxime, both in vitro and in vivo. Accordingly, oxime administration to PEG-F338A-AChE-pretreated mice enabled them to withstand repeated soman exposure (5.4 and 4 LD(50)/dose), whereas same regime treatment of non-PEGylated F338A-AChE- or PEGylated WT-AChE-pretreated mice failed to protect against the second challenge, due to rapid clearance or irreversible aging of the latter enzymes. Thus, judicious incorporation of selected mutations into the AChE mold in conjunction with its chemical modification provides means to engineer an optimal ChE-based OP bioscavenger in terms of circulatory longevity, resistance to aging, and reduced doses required for protection, even against repeated exposures to nerve agents, such as soman.

Flexibility versus "rigidity" of the functional architecture of AChE active center.

Functional architecture of the AChE active center appears to be characterized by both structural "rigidity", necessary to stabilize the catalytic triad as well as by flexibility in accommodating the different, high affinity AChE ligands. These seemingly conflicting structural properties of the active center are demonstrated through combination of structural methods with kinetic studies of the enzyme and its mutant derivatives with plethora of structurally diverse ligands and in particular with series of stereoselective covalent and noncovalent AChE ligands. Thus, steric perturbation of the acyl pocket precipitates in a pronounced stereoselectivity toward methylphosphonates by disrupting the stabilizing environment of the catalytic histidine rather than through steric exclusion demonstrating the functional importance of the "rigid" environment of the catalytic machinery. The acyl pocket, the cation-binding subsite (Trp86) and the peripheral anionic subsite were also found to be directly involved in HuAChE stereoselectivity toward charged chiral phosphonates, operating through differential positioning of the ligand cationic moiety within the active center. Residue Trp86 is also a part of the "hydrophobic patch" which seems flexible enough to accommodate the structurally diverse ligands like tacrine, galanthamine and the two diastereomers of huperzine A. Also, we have recently discovered further aspects of the role of both the unique structure and the flexibility of the "hydrophobic patch" in determining the reactivity and stereoselectivity of HuAChE toward certain carbamates including analogs of physostigmine. In these cases the ligands are accommodated mostly through hydrophobic interactions and their stereoselectivity delineates precisely the steric limits of the pocket. Hence, the HuAChE stereoselectivity provides a sensitive tool in the in depth exploration of the functional architecture of the active center. These studies suggest that the combination of "rigidity" and flexibility within the HuAChE gorge are an essential element of its molecular design.

Comparison of Collaborative Care and Colocation Treatment for Patients With Clinically Significant Depression Symptoms in Primary Care.

OBJECTIVE: The study compared clinical outcomes of depression treatment in primary care with a colocation model versus a collaborative care model (CoCM). METHODS: Patients (N=240) with Patient Health Questionnaire-9 (PHQ-9) scores of ≥10 treated for clinically significant depression symptoms in primary care sites implementing the CoCM or a colocation model were compared. PHQ-9 scores were collected at baseline and 12 weeks. RESULTS: From baseline to follow-up, reductions in PHQ-9 scores were 33% for the CoCM sites and 14% for the colocation sites, with an unadjusted mean difference in scores of 2.81 (p=.001). CONCLUSIONS: More patients treated in sites that used the CoCM experienced a significantly greater reduction in depression symptoms, compared with patients in sites with the colocation model. As greater adoption of integration models in primary care occurs, it will be important to consider potential implications of these results for promoting adoption of CoCM elements. Further replication of these findings is warranted.

Optimization and limitations of use of cryopreserved peripheral blood mononuclear cells for functional and phenotypic T-cell characterization.

The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4(+) and CD8(+) T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at -70 degrees C for < or =3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at -70 degrees C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.

Tumor-necrosis-factor blockers: differential effects on mycobacterial immunity.

BACKGROUND: Tumor necrosis factor (TNF) plays a pathogenic role in rheumatoid arthritis but is essential for antimycobacterial host defenses. The risk of reactivation of latent Mycobacterium tuberculosis infection is greater with the TNF monoclonal antibody infliximab than with the soluble TNF receptor etanercept. The basis of this difference is not known. METHODS: The effects that the monoclonal antibodies infliximab and adalimumab and the receptor etanercept have on antimycobacterial immune functions were studied by use of therapeutic drug concentrations in whole-blood culture. RESULTS: Infliximab and adalimumab reduced the proportion of tuberculosis-responsive (CD69(+)) CD4 cells by 70% and 49%, respectively (P<.05), and suppressed antigen-induced interferon (IFN)- gamma production by 70% and 64% (P<.05), respectively; in contrast, etanercept produced no significant effect. Interleukin-10 production was equally suppressed by all 3 drugs. Adalimumab and etanercept had divergent, concentration-dependent effects on control of intracellular growth of M. tuberculosis. None of the drugs induced significant levels of apoptosis or necrosis, in either monocytes or T cells. CONCLUSIONS: The tuberculosis risk posed by infliximab may reflect its combined effects on TNF and IFN- gamma .

Search for Bacillus anthracis potential vaccine candidates by a functional genomic-serologic screen.

Bacillus anthracis proteins that possess antigenic properties and are able to evoke an immune response were identified by a reductive genomic-serologic screen of a set of in silico-preselected open reading frames (ORFs). The screen included in vitro expression of the selected ORFs by coupled transcription and translation of linear PCR-generated DNA fragments, followed by immunoprecipitation with antisera from B. anthracis-infected animals. Of the 197 selected ORFs, 161 were chromosomal and 36 were on plasmids pXO1 and pXO2, and 138 of the 197 ORFs had putative functional annotations (known ORFs) and 59 had no assigned functions (unknown ORFs). A total of 129 of the known ORFs (93%) could be expressed, whereas only 38 (64%) of the unknown ORFs were successfully expressed. All 167 expressed polypeptides were subjected to immunoprecipitation with the anti-B. anthracis antisera, which revealed 52 seroreactive immunogens, only 1 of which was encoded by an unknown ORF. The high percentage of seroreactive ORFs among the functionally annotated ORFs (37%; 51/129) attests to the predictive value of the bioinformatic strategy used for vaccine candidate selection. Furthermore, the experimental findings suggest that surface-anchored proteins and adhesins or transporters, such as cell wall hydrolases, proteins involved in iron acquisition, and amino acid and oligopeptide transporters, have great potential to be immunogenic. Most of the seroreactive ORFs that were tested as DNA vaccines indeed appeared to induce a humoral response in mice. We list more than 30 novel B. anthracis immunoreactive virulence-related proteins which could be useful in diagnosis, pathogenesis studies, and future anthrax vaccine development.