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The Andrews Lab
Control of Infectious Diseases in Resource-Limited Settings
Our laboratory aims to develop innovative approaches to the control of infectious diseases in resource-limited settings. Drawing upon the fields of epidemiology, microbiology and engineering, we strive to find solutions to extend the technologies that underlie diagnosis and treatment of infectious diseases to "last-mile" communities.
Publications
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Mass incarceration as a driver of the tuberculosis epidemic in Latin America and projected effects of policy alternatives: a mathematical modelling study.
The Lancet. Public health
Liu, Y. E., Mabene, Y., Camelo, S., Rueda, Z. V., Pelissari, D. M., Dockhorn Costa Johansen, F., Huaman, M. A., Avalos-Cruz, T., Alarcón, V. A., Ladutke, L. M., Bergman, M., Cohen, T., Goldhaber-Fiebert, J. D., Croda, J., Andrews, J. R.
2024
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Abstract
Tuberculosis incidence is increasing in Latin America, where the incarcerated population has nearly quadrupled since 1990. We aimed to quantify the impact of historical and future incarceration policies on the tuberculosis epidemic, accounting for effects in and beyond prisons.In this modelling study, we calibrated dynamic compartmental transmission models to historical and contemporary data from Argentina, Brazil, Colombia, El Salvador, Mexico, and Peru, which comprise approximately 80% of the region's incarcerated population and tuberculosis burden. The model was fit independently for each country to incarceration and tuberculosis data from 1990 to 2023 (specific dates were country dependent). The model does not include HIV, drug resistance, gender or sex, or age structure. Using historical counterfactual scenarios, we estimated the transmission population attributable fraction (tPAF) for incarceration and the excess population-level burden attributable to increasing incarceration prevalence since 1990. We additionally projected the effect of alternative incarceration policies on future population tuberculosis incidence.Population tuberculosis incidence in 2019 was 29·4% (95% uncertainty interval [UI] 23·9-36·8) higher than expected without the rise in incarceration since 1990, corresponding to 34 393 (28 295-42 579) excess incident cases across countries. The incarceration tPAF in 2019 was 27·2% (20·9-35·8), exceeding estimates for other risk factors like HIV, alcohol use disorder, and undernutrition. Compared with a scenario where incarceration rates remain stable at current levels, a gradual 50% reduction in prison admissions and duration of incarceration by 2034 would reduce population tuberculosis incidence by over 10% in all countries except Mexico.The historical rise in incarceration in Latin America has resulted in a large excess tuberculosis burden that has been under-recognised to date. International health agencies, ministries of justice, and national tuberculosis programmes should collaborate to address this health crisis with comprehensive strategies, including decarceration.National Institutes of Health.
View details for DOI 10.1016/S2468-2667(24)00192-0
View details for PubMedID 39419058
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A Nanopore Sequencing-based Pharmacogenomic Panel to Personalize Tuberculosis Drug Dosing.
American journal of respiratory and critical care medicine
Verma, R., Silva, K. E., Rockwood, N., Wasmann, R. E., Yende, N., Song, T., Kim, E., Denti, P., Wilkinson, R. J., Andrews, J. R.
2024
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Abstract
Standardized dosing of anti-tubercular (TB) drugs leads to variable plasma drug levels, which are associated with adverse drug reactions, delayed treatment response, and relapse. Mutations in genes affecting drug metabolism explain considerable interindividual pharmacokinetic variability; however, pharmacogenomic (PGx) assays that predict metabolism of anti-TB drugs have been lacking.To develop a Nanopore sequencing panel and validate its performance in active TB patients to personalize treatment dosing.We developed a Nanopore sequencing panel targeting 15 single nucleotide polymorphisms (SNP) in 5 genes affecting the metabolism of anti-tuberculous drugs. For validation, we sequenced DNA samples (n=48) from the 1000 genomes project and compared variant calling accuracy with Illumina genome sequencing. We then sequenced DNA samples from patients with active TB (n=100) from South Africa on a MinION Mk1C and evaluated the relationship between genotypes and pharmacokinetic parameters for INH and RIF.The PGx panel achieved 100% concordance with Illumina sequencing in variant identification for the samples from the 1000 Genomes Project. In the clinical cohort, coverage was >100x for 1498/1500 (99.8%) amplicons across the 100 samples. One third (33%) of participants were identified as slow, 47% were intermediate and 20% were rapid isoniazid acetylators. Isoniazid clearance was 2.2 times higher among intermediate acetylators and 3.8 times higher among rapid acetylators compared with slow acetylators (p<0.0001).. Rifampin clearance was 17.3% (2.50-29.9) lower in individuals with homozygous AADAC rs1803155 G>A substitutions (p=0.0015).Targeted sequencing can enable detection of polymorphisms influencing TB drug metabolism on a low-cost, portable instrument to personalize dosing for TB treatment or prevention. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
View details for DOI 10.1164/rccm.202309-1583OC
View details for PubMedID 38647526
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Effect of BCG vaccination against Mycobacterium tuberculosis infection in adult Brazilian health-care workers: a nested clinical trial.
The Lancet. Infectious diseases
Dos Santos, P. C., Messina, N. L., de Oliveira, R. D., da Silva, P. V., Puga, M. A., Dalcolmo, M., Dos Santos, G., de Lacerda, M. V., Jardim, B. A., de Almeida E Val, F. F., Curtis, N., Andrews, J. R., Croda, J.
2024
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Abstract
The effectiveness of BCG vaccine for adult pulmonary tuberculosis remains uncertain. In this study, we aimed to evaluate the effect of vaccination with BCG-Denmark to prevent initial and sustained interferon-γ release assay conversion in Brazilian health-care workers.This substudy is a nested randomised controlled trial embedded within the BRACE trial (NCT04327206). Specifically, this substudy enrolled Brazilian health-care workers (aged ≥18 years) from three sites in Brazil (Manaus, Campo Grande, and Rio de Janeiro) irrespective of previously receiving BCG vaccination. Participants were excluded if they had contraindications to BCG vaccination, more than 1 month of treatment with specific tuberculosis treatment drugs, previous adverse reactions to BCG, recent BCG vaccination, or non-compliance with assigned interventions. Those eligible were randomly assigned (1:1) to either the BCG group (0·1 mL intradermal injection of BCG-Denmark [Danish strain 1331; AJ Vaccines, Copenhagen]) or the placebo group (intradermal injection of 0·9% saline) using a web-based randomisation process in variable-length blocks (2, 4, or 6), and were stratified based on the study site, age (<40, ≥40 to <60, ≥60 years), and comorbidity presence (diabetes, chronic respiratory disease, cardiac condition, hypertension). Sealed syringes were used to prevent inadvertent disclosure of group assignments. The QuantiFERON-TB Gold (QFT) Plus test (Qiagen; Hilden, Germany) was used for baseline and 12-month tuberculosis infection assessments. The primary efficacy outcome was QFT Plus conversion (≥0·35 IU/mL) by 12 months following vaccination in participants who had a negative baseline result (<0·35 IU/mL).Between Oct 7, 2020, and April 12, 2021, 1985 (77·3%) of 2568 participants were eligible for QFT Plus assessment at 12 months and were included in this substudy; 996 (50·2%) of 1985 were in the BCG group and 989 (49·8%) were in the placebo group. Overall, 1475 (74·3%) of 1985 participants were women and 510 (25·7%) were men, and the median age was 39 years (IQR 32-47). During the first 12 months, QFT Plus conversion occurred in 66 (3·3%) of 1985 participants, with no significant differences by study site (p=0·897). Specifically, 34 (3·4%) of 996 participants had initial QFT conversion in the BCG group compared with 32 (3·2%) of 989 in the placebo group (risk ratio 1·09 [95% CI 0·67-1·77]; p=0·791).BCG-Denmark vaccination did not reduce initial QFT Plus conversion risk in Brazilian health-care workers. This finding underscores the need to better understand tuberculosis prevention in populations at high risk.Bill & Melinda Gates Foundation, the Minderoo Foundation, Sarah and Lachlan Murdoch, the Royal Children's Hospital Foundation, Health Services Union NSW, the Peter Sowerby Foundation, SA Health, the Insurance Advisernet Foundation, the NAB Foundation, the Calvert-Jones Foundation, the Modara Pines Charitable Foundation, the United Health Group Foundation, Epworth Healthcare, and individual donors.For the Portuguese translation of the abstract see Supplementary Materials section.
View details for DOI 10.1016/S1473-3099(23)00818-6
View details for PubMedID 38423021
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Global, regional, and national estimates of tuberculosis incidence and case detection among incarcerated individuals from 2000 to 2019: a systematic analysis.
The Lancet. Public health
Martinez, L., Warren, J. L., Harries, A. D., Croda, J., Espinal, M. A., Olarte, R. A., Avedillo, P., Lienhardt, C., Bhatia, V., Liu, Q., Chakaya, J., Denholm, J. T., Lin, Y., Kawatsu, L., Zhu, L., Horsburgh, C. R., Cohen, T., Andrews, J. R.
2023; 8 (7): e511-e519
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Abstract
People who are incarcerated are at high risk of developing tuberculosis. We aimed to estimate the annual global, regional, and national incidence of tuberculosis among incarcerated populations from 2000 to 2019.We collected and aggregated data for tuberculosis incidence and prevalence estimates among incarcerated individuals in published and unpublished literature, annual tuberculosis notifications among incarcerated individuals at the country level, and the annual number of incarcerated individuals at the country level. We developed a joint hierarchical Bayesian meta-regression framework to simultaneously model tuberculosis incidence, notifications, and prevalence from 2000 to 2019. Using this model, we estimated trends in absolute tuberculosis incidence and notifications, the incidence and notification rates, and the case detection ratio by year, country, region, and globally.In 2019, we estimated a total of 125 105 (95% credible interval [CrI] 93 736-165 318) incident tuberculosis cases among incarcerated individuals globally. The estimated incidence rate per 100 000 person-years overall was 1148 (95% CrI 860-1517) but varied greatly by WHO region, from 793 (95% CrI 430-1342) in the Eastern Mediterranean region to 2242 (1515-3216) in the African region. Global incidence per 100 000 person-years between 2000 and 2012 among incarcerated individuals decreased from 1884 (95% CrI 1394-2616) to 1205 (910-1615); however, from 2013 onwards, tuberculosis incidence per 100 000 person-years was stable, from 1183 (95% CrI 876-1596) in 2013 to 1148 (860-1517) in 2019. In 2019, the global case detection ratio was estimated to be 53% (95% CrI 42-64), the lowest over the study period.Our estimates suggest a high tuberculosis incidence rate among incarcerated individuals globally with large gaps in tuberculosis case detection. Tuberculosis in incarcerated populations must be addressed with interventions specifically tailored to improve diagnoses and prevent transmission as a part of the broader global tuberculosis control effort.National Institutes of Health.
View details for DOI 10.1016/S2468-2667(23)00097-X
View details for PubMedID 37393090
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Infant BCG vaccination and risk of pulmonary and extrapulmonary tuberculosis throughout the life course: a systematic review and individual participant data meta-analysis.
The Lancet. Global health
Martinez, L., Cords, O., Liu, Q., Acuna-Villaorduna, C., Bonnet, M., Fox, G. J., Carvalho, A. C., Chan, P. C., Croda, J., Hill, P. C., Lopez-Varela, E., Donkor, S., Fielding, K., Graham, S. M., Espinal, M. A., Kampmann, B., Reingold, A., Huerga, H., Villalba, J. A., Grandjean, L., Sotgiu, G., Egere, U., Singh, S., Zhu, L., Lienhardt, C., Denholm, J. T., Seddon, J. A., Whalen, C. C., García-Basteiro, A. L., Triasih, R., Chen, C., Singh, J., Huang, L. M., Sharma, S., Hannoun, D., Del Corral, H., Mandalakas, A. M., Malone, L. L., Ling, D. L., Kritski, A., Stein, C. M., Vashishtha, R., Boulahbal, F., Fang, C. T., Boom, W. H., Netto, E. M., Lemos, A. C., Hesseling, A. C., Kay, A., Jones-López, E. C., Horsburgh, C. R., Lange, C., Andrews, J. R.
2022; 10 (9): e1307-e1316
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Abstract
BCG vaccines are given to more than 100 million children every year, but there is considerable debate regarding the effectiveness of BCG vaccination in preventing tuberculosis and death, particularly among older children and adults. We therefore aimed to investigate the age-specific impact of infant BCG vaccination on tuberculosis (pulmonary and extrapulmonary) development and mortality.In this systematic review and individual participant data meta-analysis, we searched MEDLINE, Web of Science, BIOSIS, and Embase without language restrictions for case-contact cohort studies of tuberculosis contacts published between Jan 1, 1998, and April 7, 2018. Search terms included "mycobacterium tuberculosis", "TB", "tuberculosis", and "contact". We excluded cohort studies that did not provide information on BCG vaccination or were done in countries that did not recommend BCG vaccination at birth. Individual-level participant data for a prespecified list of variables, including the characteristics of the exposed participant (contact), the index case, and the environment, were requested from authors of all eligible studies. Our primary outcome was a composite of prevalent (diagnosed at or within 90 days of baseline) and incident (diagnosed more than 90 days after baseline) tuberculosis in contacts exposed to tuberculosis. Secondary outcomes were pulmonary tuberculosis, extrapulmonary tuberculosis, and mortality. We derived adjusted odds ratios (aORs) using mixed-effects, binary, multivariable logistic regression analyses with study-level random effects, adjusting for the variable of interest, baseline age, sex, previous tuberculosis, and whether data were collected prospectively or retrospectively. We stratified our results by contact age and Mycobacterium tuberculosis infection status. This study is registered with PROSPERO, CRD42020180512.We identified 14 927 original records from our database searches. We included participant-level data from 26 cohort studies done in 17 countries in our meta-analysis. Among 68 552 participants, 1782 (2·6%) developed tuberculosis (1309 [2·6%] of 49 686 BCG-vaccinated participants vs 473 [2·5%] of 18 866 unvaccinated participants). The overall effectiveness of BCG vaccination against all tuberculosis was 18% (aOR 0·82, 95% CI 0·74-0·91). When stratified by age, BCG vaccination only significantly protected against all tuberculosis in children younger than 5 years (aOR 0·63, 95% CI 0·49-0·81). Among contacts with a positive tuberculin skin test or IFNγ release assay, BCG vaccination significantly protected against tuberculosis among all participants (aOR 0·81, 95% CI 0·69-0·96), participants younger than 5 years (0·68, 0·47-0·97), and participants aged 5-9 years (0·62, 0·38-0·99). There was no protective effect among those with negative tests, unless they were younger than 5 years (0·54, 0·32-0·90). 14 cohorts reported on whether tuberculosis was pulmonary or extrapulmonary (n=57 421). BCG vaccination significantly protected against pulmonary tuberculosis among all participants (916 [2·2%] in 41 119 vaccinated participants vs 334 [2·1%] in 16 161 unvaccinated participants; aOR 0·81, 0·70-0·94) but not against extrapulmonary tuberculosis (106 [0·3%] in 40 318 vaccinated participants vs 38 [0·2%] in 15 865 unvaccinated participants; 0·96, 0·65-1·41). In the four studies with mortality data, BCG vaccination was significantly protective against death (0·25, 0·13-0·49).Our results suggest that BCG vaccination at birth is effective at preventing tuberculosis in young children but is ineffective in adolescents and adults. Immunoprotection therefore needs to be boosted in older populations.National Institutes of Health.
View details for DOI 10.1016/S2214-109X(22)00283-2
View details for PubMedID 35961354
The Blish Lab
Defining Natural Immunity in Viral Disease
The Blish laboratory is in the Department of Medicine, Division of Infectious Diseases and Geographic Medicine and in the interdisciplinary Stanford Immunology program. Our goal is to develop new methods to prevent and control infectious diseases through better understanding of human immunology. We have several major areas of ongoing investigation.
Publications
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Comparative analysis of cell-cell communication at single-cell resolution.
Nature biotechnology
Wilk, A. J., Shalek, A. K., Holmes, S., Blish, C. A.
2023
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Abstract
Inference of cell-cell communication from single-cell RNA sequencing data is a powerful technique to uncover intercellular communication pathways, yet existing methods perform this analysis at the level of the cell type or cluster, discarding single-cell-level information. Here we present Scriabin, a flexible and scalable framework for comparative analysis of cell-cell communication at single-cell resolution that is performed without cell aggregation or downsampling. We use multiple published atlas-scale datasets, genetic perturbation screens and direct experimental validation to show that Scriabin accurately recovers expected cell-cell communication edges and identifies communication networks that can be obscured by agglomerative methods. Additionally, we use spatial transcriptomic data to show that Scriabin can uncover spatial features of interaction from dissociated data alone. Finally, we demonstrate applications to longitudinal datasets to follow communication pathways operating between timepoints. Our approach represents a broadly applicable strategy to reveal the full structure of niche-phenotype relationships in health and disease.
View details for DOI 10.1038/s41587-023-01782-z
View details for PubMedID 37169965
View details for PubMedCentralID 8104132
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SARS-CoV-2 escapes direct NK cell killing through Nsp1-mediated downregulation of ligands for NKG2D.
Cell reports
Lee, M. J., Leong, M. W., Rustagi, A., Beck, A., Zeng, L., Holmes, S., Qi, L. S., Blish, C. A.
2022: 111892
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Abstract
Natural killer (NK) cells are cytotoxic effector cells that target and lyse virally infected cells; many viruses therefore encode mechanisms to escape such NK cell killing. Here, we interrogate the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We find that NK cells exhibit poor cytotoxic responses against SARS-CoV-2-infected targets, preferentially killing uninfected bystander cells. We demonstrate that this escape is driven by downregulation of ligands for the activating receptor NKG2D (NKG2D-L). Indeed, early in viral infection, prior to NKG2D-L downregulation, NK cells are able to target and kill infected cells; however, this ability is lost as viral proteins are expressed. Finally, we find that SARS-CoV-2 non-structural protein 1 (Nsp1) mediates downregulation of NKG2D-L and that Nsp1 alone is sufficient to confer resistance to NK cell killing. Collectively, our work demonstrates that SARS-CoV-2 evades direct NK cell cytotoxicity and describes a mechanism by which this occurs.
View details for DOI 10.1016/j.celrep.2022.111892
View details for PubMedID 36543165
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SARS-CoV-2 infection drives an inflammatory response in human adipose tissue through infection of adipocytes and macrophages.
Science translational medicine
Martínez-Colón, G. J., Ratnasiri, K., Chen, H., Jiang, S., Zanley, E., Rustagi, A., Verma, R., Chen, H., Andrews, J. R., Mertz, K. D., Tzankov, A., Azagury, D., Boyd, J., Nolan, G. P., Schürch, C. M., Matter, M. S., Blish, C. A., McLaughlin, T. L.
2022: eabm9151
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Abstract
Obesity, characterized by chronic low-grade inflammation of the adipose tissue, is associated with adverse coronavirus disease 2019 (COVID-19) outcomes, yet the underlying mechanism is unknown. To explore whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of adipose tissue contributes to pathogenesis, we evaluated COVID-19 autopsy cases and deeply profiled the response of adipose tissue to SARS-CoV-2 infection in vitro. In COVID-19 autopsy cases, we identified SARS-CoV-2 RNA in adipocytes with an associated inflammatory infiltrate. We identified two distinct cellular targets of infection: adipocytes and a subset of inflammatory adipose tissue-resident macrophages. Mature adipocytes were permissive to SARS-CoV-2 infection; although macrophages were abortively infected, SARS-CoV-2 initiated inflammatory responses within both the infected macrophages and bystander preadipocytes. These data suggest that SARS-CoV-2 infection of adipose tissue could contribute to COVID-19 severity through replication of virus within adipocytes and through induction of local and systemic inflammation driven by infection of adipose tissue-resident macrophages.
View details for DOI 10.1126/scitranslmed.abm9151
View details for PubMedID 36137009
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Multi-omic profiling reveals widespread dysregulation of innate immunity and hematopoiesis in COVID-19.
The Journal of experimental medicine
Wilk, A. J., Lee, M. J., Wei, B., Parks, B., Pi, R., Martinez-Colon, G. J., Ranganath, T., Zhao, N. Q., Taylor, S., Becker, W., Stanford COVID-19 Biobank, Jimenez-Morales, D., Blomkalns, A. L., O'Hara, R., Ashley, E. A., Nadeau, K. C., Yang, S., Holmes, S., Rabinovitch, M., Rogers, A. J., Greenleaf, W. J., Blish, C. A.
2021; 218 (8)
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Abstract
Our understanding of protective versus pathological immune responses to SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is limited by inadequate profiling of patients at the extremes of the disease severity spectrum. Here, we performed multi-omic single-cell immune profiling of 64 COVID-19 patients across the full range of disease severity, from outpatients with mild disease to fatal cases. Our transcriptomic, epigenomic, and proteomic analyses revealed widespread dysfunction of peripheral innate immunity in severe and fatal COVID-19, including prominent hyperactivation signatures in neutrophils and NK cells. We also identified chromatin accessibility changes at NF-kappaB binding sites within cytokine gene loci as a potential mechanism for the striking lack of pro-inflammatory cytokine production observed in monocytes in severe and fatal COVID-19. We further demonstrated that emergency myelopoiesis is a prominent feature of fatal COVID-19. Collectively, our results reveal disease severity-associated immune phenotypes in COVID-19 and identify pathogenesis-associated pathways that are potential targets for therapeutic intervention.
View details for DOI 10.1084/jem.20210582
View details for PubMedID 34128959
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A single-cell atlas of the peripheral immune response in patients with severe COVID-19.
Nature medicine
Wilk, A. J., Rustagi, A., Zhao, N. Q., Roque, J., Martinez-Colon, G. J., McKechnie, J. L., Ivison, G. T., Ranganath, T., Vergara, R., Hollis, T., Simpson, L. J., Grant, P., Subramanian, A., Rogers, A. J., Blish, C. A.
2020
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Abstract
There is an urgent need to better understand the pathophysiology of Coronavirus disease 2019 (COVID-19), the global pandemic caused by SARS-CoV-2, which has infected more than three million people worldwide1. Approximately 20% of patients with COVID-19 develop severe disease and 5% of patients require intensive care2. Severe disease has been associated with changes in peripheral immune activity, including increased levels of pro-inflammatory cytokines3,4 that may be produced by a subset of inflammatory monocytes5,6, lymphopenia7,8 and T cell exhaustion9,10. To elucidate pathways in peripheral immune cells that might lead to immunopathology or protective immunity in severe COVID-19, we applied single-cell RNA sequencing (scRNA-seq) to profile peripheral blood mononuclear cells (PBMCs) from seven patients hospitalized for COVID-19, four of whom had acute respiratory distress syndrome, and six healthy controls. We identify reconfiguration of peripheral immune cell phenotype in COVID-19, including a heterogeneous interferon-stimulated gene signature, HLA class II downregulation and a developing neutrophil population that appears closely related to plasmablasts appearing in patients with acute respiratory failure requiring mechanical ventilation. Importantly, we found that peripheral monocytes and lymphocytes do not express substantial amounts of pro-inflammatory cytokines. Collectively, we provide a cell atlas of the peripheral immune response to severe COVID-19.
View details for DOI 10.1038/s41591-020-0944-y
View details for PubMedID 32514174
The Bollyky Lab
Immune Responses in Injured and Infected Tissues
Our lab studies how immune responses are regulated within injured and infected tissues. We work at the intersection of immunology, structural biology, bioengineering, and microbiology. Our goals are to understand the factors that drive chronic inflammation and to develop novel therapeutics to promote wound healing and immune tolerance.
Publications
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A blueprint for broadly effective bacteriophage-antibiotic cocktails against bacterial infections.
Nature communications
Kim, M. K., Chen, Q., Echterhof, A., Pennetzdorfer, N., McBride, R. C., Banaei, N., Burgener, E. B., Milla, C. E., Bollyky, P. L.
2024; 15 (1): 9987
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Abstract
Bacteriophage (phage) therapy is a promising therapeutic modality for multidrug-resistant bacterial infections, but its application is mainly limited to personalized therapy due to the narrow host range of individual phages. While phage cocktails targeting all possible bacterial receptors could theoretically confer broad coverage, the extensive diversity of bacteria and the complexity of phage-phage interactions render this approach challenging. Here, using screening protocols for identifying "complementarity groups" of phages using non-redundant receptors, we generate effective, broad-range phage cocktails that prevent the emergence of bacterial resistance. We also discover characteristic interactions between phage complementarity groups and particular antibiotic classes, facilitating the prediction of phage-antibiotic as well as phage-phage interactions. Using this strategy, we create three phage-antibiotic cocktails, each demonstrating efficacy against ≥96% of 153 Pseudomonas aeruginosa clinical isolates, including biofilm cultures, and demonstrate comparable efficacy in an in vivo wound infection model. We similarly develop effective Staphylococcus aureus phage-antibiotic cocktails and demonstrate their utility of combined cocktails against polymicrobial (mixed P. aeruginosa/S. aureus) cultures, highlighting the broad applicability of this approach. These studies establish a blueprint for the development of effective, broad-spectrum phage-antibiotic cocktails, paving the way for off-the-shelf phage-based therapeutics to combat multidrug-resistant bacterial infections.
View details for DOI 10.1038/s41467-024-53994-9
View details for PubMedID 39609398
View details for PubMedCentralID 11153159
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Phage diversity in cell-free DNA identifies bacterial pathogens in human sepsis cases.
Nature microbiology
Haddock, N. L., Barkal, L. J., Ram-Mohan, N., Kaber, G., Chiu, C. Y., Bhatt, A. S., Yang, S., Bollyky, P. L.
2023
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Abstract
Bacteriophages, viruses that infect bacteria, have great specificity for their bacterial hosts at the strain and species level. However, the relationship between the phageome and associated bacterial population dynamics is unclear. Here we generated a computational pipeline to identify sequences associated with bacteriophages and their bacterial hosts in cell-free DNA from plasma samples. Analysis of two independent cohorts, including a Stanford Cohort of 61 septic patients and 10 controls and the SeqStudy cohort of 224 septic patients and 167 controls, reveals a circulating phageome in the plasma of all sampled individuals. Moreover, infection is associated with overrepresentation of pathogen-specific phages, allowing for identification of bacterial pathogens. We find that information on phage diversity enables identification of the bacteria that produced these phages, including pathovariant strains of Escherichia coli. Phage sequences can likewise be used to distinguish between closely related bacterial species such as Staphylococcus aureus, a frequent pathogen, and coagulase-negative Staphylococcus, a frequent contaminant. Phage cell-free DNA may have utility in studying bacterial infections.
View details for DOI 10.1038/s41564-023-01406-x
View details for PubMedID 37308590
View details for PubMedCentralID 5594678
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Filamentous bacteriophage delays healing of Pseudomonas-infected wounds.
Cell reports. Medicine
Bach, M. S., de Vries, C. R., Khosravi, A., Sweere, J. M., Popescu, M. C., Chen, Q., Demirdjian, S., Hargil, A., Van Belleghem, J. D., Kaber, G., Hajfathalian, M., Burgener, E. B., Liu, D., Tran, Q., Dharmaraj, T., Birukova, M., Sunkari, V., Balaji, S., Ghosh, N., Mathew-Steiner, S. S., El Masry, M. S., Keswani, S. G., Banaei, N., Nedelec, L., Sen, C. K., Chandra, V., Secor, P. R., Suh, G. A., Bollyky, P. L.
2022; 3 (6): 100656
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Abstract
Chronic wounds infected by Pseudomonas aeruginosa (Pa) are characterized by disease progression and increased mortality. We reveal Pf, a bacteriophage produced by Pa that delays healing of chronically infected wounds in human subjects and animal models of disease. Interestingly, impairment of wound closure by Pf is independent of its effects on Pa pathogenesis. Rather, Pf impedes keratinocyte migration, which is essential for wound healing, through direct inhibition of CXCL1 signaling. In support of these findings, a prospective cohort study of 36 human patients with chronic Pa wound infections reveals that wounds infected with Pf-positive strains of Pa are more likely to progress in size compared with wounds infected with Pf-negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian cells. These findings suggest Pf may have potential as a biomarker and therapeutic target in chronic wounds.
View details for DOI 10.1016/j.xcrm.2022.100656
View details for PubMedID 35732145
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Filamentous bacteriophages are associated with chronic Pseudomonas lung infections and antibiotic resistance in cystic fibrosis
SCIENCE TRANSLATIONAL MEDICINE
Burgener, E. B., Sweere, J. M., Bach, M. S., Secor, P. R., Haddock, N., Jennings, L. K., Marvig, R. L., Johansen, H., Rossi, E., Cao, X., Tian, L., Nedelec, L., Molin, S., Bollyky, P. L., Milla, C. E.
2019; 11 (488)
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View details for DOI 10.1126/scitranslmed.aau9748
View details for Web of Science ID 000465116400003
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Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection.
Science (New York, N.Y.)
Sweere, J. M., Van Belleghem, J. D., Ishak, H. n., Bach, M. S., Popescu, M. n., Sunkari, V. n., Kaber, G. n., Manasherob, R. n., Suh, G. A., Cao, X. n., de Vries, C. R., Lam, D. N., Marshall, P. L., Birukova, M. n., Katznelson, E. n., Lazzareschi, D. V., Balaji, S. n., Keswani, S. G., Hawn, T. R., Secor, P. R., Bollyky, P. L.
2019; 363 (6434)
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Abstract
Bacteriophage are abundant at sites of bacterial infection, but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by Pseudomonas aeruginosa (Pa) in suppression of immunity against bacterial infection. Pf promote Pa wound infection in mice and are associated with chronic human Pa wound infections. Murine and human leukocytes endocytose Pf, and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)- and TIR domain-containing adapter-inducing interferon-β (TRIF)-dependent type I interferon production, inhibition of tumor necrosis factor (TNF), and the suppression of phagocytosis. Conversely, immunization of mice against Pf prevents Pa wound infection. Thus, Pf triggers maladaptive innate viral pattern-recognition responses, which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection.
View details for PubMedID 30923196
The Einav Lab
Understanding Virus-Host Protein Interactions
The goals of our lab are to better understand virus-host protein interactions, identify host proteins or pathways required by multiple viruses, and translate this knowledge into the development of novel, broad-spectrum, host-centered antiviral approaches with a high genetic barrier for resistance.
Publications
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Replication Capacity and Susceptibility of Nirmatrelvir-Resistant Mutants to Next-Generation Mpro Inhibitors in a SARS-CoV-2 Replicon System.
Antiviral research
Lo, C., Kariv, O., Hao, C., Gammeltoft, K. A., Bukh, J., Gottwein, J., Westberg, M., Lin, M. Z., Einav, S.
2024: 106022
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Abstract
There is an ongoing need to expand the anti-SARS-CoV-2 armamentarium to include agents capable of suppressing replication of drug-resistant mutants emerging during monotherapy with approved direct-acting antivirals. Using a subgenomic SARS-CoV-2 replicon system, we studied the RNA replication capacity of nirmatrelvir (NTV)-resistant mutants and their susceptibility to next-generation Mpro inhibitors, including ibuzatrelvir (ITV), ensitrelvir (ETV), and ML2006a4. Our findings revealed that E166V Mpro mutants reduced viral RNA replication, whereas other Mpro mutations retained or increased the replication capacity, suggesting the potential of the latter to dominate under NTV selective pressure. Except for having an advantage against E166A mutants, ITV largely showed the same mutational sensitivity as NTV. ETV was more effective than NTV against E166V mutants but less effective against S144A, E166A, and L167F mutants. ML2006a4 demonstrated the most effective suppression across most mutants (S144A, E166V, S144A+L50F, E166A/V+L50F, L167F+L50F, and E166A+L167F+L50F). Thus, ML2006a4 represents an attractive investigational candidate against clinically relevant NTV-resistant SARS-CoV-2 mutants.
View details for DOI 10.1016/j.antiviral.2024.106022
View details for PubMedID 39424074
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Synthesis of 3-heteroaryl-pyrrolo[2,3-b]pyridines as potent inhibitors of AP-2-associated protein kinase 1 (AAK1) with antiviral activity.
European journal of medicinal chemistry
Ravi, N. P., Van Eynde, W., Karim, M., Nhu Tran, D. H., Agrawal, A., Schols, D., Voet, A., Einav, S., Dehaen, W., De Jonghe, S.
2024; 280: 116967
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Abstract
Inhibition of AP-2-associated protein kinase 1 (AAK1) has been shown to be a promising avenue for the development of broad-spectrum antiviral agents. On a previously described AAK1 inhibitor based on a pyrrolo[2,3-b]pyridine scaffold, the concept of isosterism was applied, by replacing a carboxamide linker by various five-membered heterocycles. It led to the discovery of a novel series of AAK1 inhibitors with IC50 values in the low nM range, that also displayed antiviral activity against the dengue virus and Venezuelan equine encephalitis virus.
View details for DOI 10.1016/j.ejmech.2024.116967
View details for PubMedID 39427517
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Back-Pocket Optimization of 2-Aminopyrimidine-Based Macrocycles Leads to Potent EPHA2/GAK Kinase Inhibitors.
Journal of medicinal chemistry
Gerninghaus, J., Zhubi, R., Krämer, A., Karim, M., Tran, D. H., Joerger, A. C., Schreiber, C., Berger, L. M., Berger, B. T., Ehret, T. A., Elson, L., Lenz, C., Saxena, K., Müller, S., Einav, S., Knapp, S., Hanke, T.
2024
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Abstract
Macrocyclization of acyclic compounds is a powerful strategy for improving inhibitor potency and selectivity. Here we have optimized 2-aminopyrimidine-based macrocycles to use these compounds as chemical tools for the ephrin kinase family. Starting with a promiscuous macrocyclic inhibitor, 6, we performed a structure-guided activity relationship and selectivity study using a panel of over 100 kinases. The crystal structure of EPHA2 in complex with the developed macrocycle 23 provided a basis for further optimization by specifically targeting the back pocket, resulting in compound 55, a potent inhibitor of EPHA2/A4 and GAK. Subsequent front-pocket derivatization resulted in an interesting in cellulo selectivity profile, favoring EPHA4 over the other ephrin receptor kinase family members. The dual EPHA2/A4 and GAK inhibitor 55 prevented dengue virus infection of Huh7 liver cells. However, further investigations are needed to determine whether this was a compound-specific effect or target-related.
View details for DOI 10.1021/acs.jmedchem.4c00411
View details for PubMedID 39028937
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Numb-associated kinases regulate sandfly-borne Toscana virus entry.
Emerging microbes & infections
Moalem, Y., Katz, R., Subramaniam, A. G., Malis, Y., Yaffe, Y., Borenstein-Auerbach, N., Tadmor, K., Raved, R., Maoz, B. M., Yoo, J. S., Lustig, Y., Luxenburg, C., Perlson, E., Einav, S., Sklan, E. H.
2024: 2382237
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Abstract
Sandfly-borne Toscana virus (TOSV) is an enveloped tri-segmented negative single-strand RNA Phlebovirus. It is an emerging virus predominantly endemic in southwestern Europe and Northern Africa. Although TOSV infection is typically asymptomatic or results in mild febrile disease, it is neurovirulent and ranks among the three most common causes of summer meningitis in certain regions. Despite this clinical significance, our understanding of the molecular aspects and host factors regulating phlebovirus infection is limited.This study characterized the early steps of TOSV infection. Our findings reveal that two members of the Numb-associated kinases family of Ser/Thr kinases, namely adaptor-associated kinase 1 (AAK1) and cyclin G-associated kinase (GAK), play a role in regulating the early stages of TOSV entry. FDA-approved inhibitors targeting these kinases demonstrated significant inhibition of TOSV infection. This study suggests that AAK1 and GAK represent druggable targets for inhibiting TOSV infection and, potentially, related Phleboviruses.
View details for DOI 10.1080/22221751.2024.2382237
View details for PubMedID 39017647
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PIP4K2C inhibition reverses autophagic flux impairment induced by SARS-CoV-2.
bioRxiv : the preprint server for biology
Karim, M., Mishra, M., Lo, C. W., Saul, S., Cagirici, H. B., Tran, D. H., Agrawal, A., Ghita, L., Ojha, A., East, M. P., Gammeltoft, K. A., Sahoo, M. K., Johnson, G. L., Das, S., Jochmans, D., Cohen, C. A., Gottwein, J., Dye, J., Neff, N., Pinsky, B. A., Laitinen, T., Pantsar, T., Poso, A., Zanini, F., Jonghe, S. D., Asquith, C. R., Einav, S.
2024
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Abstract
In search for broad-spectrum antivirals, we discovered a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrated selective dual inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Advanced lipidomics revealed alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and linked its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We discovered PIP4K2C's roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C and PIKfyve as a candidate strategy to combat emerging viruses.
View details for DOI 10.1101/2024.04.15.589676
View details for PubMedID 38659941
View details for PubMedCentralID PMC11042293
Jagannathan Lab
Translational Immunology Focused on Malaria-Specific Immune Responses
The goals of this laboratory are to further our understanding of the correlates and mechanisms of clinical immunity to malaria through field-based studies, and to better understand the immunologic consequences of malaria control interventions.
These studies bridge immune profiling techniques including multiparameter flow cytometry, transcriptomics, epigenetics, and multiplex antibody profiling to epidemiologic studies of antimalarial immunity in children.
Publications
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Natural killer cell antibody-dependent cellular cytotoxicity to Plasmodium falciparum is impacted by cellular phenotypes, erythrocyte polymorphisms, parasite diversity and intensity of transmission.
Clinical & translational immunology
Tukwasibwe, S., Lewis, S. N., Taremwa, Y., van der Ploeg, K., Press, K. D., Ty, M., Namirimu Nankya, F., Musinguzi, K., Nansubuga, E., Bach, F., Chamai, M., Okitwi, M., Tumusiime, G., Nakimuli, A., Colucci, F., Kamya, M. R., Nankabirwa, J. I., Arinaitwe, E., Greenhouse, B., Dorsey, G., Rosenthal, P. J., Ssewanyana, I., Jagannathan, P.
2024; 13 (11): e70005
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Abstract
Natural killer (NK) cells make important contributions to anti-malarial immunity through antibody-dependent cellular cytotoxicity (ADCC), but the role of different components of this pathway in promoting NK cell activation remains unclear.We compared the functions and phenotypes of NK cells from malaria-exposed and malaria-naive donors, and then varied the erythrocyte genetic background, Plasmodium falciparum strain and opsonising plasma used in ADCC to observe their impacts on NK cell degranulation as measured by CD107a mobilisation.Natural killer cells from malaria-exposed adult Ugandan donors had enhanced ADCC, but an impaired pro-inflammatory response to cytokine stimulation, compared to NK cells obtained from malaria-naive adult North American donors. Cellular phenotypes from malaria-exposed donors reflected this specialisation for ADCC, with a compartment-wide downregulation of the Fc receptor γ-chain and enrichment of highly differentiated CD56dim and CD56neg populations. NK cell degranulation was enhanced in response to opsonised P. falciparum schizonts cultured in sickle cell heterozygous erythrocytes relative to wild-type erythrocytes, and when using opsonising plasma collected from donors living in a high transmission area compared to a lower transmission area despite similar levels of 3D7 schizont-specific IgG levels. However, degranulation was lowered in response to opsonised field isolate P. falciparum schizonts isolated from clinical malaria infections, compared to the 3D7 laboratory strain typically used in these assays.This work highlights important host and parasite factors that contribute to ADCC efficacy that should be considered in the design of ADCC assays.
View details for DOI 10.1002/cti2.70005
View details for PubMedID 39493859
View details for PubMedCentralID PMC11528551
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Clinical immunity to malaria involves epigenetic reprogramming of innate immune cells.
PNAS nexus
Nideffer, J., Ty, M., Donato, M., John, R., Kajubi, R., Ji, X., Nankya, F., Musinguzi, K., Press, K. D., Yang, N., Camanag, K., Greenhouse, B., Kamya, M., Feeney, M. E., Dorsey, G., Utz, P. J., Pulendran, B., Khatri, P., Jagannathan, P.
2024; 3 (8): pgae325
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Abstract
The regulation of inflammation is a critical aspect of disease tolerance and naturally acquired clinical immunity to malaria. Here, we demonstrate using RNA sequencing and epigenetic landscape profiling by cytometry by time-of-flight, that the regulation of inflammatory pathways during asymptomatic parasitemia occurs downstream of pathogen sensing-at the epigenetic level. The abundance of certain epigenetic markers (methylation of H3K27 and dimethylation of arginine residues) and decreased prevalence of histone variant H3.3 correlated with suppressed cytokine responses among monocytes of Ugandan children. Such an epigenetic signature was observed across diverse immune cell populations and not only characterized active asymptomatic parasitemia but also correlated with future long-term disease tolerance and clinical immunity when observed in uninfected children. Pseudotime analyses revealed a potential trajectory of epigenetic change that correlated with a child's age and recent parasite exposure and paralleled the acquisition of clinical immunity. Thus, our data support a model whereby exposure to Plasmodium falciparum induces epigenetic changes that regulate excessive inflammation and contribute to naturally acquire clinical immunity to malaria.
View details for DOI 10.1093/pnasnexus/pgae325
View details for PubMedID 39161730
View details for PubMedCentralID PMC11331423
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The impact of Plasmodium-driven immunoregulatory networks on immunity to malaria.
Nature reviews. Immunology
Boyle, M. J., Engwerda, C. R., Jagannathan, P.
2024
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Abstract
Malaria, caused by infection with Plasmodium parasites, drives multiple regulatory responses across the immune landscape. These regulatory responses help to protect against inflammatory disease but may in some situations hamper the acquisition of adaptive immune responses that clear parasites. In addition, the regulatory responses that occur during Plasmodium infection may negatively affect malaria vaccine efficacy in the most at-risk populations. Here, we discuss the specific cellular mechanisms of immunoregulatory networks that develop during malaria, with a focus on knowledge gained from human studies and studies that involve the main malaria parasite to affect humans, Plasmodium falciparum. Leveraging this knowledge may lead to the development of new therapeutic approaches to increase protective immunity to malaria during infection or after vaccination.
View details for DOI 10.1038/s41577-024-01041-5
View details for PubMedID 38862638
View details for PubMedCentralID 8191919
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Malaria-specific Type 1 regulatory T cells are more abundant in first pregnancies and associated with placental malaria.
EBioMedicine
Kirosingh, A. S., Delmastro, A., Kakuru, A., van der Ploeg, K., Bhattacharya, S., Press, K. D., Ty, M., Parte, L., Kizza, J., Muhindo, M., Devachanne, S., Gamain, B., Nankya, F., Musinguzi, K., Rosenthal, P. J., Feeney, M. E., Kamya, M., Dorsey, G., Jagannathan, P.
2023; 95: 104772
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Abstract
Malaria in pregnancy (MIP) causes higher morbidity in primigravid compared to multigravid women; however, the correlates and mechanisms underlying this gravidity-dependent protection remain incompletely understood. We aimed to compare the cellular immune response between primigravid and multigravid women living in a malaria-endemic region and assess for correlates of protection against MIP.We characterised the second trimester cellular immune response among 203 primigravid and multigravid pregnant women enrolled in two clinical trials of chemoprevention in eastern Uganda, utilizing RNA sequencing, flow cytometry, and functional assays. We compared responses across gravidity and determined associations with parasitaemia during pregnancy and placental malaria.Using whole blood RNA sequencing, no significant differentially expressed genes were identified between primigravid (n = 12) and multigravid (n = 11) women overall (log 2(FC) > 2, FDR < 0.1). However, primigravid (n = 49) women had higher percentages of malaria-specific, non-naïve CD4+ T cells that co-expressed IL-10 and IFNγ compared with multigravid (n = 85) women (p = 0.000023), and higher percentages of these CD4+ T cells were associated with greater risks of parasitaemia in pregnancy (Rs = 0.49, p = 0.001) and placental malaria (p = 0.0073). These IL-10 and IFNγ co-producing CD4+ T cells had a genomic signature of Tr1 cells, including expression of transcription factors cMAF and BATF and cell surface makers CTLA4 and LAG-3.Malaria-specific Tr1 cells were highly prevalent in primigravid Ugandan women, and their presence correlated with a higher risk of malaria in pregnancy. Understanding whether suppression of Tr1 cells plays a role in naturally acquired gravidity-dependent immunity may aid the development of new vaccines or treatments for MIP.This work was funded by NIH (PO1 HD059454, U01 AI141308, U19 AI089674, U01 AI155325, U01 AI150741), the March of Dimes (Basil O'Connor award), and the Bill and Melinda Gates Foundation (OPP 1113682).
View details for DOI 10.1016/j.ebiom.2023.104772
View details for PubMedID 37634385
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Malaria-driven expansion of adaptive-like functional CD56-negative NK cells correlates with clinical immunity to malaria.
Science translational medicine
Ty, M., Sun, S., Callaway, P. C., Rek, J., Press, K. D., van der Ploeg, K., Nideffer, J., Hu, Z., Klemm, S., Greenleaf, W., Donato, M., Tukwasibwe, S., Arinaitwe, E., Nankya, F., Musinguzi, K., Andrew, D., de la Parte, L., Mori, D. M., Lewis, S. N., Takahashi, S., Rodriguez-Barraquer, I., Greenhouse, B., Blish, C., Utz, P. J., Khatri, P., Dorsey, G., Kamya, M., Boyle, M., Feeney, M., Ssewanyana, I., Jagannathan, P.
2023; 15 (680): eadd9012
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Abstract
Natural killer (NK) cells likely play an important role in immunity to malaria, but the effect of repeated malaria on NK cell responses remains unclear. Here, we comprehensively profiled the NK cell response in a cohort of 264 Ugandan children. Repeated malaria exposure was associated with expansion of an atypical, CD56neg population of NK cells that differed transcriptionally, epigenetically, and phenotypically from CD56dim NK cells, including decreased expression of PLZF and the Fc receptor γ-chain, increased histone methylation, and increased protein expression of LAG-3, KIR, and LILRB1. CD56neg NK cells were highly functional and displayed greater antibody-dependent cellular cytotoxicity than CD56dim NK cells. Higher frequencies of CD56neg NK cells were associated with protection against symptomatic malaria and high parasite densities. After marked reductions in malaria transmission, frequencies of these cells rapidly declined, suggesting that continuous exposure to Plasmodium falciparum is required to maintain this modified, adaptive-like NK cell subset.
View details for DOI 10.1126/scitranslmed.add9012
View details for PubMedID 36696483
Lo Lab
Modeling transmission of infectious diseases and public health control strategies.
Our research group studies the transmission of infectious diseases and
impact of public health strategies with an ultimate goal of informing public health policy. Our group applies diverse computational methodologies, including tools of epidemiology, modeling, pathogen genomics, and policy analysis. We study a broad set of pathogens including neglected tropical diseases, vaccine-preventable infections, and COVID-19.
The Parsonnet Lab
Investigating Chronic Disease-Infection Links
The laboratory's primary research interest is investigating the role of infectious agents in chronic diseases. Much of this work has revolved around Helicobacter pylori infection as a cause of adenocarcinomas and lymphomas of the stomach.
The Relman Lab
Host-Microbe Interactions & Human Microbial Ecology
David Relman's investigative program falls within the general themes of host-pathogen interactions and human microbial ecology, and is divided into two research areas:
- Ecology of microbial communities indigenous to humans and other mammalian hosts
- Genome-wide host response patterns in systemic infectious disease
The Shafer Lab
Virus Evolution focused on HIV Therapy and Drug Resistance
My group’s research is on the mechanisms and consequences of virus evolution with a focus on HIV therapy and drug resistance. We maintain a public HIV drug resistance database (http://hivdb.stanford.edu) as a resource for HIV drug resistance surveillance, interpreting HIV drug resistance tests, and HIV drug development. These three disciplines – epidemiology, clinical management, and basic science – reflect the interdisciplinary nature of antiviral drug resistance research and represent the range of our group’s activities.
Publications
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Combating antimicrobial resistance in malaria, HIV and tuberculosis.
Nature reviews. Drug discovery
Duffey, M., Shafer, R. W., Timm, J., Burrows, J. N., Fotouhi, N., Cockett, M., Leroy, D.
2024
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Abstract
Antimicrobial resistance poses a significant threat to the sustainability of effective treatments against the three most prevalent infectious diseases: malaria, human immunodeficiency virus (HIV) infection and tuberculosis. Therefore, there is an urgent need to develop novel drugs and treatment protocols capable of reducing the emergence of resistance and combating it when it does occur. In this Review, we present an overview of the status and underlying molecular mechanisms of drug resistance in these three diseases. We also discuss current strategies to address resistance during the research and development of next-generation therapies. These strategies vary depending on the infectious agent and the array of resistance mechanisms involved. Furthermore, we explore the potential for cross-fertilization of knowledge and technology among these diseases to create innovative approaches for minimizing drug resistance and advancing the discovery and development of new anti-infective treatments. In conclusion, we advocate for the implementation of well-defined strategies to effectively mitigate and manage resistance in all interventions against infectious diseases.
View details for DOI 10.1038/s41573-024-00933-4
View details for PubMedID 38750260
View details for PubMedCentralID 9490302
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Prevalence of Emergent Dolutegravir Resistance Mutations in People Living with HIV: A Rapid Scoping Review.
Viruses
Chu, C., Tao, K., Kouamou, V., Avalos, A., Scott, J., Grant, P. M., Rhee, S. Y., McCluskey, S. M., Jordan, M. R., Morgan, R. L., Shafer, R. W.
2024; 16 (3)
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Abstract
Dolutegravir (DTG) is a cornerstone of global antiretroviral (ARV) therapy (ART) due to its high efficacy and favorable tolerability. However, limited data exist regarding the risk of emergent integrase strand transfer inhibitor (INSTI) drug-resistance mutations (DRMs) in individuals receiving DTG-containing ART.We performed a PubMed search using the term "Dolutegravir", last updated 18 December 2023, to estimate the prevalence of VF with emergent INSTI DRMs in people living with HIV (PLWH) without previous VF on an INSTI who received DTG-containing ART.Of 2131 retrieved records, 43 clinical trials, 39 cohorts, and 6 cross-sectional studies provided data across 6 clinical scenarios based on ART history, virological status, and co-administered ARVs: (1) ART-naïve PLWH receiving DTG plus two NRTIs; (2) ART-naïve PLWH receiving DTG plus lamivudine; (3) ART-experienced PLWH with VF on a previous regimen receiving DTG plus two NRTIs; (4) ART-experienced PLWH with virological suppression receiving DTG plus two NRTIs; (5) ART-experienced PLWH with virological suppression receiving DTG and a second ARV; and (6) ART-experienced PLWH with virological suppression receiving DTG monotherapy. The median proportion of PLWH in clinical trials with emergent INSTI DRMs was 1.5% for scenario 3 and 3.4% for scenario 6. In the remaining four trial scenarios, VF prevalence with emergent INSTI DRMs was ≤0.1%. Data from cohort studies minimally influenced prevalence estimates from clinical trials, whereas cross-sectional studies yielded prevalence data lacking denominator details.In clinical trials, the prevalence of VF with emergent INSTI DRMs in PLWH receiving DTG-containing regimens has been low. Novel approaches are required to assess VF prevalence with emergent INSTI DRMs in PLWH receiving DTG in real-world settings.
View details for DOI 10.3390/v16030399
View details for PubMedID 38543764
View details for PubMedCentralID PMC10975848
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Recommendations on data sharing in HIV drug resistance research.
PLoS medicine
Inzaule, S. C., Siedner, M. J., Little, S. J., Avila-Rios, S., Ayitewala, A., Bosch, R. J., Calvez, V., Ceccherini-Silberstein, F., Charpentier, C., Descamps, D., Eshleman, S. H., Fokam, J., Frenkel, L. M., Gupta, R. K., Ioannidis, J. P., Kaleebu, P., Kantor, R., Kassaye, S. G., Kosakovsky Pond, S. L., Kouamou, V., Kouyos, R. D., Kuritzkes, D. R., Lessells, R., Marcelin, A., Mbuagbaw, L., Minalga, B., Ndembi, N., Neher, R. A., Paredes, R., Pillay, D., Raizes, E. G., Rhee, S., Richman, D. D., Ruxrungtham, K., Sabeti, P. C., Schapiro, J. M., Sirivichayakul, S., Steegen, K., Sugiura, W., van Zyl, G. U., Vandamme, A., Wensing, A. M., Wertheim, J. O., Gunthard, H. F., Jordan, M. R., Shafer, R. W.
2023; 20 (9): e1004293
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Abstract
Author summary Human immunodeficiency virus (HIV) drug resistance has implications for antiretroviral treatment strategies and for containing the HIV pandemic because the development of HIV drug resistance leads to the requirement for antiretroviral drugs that may be less effective, less well-tolerated, and more expensive than those used in first-line regimens. HIV drug resistance studies are designed to determine which HIV mutations are selected by antiretroviral drugs and, in turn, how these mutations affect antiretroviral drug susceptibility and response to future antiretroviral treatment regimens. Such studies collectively form a vital knowledge base essential for monitoring global HIV drug resistance trends, interpreting HIV genotypic tests, and updating HIV treatment guidelines. Although HIV drug resistance data are collected in many studies, such data are often not publicly shared, prompting the need to recommend best practices to encourage and standardize HIV drug resistance data sharing. In contrast to other viruses, sharing HIV sequences from phylogenetic studies of transmission dynamics requires additional precautions as HIV transmission is criminalized in many countries and regions. Our recommendations are designed to ensure that the data that contribute to HIV drug resistance knowledge will be available without undue hardship to those publishing HIV drug resistance studies and without risk to people living with HIV.
View details for DOI 10.1371/journal.pmed.1004293
View details for PubMedID 37738247
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Treatment Emergent Dolutegravir Resistance Mutations in Individuals Naïve to HIV-1 Integrase Inhibitors: A Rapid Scoping Review.
Viruses
Tao, K., Rhee, S. Y., Chu, C., Avalos, A., Ahluwalia, A. K., Gupta, R. K., Jordan, M. R., Shafer, R. W.
2023; 15 (9)
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Abstract
Background: Dolutegravir (DTG)-based antiretroviral therapy (ART) rarely leads to virological failure (VF) and drug resistance in integrase strand transfer inhibitor (INSTI)-naïve persons living with HIV (PLWH). As a result, limited data are available on INSTI-associated drug resistance mutations (DRMs) selected by DTG-containing ART regimens. Methods: We reviewed studies published through July 2023 to identify those reporting emergent major INSTI-associated DRMs in INSTI-naïve PLWH receiving DTG and those containing in vitro DTG susceptibility results using a standardized assay. Results: We identified 36 publications reporting 99 PLWH in whom major nonpolymorphic INSTI-associated DRMs developed on a DTG-containing regimen and 21 publications containing 269 in vitro DTG susceptibility results. DTG-selected DRMs clustered into four largely non-overlapping mutational pathways characterized by mutations at four signature positions: R263K, G118R, N155H, and Q148H/R/K. Eighty-two (82.8%) viruses contained just one signature DRM, including R263K (n = 40), G118R (n = 24), N155H (n = 9), and Q148H/R/K (n = 9). Nine (9.1%) contained ≥1 signature DRM, and eight (8.1%) contained just other DRMs. R263K and G118R were negatively associated with one another and with N155H and Q148H/K/R. R263K alone conferred a median 2.0-fold (IQR: 1.8-2.2) reduction in DTG susceptibility. G118R alone conferred a median 18.8-fold (IQR:14.2-23.4) reduction in DTG susceptibility. N155H alone conferred a median 1.4-fold (IQR: 1.2-1.6) reduction in DTG susceptibility. Q148H/R/K alone conferred a median 0.8-fold (IQR: 0.7-1.1) reduction in DTG susceptibility. Considerably higher levels of reduced susceptibility often occurred when signature DRMs occurred with additional INSTI-associated DRMs. Conclusions: Among INSTI-naïve PLWH with VF and treatment emergent INSTI-associated DRMs, most developed one of four signature DRMs, most commonly R263K or G118R. G118R was associated with a much greater reduction in DTG susceptibility than R263K.
View details for DOI 10.3390/v15091932
View details for PubMedID 37766338
View details for PubMedCentralID PMC10536831
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Genotypic Resistance Testing of HIV-1 DNA in Peripheral Blood Mononuclear Cells.
Clinical microbiology reviews
Chu, C., Armenia, D., Walworth, C., Santoro, M. M., Shafer, R. W.
2022: e0005222
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Abstract
HIV-1 DNA exists in nonintegrated linear and circular episomal forms and as integrated proviruses. In patients with plasma viremia, most peripheral blood mononuclear cell (PBMC) HIV-1 DNA consists of recently produced nonintegrated virus DNA while in patients with prolonged virological suppression (VS) on antiretroviral therapy (ART), most PBMC HIV-1 DNA consists of proviral DNA produced months to years earlier. Drug-resistance mutations (DRMs) in PBMCs are more likely to coexist with ancestral wild-type virus populations than they are in plasma, explaining why next-generation sequencing is particularly useful for the detection of PBMC-associated DRMs. In patients with ongoing high levels of active virus replication, the DRMs detected in PBMCs and in plasma are usually highly concordant. However, in patients with lower levels of virus replication, it may take several months for plasma virus DRMs to reach detectable levels in PBMCs. This time lag explains why, in patients with VS, PBMC genotypic resistance testing (GRT) is less sensitive than historical plasma virus GRT, if previous episodes of virological failure and emergent DRMs were either not prolonged or not associated with high levels of plasma viremia. Despite the increasing use of PBMC GRT in patients with VS, few studies have examined the predictive value of DRMs on the response to a simplified ART regimen. In this review, we summarize what is known about PBMC HIV-1 DNA dynamics, particularly in patients with suppressed plasma viremia, the methods used for PBMC HIV-1 GRT, and the scenarios in which PBMC GRT has been used clinically.
View details for DOI 10.1128/cmr.00052-22
View details for PubMedID 36102816
The Singh Lab
Identifying Virulence Mechanisms Parasites Develop to Cause Disease
Our lab studies the molecular basis of pathogenesis of two medically important parasites, Toxoplasma gondii and Entamoeba histolytica. The work is aimed at understanding the virulence determinant that each parasite uses in causing disease, specifically how T. gondii evades the human immune response by converting to a dormant bradyzoite stage and how E. histolyticacauses invasive colonic and hepatic disease.
Publications
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Role of tRNA-Derived Fragments in Protozoan Parasite Biology.
Cells
Sharma, M., Singh, U.
2025; 14 (2)
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Abstract
tRNA molecules are among the most fundamental and evolutionarily conserved RNA types, primarily facilitating the translation of genetic information from mRNA into proteins. Beyond their canonical role as adaptor molecules during protein synthesis, tRNAs have evolved to perform additional functions. One such non-canonical role for tRNAs is through the generation of tRNA-derived fragments via specific cleavage processes. These tRNA-derived small RNAs (tsRNAs) are present across all three domains of life, including in protozoan parasites. They are formed through the cleavage of the parent tRNA molecules at different sites, resulting in either tRNA halves or smaller fragments. The precise mechanisms underlying the synthesis of various tRNA-derived fragments, including the specific RNases involved, as well as their distinct functions and roles in parasite physiology, are not yet fully understood and remain an active area of ongoing research. However, their role in modulating gene expression, particularly during stress responses, is becoming increasingly evident. In this context, we discuss recent findings on the roles of tRNA-derived small RNA in various protozoan parasites. Furthermore, we investigate how these tsRNAs either modulate gene expression within the parasite itself or are packaged into extracellular vesicles to alter host gene expression, thereby promoting parasite survival and adaptation.
View details for DOI 10.3390/cells14020115
View details for PubMedID 39851543
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Sex Differences in Long COVID.
JAMA network open
Shah, D. P., Thaweethai, T., Karlson, E. W., Bonilla, H., Horne, B. D., Mullington, J. M., Wisnivesky, J. P., Hornig, M., Shinnick, D. J., Klein, J. D., Erdmann, N. B., Brosnahan, S. B., Lee-Iannotti, J. K., Metz, T. D., Maughan, C., Ofotokun, I., Reeder, H. T., Stiles, L. E., Shaukat, A., Hess, R., Ashktorab, H., Bartram, L., Bassett, I. V., Becker, J. H., Brim, H., Charney, A. W., Chopra, T., Clifton, R. G., Deeks, S. G., Erlandson, K. M., Fierer, D. S., Flaherman, V. J., Fonseca, V., Gander, J. C., Hodder, S. L., Jacoby, V. L., Kotini-Shah, P., Krishnan, J. A., Kumar, A., Levy, B. D., Lieberman, D., Lin, J. J., Martin, J. N., McComsey, G. A., Moukabary, T., Okumura, M. J., Peluso, M. J., Rosen, C. J., Saade, G., Shah, P. K., Sherif, Z. A., Taylor, B. S., Tuttle, K. R., Urdaneta, A. E., Wallick, J. A., Wiley, Z., Zhang, D., Horwitz, L. I., Foulkes, A. S., Singer, N. G.
2025; 8 (1): e2455430
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Abstract
A substantial number of individuals worldwide experience long COVID, or post-COVID condition. Other postviral and autoimmune conditions have a female predominance, but whether the same is true for long COVID, especially within different subgroups, is uncertain.To evaluate sex differences in the risk of developing long COVID among adults with SARS-CoV-2 infection.This cohort study used data from the National Institutes of Health (NIH) Researching COVID to Enhance Recovery (RECOVER)-Adult cohort, which consists of individuals enrolled in and prospectively followed up at 83 sites in 33 US states plus Washington, DC, and Puerto Rico. Data were examined from all participants enrolled between October 29, 2021, and July 5, 2024, who had a qualifying study visit 6 months or more after their initial SARS-CoV-2 infection.Self-reported sex (male, female) assigned at birth.Development of long COVID, measured using a self-reported symptom-based questionnaire and scoring guideline at the first study visit that occurred at least 6 months after infection. Propensity score matching was used to estimate risk ratios (RRs) and risk differences (95% CIs). The full model included demographic and clinical characteristics and social determinants of health, and the reduced model included only age, race, and ethnicity.Among 12 276 participants who had experienced SARS-CoV-2 infection (8969 [73%] female; mean [SD] age at infection, 46 [15] years), female sex was associated with higher risk of long COVID in the primary full (RR, 1.31; 95% CI, 1.06-1.62) and reduced (RR, 1.44; 95% CI, 1.17-1.77) models. This finding was observed across all age groups except 18 to 39 years (RR, 1.04; 95% CI, 0.72-1.49). Female sex was associated with significantly higher overall long COVID risk when the analysis was restricted to nonpregnant participants (RR, 1.50; 95%: CI, 1.27-1.77). Among participants aged 40 to 54 years, the risk ratio was 1.42 (95% CI, 0.99-2.03) in menopausal female participants and 1.45 (95% CI, 1.15-1.83) in nonmenopausal female participants compared with male participants.In this prospective cohort study of the NIH RECOVER-Adult cohort, female sex was associated with an increased risk of long COVID compared with male sex, and this association was age, pregnancy, and menopausal status dependent. These findings highlight the need to identify biological mechanisms contributing to sex specificity to facilitate risk stratification, targeted drug development, and improved management of long COVID.
View details for DOI 10.1001/jamanetworkopen.2024.55430
View details for PubMedID 39841477
View details for PubMedCentralID PMC11755195
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2024 Update of the RECOVER-Adult Long COVID Research Index.
JAMA
Geng, L. N., Erlandson, K. M., Hornig, M., Letts, R., Selvaggi, C., Ashktorab, H., Atieh, O., Bartram, L., Brim, H., Brosnahan, S. B., Brown, J., Castro, M., Charney, A., Chen, P., Deeks, S. G., Erdmann, N., Flaherman, V. J., Ghamloush, M. A., Goepfert, P., Goldman, J. D., Han, J. E., Hess, R., Hirshberg, E., Hoover, S. E., Katz, S. D., Kelly, J. D., Klein, J. D., Krishnan, J. A., Lee-Iannotti, J., Levitan, E. B., Marconi, V. C., Metz, T. D., Modes, M. E., Nikolich, J. Ž., Novak, R. M., Ofotokun, I., Okumura, M. J., Parthasarathy, S., Patterson, T. F., Peluso, M. J., Poppas, A., Quintero Cardona, O., Scott, J., Shellito, J., Sherif, Z. A., Singer, N. G., Taylor, B. S., Thaweethai, T., Verduzco-Gutierrez, M., Wisnivesky, J., McComsey, G. A., Horwitz, L. I., Foulkes, A. S.
2024
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Abstract
Classification of persons with long COVID (LC) or post-COVID-19 condition must encompass the complexity and heterogeneity of the condition. Iterative refinement of the classification index for research is needed to incorporate newly available data as the field rapidly evolves.To update the 2023 research index for adults with LC using additional participant data from the Researching COVID to Enhance Recovery (RECOVER-Adult) study and an expanded symptom list based on input from patient communities.Prospective, observational cohort study including adults 18 years or older with or without known prior SARS-CoV-2 infection who were enrolled at 83 sites in the US and Puerto Rico. Included participants had at least 1 study visit taking place 4.5 months after first SARS-CoV-2 infection or later, and not within 30 days of a reinfection. The study visits took place between October 2021 and March 2024.SARS-CoV-2 infection.Presence of LC and participant-reported symptoms.A total of 13 647 participants (11 743 with known SARS-CoV-2 infection and 1904 without known prior SARS-CoV-2 infection; median age, 45 years [IQR, 34-69 years]; and 73% were female) were included. Using the least absolute shrinkage and selection operator analysis regression approach from the 2023 model, symptoms contributing to the updated 2024 index included postexertional malaise, fatigue, brain fog, dizziness, palpitations, change in smell or taste, thirst, chronic cough, chest pain, shortness of breath, and sleep apnea. For the 2024 LC research index, the optimal threshold to identify participants with highly symptomatic LC was a score of 11 or greater. The 2024 index classified 20% of participants with known prior SARS-CoV-2 infection and 4% of those without known prior SARS-CoV-2 infection as having likely LC (vs 21% and 5%, respectively, using the 2023 index) and 39% of participants with known prior SARS-CoV-2 infection as having possible LC, which is a new category for the 2024 model. Cluster analysis identified 5 LC subtypes that tracked quality-of-life measures.The 2024 LC research index for adults builds on the 2023 index with additional data and symptoms to help researchers classify symptomatic LC and its symptom subtypes. Continued future refinement of the index will be needed as the understanding of LC evolves.
View details for DOI 10.1001/jama.2024.24184
View details for PubMedID 39693079
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Identification of two transcription factors that work coordinately to regulate early development inEntamoeba.
mBio
Lozano-Amado, D., Singh, U.
2024: e0225024
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Abstract
The protozoan parasite Entamoeba has a life cycle that switches between infective cysts and invasive trophozoites. Encystation, a crucial process in parasite biology, is controlled by different mechanisms including transcriptional control. We identified two nuclear proteins in Entamoeba invadens, EIN_066100 and EIN_085620, that regulate parasite development by binding to a DNA motif (TCACTTTC) in the promoter regions of genes upregulated in the first 8 h of stage conversion. Overexpression of EIN_066100, a homolog of MAK16 protein, resulted in reduced amoebic proliferation without affecting encystation efficiency. Overexpression of EIN_085620, a protein with an RNA-recognition motif (RRM), led to increased encystation efficiency. Glutathione S-transferase (GST) pull down assays revealed that EIN_066100 interacts with EIN_085620 both in vivo and in vitro, and this interaction is mediated by the EIN_085620 RRM domain. By evaluating truncated proteins with deletions at either the N-terminal or C-terminal regions of EIN_066100, we elucidated the importance of its N-terminal region in proper protein localization, proliferation, encystation, and interaction with EIN_085620. Taken together, these results indicate a coordinated role of EIN_066100 and EIN_085620 in regulating Entamoeba development. This work sheds light on the molecular mechanisms in the earliest stages of Entamoeba encystation.IMPORTANCEAn important biological process in the biology of Entamoeba is stage conversion, which plays a crucial role in disease propagation, facilitating parasite survival outside the host and spreading to new hosts. Multiple mechanisms contribute to controlling the expression of amebic stage-specific genes such as epigenetic and transcriptional control. Identification of early transcriptional control regulators is crucial to understanding the initiation of the encystation cascade. We identified two nuclear proteins, EIN_066100 and EIN_085620, involved in the proliferation and developmental regulation of E. invadens. These proteins work by direct binding to each other and mediating encystation efficiency. Study of new regulators involved in Entamoeba development represents an important advance in a critical aspect of parasite biology.
View details for DOI 10.1128/mbio.02250-24
View details for PubMedID 39540742
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Sex differences and immune correlates of Long Covid development, symptom persistence, and resolution.
Science translational medicine
Hamlin, R. E., Pienkos, S. M., Chan, L., Stabile, M. A., Pinedo, K., Rao, M., Grant, P., Bonilla, H., Holubar, M., Singh, U., Jacobson, K. B., Jagannathan, P., Maldonado, Y., Holmes, S. P., Subramanian, A., Blish, C. A.
2024; 16 (773): eadr1032
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Sex differences have been observed in acute coronavirus disease 2019 (COVID-19) and Long Covid (LC) outcomes, with greater disease severity and mortality during acute infection in males and greater proportions of females developing LC. We hypothesized that sex-specific immune dysregulation contributes to LC pathogenesis. To investigate the immunologic underpinnings of LC development and symptom persistence, we performed multiomic analyses on blood samples obtained during acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and 3 and 12 months after infection in a cohort of 45 participants who either developed LC or recovered. Several sex-specific immune pathways were associated with LC. Males who would later develop LC exhibited increases in transforming growth factor-β (TGF-β) signaling during acute infection, whereas females who would go on to develop LC had reduced TGFB1 expression. Females who developed LC demonstrated increased expression of XIST, an RNA gene implicated in autoimmunity, during acute infection compared with females who recovered. Many immune features of LC were also conserved across sexes, such as alterations in monocyte phenotype and activation state. Nuclear factor κB (NF-κB) transcription factors were up-regulated in many cell types at acute and convalescent time points. Those with ongoing LC demonstrated reduced ETS1 expression across lymphocyte subsets and elevated intracellular IL-4 in T cell subsets, suggesting that ETS1 alterations may drive aberrantly elevated T helper cell 2-like responses in LC. Altogether, this study describes multiple innate and adaptive immune correlates of LC, some of which differ by sex, and offers insights toward the pursuit of tailored therapeutics.
View details for DOI 10.1126/scitranslmed.adr1032
View details for PubMedID 39536117
The Wang Lab
Human Immune Functions & Susceptibility to Diseases
Taia Wang’s laboratory studies mechanisms underlying the heterogeneity in human immune function during vaccination and viral infection. We are particularly interested in antibody-mediated immunity and determinants of susceptibility to antibody-mediated diseases.