Biology subjects

Ismail, N.

Publications and source records attributed to Ismail, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Duration of Initial Viremia Modulates Functional Properties of HIV-specific T Cell Receptors

Virus-specific CD8+ T cells are crucial in controlling chronic human viral infections such as HIV-1, but the effect of persistent antigen exposure on T cell repertoire formation is not well understood. In this study, we examined epitope-specific CD8+ T cell repertoires in people living with HIV-1, where duration of viremia following hyperacute infection was modulated by the time of initiation of continuous suppressive antiretroviral therapy (ART). After ART-induced undetectable viremia in persons expressing the same HLA class I allele, we analyzed the impact of early (n=6) versus delayed (n=6) ART initiation on the clonotypic composition, clonotypic cross-reactivity, functional avidity and memory differentiation profile of the HIV-specific T cell repertoire restricted by HLA-B*58:01. Using a panel of barcoded tetramers, we mapped T cell receptor (TCR) clonotypes specific for three dominant epitopes and their variants. Both groups exhibited polyclonal TCR repertoires with evidence of cross-reactivity, which was significantly enriched in donors with prolonged antigen exposure. Within this cohort, broadly cross-reactive clonotypes capable of recognizing all autologous variants were identified, but these were rare (<1%). Early ART initiation preserved repertoires characterized by higher-avidity TCRs and a relative enrichment of transitional memory CD8+ T cell subsets. These functional differences were not associated with differences in TRBV gene sharing, indicating that ART timing shapes repertoire quality and memory differentiation without altering TRBV gene bias. These findings demonstrate how antigen suppression dynamics differentially shape the breadth, functional sensitivity, and memory composition of the HIV-specific TCR repertoire, with implications for T cell-directed immunotherapies and HIV cure strategies. One Sentence SummaryThe duration of viral antigen exposure during early HIV infection shapes the functional quality, breadth, and memory composition of virus-specific CD8 T cell receptor repertoires. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/702605v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@9a5a6dorg.highwire.dtl.DTLVardef@1a6bbdaorg.highwire.dtl.DTLVardef@17707e8org.highwire.dtl.DTLVardef@1a84d9e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Rapidly and reproducibly building a comprehensive catalogueof resistance-associated variants for M. tuberculosis

BackgroundCatalogues of genetic variants associated with resistance underpin whole-genome sequencing (WGS)-based predictions of drug susceptibility in Mycobacterium tuberculosis, and are essential for molecular diagnostics and surveillance. The current gold standard catalogues are those released by the WHO but the underlying data are not fully released and they are difficult to interpret. Open and reproducible methods would help address these problems, extending the important work already done. MethodsWe have developed an automated method, catomatic, that uses a binomial test to associate informative isolates with resistance or susceptibility, and built a catalogue (catomatic-1) from the same 39,358 samples used to construct the first edition of the WHO catalogue (WHOv1). We performed a sensitivity analysis to optimise statistical and bioinformatic parameters for each drug, and benchmarked catomatic-1 against WHOv1 using an independent Validation Dataset of 14,380 isolates. FindingsBy using simpler statistics, catomatic-1 algorithmically classified 1,329 genetic variants, ranging from five for linezolid to 440 for pyrazinamide. WHOv1 included generalisable rules added by a panel of experts, increasing its predictive coverage, but at the cost of reproducibility. Despite not including such expert rules, catomatic-1 achieves comparable performance for all drugs, with sensitivities for first-line agents above 88% on the independent Validation Dataset. The automated process allowed us to efficiently explore parameter space; for instance, detecting resistant variants with low read support improved the sensitivity for all drugs. InterpretationPerformant resistance catalogues for M. tuberculosis can be built automatically using transparent and reproducible statistical methods. As more data are collected, catalogue content and performance will evolve, highlighting the need for proper versioning, machine/human readability, and open access. This approach demonstrates resistance catalogues used in surveillance and diagnostics can be rapidly and reproducibily updated. FundingThe National Institute for Health and Care Research (NIHR), Engineering and Physics Sciences Research Council (EPSRC) and ORACLE Corporation. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed and preprint servers (bioRxiv, medRxiv), and publicly available mutation catalogues for studies linking Mycobacterium tuberculosis genomic variants with drug resistance using whole-genome or targeted sequencing and phenotypic drug-susceptibility testing (pDST). Search terms combined "Mycobacterium tuberculosis", "genome sequencing", "mutation catalogue", "mutation effects", "drug resistance", and individual drug names, with no language or date restriction. We included studies providing paired, clinical genomic and pDST or MIC data, excluding purely in-silico or case-only reports. This work directly builds on methodologies and data published by five prior studies, and makes primary comparisons with the First (WHOv1) and Second (WHOv2) Editions of the WHO Catalogue of mutations in Mycobacterium tuberculosis. Added value of this studyWe developed catomatic, a transparent, reproducible tool for building catalogues of resistance- and susceptibility-associated genetic variants. Trained on the same samples used to build WHOv1 and benchmarked on an independent Validation Dataset, catomatic achieves comparable sensitivity, specificity, and definitive prediction rates to WHOv1 without expert-rule augmentation and despite using simpler statistics. It optimises parameters per drug, produces machine-readable outputs (CSV/JSON), and demonstrates that adjusting read-support thresholds can improve detection of minor resistance subpopulations. Implications of all the available evidenceCatalogues of resistance-associated variants for M. tuberculosis can be rapidly and transparently constructed. Making catalogues available in human/machine-readable formats with uncertainty estimates will improve uptake of WGS for M. tuberculosis surveillance and diagnostics; using a reproducible process permits diagnostic test manufacturers, researchers, clinical and public health laboratories to select the level of statistical support necessitated by their specific use-case, Policymakers should balance the benefits of expert rules against loss of reproducibility. Future work will expand the size of the datasets used, integrate minimum inhibitory concentration data, and establish consensus workflows for routine, transparent catalogue updates.

microbiology↗

bioRxiv2025

Data-optimal scaling of paired antibody language models

Scaling laws for large language models in natural language domains are typically derived under the assumption that performance is primarily compute-constrained. In contrast, antibody language models (AbLMs) trained on paired sequences are primarily data-limited, thus requiring different considerations. To explore how model size and data scale affect AbLM performance, we trained 15 AbLMs across all pairwise combinations of five model sizes and three training data sizes. From these experiments, we derive an AbLM-specific scaling law and estimate that training a data-optimal AbLM equivalent of the highly performant 650M-parameter ESM-2 protein language model would require [~]5.5 million paired antibody sequences. Evaluation on multiple downstream classification tasks revealed that significant performance gains emerged only with sufficiently large model size, suggesting that in data-limited domains, improved performance depends jointly on both model scale and data volume.

immunology↗

bioRxiv2025

Unpacking bedaquiline hetero-resistance: the importance of intermediate profiles for phenotypic drug-susceptibility testing

Phenotypic drug susceptibility testing (pDST) remains the gold standard for Mycobacterium tuberculosis complex drug resistance determination. Next generation sequencing technologies can identify heteroresistant populations at low frequencies, but little is known about the impact of heteroresistance on bedaquiline (BDQ) pDST results. We simulated heteroresistance using in vitro generated MmpR5 mutants mixed with the progenitor strain at various percentages (1-20%) and did pDST using MGIT960 culture (1 and 2 {micro}g/mL BDQ concentrations). Targeted Next Generation Sequencing (tNGS) was used to quantify the mutant sub-population in growth control tubes, which were expected to maintain the mutant: wild type proportion throughout the assay. Growth units of these growth control tubes were also comparable with minor differences in time-to-positivity between ratio mixtures. Only when intermediate results were considered could BDQ heteroresistance be detected at frequencies of approximately 1% by pDST at a critical concentration of 1 {micro}g/mL using BACTEC MGIT960 coupled with EpiCenter TBeXiST software. The ability of pDST, a widely available DST technique, to reveal the presence of BDQ-resistant subpopulations at the phenotypic testing stage could improve resistance determination and potentially reduce time to effective treatment. ImportanceMultidrug resistant tuberculosis (MDR-TB) is estimated to cause up to 19% of all antimicrobial resistance-attributable deaths worldwide. Further, the success rate for the treatment of drug-resistant TB, in the presence of adherence, is poor at only 68%. The advent of bedaquiline (BDQ) has revolutionized MDR-TB care, but BDQ resistance determination is hampered by several obstacles facing both phenotypic and genotypic testing. Specifically for phenotypic susceptibility testing, BDQ-resistant Mycobacterium tuberculosis isolates with variants in MmpR5, which may display minimal inhibitory concentration values just below the critical concentration or are present at low frequencies (heteroresistance; the presence of mixed mutant and wild-type populations within a specimen), are typically designated as susceptible. This may lead to prescription of an ineffective regimen and amplification of resistance. The BACTEC MGIT960 platform coupled with EpiCenter TBeXiST software for phenotypic testing, which is currently the only routinely used method of BDQ DST, can be used to derive more information about underlying resistant populations. We demonstrate how this is possible through the consideration of intermediate results (i.e., when growth units in a drug-containing tube reach the threshold for resistance but only after a further week of incubation). These intermediate results, commonly disregarded by TB laboratories, could lead to earlier detection of BDQ resistance. This is especially crucial when the genetic mechanism of resistance is unknown, a variant has not been associated with resistance in the interim, and in cases of heteroresistance.

microbiology↗

bioRxiv2024

Integrative in vivo analysis of the ethanolamine utilization bacterial microcompartment in Escherichia coli.

Bacterial microcompartments (BMCs) are self-assembling protein megacomplexes that encapsulate metabolic pathways. Although approximately 20% of sequenced bacterial genomes contain operons encoding putative BMCs, few have been thoroughly characterized, nor any in the most studied Escherichia coli strains. We used an interdisciplinary approach to gain deep molecular and functional insights into the ethanolamine utilization (Eut) BMC system encoded by the eut operon in E. coli K-12. The eut genotype was linked with the ethanolamine utilization phenotype using deletion and overexpression mutants. The subcellular dynamics and morphology of the E. coli Eut BMC were characterized in cellula by fluorescence microscopy and electron (cryo)microscopy. The minimal proteome reorganization required for ethanolamine utilization and the in vivo stochiometric composition of the Eut BMC were determined by quantitative proteomics. Finally, the first flux map connecting the Eut BMC with central metabolism in cellula was obtained by genome scale modelling and 13C-fluxomics. Our results reveal that, contrary to previous suggestions, ethanolamine serves both as a nitrogen and a carbon source in E. coli K-12, while also contributing significant metabolic overflow. Overall, this study provides a quantitative molecular and functional understanding of the BMCs involved in ethanolamine assimilation by E. coli. ImportanceThe properties of BMCs make them an ideal tool to build orthogonal network structures with minimal interactions with native metabolic and regulatory networks. However, this requires an understanding of how BMCs work natively. In this study, we combined genetic manipulation, multi-omics, modelling and microscopy to address this issue for Eut BMCs. We show that the Eut BMC in E. coli turns ethanolamine into usable carbon and nitrogen substrates to sustain growth. These results improve our understanding of compartmentalization in a widely used bacterial chassis.

microbiology↗

bioRxiv2022

Leishmania major centrin knock-out parasites alter the Kynurenine- aryl hydrocarbon receptor signaling to produce a pro-inflammatory response

Leishmaniasis is a parasitic disease that is prevalent in approximately 88 countries, and yet no licensed human vaccine exists against it. Towards control of leishmaniasis, we have developed Leishmania major centrin gene deletion mutant strains (LmCen-/-) as a live attenuated vaccine, which induces a strong Th1 response to provide IFN-{gamma}-mediated protection to the host. However, the immune mechanisms of such protection remain to be understood. Metabolomic reprogramming of the host cells following Leishmania-infection has been shown to play a critical role in pathogenicity and shaping the immune response following infection. Here, we applied untargeted mass spectrometric analysis to study the metabolic changes induced by infection with LmCen-/- and compared those with virulent L. major parasite infection to identify the immune mechanism of protection. Our data shows that immunization with LmCen-/- parasites, in contrast to virulent L. major infection, alters tryptophan metabolism to down-regulate kynurenine-AhR signaling and promote a pro-inflammatory response.

systems biology↗

bioRxiv2022

Centrin-deficient Leishmania mexicana confers protection against Old World visceral leishmaniasis

Leishmaniasis is one of the top neglected tropical diseases with significant morbidity and mortality in low and middle-income countries (LMIC). However, this disease is also spreading in the developed world. Currently, there is a lack of effective strategies to control this disease. Vaccination can be an effective measure to control leishmaniasis and has the potential to achieve disease elimination. Recently, we have generated centrin gene-deleted new world L. mexicana (LmexCen-/-) parasites using CRISPR/Cas9 and showed that they protect mice against a homologous L. mexicana infection that causes cutaneous disease. In this study, we tested whether LmexCen-/- parasites can also protect against visceral leishmaniasis caused by L. donovani in a hamster model. We show that immunization with LmexCen-/- parasites is safe and does not cause lesions. Furthermore, such immunization conferred protection against visceral leishmaniasis caused by a needle-initiated L. donovani challenge, as indicated by a significant reduction in the parasite burdens in the spleen and liver and lack of mortality. Similar control of parasite burden was also observed against a sand fly mediated L. donovani challenge. Importantly, immunization with LmexCen-/- down-regulated the Th2 response as indicated by a significant reduction in the anti-inflammatory cytokines such as IL-10 and IL-4 and increased pro-inflammatory cytokine IFN-{gamma} resulting in higher IFN-{gamma}/IL-10 and IFN-{gamma}/IL4 ratios compared to non-immunized animals. This contrasts with our studies with L. major centrin deletion mutants that showed a dominant Th1 response compared to L. major wild-type infection suggesting the divergent mechanisms of protection in the two mutant parasites. LmexCen-/- immunization resulted in long-lasting protection against L. donovani infection. Further, since the efficacy of LmCen-/- has not been determined against Leishmania strains prevalent in the Americas, LmexCen-/- may be a viable alternative. Taken together, our study demonstrates that immunization with LmexCen-/- parasites is safe and efficacious against old world visceral leishmaniasis.

immunology↗

bioRxiv2021

Skin resident memory cells generated by the L. major centrin gene deleted parasites mediate protective immune response analogous to leishmanization

Leishmaniasis is a vector-borne parasitic disease transmitted through the bite of a sand fly with no available vaccine for humans. Recently, we have developed a live attenuated Leishmania major centrin gene deleted parasite strain (LmCen-/-) that induced protection against a homologous and heterologous challenges. The protection is mediated by IFN{gamma} secreting CD4+ T effector cells and multifunctional T cells, which is analogous to leishmanization. Previously, skin tissue resident memory T cells (TRM cells) were shown to be crucial for host protection in a leishmanization model. In this study, we evaluated generation and function of skin TRM cells following immunization with LmCen-/- parasites and compared those with leishmanization. In the absence of recoverable LmCen-/- parasites, the skin of immunized mice showed functional TRM cells comparable to leishmanized mice. The generation of the skin TRM cells was supported by the induction of cytokines and chemokines essential for their production and survival. Following challenge infection with wild type L. major, TRM cells specific to L. major were rapidly recruited and proliferated at the site of infection in the immunized mice which was similar to leishmanization. Further, upon challenge, CD4+ TRM cells induced higher levels of IFN{gamma} and Granzyme B in the immunized and leishmanized mice than non-immunized mice. Taken together, our studies demonstrate that a genetically modified live attenuated Leishmania vaccine generates functional CD4+ TRM cells that mediate protection and can be a safer alternative to leishmanization.

immunology↗

bioRxiv2021

Cysteine dependence in Lactobacillus iners constitutes a novel therapeutic target to modify the vaginal microbiota

Vaginal microbiota composition affects several important reproductive health outcomes. Lactobacillus crispatus-dominant bacterial communities have favorable associations whereas anaerobe-dominant communities deficient of lactobacilli are linked to poor outcomes, including bacterial vaginosis (BV). Lactobacillus iners, the most abundant vaginal species worldwide, has adverse associations compared to L. crispatus, but standard metronidazole treatment for BV promotes L. iners-dominance, likely contributing to post-treatment relapse. L. iners is under-studied because it fails to grow in standard Lactobacillus media in vitro. Here we trace this in vitro phenotype to a species-specific cysteine requirement associated with limitations in cysteine-related transport mechanisms and show that vaginal cysteine concentrations correlate with Lactobacillus abundance in vivo. We demonstrate that cystine uptake inhibitors selectively impede L. iners growth and that combining an inhibitor with metronidazole thus promotes L. crispatus dominance of defined BV-like communities. These findings identify a novel target for therapeutic vaginal microbiota modulation to improve reproductive health.

microbiology↗

bioRxiv2021

Cotranslational folding of a periplasmic protein domain in Escherichia coli

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) - a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide - to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepBs two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45-50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.

molecular biology↗

bioRxiv2020

Rapid SARS-CoV-2 Detection and Classification Using Phase Imaging with Computational Specificity

Efforts to mitigate the COVID-19 crisis revealed that fast, accurate, and scalable testing is crucial for curbing the current impact and that of future pandemics. We propose an optical method for directly imaging unlabeled viral particles and using deep learning for detection and classification. An ultrasensitive interferometric method was used to image four virus types with nanoscale optical pathlength sensitivity. Pairing these data with fluorescence images for ground truth, we trained semantic segmentation models based on U-Net, a particular type of convolutional neural network. The trained network was applied to classify the viruses from the interferometric images only, containing simultaneously SARS-CoV-2, H1N1 (influenza-A), HAdV (adenovirus), and ZIKV (Zika). Remarkably, due to the nanoscale sensitivity in the input data, the neural network was able to identify SARS-CoV-2 vs. the other viruses with 96% accuracy. The inference time for each image is 60 ms, on a common graphic processing unit. This approach of directly imaging unlabeled viral particles may provide an extremely fast test, of less than a minute per patient. As the imaging instrument operates on regular glass slides, we envision this method as potentially testing on patient breath condensates. The necessary high throughput can be achieved by translating concepts from digital pathology, where a microscope can scan hundreds of slides automatically. One Sentence SummaryThis work proposes a rapid (<1 min.), label-free testing method for SARS-CoV-2 detection, using quantitative phase imaging and deep learning.

bioengineering↗

bioRxiv2020

Preclinical validation of a second generation leishmanization vaccine against vector transmitted fatal visceral leishmaniasis

Visceral Leishmaniasis (VL) is fatal if untreated. There is no licensed vaccine available against human leishmaniasis. We recently demonstrated protection in mice against L. major infection using a CRISPR genome edited attenuated Leishmania major strain (LmCen-/-). Here, as a pre-clinical step, we evaluated the protective efficacy of LmCen-/- against VL induced by sand fly transmitted Leishmania donovani in hamsters. Intradermal immunization of hamsters with LmCen-/- did not develop any lesion; while still priming a pro-inflammatory immune response. When challenged with L. donovani either by intradermal needle injection or by infected sand flies, LmCen-/--immunized hamsters were protected, not showing spleen or liver pathology averting VL fatality compared to control animals. Spleen cells from LmCen-/- immunized and infected sand fly challenged hamsters produced significantly higher Th1-associated cytokines and chemokines including IFN-{gamma} and TNF-, and significantly reduced expression of the anti-inflammatory cytokines IL-10 and IL-21, compared to non-immunized challenged animals. We further developed a GLP-grade LmCen-/- which showed equal protection as laboratory-grade LmCen-/- parasites in hamsters. Importantly, GLP-grade LmCen-/- parasites also induced a proinflammatory immune response in the PBMCs isolated from healthy people living in non-endemic and endemic for VL as well as cured VL people living in endemic region. Together, this study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and it is a strong candidate vaccine to be tested in a human clinical trial.

immunology↗

bioRxiv2020

Population structure, biogeography and transmissibility of Mycobacterium tuberculosis

Mycobacterium tuberculosis is a clonal pathogen proposed to have co-evolved with its human host for millennia, yet our understanding of its genomic diversity and biogeography remains incomplete. Here we use a combination of phylogenetics and dimensionality reduction to reevaluate the population structure of M. tuberculosis, providing the first in-depth analysis of the ancient East African Indian Lineage 1 and the modern Central Asian Lineage 3 and expanding our understanding of Lineages 2 and 4. We assess sub-lineages using genomic sequences from 4,939 pan-susceptible strains and find 30 new genetically distinct clades that we validate in a dataset of 4,645 independent isolates. We characterize sub-lineage geographic distributions and demonstrate a consistent geographically restricted and unrestricted pattern for 20 groups, including three groups of Lineage 1. We assess the transmissibility of the four major lineages by examining the distribution of terminal branch lengths across the M. tuberculosis phylogeny and identify evidence supporting higher transmissibility in Lineages 2 and 4 than 3 and 1 on a global scale. We define a robust expanded barcode of 95 single nucleotide substitutions (SNS) that allows for the rapid identification of 69 Mtb sub-lineages and 26 additional internal groups. Our results paint a higher resolution picture of the Mtb phylogeny and biogeography.

microbiology↗

bioRxiv2020

Characterization of genomic variants associated with resistance to bedaquiline and delamanid in naïve Mycobacterium tuberculosis clinical strains

The role of genetic mutations in genes associated to phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly understood. However, a clear understanding of the role of each genetic variant is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analysed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole genome sequencing (WGS) dataset from a collection of 4795 MTBc clinical isolates from six high-burden countries of tuberculosis (TB). From WGS analysis, we identified 61 and 158 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to the medicines. In order to characterize the role of mutations, we performed an energetic in silico analysis to evaluate their effect in the available protein structures Ddn (DLM), Fgd1 (DLM) and Rv0678 (BDQ), and minimum inhibitory concentration (MIC) assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural protein information and phenotypic data allowed for cataloging the mutations clearly associated with resistance to BDQ (n= 4) and DLM (n= 35), as well as about a hundred genetic variants without any correlation with resistance. Importantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance to the development of comprehensive molecular diagnostic tools. ImportancePhenotypic drug susceptibility tests (DST) are too slow to provide an early indication of drug susceptibility status at the time of treatment initiation and very demanding in terms of specimens handling and biosafety. The development of molecular assays to detect resistance to bedaquiline (BDQ) and delamanid (DLM) requires accurate categorization of genetic variants according to their association with phenotypic resistance. We have evaluated a large multi-country set of clinical isolates to identify mutations associated with increased minimum inhibitory concentrations (MICs) and used an in silico protein structure analysis to further unravel the potential role of these mutations in drug resistance mechanisms. The results of this study are an important source of information for the development of molecular diagnostic tests to improve the provision of appropriate treatment and care to TB patients.

microbiology↗

bioRxiv2020

A Second Generation Leishmanization Vaccine with a Markerless Attenuated Leishmania major Strain using CRISPR gene editing

Leishmaniasis is a debilitating and often fatal neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in unacceptable skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is the first genetically engineered gene deleted Leishmania strain that is antibiotic resistant marker free and does not have any off-target mutations. Mice immunized with LmCen-/- had virtually no visible lesions following challenge with L. major-infected sand flies while non-immunized animals developed large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization showed protection and an immune response comparable to leishmanization. LmCen-/- is safe since it was unable to cause disease even in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.

microbiology↗

bioRxiv2020

Horseshoe crab genomes reveal the evolutionary fates of genes and microRNAs after three rounds (3R) of whole genome duplication

Whole genome duplication (WGD) has occurred in relatively few sexually reproducing invertebrates. Consequently, the WGD that occurred in the common ancestor of horseshoe crabs ~135 million years ago provides a rare opportunity to decipher the evolutionary consequences of a duplicated invertebrate genome. Here, we present a high-quality genome assembly for the mangrove horseshoe crab Carcinoscorpius rotundicauda (1.7Gb, N50 = 90.2Mb, with 89.8% sequences anchored to 16 pseudomolecules, 2n = 32), and a resequenced genome of the tri-spine horseshoe crab Tachypleus tridentatus (1.7Gb, N50 = 109.7Mb). Analyses of gene families, microRNAs, and synteny show that horseshoe crabs have undergone three rounds (3R) of WGD, and that these WGD events are shared with spiders. Comparison of the genomes of C. rotundicauda and T. tridentatus populations from several geographic locations further elucidates the diverse fates of both coding and noncoding genes. Together, the present study represents a cornerstone for a better understanding of the consequences of invertebrate WGD events on evolutionary fates of genes and microRNAs at individual and population levels, and highlights the genetic diversity with practical values for breeding programs and conservation of horseshoe crabs.

evolutionary biology↗

bioRxiv2019

Modeling missing cases and transmission links in networks of extensively drug-resistant tuberculosis in KwaZulu-Natal, South Africa

The transmission patterns of drug-resistant tuberculosis (TB) remain poorly understood, despite over half a million incident cases in 2017. Modeling TB transmission networks can provide insight into the nature and drivers of transmission, but incomplete and non-random sampling of TB cases can pose challenges to making inferences from epidemiologic and molecular data. We conducted a quantitative bias analysis to assess the effect of missing cases on a transmission network inferred from Mtb sequencing data on extensively drug-resistant (XDR) TB cases in South Africa. We tested scenarios in which cases were missing at random, differentially by clinical characteristics, or differentially by transmission (i.e., cases with many links were under or over-sampled). Under the assumption cases were missing at random, cases in the complete, modeled network would have had a mean of 20 or more transmission links, which is far higher than expected, in order to reproduce the observed, partial network. Instead, we found that the most likely scenario involved undersampling of high-transmitting cases, and further models provided evidence for superspreading behavior. This is, to our knowledge, the first study to define and assess the support for different mechanisms of missingness in a study of TB transmission. Our findings should caution interpretation of results of future studies of TB transmission in high-incidence settings, given the potential for biased sampling, and should motivate further research aimed at identifying the specific host, pathogen, or environmental factors contributing to superspreading.

epidemiology↗

bioRxiv2019

Integrated Single-Cell Analysis of Multicellular Immune Dynamics during Hyper-Acute HIV-1 Infection

Cellular immunity is critical for controlling intracellular pathogens, but the dynamics and cooperativity of the evolving host response to infection are not well defined. Here, we apply single-cell RNA-sequencing to longitudinally profile pre- and immediately post-HIV infection peripheral immune responses of multiple cell types in four untreated individuals. Onset of viremia induces a strong transcriptional interferon response integrated across most cell types, with subsequent pro-inflammatory T cell differentiation, monocyte MHC-II upregulation, and cytolytic killing. With longitudinal sampling, we nominate key intra- and extracellular drivers that induce these programs, and assign their multi-cellular targets, temporal ordering, and duration in acute infection. Two individuals studied developed spontaneous viral control, associated with initial elevated frequencies of proliferating cytotoxic cells, inclusive of a previously unappreciated proliferating natural killer (NK) cell subset. Our study presents a unified framework for characterizing immune evolution during a persistent human viral infection at single-cell resolution, and highlights programs that may drive response coordination and influence clinical trajectory.

immunology↗