Immunoproteomics Analysis of the Murine Antibody Response to Vaccination with an Improved Francisella tularensis Live Vaccine Strain (LVS)

Background: Francisella tularensis subspecies tularensis is the causative agent of a spectrum of diseases collectively known as tularemia. An attenuated live vaccine strain (LVS) has been shown to be efficacious in humans, but safety concerns have prevented its licensure by the FDA. Recently, F. tularensis LVS has been produced under Current Good Manufacturing Practice (CGMP guidelines). Little is known about the immunogenicity of this new vaccine preparation in comparison with extensive studies conducted with laboratory passaged strains of LVS. Thus, the aim of the current work was to evaluate the repertoire of antibodies produced in mouse strains vaccinated with the new LVS vaccine preparation. Methodology/Principal Findings: In the current study, we used an immunoproteomics approach to examine the repertoire of antibodies induced following successful immunization of BALB/c versus unsuccessful vaccination of C57BL/6 mice with the new preparation of F. tularensis LVS. Successful vaccination of BALB/c mice elicited antibodies to nine identified proteins that were not recognized by antisera from vaccinated but unprotected C57BL/6 mice. In addition, the CGMP formulation of LVS stimulated a greater repertoire of antibodies following vaccination compared to vaccination with laboratory passaged ATCC LVS strain. A total of 15 immunoreactive proteins were identified in both studies, however, 16 immunoreactive proteins were uniquely reactive with sera from the new formulation of LVS. Conclusions/Significance: This is the first report characterising the antibody based immune response of the new formulation of LVS in the widely used murine model of tularemia. Using two mouse strains, we show that successfully vaccinated mice can be distinguished from unsuccessfully vaccinated mice based upon the repertoire of antibodies generated. This opens the door towards downselection of antigens for incorporation into tularemia subunit vaccines. In addition, this work also highlights differences in the humoral immune response to vaccination with the commonly used laboratory LVS strain and the new vaccine formulation of LVS.Peer reviewed: YesNRC publication: Ye

Prospecting environmental mycobacteria: combined molecular approaches reveal unprecedented diversity

Background: Environmental mycobacteria (EM) include species commonly found in various terrestrial and aquatic environments, encompassing animal and human pathogens in addition to saprophytes. Approximately 150 EM species can be separated into fast and slow growers based on sequence and copy number differences of their 16S rRNA genes. Cultivation methods are not appropriate for diversity studies; few studies have investigated EM diversity in soil despite their importance as potential reservoirs of pathogens and their hypothesized role in masking or blocking M. bovis BCG vaccine. Methods: We report here the development, optimization and validation of molecular assays targeting the 16S rRNA gene to assess diversity and prevalence of fast and slow growing EM in representative soils from semi tropical and temperate areas. New primer sets were designed also to target uniquely slow growing mycobacteria and used with PCR-DGGE, tag-encoded Titanium amplicon pyrosequencing and quantitative PCR. Results: PCR-DGGE and pyrosequencing provided a consensus of EM diversity; for example, a high abundance of pyrosequencing reads and DGGE bands corresponded to M. moriokaense, M. colombiense and M. riyadhense. As expected pyrosequencing provided more comprehensive information; additional prevalent species included M. chlorophenolicum, M. neglectum, M. gordonae, M. aemonae. Prevalence of the total Mycobacterium genus in the soil samples ranged from 2.3×107 to 2.7×108 gene targets g−1; slow growers prevalence from 2.9×105 to 1.2×107 cells g−1. Conclusions: This combined molecular approach enabled an unprecedented qualitative and quantitative assessment of EM across soil samples. Good concordance was found between methods and the bioinformatics analysis was validated by random resampling. Sequences from most pathogenic groups associated with slow growth were identified in extenso in all soils tested with a specific assay, allowing to unmask them from the Mycobacterium whole genus, in which, as minority members, they would have remained undetected

Positive selection inhibits gene mobilization and transfer in soil bacterial communities

Horizontal gene transfer (HGT) between bacterial lineages is a fundamental evolutionary process that accelerates adaptation. Sequence analyses show that conjugative plasmids are principal agents of HGT in natural communities. However, we lack understanding of how the ecology of bacterial communities and their environments affect the dynamics of plasmid-mediated gene mobilization and transfer. Here we show, in simple experimental soil bacterial communities containing a conjugative mercury resistance plasmid, the repeated, independent mobilization of transposon-borne genes from chromosome to plasmid, plasmid to chromosome and, in the absence of mercury selection, interspecific gene transfers from the chromosome of one species to the other via the plasmid. By reducing conjugation, positive selection for plasmid-encoded traits, like mercury resistance, can consequently inhibit HGT. Our results suggest that interspecific plasmid-mediated gene mobilization is most likely to occur in environments where plasmids are infectious, parasitic elements rather than those where plasmids are positively selected, beneficial elements

Patellofemoral pain syndrome (PFPS): a systematic review of anatomy and potential risk factors

Patellofemoral Pain Syndrome (PFPS), a common cause of anterior knee pain, is successfully treated in over 2/3 of patients through rehabilitation protocols designed to reduce pain and return function to the individual. Applying preventive medicine strategies, the majority of cases of PFPS may be avoided if a pre-diagnosis can be made by clinician or certified athletic trainer testing the current researched potential risk factors during a Preparticipation Screening Evaluation (PPSE). We provide a detailed and comprehensive review of the soft tissue, arterial system, and innervation to the patellofemoral joint in order to supply the clinician with the knowledge required to assess the anatomy and make recommendations to patients identified as potentially at risk. The purpose of this article is to review knee anatomy and the literature regarding potential risk factors associated with patellofemoral pain syndrome and prehabilitation strategies. A comprehensive review of knee anatomy will present the relationships of arterial collateralization, innervations, and soft tissue alignment to the possible multifactoral mechanism involved in PFPS, while attempting to advocate future use of different treatments aimed at non-soft tissue causes of PFPS

Host-Adaptation of Francisella tularensis Alters the Bacterium's Surface-Carbohydrates to Hinder Effectors of Innate and Adaptive Immunity

The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase.SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host-adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.F. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development

Systematic evaluation of immune regulation and modulation

Cancer immunotherapies are showing promising clinical results in a variety of malignancies. Monitoring the immune as well as the tumor response following these therapies has led to significant advancements in the field. Moreover, the identification and assessment of both predictive and prognostic biomarkers has become a key component to advancing these therapies. Thus, it is critical to develop systematic approaches to monitor the immune response and to interpret the data obtained from these assays. In order to address these issues and make recommendations to the field, the Society for Immunotherapy of Cancer reconvened the Immune Biomarkers Task Force. As a part of this Task Force, Working Group 3 (WG3) consisting of multidisciplinary experts from industry, academia, and government focused on the systematic assessment of immune regulation and modulation. In this review, the tumor microenvironment, microbiome, bone marrow, and adoptively transferred T cells will be used as examples to discuss the type and timing of sample collection. In addition, potential types of measurements, assays, and analyses will be discussed for each sample. Specifically, these recommendations will focus on the unique collection and assay requirements for the analysis of various samples as well as the high-throughput assays to evaluate potential biomarkers

The importance of climatic factors and outliers in predicting regional monthly campylobacteriosis risk in Georgia, USA

Incidence of Campylobacter infection exhibits a strong seasonal component and regional variations in temperate climate zones. Forecasting the risk of infection regionally may provide clues to identify sources of transmission affected by temperature and precipitation. The objectives of this study were to (1) assess temporal patterns and differences in campylobacteriosis risk among nine climatic divisions of Georgia, USA, (2) compare univariate forecasting models that analyze campylobacteriosis risk over time with those that incorporate temperature and/or precipitation, and (3) investigate alternatives to supposedly random walk series and non-random occurrences that could be outliers. Temporal patterns of campylobacteriosis risk in Georgia were visually and statistically assessed. Univariate and multivariable forecasting models were used to predict the risk of campylobacteriosis and the coefficient of determination (R(2)) was used for evaluating training (1999–2007) and holdout (2008) samples. Statistical control charting and rolling holdout periods were investigated to better understand the effect of outliers and improve forecasts. State and division level campylobacteriosis risk exhibited seasonal patterns with peaks occurring between June and August, and there were significant associations between campylobacteriosis risk, precipitation, and temperature. State and combined division forecasts were better than divisions alone, and models that included climate variables were comparable to univariate models. While rolling holdout techniques did not improve predictive ability, control charting identified high-risk time periods that require further investigation. These findings are important in (1) determining how climatic factors affect environmental sources and reservoirs of Campylobacter spp. and (2) identifying regional spikes in the risk of human Campylobacter infection and their underlying causes