19 research outputs found
Analysis Of Host Factors Involved In Regulating Hiv-1-Induced Syncytium Formation
Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus and the causative agent of Acquired Immunodeficiency Syndrome (AIDS). HIV-1 can spread through multiple modes of transmission including cell-to-cell transmission between CD4+ T cells at a transient junction known as the virological synapse (VS). The VS forms upon HIV-1 Envelope (Env) on the surface of an infected (producer) cell binding CD4 on an uninfected (target) cell. While the VS typically resolves with complete cell separation and transfer of virus particles, Env can occasionally facilitate cell-cell fusion at this site, forming a multinucleated infected cell (syncytium). Excessive syncytium formation is prevented by viral and host factors, though this subpopulation of infected cells can still comprise ~20% of all infected cells in vivo. T cell-based syncytia detected in vivo are unique from mononucleated infected cells as they contain 2-4 nuclei, can have an elongated morphology, and appear highly motile. Despite such significant presence of syncytia, little is known about how these multinucleated infected entities contribute to HIV-1 spread and pathogenesis.
During cell-to-cell transmission at the VS, viral and host factors are enriched at this site to support virus spread (reviewed in Chapter 2). This thesis focused on fusion inhibitory factors HIV-1 Gag and several host proteins, including tetraspanins, ezrin, and EWI-2. We determined that EWI-2 is recruited specifically to the producer cell side of the VS (the presynapse) where it inhibits HIV-1-induced cell-cell fusion in a dose-dependent manner (Chapter 3). Although both EWI-2 and tetraspanins are typically downregulated upon infection, both tetraspanin CD81 and EWI-2 surface levels are partially restored on HIV-1-induced CD4+ primary T cell-based syncytia compared to mononucleated infected cells.
We sought to determine whether target cells influence the surface profile upon fusion and whether the altered protein levels are maintained for the lifetime of a syncytium (Chapter 4). We demonstrated that EWI-2 surface levels on syncytia correlate with levels of the target cell population, suggesting that EWI-2 brought along by target cells at least partially restores surface expression in syncytia. Further, we determined that newly formed, “young” syncytia, have higher levels of EWI-2 than older ones, suggesting that downregulation of EWI-2 continues in syncytia. We expect that higher levels of EWI-2 on young syncytia will render them less susceptible to continued cell-cell fusion than mononucleated infected cells and may also reduce virus particle infectivity. This will be tested by analysis of a purified syncytia population to measure fusogenicity and particle infectivity relative to fusogenicity and particle infectivity of mononucleated infected cells. Those data will be included in a future manuscript.
Collectively, the work presented in this dissertation has furthered our understanding of HIV-1-induced cell-cell fusion regulation and allowed us to characterize distinct differences in protein expression between syncytia and mononucleated infected cells. These findings open the door to future investigations aimed at understanding how syncytia contribute to virus transmission and pathogenesis
Characterizing EWI-2 mediated fusion inhibition at the HIV Presynapse
HIV can be as a cell free viral particle or through a virological synapse (VS), a highly efficient more of transmission. The VS is formed through cell-cell contact mediated by HIV envelope glycoprotein (Env) on the surface of an infected cell binding the viral receptor CD4 on the surface of an uninfected target cell. At first, it may seem likely that Env, because it is fusogenic at neutral pH, would likely facilitate frequent cell-cell fusion at the VS, thus resulting in the formation of multinucleated HIV infected cells (syncytia). However, while small, T cell-based syncytia are now recognized as a feature of early HIV infection, the majority of VSs ultimately resolve with complete cell separation. The VS is tightly regulated by both viral (Gag) and host proteins (ezrin, tetraspanins, and EWI-2). The viral structural protein, Gag, regulates fusion at the VS by binding and trapping Env in a non-fusogenic state. Cellular factors, including EWI-2 interacting partners, tetraspanins and ezrin, also contribute to efficient inhibition of syncytia formation at the HIV VS.
We have identified EWI-2, a known interacting partner of both tetraspanins and ezrin, as a member of the host fusion inhibitory complex partially responsible for efficiently preventing syncytia formation. Using microscopy and flow cytometry, we showed that EWI-2, while overall downregulated from the surface of HIV infected cells, co-accumulates with Gag at the producer cell side of the VS (i.e. the presynapse) to prevent cell-cell fusion. We now seek to determine the fusion inhibitory mechanism of EWI-2 by characterizing the domains required for EWI-2 mediated fusion inhibition, and whether tetraspanin-EWI-2 interactions are necessary for EWI-2 localization and fusion prevention at the HIV VS
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Efficacy of interferon beta-1a plus remdesivir compared with remdesivir alone in hospitalised adults with COVID-19: a double-bind, randomised, placebo-controlled, phase 3 trial.
BACKGROUND: Functional impairment of interferon, a natural antiviral component of the immune system, is associated with the pathogenesis and severity of COVID-19. We aimed to compare the efficacy of interferon beta-1a in combination with remdesivir compared with remdesivir alone in hospitalised patients with COVID-19.
METHODS: We did a double-blind, randomised, placebo-controlled trial at 63 hospitals across five countries (Japan, Mexico, Singapore, South Korea, and the USA). Eligible patients were hospitalised adults (aged ≥18 years) with SARS-CoV-2 infection, as confirmed by a positive RT-PCR test, and who met one of the following criteria suggestive of lower respiratory tract infection: the presence of radiographic infiltrates on imaging, a peripheral oxygen saturation on room air of 94% or less, or requiring supplemental oxygen. Patients were excluded if they had either an alanine aminotransferase or an aspartate aminotransferase concentration more than five times the upper limit of normal; had impaired renal function; were allergic to the study product; were pregnant or breast feeding; were already on mechanical ventilation; or were anticipating discharge from the hospital or transfer to another hospital within 72 h of enrolment. Patients were randomly assigned (1:1) to receive intravenous remdesivir as a 200 mg loading dose on day 1 followed by a 100 mg maintenance dose administered daily for up to 9 days and up to four doses of either 44 μg interferon beta-1a (interferon beta-1a group plus remdesivir group) or placebo (placebo plus remdesivir group) administered subcutaneously every other day. Randomisation was stratified by study site and disease severity at enrolment. Patients, investigators, and site staff were masked to interferon beta-1a and placebo treatment; remdesivir treatment was given to all patients without masking. The primary outcome was time to recovery, defined as the first day that a patient attained a category 1, 2, or 3 score on the eight-category ordinal scale within 28 days, assessed in the modified intention-to-treat population, defined as all randomised patients who were classified according to actual clinical severity. Safety was assessed in the as-treated population, defined as all patients who received at least one dose of the assigned treatment. This trial is registered with ClinicalTrials.gov, NCT04492475.
FINDINGS: Between Aug 5, 2020, and Nov 11, 2020, 969 patients were enrolled and randomly assigned to the interferon beta-1a plus remdesivir group (n=487) or to the placebo plus remdesivir group (n=482). The mean duration of symptoms before enrolment was 8·7 days (SD 4·4) in the interferon beta-1a plus remdesivir group and 8·5 days (SD 4·3) days in the placebo plus remdesivir group. Patients in both groups had a time to recovery of 5 days (95% CI not estimable) (rate ratio of interferon beta-1a plus remdesivir group vs placebo plus remdesivir 0·99 [95% CI 0·87-1·13]; p=0·88). The Kaplan-Meier estimate of mortality at 28 days was 5% (95% CI 3-7%) in the interferon beta-1a plus remdesivir group and 3% (2-6%) in the placebo plus remdesivir group (hazard ratio 1·33 [95% CI 0·69-2·55]; p=0·39). Patients who did not require high-flow oxygen at baseline were more likely to have at least one related adverse event in the interferon beta-1a plus remdesivir group (33 [7%] of 442 patients) than in the placebo plus remdesivir group (15 [3%] of 435). In patients who required high-flow oxygen at baseline, 24 (69%) of 35 had an adverse event and 21 (60%) had a serious adverse event in the interferon beta-1a plus remdesivir group compared with 13 (39%) of 33 who had an adverse event and eight (24%) who had a serious adverse event in the placebo plus remdesivir group.
INTERPRETATION: Interferon beta-1a plus remdesivir was not superior to remdesivir alone in hospitalised patients with COVID-19 pneumonia. Patients who required high-flow oxygen at baseline had worse outcomes after treatment with interferon beta-1a compared with those given placebo.
FUNDING: The National Institute of Allergy and Infectious Diseases (USA)
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Efficacy of interferon beta-1a plus remdesivir compared with remdesivir alone in hospitalised adults with COVID-19: a double-bind, randomised, placebo-controlled, phase 3 trial.
BackgroundFunctional impairment of interferon, a natural antiviral component of the immune system, is associated with the pathogenesis and severity of COVID-19. We aimed to compare the efficacy of interferon beta-1a in combination with remdesivir compared with remdesivir alone in hospitalised patients with COVID-19.MethodsWe did a double-blind, randomised, placebo-controlled trial at 63 hospitals across five countries (Japan, Mexico, Singapore, South Korea, and the USA). Eligible patients were hospitalised adults (aged ≥18 years) with SARS-CoV-2 infection, as confirmed by a positive RT-PCR test, and who met one of the following criteria suggestive of lower respiratory tract infection: the presence of radiographic infiltrates on imaging, a peripheral oxygen saturation on room air of 94% or less, or requiring supplemental oxygen. Patients were excluded if they had either an alanine aminotransferase or an aspartate aminotransferase concentration more than five times the upper limit of normal; had impaired renal function; were allergic to the study product; were pregnant or breast feeding; were already on mechanical ventilation; or were anticipating discharge from the hospital or transfer to another hospital within 72 h of enrolment. Patients were randomly assigned (1:1) to receive intravenous remdesivir as a 200 mg loading dose on day 1 followed by a 100 mg maintenance dose administered daily for up to 9 days and up to four doses of either 44 μg interferon beta-1a (interferon beta-1a group plus remdesivir group) or placebo (placebo plus remdesivir group) administered subcutaneously every other day. Randomisation was stratified by study site and disease severity at enrolment. Patients, investigators, and site staff were masked to interferon beta-1a and placebo treatment; remdesivir treatment was given to all patients without masking. The primary outcome was time to recovery, defined as the first day that a patient attained a category 1, 2, or 3 score on the eight-category ordinal scale within 28 days, assessed in the modified intention-to-treat population, defined as all randomised patients who were classified according to actual clinical severity. Safety was assessed in the as-treated population, defined as all patients who received at least one dose of the assigned treatment. This trial is registered with ClinicalTrials.gov, NCT04492475.FindingsBetween Aug 5, 2020, and Nov 11, 2020, 969 patients were enrolled and randomly assigned to the interferon beta-1a plus remdesivir group (n=487) or to the placebo plus remdesivir group (n=482). The mean duration of symptoms before enrolment was 8·7 days (SD 4·4) in the interferon beta-1a plus remdesivir group and 8·5 days (SD 4·3) days in the placebo plus remdesivir group. Patients in both groups had a time to recovery of 5 days (95% CI not estimable) (rate ratio of interferon beta-1a plus remdesivir group vs placebo plus remdesivir 0·99 [95% CI 0·87-1·13]; p=0·88). The Kaplan-Meier estimate of mortality at 28 days was 5% (95% CI 3-7%) in the interferon beta-1a plus remdesivir group and 3% (2-6%) in the placebo plus remdesivir group (hazard ratio 1·33 [95% CI 0·69-2·55]; p=0·39). Patients who did not require high-flow oxygen at baseline were more likely to have at least one related adverse event in the interferon beta-1a plus remdesivir group (33 [7%] of 442 patients) than in the placebo plus remdesivir group (15 [3%] of 435). In patients who required high-flow oxygen at baseline, 24 (69%) of 35 had an adverse event and 21 (60%) had a serious adverse event in the interferon beta-1a plus remdesivir group compared with 13 (39%) of 33 who had an adverse event and eight (24%) who had a serious adverse event in the placebo plus remdesivir group.InterpretationInterferon beta-1a plus remdesivir was not superior to remdesivir alone in hospitalised patients with COVID-19 pneumonia. Patients who required high-flow oxygen at baseline had worse outcomes after treatment with interferon beta-1a compared with those given placebo.FundingThe National Institute of Allergy and Infectious Diseases (USA)
Brainhack: Developing a culture of open, inclusive, community-driven neuroscience
Brainhack is an innovative meeting format that promotes scientific collaboration and education in an open, inclusive environment. This NeuroView describes the myriad benefits for participants and the research community and how Brainhacks complement conventional formats to augment scientific progress