PLoS Pathogens Research Articles

RNA-seq research landscape in Africa: systematic review reveals disparities and opportunities

RNA sequencing has emerged as the standard method for transcriptome profiling of several human diseases. We performed a systematic review detailing the state of RNA-seq analyses in Africa from its inception till February 2022. Our goal was to provide an update on the state of RNA-seq analyses in Africa, including research gaps, funding information, participants information, authorship and collaborations. Following the PRISMA guidelines, we performed an exhaustive literature search for RNA-seq studies conducted in Africa, using PubMed, Scopus and Academic Search Complete (EBSCOhost). The output was exported to Endnote X9 for analyses. The initial literature search yielded 10,369 articles spread across PubMed (4916), Scopus (4847) and EBSCOhost (580). By applying our exclusion criteria, 28 full-text articles remained and were thoroughly analyzed. Overall, 17 human diseases were studied, including cancers (10/28), infectious disease (4/28), parasitic disease (4/28), autoimmune disorders (2/28) and neglected tropical diseases (2/28). Majority of the articles were published in PLoS Pathogens, BioMed Central and Nature. The National Institutes of Health (42.4%), the Bill & Melinda Gates Foundation (7.5%) and the Wellcome Trust (7.5%) were the top funders of the research studies. Eleven African countries contributed to the participant group, with 57% located in Eastern Africa, 23.1% from Western and 16.7% from Southern Africa. The extremely low number of RNA-seq research studies in Africa is worrying and calls for an immediate investment in research by the African governments. The funding agencies and institutional review boards should also ensure that African collaborators are treated equitably in the course of the research projects.

Female Dynamics in Authorship of Scientific Publications in the Public Library of Science: A 10-year Bibliometric Analysis of Biomedical Research.

Women are generally underrepresented in science, technology, engineering, and mathematics (STEM). As scientific production reflects scholarly impact and participation in the scientific process, the number of journal publications forms a pertinent measure of academic productivity. This study examined the prevalence and evolution of female representation in prominent author positions across multidisciplinary biomedical research. Publications from seven exemplar cross-specialty journals of the Public Library of Science (PLoS Medicine, PLoS Biology, PLoS One, PLoS Computational Biology, PLoS Genetics, PLoS Pathogens, and PLoS Neglected Tropical Diseases) between January 2010 and December 2020 were extracted from Web of Science. Using Genderize.io, the gender of authors from their first names was estimated using a 75% threshold. The association between female prevalence in first and last authorship and journal was evaluated using a binary logistic regression, and odds ratios were estimated against a 50:50 reference on gender. In 266,739 publications, 43.3% of first authors and 26.7% of last authors were females. Across the ten-year period, female first authorship increased by 19.6% and last authorship by 3.2%. Among all journals, PLoS Neglected Tropical Diseases had the greatest total proportion of female first authors (45.7%) and PLoS Medicine of female last authors (32%), while PLoS Computational Biology had the lowest proportion in these categories (23.7% and 17.2%). First authors were less likely to be females in all PLoS journals (p < 0.05) except for PLoS Neglected Tropical Diseases (odds ratio: 0.84, 95% confidence interval: 0.71-1.00), where the odds of female authorship were not significantly different (p = 0.054). Last authors were not more likely to be females in all PLoS journals (p < 0.001). Overall, women still appear underrepresented as first authors in biomedical publications and their representation as last authors has severely lagged. Efforts towards gender equality in scholarly authorship will contribute to the representation of women in biomedical research and ensure that their potential is not lost.

P381 Kinetics of Titan cells generation and transcriptome modifications comparing three in vitro protocols

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesTo face and escape his environment and the host immune response, Candida neoformans is able to change his morphology (Titan cells) and his metabolism (dormancy, quiescence). Titan cells (TC) have been observed in lungs and brains in experimental mouse models of cryptococcosis and in patients. In 2018, three independent teams published three protocols allowing the reproducible generation of TC in vitro [Hommel B et al., (AA); Trevijano-Contador N, et al., (OZ), and Dambuza IM et al., (EB), in PLoS Pathogens 2018]. TC generated in these protocols exhibited the same features as the TC described in vivo. Our objective was to compare and describe the three protocols in parallel to highlight common and different features that can impact further study using those specific protocols.MethodsA total of 22 h before starting the kinetics of the TC generation, the three protocols requested a pre-culture at 30°C in three different liquid media. The medium for TC production for the three labs was also different but a common factor is the addition of fetal calf serum (FBS) for OZ and EB. This kinetics was evaluated for size and quantity (%) of TC produced over a 72 h period (H0, H18, H24, H48, H72) at 30°C under shaking for AA while OZ and EB protocols incubate the cells at 37°C and 5% of CO2, while the whole transcriptome was analyzed at H0, H3, H7, and H18 in triplicates.ResultsOZ generated the highest percentage of TC, 63.1% and 58.2% at H18 and H24, and decreased drastically down to 6.7% at H48. EB reached a high percentage of TC at H24 for 46.7% and dropdown <10% until the end of the kinetics. AA did not reach a quantity of TC as high as the two other protocols but it remained constant over a period of H72 (Table 1). RNA sequencing preliminary analysis showed some differences in genes expressed at the different time points analyzed. The PCA analysis revealed that the triplicates of each protocol for the 4-time points analyzed are closed to each other, related to the good quality of our experiments. The differential gene expression (DGE) showed significant (P <.01 and Log2 fold change >1) differences at H0 which highlights the impact of the preculture on the TC process. The highest numbers of DGE are observed between H0 and H7 for the three protocols, where about two 450 DGE, two 000 DGE, and two 300 AA, OZ, and EB, respectively. After analysis of the PCA plot during the kinetics, EB and OZ are grouped while AA is not. That could be explained by the presence of FBS in OZ and EB protocols.ConclusionBy running the three protocols in parallel, we showed here that the kinetics of TC generation differed between each other with a significant variation of the transcriptome. This is an important finding that paves the way to compare more deeply the transcriptome of C. neoformans during TC generation with the final goal is to identify the genes associated with TC generation.

Preprint citation practice in PLOS

The role of preprints in the scientific production and their part in citations have been growing over the past 10 years. In this paper we study preprint citations in several different aspects: the progression of preprint citations over time, their relative frequencies in relation to the IMRaD structure of articles, their distributions over time, per preprint database and per PLOS journal. We have processed the PLOS corpus that covers 7 journals and a total of about 240,000 articles up to January 2021, and produced a dataset of 8460 preprint citation contexts that cite 12 different preprint databases. Our results show that preprint citations are found with the highest frequency in the Method section of articles, though small variations exist with respect to journals. The PLOS Computational Biology journal stands out as it contains more than three times more preprint citations than any other PLOS journal. The relative parts of the different preprint databases are also examined. While ArXiv and bioRxiv are the most frequent citation sources, bioRxiv’s disciplinary nature can be observed as it is the source of more than 70% of preprint citations in PLOS Biology, PLOS Genetics and PLOS Pathogens. We have also compared the lexical content of preprint citation contexts to the citation content to peer-reviewed publications. Finally, by performing a lexicometric analysis, we have shown that preprint citation contexts differ significantly from citation contexts of peer-reviewed publications. This confirms that authors make use of different lexical content when citing preprints compared to the rest of citations.

A Comprehensive Review of Vaccines against Covid-19

A Comprehensive Review of Vaccines against Covid-19

A Message to Our Readers

Previous articleNext article FreeA Message to Our ReadersPDFPDF PLUSFull Text Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinked InRedditEmailQR Code SectionsMoreWe are proud to announce that Dr. Daniel E. Dykhuizen has been promoted to Editor Emeritus of The Quarterly Review of Biology, effective 1 September 2021. With Dr. Dykhuizen’s designation as Editor Emeritus, Dr. Liliana M. Dávalos will assume the responsibilities of Editor-in-Chief, joined with James D. Thomson, Gregory A. Wray, and John J. Wiens as Editors.Dr. Dykhuizen has been associated with the QRB since 1992, first as a Book Review Consultant (1992–2002), second as an Editor (2002–2005 and 2007–2010), and then as Editor-in-Chief (2010–2021).Dr. Dykhuizen is an expert in population genetics, having received a Ph.D. under the mentorship of Professor Richard Lewontin from the University of Chicago in 1970. He joined the Department of Ecology and Evolution at the State University of New York at Stony Brook, now Stony Brook University, in 1987. He has served as Editor of Genetica, Molecular Phylogenetics and Evolution, and Journal of Bacteriology, and was on the editorial board of Infection, Genetics and Evolution, and PLOS Pathogens. He has also served on numerous grant panels. Dr. Dykhuizen is a Fellow of the American Association for the Advancement of Science and the American Academy of Microbiology. In 2009, the State University of New York Board of Trustees appointed Dr. Dykhuizen to the rank of Distinguished Professor. He was also the winner of the Award for Faculty Mentoring, presented by the University President in 2011. A symposium entitled “Experimental Evolution and DNA” was held in honor of Dr. Dykhuizen’s professional scientific achievements in 2012.It gives us great pleasure to thank Dr. Dykhuizen for his long service to The Quarterly Review of Biology. We wish him health and happiness in his future endeavors.The Editors and Staff of The Quarterly Review of Biology Previous articleNext article DetailsFiguresReferencesCited by The Quarterly Review of Biology Volume 96, Number 3September 2021 Published in association with Stony Brook University Article DOIhttps://doi.org/10.1086/716164 Views: 235 © 2021 The University of Chicago. All rights reserved.PDF download Crossref reports no articles citing this article.

Abstract LB105: In vivo CAR T therapy: targeted in vivo gene delivery of a CAR using a CD8-specific fusogen results in tumor eradication

Abstract CAR T cells are highly effective at inducing remissions in patients with refractory B cell malignancies. The ex vivo process used to manufacture these therapies limits patient access and exposes T cells to non-physiologic conditions in culture. Viral vectors are efficient at genetically modifying T cells and are used in the ex vivo manufacture of most CAR T cell products but lack target-cell specificity. Here we describe how viral envelope glycoproteins, termed fusogens, engineered to target the CD8 co-receptor can efficiently and specifically deliver a chimeric antigen receptor (CAR) to human T cells in vivo that support target cell killing and tumor eradication. Modification of viral envelope proteins to include antibody-based binding domains (CD8 binders) can produce fusogen-viral vector compositions that deliver genetic payloads to CD8 T cells with therapeutic effect (1, 2). Screening CD8-targeted binders identified fusogens with a range of on-target cell transduction efficiencies and off-target cell line specificities. CD8-targeted fusogens with the highest efficiency were comparable to VSVg-pseudotyped lentiviruses at transducing CD8 T cells, but unlike VSVg showed CD8 T cell specific transduction. Interestingly, CD8-targeted fusogens were superior to VSVg-pseudotyped lentivirus at transducing non-activated human T cells. In vivo fusogen-mediated delivery showed efficient and specific CD8 T cell transduction and expression of a GFP reporter gene. Second-generation CARs containing a CD19 binding domain and a 41BB costimulatory domain delivered by targeted fusogen demonstrated CD8 specific expression and functional activity against non-malignant B cells and CD19+ Nalm6 leukemia cells. To demonstrate targeted fusogen-mediated in vivo CAR delivery and activity, we established Nalm6 xenografts in mice into which unmodified activated human PBMCs were infused. Activated human PBMCs alone were unable to control tumor growth. A single intravenous delivery of a CD8 fusogen containing a second-generation CD19 CAR transgene across a range of functional titer doses resulted in the generation of CD8 CAR Ts that eradicated the CD19+ tumor xenografts. A high percentage of T cells demonstrated CAR expression after fusogen delivery with clear specificity for the CD8+ cells. Importantly, the fusogen was able to generate a functional CAR response regardless of prior activation status of the T cells. Targeted in vivo delivery of CAR into T cell subsets may represent a novel therapeutic approach for human B cell malignancies. The ability to specifically deliver a CAR gene to a T cell in vivo would be transformative in terms of patient access and is likely to generate a qualitatively superior therapeutic T cell.

A systematic pipeline for classifying bacterial operons reveals the evolutionary landscape of biofilm machineries.

In bacteria functionally related genes comprising metabolic pathways and protein complexes are frequently encoded in operons and are widely conserved across phylogenetically diverse species. The evolution of these operon-encoded processes is affected by diverse mechanisms such as gene duplication, loss, rearrangement, and horizontal transfer. These mechanisms can result in functional diversification, increasing the potential evolution of novel biological pathways, and enabling pre-existing pathways to adapt to the requirements of particular environments. Despite the fundamental importance that these mechanisms play in bacterial environmental adaptation, a systematic approach for studying the evolution of operon organization is lacking. Herein, we present a novel method to study the evolution of operons based on phylogenetic clustering of operon-encoded protein families and genomic-proximity network visualizations of operon architectures. We applied this approach to study the evolution of the synthase dependent exopolysaccharide (EPS) biosynthetic systems: cellulose, acetylated cellulose, poly-β-1,6-N-acetyl-D-glucosamine (PNAG), Pel, and alginate. These polymers have important roles in biofilm formation, antibiotic tolerance, and as virulence factors in opportunistic pathogens. Our approach revealed the complex evolutionary landscape of EPS machineries, and enabled operons to be classified into evolutionarily distinct lineages. Cellulose operons show phyla-specific operon lineages resulting from gene loss, rearrangement, and the acquisition of accessory loci, and the occurrence of whole-operon duplications arising through horizonal gene transfer. Our evolution-based classification also distinguishes between PNAG production from Gram-negative and Gram-positive bacteria on the basis of structural and functional evolution of the acetylation modification domains shared by PgaB and IcaB loci, respectively. We also predict several pel-like operon lineages in Gram-positive bacteria and demonstrate in our companion paper (Whitfield et al PLoS Pathogens, in press) that Bacillus cereus produces a Pel-dependent biofilm that is regulated by cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP).

Understanding the Mechanisms Underlying Thrombocytopenia in Visceral Leishmaniasis

Visceral leishmaniasis (VL) is a neglected tropical parasitic disease caused by Leishmania parasites and only second to malaria in terms of worldwide morbidity and mortality. According to recent WHO report, there are 500,000 cases of VL worldwide leading to ~30,000 deaths per year. VL is endemic in 98 countries but the major disease burden is contributed by Brazil, India and Sudan. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulations and extensive hematological complications. We have shown previously using experimental models of infection that the infiltration of CD4+ T cells results in disruption to the bone marrow environment, resulting in dysfunctional hematopoietic stem and progenitor cells self-renewal (Pinto et al, PLOS Pathogens, 2017) and aberrant medullary erythropoiesis causing pathological anemia (Preham et al, Frontiers in Immunology, 2018). Thrombocytopenia is also dominant hematological feature seen in both human and experimental models that may reflect either reduced platelet production or enhanced clearance. However, the mechanisms of VL-driven thrombocytopenia remain poorly understood. The aim of this study is to explore the possible underlying mechanisms from platelet production to phagocytic cells dependent clearance. Using a murine experimental model of VL, we demonstrate a steady decrease in the platelet count from d14 onwards in infected mice culminating in severe thrombocytopenia on d28 of infection (infected: 225.9 ±35.7 vs naïve: 1005 ±90.6, x 106/µl). Critically, thrombocytopenia is completely reversible after a single dose of liposomal amphotericin B (Ambisome @ 8mg/kg bodyweight, IV) which clears parasites by delivering the drug directly to parasite harbouring tissue macrophages, thereby improving parasite clearance and reducing toxicity. Despite significant thrombocytopenia, the number and gross morphology of bone marrow megakaryocytes (MKs) were not altered, but MK ultrastructure studies using transmission electron microscopy identified significantly reduced demarcation membranes in infected mice compared to naïve. Levels of plasma thrombopoietin (TPO), the key regulator of MK differentiation and platelet production, were decreased in infected vs naïve mice (1254 ± 95.49 vs 3249 ± 125.1 pg/ml) and administration of exogenous TPO resulted in complete recovery of platelet counts. Given that the majority of TPO is produced by the liver, reduction in the levels of circulating TPO during infection is likely due to destruction of liver architecture by parasite loaded hepatic granulomas. Together, these data suggest that despite some changes in MK cytoplasmic maturation, the bone marrow microenvironment remains supportive of MK differentiation capacity during VL. As platelet production is not significantly altered by VL, we next determined effects on platelet clearance. Large number of highly active splenic macrophages are common in VL and are known for their phagocytic properties. Experiments conducted on VL-infected splenectomised mice demonstrated a reduction in thrombocytopenia compared to sham-operated infected mice (685 ±32 vs 297± 16, x 106/µl) and showed a great response to exogenous TPO, implying splenic clearance may be involved in thrombocytopenia. Partial depletion of splenic macrophages in infected mice using clodronate liposomes did not alter platelet count, whereas neutrophil deletion (anti-Gr1 mAb @ 250ug/g IP) in infected mice resulted in a near 2-fold increase in platelet counts. Furthermore, circulating platelets in VL infected mice were IgG coated compared to naive which is likely to further enhance autoimmune platelet clearance. Severe thrombocytopenia and bleeding are important clinical manifestations of VL. Our findings clearly demonstrate that the mechanisms of thrombocytopenia in VL are multifactorial but do not cause permanent long term damage to the BM microenvironment. Critically, these changes could be reversed rapidly by clearing parasitemia, using TPO agonists to increase numbers of circulating platelets and/or by reducing platelet clearance. This highlights the possibility of re-evaluating the current treatment regimen in VL endemic countries by including therapeutic interventions aimed at reversing severe thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

A high throughput genetic screen to uncover novel antiviral mechanisms of the interferon system

Abstract The innate immune responses provide the first line of host defense against virus infection. Type I interferon (IFN) system is a well-known mediator of the host innate antiviral responses. Virus infection is recognized by the cellular sensors, also known as pattern recognition receptors, which via a series of signaling pathways, induce the synthesis of IFNs and the IFN-stimulated genes (ISGs). The ISG-encoded protein products function as viral restriction factors, either by interfering with the stages of viral life cycle or by promoting cell death. We have shown that the IRF3, a key transcription factor responsible for inducing IFNs and ISGs, also provides antiviral responses by triggering apoptosis of the infected cells (Chattopadhyay et al Immunity, 2016). To investigate how the IRF3-inducible genes provide antiviral protection, we performed a high throughput genetic screen of human ISGs. Using an shRNA library of the human ISGs, we have identified a novel antiviral ISG, tudor domain containing 7 (TDRD7). TDRD7 inhibits virus replication by interfering with the cellular autophagy pathway, which is required for the replication a number of viruses. Mechanistically, TDRD7 inhibits the autophagy-initiating kinase, AMPK to suppress virus-induced autophagy pathway. We reported that multiple paramyxoviruses, including the clinically relevant human pathogens, respiratory syncytial virus (RSV) and human parainfluenza virus (HPIV3), are inhibited by the new antiviral mechanism of TDRD7 (Subramanian et al PLOS Pathogens, 2018). Using additional RNA and DNA viruses, which activate autophagy during infection, we are investigating whether the new antiviral mechanism similarly inhibits them.

The most influential articles in Helicobacter pylori research: A bibliometric analysis.

The number of articles that researchers must be familiar with is increasing, along with the importance of selective searching and summarization. This study aimed to assess and characterize the most influential articles in Helicobacter pylori research. We performed a search of the top-100 cited articles using the Web of Science Core Collection (WoSCC) and Google Scholar from their inception to 2018. The top-100 Altmetric Attention Score (AAS) articles based on online media mentions were also searched using the term Hpylori. Each article was evaluated for the following characteristics: citation number, title, journal, publication year, and authorship. The citation number for the top-100 WoSCC articles ranged from 44 to 367. Gut published the largest number of articles (11%). In the top-100 Google Scholar articles, Lancet had the largest number of articles (13%); however, among the top-1000 cited articles published after 2012, Helicobacter published the largest number (46%). The largest number of top-100 AAS articles was published by PLOS Pathogens (6%). PubMed Central articles' citations in WoSCC or Google Scholar showed significant correlation with those from each metric; however, AAS showed no correlation. The proportion of basic research was 36%-37% in top-cited articles; but, 52% in the top-100 AAS articles. No time trend in the number of publications or citations of basic/clinical research in the top-100 bibliometrics was found. "Meta-analysis/systematic review," "gastric cancer," "eradication," and "association" were the most influential title words. This study presents a detailed list of top-100 articles, journals, authors, and topic title words.

Transcriptome and Somatic Mutation Associated with Non-Malignant Human T Lymphotropic Virus Type 1 Infection and Adult T-Cell Leukemia/Lymphoma

Aggressive Adult T-cell leukemia/lymphoma (ATL), a human T lymphotropic virus type 1 (HTLV-1) -associated disease, has a poor prognosis. There is an urgent need for effective prevention and treatment. A large number of genomic aberrations including hundreds of somatic mutations and copy number changes are typically observed in ATL tumours, with certain genes, PLCG1, PRKCB, CCR4, CARD11, STAT3, TP53, VAV1, TBL1XR1, NOTCH1, GATA3 and IRF4 mutated in over 10% of cases(1). CD4+CCR4+CD26-CD7- is the dominant immunophenotype of lymphocytes in patients with ATL (ATL cells) but infected cells with similar immunophenotype (‘ATL-like’ cells) are also present in patients with non-malignant HTLV-1 infection(2, 3). Aggressive ATL develops in patients with non-malignant HTLV-1 (asymptomatic carriers (AC) and patients with HTLV-1 associated myelopathy (HAM)) over decades with evolution from high proviral load (PVL) and non-dominant clonal growth through emergence of dominant clones and indolent to aggressive ATL. HTLV-1 infected clones have been shown to have cells with mixed immunophenotype. The aim was to investigate specific genomic aberration associated with non-malignant HTLV-1 infection, dominant clonal growth and ATL.RNA sequencing followed by differential gene expression and mutational analysis of HTLV-1 and human genes was performed on sorted CD4+CCR4+CD26-CD7- cells from nine patients with ATL (four with indolent, four with aggressive ATL and one with indolent to aggressive transformation; ATL cells); and 18 patients with high PVL non-malignant HTLV-1 infection (three with dominant clones and 15 with non-dominant clones, ‘ATL-like’ infected cells). Seven antisense transcript of HTLV-1 genome was detected. A spliced antisense transcript spanning the whole HTLV-1 genome was detected in all samples whilst two novel transcripts were detected in > 2 samples. There was no significant difference in viral transcriptome expression between ATL and ‘ATL-like’ cells. A total of 13637 including 7952 well annotated human genes were detected within which 400 genes were significantly differentially expressed ( > 2 fold change and false discover rate < 0.1) between ATL and ‘ATL-like’ cells as shown in figure 1. Small nuclear RNA and endothelial cancer associated genes were upregulated in patients with ATL whilst T-cell, inflammatory, apoptosis and proliferation related genes were upregulated in patients with non-malignant HTLV-1 infection respectively. Principle component analysis did not showed any significant cluster but hierarchical analysis using differentially expressed genes showed clustering of ATL cells from patient with aggressive ATL, indolent ATL and ‘ATL-like’ cells from patients with non-malignant HTLV-1 infection (AC and HAM) of ATL as shown in figure 2. One out of three patient with high PVL non-malignant HTLV-1 infection and dominant clones (HKU) clustered with ATL and this patient progressed to ATL 12 months from sample data. Hallmark pathway analysis showed upregulation cluster in ‘ATL-like’ cells with only metabolism associated pathways clustered in ATL cells as shown in table 1. Expression of all recurrently mutated genes was detected and mutation analysis is currently underway.In summary, there is a major overlap of infected cell transcriptome in patient with non-malignant HTLV-1 infection and ATL. ATL cells have downregulation of T-cell, inflammatory, apoptosis and proliferation related genes compared to ‘ATL-like’ infected cells whilst upregulation of small nuclear RNAs. Transcriptome analysis in patient with high PVL non-malignant HTLV-1 infection might help in further prognostication in malignant risk.Kataoka K, Nagata Y, Kitanaka A, Shiraishi Y, Shimamura T, Yasunaga J, et al. Integrated molecular analysis of adult T cell leukemia/lymphoma. Nature genetics. 2015;47(11):1304-15.Kagdi H, Demontis MA, Ramos JC, Taylor GP. Switching and loss of cellular cytokine producing capacity characterize in vivo viral infection and malignant transformation in human T- lymphotropic virus type 1 infection. PLoS pathogens. 2018;14(2):e1006861.Kagdi HH, Demontis MA, Fields PA, Ramos JC, Bangham CR, Taylor GP. Risk stratification of adult T-cell leukemia/lymphoma using immunophenotyping. Cancer medicine. 2017;6(1):298-309. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The In Vivo infected Clones Have Mixed Immunophenotype and Malignant Transformation Is Characterized By Dominant Growth of CD4+CCR4+CD26-CD7- Cells in Human T Lymphotropic Virus Type 1 Infection

Aggressive Adult T cell leukemia/lymphoma (ATL), a human T lymphotropic virus type 1 (HTLV-1) -associated disease, has a poor prognosis. There is an urgent need for effective prevention and treatment. Aggressive ATL develops in patients with non-malignant HTLV-1 (asymptomatic carriers (AC) and patients with HTLV-1 associated myelopathy (HAM)) over decades with evolution from high proviral load (PVL) and non-dominant clonal growth through emergence of dominant clones and indolent to aggressive ATL. A single dominant clone is a major contributor to HTLV-1 infection burden in ATL despite a polyclonal background whilst hundreds of small clones contribute relatively equally to infection burden in non-malignant HTLV-1 infection(1). CD4+CCR4+CD26-CD7- is the dominant immunophenotype of lymphocytes in patients with ATL (ATL cells) but infected cells with similar immunophenotype (‘ATL-like’ cells) are also present in patients with non-malignant HTLV-1 infection(2). We hypothesize that ‘ATL-like’ infected cells harbor: larger HTLV-1 infected clones detected in total PBMC and are more oligo-clonal compared to total PBMC in patients with high PVL non-malignant HTLV-1 infection and; only the dominant clone detected in total PBMC in patients with ATL. The aim is to study the relationship between clonality and immunophenotype in patients with high PVL non-malignant HTLV-1 infection and ATL.Clonality analysis was performed on total PBMC by ligation mediated PCR followed by high throughput sequencing (LMPCR-HTS) and on sorted CD4+CCR4+CD26-CD7- cells by LMPCR-HTS and T-cell receptor (TCR) sequencing in ACs (four and nine respectively), patients with HAM (four and nine respectively) and patients with ATL (four and nine respectively). Clonality analysis within CD4+CCR4+CD26-CD7- cells, similar to PBMC, showed hundreds of non-dominant clones (relative abundance of largest clone ≤ 25%) in 83% of patients with high PVL non-malignant HTLV-1 infection and a dominant clone (relative abundance of largest clone ≥ 25%) over a background of non-dominant clones in 17% and 100% of patients with non-malignant HTLV-1 infection and ATL respectively. CD4+CCR4+CD26-CD7- cells contributed a median of 95% of the largest PBMC clone in patients with ATL and a median of 23% of the largest PBMC clone in 63% of patients with non-malignant HTLV-1 infection. Cells which do not have ‘ATL-like’ immunophenotype (CD4+CCR4- and CD4+CCR4+CD26-CD7+) were also detected alongside CD4+CCR4+CD26-CD7- cells within individual clones in patients with non-malignant HTLV-1 infection as shown in figure 1. Three out of eighteen patients with high PVL non-malignant HTLV-1 infection had two or more dominant clones, one of which showed signs of overt ATL 12 months after the sample data. Clonality analysis in an patient with incident aggressive ATL showed presence of two dominant clones with two integrated provirus each 3 months prior to diagnosis and single dominant clone at presentation, remission and relapse as shown in figure 2. There was no significant difference in distribution of viral integration site by LMPCR-HTS or TCR repertoire in patients with non-malignant HTLV-1 infection and ATL.In summary, ‘ATL-like’ cells are derived from relatively small non-dominant clones in the majority of patient with high PVL non-malignant HTLV-1 infection and from a dominant clone over a non-dominant background in patients with ATL. The dominant clone in patients with ATL was comprised almost universally of ‘ATL-like’ cells whilst the large PBMC clones in patients with non-malignant HTLV-1 infection had cells with a mixed immunophenotype including ‘ATL-like’ and non- ‘ATL-like’ cells. Oligoclonal proliferation of ‘ATL-like’ cells precedes monoclonal proliferation at clinical presentation of ATL. Clonality analysis in patient with high PVL non-malignant HTLV-1 infection might help in further prognostication in malignant risk.Cook LB, Melamed A, Niederer H, Valganon M, Laydon D, Foroni L, et al. The role of HTLV-1 clonality, proviral structure, and genomic integration site in adult T-cell leukemia/lymphoma. Blood. 2014;123(25):3925-31.Kagdi H, Demontis MA, Ramos JC, Taylor GP. Switching and loss of cellular cytokine producing capacity characterize in vivo viral infection and malignant transformation in human T- lymphotropic virus type 1 infection. PLoS pathogens. 2018;14(2):e1006861. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

LUNG NODULES: VALLEY FEVER OR CANCER IN HIV

SESSION TITLE: Medical Student/Resident Pulmonary Manifestations of Systemic Disease 3 SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/09/2018 01:15 PM - 02:15 PM INTRODUCTION: The Human Immunodeficiency Virus (HIV) epidemic has vastly changed the prevalence and epidemiological burden of fungal infections. With worldwide travel, diseases such as coccidioidomycosis, while rare in non endemic parts of the United States of America, <1%, have been increasing in incidence. Coccidioidomycosis may present with a constellation of clinical and radiological findings that often confound diagnosticians, further emphasizing the need for more sensitive risk calculators. CASE PRESENTATION: A 45 year old caucasian male with HIV, last CD4 845, on highly active antiretroviral therapy (HAART) presents with a six week history of cough. He reports a persistent dry cough associated with right-sided chest pain now resolved and constitutional symptoms. He also has shortness of breath, treated with Albuterol inhaler. He is a current smoker with 16 pack years. Prior to onset of his symptoms, he completed a cross-country road trip from California to the east coast. He denied any ill-contacts or limitations in activity. On exam, he is afebrile in no respiratory distress with an unremarkable exam. His white blood cell count was 9.7 (13% eosinophils) along with positive coccidioidomycosis antibody complement fixation 1:4 and elevated IgG and IgM. Notably tuberculosis quantiferon gold, histoplasmosis antibodies and urine antigen were within normal limits. Computerized Tomography chest with contrast revealed 1.9x1.4x1.1 cm spiculated solid upper lobe nodule along with scattered subcentimeter, noncalcified solid round nodules in all lobes bilaterally and mediastinal lymphadenopathy. He was commenced on oral fluconazole for pulmonary coccidioidomycosis and scheduled for follow-up. DISCUSSION: Numerous advances have been made in the management of HIV, however aspects of the pathophysiology still remain unknown. ATS (2010) recommended treatment of coccidioidomycosis in all immunocompromised patients e.g. HIV AIDS as well as treatment in immunocompetent patients with HIV (BII). Nathan et al, later highlighted not only the role of a decreased number of CD4 cells as a component of immunosuppression in HIV patients but also the progressive loss of T cell function due to neutrophil immunosuppression in chronic HIV infection. As HIV places patients at higher risk for disseminated disease, it further confounds differentiating lung nodules due to coccidioidomycosis from lung cancer. Ronaghi et al, found significant radiographic overlap between the aforementioned. A study by Peterson et al, evaluated three existing online risk calculators and found they performed well in patients with lung cancer but poorly in patients with coccidioidomycosis. No serological marker has been validated. CONCLUSIONS: The increasing prevalence of HIV along with worldwide travel will mandate the need for more reliable risk assessment tools to prevent unnecessary invasive procedures and treatment for decades to come. Reference #1: Nathan L. Bowers, E. Scott Helton, Richard P. H. Huijbregts, Paul A. Goepfert, Sonya L. Heath, Zdenek Hel. Immune Suppression by Neutrophils in HIV-1 Infection: Role of PD-L1/PD-1 Pathway. PLOS Pathogens. 2014 March; 10(3). E10003993 Reference #2: Reza Ronaghi, MD; Ali Rashidian, MD; Paul Mills, PhD; Kurt Hildebrandt, MD; Keith Carson, MD; Michael Peterson, MD. Radiographic Features Differentiating Lung Nodules Caused by Coccidioidomycosis From Lung Cancer. CHEST. 2015 October; 148(4_MeetingAbstracts): 509A. https://doi.org/10.1378/chest.2248593 Reference #3: Michael Peterson, Reza Ronagi, Paul Mills. Differentiating incidental lung nodules due to lung cancer from those due to coccidioidomycosis. European Respiratory Journal. 2015; 46. https://doi.org/10.1183/13993003.congress-2015.PA2996 DISCLOSURES: No relevant relationships by Sahai Donaldson, source=Web Response No relevant relationships by Karlene Williams, source=Web Response

RNA-based disease control as a complementary measure to fight Fusarium fungi through silencing of the azole target Cytochrome P450 Lanosterol C-14 \u03b1-Demethylase

RNA-based disease control has shown great potential for controlling pest and diseases in crop plants. While delivery of inhibitory noncoding double-stranded (ds)RNA by transgenic expression is a promising concept, it requires the generation of transgenic crop plants, which may cause substantial delay for application strategies depending on the transformability and genetic stability of the crop plant species. Focusing on agronomic important barley - Fusarium spec. pathosystems, we have sought for alternative strategies to apply dsRNAs for fungal control. Recently, we have demonstrated that a spray application of a long noncoding dsRNA termed CYP3RNA, which targets the three fungal Cytochrome P450 lanosterol C-14α-demethylase genes FgCYP51A, FgCYP51B, and FgCYP51C, inhibits Fusarium graminearum (Fg) on barley leaves (Koch et al., PLoS Pathogens, 12, e1005901, 2016). Here we show that another Fusarium species, F. culmorum (Fc), also is sensitive to CYP51-derived dsRNAs. Treating Fc with various dsRNAs targeting the genes FcCYP51A, FcCYP51B and FcCYP51C was destructive to the fungus and resulted in growth retardation in in vitro cultures. We discuss important consequences of this finding on future RNA-based disease control strategies. Given the ease of design, high specificity, and applicability to diverse pathogens, the use of target-specific dsRNA as an anti-fungal agent offers unprecedented potential for novel plant protection strategies.

D-103 HIV Cure: Time to rethink the “Shock and Kill” strategy?

The favored initial approach by many to depleting HIV reservoirs and achieving an HIV cure was the “shock and kill” strategy, consisting of “shocking” proviruses out of latency and killing the cells with newly expressed viral antigens, either by direct viral-mediated cytotoxicity or immune-mediated clearance. However, in vitro models showed that proviral latency reversal alone did not result in death of infected cells (Shan, et al. Immunity 2012). Ex vivo experiments revealed that only a small fraction of proviruses (~1.5%) can be reactivated to produce virions with maximum CD4+T-cell activation, and that &lt;&lt;1% are reactivated with current, small molecule latency reversing agents (Cillo, et al. PNAS 2014). In addition, maximum CD4+T-cell activation can cause the expansion of clones carrying intact proviruses capable of sustained production of infectious virus (Bui, et al PLoS Pathogens 2017). With regard to immune-mediated killing of infected cells, increasing antibody-dependent effector functions including ADCC, ADCP, and ADCF is a popular approach, but a recent trial (ACTG A5342) of two doses (40 mg/kg) of the bnMAb VRC01 revealed that is had no effect on persistent viremia and did not decrease the number infected cells in blood or the subset expressing viral RNA. Although many more approaches to “shock and kill” are in development and being tested alone and in combination, these initial data summarized above suggest that the goal of reservoir depletion may be more difficult to achieve than anticipated because of barriers to latency reversal and resistance of infected cells to immune-mediated clearance.

Flying Fox: The Animal Origin of SARS-CoV

David Cyranoski (1 December, 2017)1 reported in Nature (news) that ”Bat cave solves mystery of deadly SARS virus — and suggests new outbreak could occur”, based on a paper (30 November, 2017)2 of some Chinese scientists in PLoS Pathogens. However, I don’t agree with their conclusion that Yunnan bat is the culprit of SARS-CoV duo to their many doubtful points, logic errors and disregarding some existing evidences. The animal origin of SARS-CoV is a cosmopolitan difficult question. Its solution must meet all suspicion, evidences and logic simultaneously. A wrong scientific conclusion can lead to terrible behavior, for example, leading the masked palm civets (Paguma larvata) to be massacred during 2002-2003 in China, as the culprit of SARS. For this reason, I make important comments and clarifications on their above conclusions and provide an alternative way of thinking for the academic community to discuss. Here I show: (1) the animal origin of SARS-CoV might be a flying fox, whose crucial genes of SARS virus - for a protein that allows the virus to latch onto and infect cells — had been activated by an underground uranium mine in the Panxi Rift 1,000 km away from Guangdong; (2) unless so complex process above is repeated, another SARS-CoV event will not erupt again within Guangdong; (3) SARS was a much extremely accidental event. Finally, I restore the entire process of SARSCoV event, using all the evidence chain and whole logic, from the point of view of system theory, based on the correlation between biology and geology. That might provide an impetus to similar investigation elsewhere.

Macrophages as drivers of an opportunistic infection

Opportunistic pathogens are a worldwide cause of mortality and morbidity, and infections with intrinsically antibiotic-resistant pathogens have a large clinical, social and economic impact. Bacteria belonging to the Burkholderia cepacia complex (Bcc), ubiquitous in natural and industrial environments, are notorious pathogens for individuals with cystic fibrosis (CF). In addition, Burkholderia cenocepacia is emerging as the culprit of non-CF related, sometimes fatal infections. Knowledge of the underlying infection mechanism of these pathogens is important for efficient treatment, however, to date not much is known about the lifestyle of Bcc bacteria during infection. In our recent study published in PLoS Pathogens, we provide experimental evidence that macrophages are critically important for proliferation of B. cenocepacia, and are major drivers of fatal pro-inflammatory infections in zebrafish larvae. This is in agreement with recent clinical information showing that B. cenocepacia is mainly localised in phagocytes in infected CF lungs. A predominant intramacrophage stage and a host-detrimental role for macrophages have major implications for treatment strategies of both CF and non-CF infections. Intracellular survival of bacteria traditionally classified as extracellular, including Staphylococcus aureus and Pseudomonas aeruginosa, is an emerging theme. Our finding that macrophages are essential for proliferation of B. cenocepacia in the host suggests a new paradigm for Bcc infections and urges the development of novel anti-infectious therapies to efficiently disarm these intrinsically antibiotic resistant facultative intracellular pathogens.

Cytomegalovirus Pneumonia in a Patient Exposed to Vedolizumab

SESSION TITLE: Lung Pathology 2 SESSION TYPE: Affiliate Case Report Poster PRESENTED ON: Tuesday, October 31, 2017 at 01:30 PM - 02:30 PM INTRODUCTION: Cytomegalovirus (CMV) requires a repeated or prolonged exposure for transmission. In adulthood, it is usually transmitted sexually. Once infected, an individual becomes a carrier. Reactivation develops in immunosuppressed patients. CASE PRESENTATION: A 19-year-old female with a history of Crohn’s disease presented to the hospital in December 2015 with a one week history of fever, dry cough, nausea, and watery diarrhea. Her medical history was significant for refractory Crohn’s disease, previously treated with 6-mercaptopurine that was switched to vedolizumab four months prior to hospitalization. On admission, the patient was hemodynamically stable. Her physical exam was unremarkable except for faint crackles in right lung base. Laboratory data demonstrated leukopenia (2.4 μl) with 33% band cells. Chest x-ray revealed bibasilar alveolar opacities (Fig.1). The patient was started on broad spectrum antibiotics. Her early hospital course was complicated with hypoxemia. Subsequently, she became hypotensive requiring medical intensive care. A non-contrast CT scan of her chest revealed bilateral consolidations (Fig.2). A bronchoscopic examination with a bronchoalveolar lavage (BAL) and transbronchial biopsies were performed to determine the etiology of her pneumonia (PNA). BAL returned positive for CMV and pathology revealed positive CMV immunostain uptake within the alveolar cells. The sample was negative for bacterial or fungal infections. Her blood microbiology was positive for CMV, with a viral load of 27,3000 IU/mL. The patient was started on Gancyclovir for disseminated CMV disease and PNA with gradual clinical improvement. She was discharged after two weeks. DISCUSSION: This is the first case reported of CMV pneumonia in a patient taking vedolizumab. Vedolizumab is a bowel specific monoclonal antibody that inhibits the interaction of α4β7 integrin on the lymphocyte surface with cell adhesion molecules present on the intestinal epithelium. As a consequence, the infiltration of leukocytes is reduced in the inflamed tissue. There are reported cases of CMV colitis occurring in patient’s exposed to vedolizumab. We hypothesize that the patient had CMV colitis and developed disseminated disease through viral translocation. CMV is known to disrupt tight junctions in the intestinal epithelial cells, leading to translocation. Moreover, prevention of memory T-lymphocyte aggregation from immunosuppression likely contributed to her viremia and pneumonia. Per the FDA’s Advisory Committee Briefing Document upon drug approval, there were no reports of opportunistic infections, including CMV, outside the gastrointestinal tract. CONCLUSIONS: We strongly suggest to pursue aggressive work up for patient’s on vedolizumab who present with hypoxia, fever and parenchymal lung consolidation. Reference #1: Maidji E, et al. (2017) Replication of CMV in the gut of HIV-infected individuals and epithelial barrier dysfunction. PLOS Pathogens 13(2): e1006202. doi: 10.1371/journal.ppat.1006202 DISCLOSURE: The following authors have nothing to disclose: Alireza Nathani, Aakash Modi, Scott Beegle, Kaitlyn Brennan No Product/Research Disclosure Information

Cationic bactericidal peptide 1018 does not specifically target the stringent response alarmone (p)ppGpp.

The bacterial stringent response is a key regulator of bacterial virulence, biofilm formation and antibiotic tolerance, and is a promising target for the development of new antibacterial compounds. The intracellular nucleotide (p)ppGpp acts as a messenger orchestrating the stringent response. A synthetic peptide 1018 was recently proposed to specifically disrupt biofilms by inhibiting the stringent response via direct interaction with (p)ppGpp (de la Fuente-Núñez et al. (2014) PLoS Pathogens). We have interrogated the specificity of the proposed molecular mechanism. When inhibition of Pseudomonas aeruginosa planktonic and biofilm growth is tested simultaneously in the same assay, peptides 1018 and the control peptide 8101 generated by an inversion of the amino acid sequence of 1018 are equally potent, and, importantly, do not display a preferential activity against biofilm. 1018 inhibits planktonic growth of Escherichia coli equally efficiently either when the alleged target, (p)ppGpp, is essential (MOPS media lacking amino acid L-valine), or dispensable for growth (MOPS media supplemented with L-valine). Genetic disruption of the genes relA and spoT responsible for (p)ppGpp synthesis moderately sensitizes – rather than protects – E. coli to 1018. We suggest that the antimicrobial activity of 1018 does not rely on specific recognition of the stringent response messenger (p)ppGpp.