Multivariate time-series analysis of biomarkers from a dengue cohort offers new approaches for diagnosis and prognosis

Dengue is a major public health problem worldwide with distinct clinical manifestations: an acute presentation (dengue fever, DF) similar to other febrile illnesses (OFI) and a more severe, life-threatening form (severe dengue, SD). Due to nonspecific clinical presentation during the early phase of dengue infection, differentiating DF from OFI has remained a chal-lenge, and current methods to determine severity of dengue remain poor early predictors. We present a prospective clinical cohort study conducted in Caracas, Venezuela from 2001–2005, designed to determine whether clinical and hematological parameters could distinguish DF from OFI, and identify early prognostic biomarkers of SD. From 204 enrolled suspected dengue patients, there were 111 confirmed dengue cases. Piecewise mixed effects regression and nonparametric statistics were used to analyze longitudinal records. Decreased serum albumin and fibrinogen along with increased D-dimer, thrombin-anti-thrombin complex, activated partial thromboplastin time and thrombin time were prognostic of SD on the day of defervescence. In the febrile phase, the day-to-day rates of change in serum albumin and fibrinogen concentration, along with platelet counts, were significantly decreased in dengue patients compared to OFI, while the day-to-day rates of change of lym-phocytes (%) and thrombin time were increased. In dengue patients, the absolute lympho-cytes to neutrophils ratio showed specific temporal increase, enabling classification of dengue patients entering the critical phase with an area under the ROC curve of 0.79. Secondary dengue patients had elongation of Thrombin time compared to primary cases while the D-dimer formation (fibrinolysis marker) remained always lower for secondary compared to primary cases. Based on partial analysis of 31 viral complete genomes, a high frequency of C-to-T transitions located at the third codon position was observed, suggesting deamina-tion events with five major hot spots of amino acid polymorphic sites outside in non-structural proteins. No association of severe outcome was statistically significant for any of the five major polymorphic sites found. This study offers an improved understanding of dengue hemostasis and a novel way of approaching dengue diagnosis and disease prognosis using piecewise mixed effect regression modeling. It also suggests that a better discrimination of the day of disease can improve the diagnostic and prognostic classification power of clinical variables using ROC curve analysis. The piecewise mixed effect regression model corroborated key early clinical determinants of disease, and offers a time-series approach for future vaccine and pathogenesis clinical studies

A modified anthrax toxin-based enzyme-linked immunospot assay reveals robust T cell responses in symptomatic and asymptomatic Ebola virus exposed individuals

Background Ebola virus (EBOV) caused more than 11,000 deaths during the 2013–2016 epidemic in West Africa without approved vaccines or immunotherapeutics. Despite its high lethality in some individuals, EBOV infection can produce little to no symptoms in others. A better understanding of the immune responses in individuals who experienced minimally symptomatic and asymptomatic infection could aid the development of more effective vaccines and antivirals against EBOV and related filoviruses. Methodology/Principle findings Between August and November 2017, blood samples were collected from 19 study participants in Lagos, Nigeria, including 3 Ebola virus disease (EVD) survivors, 10 individuals with documented close contact with symptomatic EVD patients, and 6 control healthcare workers for a cross-sectional serosurvey and T cell analysis. The Lagos samples, as well as archived serum collected from healthy individuals living in surrounding areas of the 1976 Democratic Republic of Congo (DRC) epidemic, were tested for EBOV IgG using commercial enzyme-linked immunosorbent assays (ELISAs) and Western blots. We detected antibodies in 3 out of 3 Lagos survivors and identified 2 seropositive individuals not known to have ever been infected. Of the DRC samples tested, we detected antibodies in 9 out of 71 (12.7%). To characterize the T cell responses in the Lagos samples, we developed an anthrax toxin-based enzyme-linked immunospot (ELISPOT) assay. The seropositive asymptomatic individuals had T cell responses against EBOV nucleoprotein, matrix protein, and glycoprotein 1 that were stronger in magnitude compared to the survivors. Conclusion/Significance Our data provide further evidence of EBOV exposure in individuals without EVD-like illness and, for the first time, demonstrate that these individuals have T cell responses that are stronger in magnitude compared to severe cases. These findings suggest that T cell immunity may protect against severe EVD, which has important implications for vaccine development

Intermediate filaments enable pathogen docking to trigger type 3 effector translocation

Type 3 secretion systems (T3SSs) of bacterial pathogens translocate bacterial effector proteins that mediate disease into the eukaryotic cytosol. Effectors traverse the plasma membrane through a translocon pore formed by T3SS proteins. In a genome-wide selection, we identified the intermediate filament vimentin as required for infection by the T3SS-dependent pathogen Shigella flexneri. We found that vimentin is required for efficient T3SS translocation of effectors by S. flexneri and other pathogens that use T3SS, Salmonella Typhimurium and Yersinia pseudotuberculosis. Vimentin and the intestinal epithelial intermediate filament keratin 18 interact with the C-terminus of the Shigella translocon pore protein IpaC. Vimentin and its interaction with IpaC are dispensable for pore formation, but are required for stable docking of S. flexneri to cells; moreover, stable docking triggers effector secretion. These findings establish that stable docking of the bacterium specifically requires intermediate filaments, is a process distinct from pore formation, and is a prerequisite for effector secretion

Development and Validation of a Rapid Screening Test for HTLV-I IgG Antibodies

Initial diagnosis of human T cell lymphotropic virus (HTLV) infections is mainly based by detecting antibodies in plasma or serum using laboratory-based methods. The aim of this study was to develop and evaluate a rapid screening test for HTLV-I antibodies. Our rapid screening test uses HTLV-I p24 antigen conjugated to gold nanoparticles and an anti-human IgG antibody immobilized to a nitrocellulose strip to detect human HTLV-I p24-specific IgG antibodies via immunochromatography. Performance of the rapid screening test for HTLV-I was conducted on a total of 118 serum specimens collected in Salvador, Bahia, the epicenter for HTLV-1 infection in Brazil. Using a Western blot test as the comparator, 55 serum specimens were HTLV-I positive, 5 were HTLV-I and HTLV-II positive, and 58 were negative. The sensitivity of the rapid screening test for HTLV-1 was 96.7% and the specificity was 100%. The rapid screening test did not show cross-reaction with serum specimens from individuals with potentially interfering infections including those caused by HTLV-II, HIV-I, HIV-II, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus, Epstein–Barr virus, SARS-CoV-2, Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Toxoplasma gondii, and Plasmodium falciparum. The rapid screening test also did not show cross-reaction with potentially interfering substances. Strategies for HTLV diagnosis in non- and high-endemic areas can be improved with low-cost, rapid screening tests

T Cell Responses to Nonstructural Protein 3 Distinguish Infections by Dengue and Zika Viruses

The 2015–2016 Zika virus (ZIKV) epidemic in the Americas and the Caribbean demonstrated that clinical assays to detect, distinguish, and characterize immune responses to flaviviral infections are needed. ZIKV and dengue virus (DENV) are mosquito-transmitted flaviviruses sharing overlapping geographic distributions and have significant sequence similarities that can increase the potential for antibody and T cell cross-reaction. Using nonstructural protein 1-based enzyme-linked immunosorbent assays (ELISAs), we determined the serostatus of individuals living in a region of DENV and ZIKV endemicity in Brazil, identifying individuals with primary DENV (pDENV) and primary ZIKV (pZIKV), ZIKV with primary DENV (ZIKVwpDENV), and secondary DENV (sDENV) infections; the presence of pDENV and pZIKV was further confirmed by neutralization tests. Development of an enzyme-linked immunosorbent spot (ELISPOT) assay for DENV and ZIKV structural and nonstructural (NS) protein antigens enabled us to distinguish infections by these viruses based on T cell responses and to characterize those responses. We found that gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) T cell responses to NS3 differentiated DENV and ZIKV infections with 94% sensitivity and 92% specificity. In general, we also showed that pDENV and sDENV cases and pZIKV and ZIKVwpDENV cases elicit similar T cell response patterns and that HIV-infected individuals show T cell responses that are lower than those shown by HIV-negative individuals. These results have important implications for DENV and ZIKV diagnostic and vaccine development and provide critical insights into the T cell response in individuals with multiple flaviviral infections.The potential for antibody and T cell cross-reactions to DENV and ZIKV, flaviviruses that cocirculate and can sequentially infect individuals, has complicated diagnostic and vaccine development. Our serological data show that antibodies to nonstructural protein 1 can distinguish sequential human infections by DENV and ZIKV. The development of a simple and inexpensive assay also enables the differentiation of DENV and ZIKV infections based on characterization of T cell responses. Our T cell data reveal strong response patterns that are similar in nature to those seen with individuals with one or multiple DENV infections and with individuals with only primary ZIKV infection and ZIKV-infected individuals with previous DENV exposure. The characterization of T cell responses in a serologically validated group of individuals is of relevance to the development of vaccines and immunotherapeutics against these global threats

<i>Shigella</i> Effector OspB Activates mTORC1 in a Manner That Depends on IQGAP1 and Promotes Cell Proliferation

<div><p>The intracellular bacterial pathogen <i>Shigella</i> infects and spreads through the human intestinal epithelium. Effector proteins delivered by <i>Shigella</i> into cells promote infection by modulating diverse host functions. We demonstrate that the effector protein OspB interacts directly with the scaffolding protein IQGAP1, and that the absence of either OspB or IQGAP1 during infection leads to larger areas of <i>S</i>. <i>flexneri</i> spread through cell monolayers. We show that the effect on the area of bacterial spread is due to OspB triggering increased cell proliferation at the periphery of infected foci, thereby replacing some of the cells that die within infected foci and restricting the area of bacterial spread. We demonstrate that OspB enhancement of cell proliferation results from activation of mTORC1, a master regulator of cell growth, and is blocked by the mTORC1-specific inhibitor rapamycin. OspB activation of mTORC1, and its effects on cell proliferation and bacterial spread, depends on IQGAP1. Our results identify OspB as a regulator of mTORC1 and mTORC1-dependent cell proliferation early during <i>S</i>. <i>flexneri</i> infection and establish a role for IQGAP1 in mTORC1 signaling. They also raise the possibility that IQGAP1 serves as a scaffold for the assembly of an OspB-mTORC1 signaling complex.</p></div

OspB limits <i>S</i>. <i>flexneri</i> spread dependent on IQGAP1.

<p>(A) Area of spread of GFP-producing wild type <i>S</i>. <i>flexneri</i> in IQGAP1^-/- and IQGAP1^+/+ MEFs, transfected or not with Myc-IQGAP1. a.u., arbitrary units. (B) Area of spread of GFP-producing wild type (WT) or <i>ospB S</i>. <i>flexneri</i> complemented or not with <i>ospB</i> in IQGAP1^-/- or IQGAP1^+/+ cells. (C) Area of spread of GFP-producing WT or <i>ospB S</i>. <i>flexneri</i> in IQGAP1^-/- or IQGAP1^+/+ cells transfected with a plasmid carrying either <i>ospB gfp</i> or <i>gfp</i> alone. Mean ± S.D. Data represent three or more independent experiments. *, p < 0.05, Student’s two-tailed t test.</p

OspB activation of mTORC1.

<p>(A-B) Phosphorylation of mTOR substrate S6K in the presence of OspB and dependent on IQGAP1. Representative western blot (A) and band densitometry of phospho-S6K signal normalized to total S6K (B). (C) Inhibition by rapamycin of OspB-induced and IQGAP1-dependent phosphorylation of S6K. (D-E) Reduced phosphorylation of Akt on Thr-308 in the presence of OspB and dependent on IQGAP1. Representative western blot (D), and band densitometry of phospho-Akt Thr-308 signal normalized to total Akt (E). Apparent MWs are indicated in Kd. Densitometry is mean ± S.D. Data represent three or more independent experiments. *, p < 0.05, Student’s two-tailed t test.</p

OspB enhances cell proliferation dependent on IQGAP1 and inhibited by rapamycin.

<p>(A) Impact of insulin-like growth factor on area of spread of <i>S</i>. <i>flexneri</i> strains producing or not producing OspB. IGF, insulin-like growth factor. *, p < 0.05, Student’s two-tailed t test. (B) Relative cell density at the edge of infectious foci (normalized to monolayer background) for IQGAP1+/+ cells infected with the <i>ospB</i> mutant complemented or not with a plasmid expressing OspB. *, p < 0.05, Student’s one-tailed t test. (C) Proliferation rate of IQGAP1^+/+ and IQGAP1^-/- MEFs transiently transfected with OspB GFP or GFP alone. *, p < 0.05 compared with all other conditions at the same time point, 2-way ANOVA. (D) Saturation density of IQGAP1^+/+ and IQGAP1^-/- MEFs transiently transfected with OspB GFP or GFP alone. *, p < 0.05, Student’s two-tailed t test. (E) Proliferation rate of IQGAP1^+/+ MEFs transiently transfected with OspB GFP or GFP alone and treated with 10 nM rapamycin or DMSO carrier alone. *, **, p < 0.05 compared with cells transfected with GFP alone at the same time point; ***, p < 0.05 compared with cells treated with rapamycin at the same time point; 2-way ANOVA. (F) Representative images of cells on day 3 of experiment shown in panel E. Data represent mean ± S.D. of three or more independent experiments.</p