Repeated mRNA vaccination sequentially boosts SARS-CoV-2-specific CD8+ T cells in persons with previous COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hybrid immunity is more protective than vaccination or previous infection alone. To investigate the kinetics of spike-reactive T (TS) cells from SARS-CoV-2 infection through messenger RNA vaccination in persons with hybrid immunity, we identified the T cell receptor (TCR) sequences of thousands of index TS cells and tracked their frequency in bulk TCRß repertoires sampled longitudinally from the peripheral blood of persons who had recovered from coronavirus disease 2019 (COVID-19). Vaccinations led to large expansions in memory TS cell clonotypes, most of which were CD8+ T cells, while also eliciting diverse TS cell clonotypes not observed before vaccination. TCR sequence similarity clustering identified public CD8+ and CD4+ TCR motifs associated with spike (S) specificity. Synthesis of longitudinal bulk ex vivo single-chain TCRß repertoires and paired-chain TCRÉ‘ß sequences from droplet sequencing of TS cells provides a roadmap for the rapid assessment of T cell responses to vaccines and emerging pathogens.

The Regulation of Nucleic Acid Vaccine Responses by the Microbiome.

Nucleic acid vaccines, including both RNA and DNA platforms, are key technologies that have considerable promise in combating both infectious disease and cancer. However, little is known about the extrinsic factors that regulate nucleic acid vaccine responses and which may determine their effectiveness. The microbiome is recognized as a significant regulator of immune development and response, whose role in regulating some traditional vaccine platforms has recently been discovered. Using germ-free and specific pathogen-free mouse models in combination with different protein, DNA, and mRNA vaccine regimens, we demonstrate that the microbiome is a significant regulator of nucleic acid vaccine immunogenicity. Although the presence of the microbiome enhances CD8+ T cell responses to mRNA lipid nanoparticle immunization, the microbiome suppresses Ig and CD4+ T cell responses to DNA-prime, DNA-protein-boost immunization, indicating contrasting roles for the microbiome in the regulation of these different nucleic acid vaccine platforms. In the case of mRNA lipid nanoparticle vaccination, germ-free mice display reduced dendritic cell/macrophage activation that may underlie the deficient vaccine response. Our study identifies the microbiome as a relevant determinant of nucleic acid vaccine response with implications for continued therapeutic development and deployment of these vaccines.

Survival of Vinyl Chloride Respiring Dehalococcoides mccartyi under Long-Term Electron Donor Limitation.

In anoxic groundwater aquifers, the long-term survival of Dehalococcoides mccartyi populations expressing the gene vcrA (or bvcA) encoding reductive vinyl chloride dehalogenases are important to achieve complete dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) to nonchlorinated ethene. The absence or inactivity of vcrA-containing Dehalococcoides results in the accumulation of the harmful chlorinated intermediates dichloroethene (DCE) and vinyl chloride (VC). Although vcrA-containing Dehalococcoides subpopulations depend on synergistic interaction with other organohalide-respiring populations generating their metabolic electron acceptors (DCE and VC), their survival requires successful competition for electron donor within the entire organohalide-respiring microbial community. To understand this dualism of synergy and competition under growth conditions relevant in contaminated aquifers, we investigated Dehalococcoides-level population structure when subjected to a change in the ratio of electron donor to chlorinated electron acceptor in continuously stirred tank reactors (CSTRs) operated over 7 years. When the electron donor formate was supplied in stoichiometric excess to TCE, both tceA-containing and vcrA-containing Dehalococcoides populations persisted, and near-complete dechlorination to ethene was stably maintained. When the electron donor formate was supplied at substoichiometric concentrations, the interactions between tceA-containing and vcrA-containing populations shifted toward direct competition for the same limiting catabolic electron donor substrate with subsequent niche exclusion of the vcrA-containing population. After more than 2000 days of operation under electron donor limitation, increasing the electron donor to TCE ratio facilitated a recovery of the vcrA-containing Dehalococoides population to its original frequency. We demonstrate that electron donor scarcity alone, in the absence of competing metabolic processes or inhibitory dechlorination intermediate products, is sufficient to alter the Dehalococcoides population structure. These results underscore the importance of electron donor and chloroethene stoichiometry in maintaining balanced functional performance within consortia composed of multiple D. mccartyi subpopulations, even when other competing electron acceptor processes are absent.

1,2-Dichloroethane Exposure Alters the Population Structure, Metabolism, and Kinetics of a Trichloroethene-Dechlorinating Dehalococcoides mccartyi Consortium.

Bioremediation of groundwater contaminated with chlorinated aliphatic hydrocarbons such as perchloroethene and trichloroethene can result in the accumulation of the undesirable intermediate vinyl chloride. Such accumulation can either be due to the absence of specific vinyl chloride respiring Dehalococcoides mccartyi or to the inhibition of such strains by the metabolism of other microorganisms. The fitness of vinyl chloride respiring Dehalococcoides mccartyi subpopulations is particularly uncertain in the presence of chloroethene/chloroethane cocontaminant mixtures, which are commonly found in contaminated groundwater. Therefore, we investigated the structure of Dehalococcoides populations in a continuously fed reactor system under changing chloroethene/ethane influent conditions. We observed that increasing the influent ratio of 1,2-dichloroethane to trichloroethene was associated with ecological selection of a tceA-containing Dehalococcoides population relative to a vcrA-containing Dehalococcoides population. Although both vinyl chloride and 1,2-dichloroethane could be simultaneously transformed to ethene, prolonged exposure to 1,2-dichloroethane diminished the vinyl chloride transforming capacity of the culture. Kinetic tests revealed that dechlorination of 1,2-dichloroethane by the consortium was strongly inhibited by cis-dichloroethene but not vinyl chloride. Native polyacrylamide gel electrophoresis and mass spectrometry revealed that a trichloroethene reductive dehalogenase (TceA) homologue was the most consistently expressed of four detectable reductive dehalogenases during 1,2-dichloroethane exposure, suggesting that it catalyzes the reductive dihaloelimination of 1,2-dichloroethane to ethene.

Nanoliter qPCR platform for highly parallel, quantitative assessment of reductive dehalogenase genes and populations of dehalogenating microorganisms in complex environments.

Idiosyncratic combinations of reductive dehalogenase (rdh) genes are a distinguishing genomic feature of closely related organohalogen-respiring bacteria. This feature can be used to deconvolute the population structure of organohalogen-respiring bacteria in complex environments and to identify relevant subpopulations, which is important for tracking interspecies dynamics needed for successful site remediation. Here we report the development of a nanoliter qPCR platform to identify organohalogen-respiring bacteria and populations by quantifying major orthologous reductive dehalogenase gene groups. The qPCR assays can be operated in parallel within a 5184-well nanoliter qPCR (nL-qPCR) chip at a single annealing temperature and buffer condition. We developed a robust bioinformatics approach to select from thousands of computationally proposed primer pairs those that are specific to individual rdh gene groups and compatible with a single amplification condition. We validated hundreds of the most selective qPCR assays and examined their performance in a trichloroethene-degrading bioreactor, revealing population structures as well as their unexpected shifts in abundance and community dynamics.

Merkel cell polyomavirus-specific and CD39+CLA+ CD8 T cells as blood-based predictive biomarkers for PD-1 blockade in Merkel cell carcinoma.

Merkel cell carcinoma is a skin cancer often driven by Merkel cell polyomavirus (MCPyV) with high rates of response to anti-PD-1 therapy despite low mutational burden. MCPyV-specific CD8 T cells are implicated in anti-PD-1-associated immune responses and provide a means to directly study tumor-specific T cell responses to treatment. Using mass cytometry and combinatorial tetramer staining, we find that baseline frequencies of blood MCPyV-specific cells correlated with response and survival. Frequencies of these cells decrease markedly during response to therapy. Phenotypes of MCPyV-specific CD8 T cells have distinct expression patterns of CD39, cutaneous lymphocyte-associated antigen (CLA), and CD103. Correspondingly, overall bulk CD39+CLA+ CD8 T cell frequencies in blood correlate with MCPyV-specific cell frequencies and similarly predicted favorable clinical outcomes. Conversely, frequencies of CD39+CD103+ CD8 T cells are associated with tumor burden and worse outcomes. These cell subsets can be useful as biomarkers and to isolate blood-derived tumor-specific T cells.

Multi-omic profiling reveals the endogenous and neoplastic responses to immunotherapies in cutaneous T cell lymphoma.

Cutaneous T cell lymphomas (CTCLs) are skin cancers with poor survival rates and limited treatments. While immunotherapies have shown some efficacy, the immunological consequences of administering immune-activating agents to CTCL patients have not been systematically characterized. We apply a suite of high-dimensional technologies to investigate the local, cellular, and systemic responses in CTCL patients receiving either mono- or combination anti-PD-1 plus interferon-gamma (IFN-γ) therapy. Neoplastic T cells display no evidence of activation after immunotherapy. IFN-γ induces muted endogenous immunological responses, while anti-PD-1 elicits broader changes, including increased abundance of CLA+CD39+ T cells. We develop an unbiased multi-omic profiling approach enabling discovery of immune modules stratifying patients. We identify an enrichment of activated regulatory CLA+CD39+ T cells in non-responders and activated cytotoxic CLA+CD39+ T cells in leukemic patients. Our results provide insights into the effects of immunotherapy in CTCL patients and a generalizable framework for multi-omic analysis of clinical trials.

Expansion of the HSV-2-specific T cell repertoire in skin after immunotherapeutic HSV-2 vaccine.

The skin at the site of HSV-2 reactivation is enriched for HSV-2-specific T cells. To evaluate whether an immunotherapeutic vaccine could elicit skin-based memory T cells, we studied skin biopsies and HSV-2-reactive CD4+ T cells from PBMCs by T cell receptor (TCR) ß chain (TRB) sequencing before and after vaccination with a replication-incompetent whole-virus HSV-2 vaccine candidate (HSV529). The representation of HSV-2-reactive CD4+ TRB sequences from PBMCs in the skin TRB repertoire increased after the first vaccine dose. We found sustained expansion after vaccination of unique, skin-based T cell clonotypes that were not detected in HSV-2-reactive CD4+ T cells isolated from PBMCs. In one participant, a switch in immunodominance occurred with the emergence of a TCR αß pair after vaccination that was not detected in blood. This TCRαß was shown to be HSV-2 reactive by expression of a synthetic TCR in a Jurkat-based NR4A1 reporter system. The skin in areas of HSV-2 reactivation possessed an oligoclonal TRB repertoire that was distinct from the circulation. Defining the influence of therapeutic vaccination on the HSV-2-specific TRB repertoire requires tissue-based evaluation.

Probing Dermal Immunity to Mycobacteria through a Controlled Human Infection Model.

Cutaneous mycobacterial infections cause substantial morbidity and are challenging to diagnose and treat. An improved understanding of the dermal immune response to mycobacteria may inspire new therapeutic approaches. We conducted a controlled human infection study with 10 participants who received 2 × 106 CFUs of Mycobacterium bovis bacillus Calmette-Guérin (Tice strain) intradermally and were randomized to receive isoniazid or no treatment. Peripheral blood was collected at multiple time points for flow cytometry, bulk RNA sequencing (RNA-seq), and serum Ab assessments. Systemic immune responses were detected as early as 8 d postchallenge in this M. bovis bacillus Calmette-Guérin-naive population. Injection-site skin biopsies were performed at days 3 and 15 postchallenge and underwent immune profiling using mass cytometry and single-cell RNA-seq, as well as quantitative assessments of bacterial viability and burden. Molecular viability testing and standard culture results correlated well, although no differences were observed between treatment arms. Single-cell RNA-seq revealed various immune and nonimmune cell types in the skin, and communication between them was inferred by ligand-receptor gene expression. Day 3 communication was predominantly directed toward monocytes from keratinocyte, muscle, epithelial, and endothelial cells, largely via the migration inhibitory factor pathway and HLA-E-KLRK1 interaction. At day 15, communication was more balanced between cell types. These data reveal the potential role of nonimmune cells in the dermal immune response to mycobacteria and the utility of human challenge studies to augment our understanding of mycobacterial infections.

The Regulation of Nucleic Acid Vaccine Responses by the Microbiome.

Nucleic acid vaccines, including both RNA and DNA platforms, are key technologies that have considerable promise in combating both infectious disease and cancer. However, little is known about the extrinsic factors that regulate nucleic acid vaccine responses and which may determine their effectiveness. The microbiome is recognized as a significant regulator of immune development and response, whose role in regulating some traditional vaccine platforms has recently been discovered. Using germ-free and specific-pathogen-free mouse models in combination with different protein, DNA, and mRNA vaccine regimens, we demonstrate that the microbiome is a significant regulator of nucleic acid vaccine immunogenicity. While the presence of the microbiome enhances CD8+ T cell responses to mRNA lipid nanoparticle (LNP) immunization, the microbiome suppresses immunoglobulin and CD4+ T cell responses to DNA-prime, DNA-protein-boost immunization, indicating contrasting roles for the microbiome in the regulation of these different nucleic acid vaccine platforms. In the case of mRNA-LNP vaccination, germ-free mice display reduced dendritic cell/macrophage activation that may underlie the deficient vaccine response. Our study identifies the microbiome as a relevant determinant of nucleic acid vaccine response with implications for their continued therapeutic development and deployment.

Strain-level characterization of health-associated bacterial consortia that colonize the human gut during infancy.

Background: The human gut microbiome develops rapidly during infancy, a key window of development coinciding with maturation of the adaptive immune system. However, little is known of the microbiome growth dynamics over the first few months of life and whether there are any generalizable patterns across human populations. We performed metagenomic sequencing on stool samples (n=94) from a cohort of infants (n=15) at monthly intervals in the first six months of life, augmenting our dataset with seven published studies for a total of 4,441 metagenomes from 1,162 infants. Results: Strain-level de novo analysis was used to identify 592 of the most abundant organisms in the infant gut microbiome. Previously unrecognized consortia were identified which exhibited highly correlated abundances across samples and were composed of diverse species spanning multiple genera. Analysis of a cohort of infants with cystic fibrosis identified one such novel consortium of diverse Enterobacterales which was positively correlated with weight gain. While all studies showed an increased community stability during the first year of life, microbial dynamics varied widely in the first few months of life, both by study and by individual. Conclusion: By augmenting published metagenomic datasets with data from a newly established cohort we were able to identify novel groups of organisms that are correlated with measures of robust human development. We hypothesize that the presence of these groups may impact human health in aggregate in ways that individual species may not in isolation.

mRNA vaccination boosts S-specific T cell memory and promotes expansion of CD45RAint TEMRA-like CD8+ T cells in COVID-19 recovered individuals.

SARS-CoV-2 infection and mRNA vaccination both elicit spike (S)-specific T cell responses. To analyze how T cell memory from prior infection influences T cell responses to vaccination, we evaluated functional T cell responses in naive and previously infected vaccine recipients. Pre-vaccine S-specific responses are predictive of subsequent CD8+ T cell vaccine-response magnitudes. Comparing baseline with post-vaccination TCRß repertoires, we observed large clonotypic expansions correlated with the frequency of spike-specific T cells. Epitope mapping the largest CD8+ T cell responses confirms that an HLA-A∗03:01 epitope was highly immunodominant. Peptide-MHC tetramer staining together with mass cytometry and single-cell sequencing permit detailed phenotyping and clonotypic tracking of these S-specific CD8+ T cells. Our results demonstrate that infection-induced S-specific CD8+ T cell memory plays a significant role in shaping the magnitude and clonal composition of the circulating T cell repertoire after vaccination, with mRNA vaccination promoting CD8+ memory T cells to a TEMRA-like phenotype.

Flexible Distance-Based TCR Analysis in Python with tcrdist3.

Paired- and single-chain T cell receptor (TCR) sequencing are now commonly used techniques for interrogating adaptive immune responses. TCRs targeting the same epitope frequently share motifs consisting of critical contact residues. Here we illustrate the key features of tcrdist3, a new Python package for distance-based TCR analysis through a series of three interactive examples. In the first example, we illustrate how tcrdist3 can integrate sequence similarity networks, gene-usage plots, and background-adjusted CDR3 logos to identify TCR sequence features conferring antigen specificity among sets of peptide-MHC-multimer sorted receptors. In the second example, we show how the TCRjoin feature in tcrdist3 can be used to flexibly query receptor sequences of interest against bulk repertoires or libraries of previously annotated TCRs based on matching of similar sequences. In the third example, we show how the TCRdist metric can be leveraged to identify candidate polyclonal receptors under antigenic selection in bulk repertoires based on sequence neighbor enrichment testing, a statistical approach similar to TCRNET and ALICE algorithms, but with added flexibility in how the neighborhood can be defined.

Durable Expansion of TCR-δ Meta-Clonotypes After BCG Revaccination in Humans.

Mycobacterium bovis bacille Calmette-Guérin (BCG) has been used for 100 years and prevents disseminated tuberculosis and death in young children. However, it shows only partial efficacy against pulmonary tuberculosis (TB) in adults, so new vaccines are urgently needed. The protective efficacy of BCG depends on T cells, which are typically activated by pathogen-derived protein antigens that bind to highly polymorphic major histocompatibility complex (MHC) molecules. Some T cells recognize non-protein antigens via antigen presenting systems that are independent of genetic background, leading to their designation as donor-unrestricted T (DURT) cells. Whether live whole cell vaccines, like BCG, can induce durable expansions of DURT cells in humans is not known. We used combinatorial tetramer staining, multi-parameter flow cytometry, and immunosequencing to comprehensively characterize the effect of BCG on activation and expansion of DURT cell subsets. We examined peripheral blood mononuclear cells (PBMC) derived from a Phase I study of South African adults in which samples were archived at baseline, 3 weeks, and 52 weeks post-BCG revaccination. We did not observe a change in the frequency of total mucosal-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells, germline encoded mycolyl-reactive (GEM) T cells, or γδ T cells at 52 weeks post-BCG. However, immunosequencing revealed a set of TCR-δ clonotypes that were expanded at 52 weeks post-BCG revaccination. These expanded clones expressed the Vδ2 gene segment and could be further defined on the basis of biochemical similarity into several 'meta-clonotypes' that likely recognize similar epitopes. Our data reveal that BCG vaccination leads to durable expansion of DURT cell clonotypes despite a limited effect on total circulating frequencies in the blood and have implications for defining the immunogenicity of candidate whole cell TB vaccines.

Multi-trial analysis of HIV-1 envelope gp41-reactive antibodies among global recipients of candidate HIV-1 vaccines.

Many participants in HIV-1 vaccine trials, who have not previously been exposed to or vaccinated against HIV-1, display serum immunoglobulin antibodies that bind the gp41 region of HIV-1 envelope prior to vaccination. Previous studies have hypothesized that these pre-existing antibodies may be cross-reactive and may skew future vaccine responses. In 12 large studies conducted by the HIV Vaccine Trial Network (HVTN) (n=1470 individuals), we find wide variation among participants in the pre-vaccine levels of gp41-reactive antibodies as measured by the binding antibody multiplex assay (BAMA). In the absence of exposure to the gp41 immunogen, anti-gp41 IgG levels were temporally stable over 26-52 weeks in repeated measures of placebo recipients. The analysis revealed that the geometric mean of pre-vaccine anti-gp41 IgG response was greater among participants in South Africa compared with participants in the United States. With gene-level metagenomic sequencing of pre-vaccination fecal samples collected from participants in one trial (HVTN 106), we detected positive associations between pre-vaccine anti-gp41 IgG and abundance of genes from multiple taxa in the Eubacteriales order. The genes most strongly associated with higher baseline anti-gp41 IgG mapped to a clade containing Blautia wexlerae and closely related strains. In trials with vaccine products containing the full or partial portion of gp41 immunogen alongside a gp120 immunogen, we did not find evidence that individuals with higher baseline anti-gp41 IgG had different levels of anti-gp120 IgG after vaccination compared to individuals with lower pre-vaccine anti-gp41 levels (pooled estimate of standardized mean difference -0.01 with a 95% CI [-0.37; 0.34]).

CD8+ T cell clonotypes from prior SARS-CoV-2 infection predominate during the cellular immune response to mRNA vaccination.

Almost three years into the SARS-CoV-2 pandemic, hybrid immunity is highly prevalent worldwide and more protective than vaccination or prior infection alone. Given emerging resistance of variant strains to neutralizing antibodies (nAb), it is likely that T cells contribute to this protection. To understand how sequential SARS-CoV-2 infection and mRNA-vectored SARS-CoV-2 spike (S) vaccines affect T cell clonotype-level expansion kinetics, we identified and cross-referenced TCR sequences from thousands of S-reactive single cells against deeply sequenced peripheral blood TCR repertoires longitudinally collected from persons during COVID-19 convalescence through booster vaccination. Successive vaccinations recalled memory T cells and elicited antigen-specific T cell clonotypes not detected after infection. Vaccine-related recruitment of novel clonotypes and the expansion of S-specific clones were most strongly observed for CD8+ T cells. Severe COVID-19 illness was associated with a more diverse CD4+ T cell response to SARS-CoV-2 both prior to and after mRNA vaccination, suggesting imprinting of CD4+ T cells by severe infection. TCR sequence similarity search algorithms revealed myriad public TCR clusters correlating with human leukocyte antigen (HLA) alleles. Selected TCRs from distinct clusters functionally recognized S in the predicted HLA context, with fine viral peptide requirements differing between TCRs. Most subjects tested had S-specific T cells in the nasal mucosa after a 3rd mRNA vaccine dose. The blood and nasal T cell responses to vaccination revealed by clonal tracking were more heterogeneous than nAb boosts. Analysis of bulk and single cell TCR sequences reveals T cell kinetics and diversity at the clonotype level, without requiring prior knowledge of T cell epitopes or HLA restriction, providing a roadmap for rapid assessment of T cell responses to emerging pathogens.

TCR meta-clonotypes for biomarker discovery with tcrdist3 enabled identification of public, HLA-restricted clusters of SARS-CoV-2 TCRs.

T-cell receptors (TCRs) encode clinically valuable information that reflects prior antigen exposure and potential future response. However, despite advances in deep repertoire sequencing, enormous TCR diversity complicates the use of TCR clonotypes as clinical biomarkers. We propose a new framework that leverages experimentally inferred antigen-associated TCRs to form meta-clonotypes - groups of biochemically similar TCRs - that can be used to robustly quantify functionally similar TCRs in bulk repertoires across individuals. We apply the framework to TCR data from COVID-19 patients, generating 1831 public TCR meta-clonotypes from the SARS-CoV-2 antigen-associated TCRs that have strong evidence of restriction to patients with a specific human leukocyte antigen (HLA) genotype. Applied to independent cohorts, meta-clonotypes targeting these specific epitopes were more frequently detected in bulk repertoires compared to exact amino acid matches, and 59.7% (1093/1831) were more abundant among COVID-19 patients that expressed the putative restricting HLA allele (false discovery rate [FDR]<0.01), demonstrating the potential utility of meta-clonotypes as antigen-specific features for biomarker development. To enable further applications, we developed an open-source software package, tcrdist3, that implements this framework and facilitates flexible workflows for distance-based TCR repertoire analysis.

TCR meta-clonotypes for biomarker discovery with tcrdist3: identification of public, HLA-restricted SARS-CoV-2 associated TCR features.

As the mechanistic basis of adaptive cellular antigen recognition, T cell receptors (TCRs) encode clinically valuable information that reflects prior antigen exposure and potential future response. However, despite advances in deep repertoire sequencing, enormous TCR diversity complicates the use of TCR clonotypes as clinical biomarkers. We propose a new framework that leverages antigen-enriched repertoires to form meta-clonotypes - groups of biochemically similar TCRs - that can be used to robustly identify and quantify functionally similar TCRs in bulk repertoires. We apply the framework to TCR data from COVID-19 patients, generating 1831 public TCR meta-clonotypes from the 17 SARS-CoV-2 antigen-enriched repertoires with the strongest evidence of HLA-restriction. Applied to independent cohorts, meta-clonotypes targeting these specific epitopes were more frequently detected in bulk repertoires compared to exact amino acid matches, and 59.7% (1093/1831) were more abundant among COVID-19 patients that expressed the putative restricting HLA allele (FDR < 0.01), demonstrating the potential utility of meta-clonotypes as antigen-specific features for biomarker development. To enable further applications, we developed an open-source software package, tcrdist3, that implements this framework and facilitates flexible workflows for distance-based TCR repertoire analysis.

High-Throughput Multiparallel Enteropathogen Detection via Nano-Liter qPCR.

Quantitative molecular diagnostic methods can effectively detect pathogen-specific nucleic acid sequences, but costs associated with multi-pathogen panels hinder their widespread use in research trials. Nano-liter qPCR (nL-qPCR) is a miniaturized tool for quantification of multiple targets in large numbers of samples based on assay parallelization on a single chip, with potentially significant cost-savings due to rapid throughput and reduced reagent volumes. We evaluated a suite of novel and published assays to detect 17 enteric pathogens using a commercially available nL-qPCR technology. Amplification efficiencies ranged from 88 to 98% (mean 91%) and were reproducible across four operators at two separate facilities. When applied to fecal material, assays were sensitive and selective (99.8% of DNA amplified were genes from the target organism). Due to nanofluidic volumes, detection limits were 1-2 orders of magnitude less sensitive for nL-qPCR than an enteric TaqMan Array Card (TAC). However, higher detection limits do not hinder detection of diarrhea-causing pathogen concentrations. Compared to TAC, nL-qPCR displayed 99% (95% CI 0.98, 0.99) negative percent agreement and 62% (95% CI 0.59, 0.65) overall positive percent agreement for presence of pathogens across diarrheal and non-diarrheal fecal samples. Positive percent agreement was 89% among samples with concentrations above the nL-qPCR detection limits. nL-qPCR assays showed an underestimation bias of 0.34 log10 copies/gram of stool [IQR -0.40, -0.28] compared with TAC. With 12 times higher throughput for a sixth of the per-sample cost of the enteric TAC, the nL-qPCR chip is a viable alternative for enteropathogen quantification for studies where other technologies are cost-prohibitive.

Single cell genomic study of Dehalococcoidetes species from deep-sea sediments of the Peruvian Margin.

The phylum Chloroflexi is one of the most frequently detected phyla in the subseafloor of the Pacific Ocean margins. Dehalogenating Chloroflexi (Dehalococcoidetes) was originally discovered as the key microorganisms mediating reductive dehalogenation via their key enzymes reductive dehalogenases (Rdh) as sole mode of energy conservation in terrestrial environments. The frequent detection of Dehalococcoidetes-related 16S rRNA and rdh genes in the marine subsurface implies a role for dissimilatory dehalorespiration in this environment; however, the two genes have never been linked to each other. To provide fundamental insights into the metabolism, genomic population structure and evolution of marine subsurface Dehalococcoidetes sp., we analyzed a non-contaminated deep-sea sediment core sample from the Peruvian Margin Ocean Drilling Program (ODP) site 1230, collected 7.3 m below the seafloor by a single cell genomic approach. We present for the first time single cell genomic data on three deep-sea Chloroflexi (Dsc) single cells from a marine subsurface environment. Two of the single cells were considered to be part of a local Dehalococcoidetes population and assembled together into a 1.38-Mb genome, which appears to be at least 85% complete. Despite a high degree of sequence-level similarity between the shared proteins in the Dsc and terrestrial Dehalococcoidetes, no evidence for catabolic reductive dehalogenation was found in Dsc. The genome content is however consistent with a strictly anaerobic organotrophic or lithotrophic lifestyle.