Molecular determinants of cross-reactivity and potency by VH3-33 antibodies against the Plasmodium falciparum circumsporozoite protein.

IGHV3-33-encoded antibodies are prevalent in the human humoral response against the Plasmodium falciparum circumsporozoite protein (PfCSP). Among VH3-33 antibodies, cross-reactivity between PfCSP major repeat (NANP), minor (NVDP), and junctional (NPDP) motifs is associated with high affinity and potent parasite inhibition. However, the molecular basis of antibody cross-reactivity and the relationship with efficacy remain unresolved. Here, we perform an extensive structure-function characterization of 12 VH3-33 anti-PfCSP monoclonal antibodies (mAbs) with varying degrees of cross-reactivity induced by immunization of mice expressing a human immunoglobulin gene repertoire. We identify residues in the antibody paratope that mediate cross-reactive binding and delineate four distinct epitope conformations induced by antibody binding, with one consistently associated with high protective efficacy and another that confers comparably potent inhibition of parasite liver invasion. Our data show a link between molecular features of cross-reactive VH3-33 mAb binding to PfCSP and mAb potency, relevant for the development of antibody-based interventions against malaria.

Glycosylated nanoparticle-based PfCSP vaccine confers long-lasting antibody responses and sterile protection in mouse malaria model.

The development of an effective and durable vaccine remains a central goal in the fight against malaria. Circumsporozoite protein (CSP) is the major surface protein of sporozoites and the target of the only licensed Plasmodium falciparum (Pf) malaria vaccine, RTS,S/AS01. However, vaccine efficacy is low and short-lived, highlighting the need for a second-generation vaccine with superior efficacy and durability. Here, we report a Helicobacter pylori apoferritin-based nanoparticle immunogen that elicits strong B cell responses against PfCSP epitopes that are targeted by the most potent human monoclonal antibodies. Glycan engineering of the scaffold and fusion of an exogenous T cell epitope enhanced the anti-PfCSP B cell response eliciting strong, long-lived and protective humoral immunity in mice. Our study highlights the power of rational vaccine design to generate a highly efficacious second-generation anti-infective malaria vaccine candidate and provides the basis for its further development.

Molecular and functional properties of human Plasmodium falciparum CSP C-terminus antibodies.

Human monoclonal antibodies (mAbs) against the central repeat and junction domain of Plasmodium falciparum circumsporozoite protein (PfCSP) have been studied extensively to guide malaria vaccine design compared to antibodies against the PfCSP C terminus. Here, we describe the molecular characteristics and protective potential of 73 germline and mutated human mAbs against the highly immunogenic PfCSP C-terminal domain. Two mAbs recognized linear epitopes in the C-terminal linker with sequence similarity to repeat and junction motifs, whereas all others targeted conformational epitopes in the α-thrombospondin repeat (α-TSR) domain. Specificity for the polymorphic Th2R/Th3R but not the conserved RII+/CS.T3 region in the α-TSR was associated with IGHV3-21/IGVL3-21 or IGLV3-1 gene usage. Although the C terminus specific mAbs showed signs of more efficient affinity maturation and class-switching compared to anti-repeat mAbs, live sporozoite binding and inhibitory activity was limited to a single C-linker reactive mAb with cross-reactivity to the central repeat and junction. The data provide novel insights in the human anti-C-linker and anti-α-TSR antibody response that support exclusion of the PfCSP C terminus from malaria vaccine designs.

Light-Trapping Electrode for the Efficiency Enhancement of Bifacial Perovskite Solar Cells.

Antireflection and light-trapping coatings are important parts of photovoltaic architectures, which enable the reduction of parasitic optical losses, and therefore increase the power conversion efficiency (PCE). Here, we propose a novel approach to enhance the efficiency of perovskite solar cells using a light-trapping electrode (LTE) with non-reciprocal optical transmission, consisting of a perforated metal film covered with a densely packed array of nanospheres. Our LTE combines charge collection and light trapping, and it can replace classical transparent conducting oxides (TCOs) such as ITO or FTO, providing better optical transmission and conductivity. One of the most promising applications of our original LTE is the optimization of efficient bifacial perovskite solar cells. We demonstrate that with our LTE, the short-circuit current density and fill factor are improved for both front and back illumination of the solar cells. Thus, we observe an 11% improvement in the light absorption for the monofacial PSCs, and a 15% for the bifacial PSCs. The best theoretical results of efficiency for our PSCs are 27.9% (monofacial) and 33.4% (bifacial). Our study opens new prospects for the further efficiency enhancement for perovskite solar cells.

Clonal evolution and TCR specificity of the human TFH cell response to Plasmodium falciparum CSP.

T follicular helper (TFH) cells play a crucial role in the development of long-lived, high-quality B cell responses after infection and vaccination. However, little is known about how antigen-specific TFH cells clonally evolve in response to complex pathogens and what guides the targeting of different epitopes. Here, we assessed the cell phenotype, clonal dynamics, and T cell receptor (TCR) specificity of human circulating TFH (cTFH) cells during successive malaria immunizations with radiation-attenuated Plasmodium falciparum (Pf) sporozoites. Repeated parasite exposures induced a dynamic, polyclonal cTFH response with high frequency of cells specific to a small number of epitopes in Pf circumsporozoite protein (PfCSP), the primary sporozoite surface protein and well-defined vaccine target. Human leukocyte antigen (HLA) restrictions and differences in TCR generation probability were associated with differences in the epitope targeting frequency and indicated the potential of amino acids 311 to 333 in the Th2R/T* region as a T cell supertope. But most of vaccine-induced anti-amino acid 311 to 333 TCRs, including convergent TCRs with high sequence similarity, failed to tolerate natural polymorphisms in their target peptide sequence, thus demonstrating that the TFH cell response was limited to the vaccine strain. These data suggest that the high parasite diversity in endemic areas will limit boosting of the vaccine-induced TFH cell response by natural infections. Our findings may guide the further design of PfCSP-based malaria vaccines able to induce potent T helper cell responses for broad, long-lasting antibody responses.

The Interplay between CD27dull and CD27bright B Cells Ensures the Flexibility, Stability, and Resilience of Human B Cell Memory.

Memory B cells (MBCs) epitomize the adaptation of the immune system to the environment. We identify two MBC subsets in peripheral blood, CD27dull and CD27bright MBCs, whose frequency changes with age. Heavy chain variable region (VH) usage, somatic mutation frequency replacement-to-silent ratio, and CDR3 property changes, reflecting consecutive selection of highly antigen-specific, low cross-reactive antibody variants, all demonstrate that CD27dull and CD27bright MBCs represent sequential MBC developmental stages, and stringent antigen-driven pressure selects CD27dull into the CD27bright MBC pool. Dynamics of human MBCs are exploited in pregnancy, when 50% of maternal MBCs are lost and CD27dull MBCs transit to the more differentiated CD27bright stage. In the postpartum period, the maternal MBC pool is replenished by the expansion of persistent CD27dull clones. Thus, the stability and flexibility of human B cell memory is ensured by CD27dull MBCs that expand and differentiate in response to change.

Impact of sediment parameters in the prediction of benthic microbial fuel cell performance.

The benthic microbial fuel cell (BMFC) is a promising technology for harvesting renewable energy from marine littoral environments. The scientific community has researched BMFC technology for well over a decade, but the in situ performance remains challenging. To address this challenge, BMFC power experiments were performed on sediment collected from San Diego Bay (CA, USA), La Spezia (Italy) and Honolulu (HI, USA) in the ever-changing littoral environment. Analysis of BMFC laboratory data found the power density varied substantially across 11 sites in San Diego Bay. In addition, data from experiments repeated at four locations in San Diego Bay showed significant differences between experiments performed in 2014, 2016 and 2019. Multivariable linear analysis showed BMFC 90 day cumulative power density was positively correlated with the total organic carbon (p < 0.05) and negatively correlated with the black carbon in the sediment (p < 0.05). Regression coefficients trained on the San Diego Bay data from 2014 facilitated accurate predictions of BMFC performance in 2016 and 2019. The modeling paradigm accurately explained variations in BMFC power performance in La Spezia and showed sediment parameters can impact BMFC performance differently across geographic regions. The results demonstrate a great potential to use sediment parameters and statistical modeling to predict BMFC power performance prior to deployment in oceanographic environments, thereby reducing cost, work force and resources.

sumrep: A Summary Statistic Framework for Immune Receptor Repertoire Comparison and Model Validation.

The adaptive immune system generates an incredible diversity of antigen receptors for B and T cells to keep dangerous pathogens at bay. The DNA sequences coding for these receptors arise by a complex recombination process followed by a series of productivity-based filters, as well as affinity maturation for B cells, giving considerable diversity to the circulating pool of receptor sequences. Although these datasets hold considerable promise for medical and public health applications, the complex structure of the resulting adaptive immune receptor repertoire sequencing (AIRR-seq) datasets makes analysis difficult. In this paper we introduce sumrep, an R package that efficiently performs a wide variety of repertoire summaries and comparisons, and show how sumrep can be used to perform model validation. We find that summaries vary in their ability to differentiate between datasets, although many are able to distinguish between covariates such as donor, timepoint, and cell type for BCR and TCR repertoires. We show that deletion and insertion lengths resulting from V(D)J recombination tend to be more discriminative characterizations of a repertoire than summaries that describe the amino acid composition of the CDR3 region. We also find that state-of-the-art generative models excel at recapitulating gene usage and recombination statistics in a given experimental repertoire, but struggle to capture many physiochemical properties of real repertoires.

Comparative Analysis of B-Cell Receptor Repertoires Induced by Live Yellow Fever Vaccine in Young and Middle-Age Donors.

Age-related changes can significantly alter the state of adaptive immune system and often lead to attenuated response to novel pathogens and vaccination. In present study we employed 5'RACE UMI-based full length and nearly error-free immunoglobulin profiling to compare plasma cell antibody repertoires in young (19-26 years) and middle-age (45-58 years) individuals vaccinated with a live yellow fever vaccine, modeling a newly encountered pathogen. Our analysis has revealed age-related differences in the responding antibody repertoire ranging from distinct IGH CDR3 repertoire properties to differences in somatic hypermutation intensity and efficiency and antibody lineage tree structure. Overall, our findings suggest that younger individuals respond with a more diverse antibody repertoire and employ a more efficient somatic hypermutation process than elder individuals in response to a newly encountered pathogen.

Marine microbial community response to inorganic and organic sediment amendments in laboratory mesocosms.

Sediment amendments provide promising strategies of enhancing sequestration of heavy metals and degradation of organic contaminants. The impacts of sediment amendments for metal and organic remediation including apatite, organoclay (and apatite and organoclay in geotextile mats), acetate, and chitin on environmental microbial communities in overlying water and sediment profiles are reported here. These experiments were performed concurrent with an ecotoxicity evaluation (data submitted in companion paper) and X-ray absorption spectroscopy of zinc speciation post apatite amendments. X-ray absorption spectra showed that a modest modification of zinc speciation occurred in amended treatments. Significant changes in both bacterial cell densities and populations were observed in response to amendments of apatite+organoclay, chitin, and acetate. The enriched bacteria and breakdown of these amendments were likely attributed to water quality degradation (e.g. ammonia and dissolved oxygen). Molecular fingerprints of bacterial communities by denaturant gradient gel electrophoresis (DGGE) showed that distinct bacterial populations occurred in overlying waters from different amendments: apatite+organoclay led to the dominance of Gammaproteobacteria, acetate enriched Alphaproteobacteria, and chitin treatment led to a dominance of Bacteroidetes and Alphaproteobacteria. In amended sediments, Firmicutes, Bacteroidetes, and Deltaproteobacteria (Desulfovibrio) were commonly found with chitin and apatite+chitin treatments. Finally, sulfate-reducing bacteria (e.g. Desulfovibrio) and metal-reducing bacteria were also recovered with most probable number (MPN) analyses in treatments with acetate, chitin, and apatite+chitin. These geochemically important bacteria were stimulated by amendments and may play critical functional roles in the metal and organic contaminant remediation process for future investigations of contaminated sediments.

Genomic encyclopedia of sugar utilization pathways in the Shewanella genus.

BACKGROUND: Carbohydrates are a primary source of carbon and energy for many bacteria. Accurate projection of known carbohydrate catabolic pathways across diverse bacteria with complete genomes constitutes a substantial challenge due to frequent variations in components of these pathways. To address a practically and fundamentally important challenge of reconstruction of carbohydrate utilization machinery in any microorganism directly from its genomic sequence, we combined a subsystems-based comparative genomic approach with experimental validation of selected bioinformatic predictions by a combination of biochemical, genetic and physiological experiments. RESULTS: We applied this integrated approach to systematically map carbohydrate utilization pathways in 19 genomes from the Shewanella genus. The obtained genomic encyclopedia of sugar utilization includes ~170 protein families (mostly metabolic enzymes, transporters and transcriptional regulators) spanning 17 distinct pathways with a mosaic distribution across Shewanella species providing insights into their ecophysiology and adaptive evolution. Phenotypic assays revealed a remarkable consistency between predicted and observed phenotype, an ability to utilize an individual sugar as a sole source of carbon and energy, over the entire matrix of tested strains and sugars.Comparison of the reconstructed catabolic pathways with E. coli identified multiple differences that are manifested at various levels, from the presence or absence of certain sugar catabolic pathways, nonorthologous gene replacements and alternative biochemical routes to a different organization of transcription regulatory networks. CONCLUSIONS: The reconstructed sugar catabolome in Shewanella spp includes 62 novel isofunctional families of enzymes, transporters, and regulators. In addition to improving our knowledge of genomics and functional organization of carbohydrate utilization in Shewanella, this study led to a substantial expansion of our current version of the Genomic Encyclopedia of Carbohydrate Utilization. A systematic and iterative application of this approach to multiple taxonomic groups of bacteria will further enhance it, creating a knowledge base adequate for the efficient analysis of any newly sequenced genome as well as of the emerging metagenomic data.

Mercury speciation in marine sediments under sulfate-limited conditions.

Sediment profiles of total mercury (Hg) and monomethylmercury (MMHg) were determined from a 30-m drill hole located north of Venice, Italy. While the sediment profile of total Hg concentration was fairly constant between 1 and 10 m, that of the MMHg concentration showed an unexpected peak at a depth of 6 m. Due to the limited sulfate content (<1 mM) at the depth of 6 m, we hypothesized that the methylation of inorganic Hg(II) at this depth is associated with the syntrophic processes occurring between methanogens and sulfidogens. To test this hypothesis, anoxic sediment slurries were prepared using buried Venice Lagoon sediments amended with HgCl(2), and we monitored MMHg concentration in sediment slurries over time under two geochemical conditions: high sulfate (1-16 mM) and limited sulfate concentrations (<100 microM). After day 52 and onward from the addition of inorganic Hg(II), the MMHg concentrations were higher in sulfate-limited slurries compared to high sulfate slurries, along with methane production in both slurries. On the basis of these results, we argue that active methylation of inorganic Hg(II) occurs under sulfate-limited conditions possibly by syntrophic processes occurring between methanogens and sulfidogens. The environmental significance of syntrophic Hg(II) methylation should be further studied.

Conserved synteny at the protein family level reveals genes underlying Shewanella species' cold tolerance and predicts their novel phenotypes.

Bacteria of the genus Shewanella can thrive in different environments and demonstrate significant variability in their metabolic and ecophysiological capabilities including cold and salt tolerance. Genomic characteristics underlying this variability across species are largely unknown. In this study, we address the problem by a comparison of the physiological, metabolic, and genomic characteristics of 19 sequenced Shewanella species. We have employed two novel approaches based on association of a phenotypic trait with the number of the trait-specific protein families (Pfam domains) and on the conservation of synteny (order in the genome) of the trait-related genes. Our first approach is top-down and involves experimental evaluation and quantification of the species' cold tolerance followed by identification of the correlated Pfam domains and genes with a conserved synteny. The second, a bottom-up approach, predicts novel phenotypes of the species by calculating profiles of each Pfam domain among their genomes and following pair-wise correlation of the profiles and their network clustering. Using the first approach, we find a link between cold and salt tolerance of the species and the presence in the genome of a Na(+)/H(+) antiporter gene cluster. Other cold-tolerance-related genes include peptidases, chemotaxis sensory transducer proteins, a cysteine exporter, and helicases. Using the bottom-up approach, we found several novel phenotypes in the newly sequenced Shewanella species, including degradation of aromatic compounds by an aerobic hybrid pathway in Shewanella woodyi, degradation of ethanolamine by Shewanella benthica, and propanediol degradation by Shewanella putrefaciens CN32 and Shewanella sp. W3-18-1.

Comparative systems biology across an evolutionary gradient within the Shewanella genus.

To what extent genotypic differences translate to phenotypic variation remains a poorly understood issue of paramount importance for several cornerstone concepts of microbiology including the species definition. Here, we take advantage of the completed genomic sequences, expressed proteomic profiles, and physiological studies of 10 closely related Shewanella strains and species to provide quantitative insights into this issue. Our analyses revealed that, despite extensive horizontal gene transfer within these genomes, the genotypic and phenotypic similarities among the organisms were generally predictable from their evolutionary relatedness. The power of the predictions depended on the degree of ecological specialization of the organisms evaluated. Using the gradient of evolutionary relatedness formed by these genomes, we were able to partly isolate the effect of ecology from that of evolutionary divergence and to rank the different cellular functions in terms of their rates of evolution. Our ranking also revealed that whole-cell protein expression differences among these organisms, when the organisms were grown under identical conditions, were relatively larger than differences at the genome level, suggesting that similarity in gene regulation and expression should constitute another important parameter for (new) species description. Collectively, our results provide important new information toward beginning a systems-level understanding of bacterial species and genera.

Current production and metal oxide reduction by Shewanella oneidensis MR-1 wild type and mutants.

Shewanella oneidensis MR-1 is a gram-negative facultative anaerobe capable of utilizing a broad range of electron acceptors, including several solid substrates. S. oneidensis MR-1 can reduce Mn(IV) and Fe(III) oxides and can produce current in microbial fuel cells. The mechanisms that are employed by S. oneidensis MR-1 to execute these processes have not yet been fully elucidated. Several different S. oneidensis MR-1 deletion mutants were generated and tested for current production and metal oxide reduction. The results showed that a few key cytochromes play a role in all of the processes but that their degrees of participation in each process are very different. Overall, these data suggest a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1.

Biogeochemical factors affecting mercury methylation in sediments of the Venice Lagoon, Italy.

Mercury methylation and sulfate reduction rates, total Hg, and monomethyl Hg in the sediments of the Venice Lagoon (Italy) were measured in June 2005 in order to identify the factors affecting the methylation of inorganic Hg. While the rates of Hg methylation and sulfate reduction were generally higher in the surface layers (0-2.5 cm), the correlation between Hg methylation and sulfate reduction rates was not significant when considering all depths and sites. This discrepancy is discussed considering two factors: the activity of sulfate-reducing bacteria and Hg solubility. The former factor is important in determining the Hg methylation rate in comparable geochemical conditions as evidenced by similar vertical profiles of Hg methylation and sulfate reduction rates in each sediment core. The latter factor was assessed by comparing the Hg methylation rate with the particle-water partition coefficient of Hg. The Hg methylation rates normalized to sulfate reduction rates showed a negative linear correlation with the logarithm of the particle-water partition coefficient of Hg, suggesting that the availability of dissolved Hg is a critical factor affecting Hg methylation. Solid FeS seems to play an important role in controlling the solubility of Hg in Venice Lagoon sediments, where sulfate and iron reductions are the dominant electron-accepting processes. Overall, the production of monomethyl Hg in the Venice Lagoon is controlled by a fine balance between microbial and geochemical processes with key factors being the microbial sulfate reduction rate and the availability of dissolved Hg.

Toxicity of Cr(lll) to Shewanella sp. strain MR-4 during Cr(VI) reduction.

Bioremediation of chromium through the reduction of hexavalent chromium (as the chromate ion, CrO42-) is based on the notion that the product, trivalent chromium (Cr(III)), is less toxic than chromate. In this study, we show that soluble Cr(III), present at pH 6-8 as the Cr3+ ion and/or hydroxyl complexes (henceforth referred to as uncomplexed Cr(III)), can be found transiently in significant concentrations and has a deleterious effect on Shewanella sp. MR-4. However, Cr(lll) complexed to an organic ligand or precipitated as Cr(OH)3(s) has little or no effect on cells. Similarly, during the reduction of Cr(VI) by strain MR-4, complexation of the product, Cr(lll), results in increased cell survival and extended Cr(VI) reduction activity. These results and gene expression data obtained by qRT-PCR (quantitative reverse transcription-PCR) suggestthatthe observed toxic effect of Cr(II) formed during Cr(VI) reduction or added as an uncomplexed species is due to the interference with basic cell activities such as DNA transcription and/or replication. Important implications for the bioremediation of Cr(VI)-contaminated sites emerge from this study: Cr(VI) reduction by Shewanella sp. MR-4 is enhanced and sustained by the presence of compounds able to complex Cr(III) as it is being formed but, in turn, the complexation of Cr(III) precludes its precipitation and immobilization.

Comparative genomics and experimental characterization of N-acetylglucosamine utilization pathway of Shewanella oneidensis.

We used a comparative genomics approach implemented in the SEED annotation environment to reconstruct the chitin and GlcNAc utilization subsystem and regulatory network in most proteobacteria, including 11 species of Shewanella with completely sequenced genomes. Comparative analysis of candidate regulatory sites allowed us to characterize three different GlcNAc-specific regulons, NagC, NagR, and NagQ, in various proteobacteria and to tentatively assign a number of novel genes with specific functional roles, in particular new GlcNAc-related transport systems, to this subsystem. Genes SO3506 and SO3507, originally annotated as hypothetical in Shewanella oneidensis MR-1, were suggested to encode novel variants of GlcN-6-P deaminase and GlcNAc kinase, respectively. Reconstitution of the GlcNAc catabolic pathway in vitro using these purified recombinant proteins and GlcNAc-6-P deacetylase (SO3505) validated the entire pathway. Kinetic characterization of GlcN-6-P deaminase demonstrated that it is the subject of allosteric activation by GlcNAc-6-P. Consistent with genomic data, all tested Shewanella strains except S. frigidimarina, which lacked representative genes for the GlcNAc metabolism, were capable of utilizing GlcNAc as the sole source of carbon and energy. This study expands the range of carbon substrates utilized by Shewanella spp., unambiguously identifies several genes involved in chitin metabolism, and describes a novel variant of the classical three-step biochemical conversion of GlcNAc to fructose 6-phosphate first described in Escherichia coli.