Differential peripheral immune signatures elicited by vegan versus ketogenic diets in humans.

Nutrition has broad impacts on all physiological processes. However, how nutrition affects human immunity remains largely unknown. Here we explored the impact of a dietary intervention on both immunity and the microbiota by performing a post hoc analysis of a clinical trial in which each of the 20 participants sequentially consumed vegan or ketogenic diets for 2 weeks ( NCT03878108 ). Using a multiomics approach including multidimensional flow cytometry, transcriptomic, proteomic, metabolomic and metagenomic datasets, we assessed the impact of each diet, and dietary switch, on host immunity and the microbiota. Our data revealed that overall, a ketogenic diet was associated with a significant upregulation of pathways and enrichment in cells associated with the adaptive immune system. In contrast, a vegan diet had a significant impact on the innate immune system, including upregulation of pathways associated with antiviral immunity. Both diets significantly and differentially impacted the microbiome and host-associated amino acid metabolism, with a strong downregulation of most microbial pathways following ketogenic diet compared with baseline and vegan diet. Despite the diversity of participants, we also observed a tightly connected network between datasets driven by compounds associated with amino acids, lipids and the immune system. Collectively, this work demonstrates that in diverse participants 2 weeks of controlled dietary intervention is sufficient to significantly and divergently impact host immunity, which could have implications for precision nutritional interventions. ClinicalTrials.gov registration: NCT03878108 .

Influenza vaccination reveals sex dimorphic imprints of prior mild COVID-19.

Acute viral infections can have durable functional impacts on the immune system long after recovery, but how they affect homeostatic immune states and responses to future perturbations remain poorly understood1-4. Here we use systems immunology approaches, including longitudinal multimodal single-cell analysis (surface proteins, transcriptome and V(D)J sequences) to comparatively assess baseline immune statuses and responses to influenza vaccination in 33 healthy individuals after recovery from mild, non-hospitalized COVID-19 (mean, 151 days after diagnosis) and 40 age- and sex-matched control individuals who had never had COVID-19. At the baseline and independent of time after COVID-19, recoverees had elevated T cell activation signatures and lower expression of innate immune genes including Toll-like receptors in monocytes. Male individuals who had recovered from COVID-19 had coordinately higher innate, influenza-specific plasmablast, and antibody responses after vaccination compared with healthy male individuals and female individuals who had recovered from COVID-19, in part because male recoverees had monocytes with higher IL-15 responses early after vaccination coupled with elevated prevaccination frequencies of 'virtual memory'-like CD8+ T cells poised to produce more IFNγ after IL-15 stimulation. Moreover, the expression of the repressed innate immune genes in monocytes increased by day 1 to day 28 after vaccination in recoverees, therefore moving towards the prevaccination baseline of the healthy control individuals. By contrast, these genes decreased on day 1 and returned to the baseline by day 28 in the control individuals. Our study reveals sex-dimorphic effects of previous mild COVID-19 and suggests that viral infections in humans can establish new immunological set-points that affect future immune responses in an antigen-agnostic manner.

Adaptive immune responses to SARS-CoV-2 persist in the pharyngeal lymphoid tissue of children.

Most studies of adaptive immunity to SARS-CoV-2 infection focus on peripheral blood, which may not fully reflect immune responses at the site of infection. Using samples from 110 children undergoing tonsillectomy and adenoidectomy during the COVID-19 pandemic, we identified 24 samples with evidence of previous SARS-CoV-2 infection, including neutralizing antibodies in serum and SARS-CoV-2-specific germinal center and memory B cells in the tonsils and adenoids. Single-cell B cell receptor (BCR) sequencing indicated virus-specific BCRs were class-switched and somatically hypermutated, with overlapping clones in the two tissues. Expanded T cell clonotypes were found in tonsils, adenoids and blood post-COVID-19, some with CDR3 sequences identical to previously reported SARS-CoV-2-reactive T cell receptors (TCRs). Pharyngeal tissues from COVID-19-convalescent children showed persistent expansion of germinal center and antiviral lymphocyte populations associated with interferon (IFN)-γ-type responses, particularly in the adenoids, and viral RNA in both tissues. Our results provide evidence for persistent tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract of children after infection.

Peroxisome proliferator activated receptor-γ agonist pioglitazone improves vascular and metabolic dysfunction in systemic lupus erythematosus.

OBJECTIVES: Premature cardiovascular events in systemic lupus erythematosus (SLE) contribute to morbidity and mortality, with no effective preventive strategies described to date. Immune dysregulation and metabolic disturbances appear to play prominent roles in the induction of vascular disease in SLE. The peroxisome proliferator activated receptor-gamma agonist pioglitazone (PGZ suppresses vascular damage and immune dysregulation in murine lupus and improves endothelial dysfunction in other inflammatory diseases. We hypothesised that PGZ could improve vascular dysfunction and cardiometabolic parameters in SLE. METHODS: Eighty SLE subjects with mild to severe disease activity were randomised to a sequence of PGZ followed by placebo for 3 months, or vice versa, in a double-blind, cross-over design with a 2-month wash-out period. Primary endpoints were parameters of endothelial function and arterial inflammation, measured by multimodal assessments. Additional outcome measures of disease activity, neutrophil dysregulation, metabolic disturbances and gene expression studies were performed. RESULTS: Seventy-two subjects completed the study. PGZ was associated with a significant reduction in Cardio-Ankle Vascular Index (a measure of arterial stiffness) compared with placebo. Various metabolic parameters improved with PGZ, including insulin resistance and lipoprotein profiles. Circulating neutrophil extracellular trap levels also significantly decreased with PGZ compared with placebo. Most adverse events experienced while on PGZ were mild and resolved with reduction in PGZ dose. CONCLUSION: PGZ was well tolerated and induced significant improvement in vascular stiffness and cardiometabolic parameters in SLE. The results suggest that PGZ should be further explored as a modulator of cardiovascular disease risk in SLE. TRIAL REGISTRATION NUMBER: NCT02338999.

Robust, persistent adaptive immune responses to SARS-CoV-2 in the oropharyngeal lymphoid tissue of children.

SARS-CoV-2 infection triggers adaptive immune responses from both T and B cells. However, most studies focus on peripheral blood, which may not fully reflect immune responses in lymphoid tissues at the site of infection. To evaluate both local and systemic adaptive immune responses to SARS-CoV-2, we collected peripheral blood, tonsils, and adenoids from 110 children undergoing tonsillectomy/adenoidectomy during the COVID-19 pandemic and found 24 with evidence of prior SARS-CoV-2 infection, including detectable neutralizing antibodies against multiple viral variants. We identified SARS-CoV-2-specific germinal center (GC) and memory B cells; single cell BCR sequencing showed that these virus-specific B cells were class-switched and somatically hypermutated, with overlapping clones in the adenoids and tonsils. Oropharyngeal tissues from COVID-19-convalescent children showed persistent expansion of GC and anti-viral lymphocyte populations associated with an IFN-γ-type response, with particularly prominent changes in the adenoids, as well as evidence of persistent viral RNA in both tonsil and adenoid tissues of many participants. Our results show robust, tissue-specific adaptive immune responses to SARS-CoV-2 in the upper respiratory tract of children weeks to months after acute infection, providing evidence of persistent localized immunity to this respiratory virus.

Influenza vaccination and single cell multiomics reveal sex dimorphic immune imprints of prior mild COVID-19.

Viral infections can have profound and durable functional impacts on the immune system. There is an urgent need to characterize the long-term immune effects of SARS-CoV-2 infection given the persistence of symptoms in some individuals and the continued threat of novel variants. Here we use systems immunology, including longitudinal multimodal single cell analysis (surface proteins, transcriptome, and V(D)J sequences) from 33 previously healthy individuals after recovery from mild, non-hospitalized COVID-19 and 40 age- and sex-matched healthy controls with no history of COVID-19 to comparatively assess the post-infection immune status (mean: 151 days after diagnosis) and subsequent innate and adaptive responses to seasonal influenza vaccination. Identification of both sex-specific and -independent temporally stable changes, including signatures of T-cell activation and repression of innate defense/immune receptor genes (e.g., Toll-like receptors) in monocytes, suggest that mild COVID-19 can establish new post-recovery immunological set-points. COVID-19-recovered males had higher innate, influenza-specific plasmablast, and antibody responses after vaccination compared to healthy males and COVID-19-recovered females, partly attributable to elevated pre-vaccination frequencies of a GPR56 expressing CD8+ T-cell subset in male recoverees that are "poised" to produce higher levels of IFNγ upon inflammatory stimulation. Intriguingly, by day 1 post-vaccination in COVID-19-recovered subjects, the expression of the repressed genes in monocytes increased and moved towards the pre-vaccination baseline of healthy controls, suggesting that the acute inflammation induced by vaccination could partly reset the immune states established by mild COVID-19. Our study reveals sex-dimorphic immune imprints and in vivo functional impacts of mild COVID-19 in humans, suggesting that prior COVID-19, and possibly respiratory viral infections in general, could change future responses to vaccination and in turn, vaccines could help reset the immune system after COVID-19, both in an antigen-agnostic manner.

Comparative Genomics Analyses Support the Reclassification of Bisgaard Taxon 40 as Mergibacter gen. nov., With Mergibacter septicus sp. nov. as Type Species: Novel Insights Into the Phylogeny and Virulence Factors of a Pasteurellaceae Family Member Associated With Mortality Events in Seabirds.

The Pasteurellaceae family has been associated with fatal diseases in numerous avian species. Several new taxa within this family, including Bisgaard taxon 40, have been recently described in wild birds, but their genomic characteristics and pathogenicity are not well understood. We isolated Bisgaard taxon 40 from four species of seabirds, including one sampled during a mass, multi-species mortality event in Florida, United States. Here, we present a comprehensive phenotypic and genetic characterization of Bisgaard taxon 40 and comparative genomic analysis with reference strains from the Pasteurellaceae family, aiming at determining its phylogenetic position, antimicrobial susceptibility profile, and identifying putative virulence factors. In silico multilocus sequence-based and whole-genome-based phylogenetic analysis clustered all Bisgaard taxon 40 strains together on a distinct branch separated from the other members of the Pasteurellaceae family, indicating that Bisgaard taxon 40 could represent a new genus. These findings were further supported by protein similarity analyses using the concatenation of 31 conserved proteins and other taxonomic approaches such as the percentage of conserved protein test. Additionally, several putative virulence factors were identified, including those associated with adhesion (capsule, ompA, ompH) and colonization (exbD, fur, galU, galE, lpxA, lpxC, and kdsA) of the host and a cytolethal distending toxin (cdt), which may have played a role in disease development leading to the mortality event. Considerably low minimum inhibitory concentrations (MICs) were found for all the drugs tested, in concordance with the absence of antimicrobial resistance genes in these genomes. The novel findings of this study highlight genomic and phenotypic characteristics of this bacterium, providing insights into genome evolution and pathogenicity. We propose a reclassification of these organisms within the Pasteurellaceae family, designated as Mergibacter gen. nov., with Mergibacter septicus sp. nov. as the type species. The type strain is Mergibacter septicus A25201T (=DSM 112696).

Endogenous retroviruses promote homeostatic and inflammatory responses to the microbiota.

The microbiota plays a fundamental role in regulating host immunity. However, the processes involved in the initiation and regulation of immunity to the microbiota remain largely unknown. Here, we show that the skin microbiota promotes the discrete expression of defined endogenous retroviruses (ERVs). Keratinocyte-intrinsic responses to ERVs depended on cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes protein (STING) signaling and promoted the induction of commensal-specific T cells. Inhibition of ERV reverse transcription significantly impacted these responses, resulting in impaired immunity to the microbiota and its associated tissue repair function. Conversely, a lipid-enriched diet primed the skin for heightened ERV- expression in response to commensal colonization, leading to increased immune responses and tissue inflammation. Together, our results support the idea that the host may have co-opted its endogenous virome as a means to communicate with the exogenous microbiota, resulting in a multi-kingdom dialog that controls both tissue homeostasis and inflammation.

Evolution of Antibiotic Resistance in Surrogates of Francisella tularensis (LVS and Francisella novicida): Effects on Biofilm Formation and Fitness.

Francisella tularensis, the causative agent of tularemia, is capable of causing disease in a multitude of mammals and remains a formidable human pathogen due to a high morbidity, low infectious dose, lack of a FDA approved vaccine, and ease of aerosolization. For these reasons, there is concern over the use of F. tularensis as a biological weapon, and, therefore, it has been classified as a Tier 1 select agent. Fluoroquinolones and aminoglycosides often serve as the first line of defense for treatment of tularemia. However, high levels of resistance to these antibiotics has been observed in gram-negative bacteria in recent years, and naturally derived resistant Francisella strains have been described in the literature. The acquisition of antibiotic resistance, either natural or engineered, presents a challenge for the development of medical countermeasures. In this study, we generated a surrogate panel of antibiotic resistant F. novicida and Live Vaccine Strain (LVS) by selection in the presence of antibiotics and characterized their growth, biofilm capacity, and fitness. These experiments were carried out in an effort to (1) assess the fitness of resistant strains; and (2) identify new targets to investigate for the development of vaccines or therapeutics. All strains exhibited a high level of resistance to either ciprofloxacin or streptomycin, a fluoroquinolone and aminoglycoside, respectively. Whole genome sequencing of this panel revealed both on-pathway and off-pathway mutations, with more mutations arising in LVS. For F. novicida, we observed decreased biofilm formation for all ciprofloxacin resistant strains compared to wild-type, while streptomycin resistant isolates were unaffected in biofilm capacity. The fitness of representative antibiotic resistant strains was assessed in vitro in murine macrophage-like cell lines, and also in vivo in a murine model of pneumonic infection. These experiments revealed that mutations obtained by these methods led to nearly all ciprofloxacin resistant Francisella strains tested being completely attenuated while mild attenuation was observed in streptomycin resistant strains. This study is one of the few to examine the link between acquired antibiotic resistance and fitness in Francisella spp., as well as enable the discovery of new targets for medical countermeasure development.

Bacteriophage-associated genes responsible for the widely divergent phenotypes of variants of Burkholderia pseudomallei strain MSHR5848.

PURPOSE: Burkholderia pseudomallei, the tier 1 agent of melioidosis, is a saprophytic microbe that causes endemic infections in tropical regions such as South-East Asia and Northern Australia. It is globally distributed, challenging to diagnose and treat, infectious by several routes including inhalation, and has potential for adversarial use. B. pseudomallei strain MSHR5848 produces two colony variants, smooth (S) and rough (R), which exhibit a divergent range of morphological, biochemical and metabolic phenotypes, and differ in macrophage and animal infectivity. We aimed to characterize two major phenotypic differences, analyse gene expression and study the regulatory basis of the variation. METHODOLOGY: Phenotypic expression was characterized by DNA and RNA sequencing, microscopy, and differential bacteriology. Regulatory genes were identified by cloning and bioinformatics.Results/Key findings. Whereas S produced larger quantities of extracellular DNA, R was upregulated in the production of a unique chromosome 1-encoded Siphoviridae-like bacteriophage, φMSHR5848. Exploratory transcriptional analyses revealed significant differences in variant expression of genes encoding siderophores, pili assembly, type VI secretion system cluster 4 (T6SS-4) proteins, several exopolysaccharides and secondary metabolites. A single 3 base duplication in S was the only difference that separated the variants genetically. It occurred upstream of a cluster of bacteriophage-associated genes on chromosome 2 that were upregulated in S. The first two genes were involved in regulating expression of the multiple phenotypes distinguishing S and R. CONCLUSION: Bacteriophage-associated proteins have a major role in the phenotypic expression of MSHR5848. The goals are to determine the regulatory basis of this phenotypic variation and its role in pathogenesis and environmental persistence of B. pseudomallei.

Functionality of Two Origins of Replication in Vibrio cholerae Strains With a Single Chromosome.

Chromosomal inheritance in bacteria usually entails bidirectional replication of a single chromosome from a single origin into two copies and subsequent partitioning of one copy each into daughter cells upon cell division. However, the human pathogen Vibrio cholerae and other Vibrionaceae harbor two chromosomes, a large Chr1 and a small Chr2. Chr1 and Chr2 have different origins, an oriC-type origin and a P1 plasmid-type origin, respectively, driving the replication of respective chromosomes. Recently, we described naturally occurring exceptions to the two-chromosome rule of Vibrionaceae: i.e., Chr1 and Chr2 fused single chromosome V. cholerae strains, NSCV1 and NSCV2, in which both origins of replication are present. Using NSCV1 and NSCV2, here we tested whether two types of origins of replication can function simultaneously on the same chromosome or one or the other origin is silenced. We found that in NSCV1, both origins are active whereas in NSCV2 ori2 is silenced despite the fact that it is functional in an isolated context. The ori2 activity appears to be primarily determined by the copy number of the triggering site, crtS, which in turn is determined by its location with respect to ori1 and ori2 on the fused chromosome.

The Egyptian Rousette Genome Reveals Unexpected Features of Bat Antiviral Immunity.

Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection, rather than enhanced potency of antiviral defenses, may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans.

A spontaneous mutation in kdsD, a biosynthesis gene for 3 Deoxy-D-manno-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of Francisella tularensis and caused a high level of attenuation in murine models of tularemia.

Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures.

Burkholderia humptydooensis sp. nov., a New Species Related to Burkholderia thailandensis and the Fifth Member of the Burkholderia pseudomallei Complex.

During routine screening for Burkholderia pseudomallei from water wells in northern Australia in areas where it is endemic, Gram-negative bacteria (strains MSMB43T, MSMB121, and MSMB122) with a similar morphology and biochemical pattern to B. pseudomallei and B. thailandensis were coisolated with B. pseudomallei on Ashdown's selective agar. To determine the exact taxonomic position of these strains and to distinguish them from B. pseudomallei and B. thailandensis, they were subjected to a series of phenotypic and molecular analyses. Biochemical and fatty acid methyl ester analysis was unable to distinguish B. humptydooensis sp. nov. from closely related species. With matrix-assisted laser desorption ionization-time of flight analysis, all isolates grouped together in a cluster separate from other Burkholderia spp. 16S rRNA and recA sequence analyses demonstrated phylogenetic placement for B. humptydooensis sp. nov. in a novel clade within the B. pseudomallei group. Multilocus sequence typing (MLST) analysis of the three isolates in comparison with MLST data from 3,340 B. pseudomallei strains and related taxa revealed a new sequence type (ST318). Genome-to-genome distance calculations and the average nucleotide identity of all isolates to both B. thailandensis and B. pseudomallei, based on whole-genome sequences, also confirmed B. humptydooensis sp. nov. as a novel Burkholderia species within the B. pseudomallei complex. Molecular analyses clearly demonstrated that strains MSMB43T, MSMB121, and MSMB122 belong to a novel Burkholderia species for which the name Burkholderia humptydooensis sp. nov. is proposed, with the type strain MSMB43T (American Type Culture Collection BAA-2767; Belgian Co-ordinated Collections of Microorganisms LMG 29471; DDBJ accession numbers CP013380 to CP013382).IMPORTANCEBurkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The genus Burkholderia consists of a diverse group of species, with the closest relatives of B. pseudomallei referred to as the B. pseudomallei complex. A proposed novel species, B. humptydooensis sp. nov., was isolated from a bore water sample from the Northern Territory in Australia. B. humptydooensis sp. nov. is phylogenetically distinct from B. pseudomallei and other members of the B. pseudomallei complex, making it the fifth member of this important group of bacteria.

Complete Genome Sequence of Pigmentation-Negative Yersinia pestis Strain Cadman.

Here, we report the genome sequence of Yersinia pestis strain Cadman, an attenuated strain lacking the pgm locus. Y. pestis is the causative agent of plague and generally must be worked with under biosafety level 3 (BSL-3) conditions. However, strains lacking the pgm locus are considered safe to work with under BSL-2 conditions.

The Effects of Signal Erosion and Core Genome Reduction on the Identification of Diagnostic Markers.

UNLABELLED: Whole-genome sequence (WGS) data are commonly used to design diagnostic targets for the identification of bacterial pathogens. To do this effectively, genomics databases must be comprehensive to identify the strict core genome that is specific to the target pathogen. As additional genomes are analyzed, the core genome size is reduced and there is erosion of the target-specific regions due to commonality with related species, potentially resulting in the identification of false positives and/or false negatives. IMPORTANCE: A comparative analysis of 1,130 Burkholderia genomes identified unique markers for many named species, including the human pathogens B. pseudomallei and B. mallei Due to core genome reduction and signature erosion, only 38 targets specific to B. pseudomallei/mallei were identified. By using only public genomes, a larger number of markers were identified, due to undersampling, and this larger number represents the potential for false positives. This analysis has implications for the design of diagnostics for other species where the genomic space of the target and/or closely related species is not well defined.

No assembly required: Full-length MHC class I allele discovery by PacBio circular consensus sequencing.

Single-molecule real-time (SMRT) sequencing technology with the Pacific Biosciences (PacBio) RS II platform offers the potential to obtain full-length coding regions (∼1100-bp) from MHC class I cDNAs. Despite the relatively high error rate associated with SMRT technology, high quality sequences can be obtained by circular consensus sequencing (CCS) due to the random nature of the error profile. In the present study we first validated the ability of SMRT-CCS to accurately identify class I transcripts in Mauritian-origin cynomolgus macaques (Macaca fascicularis) that have been characterized previously by cloning and Sanger-based sequencing as well as pyrosequencing approaches. We then applied this SMRT-CCS method to characterize 60 novel full-length class I transcript sequences expressed by a cohort of cynomolgus macaques from China. The SMRT-CCS method described here provides a straightforward protocol for characterization of unfragmented single-molecule cDNA transcripts that will potentially revolutionize MHC class I allele discovery in nonhuman primates and other species.