Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

The CD225 Domain of IFITM3 is Required for both IFITM Protein Association and Inhibition of Influenza A Virus and Dengue Virus Replication

IFITM3 is an interferon stimulated gene which inhibits the replication of multiple pathogenic viruses in vitro and in vivo. IFITM3 is a member of a large protein super family, whose members share a functionally-undefined area of high amino acid conservation, the CD225 domain. We performed mutational analyses of IFITM3 and identified multiple residues within the CD225 domain, consisting of the first intramembrane domain (IM1) and a conserved intracellular loop (CIL), that are required for restriction of both influenza A virus (IAV) and dengue virus (DENV) infection in vitro. Two phenylalanines within IM1 (F75 and F78) also mediate a physical association between IFITM proteins, and the loss of this interaction decreases IFITM3-mediated restriction. By extension, similar IM1-mediated associations may contribute to the functions of additional members of the CD225 domain family. IFITM3\u27s distal N-terminal domain is also needed for full anti-viral activity, including a tyrosine (Y20), whose alteration results in mislocalization of a portion of IFITM3 to the cell periphery and surface. Comparative analyses demonstrate that similar molecular determinants are needed for IFITM3\u27s restriction of both IAV and DENV. However, a portion of the CIL including Y99 and R87 is preferentially needed for inhibition of the orthomyxovirus. Several IFITM3 proteins engineered with rare single nucleotide polymorphisms demonstrated reduced expression or mislocalization, and these events were associated with enhanced viral replication in vitro, suggesting that possessing such alleles may impact an individual\u27s risk for viral infection. Based on this and other data, we propose a model for IFITM3-mediated restriction

Defining the range of pathogens susceptible to ifitm3 restriction using a knockout mouse model

The interferon-inducible transmembrane (IFITM) family of proteins has been shown to restrict a broad range of viruses in vitro and in vivo by halting progress through the late endosomal pathway. Further, single nucleotide polymorphisms (SNPs) in its sequence have been linked with risk of developing severe influenza virus infections in humans. The number of viruses restricted by this host protein has continued to grow since it was first demonstrated as playing an antiviral role; all of which enter cells via the endosomal pathway. We therefore sought to test the limits of antimicrobial restriction by Ifitm3 using a knockout mouse model. We showed that Ifitm3 does not impact on the restriction or pathogenesis of bacterial (Salmonella typhimurium, Citrobacter rodentium , Mycobacterium tuberculosis) or protozoan (Plasmodium berghei ) pathogens, despite in vitro evidence. However, Ifitm3 is capable of restricting respiratory syncytial virus (RSV) in vivo either through directly restricting RSV cell infection, or by exerting a previously uncharacterised function controlling disease pathogenesis. This represents the first demonstration of a virus that enters directly through the plasma membrane, without the need for the endosomal pathway, being restricted by the IFITM family; therefore further defining the role of these antiviral proteins

<i>Citrobacter rodentium</i> challenge of wild type and Ifitm3^-/- mice.

<p>Mice were orally infected with <i>C. rodentium</i> and weighed daily to monitor morbidity (<b>A</b>). Faecal samples were taken over the course of infection (<b>B</b>), and were homogenised, diluted and plated to count the number of colony forming units (CFU) shed over the course of the challenge. Mice were also killed on days 14 and 28 pi and CFU of <i>C. rodentium</i> were counted from caecal patch (<b>C</b>), caecum (<b>D</b>), colon (<b>E</b>), caecal contents (<b>F</b>), liver (<b>G</b>) and spleen (<b>H</b>). ■: wild type, □: Ifitm3^-/-. Results show means ± S.D. (n > 4).</p

Comparison of RSV infection in Ifitm3^-/- mice to wild type mice.

<p>Ifitm3^-/- or wild type littermate control mice were infected i.n. with 5x10⁵ RSV A2. Mice were weighed daily and weight changes recorded as a percentage of original weight (<b>A</b>). Lungs were excised and viral load calculated by qPCR on days 4 and 7 pi (<b>B</b>). Total cell counts from lung (<b>C</b>) and BAL (<b>D</b>) were calculated, along with totals of CD3, CD4 and CD8 (T cells), CD19 (B cells) and DX5+ (NK cells) (<b>E</b>) measured in lung by flow cytometry on day 7 post infection. Granulocyte numbers were also calculated in the BAL on day 7 post infection (F) Levels of the inflammatory cytokines IFNγ and IL-1β in lung (<b>G</b>) and IFNγ and IL-6 in BAL (H) were measured by ELISA on day 7 post infection. ■: wild type, □: Ifitm3^-/-. Results show means ± S.D. (n > 5). Statistical significance was assessed by Student’s <i>t</i>-test, or ANOVA followed by Bonferroni's Multiple Comparison Test when there were more than two groups (* <i>p</i><0.05, ** <i>p</i><0.01, *** p<0.001).</p

<i>Salmonella</i> Typhimurium challenge of wild type and Ifitm3^-/- mice.

<p>Mice were intravenously injected with <i>S</i>. Typhimurium and observed for weight loss for 28 days pi (<b>A</b>). Mice were killed on day 28 pi to assess neutralising antibody titre against <i>S</i>. Typhimurium (<b>B</b>). Bacterial contents from spleen (<b>C</b>), liver (<b>D</b>) and caecal contents (<b>E</b>) were titred on days 14 and 28 pi to assess the bacterial colonisation. ■: wild type, □: Ifitm3^-/-. Results show means ± S.D. (n > 3).</p

Malarial challenge of wild type and Ifitm3^-/- mice with <i>P. berghei</i> ANKA.

<p>Mice were intraperitoneally injected with red blood cells containing <i>P. berghei</i> ANKA and were monitored for survival for 12 days pi. n = 9 for wild type, n = 2 for each of Ifitm3^-/- and Ifngr^-/- mutants (<b>A</b>). Parasite biomass was monitored by measuring the activity of a luciferase reporter gene constitutively expressed by the parasite and expressed as relative light units (RLU) (<b>B</b>), and cytokine dysregulation was measured from the sera on day three pi by cytometric bead assay (<b>C</b>). ■: wild type, □: Ifitm3^-/-, ○: Ifngr^-/-. Results show means ± S.D. (n > 2).</p

Bacterial growth kinetics of <i>M. tuberculosis</i> in the lungs of wild type and Ifitm3^-/- mice.

<p>Mice were killed over the course of infection with H37Rv <i>M. tuberculosis</i> to determine the bacterial load within their lungs. ■: wild type, □: Ifitm3^-/-. Results show means ± S.D. (n > 5).</p

Expression of Ifitm3 at the predominant sites of pathogen infection.

<p>Paraffin-embedded sections from wild type and Ifitm3^-/- mice were cut and stained for expression of Ifitm3 (brown), and counterstained with hematoxylin (blue). Original magnification of lymph node and spleen 10×; lung and intestine 20×; liver 40×.</p