Treatment of W. bancrofti (Wb) in HIV/Wb Coinfections in South India

Background: The disease course of human immunodeficiency virus (HIV) is often altered by existing or newly acquired coincident infections. Methodology/Principal Findings: To assess the influence of pre-existing Wuchereria bancrofti infection on HIV progression, we performed a case-controlled treatment study of HIV positive individuals with (FIL+) or without (FIL-) W. bancrofti infection. Twenty-eight HIV+/FIL+ and 51 matched HIV+/FIL- subjects were treated with a single dose of diethylcarbamazine and albendazole (DEC/Alb) and followed for a year at regular intervals. Sixteen of the HIV+/FIL+ subjects (54%) and 28 of the HIV+/FIL- controls (57%) were on antiretroviral therapy (ART) during the study. Following treatment, no differences were noted in clinical outcomes between the 2 groups. There also was no significant difference between the groups in the HIV viral load at 12 months as a percentage of baseline viral load (HIV+/FIL+ group had on average 0.97 times the response of the HIV+/FIL- group, 95% CI 0.88, 1.07) between the groups. Furthermore, there were no significant differences found in either the change in viral load at 1, 3, or 6 months or in the change in CD4 count at 3, 6, or 12 months between the 2 groups. Conclusions/Significance: We were unable to find a significant effect of W. bancrofti infection or its treatment on HIV clinical course or surrogate markers of HIV disease progression though we recognized that our study was limited by the smaller than predicted sample size and by the use of ART in half of the patients. Treatment of W. bancrofti coinfection in HIV positive subjects (as is usual in mass drug administration campaigns) did not represent an increased risk to the subjects, and should therefore be considered for PLWHA living in W. bancrofti endemic areas

A site assessment tool for inpatient controlled human infection models for enteric disease pathogens

The use of the controlled human infection model to facilitate product development and to advance understanding of host-pathogen interactions is of increasing interest. While administering a virulent (or infective) organism to a susceptible host necessitates an ongoing evaluation of safety and ethical considerations, a central theme in conducting these studies in a safe and ethical manner that yields actionable data is their conduct in facilities well-suited to address their unique attributes. To that end, we have developed a framework for evaluating potential sites in which to conduct inpatient enteric controlled human infection model to ensure consistency and increase the likelihood of success.publishedVersio

The way forward for ETEC controlled human infection models (CHIMs)

In the absence of good animal models, Controlled Human Infection Models (CHIMs) are useful to assess efficacy of new vaccine candidates against Enterotoxic Escherichia coli (ETEC), as well as other preventive or therapeutic interventions. At the 2018 Vaccines Against Shigella and ETEC (VASE) conference, a workshop was held to further review and discuss new challenge model developments and key issues related to further model standardization. During the workshop, invited speakers briefly summarized for attendees recent developments and main agenda issues before workshop participants were divided into four groups for more focused discussions. The main issues discussed were: (1) whether there is a need for more ETEC strains to test a diversity of vaccine candidates, and if so, what criteria/qualities are desirable in strain selection; (2) how ETEC CHIMs could be more standardized to better support ETEC vaccine development; (3) how volunteer selection criteria and screening should be performed, and; (4) how an expanded sample collection schema and collaborative analysis plan may facilitate a more in-depth assessment of the role of antigen-specific humoral and cellular immune responses in ETEC infection, and provide better insights into ETEC pathogenesis and correlates of protection. The workshop concluded that additional challenge strains may need to be developed to better support new vaccines and therapeutics that are advancing in the development pipeline. In this regard, the need for a well characterized ST-only expressing ETEC strain was highlighted as a priority given that promising new heat stable toxoid based vaccine candidates are on the horizon. In addition, further standardization of the ETEC CHIMs was strongly encouraged, noting that it may not be realistic to standardize across all strains. Also, intensified volunteer screening may result in higher attack rates, although more stringent eligibility criteria may contribute to a more limited application of the model and diminish its representativeness. Finally, a sampling schedule and priority list for minimum set of samples was also proposed. Future workshops could be held to further refine standards for ETEC CHIMS and to facilitate more collaborative work on stored sample sets from previous and future ETEC CHIMs to maximize the contribution of these trials to our understanding of ETEC pathogenesis and our development of better prevention and control measures for this important pathogen

The way forward for ETEC controlled human infection models (CHIMs)

In the absence of good animal models, Controlled Human Infection Models (CHIMs) are useful to assess efficacy of new vaccine candidates against Enterotoxic Escherichia coli (ETEC), as well as other preventive or therapeutic interventions. At the 2018 Vaccines Against Shigella and ETEC (VASE) conference, a workshop was held to further review and discuss new challenge model developments and key issues related to further model standardization. During the workshop, invited speakers briefly summarized for attendees recent developments and main agenda issues before workshop participants were divided into four groups for more focused discussions. The main issues discussed were: (1) whether there is a need for more ETEC strains to test a diversity of vaccine candidates, and if so, what criteria/qualities are desirable in strain selection; (2) how ETEC CHIMs could be more standardized to better support ETEC vaccine development; (3) how volunteer selection criteria and screening should be performed, and; (4) how an expanded sample collection schema and collaborative analysis plan may facilitate a more in-depth assessment of the role of antigen-specific humoral and cellular immune responses in ETEC infection, and provide better insights into ETEC pathogenesis and correlates of protection. The workshop concluded that additional challenge strains may need to be developed to better support new vaccines and therapeutics that are advancing in the development pipeline. In this regard, the need for a well characterized ST-only expressing ETEC strain was highlighted as a priority given that promising new heat stable toxoid based vaccine candidates are on the horizon. In addition, further standardization of the ETEC CHIMs was strongly encouraged, noting that it may not be realistic to standardize across all strains. Also, intensified volunteer screening may result in higher attack rates, although more stringent eligibility criteria may contribute to a more limited application of the model and diminish its representativeness. Finally, a sampling schedule and priority list for minimum set of samples was also proposed. Future workshops could be held to further refine standards for ETEC CHIMS and to facilitate more collaborative work on stored sample sets from previous and future ETEC CHIMs to maximize the contribution of these trials to our understanding of ETEC pathogenesis and our development of better prevention and control measures for this important pathogen

Mycobacteria induce TPL-2 mediated IL-10 in IL-4-generated alternatively activated macrophages.

IL-4 drives expansion of Th2 cells that cause generation of alternatively activated macrophages (AAMs). Filarial infections are established early in life, induce increased IL-4 production are co-endemic with tuberculosis (TB). We sought to understand, therefore, how mycobacteria are handled in the context of IL-4-induced AAM. Comparing IL-4 generated in vitro monocyte derived human AAMs to LPS and IFN-γ generated classically macrophages (CAMs), both infected with mycobacteria (BCG), we demonstrated increased early BCG uptake and increased IL-10 production in AAMs compared to CAMs. We further demonstrated that increased IL-10 production is mediated by upregulation of tumor progression locus 2 (TPL-2), an upstream activator of extracellular signal related kinases (ERKs) in AAMs but not in CAMs, both at the transcript as well as the protein level. Pharmacologic inhibition of TPL-2 significantly diminished IL-10 production only in BCG-infected AAMs. Finally, we validated our findings in an in vivo C57Bl/6 model of filarial infection, where an exaggerated Th2 induced lung-specific alternative activation led to TPL-2 and IL-10 upregulation on subsequent TB infection. These data show that in response to mycobacterial infection, IL-4 generated AAMs in chronic filarial infections have impaired immune responses to TB infection by increasing IL-10 production in a TPL-2 mediated manner

AAMs compared to CAMs show decreased CCL13 expression and increased IL-10 production post BCG infection.

<p><b>Increased post-BCG IL-10 production was not seen in <i>B</i>. <i>malayi</i> microfilaria (mf) polarized macrophages. Panel a</b> shows change in relative mRNA expression of CCL13 from baseline to 24 hrs. post BCG infection expressed as 1/ΔCt in AAMs on the left panel and CAMs on the right panel. Individual lines represent each subject. <b>Panel b</b>: Net cytokine production (IL-6, IL-10, IL-12p40, IL-1α, IL-1β, and TNF-α) post BCG infection in pg/ml is compared between AAM and CAM conditions. Box and whisker plots represent median with 95% confidence intervals and individual dots representing each subject. <b>Panel c</b>: Net IL-10 production post BCG infection (measured in pg/ml) compared between mf polarized, mf+IL-4 polarized and CAMs. Box and whisker plots represent median with 95% confidence intervals and individual dots representing each subject.</p

Increased IL-10 production in AAMs but not CAMs in is mediated by TPL-2.

<p><b>Panel a</b>: Left panel (top and bottom) shows kinetics of net IL-10 production in AAMs and CAMs (in pg/ml) starting from pre BCG infection time point (pre-Inf) to 1, 4,6,18 and 24 hrs. post BCG infection. Middle panel shows relative mRNA expression of TPL-2 at the same time points, expressed as 1/ΔCt in AAMs and CAMs. Right panel shows relative mRNA expression of STAT3, expressed as 1/ΔCt in AAMs and CAMs. Vertical bars represent median relative expression Panel <b>b</b>: Whole cell extracts of AAMs and CAMs, generated at pre BCG infection and at 24hrs post infection were analyzed by immunoblotting with anti-TPL-2 antibodies utilizing anti-GAPDH as control (top panel). Bottom pane shows change in relative TPL-2 band density in AAMs vs. CAMs as assessed by ImageJ software comparing pre BCG infection time point (<b>Preinf</b>) with 24 hrs. post BCG infection (<b>24hr</b>). <b>Panel c</b>: Net IL-10 production (in pg/ml) is shown in AAMs and CAMs cultured with BCG alone for 24 hrs. or BCG and TPL-2 inhibitor (C₂₁H₁₄ClFN₆) at 500nM (IC⁵⁰). Individual dots representing each subject. A total of 12 subjects were analyzed for experiments in 4a, b and c.</p

AAMs show increased BCG uptake compared to CAMs using ImageStream^X.

<p><b>Panel a</b> shows sequential gating strategy for assessing bacterial internalization within the cell. Cells that were in the field of focus were defined using Gradient RMS vs. Area features; next, plotting symmetry vs. circularity distinguished macrophages from free bacteria. Gating was then performed on RFP positive cells to identify macrophages associated with bacteria. Delta Centroid feature was used to assess the distance between the center of the cell and the RFP labeled bacteria to assess internalization. <b>Panel b</b>: representative images showing reproducibility and accuracy of internalization assessment using the above gating strategy. Three different examples of RFP labeled bacteria internalized (left panel) within macrophages vs. remaining external (right panel) are shown. <b>Panel c</b>: Graph comparing median percentage of cells internalizing bacteria (for n = 12 donors) using above strategy in AAMs (grey column) vs. CAMs (black column).</p

At baseline AAMs have increased CD206 (Mannose receptor) expression compared to CAMs.

<p><b>Panel a</b>: Relative mRNA expression of previously defined AAM-specific marker (CCL13) expressed as 1/ΔCt, was compared between alternatively activated macrophages (AAMs) and classically activated macrophages (CAMs). Individual dots representing each subject. Horizontal bars represent median. <b>Panel b</b>: Representative plots showing CD206 (Mannose Receptor) expression (y-axis) compared on CD14+ cells (x-axis) between M-CSF,IL-4 (AAM) and LPS/IFN-γ (CAM) by flow cytometry <b>Panel c</b>:total cytokine production (IL-6, IL-10, IL-12p40, IL-1α, IL-1β, and tumor necrosis factor alpha [TNF-α]) in pg/ml is compared between AAM and CAM conditions. Cells were cultured with recombinant human IL-4 (rhIL-4; 50 ng/ml) for AAMs) or with LPS (1 μg/ml)/IFN-γ (20 ng/ml) for CAMs for 48 hours. Individual dots representing each subject. Horizontal bars represent median.</p