Dendritic cell therapy with CD137L-DC-EBV-VAX in locally recurrent or metastatic nasopharyngeal carcinoma is safe and confers clinical benefit.

INTRODUCTION: Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC), and provides a target for a dendritic cell (DC) vaccine. CD137 ligand (CD137L) expressed on antigen presenting cells, costimulates CD137-expressing T cells, and reverse CD137L signaling differentiates monocytes to CD137L-DC, a type of DC, which is more potent than classical DC in stimulating T cells. METHODS: In this phase I study, patients with locally recurrent or metastatic NPC were administered CD137L-DC pulsed with EBV antigens (CD137L-DC-EBV-VAX). RESULTS: Of the 12 patients treated, 9 received full 7 vaccine doses with a mean administered cell count of 23.9 × 106 per dose. Treatment was well tolerated with only 4 cases of grade 1 related adverse events. A partial response was obtained in 1 patient, and 4 patients are still benefitting from a progression free survival (PFS) of currently 2-3 years. The mean pre-treatment neutrophil: lymphocyte ratio was 3.4 and a value of less than 3 was associated with prolonged median PFS. Progressors were characterized by a high frequency of naïve T cells but a low frequency of CD8+ effector T cells while patients with a clinical benefit (CB) had a high frequency of memory T cells. Patients with CB had lower plasma EBV DNA levels, and a reduction after vaccination. CONCLUSION: CD137L-DC-EBV-VAX was well tolerated. The use of CD137L-DC-EBV-VAX is demonstrated to be safe. Consistent results were obtained from all 12 patients, indicating that CD137L-DC-EBV-VAX induces an anti-EBV and anti-NPC immune response, and warranting further studies in patients post effective chemotherapy. PRECIS: The first clinical testing of CD137L-DC, a new type of monocyte-derived DC, finds that CD137L-DC are safe, and that they can induce an immune response against Epstein-Barr virus-associated nasopharyngeal carcinoma that leads to tumor regression or prevents tumor progression.

Longitudinal Changes in Diet Cause Repeatable and Largely Reversible Shifts in Gut Microbial Communities of Laboratory Mice and Are Observed across Segments of the Entire Intestinal Tract.

Dietary changes are known to alter the composition of the gut microbiome. However, it is less understood how repeatable and reversible these changes are and how diet switches affect the microbiota in the various segments of the gastrointestinal tract. Here, a treatment group of conventionally raised laboratory mice is subjected to two periods of western diet (WD) interrupted by a period of standard diet (SD) of the same duration. Beta-diversity analyses show that diet-induced microbiota changes are largely reversible (q = 0.1501; PERMANOVA, weighted-UniFrac comparison of the treatment-SD group to the control-SD group) and repeatable (q = 0.032; PERMANOVA, weighted-UniFrac comparison of both WD treatments). Furthermore, we report that diet switches alter the gut microbiota composition along the length of the intestinal tract in a segment-specific manner, leading to gut segment-specific Firmicutes/Bacteroidota ratios. We identified prevalent and distinct Amplicon Sequencing Variants (ASVs), particularly in genera of the recently described Muribaculaceae, along the gut as well as ASVs that are differentially abundant between segments of treatment and control groups. Overall, this study provides insights into the reversibility of diet-induced microbiota changes and highlights the importance of expanding sampling efforts beyond the collections of fecal samples to characterize diet-dependent and segment-specific microbiome differences.

Muribaculum gordoncarteri sp. nov., an anaerobic bacterium from the faeces of C57BL/6J mice.

An anaerobic bacterial strain, named TLL-A4T, was isolated from fecal pellets of conventionally raised C57BL/6J mice. Analysis of the 16S rRNA gene indicated that the strain belongs to the phylum Bacteroidetes and, more specifically, to the recently proposed Muribaculaceae (also known as S24-7 clade or Candidatus Homeothermaceae). Strain TLL-A4T's 16S rRNA gene shared 92.8 % sequence identity with the type strain of the only published species of the genus Muribaculum, Muribaculum intestinale DSM 28989T. Genome-sequencing of TLL-A4T was performed to compare average amino acid identity (AAI) value and percentage of conserved proteins (POCP) between both strains. The AAI analysis revealed that strain TLL-A4T had high identity (69.8 %) with M. intestinale DSM 28989T, while the POCP was 56 %. These values indicate that strain TLL-A4T could be considered a member of the genus Muribaculum but not belonging to the species M. intestinale. Quinone analysis indicated MK10 (63 %) and MK11 (32 %) as major quinones in the membrane, while MK9 was only present as a minor component (5 %). The main cellular fatty acid was anteiso-C15 : 0 (42.8 %); summed feature 11 (17.5 %), C15 : 0 iso (13.4 %), C18 : 1 ω9c (5.6 %), C16.0 3-OH (4.5 %) and C15 : 0 (4.2 %) were detected in minor amounts. Analysis of enzyme activities using the API 32A and API 20A kits indicated major differences between strain TLL-A4T and Muribaculum intestinale DSM 28989T. Based on genotypic, phylogenetic and phenotypic differences, strain TLL-A4T is considered to represent a novel species of the genus Muribaculum, for which the name Muribaculum gordoncarteri sp. nov. is proposed. The type strain is TLL-A4T (=DSM 108194T=KCTC 15770T).

Cultivation and description of Duncaniella dubosii sp. nov., Duncaniella freteri sp. nov. and emended description of the species Duncaniella muris.

Three bacterial strains, C9, H5 and TLL-A3, were isolated from fecal pellets of conventionally raised C57BL/6J mice. Analysis of 16S rRNA genes indicated that the strains belonged to the Muribaculaceae, and shared 91.6-99.9 % sequence identity with the recently described Duncaniella muris DSM 103720T. Genome-sequencing of the isolates was performed to compare average nucleotide identities (ANI) between strains. The ANI analysis revealed that all isolates shared highest ANI with D. muris DSM 103720T, with strain C9 being most similar (ANI: 98.0 %) followed by strains H5 (ANI: 76.4 %) and TLL-A3 (ANI: 74.4 %). Likewise, digital DNA-DNA hybridization (dDDH) indicated high similarity of strain C9 (dDDH: 86.6 %) to D. muris DSM 103720T, but strains H5 and TLL-A3 showed lower similarity (dDDH <35 %) to either of the three type species of the Muribaculaceae (Muribaculum intestinale DSM 28989T , Paramuribaculum intestinale DSM 100749T, D. muris DSM 103720T). MK-10 and MK-11 were abundant in all three isolates, but concentrations varied between species. Based on genotypic, phylogenetic and phenotypic differences, the strains TLL-A3 and H5 are considered to represent novel species of the genus Duncaniella, for which the names Duncaniella freteri sp. nov., and Duncaniella dubosii sp. nov., are proposed. The respective type strains are TLL-A3T (=DSM 108168T=KCTC 15769T), and H5T (=DSM 107170T=KCTC 15734T). Strain C9 reveals limited sequence dissimilarity and minor differences in morphological properties with Duncaniella muris DSM 103720T and is therefore proposed to belong to the same species. The respective strain is C9 (=DSM 107165=KCTC 15733).

Insights into the microbiome of farmed Asian sea bass (Lates calcarifer) with symptoms of tenacibaculosis and description of Tenacibaculum singaporense sp. nov.

Outbreaks of diseases in farmed fish remain a recurring problem despite the development of vaccines and improved hygiene standards on aquaculture farms. One commonly observed bacterial disease in tropical aquaculture of the South-East Asian region is tenacibaculosis, which is attributed to members of the genus Tenacibaculum (family Flavobacteriaceae, phylum Bacteroidetes), most notably Tenacibaculum maritimum. The impact of tenacibaculosis on the fish microbiota remains poorly understood. In this study, we analysed the microbiota of different tissues of commercially reared Asian seabass (Lates calcarifer) that showed symptoms of tenacibaculosis and compared the microbial communities to those of healthy and experimentally infected fish that were exposed to diseased farmed fish. The relative abundance of Tenacibaculum species in experimentally infected fish was significantly lower than in commercially reared diseased fish and revealed a higher prevalence of different Tenacibaculum species. One isolated strain, TLL-A2T, shares 98.7% 16S rRNA gene identity with Tenacibaculum mesophilum DSM 13764T. The genome of strain TLL-A2T was sequenced and compared to that of T. mesophilum DSM 13764T. Analysis of average nucleotide identity and comparative genome analysis revealed only 92% identity between T. mesophilum DSM 13764T and strain TLL-A2T and differences between the two strains in predicted carbohydrate activating enzymes respectively. Phenotypic comparison between strain TLL-A2T and T. mesophilum DSM 13764T indicated additional differences, such as growth response at different salt concentrations. Based on molecular and phenotypic differences, strain TLL-A2T (=DSM 106434T, KCTC 62393T) is proposed as the type strain of Tenacibaculum singaporense sp. nov.

Schaedlerella arabinosiphila gen. nov., sp. nov., a D-arabinose-utilizing bacterium isolated from faeces of C57BL/6J mice that is a close relative of Clostridium species ASF 502.

The use of gnotobiotics has attracted wide interest in recent years due to technological advances that have revealed the importance of host-associated microbiomes for host physiology and health. One of the oldest and most important gnotobiotic mouse model, the altered Schaedler flora (ASF) has been used for several decades. ASF comprises eight different bacterial strains, which have been characterized to different extent, but only a few are available through public strain collections. Here, the isolation of a close relative of one of the less-studied ASF strains, Clostridium species ASF 502, from faeces of C57BL/6J mice is reported. Isolate TLL-A1T shares 99.5 % 16S rRNA gene sequence identity with Clostridium species ASF 502 and phylogenetic analyses indicate that both strains belong to the uncultured so-called 'Lachnospiraceae UCG 006' clade. The rare sugar d-arabinose was used as a sole carbon source in the anaerobic isolation medium. Results of growth experiments with TLL-A1T on different carbon sources and analysis of its ~6.5 Gb indicate that TLL-A1T harbours a large gene repertoire that enables it to utilize a variety of carbohydrates for growth. Comparative genome analyses of TLL-A1T and Clostridium species ASF 502 reveal differences in genome content between the two strains, in particular with regards to carbohydrate-activating enzymes. Based on genomic, molecular and phenotypic differences, we propose to classify strain TLL-A1T (DSM 106076T=KCTC 15657T) as a representative of a new genus and a new species, for which we propose the name Schaedlerella arabinosiphila gen. nov., sp. nov.

Comparative Genomic Analysis of Members of the Genera Methanosphaera and Methanobrevibacter Reveals Distinct Clades with Specific Potential Metabolic Functions.

Methanobrevibacter and Methanosphaera species represent some of the most prevalent methanogenic archaea in the gastrointestinal tract of animals and humans and play an important role in this environment. The aim of this study was to identify genomic features that are shared or specific for members of each genus with a special emphasis of the analysis on the assimilation of nitrogen and acetate and the utilization of methanol and ethanol for methanogenesis. Here, draft genome sequences of Methanobrevibacter thaueri strain DSM 11995T, Methanobrevibacter woesei strain DSM 11979T, and Methanosphaera cuniculi strain 4103T are reported and compared to those of 16 other Methanobrevibacter and Methanosphaera genomes, including genomes of the 13 currently available types of strains of the two genera. The comparative genome analyses indicate that among other genes, the absence of molybdopterin cofactor biosynthesis is conserved in Methanosphaera species but reveals also that the three species share a core set of more than 300 genes that distinguishes the genus Methanosphaera from the genus Methanobrevibacter. Multilocus sequence analysis shows that the genus Methanobrevibacter can be subdivided into clades, potentially new genera, which may display characteristic specific metabolic features. These features include not only the potential ability of nitrogen fixation and acetate assimilation in a clade comprised of Methanobrevibacter species from the termite gut and Methanobrevibacter arboriphilus strains but also the potential capability to utilize ethanol and methanol in a clade comprising Methanobrevibacter wolinii strain DSM 11976T, Mbb. sp. AbM4, and Mbb. boviskoreani strain DSM 25824T.

The intestinal digesta microbiota of tropical marine fish is largely uncultured and distinct from surrounding water microbiota.

Studying the gut microbes of marine fishes is an important part of conservation as many fish species are increasingly threatened by extinction. The gut microbiota of only a small fraction of the more than 32,000 known fish species has been investigated. In this study we analysed the intestinal digesta microbiota composition of more than 50 different wild fish species from tropical waters. Our results show that the fish harbour intestinal digesta microbiota that are distinct from that of the surrounding water and that location, domestication status, and host intrinsic factors are strongly associated with the microbiota composition. Furthermore, we show that the vast majority (~97%) of the fish-associated microorganisms do not have any cultured representative. Considering the impact of the microbiota on host health and physiology, these findings underpin the call to also preserve the microbiota of host species, especially those that may be exposed to habitat destruction.

Host Age Prediction from Fecal Microbiota Composition in Male C57BL/6J Mice.

The lifelong relationship between microorganisms and hosts has a profound impact on the overall health and physiology of the holobiont. Microbiome composition throughout the life span of a host remains largely understudied. Here, the fecal microbiota of conventionally raised C57BL/6J male mice was characterized throughout almost the entire adult life span, from "maturing" (9 weeks) until "very old" (112 weeks) age. Our results suggest that microbiota changes occur throughout life but are more pronounced in maturing to middle-age mice than in mice later in life. Phylum-level analysis indicates a shift of the Bacteroidota-to-Firmicutes ratio in favor of Firmicutes in old and very old mice. More Firmicutes amplicon sequence variants (ASVs) were transient with varying successional patterns than Bacteroidota ASVs, which varied primarily during maturation. Microbiota configurations from five defined life phases were used as training sets in a Bayesian model, which effectively enabled the prediction of host age. These results suggest that age-associated compositional differences may have considerable implications for the interpretation and comparability of animal model-based microbiome studies. The sensitivity of the age prediction to dietary perturbations was tested by applying this approach to two age-matched groups of C57BL/6J mice that were fed either a standard or western diet. The predicted age for the western diet-fed animals was on average 27 ± 11 (mean ± standard deviation) weeks older than that of standard diet-fed animals. This indicates that the fecal microbiota-based predicted age may be influenced not only by the host age and physiology but also potentially by other factors such as diet. IMPORTANCE The gut microbiome of a host changes with age. Cross-sectional studies demonstrate that microbiota of different age groups are distinct but do not demonstrate the temporal change that a longitudinal study is able to show. Here, we performed a longitudinal study of adult mice for over 2 years. We identified life stages where compositional changes were more dynamic and showed temporal changes for the more abundant species. Using a Bayesian model, we could reliably predict the life stages of the mice. Application of the same training set to mice fed different dietary regimens revealed that life-stage age predictions were possible for mice fed the same diet but less so for mice fed different diets. This study sheds light on the temporal changes that occur within the gut microbiota of laboratory mice over their life span and may inform researchers on the appropriate mouse age for their research.

Complete Genome Sequence of Duncaniella muris Strain B8, Isolated from the Feces of C57/BL6 Mice.

Here, the complete genome sequence of Duncaniella muris strain B8 is presented. The anaerobic strain was isolated from the feces of C57/BL6 mice and is closely related to D. muris strain DSM 103720, which is the type strain of the recently proposed genus Duncaniella of the Muribaculaceae.

Complete Genome Sequence of Sponge-Associated Tenacibaculum mesophilum DSM 13764T.

Here, the complete genome sequence of sponge-associated Tenacibaculum mesophilum DSM 13764T is presented. T. mesophilum is a close relative of the fish pathogen T. maritimum, which causes significant fish disease outbreaks in aquaculture facilities. The T. mesophilum genome sequence will serve as an important resource for comparative genomics approaches.

Large-scale Isolation of Highly Pure "Untouched" Regulatory T Cells in a GMP Environment for Adoptive Cell Therapy.

Adoptive cell therapy is an emerging treatment strategy for a number of serious diseases. Regulatory T (Treg) cells represent 1 cell type of particular interest for therapy of inflammatory conditions, as they are responsible for controlling unwanted immune responses. Initial clinical trials of adoptive transfer of Treg cells in patients with graft-versus-host disease were shown to be safe. However, obtaining sufficient numbers of highly pure and functional Treg cells with minimal contamination remains a challenge. We developed a novel approach to isolate "untouched" human Treg cells from healthy donors on the basis of negative selection using the surface markers CD49d and CD127. This procedure, which uses an antibody cocktail and magnetic beads for separation in an automated system (RoboSep), was scaled up and adapted to be compatible with good manufacturing practice conditions. With this setup we performed 9 Treg isolations from large-scale leukapheresis samples in a good manufacturing practice facility. These runs yielded sufficient numbers of "untouched" Treg cells for immediate use in clinical applications. The cell preparations consisted of viable highly pure FoxP3-positive Treg cells that were functional in suppressing the proliferation of effector T cells. Contamination with CD4 effector T cells was <10%. All other cell types did not exceed 2% in the final product. Remaining isolation reagents were reduced to levels that are considered safe. Treg cells isolated with this procedure will be used in a phase I clinical trial of adoptive transfer into leukemia patients developing graft-versus-host disease after stem cell transplantation.